Williams Hematology, 9E (MEDICAL/DENISTRY) [9 ed.] 0071833005, 9780071833004

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Williams

Hematology

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Notice Medicine is an ever-changing science. As new research and clinical experience broaden our knowledge, changes in treatment and drug therapy are required. The authors and the publisher of this work have checked with sources believed to be reliable in their efforts to provide information that is complete and generally in accord with the standards accepted at the time of publication. However, in view of the possibility of human error or changes in medical sciences, neither the authors nor the publisher nor any other party who has been involved in the preparation or publication of this work warrants that the information contained herein is in every respect accurate or complete, and they disclaim all responsibility for any errors or omissions or for the results obtained from use of the information contained in this work. Readers are encouraged to confirm the information contained herein with other sources. For example and in particular, readers are advised to check the product information sheet included in the package of each drug they plan to administer to be certain that the information contained in this work is accurate and that changes have not been made in the recommended dose or in the contraindications for administration. This recommendation is of particular importance in connection with new or infrequently used drugs.

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Williams

Hematology Ninth Edition

Kenneth Kaushansky, MD, MACP Senior Vice President for Health Sciences Dean, School of Medicine SUNY Distinguished Professor Stony Brook University Stony Brook, New York

Josef T. Prchal, MD

Professor of Medicine, Pathology, and Genetics Hematology Division University of Utah Salt Lake City, Utah Department of Pathophysiology First Faculty of Medicine Charles University in Prague Prague, Czech Republic

Oliver W. Press, MD, PhD

Acting Director, Clinical Research Division Dr. Penny E. Peterson Memorial Chair for Lymphoma Research Fred Hutchinson Cancer Research Center Professor of Medicine and Bioengineering University of Washington Seattle, Washington

Marshall A. Lichtman, MD

Professor of Medicine and of Biochemistry and Biophysics University of Rochester Medical Center Rochester, New York

Marcel Levi, MD, PhD

Professor of Medicine Dean, Faculty of Medicine Academic Medical Center University of Amsterdam Amsterdam, The Netherlands

Linda J. Burns, MD

Professor of Medicine Division of Hematology, Oncology and Transplantation University of Minnesota Minneapolis, Minnesota

Michael A. Caligiuri, MD

Director, Comprehensive Cancer Center CEO, James Cancer Hospital and Solove Research Institute Professor of Medicine The Ohio State University Columbus, Ohio

New York Chicago San Francisco Athens London Madrid Mexico City Milan New Delhi Singapore Sydney Toronto

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Copyright © 2016, by McGraw-Hill Education. All rights reserved. Except as permitted under the United States Copyright Act of 1976, no part of this publication may be reproduced or distributed in any form or by any means, or stored in a database or retrieval system, without the prior written permission of the publisher, with the exception that the program listings may be entered, stored, and executed in a computer system, but they may not be reproduced for publication. ISBN: 978-0-07-183301-1 MHID: 0-07-183301-3 The material in this eBook also appears in the print version of this title: ISBN: 978-0-07-183300-4, MHID: 0-07-183300-5. eBook conversion by codeMantra Version 1.0 All trademarks are trademarks of their respective owners. Rather than put a trademark symbol after every occurrence of a trademarked name, we use names in an editorial fashion only, and to the benefit of the trademark owner, with no intention of infringement of the trademark. Where such designations appear in this book, they have been printed with initial caps. McGraw-Hill Education eBooks are available at special quantity discounts to use as premiums and sales promotions or for use in corporate training programs. To contact a representative, please visit the Contact Us page at www.mhprofessional.com. TERMS OF USE This is a copyrighted work and McGraw-Hill Education and its licensors reserve all rights in and to the work. Use of this work is subject to these terms. Except as permitted under the Copyright Act of 1976 and the right to store and retrieve one copy of the work, you may not decompile, disassemble, reverse engineer, reproduce, modify, create derivative works based upon, transmit, distribute, disseminate, sell, publish or sublicense the work or any part of it without McGraw-Hill Education’s prior consent. You may use the work for your own noncommercial and personal use; any other use of the work is strictly prohibited. Your right to use the work may be terminated if you fail to comply with these terms. THE WORK IS PROVIDED “AS IS.” McGRAW-HILL EDUCATION AND ITS LICENSORS MAKE NO GUARANTEES OR WARRANTIES AS TO THE ACCURACY, ADEQUACY OR COMPLETENESS OF OR RESULTS TO BE OBTAINED FROM USING THE WORK, INCLUDING ANY INFORMATION THAT CAN BE ACCESSED THROUGH THE WORK VIA HYPERLINK OR OTHERWISE, AND EXPRESSLY DISCLAIM ANY WARRANTY, EXPRESS OR IMPLIED, INCLUDING BUT NOT LIMITED TO IMPLIED WARRANTIES OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. McGraw-Hill Education and its licensors do not warrant or guarantee that the functions contained in the work will meet your requirements or that its operation will be uninterrupted or error free. Neither McGraw-Hill Education nor its licensors shall be liable to you or anyone else for any inaccuracy, error or omission, regardless of cause, in the work or for any damages resulting therefrom. McGraw-Hill Education has no responsibility for the content of any information accessed through the work. Under no circumstances shall McGraw-Hill Education and/or its licensors be liable for any indirect, incidental, special, punitive, consequential or similar damages that result from the use of or inability to use the work, even if any of them has been advised of the possibility of such damages. This limitation of liability shall apply to any claim or cause whatsoever whether such claim or cause arises in contract, tort or otherwise.

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CONTENTS Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxi PART I Clinical Evaluation of the Patient 1. Initial Approach to the Patient: History and Physical Examination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Marshall A. Lichtman and Linda J. Burns

2. Examination of Blood Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Daniel H. Ryan

3. Examination of The Marrow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 Daniel H. Ryan

4. Consultative Hematology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 Rondeep S. Brar and Stanley L. Schrier

PART II The Organization of the Lymphohematopoietic Tissues 5. Structure of the Marrow and the Hematopoietic Microenvironment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 Utpal P. Davé and Mark J. Koury

6. The Organization and Structure of Lymphoid Tissues . . . . . . . . 85 Aharon G. Freud and Michael A. Caligiuri

16. Cell-Cycle Regulation and Hematologic Disorders . . . . . . . . . 213 Yun Dai, Prithviraj Bose, and Steven Grant

17. Signal Transduction Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . 247 Kenneth Kaushansky

18. Hematopoietic Stem Cells, Progenitors, and Cytokines . . . . . . 257 Kenneth Kaushansky

19. The Inflammatory Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279 Jeffrey S. Warren and Peter A. Ward

20. Innate Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293 Bruce Beutler

21. Dendritic Cells and Adaptive Immunity . . . . . . . . . . . . . . . . . . 307 Madhav Dhodapkar, Crystal L. Mackall, and Ralph M. Steinman

PART V Therapeutic Principles 22. Pharmacology and Toxicity of Antineoplastic Drugs . . . . . . . . 315 Benjamin Izar, Dustin Dzube, James M. Cleary, Constantine S. Mitsiades, Paul G. Richardson, Jeffrey A. Barnes, and Bruce A. Chabner

23. Hematopoietic Cell Transplantation . . . . . . . . . . . . . . . . . . . . . . 353 Andrew R. Rezvani, Robert Lowsky, and Robert S. Negrin

24. Treatment of Infections in The Immunocompromised Host . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383 Lisa Beutler and Jennifer Babik

PART III Epochal Hematology

25. Antithrombotic Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393 Gregory C. Connolly and Charles W. Francis

7. Hematology of the Fetus and Newborn . . . . . . . . . . . . . . . . . . . . 99

26. Immune Cell Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 409

8. Hematology during Pregnancy . . . . . . . . . . . . . . . . . . . . . . . . . . 119

27. Vaccine Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 421

9. Hematology in Older Persons . . . . . . . . . . . . . . . . . . . . . . . . . . . 129

28. Therapeutic Apheresis: Indications, Efficacy, and Complications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 427

James Palis and George B. Segel Martha P. Mims

William B. Ershler, Andrew S. Artz, and Bindu Kanapuru

PART IV Molecular and Cellular Hematology 10. Genetic Principles and Molecular Biology . . . . . . . . . . . . . . . . . 145

Lynn B. Jorde

11. Genomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155

Carolina Berger and Stanley R. Riddell

Katayoun Rezvani and Jeffrey J. Molldrem

Robert Weinstein

29. Gene Therapy for Hematologic Diseases . . . . . . . . . . . . . . . . . . 437 Hua Fung and Stanton Gerson

30. Regenerative Medicine: Multipotential Cell Therapy for Tissue Repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 447 Jakub Tolar, Mark J Osborn, Randy Daughters, Anannya Banga, and John Wagner

Lukas D. Wartman and Elaine R. Mardis

12. Epigenetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165 Bradley R. Cairns

13. Cytogenetics and Genetic Abnormalities . . . . . . . . . . . . . . . . . . 173 Lucy A. Godley, Madina Sukhanova, Gordana Raca, and Michelle M. Le Beau

14. Metabolism of Hematologic Neoplastic Cells . . . . . . . . . . . . . . 191 Zandra E. Walton, Annie L. Hsieh, and Chi V. Dang

15. Apoptosis Mechanisms: Relevance to the Hematopoietic System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203 John C. Reed

PART VI The Erythrocyte 31. Structure and Composition of the Erythrocyte . . . . . . . . . . . . 461 Narla Mohandas

32. Erythropoiesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 479 Josef T. Prchal and Perumal Thiagarajan

33. Erythrocyte Turnover . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 495 Perumal Thiagarajan and Josef Prchal

34. Clinical Manifestations and Classification of Erythrocyte Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 503 Josef T. Prchal

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Contents

35. Aplastic Anemia: Acquired and Inherited . . . . . . . . . . . . . . . . . 513 George B. Segel and Marshall A. Lichtman

36. Pure Red Cell Aplasia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 539 Neal S. Young

37. Anemia of Chronic Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 549 Tomas Ganz 38. Erythropoietic Effects of Endocrine Disorders . . . . . . . . . . . . . 559 Xylina T. Gregg 39. The Congenital Dyserythropoietic Anemias . . . . . . . . . . . . . . . 563 Achille Iolascon

40. Paroxysmal Nocturnal Hemoglobinuria . . . . . . . . . . . . . . . . . . . 571 Charles J. Parker

41. Folate, Cobalamin, and Megaloblastic Anemias . . . . . . . . . . . . 583 Ralph Green

42. Iron Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 617 Tomas Ganz

43. Iron Deficiency and Overload . . . . . . . . . . . . . . . . . . . . . . . . . . . 627 Tomas Ganz

44. Anemia Resulting from Other Nutritional Deficiencies . . . . . 651 Ralph Green

45. Anemia Associated with Marrow Infiltration . . . . . . . . . . . . . . 657 Vishnu VB Reddy and Josef T. Prchal

46. Erythrocyte Membrane Disorders . . . . . . . . . . . . . . . . . . . . . . . . 661 Theresa L Coetzer

47. Erythrocyte Enzyme Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . 689 Wouter W. van Solinge and Richard van Wijk

48. The Thalassemias: Disorders of Globin Synthesis . . . . . . . . . . . 725 David J. Weatherall

49. Disorders of Hemoglobin Structure: Sickle Cell Anemia and Related Abnormalities . . . . . . . . . . . . . . . . . . . . . . 759 Kavita Natrajan and Abdullah Kutlar

50. Methemoglobinemia and Other Dyshemoglobinemias . . . . . . 789 Archana M. Agarwal and Josef T. Prchal

51. Fragmentation Hemolytic Anemia . . . . . . . . . . . . . . . . . . . . . . . 801 Kelty R. Baker and Joel Moake

52. Erythrocyte Disorders as a Result of Chemical and Physical Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 809 Paul C. Herrmann

53. Hemolytic Anemia Resulting from Infections with Microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 815 Marshall A. Lichtman

PART VII Neutrophils, Eosinophils, Basophils, and Mast Cells 60. Structure and Composition of Neutrophils, Eosinophils, and Basophils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 925 C. Wayne Smith

61. Production, Distribution, and Fate of Neutrophils . . . . . . . . . . 939 C. Wayne Smith

62. Eosinophils and Related Disorders . . . . . . . . . . . . . . . . . . . . . . . 947 Andrew J. Wardlaw

63. Basophils, Mast Cells, and Related Disorders . . . . . . . . . . . . . . 965 Stephen J. Galli, Dean D. Metcalfe, Daniel A. Arber, and Ann M. Dvorak

64. Classification and Clinical Manifestations of Neutrophil Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 983 Marshall A. Lichtman

65. Neutropenia and Neutrophilia . . . . . . . . . . . . . . . . . . . . . . . . . . . 991 David C. Dale and Karl Welte

66. Disorders of Neutrophil Function . . . . . . . . . . . . . . . . . . . . . . . 1005 Niels Borregaard

PART VIII Monocytes and Macrophages 67. Structure, Receptors, and Functions of Monocytes and Macrophages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1045 Steven D. Douglas and Anne G. Douglas

68. Production, Distribution, and Activation of Monocytes and Macrophages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1075 Steven D. Douglas and Anne G. Douglas

69. Classification and Clinical Manifestations of Disorders of Monocytes and Macrophages . . . . . . . . . . . . . . . . . . . . . . . . 1089 Marshall A. Lichtman

70. Monocytosis and Monocytopenia . . . . . . . . . . . . . . . . . . . . . . . 1095 Marshall A. Lichtman

71. Inflammatory and Malignant Histiocytosis . . . . . . . . . . . . . . . 1101 Kenneth L. McClain and Carl E. Allen

72. Gaucher Disease and Related Lysosomal Storage Diseases . . 1121 Ari Zimran and Deborah Elstein

PART IX Lymphocytes and Plasma Cells 73. The Structure of Lymphocytes and Plasma Cells . . . . . . . . . . . 1137

54. Hemolytic Anemia Resulting from Immune Injury . . . . . . . . . 823

Natarajan Muthusamy and Michael A. Caligiuri

55. Alloimmune Hemolytic Disease of the Fetus and Newborn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 847

Christopher S. Seet and Gay M. Crooks

56. Hypersplenism and Hyposplenism . . . . . . . . . . . . . . . . . . . . . . . 863

Thomas J. Kipps

Charles H. Packman

Ross M. Fasano, Jeanne E. Hendrickson, and Naomi L. C. Luban

74. Lymphopoiesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1149 75. Functions of B Lymphocytes and Plasma Cells in Immunoglobulin Production . . . . . . . . . . . . . . . . . . . . . . . . . . . 1159

57. Primary and Secondary Erythrocytoses . . . . . . . . . . . . . . . . . . . 871

76. Functions of T Lymphocytes: T-Cell Receptors for Antigen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1175

58. The Porphyrias . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 889

77. Functions of Natural Killer Cells . . . . . . . . . . . . . . . . . . . . . . . . 1189

59. Polyclonal and Hereditary Sideroblastic Anemias . . . . . . . . . . 915

78. Classification and Clinical Manifestations of Lymphocyte and Plasma Cell Disorders . . . . . . . . . . . . . . . . . . 1195 Yvonne A. Efebera and Michael A. Caligiuri

Jaime Caro and Srikanth Nagalla Josef T. Prchal

John D. Phillips and Karl E. Anderson Prem Ponka and Josef T. Prchal

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Giorgio Trinchieri, Richard W. Childs, and Lewis L. Lanier

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79. Lymphocytosis and Lymphocytopenia . . . . . . . . . . . . . . . . . . 1199

101. Marginal Zone B-Cell Lymphomas . . . . . . . . . . . . . . . . . . . . . . 1663

80. Immunodeficiency Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1211

102. Burkitt Lymphoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1671

81. Hematologic Manifestations of Acquired Immunodeficiency Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1239

103. Cutaneous T-Cell Lymphoma (Mycosis Fungoides and Sézary Syndrome) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1679

82. Mononucleosis Syndromes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1261

104. Mature T-Cell and Natural Killer Cell Lymphomas . . . . . . . . 1693

Sumithira Vasu and Michael A. Caligiuri Hans D. Ochs and Luigi D. Notarangelo

Virginia C. Broudy, Robert D. Harrington Robert F. Betts

PART X Malignant Myeloid Diseases 83. Classification and Clinical Manifestations of the Clonal Myeloid Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1275 Marshall A. Lichtman

84. Polycythemia Vera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1291 Jaroslav F. Prchal and Josef T. Prchal

85. Essential Thrombocythemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1307

Pier Luigi Zinzani and Alessandro Broccoli

Andrew G. Evans and Jonathan W. Friedberg

Larisa J. Geskin

Neha Mehta, Alison Moskowitz, and Steven Horwitz

105. Plasma Cell Neoplasms: General Considerations . . . . . . . . . . 1707 Guido Tricot, Siegfried Janz, Kalyan Nadiminti, Erik Wendlandt, and Fenghuang Zhan

106. Essential Monoclonal Gammopathy . . . . . . . . . . . . . . . . . . . . . 1721 Marshall A. Lichtman

107. Myeloma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1733 Elizabeth O’Donnell, Francesca Cottini, Noopur Raje, and Kenneth Anderson

108. Immunoglobulin Light-Chain Amyloidosis . . . . . . . . . . . . . . . 1773

Philip A. Beer and Anthony R. Green

Morie A. Gertz, Taimur Sher, Angela Dispenzieri, and Francis K. Buadi

86. Primary Myelofibrosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1319

109. Macroglobulinemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1785

Marshall A. Lichtman and Josef T. Prchal

87. Myelodysplastic Syndromes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1341 Rafael Bejar and David P. Steensma

88. Acute Myelogenous Leukemia . . . . . . . . . . . . . . . . . . . . . . . . . . 1373 Jane L. Liesveld and Marshall A. Lichtman

89. Chronic Myelogenous Leukemia and Related Disorders . . . . 1437 Jane L. Liesveld and Marshall A. Lichtman

PART XI Malignant Lymphoid Diseases

Steven P. Treon, Jorge J. Castillo, Zachary R. Hunter, and Giampaolo Merlini

110. Heavy-Chain Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1803 Dietlind L. Wahner-Roedler and Robert A. Kyle

PART XII Hemostasis and Thrombosis 111. Megakaryopoiesis and Thrombopoiesis . . . . . . . . . . . . . . . . . . 1815 Kenneth Kaushansky

112. Platelet Morphology, Biochemistry, and Function . . . . . . . . . 1829

90. Classification of Malignant Lymphoid Disorders . . . . . . . . . . 1493

Susan S. Smyth, Sidney Whiteheart, Joseph E. Italiano Jr., Paul Bray, and Barry S. Coller

91. Acute Lymphoblastic Leukemia . . . . . . . . . . . . . . . . . . . . . . . . . 1505

113. Molecular Biology and Biochemistry of the Coagulation Factors and Pathways of Hemostasis . . . . . . . . . 1915

Robert A. Baiocchi Richard A. Larson

92. Chronic Lymphocytic Leukemia . . . . . . . . . . . . . . . . . . . . . . . . 1527 Farrukh T. Awan and John C. Byrd

93. Hairy Cell Leukemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1553 Michael R. Grever and Gerard Lozanski

Mettine H. A. Bos, Cornelis van ‘t Veer, and Pieter H. Reitsma

114. Control of Coagulation Reactions . . . . . . . . . . . . . . . . . . . . . . . 1949  Laurent O. Mosnier and John H. Griffin

115. Vascular Function in Hemostasis . . . . . . . . . . . . . . . . . . . . . . . 1967

94. Large Granular Lymphocytic Leukemia . . . . . . . . . . . . . . . . . . 1563

Katherine A. Hajjar, Aaron J. Marcus, and William Muller

95. General Considerations for Lymphomas: Epidemiology, Etiology, Heterogeneity, and Primary Extranodal Disease . . 1569

Marcel Levi, Uri Seligsohn, and Kenneth Kaushansky

Pierluigi Porcu and Aharon G. Freud

Oliver W. Press and Marshall A. Lichtman

116. Classification, Clinical Manifestations, and Evaluation of Disorders of Hemostasis . . . . . . . . . . . . . . . . . . . 1985 117. Thrombocytopenia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1993

96. Pathology of Lymphomas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1587

Reyhan Diz-Küçükkaya and José A. López

97. Hodgkin Lymphoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1603

Adam Cuker and Mortimer Poncz

98. Diffuse Large B-Cell Lymphoma and Related Diseases . . . . . 1625

Kenneth Kaushansky

99. Follicular Lymphoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1641

A. Koneti Rao and Barry S. Coller

100. Mantle Cell Lymphoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1653

Charles S. Abrams, Sanford J. Shattil, and Joel S. Bennett

Randy D. Gascoyne and Brian F. Skinnider Oliver W. Press

Stephen D. Smith and Oliver W. Press Oliver W. Press

Martin Dreyling

118. Heparin-Induced Thrombocytopenia . . . . . . . . . . . . . . . . . . . . 2025 119. Reactive Thrombocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2035 120. Hereditary Qualitative Platelet Disorders . . . . . . . . . . . . . . . . . 2039 121. Acquired Qualitative Platelet Disorders . . . . . . . . . . . . . . . . . . 2073 122. The Vascular Purpuras . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2097 Doru T. Alexandrescu and Marcel Levi

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Contents

123. Hemophilia A and Hemophilia B . . . . . . . . . . . . . . . . . . . . . . . 2113

133. Venous Thrombosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2267

124. Inherited Deficiencies of Coagulation Factors II, V, V+VIII, VII, X, XI, and XIII . . . . . . . . . . . . . . . . . . . . . . . . . . . 2133

134. Atherothrombosis: Disease Initiation, Progression, and Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2281

125. Hereditary Fibrinogen Abnormalities . . . . . . . . . . . . . . . . . . . . 2151

135. Fibrinolysis and Thrombolysis . . . . . . . . . . . . . . . . . . . . . . . . . . 2303

Miguel A. Escobar and Nigel S. Key

Flora Peyvandi and Marzia Menegatti

Marguerite Neerman-Arbez and Philippe de Moerloose

126. von Willebrand Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2163 Jill Johnsen and David Ginsburg

127. Antibody-Mediated Coagulation Factor Deficiencies . . . . . . 2183 Sean R. Stowell, John S. (Pete) Lollar, and Shannon L. Meeks

128. Hemostatic Alterations in Liver Disease and Liver Transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2191 Frank W.G. Leebeek and Ton Lisman

129. Disseminated Intravascular Coagulation . . . . . . . . . . . . . . . . . 2199 Marcel Levi and Uri Seligsohn

130. Hereditary Thrombophilia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2221 Saskia Middeldorp and Michiel Coppens

131. The Antiphospholipid Syndrome . . . . . . . . . . . . . . . . . . . . . . . . 2233 Jacob H. Rand and Lucia Wolgast

Gary E. Raskob, Russell D. Hull, and Harry R. Buller

Emile R. Mohler III and Andrew I. Schafer Katherine A. Hajjar and Jia Ruan

PART XIII Transfusion Medicine 136. Erythrocyte Antigens and Antibodies . . . . . . . . . . . . . . . . . . . . 2329 Marion E. Reid and Christine Lomas-Francis

137. Human Leukocyte and Platelet Antigens . . . . . . . . . . . . . . . . . 2353 Myra Coppage, David Stroncek, Janice McFarland, and Neil Blumberg

138. Blood Procurement and Red Cell Transfusion . . . . . . . . . . . . 2365 Jeffrey McCullough, Majed A. Refaai, and Claudia S. Cohn

139. Preservation and Clinical Use of Platelets . . . . . . . . . . . . . . . . 2381 Terry Gernsheimer and Sherrill Slichter

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2393

132. Thrombotic Microangiopathies . . . . . . . . . . . . . . . . . . . . . . . . . 2253 J. Evan Sadler

Kaushansky_FM_pi_xxii.indd 8

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ix ix

CONTRIBUTORS Charles S. Abrams, MD [121]

Professor of Medicine, Pathology and Laboratory Medicine Vice Chair for Research & Chief Scientific Officer Department of Medicine University of Pennsylvania School of Medicine Philadelphia, Pennsylvania

Archana M. Agarwal, MD [50]

Department of Pathology University of Utah/ARUP Laboratories Salt Lake City, Utah

Doru T. Alexandrescu, MD [122]

Jennifer Babik, MD, PhD [24] Division of Infectious Diseases Department of Medicine University of California San Francisco, California

Robert A. Baiocchi, MD, PhD [90] Associate Professor of Medicine Division of Hematology Department of Internal Medicine The Ohio State University Columbus, Ohio

Department of Medicine Division of Dermatology University of California, San Diego VA San Diego Health Care System San Diego, California

Kelty R. Baker, MD [51]

Carl E. Allen, MD, PhD [71]

Associate Professor of Pediatrics Texas Children’s Cancer Center/Hematology Baylor College of Medicine Houston, Texas

Assistant Professor Department of Genetics Cell Biology, and Development, Stem Cell Institute University of Minnesota Minneapolis, Minnesota

Karl E. Anderson, MD, FACP [58]

Jeffrey A. Barnes [22]

Kenneth Anderson, MD [107]

Philip A. Beer, MRCP, FRCPath, PhD [85]

Professor, Departments of Preventative Medicine and Community Health, Internal Medicine, and Pharmacology and Toxicology University of Texas Medical Branch Galveston, Texas

Dana-Farber Cancer Institute Boston, Massachusetts

Daniel A. Arber, MD [63]

Clinical Assistant Professor Baylor College of Medicine Houston, Texas

Anannya Banga, PhD, [30]

Instructor in Medicine Dana-Farber Cancer Institute Harvard Medical School Boston, Massachusetts Wellcome Trust Sanger Institute Wellcome Trust Genome Campus, Hinxton Cambridge, United Kingdom

Ronald F. Dorfman, MBBch, FRCPath Professor in Hematopathology Professor of Pathology Stanford University School of Medicine Stanford University Medical Center Stanford, California

Rafael Bejar, MD, PhD [87]

Andrew S. Artz, MD, MS [9]

Joel S. Bennett, MD [121]

Associate Professor of Medicine University of Chicago Chicago, Illinois

Farrukh T. Awan, MD [92]

Associate Professor of Internal Medicine Division of Hematology Department of internal Medicine The Ohio State University Comprehensive Cancer Center Columbus, Ohio

Kaushansky_FM_pi_xxii.indd 9

Division of Hematology and Oncology Moores Cancer Center University of California San Diego La Jolla, California Professor of Medicine Division of Hematology-Oncology University of Pennsylvania School of Medicine Philadelphia, Pennsylvania

Carolina Berger, MD [26]

Fred Hutchinson Cancer Research Center Seattle, Washington

9/21/15 4:40 PM

x

Contributors

Robert F. Betts, MD [82]

Professor of Medicine, Emeritus Division of Infectious Diseases University of Rochester Medical Center Rochester, New York

Bruce Beutler, MD [20]

Regental Professor and Director Center for the Genetics of Host Defense Raymond and Ellen Willie Distinguished Chair in Cancer Research in Honor of Laverne and Raymond Willie Sr. University of Texas Southwestern Medical Center Dallas, Texas

Lisa Beutler, MD, PhD [24] Department of Medicine UCSF School of Medicine San Francisco, California

Francis K. Buadi, MD [108] Division of Hematology Mayo Clinic Rochester, Minnesota

Harry R. Buller, MD [133]

Professor of Medicine, Department of Vascular Medicine Academic Medical Center Amsterdam, The Netherlands

Linda J. Burns, MD [1]

National Marrow Donor Program/Be The Match Vice President and Medical Director Health Services Research Minneapolis, Minnosata

John C. Byrd, MD [92]

Professor and Director, Clinical Laboratories and Transfusion Medicine Department of Pathology and Laboratory Medicine University of Rochester Rochester, New York

D. Warren Brown Chair of Leukemia Research Professor of Medicine, Medicinal Chemistry, and Veterinary Biosciences Director, Division of Hematology Department of Medicine The Ohio State University Columbus, Ohio

Niels Borregaard, MD, PhD [66]

Bradley R. Cairns, PhD [12]

Neil Blumberg, MD [137]

Professor of Hematology Department of Hematology University of Copenhagen Copenhagen, Denmark

Prithviraj Bose, MD [16]

Assistant Professor Department of Leukemia University of Texas MD Anderson Cancer Center Houston, Texas

Rondeep S. Brar, MD [4]

Clinical Assistant Professor of Medicine (Hematology and Oncology) Stanford University School of Medicine Stanford, California

Paul Bray, MD [112]

Professor Director, Division of Hematology Jefferson University Philadelphia, Pennsylvania

Alessandro Broccoli, MD [101]

Howard Hughes Medical Institute Professor and Chair Department of Oncological Sciences Huntsman Cancer Institute University of Utah School of Medicine Salt Lake City, Utah

Michael A. Caligiuri, MD [6, 73, 78, 79]

Professor and Director, The Ohio State University Comprehensive Cancer Center CEO, James Cancer Hospital and Solove Research Institute The Ohio State University Columbus, Ohio

Jaime Caro, MD [56]

Professor of Medicine Department of Medicine Thomas Jefferson University Cardeza Foundation for Hematologic Research Philadelphia, Pennsylvania

Jorge J. Castillo, MD [109]

Institute of Hematology “L. e A. Seràgnoli” University of Bologna Bologna, Italy

Assistant Professor of Medicine Dana-Farber Cancer Institute Harvard Medical School Boston, Massachusetts

Virginia C. Broudy, MD [81]

Bruce A. Chabner, MD [22]

Professor of Medicine Scripps Professor of Hematology University of Washington Seattle, Washington

Kaushansky_FM_pi_xxii.indd 10

Professor of Medicine Massachusetts General Hospital Cancer Center Harvard Medical School Boston, Massachusetts

9/21/15 4:40 PM

Contributors

Richard W. Childs, MD [77]

Adam Cuker, MD, MS [118]

James M. Cleary, MD, PhD [22]

Yun Dai, MD [16]

Clinical Director, NHLBI Chief, Section of Transplantation Immunotherapy National Heart, Lung, and Blood Institute, NIH Bethesda, Maryland Instructor in Medicine Dana-Farber Cancer Institute Harvard Medical School Boston, Massachusetts

Theresa L Coetzer, MD [46]

Assistant Professor of Medicine & of Pathology and Laboratory Medicine Perelman School of Medicine at the University of Pennsylvania Philadelphia, Pennsylvania Associate Professor of Medicine Department of Medicine Massey Cancer Center Virginia Commonwealth University Richmond, Virginia

Head: Red Cell Membrane Unit Department of Molecular Medicine and Haematology National Health Laboratory Service University of the Witwatersrand Wits Medical School Johannesburg, South Africa

David C. Dale, MD [65]

Claudia S. Cohn, MD [138]

Assistant Professor, Laboratory Medicine and Pathology University of Minnesota Minneapolis, Minnesota

Professor and Director Abramson Cancer Center University of Pennsylvania Philadelphia, Pennsylvania

Barry S. Coller, MD [112, 120]

Utpal P. Davé, MD [5]

Head Allen and Frances Adler Laboratory of Blood and Vascular Biology Physician-in-Chief Vice President for Medical Affairs The Rockefeller University New York, New York

Gregory C. Connolly, MD [25] Department of Medicine Lipson Cancer Center Rochester Regional Health System Rochester, New York

Myra Coppage [137]

Associate Professor of Laboratory Medicine Department of Pathology and Laboratory Medicine University of Rochester Rochester, New York

Michiel Coppens, MD, PhD [130] Department of Vascular Medicine Academic Medical Center Amsterdam, The Netherlands

Francesca Cottini, MD [107] Dana-Farber Cancer Institute Boston, Massachusetts

Gay M. Crooks, MB, BS, FRACP [74]

Professor Departments of Pathology & Laboratory Medicine and Pediatrics David Geffen School of Medicine University of California, Los Angeles Los Angeles, California

Kaushansky_FM_pi_xxii.indd 11

xi

Professor of Medicine Department of Medicine University of Washington Seattle, Washington

Chi V. Dang, MD, PhD [14]

Division of Hematology/Oncology Department of Medicine Vanderbilt University Medical Center Nashville, Tennessee

Randy Daughters, PhD [30]

Assistant Professor Department of Genetics Cell Biology, and Development, Stem Cell Institute University of Minnesota Minneapolis, Minnesota

Philippe de Moerloose, MD [125]

Professor Division of Angiology and Haemostasis University of Geneva Faculty of Medicine Geneva, Switzerland

Madhav Dhodapkar, MBBS [21]

Arthur H. and Isabel Bunker Professor of Medicine (Hematology) and Professor of Immunobiology Chief, Section of Hematology, Department of Internal Medicine Clinical Research Program Leader, Hematology Program Yale Cancer Center New Haven, Connecticut

Angela Dispenzieri, MD [108] Division of Hematology Mayo Clinic Rochester, Minnesota

9/21/15 4:40 PM

xii

Contributors

Reyhan Diz-Küçükkaya, MD [117] Associate Professor Department of Internal Medicine Division of Hematology Istanbul University Istanbul Faculty of Medicine Istanbul, Turkey

Anne G. Douglas, BA [67, 68]

Student, Perelman School of Medicine University of Pennsylvania (Class of 2017) Philadelphia, Pennsylvania

Steven D. Douglas, MD [67, 68] Professor and Associate Chair Department of Pediatrics Perelman School of Medicine University of Pennsylvania Children’s Hospital of Philadelphia Philadelphia, Pennsylvania

Martin Dreyling, MD [100]

Andrew G. Evans, MD, PhD [102]

Assistant Professor Department of Pathology and Laboratory Medicine University of Rochester Medical Center Rochester, New York

Ross M. Fasano, MD [55]

Assistant Professor Emory University School of Medicine Departments of Pathology and Pediatric Hematology Assistant Director, Children’s Healthcare of Atlanta Transfusion Services Associate Director, Grady Health System Transfusion Service Atlanta, Georgia

Charles W. Francis, MD [25]

Hematology/Oncology Division University of Rochester Medical Center Rochester, New York

Aharon G. Freud, MD, PhD [6, 94]

Department of Internal Medicine III Medical Center of the University of Munich Munich, Germany

Assistant Professor Department of Pathology The Ohio State University Columbus, Ohio

Ann M. Dvorak, MD [63]

Jonathan W. Friedberg, MD [102]

Senior Pathologist, Professor of Pathology Department of Pathology Beth Israel Deaconess Medical Center Harvard Medical School Boston, Massachusetts

Dustin Dzube, MD [22]

Resident Physician Massachusetts General Hospital Harvard Medical School Boston, Massachusetts

Yvonne A. Efebera, MD, MPH [78]

Associate Professor of Internal Medicine Division of Hematology Department of Internal Medicine The Ohio State University Columbus, Ohio

Deborah Elstein, PhD [72]

Samuel Durand Professor of Medicine Director, Wilmot Cancer Institute University of Rochester Medical Center Rochester, New York

Hua Fung, MD [29]

Case Western Reserve University University Hospital of Cleveland Cleveland, Ohio

Stephen J. Galli, MD [63]

Mary Hewitt Loveless, MD, Professor Professor of Pathology and of Microbiology and Immunology Chair, Department of Pathology Stanford University School of Medicine Stanford University Medical Center Stanford, California

Tomas Ganz, MD, PhD [37, 42, 43]

Gaucher Clinic Shaare Zedek Medical Center Jerusalem, Israel

Departments of Medicine and Pathology, David Geffen School of Medicine University of California, Los Angeles Los Angeles, California

William B. Ershler, MD [9]

Randy D. Gascoyne, MD, FRCPC [96]

Scientific Director Institute for Advanced Studies in Aging and Geriatrics Falls Church, Virginia

Miguel A. Escobar, MD [123]

Professor of Medicine and Pediatrics Division of Hematology University of Texas Health Science Center at Houston Director, Gulf States Hemophilia and Thrombophilia Center Houston, Texas

Kaushansky_FM_pi_xxii.indd 12

Clinical Professor of Pathology Research Director, Centre for Lymphoid Cancers Departments of Pathology and Advanced Therapeutics British Columbia Cancer Agency, the BC Centre Research Center and University of British Columbia Vancouver, British Columbia, Canada

9/21/15 4:40 PM

Contributors

Terry B. Gernsheimer, MD [139]

Professor of Medicine Department of Medicine, Division of Hematology University of Washington School of Medicine Seattle Cancer Care Alliance Seattle, Washington

Stanton Gerson, MD [29]

Director, Case Comprehensive Cancer, Seidman Cancer Center & National Center for Regenerative Medicine Distinguished University Professor Case Western Reserve University University Hospital of Cleveland Cleveland, Ohio

Morie A. Gertz, MD, MACP [108] Division of Hematology Mayo Clinic Rochester, Minnesota

Larisa J. Geskin, MD, FAAD [103, 105]

Associate Professor of Dermatology and Medicine Director, Division of Cutaneous Oncology and Comprehensive Skin Cancer Center Department of Dermatology Columbia University New York, New York

David Ginsburg, MD [126]

Professor, Department of Internal Medicine, Human Genetics and Pediatrics Investigator, Howard Hughes Medical Institute Life Sciences Institute University of Michigan Ann Arbor, Michigan

Lucy A. Godley, MD, PhD [13]

Section of Hematology/Oncology Department of Medicine and The University of Chicago Comprehensive Cancer Center The University of Chicago Chicago, Illinois

Steven Grant, MD [16]

Professor of Medicine and Biochemistry Shirley and Sture Gordon Olsson Professor of Oncology Associate Director Translational Research, Massey Cancer Center Virginia Commonwealth University Health Sciences Center Richmond, Virginia

Anthony R. Green, PhD, FRCP, FRCPath, FMedSci [85]

Xylina T. Gregg, MD [38]

Director of Laboratory Services Utah Cancer Specialists Salt Lake City, Utah

Michael R. Grever, MD [93]

Chair and Professor Department of Internal Medicine Bertha Bouroncle MD and Andrew Pereny Chair in Medicine The Ohio State University Columbus, Ohio

John Gribben, MD, DSc, FRCP, FRCPath, FMedSci [76] Chair of Medical Oncology Barts Cancer Institute Centre for Haemato-Oncology Queen Mary University of London London, United Kingdom

John H. Griffin, PhD [114]

Professor Department of Molecular and Experimental Medicine The Scripps Research Institute La Jolla, California

Katherine A. Hajjar, MD [115, 135]

Professor of Pediatrics Brine Family Professor, Department of Cell and Developmental Biology Professor of Medicine Well Cornell Medical College Attending Pediatrician New York Presbyterian Hospital New York, New York

Robert D. Harrington, MD [81] Professor of Medicine University of Washington Seattle, Washington

Jeanne E. Hendrickson, MD [55]

Associate Professor Departments of Laboratory Medicine and Pediatrics Yale University School of Medicine New Haven, Connecticut

Paul C. Herrmann, MD, PhD [52]

Associate Professor and Chair Department of Pathology and Human Anatomy Loma Linda University School of Medicine Loma Linda, California

Professor of Haematology Cambridge Institute for Medical Research and Stem Cell Institute University of Cambridge Cambridge, United Kingdom

Steven Horwitz, MD [104]

Ralph Green, MD, PhD, FRCPath [41, 44]

Annie L. Hsieh, MD [14]

Professor of Pathology and Medicine University of California Davis Medical Center Sacramento, California

Kaushansky_FM_pi_xxii.indd 13

xiii

Department of Medicine Memorial Sloan Kettering Cancer Center New York, New York Department of Pathology Johns Hopkins University, School of Medicine Baltimore, Maryland

9/21/15 4:40 PM

xiv

Contributors

Zachary R. Hunter, PhD [109]

Bing Center for Waldenstrom’s Macroglobulinemia Dana-Farber Cancer Institute Instructor of Medicine, Harvard Medical School Boston, Massachusetts

Russell D. Hull, MD [133]

Professor Department of Medicine University of Calgary Active Staff Department of Internal Medicine Foothills Hospital Calgary, Alberta, Canada

Achille Iolascon, MD, PhD [39]

Professor of Medical Genetics Dept. of Molecular Medicine and Medical Biotechnologies University Federico II of Naples Naples, Italy

Joseph E. Italiano Jr., PhD [112] Associate Professor of Medicine Brigham and Women’s Hospital Harvard Medical School Boston, Massachusetts

Benjamin Izar, MD, PhD [22]

Post-doctoral Scientist Dana-Farber Cancer Institute and Broad Institute Associate Physician, Brigham and Women’s Hospital Harvard Medical School Boston, Massachusetts

Siegfried Janz, MD, DSc [105]

Division of Hematology/Oncology & Blood and Marrow Transplantation Department of Pathology, Carver College of Medicine University of Iowa Health Care Iowa City, Iowa

Jill M. Johnsen, MD [126]

Assistant Member, Research Institute Bloodworks Northwest Puget Sound Blood Center Assistant Professor, Division of Hematology Department of Medicine University of Washington Seattle, Washington

Lynn B. Jorde, PhD [10]

H. A. and Edna Benning Presidential Professor Department of Human Genetics University of Utah School of Medicine Salt Lake City, Utah

Bindu Kanapuru, MD [9]

Institute for Advanced Studies in Aging and Geriatrics Falls Church, Virginia

Kaushansky_FM_pi_xxii.indd 14

Kenneth Kaushansky, MD, MACP [17, 18, 111, 116, 119] Senior Vice President, Health Sciences Dean, School of Medicine SUNY Distinguished Professor Stony Brook Medicine State University of New York Stony Brook, New York

Nigel S. Key, MB, ChB, FRCP [123]

Harold R. Roberts Distinguished Professor of Medicine Director, University of North Carolina Hemophilia and Thrombosis Center Chapel Hill, North Carolina

Thomas J. Kipps, MD, PhD [75]

Evelyn and Edwin Tasch Chair in Cancer Research Professor of Medicine Division of Hematology/Oncology Deputy Director for Research Operations Moores UCSD Cancer Center University of California, San Diego La Jolla, California

Mark J. Koury, MD [5]

Division of Hematology/Oncology Department of Medicine Vanderbilt University Medical Center Nashville, Tennessee

Abdullah Kutlar, MD [49] Professor of Medicine Georgia Sickle Cell Center Medical College of Georgia Sickle Cell Center Augusta, Georgia

Robert A. Kyle, MD [110,]

Professor of Medicine Laboratory Medicine and Pathology Mayo Clinic Rochester, Minnesota

Lewis L. Lanier, PhD [77]

Professor Department of Microbiology and Immunology University of California, San Francisco San Francisco, California

Richard A. Larson, MD [91]

Section of Hematology/Oncology Department of Medicine and the Comprehensive Cancer Center University of Chicago Chicago, Illinois

Michelle M. Le Beau, PhD [13]

Section of Hematology/Oncology Department of Medicine and the Center Research Center University of Chicago Chicago, Illinois

9/21/15 4:40 PM

Contributors

Frank W.G. Leebeek, MD, PhD [128] Professor of Hematology Department of Hematology Erasmus University Medical Center Rotterdam, The Netherlands

Marcel Levi, MD, PhD [116, 122, 129 ]

Department of Medicine/Vascular Medicine Academic Medical Center University of Amsterdam Amsterdam, The Netherlands

Marshall A. Lichtman, MD [1, 35, 53, 64, 69, 70, 83, 86, 88, 89, 95, 106] Professor of Medicine and of Biochemistry and Biophysics University of Rochester Medical Center Rochester, New York

Jane L. Liesveld, MD [88, 89]

Professor of Medicine (Hematology-Oncology) James P. Wilmot Cancer Institute University of Rochester Medical Center Rochester, New York

Ton Lisman, PhD [128]

Professor of Experimental Surgery Surgical Research Laboratory and Section of Hepatobiliary Surgery and Liver Transplantation Department of Surgery University Medical Center, Groningen Groningen, The Netherlands

John S. (Pete) Lollar III, MD [127]

Aflac Cancer Center and Blood Disorders Services Department of Pediatrics Emory University Atlanta, Georgia

Naomi L. C. Luban, MD [55]

Professor, Pediatrics and Pathology George Washington University Medical Center Division Chief, Laboratory Medicine Director, Transfusion Medicine/Donor Center Children’s National Medical Center Washington, D.C.

Crystal L. Mackall, MH [21]

Head, Immunology Section and Chief, Pediatric Oncology Branch National Cancer Institute Bethesda, Maryland

Aaron J. Marcus, MD* [115]

Professor of Medicine Weill Cornell Medical College Attending Physician New York Harbor Healthcare System New York, New York

Elaine R. Mardis, PhD [11]

Robert E. and Louise F. Dunn Distinguished Professor of Medicine Co-director, The Genome Institute, Division of Genomics and Bioinformatics, Department of Medicine, Washington University School of Medicine Siteman Cancer Center, Washington University School of Medicine Saint Louis, Missouri

Fabienne McClanahan, MD, PhD [76] Barts Cancer Institute Centre for Haemato-Oncology Queen Mary University of London London, United Kingdom

Christine Lomas-Francis, MSc, FIBMS [136]

Kenneth L. McClain, MD, PhD [71]

Technical Director Laboratory of Immunohematology and Genomics New York Blood center New York, New York

Professor of Pediatrics Texas Children’s Cancer Center/Hematology Baylor College of Medicine Houston, Texas

José A. Lópéz, MD [117]

Jeffrey McCullough, MD [138]

Chief Scientific Officer Bloodworks Northwest Professor of Medicine and Biochemistry University of Washington Seattle, Washington

Robert Lowsky, MD [23]

Division of Blood and Marrow Transplantation Stanford University Stanford, California

Gerard Lozanski, MD [93] Director, Hematopathology Medical Director Flow Cytometry Laboratory Associate Professor—Clinical Department of Pathology The Ohio State University Columbus, Ohio

xv

Professor Department of Laboratory Medicine and Pathology American Red Cross Professor, Transfusion Medicine University of Minnesota Medical School Minneapolis, Minnesota

Janice McFarland, MD [137]

Blood Center of Southeast Wisconsin Milwaukee, Wisconsin

Shannon L. Meeks, MD [127]

Aflac Cancer Center and Blood Disorders Services Department of Pediatrics Emory University Atlanta, Georgia

Deceased

*

Kaushansky_FM_pi_xxii.indd 15

9/21/15 4:40 PM

xvi

Contributors

Neha Mehta, MD [104]

Department of Medicine Memorial Sloan Kettering Cancer Center New York, New York

Marzia Menegatti, MD [124]

Angelo Bianchi Bonomi Hemophilia and Thrombosis Center Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico University of Milan Milan, Italy

Giampaolo Merlini, MD [109]

Director, Center for Research and Treatment of Systematic Amyloidoses University Hospital Policlinico San Matteo Professor, Department of Medicine University of Pavia Pavia, Italy

Dean D. Metcalfe, MD [63]

Chief, Laboratory of Allergic Diseases Chief, MCBS/LAD NAID/National Institute of Health Bethesda, Maryland

H. A. Mettine Bos, HA, PhD [113]

Assistant Professor Division of Thrombosis and Hemostasis Einthoven Laboratory for Experimental Vascular Medicine Leiden University Medical Center Leiden, The Netherlands

Saskia Middeldorp, MD, PhD [130] Department of Vascular Medicine Academic Medical Center Amsterdam, The Netherlands

Martha P. Mims, MD, PhD [8]

Professor of Medicine Section Chief, Section of Hematology/Oncology Baylor College of Medicine Houston, Texas

Constantine S. Mitsiades, MD, PhD [22] Assistant Professor of Medicine Dana-Farber Cancer Institute Harvard Medical School Boston, Massachusetts

Joel Moake, MD [51]

Senior Research Scientist and Associate Director Biomedical Engineering Laboratory Rice University Houston, Texas

Narla Mohandas, D.Sc [31] Red Cell Physiology Laboratory New York Blood Center New York, New York

Kaushansky_FM_pi_xxii.indd 16

Emile R. Mohler III, MD [134]

Director, Vascular Medicine Professor of Medicine Division of Cardiovascular Medicine Perelman School of Medicine at the University of Pennsylvania Philadelphia, Pennsylvania

Jeffrey J. Molldrem, MD [27]

Professor of Medicine Stem Cell Transplantation and Cellular Therapy, MD Anderson Cancer Center Houston, Texas

Alison Moskowitz, MD [104]

Department of Medicine Memorial Sloan Kettering Cancer Center New York, New York

Laurent O. Mosnier, PhD [114]

Associate Professor Department of Molecular and Experimental Medicine The Scripps Research Institute La Jolla, California

William A. Muller, MD, PhD [115] Magerstadt Professor and Chair Department of Pathology Feinberg School of Medicine Northwestern University Chicago, Illinois

Natarajan Muthusamy, DVM, PhD [73] Professor of Medicine Division of Hematology Department of Internal Medicine The Ohio State University Columbus, Ohio

Kalyan Nadiminti, MD [105]

Division of Hematology/Oncology & Blood and Marrow Transplantation Department of Pathology, Carver College of Medicine University of Iowa Health Care Iowa City, Iowa

Srikanth Nagalla, MBBS, MS [56]

Assistant Professor of Medicine Division of Hematology Cardeza Foundation for Hematologic Research Thomas Jefferson University Philadelphia, Pennsylvania

Kavita Natrajan, MBBS [49]

Associate Professor of Medicine Division of Hematology/Oncology Georgia Regents University Augusta, Georgia

Marguerite Neerman-Arbez, PhD [125]

Professor Department of Genetic Medicine and Development University of Geneva Faculty of Medicine Geneva, Switzerland

9/21/15 4:40 PM

Contributors

Robert S. Negrin, MD [23]

Division of Blood and Marrow Transplantation Stanford University Stanford, California

Luigi D. Notarangelo, MD [80]

Mortimer Poncz, MD [118]

Jane Fishman Grinberg Professor of Pediatrics Perelman School of Medicine at the University of Pennsylvania Children’s Hospital of Philadelphia Philadelphia, Pennsylvania

Professor of Pediatrics and Pathology Harvard Medical School Jeffrey Modell Chair of Pediatric Immunology Research Division of Immunology, Children’s Hospital Boston Boston, Massachusetts

Prem Ponka, MD [59]

Hans D. Ochs, MD [80]

Pierluigi Porcu, MD [94]

Professor of Pediatrics Jeffrey Modell Chair of Pediatric Immunology Research Division of Immunology Seattle Children’s Research Hospital Department of Pediatrics University of Washington Seattle, Washington

Elizabeth O’Donnell, MD [107] Massachusetts General Hospital Boston, Massachusetts

Mark J. Osborn, PhD [30]

Assistant Professor Pediatrics Blood and Marrow Transplantation, Stem Cell Institute University of Minnesota Minneapolis, Minnesota

Charles H. Packman, MD [54]

Professor of Medicine University of North Carolina School of Medicine Levine Cancer Institute, Hematologic Oncology and Blood Disorders Charlotte, North Carolina

Professor of Physiology and Medicine Lady Davis Institute McGill University Montreal, Quebec, Canada Professor of Internal Medicine Division of Hematology, and Comprehensive Cancer Center The Ohio State University Columbus, Ohio

Jaroslav F. Prchal, MD [84]

Director, Department of Oncology St. Mary’s Hospital Montreal, Quebec, Canada

Josef T. Prchal, MD [ 32, 33, 34, 45, 50, 57, 59, 84, 86]

The Charles A. Nugent, M.D., and Margaret Nugent Professor Division of Hematology, Pathology, and Genetics University of Utah Salt Lake City, Utah Department of Pathophysiology First Faculty of Medicine Charles University Prague, Czech Republic

Oliver W. Press, MD, PhD [ 95, 97, 98, 99]

Professor of Pediatrics University of Rochester Medical Center Rochester, New York

Acting Senior Vice President, Fred Hutchinson Cancer Research Center Acting Director, Clinical Research Division, FHCRC Recipient, Dr. Penny E. Peterson Memorial Chair for Lymphoma Research Professor of Medicine and Bioengineering University of Washington Seattle, Washington

Charles J. Parker, MD [40]

Gordana Raca, MD, PhD [13]

James Palis, MD [7]

Professor of Medicine Division of Hematology and Bone Marrow Transplantation University of Utah School of Medicine Salt Lake City, Utah

Flora Peyvandi, MD [124]

xvii

Section of Hematology/Oncology Department of Medicine and The University of Chicago Comprehensive Cancer Center University of Chicago Chicago, Illinois

Angelo Bianchi Bonomi Hemophilia and Thrombosis Center Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico University of Milan Milan, Italy

Noopur Raje, MD [107]

John D. Phillips, PhD [58]

Professor of Pathology and Medicine Director of Hematology Laboratory Montefiore Medical Center The University Hospital for the Albert Einstein College of Medicine Bronx, New York

Associate Professor of Medicine Division of Hematology University of Utah School of Medicine Salt Lake City, Utah

Kaushansky_FM_pi_xxii.indd 17

Massachusetts General Hospital Boston, Massachusetts

Jacob H. Rand, MD [131]

9/21/15 4:40 PM

xviii

Contributors

A. Koneti Rao, MD [120]

Sol Sherry Professor of Medicine Director of Benign Hematology, Hemostasis and Thrombosis Co-Director, Sol Sherry Thrombosis Research Center Temple University School of Medicine Philadelphia, Pennsylvania

Gary E. Raskob, PhD [133]

Dean, College of Public Health Regents Professor, Epidemiology and Medicine The University of Oklahoma Health Science Center Oklahoma City, Oklahoma

Vishnu VB Reddy, MD [45]

Department of Pathology, University of Alabama in Birmingham, Birmingham, Alabama

John C. Reed, MD, PhD [15]

Pharmaceutical Research & Early Development Roche Innovation Center-Basel Basel, Switzerland

Majed A. Refaai, MD [138]

Associate Professor Department of Pathology and Laboratory Medicine University of Rochester Medical Center Rochester, New York

Marion E. Reid, PhD, DSc (Hon.) [136] (Retired) New York Blood Center New York, New York

Pieter H. Reitsma, PhD [113]

Professor in Experimental Molecular Medicine Division of Thrombosis and Hemostasis Einthoven Laboratory for Experimental Vascular Medicine Leiden University Medical Center Leiden, The Netherlands

Andrew R. Rezvani, MD [23]

Jia Ruan, MD, PhD [135]

Associate Professor Department of Medicine Weill Cornell Medical College Associate Attending Physician New York Presbyterian Hospital New York, New York

Daniel H. Ryan, MD [2, 3]

Professor Emeritus Department of Pathology and Laboratory Medicine University of Rochester Medical Center Rochester, New York

J. Evan Sadler, MD, PhD [132]

Ira M. Lang Professor of Medicine Washington University School of Medicine St. Louis, Missouri

Andrew I. Schafer, MD [134]

Professor of Medicine, Director, The Richard T. Silver Center for Myeloproliferative Neoplasms, Weill Cornell Medical College New York, New York

Stanley L. Schrier, MD [4]

Professor of Medicine (Hematology) Active emeritus Division of Hematology Stanford University School of Medicine Stanford, California

Christopher S. Seet, MD [74]

Department of Medicine Division of Hematology/Oncology David Geffen School of Medicine University of California Los Angeles, California

George B. Segel, MD [7, 35]

Division of Blood and Marrow Transplantation Stanford University Stanford, California

Professor of Pediatrics, Emeritus Professor of Medicine University of Rochester Medical Center Rochester, New York

Katayoun Rezvani, MD [27]

Uri Seligsohn, MD [116, 129]

Professor of Medicine Stem Cell Transplantation and Cellular Therapy MD Anderson Cancer Center Houston, Texas

Paul G. Richardson, MD [22] Professor of Medicine Dana-Farber Cancer Institute Harvard Medical School Boston, Massachusetts

Stanley R. Riddell, MD [26]

Member, Clinical Research Division Fred Hutchinson Cancer Research Center Seattle, Washington

Kaushansky_FM_pi_xxii.indd 18

Professor of Hematology and Director Amalia Biron Research Institute of Thrombosis and Hemostasis Sheba Medical Center Tel-Hashomer and Sackler Faculty of Medicine Tel Aviv University Tel Aviv, Israel

Sanford J. Shattil, MD [121]

Professor and Chief, Division of Hematology-Oncology Department of Medicine University of California, San Diego Adjunct Professor of Molecular and Experimental Medicine The Scripps Research Institute La Jolla, California

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Contributors

Taimur Sher, MD [108]

Division of Hematology/Oncology Mayo Clinic Jacksonville, Florida

Brian F. Skinnider, MD [96]

Clinical Associate Professor Department of Pathology Vancouver General Hospital, British Columbia Cancer Agency, and University of British Columbia Vancouver, British Columbia, Canada

Sherrill J. Slichter, MD [139]

Perumal Thiagarajan, MD [32, 33]

Professor of Medicine and Pathology Baylor College of Medicine Director, Blood Bank and Hematology Laboratory Michael E. DeBakey VA Medical Center Houston, Texas

Jakub Tolar, MD, PhD [30]

Professor, Department of Pediatrics Blood and Marrow Transplantation, Stem Cell Institute University of Minnesota Minneapolis, Minnesota

Professor of Medicine Department of Medicine, Division of Hematology University of Washington School of Medicine Bloodworks Northwest Seattle, Washington

Steven P. Treon [109]

C. Wayne Smith, MD [60, 61]

Guido Tricot, MD, PhD [105]

Professor and Head, Section of Leukocyte Biology Department of Pediatrics Baylor College of Medicine Houston, Texas

Stephen D. Smith, MD [98]

Associate Professor, Internal Medicine Division of Medical Oncology University of Washington Seattle, Washington

Susan S. Smyth, MD, PhD [112]

Jeff Gill Professor of Cardiology Chief, Division of Cardiovascular Medicine Medical Director, Gill Heart Institute University of Kentucky Lexington, Kentucky

David P. Steensma, MD [87]

Department of Medical Oncology Division of Hematological Malignancies Dana-Farber Cancer Institute Boston, Massachusetts

Sean R. Stowell, MD, PhD [127]

Department of Pathology and Laboratory Medicine Emory University Atlanta, Georgia

Director, Bing Center for Waldenstrom’s Macroglobulinemia Dana-Farber Cancer Institute Associate Professor, Harvard Medical School Boston, Massachusetts Division of Hematology/Oncology & Blood and Marrow Transplantation Department of Pathology, Carver College of Medicine University of Iowa Health Care Iowa City, Iowa

Giorgio Trinchieri, MD [77]

Director, Cancer and Inflammation Program Chief, Laboratory of Experimental Immunology Center for Cancer Research, NCI, NIH Bethesda, Maryland

Wouter W. van Solinge, PhD [47]

Professor of Laboratory Medicine Head of Department Chair and Medical Director Division Laboratories and Pharmacy Department of Clinical Chemistry and Haematology University Medical Center Utrecht Utrecht, The Netherlands

Cornelis van ‘t Veer, PhD [113]

Associate Professor Center for Experimental and Molecular Medicine Academic Medical Center Amsterdam, The Netherlands

Richard van Wijk, PhD [47]

Department of Transfusion Medicine National Institutes of Health Bethesda, Maryland

Associate professor Department of Clinical Chemistry and Haematology Division Laboratories and Pharmacy University Medical Center Utrecht Utrecht, The Netherlands

Madina Sukhanova, PhD [13]

Sumithira Vasu, MBBS [79]

David Stroncek [137]

Section of Hematology/Oncology Department of Medicine and the Center Research Center University of Chicago Chicago, Illinois

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xix

Assistant Professor Medical Director, Cell Therapy Lab Blood and Marrow Transplantation Section Division of Hematology The Ohio State University Columbus, Ohio

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xx

Contributors

John Wagner, MD [30]

Karl Welte, MD [65]

Dietlind L. Wahner-Roedler, MD [110]

Erik Wendlandt, PhD [105]

Professor, Department of Pediatrics Blood and Marrow Transplantation, Stem Cell Institute University of Minnesota Minneapolis, Minnesota Professor of Medicine Mayo Clinic Rochester, Minnesota

Zandra E. Walton [14]

Abramson Family Cancer Research Institute Perelman School of Medicine University of Pennsylvania Philadelphia, Pennsylvania

Peter A. Ward, MD [19]

Godfrey D. Stobbe Professor of Pathology Department of Pathology University of Michigan Medical School Ann Arbor, Michigan

Andrew J. Wardlaw, MD, PhD [62] Institute for Lung Health Department of Infection Immunity and Inflammation Leicester University Medical School Leicester, United Kingdom

Jeffrey S. Warren, MD [19]

Aldred S. Warthin Professor of Pathology Department of Pathology University of Michigan Medical School Ann Arbor, Michigan

Lukas D. Wartman, MD [11]

Assistant Professor, Section of Stem Cell Biology Division of Oncology, Department of Medicine, Washington University School of Medicine Siteman Cancer Center, Washington University School of Medicine Assistant Director, Section of Cancer Genomics The Genome Institute, Washington University School of Medicine St. Louis, Missouri

Sir David J. Weatherall, MD [48]

Professor Weatherall Institute of Molecular Medicine John Radcliffe Hospital Headington, Oxford, United Kingdom

Senior-Professor Department of Pediatrics University of Tübingen Tübingen, Germany Division of Hematology/Oncology & Blood and Marrow Transplantation Department of Pathology, Carver College of Medicine University of Iowa Health Care Iowa City, Iowa

Sidney Whiteheart, PhD [112]

Professor Molecular and Cellular Biochemistry University of Kentucky College of Medicine Lexington, Kentucky

Lucia Wolgast, MD [131]

Assistant Professor of Pathology (Clinical) Director, Clinical Laboratories, Moses Division Associate Director, Hematology Laboratories Montefiore Medical Center/Albert Einstein College of Medicine Department of Pathology Bronx, New York

Neal S. Young, MD [36]

Hematology Branch National Heart, Lung, and Blood National Institutes of Health Bethesda, Maryland

Fenghuang Zhan, MD, PhD [105]

Division of Hematology/Oncology & Blood and Marrow Transplantation Department of Pathology, Carver College of Medicine University of Iowa Health Care Iowa City, Iowa

Ari Zimran, MD [72]

Gaucher Clinic Shaare Zedek Medical Center Jerusalem, Israel

Pier Luigi Zinzani, MD, PhD [101]

Professor Institute of Hematology “L. e A. Seràgnoli” University of Bologna Bologna, Italy

Robert Weinstein, MD [28]

Professor of Medicine and Pathology University of Massachusetts Medical School Chief, Division of Transfusion Medicine UMass Memorial Medical Center Worcester, Massachusetts

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xxi xxi

PREFACE The first edition of Williams Hematology (né Hematology) was published in 1972. This, our 9th edition, will represent our continued efforts over nearly one-half century to provide the most current concepts of the pathophysiology and treatment of hematologic diseases. The rate of growth in our understanding of diseases of blood cells and coagulation pathways provides a challenge for editors of a comprehensive textbook of hematology. The sequencing of individual genomes, analysis of the “dark DNA” and noncoding RNAs, advances in knowledge in proteomics, metabolomics, and other “-omics” fields, as applied to hematologic disorders, have accelerated the understanding of the pathogenesis of the diseases of our interest. The rate at which basic knowledge in molecular and cellular biology and immunology has been translated into improved diagnostic and therapeutic methods is equally impressive. Specific molecular targets for therapy in several hematologic disorders have become reality, and it is not hyperbole to state that hematology is the poster child for the rational design of therapeutics applicable to other fields of medicine. This edition of Williams Hematology includes changes designed to facilitate ease of access to information, both within the book and its associated links, and has been modestly reorganized to reflect our greater understanding of the origins of hematologic disorders. Each chapter has been revised or rewritten to provide current information. Four new chapters have been added and other notable changes have been made. Chapter 4 “Consultative Hematology” is new to this edition. The chapter “Epigenetics and Genomics” has been divided into separate chapters to reflect the growth of knowledge in those disciplines. Chapter 14, “Metabolism of Hematologic Neoplastic Cells” is new, as this topic has become the basis of multiple potential drug targets for hematologic disease. A section on “Autophagy” has been added to Chap 15 “Apoptosis Mechanisms: Relevance to the Hematopoietic System,” as the topic is becoming increasingly important for understanding of the physiology of blood cell development; and an independent chapter “Heparin-Induced Thrombocytopenia” (Chap 118) has been created to reflect both its pathophysiologic and clinical importance. Recognizing that at the heart of diagnostic hematology is blood and marrow cell morphology, we have continued our incorporation of informative color images of the relevant disease topics in each chapter, allowing easy access to illustrations of cell morphology important to diagnosis. The 9th edition of Williams Hematology is also available online, as part of the excellent www.accessmedicine.com website. With direct links to a comprehensive drug therapy database and to other important medical texts, including Harrison’s Principles of Internal Medicine and Goodman and Gilman’s The Pharmacological Basis of Therapeutics, Williams Hematology Online is part of a powerful resource covering all disciplines within medical education and practice. The online edition

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of Williams Hematology also includes PubMed links to journal articles cited in the references. In addition, Williams Manual of Hematology will be revised to reflect the diagnostic and therapeutic advances incorporated in the 9th edition of Williams Hematology. The convenient Manual features the most clinically salient content from the parent text, and is useful in time-restricted clinical situations. The Manual will be available for iPhone™ and other mobile formats. The readers of the 9th edition of Williams Hematology will note a “changing of (some of) the guard” of our editorial group; Drs. Marcel Levi (a member of the 8th edition of Williams Manual of Hematology editorial group), Oliver Press, Linda Burns, and Michael Caligiuri have joined continuing editors Drs. Kenneth Kaushansky, Marshall Lichtman, and Josef Prchal in the 9th edition. The production of this book required the timely cooperation of 101 contributors for the production of 139 chapters. We are grateful for their work in providing this comprehensive and up-to-date text. Despite the growth of both basic and clinical knowledge and the passion that each of our contributors brings to the topic of their chapter, we have been able to maintain the text in a single volume through scrupulous attention to chapter length. Each editor has had expert administrative assistance in the management of the manuscripts for which they were primarily responsible. We thank Susan Madden in Salt Lake City, Utah; Nancy Press and Deborah Lemon in Seattle, Washington; and Annie Thompson, Rebecca Posey, and Kimberly Morley in Columbus, Ohio for their very helpful participation in the production of the book. Special thanks go to Susan Daley in Rochester, New York, and Marie Brito in Stony Brook, New York, who were responsible for coordinating the management of 139 chapters, including many new figures and tables, and managing other administrative matters, a challenging task that Ms. Daley and Ms. Brito performed with skill and good humor. The editors also acknowledge the interest and support of our colleagues at McGraw-Hill, including James F. Shanahan, Publisher, Medical Publishing; Karen Edmonson, Senior Editor for Williams Hematology; and Harriet Lebowitz, Senior Project Development Editor for Williams Hematology. Kenneth Kaushansky Marshall A. Lichtman Joseph T. Prchal Marcel Levi Oliver W. Press Linda J. Burns Michael A. Caligiuri

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Part I  Clinical Evaluation of the Patient 1. Initial Approach to the Patient: History and Physical Examination . . . . . . . . . . . . . . . . . . . 3 2. Examination of Blood Cells . . . . . . . . . . . . . . 11

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3. Examination of the Marrow . . . . . . . . . . . . . 27 4. Consultative Hematology . . . . . . . . . . . . . . . 41

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CHAPTER 1

INITIAL APPROACH TO THE PATIENT: HISTORY AND PHYSICAL EXAMINATION

Marshall A. Lichtman and Linda J. Burns

SUMMARY The care of a patient with a suspected hematologic abnormality begins with a systematic attempt to determine the nature of the illness by eliciting an in-depth medical history and performing a thorough physical examination. The physician should identify the patient’s symptoms systematically and obtain as much relevant information as possible about their origin and evolution and about the general health of the patient by appropriate questions designed to explore the patient’s recent and remote experience. Reviewing previous records may add important data for understanding the onset or progression of illness. Hereditary and environmental factors should be carefully sought and evaluated. The use of drugs and medications, nutritional patterns, and sexual behavior should be considered. The physician follows the medical history with a physical examination to obtain evidence for tissue and organ abnormalities that can be assessed through bedside observation to permit a careful search for signs of the illnesses suggested by the history. Skin changes and hepatic, splenic, or lymph nodal enlargement are a few findings that may be of considerable help in pointing toward a diagnosis. Additional history is obtained during the physical examination, as findings suggest an additional or alternative consideration. Thus, the history and physical examination should be considered as a unit, providing the basic information with which further diagnostic information is integrated: blood and marrow studies, imaging studies, and biopsies.   Primary hematologic diseases are common in the aggregate, but hematologic manifestations secondary to other diseases occur even more frequently. For example, the signs and symptoms of anemia and the presence of enlarged lymph nodes are common clinical findings that may be related to a hematologic disease but occur frequently as secondary manifestations of disorders not considered primarily hematologic. A wide variety of diseases may produce signs or symptoms of hematologic illness. Thus, in patients with a connective tissue disease, all the signs and symptoms of anemia may be elicited and lymphadenopathy may be notable, but additional findings are usually present that indicate primary involvement of some system besides the hematopoietic (marrow) or lymphopoietic (lymph nodes or other lymphatic sites). In this discussion, emphasis is placed on the clinical findings resulting from either primary hematologic disease or the complications of hematologic disorders so as to avoid presenting an extensive catalog of signs and symptoms encountered in general clinical medicine.

Acronyms and Abbreviations: Ig, immunoglobulin; IL, interleukin; POEMS, polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes; PS, performance status.

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  In each discussion of specific diseases in subsequent chapters, the signs and symptoms that accompany the particular disorder are presented, and the clinical findings are covered in detail. In this chapter, a more general systematic approach is taken.

THE HEMATOLOGY CONSULTATION Table 1–1 lists the major abnormalities that result in the evaluation of the patient by the hematologist. The signs indicated in Table  1–1 may reflect a primary or secondary hematologic problem. For example, immature granulocytes in the blood may be signs of myeloid diseases such as myelogenous leukemia, or, depending on the frequency of these cells and the level of immaturity, the dislodgment of cells resulting from marrow metastases of a carcinoma. Nucleated red cells in the blood may reflect the breakdown in the marrow–blood interface seen in primary myelofibrosis or the hypoxia of congestive heart failure. Certain disorders have a propensity for secondary hematologic abnormalities; renal, liver, and connective tissue diseases are prominent among such abnormalities. Chronic alcoholism, nutritional fetishes, and the use of certain medications may be causal factors in blood cell or coagulation protein disorders. Pregnant women and persons of older age are prone to certain hematologic disorders: anemia, thrombocytopenia, or intravascular coagulation in the former case, and hematologic malignancies, pernicious anemia and the anemia of aging in the latter. The history and physical examination can provide vital clues to the possible diagnosis and also to the rationale choice of laboratory tests.

THE HISTORY In today’s technology- and procedure-driven medical environment, the importance of carefully gathering information from patient inquiry and examination is at risk of losing its primacy. The history (and physical examination) remains the vital starting point for the evaluation of any clinical problem.1–3

GENERAL SYMPTOMS AND SIGNS Performance status (PS) is used to establish semiquantitatively the extent of a patient’s disability. This status is important in evaluating patient comparability in clinical trials, in determining the likely tolerance to cytotoxic therapy, and in evaluating the effects of therapy. Table 1–2 presents a well-founded set of criteria for measuring PS.4 An abbreviated version sometimes is used, as proposed by the Eastern Cooperative Oncology Group (Table 1–3).5 Weight loss is a frequent accompaniment of many serious diseases, including primary hematologic malignancies, but it is not a prominent accompaniment of most hematologic diseases. Many “wasting” diseases, such as disseminated carcinoma and tuberculosis, cause anemia, and pronounced emaciation should suggest one of these diseases rather than anemia as the primary disorder. Fever is a common early manifestation of the aggressive lymphomas or acute leukemias as a result of pyrogenic cytokines (e.g., interleukin [IL]-1, IL-6, and IL-8) released as a reflection of the disease itself. After chemotherapy-induced cytopenias or in the face of accompanying immunodeficiency, infection is usually the cause of fever. In patients with “fever of unknown origin,” lymphoma, particularly Hodgkin lymphoma, should be considered. Occasionally, primary myelofibrosis, acute leukemia, advanced myelodysplastic syndrome, and other lymphomas may also cause fever. In rare patients with severe pernicious

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4

Part I: Clinical Evaluation of the Patient

TABLE 1–1.  Findings That May Lead to a Hematology Consultation

TABLE 1–3.  Eastern Cooperative Oncology Group Performance Status5

Decreased hemoglobin concentration (anemia) Increased hemoglobin concentration (polycythemia) Elevated serum ferritin level Leukopenia or neutropenia Immature granulocytes or nucleated red cells in the blood Pancytopenia Granulocytosis: neutrophilia, eosinophilia, basophilia, or mastocytosis Monocytosis Lymphocytosis Lymphadenopathy Splenomegaly Hypergammaglobulinemia: monoclonal or polyclonal Purpura Thrombocytopenia Thrombocytosis Exaggerated bleeding: spontaneous or trauma related Prolonged partial thromboplastin or prothrombin coagulation times Venous thromboembolism Thrombophilia Obstetrical adverse events (e.g., recurrent fetal loss, stillbirth, and HELLP syndrome)

Grade

Activity

0

Fully active, able to carry on all predisease performance without restriction

1

Restricted in physically strenuous activity but ambulatory and able to carry out work of a light or sedentary nature, e.g., light housework, office work

2

Ambulatory and capable of all self-care but unable to carry out any work activities; up and about more than 50% of waking hours

3

Capable of only limited self-care, confined to bed or chair more than 50% of waking hours

4

Completely disabled; cannot carry on any self-care; totally confined to bed or chair

5

Dead

HELLP, hemolytic anemia, elevated liver enzymes, and low platelet count.

TABLE 1–2.  Criteria of Performance Status (Karnofsky Scale)4 Able to carry on normal activity; no special care is needed. 100%

Normal; no complaints, no evidence of disease

90%

Able to carry on normal activity; minor signs or symptoms of disease 80% Normal activity with effort; some signs or symptoms of disease Unable to work; able to live at home, care for most personal needs; a varying amount of assistance is needed. 70% Cares for self; unable to carry on normal activity or to do active work 60% Requires occasional assistance but is able to care for most personal needs 50% Requires considerable assistance and frequent medical care Unable to care for self; requires equivalent of institutional or hospital care; disease may be progressing rapidly. 40% Disabled; requires special care and assistance 30% Severely disabled; hospitalization is indicated though death not imminent 20% Very sick; hospitalization necessary; active supportive treatment necessary 10% Moribund; fatal processes progressing rapidly 0%

Dead

Adapted with permission from Mor V, Laliberte L, Morris JN, Wiemann M: The Karnofsky performance status scale: An examination of its reliability and validity in a research setting Cancer 1984 May 1; 53(9):2002–2007.

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Oken MM, Creech RH, Tormey DC, et al: Toxicity and response criteria of the Eastern Cooperative Oncology Group. Am J Clin Oncol. anemia or hemolytic anemia, fever may be present. Chills may accompany severe hemolytic processes and the bacteremia that may complicate the immunocompromised or neutropenic patient. Night sweats suggest the presence of low-grade fever and may occur in patients with lymphoma or leukemia. Fatigue, malaise, and lassitude are such common accompaniments of both physical and emotional disorders that their evaluation is complex and often difficult. In patients with serious disease, these symptoms may be readily explained by fever, muscle wasting, or other associated findings. Patients with moderate or severe anemia frequently complain of fatigue, malaise, or lassitude and these symptoms may accompany the hematologic malignancies. Fatigue or lassitude may occur also with iron deficiency even in the absence of sufficient anemia to account for the symptom. In slowly developing chronic anemias, the patient may not recognize reduced exercise tolerance, or other loss of physical capabilities except in retrospect, after a remission or a cure has been induced by appropriate therapy. Anemia may be responsible for more symptoms than has been traditionally recognized, as suggested by the remarkable improvement in quality of life of most uremic patients treated with erythropoietin. Weakness may accompany anemia or the wasting of malignant processes, in which cases it is manifest as a general loss of strength or reduced capacity for exercise. The weakness may be localized as a result of neurologic complications of hematologic disease. In vitamin B12 deficiency (e.g., pernicious anemia), there may be weakness of the lower extremities, accompanied by numbness, tingling, and unsteadiness of gait. Peripheral neuropathy also occurs with monoclonal immunoglobulinemias. Weakness of one or more extremities in patients with leukemia, myeloma, or lymphoma may signify central or peripheral nervous system invasion or compression as a result of vertebral collapse, a paraneoplastic syndrome (e.g., encephalitis), or brain or meningeal involvement. Myopathy secondary to malignancy occurs with the hematologic malignancies and is usually manifest as weakness of proximal muscle groups. Foot drop or wrist drop may occur in lead poisoning, amyloidosis, systemic autoimmune diseases, or as a complication of vincristine therapy. Paralysis may occur in acute intermittent porphyria.

SPECIFIC SYMPTOMS OR SIGNS Nervous System

Headache may be the result of a number of causes related to hematologic diseases. Anemia or polycythemia may cause mild to severe headache.

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Chapter 1: Initial Approach to the Patient: History and Physical Examination

Invasion or compression of the brain by leukemia or lymphoma, or opportunistic infection of the central nervous system by Cryptococcus or Mycobacterium species, may also cause headache in patients with hematologic malignancies. Hemorrhage into the brain or subarachnoid space in patients with thrombocytopenia or other bleeding disorders may cause sudden, severe headache. Paresthesias may occur because of peripheral neuropathy in pernicious anemia or secondary to hematologic malignancy or amyloidosis. They may also result from therapy with vincristine. Confusion may accompany malignant or infectious processes involving the brain, sometimes as a result of the accompanying fever. Confusion may also occur with severe anemia, hypercalcemia (e.g., myeloma), thrombotic thrombocytopenic purpura, or high-dose glucocorticoid therapy. Confusion or apparent senility may be a manifestation of pernicious anemia. Frank psychosis may develop in acute intermittent porphyria or with high-dose glucocorticoid therapy. Impairment of consciousness may be a result of increased intracranial pressure secondary to hemorrhage or leukemia or lymphoma in the central nervous system. It may also accompany severe anemia, polycythemia, hyperviscosity secondary, usually, to an immunoglobulin (Ig) M monoclonal protein (uncommonly IgA or IgG) in the plasma, or a leukemic hyperleukocytosis syndrome, especially in chronic myelogenous leukemia.

Eyes

Conjunctival plethora is a feature of polycythemia and pallor a result of anemia. Occasionally blindness may result from retinal hemorrhages secondary to severe anemia and thrombocytopenia or blurred vision resulting from severe hyperviscosity resulting from macroglobulinemia or extreme hyperleukocytosis of leukemia. Partial or complete visual loss can stem from retinal vein or artery thrombosis. Diplopia or disturbances of ocular movement may occur with orbital tumors or paralysis of the third, fourth, or sixth cranial nerves because of compression by tumor, especially extranodal lymphoma, extramedullary myeloma, or myeloid (granulocytic) sarcoma.

Ears

Vertigo, tinnitus, and “roaring” in the ears may occur with marked anemia, polycythemia, hyperleukocytic leukemia, or macroglobulinemia-induced hyperviscosity. Ménière disease was first described in a patient with acute leukemia and inner ear hemorrhage.

Nasopharynx, Oropharynx, and Oral Cavity

Epistaxis may occur in patients with thrombocytopenia, acquired or inherited platelet function disorders, and von Willebrand disease. Anosmia or olfactory hallucinations occur in pernicious anemia. The nasopharynx may be invaded by a granulocytic sarcoma or extranodal lymphoma; the symptoms are dependent on the structures invaded. The paranasal sinuses may be involved by opportunistic organisms, such as fungus in patients with severe, prolonged neutropenia. Pain or tingling in the tongue occurs in pernicious anemia and may accompany severe iron deficiency or vitamin deficiencies. Macroglossia occurs in amyloidosis. Bleeding gums may occur with bleeding disorders. Infiltration of the gingiva with leukemic cells occurs notably in acute monocytic leukemia. Ulceration of the tongue or oral mucosa may be severe in the acute leukemias or in patients with severe neutropenia. Dryness of the mouth may be caused by hypercalcemia, secondary, for example, to myeloma. Dysphagia may be seen in patients with severe mucous membrane atrophy associated with chronic iron-deficiency anemia.

Neck

Painless swelling in the neck is characteristic of lymphoma but may be caused by a number of other diseases as well. Occasionally, the enlarged

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5

lymph nodes of lymphomas may be tender or painful because of secondary infection or rapid growth. Painful or tender lymphadenopathy is usually associated with inflammatory reactions, such as infectious mononucleosis or suppurative adenitis. Diffuse swelling of the neck and face may occur with obstruction of the superior vena cava due to lymphomatous compression.

Chest and Heart

Both dyspnea and palpitations, usually on effort but occasionally at rest, may occur because of anemia or pulmonary embolism. Congestive heart failure may supervene, and angina pectoris may become manifest in anemic patients. The impact of anemia on the circulatory system depends in part on the rapidity with which it develops, and chronic anemia may become severe without producing major symptoms; with severe acute blood loss, the patient may develop shock with a nearly normal hemoglobin level, prior to compensatory hemodilution. Cough may result from enlarged mediastinal nodes compressing the trachea or bronchi. Chest pain may arise from involvement of the ribs or sternum with lymphoma or multiple myeloma, nerve-root invasion or compression, or herpes zoster; the pain of herpes zoster usually precedes the skin lesions by several days. Chest pain with inspiration suggests a pulmonary infarct, as does hemoptysis. Tenderness of the sternum may be quite pronounced in chronic myelogenous or acute leukemia, and occasionally in primary myelofibrosis, or if intramedullary lymphoma or myeloma proliferation is rapidly progressive.

Gastrointestinal System

Dysphagia has already been mentioned under “Nasopharynx, Oropharynx, and Oral Cavity” above. Anorexia frequently occurs but usually has no specific diagnostic significance. Hypercalcemia and azotemia cause anorexia, nausea, and vomiting. A variety of ill-defined gastrointestinal complaints grouped under the heading “indigestion” may occur with hematologic diseases. Abdominal fullness, premature satiety, belching, or discomfort may occur because of a greatly enlarged spleen, but such splenomegaly may also be entirely asymptomatic. Abdominal pain may arise from intestinal obstruction by lymphoma, retroperitoneal bleeding, lead poisoning, ileus secondary to therapy with the vinca alkaloids, acute hemolysis, allergic purpura, the abdominal crises of sickle cell disease, or acute intermittent porphyria. Diarrhea may occur in pernicious anemia. It also may be prominent in the various forms of intestinal malabsorption, although significant malabsorption may occur without diarrhea. In small-bowel malabsorption, steatorrhea may be a notable feature. Malabsorption may be a manifestation of small-bowel lymphoma. Gastrointestinal bleeding related to thrombocytopenia or other bleeding disorder may be occult but often is manifest as hematemesis or melena. Hematochezia can occur if a bleeding disorder is associated with a colonic lesion. Constipation may occur in the patient with hypercalcemia or in one receiving treatment with the vinca alkaloids.

Genitourinary and Reproductive Systems

Impotence or bladder dysfunction may occur with spinal cord or peripheral nerve damage caused by one of the hematologic malignancies or with pernicious anemia. Priapism may occur in hyperleukocytic leukemia, essential thrombocythemia, or sickle cell disease. Hematuria may be a manifestation of hemophilia A or B. Red urine may also occur with intravascular hemolysis (hemoglobinuria), myoglobinuria, or porphyrinuria. Injection of anthracycline drugs or ingestion of drugs such as phenazopyridine (Pyridium) regularly causes the urine to turn red. The use of deferoxamine mesylate (Desferal) may result in rust colored urine. Amenorrhea may also be induced by certain drugs, such as antimetabolites or alkylating agents. Menorrhagia is a common cause of iron deficiency, and care must be taken to obtain a history of the

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6

Part I: Clinical Evaluation of the Patient

number of prior pregnancies and an accurate assessment of the extent of menstrual blood loss. Semiquantification can be obtained from estimates of the number of days of heavy bleeding (usually 1500 cells/μL) or greater eosinophilia, or those with evidence of end organ damage, as these groups are more likely to have serious pathology. The history should include assessment of B symptoms, rash, diarrhea, allergic symptoms, travel history, and food intake. Ingestion of raw or undercooked meat, especially pork, increases the chance of parasitic infection with Trichinella spiralis, which may be accompanied by significant eosinophilia, periorbital edema, myositis, and fever. This infestation usually occurs at festivities where a pig is roasted and served. Pork from abattoirs involves mixing of meat from a large number of pigs, diluting the Trichinella organisms that might have infected a rare animal. The geographic location and lifestyle of the patient determines if consideration of another parasitic infestation is a high probability. In underdeveloped countries, helminthic infections are the most common cause of eosinophilia (see Chap. 62, Table  62–5 for causes of helminthic-induced eosinophilia). Signs of adrenal insufficiency (fatigue, hypotension, hyperpigmentation) a rare cause of eosinophilia, may be subtle. Rhinosinusitis, asthma, and eosinophilia should trigger screening for eosinophilic granulomatosis with polyangiitis (Churg-Strauss syndrome). Mast cell disorders are also associated with eosinophilia, and should be kept in mind in patients with a rash suggestive of urticaria pigmentosa or symptoms of mediator release with identified triggers. Patients with extreme eosinophilia are often critically ill and nearly always require hospitalization because of the high probability of malignancy or infection, in addition to risks for life-threatening damage to the cardiac, respiratory, nervous, and gastrointestinal systems.

BASOPHILIA AND MONOCYTOSIS Disorders associated with basophilia and monocytosis are more limited, and are listed in Chaps. 63 and 70, respectively. In the absence of an obvious infectious/inflammatory insult, basophilia should always trigger evaluation for CML and PV. Unexplained monocytosis, particularly in elderly patients with other cytopenias, should reflex concern for myeloid malignancies such as MDS and chronic myelomonocytic leukemia (CMML) and generally warrants examination of the marrow.

ERYTHROCYTOSIS/POLYCYTHEMIA As opposed to hematologic consultation for the cytopenias, evaluation for polycythemia generally has a more limited differential diagnosis (Chaps. 57 and 84). Technically, “polycythemia” refers to increases in RBC, WBC, and platelets, while “erythrocytosis” more specifically refers to increases in RBCs alone. We are aware, however, that in common hematologic parlance the term polycythemia is frequently used to indicate erythrocytosis and here we use them interchangeably. The disorders that may cause polycythemia are diverse and have widely varying treatments. Attention to detail is critical. First, the distinction between absolute and relative polycythemia should be made. The former refers to a true elevation of the red cell mass, whereas the latter refers to an apparent increase in hemoglobin caused by a contracted plasma volume. Reduced plasma volumes might be seen in patients who are dehydrated and are also reported in chronic smokers. However, smokers are more often polycythemic by virtue of their cardiopulmonary disease, so this distinction is difficult to make. When evaluating a referral for elevated RBC, hemoglobin, or hematocrit, one begins by determining which measure is elevated. Although definitions vary, one may assume polycythemia is present if the hemoglobin is greater than 18.5 g/dL in men or greater than 16.5 g/dL in women.

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Chapter 4: Consultative Hematology

If a referral is received for an elevated RBC count in the absence of true erythrocytosis, a major consideration is thalassemic trait. Additional clues for this would include a normal or low hemoglobin in addition to severe microcytosis, and a blood film showing targets, as well as hypochromia and microcytosis. The history is critical in evaluation of erythrocytosis (Chap. 57). Key items after determining if the abnormality is acquired or congenital include: • • • • • •

History of pulmonary disease or chronic hypoxia Risk factors for renal, hepatic, or CNS tumors History of obstructive sleep apnea Family history of polycythemia Use of androgens or anabolic steroids Surreptitious erythropoietin (EPO) injection (particularly in competitive athletes) • Presence of symptoms related to polycythemia Primary polycythemia refers to autonomous marrow production of erythrocytes, as in PV, whereas secondary polycythemia refers to increased erythrocyte production from stimulation by EPO (Chap. 57). Elevated EPO levels may be a compensatory response to hypoxia or produced in excess by malignancy. Secondary causes of polycythemia are discussed at length in Chap. 57. Historical symptoms related to polycythemia should also be elicited, including headache, fatigue, visual changes, and shortness of breath. Symptoms of pruritus, erythromelalgia, or intolerance of hot water might be more suggestive of PV. Clues during the physical exam include: 1. Digital clubbing, which might suggest pulmonary disease 2. Splenomegaly, which might suggest PV 3. Hepatomegaly, which might suggest PV or a hepatic tumor 4. Facial plethora, which is often seen in PV, although can be seen in erythrocytosis of any cause The laboratory evaluation should include a CBC, EPO level, and venous blood gas measurement. The latter is useful in that it allows indirect calculation of the partial pressure required to achieve 50 percent saturation (p50) (reduced in high-affinity hemoglobinopathies, which should be considered in cases of familial polycythemia) and also provides information regarding carboxyhemoglobin (elevated in smokers or in carbon monoxide poisoning) and methemoglobin levels. The combination of polycythemia and a low EPO level is highly suggestive of PV. This should trigger reflex mutational testing for JAK2V617F (exon 14), and, if negative, Janus kinase 2 (JAK2) exon 12 mutation. These two mutations capture nearly all cases of PV, and if negative, should trigger the diagnosis to be reconsidered and/or tertiary referral. More commonly, the EPO level is found to be normal or elevated. This makes PV less likely, although certainly not exclusionary. If the patient has symptoms suggestive of PV or otherwise unexplained polycythemia, JAK2 mutational testing should still be obtained. If the EPO level is high normal/elevated and there are no PVrelated symptoms, a thorough evaluation for secondary polycythemia should be performed. In the absence of cardiopulmonary disease or obvious offending medications, polycythemia with a significantly elevated EPO level should trigger evaluation for malignancy. Although rare, familial polycythemia should always be in the differential, particularly if the family history is suggestive. The various mutations in regulators of erythropoiesis and hypoxia-sensing, and are discussed in detail in Chap. 57. A diagnostic algorithm for erythrocytosis is shown in Chap. 57, Fig.  57–6.

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THROMBOCYTOSIS When evaluating thrombocytosis, one must generally determine whether it is reactive or the manifestation of a MPN (Chaps. 84 to 86). Historical details should include: When were the platelets first elevated? Is the elevation intermittent or constant? Have the platelets ever exceeded 1,000,000/μL? Has there been any thrombosis? Has there been paradoxical bleeding suggestive of acquired von Willebrand disease (VWD)? Causes of reactive thrombocytosis (Chap. 119), such as inflammatory disease, infection, recent splenectomy, iron deficiency, and malignancy, should be explored. The physical exam should include evaluation for organomegaly, given this may be seen in a variety of MPNs. In reviewing the CBC, a concomitant elevation of the hemoglobin and platelets might suggest PV. Neutrophilia, myeloid immaturity, or basophilia might suggest CML or myelofibrosis. If there is significant suspicion for a MPN, such as organomegaly, persistent thrombocytosis, polycythemia, or neutrophilia, additional evaluation might include molecular testing for BCR-Abl, JAK2, and/or calreticulin mutations (Chaps. 84–86).

PREGNANCY Hematologic issues arising during pregnancy are a common cause for consultation. In contrast to the nonpregnant patient, the consultant must consider both the patient and the fetus (Chaps. 7 and 8). In this section we will not consider hematologic disorders of the fetus such as hemolytic disease of the newborn and neonatal alloimmune thrombocytopenia (Chaps. 8 and 55). During the transition from the second to third trimester, the plasma volume increases by approximately 1.0 L while the RBC mass increases by approximately 0.25 L. This partial hemodilution can cause a drop in hemoglobin values below the normal 12 g/dL for women. There are further complicating issues. The growing fetus requires approximately 500 mg of iron from the mother, and if the mother is already iron deficient and/or not taking adequate iron supplements, iron-deficiency anemia will develop. In mothers with thalassemia intermedia, the marrow is already stressed and providing close to maximum compensatory erythropoiesis at baseline. The marrow cannot provide the additional 0.25 L of RBC mass during pregnancy, causing hemoglobin levels to fall where both the patient and fetus may be stressed. Supportive transfusions are often necessary in this case. One is often asked to consult because of a neutrophilic leukocytosis occurring during the second and third trimesters. This is typically physiologic and the film may show myeloid immaturity with bands, metamyelocytes, and even myelocytes. Observation is recommended. Other than the variations noted above, the approach to anemia and WBC abnormalities is essentially identical to the nonpregnant patient. Thrombocytopenia as low as 50,000 to 70,000/μL may be seen as a normal consequence of pregnancy and is termed gestational thrombocytopenia. It requires no specific management. Nongestational thrombocytopenia, however, requires particular care because the differential is broader in pregnancy and because of the risk of both maternal and fetal hemorrhage. For example, when treating patients with ITP, there is concern that the causative antiplatelet antibody will cross the placenta and cause fetal thrombocytopenia. The distortion of the fetus, particularly of the cranium during delivery, raises concern about intracranial hemorrhage if the neonate is thrombocytopenic. Newborns of mothers with ITP should generally have serial platelet counts over the first week of life as thrombocytopenia may be delayed as splenic function develops. The appearance of low platelets along with hypertension in the third trimester raises concern for preeclampsia. Perhaps a more-severe

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Part I: Clinical Evaluation of the Patient

variant of preeclampsia is the HELLP syndrome (Chaps. 8 and 51), an emergency where low platelets are accompanied by microangiopathic hemolysis and hepatic dysfunction. The blood film and liver function tests are essential in making this diagnosis. Disorders of hemostasis/coagulation include placental abruption and retained products of conception. These two conditions are unique to pregnancy and release large amounts of necrotic tissue into the circulation, leading to DIC (Chap. 129). The consultant is often called because of severe bleeding from many sites, including the vagina, vascular access sites, and surgical incisions. Laboratory findings often demonstrate profound anemia and thrombocytopenia, and the blood film shows microangiopathy. The coagulation panel demonstrates a prolonged prothrombin time (PT) and partial thromboplastin time (PTT), low fibrinogen, and significantly elevated D-dimer. If assayed, virtually all coagulation factors will be low. The key to management is prompt recognition, establishment of adequate IV access, and massive replacement of RBCs, platelets, and coagulation factors, along with removal of the uterine contents. Two disorders have a specific predilection for the immediate postpartum period: appearance of a factor VIII inhibitor and postpartum TTP. Patients with a factor VIII inhibitor will show a long PTT and mixing studies will identify and quantify the inhibitor. Patients with postpartum TTP will have a clinical presentation like other TTP patients: microangiopathic hemolysis, thrombocytopenia, and potentially CNS and renal involvement.

BLEEDING Clinical bleeding is a common request for hematologic consultation. The setting and context provide important clues about the diagnosis. Requests arising from the ICU or emergency room (ER) usually relate to a specific event like trauma or surgery. These requests may involve hemodynamic compromise and often mandate a rapid response. Conversely, complaints about “easy bruising” can be equally ominous, but often don’t rise to the same level of acuity. The conventional wisdom is that mucosal and skin bleeding is likely to be caused by platelet abnormalities (qualitative or quantitative), vascular disorders, or VWD, whereas deep tissue or joint bleeding is caused by coagulation factor deficiencies. This is generally accurate, although there is substantial overlap caused by factors like age and comorbid medications (including aspirin, nonsteroidal antiinflammatory drugs [NSAIDs], and anticoagulants). As expected, the history is critical. One must inquire about bleeding length, duration, and context. A long duration and early onset suggest a hereditary disorder. Bleedings that occurs after dental extractions or surgery are clues for mild hemophilia or VWD. If the bleeding always occurs at one site, there may well be a local issue, whereas bleeding at multiple sites points to a systemic disorder. Drug history is critical, not only for anticoagulant drugs, but also for the many agents that cause drug-induced platelet functional abnormalities (Chap. 121). A history of alcohol abuse, hepatic disease, or renal disease is relevant. The exam should be used to identify petechiae, oral blood blisters, ecchymoses, hematomas, giant hemangiomas, hemarthroses, and liver/ spleen size. Laboratory analysis should include a blood film, CBC, and chemistry panel. A basic coagulation panel containing a PT, PTT, fibrinogen, D-dimer, thrombin time (TT), and reptilase time (RT) is also important (Chaps. 114 and 116). The PT and PTT screen both the intrinsic and extrinsic pathways. The fibrinogen and D-dimer provide useful information about DIC and fibrinolysis, while a discrepancy between the RT and TT can determine whether there is in vivo presence of heparin.

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Before deciding that a prolonged PT or PTT is because of a factor deficiency, consider the possibility of an inhibitor by ordering a mixing study. If an inhibitor is ruled out, then one can determine whether specific factor assays are warranted. A prolonged PTT might also be the result of a lupus anticoagulant, which generally poses a risk for thrombosis rather than hemorrhage. If there is recurrent mucosal bleeding and a family history of bleeding, we generally obtain screening for VWD. A platelet function analysis (PFA) is a highly sensitive test for VWD. Additional tests, such as a von Willebrand antigen, ristocetin cofactor activity, and factor VIII levels can be used to confirm the diagnosis. More expensive studies, such as multimer analysis and ristocetin-induced platelet aggregation (RIPA) are generally not needed if screening studies are negative. A variety of disorders are associated with acquired VWD, including cardiac valvular disorders, extreme thrombocytosis, paraproteinemias, and autoimmune disorders (Chap. 126). Mucosal or posttraumatic bleeding might also suggest a platelet functional defect. Screening with a PFA is often useful before launching into more detailed studies, such as formal platelet aggregometry. A low fibrinogen and markedly elevated D-dimer points to DIC, which can be seen in a variety of systemic illnesses and is associated with both bleeding and thrombosis. Of course, not all bleeding is related to disorders of coagulation factors and platelets. One should not forget vasculitis or other vascular defects, such as hereditary hemorrhagic telangiectasia (HHT), when evaluating a patient with recurrent mucosal bleeding and epistaxis. Vitamin C deficiency (scurvy) can rarely be encountered in alcoholics or severely malnourished individuals and results in bruising and gingival bleeding because of defective collagen synthesis. Pathologic fibrinolysis may also result in bleeding and is not easily assessed by standard coagulation panels (Chap. 135). We have used thromboelastography to evaluate global hemostasis on an individualized basis, although this study lacks broad clinical utility. Finally, preoperative consultations for bleeding can be frustrating because there are no generally accepted guidelines. The most important tool is the history including family and personal history of bleeding. The type, site, and timing of prior episodes of bleeding, whether postoperative, traumatic, or spontaneous, provide the requisite information upon which to base further testing as discussed above.

THROMBOSIS VENOUS THROMBOSIS Consultations regarding deep venous thrombosis (DVT) may be daunting. The decision to commit a young patient to indefinite anticoagulation (AC), or to cease AC in an older patient at increased risk for recurrence often gives the physician pause. Here we discuss some of the highlights of our approach to venous thromboembolism. The individual components are discussed at length in other chapters, including principles of AC (Chap. 25), DVT (Chap. 133), hereditary thrombophilia (Chap. 130), and the antiphospholipid antibody syndrome (APS; Chap. 131). Patients often have significant anxiety after a thrombotic event. Although considerable progress has been made in the safety and convenience of AC, the process still poses serious risk and may impair the patient’s quality of life. It is important to communicate that AC management is inherently complex, therapeutic approaches must be individualized, and, ultimately, no approach is without risk, including life-threatening bleeding and/or thrombosis.

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Chapter 4: Consultative Hematology

One should begin the evaluation of a new DVT by defining the context of the event. Critical questions include: 1. Were there any risk factors, such as surgery, immobility, trauma, indwelling catheters, cirrhosis, nephrotic syndrome, inflammatory disorders, or systemic estrogen therapy (Chap. 133)? 2. If present, are those risk factors modifiable and might they have reasonably provoked the event? 3. If dealing with a reoccurrence while on AC, had the patient been compliant with therapy? 4. Are there historical or physical clues that might suggest malignancy, APS, or a hereditary thrombophilia? When probing hard enough, one is often able to identify a “risk factor” for thrombosis. This alone is insufficient to label an event “provoked”; rather, that risk factor must be thought to have reasonably caused the event. Attribution of causality is arduous and ultimately subjective. When dealing with recurrent events in patients on AC, compliance must be probed at length. Labeling a patient as having “failed” therapy has significant consequences. First, this might suggest an underlying thrombophilia, such as malignancy or APS. Second, the lack of clear superiority data of one agent over another makes management decisions murky. The physical exam must of necessity focus on the affected extremity. However, a comprehensive exam may provide valuable clues. Adenopathy or temporal wasting might suggest malignancy. Arthritis and malar rash might suggest an autoimmune diathesis such as lupus. Organomegaly and erythromelalgia should trigger concern for an MPN such as PV or ET. Spontaneous upper-extremity events might suggest thoracic outlet syndrome whereas unprovoked left iliofemoral DVT, particularly in young women, may represent May-Thurner syndrome. Screening for underlying hereditary thrombophilias is often pursued although the general utility of this approach is not clear. This is discussed at length in Chap. 130. In general, such screening rarely changes management and has the potential for error if performed at the incorrect time. For example, levels of antithrombin III, protein C, and protein S may be falsely low in the acute setting. Functional protein S levels might be reduced when the factor V Leiden mutation is present, leading to erroneous diagnosis. Anticardiolipin antibodies require sustained elevation over 12 weeks to satisfy criteria for APS. Pregnancy and hepatic disease can also affect the serum levels of various pro- and anticoagulants and confound diagnosis. The decision regarding the duration of therapy is complex (Chap. 133). These decisions must incorporate the risk of recurrence, risk of bleeding, and patient preferences. Generally, patients with either a provoked or distal DVT may be treated for a finite course, generally three months. Patients with unprovoked DVT/pulmonary embolism (PE), APS, recurrent thromboses, or active malignancy are often considered for indefinite therapy should the bleeding risk be acceptable. In patients with unprovoked events who discontinue AC after a finite course, efforts aimed at risk stratification via D-dimer assays appear to be useful. Thromboprophylaxis with aspirin, has shown promise. However these approaches are not standardized. The American College of Chest Physicians publishes evidence-based antithrombotic guidelines that provide specific recommendations for a variety of scenarios and are a useful resource. The availability of new, oral anticoagulants has dramatically changed AC management from both the patient and physician perspectives. Dabigatran, a direct thrombin inhibitor, and rivaroxaban, a factor Xa inhibitor, are FDA approved for the treatment of venous thromboembolism, with the former requiring an initial 5 to 10 days of parenteral

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AC. Both agents are oral, require adequate renal function, and produce a reliable anticoagulant effect that need not be monitored or titrated. They lack reliable antidotes in the event of bleeding, although such products are in development. It should be noted that patients with poor warfarin compliance are equally poor candidates for these agents. Because of their short half-life, skipped doses will result in a prompt loss of AC and increased risk of recurrent thrombosis. In general, there is insufficient information regarding the superiority of one anticoagulant over another. There is abundant experience with warfarin (Coumadin) and LMWH. The latter is often preferred in patients with malignancy, although high-quality evidence in its support is lacking. Dabigatran and rivaroxaban lack specific data in hereditary thrombophilias, malignancy, and APS, and therefore caution should be exercised in these settings. Dabigatran showed an excess risk of bleeding and thrombosis when compared to Coumadin in patients with mechanical heart valves, exemplifying the potential peril in assuming anticoagulants are of equal efficacy in different settings. Decisions regarding AC touch upon every aspect of a patient’s life and are not taken lightly. They are not as black and white as standardized chemotherapy regimens and require an understanding of the patient’s lifestyle, values, and risk of recurrence. Such assessment is difficult in the modern time-constrained environment. Furthermore, such decisions should not be made in a single-instance and then followed indefinitely. Rather, the decision to continue (or withdraw) AC is dynamic and should be revisited serially depending on the tolerance of therapy and other medical comorbidities.

ARTERIAL THROMBOSIS Consultation is often requested in patients with arterial thrombosis, such as myocardial infarction, cerebrovascular accident, or acute limb ischemia. In the vast majority of cases, however, this is related to underlying atherosclerosis with local inflammation rather than a primary hypercoagulable state. Risk factors, mechanisms, and treatment of atherothrombosis are discussed in Chap. 134. In rare cases where underlying risk factors for atherothrombosis are absent or there is a strong family history of thrombosis, particularly at young ages, we perform a limited hypercoagulable evaluation, including studies for the APS. Paroxysmal nocturnal hemoglobinuria (PNH) and MPNs may rarely result in arterial thromboses. We rarely find it helpful to obtain studies for protein C, protein S, or antithrombin III deficiency, and do not obtain studies for factor V Leiden or prothrombin 20210A mutations as these do not have a meaningful effect on management. Furthermore, routine screening for the thermolabile variant of the methylenetetrahydrofolate reductase (MTHFR) should be discouraged as there is no evidence of benefit in reducing plasma homocysteine levels. Hence, broad hypercoagulable evaluations are not useful in isolated arterial thrombosis, as most findings are likely incidental rather than causal, and do not have a direct impact on patient management.

IMMATURE CELLS ON THE BLOOD FILM Consultations may arise from the discovery of incidental abnormalities on the blood film. Nucleated RBCs (nRBCs) and immature myeloid cells are relatively common.

NUCLEATED RED BLOOD CELLS The clinical lab may report the finding of nRBCs, which is often reported in the differential as #nRBC/100 WBC.

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Although there are several causes for the appearance of nRBCs, two predominate: stress erythropoiesis and extramedullary hematopoiesis. Stress erythropoiesis occurs as a response to severe anemia. The marrow attempts to compensate by increasing erythropoiesis (Chaps. 32 and 33) and discharges reticulocytes of increasingly younger age to the blood. If the anemic stress is not ameliorated or deepens further, nRBCs, usually late normoblasts, leave the marrow. The blood film often shows abundant polychromasia, skip macrocytes, and late normoblasts. Extramedullary hematopoiesis can occur if the normal marrow is replaced by fibrosis or cancer or if somatic mutation such as seen in primary myelofibrosis decreases stem cell adherence (Chap. 86). The hematopoietic stem cells travel through the blood to find suitable alternative sites for growth, often settling in the spleen. However, the sinusoidal structure there is not identical to that of the marrow. Hence, blood cells, particularly nRBCs, are released in an uncoordinated manner. A leukoerythroblastic smear (nRBCs, teardrop RBCs, myeloid immaturity, giant platelets) is a significant clue. If clinically indicated, biopsy of the marrow will usually confirm the diagnosis.

Critical diagnostic elements include personal history, family history, CBC, and the blood film. Has the diagnosis been made on physical exam or was it detected incidentally on an imaging procedure? If prior studies are available for comparison, one may be able to use the EMR to determine how long the spleen has been enlarged. On exam, is the spleen tip barely palpable or does it extend into the pelvis and cross the midline as might be the case with primary myelofibrosis (Chap. 86)? Evidence of cirrhosis or heart failure might suggest congestive splenomegaly. Erythrocytosis and pruritus would raise concern for PV. A blood film showing basophilia and myeloid immaturity might indicate CML. If risk factors are present, HIV testing is appropriate. Thalassemia can readily be identified via the blood film. Because the diagnostic possibilities are innumerable, it is critical to avoid a shotgun approach, identify the likely diagnostic possibilities, and focus the workup accordingly. It is almost never necessary to do a diagnostic splenectomy.

IMMATURE MYELOID CELLS

Monoclonal gammopathies are increasingly detected given the wide availability of comprehensive metabolic panels and the subsequent use of serum protein electrophoresis (SPEP) as a screening tool. Referring physicians often have already requested a SPEP in cases of suspected myeloma, anemia, unexplained renal failure, or neuropathy. The finding of elevated serum protein or globulin fractions, or the report of extensive rouleaux formation on blood film may also trigger a request for SPEP. The SPEP can demonstrate the presence of a monoclonal protein whereas immunofixation defines the heavy-chain isotype and lightchain restriction. Rouleaux formation reported on a CBC is not synonymous with a monoclonal protein. Rouleaux simply describes visible stacked red cells, either as a result of poor smear preparation, inappropriate viewing in the “thick” area of the slide, or as a consequence of increased plasma proteins, such as immunoglobulins and fibrinogen. Although rouleaux may result from a monoclonal gammopathy, it may also be seen in chronic infections, autoimmune disorders, and liver disease. When seeing referrals for monoclonal gammopathies, one should obtain a CBC, renal and liver function studies, blood film, immunoglobulin panel, free light-chain ratio, urine protein electrophoresis (UPEP) with immunofixation, and skeletal survey. This analysis seeks to identify any evidence of end organ damage attributable to the monoclonal population, such as hypercalcemia, anemia, renal dysfunction, or lytic bone disease. The physical exam focuses on the presence of adenopathy or organomegaly that might suggest a lymphoproliferative disorder. If there is no clear evidence of end organ damage, the term monoclonal gammopathy of undetermined significance (MGUS) is often applied, and observation is pursued. The natural history, risk stratification, and prognosis of such patients in discussed in Chaps. 106 and 107. Monoclonal proteins may also be seen in association with several disorders, including plasma cell dyscrasias, lymphoproliferative disorders, infections, and various autoimmune diseases. Notably, it is important to identify patients with signs concerning for immunoglobulin light-chain amyloidosis (nephrotic range proteinuria, macroglossia, neuropathy). In cases of immunoglobulin (Ig) M paraproteins, look for evidence of Waldenström macroglobulinemia (adenopathy, constitutional symptoms, organomegaly, bleeding). Although rare, in patients with anemia and IgM κ paraproteins, one should inquire about cold sensitivity and seek to exclude cold agglutinin hemolysis. One is often faced with an elderly patient with multiple comorbidities, such as chronic renal dysfunction, peripheral neuropathy, diabetes mellitus, and osteoporosis in conjunction with a systemic paraprotein.

The clinical lab may report the presence of metamyelocytes, myelocytes, promyelocytes, or blasts on the blood film. The differential is enormous, ranging from normal pregnancy to myeloid malignancies (Chaps. 61–63). One begins with a thorough history, CBC, and blood film. We inquire about recent stressors and the use of glucocorticoids. In patients with normal blood counts and rare metamyelocytes, observation is generally appropriate. The presence of peripheral blasts, particularly in association with cytopenias, is never normal and mandates examination of the marrow. A full spectrum of myeloid maturity, particularly in association with basophilia, should raise concern for CML.

LYMPHADENOPATHY On occasion, consultation is requested for lymphadenopathy without a tissue diagnosis. The differential diagnosis is vast, including benign adenopathy, viral infections, autoimmune disorders, and malignant lymphomas. A thorough history is critical (Chap. 1). Laboratory studies should include a CBC and blood film. Generally with this information, the diagnostic studies can be focused and lymph node biopsy is not always required. For example, in adolescents with fever, pharyngitis, and cervical adenopathy, tests for infectious mononucleosis are pursued. If there is an accompanying lymphocytosis, the diagnosis might be established via flow cytometry of the blood, as in CLL. If the patient is well appearing, asymptomatic, and the nodes are minimally enlarged, a short period of observation is often prudent. In patients with cytopenias, B symptoms, and organomegaly without obvious infections, lymph node biopsy is nearly always indicated. Fine-needle aspirates, although often more convenient, are discouraged because the lack of lymph node architecture impairs pathologic analysis. If a diagnosis of malignant lymphoma is suspected, treatment with glucocorticoids prior to tissue diagnosis is also discouraged as it may impair diagnostic sensitivity.

SPLENOMEGALY Referrals for splenomegaly open up an immense differential, including hemoglobinopathies, infection, liver disease, heart failure, autoimmune disorders, and malignant leukemia or lymphoma (Chap. 56).

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MONOCLONAL GAMMOPATHY

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Chapter 4: Consultative Hematology

In most cases, if these findings have been chronic and longstanding, they are thought to be unrelated to the paraprotein, and ongoing observation is appropriate rather than cytotoxic chemotherapy. In some cases, particularly those in which historical labs are unavailable, this distinction is more difficult to make, and marrow exam may be useful to assess the degree of marrow effacement. In younger patients with monoclonal proteins greater than 1.5 g/dL, non-IgG isotypes, or abnormal free light-chain ratios, we often obtain marrow biopsies given the higher likelihood of progression and potential intervention for patients with high-risk smoldering myeloma.

ADVICE TO REFERRING PHYSICIANS A good relationship and open line of communication between hematologists and referring physicians are imperative. A few points to keep in mind: • The clinical history is invaluable. If there is lack of clarity, we recommend a quick phone call to the referring physician focusing on the salient features of the patient’s medical history and the reason for consultation. Much like pathologists, this information helps us place the labs and blood film in appropriate context and aids the diagnostic evaluation, particularly in cases with broad differentials such as anemia or leukopenia. The importance of the history and physical exam also reinforces the need for the attending hematologist to personally review the blood film, rather than relying solely on hematopathologists or laboratory technicians. • Avoid the laboratory “shotgun” approach. For example, exhaustive hypercoagulable studies in patients with provoked thromboses are

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not particularly useful and can create patient anxiety. Patients have concerns about their “genetic disease,” and hematologists have a hard time explaining the implications of tests they would not typically order. The consulting hematologist should direct the laboratory evaluation to avoid unnecessary, duplicate, and/or costly tests. • The increasing variety of molecular and genetic diagnostics, in addition to the evolving complexity of hematopathology, mandates one be aware of the resources of their local hematologist. For example, rare disorders such as systemic mastocytosis, CNL, severe eosinophilia, and atypical CML are often best evaluated in a tertiary center. Once the diagnosis is made and a treatment plan established, care should then be transitioned to local physicians, with intermittent input from an academic center if required. Value should always be placed on avoiding repeat marrow examinations. • With rare exception, diagnoses should not be made off scant marrow specimens. Terms such as “aspiculate aspirate” and “subcortical biopsy” should trigger concern for an inadequate specimen. In such cases, a repeat biopsy should be obtained by an experienced provider rather than making diagnostic assumptions from a poor specimen. • A referral to a hematologist, “cancer center,” or hematologist/oncologist often generates considerable patient anxiety, even if not verbalized. The waiting period of several days to weeks to see such a provider can cause significant distress. Unless the diagnosis is clear, it is useful to counsel patients that such a referral does not imply the presence of “cancer” or “leukemia” but rather a request for more information.

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Part II  The Organization of the Lymphohematopoietic Tissues 5. Structure of the Marrow and the Hematopoietic Microenvironment . . . . . . 53

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6. The Organization and Structure of Lymphoid Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

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CHAPTER 5

STRUCTURE OF THE MARROW AND THE HEMATOPOIETIC MICROENVIRONMENT

Utpal P. Davé and Mark J. Koury*

  The marrow stroma consists principally of a network of sinuses that originate at the endosteum from cortical capillaries and terminate in collecting vessels that enter the systemic venous circulation. The trilaminar sinus wall is composed of endothelial cells; a thin basement membrane; and adventitial reticular cells that are progenitors of chondrocytes, osteoblasts and adipocytes. Stem cells can leave and reenter marrow as part of their normal circulation.   Hematopoiesis, the proliferation and differentiation of stem cells and their progeny in the intersinus spaces, is controlled by a complex array of stimulatory and inhibitory cytokines, cell–cell contacts, and interactions with the extracellular matrix. In this unique environment, lymphohematopoietic stem cells differentiate into all the blood cell lineages. Mature cells are produced and released to maintain steady-state blood cell levels. The hematopoietic system also can respond to meet increased demands for additional cells as a result of blood loss, hemolysis, inflammation, immune cytopenias, and other causes.

SUMMARY The marrow, located in the medullary cavity of bone, is the site of hematopoiesis in humans. The marrow produces approximately 6 billion cells per kilogram of body weight per day. Hematopoietically active (red) marrow regresses after birth until late adolescence, after which it is focused in the lower skull, vertebrae, shoulder and pelvic girdles, ribs, and sternum. Fat cells (yellow marrow) replace hematopoietic cells in the bones of the hands, feet, legs, and arms. Fat comprises approximately 50 percent of red marrow in the adult. Further fatty replacement of the red marrow continues slowly with aging, but hematopoiesis can be expanded when demand for blood cells is increased.

Acronyms and Abbreviations: AGM, aorta-gonad-mesonephros; ALCAM, activated leukocyte adhesion molecule; bFGF, basic fibroblast growth factor; BFU-E, burstforming unit–erythroid; BMP, bone morphogenetic protein; CAR, CXCL 12–abundant reticular cells; CD, cluster of differentiation; C/EBP, CCAAT/enhancer-binding protein; CFU-E, colony forming unit–erythroid; CFC-G, colony-forming cell-granulocyte; CXCL12/SDF1, stromal cell-derived factor; dpc, days postcoitum; EBI, erythroblastic island; ECM, extracellular matrix; ELAM, endothelial leukocyte adhesion molecule; EPO, erythropoietin; FN, fibronectin; GAG, glycosaminoglycan; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; GMP, granulocyte-macrophage progenitor; HGF, hepatocyte growth factor; HIF, hypoxia-inducible factor; HSC, pluripotent hematopoietic stem cell; ICAM, intercellular adhesion molecule; IHH, Indian hedgehog family of proteins; IL, interleukin; LFA, lymphocyte function antigen; MAdCAM, mucosal addressin cell adhesion molecule; M-CSF, macrophage colony-stimulating factor; MEP, megakaryocytic-erythroid progenitor; MIP, macrophage inflammatory protein; MMP, matrix metalloproteinase; MPP, multipotential pluripotential progenitor; MSC, mesenchymal stem cell; NFAT, nuclear factor of activated T cells; NK, natural killer; OPG, osteoprotegerin; PDGF, platelet-derived growth factor; PECAM, platelet endothelial cell adhesion molecule; PPAR, peroxisome proliferator-activated receptor; ProEBs, proerythroblasts; PSGL, P-selectin glycoprotein ligand; RANK, receptor activator of nuclear factor-κB; Rb, retinoblastoma tumor-suppressor protein; SCF, stem cell factor; Siglecs, sialic acid-binding immunoglobulin (Ig)-like lectins; SP, side population; TGF-β, transforming growth factor-β; TLR, toll-like receptor; TNF-α, tumor necrosis factor-α; TPO, thrombopoietin; TRAP, tartrate-resistant acid phosphatase; TSP, thrombospondin; VCAM, vascular cell adhesion molecule; VEGF, vascular endothelial growth factor; VLA, very-late antigen. Marshall A. Lichtman was an author of this chapter in the previous six editions, and some material from those editions, including all illustrations, has been retained.

*

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HISTORY AND GENERAL CONSIDERATIONS The marrow, one of the largest organs in the human body, is the principal site for blood cell formation. In the normal adult, daily marrow production amounts to approximately 2.5 billion red cells, 2.5 billion platelets, and 1 billion granulocytes per kilogram of body weight. The rate of production adjusts to actual needs and can vary from a basal rate to several times normal. Until the late 19th century, blood cell formation was thought to be the prerogative of the lymph nodes or the liver and spleen. In 1868, Neuman1 and Bizzozero2 independently observed nucleated blood cells in material squeezed from the ribs of human cadavers and proposed that the marrow is the major source of blood cells.3 The first in vivo marrow biopsy probably was done in 1876 by Mosler,4 who used a wood drill to obtain marrow particles from a patient with leukemia. Studies by Arinkin5 in 1929 established marrow aspiration as a safe, easy, and useful technique (Chap. 3). Kinetic studies of marrow cells, using radioisotopes and in vitro cultures, have shown that cell lineages consist mainly of maturing cells with a finite functional life span. On the other hand, sustained cellular production depends on pools of primordial cells capable of both differentiation and self-replication.6 The most primitive pool consists of pluripotential lymphohematopoietic stem cells with the capacity for continuous self-renewal, that is, hematopoietic stem cells (HSCs). The more mature pools consist of differentiating progenitor cells, with their maturation restricted to single or limited numbers of cell lineages and more restricted capacities for self-renewal (Chap. 18). The proliferative activity of these pools involves humoral feedback from peripheral target tissues7 and cell–cell and cell–matrix interactions within the microenvironment of the marrow.8 The marrow stroma and nearby hematopoietic cells provide unique structural and chemical environments (niches) that support the survival, differentiation, and proliferation of pluripotential HSCs. HSC interactive niches9 have been identified at the structural and molecular10 levels and are dynamically controlled by bone morphogenetic proteins (BMPs)11 and factors regulating intramedullary osteoblastic cells and their progenitors.12 Early stem cells can be identified and isolated using a unique array of surface antigen-receptor expressions (CD34+/−, Thy1,lo KIT+, CD38−, CD33−, vascular endothelial [VE]-cadherin+, KDR/FLK1+, FLK2−/FLT3−, CD133+/−)13–18 and have a unique molecular signature.19,20 The ability to efflux specific chemical dyes has also been used to provide enriched populations of HSC.21–24 Isolated cell populations enriched in HSC can be quantified

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Cellularity (%) 100 80

Prenatal

Postnatal

Bone marrow

Yolk sac

Vertebra

Liver

60

Sternum

40 20 0

Spleen

1 2 3 4 5 6 7 8 9 Fetal months

Tibia

10 Birth

Rib

Femur

20

30

40

50

60

70

Age in years

Figure 5–1.  Expansion and recession of hematopoietic activity in extramedullary and medullary sites. For details regarding the nature of yolk sac and hepatic hematopoiesis, see “Sites of Hematopoiesis: Embryogenesis and Early Stem Cell Development.” Chapter 7 provides a more comprehensive treatment of this topic (see Fig. 7–1 in Chap. 7). using in vitro long-term progenitor assays and surrogate in vivo repopulating assays in severely immunodeficient mice and xenogeneic animal models (Chap. 18).

SITES OF HEMATOPOIESIS EMBRYOGENESIS AND EARLY STEM CELL DEVELOPMENT As shown in Fig. 5–1, the marrow is the last in a series of anatomical sites of hematopoiesis that change several times during embryonic and fetal development.25–28 The earliest hematopoietic cells develop in the blood islands of the extraembryonic yolk sac during late gastrulation and form the primitive hematopoietic system. This primitive hematopoiesis is transient, lasting from the appearance of the blood islands at embryonic days 7.5 days postcoitum (dpc) in mice and 19 dpc in humans through the final cellular divisions in the circulating embryonic blood at 13.5 dpc in mice and week 6 in humans.28,29 The large majority of primitive blood cells produced are erythrocytes that enucleate after release into the circulation, and their hemoglobin contains the embryonic α- and β-globin chains. Primitive hematopoietic cells also give rise to macrophages and megakaryocytes. Overlapping with this transient primitive hematopoiesis is definitive hematopoiesis that gives rise to all of the blood cell types found in the adult (Chap. 7). Transplantation experiments in hematopoietically ablated mice have demonstrated that definitive hematopoietic cells arise on 8.5 to 11.5 dpc in mice and weeks 4 to 6 in humans in three different embryonic locations: the yolk sac blood islands, the anterior portion of the aorta-gonad-mesonephros (AGM) region, and the allantoic portion of the developing placenta.26–28 The definitive murine erythroid cells circulating on 8.5 to 11.5 dpc appear to be descendent from a transient population of erythroid/myeloid progenitors derived from the yolk sac, rather than being derived from HSCs that arise in the placenta and AGM as occurs at later times in the fetus and adult.30 Serial transplantation in irradiated mice demonstrated that the earliest appearance of the intraembryonic human HSCs is in the AGM at week 5.31 HSCs migrate through the blood to the fetal liver where they seed and mature into all of the cellular elements of the blood.25–28 Erythrocytes, the predominant cell produced by definitive hematopoiesis during prenatal development, are smaller than the primitive erythrocytes, and their hemoglobin contains the fetal and adult globin chains. In mid-gestation, the HSCs that have migrated to the fetal liver undergo an exponential expansion and

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display a specific integrin (Mac-1) that is not found in marrow HSCs.32 In the last third of gestation, the HSCs and early hematopoietic progenitor cells migrate from the fetal liver through the circulation seeding the spleen and marrow. Fetal liver hematopoiesis declines steadily as the spleen and marrow become the major hematopoietic sites. At birth, the marrow is the major hematopoietic site in humans, while the spleen remains a prominent but decreasing site in the mouse (Chap. 7). Visceral endoderm is in close proximity to the mesoderm formed by gastrulation in those sites where HSCs are generated in the embryo. This proximity is important in that the endoderm appears to induce both endothelial and blood cell development in the adjacent mesoderm through secretion of Indian hedgehog (IHH), a member of the hedgehog family of proteins.33 IHH, in turn, upregulates the expression of BMP4 in the developing mesodermal cells.33 BMP4 upregulation is important for the development of both the endothelial cells that form blood vessels and the HSCs located within these vessels.33,34 Developing endothelial cells and hematopoietic cells in the vessels formed by these endothelial cells are found in each site of primitive and definitive hematopoiesis. The close association of these two cell types in the developing embryo has led to the proposal for their having a common precursor, the hemangioblast.35,36 Important proteins involved in the development of the hemangioblasts are BMP4, VE growth factor receptor KDR/Flk-1, transcription factor TAL1, and TAL1’s binding partner LMO2.35,36 Marked endothelial cells in mice give rise to the HSCs.37 Imaging studies in zebrafish38,39 and mice40 indicate that specialized hemogenic endothelial cells in the ventral part of the aorta can transform without mitosis into HSCs. The differentiation of HSCs from hemangioblasts and/or hemogenic endothelium requires the signaling protein Notch1 and the transcription factors GATA-2, MYB, and Runx1.35,36,41,42 The mechanism driving this earliest expansion of HSC is not well-defined, but two factors that also play roles later, KIT ligand/stem cell factor (SCF) and interleukin (IL)-3, are important in the embryo. BMP4, in addition to its role in the induction of hematopoietic and endothelial differentiation, increases proliferative and self-renewal of HSCs33,34 as it differentially upregulates KIT (SCF receptor) in the HSCs, but not in adjacent endothelial cells.43 Expansion of the earliest definitive HSC is also mediated by Notch signaling as it induces the Runx1 transcription factor41,42 and one of its targets, the IL-3 gene.44

STEM CELL AND MESENCHYMAL CELL PLASTICITY Primitive stem cells from human fetal liver or marrow reconstitute all lymphohematopoietic-derived cells and part of the stromal microenvironment in in vivo repopulation assays.45 These observations are consistent with the early derivation of hematopoietic, vascular, and stromal cells from a CD34−, KDR/Flk-1+, multipotential mesenchymal stem cell.14–16,46 Identification of AC133+, CD34−, CD7− HSCs47 and demonstration of endothelial precursors in AC133+ progenitor cells48 underscore the crosstalk between hematopoiesis and angiogenesis signaling pathways and establish the functional role of hemangioblasts in ontogeny.49–51 As early fetal hematopoiesis is established, the yolk sac vascular networks remain active sites of progenitor production and hematopoiesis.28 Long-term reconstituting HSCs express two members of the ATP-binding cassette genes (ABCG-2 and P-glycoprotein), allowing the efflux of mitochondrial vital dyes such as Hoechst 33342 and rhodamine 123 and their isolation by multiparameter flow cytometry based on their low side scatter (side population [SP] cells).21–24 Enrichment of the SP population for HSC has been achieved in both adult marrow52 and fetal liver53 populations by using the signaling lymphocyte and activation markers (SLAMs) to select cells with the specific phenotype

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(CD150+, CD244−, CD48−). Nearly half of the individual cells in the CD150+, CD244−, CD48− population provide long-term hematopoietic reconstitution in irradiated mice.52 Derivation of hematopoietic cells from adult tissue (muscle, liver) is attributed to resident marrow-derived stem cells in these tissues.54,55 A role for adult marrow-derived mesenchymal stem cells in the repair and regeneration of nonmarrow organs has been described, including cardiac and smooth muscle, liver, and brain.56,57 However, these marrow-derived mesenchymal stem cells function mainly by providing a microenvironment through various cytokines that induce cell growth and stimulate vascularization or by fusing with local cells, rather than by transdifferentiation into specific differentiated cells of the organ undergoing repair (Chap. 18).56,57

HISTOGENESIS Stroma and Hematopoietic Tissue

The formation of the marrow in the third trimester of mammalian prenatal development involves the circulation and chemotaxis of HSCs, which have greatly expanded their numbers in the fetal liver, to the newly developed marrow niche (see “Marrow Structure” below). The release of HSC from the murine fetal liver coincides with the progressive loss of two adhesion proteins, CD144 (VE-cadherin) and CD41 (integrin α2b).58,59 In mice, the seeding of the marrow with HSCs is first detected at 17.5 dpc,60 but the formation of the marrow niches for the HSCs and their progeny occurs in the preceding 3 days in sites of endochondral bone formation.61 Differentiation of a clonal skeletal progenitor stem cell results in cell populations that form cartilage, bone, or marrow niches that either support HSCs or the differentiating progeny of HSCs.62 The specific cells supported by a niche depend upon the expressions of endoglin, Thy1, and aminopeptidase A by the mesenchymal descendants of the skeletal progenitor stem cell. The migration of the circulating HSCs to their supporting marrow niches, which are formed by cells expressing aminopeptidase A but not endoglin or Thy1,62 is directed by the synergistic action of the chemokines CXCL12 and SCF for which the HSCs display the respective receptors, CXCR4 and KIT.60 Other chemotactic factors and adhesion molecules contribute to HSC migration from the fetal liver to the developing bone where their seeding and differentiation initiates marrow hematopoietic function in mammals.58–60 Cavities within bone occur in the human being at about the fifth fetal month and soon become the exclusive site for granulocytic and megakaryocytic proliferation. Erythropoietic activity at the time is confined to the liver. The microenvironment in the marrow becomes supportive of erythroblasts only toward the end of the last trimester (see Fig. 5–1). The formation of the marrow cavities in the developing mouse bones appear at a relatively later time in the prenatal life of mice than humans, and it involves an IHH-regulated63 synchronized maturation of osteoblast progenitors arising from mesenchymal stem cells and osteoclast progenitors arising from HSCs in the areas of mineralized cartilage of the fetal bones.64 Most of the marrow spaces form in the endochondral bones but some marrow develops in the intramembranous bones of the cranium and scapulae. As these respective progenitors differentiate in situ they acquire the phenotype of osteoblasts with expression of osteopontin, osteonectin, bone sialoprotein, and macrophage colony-stimulating factor (M-CSF), and of osteoclasts with expression of tartrate-resistant acid phosphatase (TRAP), calcitonin receptors, and c-FMS (M-CSF receptor).64 In the human, marrow hematopoiesis begins at the 11th week of gestation in specialized mesodermal structures termed primary logettes.65 The logettes are composed of mesenchymal cells and fibers that surround a central artery and protrude into the venous sinuses of the developing marrow cavities. The myeloid and

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55

erythroid hematopoietic cells that populate the logettes are derived not from HSCs but rather from later-committed progenitors.65 Just after birth the HSCs are found in the marrow, and hematopoiesis is evident throughout the marrow cavity.

Adipose Tissue

By the fourth year of life, a significant number of fat cells have appeared in the diaphysis of the human long bones.66 These cells slowly replace hematopoietic elements and expand centripetally until, at approximately 18 years of age, hematopoietic marrow is found only in the vertebrae, ribs, skull, pelvis, and proximal epiphyses of the femora and humeri. Direct measurements of the volume of bone cavities reveal increases from 1.4 percent of body weight at birth to 4.8 percent in the adult,66 whereas blood volume decreases from 8 percent of body weight in the newborn to approximately 7 percent in the adult.67 Expansion of marrow space continues throughout life, resulting in a further gradual increase in the amount of fatty tissue in all bone cavities, especially in the long bones.68,69 Although the quantity of adipose tissue in the head and trochanteric parts of the femur varies in individuals, the fat content of this area of hematopoietic marrow progressively increases as adult humans age.70 The preference of hematopoietic tissue for centrally located bones has been ascribed to higher central tissue temperature with greater vascularity.71 In mice, an increased prevalence of adipose tissue in tail vertebrae as opposed to the more central thoracic vertebrae is associated with fewer HSCs and hematopoietic progenitors.72 Genetic absence of adipose tissue or chemical inhibition of adipocyte generation was associated with improved posttransplant hematopoietic regeneration, suggesting that marrow adipocytes are negative regulators in the hematopoietic microenvironment.72

MARROW STRUCTURE VASCULATURE The blood supply to the marrow comes from two major sources. The nutrient artery, the principal source, penetrates the cortex through the nutrient canal. In the marrow cavity, the nutrient artery bifurcates into ascending and descending central or medullary arteries from which radial branches travel to the inner face of the cortex. After repenetrating the endosteum, the radial vessels diminish in caliber to structures of capillary size that course within the canalicular system of the cortex. In the canalicular system, arterial blood from the nutrient artery mixes with blood that enters the cortical capillary system from the periosteal capillaries derived from muscular arteries.73 After reentering the marrow cavity, the cortical capillaries form a sinusoidal network (Fig. 5–2). Hematopoietic cells are located in the intersinusoidal tissue spaces. Some arteries have specialized, thin-walled segments that arise abruptly as continuations of arteries with walls of normal thickness.74 These vessels give off nearly perpendicular branches analogous to the arterial branching observed in the spleen and kidney, permitting volume compensation for changes in intramedullary pressure. In the marrow cavity, blood flows through a highly branching network of medullary sinuses. These sinuses collect into a large central sinus from which the blood enters the systemic venous circulation through emissary veins. Histomorphic studies of normal murine marrow demonstrate that all hematopoietic cells are within 18 μm or less than 3 cell diameters of a blood vessel.75 Vascular networks consisting of cells expressing CD31, CD34, and CD105 (endoglin) but lacking intercellular adhesion molecule (ICAM)-1, ICAM-2, ICAM-3, or endothelial leukocyte adhesion molecule (ELAM)-1 (E-selectin) can form within the stroma of long-term marrow cultures. These findings underscore the intimate relationship of

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Periosteal artery Periosteal capillaries

Cortex Venous sinuses

Radial branches

Hemopoietic spaces Central sinus

Central artery

Emissary vein

Nutrient artery

Figure 5–2.  Schematic of the marrow circulation (see “Marrow Struc-

ture” for further explanation). (Used with permission of Lichtman MA, University of Rochester.)

blood vessels to hematopoietic activity.76 A study of early hematopoiesis of human marrow from long bones (ages 6–28 weeks) has shown an absence of CD34+ hematopoietic progenitors before onset of hematopoiesis, a predominance of CD68+ cells mediating chondrolysis, and CD34+ endothelial cells developing into specific vascular structures organized by endothelial cells and myoid cells.77 Vascular endothelial growth factor (VEGF) receptors found on CD34+ cells16 and AGM primitive stem cells underscore the common ontogeny.78 Subsets of CD34+ cells expressing the AC133 antigen and the human VEGF receptor-2 (KDR/FLK1) define the functional endothelial precursor phenotype.79 Endothelial progenitors residing in the CD34+, CD11b+ subsets are capable of producing and binding angiopoietins,80 and fibronectin (FN) enhances VEGF-induced CD34 cell differentiation into endothelial cells.81 Growth and remodeling of bone, marrow space, and the vasculature that supplies them with nutrients and oxygen are closely linked by the relative hypoxia of the marrow and surrounding bone.82 The transcription factors, hypoxia-inducible factor (HIF)-1α and -2α, are stabilized by hypoxia and increase VEGF expression in osteoblasts, and lead to regulated, coupled growth by endothelial cells and osteoblasts, both of which have VEGF receptors.82 The expansion of erythropoiesis in response to erythropoietin (EPO) in mice is associated with a reciprocal decrease in the vasculature.83

INNERVATION Myelinated and nonmyelinated nerve fibers are present in periarterial sheaths in the marrow,84 where they are believed to regulate arterial vessel tone. Nerve terminals are distributed between layers of periarterial adventitial cells or localize next to arterial smooth muscle cells.85 Nonmyelinated fibers terminate in the hematopoietic spaces, implying that neurohumors elaborated from free-nerve terminals affect hematopoiesis. Intimate cell–cell communication between sympathetic nerve cells and structural elements within the marrow sinuses occurs at less than 5 percent of nerve terminals that terminate within the hematopoietic parenchyma or on sinus walls. This anatomical unit, termed a neuroreticular complex, consists of efferent (autonomic) nerves and marrow stromal cells connected by gap junctions.85 The marrow is

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highly innervated along the arterioles and less frequently along capillaries, where neurologic control of blood flow and angiogenesis appear to be mediated via neurokinin A and substance P.86

SINUS ARCHITECTURE, NONHEMATOPOIETIC CELL ORGANIZATION AND NICHE FORMATION In mammals, hematopoiesis occurs in the extravascular spaces between marrow sinuses. The marrow sinus wall is composed of a luminal layer of endothelial cells and an abluminal coat of adventitial reticular cells, which forms an incomplete outer lining (Fig. 5–3). A thin, interrupted basement lamina is present between the cell layers. Circulating HSCs move across the sinus endothelium into the extravascular space where they proliferate and differentiate into mature cells, which move across the sinus endothelium and circulate in the blood. Nonhematopoietic cells and extracellular matrix in the extravascular space form the marrow stroma. Stromal cells obtained from animal or human marrow can be studied in cultures,87 are derived from fibroblasts, and have unique phenotypic and functional characteristics that allow them to nurture hematopoietic development in highly specialized microenvironmental niches.88 However, newer studies with mutant mice and mice with specific cells that can be identified by direct fluorescence microscopy89 have led to an understanding of the spatial orientation of the stroma and the localization of hematopoietic niches that they form in the marrow. The hematopoietic niche concept was originally described for an operationally defined murine multipotential pluripotential progenitor (MPP) in the spleen,90 but it has been extended to various marrow hematopoietic subpopulations, including physically demonstrated niches of HSCs,91 lymphoid cells,92,93 and erythroid cells.93,94 The cellular components of these hematopoietic areas of the marrow include several types of nonhematopoietic cells including: (1) the sinus endothelial cells, (2) mesenchymal stem cells (MSCs) that form the skeletal elements of bone and marrow space such as chondrocytes, osteoblasts, osteocytes, fibroblasts, and adipocytes, and (3) terminally differentiated cells of hematopoietic origin such as macrophages, lymphocytes, and plasma cells. Experiments in both mice61 and humans95 have demonstrated by heterotopic bone formation that host marrow sinusoidal endothelial cells and hematopoietic cells will infiltrate and develop within microenvironment provided by a transplanted MSCs and its progeny. In mice, these MSCs are identified by a CD105+, Thy1−, 6C3− phenotype, which can support specific hematopoietic populations as their progeny develop Thy1 and 6C3 expression.62 In humans, these MSCs are identified as CD45−, CD146+ adventitial reticular cells with fibroblast colony forming capacity that can interconvert between this MSC status and CD146− chondrocytes.96 Studies localizing marrow areas that support murine HSCs and their early progeny the hematopoietic progenitor cells (HPCs) have led to the concept of two niches for these hematopoietic cells: an endosteal niche that promotes HSC quiescence and a vascular/perivascular niche that is associated with self-replicating HSCs.97 Studies combining vascular and endosteal imaging demonstrate that HSC/HPCs localized in the endosteal areas were also within a few cell diameters of VE cells.75,98 The hypoxic status of HSC/HPCs, in terms of HIF expression, is unrelated to their proximity to blood vessels,98 the flow rate of blood in the marrow vessels in the vicinity of HSCs appears to be very low,99 and the lowest oxygen tension directly measured in the marrow is in the perivascular areas of microvessels.100 These results suggest that the functional status of microvessels has a larger role in HSC niche activity than the proximity of the potential niche to its vascular supply.

Endothelial Cells

Endothelial cells are broad flat cells that completely cover the inner surface of the sinus.101 They form a major barrier and control the system

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Figure 5–3. Transmission electron micrograph of a

mouse marrow sinus. The small arrow in the sinus lumen (L) indicates the perikaryon of an endothelial cell. Several endothelial cell junctions are present along the circumference of the sinus endothelial wall. Thus, the wall is composed of the cytoplasm of endothelial cells that overlap or interdigitate. Two adventitial reticular cell bodies are identified by arrows at the top and upper left of the sinus. The cytoplasm of the adventitial reticular cells is discontinuous as it is followed around the sinus. Three cytoplasmic processes of adventitial reticular cells are indicated by arrows. Other, smaller processes of reticular cell cytoplasm are found upon close inspection of the sinus periphery and the hematopoietic spaces. The scattered rough endoplasmic reticulum and dense bodies are characteristic of the reticular cell cytoplasm. (Reproduced with permission from Lichtman MA: The ultrastructure of the hemopoietic environment of the marrow: A review. Exp Hematol 9:391, 1981.)

L

1.0 µm

for chemicals and particles entering and leaving the hematopoietic spaces, with overlapping or interdigitating unions permitting volume expansion.102 The endothelium of marrow sinusoids is actively endocytic and contains clathrin-coated pits, clathrin-coated vesicles, lysosomes, phagosomes, transfer tubules, and diaphragmed fenestrae.103,104 Marrow endothelial cells express von Willebrand factor protein,105 type IV collagen, and laminin.106 They also constitutively express adhesion molecules: ICAM-3,107 vascular cell adhesion molecule (VCAM)-1, and E-selectin,108 all of which regulate HSC proliferation.109 The distribution of sialic acid and other carbohydrates on the luminal surface of marrow sinus endothelium is discontinued at diaphragmed fenestrae and coated pits, suggesting such glycosylation plays a role in endothelial membrane function and cellular interactions.110 In vivo, the conditional deletion in endothelial cells of gp130, the common receptor component for several cytokines, including IL-6, leads to a hypocellular marrow as mice age.111 The loss of gp130 from marrow endothelial cells affects the progenitor cell populations rather than the HSC leading to a lethal anemia, a leukocytosis, but normal platelets.108 Marrow endothelial cells via direct cell–cell contacts and secreted peptides uniquely influence osteoprogenitor cell differentiation112 and regulate hematopoiesis.113 Marrow microvascular endothelium has major roles in osteogenesis through its physiologic production of the

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VEGF164 isoform as well as multiple cytokines that are usually associated with inflammation.114 Other marrow endothelial cell cytokines that affect hematopoiesis include SCF,115 angiopoietin-like protein 3,116 IL-5,117 thymosin β4, AcSDKP,118 and B-type natriuretic peptide.119 Endothelial cells also regulate cellular trafficking into and out of the marrow sinusoidal spaces by altering their permeability and reorganizing their cytoskeleton by ICAM-3, by VE-cadherin–mediated cell–cell contacts,107,120 and via specialized heparin sulfate proteoglycans,121 CXCL12 bound to surface proteoglycans,122 and other chemokines/chemokine receptors,123,124 such as fractalkine, a membrane-bound chemokine with a mucin stalk expressed in activated vascular beds.125 Marrow sinusoidal endothelium specifically expresses hyaluron and sialylated CD22 ligands, which are homing receptors for recirculating HSCs75 and B lymphocytes,126 respectively.

Adventitial Reticular Cells

The abluminal or adventitial surface of the vascular sinus is composed of reticular cells.101,127,128 The reticular cell bodies are contiguous with the sinus, forming part of its adventitial coat (see Fig. 5–3). Their extensive branching cytoplasmic processes envelop the outer wall of the sinus to form an adventitial sheath. The sheath is interrupted and is estimated to cover approximately two-thirds of the abluminal surface area of sinuses.

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Figure 5–4.  Scanning electron micrograph of rat marrow sinus. The floor of the lumen (L) is indicated. The arrow on the left indicates the cell body of an adventitial reticular cell, which is just beneath the endothelial cell layer. Reticular cell processes can be seen coursing between the sinus wall and the hematopoietic compartment (small arrows).(Reproduced with permission from Lichtman MA: The ultrastructure of the hemopoietic environment of the marrow: A review. Exp Hematol 9:391, 1981.)

L

The reticular cells synthesize reticular (argentophilic) fibers that, with their cytoplasmic processes, extend into the hematopoietic compartments and form a meshwork on which hematopoietic cells rest (Figs. 5–4 and 5–5). The cell bodies, their broad processes, and their fibers constitute the reticulum of the marrow. Adventitial reticular cells can differentiate along the smooth muscle pathway and contain α smooth-muscle actin, vimentin, laminin, FN, and collagens I, III, and IV.129,130 More specialized contractile reticular “barrier cells” have been described in mouse marrow after hematopoietic stress.131 Barrier cells increase in number and seem to enclose small

vessels and extend the venous sinuses so that release of precursors is restrained while accommodating an increased entry of mature cells into the circulation.131 Studies in both mice and humans have identified subsets of adventitial reticular cells as MSCs with adipocytic-osteogenic potential that in mice appear to have significant overlap: (1) CXCL12–abundant reticular (CAR) cells,132 (2) adventitial reticular cells expressing the intermediate filament protein Nestin and displaying the surface proteins platelet-derived growth factor (PDGF) receptor-α and CD51 (Nestin+, PDGFRα+, CD51+),133 and (3) adventitial reticular cells

Figure 5–5.  Scanning electron micrograph

L

∗

∗∗

Kaushansky_chapter 05_p0051-0084.indd 58

of rat femoral marrow sinus. The lumen (L) of an exposed sinus that has been cut open is indicated. The single asterisk indicates the process of an adventitial reticular cell and the intimate contact it makes with a hematopoietic cell. To the left of this process are adventitial reticular cell fibers, which form a scaffold for hematopoietic cells. The double asterisk identifies a portion of a reticular cell. The hole in the sinus floor is an artifact of preparation or a migration channel bereft of the emigrating cell. Empty spaces between cells and fibers are artifacts of preparation. The arrow to the left points to thin-walled fenestrae in the endothelial cytoplasm. The arrow to the right identifies the portion of a reticulocyte that may be penetrating the sinus wall, early in egress (see Fig. 5–8A). (Used Lichtman MA, University of Rochester.)

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expressing leptin receptors.115,134 The human equivalents are a population of CD45−, CD146+ adventitial reticular cells that have smaller subsets that express Nestin, PDGFRα, and CD51.133 A major proportion of these subsets are restricted to the perivascular area, but have some cells scattered throughout the hematopoietic marrow. However, because these adventitial reticular subsets are also the major sources of CXCL12 and SCF in the marrow, they have important roles in establishing the HSC niche near the marrow microvasculature while their progeny establish the endosteum and its associated hematopoietic niche in the marrow. The majority of CAR cells are in close association with the sinusoidal endothelial cells but some are also associated with the endosteum. Like the Nestin+ MSCs, CAR cells appear to be progenitors of osteoblasts and adipocytes while producing major amounts of CXCL12 and SCF.135 Development of CAR cells and their production of CXCL12 and SCF is associated with the expression of the transcription factor Fox1c.136 CAR cells and the niches that they create in the marrow are required for the normal development of HSC, various differentiation stages of Blymphocytes, natural killer cells, and the plasmacytoid dendritic cells that are all found in close physical association with CAR cells.137 Autonomic neurons innervate the perivascular Nestin+, PDGFRα+, CD51+ adventitial reticular cells which maintain the HSC niche by several surface-displayed and/or secreted products including IL-7 and VCAM-1, in addition to SCF and CXCL12.138 β-Adrenergic neurotransmission inhibits the expression of these proteins so that mice with specific denervation have decreased marrow cellularity and increased circulating hematopoietic progenitors.139 The sympathetic nervous system controls circadian fluctuations in circulating HSC numbers though its effect on MSC expression of the chemokine CXCL12 in the marrow.140 Studies in mice with defective myelinization and mice treated with adrenergic antagonists or agonists indicate that the adrenergic nervous system in the marrow also regulates mobilization of HSCs by granulocyte colony-stimulating factor (G-CSF).141 However, the nonmyelinating Schwann cells associated with the autonomic nerves of the marrow secrete transforming growth factor-β (TGF-β) and thereby maintain the HSC quiescence.142

59

Adipocytes

Adipocytes in the marrow develop by lipogenesis in fibroblast-like cells (Fig. 5–6). Reticular cells in mouse and human marrow can undergo transformation to fat cells in vitro and can revert into fibroblasts in culture by lipolysis,101,143 and the Nestin+ MSCs138 and CAR MSCs can differentiate into adipocytes. A reciprocal relationship between adipocyte and osteoblast differentiation of MSCs appears to be controlled by multiple transcription factors, with both peroxisome proliferator-activated receptor-γ2 (PPARγ2)144 and CCAAT/enhancer binding protein (C/EBP)145 promoting adipocyte differentiation. Marrow fat cells are relatively resistant to lipolysis during starvation, and their phenotype is consistent with both white and brown fat.146,147 Although the proportion of saturated fatty acids is lower than in other fat deposits, marrow fat composition depends on whether it is located in the red, hematopoietically active, or the yellow, hematopoietically inactive, marrow. Human marrow adipocytes support the differentiation of late-stage, committed, myeloid and lymphoid hematopoietic cells, but they are unable to support earlier progenitor stages.148 Quantification of immature hematopoietic cells including HSCs shows reduced numbers in human marrows with increased fat,70 and in vivo studies in mice confirm that marrow adipocytes create a negative hematopoietic microenvironment that reduces development of HSCs and early-stage common hematopoietic progenitors.72

Bone Cells

Osteoblasts, osteoclasts, and elongated flat cells with a spindle-shaped nucleus form the marrow endosteal lining.149 These endosteal cells and the closely associated microvascular cells participate in a dynamic process in which endochondral bone formation proceeds with removal of calcified cartilage and connective tissues by macrophages while new bone is formed by osteoblasts and remodeled by specialized osteoclasts.114,150 Osteoblasts that become embedded in the bone matrix proteins are termed osteocytes, a terminally differentiated cell that has secretory capacity and influences the activities of osteoblasts, osteoclasts, and hematopoietic cells. Resting endosteal cells express vimentin, tenascin, α smooth-muscle actin, osteocalcin, CD51, and CD56. They

Figure 5–6.  Scanning electron micrograph of rat femoral marrow. Several sinuses and the intervening hematopoietic cords are evident. The exposed lumen (L) of one branching sinus is indicated. The sinus, just above the L, contains a bean-shaped proplatelet with an attenuated strand connected to a separating smaller proplatelet fragment. Smaller proplatelet fragments are below the L. The short horizontal arrow points to the cytoplasm of a transected megakaryocyte. The lower arrow points to a fat cell. The rat femoral marrow contains a modest number of fat cells. Spaces in the hematopoietic cords are artifacts resulting from transecting the femur. (Reproduced with permission from Lichtman MA: The ultrastructure of the hemopoietic environment of the marrow: A review. Exp Hematol 9:391, 1981.)

L

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do not react with antibodies to CD3, CD15, CD20, CD34, CD45, CD68, or CD117.151 Enriched CD56+, CD45−, CD34− endosteal cells grown in the presence of cytokines (insulin growth factor I, basic fibroblast growth factor [bFGF], SCF, IL-3, granulocyte-macrophage colony-stimulating factor [GM-CSF]) do not give rise to hematopoietic cells, which suggests they are not totipotent MSCs.151 In the next sections, the two major types of cells responsible for endosteal activity, osteoblasts and osteoclasts, are considered in terms of their potential roles in maintaining the hematopoietic niche.

Osteoblasts

Osteoblasts have three major functions: formation of new bone by regulating the secretion of the bone matrix proteins, regulation of bone resorption via osteoclast activity, and regulation of the hematopoietic environment mainly by secretion of cytokines. Bone-forming osteoblast progenitor cells, like stromal precursors, reside in the CD34−, STRO1+ nonadherent marrow cell population.152,153 The differentiation of mesenchymal cells into either osteoblasts or adipocytes is related to the relative activities of Runx2 and PPARγ, respectively.154 With aging, the sensitivity to PPARγ appears to increase, contributing to the increase in adipose tissue in the marrow found with older age.154 BMP2,155 bFGF,156 hepatocyte growth factor (HGF),157 parathyroid hormone12 and endothelin-1158 promote osteoblast growth, whereas the cytokine TGF-β159 and the transcription factor osterix160 promote differentiation. Osteoblasts increase early hematopoietic progenitor survival in long-term cultures and secrete hematopoietic growth factors such as M-CSF, G-CSF, GM-CSF, IL-1, and IL-6.161,162 Osteoblasts also produce various cytokines such as hematopoietic cell-cycle inhibitory factors TGF-β,163 osteopontin,164 and CXCL12,12 as well as cell-cycle stimulatory factor Dickkopf-1,165 all of which contribute to stem cell regulation within the marrow microenvironment. Direct cell–cell communication has been shown in the marrow and in osteoblastic cell networks,166 indicating a potential regulatory role for anatomical gap junctions in hematopoiesis.167,168 The size of stem cell niches increases after osteoblastic expansion and Notch activation in transgenic models.11,12 In another model, intramedullary hematopoiesis and stem cell numbers are severely diminished following in vivo ablation of osteoblasts,169 underscoring the importance of this cell type to the marrow hematopoietic inductive microenvironment. The lymphoid niches for early lymphoid progenitors and differentiating B cells are located adjacent to the endosteal surface.92,93 Osteocytes, which are terminally differentiated osteoblasts trapped in the bony matrix, secrete cytokines into the marrow space that act in a negative feedback manner on new bone formation. Specifically, the osteoblast and stromal cell surface protein receptor activator of nuclear factor-κB ligand (RANKL) activates osteoclasts,170 while the cytokine sclerostin suppresses osteoblast activity.171 Disruption of the signaling mechanism of G-protein receptors in osteocytes leads to an expansion of myelopoiesis that is mediated by secreted myelopoietic cytokines, most likely G-CSF.172

Osteoclasts

Mature osteoclasts are multinucleated giant cells derived from fusion of progenitor cells of the monocyte/macrophage lineage of the HSC.173 The mature osteoclasts resorb and remodel bone, regulate osteoblast activity, and help control the HSC entry into and exit from the marrow.174,175 The osteoclasts have motile and resorptive phases. They require the Wiskott-Aldrich syndrome protein during clustering and fusion of actin-based adhesion structures named podosomes.176 Podosomes are involved in the formation of specific structures termed sealing zones in which actin rings surround an area of ruffled plasma membrane at the face of the endosteal bone. Within these sealing zones, osteoclasts secrete hydrochloric acid and digestive enzymes that resorb bone.

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Osteoclasts also can be derived from pro-B cells, as shown by Pax-5 knockout mice, which have increased osteoclasts and severe osteopenia.177 When osteoclast activity or number are reduced or eliminated in mice through null mutations or homologous recombination, the marrow cavities fail to form resulting in osteopetrosis. Based on studies of osteopetrotic mice, proteins required for osteoclast differentiation include the macrophage transcription factor PU.1; the secreted and surface displayed cytokine M-CSF of stromal cells and its receptor c-FMS on osteoclasts; the transcription factor c-FOS; the cytokine RANKL; its osteoclast receptor RANK, the signaling transducer tumor necrosis factor-α (TNF-α) receptor-associated factor 6 (TRAF 6); the downstream transcription factor nuclear factor (NF)-κB, and nuclear factor of activated T cells (NFAT).175,178,179 Other osteopetrotic mice strains have deficiency of proteins required for the bone resorption function of osteoclasts. These proteins include the β3 component of the αvβ3 integrin (vitronectin receptor) required for binding of the osteoclast sealing zone to bone; c-Src signaling protein; the proton transporting H+ adenosine triphosphatase (ATPase) and chloride channel protein required for HCl secretion; and the secreted osteoclast proteins cathepsin K, matrix metalloproteinases, and TRAP that digest the bone matrix.174,175,179 Osteoblast/stromal cells regulate differentiation of osteoclasts through intimate cell–cell contacts. They are found in direct apposition to osteoclasts with coated pit formation, suggesting accumulation of receptor–ligand complexes in endocytic vesicles.180,181 The recruitment of the osteoblasts and osteoclasts appears to be through capillaries associated with the remodeling compartment.182 A major regulatory mechanism by which osteoblasts and osteoclasts interact is the RANK/ RANKL/osteoprotegerin (OPG) system of signaling.182 Osteoclast differentiation and maturation require the signaling cascade from RANK on the cell surface through TRAF 6, NF-κB, and NFAT.180 Osteoblasts and their progenitor cells display RANKL on their surfaces, and binding of RANKL to the RANK on the osteoclasts and their progenitors promotes differentiation and activation of the osteoclasts. Osteoblasts also secrete OPG, a decoy receptor for RANKL, which inactivates RANKL by binding to the active site of RANKL, thereby preventing its binding to RANK. As a result, osteoclastic activity is decreased when OPG concentrations are high and increased when they are low.183 Another signaling mechanism by which osteoclasts and osteoblasts reciprocally regulate the differentiation and activities of each other is the ephrinB2-EphB4 signaling system.184 Osteoclasts express ephrinB2 on their surfaces while the osteoblasts express EphB4, a member of the receptor tyrosine kinase (RTK) family, which is the receptor for ephrinB2. Binding of ephrinB2-EphB4 results in bidirectional signaling in which osteoclast differentiation is decreased though suppression of the c-FOS–NFATc1 activity, whereas osteoblast differentiation is increased by EphB4 signaling.184 Osteoclasts produce HGF and express c-Met, the HGF receptor, implying a paracrine and autocrine regulatory pathway between them and adjoining osteoblasts.157,185 Similarly, blocking expression of cadherin-6 interferes with heterotypic interactions between osteoclasts and stromal cells, impairing their ability to support osteoclast formation.186 CD9, a tetraspanin transmembrane adhesion protein on stromal cells,187 influences myelopoiesis in long-term marrow cultures.188 Inhibition of stromal cell CD9-mediated signaling by a blocking antibody reduces osteoclast differentiation factor transcription, leading to reduced osteoclastogenesis.189 Macrophage-stimulating protein, a HGF-like protein, signals through the stem cell-derived tyrosine kinase, a member of the HGF receptor family. It also stimulates osteoclast bone-resorbing activity by enhanced cytoskeletal reorganization without affecting proliferation of osteoclast precursors.190,191 Osteoclast differentiation is influenced by monocytes expressing ADAM-8 (CD156), a protein of the disintegrin and metalloproteinase family,192 and eosinophil chemotactic

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Chapter 5: Structure of the Marrow and the Hematopoietic Microenvironment

factor-L (ECFL),193 characterizing complex cell–cell, cell adhesion protein, stromal cell cytokine, and chemokine signals within the marrow microenvironment.

LYMPHOCYTES Lymphocytes, including T, natural killer (NK), B, and plasma cells, and macrophages, including monocyte-derived, antigen-presenting dendritic cells, arise from the HSCs and undergo part of their differentiation in the marrow. They then circulate and, in the case of the lymphoid cells, reside and further differentiate in other organs such as the thymus, spleen or lymph nodes, before returning to the marrow, where they terminally differentiate and form part of the marrow microenvironment by producing growth factors (IL-3, CCL3) and participating in cell–cell interactions with developing progenitors.84,101,194 Monocytic/ macrophage progenitor cells can enter the circulation and later enter many different tissues where they differentiate into macrophages. In the marrow, the monocytic/macrophage progenitors can differentiate into macrophages or fuse and become osteoclasts. Lymphocytes and macrophages concentrate around arterial vessels, near the center of the hematopoietic cords. B cells also cluster near the osteal surface.92,93 Mature B and T lymphocytes in the marrow are in contact with a specific set of monocyte-derived, antigen-presenting dendritic cells that are clustered around the blood vessels.195 Lymphocytic differentiation begins as HSCs that have committed to differentiation as multipotent HPCs (MPPs) lose their potential to become megakaryocytic-erythroid progenitors (MEPs) and granulocyte-macrophage (GM) progenitors; this change in differentiation potential is detectable as the upregulation of lymphoid-specific transcripts, that is, they are lymphoid-primed multipotent progenitors (LMPPs). The commitment of LMPPs to lymphoid differentiation in these early-stage HPCs is reinforced by progressive expression of FMSlike tyrosine kinase 3 (Flt-3), IL-7 receptors (IL-7R), and recombination activating genes-1/2 (Rag-1/2) proteins.196,197 These early lymphoid progenitors (ELPs) require a microenvironment provided by MSCs and their osteogenic progeny which supplies VCAM-1, CXCL12, Flt-3 ligand, and IL-7.198,199 The ELPs enter the blood with transit to the thymus where they undergo differentiation into T cells. In addition, to its role as site of early T-lymphocyte development, the marrow acts a secondary organ for the proliferation of mature CD8 and CD4 memory T lymphocytes. Although no specific organized structure or niche has been found for these T lymphocytes, they can represent up to 4 percent of nucleated cells in the marrow that they reenter by migrating through the sinusoidal endothelium from the blood.200 Alternatively, LMPPs can remain in the marrow and differentiate into common lymphocyte progenitors (CLPs) that give rise to NK progenitor cells, which differentiate in the marrow, or prepro-B cells that mature to the pro-B cells, which migrate from the marrow to the lymph nodes or spleen where they differentiate further.196,197 Marrow stromal cells facilitate the maturation of NK cells,201 an effect likely mediated by stromal-derived Flt-3 ligand and IL-15.202 Within the marrow, both NK cells and CD8+ memory T cells require the coordinated expression of secreted IL-15 and surface IL-15 receptors by other marrow cells for their survival and development.203 The marrow MSCs and their osteogenic progeny also create a microenvironment for proliferation and differentiation of ELPs through the later sequential lymphoid stages of CLPs, prepro-B cells, pro-B cells, and pre-B cells via the provision of osterix and galectin-1.198 The differentiation stages subsequent to the pro-B cells occur after the cells enter the blood and seed the lymphoid follicles of the secondary lymphoid organs, mainly spleen and lymph nodes. From these lymphoid organs, the cells then reenter the blood as B lymphocytes

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or immature plasma cells. The immature plasma cells that have differentiated in the spleen and will become the long-lived plasma cells return home to the marrow, where they are located in contact with CXCL12-producing stromal cells. A negative feedback is completed as the mature plasma cells either compete with the prepro-B cells for sites on the CXCL12-producing stromal cells or directly induce apoptosis of the prepro-B cells.204 Marrow blood vessel-associated dendritic cells produce macrophage migration-inhibition factor, a cytokine required for survival of mature B lymphocytes that have matured in secondary lymphoid organs and recirculated to the marrow.195

MACROPHAGES Hematopoietic progenitors restricted to monocyte/macrophage differentiation are characterized by expression of M-CSF receptors (FMS), membrane-activating complex-1 (CD11b), and F4/80 antigen, and give rise to monocytes that enter the blood.205 These nondividing monocytes can then enter various organs, including a subset with high Ly6C that reenter the marrow where they become macrophages and antigen-presenting dendritic cells.205,206 Although they are both descendants of similar M-CSF–dependent monocytic progenitors, macrophages differ from osteoclasts by their single nucleus and, in mice, expression of F4/80 antigen as well as lack of TRAP and calcitonin receptors.206 Marrow macrophage phenotype207 is regulated by adjoining stromal cell–accessory cell–derived colony-stimulating factors and cytokines,208 such as M-CSF–induced upregulation of α4β1- and α5β1-integrin expression209 and Flt-3 ligand-promoting macrophage outgrowth with B-cell– associated antigens.210 Macrophages are an integral component of the local microenvironment and regulate hematopoiesis via a complex array of dual-acting stem cell stimulatory and inhibitory factors, such as IL-1, CCL3, TNF-α, and TGF-β.211–213 Macrophages respond to PDGF by upregulating IL-1 secretion and thereby activating primitive hematopoietic cells.214 Macrophages also modulate the structure and composition of the extracellular matrix (ECM) and its FN content.215 Specialized macrophages termed osteomacs form a canopy over the active osteoblasts and osteoclasts on the endosteal surface, where the macrophages coordinate the bone-forming activity of osteoblasts and bone-resorbing activity of osteoclasts.206 Another subset of macrophages, which are identified by CD169 (sialoadhesin/Siglec-1 [sialic acid-binding immunoglobulin-like lectin-1]), act to retain in the marrow those HSCs and early progenitor cells that are capable of circulation in the blood.216 CD169-expressing macrophages also comprise the central macrophages of erythroblastic islands that interact directly with erythroid cells,217 enhancing their proliferation and differentiation. Similarly, mature B and T lymphocytes in the marrow are supported in the specific microenvironment provided by monocyte-derived, antigenpresenting dendritic cells that are clustered around the blood vessels.195

EXTRACELLULAR MATRIX Mesenchymal cells forming the cellular stroma in the marrow create a network of ECM proteins, such as proteoglycans or glycosaminoglycans (GAGs), FN, tenascin, collagen, laminin, and thrombospondin (TSP).218–221 Localizing signals are provided by stromal–ECM and hematopoietic cell adhesive interactions222,223 in concert with chemokines224 and cytokines bound to heparin-like structures in the GAGs.225 The binding of specific cytokines may enhance the activity of a cytokine if the GAG-binding site does not interfere with the site that binds the cytokine receptor, whereas GAG-binding sites that overlap or interfere with a cytokine receptor-binding site can inhibit the cytokine function.225 Table 5–1 lists the cytokines that are presented on the surface of stromal cells and matrix-binding chemokines and cytokines.225–238

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TABLE 5–1.  Cell Membrane Presentation and Matrix Association of Cytokines and Chemokines Cell Membrane

Matrix Association

Chemokine

Chemokine

Fractalkine

RANTES, PF-4, IP-10, IL-8 Macrophage inflammatory proteins (MIP-1α, MIP-1β) CXCL12/stromal cell-derived growth factor-1 (SDF-1α, SDF-1β) Monocyte chemoattractant protein-1 (MCP-1)

Cytokine

Cytokine

c-KIT ligand

Granulocyte-macrophage colonystimulating factor

Tumor necrosis factor-α (TNF-α)

Interferon-γ (IFN-γ)

Interleukin-1 (IL-1)

Leukemia inhibitory factor (LIF)

Macrophage colonystimulating factor (M-CSF)

Interleukins (IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-12) Basic fibroblast growth factor (bFGF)

Transforming growth factor-α (TGF-α)

Hepatocyte growth factor (HGF) Transforming growth factor-β (TGFβ; binding to endoglin and heparan sulfate)

IP-10, interferon-inducible protein 10; PF-4, platelet factor 4; RANTES, regulated upon activation normal T-cell expressed and secreted.

In addition to its supply of hematopoietic growth factors, the ECM provides noncellular binding partners for surface adhesion molecules of the hematopoietic and mesenchymal cells. The marrow microelasticity, which is a function of cellular density and ECM composition, varies more than 100-fold from the soft central areas to the much stiffer endosteal areas.239 This microelasticity determines the differentiation of MSCs,240 and the fate of HSCs and committed hematopoietic cells.241 In HSCs and HPCs, the activities of two nonmuscle myosin isozymes are regulated in response to the elasticity of the ECM. The increased relative activity of nonmuscle myosin II B that mediates asymmetric cell division and self-renewal is greatest in the stiffer ECM of endosteal areas, whereas increased relative activity of non-muscle myosin IIA in the areas of softer ECM mediates symmetric cell division and differentiation.239

Proteoglycans

Proteoglycans are polyanionic macromolecules (heparan sulfate, dermatan, chondroitin sulfate, hyaluronic acid) that are distributed on the surface of adventitial reticular cells and within the ECM.218,242 Heparan sulfate is the main cell-surface GAG in long-term marrow cultures, and chondroitin sulfate is the major secreted species.243 D-xylosides, which stimulate artificial sulfated GAG synthesis, increase chondroitin sulfate synthesis and hematopoietic cell production.243 Hyaluronic acid and chondroitin sulfate-containing proteoglycans are prominent in the adherent and nonadherent compartments of long-term marrow cultures.242 Heparin-containing and heparan sulfate-containing proteoglycans interact with laminin and type IV collagen244 and may play a role in cell–cell interactions, cytokine presentation, and cell

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differentiation.245–247 They also mediate progenitor cell binding to other ECM molecules such as FN.248–250 Agrin, a proteoglycan associated with neuromuscular junctions, is produced in the marrow by MSCs, osteoblasts and monocytes, and interacts through α-dystroglycan receptors on HSCs251 and their progeny as they differentiate along the monocyte/macrophage lineage.252 Agrin-deficient mice have hypocellular marrows as a result of decrease in all marrow hematopoietic cell lineages251 as well as specific inhibition of numbers and phagocytic function of monocytes and macrophages.252 An important hematopoietic cell proteoglycan, CD44, has hyaluronate as a major matrix ligand. The CD44–hyaluronate interaction is greatly enhanced by various cytokines, and it promotes other matrix and cellular interactions by hematopoietic cells.253 Cytokines (GM-CSF, IL-3, SCF) rapidly induce CD44 expression and increase CD44-mediated adhesion of CD34+ hematopoietic progenitors to hyaluronan.254 Lymphocyte CD44 has a binding site on the carboxy-terminal heparinbinding domain of FN,255 and neutralizing antibodies to CD44 inhibit hematopoiesis in long-term marrow cultures.256 Chondroitin sulfates A and B mediate monocyte and B-cell activation via a CD44-dependent pathway.257 Hyaluronate binding enhances hematopoiesis by releasing IL-1 in a CD44-dependent manner and IL-6 by a CD44-independent pathway.258 In humans, a specific CD44 isoform displayed on hematopoietic cells is a ligand for E- and L-selectins and plays a role in HSC homing and integrin-mediated transendothelial migration in the marrow.259 Heparan sulfate mediates IL-7–dependent lymphopoiesis235 and modulates hematopoiesis and stromal cell–matrix remodeling260 by anchoring HGF236,261 and bFGF.260,262,263 On the surface of marrow stromal cells, the main heparan sulfate-containing proteoglycans are syndecan-3, syndecan-4, and glypican-1. In the ECM, the most prevalent form is perlecan.264 Syndecan-3 is expressed by marrow stromal cells as a variant form with a core protein of 50 to 55 kDa, suggesting syndecan-3 plays a role in hematopoiesis.264 Perlecan promotes bFGF receptor binding and mitogenesis, and can bind GM-CSF.257,265 Heparan sulfate expression is induced on the cell surface in early erythroid differentiation of multipotential HSC.266 Glypican-4, another member of this family, is found on marrow stromal cells and progenitor cells.267 Syndecan-1 expression in B lymphoid cells is reduced by IL-6, which implies similar regulatory pathways in other cell types.268 Biglycan, a matrix glycoprotein, with homology to osteonectin, and the molecule SIM, a transmembrane protein, selectively increase IL-7–dependent proliferation of B cells.269 Interactions of B cells with other components of the immune system are mediated by syndecan-4, which facilitates the formation of dendritic processes270 and regulates focal adhesion, stress fiber formation, and cell migration.271

Fibronectin

FN localizes at sites of attachment of hematopoietic cells and marrow stromal cells in vitro219,272 and at sites of interaction between these cells and developing granulocytes or monocytes.273 Early erythroid progenitors bind FN through their integrin receptors α5β1 and α4β1.274,275 Adhesion of HPCs to stroma is partly mediated by FN.248,276 The alternatively spliced form of FN (type III connecting segment [IIICS]), which is expressed uniquely within the marrow microenvironment, associates with the α4β1-integrin receptor on HSC.277 Additional IIICS FN variants have been detected in marrow stroma, providing for finely controlled progenitor–stem cell interactions based on messenger RNA splicing.278 FN adhesion to peptide domains, such as the CS1 domain (which activates α4 integrins) or stromal cells, can have dual effects of stimulation and inhibition of hematopoietic progenitor growth.279–282 The very-late antigens (VLA)-4 and VLA-5 (α4β1 and α5β1) and CD44 cooperate to promote FN adhesive interactions.279,282–284 Cytokines

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such as IL-3, SCF, and thrombopoietin (TPO) augment the magnitude of FN-mediated HPC adhesion and migration.285–288 Functional effects of FN within the marrow ECM include decreased erythroblast FN adhesion as differentiation progresses274,283 with modulation of erythroid cell differentiation dependent upon competing binding of α4β1 integrin with FN in the ECM and with central macrophages in erythroblastic islands.289 Binding of collagen I in the marrow ECM by megakaryocytes leads to their spreading and inhibition of proplatelet formation by a mechanism involving FN induction and secretion with polymerization via cosecreted factor XIII-A.290 FN is required for expression of gelatinase in macrophages291 and regulates cytokine release by M-CSF– activated macrophages292 and chondrocytes.293

Tenascin

The fibrillar glycoprotein tenascin-C is found in the microenvironment surrounding maturing hematopoietic cells.218,294 Tenascin-C has distinct functional domains that promote hematopoietic cell adhesion to ECM proteins or mediate a strong mitogenic signal to marrow mononuclear cells.295 Although tenascin-C–deficient mutant mice appear to have normal steady-state hematopoiesis, colony-forming capacity of marrow is markedly decreased,296 marrow regeneration capacity after cytoreductive agents is decreased,297 and retention of T-lymphocyte progenitors is impaired.297 This last effect is mediated through the α9β1 integrin on T-lymphocytes progenitors, but effects on HSCs and early hematopoietic progenitors is mediated by a different mechanism.297 Mutant tenascin-C–deficient animals also display decreased FN in their marrow, suggesting a possible mechanistic interaction between tenascin-C and FN in the marrow microenvironment.298

Collagen

Collagen types I and III are associated with microvascular walls, whereas collagen type IV is confined to basal lamina beneath endothelial cells.160,299 Marrow-derived capillary networks grow in collagen gel cultures,300 inhibition of collagen synthesis reduces hematopoiesis in vitro,301 and collagen-based scaffolds are most effective for in vitro three-dimensional models of the hematopoietic microenvironment.302 Marrow-derived fibroblasts and stromal cells synthesize collagens I, III, IV, V, and VI.303 Collagen VI binds von Willebrand factor and is a strong cytoadhesive component of the marrow microenvironment.304 Erythroid and granulocytic progenitors adhere to collagen type I in vitro.305 Collagen type XIV, another fibril-associated collagen, promotes hematopoietic cell adhesion of myeloid and lymphoid cell lines.306 In situ immunolocalization of ECM proteins in murine marrow shows that collagen types I and IV and FN localize to the endosteum.307 Megakaryocyte binding to collagen I that induces FN secretion and polymerization290 enhances the α2β1-mediated collagen binding by megakaryocytes, permitting increased megakaryocyte adhesion and migration,308 which are also mediated by other megakaryocytic collagen receptors including glycoprotein VI and discoid domain receptor 1(DDR1).309

Laminin

Laminins, multidomain glycoproteins with mitogenic and adhesive sites, are major components of the ECM and basement membranes.310 Laminin interactions with collagen type IV and basement membrane components such as proteoglycans and entactin311 regulate leukocyte chemotaxis.312,313 CD34+ granulocytic progenitors,314 mature monocytes,315 and neutrophils316 adhere to laminins. The role of laminins within the cytomatrix may be to strengthen adhesive interactions with α5β1 (VLA-5) and α6β1 (VLA-6) on hematopoietic cells.316 In combination with FN in vitro, laminins can expand both HSCs and several more differentiated progenitors.317 Laminins are composed of α, β, and γ polypeptides with expression of laminin-2 (α2β1γ1), laminin-8 (α4β1γ1),

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and laminin-10 (α5β1γ1) in the marrow.318 Stromal cells in cultures and cytokine-expanded CD34+cells also express laminin β2, which is found in the pericellular space in marrow and intracellularly in megakaryocytes.319 Laminin-γ2 chain expression, which is unique to marrowderived stromal cells, colocalizes with α smooth-muscle actin in marrow and is not expressed in endothelial cells or megakaryocytes.320 Integrins α6β1 and α6β4 are receptors for laminin-10/11 and laminin-8.314 Laminin-10/11 (α5β1γ1/γ5β2γ1) and FN bind CD34+ and CD34+ CD38–progenitors, whereas laminin-8 (α4β1γ1) and laminin-10/11 facilitate CXCL12–mediated transmigration of CD34+ cells.314 In mouse repopulation studies, antibodies that block the α6 components of laminin receptors decreased the homing of HSCs and colony-forming units–granulocyte-macrophage (CFU-GM).321 When combined with antibodies to the α4 component of integrins, antibodies that block the α6 components synergistically decreased marrow homing of the short-term, multipotent repopulating cells. In contrast to this role of these integrin receptors in the homing of HSCs, a 67-kDa, nonintegrin laminin receptor is upregulated in HSCs following G-CSF stimulation and plays a significant role in their mobilization.322 This 67-kDa nonintegrin receptor for laminin also has a role in the marrow homing of burst-forming units–erythroid (BFU-Es), early-stage erythroid progenitors that circulate in the blood.323 On the other hand, the Lutheran blood group glycoproteins serve as receptors for the α5 integrin component of laminins on the late-stage erythroid cells.324 Laminin promotes the M-CSF–dependent proliferation of marrow-derived macrophages via the α6-integrin subunit,325 and α6β1 mediates mast cell adhesion to laminin.326

Thrombospondin

The TSPs are secreted matrix glycoproteins that modulate cell function by altering cell–matrix interactions.327 TSP1, a multifunctional ECM protein initially identified in platelet α granules, has domains that interact with collagen and FN and may participate in HSC lodgment.328 TSP activates TGF-β329 which results in a stimulatory effect on NK cells.330 TSP binds to matrix heparan sulfates178 and inhibits osteogenic differentiation.331,332 Receptors on hematopoietic and nonhematopoietic cells can interact with TSP, including CD36333 and the cutaneous lymphocyte antigen-1 protein of the CD36/LIMP II gene family.334 CD36 is expressed during erythroid and megakaryocytic maturation.335 TSP stimulates matrix metalloproteinase-9 activity in endothelial cells336 and is chemotactic to monocytes.337 A 140-kDa fragment of TSP1 binds bFGF, and TSP1 acts as a scavenger for matrix-associated angiogenic factors (fibroblast growth factor 2, VEGF, HGF), underscoring its antiangiogenic properties.338,339 Mice deficient in TSP2 demonstrate that TSP2 is taken up in an integrin-dependent manner within the marrow and is necessary for the release of functionally competent platelets by megakaryocytes.340 The 21-amino acid, C-terminal peptide of TSP4 stimulates proliferation of multiple types of early hematopoietic progenitors through the regulator of differentiation 1 (ROD1) nuclear receptor and increases erythropoiesis in mice.341

Vitronectin

Vitronectin, a major cytoadhesive glycoprotein, is present in plasma and the interstitial matrix of tissues. It interacts with a vast number of ECM components, cytokines, growth factors and proteolytic enzymes in vitro and in vivo.342 Vitronectin also binds to several αv-containing integrins,342 including the integrin αvβ3 receptor (CD51) on fibroblasts, endothelial cells, osteoclasts,343,344 and mature hematopoietic cells, including megakaryocytes, platelets,345 and mast cells.346 The integrin αvβ3 is expressed on monocyte-macrophages and neutrophils and mediates their transendothelial migration.347,348 The vitronectin receptor cooperates with TSP and CD36 in the recognition and phagocytosis of

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apoptotic cells by neutrophils, macrophages, and dendritic cells.349–351 Vitronectin-deficient mice have normal blood cell counts,352 but thrombogenesis, new microvessel formation and tissue repair capacity are impaired,353 most likely due to failure of inflammatory and thrombotic mechanisms. Thus, in the marrow ECM, vitronectin functions mainly in the coordination of apoptotic cell clearance, cellular migration, bone remodeling, and angiogenesis.

Other Matrix Proteins

Osteopontin, a glycoprotein produced by osteoblasts and hematopoietic cells in the marrow, binds to FN and collagen.354,355 The predominant form of osteopontin in the marrow is thrombin-cleaved, and its N-terminal peptide is the active ligand for the α9β1 and α4β1 integrins on HSCs and circulating hematopoietic progenitors that plays a role in their attraction to and binding in the marrow.356 Osteopontin can bind numerous integrins and CD44, and its binding through β1-integrin results in suppression of proliferation and maintenance of quiescence in HSCs.354,355 Conversely, the same osteopontin–β1-integrin pathway induces proliferation in erythroblasts.357 Osteopontin also plays a role in the development of NK cells358,359 and T lymphocytes.355 The fibulins are proteins secreted by the stromal cells of marrow, including osteoblasts and endothelial cells.360,361 The metalloproteinase-resistant fibulin-1 accumulates in the ECM where it binds to a specific site on FN,360,361 thereby disrupting HSC binding to FN with resultant decreases in HSC proliferation and differentiation.361 Thus, fibulin-1 can act as a negative regulator that can maintain the quiescence of HSCs in the marrow.

HEMATOPOIETIC CELL ORGANIZATION Erythroblasts

Erythroid progenitor cells arise from MPPs via the activity of the transcription factor GATA-1, which promotes differentiation toward the bipotent MEP that can subsequently differentiate into either erythroblasts or megakaryocytes (Chap. 32).362 MEP fate is determined by the relative activities of two competing transcription factors, KLF-1, which directs differentiation toward the erythroid lineage, and Fli-1, which directs differentiation toward the megakaryocytic lineage.362,363 The earliest progenitor cells committed solely to erythroid differentiation, BFU-Es,364 which are defined by production of large colonies or bursts of erythroblasts after weeks in tissue culture, can circulate in the blood and reenter the marrow. When a BFU-E or one of its progeny, the colony-forming units–erythroid (CFU-Es),364 associates with a marrow macrophage, they form the precursor of the basic unit of terminal erythropoiesis, the erythroblastic island (EBI).94 Under the influence of the KLF-1 in both the macrophage and the erythroid cells,365,366 an EBI develops as a central macrophage surrounded by as many as 30 adherent erythroblasts at various stages of differentiation from CFU-E through enucleating orthochromatic erythroblast. At least five cell-surface protein pairs contribute to adherence between macrophages and erythroblasts in EBIs94: (1) VCAM-1 on macrophages and α4β1 integrin (VLA-4) on erythroblasts; (2) αv component of integrins on macrophages and ICAM-4 on erythroblasts; (3) erythroblast-macrophage protein (EMP), on both erythroblasts and macrophages via a homophilic reaction; (4) CD169/Siglec-1 on macrophages and sialylated glycoproteins on erythroblasts; and (5) hemoglobin-haptoglobin receptor (CD163) on macrophages and an unknown binding partner on erythroblasts. Differentiating erythroblasts are defined as basophilic, polychromatophilic, and orthochromatic erythroblasts by their morphologic appearances in Giemsa-stained films of aspirated marrows. However, CFU-Es and their immediate progeny, the proerythroblasts (ProEBs), as well as the morphologically defined, later erythroblast stages can be purified and defined by flow cytometry. Murine erythroid cells from

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CFU-Es through ProEBs, are identified by flow cytometric expression patterns of transferrin receptor (CD71) and the erythroid-specific membrane glycoprotein Ter119,367 or of CD44 and forward light scatter.368 Likewise, expression patterns of glycophorin A, Band 3, and the α4 component of integrin permit identification of the same stages in human erythroid differentiation.369 In EBIs, CFU-Es lose SCF dependence that had been present throughout their differentiation from HSCs, and CFU-Es, ProEBs, and early basophilic erythroblasts develop a dependence upon EPO to prevent apoptosis.370 The level of EPO, the principal regulator of erythropoiesis, is regulated by tissue oxygen delivery in the kidney, and is dependent on both blood oxygen levels and red cell numbers.370 However, during hypoxic stress, CFU-Es and ProEBs can be increased without differentiation in response to circulating glucocorticoid hormones371,372 and BMP4 from central macrophages of EBIs.373 EPO prevents apoptosis by decreasing expression of Fas, a membrane protein of the TNF-α receptor family that is prominently expressed on CFU-E, ProEBs and early basophilic stage erythroblasts. Fas activation triggers a series of caspases, a family of intracellular proteases that cleave other caspase members in sequential fashion, ultimately inducing apoptosis.374 Fasligand, which binds and activates Fas, is produced mainly by immature erythroblasts in mice375 and by mature erythroblasts in humans.376 EPO also suppresses apoptosis in late-stage erythroblasts by inducing the antiapoptotic protein Bcl-XL, which stabilizes mitochondria, preventing the activation of caspases other than those activated by Fas.377 As a result of the Fas/Fas-ligand negative feedback within the EBI, differentiating erythroblasts can modulate the rate of CFU-E/ProEB apoptosis and provide regulated control rates of erythrocyte production commensurate with erythropoietic demand. In EBIs, differentiation events include: (1) hemoglobin production in differentiating erythroblasts, (2) formation of the erythrocyte plasma membrane and underlying membrane skeleton, (3) cell size decrease associated with the terminal 4 to 5 cell divisions being a result of decreased duration of the G1 phase of erythroblasts attached to central macrophages,378 and (4) nuclear condensation,379 stiffening,380 and extrusion.381 Erythroblast enucleation requires nonmuscle myosin IIB382 and filamentous actin381 to produce a membrane-enveloped nucleus and a nascent reticulocyte. The central macrophage sends out extensive slender membranous processes that envelop each erythroblast and phagocytize defective erythroblasts and extruded nuclei.383 The extruded nuclei display phosphatidylserine on their plasma membranes that leads to rapid phagocytosis by the central macrophage.384 Phagocytosis of extruded nuclei with recycling of the DNA components is essential in that deoxyribonuclease II–deficient mice die from an underproduction anemia with fetal liver macrophages filled with extruded erythroid nuclei.385 The irregularly shaped, maturing reticulocytes can interact directly with the central macrophages before entering the blood through the venous sinuses.94

Megakaryocytes

During thrombopoiesis, HSC in the subcortical regions of the hematopoietic cords generate megakaryocytes by sequential, overlapping expressions of specific transcription factors. First HSCs differentiate to common myeloid progenitors (CMPs) via the influence of PU.1 and GATA-1, next to MEPs via GATA-1/FOG, then to megakaryocytic progenitors via Fli-1, and finally to megakaryocytes via NF-E2 (Chap. 111).362,386 The microenvironmental factors that control survival and differentiation of megakaryocytes and their progenitors include a similar pattern of dependence to that of erythroid progenitors, with an overlapping decrease in dependence on SCF and an increasing dependence upon a physiologically regulated cytokine, TPO in the case of megakaryocytes, which ceases before the completion of differentiation.386,387

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TPO concentrations are reciprocally related to the circulating platelet mass, which is the major site of metabolism of the hormone.388 As the major regulator of megakaryocyte development, TPO acts in concert with several synergistic cytokines, including IL-11, IL-3, and IL-6.386,387 TPO induces endomitosis in terminally differentiating megakaryocytes by inhibiting cytokinesis through reduced function of the contractile ring of filamentous actin and suppression of nonmuscle myosin expression.389,390 However, DNA replication and accumulation of cytoplasmic proteins continues during six to seven of these endomitotic cell cycles. The resultant polyploid nucleus and abundant cytoplasm characterize the mature megakaryocyte which can account for 2 percent of marrow hematopoietic cell volume.93 Mature megakaryocytes lie directly outside the marrow vascular sinus wall391 because of their translocation during differentiation under the influence of platelet endothelial cell adhesion molecule (PECAM)-1 (CD31) expressed on endothelial cells392,393 and an autocrine pathway of VEGF-A and its receptor Flt-1 stimulating CXCR4 (receptor for CXCL12) expression on megakaryocytes.394 This migration of maturing megakaryocytes is associated with the development of podosomes, actin-based extensions that bind to and remodel the local ECM.395 The podosomes not only direct the megakaryocytes through the marrow to the sinus wall, but they also extend through the sinus basement membrane into the circulation.395 Terminal differentiation of megakaryocytes involves the development of branching cytoplasmic protrusions, the proplatelets. Proplatelets are formed around a microtubular core that both provides a sliding mechanism that elongates and extends them into the vascular sinus lumen, but also provides a conduit for the redistribution of cytoplasmic granules from the megakaryocytes to bulbous formations at the distal ends of the proplatelets.389

Granulocytes

Granulocytes are mature myeloid cells comprised of neutrophils, eosinophils, and basophils originating from stem cells and myeloid progenitor cells concentrated in the subcortical regions of the hematopoietic cords (Chap. 18).396 Granulocytes are terminally differentiated from common granulocyte-macrophage progenitor (GMP) cells which arise from MPPs through the expression of multiple transcription factors (Chap. 61). The transcription factor PU.1 promotes the development of the GMP phenotype and antagonizes the activity of GATA-1, which promotes MEP differentiation.397,398 The myeloid commitment of GMPs is reinforced by C/EBPα, which promotes myeloid differentiation while suppressing the B-lymphoid transcription factor Pax5.398,399 The further activity of C/EBPα is associated with granulocytic differentiation, whereas increased PU.1 activity is associated with monocytic differentiation.400 The progression of myeloid differentiation beyond the promyelocyte stage, including the formation of secondary and tertiary granules, requires both C/EBP and the GFI-1 transcription factors.400,401 GFI-1 also antagonizes the activity of the Egr-1 and Egr-2 transcription factors that are associated with monocytic differentiation.400 The timing of expression and relative ratios of C/EBPα and GATA-2 transcription factors regulate differentiation of the GMP into a mature neutrophil, eosinophil, basophil, or mast cell.399 Increased C/EBPα activity at this stage promotes a differentiation pathway toward neutrophils and eosinophils, whereas increased GATA-2 activity promotes differentiation toward basophils and mast cells.399 Cells differentiating along the neutrophil and/or eosinophil pathway will follow a terminal neutrophil path when only C/EBPα is expressed, and a terminal eosinophil path when both C/EBPα and GATA-2 are expressed. Those cells differentiating along the basophil/mast cell pathway will follow a terminal mast cell path when only GATA-2 is active and a terminal basophil path when both GATA-2 and C/EBPα are active.

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A group of hematopoietic growth factors, including SCF, GM-CSF, G-CSF, IL-6, and IL-5, support granulocytic progenitor/precursor viability and proliferation. In some cases, these growth factors can mobilize of these progenitors/precursors and their mature progeny from the marrow. These growth factors are produced in sites of inflammation in peripheral tissues, although some such as SCF and M-CSF are normally produced in the marrow stroma. Two hematopoietic growth factors have lineage-specific late-stage granulocytic cells as targets: IL-5 for eosinophil progenitors and G-CSF for neutrophilic progenitors. IL-5 is produced mainly by the T-helper type 2 (Th2) lymphocytes in response to allergens (Chap. 62).402,403 Eosinophilic progenitor cells display an IL-5α receptor protein that when associated with the common β receptor (CSF2RB), binds IL-5, leading to their survival and proliferation.402 Mature eosinophils have survival and chemotactic responses to IL-5, which mediates their entry into the circulation and accumulation in sites of allergic inflammation. GM-CSF, G-CSF, IL-3, and IL-6 all stimulate granulopoiesis in vivo, but only the deficiency of G-CSF results in severe neutropenia, making it the likely regulator of normal circulating granulocyte numbers.404 Under normal steady-state conditions, 1 to 2 percent of neutrophils circulate transiently in the blood, while the majority remains in the marrow unless mobilized by inflammation in other areas of the body. Models of G-CSF regulation of granulopoiesis and circulating neutrophils under normal conditions and during inflammatory states have been proposed.405,406 Newly formed neutrophils have low expression of CXCR4 and can exit the marrow by migration through sinusoidal endothelial cells. As they age in the circulation they express more CXCR4 and are attracted back to the marrow by stromal CXCL12, the CXCR4 ligand.405 After reentering the marrow, the senescent neutrophils undergo apoptosis and are phagocytosed by macrophages that, in turn, produce G-CSF stimulating more granulopoiesis.405 Cells at sites of inflammation produce both G-CSF and chemokines, including KC chemokine (CXCL1), and macrophage inhibitory protein (MIP)-2 (CXCL2). The secreted G-CSF acts on the marrow mobilizing neutrophils by its ability to reduce both marrow CXCL12 production and neutrophilic CXCR4 expression. G-CSF, however, does not recruit the neutrophils to sites of inflammation from the blood.405 By their chemotactic properties, CXCL1 and CXCL2 also induce rapid mobilization from the marrow into the blood and to sites of inflammation.405 Another model involves similar migration of neutrophils from the marrow that depends on G-CSF downregulating CXCL12 production and neutrophilic CXCR4 expression, but the feedback that decreases G-CSF occurs in the peripheral tissues.406 In this model, macrophages that phagocytose apoptotic neutrophils in the peripheral tissues decrease IL-23 production, which decreases IL-17 production by a subset of T-lymphocytes that, in turn, results in decreased G-CSF in the marrow.

CELL ADHESION AND HOMING After their initial migration from the yolk sac, AGM, or placenta to the marrow, the HSCs are located in specific sites in the marrow through interactions with other types of cells and with matrix proteins. HSCs do not remain permanently in the marrow because a small percentage of them are continuously entering the blood through the venous sinusoids, circulating briefly, and then reentering the marrow.407,408 In addition to the HSCs, the more differentiated progenitor cells, such as the short-term repopulating cells and the primitive BFU-Es, can circulate in a similar manner. When circulating, the HSCs can either reenter the marrow or they can enter other organs. After entering the interstitium of a peripheral organ, the HSCs can give rise to myeloid progeny and/or they enter the lymphatic drainage of the organ and circulate through lymphatic vessels and thoracic duct before reentering

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the blood.409 HSCs display multiple adhesion and cytokine receptors that allow them to attach to cellular and matrix components within the marrow sinusoidal spaces.275,277,410–412 Such attachments facilitate HSC homing and lodgment in the marrow and provide the cell–cell contacts required for HSC survival and steady-state proliferation,413 as shown by membrane-bound SCF regulation of HSC lodgment in the endosteal marrow region.414 In most lineages, differentiated cells are released from the marrow, circulate in the blood, and eventually home to the marrow. In some cell types, the circulating cells will differentiate further in peripheral organs such as B lymphocytes in the lymph nodes and spleen, monocytes in the tissues, and T lymphocytes in the thymus. After a period of residence in these secondary lymphoid organs, some lymphocytes travel through the lymph and blood, homing to the marrow, where they become functioning mature cells, such as plasma cells and CD4 and CD8 mature T lymphocytes.199,200,204 Mature and band forms of neutrophils exit the marrow, circulate in the blood and, if not recruited to a site of inflammation, home as senescent cells to the marrow by the CXCL12/CXCR4 mechanism described in the preceding “Granulocytes” section.405 Senescent erythrocytes are also removed from circulation through a mechanism that involves binding surface ICAM-4 to integrin αLβ2 (lymphocyte function-associated antigen [LFA]-1) on macrophages in the spleen and marrow.415 Mature leukocytes that participate in inflammatory reactions, such as the lymphocytes, monocytes/macrophages, and eosinophils, exit the circulation in areas of infection, allergic reactions, or injury. Table 5–2 lists the adhesive receptors and their ligands present on HSCs, progenitor cells, and components of the hematopoietic microenvironment, but receptor–ligand interactions that regulate the trafficking of mature leukocytes are not exhaustively listed.416,417

INTEGRINS Integrins mediate important cellular functions, including embryonic development, cell differentiation, and adhesive interactions between hematopoietic cells and inflammatory cells and surrounding vascular and stromal microenvironments.411,412,418 Integrins are divalent cation-requiring heterodimeric proteins (18 α subunits and 8 β subunits) subdivided by the β-chain component. Table  5–2 indicates that α-chains can associate with more than one β-chain subunit. The principal integrin receptors of the β1 subgroup involved in HSC-endothelial and HSC-stromal interactions are α4β1 (VLA-4), α5β1 (VLA-5), and αLβ2 (LFA-1) of the β2 subgroup. α4β1-based stromal adhesion events regulate erythropoiesis in the stages after EPO dependence.419 Granulopoiesis is stimulated by α4β1 activation by marrow stromal cells in cooperation with PECAM-1 (CD31), an immunoglobulin superfamily member.420 Antibodies against α4 or small molecule antagonists can mobilize hematopoietic stem and progenitor cells into the peripheral circulation.421 The high expression of α4β1 in granulocytic precursor cells and newly formed granulocytes has an important role in their adherence to VCAM-1 in the marrow, whereas the downregulation of α4β1 in the more mature neutrophils works in concert with CXCL12/ CXCR4 for their release into the blood.422 The α4β1 integrin on B lymphocytes is important for interactions with the VCAM-1 on the stromal cells in the B-lymphocyte niche, both in early B-lymphocyte development prior to migration out of the marrow and in later development of plasma cell precursors that have reentered the marrow.199 An acquired defect in stromal function, characterized by a deficiency in VCAM-1 and IL-7 expression,423–425 accounts for the delayed B-lymphoid reconstitution seen after marrow transplantation. During thrombopoiesis, CXCL12 induces VCAM-1 in the marrow sinusoid endothelial cells426 that mediates the binding of the megakaryocytes to the endothelium.427 Integrin α4β7 and its receptor, mucosal addressin cell adhesion molecule

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(MAdCAM)-1, like the integrin α4β1/VCAM-1 receptor pair, contribute equally to the homing of HSC to the marrow.428,429 Integrins are signaling molecules.430 After engaging their ligands, or subsequent to activation by monoclonal antibodies, multiple events (tyrosine phosphorylation of focal adhesion kinase, paxillin, and ERK-2) are triggered (outside–in signaling), culminating with Ras activation.431,432 Integrin receptor crosstalk433 with other adhesive receptor members, such as the immunoglobulin superfamily (NK cell–T cell [αLβ2/DYNAM-1], CD34+endothelial cell PECAM-1,434–436 or selectins,437 also results from outside–in signaling events that regulate receptor-binding affinity438,439 and mediates inhibitory signals for erythroid, myeloid, and lymphoid progenitor growth.440–443 Integrin binding of their respective receptors, such as α4β1/VCAM-1 or α4β1/FN, in early CD34+ progenitors enhances viability and preserves their long-term repopulating ability.444 In studies of isolated SP cells, high expression of the vitronectin receptor αvβ3 (CD51/CD61) is associated with quiescence and long-term repopulating ability.445 Conversely, expression of the α2 integrin is associated with only short-term repopulating capacity.446

IMMUNOGLOBULIN SUPERFAMILY The immunoglobulin superfamily designates a group of molecules containing one or more amino acid repeats also found in immunoglobulins and includes PECAM-1 (CD31), ICAM-3/R (CD50) and ICAM-1 (CD54), LFA-3 (CD58), ICAM-2 (CD102), VCAM-1 (CD106), KIT (CD117), and LW/ICAM-4 (CD242) (see Table  5–2).447–461 VCAM-1 is upregulated by inflammatory cytokines, IL-4 and IL-13.462,463 Immunoglobulin-like adhesion molecules also include NCAM, a neural cell-adhesion molecule that binds lymphocytes but not hematopoietic progenitors; Thy1, a stem cell antigen; major histocompatibility complex classes I and II; and CD2, CD4, and CD8 (see Table  5–2).247 LW/ ICAM-4 on erythroblasts binds the αv component of integrins on macrophages in EBIs,94 whereas the normal function of Lutheran red blood cell antigen, Lu/B-CAM (CD239), which binds the α5 component of laminin and is expressed late in erythroblast differentiation, is uncertain.461 The sialic acid-binding immunoglobulin-like lectins (Siglecs) are a family of surface proteins found on lymphocytes and myeloid cells that bind sialic acid residues of glycoproteins.464 Some Siglecs are evolutionarily conserved, such as Siglec-1 (sialoadhesin), which is highly expressed on macrophages, including the central macrophages of EBIs, and CD22, a coreceptor on B-lymphocytes. The remaining Siglecs, which are phylogenetically evolving rapidly, include CD33, which is expressed in lymphocytes and in all stages of myeloid cells where it is a commonly used marker for acute myeloid leukemia.

LECTINS (SELECTINS) Homing of stem cells requires lectin receptors with galactosyl and mannosyl specificities.465,466 The selectins are a family of adhesion molecules, each containing type C lectin structures.467 The leukocyte selectin (L-selectin, CD62L) is expressed on hematopoietic stem and progenitor cells and mediates adhesive interactions with other receptors (addressins), such as the CD34 sialomucin present on specialized endothelium, using sialylated fucosyl-glucoconjugates (see Table   5–2).259 The CD34 receptor on stem cells, however, does not bind L-selectin,259 as a putative L-selectin ligand may exist on these cells but is yet to be defined. The selectin family also contains CD62E, an E-selectin constitutively expressed on the marrow sinusoidal endothelium that regulates transmigration of leukocytes and CD34+ stem cell homing. The third member of this family is P-selectin, which is found on platelets. Pselectin can bind HSCs using a mucin receptor, CD162 also known as

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TABLE 5–2.  Hematopoietic and Microenvironment Adhesion Receptors and Their Ligands Receptor Subgroups

Receptor

Cellular Distribution

Ligand

CD49d, α4β1 (VLA-4)

CD34+ cells (erythroid, and lymphomyeloid progenitors)

VCAM-1 (CD106), FN, TSP

CD49e, α5β1 (VLA-5)

CD34+ cells, bone cells

FN, laminin

CD49f, α6β1 (VLA-6)

Rare CD34+ cells, monocytes

Collagen, laminin

CD11a/CD18, αLβ2 (LFA-1)

CD34+ cell subsets, not on repopulating stem cells

ICAM-1, ICAM-2, ICAM-3, DNAM-1

CD11b/CD18, αMβ2 (Mac-1)

CD34+ subsets, monocytes

ICAM-1, ICAM-2, iC3b, fibrinogen

β3 subgroup

Vβ3 (VNR)

Megakaryocytes, osteoclast

FN, TSP, CD31

β7 subgroup

α4β7 (LPAM-1)

Lymphoid and myeloid progenitor cells, mature myeloid cells

MAdCAM-1, VCAM-1, FN

CD31 (PECAM-1)

ECs, CD34+ cells, monocytes

CD31 homophilic adhesion, αVβ3 (VNR), CD38

CD50 (ICAM-3, ICAM-R)

CD34+ cells, monocytes

αLβ2 (LFA-1), CD11d/CD18 (αDβ2)

INTEGRINS β1 subgroup (CD29)

β2 subgroup (CD18)

IMMUNOGLOBULINS

CD54 (ICAM-1)

CD34+ cells, stroma, activated ECs

αLβ2 (LFA-1), αMβ2 (Mac-1)

CD58 (LFA-3)

CD34+ progenitors, stroma, ECs

CD2

CD102 (ICAM-2)

ECs, monocytes

αLβ2 (LFA-1)

CD106 (VCAM-1)

Stroma, activated ECs

α4β1 (VLA-4), α4β7 (LPAM-1)

CD117 (c-KIT)

CD34+ progenitors

Membrane KIT ligand

CD242 (ICAM-4)

Erythroid cells

αV-Integrins

PRR2 (related to CD155, the poliovirus receptor)

CD34+, CD33+, CD41+, myelomonocytic cells, megakaryocytic cells, ECs

PRR2 homophilic adhesion

CD62L (L-selectin)

Stroma, CD34+ cells

GlyCAM-1, MAdCAM-1, CD162, CD34, sLex, PCLP1

CD62E (E-selectin)

Activated ECs, (marrow ECs express CD62E constitutively)

CD15, sLea, CD162, CLA, sLex

CD62P (P-selectin)

Activated ECs

CD162, sLex, CD24 (HSA)

CD34

CD34+ cells, ECs

Selectins, other ligands?

CD43

CD34+, monocytes, NK cells

CD54 (ICAM-1)

CD162 (PSGL-1)

CD34+ cells, ECs

CD62L, CD62E, CD62P

CD164 (MGC-24v)

CD34+ cells, stroma, monocytes

Unknown

CD166 (HCA, ALCAM)

CD34+ cells, stromal cells, ECs

CD6, CD166

CD44

CD34+ cells, broad distribution

Hyaluronan, bFGF, HGF

CD38

CD34+ subsets, early T and B cells, plasma cells, thymocytes

CD31, hyaluronan

CD144 (VE-cadherin)

CFU-E, stromal cells, ECs

E-cadherin

CD157 (BST-1)

Stroma, T and B cells, myeloid cells

Unknown

LECTINS

SIALOMUCINS

HYALADHERIN OTHER

ALCAM, activated leukocyte adhesion molecule; bFGF, basic fibroblast growth factor; BST, bone marrow stroma; CD, cluster designation; CFU-E, colony forming unit–erythroid; CLA, cutaneous lymphocyte antigen; DNAM-1, DNAX accessory Molecule-1; EC, endothelial cell; FN, fibronectin; GlyCAM, glycosylation-dependent cell adhesion molecule; HCA, hematopoietic cell antigen; HGF, hepatocyte growth factor; HSA, heatstable antigen; ICAM, intercellular adhesion molecule; iC3b, inactive complement 3b complex; KIT, tyrosine-protein kinase; LFA, lymphocyte function-associated antigen; LPAM, lymphocyte Peyer patch-specific adhesion molecule; MAdCAM, mucosal addressin cell adhesion molecule; MGC-24, multiglycosylated core of 24 kDa; NK, natural killer; PCLP, podocalyxin-like protein; PECAM, platelet/endothelial cell adhesion molecule; PRR2, poliovirus receptor-related protein 2; PSGL-P, selectin glycoprotein ligand; sLe, sialyl Lewis; TSP, thrombospondin; VCAM, vascular cell adhesion molecule; VE, vascular endothelial; VLA, very-late antigen; VNR, vitronectin receptor.

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the P-selectin glycoprotein ligand (PSGL)-1, which binds to all three selectins (see Table  5–2). These proteins are responsible for leukocyte rolling over endothelial surfaces and tethering, thereby allowing integrin-mediated firm adhesion to the endothelium and mediating cellular homing events using specialized high endothelial venule lymphocyte homing sites.140,467,468 In addition to their role in HSC homing in the marrow, E-selectin and P-selectin can promote quiescence in HSC and induce apoptosis of late-stage myeloid progenitors while promoting the expansion (P-selectin) or differentiation (E-selectin) of short-term repopulating cells.469

SIALOMUCINS Three members of the CD34 family—CD34, podocalyxin, and endoglycan—are expressed on vascular endothelium, HSCs, and various hematopoietic cell lineages.470 When expressed on lymphoid high endothelial venules, these sialomucins are receptors for L-selectin, but their differential glycosylation in hematopoietic cells prevents Lselectin binding and results in their reducing nonspecific adhesion and potentially enhancing mobility. Although its function has not been determined, endomucin is another CD34-like sialomucin expressed in endothelium and in HSCs.471 In T lymphocytes, where it affects mobility, CD43 (leukosialin) acts in concert with PSGL-1 and binds both P-selectin and E-selectin.472,473 CD43 in neutrophils can foster adhesion when binding to E-selectin on endothelial cells, but it inhibits adhesion in most instances.473 CD43 can also regulate hematopoietic progenitor survival.474 CD162 (PSGL-1), a sialomucin that binds all three selectins, is important in leukocyte trafficking and stem cell homing.467,468,470 CD164 (endolyn), another sialomucin receptor displayed on HSCs, forms a complex with CXCR4, VLA-4, and VLA-5 on the leading edge of migrating HSCs after exposure to FN-bound CXCL12, indicating a role for CD164 in the homing of HSCs.475 CD166 (hematopoietic cell antigen [HCA], activated leukocyte adhesion molecule [ALCAM]) is expressed on HSCs and osteoblasts and is required for long-term engraftment potential of donor HSCs in murine transplantation models, probably through homophilic interaction.476–478 CD166’s only other ligand is CD6.477,479

HYALADHERINS The fifth subgroup listed in Table  5–2 is the cartilage-related proteoglycan, CD44, also known as the lymphocyte homing cell adhesion molecule (HCAM). This adhesion receptor, which binds the hyaluronic acid in the marrow matrix and can be a receptor for E-selectin, is expressed on neutrophils, lymphocytes, erythroblasts, and HSC.467,468 CD44 displayed on HSCs facilitates their homing and adhesion to the marrow and plays a role in their mobilization in response to G-CSF.467,468,480 Studies with CD44-deficient mice show no defects in HSC homing and growth, and no decrease in hematopoiesis, suggesting that another hyaladherin receptor may compensate for the absence of CD44.481 The other hyaladherin receptor on HSC is the receptor for hyaluron-mediated mobility (CD168/RHAMM),467,481 which does provide hyaluronic acid binding by neutrophils under inflammatory conditions in CD44 deficiency.482 Thus, CD44 and CD168/RHAMM may provide redundant hyaluronic acid binding in HSC.

OTHER ADHESION MOLECULES CD38 is an adhesion receptor that binds the CD31 receptor and matrix hyaluronan. It is expressed on early T and B cells and subsets of CD34+ hematopoietic progenitors.483 Similar to CD38, the stromal adhesion receptor BST-1 (CD157) is an adenosine diphosphate-ribosyl cyclase that is involved in regulation of intracellular calcium concentrations.

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CD157 is expressed on marrow stroma, T and B cells, and myeloid cells. It promotes pre–B-cell adhesion and growth.483 Cadherins are large molecules involved in cell–cell junctions and vascular integrity. CD144 (VE-cadherin) is expressed on CD34+ hematopoietic and endothelial progenitor cells and is an important molecule for trafficking of HSCs in fetal tissues and for the maintenance of HSC self-renewal.115,484,485 Downregulation of VE-cadherin is associated with crosslinking of VCAM-1, resulting in enhanced transendothelial migration of CD34+ cells in response to CXCL12.120 Although N-cadherin expression by both HSC and osteoblasts has been proposed to play a role in their interactions, experimental results in knockout mice do not support such a role.11,486

CELLULAR HOMING Leukocyte trafficking and migration have been central to understanding mechanisms of tissue homing. One of the best studied processes is lymphocyte homing to secondary lymphoid organs via specialized high endothelial venules (HEVs). Generally, leukocytes home to areas of inflammation by adhering to the endothelium and migrating between intercellular spaces by a sequence of specific events that begins with tethering of the leukocytes to the luminal surface of the endothelial cells.487 In the secondary lymphoid organs, tethering is mediated by L-selectin/CD62L receptor on the surface of naïve lymphocytes that binds a complex carbohydrate determinant, 6-sulfo-sialyl Lewis X, on glycoproteins called peripheral node addressins, such as CD34, podocalyxin, and endomucin.488,489 P-selectin and E-selectin are upregulated on the endothelial cell surface in response to various inflammatory cytokines, where they bind their respective receptors, PSGL-1 and CD44 on leukocytes.466,490 Tethering results in rolling of the leukocytes along the endothelial surface. Interactions of VLA-4 and α4β7 integrin on the surface of lymphocytes with their respective ligands VCAM-1 and MAdCAM-1 on HEVs may also mediate rolling.467 Rolling of neutrophils is slowed further by PSGL-1 and L-selectin activation of other adhesion molecules that include the β2 integrins αLβ2 (LFA-1) and αMβ2 (Mac-1).490–492 These β2 integrins, in turn, bind ICAM-1 on endothelial cells. The rolling leukocytes also receive signals through surface Gprotein–coupled receptors that bind chemokines in the heparan sulfate proteoglycans on the endothelial cells.490–492 The interaction of PSGL-1, L-selectin, integrins, and G-protein– coupled receptors with their endothelial ligands leads to cytoskeletal changes with arrest of rolling and adhesion to the endothelium. The adherent leukocytes undergo a rapid diapedesis, with migration either through or between the endothelial cells into the abluminal interstitium. At the interface with the adherent leukocyte, ICAM-1 and VCAM-1 in the endothelial cell are concentrated in a cup-like, caveolin-rich structure that internalizes ICAM-1.492–494 This caveolin-rich structure is linked to the endothelial cell cytoskeleton through vimentin. The internalization of the ICAM caveolae leads to the formation of a channel through the cell to the abluminal surface. When leukocytes follow a paracellular route through the endothelium, they require the coordinated activity of multiple adhesion proteins. These include PECAM-1, CD99, JAM proteins, and VE-cadherin, each of which mediates homophilic interactions at intercellular junctions between endothelial cells, and ICAM2.492–494 Although the roles of these proteins are uncertain, antibody inhibition and knockout mice demonstrate that they are necessary for the unidirectional migration of the leukocyte through the endothelium. PECAM-1, CD99, and JAM-C are expressed on leukocytes and may be involved in homophilic interactions between the migrating leukocyte and the endothelial junction. LFA-1 and Mac-1 on leukocytes can bind and interact with ICAM-2 and JAM-A on endothelium, whereas leukocyte VLA-4 can interact with endothelial JAM-B.

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The driving force for the migration and homing of leukocytes is the expression of chemoattractants at the site of inflammation or areas of constitutive production, such as the secondary lymphoid organs or the marrow. Bacterial peptides, complement components, and cytokines are produced in inflammatory sites. More than 40 different, but structurally related, chemotactic cytokines (chemokines) can be produced by leukocytes in inflammatory sites.495,496 Chemokines accumulate on cell surfaces or in extracellular matrices through their binding to GAGs.495–497 Concentrations and chemotactic activities of each cytokine are related to production rate, binding affinities to GAGs, presence of decoy chemokines that can compete with chemotactic activity, and modulation by metalloproteinases that enhance or diminish activities of substrate chemokines.495 Based on the location of one or two cysteine residues in the amino terminus, chemokines are divided into four subfamilies.224,495,496 One large subfamily comprises the CXC ligand (CXCL) chemokines (e.g., platelet factor 4, IL-8, melanocyte growth-stimulating activity/GROα, neutrophil activating protein-2, granulocyte chemotactic protein-2), which mediate neutrophil migration and activation. The other large subfamily comprises the CC ligand (CCL) chemokines (e.g., CCL3 [MIP-1α], CCL4 [MIP-1β], [CCL5] RANTES [regulated on activation, normal T-cell expressed, presumed secreted], MCP-1 through MCP-5), which mediate mostly monocyte, and in some cases lymphocyte, chemotaxis.497 A chemokine with CXXXCL structure is fractalkine, an endothelial transmembrane mucin–chemokine hybrid molecule that mediates the rapid capture, firm adhesion, and activation under physiologic flow of circulating monocytes, resting or IL-2–activated CD8 lymphocytes, and NK cells.498 The cytokines TNF-α and IL-1 upregulate fractalkine, in keeping with the need to rapidly recruit effector cells at sites of inflammation. The chemokine receptors on the surface of leukocytes are coupled to G proteins that initiate signaling for chemotaxis upon chemokine ligand binding.495,496 The chemokine receptors for the two large subfamilies bind those members such that CXCLs bind CXCRs and CCLs bind CCRs. However, within these two subfamilies is significant redundancy and promiscuity in chemokinereceptor binding. Table 5–3 gives a detailed listing of chemokine receptors and the cellular targets and ligands interacting with each receptor subgroup. A major exception to this redundant and promiscuous chemokinereceptor interaction is the specific binding of CXCL12/stromal cell-derived factor (SDF)-1α to its receptor CXCR4, which is associated with homeostatic maintenance of cell populations, including HSCs and their progeny in the marrow.496,499 CXCL12 can bind to one other chemokine receptor (CXCR7), but mouse knockout experiments show that CXCL12 null and CXCR4 null mice have embryonic lethal phenotypes that are markedly similar whereas CXCR7 null mice have postnatal lethality due to cardiovascular defects; CXCR7 may have a role in ligand sequestration but not in hematopoiesis.496,500–502 CXCL12 is produced by the bone, endothelial, perivascular reticular cells and some hematopoietic cells in the marrow, and its receptor CXCR4 is expressed on various hematopoietic and mature blood cells.468,499,503 The murine gene Cxcl12 was floxed, allowing conditional deletion by various Cre transgenics expressed in mesenchymal progenitor cells. Conditional deletion of Cxcl12 in mineralizing osteoblasts resulted in no obvious phenotype whereas deletion in Osterix-Cre–expressing reticular (CAR cells) and osteoblast cells resulted in constitutive HSC mobilization and loss of B-lymphoid progenitor cells.504,505 The Cre transgenics that delete floxed Cxcl12 alleles have complicated patterns of expression and current evidence supports a more important role for the perivascular niche in the homing of HSCs.505 Hence, mouse genetics and pharmacologic inhibition show that CXCL12 and CXCR4 are involved in the trafficking of HSCs, committed progenitor cells, and mature cells,

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including neutrophils, dendritic cells, NK cells, and T and B lymphocytes.404,405,421,499,503 The cellular specificity of the homing, localization, and mobilization that are driven by CXCL12 and CXCR4 are regulated by additional chemokines, adhesion proteins, and metalloproteinases associated with specific hematopoietic cell types and/or the organs to which they home, in which they reside, and from which they are mobilized.499,503 In the case of HSCs homing from the peripheral tissues through which they migrate, their initial entry into the lymphatic vessels is driven by the lipid chemoattractant sphingosine-1-phosphate (S-1-P).409 HSC display S-1-P receptors that respond to high levels in the lymph compared to the peripheral tissues where S-1-P is degraded. For the HSCs and the marrow, multiple experiments using inhibitors and antibodies with stem cell transplantation in mice and humans, parabiotic experiments with mice, and transplantation of human HSCs into immunocompromised mice (e.g., nonobese diabetic [NOD]/severe combined immunodeficiency [SCID] strains) have contributed to an understanding of some interactions of these multiple factors that influence HSCs within the marrow.506 Two adhesion mechanisms that play major roles in CXCL12-mediated HSCs homing to the marrow are the binding and activation of α4β1 integrin and selectin ligands, particularly PSGL-1,140,468,507 on HSCs to their respective receptors, VCAM-1, and Pand E-selectins on the marrow sinusoidal endothelium.428,508 Although α4β1 integrin appears to be the major integrin on HSCs involved in the first step of homing, other integrins have been implicated as having supporting roles, including α5β1, α4β7, and α6β1 or α6β4 integrin that bind to FN, MAdCAM-1, and laminins in the marrow.321,421 Similarly, a coordinated action between CXCR4 that has bound CXCL12 and the CD44 isoform on HSCs,509 or another hyaladherin such as RHAMM,481 may provide a source of adhesion for HSCs to hyaluronic acid on marrow endothelial cells in the homing process. In cord blood cells enriched for HSCs, the colocalization and cooperative activity of the endolyn with CXCR4, α4β1, and α5β1 integrin appears to enhance HSCs homing to the marrow in response to CXCL12.475 CXCR4 has also been colocalized in lipid rafts on HSCs with Rac-1, a member of the receptor-associated RhoGTPases.510 The RhoGTPases have two members, Rac-2 and RhoH, that are hematopoietic specific and, with other more widely expressed members such as Rac-1, Cdc42, and Rho A, are downstream effectors of CXCR4, β1-integrin, and KIT signaling in HSCs.511 The various RhoGTPases modulate actin polymerization and lead to cytoskeletal changes that are required for survival, proliferation, homing, and mobilization of HSCs and their progeny. In the homing of HSCs, the RhoGTPasemediated signaling provided by the coordinated action of CXCR4, β1 integrins, and CD44 leads to the rolling, arrest, and transmigration of the marrow sinus endothelial cells. Once the HSCs have migrated across the sinusoidal endothelial cells, they migrate further within the marrow in response to CXCL12. Using fluorescent SLAM-labeled markers for the identification of HSC in murine transplantation experiments, the homing of HSCs in the marrow cavity is associated with reticular cells that harbor the highest numbers of CAR cells in the marrow.132 The majority of CAR cells are in the perivascular areas to which the HSCs home.52 Another factor that may contribute to perivascular homing, especially following stress, such as lethal irradiation, is the ability of the marrow sinusoidal endothelial cells that express CXCR4 that binds circulating CXCL12 and transports it into the perivascular areas of the marrow.503,512 A second area in the marrow to which HSC home is the endosteal niche because of the proximity of these endosteal areas to perivascular areas,512 as well as the abundant CXCL12 production by osteoblasts and osteoclasts.134,165 Thus, two HSC niches are recognized in the marrow—perivascular and endosteal—with HSCs in the perivascular areas more likely to proliferate, differentiate, and mobilize into the blood than HSCs in the endosteal areas.75,150,512

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TABLE 5–3.  Chemokine Receptors, Interacting Chemokine Ligands, and Cellular Specificity Receptors

Receptor Expression

Chemokine Ligands

CXCR1

Neutrophils, monocytes

CXCL2 (GROβ), CXCL3 (GROγ), CXCL5 (ENA78), CXCL6 (GCP-2), CXCL8 (IL-8)

CXCR2

Neutrophils, IL-5–primed Eos, monocytes

CXCL1,2,3 (GROα/β/γ), CXCL5 (ENA78), CXCL6, CXCL7 (NAP-2), CXCL8(IL-8),

CXCR3

Activated memory and naïve T cells, NK cells; T (preferentially Th1) cells, B cells

CXCL9 (MIG), CXCL10 (IP-10), CXCL11 (I-TAC)

CXCR4

Neutrophils, monocytes, megakaryocytes, CD34+ and pre–B-cell CXCL12 (SDF-1α, SDF-1β) precursors, resting and activated T cells, DCs

CXCR5

B lymphocytes, T lymphocytes

CXCL13 (BCA-1/BLC)

CXCR6

T lymphocytes

CXCL16 (SR-PSOX)

CXCR7

B lymphocyte, T lymphocytes, Basos, monocytes, NK cells

CXCL11 (I-TAC), CXCL12 (SDF-1α)

CX3CR1

Monocytes, DCs, CD34+ cells, NK cells; in nodal tissues activated T-helper lymphocytes, activated B cells, and follicular DCs

CX3CL1 (fractalkine/neurotactin)

XCR1

Resting T cells, NK cells

XCL1 (lymphotactin/SCM-1α/ATAC), XCL2 (SCM-1β)

CCR1

Monocytes, EOS, basophils, activated Neu and T cells, CD34+ cells, immature DCs

CCL3 (MIP-1α), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (MCP-2), CCL13 (MCP-4), CCL22 (MDC), CCL23 (MPIF-1)

Monocytes, T cells (not Neu, EOS, or B cells)

CCL14 (HCC-1), CCL15 (HCC-2/MIP-5), CCL16 (HCC-4/LEC)

CCR2

Monocytes, basophils, DCs, T cells, activated memory CD4 T cells, NK cells

CCL2(MCP-1), CCL7 (MCP-3), CCL8 (MCP-2), CCL13 (MCP-4)

CCR3

Eos, thymocytes, basophils, DCs, activated memory CD4 T cells

CCL5 (RANTES), CCL7 (MCP-3), CCL8 (MCP-2), CCL11 (Eotaxin-1), CCL13 (MCP-4), CCL15 (HCC-2/MIP-5), CCL24 (Eotaxin-2/MPIF-2), CCL26 (Eotaxin-3)

CCR4

Activated T cells, immature DCs

CCL17 (TARC)

Monocyte-derived DCs, activated NK cells

CCL22 (MDC)

Thymocytes (CD3+, CD4+, CD8 )

CCL22 (MDC)

Monocytes, activated memory CD4 T cells

CCL5 (RANTES), CCL8 (MCP-2), CCL13 (MCP-4), CCL14 (HCC-1)

Immature DCs, CD34+ cells, NK cells

CCL3 (MIP-1α), CCL4 (MIP-1β)

Human thymocytes

CCL4 (MIP-1β)

CCR6

T cells, CD34+–derived dendritic cells

CCL20 (MIP-3α/LARC/exodus-1)

CCR7

Activated T (naïve and memory T cells) > B lymphocytes, NK cells CCL19 (MIP-3β/ELC/exodus-3), CCL21 (SLC/exodus-2/ subsets, CD34+ macrophage progenitors, mature DCs 6Ckine) (6Ckine inactive on B cells)

CCR8

Monocytes, T (Th2) cells, NK cells

CCL1 (I309), CCL17 (TARC)

CCR9

Thymocytes (CD4+/CD8+, CD4+/CD8–), activated macrophages

CCL25 (TECK)

CCR10

Skin-homing memory T cells, CD4/CD8 cells

CCL26 (Eotaxin-3), CCL27 (CTACK/ILC/ESkine), CCL28 (MEC)

CCR1 and CCR3

Neutrophils, monocytes, lymphocytes

CCL15 (HCC-2/MIP-5)

Not known

Resting T cells

CCL18 (DC-CK1/PARC)

low

CCR5

CCR3/CCR10 Memory lymphocytes, Eos, IgA plasmablasts

CCL28 (MEC)

6Ckine, chemokine with 6 cysteines; ATAC, activation-induced, chemokine-related molecule; Baso, basophil; BCA, B-cell attracting chemokine; BLC, B-cell homing chemokine that activates Burkitt lymphoma receptor 1 (BLR1); CTACK, cutaneous T-cell–attracting chemokine; DC, dendritic cell; ELC, EBI1-ligand chemokine; ENA, epithelial neutrophil-activating protein; EOS, eosinophil; ESkine, embryonal stem cell chemokine; GCP, granulocyte chemotactic protein; GRO, growth-related oncogene; HCC, human C-C chemokine; IgA, immunoglobulin A; IL-8 is also chemotactic for a specific subset of (CD3+, CD8+, CD56+, CD26−) T cells; IP, interferon-inducible protein; I-TAC, interferon-inducible T-cell α chemoattractant; LARC, liver and activation-regulated chemokine; LEC, liver-expressed chemokine; MCP, monocyte chemoattractant protein; MDC, macrophage-derived chemokine, MDC is chemotactic to eosinophils, in a CCR3- and CCR4-independent manner; MEC, mucosae-associated epithelial chemokine; MIG, monokine induced by interferon-γ; MIP, macrophage inflammatory protein; MPIF, myeloid progenitor inhibitory factor; NAP, neutrophil-activating peptide; NK, natural killer; PARC, pulmonary and activation-regulated chemokine; RANTES, regulated on activation, normal T-cell expressed and secreted; SCM, single-C motif; SDF, stromal cell-derived factor; SLC, secondary lymphoid tissue chemokine, also known as exodus-2 and 6Ckine; SR-PSOX, scavenger receptor for phosphatidylserine and oxidized lipoprotein; TARC, thymus and activation-regulated chemokine; TECK, thymus-expressed chemokine; Th2, T-helper cell type 2.

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In the marrow, multiple mechanisms act to stabilize and reinforce the lodgment of HSC, that is, to maintain the HSC in niches. One prominent mechanism is the binding of SCF, either secreted in and adherent to the marrow matrix or displayed on stromal cells. The absence of either KIT or SCF results in embryonic failure of hematopoiesis as a result of impaired homing of HSCs to the fetal liver where SCF acts cooperatively with CXCL12 as a chemoattractant, and to impaired retention of HSCs in the marrow513 where KIT upregulates HSC expression of integrins α4β1 and α5β1.514 The β1 integrins of the HSCs also bind osteopontin, which, in turn, is bound to other matrix proteins, such as FN and collagen. Similarly, CD44 on HSCs binds to hyaluronic acid, FN, and collagen the marrow matrix.164 Two receptors on HSCs that contribute specifically to endosteal niche retention are the calcium-sensing receptor,515 which is needed for effective binding to collagen, and the Tie family receptor kinases, specifically Tie-2 receptor, which mediates HSC integrin binding to FN after engaging its ligand, angiopoitein-1, that is expressed by osteoblasts.516,517 Marrow SP cells enriched with longterm repopulating quiescent HSCs display high expression of β3-integrin, most likely as the vitronectin receptor αvβ3, suggesting another integrin–matrix protein interaction that supports HSC retention.445,518 One mechanism of retention in the endosteal niche is the long-term maintenance of HSCs by TPO produced by adjacent osteoblasts.519,520 The binding of TPO by its receptor induces HSC quiescence, whereas the absence of TPO leads to active cell cycling and to a protracted and progressive depletion of HSCs.519,520

CELLULAR RELEASE Cell migration from the marrow occurs between adventitial cells and through endothelial cell channels that develop at the time of cell transit. Electron micrographs of leukocytes partially translocated across endothelium indicate that marked deformation of these cells occurs as

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they penetrate the cytoplasm of the endothelial cell and enter the sinus lumen (Fig. 5–7).391 As with reticulocytes, egress occurs adjacent to junctions of endothelial cells.383 The nucleus of the granulocyte, usually segmented, does not require as marked a deformation to traverse the migration pore as do the nuclei of monocytes and lymphocytes.391 This transendothelial migration is likely to be related to leukocyte migration from the blood and into areas of inflammation described in the section on adhesion and homing because the marrow sinusoidal endothelial cells constitutively express adhesion proteins that are upregulated in inflammation, including VCAM-1, ICAM-1, and E- and P-selectin.405 Immature granulocytes in the marrow are anchored to adventitial reticular cells through lectin-like adhesion molecules. Gradual loss of these molecules (e.g., shedding of L-selectin) during maturation or after activation could permit movement toward the sinus wall.521 Transient changes in surface glycoproteins (upregulation of α-2,6-sialylation of CD11b and CD18) of maturing marrow myeloid cells lead to decreased stromal and FN adhesion and may favor contact with endothelium and cell egress.522 The complement component C5a and G-CSF administration recruit neutrophils by altering integrins (low CD11a with G-CSF) and decreased L-selectin expression (with both agents).523,524 Similar findings obtained in mice lacking two or all three selectins underscore the essential role of selectins in neutrophil recruitment.525 Mature leukocytes retain their nuclei as they enter the marrow venous sinuses and circulate in the blood, but erythroid and megakaryocytic cells release anucleate cells and their residual nuclei are rapidly phagocytosed by marrow macrophages.94,384,526 Occasional immature granulocytes and megakaryocyte nuclei or whole megakaryocytes are present in cell concentrates of normal blood.527 Restrictions on the release of immature myeloid cells, erythroblasts, and megakaryocytes are associated with the relative stiffness of their nuclei because of the ratio of nuclear lamin isotypes in erythroid and immature myeloid precursors and increased total lamins in megakaryocytes.380

Figure 5–7.  Transmission electron micrograph of mouse femoral marrow. The lumen (L) of a sinus is indicated. Endothelial cell cytoplasm separates the sinus lumen from the hematopoietic spaces (arrow). Two neutrophils are evident traversing the sinus wall. Note deformation of the cell producing a narrow waist where the cell passes through endothelium. The luminal portion of the migrating cells is granule-poor. The remainder of the cytoplasm is granule-rich, possibly reflecting gel-sol transformation during pseudopod formation. (Used with permission of Lichtman MA, University of Rochester.)

L

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A number of releasing factors are implicated in the initiation of marrow granulocyte egress, including G-CSF,528,529 GM-CSF,530 the C3e component of complement,531 zymosan-activated plasma-containing complement fragments,532 glucocorticoid hormones,533 androgenic steroids,534 and endotoxin.535 Neutrophils residing in the marrow venous sinusoids are rapidly released into the circulation by IL-8.536 In a rat model in which releasing factors can be given through the femoral artery and neutrophils collected from the femoral vein, chemokines CXCL2 (MIP-2) and CXCL1 (KC) that are produced at sites of inflammation induce rapid, selective neutrophil migration from the marrow compartment into the blood.537,538 Blocking or inhibiting the α4-integrin component, β2-integrin component, or the sheddase that catalyzes the proteolysis of L-selectin on migrating HSCs indicates that the interaction of the highly expressed VLA-4 on neutrophils with VCAM-1 on the sinusoidal endothelial cells is required for transendothelial migration, whereas shedding of L-selectin has no effect, and β2-integrin binding helps retain the neutrophils in the marrow.537 Blocking the neutrophil enzyme matrix metalloproteinase-9 (MMP-9) had no effect on the chemokine-induced neutrophil migration.538 CXCL2- and CXCL1induced migration is synergistic with the rapid, selective neutrophil migration from the marrow induced by G-CSF,539 which is mediated by interrupting the interaction of CXCL12 in the marrow and CXC4R on neutrophils.540 In a similar hind-leg model in guinea pigs, IL-5 and eotaxin, both of which are produced in sites of allergic inflammation, induce the rapid, selective migration of eosinophils from the marrow to blood with a synergistic effect when both are administered.541 CCL11 (eotaxin) alone induces the migration of both eosinophil progenitor cells and mature eosinophils.541 The route of migration is transendothelial, and blocking experiments demonstrate that β2-integrin binding enhances eosinophil migration from the marrow to the blood, whereas α4-integrin binding helps retain eosinophils in the marrow.542 Prostaglandin D2 (PGD2 is produced by mast cells in sites of allergic inflammation, and it induces rapid, selective migration of eosinophils from the marrow to the blood in the guinea pig model.543 The eosinophils respond via two PGD2 receptors, chemoattractant receptor-homologous molecule on Th2 (CRHTH2) and D-type prostanoid (DP) receptors.543

Releasing factors for reticulocytes have been difficult to identify. Adventitial reticular cell cytoplasm is a barrier to the reticulocytes on the abluminal surface of the endothelium.544 Phlebotomy, phenylhydrazine-induced hemolytic anemia, and EPO result in marked reduction of the adventitial cell cover of the sinus, a process that is thought to facilitate cell egress through the endothelium.545 Immature reticulocytes have much less deformability than more mature ones,546 suggesting that active migration by nascent reticulocytes through the endothelial cells is relatively unlikely, and release is via a passive mechanism. Thus, reticulocytes appear to require a pressure gradient to cross the venous endothelium and enter the blood as shown in Fig. 5–8.544,545 The pressures within the marrow sinuses are pulsatile, and pressures sufficient to cause egress may be transient.547 Another force that may contribute to reticulocyte egress is provided by the increasing numbers of erythroblasts proliferating in the EBIs that displace the more mature reticulocytes peripherally toward the venous sinuses.548 Platelet release by the megakaryocyte requires both actin-based podosomes and microtubulin-based proplatelets that extend through of the marrow sinus endothelium into the blood as described in the “Megakaryocytes” section of this chapter. The proplatelets can be separated from the megakaryocyte in the marrow, but the fate of these separated proplatelets is not certain, and they may not give rise to platelets.549 In normal thrombopoiesis, increased concentrations of S-1-P in the circulating blood activate the S-1-P receptor on the megakaryocytes, thereby, promoting proplatelet extension into the vascular sinus.550 The proplatelets extend through the endothelium (Fig. 5–9) and into the lumen of the venous sinus (see Figs. 5-6 and 5–10) producing elongated bean-shaped proplatelets.389,391 The formation of platelets also requires S-1-P and its receptor550 combined with the shear force of the blood flow,549 which releases both individual platelets or proplatelets themselves that later fragment in the circulation. Under homeostatic conditions, the migration of HSCs from the marrow into the blood is a rare but steady process.408,409,551 With the stress of chemotherapeutic agents or pharmacologic doses of G-CSF administration, many HSCs are recruited into active cell cycle,551 and they migrate into the blood before homing again to the marrow.408 The

L L

L

1.0 µm

A

B

C

Figure 5–8.  Transmission electron micrograph of mouse femoral marrow. Composite of reticulocytes in egress. A. Small protrusion of marrow reticulocyte into sinus lumen (L). B. Reticulocyte in egress, with approximately half the cell in the sinus lumen. C. Reticulocyte virtually in the sinus. Egress occurs through a migration pore that is parajunctional in position (arrows point to endothelial cell junctions).

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Figure 5–9.  Transmission electron micrograph of mouse

femoral marrow. A. The lumen (L) of a marrow sinus is indicated. The arrow points to the thin endothelial cytoplasmic lining of the sinus. The nucleus of a megakaryocyte (N) is indicated, with the cytoplasm of the megakaryocyte invaginating the endothelial cell cytoplasm in three places below the lumen. B. The arrow indicates the thin endothelial cell cytoplasmic lining of the sinus. The endothelium is attenuated to a double membrane in two places. A small process of megakaryocyte cytoplasm has formed a pore in the endothelial cell and has entered the sinus lumen (L). Cytoplasm flows through such pores and delivers proplatelets to the sinus lumen. (Used with permission of MA Lichtman, University of Rochester.)

L

L

N

A

B

stress of moderate blood loss also increases the cell cycling of the HSCs, but those cycling HSCs cannot be detected in the blood,552 indicating that the migration of HSCs in response to stress is very likely related to the inflammatory/injury component of the stress. This relationship between inflammation/injury and HSC migration has been used experimentally to understand the mechanisms of HSC migration into the blood and clinically to mobilize the HSCs into the blood for collection for use in stem cell transplantation. Not surprisingly, these studies demonstrate that much of the regulation of HSC migration involves the reversal or inhibition of the mechanisms by which the HSCs home to the marrow and develop quiescence. Many hematopoietic growth factors can mobilize HSCs from the marrow to the blood, but the best understood and most used clinically is G-CSF.480,506,553 Similar to other growth factors, the G-CSF mobilization of HSCs requires several days for maximal effect. A major determinant in both the homing to and migration from the marrow is the interaction of CXCR4 on HSCs with its ligand CXCL12 in the marrow. G-CSF induces stem cell mobilization by decreasing CXCL12 signaling.554 CXCR4 knockout mice do not mobilize HSCs with G-CSF, but they mobilize HSCs in response to VLA-4 (α4β1 integrin) antagonists.555 Inhibitor studies originally identified the mobilization mechanism as

the degradation of CXCL12 by neutrophil-associated enzymes such as neutrophil elastase, cathepsin G, and MMP-9 or the HSC enzyme CD26/dipeptidylpeptidase, but mice genetically null for the proteases or treated with other protease inhibitors still show the G-CSF–induced decrease of CXCL12 mRNA and protein.480,553,556,557 Multiple mechanisms for CXCL12 modulation have been proposed, including the adrenergic nervous system suppressing MSC production of CXCL12 and direct G-CSF suppression of osteoblast lineage cells in the marrow.140,141,557,558 The successful development of small antagonists of CXCR4, such as plerixafor (formerly AMD3100), has provided a rapid means to mobilize HSCs and is used clinically for those patients that fail to mobilize with G-CSF.421 Similarly, blocking α4-integrin binding or genetic deletion of the α4-integrin component leads to HSC mobilization within 1 or 2 days under both homeostatic or G-CSF–induced conditions.421 This mobilization appears to be mainly mediated through disruption of VLA-4 activity and is further enhanced by blocking other adhesion mediators such as the β2-integrins or E-selectin, neither of which has an effect when used alone.421,559 Some of β2-integrin’s synergistic effects may be indirect through the action on other cells.560 HSC mobilization with antibodies against the α4 component of integrin561 is replicated by potent and selective small molecule antagonists.562 The results of interfering

L

∗

N

∗ ∗

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∗

Figure 5–10. Transmission electron micrograph of mouse femoral marrow. The marrow sinus lumen (L) and a megakaryocyte nucleus (N) virtually denuded of cytoplasm are indicated. The megakaryocyte nucleus abuts the nucleus of an adventitial reticular cell; the latter is separated from the lumen by the very thin endothelial cell cytoplasm. A portion of residual megakaryocyte cytoplasm (proplatelet) can be seen streaming into the lumen (arrow). The lumen contains several proplatelets (asterisks). Compare the size of the proplatelets to that of lymphocyte in the sinus. The bean-shaped, three-dimensional appearance of the proplatelets can be seen in the scanning micrograph shown in Fig. 5–6. (Used with permission of MA Lichtman, University of Rochester.)

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with two other adhesion mediators of HSC homing, CD44 and SCF, are unclear in that antibodies to CD44 or administration of SCF induced HSC mobilization while genetic deficiencies of CD44 or KIT resulted in decreased G-CSF mobilization.480 Two chemokine ligands of the CXCR2 receptor, IL-8 and GRO-β (KC in mice), induce HSC mobilization within minutes to hours and can synergize with G-CSF, but their action is more complex in that it is mediated through neutrophils and their enzymes including MMP-9.480,563

CELL PROLIFERATION, APOPTOSIS, AND MATURATION Irrespective of their location during the postnatal period, HSCs undergo continued self-renewal divisions, but at 3 to 4 weeks of age in mice (corresponding to 2 to 4 years in humans), they switch to their characteristic cell-cycle quiescence found in adult HSCs.564,565 This switch appears to be an intrinsic event that also decreases the myeloid differentiation potential of the HSCs.564 In the marrow endosteal niche, HSCs have multiple stimuli that induce cell-cycle quiescence. These stimuli include high concentrations of CXCL12 binding CXCR4499,503; low concentrations of CD34, podocalyxin, and endoglycan; TPO binding by MPL519,520; variable binding to matrix proteins such as osteopontin, FN, and fibulin, that depend upon angiopoetin-1/Tie-2, and SCF/KIT activities.355,514,516 Compared to HSCs located outside the endosteal niche, HSCs that are closely associated with to the endosteum have greater quiescence, marrow homing, and long-term reconstitution capacity.566 In murine transplantation studies, cell-cycle status significantly impacts the rate of engraftment and donor hematopoiesis with HSCs in G0 phase providing maximal long-term reconstitution, whereas HSCs in G1, S, G2, or M phases provide minimal engraftment or long-term reconstitution.551,564,567 In long-term in vivo labeling with bromodeoxyuridine (BrdU), murine HSC immunophenotypically defined as Lin−, Sca-1+, KIT+, CD150+, CD48−, and CD34− have the greatest reconstitution capacity and are located in both endosteal and central areas of the marrow.551 These HSCs are extremely quiescent, dormant, with an estimated division rate of only four or five times over the life of the adult mouse. However, the large majority of them are able to enter cell cycle and are mobilized within a day or two of stressful stimuli, including G-CSF or 5-fluorouracil (5-FU) administration.551 Dormancy or quiescence is resumed upon homing and reestablishing marrow residence, indicating that the long-term reconstituting HSCs provide a large reserve that is able to respond, but only under situations of stress.551 Dormant or quiescent HSCs are determined to be in G0 based on lower RNA content and diploid genomic DNA content.567,568 Entry into the cell cycle induces cells into the G1 phase where a restriction (R) point is encountered beyond which further progression to S phase and subsequent transit through G2 to M phases is irreversible. The sequence of events and in particular transit through the R point is tightly regulated by the retinoblastoma tumor-suppressor protein (Rb) and its paralogs (p107, and p130).569,570 Rb is regulated by phosphorylation that is catalyzed by cyclin-dependent kinases, Cdk2, Cdk4, and Cdk6. Cdk4 and Cdk6 are regulated by D-type cyclins (D1, D2, D3), and Cdk2 is regulated by E-type cyclins (E1 and E2), at early and late stages, respectively, of the G1 phase. Hyperphosphorylated Rb releases E2F transcription factors that promote entry into S phase by transcription of multiple genes required for replication.571,572 MPPs, the short-term repopulating cells and the colony-forming unit–granulocyte-erythroidmonocyte-macrophage (CFU-GEMM), have relatively low rates of proliferation, but they are greatly increased compared to the very infrequent cell divisions of HSC. The D cyclins and Cdk4 and Cdk6 kinases are important in these early progenitor cells because knockout mice that

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lack all three D cyclins573 or lack both Cdk4 and Cdk6 kinases574 have specific, lethal hematopoietic failures at the fetal liver stage of definitive hematopoiesis.572 In both of these knockout models, the HSC populations have little or no loss of numbers, but the multipotent progenitors are severely reduced, indicating these cell cycle regulators are required for the process that commits the HSC to increased proliferation during differentiation.573,574 As they divide, MPPs have progressively restricted lineage potential, which is regulated by various transcription factors as described above in the sections on the individual cell types in the marrow. The single-lineage progenitors further increase the percentages of their populations in active cell cycle so that by the later stages of CFU-E, CFC-G, and more mature hematopoietic precursor cell development, the majority are in the S, G2, and M phases.575 The two potential sources of extracellular stimuli that increase hematopoietic cell division are soluble hematopoietic cytokines and local interactions of the progenitors with other cells and matrix in the marrow. Hematopoietic cytokines include those produced either in remote organs, such as EPO, or those produced in a wide variety of organs, including the marrow, such as TPO, GM-CSF, and G-CSF.576 These latter hematopoietic cytokines have multiple effects on their target progenitor cells, including the promotion of survival, maturation, and migration, that are important for the increased production and recruitment of the mature cells to sites of inflammation.576 Among the cytokines, M-CSF is mitogenic, that is, it promotes progression from G1 to S phase, in macrophages and their precursors.577 The signaling from FMS (CSF1R), the M-CSF receptor, which leads to S-phase progression, is mediated by both cyclin D1 and the transcription factor MYC.578 Among the various cellular interactions of late progenitors and precursors, attachment to central macrophages of EBIs promotes the G1 to S phase transition in erythroid progenitors/precursors.378 This mitogenic effect of macrophage-erythroid cell interaction is unrelated to the antiapoptotic effect of EPO on the erythroid cells during these stages of erythroid differentiation.378 Mature hematopoietic cells cease cell division prior to their release from the marrow, but the mechanisms that signal cell cycle arrest in hematopoietic cells as they mature are uncertain. Among the potential mediators of this cell-cycle arrest are Rb and several intracellular inhibitors of the cyclin-dependent kinases, specifically the INK4 proteins (p15, p16, p18, p19) that inhibit Cdk4 and Cdk6 and the CIP/KIP family of Cdk2 inhibitors (p21, p27, and p57).575 Rb knockout mice have a lethal anemia during fetal liver hematopoiesis that is associated with persistent progression through cell cycle, but the erythroblast apoptosis appears to be related to failure of mitochondrial biogenesis.570 Understanding the activity of p16INK4a in regulating cell cycle is complicated by its potential role in senescence and apoptosis of HSC.579 Although p21 and p27 proteins are proposed as having roles in the TGF-β–induced HSC quiescence and in the increased proliferation of later progenitor stages, Cdk2 knockout mice do not have impaired hematopoiesis,580 indicating that other cell-cycle mediators are required for the cessation of proliferation that accompanies terminal differentiation. Apoptosis is the major regulator of cellular populations in the marrow. Because of the exponential expansion of cells in a proliferating population, cell death has a dramatic effect on the numbers of cells in subsequent generations.581 Thus, the regulation of hematopoietic cell populations by apoptosis provides a mechanism for dramatic and prompt changes in blood cell production. During various stages of differentiation, hematopoietic cells depend upon specific cytokines to prevent apoptosis.576,581 A wide range of sensitivities to the hematopoietic cytokines among the dependent cells, as has been demonstrated for erythroid cells and EPO,582 results in differential survival that allows for a graded response. Experiments in knockout mice have identified specific proteins in the Bcl-2 family as principal regulators of the intrinsic or

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mitochondrial apoptosis pathway in the homeostasis of the hematopoietic cells populations in the marrow.583,584 Antiapoptotic members of the Bcl-2 family (Bcl-2, Bcl-XL, Mcl-1, and A1) stabilize the mitochondrial membranes by preventing mitochondrial depolarization by the poreforming family members, Bax and Bak.585 The antiapoptotic members are also opposed by the proapoptotic, regulatory family members that consist of the BH3-only domain, such as Bim, Bid, Nix, and Puma. In HSC and multipotent progenitors, Mcl-1 is required to prevent apoptosis, and SCF stimulation increases the Mcl-1 expression.586,587 In the later stages of single-lineage progenitors, Mcl-1 continues to be required for survival of neutrophil and B and T lymphocytes, but it is antagonized by the expression of Bim and Puma in these progenitors, providing a means to eliminate specific cells, such as autoreactive B and T lymphocytes.583,584 A1 is required for normal neutrophil survival.588 In the erythroid lineage, Bcl-XL is required to prevent apoptosis at the late erythroblast stage,589 and the proapoptotic Nix protein is also expressed.590 The sequential proapoptotic and antiapoptotic stimuli that regulate erythropoiesis demonstrate overlapping and cooperative interactions that affect erythroid cell homeostasis by both survival and differentiation. Following moderate blood loss, an increased percentage of HSC enter cell cycle and self-renewal.552 In the BFU-E through CFU-E stages, SCF and glucocorticoids act in concert to upregulate proliferation according to the erythropoietic requirements.591 However, because CFU-E depends upon EPO, SCF and EPO act together, enhancing the proliferation and survival, respectively, of CFU-E.592 EPO prevents apoptosis of CFU-E through basophilic erythroblast stages by decreasing Fas expression,374,375 but its upregulation of Bcl-XL prevents the apoptosis of the late-stage hemoglobin-producing erythroblasts.589 Expression of proapoptotic Nix in very late erythroblasts and reticulocytes plays a major role in targeting mitochondria for nontoxic elimination by autophagy.593,594 Various mathematical models have been constructed to explain the production rates for each cell type during homeostasis and during periods of increased and decreased production. A model of homeostatic human marrow has been based upon marrow films and sections relating differential counts of marrow samples to their content of injected radioactive iron. A number of assumptions and approximations are made,595 but the summary data (Table 5–4) agree well with many other observations on the cellular content and kinetics of normal marrows. Under pathologic conditions such as infection, inflammation, or hematopoietic dysplasia, the proliferation and differentiation of hematopoietic progenitors may be affected by microbial products, cytokines, and cellular interactions that do not have a role in normal hematopoietic development. Infections, for example, can lead to increased myelopoiesis

TABLE 5–4.  Normal Precursor Cell Kinetics Marrow Number (cells/kg)

Transit Production Rate Time (days) (cells/kg/day)

Erythroblasts

5.3 × 109

~5.0

3.0 × 109

Reticulocytes

8.2 × 10

2.8

3.0 × 109

~7.0

2.0 × 106

~5.0

0.85 × 109

6.6

0.85 × 109

Cell Type I.  Red cells

II. Megakaryocytes

9

15.0 × 106

III. Granulocytes Proliferation pool 2.1 × 109 Postmitotic pool

5.6 × 109

Reproduced with permission from Finch CA, Harker LA, Cook JD: Kinetics of the formed elements of human blood. Blood 50(4): 699–707, 1977.

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without the involvement of the hematopoietic cytokines. HSC and their myeloid and lymphoid progeny have multiple toll-like receptors (TLRs) which bind specific bacterial or viral molecules.596,597 The activation of TLRs leads to increased myelopoietic proliferation and differentiation, especially of the monocyte/macrophage lineage, and differentiation of lymphoid cells toward the dendritic cell phenotype.596,598 Although increased hematopoietic cytokines are produced by TLR activation, a direct response to TLR activation in hematopoietic cells changes the prevalent myeloid transcription factor from C/EBPα, which mediates homeostasis by hematopoietic cytokines, to C/EBPβ, which mediates the emergency responses to TLR activation.599 In response to the activation of TLRs, mature neutrophils have decreased apoptosis as a result of increased Mcl-1 and decreased Bad activity.600 This may be a result of direct ligation of TLR receptors on LT-HSC, ST-HSC, and MPP that are then stimulated to secrete cytokines such as IL-6, GM-CSF, and TNF-α.601,602 An alternative path to apoptosis in hematopoietic cells is the activation of specific death-domain receptors for the ligands such as FAS ligand, TNF-α, and TRAIL (tumor necrosis factor–related apoptosis-inducing ligand). Although these ligands are most commonly associated with pathologic states where they may play a role in the anemias of chronic disease, they have also been proposed to have a regulatory role in normal erythropoiesis.603

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578. Roussel MF, Theodoras AM, Pagano M, Sherr CJ: Rescue of defective mitogenic signaling by D-type cyclins. Proc Natl Acad Sci U S A 92:6837, 1995. 579. Oguro H, Iwama A: Life and death in hematopoietic stem cells. Curr Opin Immunol 19:503, 2007. 580. Berthet C, Rodriguez-Galan MC, Hodge DL, et al: Hematopoiesis and thymic apoptosis are not affected by the loss of Cdk2. Mol Cell Biol 27:5079, 2007. 581. Koury MJ: Programmed cell death (apoptosis) in hematopoiesis. Exp Hematol 20:391, 1992. 582. Kelley LL, Koury MJ, Bondurant MC, et al: Survival or death of individual proerythroblasts results from differing erythropoietin sensitivities: A mechanism for controlled rates of erythrocyte production. Blood 82:2340, 1993. 583. Opferman JT: Life and death during hematopoietic differentiation. Curr Opin Immunol 19:497, 2007. 584. Reed JC: Bcl-2-family proteins and hematologic malignancies: History and future prospects. Blood 111:3322, 2008. 585. Llambi F, Green DR: Apoptosis and oncogenesis: Give and take in the BCL-2 family. Curr Opin Genet Dev 21:12, 2011. 586. Kaisho T, Ishikawa J, Oritani K, et al: BST-1, a surface molecule of bone marrow stromal cell lines that facilitates pre-B-cell growth. Proc Natl Acad Sci U S A 91:5325, 1994. 587. Opferman JT, Iwasaki H, Ong CC, et al: Obligate role of anti-apoptotic MCL-1 in the survival of hematopoietic stem cells. Science 307:1101, 2005. 588. Hamasaki A, Sendo F, Nakayama K, et al: Accelerated neutrophil apoptosis in mice lacking A1-a, a subtype of the bcl-2-related A1 gene. J Exp Med 188:1985, 1998. 589. Rhodes MM, Kopsombut P, Bondurant MC, et al: Bcl-x(L) prevents apoptosis of latestage erythroblasts but does not mediate the antiapoptotic effect of erythropoietin. Blood 106:1857, 2005. 590. Aerbajinai W, Giattina M, Lee YT, et al: The proapoptotic factor Nix is coexpressed with Bcl-xL during terminal erythroid differentiation. Blood 102:712, 2003. 591. von Lindern M, Schmidt U, Beug H: Control of erythropoiesis by erythropoietin and stem cell factor: A novel role for Bruton’s tyrosine kinase. Cell Cycle 3:876, 2004. 592. Muta K, Krantz SB, Bondurant MC, Wickrema A: Distinct roles of erythropoietin, insulin-like growth factor I, and stem cell factor in the development of erythroid progenitor cells. J Clin Invest 94:34, 1994. 593. Sandoval H, Thiagarajan P, Dasgupta SK, et al: Essential role for Nix in autophagic maturation of erythroid cells. Nature 454:232, 2008. 594. Schweers RL, Zhang J, Randall MS, et al: NIX is required for programmed mitochondrial clearance during reticulocyte maturation. Proc Natl Acad Sci U S A 104:19500, 2007. 595. Finch CA, Harker LA, Cook JD: Kinetics of the formed elements of human blood. Blood 50:699, 1977. 596. Nagai Y, Garrett KP, Ohta S, et al: Toll-like receptors on hematopoietic progenitor cells stimulate innate immune system replenishment. Immunity 24:801, 2006. 597. Yáñez A, Goodridge HS, Gozalbo D, Gil ML: TLRs control hematopoiesis during infection. Eur J Immunol 43:2526, 2013. 598. Sioud M, Fløisand Y, Forfang L, Lund-Johansen F: Signaling through toll-like receptor 7/8 induces the differentiation of human bone marrow CD34+ progenitor cells along the myeloid lineage. J Mol Biol 364:945, 2006. 599. Hirai H, Zhang P, Dayaram T, et al: C/EBPbeta is required for “emergency” granulopoiesis. Nat Immunol 7:732, 2006. 600. McGettrick AF, O’Neill LAJ: Toll-like receptors: Key activators of leucocytes and regulator of haematopoiesis. Br J Haematol 139:185, 2007. 601. Zhao JL, Ma C, O’Connell RM, et al: Conversion of danger signals into cytokine signals by hematopoietic stem and progenitor cells for regulation of stress-induced hematopoiesis. Cell Stem Cell 14:445, 2014. 602. Welner Robert S, Kincade Paul W: 9-1-1: HSCs respond to emergency calls. Cell Stem Cell 14:415, 2014. 603. Testa U: Apoptotic mechanisms in the control of erythropoiesis. Leukemia 18:1176, 2004.

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CHAPTER 6

THE ORGANIZATION AND STRUCTURE OF LYMPHOID TISSUES

Aharon G. Freud and Michael A. Caligiuri*

SUMMARY The lymphoid tissues can be divided into primary and secondary lymphoid organs. Primary lymphoid tissues are sites where lymphocytes develop from progenitor cells into functional and mature lymphocytes. The major primary lymphoid tissue is the marrow, the site where all lymphocyte progenitor cells reside and initially differentiate. This organ is discussed in Chap. 5. The other primary lymphoid tissue is the thymus, the site where progenitor cells derived from the marrow differentiate into mature thymus-derived (T) cells. Secondary lymphoid tissues are sites where lymphocytes undergo additional maturation and also interact with each other and with nonlymphoid cells to generate immune responses to antigens. These tissues include the spleen, lymph nodes, and mucosa-associated lymphoid tissues such as tonsils. The structure of these tissues provides insight into how the immune system discriminates between self-antigens and foreign antigens and develops the capacity to orchestrate a variety of specific and nonspecific defenses against invading pathogens.

THYMIC ANATOMY The thymus is located in the superior mediastinum, overlying, in order, the left brachiocephalic (or innominate) vein, the innominate artery, the left common carotid artery, and the trachea. It overlaps the upper limit of the pericardial sac below and extends into the neck beneath the upper anterior ribs. It receives its blood supply from the internal thoracic arteries. Venous blood from the thymus drains into the brachiocephalic and internal thoracic veins, which communicate above with the inferior thyroid veins. Arising from the third and fourth branchial pouches as an epithelial organ populated by lymphoid cells and endoderm-derived thymic epithelial cells, the thymus develops at about the eighth week of gestation.2 The thymus increases in size through fetal and postnatal life and remains ample into puberty,3 when it weighs approximately 40 g. Thereafter, the size progressively decreases with aging as a consequence of thymic involution.4 The cause of thymic involution is likely in part a result of the influence of glucocorticoid hormones.5 Nonetheless, there is evidence that T lymphocytes continue to develop throughout life, potentially including in some extrathymic sites.6 The volume of the thymus can be estimated by sonography. In one study of 149 healthy term infants within 1 week of birth, there was a significant correlation between the estimated thymic volume and the weight of the infant.3,7 However, no correlation was apparent between the estimated thymic volume and the infant’s sex, length, or gestational age. Also, there was no apparent correlation between estimated volume and the proportions of CD4+ T cells or CD8+ T cells found in the blood. The estimated thymic volume of healthy infants increases from birth to 4 and 8 months of age and then decreases.3 Most of the individual variation at 4 and 10 months of age appears to correlate with breastfeeding status, body size, and, to a lesser extent, illness. Breastfed infants at 4 months of age have significantly larger estimated thymic volumes than do age-matched formula-fed infants with similar thymic volumes at birth.8

THYMIC ARCHITECTURE

THE THYMUS The thymus is the site for development of thymic-derived lymphocytes, or T cells. In this organ, developing T cells, called thymocytes, differentiate from lymphoid stem cells derived from the marrow into functional, mature T cells.1 It is here that T cells acquire their repertoire of specific antigen receptors to cope with the antigenic challenges received throughout one’s life span. Once they have completed their maturation, the T cells leave the thymus and circulate in the blood and through secondary lymphoid tissues.

Acronyms and Abbreviations: AIRE, autoimmune regulatory gene; APECED, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy; CT, computed tomography; GALT, gastrointestinal-associated lymphoid tissue; Ig, immunoglobulin; IL, interleukin; ILC, innate lymphoid cell; MALT, mucosa-associated lymphoid tissue; MHC, major histocompatibility complex; NK, natural killer; PALS, periarteriolar lymphoid sheath; PGA syndrome, polyglandular autoimmune syndrome; r, correlation coefficient; T, thymus-derived; TCR, T-cell receptor.

This chapter was prepared by Thomas J. Kipps in the 8th edition and much of the text has been retained.

*

Kaushansky_chapter 06_p0085-0096.indd 85

A longitudinal fissure divides the thymus into two asymmetrical lobes, a larger right and a smaller left, that are derived from the right and left branchial pouches, respectively. These two developmentally separate parts of the thymus are easily separated from each other by blunt dissection. Each lobe of the thymus is divided into multiple lobules by fibrous septa that extend inward from an outer capsule. Each lobule consists of an outer cortex and an inner medulla (Fig. 6–1). The cortex contains dense collections of thymocytes (developing immature T cells) that cytologically appear as lymphocytes of slightly variable size with scattered, rare mitoses. The lighter-staining medulla is loosely arranged and more sparsely populated by mature thymocytes and characteristic tightly packed whorls of squamous-appearing epithelial cells, called thymic or Hassall corpuscles (Fig. 6–2). These appear to be remnants of degenerating cells and are rich in high-molecular-weight cytokeratins. Hassall’s corpuscles are thought to serve a critical role in the development of regulatory T cells.9 The thymus contains several important cell types that serve a variety of functions including supporting the maturation of thymocytes into mature T cells. There are several types of specialized epithelial cells within the thymus.10 The three main categories of thymic epithelial cells are the medullary epithelial cells, which are organized into clusters; the cortical epithelial cells, which form an epithelial network; and the epithelial cells of the outer cortex. The epithelial cells in the cortex and medulla often have a stellate shape, display desmosomal intercellular connections, and likely function as support cells to developing thymocytes by providing important growth factors such

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Figure 6–1.  Normal human infant thymus.

M

The thymus is surrounded by dense connective tissue capsule (Cap). It is organized into adjacent lobules separated by capsular connective tissue extensions or trabeculae. The lobules each have a dense cortex (C) and a lighter staining medulla (M). The medulla is a continuous tissue surrounded by the cortex that extends throughout the thymus, and it cannot be appreciated in a single section. (Reproduced with permission from Lichtman’s Atlas of Hematology, www.accessmedicine. com.)

C Cap

as interleukin (IL)-7.11 In addition, at primarily the corticomedullary junction, the thymus contains marrow-derived antigen-presenting cells, mostly interdigitating dendritic cells and macrophages. Scattered B cells are also present in the thymus, and these interact with maturing thymocytes and potentially regulate T-cell development.12,13 After puberty, thymic involution begins within the cortex. This region may disappear completely with aging, while medullary remnants persist throughout life. Glucocorticoids also may induce atrophy of the cortex secondary to glucocorticoid-induced apoptosis of cortical

thymocytes.5 This also may be seen in conditions that are associated with increases in circulating glucocorticoid hormones, for example, pregnancy or stress.14,15

THYMIC IMMUNE FUNCTION The thymus is the site of T-cell development. The importance of the thymus is underscored by patients with DiGeorge syndrome, or chromosome 22q11.2 deletion syndrome, who lack the genes required for

Figure 6–2.  Normal human infant thymus.

Higher magnification. Medulla. The arrows indicate thymic corpuscles (synonymous with Hassall corpuscles). They are composed of tightly packed, concentrically arranged, type IV endothelioreticular cells with flattened nuclei. The central mass is composed of keratinized cells. In addition to thymic corpuscles and the mass of small densely stained T lymphocytes, the medulla contains scattered, larger, type V epithelioreticular cells with their light nuclei, dark nucleolus, and eosinophilic cytoplasm, evident on this section. (Reproduced with permission from Lichtman’s Atlas of Hematology, www.accessmedicine.com.)

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Chapter 6: The Organization and Structure of Lymphoid Tissues

Figure 6–3.  Structure of the thymus. The top half of the

Hassall corpuscle

Capsule Trabeculum Thymocytes

T lymphocytes (mature)

Dendritic cell

Epithelial cell Cortex Stem cell

{

CD4– CD8– TCR– (Common precursor pre-T cell)

Precursor

{

figure provides a cross section of a thymic lobule, indicating the outer cortex (left), inner medulla (center), and periphery (far right). The arrows indicate various structures and cell types. As thymocytes mature, they migrate from the cortex toward the medullary region and acquire phenotypic features that are outlined at the bottom of the figure, as described in the text (Chap. 74).

Periphery

Medulla

CD4+ CD8+ TCRαβ (low)

{ {

CD4+ TH CD8– TCRαβ (high) CD4– TC CD8+ TCRαβ (high)

Rearrangement (TCR), positive/negative selection

Functional T lymphocytes Helper (Th), cytolytic (TC)

thymic development.16 These patients do not develop T cells and hence have profound immune deficiency. Prothymocytes originate in the marrow and migrate to the thymus, where they mature into T cells (Chap. 76). Maturation of T cells is accompanied by the sequential acquisition of various T-cell markers including CD2, CD3, CD4 or CD8, CD5, and the T-cell receptor (TCR) (Fig. 6–3).17 Terminal deoxynucleotidyl transferase (TdT) is found in prothymocytes and immature thymocytes but is absent in mature T cells. TdT facilitates the successful rearrangement of TCR genes in immature thymocytes.18 T-cell precursors can be found in distinct microenvironments within the thymus. Marrow-derived CD34+ pre-T cells enter the cortex via small blood vessels and are double-negative for CD4 and CD8 antigens.1 One of the earliest identifiable T-cell membrane antigens is CD2. As the thymocytes proliferate and differentiate in the cortex, they acquire CD4 and CD8 antigens. They subsequently acquire the CD3 antigen and the TCR for antigen as they migrate toward the medulla. In the cortex, the thymocytes are induced to express the chemokine receptor, CCR7, which directs their migration to CCL19- and CCL21producing cells in the thymic medulla where they undergo further maturation.19 Positive and negative selection of maturing T cells takes place in the thymus.20 Double-positive (CD4+ and CD8+) thymocytes undergo an initial positive selection step that is mediated exclusively by thymic cortical epithelium to ensure that developing T cells can recognize peptides in the context of self major histocompatibility complex (MHC) molecules.21 Thymocytes that have TCRs capable of interacting with self MHC molecules expressed by thymic cortical epithelial cells undergo expansion, whereas thymocytes with defective TCR undergo apoptosis.22–24 As these positively selected cells migrate toward the medulla, they experience negative selection through their interaction with thymic medullary epithelial cells in order to ensure that any T cells that react too strongly to self MHC molecules are deleted. These thymic medullary epithelial cells uniquely express the autoimmune regulatory

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87

gene (AIRE). AIRE encodes a transcriptional regulator that promotes ectopic expression of a large repertoire of transcripts encoding proteins that ordinarily are restricted to differentiated organs residing in the periphery.25 This allows the thymic medullary epithelial cells to express many different self-antigens, which are presented to developing thymocytes. Those thymocytes that have TCR that react too vigorously with the MHC molecules of the medullary epithelium will undergo apoptosis.23 Most of the developing thymocytes are destroyed. In this way, only those T cells that have the appropriate level of affinity for selfMHC molecules yet are not reactive against self antigens will reach the medulla to undergo the final maturation stages and eventually exit the thymus via efferent lymphatics as functionally competent naïve CD4+ and CD8+ single-positive T cells. Patients with the rare disease autoimmune polyendocrinopathycandidiasis-ectodermal dystrophy (APECED) or polyglandular autoimmune (PGA) syndrome type I (PGA I) underscore the importance of negative selection of thymocytes by thymic medullary epithelial cells. APECED, or PGA I, is characterized by chronic mucocutaneous candidiasis, hypoparathyroidism, and adrenal insufficiency. In addition, most patients also have a number of other autoimmune manifestations, including thyroiditis, type 1 diabetes, ovarian failure, alopecia, and/or hepatitis.26 These patients have genetic defects in AIRE,27 which precludes their thymic epithelial cells from expressing the large variety of tissue differentiation self-antigens required for the negative selection of self-reactive thymocytes and the generation of central T-cell tolerance.25,28

THE SPLEEN The spleen is a specialized abdominal organ serving multiple functions in erythrocyte clearance, innate and adaptive immunity, and the regulation of blood volume. In general the spleen contains two structurally and functionally distinct components: white and red pulp. The white pulp of the spleen consists of secondary lymphoid tissue that provides

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an environment in which the cells of the immune system can interact with one another to mount adaptive immune responses to bloodborne antigens. The splenic red pulp contains macrophages that are responsible for clearing the blood of unwanted foreign substances and senescent erythrocytes, even in the absence of specific immunity. Thus, it acts as a filter for the blood.

SPLENIC ANATOMY The spleen is located within the peritoneum in the left upper quadrant of the abdomen between the fundus of the stomach and the diaphragm. It receives its blood supply from the systemic circulation via the splenic artery, which branches off the celiac trunk, and the left gastroepiploic artery.29 The blood returning from the spleen drains into the portal circulation via the splenic vein. Therefore, the spleen can become congested with blood and increase in size when there is portal vein hypertension (Chap. 56). Approximately 10 percent of individuals have one or more accessory spleens. Accessory spleens are usually 1 cm in diameter and resemble lymph nodes. However, they usually are covered with peritoneum, as is the spleen itself. Accessory spleens typically lie along the course of the splenic artery or its gastroepiploic branch, but they may be elsewhere.30 The commonest location is near the hilus of the spleen, but approximately 1 in 6 accessory spleens can be found embedded in the tail of the pancreas, where they may be occasionally mistaken for a pancreatic mass lesion.31 The average weight of the spleen in the adult human is 135 g (range: 100 to 250 g). However, when emptied of blood it weighs only approximately 80 g. On autopsy of 539 subjects with normal spleens, there was a positive correlation between the spleen weight and both the degree of acute splenic congestion and the subject’s height and weight, but not with the subject’s sex or age.32 The splenic volume can be estimated by computed tomography (CT) of the abdomen.33 In one study, the splenic volume was calculated from the linear and the maximal cross-sectional area measurements of the spleen, using the following formula: splenic volume = 30 cm3 + 0.58 (the product of the width, length, and thickness of the spleen measured in centimeters).34 Using this formula, the mean value of the calculated splenic volume for 47 normal subjects was 214.6 cm3, with a range of 107.2 to 314.5 cm3. The calculated splenic volume did not appear to vary significantly with the subject’s age, gender, height, weight, body mass index, or the diameter of the first lumbar vertebra, the latter being considered representative of body habitus on CT. The splenic volume also can be estimated by sonography, which provides good correlation with volumes measured by helical abdominal CT or actual volume displaced by the excised organ. In one study of 50 patients, the linear measurement by sonography that correlated most closely with CT volume was the spleen width measured on a longitudinal section with the patient in the right lateral decubitus position (correlation coefficient [r] = 0.89, p T or c.968T>C).273 Thus G6PD A– arose in an individual who already had the G6PD A+ mutation. However, the ancestral human sequence has been deduced to be that of G6PD B, both by showing that this is the sequence of the chimpanzee,274 our nearest relative, and by analysis of linkage dysequilibrium.275 Although it has been suggested that only the interaction of p.Val68Met and p.Asn126Asp invariably results in G6PD A– deficiency,276 the

Kaushansky_chapter 47_p0689-0724.indd 702

c.202G>A mutation has been found in a patient to cause deficiency without the presence of the mutation at cDNA nucleotide (nt) 376.274 Variants in the Mediterranean Region Among white populations, G6PD deficiency is most common in Mediterranean countries. The most common enzyme variant in this region is G6PD Mediterranean.270,277 The enzyme activity of the red cells of individuals who have inherited this abnormal gene is barely detectable. Other variants are also prevalent in the Mediterranean region, including G6PD A– and G6PD Seattle (see Table  47–4). Variants in Asia A great many different variants have been described in Asian populations. Some of these proved to be identical at a molecular level (e.g., G6PD Gifu-like, Canton, Agrigento-like, and Taiwan-Hakka all have the same mutation at cDNA nt 1376 [see Table  47–4]). DNA analysis shows that more than 100 different mutations are found in various Asian populations.160,278 Variants Producing Hereditary Nonspherocytic Hemolytic Anemia Some mutations of G6PD result in chronic hemolysis without, but exacerbated by, precipitating causes. These variants are class I mutants (World Health Organization [WHO] class 1).265 From a functional point of view, these mutations are more severe than the more commonly occurring polymorphic forms of the enzyme, such as G6PD Mediterranean and G6PD A–. On a molecular level, such variants are often caused by mutations located in exons 10 and 11, encoding the subunit interface, or affect residues that bind the structural NADP molecule.143,158 There are, however, exceptions to this rule.28,279–281 The clinical severity of these variants can be quite variable.282 G6PD deficiency has also been encountered in the rat, dog,283 mouse,284 and horse.285 G6PD deficiency in mice has been rescued by stable in vivo expression of the human G6PD gene in hematopoietic tissues by a gene transfer approach.271,286

Pyruvate Kinase

PK deficiency is the second most common enzyme disorder in glycolysis and the most common cause of nonspherocytic hemolytic anemia.287 Like G6PD deficiency, the disease is genetically heterogeneous, with different mutations causing different kinetic changes in the enzyme that is formed. There are even cases in which the activity of PK as measured in vitro is higher than normal, but a kinetically abnormal enzyme is responsible for the occurrence of hemolytic anemia.288 Kinetic characterization and analysis of PK mutants is considerably more complex than analysis of G6PD mutants. Most PK-deficient patients are compound heterozygous for two different (missense) mutations, rather than homozygous for one. Assuming that stable mutant monomers are synthesized, up to seven different tetrameric forms of PK may be present in compound heterozygous individuals, each with distinct structural and kinetic properties. This complicates genotype-to-phenotype correlations in these individuals as it is difficult to infer which mutation is primarily responsible for deficient enzyme function and the clinical phenotype.289,290 More than 230 mutations in the PKLR gene encoding the red cell PK have been identified. Seventy percent of these mutations are missense mutations affecting conserved residues in structurally and functionally important domains of PK. There appears to be no direct relationship between the nature and location of the substituted amino acid and the type of molecular perturbation.124 Hence, the nature of the mutation has relatively little predictive value with respect to the severity of the clinical course and the phenotypic expression of identical mutations can be strikingly different in patients.29,289–291 Apart from decreased red blood cell survival ineffective erythropoiesis because of increased numbers of apoptotic cells is implicated as one of the pathophysiologic features of PK deficiency.292,293 In particular, glycolytic inhibition by mutation of PKLR has been suggested to augment oxidative stress, leading to proapoptotic gene expression.293

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Chapter 47: Erythrocyte Enzyme Disorders

703

TABLE 47–4.  Major Polymorphic G6PD Variants Variant

Nucleotide Substitution

Amino Acid Substitution

WHO Class†

Distribution

Reference

Gaohe

c.95A>G

p.(His32Arg)

III

Chinese

673

Honiara

c.99A>G c.1360C>T

p.(Ile33Met) p.(Arg454Cys)

I

Solomon Islands

674

Orissa

c.131C>G

p.(Ala44Gly)

III

India, Italy

675, 676

Aures

c.143T>C

p.(Ile48Thr)

III

Algeria, Tunisia

677, 678

Metaponto

c.172G>A

p.(Asp58Asn)

III

Italy

679

A–

c.202G>A c.376A>G

p.(Val68Met) p.(Asn126Asp)

III

Africa

277

Namoru

c.208T>C

p.(Tyr70His)

II

Vanuatu Archipelago

680

Ube-Konan

c.241C>T

p.(Arg81Cys)

III

Japan, Italy

676, 681

A+

c.376A>G

p.(Asn126Asp)

III-IV

Africa, Mediterranean

272

Vanua Lava

c.383T>C

p.(Leu128Pro)

II

Southwestern Pacific

680

Quing Yan

c.392G>T

p.(Gly131Val)

III

China

682

Mahidol

c.487G>A

p.(Gly163Ser)

III

Southeast Asia

683

Santamaria

c.542A>T c.376A>G

p.(Asp181Val) p.(Asn126Asp)

II

Costa Rica, Italy

684, 685

Mediterranean, Dallas, Panama, Sassari

c.563C>T

p.(Ser188Phe)

II

Mediterranean

277, 686

Coimbra

c.592C>T

p.(Arg198Cys)

II

India, Portugal

687

A–

c.680G>T c.376A>G

p.(Arg227Leu) p.(Asn126Asp)

III

Africa

274

p.(Asp282His)

III

United States, Italy

688–690

p.(Arg285His)

III

Italy

691

Seattle, Lodi, Modena, Ferrara II, Athens-like Montalbano

c.854G>A

Viangchan, Jammu

c.871G>A

p.(Val291Met)

II

China

692, 693

Kalyan, Kerala, Jamnaga, Rohini

c.949G>A

p.(Glu317Lys)

III

India

694, 695

A–, Betica, Selma, Guantanamo

c.968T>C c.376A>G

p.(Leu323Pro) p.(Asn126Asp)

III

Africa, Spain

274

Chatham

c.1003G>A

p.(Ala335Thr)

II

Italy, Asia, Africa

277

Chinese-5

c.1024C>T

p.(Leu342Phe)

III

China

682

Ierapetra

c.1057C>T

p.(Pro353Ser)

II

Greece

696

Cassano

c.1347G>C

p.(Gln449His)

II

Italy, Greece

697, 698

Union, Maewo, Chinese-2, Kalo

c.1360C>T

p.(Arg454Cys)

II

Italy, Spain, China, Japan

697, 699, 700

Canton, Taiwan-Hakka, Gifu-like, Agrigento-like

c.1376G>T

p.(Arg459Leu)

II

Japan, Italy

701, 702

Cosenza

c.1376G>C

p.(Arg459Pro)

II

Italy

697

Kaiping, Anant, Dhon, Sapporo-like, Wosera

c.1388G>A

p.(Arg463His)

II

China

700, 702

 †Class 1, severely deficient, associated with nonspherocytic hemolytic anemia; class 2, severe deficiency (1 to 10 percent residual activity), associated with acute hemolytic anemia; class 3, moderate deficiency (10 to 60 percent residual activity); class 4, not deficient (60 to 150 percent activity); class 5, increased activity (>150 percent). Adapted from PJ Mason, JM Bautista, F Gilsanz158 and A Minucci, K Moradkhani, MJ Hwang, et al.160 Data from Mason, P. J., Bautista, J. M., and Gilsanz, F. G6PD deficiency: The genotype-phenotype association. Blood reviews. 21: 267–283, 2007 and Minucci, A., Moradkhani, K., Hwang, M. J., et al. Glucose-6-phosphate dehydrogenase (G6PD) mutations database: Review of the “old” and update of the new mutations. Blood cells, molecules & diseases 48: 154–165, 2012.

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PK deficiency may be also caused by mutations not directly involving the PKLR gene as demonstrated by a deficiency of PK being one of the key features of severe congenital hemolytic anemia caused by mutations in the key erythroid transcription factor KLF1.294 There is evidence that PK deficiency provides protection against infection and replication of Plasmodium falciparum in human erythrocytes,295,296 an effect possibly mediated by reduced ATP levels in PKdeficient red blood cells.297 This suggests that PK deficiency may confer a protective advantage against malaria in human populations in areas where this disease is endemic. In agreement with this, population studies on sub-Saharan African populations indicate that malaria is acting as a selective force in the PKLR genomic region.298–300 PK deficiency has also been recognized in mice, dogs, and multiple breeds of domestic cats.301 In all these animals, the deficiency causes severe anemia and marked reticulocytosis, closely resembling human PK deficiency. Basenji dogs with PK deficiency completely lack PKLR enzymatic activity and, instead, only the PK-M2 isozyme is expressed in their red blood cells.302 A unique feature of PK deficiency in dogs is the progressive development of myelofibrosis and osteosclerosis. Marrow fibrosis may occur in response to damage caused by iron overload,303 although factors associated with marked erythropoiesis have also been proposed to play a role.304 Gene therapy approaches have been employed to cure PK deficiency in dogs.305 PK-deficient mice show delayed switching from PK-M2 to PK-R, resulting in delayed onset of the hemolytic anemia.306 PK deficiency in mice has been rescued by expression of the human PK-R isozyme in murine hematopoietic stem cells.307,308

Other Enzyme Deficiencies

Hexokinase Deficiency Nineteen families with HK deficiency have been described as of the time of this writing309–311 and only four patients have been characterized at the molecular level.310–313 Two of these patients were homozygous, either for a highly conserved substitution in the enzyme’s active site313 or a lethal out-of-frame deletion of exons 5 to 8 of HK1.310 In one patient a regulatory mutation was identified in the putative erythroid-specific promoter. In vitro, this mutation disrupted binding of the AP-1 transcription factor complex, leading to strongly decreased gene expression.311 In mice, a mutation designated downeast anemia causes severe hemolytic anemia with extensive tissue iron deposition and marked reticulocytosis, representing a mouse model of generalized HK deficiency.314 Glucosephosphate Isomerase Deficiency Glucosephosphate isomerase deficiency is second to PK deficiency in frequency, with respect to glycolytic enzymopathies. To date, approximately 55 families with glucosephosphate isomerase deficiency have been described worldwide.315–320 Most of these patients are compound heterozygous for mutations that partially inactivate the enzyme. Most of the 31 GPI mutations reported to date are missense mutations. Mapping of these mutations to the crystal structure of the human enzyme and recombinant expression of genetic variants has provided considerable insight in the molecular mechanisms causing hemolytic anemia in this disorder.321,322 The majority of the mutations disrupt key interactions that contribute directly or indirectly to the architecture of the enzyme’s active site.321 In rare cases, GPI deficiency also affects nonerythroid tissues, causing severe neuromuscular symptoms and granulocyte dysfunction.323–328 The finding that GPI also functions as a neuroleukin,329 an autocrine motility factor,330 a nerve growth factor,331 and a differentiation and maturation mediator332 has led to the hypothesis that the mutation-dependent loss of cytokine function of GPI could account for the neuromuscular symptoms.333 An alternative explanation involves disturbed glycerolipid biosynthesis in GPI deficiency, which could have significant effects on membrane formation, membrane function, and axonal migration.334,335

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Homozygous GPI-deficient mice exhibit hematologic features resembling that of the human enzymopathies. In addition, other tissues are also affected, indicating a reduced glycolytic capability of the whole organism.336 Complete loss of GPI in mice is embryonically lethal.337 Phosphofructokinase Deficiency Because red cells contain both PFK M and L subunits, mutations affecting either gene (PFKM or PFKL) will lead to a partially reduced red cell enzyme activity in PFK deficiency. Mutations in the PFKM gene cause PFKM deficiency or glycogen storage disease VII (Tarui disease).338 The disease is characterized predominantly by mild to severe myopathy, in particular exercise intolerance, cramps, and myoglobinuria. The associated hemolysis is usually mild but may be absent. As of this writing, there has been only one reported case in which an unstable L subunit was identified. This patient exhibited no signs of myopathy or hemolysis.75 Approximately 100 cases of PFK deficiency have been reported as of this writing, and 23 mutant PFKM alleles are reported. Approximately half of the reported mutations are missense mutations, the remaining mutations mostly affect splicing. Intriguingly, PFK-deficient Ashkenazi Jews share two common mutations: a G>A base change affecting the donor splice site of intron 5 (c.237+1G>A) and a single base deletion in exon 2 (c.2003delC).33,339 The mode of action by which missense mutations cause disease is largely unknown.33,340–347 PFK deficiency in dogs301 is characterized by the association of hemolytic crises with strenuous exercise.348 Pfkm null mice show exercise intolerance, reduced life span, and progressive cardiac hypertrophy, suggesting that Tarui disease should be considered as a complex systemic disorder rather than a muscle glycogenosis.349,350 Aldolase Deficiency At the time of this writing, only six patients with red cell aldolase deficiency had been described, four of whom were characterized at the DNA level. All displayed moderate chronic hemolytic anemia, either by itself351 or accompanied by myopathy,352,353 rhabdomyolysis,354 psychomotor retardation,352 or mental retardation.77,352 Triosephosphate Isomerase Deficiency TPI deficiency is characterized by hemolytic anemia, often accompanied by neonatal hyperbilirubinemia requiring exchange transfusion. In addition, patients display progressive neurologic dysfunction, increased susceptibility to infection, and cardiomyopathy.355 Most affected individuals die before the age of 6 years, but there are remarkable exceptions.356 TPI deficiency is the most severe disorder of glycolysis. Key in the pathophysiology of the severe neuromuscular disease is the formation of toxic protein aggregates: accumulation of the substrate dihydroxyacetone phosphate results in elevated levels of the toxic methylglyoxal, leading to the formation of terminal glycation of proteins, whereas mutation-induced changes in the quaternary structure of TPI lead to the formation of an aggregation-prone protein.357,358 Therefore, it has been suggested that TPI deficiency represents a conformational rather than a metabolic disease.357 Approximately 40 patients and 19 different mutations have been reported in TPI deficiency.355,358–363 The most common mutation is the p.(Glu104Asp) amino acid change which is detected in approximately 80 percent of patients, all descendants from a common ancestor.364 Studies on recombinant mutant TPI show that the p.(Glu104Asp) does not affect catalysis. Instead, the mutation disrupts a conserved network of buried water molecules, which prevents efficient formation of the active TPI dimer, causing its dissociation in inactive monomers.85 TPI-null mice die at an early stage of development.365 Hemolytic anemia characterizes the only viable mouse model of TPI deficiency.366 Studies on a Drosophila model recapitulating the neurologic phenotype of TPI deficiency367 suggests that loss of an isomerase-independent function of TPI underlies the neuropathogenesis in TPI deficiency.368 Phosphoglycerate Kinase Deficiency PGK deficiency is one of the relatively uncommon causes of hereditary nonspherocytic

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hemolytic anemia. Mutations in the X chromosome-linked gene may cause mild to severe chronic hemolysis, neurologic dysfunction, and myopathy.369 Approximately 40 patients with PGK deficiency have been reported.369,370 Most patients manifest either hemolytic anemia in combination with neurologic symptoms, including mental retardation, seizures, progressive decline of motor function, and developmental delay, or isolated myopathy.370–372 The combination of all clinical manifestations is a rare event, described in only 2 families.373,374 Splenectomy has been reported to be beneficial but does not correct the hemolytic process.341,369 Marrow transplantation has been performed to prevent the manifestation of severe neurologic symptoms.375 Twenty-two unique mutations have been identified.370,371 Most of these mutations (80 percent) are missense mutations. Most of the encoded amino acid changes heavily affect the protein’s thermal stability and to a different extent catalytic efficiency.371,376 In an attempt to correlate the genotype to the phenotype, it was found that amino acid changes grossly impairing protein stability but moderately affecting kinetic properties were associated mostly with hemolytic anemia and neurological symptoms. Mutations perturbing both catalysis and heat stability were associated with myopathy alone, whereas mutations faintly affecting molecular properties of PGK correlated with a wide range of clinical symptoms.376 Yet, the precise reason for the different clinical manifestations of mutations of the same gene remains unknown, suggesting the involvement of yet unknown alternate function of this enzyme, environmental, metabolic, genetic and/or epigenetic factors.372,376 Bisphosphoglycerate Mutase Deficiency Bisphosphoglycerate mutase deficiency is a very rare disorder. Only three affected families have been characterized. Bisphosphoglycerate mutase deficiency appears to be inherited as an autosomal recessive disorder. However, some heterozygous relatives have had a borderline high hemoglobin concentration,377,378 and in one single affected patient only one mutation was identified.379 Erythrocytosis was the predominant feature of the clinically normal probands, likely resulting from reduced 2,3-BPG levels380 and, consequently, the increased oxygen affinity of hemoglobin (Chap. 57). Glutamate Cysteine Ligase Deficiency GCL deficiency is associated with mild hereditary nonspherocytic hemolytic anemia that may be fully compensated. Drug- and infection-induced hemolytic crises may occur as a consequence of strongly reduced GSH levels. As of this writing, eight cases of GCL deficiency had been described, belonging to six unrelated families.381–388 In approximately half of the patients with GCL deficiency, the hemolytic anemia was accompanied by impaired neurological function.388 Six patients have been characterized at the molecular level and five different mutations have been reported.385–388 In all these cases, the causative mutation affected the catalytic subunit of GCL. The clinically observed mutations have been mapped to a homology model of the human enzyme, based on the crystal structure of GCL of Saccharomyces cerevisiae, thus explaining the molecular basis of GSH depletion as a result of GCL deficiency.192 Complementary expression studies in mice showed that these GCL mutations impair glutathione production by reducing the activity of the catalytic subunit of GCL. Addition of the modifier subunit was able to largely restore enzymatic activity, thereby underscoring the critical role of GCLM.389 Complete deficiency of GCLC has shown to be lethal in mice,390,391 whereas GCLM-null mice are viable and show no overt phenotype despite strongly reduced GSH levels, including a reduction of more than 90 percent in red blood cells.392 Upon exposure to oxidative stress, however, red blood cells from such mice undergo massive hemolysis with fatal outcome.393 Glutathione Synthetase Deficiency GS deficiency394 is the most common abnormality of red cell glutathione metabolism. Three distinct clinical forms of GS deficiency can be distinguished,395 most likely reflecting different mutations or epigenetic modifications in the GS

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gene.396 Patients with mild GS deficiency display mild hemolytic anemia as their only symptom. In contrast, patients with a moderate deficiency usually present in the neonatal period with metabolic acidosis, 5oxoprolinuria, and mild to moderate hemolytic anemia. In addition to these symptoms, patients with the third and most severe type develop progressive neurologic symptoms such as psychomotor retardation, mental retardation, seizures, ataxia, and spasticity. 5-Oxoprolinuria results from accumulation of γ-glutamylcysteine because of decreased feedback inhibition of GCL by the decreased levels of GSH.397 Importantly, 5-oxoprolinuria may have other causes.398,399 Experiments in rats show that acute administration of 5-oxoproline induces oxidative damage in the brain, a mechanism that may be involved in the neurologic symptoms of severe GS deficiency.400 The diagnosis of GS deficiency has been established in more than 70 patients from 50 families,396,397,401,402 of whom approximately 25 percent died in childhood.401 Thirty-two mutations are identified as being associated with GS deficiency. Based on the nature of the mutation, and taking into account GS activity and GSH levels it seems possible to predict a mild versus a more severe phenotype.396 The structural effects of a number of missense mutations have been determined.197 A long-term followup study showed that early diagnosis, correction of acidosis, and early supplementation with antioxidants vitamins C and E improve survival and long-term outcome.395 For these reasons it has been argued that GS deficiency should be included in the newborn screening program.401 Complete deficiency of GS has shown to be lethal in mice, whereas heterozygous animals survive with no distinct phenotype.403 Glutathione Reductase Deficiency Only two families with hereditary GR deficiency have been described and characterized.404,405 The complete absence of GR in the red cells of members of one family was associated with only rare episodes of hemolysis, possibly caused by fava beans. GR deficiency was caused by homozygosity for a large genomic deletion. GR deficiency in the other family was caused by compound heterozygosity for a nonsense mutation, and a missense mutation affecting a highly conserved residue. GR in red cells was undetectable, but some residual activity was found in the patient’s leucocytes.404 In vitro studies on members of one of the GR deficiency families has provided experimental evidence that GR deficiency may protect from malarial infection by enhancing phagocytosis of ring-infected red blood cells.406 Adenylate Kinase Deficiency AK deficiency has been reported in 12 unrelated families and 7 different mutations have been identified.263,407–412 In all but one case,263,413 the deficiency was associated with moderate to severe hemolytic anemia. In some of the patients, mental retardation and psychomotor impairment was also observed.410,414 Studies on a number of recombinant proteins revealed strongly altered catalytic properties or protein stability resulting from mutation.241 In contrast, patient’s cells sometimes displayed considerable residual enzymatic activity. The activation of expression of other isozymes, that is, AK2 and AK3, has been proposed as one of the factors contributing to this apparent discrepancy.412 Adenosine Deaminase Hyperactivity An increased activity of ADA is associated with hereditary nonspherocytic hemolytic anemia. It is the only red cell enzyme disorder that is inherited in an autosomal dominant disorder.415 Adenosine deaminase hyperactivity results in depletion of red cell ATP.415,416 Few cases with a 30- to 70-fold increase in activity have been described. The molecular mechanism of this disorder has not been identified but the markedly increased amounts of ADA mRNA in affected individuals indicate that the red blood cell–specific overexpression occurs at the mRNA level,417 causing an overproduction of a structurally normal enzyme.418 ADA hyperactivity probably results from a cis-acting mutation in the vicinity of the ADA gene.419

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reported missense mutations affect residues of the catalytic site, suggesting that the reduced catalytic efficiency and/or instability result from secondary effects related to conformational changes.248 Acquired deficiency of P5′N1 may result from lead poisoning. Structural studies have shown that Pb2+ specifically binds within the active site, in a different position than Mg2+ but with much higher affinity.248 Because simultaneous binding of Mg2+ and Pb2+ is not possible, Pb2+ outcompetes Mg2+, thereby preventing this essential cofactor from binding, thus abolishing catalytic activity. P5′N1 activity is also inhibited in β-thalassemia and related disorders that result in excess α-globin chains, such as hemoglobin E, probably from oxidative damage induced by excess α-globin chains.436,437

MECHANISM OF HEMOLYSIS G6PD Deficiency

The life span of G6PD-deficient red cells is shortened under many circumstances, particularly during drug administration and infection. The exact reason for this is not known.

Figure 47–6. Prominent basophilic stippling in pyrimidine-5′nucleotidase-1 (P5′N1) deficiency.

For reasons that are not understood, milder elevations of red cell ADA activity (two- to sixfold) are also increased in most, but not all, patients with Diamond-Blackfan anemia.186 Deficiency of ADA is associated with severe combined immunodeficiency (Chap. 80). In this disorder, large quantities of deoxyadenine nucleotides, not normally present in erythrocytes, accumulate. Pyrimidine 5′-Nucleotidase Deficiency Pyrimidine 5′-nucleotidase deficiency is the most frequent disorder of red cell nucleotide metabolism and a relatively common cause of mild-to-moderate hemolytic anemia.420–422 More than 100 patients have been reported, but because of the relatively mild phenotype many patients may remain undetected. Deficient enzyme function leads to the accumulation of pyrimidine nucleotides. This results in prominent stippling on the blood film, the hallmark of this disorder (Fig. 47–6).244 Hence, P5′N1 deficiency is the only red cell enzyme deficiency in which red cell morphology is helpful in establishing the diagnosis. The precise mechanism leading to premature destruction of P5′N1-deficient red cells is unknown. Some proposed pathophysiologic mechanisms have related the accumulation of pyrimidine nucleotide to alterations of the red cell membrane due to increased levels of cytidine diphosphate (CDP)-choline and CDP-ethanolamide,423 decreased pentose phosphate shunt activity,424–426 chelation of Mg2+ ions that serves as a cofactor for a number of enzymes,427 decreased phosphoribosyl pyrophosphate synthetase activity,428,429 increased activity of pyrimidine nucleoside monophosphate kinase,430 increased levels of GSH,431 and competition with reactions that require ADP or ATP.432 However, clear cause-and-effect relationships have not been established. As of this writing, 27 different mutations have been reported in NT5C3A in association with P5′N1 deficiency.420,433,434 Most patients were found to be homozygous for a specific mutation. The majority of mutations concern frameshift or nonsense mutations, deletions, or mutations that affect splicing. Functional analysis of reported missense mutations was studied using recombinant mutant proteins. These rendered contrasting results between the substantial changes in kinetic behavior and thermostability and the actual residual enzymatic activity in patient’s red cells, probably due to compensation by upregulation of other nucleotidases.435 Of interest is the observation that none of the

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Drug-Induced Hemolysis

Drug-induced hemolysis in G6PD-deficient cells is generally accompanied by the formation of Heinz bodies, particles of denatured hemoglobin, and stromal protein (Chap. 49), formed only in the presence of oxygen.438 Together with the inability to protect their GSH against drug challenge, this suggests that a major component of the hemolytic process is the inability of G6PD-deficient cells to protect sulfhydryl groups against oxidative damage.2 The mechanism by which Heinz bodies are formed and become attached to red cell stroma has been the subject of considerable investigation and speculation. Exposure of red cells to certain drugs results in the formation of low levels of hydrogen peroxide as the drug interacts with hemoglobin.439 In addition, some drugs may form free radicals that oxidize GSH without the formation of peroxide as an intermediate.440 The formation of free radicals of GSH through the action of peroxide or by the direct action of drugs may be followed either by oxidation of GSH to the disulfide form (GSSG) or complexing of the glutathione with hemoglobin to form a mixed disulfide. Such mixed disulfides are believed to form initially with the sulfhydryl group of the β-93 position of β-globin.441 The mixed disulfide of GSH and hemoglobin is probably unstable and undergoes conformational changes exposing interior sulfhydryl groups to oxidation and mixed disulfide formation. Globin chain separation into free α and β chains also occurs.442 Once such oxidation has occurred, hemoglobin is denatured irreversibly and will precipitate as Heinz bodies. Normal red cells can defend themselves to a considerable extent against such changes by reducing GSSG to GSH and by reducing the mixed disulfides of GSH and hemoglobin through the GR reaction.42 However, the reduction of these disulfide bonds requires a source of NADPH. Because G6PD-deficient red cells are unable to reduce NADP+ to NADPH at a normal rate, they are unable to reduce hydrogen peroxide or the mixed disulfides of hemoglobin and GSH. Moreover, because catalase contains tightly bound NADPH443 that is required for activity, the lack of freely available NADPH generation may, in addition, impede disposal of hydrogen peroxide by the catalase-dependent pathway.444 When such cells are challenged by drugs, they form Heinz bodies more readily than do normal cells. Cells containing Heinz bodies encounter difficulty in traversing the splenic pulp445 and are eliminated relatively rapidly from the circulation. Figure 47–7 summarizes a plausible scenario of the metabolic events that leads to red cell damage and eventually destruction. However, it has been shown that in mice, targeted disruption of the gene encoding glutathione peroxidase has little effect on oxidation of hemoglobin of murine red cells challenged with peroxides.199 In addition,

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Sup Dismut

O–2 DRUG + HbO2

H2O2

GSH-Px

707

of the acid phosphatase gene have been attributed to a decrease in the f isoforms of this tyrosine phosphatase and, consequently, low GSH levels.455 Immunologic factors do not seem to play a role in favism.456 Increased levels of red cell calcium457,458 and consequent “crossbonding” of membranes may occur. Other membrane alterations that have been described are oxidation and clustering of membrane proteins, hemichrome binding to the internal face of the membrane, destabilization of the membrane, and the release of microvesicles.459–462

Icterus Neonatorum

GSSG

GSH

+

+

NADPH

GR

NADP

G-6-PD

6–Phosphogluconate

Glucose-6-P

Figure 47–7.  Reactions through which hydrogen peroxide is gen-

erated and detoxified in the erythrocyte. In glucose-6-phosphate dehydrogenase (G6PD) deficiency and related disorders, inadequate generation of nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH) results in accumulation of GSSG and probably of hydrogen peroxide (H2O2). The accumulation of these substances leads to hemoglobin denaturation, Heinz body formation, and, consequently, to decreased red cell survival. GR, glutathione reductase; GSH, reduced glutathione; GSH-Px, glutathione peroxidase; GSSG, glutathione disulfide (oxidized glutathione); HbO2, oxyhemoglobin; NADP, nicotinamide adenine dinucleotide phosphate; Sup Dismut, superoxide dismutase.

catalase-null mice show negligible antioxidant function of catalase in oxidant injury.446 If such murine models reflect the situation in man, then different pathways requiring GSH, such as the thioredoxin, and/or peroxiredoxin reactions, may be important.446,447 The formation of methemoglobin frequently accompanies the administration of drugs that have the capacity to produce hemolysis of G6PD-deficient cells.448 The heme groups of methemoglobin become detached from the globin more readily than do the heme groups of oxyhemoglobin.449 It is not clear whether methemoglobin formation plays an important role in the oxidative degradation of hemoglobin to Heinz bodies or whether formation of methemoglobin is merely an incidental side effect of oxidative drugs.450,451

Infection-Induced Hemolysis

The mechanism of hemolysis induced by infection or occurring spontaneously in G6PD-deficient subjects is not well understood. The generation of hydrogen peroxide by phagocytizing leukocytes may play a role in this type of hemolytic reaction.451

Favism

Substances capable of destroying red cell GSH have been isolated from fava beans,452 but scientific evidence that these components (i.e., divicine and isouramil) are indeed responsible for hemolysis is lacking. Favism occurs only in G6PD-deficient subjects, but not all individuals in a particular family may be sensitive to the hemolytic effect of the beans. Nonetheless, some tendency toward familial occurrence has suggested that an additional genetic factor may be important.453 The observation of increased excretion of glucaric acid454 led to the suggestion that a defect in glucuronide formation might be present. Specific genotypes

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G6PD-deficient neonates are at increased risk of developing severe icterus neonatorum. The icterus is frequently unaccompanied by changes in hematologic indices reflective of a hemolytic process.463–465 The reason for this discrepancy is unclear. Icterus probably results principally from inadequate processing of bilirubin by the immature liver of G6PDdeficient infants. The demonstrated increase in carboxyhemoglobin levels, indicative of increased heme catabolism, suggests, however, that shortening of red cell life span also plays a role.466 A predisposing factor for severe jaundice in G6PD deficiency is mutation of the uridine diphosphoglucuronate glucuronosyltransferase-1 gene (UGT1A1) promoter,467 or, in Asia, the c.211G>A coding mutation.468 In adults, these mutations are associated with Gilbert syndrome. The limited data available on liver G6PD in deficient adults469 suggest that a considerable degree of deficiency may be present. If such a deficiency also is present in infants, it may play a role in impairing the borderline ability of infant livers with the UGT1A1 promoter defect to catabolize bilirubin, in particular when a hemolytic process is set off because of contact with environmental factors, for example, neocytolysis (Chap. 33) certain drugs, naphthalene containing mothballs, etc. However, it is becoming apparent that modulation of bilirubin metabolism and serum bilirubin levels is under complex genetic control,470 and coexpressing of mutations in other genes, for example, SLCO1B3,471 may contribute further to the bilirubin production-conjugation imbalance in G6PD-deficient individuals.472

Deficiencies of Other Enzymes of the Hexose Monophosphate Shunt and of Glutathione Metabolism

Deficiencies of glutamate cysteine synthetase, GS, and GR are associated with a decrease in red cell GSH levels. The generally mild hemolysis that occurs in these disorders probably has a pathogenesis similar to the hemolysis that occurs in G6PD deficiency. Other defects of the hexose monophosphate shunt and associated metabolic pathways are not associated with hemolysis (see Table  47–3).

Other Enzyme Deficiencies

How deficiencies of enzymes other than those of the hexose monophosphate pathway result in shortening of red cell life-span remains unknown, although it has been the object of much experimental work and of speculation. It is often believed that ATP depletion is a common pathway in producing damage to the cell leading to its destruction,473 but the evidence that this is the case is not always compelling.474 Nevertheless, it seems reasonable to assume that a red cell, deprived of a source of energy becomes sodium and calcium logged and potassium depleted, and the red cell shape changes from a flexible biconcave disk. Such a cell is quickly removed from the circulation by the filtering action of the spleen and the monocyte-macrophage system. Even if it survived, such an energy-deprived cell would gradually turn brown as hemoglobin is oxidized to methemoglobin by the very high concentrations of oxygen within the erythrocyte. Calcium has been proposed to play a central role. In particular, malfunction of ATP-dependent calcium transporters could lead to increased intracellular calcium levels that could affect red cell membrane proteins (i.e., protein 4.1), the lipid bilayer, volume regulation, metabolism, and redox state preservation, consequently leading

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to proteolysis, oxidation, irreversible cellular shrinkage, phosphatidyl exposure and premature clearance.475 In agreement with this, PFK deficiency has been shown to result in increased calcium levels, accompanied by volume loss and metabolic dysregulation.476,477 It is possible that, at least in some cases, alteration of the levels of red cell intermediate metabolites interferes with synthesis of cell components in early stages of development of the cell. In agreement with this, the lack of pyruvate has been implicated in the ineffective maturation of erythroid progenitors in PK-deficient mice.478

CLINICAL FEATURES COMMON FORMS OF GLUCOSE-6-PHOSPHATE DEHYDROGENASE DEFICIENCY Individuals who inherit the common (polymorphic) forms of G6PD deficiency, such as G6PD A– or G6PD Mediterranean, usually have no clinical manifestations. The major clinical consequence of G6PD deficiency is hemolytic anemia in adults and neonatal icterus in infants. Usually the anemia is episodic, but some of the unusual variants of G6PD may cause nonspherocytic congenital hemolytic disease (see “Variants Producing Hereditary Nonspherocytic Hemolytic Anemia” above). In general, hemolysis is associated with stress, most notably drug administration, infection, and, in certain individuals, exposure to fava beans.

Drug-Induced Hemolytic Anemia

Table 47–5 is an evidence-based3,479 list of drugs and other chemicals that are predicted to precipitate hemolytic reactions in G6PD-deficient individuals, and drugs that are innocuous when given in normal doses,

TABLE 47–5.  Drugs That Can Trigger Hemolysis in G6PD-Deficient Individuals Category of Drug

Predictable Hemolysis

Possible Hemolysis

Antimalarials

Dapsone

Chloroquine

Primaquine

Quinine

Methylene blue Analgesics/ Antipyretic

Phenazopyridine

Aspirin (high doses) Paracetamol (Acetaminophen)

Antibacterials

Cotrimoxazole

Sulfasalazine

Sulfadiazine Quinolones (including nalidixic acid, ciprofloxacin, ofloxacin)

Hemolytic Anemia Occurring During Infection

Nitrofurantoin Other

Rasburicase

Chloramphenicol

Toluidine blue

Isoniazid Ascorbic acid Glibenclamide Vitamin K Isosorbide dinitrate

Reproduced with pemission from Luzzatto L, Seneca E: G6PD deficiency: A classic example of pharmacogenetics with on-going clinical implications. Br J Haematol 164(4):469–480, 2014.

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but may be hemolytic when given in excessive doses. A case in point is ascorbic acid, which does not cause hemolytic anemia in normal doses but which can produce severe, even fatal, hemolysis at doses of 80 g or more intravenously.480–482 Some drugs, such as chloramphenicol, may induce mild hemolysis in a person with severe, Mediterranean-type G6PD deficiency,484 but not in those with the milder A– or Canton485 types of deficiency. Furthermore, there appears, to be a difference in the severity of the reaction to the same drug of different individuals with the same G6PD variant. For example, red cells from a single G6PDdeficient individual were hemolyzed in the circulation of some recipients who were given thiazolsulfone, but their survival was normal in the circulation of others.438 Sulfamethoxazole, which was clearly hemolytic in experimental studies, does not appear to be a common cause of hemolysis in a clinical setting.486 Undoubtedly, individual differences in the metabolism and excretion of drugs influence the extent to which G6PD-deficient red cells are destroyed.487,488 Several animal models have been developed to serve as a screening platform for the determination of hemolytic toxicity of pharmacologic agents in G6PD deficiency.489,490–492 Typically, an episode of drug-induced hemolysis in G6PD-deficient individuals begins 1 to 3 days after drug administration is initiated.493 Heinz bodies appear in the red cells, and the hemoglobin concentration begins to decline rapidly.494 As hemolysis progresses, Heinz bodies disappear from the circulation, presumably as they or the erythrocytes that contain them are removed by the spleen. In severe cases, abdominal or back pain may occur. The urine may turn dark or even black. Within 4 to 6 days, there is generally an increase in the reticulocyte count, except in instances in which the patient has received the offending drug for treatment of an active infection as infection depresses erythropoiesis (Chap. 37). Because of the tendency of infections and certain other stressful situations to precipitate hemolysis in G6PD-deficient individuals, many drugs have been incorrectly implicated as a cause. Other drugs, such as aspirin, have appeared on many lists of proscribed medications because very large doses could slightly reduce the red cell life span. It is important to recognize that such drugs do not produce clinically significant hemolytic anemia. Advising patients not to ingest these drugs may not only deprive patients of potentially helpful medications, but will also weaken their confidence in the advice that they have received. Most G6PD-deficient patients, after all, have taken aspirin without untoward effect and are likely to distrust an advisor who counsels them that the ingestion of aspirin will have catastrophic effects. In the A– type of G6PD deficiency, the hemolytic anemia is self-limited493 because the young red cells produced in response to hemolysis have nearly normal G6PD levels and are relatively resistant to hemolysis.495 The hemoglobin level may return to normal even while the same dose of drug that initially precipitated hemolysis is administered. In contrast, hemolysis is not self-limited in the more severe types such as Mediterranean deficiency.496

Anemia often develops rather suddenly in G6PD-deficient individuals within a few days of onset of a febrile illness. The anemia is usually relatively mild, with a decline in the hemoglobin concentration of 3 or 4 g/dL. Hemolysis has been noted particularly in patients suffering from hepatitides A and B, cytomegalovirus, and pneumonia, and in those with typhoid fever.497–499 The fulminating form of the disease occurs particularly frequently among G6PD-deficient patients who are infected with Rocky Mountain spotted fever.500 Jaundice is not a prominent part of the clinical picture, except where hemolysis occurs in association with infectious hepatitis.501,502 In that case, it can be quite intense. Presumably because of the effect of the infection, reticulocytosis is usually absent, and recovery from the anemia is generally delayed until after the

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active infection has abated. In rare cases, G6PD deficiency may present as transient aplastic crisis caused by viral infection.503,504

Favism

Favism is potentially one of the gravest clinical consequences of G6PD deficiency. It occurs much more commonly in children than in adults, and occurs almost exclusively in persons who have inherited variants of G6PD that cause severe deficiency (most frequently associated with the Mediterranean variant), but rarely has the disorder been noted in patients with G6PD A–.505 The onset of hemolysis may be quite sudden, having been reported to occur within the first hours after exposure to fava beans. More commonly, the onset is gradual, hemolysis being noticed 1 to 2 days after ingestion of the beans.506 The urine becomes red or quite dark, and in severe cases shock may develop within a short time. Care should be taken to avoid acute renal failure. The oxidative stress causes membrane changes in erythrocytes, leading to extravascular hemolysis (in addition to the intravascular destruction).3 Sometimes the patient or parent does not realize that fava beans have been ingested, as they may be incorporated into foods such as Yew Dow, eaten by the Chinese,507 or falafel, eaten in the Middle East. Occasionally ingestion of other foodstuffs, such as unripe peaches508 or a spiced Nigerian barbecued meat known as red suya,509 has been reported to precipitate hemolysis. The toxic constituents of the fava beans are transmitted into the milk of breastfeeding mothers, putting affected babies at risk.510

Neonatal Icterus

Although serious, the clinical consequences of drug-induced hemolysis, favism, or chronic hemolytic anemia are usually not devastating, and death from favism is a very rare event. The most serious consequence of G6PD deficiency is icterus neonatorum.463 G6PD-deficient neonates are an estimated three to four times more at risk for hyperbilirubinemia and phototherapy than G6PD-adequate neonates,511 depending on population groups and geographic area.512 Jaundice commences in the immediate perinatal period, and is usually evident by 1 to 4 days of age, similar to physiologic jaundice, but is seen at a later time than in blood group alloimmunization.513 The jaundice may be quite severe and, if untreated, may result in kernicterus. Reports indicate an overrepresentation of G6PD deficiency among cases of kernicterus relative to the frequency of in the background population, also in countries with a low overall frequency of G6PD deficiency.472 Thus, G6PD deficiency is a preventable cause of mental retardation,514–516 and this aspect of the disorder has considerable public health significance. Neonatal screening for G6PD deficiency has been associated with a decrease in the number of cases of kernicterus.472

Nonspherocytic Hemolytic Anemia

As described, the anemia in G6PD deficiency is usually episodic and acute, but some sporadic variants of G6PD may cause nonspherocytic congenital hemolytic disease, exacerbated by oxidative stress. Affected individuals have a history of severe neonatal jaundice, and features of chronic hemolysis (see “Variants Producing Hereditary Nonspherocytic Hemolytic Anemia” above). The hemolysis is mainly extravascular.

Effects on Other Tissues

In the common variants of G6PD, such as G6PD A– and Mediterranean, and even in most of the severely deficient variants, there is usually no demonstrated defect in leukocyte number or function.517 However, there have been reports of isolated instances of leukocyte dysfunction associated with rare, severely deficient variants of G6PD.280,281,518–522 Patients with G6PD deficiency do not have a bleeding tendency, and studies of platelet function have yielded conflicting results.523,524 Occasionally, cataracts have been observed in patients with variants of G6PD

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that produce nonspherocytic hemolytic anemia,525–527 or in neonatal patients.528 The incidence of senile cataracts may be increased in G6PD deficiency,529,530 but this remains controversial.531,532 Small studies from the Middle East are suggestive that decreased G6PD activity may predispose to the development of diabetes.533–535 A number of studies reported on acute rhabdomyolysis in patients with G6PD deficiency, suggesting that this condition could predispose to muscle damage,535–540 probably through the depletion of NADPH.541 Others however, have demonstrated that G6PD-deficient individuals can participate in various physical activities, even high-intensity muscle damaging activities542 without a negative impact on muscle function and redox status.543,544 Although claims have been made that an association exists between various kinds of G6PD deficiency and cancer,545,546 the relationship between G6PD status and cancer is not clear as epidemiologic studies have not demonstrated any difference in risk for cancers between G6PD-deficient and normal patients.547–549 Some role for G6PD in carcinogenesis may be conceivable, though, given the finding that mutation of p53 abolishes the direct binding of this major tumor-suppressor gene to G6PD, thereby enhancing hexose monophosphate shunt flux and tumor cell biosynthesis.550 Population studies are needed to better elucidate the postulated effects of G6PD deficiency on the development of cardiovascular disease.278,551

ENZYME DEFICIENCIES OTHER THAN GLUCOSE-6-PHOSPHATE DEHYDROGENASE Most patients with hereditary nonspherocytic hemolytic anemia manifest only the usual clinical signs and symptoms of chronic hemolysis. The degree of anemia in this group of disorders varies widely. In some cases of very severe PK deficiency, scarcely any deficient cells survive in the circulation, and only transfused cells are found or steady-state hemoglobin levels as low as 5 g/dL are encountered. Other patients with hereditary nonspherocytic hemolytic anemia may manifest compensated hemolysis with a normal steady-state hemoglobin concentration. Chronic jaundice is a common finding, and splenomegaly is often present. Gallstones are common. As in other forms of chronic hemolytic anemia, ankle ulcers may be present.552,553 Pregnancy has been thought to precipitate hemolysis in patients with PK deficiency, perhaps even in heterozygotes.554–556 In PK deficiency, the increased 2,3-BPG levels may ameliorate the anemia by lowering the oxygen-affinity of hemoglobin. Some PK-deficient patients present with hydrops fetalis.557 In the case of some enzyme defects, characteristic nonhematologic systemic manifestations may be present, and these may be the only sign of the enzyme deficiency. For example, patients with PFK deficiency may have type VII muscle glycogen storage disease. In some patients with this defect, hemolysis is present without muscle manifestations, but in others both muscle abnormalities and hemolysis occur.558 Glutathione synthetase deficiency may be associated with 5-oxoprolinuria and neuromuscular disturbances, and such abnormalities may occur either with559 or without hematologic abnormalities.262 On the other hand, some patients with GS deficiency manifest only the hematologic abnormalities.382 Spinocerebellar degeneration was documented in the first case of glutamate cysteine synthetase described,381,384 but was not present in subsequently investigated patients.382,383 Patients with TPI deficiency nearly always manifest serious neuromuscular disease, and most of the patients who inherit this abnormality die in the first decade of life,560,561 but there are exceptions, as only one of two brothers with the same genotype manifested neurologic disease (see “Genetic Modifiers of the Phenotypes” below).562,563 Neurologic symptoms have also been noted in patients with deficiencies of glucosephosphate isomerase

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and PGK.333,564 Myoglobinuria has been encountered in patients with PGK,261,565 aldolase,352 and G6PD deficiency.539 Table  47–2 summarizes the clinical features of enzyme deficiencies causing nonspherocytic hemolytic anemia.

GENETIC MODIFIERS OF THE PHENOTYPES The clinical phenotype of both acute and chronic hemolysis can be modified by coinherited (although unrelated) other defects of the red cells. Combined deficiencies of, for example, GPI and G6PD,316 of PK and band 3,566–568 of PK and α-thalassemia,569 and of PK and G6PD570 have been documented. The inheritance of polymorphic UGT1A1 promoter alleles exacerbates the icterus both in neonates and in adults with G6PD deficiency (see also “Mechanism of Hemolysis” above).472 Overt iron overload and iron-related morbidity in PK deficiency has been attributed to coinheritance of mutations in HFE, the gene associated with hereditary hemochromatosis.571 A striking example of complex interplay defining the differences between the genotype and the phenotype was described in a Hungarian family with TPI deficiency. Two adult germline-identical compound heterozygous brothers displayed strikingly different phenotypes. Both had the same severe decrease in TPI activity and congenital hemolytic anemia, but only one suffered from severe neurologic disorder. Studies aimed at the pathogenesis of this differing phenotype indicated functional differences between the two brothers in lipid environment of the red cell membrane proteins influencing the enzyme activities,562 as well as differences in TPI1 mRNA expression, and protein expression levels of prolyl oligopeptidase, the activity decrease of which has been reported in well-characterized neurodegenerative diseases.572 The variety of clinical features associated with the various enzymopathies, regardless of the underlying molecular mechanism, do unequivocally demonstrate that the phenotype of hereditary red blood cell enzymopathies, is not solely dependent on the molecular properties of mutant proteins but rather reflects a complex interplay between physiologic, environmental, and other (genetic) factors. Putative phenotypic modifiers include differences in genetic background, concomitant functional polymorphisms of other glycolytic enzymes (many enzymes are regulated by their product or other metabolites), posttranslational modification, ineffective erythropoiesis, and different splenic function. As an example, persistent expression of the PK-M2 isozyme has been reported in the red blood cells of patients (and animals) with severe PK deficiency.29,573 The survival of these patients, though not in all cases may be enabled by this compensatory increase in PK activity.574

LABORATORY FEATURES Varying degrees of anemia and reticulocytosis are the main hematologic laboratory features of patients with hereditary nonspherocytic hemolytic anemia. Heinz bodies often are found in the erythrocytes of G6PD-deficient patients undergoing drug-induced hemolysis. In the absence of hemolysis, the light-microscopic morphology of G6PDdeficient red cells appears to be normal. Differences in the texture of the membrane of the cells have, however, been observed under electron microscopy.575 When a hemolytic drug is administered to a G6PDdeficient patient, Heinz bodies (Chap. 31) develop in the erythrocytes immediately preceding and in the early phases of the hemolytic episode. If the hemolytic anemia is very severe, spherocytosis and red cell fragmentation may be seen in the stained film. Despite the fact that “bite cells” may be noted in the blood of a G6PD-deficient patient undergoing drug-induced hemolysis, the association with G6PD deficiency is doubtful because such cells are usually lacking in acute hemolytic

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states of patients with common G6PD variants or in G6PD-deficient patients with chronic hemolysis. Moreover, “bite cells” have been noted in G6PD-replete patients.576,577 The presence of small, densely staining cells has often been noted in the blood films of patients with hereditary nonspherocytic hemolytic anemia with defects other than G6PD deficiency. Particularly when manifesting an echinocytic appearance, such cells have been thought to be common in PK deficiency. In one reported case,578 spectacular numbers of such cells were observed. However, cells of this type are seen in many blood films both from patients with other glycolytic enzyme deficiencies and from those with other disorders and it is hazardous to attempt to make an enzymatic diagnosis on the basis of such findings. Basophilic stippling of the erythrocytes is prominent in most patients with pyrimidine 5′-nucleotidase deficiency but is on itself an unspecific finding, and may not be apparent in blood that has been collected in ethylenediaminetetraacetic acid anticoagulant. Leukopenia occasionally is observed in patients with hereditary nonspherocytic hemolytic anemia, possibly secondary to splenic enlargement. Other laboratory stigmata of increased hemolysis may include increased levels of serum bilirubin, decreased haptoglobin levels, and increased serum LDH activity (Chap. 33). Reticulocytosis is frequently observed, which may result in increased mean corpuscular volume of erythrocytes. In PK deficiency, splenectomy increases reticulocyte counts even further because in particular the younger PK-deficient red blood cells are preferentially sequestered by the spleen.579 Also in P5′N1 deficiency reticulocytes tend to be higher in splenectomized patients compared to non-splenectomized patients.420 Diagnosis of red cell enzyme deficiencies usually depends on the demonstration of decreased enzyme activity either through a quantitative assay or a screening test.580–583 Assay of most of the enzymes generally is carried out by measuring the rate of reduction or oxidation of nicotinamide adenine nucleotides in an ultraviolet spectrophotometer, and a number of screening tests that depend upon the development or loss of fluorescence have been devised.584 However, difficulties arise when the patient has been transfused so that the blood drawn represents a mixture of the patient’s own cells and those obtained from the blood bank. Under the circumstances, DNA analysis may prove invaluable, because the DNA is extracted from blood leukocytes and transfused leukocytes do not persist in the circulation. Alternatively, density fractionation has been applied to isolate fractions of patient’s red cells, in which an enzyme deficiency can be detected.585 Although detection of G6PD deficiency in the healthy, fully affected (hemizygous) male can be achieved readily through either assay or screening tests, difficulties arise when a patient with G6PD deficiency of the A– type has undergone a hemolytic episode. As the older, more enzyme-deficient cells are removed from the circulation and are replaced by young cells, the level of the enzyme begins to increase toward normal. Under such circumstances, suspicion that the patient may be G6PD deficient should be raised by the fact that enzyme activity is not increased, even though the reticulocytes count is elevated.586,587 It is helpful to perform DNA mutational analyses, carry out family studies, or to wait until the circulating red cells have aged sufficiently to betray their lack of enzyme. Even greater difficulties are encountered in attempting to diagnose heterozygotes for G6PD deficiency.588 Because the gene is X linked, a population of normal red cells coexists with the deficient cells. This may mask the enzyme deficiency when screening tests are used. Even enzyme assays carried out on erythrocytes of heterozygous females frequently may be in the normal range. Here DNA mutational analyses and histochemical methods that depend upon individual red cell enzyme activity may be useful.589,590 In addition, the ascorbate cyanide test,591 in which screening is carried out on a whole-cell population rather than

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on a lysate, may be more sensitive than the other screening procedures. Prenatal diagnosis of G6PD deficiency is also possible using DNA mutational analyses approach. Testing for red cells enzyme deficiencies is best done in specialized laboratories. Specimens can be shipped by mail to reference laboratories. As a rule, whole-blood specimens are suitable and can best be sent at 4°C as some enzymes, notably PFK, are relatively unstable.580 Blood from a healthy volunteer should be shipped with the patient sample to serve as a shipping control. Exceptions are assays for phosphorylated sugar intermediates, 2,3-BPG, and nucleotide intermediates, which are unstable in freshly drawn blood and require immediate deproteinization in perchloric acid. Several aspects should be kept in mind when interpreting test results. First, care must be taken to remove leukocytes and platelets in assays such as for PK, as these cells contain PK activity, obscuring a deficiency in the red cells. Second, one should be aware of the already mentioned red cell age dependency of, for example, PK, HK, and G6PD. The measurement of these enzymes simultaneously can give an idea about red cell age and relative deficiencies. If patients received blood transfusions, interpreting results from red cell enzyme assays is generally not possible because the presence of donor erythrocytes will obscure any deficiencies. Some mutant enzymes also display a normal activity in vitro, whereas in vivo severe hemolysis can occur, reflecting the differences between optimal circumstances in vitro and the in vivo cellular environment. More sophisticated assays to measure, for example, heat instability and kinetics, have to be used in those cases. Interpretation can be particularly challenging in newborn patients given the differences in red cell energy metabolism and enzymatic activities between adults and newborn infants.592–596 Molecular diagnosis is now available for all red cell enzyme deficiencies.

DIFFERENTIAL DIAGNOSIS Drug-induced hemolytic anemia resulting from G6PD deficiency is similar in its clinical features and in certain laboratory features, to drug-induced hemolytic anemia associated with unstable hemoglobins (Chap. 49). Other enzyme defects affecting the pentose-phosphate shunt, such as a deficiency of GS, also may mimic G6PD deficiency. The diagnosis of hemoglobinopathies can be excluded by performing a stability test,597 hemoglobin electrophoresis or DNA sequence analysis. These are normal in G6PD deficiency. Some of the screening tests, particularly the ascorbate cyanide test,591 may give positive results in the above-named disorders, but a G6PD assay or the fluorescent screening test will be positive only in G6PD deficiency. In addition, defects of the erythrocyte membrane should be excluded (Chap. 46), but these cytoskeletal and other membrane defects are generally associated with characteristic morphologic abnormalities, that makes them easy to differentiate from hemolysis because of enzyme defects. Physicians often attempt to establish the cause of hereditary nonspherocytic hemolytic anemia on the basis of the appearance of red cells on a blood film. In reality, red cell morphology is helpful only in the diagnosis of pyrimidine 5′-nucleotidase deficiency because of the characteristic stippling of the red cells that is observed in that disorder. The appearance of Heinz bodies suggests the possible presence of an unstable hemoglobin, or defective GSH metabolism. They are more likely to be present after splenectomy. Because the laboratory diagnosis of these disorders may entail considerable expenditure of time and effort, it is prudent to perform the simplest tests for the most common causes of hereditary nonspherocytic hemolytic anemia first. Accordingly, it is useful to carry out screening tests580,582 for G6PD and PK activity and an isopropanol stability test to detect an unstable hemoglobin (Chap. 49). If prominent

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stippling of erythrocytes is present, examination of the ultraviolet spectrum of a perchloric acid extract of the erythrocytes, reflecting the ratio between pyrimidine and purine nucleotide content, may help to establish the diagnosis of pyrimidine 5′-nucleotidase deficiency.598 Beyond these relatively simple procedures it is probably rarely useful to pick and choose individual enzyme assays on the basis of family history or clinical manifestations. Rather, it is usually appropriate to submit a blood sample to a reference laboratory that has the capability of performing all the enzyme assays listed in Table  47–3. Preferably, the suspicion of a specific enzyme disorder causing hereditary nonspherocytic hemolytic anemia is confirmed by DNA sequence analysis. This also enables prenatal diagnosis which has already been achieved for some of enzymatic defects.599–607 Notably, in an estimated 70 percent of cases of suspected hereditary nonspherocytic hemolytic anemia no enzymatic abnormality is found.608,609 Current promising approaches such as red cell proteome analysis610–612 and/or the use of next-generation sequencing technologies613 may aid in a better and more comprehensive understanding of the etiology of this disorder.

THERAPY GLUCOSE-6-PHOSPHATE DEHYDROGENASE DEFICIENCY G6PD-deficient individuals should avoid drugs that are predicted to induce hemolytic episodes (see Table  47–5). However, it is important to realize that such patients are able to tolerate most drugs. Unfortunately, in the past, a number of case reports incorrectly suggested that some drugs had hemolytic potential that subsequently were shown to be safe (see Table  47–5, possible hemolysis). Although it is possible that some of these may be hemolytic in some patients or under some circumstances, this is unlikely, and G6PD-deficient patients should not be deprived of the possible benefit of these drugs. If hemolysis occurs as a result of drug ingestion or infection, particularly in the milder A– type of deficiency, transfusion usually is not required. If, however, the rate of hemolysis is very rapid, as may occur, for example, in favism, transfusions of packed cells may be useful. Good urine flow should be maintained in patients with hemoglobinuria to avert renal damage. Infants with neonatal jaundice resulting from G6PD deficiency may require phototherapy or exchange transfusion; in areas in which G6PD deficiency is prevalent, care must be taken not to give G6PD-deficient blood to such newborns.614 A single dose of Sn-mesoporphyrin, a potent inhibitor of heme oxygenase, has been advocated to eliminate the need for phototherapy.615 Patients with hereditary nonspherocytic hemolytic anemia resulting from G6PD deficiency usually do not require any therapy. Splenectomy is often ineffective, although some improvement has been reported in a number of cases following removal of the spleen.264,616 In most cases, the anemia is not very severe, but in some instances frequent transfusions have been necessary.617,618 The antioxidant properties of vitamin E have been tested in G6PD-deficient subjects, and a slight but statistically significant reduction in hemolysis was observed.619,620 These results could not be confirmed in other studies.621,622 It has been suggested that desferrioxamine decreases hemolysis.623,624 Inhibition of histone acetylation by histone deacetylase inhibitors has been shown to increase G6PD gene transcription in erythroid progenitor cells and restore G6PD deficiency.625

OTHER ENZYME DEFICIENCIES Most patients with hereditary nonspherocytic hemolytic anemia secondary to red cell enzymopathies do not require therapy, other than blood transfusion during hemolytic periods, if the anemia needs

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clinically to be corrected. There are patients with PK deficiency who need to be transfused continually. Chronic transfusion therapy usually requires iron chelation if of sufficient iron load. Patients with TPI deficiency generally die as children, not because of the severity of the anemia but because of the severe neuromuscular effects of the enzyme deficiency. It has been proposed that the exogenous replacement of TPI might be useful for the treatment of this deficiency,626 but no clinical trials have been carried out. PK deficiency627 and PGK deficiency375 have been treated successfully by stem cell transplantation, but this is still only very rarely done. Studies are underway to improve gene therapy in PK deficiency.305,307,308 In PK deficiency, erythroid cells have been treated ex vivo with glycolytic intermediates to correct for metabolic dysfunction.628 Preliminary evidence indicates that small molecule activation of mutant PK may be able to restore glycolytic pathway activity and normalize red cell metabolism in PK deficiency.629 The jaundice of glucosephosphate isomerase deficiency has been treated by the administration of phenobarbital.630 The principal decision that the physician must make regarding patients with hereditary nonspherocytic hemolytic anemia is whether or not they require a splenectomy. This decision is not made easily as the response is unpredictable, and some patients who fail to respond may develop serious thrombotic complications, resulting thrombocytosis is often exaggerated when splenectomy does not ameliorate the hemolysis. The recommendation that is made should be based upon the following considerations: (1) severity of the disease, (2) family history of response to splenectomy, (3) the underlying defect, and (4) perhaps the need for cholecystectomy. Because it is unusual to obtain more than a partial response to splenectomy, this procedure should probably be reserved for patients whose quality of life is impaired by their anemia. The operation needs to be particularly considered for patients who need frequent transfusion and for those who require gallbladder surgery, in which splenectomy might be carried out as part of the same procedure. The best guide to the likely efficacy of splenectomy is probably the response to splenectomy of other affected family members. Unfortunately, such information is only occasionally available. The physician must therefore rely upon the experience of other patients with hereditary nonspherocytic hemolytic anemia of similar etiology to serve as a guide. However, even as the large group of patients with hereditary nonspherocytic hemolytic anemia represents a heterogeneous population, so individuals with a single enzymatic lesion, such as PK deficiency, are heterogeneous. Each family is likely to be afflicted with a distinct mutant enzyme, and the various mutants may differ both with respect to clinical manifestations and with respect to response to splenectomy. Some of the available information regarding response to splenectomy of patients with hereditary nonspherocytic hemolytic anemia has been reviewed264 and is summarized in Table  47–2. Glucocorticoids are of no known value in this group of disorders. Folic acid is often given, as in other patients with increased marrow activity, but without proven hematologic benefit. In the absence of iron deficiency, iron is contraindicated. Iron overload is a complication in this group of disorders, particularly in connection with PK deficie ncy,289,571,631,632 even in nontransfused patients.633 The iron overload is probably multifactorial (Chap. 43), involving chronic hemolysis, ineffective erythropoiesis, splenectomy, coinheritance of hereditary hemochromatosis gene (HFE) mutations, growth differentiation factor-15, and hepcidin levels.571,634,635

COURSE AND PROGNOSIS Hemolytic episodes in the A– type of deficiency are usually self-limited, even if drug administration is continued. This is not the case in the more severe Mediterranean type of deficiency.636 In patients with hereditary

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nonspherocytic hemolytic anemia resulting from G6PD deficiency, gallstones may occur.637 During periods of infections or drug administration, anemia may increase in severity. Otherwise, the hemoglobin level of affected subjects remains relatively stable. Nearly all patients with drug- or infection-induced hemolysis recover uneventfully. Favism must be considered, by comparison, a relatively dangerous disease. The most serious complication of G6PD deficiency is neonatal icterus. If not recognized early and properly treated, it can lead to kernicterus (see “Clinical Features” above). In one large population study, a decreasing incidence of G6PD deficiency was noted with increasing age of the population,638 but no such change was observed in another.22 Although age stratification might represent evidence of a shorter life span for individuals with the A– deficiency, other factors are more likely explanations. Examination of the health records of more than 65,000 U.S. Veterans Administration males failed to reveal any higher frequency of any illness in G6PD-deficient compared to nondeficient subjects.639 Furthermore, it appears that there are no indications that G6PD-deficient individuals should systematically be excluded from serving as blood donors,640 or hematopoietic stem cell donor.641 In view of the benign nature of the common types of G6PD deficiency, community-based population screening is not recommended. However, screening for G6PD deficiency of all patients admitted to the hospital may be useful in anticipating hemolytic reactions and in understanding them if they occur; however, this recommendation has not been submitted to rigorous analysis and is controversial because of low likelihood of any preventable hemolysis. This is particularly prudent if a drug such as dapsone or rasburicase, known to cause hemolysis in G6PD-deficient individuals, is to be given.483,642 Study of family members of patients with this X chromosome-linked enzyme deficiency can be helpful in providing appropriate counseling to affected individuals. The diagnosis of hereditary nonspherocytic hemolytic anemia has been made as late as the seventh decade,202 and the disease can be fatal in the first few years of life. TPI deficiency appears to have the worst prognosis of all of the known defects that cause this disorder. With few exceptions, patients with this deficiency have died by the fifth or sixth year of life, usually of cardiopulmonary failure. PK deficiency, too, can be fatal in early childhood; the PK mutation prevalent among the Amish of Pennsylvania produces particularly severe disease.643 Unless the affected homozygous children have their spleens removed, the disorder is commonly lethal. In PK deficiency, compound heterozygotes and homozygotes can suffer of major side effects as a result of the chronic hemolysis and the burden of repeated transfusions and iron chelation. In general, however, hereditary nonspherocytic hemolytic anemia is a relatively mild disease and most affected individuals lead a relatively normal life, apparently without much compromise of life span.

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Rider NL, Strauss KA, Brown K, et al: Erythrocyte pyruvate kinase deficiency in an old-order Amish cohort: Longitudinal risk and disease management. Am J Hematol 86:827–834, 2011. 633. Zanella A, Berzuini A, Colombo MB, et al: Iron status in red cell pyruvate kinase deficiency: Study of Italian cases. Br J Haematol 83:485–490, 1993. 634. Finkenstedt A, Bianchi P, Theurl I, et al: Regulation of iron metabolism through GDF15 and hepcidin in pyruvate kinase deficiency. Br J Haematol 144:789–793, 2009. 635. Mojzikova R, Koralkova P, Holub D, et al: Iron status in patients with pyruvate kinase deficiency: Neonatal hyperferritinaemia associated with a novel frameshift deletion in the PKLR gene (p.Arg518fs), and low hepcidin to ferritin ratios. Br J Haematol 165: 556–563, 2014. 636. Pannacciulli I, Tizianello A, Ajmar F, et al: The course of experimentally-induced hemolytic anemia in a primaquine- sensitive Caucasian. A case study. Blood 25:92–95, 1965. 637. Meloni T, Forteleoni G, Noja G, et al: Increased prevalence of glucose-6-phosphate dehydrogenase deficiency in patients with cholelithiasis. Acta Haematol 85:76–78, 1991. 638. Petrakis NL, Wiesenfeld SL, Sams BJ, et al: Prevalence of sickle-cell trait and glucose-6phosphate dehydrogenase deficiency. N Engl J Med 282:767–770, 1970. 639. Heller P, Best WR, Nelson RB, et al: Clinical implications of sickle-cell trait and glucose-6-phosphate dehydrogenase deficiency in hospitalized black male patients. N Engl J Med 300:1001–1005, 1979. 640. Renzaho AM, Husser E, Polonsky M: Should blood donors be routinely screened for glucose-6-phosphate dehydrogenase deficiency? A systematic review of clinical studies focusing on patients transfused with glucose-6-phosphate dehydrogenase-deficient red cells. Transfus Med Rev 28:7–17, 2014. 641. Pilo F, Baronciani D, Depau C, et al: Safety of hematopoietic stem cell donation in glucose 6 phosphate dehydrogenase-deficient donors. Bone Marrow Transplant 48:36–39, 2013. 642. Pamba A, Richardson ND, Carter N, et al: Clinical spectrum and severity of hemolytic anemia in glucose 6-phosphate dehydrogenase-deficient children receiving dapsone [in process citation]. Blood 120:4123–4133, 2012. 643. Bowman HS, McKusick VA, Dronamraju KR: Pyruvate kinase deficient hemolytic anemia in an Amish isolate. Hum GenetAm J Hum Genet 17:1–8, 1965. 644. Beutler E, Duron O, Kelly BM: Improved method for the determination of blood glutathione. J Lab Clin Med 61:882–890, 1963. 645. Beutler E, Gelbart T: Improved assay of the enzymes of glutathione synthesis: Gamma-glutamylcysteine synthetase and glutathione synthetase. Clin Chim Acta 158: 115–123, 1986. 646. Valentine WN, Fink K, Paglia DE, et al: Hereditary hemolytic anemia with human erythrocyte pyrimidine 5′-nucleotidase deficiency. J Clin Invest 54:866–879, 1974. 647. Torrance J, West C, Beutler E: A simple rapid radiometric assay for pyrimidine-5′nucleotidase. J Lab Clin Med 90:563–568, 1977. 648. Beutler E, Kuhl W, Gelbart T: Blood cell phosphogluconolactonase: Assay and properties. Br J Haematol. 62:577–586, 1986. 649. Bird TD, Hamernyik P, Nutter JY, et al: Inherited deficiency of delta-aminolevulinic acid dehydratase. Am J Hum Genet 31:662–668, 1979. 650. Kamatani N, Hakoda M, Otsuka S, et al: Only three mutations account for almost all defective alleles causing adenine phosphoribosyltransferase deficiency in Japanese patients. J Clin Invest 90:130–135, 1992. 651. Hidaka Y, Palella TD, O’Toole TE, et al: Human adenine phosphoribosyltransferase. Identification of allelic mutations at the nucleotide level as a cause of complete deficiency of the enzyme. J Clin Invest 80:1409–1415, 1987. 652. Resta R, Thompson LF: SCID: The role of adenosine deaminase deficiency. Immunol Today 18:371–374, 1997.

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653. Ogasawara N, Goto H, Yamada Y, et al: Distribution of AMP-deaminase isozymes in rat tissues. Eur J Biochem 87:297–304, 1978. 654. Yamada Y, Goto H, Wakamatsu N, et al: A rare case of complete human erythrocyte AMP deaminase deficiency due to two novel missense mutations in AMPD3. Hum Mutat 17:78, 2001. 655. Armstrong JM, Myers DV, Verpoorte JA, et al: Purification and properties of human erythrocyte carbonic anhydrases. J Biol Chem 241:5137–5149, 1966. 656. Kendall AG, Tashian RE: Erythrocyte carbonic anhydrase I: Inherited deficiency in humans. Science 197:471–472, 1977. 657. Roth DE, Venta PJ, Tashian RE, et al: Molecular basis of human carbonic anhydrase II deficiency. Proc Natl Acad Sci U S A 89:1804–1808, 1992. 658. Goth L, Rass P, Pay A: Catalase enzyme mutations and their association with diseases. Mol Diagn 8:141–149, 2004. 659. Percy MJ, Lappin TR: Recessive congenital methaemoglobinaemia: Cytochrome b(5) reductase deficiency. Br J Haematol 141:298–308, 2008. 660. Simonelli F, Giovane A, Frunzio S, et al: Galactokinase activity in patients with idiopathic presenile and senile cataract. Metab Pediatr Syst Ophthalmol 15:53–56, 1992. 661. Karas N, Gobec L, Pfeifer V, et al: Mutations in galactose-1-phosphate uridyltransferase gene in patients with idiopathic presenile cataract. J Inherit Metab Dis 26:699–704, 2003. 662. Beutler E: Red cell enzyme defects as non-diseases and as diseases. Blood 54:1–7, 1979. 663. Beutler E: Effect of flavin compounds on glutathione reductase activity: In vivo and in vitro studies. J Clin Invest 48:1957–1966, 1969. 664. Valentine WN, Paglia DE, Neerhout RC, et al: Erythrocyte glyoxalase II deficiency with coincidental hereditary elliptocytosis. Blood 36:797–808, 1970. 665. Johnson LA, Gordon RB, Emmerson BT: Hypoxanthine-guanine phosphoribosyltransferase: A simple spectrophotometric assay. Clin Chim Acta 80:203–207, 1977. 666. Larovere LE, Romero N, Fairbanks LD, et al: A novel missense mutation, c.584A > C (Y195S), in two unrelated Argentine patients with hypoxanthine-guanine phosphoribosyl-transferase deficiency, neurological variant. Mol Genet Metab 81:352–354, 2004. 667. Sumi S, Marinaki AM, Arenas M, et al: Genetic basis of inosine triphosphate pyrophosphohydrolase deficiency. Hum Genet 111:360–367, 2002. 668. Sass MD, Caruso CJ, Farhangi M: TPNH-methemoglobin reductase deficiency: A new red-cell enzyme defect. J Lab Clin Med 70:760–767, 1967. 669. Ferrell RE, Escallon M, Aguilar L, et al: Erythrocyte phosphoglucomutase: A family study of a PGM1 deficient allele. Hum Genet 67:306–308, 1984. 670. Banki K, Hutter E, Colombo E, et al: Glutathione levels and sensitivity to apoptosis are regulated by changes in transaldolase expression. J Biol Chem 271:32994–33001, 1996. 671. Chamberlain BR, Buttery JE: Reappraisal of the uroporphyrinogen I synthase assay, and a proposed modified method. Clin Chem 26:1346–1347, 1980. 672. Strand LJ, Meyer UA, Felsher BF, et al: Decreased red cell uroporphyrinogen I synthetase activity in intermittent acute porphyria. J Clin Invest 51:2530–2536, 1972. 673. Chao LT, Du CS, Louie E, et al: A to G substitution identified in exon 2 of the G6PD gene among G6PD deficient Chinese. Nucleic Acids Res 19:6056, 1991. 674. Hirono A, Ishii A, Kere N, et al: Molecular analysis of glucose-6-phosphate dehydrogenase variants in the Solomon Islands. Am J Hum Genet 56:1243–1245, 1995. 675. Kaeda JS, Chhotray GP, Ranjit MR, et al: A new glucose-6-phosphate dehydrogenase variant, G6PD Orissa (44 Ala—>Gly), is the major polymorphic variant in tribal populations in India. Am J Hum Genet 57:1335–1341, 1995. 676. Minucci A, Antenucci M, Giardina B, et al: G6PD Murcia, G6PD Ube and G6PD Orissa: Report of three G6PD mutations unusual for Italian population. Clin Biochem 43:1180–1181, 2010. 677. Nafa K, Reghis A, Osmani N, et al: G6PD Aures: A new mutation (48 Ile—>Thr) causing mild G6PD deficiency is associated with favism. Hum Mol Genet. 2:81–82, 1993. 678. Daoud BB, Mosbehi I, Prehu C, et al: Molecular characterization of erythrocyte glucose-6-phosphate dehydrogenase deficiency in Tunisia. Pathol Biol (Paris) 56:260– 267, 2008. 679. Calabro V, Giacobbe A, Vallone D, et al: Genetic heterogeneity at the glucose-6phosphate dehydrogenase locus in southern Italy: A study on a population from the Matera district. Hum Genet 86:49–53, 1990.

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680. Ganczakowski M, Town M, Bowden DK, et al: Multiple glucose 6-phosphate dehydrogenase-deficient variants correlate with malaria endemicity in the Vanuatu archipelago (southwestern Pacific). Am J Hum Genet 56:294–301, 1995. 681. Nakatsuji T, Miwa S: Incidence and characteristics of glucose-6-phosphate dehydrogenase variants in Japan. Hum Genet 51:297–305, 1979. 682. Chiu DT, Zuo L, Chao L, et al: Molecular characterization of glucose-6-phosphate dehydrogenase (G6PD) deficiency in patients of Chinese descent and identification of new base substitutions in the human G6PD gene. Blood 81:2150–2154, 1993. 683. Vulliamy TJ, Wanachiwanawin W, Mason PJ, et al: G6PD Mahidol, a common deficient variant in South East Asia is caused by a (163)glycine—serine mutation. Nucleic Acids Res 17: 5868, 1989. 684. Beutler E, Kuhl W, Saenz GF, et al: Mutation analysis of glucose-6-phosphate dehydrogenase (G6PD) variants in Costa Rica. Hum Genet 87:462–464, 1991. 685. Cittadella R, Civitelli D, Manna I, et al: Genetic heterogeneity of glucose-6-phosphate dehydrogenase deficiency in south-east Sicily. Ann Hum Genet 61:229–234, 1997. 686. De Vita G, Alcalay M, Sampietro M, et al: Two point mutations are responsible for G6PD polymorphism in Sardinia. Am J Hum Genet 44:233–240, 1989. 687. Corcoran CM, Calabro V, Tamagnini G, et al: Molecular heterogeneity underlying the G6PD Mediterranean phenotype. Hum Genet 88:688–690, 1992. 688. Kirkman HN, Simon ER, Pickard BM: Seattle variant of glucose-6-phosphate dehydrogenase. J Lab Clin Med 66:834–840, 1965. 689. Cappellini MD, Sampietro M, Toniolo D, et al: Biochemical and molecular characterization of a new sporadic glucose-6-phosphate dehydrogenase variant described in Italy: G6PD Modena. Br J Haematol 87:209–211, 1994. 690. Cappellini MD, Martinez di Montemuros F, Dotti C, et al: Molecular characterisation of the glucose-6-phosphate dehydrogenase (G6PD) Ferrara II variant. Hum Genet 95:440–442, 1995. 691. Viglietto G, Montanaro V, Calabro V, et al: Common glucose-6-phosphate dehydrogenase (G6PD) variants from the Italian population: Biochemical and molecular characterization. Ann Hum Genet 54:1–15, 1990. 692. Beutler E, Westwood B, Kuhl W: Definition of the mutations of G6PD Wayne, G6PD Viangchan, G6PD Jammu, and G6PD “LeJeune”. Acta Haematol 86:179–182, 1991. 693. Poon MC, Hall K, Scott CW, et al: G6PD Viangchan: A new glucose 6-phosphate dehydrogenase variant from Laos. Hum Genet 78:98–99, 1988. 694. Ahluwalia A, Corcoran CM, Vulliamy TJ, et al: G6PD Kalyan and G6PD Kerala; two deficient variants in India caused by the same 317 Glu—>Lys mutation. Hum Mol Genet 1:209–210, 1992. 695. Sukumar S, Mukherjee MB, Colah RB, et al: Two distinct Indian G6PD variants G6PD Jamnagar and G6PD Rohini caused by the same 949 G—>A mutation. Blood Cells Mol Dis 35:193–195, 2005. 696. Beutler E, Westwood B, Prchal JT, et al: New glucose-6-phosphate dehydrogenase mutations from various ethnic groups. Blood 80:255–256, 1992. 697. Calabro V, Mason PJ, Filosa S, et al: Genetic heterogeneity of glucose-6-phosphate dehydrogenase deficiency revealed by single-strand conformation and sequence analysis. Am J Hum Genet 52:527–536, 1993. 698. Menounos P, Zervas C, Garinis G, et al: Molecular heterogeneity of the glucose-6-phosphate dehydrogenase deficiency in the Hellenic population. Hum Hered 50:237–241, 2000. 699. Perng LI, Chiou SS, Liu TC, et al: A novel C to T substitution at nucleotide 1360 of cDNA which abolishes a natural Hha I site accounts for a new G6PD deficiency gene in Chinese. Hum Mol Genet 1:205, 1992. 700. Wagner G, Bhatia K, Board P: Glucose-6-phosphate dehydrogenase deficiency mutations in Papua New Guinea. Hum Biol 68:383–394, 1996. 701. Stevens DJ, Wanachiwanawin W, Mason PJ, et al: G6PD Canton a common deficient variant in South East Asia caused by a 459 Arg—Leu mutation. Nucleic Acids Res 18:7190, 1990. 702. Chiu DT, Zuo L, Chen E, et al: Two commonly occurring nucleotide base substitutions in Chinese G6PD variants. Biochem Biophys Res Commun 180:988–993, 1991.

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CHAPTER 48

THE THALASSEMIAS: DISORDERS OF GLOBIN SYNTHESIS

David J. Weatherall

SUMMARY The thalassemias are the commonest monogenic diseases in man. They occur at a high gene frequency throughout the Mediterranean populations, the Middle East, the Indian subcontinent, and Myanmar, and in a line stretching from southern China through Thailand and the Malay peninsula into the island populations of the Pacific. They are also seen commonly in countries in which there has been immigration from these high-frequency populations.   There are two main classes of thalassemias, α and β, in which the α- and β-globin genes are involved, and rarer forms caused by abnormalities of other globin genes. Some extremely rare congenital and acquired thalassemia that have intact globin genes are caused by either mutations of nonglobin genes or factors yet to be elucidated. All thalassemias have in common an imbalanced rate of production of the globin chains of adult hemoglobin, excess α chains in β-thalassemia and excess β chains in α-thalassemia. Several hundred different mutations at the α- and β-globin loci have been defined as the cause of the reduced or absent output of α or β chains. The high frequency and genetic diversity of the thalassemias is related to past or present heterozygote resistance to malaria.   The pathophysiology of the thalassemias can be traced to the deleterious effects of the globin-chain subunits that are produced in excess. In βthalassemia, excess α chains cause damage to the red cell precursors and red

Acronyms and Abbreviations: AATAAA, the polyadenylation signal site; ATR-16, α-thalassemia chromosome 16-linked mental retardation syndrome; ATR-X, αthalassemia X-linked mental retardation syndrome; BCL11A, B-cell lymphoma/leukemia oncogene important for γ- to β-globin switching; CAP site, a DNA site located in or near a promoter; DNase I, an enzyme used to detect DNA-protein interaction; GATA-1, a transcription factor essential for productive erythropoiesis; HPFH, hereditary persistence of fetal hemoglobin; HS, hypersensitive site to DNase I treatment; IVS, intervening sequence of a gene (i.e., an intron); KLF1, erythroid Kruppel-like factor; LCR, locus control region; MCS, multispecies conserved sequences; NFE-2, “nuclear factor, erythroid 2” is a transcription factor essential for productive erythropoiesis; PHD region, known as plant homeodomain is a DNA region with zinc finger motif commonly deleted in ATR-X α-thalassemia; RFLP, restriction fragment length polymorphism; TATA box, a DNA sequence (cis-regulatory element) found in the promoter region of genes.

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cells and lead to profound anemia. This causes expansion of the ineffective marrow, with severe effects on development, bone formation, and growth. The major cause of morbidity and mortality is the effect of iron deposition in the endocrine organs, liver, and heart, which results from increased intestinal absorption and the effects of blood transfusion. The pathophysiology of the α-thalassemias is different because the excess β chains that result from defective α-chain production form β4 molecules, or hemoglobin H, which is soluble and does not precipitate in the marrow. However, it is unstable and precipitates in older red cells. Hence, the anemia of α-thalassemia is hemolytic rather than dyserythropoietic.   The clinical pictures of α- and β-thalassemia vary widely, and knowledge is gradually being amassed about some of the genetic and environmental factors that modify these phenotypes.   Because the carrier states for the thalassemias can be identified and affected fetuses can be diagnosed by DNA analysis after the ninth to tenth week of gestation, these conditions are widely amenable to prenatal diagnosis. Currently, marrow transplantation is the only way in which they can be cured. Symptomatic management is based on regular blood transfusion, iron chelation therapy, and the judicious use of splenectomy. Experimental approaches to their management include the stimulation of fetal hemoglobin synthesis and attempts at somatic cell gene therapy.

DEFINITIONS AND HISTORY In 1925, Cooley and Lee1 first described a form of severe anemia that occurred early in life and was associated with splenomegaly and bone changes. In 1932, George H. Whipple and William L. Bradford2 published a comprehensive account of the pathologic findings in this disease. Whipple coined the phrase thalassic anemia3,4 and condensed it to thalassemia, from θαλασσα (“the sea”), because early patients were all of Mediterranean background. The true genetic character of the disorder became fully appreciated after 1940. The disease described by Cooley and Lee is the homozygous state of an autosomal gene for which the heterozygous state is associated with much milder hematologic changes. The severe homozygous condition became known as thalassemia major. The heterozygous states, thalassemia trait, were designated according to their severity as thalassemia minor or minima.3,5–7 Later, the term thalassemia intermedia was used to describe disorders that were milder than the major form but more severe than the traits. Thalassemia is not a single disease but a group of disorders, each resulting from an inherited abnormality of globin production.7 The conditions form part of the spectrum of diseases known collectively as the hemoglobinopathies, which can be classified broadly into two types. The first subdivision consists of conditions, such as sickle cell anemia, that result from an inherited structural alteration in one of the globin chains. Although such abnormal hemoglobins may be synthesized less efficiently or broken down more rapidly than normal adult hemoglobin, the associated clinical abnormalities result from the physical properties of the abnormal hemoglobin (Chap. 49). The second major subdivision of the hemoglobinopathies, the thalassemias, consists of inherited defects in the rate of synthesis of one or more of the globin chains. The result is imbalanced globin chain production, ineffective erythropoiesis, hemolysis, and a variable degree of anemia. Several monographs describe the historical aspects of thalassemia in greater detail.5,7

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TABLE 48–1.  Thalassemias and Related Disorders α-Thalassemia   α0   α+   Deletion (–α)   Nondeletion (αT ) β-Thalassemia   β0   β+   Normal hemoglobin A2  Dominant   Unlinked to β-globin genes δβ-Thalassemia  (δβ)+   (δβ)0  (Aγ δβ)0 γ-Thalassemia δ-Thalassemia   δ0   δ+ εγδβ-Thalassemia Hereditary Persistence of Fetal Hemoglobin  Deletion   (δβ)0, (Aγ δβ)0  Nondeletion   Linked to β-globin genes  

γ β+, Aγ β+

G

  Unlinked to β-globin genes

DIFFERENT FORMS OF THALASSEMIA Thalassemia can be defined as a condition in which a reduced rate of synthesis of one or more of the globin chains leads to imbalanced globin-chain synthesis, defective hemoglobin production, and damage to the red cells or their precursors from the effects of the globin subunits that are produced in relative excess.7,8 Table 48–1 summarizes the main varieties of thalassemia that have been defined with certainty. The β-thalassemias are divided into two main varieties. In one form, β0-thalassemia, there is no β-chain production. In the other form, β+-thalassemia, there is a partial deficiency of β-chain production. The hallmark of the common forms of β-thalassemia is an elevated level of hemoglobin A2 in heterozygotes. In a less-common class of β-thalassemias, heterozygotes have normal hemoglobin A2 levels. Other rare forms include varieties of β-thalassemia intermedia that are inherited in a dominant fashion, that is, heterozygotes are severely affected, and there is a variety in which the genetic determinants are not linked to the β-globin gene cluster.7,9,10 The δβ-thalassemias are heterogeneous. In some cases, no δ or β chains are synthesized. Originally, these disorders were classified according to the structure of the hemoglobin F produced, that is, G A γ γ(δβ)0- and Gγ(δβ)0-thalassemia. This classification is illogical. The

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conditions are best described by the globin chains that are defectively synthesized, that is, simply (δβ)+-, (δβ)0-, and (Aγδβ)0-thalassemia.7,10 In the (δβ)+-thalassemias, an abnormal hemoglobin is produced that has normal α chains combined with non-α chains consisting of the N-terminal residues of the δ chain fused to the C-terminal residues of the β chain. These fusion variants, called the Lepore hemoglobins, show structural heterogeneity. The δ-thalassemias7,10 are characterized by reduced output of δ chains and hence reduced hemoglobin A2 levels in heterozygotes and an absence of hemoglobin A2 in homozygotes. They are of no clinical significance except that, when inherited with β-thalassemia trait, the level of hemoglobin A2 is reduced to the normal range. A disorder characterized by defective ε-, γ-, δ-, and β-chain synthesis has been defined at the clinical and molecular level.7,10 The homozygous state for this condition, εγδβ-thalassemia, presumably is not compatible with fetal survival. It has been observed only in heterozygotes. Hereditary persistence of fetal hemoglobin (HPFH) is a heterogeneous condition characterized by persistent fetal hemoglobin.7,9,10 It is classified into deletion and nondeletion forms. The deletion forms of HPFH can be classified, like δβ-thalassemia, as (δβ)0 HPFH and then subdivided according to the particular population in which this occurs and its associated molecular defect. In effect, the deletion forms of HPFH are very similar to β-thalassemia except for more efficient γ-chain synthesis and, therefore, less chain imbalance and a milder phenotype. The homozygous state is associated with mild thalassemic changes. In fact, the β-thalassemias and deletion forms of HPFH form a clinical continuum. The nondeletion forms of HPFH also are heterogeneous. In some cases, they are associated with mutations that involve the β-globin gene cluster and in which there is β-chain synthesis cis to the HPFH determinant. These conditions are subdivided into Gγβ+ HPFH and Aγβ+ HPFH. Again, they often are subclassified according to the population in which they occur, for example, Greek HPFH, British HPFH, and so on. Finally, a heterogeneous group of HPFH determinants is associated with very low levels of persistent fetal hemoglobin, the genetic loci of which, at least in some cases, are not linked to the β-globin gene cluster. Because α chains are present in both fetal and adult hemoglobins, a deficiency of α-chain production affects hemoglobin synthesis in fetal and in adult life. A reduced rate of α-chain synthesis in fetal life results in an excess of γ chains, which form γ4 tetramers, or hemoglobin Bart’s. In adult life, a deficiency of α chains results in an excess of β chains, which form β4 tetramers, or hemoglobin H. Because there are two α-globin genes per haploid genome, the genetics of α-thalassemia is more complicated than that of β-thalassemia. There are two main groups of α-thalassemia determinants.7,10 First, in the α0-thalassemias (formerly called α-thalassemia 1), no α chains are produced from an affected chromosome; that is, both linked α-globin genes are inactivated. Second, in the α+-thalassemias (formerly called α-thalassemia 2), the output of one of the linked pair of α-globin genes is defective. The α+-thalassemias are subdivided into deletion and nondeletion types. Both the α0-thalassemias and deletion and nondeletion forms of α+-thalassemia are extremely heterogeneous at the molecular level. There are two major clinical phenotypes of α-thalassemia: the hemoglobin Bart’s hydrops syndrome, which usually reflects the homozygous state for α0-thalassemia, and hemoglobin H disease, which usually results from the compound heterozygous state for α0- and α+-thalassemia. Because the structural hemoglobin variants and the thalassemias occur at a high frequency in some populations, the two types of genetic defect can be found in the same individual. The different genetic varieties of thalassemia and their combinations with the genes for abnormal hemoglobins produce a series of disorders known collectively as the thalassemia syndromes.7

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IVS 1 - 5 G→C IVS 1 - 1 G→T CODONS 41 - 42.4bp DEL CODONS 26 GAG→AAG(HbE)

CODON 6 - 1bp IVS 1 - 1G→A IVS 2 - 1G→A IVS 2 - 745C→G CODON 39 CAG→TAG IVS 1 - 6T→C IVS 1 -110 G→A

IVS 1 - 110 G→A IVS 1 - 5 G→C IVS 1 - 6 T→C CODON 39 CAG→TAG CODON 8 2bp DEL

IVS 2 - 654 C→T CODONS 41 - 42.4bp DEL. CODON 17 AAG→TAG CODON 26 GAG→AAG(HbE) –28 A→G –29A→G

IVS 1 - 5G→C

–29 A→G –88 C→T CODON 24 T→A POLY-A T→C

IVS 1 - 5 G→C 619 bp DELETION CODON 8/9 + G IVS 1 -1 G→T CODONS 41 - 42.4bp DEL.

Figure 48–1.  World distribution of β-thalassemia.

EPIDEMIOLOGY AND POPULATION GENETICS The β-thalassemias are distributed widely in Mediterranean populations, the Middle East, parts of India and Pakistan, and throughout Southeast Asia (Fig. 48–1).7,11,12 The disease is common in Tajikistan, Turkmenistan, Kyrgyzstan, and the People’s Republic of China. Because of the extensive migration from areas of high gene frequency such as the Mediterranean region (e.g., Italy, Greece), Africa, and Asia to the Americas, the α- and β-thalassemia genes and clinical disease are relatively common, especially in North, but also South, America. The β-thalassemias are rare in Africa, except for isolated pockets in West Africa, notably Liberia, and in parts of North Africa. However, β-thalassemia occurs sporadically in all racial groups and has been observed in the homozygous state in persons of pure Anglo-Saxon heritage. Thus, a patient’s racial background does not preclude the diagnosis. The δβ-thalassemias have been observed sporadically in many racial groups, although no high-frequency populations have been defined. Similarly, the hemoglobin Lepore syndromes have been found in many populations, but, with the possible exceptions of central Italy, Western Europe, and parts of Spain and Portugal, these disorders have not been found to occur at a high frequency in any particular region. The α-thalassemias occur widely throughout Africa, the Mediterranean countries, the Middle East, and Southeast Asia (Fig. 48–2).7,11,12 The α0-thalassemias are found most commonly in Mediterranean and Oriental populations, but are extremely rare in African and Middle Eastern populations. However, the deletion forms of α+-thalassemia occur at a high frequency throughout West Africa, the Mediterranean, the Middle East, and Southeast Asia. In United States, approximately 30 percent of Americans of African descent carry the gene α+-thalassemia. Up to 80 percent of the population of some parts of Papua New Guinea are carriers for the deletion form of α+-thalassemia. How common the nondeletion forms of α+-thalassemia are in any particular populations

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is uncertain, but they have been reported quite frequently in some of the Mediterranean island populations and in the Middle Eastern and Southeast Asian populations. Because the hemoglobin Bart’s hydrops syndrome and hemoglobin H disease require the action of an α0-thalassemia determinant, these disorders are found at a high frequency only in Southeast Asia and in parts of the Mediterranean region. The α-chain termination mutants, such as hemoglobin Constant Spring, seem to be particularly common in Southeast Asia. Approximately 4 percent of the population in Thailand are carriers. In 1949, J.B.S. Haldane13 suggested that thalassemia had reached its high frequency in tropical regions because heterozygotes are protected against malaria.13 Although many population studies have tested this

1–15% 5–15% 60%

5–80% 40–80%

5–40%

Figure 48–2. World distribution of α+- (hatched areas) and α0_ thalassemia (shaded areas).

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hypothesis, elucidation of some of the extremely complex population genetics underlying polymorphic systems such as the thalassemias has been possible only with the advent of recombinant DNA technology. In each of the high-frequency areas for the β-thalassemias, a few common mutations and varying numbers of rare mutations are seen (see Fig. 48–1). Furthermore, in each of these regions the pattern of mutations is different, usually found in the context of different haplotypes in the associated β-globin gene cluster.11,14,15 Similar observations have been made in the α-thalassemias (see Fig. 48–2).7,11 These studies suggest the thalassemias arose independently in different populations and then achieved their high frequency by selection. Although some movement of the thalassemia genes may have resulted from drift, independent mutation and selection undoubtedly provide the overall basis for their world distribution. Early studies in Sardinia, showing that β-thalassemia is less common in the mountainous regions where malarial transmission is low, supported Haldane’s suggestion that β-thalassemia reached its high frequency because of protection against malarial infections.16 For many years these data remained the only convincing evidence for a protective effect. However, later studies using malaria endemicity data and globin-gene mapping showed a clear altitude-related effect on the frequency of α-thalassemia in Papua New Guinea. In addition, a sharp cline (a gradual change of species phenotype over a geographical area) in the frequency of α-thalassemia has been found in the region stretching south from Papua New Guinea through the island populations of Melanesia to New Caledonia. This is mirrored by a similar gradient in the distribution of malaria.17 The effect of drift and founder effect in these island populations has been largely excluded by showing that other DNA polymorphisms have a random distribution through the region, with no evidence of a cline similar to that characterizing the distribution of α-thalassemia and malaria. Firm evidence for protection of individuals with mild forms of α+-thalassemia against Plasmodium falciparum malaria has been provided. In a case-control study performed in Papua New Guinea, the homozygous state for α+-thalassemia offered approximately 60 percent protection against hospital admittance because of serious complications of malaria, notably coma or profound anemia.18 Similar levels of protection by α-thalassemia against P. falciparum malaria have been found in several different African populations.19 However, it is becoming clear that there are complex genetic epistatic interactions between protective polymorphisms of this kind. For example, although α-thalassemia and the sickle cell trait both offer strong protection against P. falciparum malaria, in those who inherit both traits, the protection is canceled out and they are fully susceptible to the disease.20 Interactions of this type will have an important effect on the gene frequency of protective polymorphisms in countries in which more than one exists in the same population. There is growing evidence that both immune and cellular mechanisms may underlie these protective effects of different red cell polymorphisms against malarial infection. Followup studies of cohorts of babies with α-thalassemia suggest that, in the first year of life, they are more prone to Plasmodium vivax and P. falciparum malaria. Because there is evidence for cross-immunization between these two species, it is possible that this effect induces early immunization that may result in babies with α-thalassemia being more resistant to P. falciparum malaria later in life.21 At the cellular level there is no evidence that α-thalassemia has any effect on the rates of parasite invasion and growth in red cells. However, parasitized α-thalassemic red cells are more susceptible to phagocytosis in vitro, and are less able than normal cells to form rosettes, an in vitro phenomena whereby uninfected cells bind to infected cells that is strongly associated with severity of infection, and express low levels of complement receptor 1, which is required for rosette formation.22 These highly complex immune and cellular interactions are discussed in detail

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in reviews.19,23,24 Although there are less data of this kind available for the β-thalassemias, there is strong indirect evidence that their high frequency has also been maintained by protection against P. falciparum malaria.

ETIOLOGY AND PATHOGENESIS GENETIC CONTROL AND SYNTHESIS OF HEMOGLOBIN The structure and ontogeny of the hemoglobins are reviewed in Chaps. 7 and 49, respectively. Only those aspects with particular relevance to the thalassemia problem are discussed here. Human adult hemoglobin is a heterogeneous mixture of proteins consisting of the major component hemoglobin A and the minor component hemoglobin A2, which constitutes approximately 2.5 percent of the total. In intrauterine life, the main hemoglobin is hemoglobin F. The structure of these hemoglobins is similar. Each consists of two separate pairs of identical globin chains. Except for some of the embryonic hemoglobins (see below), all normal human hemoglobins have one pair of α chains. In hemoglobin A, the α chains are combined with β chains (α2β2), in hemoglobin A2 with δ chains (α2δ2), and in hemoglobin F with γ chains (α2γ2). Human hemoglobin shows further heterogeneity, particularly in fetal life, and this has important implications for understanding the thalassemias and for approaches to their prenatal diagnosis. Hemoglobin F is a mixture of molecular species with the formulas α2γ2136Gly and α2γ2136Ala. The γ chains containing glycine at position 136 are designated Gγ chains. The γ chains containing alanine are called Aγ chains. At birth, the ratio of molecules containing Gγ chains to those containing Aγ chains is approximately 3:1. The ratio varies widely in the trace amounts of hemoglobin F present in normal adults. Before week 8 of intrauterine life, three embryonic hemoglobins— Gower 1 (ξ2ε2), Gower 2 (α2ε2), and Portland (ξ2 γ2)—are present. The ξ and ε chains are the embryonic counterparts of the adult α and β and γ and δ chains, respectively. ξ-Chain synthesis persists beyond the embryonic stage of development in some of the α-thalassemias. Persistent ε-chain production has not been found in any of the thalassemia syndromes. During fetal development, an orderly switch from ξ- to α-chain and from ε- to γ-chain production occurs, followed by β- and δ-chain production after birth. Figure 48–3 shows the different human hemoglobins and the arrangements of the α-gene cluster on chromosome 16 and the β-gene cluster on chromosome 11.

Globin Gene Clusters

Although some individual variability exists, the α-gene cluster usually contains one functional ξ gene and two α genes, designated α2 and α1. It also contains four pseudogenes: ψξ1, ψα1, ψα2, and θ1.9,10 These four pseudogenes are remarkably conserved among different species. Although it appears to be expressed early in fetal life, its function is unknown. It likely does not produce a viable globin chain. Each α gene is located in a region of homology approximately 4 kb long, interrupted by two small nonhomologous regions.25–27 The homologous regions are believed to result from gene duplication, and the nonhomologous segments are believed to arise subsequently by insertion of DNA into the noncoding regions around one of the two genes. The exons of the two α-globin genes have identical sequences. The first intron in each gene is identical. The second intron of α1 is nine bases longer and differs by three bases from that in the α2 gene.27–29 Despite their high degree of homology, the sequences of the two α-globin genes diverge in their 3′ untranslated regions 13 bases beyond the TAA stop codon. These differences provide

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Figure 48–3.  Genetic control of human

hemoglobin (Hgb). The main globin gene clusters are located on chromosomes 11 and 16. At each stage of development, different genes in these clusters are activated or repressed. The different globin chains directed by individual genes are synthesized independently and combine in random fashion as indicated by the arrows.

an opportunity to assess the relative output of the genes, an important part of the analysis of the α-thalassemias.30,31 Production of α2 messenger RNA appears to exceed that of α1 by a factor of 1.5 to 3. ψξ1 and ξ2 genes also are highly homologous. The introns are much larger than those of α-globin genes. In contrast to the latter, IVS-1 is larger than IVS-2. In each ξ gene, IVS-1 contains several copies of a simple repeated 14-bp sequence that is similar to sequences located between the two ξ genes and near the human insulin gene. The coding sequence of the first exon of ψξ1 contains three base changes, one of which gives rise to a premature stop codon, thus making ψξ1 an inactive pseudogene. The regions separating and surrounding the α-like structural genes have been analyzed in detail. Of particular relevance to thalassemia is the polymorphic nature of this gene cluster.32 The cluster contains five hypervariable regions: one downstream from the α1 gene, one between the ξ and ψξ genes, one in the first intron of both the ξ and ψξ genes, and one 5′ to the cluster. These regions consist of varying numbers of tandem repeats of nucleotide sequences. Taken together with single-base restriction fragment length polymorphisms (RFLPs), the variability of the α-globin gene cluster reaches a heterozygosity level of approximately 0.95. Thus, each parental α-globin gene cluster can be identified in the majority of persons. This heterogeneity has important implications for tracing the history of the thalassemia mutations. Figure  48–3 shows the arrangement of the β-globin gene cluster on the short arm of chromosome 11. Each of the individual genes and their flanking regions have been sequenced.33–36 Like the α1 and α2 gene pairs, the Gγ and Aγ genes share a similar sequence. In fact, the Gγ and A γ genes on one chromosome are identical in the region 5′ to the center of the large intron yet show some divergence 3′ to that position. At the boundary between the conserved and divergent regions, a block of simple sequence may be a “hot spot” for initiation of recombination events that lead to unidirectional gene conversion. Like the α-globin genes, the β-gene cluster contains a series of single-point RFLPs, although in this case no hypervariable regions have been identified.37,38 The arrangement of RFLPs, or haplotypes, in the β-globin gene cluster falls into two domains. The 5′ side of the β gene, spanning approximately 32 kb from the ε gene to the 3′ end of the ψβ gene, contains three common patterns of RFLPs. The region encompassing about 18 kb to the 3′ side of the β-globin gene also contains three common patterns in different populations. Between these regions is a sequence of about 11 kb in which there is randomization of the 5′ and 3′ domains; hence, a relatively higher frequency of recombination can occur.38 The β-globin gene haplotypes are similar in most populations but differ markedly in individuals of African origin. These findings suggest the haplotype arrangements were laid down very early during evolution. The findings are consistent with data obtained from mitochondrial DNA polymorphisms pointing to the early emergence of a relatively small population from Africa with subsequent divergence into

Kaushansky_chapter 48_p0725-0758.indd 729

other racial groups.39 Again, they are extremely useful for analyzing the population genetics and history of the thalassemia mutations. The regions flanking the coding regions of the globin genes contain a number of conserved sequences essential for their expression.28,33 The first conserved sequence is the TATA box, which serves accurately to locate the site of transcription initiation at the CAP site, usually about 30 bases downstream. It also appears to influence the rate of transcription. In addition, two so-called upstream promoter elements are present. A second conserved sequence, the CCAAT box, is located 70 or 80 bp upstream. The third conserved sequence, the CACCC homology box, is located further 5′, approximately 80 to 100 bp from the CAP site. It can be either inverted or duplicated. These promoter sequences also are required for optimal transcription. Mutations in this region of the β-globin gene cause its defective expression and these findings provide the foundation for understating regulation of other human genes. The globin genes also have conserved sequences in their 3′ flanking regions, notably AATAAA, which is the polyadenylation signal site. Regulation of Globin Gene Clusters Figure 48–4 summarizes the mechanism of globin gene expression. The primary transcript is a mRNA precursor containing both intron and exon sequences. During its stay in C A C C C

C C A A T

T A T A

FLANKING

A T G NC

IVS-1

GT AG

GT

IVS-2

A A T TA AA AA AG

NC

FLANKING

Gene

3′ mRNA Precursor

5′

5′ CAP Nucleus

AAAA-A AAAA-A

Excision of introns Splicing of exons Processed mRNA

AAAA-A

Translation

Cytoplasm

A UG U AC

Ribosome

U GC U UC A C G AG A

UAA

Transfer RNA Amino acid Growing chain

Finished chain

Figure 48–4.  Expression of a human globin gene.

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the nucleus, it undergoes a good deal of processing that entails capping the 5′ end and polyadenylation of the 3′ end, both of which probably serve to stabilize the transcript (Chap. 10). The intervening sequences are removed from the mRNA precursor in a complex two-stage process that relies on certain critical sequences at the intron–exon junctions. The method by which globin gene clusters are regulated is important to understanding the pathogenesis of the thalassemias. Many details remain to be determined, but studies performed over the last few years have provided at least an outline of some of the major mechanisms of globin gene regulation.7,9,40–42 Most of the DNA within cells that is not involved in gene transcription is packaged into a compact form that is inaccessible to transcription factors and RNA polymerase. Transcriptional activity is characterized by a major change in the structure of the chromatin surrounding a particular gene. These alterations in chromatin structure can be identified by enhanced sensitivity to exogenous nucleases. Erythroid lineage-specific nuclease-hypersensitive sites are found at several locations in the β-globin gene cluster, which vary during different stages of development. In fetal life, these sites are associated with the promoter regions of all four globin genes. In adult erythroid cells, the sites associated with the γ genes are absent. The methylation state of the genes plays an important role in their ability to be expressed. In human and other animal tissues, the globin genes are extensively methylated in nonerythroid organs and are relatively undermethylated in hematopoietic tissues. Changes in chromatin configuration around the globin genes at different stages of development are reflected by alterations in their methylation state. In addition to the promoter elements, several other important regulatory sequences have been identified in the globin gene clusters. For example, several enhancer sequences thought to be involved with tissue-specific expression have been identified. Their sequences are similar to the upstream activating sequences of the promoter elements. Both consist of a number of “modules,” or motifs, that contain binding sites for transcriptional activators or repressors. The enhancer sequences are thought to act by coming into spatial apposition with the promoter sequences to increase the efficiency of transcription of particular genes. It now is clear that transcriptional regulatory proteins may bind to both the promoter region of a gene and to the enhancer. Some of these transcriptional proteins, GATA-1 and NFE-2, for example, appear to be largely restricted to hematopoietic tissues.40 These proteins may bring the promoter and the enhancer into close physical proximity, permitting transcription factors bound to the enhancer to interact with the transcriptional complex that forms near the TATA box. At least some of these hematopoietic gene transcription factors likely will be developmental-stage specific. Another set of erythroid-specific nuclease-hypersensitive sites is located upstream from the embryonic globin genes in both the α- and β-gene clusters. These sites mark the regions of particularly important control elements. In the case of the β-globin gene cluster, the region is marked by five hypersensitive sites to DNase I treatment (HS) (an enzyme used to detect DNA-protein interaction).40 The most 5′ site (HS5) does not show tissue specificity. HS1 through HS4, which together form the locus control region (LCR), are largely erythroid-specific. Each of the regions of the LCR contains a variety of binding sites for erythroid transcription factors. The precise function of the LCR is not known, but it is undoubtedly required to establish a transcriptionally active domain spanning the entire globin gene cluster. The α-globin gene cluster also has a major regulatory element of this kind, in this case HS40.41 This forms part of four highly conserved noncoding sequences, or multispecies conserved sequences (MCSs), called MSC-R1-R4; of these elements only MSC-R2, that is HS40, is essential for α-globin gene expression. Although deletions of this region inactivate the entire α-globin gene

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cluster, its action must be fundamentally different from that of the β-globin LCR because the chromatin structure of the α-gene cluster is in an open conformation in all tissues. Some forms of thalassemia result from deletions involving these regulatory regions. In addition, the phenotypic effects of deletions of these gene clusters are strongly positional, which may reflect the relative distance of particular genes from the LCR and HS40.

Developmental Changes in Globin Gene Expression

One particularly important aspect of human globin genes is regulation of the switch from fetal to adult hemoglobin. Because many of the thalassemias and related disorders of the β-globin gene cluster are associated with persistent γ-chain synthesis, a full understanding of their pathophysiology must include an explanation for this important phenomenon, which plays a considerable role in modifying their phenotypic expression. The complex topic of hemoglobin switching has been the subject of several extensive reviews.7,42 β-Globin synthesis commences early during fetal life, at approximately 8 to 10 weeks’ gestation. β-Globin synthesis continues at a low level, approximately 10 percent of the total non–α-globin chain production, up to approximately 36 weeks’ gestation, after which it is considerably augmented. At the same time, γ-globin chain synthesis starts to decline so that, at birth, approximately equal amounts of γ- and β-globin chains are produced. Over the first year of life, γ-chain synthesis gradually declines. By the end of the first year, γ-chain synthesis amounts to less than 1 percent of the total non–α-globin chain output. In adults the small amount of hemoglobin F is confined to an erythrocyte population called F cells. How this series of developmental switches is regulated is not clear. The process is not organ specific but is synchronized throughout the developing hematopoietic tissues. Although environmental factors may be involved, the bulk of experimental evidence suggests some form of “time clock” is built into the hematopoietic stem cell. At the chromosomal level, regulation appears to occur in a complex manner involving both developmental stage-specific trans-activating factors and the relative proximity of the different genes of the β-globin gene cluster to LCR. Some of the elements involved in the stage-specific regulation of human globin genes have been identified. KLF1 (erythroid Kruppellike factor), a developmental stage–enriched protein, activates human β-globin gene expression and is involved in human γ- to β-globin gene switching.43 More recently BCL11A and MYB have also been identified as being involved in this process.42 Fetal hemoglobin synthesis can be reactivated at low levels in states of hematopoietic stress and at higher levels in certain hematologic malignancies, notably juvenile myeloid leukemia. However, high levels of hemoglobin F production are seen consistently in adult life only in the hemoglobinopathies.

M  OLECULAR BASIS OF THE THALASSEMIAS Once cloning and sequencing of globin genes from patients with many different forms of thalassemia were possible, the wide spectrum of mutations underlying these conditions became clear. A picture of remarkable heterogeneity has emerged. For more extensive coverage of this topic, the reader is referred to several monographs and reviews.7,9,10,44–46

β-THALASSEMIA β-thalassemia is extremely heterogeneous at the molecular level.7 More than 200 different mutations have been found in association with the β-thalassemia phenotype.7 Broadly, they fall into deletions

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this distribution pattern, only approximately 20 alleles account for the majority of all β-thalassemia determinants (see Fig. 48–1).

TABLE 48–2.  Molecular Pathology of the β-Thalassemias β0- or β+ -Thalassemia

Gene Deletions

 Transcription

At least 17 different deletions affecting only the β genes have been described. With one exception, the deletions are rare and appear to be isolated, single events. The 619-bp deletion at the 3′ end of the β gene is more common,48 but even that is restricted to the Sind and Gujarati populations of Pakistan and India, where it accounts for approximately 50 percent of β-thalassemia alleles.48 The Indian 619-bp deletion removes the 3′ end of the β gene but leaves the 5′ end intact. Many of the other deletions remove the 5′ end of the gene and leave the δ gene intact.49–53 Homozygotes for these deletions have β0-thalassemia. Heterozygotes for the Indian deletion have increased hemoglobin A2 and F levels identical to those seen in heterozygotes for the other common forms of β-thalassemia. Heterozygotes for the other deletions all have unusually high hemoglobin A2 levels.7 Increased δ-chain production results from increased δ-gene transcription in cis to the deletion, possibly as a result of reduced competition from the deleted 5′ β gene for transcription factors.

 Deletions  Insertions  Promoter  5′-UTR Processing of mRNA  Junctional   Consensus splicing sequences   Cryptic splice sites in introns   Cryptic splice sites in exons   Poly (A) addition site Translation  Initiation  Nonsense

Other Transcriptional Mutations

 Frameshift

Several different base substitutions involve the conserved sequences upstream from the β-globin gene.7 In every case, the phenotype is β+-thalassemia, although considerable variability exists in the clinical severity associated with different mutations of this type. Several mutations, at positions –88 and –87 relative to the mRNA CAP site, for example,54,55 are close to the CCAAT box, whereas others lie within the TATA box homology.56–59 Some mutations upstream from the β-globin gene are associated with even more subtle alterations in phenotype. For example, a C→T substitution at position –101, which involves one of the upstream promoter elements, is associated with “silent” β-thalassemia, that is, a completely normal (“silent”) phenotype that can be identified only by its interaction with more severe forms of β-thalassemia in compound heterozygotes.60 A single example of an A→C substitution at the CAP site (+1) was described in an Asian Indian who, despite being homozygous for the mutation, appeared to have the phenotype of the β-thalassemia trait.61 Upstream regulatory mutations confirm the importance of the role of conserved sequences in this region as regulators of the transcription of the β-globin genes and provide the basis for some of the mildest forms of β-thalassemia, particularly those in African populations, and for some varieties of “silent” β-thalassemia.

Posttranslational stability  Unstable β-chain variants Normal hemoglobin A2 β-thalassemia   β-Thalassemia and δ-thalassemia, cis or trans  “Silent” β-thalassemia   Some promoter mutations   CAP +1, CAP +3, etc.  5′ UTR   Some splice mutations Dominant β-thalassemia   Mainly point mutations or rearrangements in exon 3   Other unstable variants UTR, untranslated region. note: A full list of mutations is given in Refs. 7 and 45.

of the β-globin gene and nondeletional mutations that may affect the transcription, processing, or translation of β-globin messenger (Table 48–2 and Fig. 48–5). Each major population group has a different set of β-thalassemia mutations, usually consisting of two or three mutations forming the bulk and large numbers of rare mutations. Because of

RNA-Processing Mutations

One surprise about β-thalassemia has been the remarkable diversity of the single-base mutations that can interfere with the intranuclear processing of mRNA.

Figure 48–5. Classes of mutations that underlie β-thalassemia. C, CAP site; FS, frameshift; I, initiation site; NS, nonsense mutation; POLY A, polyA addition site mutation; PR, promoter; SPL, splicing mutation. For a complete list see ref. 304.

Deletions

1

PR

731

IVS-1

C I FS SPL SPL NS

2

FS NS

IVS-2

SPL Point mutations

Kaushansky_chapter 48_p0725-0758.indd 731

3

SPL FS NS

POLY A 100 bp

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Figure 48–6.  Activation of cryptic splice sites in

exon 1 as the cause of β+-thalassemia, hemoglobin E, and hemoglobin Knossos. The similarities between the 5′ splice region of intron 1 and the cryptic splice region in exon 1 are shown in capitals.

The boundaries of exons and introns are marked by invariant dinucleotides, GT at the 5′ (donor) and AG at the 3′ (receptor) sites. Single-base changes that involve either of these splice junctions totally abolish normal RNA splicing and result in the β0-thalassemia phenotype.7,62–66 Highly conserved sequences involved in mRNA processing surround the invariant dinucleotides at the splice junctions. Different varieties of β-thalassemia involve single-base substitutions within the consensus sequence of the IVS-1 donor site.55,58,63–69 These mutations are particularly interesting because of the remarkable variability in their associated phenotypes. For example, substitution of the G in position 5 of IVS-1 by C or T results in severe β+-thalassemia.55 On the other hand, a T→C change at position 6, found commonly in the Mediterranean region,70 results in a very mild form of β+-thalassemia. The G→C change at position 5 has also been found in Melanesia and appears to be the most common cause of β-thalassemia in Papua New Guinea.71 RNA processing is affected by mutations that create new splice sites within either introns or exons. Again, these lesions are remarkably variable in their phenotypic effect, depending on the degree to which the new site is utilized compared with the normal splice site. For example, the G→A substitution at position 110 of IVS-1, which is one of the most common forms of β-thalassemia in the Mediterranean region, leads to only approximately 10 percent splicing at the normal site and hence results in a severe β+-thalassemia phenotype.72,73 Similarly, a mutation that produces a new acceptor site at position 116 in IVS-1 results in little or no β-globin mRNA production and the β0-thalassemia phenotype.74 Several mutations that generate new donor sites within IVS-2 of the β-globin gene have been described.55,68 Another mechanism for abnormal splicing is activation of donor sites within exons (Fig. 48–6). For example, within exon 1 is a cryptic donor site in the region of codons 24 through 27. This site contains a GT dinucleotide. An adjacent substitution that alters the site so that it more closely resembles the consensus donor splice site results in its activation, even though the normal site is active. Several mutations in this region can activate this site so that it is utilized during RNA processing, with the production of abnormal mRNAs.75–78 Three of the substitutions— A→G in codon 19, G→A in codon 26, and G→T in codon 27—result in reduced production of β-globin mRNA and an amino acid substitution so that the mRNA that is spliced normally is translated into protein. The abnormal hemoglobins produced are hemoglobins Malay, E, and Knossos, respectively, all of which are associated with a β-thalassemia phenotype, presumably as a result of reduced overall output of normal mRNA (Fig. 48–6). A variety of other cryptic splice mutations within introns and exons have been described.44 Another class of processing mutations involves the polyadenylation signal site AAUAAA in the 3′ untranslated region of β-globin

Kaushansky_chapter 48_p0725-0758.indd 732

mRNA.79–81 For example, a T→C substitution in this sequence leads to only one-tenth the normal amount of β-globin mRNA and hence the severe β+-thalassemia phenotype.79

Mutations Causing Abnormal Translation of Messenger RNA

Base substitutions that change an amino acid codon into a chain termination codon, that is, nonsense mutations, prevent translation of the mRNA and result in β0-thalassemia. Many substitutions of this type have been described.7,44 For example, a codon 17 mutation is common in Southeast Asia,82,83 and a codon 39 mutation occurs at a high frequency in the Mediterranean region.84,85 The insertion or deletion of one, two, or four nucleotides in the coding region of the β-globin gene disrupts the normal reading frame and results, upon translation of the mRNA, in the addition of anomalous amino acids until a termination codon is reached in the new reading frame. Several frameshift mutations of this type have been described.7,44 Two mutations—the insertion of one nucleotide between codons 8 and 9 and a deletion of four nucleotides in codons 41 and 42—are common in Asian Indians.63 The latter deletions are found frequently in different populations in Southeast Asia.83 An unusual β+-thalassemia was described in a patient from the Czech Republic in whom a full-length L1 transposon was inserted into the second intron of β-globin, creating a β+-thalassemia phenotype by an undefined molecular mechanism.86

Dominantly Inherited β-Thalassemia

Families in which a picture indistinguishable from moderately severe β-thalassemia has segregated in mendelian dominant fashion have been reported sporadically.87,88 Because this condition often is characterized by the presence of inclusion bodies in the red cell precursors, it has been called inclusion body β-thalassemia. However, because all severe forms of β-thalassemia have inclusions in the red cell precursors, the term dominantly inherited β-thalassemia is preferred.7,89 Sequence analysis has shown that these conditions are heterogeneous at the molecular level, but that many involve mutations of exon 3 of the β-globin gene. The mutations include frameshifts, premature chain termination mutations, and complex rearrangements that lead to synthesis of truncated or elongated and highly unstable β-globin gene products.7,89–93 The most common mutation of this type is a GAA→TAA change at codon 121 that leads to synthesis of a truncated β-globin chain.94 Although an abnormal β-chain product from loci affected by mutations of this type is unusual, many of these conditions are designated as hemoglobin variants. The reason why mutations occurring in exons 1 and 2 produce the classic form of recessive β-thalassemia whereas the bulk of the dominant thalassemias result from mutations in exon 3 has become clearer. In the former case, very little abnormal β-globin mRNA is found in the

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cytoplasm of the red cell precursors, whereas exon 3 mutations are associated with full-length but abnormal mRNA accumulation. The different phenotypes of these premature termination codons have been suggested to reflect a phenomenon called nonsense-mediated RNA decay, a surveillance system to prevent transport of mRNA coding for truncated peptides. Presumably this process is active in the case of exon 1 or 2 mutations, in which affected mRNAs are degraded, but is not active in the case of exon 3 mutations.95–97 A complete list of the mutations that underlie the dominant β-thalassemias is given in reference 44.

TABLE 48–3.  δβ-Thalassemias (δβ)+-Thalassemia   Hgb Lepore thalassemia   Hgb Lepore Washington-Boston   Hgb Lepore Hollandia   Hgb Lepore Baltimore   Phenocopies of (δβ)+-thalassemia  Sardinian δβ-thalassemia

Unstable β-Globin Variants

Some β-globin chain variants are highly unstable but are capable of forming a viable tetramer. The resulting unstable hemoglobins may precipitate in the red cell precursors or in the blood, giving rise to a spectrum of conditions ranging from dominantly inherited β-thalassemia to a hemolytic anemia similar to the anemia associated with other unstable hemoglobins. The first unstable hemoglobin to be described was hemoglobin Indianapolis.98 Its structure was characterized by DNA analysis performed on stored autopsy material; however, the original description proved to be incorrect.99

 Corfu δβ-thalassemia  Chinese δβ-thalassemia   β-Thalassemia with δ-thalassemia (δβ)°-Thalassemia  Sicilian  Indian  Japanese  Spanish

Silent β-Thalassemia

A number of extremely mild β-thalassemia alleles are either silent or almost unidentifiable in heterozygotes (see Table  48–2). Some alleles are in the region of the promoter boxes of the β-globin gene, but others involve the CAP sites or the 5′ or 3′ untranslated regions.7,44 These alleles usually are identified by finding a form of β-thalassemia intermedia in which one parent has a typical thalassemia trait and the other parent appears to be normal but, in fact, is a carrier of one of the mild β-thalassemia alleles.

 Black

β-Thalassemia Mutations Unlinked to the B-Globin Gene Cluster

 Indian

Several family studies suggest the existence of mutations that result in the β-thalassemia phenotype but do not segregate with the β-globin genes100; however, their molecular basis has not been determined. Further evidence for the existence of novel mutations of this type can be found in reference 7.

Variant Forms of β-Thalassemia

  Eastern European  Macedonian  Turkish  Laotian  Thai (Aγδβ)°-Thalassemia  German  Cantonese  Turkish   Malay 2  Belgian  Black

In several forms of β-thalassemia, the hemoglobin A2 level is normal in heterozygotes. Some cases result from “silent” β-thalassemia alleles, whereas others reflect the coinheritance of β- and δ-thalassemia.7

 Chinese

δβ-THALASSEMIA

 Italian

The δβ-thalassemias are classified into the (δβ) - and (δβ) -thalassemias (Table 48–3). The (δβ)0-thalassemias are further divided into (δβ)0-thalassemia, in which both the δ- and β-globin genes are deleted, and (Aγδβ)0-thalassemia, in which the Gγ, δ, and β genes are deleted. Because many different deletion forms of δβ-thalassemia have been described, they are further classified according to the country in which they were first identified (Table  48–3). +

(δβ)0- and (Aγδβ)0-Thalassemia

0

Nearly all these conditions result from deletions involving varying lengths of the β-globin gene cluster. Many different varieties have been described in different populations (see Table  48–3), although their heterozygous and homozygous phenotypes are very similar.7 Rare forms of these conditions result from more complex gene rearrangements. For example, one form of (Aγδβ)0-thalassemia, found in Indian populations, does not result from a simple linear deletion but rather from a complex

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 Yunnanese  Thai

Hgb, hemoglobin. note: Details of the molecular pathology of these conditions are given in Refs. 7 and 45.

rearrangement with two deletions, one affecting the Aγ gene and the other the δ and β genes. The intervening region is intact but inverted.101 Figure 48–7 illustrates some of these conditions.

(δβ)+-Thalassemia

The (δβ)+-thalassemias usually are associated with the production of structural hemoglobin variants called Lepore.102 Hemoglobin Lepore contains normal α chains and non-α chains that consist of the first 50 to 80 amino acid residues of the δ chains and the last 60 to 90 residues of the normal C-terminal amino acid sequence of the β chains. Thus,

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Figure 48–7.  Some deletions responsible for the β- and δβ-thalassemias and hereditary persistence of fetal hemoglobin. For a complete list see

reference 304.

the Lepore non-α chain is a β-fusion chain. Several different varieties of hemoglobin Lepore have been described—Washington-Boston, Baltimore, and Hollandia—in which the transition from δ to β sequences occurs at different points.7 The fusion chains probably arose by nonhomologous crossing over between part of the δ locus on one chromosome and part of the β locus on the complementary chromosome (Fig. 48–8). This event results from misalignment of chromosome pairing during meiosis so that a δ-chain gene pairs with a β-chain gene instead of with its homologous partner.103 Figure  48–8 shows such a mechanism should give rise to two abnormal chromosomes: the first, the Lepore chromosome, will have no normal δ or β loci but simply a δβ fusion gene. Opposite the homologous pairs of chromosomes should be an anti-Lepore (βδ) fusion gene and normal δ and β loci. A variety of anti–Lepore-like hemoglobins have been discovered, including hemoglobins Miyada, P-Congo, Lincoln Park, and P-Nilotic.7 All the hemoglobin Lepore disorders are characterized by a severe form of δβ-thalassemia. The output of the γ-globin genes on the chromosome with the δβ fusion gene is not increased sufficiently to compensate for the low output of the δβ fusion product. The reduced rate of production of the δβ fusion chains

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of hemoglobin Lepore presumably reflects the fact that its genetic determinant has the δ gene promoter region, which is structurally different from the β-globin gene promoter and is associated with a reduced rate of transcription of its gene product.

δβ-Thalassemia-Like Disorders Resulting from Two Mutations in the β-Globin Gene Cluster

A heterogeneous group of nondeletion δβ-thalassemias has been described, most resulting from two mutations in the εγδβ-globin gene cluster (see Table  48–3). Strictly speaking, they are not all δβ-thalassemias, but they often appear in the literature under this title because their phenotypes resemble the deletion forms of (δβ)0-thalassemia. In the Sardinian form of δβ-thalassemia, the β-globin gene has the common Mediterranean codon 39 nonsense mutation that leads to an absence of β-globin synthesis. The relatively high expression of the Aγ gene in cis gives this condition the δβ-thalassemia phenotype because of a point mutation at position –196 upstream from the Aγ gene (see “Hereditary Persistence of Fetal Hemoglobin” below). The phenotypic picture, in which heterozygotes have 15 to 20 percent hemoglobin F and

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Figure 48–8.  Mechanisms for the production of the Lepore and anti-Lepore hemoglobins. Hgb, hemoglobin.

normal hemoglobin A2 levels, is identical to that of δβ-thalassemia.103 Another condition having the β-thalassemia phenotype, with greater than 20 percent hemoglobin F in heterozygotes, has been described in a Chinese patient in whom defective β-globin chain synthesis appears to result from an A→G change in the ATA sequence in the promoter region of the β-globin gene.104 The increased γ-chain synthesis, which appears to involve both Gγ and Aγ cis to this mutation, remains unexplained. A disorder originally called δβ-thalassemia has been described in the Corfu population.105,106 The condition results from two mutations in the β-globin gene cluster: first, a 7201-bp deletion that starts in the δ-globin gene, IVS-2, position 818 to 822, and extends upstream to a 5′ breakpoint located 1719 to 1722 bp 3′ to the ψβ-gene termination codon; and second, a G→A mutation at position 5 in the donor site consensus region of IVS-1 of the β-globin gene. The output from this chromosome consists of relatively high levels of γ chains with very low levels of β chains. The condition resembles δβ-thalassemia in the homozygous state, with almost 100 percent hemoglobin F, traces of hemoglobin A, but no hemoglobin A2. Heterozygotes have only slightly elevated hemoglobin F levels, with a phenotype similar to “normal A2β-thalassemia.”

εγδβ-THALASSEMIA These rare conditions107–113 result from long deletions that begin upstream from the β-gene complex 55 kb or more 5′ to the ε gene and terminate within the cluster (see Fig. 48–7). In two cases, designated Dutch110,111 and English,112 the deletions leave the β-globin gene intact, but no β-chain production occurs even though the gene is expressed in heterologous systems. The molecular basis for inactivation of the β-globin gene cis to these deletions was clarified by the discovery of the LCR approximately 50 kb upstream from the εγδβ-globin gene cluster (see “Genetic Control and Synthesis of Hemoglobin” above). Removal of this critical regulatory region seems to completely inactivate the downstream globin gene complex. The Hispanic form of εγδβ-thalassemia113 results from a deletion that includes most of the LCR, including four of the five DNase-1hypersensitive sites. These lesions appear to close down the chromatin domain that usually is open in erythroid tissues and delay replication of the β-globin genes in the cell cycle. Thus, although they are rare, the lesions have been of considerable importance because analysis of the

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Dutch deletion first pointed to the possibility of a major control region upstream from the β-like-globin gene cluster and ultimately led to the discovery of the β-globin LCR.

HEREDITARY PERSISTENCE OF FETAL HEMOGLOBIN This heterogeneous group of conditions produces phenotypes very similar to those of the δβ-thalassemias, except that defective β-chain production appears to be almost, but in some forms not completely, compensated by persistent γ-chain production. These conditions are best classified into deletion and nondeletion forms (Table 48–4). In the past, the conditions were classified into pancellular and heterocellular varieties, depending on the intercellular distribution of fetal hemoglobin. However, this subdivision now appears to bear little relevance to their molecular basis and probably relates more to the particular level of fetal hemoglobin and how its cellular distribution is determined.7 The deletion forms of HPFH are heterogeneous (see Fig. 48–7). The two African varieties result from extensive deletions of similar length (18 kb) deletion that removes the α1 gene and the region downstream was identified in which the α2 gene remains intact but is completely inactivated, giving the α0-thalassemia phenotype. Although the inactive α2 gene retains all its local and remote cis-regulatory elements, its expression is completely silenced and its CpG island is completely methylated as a result of transcription of antisense RNA expressed from a locus that had been juxtaposed to the α2 gene because of the large deletion.133,134 In some cases, this condition results from a terminal truncation of the short arm of chromosome 16 to a site 50 kb distal to the α-globin genes.135 It is interesting that the telomeric consensus sequence (TTAGGGG)n has been added directly to the site of the break. Because this mutation is stably inherited, telomeric DNA alone appears sufficient to stabilize the broken chromosome end. This observation raises the possibility that other genetic diseases result from chromosomal truncations. Several deletions have been identified that appear to downregulate α-globin genes by removing the α-globin LCR (HS40).7,136,137 In each case, the α-globin genes are left intact, although in one the 3′ breakpoint is found between the ξ and ψξ genes, thus removing the ξ gene. These deletions appear to completely inactivate the α-globin gene complex, just as deletions of the β-globin LCR inactivate the entire β-gene complex. Such deletions have not been observed in the homozygous state, presumably because they would be lethal.

α+-Thalassemia Gene Deletions

The most common forms of α+-thalassemia (–α3.7 and –α4.2) involve deletion of one or the other of the duplicated α-globin genes (see Figs. 48–10 and 48–11). Each α gene is located within a region of homology approximately 4 kb long, interrupted by two nonhomologous regions. The homologous regions are believed to have resulted from an ancient duplication event and to have subsequently subdivided, presumably by insertions and deletions, to give three homologous subsegments referred to as X, Y, and Z (see Fig. 48–11). The duplicated Z boxes are 3.7 kb apart, and the X boxes are 4.2 kb apart. Misalignment and reciprocal crossover between these segments at meiosis can give rise to chromosomes with either single (–α) or triplicated (ααα) α-globin genes. Such an occurrence between homologous Z boxes deletes 3.7 kb of DNA (rightward deletion). A similar crossover between the two X blocks deletes 4.2 kb of DNA (leftward deletion –α4.2).138 The corresponding triplicated α-gene arrangements are referred to as αααanti–3.7 and αanti–4.2.139–141 More detailed analysis of these crossover events indicates they occur more commonly in the Z box. At least three different –α3.7 deletions have been found,

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Figure 48–10.  Some deletions of the α-globin gene cluster responsible for α0-thalassemia. Deletions: MC, initials of patient; CAL, initials of patient;

THAI, Thai; FIL, Filipino; CI, Conway Islands; BRIT, United Kingdom; SA, South Africa; MED, Mediterranean; SEA, Southeast Asian; SPAN, Spanish. The top line indicates the size of the region in kilobases (K). The second line shows the different genes that constitute the α-globin gene cluster, HS40, the major regulatory region of the cluster, and the position of other genes in the region. The lines in blue represent the size of the deletions that have been described in α0-thalassemia, while those in red below them on the right-hand side of the figure show some of the deletions that have now been reported in different forms of α+-thalassemia. The lines in yellow on the left side of the figure represent some of the deletions that have been reported upstream from the α-globin gene cluster, which, because they remove the major regulatory region, result in the phenotype of α0-thalassemia. For a more detailed list of these deletions and references to those marked in this diagram, see references 45 and 304.

depending on exactly where the crossover occurred.142 These deletions are designated –α3.7I, –α3.7II, and –α3.7III, respectively. Other, rarer deletions of a single α gene have been observed.7

Nondeletion α-Thalassemia

Figure 48–11.  Mechanisms for production of the common deletion forms of α+-thalassemia. A. Normal α-globin gene cluster showing the homology boxes X, Y, and Z. B. Rightward crossover through the Z bones, giving rise to the 3.7-kb deletion and a chromosome with three α-globin genes. C. Leftward crossover through the Z boxes, giving rise to a 4.2-kb deletion and a chromosome containing three α genes.

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Because expression of the α2 gene is two to three times greater than expression of the α1 gene, the finding that most of the nondeletion mutants discovered to date affect predominantly α2 gene expression is not surprising. Presumably this is ascertainment bias because of the greater phenotypic effect of these lesions. It also is possible that defective expression of the α2 gene has come under greater selective pressure. Like the β-thalassemia mutations, α-thalassemia mutations7 can be classified according to the level of gene expression they affect (see Table  48–5). Several processing mutations have been identified. For example, a pentanucleotide deletion includes the 5′ splice site of IVS-1 of the α2-globin gene. This mutation involves the invariant GT donor splicing sequence and thus completely inactivates the α2 gene.143 A second mutant of this type, found commonly in the Middle East, involves the poly-A addition signal site (AATAAA→AATAAG) and downregulates the α2 gene by interfering with 3′ end processing.144,145 A second group of nondeletion α-thalassemias results from mutations that interfere with translation of mRNA.7 Several mutations involve the initiation codon.146–149 In one case, for example, the initiation codon

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Chapter 48: The Thalassemias: Disorders of Globin Synthesis

Hgb Seal Rock Hgb Pakse A(Glu) (Tyr) GAA UAU

? UUA(Leu)

Hgb A UGA (terminate) UAG

HgbA UAA a142

AAA (Lys) Hgb lcaria

CAA (Gln) Hgb CS

UCA (Ser) Hgb Koya Dora

codon. Hgb, hemoglobin; Hgb CS, hemoglobin Constant Spring;

is inactivated by a T→C transition.146 In another case, efficiency of initiation is reduced by a dinucleotide deletion in the consensus sequence around the start signal.149 Five mutations that affect termination of translation and give rise to elongated α chains have been identified: hemoglobins Constant Spring, Icaria, Koya Dora, Seal Rock, and Pakse.7 Each mutation specifically changes the termination codon TAA so that an amino acid is inserted instead of the chain terminating (Fig. 48–12). This process is followed by read-through of mRNA that is not normally translated until another “in-phase” stop codon is reached. Thus, each of these variants has an elongated α chain. The “read-through” of α-globin mRNA that usually is not utilized likely reduces its stability.150 Several nonsense mutations occur, for example, one in exon 3 of the α2globin gene.151 Finally, several mutations occur that cause α-thalassemia by producing highly unstable α-globin chains, including hemoglobins Quong Sze,152 Suan Doc,153 Petah Tikvah,154 and Evanston.155 A complete list of nondeletion α-thalassemia alleles is given in reference 45.

Interactions of α-Thalassemia Haplotypes

Many α-thalassemia haplotypes have been described, and potentially more than 500 interactions are possible!7 These phenotypes result in four broad categories: (1) normal, (2) conditions characterized by mild hematologic changes but no clinical abnormality, (3) hemoglobin H disease, and (4) hemoglobin Bart’s hydrops fetalis syndrome. The heterozygous states for deletion or nondeletion forms of α+-thalassemia either cause extremely mild hematologic abnormalities or are completely silent. In populations where α-thalassemia is common, the homozygous state for α+-thalassemia (–α/–α) can produce a hematologic phenotype identical to that of the heterozygous state for α0-thalassemia (– –/αα), that is, mild anemia with reduced mean cell hemoglobin and mean cell volume values. Hemoglobin H disease usually results from the compound heterozygous state for α0-thalassemia and either deletion or non-deletion α+-thalassemia. It occurs most frequently in Southeast Asia (– –SEA/–α3.7) and the Mediterranean region (usually – –MED/–α3.7). The hemoglobin Bart’s hydrops fetalis syndrome usually results from the homozygous state for α0-thalassemia, most commonly – –SEA/– –SEA or – –MED/– –MED. A few infants with this syndrome who synthesized very low levels of α chains at birth have been reported. Gene-mapping studies suggest these cases result from interaction of α0-thalassemia with nondeletion mutations (ααND). Some unusual forms of α-thalassemia are completely unrelated to the common forms of the disease that occur in tropical populations. These

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conditions, which can occur in any racial groups, include α-thalassemia associated with mental retardation or leukemia. Their importance lies with the diagnostic problems they may present and, more importantly, the light that elucidation of the α-thalassemia pathology may shed on broader disease mechanisms.

Molecular Pathology of the α-Thalassemia Mental Retardation Syndrome

Figure 48–12.  Point mutations in the α-globin gene termination

Unusual Forms of α-Thalassemia

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The first descriptions of noninherited forms of α-thalassemia associated with mental retardation suggested the lesions involving the α-globin gene locus were acquired in the paternal germ cells and that their molecular pathology might help elucidate the associated developmental changes.156 Two separate syndromes of this type now are evident. In one group of patients, long deletions involve the α-globin gene cluster and remove at least 1 Mb.157 This condition can arise in several ways, including unbalanced translocation involving chromosome 16, truncation of the tip of chromosome 16, and loss of the α-globin gene cluster and parts of its flanking regions by other mechanisms. These findings localize a region of approximately 1.7 Mb in band 16p13.3 proximal to the α-globin genes as being causative of mental handicap.41 The second group is characterized by defective α-globin synthesis associated with severe mental retardation and a relatively homogeneous pattern of dysmorphology.158 Extensive structural studies have shown no abnormalities of the α-globin genes. These chromosomes direct the synthesis of normal amounts of α-globin in mouse erythroleukemia cells, suggesting that α-thalassemia results from deficiency of a trans-activating factor involved in regulation of the α-globin genes. This condition is encoded by a locus on the short arm of the X chromosome.159 ATR-X, the gene involved, is a DNA helicase with many features of a DNA-binding protein. Many different mutations of this gene have been identified in different families with the ATR-X (α-thalassemia X-linked mental retardation) syndrome.131,160 Studies have identified a plant homeodomain (PHD) region and an adenosine triphosphatase (ATPase)/helicase domain.161 Because patients with ATR-X syndrome show defective methylation of recombinant DNA arrays and related defects, this condition likely is one of a growing list of disorders that result from disordered chromatin remodeling.162,163

α-Thalassemia and Myelodysplasia

The hematologic findings of hemoglobin H disease or mild α-thalassemia occasionally are observed in elderly patients with myeloid leukemia or the myelodysplastic syndrome. Earlier studies suggested this finding resulted from an acquired defect of α-globin synthesis in which the α-globin genes were completely inactivated in the neoplastic hemopoietic cell line.164 The molecular basis for this observation now is known to reside in a variety of different mutations involving ATR-X.41,165 The relationship of these somatic mutations of ATR-X to the neoplastic transformation remains to be determined. The molecular defect of other cases of acquired α-thalassemia, such as that seen in variable combined immunodeficiency,166 also remains to be defined.

PATHOPHYSIOLOGY Almost all the pathophysiologic features of the thalassemias can be related to a primary imbalance of globin-chain synthesis. This phenomenon makes the thalassemias fundamentally different from all the other genetic and acquired disorders of hemoglobin production and, to a large extent, explains their extreme severity in the homozygous and compound heterozygous states (Fig. 48–13). The anemia of β-thalassemia has three major components. First, and most important, is ineffective erythropoiesis with intramedullary destruction of a variable proportion of the developing red cell

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Excess Precipitation HgbF Selective survival of HgbF-containing cells

Hemolysis

Destruction of RBC precursors

Splenomegaly (pooling, plasma volume expansion)

Ineffective erythropoiesis

High oxygen affinity of red cells

Anemia Tissue hypoxia Erythropoietin

Transfusion

Marrow expansion

Bone deformity Increased metabolic rate Wasting Gout Folate deficiency

Increased iron absorption Iron loading Endocrine deficiencies Cirrhosis Cardiac failure Death

Figure 48–13.  Pathophysiology of β-thalassemia. HgbF, hemoglobin F; RBC, red blood cell.

precursors. Second is hemolysis resulting from destruction of mature red cells containing α-chain inclusions. Third are the hypochromic and microcytic red cells that result from the overall reduction in hemoglobin synthesis. Because the primary defect in β-thalassemia involves β-chain production, synthesis of hemoglobins F and A2 should be unaffected. Fetal hemoglobin production in utero is normal. The clinical manifestations of thalassemia appear only when the neonatal switch from γ- to β-chain production occurs. However, fetal hemoglobin synthesis persists beyond the neonatal period in nearly all forms of β-thalassemia (see “Persistent Fetal Hemoglobin Production and Cellular Heterogeneity” below). β-Thalassemia heterozygotes have an elevated level of hemoglobin A2. The elevated level appears to reflect not only a relative decrease in hemoglobin A as a result of defective β-chain synthesis but also an absolute increase in the output of δ chains both cis and trans to the mutant β-globin gene.7 Because α chains are shared by hemoglobins F, A, and A2, there is no increase in hemoglobin F in the α-thalassemias. The excess γ and β chains formed as a result of defective α-chain production produce soluble homotetramers (see “Mechanisms and Consequences of Erythroid Precursor Damage and Red Cell Damage” below). Hence there is less ineffective erythropoiesis than in β-thalassemia and the major cause of anemia is hemolysis and poorly hemoglobinized red cells.

IMBALANCED GLOBIN-CHAIN SYNTHESIS Measurements of in vitro globin-chain synthesis in the blood or marrow of patients with different types of thalassemia167,168 and family studies that allow examination of the action of thalassemia genes in patients

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who also inherited α- or β-globin structural variants7,9 provide a clear picture of the action of the thalassemia determinants. In homozygous β-thalassemia, β-globin synthesis is either absent or markedly reduced. The result is excessive production of α-globin chains. α-Globin chains are incapable of forming a viable hemoglobin tetramer, so the chains precipitate in red cell precursors. The resulting inclusion bodies can be demonstrated by both light and electron microscopy.169,170 In the marrow, precipitation can be seen in the earliest hemoglobinized precursors and throughout the erythroid maturation pathway.171 These large inclusions are responsible for intramedullary destruction of red cell precursors and hence for the ineffective erythropoiesis characterizing all the β-thalassemias. A large proportion of the developing erythroblasts are destroyed within the marrow in severe cases.172 Any red cells that are released are prematurely destroyed by mechanisms that are considered below in “Mechanisms and Consequences of Erythroid Precursor and Red Cell Damage.” β-Thalassemia heterozygotes also have imbalanced globin-chain synthesis, but the magnitude of α-chain excess is much less and presumably can be resolved by the proteolytic enzymes of the red cell precursors.173 Notwithstanding, a mild degree of ineffective erythropoiesis occurs. Although there is marked globin-chain imbalance in the severe α-thalassemias,7,167 the excess γ and β chains form homotetramers that do not precipitate in the red cell precursors to the same extent as excess α chains in β-thalassemia. Hence the pathophysiology of anemia is fundamentally different between the two conditions.

MECHANISMS AND CONSEQUENCES OF ERYTHROID PRECURSOR AND RED CELL DAMAGE Damage to the red cell membrane by the globin-chain precipitation process occurs by two major routes: generation of hemichromes (Chap. 49) from excess α chains with subsequent structural damage to the red cell membrane, and similar damage mediated through the degradation products of excess α chains.7,174–176 The degradation products of free α chains—globin, heme, hemin (oxidized heme), and free iron—also play a role in damaging red cell membranes. Excess globin chains bind to different membrane proteins and alter their structure and function. Excess iron, by generating oxygen free radicals, damages several red cell membrane components (including lipids and protein) and intracellular organelles. Heme and its products can catalyze the formation of a variety of reactive oxygen species that can damage the red cell membrane. These changes are reflected in an increased rate of apoptosis of red cell precursors.177 The red cells are rigid and underhydrated, leak potassium, and have increased levels of calcium and low, unstable levels of ATP. Damage to the red cells can also be mediated by the presence of rigid inclusion bodies during passage of the red cells through the spleen. The consequences of excess non–α-chain production in the α-thalassemias are quite different. Because α chains are shared by both fetal and adult hemoglobin (Chaps. 6 and 48), defective α-chain production is manifest in both fetal and adult life. In the fetus, it leads to excess γ-chain production; in the adult, it leads to an excess of β chains. Excess γ chains form γ4 homotetramers or hemoglobin Bart’s178; excess β chains form β4 homotetramers or hemoglobin H.179 The fact that γ and β chains form homotetramers is the reason for the fundamental difference in the pathophysiology of α- and β-thalassemia. Because γ4 and β4 tetramers are soluble, they do not precipitate to any significant degree in the marrow, and therefore the α-thalassemias are not characterized by severe ineffective erythropoiesis. However, β4 tetramers precipitate as red cells age, with the formation of inclusion bodies. Thus, the anemia of the more severe forms of α-thalassemia in the adult results from a shortened

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survival of red cells consequent to their damage in the microvasculature of the spleen as a result of the presence of the inclusions. In addition, because of the defect in hemoglobin synthesis, the cells are hypochromic and microcytic. Hemoglobin Bart’s is more stable than hemoglobin H and does not form large inclusions. Although, as is the case in β-thalassemia, excess globin chains cause damage to the red cell membrane, the mechanisms are different in the two forms of the disease. As described in “Etiology and Pathogenesis” above, in β-thalassemia, excess α chains result in mechanical instability and oxidative damage to a variety of membrane proteins, notably protein 4.1. However, in α-thalassemia, the membranes are hyperstable, and no evidence of oxidation or dysfunction of this protein is present. Furthermore, the state of red cell hydration is different in α-thalassemia. Accumulation of excess β chains results in increased hydration. These differences in the pathophysiology of membrane damage between αand β-thalassemia are discussed in detail in references 7 and 174 to 176. Another factor exacerbates the tissue hypoxia of the anemia of the α-thalassemias. Both hemoglobin Bart’s and hemoglobin H show no heme–heme interaction and have almost hyperbolic oxygen dissociation curves with very high oxygen affinities. Thus, they are not able to liberate oxygen at physiologic tissue tensions; in effect, they are useless as oxygen carriers.7 As a consequence, infants with high levels of hemoglobin Bart’s have severe intrauterine hypoxia. This is the major basis for the clinical picture of homozygous α0-thalassemia, which results in the stillbirth of hydropic infants late in pregnancy or at term. Oxygen deprivation is reflected by the grossly hydropic state of the infant, presumably as a result of increased capillary permeability, and by severe erythroblastosis. Deficient fetal oxygenation probably is responsible for the enormously hypertrophied placentas and possibly for the associated developmental abnormalities that occur with the severe forms of intrauterine α-thalassemia.7

rapidly in the spleen and elsewhere, cells with a much longer survival that contain relatively more hemoglobin F, and populations of intermediate age and hemoglobin constitution.7,182 Although cell selection is probably the main reason for the increased levels of hemoglobin F in the red cells in β-thalassemia, other mechanisms may also be involved. In any form of “stress erythropoiesis,” that is, rapid erythroid proliferation, there is a tendency for a relative increase in γ-chain production. Furthermore, as discussed in “Hereditary Persistence of Fetal Hemoglobin” above, several genes or chromosomal locations have been defined in which polymorphisms are involved in the increased basal production of γ chains and a relative increase in the number of F cells in the blood. The interaction of these different loci appear to be responsible for high levels of hemoglobin F production in β-thalassemia and sickle cell anemia with the production of milder phenotypes.125–128,184 However, biosynthesis studies indicate that marrow expansion and the selective survival of F-cell precursors and their progeny are the major factors in hemoglobin F production in hemoglobin E/β-thalassemia.183 Because a reciprocal relation exists between γ- and δ-chain synthesis, the red cells of β-thalassemia homozygotes containing large amounts of hemoglobin F have relatively low hemoglobin A2 levels.7 Thus, the measured percent hemoglobin A2 in these individuals is the average of a very heterogeneous cell population. This finding probably accounts for the extreme variability in hemoglobin A2 levels found in homozygotes for this disorder. A further consequence of the persistence of hemoglobin F in β-thalassemia is the high oxygen affinity of the red cells.

PERSISTENT FETAL HEMOGLOBIN PRODUCTION AND CELLULAR HETEROGENEITY

The profound anemia of homozygous β-thalassemia and the relatively high oxygen affinity of hemoglobin F combine to cause severe tissue hypoxia. Because of the high oxygen affinity of hemoglobins Bart’s and H, a similar defect in tissue oxygenation occurs in the more severe forms of α-thalassemia. The major adaptive response to hypoxia is increased erythropoietin production. It has been found that in severely anemic children with hemoglobin E β-thalassemia, age and hemoglobin levels are independent variables in erythropoietin response and that for a given hemoglobin level there is a relatively high erythropoietin in very young children.185 These observations provide an explanation for the rather unstable phenotype of many intermediate forms of β-thalassemia during early childhood. The major effect of these very high levels of erythropoietin production is expansion of the dyserythropoietic marrow. The results are deformities of the skull and face and porosity of the long bones.7 Extramedullary hematopoietic tumors may develop in extreme cases. Apart from the production of severe skeletal deformities, marrow expansion may cause pathologic fractures and sinus and middle ear infection as a result of ineffective drainage. Another important effect of the enormous expansion of the marrow mass is the diversion of calories required for normal development to the ineffective red cell precursors. Thus, patients severely affected by thalassemia show poor development and wasting. The massive turnover of erythroid precursors may result in secondary hyperuricemia and gout and severe folate deficiency. The effects of gross intrauterine hypoxia in homozygous α0thalassemia have been described. In the symptomatic forms of αthalassemia (e.g., hemoglobin H disease) that are compatible with survival into adult life, bone changes and other consequences of erythroid expansion are seen, although less commonly than in β-thalassemia.

Children with severe thalassemia have an increased level of hemoglobin F that persists into childhood and later.7,10 In the β0-thalassemias, hemoglobin F is the only hemoglobin produced, except for small amounts of hemoglobin A2. Examination of the blood using staining methods specific for hemoglobin F shows that it is heterogeneously distributed among the red cells.7 Persistent hemoglobin F production is not a major feature of the more severe forms of α-thalassemia. The mechanism of persistent γ-chain synthesis in the thalassemias is incompletely understood. Normal adults have small quantities of hemoglobin F that are heterogeneously distributed among the red cells. Cells with demonstrable hemoglobin F are called F cells. One important mechanism for high hemoglobin F levels in the blood of patients with β-thalassemia is cell selection.7,180–183 The major cause of ineffective erythropoiesis and shortened red cell survival in β-thalassemia is the deleterious effect of excess α chains on erythroid maturation in the marrow and on the survival of red cells in the blood. Therefore, red cell precursors that produce γ chains are at a selective advantage. Excess α chains combine with γ chains to produce hemoglobin F; therefore, the magnitude of α-chain precipitation is less. Differential centrifugation experiments181–183 and in vivo labeling studies180 have shown that populations of red cells with relatively large amounts of hemoglobin F are more efficiently produced and survive longer in the blood. The blood of patients with homozygous β-thalassemia shows remarkable cellular heterogeneity with respect to red cell survival, such as populations of cells containing predominantly hemoglobin A that are destroyed very

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SPLENOMEGALY: DILUTIONAL ANEMIA Constant exposure of the spleen to red cells with inclusions consisting of precipitated globin chains gives rise to the phenomenon of “work hypertrophy.” Progressive splenomegaly occurs in both α- and β-thalassemia and may worsen the anemia.7,10 A large spleen acts as a sump for red cells, sequestering a considerable proportion of the red cell mass. Furthermore, splenomegaly may cause plasma volume expansion, a complication that can be exacerbated by massive expansion of the erythroid marrow. The combination of pooling of the red cells in the spleen and plasma volume expansion can exacerbate the anemia in both α- and β-thalassemia.

ABNORMAL IRON METABOLISM β-Thalassemia homozygotes that are anemic manifest increased intestinal iron absorption that is related to the degree of expansion of the red cell precursor population. Iron absorption is decreased by blood transfusion.7,10 Increased absorption causes a steady accumulation of iron, first in the Kupffer cells of the liver and the macrophages of the spleen and later in the parenchymal cells of the liver. Most patients homozygous for β-thalassemia require regular blood transfusion; thus, transfusional siderosis adds to the iron accumulation. Iron accumulates in the endocrine glands,7,186 particularly in the parathyroids, pituitary, pancreas, skin leading to increased pigmentation, liver, and, most important, in the myocardium.7,187 Iron accumulation in the myocardium leads to death by involving the conducting tissues or by causing intractable cardiac failure. Other consequences of iron loading include diabetes, hypoparathyroidism, hypothyroidism, and abnormalities of hypothalamic–pituitary function leading to growth retardation and hypogonadism.7,186 Recent work on the mechanisms of hepcidin downregulation in association with marrow hypertrophy provides a much better understanding of the mechanisms of iron loading in diseases like thalassemia and may provide new therapeutic options for the future (Chap. 43 and Ref. 188). Accurate information is available regarding the levels of body iron, as reflected by hepatic iron, at which patients are at risk for serious complications of iron overload.7,189 These studies, which extrapolate data obtained from patients with genetic hemochromatosis, suggest that patients with hepatic iron levels of approximately 80 μmol of iron per gram of liver, wet weight (~15 mg of iron per gram of liver, dry weight), are at increased risk for hepatic disease and endocrine organ damage. Patients with higher body iron burdens are at particular risk for cardiac disease and early death (Chap. 43). Disordered iron metabolism is less common in the adult forms of α-thalassemia. The milder degree of anemia, fewer transfusions, and the less marked erythroid expansion of the marrow are likely explanations. The mechanisms whereby iron, and in particular non–transferrinbound iron mediate tissue damage, and recent evidence about the central role of hepcidin in the abnormal regulation of iron absorption in disorders like thalassemia are discussed in Chap. 42.

INFECTION All forms of severe thalassemia appear to be associated with an increased susceptibility to bacterial infection.7 The reason is not known. The relatively high serum iron levels may favor bacterial growth. Another possible mechanism is blockade of the monocyte–macrophage system as a result of the increased rate of destruction of red cells. No consistent defects in white cell or immune function have been reported, and high serum iron levels as an important factor remain to be unequivocally demonstrated. The one exception is infection with Yersinia enterocolitica, a normally nonvirulent pathogen that can produce its

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own siderophore and hence can thrive in iron excess. Transfusiondependent patients with thalassemia are at particular risk for bloodborne infections including hepatitis B, hepatitis C, HIV/AIDS, and, in some parts of the world, malaria.

COAGULATION DEFECTS The increasing knowledge about the potential hypercoagulable state in some forms of thalassemia has been reviewed in detail.174–176,190 Evidence indicates that patients, particularly after splenectomy and with high platelet counts, may develop progressive pulmonary arterial disease as a result of platelet aggregation in the pulmonary circulation. Furthermore, using thalassemic red cells as a source of phospholipids, enhanced thrombin generation has been demonstrated in a prothrombinase assay. The procoagulant effect of thalassemia cells appears to result from increased expression of anionic phospholipids on the red cell surface (Chap. 33). Normally, neutral or negatively charged phospholipids are confined to the inner leaflet of the red cell membrane, an effect that is mediated by the action of aminophospholipid translocase, an enzyme sometimes known as flippase. In effect, this enzyme flips aminophospholipids that are diffused to the outer leaflet back to the inner leaflet (Chaps. 31 and 46). The current belief is that these aminophospholipids in thalassemic red cells are moved to the outer leaflet, thus providing a surface on which coagulation can be activated. Other nonspecific changes in the coagulation pathway and its antagonists have been observed in patients with different forms of thalassemia. There is increasing evidence that, as in the case of sickle cell anemia (Chap. 49), the hemolytic component of the anemia of β-thalassemia is associated with the release of hemoglobin and arginase resulting in impaired nitric oxide availability and endothelial dysfunction with progressive pulmonary hypertension.191 There may be other contributions to this complication including increased coagulability and local structural damage to the lungs relating to excess iron deposition.

CLINICAL HETEROGENEITY The pathophysiologic mechanisms described above provide the basis for the remarkably diverse clinical findings in the thalassemia syndromes.7,192 All the manifestations of β-thalassemia can be related to excess α-chain production. Thus, any mechanism that reduces the excess of α chains should reduce the clinical severity of the disease. Several elegant “experiments of nature” have shown that this reasoning is true and, incidentally, have confirmed that globin-chain imbalance is the major factor determining the severity of the thalassemias. Coinheritance of α-thalassemia can reduce the severity of the more severe forms of β-thalassemia.193,194 The effect is much more marked in individuals who are homozygotes or compound heterozygotes for different forms of β+-thalassemia. β0-Thalassemia homozygotes who have inherited α-thalassemia seem to be protected little, if at all. Severe β-thalassemia can be modified by the coinheritance of genetic determinants for enhanced production of γ chains. Several determinants may be involved. For example, inheritance of a particular RFLP haplotype in the region 5′ to the β-globin gene may be an important factor.195,196 This particular β-globin gene haplotype is associated with a single base change, C→T, at position –158 relative to the Gγ-globin gene, an alteration that creates a cleavage site for the restriction enzyme XmnI.121 An excess of individuals homozygous for T (XmnI+ +) with the phenotype of thalassemia intermedia exist compared with thalassemia major in different populations.196–198 Whether this polymorphism is the only factor that increases hemoglobin F production in these cases is not absolutely clear. As discussed under “Hereditary Persistence of Fetal Hemoglobin” above, it is now clear that there are

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loci on chromosomes 2, 6, and 8, and possibly the X chromosome, at which polymorphisms are involved in the elevation of fetal hemoglobin synthesis and that their coinheritance may significantly modify the phenotype of different forms of β-thalassemia. Some mutations that cause β-thalassemia are associated with a mild phenotype because they result in only modest reduction of β-chain production.7 For example, mutations at positions –29 and –88 are associated with mild β+-thalassemia in Africans. Similarly, particularly mild phenotypes are commonly found with a base substitution at position 6 in IVS-1 and at position –87 in the 5′-flanking region of the β-globin gene in Mediterranean populations. The homozygous state for the IVS-1 position 6 mutation usually produces an extremely mild form of β-thalassemia. When these “mild” mutations are coinherited with more-severe β-thalassemia determinants, the compound heterozygous states are characterized by a more severe form of thalassemia intermedia. Other forms of thalassemia intermedia are associated with the homozygous state for δβ-thalassemia, the various interactions of βthalassemia with δβ-thalassemia, and heterozygous β-thalassemia of the severe variety or in association with triplicated α-gene loci.7,10,198 These complex interactions are the subject of several extensive reviews.198–200 These mechanisms for the phenotypic variability of the β-thalassemias represent only the beginning of our understanding of the genetic diversity of these conditions. Hence, defining a series of genetic modifiers that act at different levels is useful.192 Primary modifiers represent the diversity of mutations at the β-globin gene locus. Secondary modifiers are those, such as α-thalassemia and increased hemoglobin F production, that directly modify the relative degree of the imbalanced globin chain output. However, an increasing number of tertiary modifiers, that is, genetic diversity, have an important effect on the complications of the disease. These include loci involved in iron, bone, and bilirubin metabolism and in determining resistance of susceptibility to infection. Furthermore, phenotypic diversity may reflect different degrees of adaptation to anemia and the effect of the environment. These complex issues have been reviewed192 and are illustrated in Fig. 48–14. Several extensive reviews of the pathophysiology of the intermediate forms of β-thalassemia in different populations are available.199,200 The α-thalassemias, particularly hemoglobin H disease, show considerable clinical diversity. Some of this variability can be related to particular genotypes,7,41 but the reasons for the heterogeneity of these disorders is not clear.

CLINICAL FEATURES β- AND δβ-THALASSEMIAS The most clinically severe form of β-thalassemia is thalassemia major. A milder clinical picture, characterized by a later onset and either no transfusion requirement or at least fewer transfusions than are required to treat the major form of the illnesses, is designated β-thalassemia intermedia. β-Thalassemia minor is the term used to describe the heterozygous carrier state for β-thalassemia. More extensive accounts of the clinical features of these conditions are given in two monographs.7,9

β-THALASSEMIA MAJOR The homozygous or compound heterozygous state for β-thalassemia, thalassemia major, produces the clinical picture first described by Cooley and Lee1 in 1925. Affected infants are well at birth. Anemia usually develops during the first few months of life and becomes progressively more severe. The infants fail to thrive and may have feeding problems, bouts of fever, diarrhea, and other gastrointestinal symptoms. The majority of infants who develop transfusion-dependent homozygous β-thalassemia present with these symptoms within the first year of life.

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Figure 48–14.  Different levels of modification of the β-thalassemia

phenotype. COL, various genes involved in collagen metabolism; CO-selection, indicates variable selection of genes involved in susceptibility to infection along with different thalassemia genes; HFE, gene for hereditary hemochromatosis; Hgb F, hemoglobin F; ICAM, intercellular adhesion molecule; OR, estrogen receptor; TNF, tumor necrosis factor; UGT1A1, uridine diphosphate-glucuronyltransferase; VDR, vitamin D receptor. (Adapted with permission from Weatherall DJ: Phenotype-genotype relationships in monogenic disease: lessons from the thalassaemias. Nat Rev Genet 2(4):245–255, 2001.)

A later onset suggests the condition will develop into one of the intermediate forms of β-thalassemia (see “Pathophysiology” above). The course of the disease in childhood depends almost entirely on whether the child is maintained on an adequate transfusion program.7,9 The classic textbook picture of Cooley anemia describes the disease as it was seen before these children could be maintained with relatively normal hemoglobin levels by regular blood transfusions. If adequate transfusion is possible, children grow and develop normally and have no abnormal physical signs. Few of the complications of the disorder occur during childhood. The disease presents a problem only when the effects of iron loading resulting from ineffective erythropoiesis and from repeated blood transfusions become apparent at the end of the first decade. Children who are treated with an adequate iron chelation regimen develop normally, although some of them remain short in height. An inadequately transfused child develops the typical features of Cooley anemia. Growth is stunted. With bossing of the skull and overgrowth of the maxillary region, the face gradually assumes a “mongoloid” appearance. These changes are associated with a characteristic radiologic appearance of the skull, long bones, and hands (Fig. 48–15). The diploe widens, with a “hair on end” or “sun ray” appearance and a lacy trabeculation of the long bones and phalanges. Gross skeletal deformities can occur. The liver and spleen are enlarged, and the pigmentation of the skin increases. Many features of a hypermetabolic state, as evidence by fever, wasting, and hyperuricemia, may develop. The clinical course is characterized by severe anemia with frequent complications. These children are particularly prone to infection, which

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Osteoporosis is being recognized increasingly and may, at least in part, be a reflection of hypogonadism.201

β-THALASSEMIA INTERMEDIA

Figure 48–15.  Radiologic appearances of the hands in homozygous β-thalassemia. The scattered lucent areas in the bones of the fingers reflect the marked expansion of marrow in distal areas.

is a common cause of death. Spontaneous fractures occur commonly as a result of the expansion of the marrow cavities with thinning of the long bones and skull. Maxillary deformities often lead to dental problems from malocclusion. Formation of massive deposits of extramedullary hematopoietic tissue may cause neurologic complications. With the gross splenomegaly that may occur, secondary thrombocytopenia and leukopenia frequently develop, leading to a further tendency to infection and bleeding. Splenectomy is frequently performed to reduce transfusion frequency and severe thrombocytopenia; however, postsplenectomy infections are particularly common.7 Bleeding tendency may be seen in the absence of thrombocytopenia. Epistaxis is particularly common. These hemostatic problems are associated with poor liver function in some cases. Chronic leg ulceration may occur but is more common in thalassemia intermedia. Children who have grown and developed normally throughout the first 10 years of life as a result of regular blood transfusion begin to develop the symptoms of iron loading as they enter puberty, particularly if they have not received adequate iron chelation.7,9 The first indication of iron loading usually is the absence of the pubertal growth spurt and failure of the menarche. Over the succeeding years, a variety of endocrine disturbances may develop, particularly diabetes mellitus, hypogonadotrophic hypogonadism, and growth hormone deficiency. Hypothyroidism and adrenal insufficiency also occur but are less common.7,186 Toward the end of the second decade, cardiac complications arise, and death usually occurs in the second or third decade as a result of cardiac siderosis.187–189 Cardiac siderosis may cause an acute cardiac death with arrhythmia, or intractable cardiac failure. Both of these complications can be precipitated by intercurrent infection. Even the adequately transfused child who has received chelation therapy may suffer a number of complications. Bloodborne infection, notably with hepatitis B or C,201 HIV,202 or malaria,203 is extremely common in some populations, although the frequency is decreasing with the use of widespread blood-donor screening programs. Delayed puberty and growth retardation are common and probably reflect hypogonadotrophic hypogonadism and damage to the pituitary gland.201,204

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The clinical phenotype of patients designated as having thalassemia intermedia is more severe than the usual asymptomatic thalassemia trait but milder than transfusion-dependent thalassemia major.7,199,200 The syndrome encompasses disorders with a wide spectrum of disability. At the severe end, patients present with anemia later than patients with the transfusion-dependent forms of homozygous β-thalassemia and are just able to maintain a hemoglobin level of approximately 6 g/ dL without transfusion. However, their growth and development are retarded. The patients become seriously disabled, with marked skeletal deformities, arthritis, and bone pain; progressive splenomegaly; growth retardation; and chronic ulcerations above the ankles. At the other end of the spectrum, patients remain completely asymptomatic until adult life and are transfusion independent, with hemoglobin levels as high as 10 to 12 g/dL. All varieties of intermediate severity are observed. Some patients become disabled simply from the effects of hypersplenism. Intensive studies of the molecular pathology of this condition have provided some guidelines about genotype–phenotype relationships that are useful for genetic counseling (Table 48–6). Overall, the clinical features of the intermediate forms of βthalassemia are similar to the features of β-thalassemia major. At the severe end of the spectrum, particularly in cases of growth retardation, patients should be treated with regular transfusion. However, a number of important complications, including progressive hypersplenism, occur in patients with milder forms. Clinically significant iron loading

TABLE 48–6.  Genotypes of Patients with β-Thalassemia Intermedia Mild forms of β-thalassemia   Homozygosity for mild β+-thalassemia alleles   Compound heterozygosity for two mild β+-thalassemia alleles  Compound heterozygosity for a “silent” or mild and more-severe β-thalassemia allele Inheritance of α- and β-thalassemia   β+-Thalassemia with α0-thalassemia (– –/αα) or α+-thalassemia (–α/αα or –α/–α)   β+-Thalassemia with genotype of Hgb H disease (– – /–α) β-Thalassemia with elevated γ-chain synthesis  Homozygous β-thalassemia with heterocellular HPFH  Homozygous β-thalassemia with homozygous Gγ 158 T→C change (some cases)  Compound heterozygosity for β-thalassemia and deletion forms of HPFH Compound heterozygosity for β-thalassemia and β-chain variants   Hgb E/β-thalassemia   Other interactions with rare β-chain variants Heterozygous β-thalassemia with triplicated or quadruplicated α-chain genes (ααα or αααα)   Dominant forms of β-thalassemia   Interactions of β- and (δβ)+- or (δβ)0-thalassemia Hgb, hemoglobin; HPFH, hereditary persistence of fetal hemoglobin.

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as a result of increased absorption is seen even in patients with infrequent transfusions (Chap. 43). Iron overload results in frequent diabetes and endocrine disturbances, typically by fourth decade of life. A high incidence of pigment gallstones, skeletal deformities, bone and joint disease, leg ulcers, and thrombotic tendency, particularly after splenectomy, is observed.7 Hematologists should be aware that in patients heterozygous for rare forms of β-thalassemia, a phenotype of thalassemia intermedia that results in the clinical constellation of autosomal dominant thalassemia (discussed in “Pathophysiology” above) is encountered on rare occasions.

β-THALASSEMIA MINOR The heterozygous state for β-thalassemia is usually identified during family studies of patients with more severe forms of β-thalassemia, population surveys, or, most frequently, by the chance finding of the characteristic hematologic changes during a routine study. There is an extensive literature on this condition,7 some of which suggests that affected individuals may have symptoms of anemia and, not infrequently, splenomegaly, while other studies suggest that the condition is completely symptomless and palpable splenomegaly does not occur. Surprisingly, none of these studies have been controlled. A controlled study reported that individuals with the β-thalassemia trait suffer from fatigue and other symptoms indistinguishable from those with mild anemias from other causes. There was no difference in the frequency of palpable splenomegaly between the thalassemic and control groups.205 The trait not infrequently causes a moderately severe anemia of pregnancy, in some cases requiring transfusion. Some β-thalassemia carriers have increased iron stores, although this is most often a result of inappropriate iron therapy based on a misdiagnosis. In countries where there is a relatively high frequency of genetic determinants for hemochromatosis, the possibility of their coinheritance should be borne in mind if a patient with β-thalassemia trait with an unusually high plasma iron or serum ferritin level is encountered.

α-THALASSEMIAS Hemoglobin Bart’s Hydrops Fetalis Syndrome

This disorder is a frequent cause of stillbirth in Southeast Asia. Infants either are stillborn between 34 and 40 weeks’ gestation or are born alive but die within the first few hours.7,206 Pallor, edema, and hepatosplenomegaly are seen. The clinical picture resembles hydrops fetalis as a result of Rh blood group incompatibility. Massive extramedullary hemopoiesis and enlargement of the placenta are noted at autopsy. A variety of congenital anomalies have been observed. The rescue of a few infants with this syndrome by prenatal detection and exchange transfusion has been reported. These babies have grown and developed normally, although they are blood transfusion–dependent.207,208 This condition is associated with a high incidence of maternal toxemia of pregnancy and difficulties at the time of delivery because of the massive placenta.206 The reason for placental hypertrophy is unknown, although severe intrauterine hypoxia is suspected because a similar phenomenon is observed in hydrops infants with Rh incompatibility.

Hemoglobin H Disease

Hemoglobin H disease was described independently in the United States and in Greece in 1956.209,210 The clinical findings are variable. A few patients are affected almost as severely as patients with β-thalassemia major, but most patients have a much milder course.7,211 Lifelong anemia with variable splenomegaly occurs; bone changes are unusual.

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As discussed earlier in “Etiology and Pathogenesis,” a few attempts have been made to correlate the genotype with the phenotype of hemoglobin H disease. In general, as expected, patients with a nondeletion form of α-thalassemia affecting the predominant α2 gene interacting with an α0-thalassemia determinant αNDα/– –, or αConstantSpringα/– –, for example, have higher hemoglobin H levels, a greater degree of anemia, and a more severe clinical course than patients with the – –/–α genotype.212–215

Milder Forms of α-Thalassemia

Because two α-globin genes exist per haploid genome, a wide spectrum of different conditions with overlapping phenotypes result from their various interactions.7 The carrier states for the deletion and nondeletion forms of α-thalassemia, –α/αα and αNDα/αα, are symptomless. Similarly, the homozygous states for the deletion forms of α+-thalassemia, –α/–α, and the heterozygous state for α0-thalassemia, – –/αα, are symptomless, although they are associated with mild anemia and red cell changes. On the other hand, the homozygous states for the nondeletion forms of α-thalassemia, αNDα/αNDα, are associated with an extremely diverse series of phenotypes. As mentioned in “Interactions of α-Thalassemia Haplotypes” above in “Etiology and Pathogenesis,” they sometimes result in the clinical picture of hemoglobin H disease. In other patients, they are associated with only mild hypochromic anemia.7 The homozygous states for the chain termination mutants, notably hemoglobin Constant Spring, constitute a special case because they produce a particularly characteristic phenotype. In this case, moderate hemolytic anemia with splenomegaly are seen.7,216,217

α-Thalassemia and Mental Retardation

The clinical phenotype of these conditions is heterogeneous. In cases associated with chromosomal deletion (tip of chromosome 16; ATR-16 [α-thalassemia chromosome 16-linked mental retardation syndrome]), the clinical defects vary with the extent of chromosomal defect; only α-thalassemia and mental retardation are constant.157 To some extent this clinical variation is related to the length of the associated deletions; those which extend for 2000 kb involve the genes that are involved in tuberous sclerosis and polycystic kidney disease. In these cases the latter dominate the clinical picture, but there mental retardation and αthalassemia are also associated. The clinical phenotype in the second group of these disorders, which are caused by mutations of ATR-X, includes skeletal abnormalities, dysmorphic face, neonatal hypotonus, genital abnormalities, and a variety of less-constant features, in addition to mental retardation and α-thalassemia.158

εγδβ-Thalassemia

The clinical picture varies with the stage of development.7 Neonates may be significantly anemic and require transfusions. In contrast, children and adults with this condition are asymptomatic. They have the clinical and laboratory picture of heterozygous β-thalassemia, with the exception of a normal hemoglobin A2 level. The reason for this discrepancy of developmental differences of the clinical phenotype has not been identified. The homozygous state is assumed to be lethal.

LABORATORY FEATURES β-THALASSEMIA MAJOR Hemoglobin levels at presentation may range from 2 to 3 g/dL or even lower.7 The red cells show marked anisopoikilocytosis, with hypochromia, target cell formation, and a variable degree of basophilic stippling (Fig. 48–16). The appearance of the blood film varies, depending on whether the spleen is intact. In nonsplenectomized patients, large

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A

B

C

Figure 48–16.  Blood films in β-thalassemia. A. β-Thalassemia minor. Anisocytosis, poikilocytosis, hypochromia. Occasional spherocytes and stomatocytes. B. Scanning electron micrograph of cells in (A) showing more detail of the poikilocytes. Note the knizocyte (pinch-bottle cell) at the lower right. C. β-Thalassemia major. Marked anisocytosis with many microcytes. Marked poikilocytosis. Anisochromia. Nucleated red cell on the right. Small lymphocyte on the left. (Reproduced with permission from Lichtman’s Atlas of Hematology, www.accessmedicine.com.) poikilocytes are common. After splenectomy, large, flat macrocytes and small, deformed microcytes are frequently seen. The reticulocyte count is moderately elevated, and nucleated red cells nearly always are present in the blood. These red cell forms may reach very high levels after splenectomy. The white cell and platelet counts are slightly elevated unless secondary hypersplenism occurs. Staining of the blood with methyl violet, particularly in splenectomized subjects, reveals stippling or ragged inclusion bodies in the red cells.169 These inclusions can nearly always be found in the red cell precursors in the marrow. The marrow usually shows erythroid hyperplasia with morphologic abnormalities of the erythroblasts, such as striking basophilic stippling and increased iron deposition. Iron kinetic studies indicate markedly ineffective erythropoiesis, and red cell survival usually is shortened. Populations of cells with very short survival and longer-lived populations of cells are seen. The latter contain relatively more fetal hemoglobin. An increased level of fetal hemoglobin, ranging from less than 10 percent to greater than 90 percent, is characteristic of homozygous β-thalassemia. No hemoglobin A is produced in β0-thalassemia. The fetal hemoglobin is heterogeneously distributed among the red cells. Hemoglobin A2 levels in homozygous β-thalassemia may be low, normal, or high. However, expressed as a proportion of hemoglobin A, the hemoglobin A2 level almost invariably is elevated. Differential centrifugation studies indicate some heterogeneity of hemoglobin F and A2 distribution among thalassemic red cells, but their level in whole blood gives little indication of their total rates of synthesis. In vitro hemoglobin synthesis studies using marrow or blood show a marked degree of globin-chain imbalance. Marked excess of α-chain

A

over β- and γ-chain production is always observed. Other aspects of the laboratory findings in this condition, including red cell survival, iron absorption, ferrokinetics, erythrokinetics, and the consequences of iron loading, were discussed earlier (see “Etiology and Pathogenesis” above). The examination of siblings, parents, and children can be very important in confirming the diagnosis by finding the abnormalities in other family members, and the examining physician should make every effort to obtain a complete blood count in family members. With the exception of higher hemoglobin levels, the hematological changes in β-thalassemia intermedia are similar to those in β-thalassemia major (Fig. 48–17).

β-THALASSEMIA MINOR Hemoglobin values of patients with β-thalassemia minor usually range from 9 to 11 g/dL. The most consistent finding is small, poorly hemoglobinized red cells (see Fig. 48–16), resulting in mean cell hemoglobin (MCH) values of 20 to 22 pg and mean corpuscular volume (MCV) values of 50 to 70 fL. The red cell count is usually normal or elevated and the hemoglobin and hematocrit is usually slightly below normal; however, the red cell indices are particularly useful in screening for heterozygous carriers of thalassemia in population surveys. The marrow in heterozygous β-thalassemia shows slight erythroid hyperplasia with rare red cell inclusions. Megaloblastic transformation as a result of folic acid deficiency occurs occasionally, particularly during pregnancy. A mild degree of ineffective erythropoiesis is noted, but red cell survival is normal or nearly normal. The hemoglobin A2 level is increased to

B

Figure 48–17.  A. Thalassemia intermedia. Blood films. Marked anisocytosis, poikilocytosis with elliptical, oval, tear-drop-shaped, and fragmented

red cells. Target cells. B. Postsplenectomy. Morphology similar to that in (A) but with a nucleated red cell, coarsely stippled cell in center of field, and large and numerous platelets, indicative of the changes superimposed by splenectomy. (Reproduced with permission from Lichtman's Atlas of Hematology, www.accessmedicine.com.)

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3.5 to 7.0 percent. The level of fetal hemoglobin is elevated in approximately 50 percent of cases, usually to 1 to 3 percent and rarely to greater than 5 percent.

α-THALASSEMIAS Hemoglobin Bart’s Hydrops Fetalis Syndrome

In infants with the hydrops fetalis syndrome, the blood film shows severe thalassemic changes with many nucleated red cells. The hemoglobin consists mainly of hemoglobin Bart’s, with approximately 10 to 20 percent hemoglobin Portland. Usually no hemoglobin A or F is present, although rare cases that seem to result from interaction of α0thalassemia with a severe nondeletion form of α+-thalassemia show small amounts of hemoglobin A.

Hemoglobin H Disease

The blood film shows hypochromia and anisopoikilocytosis. The reticulocyte count usually is approximately 5 percent. Incubation of the red cells with brilliant cresyl blue results in ragged inclusion bodies in almost all cells. These bodies form because of precipitation of hemoglobin H in vitro as a result of redox action of the dye. After splenectomy, large, single Heinz bodies are observed in some cells (Fig. 48–18). These bodies are formed by in vitro precipitation of the unstable hemoglobin H molecule and are seen only after splenectomy. Hemoglobin H constitutes between 5 and 40 percent of the total hemoglobin. Traces of hemoglobin Bart’s may be present, and the hemoglobin A2 level usually is slightly subnormal.

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α0-Thalassemia and α+-Thalassemia Traits

The α0-thalassemia trait is characterized by the presence of 5 to 15 percent hemoglobin Bart’s at birth.7 This hemoglobin disappears during maturation and is not replaced by a similar amount of hemoglobin H. An occasional cell with hemoglobin H inclusion bodies may appear after incubation with brilliant cresyl blue. This phenomenon is often used as a diagnostic test for the α-thalassemia trait. However, the test is difficult to standardize and requires much experience to be useful. In adult life, the red cells of heterozygotes have morphologic changes of heterozygous thalassemia with low MCH and MCV values. The electrophoretic pattern is normal. Globin-synthesis studies show a deficit of α-chain production, with an α-chain–to–β-chain production ratio of approximately 0.7. The α+-thalassemia trait (–α/αα) is characterized by a mild reduction in MCH and MCV values although in some cases there are normal values, 1 to 2 percent of hemoglobin Bart’s at birth in some but not all cases, and a slightly reduced α-chain–to–β-chain production ratio of approximately 0.8; thus, this genotype often is referred to as silent carrier. Extensive studies comparing the level of hemoglobin Bart’s at birth with a DNA analyses demonstrated that there is no detectable hemoglobin Bart’s in a significant number of newborns who are heterozygous for α+-thalassemia.218,219 Globin gene synthetic ratios can be distinguished from normal only by studying relatively large numbers of samples and comparing the mean α–to–β ratio with that of normal control subjects. This approach is not reliable for diagnosing individual cases of the α+-thalassemia trait, and, unfortunately, no reliable method of diagnosis is available except for DNA analysis.

A

B

C

D

Figure 48–18.  Hemoglobin H disease (α-thalassemia). Blood films. A. Note hypochromic red cells, anisocytosis, target cells, poikilocytes, includ-

ing tear-drop-shaped red cells. B. Wet preparation stained with crystal violet. Inclusions in red cells (Heinz bodies) usually attached to membrane. C. Postsplenectomy. Note reduction in poikilocytes and frequency of target cells, a change consistent with hemoglobin H disease and enhanced by postsplenectomy effects. A nucleated red cell is in this field, reflecting an increase in their prevalence in the blood after splenectomy. D. Blood incubated for 90 minutes with brilliant cresyl blue. Numerous hemoglobin H intracellular precipitates (precipitates of excess β-globin chains). The frequent crenation is an artifact of the incubation conditions. (Reproduced with permission from Lichtman's Atlas of Hematology, www.accessmedicine.com.)

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Homozygous State for Nondeletion Types of α-Thalassemia

The homozygous state for nondeletion forms of α-thalassemia involving the dominant (α2) globin gene causes a more severe deficit of α chains than do the deletion forms of α+-thalassemia. In some cases, the homozygous state produces hemoglobin H disease. The homozygous state for hemoglobin Constant Spring or other chain-termination mutations is associated with moderately severe hemolytic anemia in which, for reasons not explained, no hemoglobin H is present but small amounts of hemoglobin Bart’s persist into adult life. The homozygous states for the other nondeletion forms of α+thalassemia are associated with hemoglobin H disease. In the homozygous state for hemoglobin Constant Spring, the blood picture shows mild thalassemic changes with normal-size red cells.216,217 The hemoglobin consists of approximately 5 to 6 percent hemoglobin Constant Spring, normal hemoglobin A2 levels, and trace amounts of hemoglobin Bart’s. The remainder is hemoglobin A. The heterozygous state for hemoglobin Constant Spring shows no hematologic abnormality. The hemoglobin pattern is normal except for the presence of approximately 0.5 percent hemoglobin Constant Spring. The latter can be observed on alkaline starch-gel electrophoresis as a faint band migrating between hemoglobin A2 and the origin. It is best seen on heavily loaded starch gels and is easily missed if other electrophoretic techniques are used (Fig. 48–19). In the newborn, usually 1 to 3 percent hemoglobin Bart’s is present in the cord blood.

Homozygous State for Deletion Forms of α+-Thalassemia

The homozygous state for deletion forms of α+-thalassemia is characterized by a thalassemic blood picture with 5 to 10 percent hemoglobin Bart’s at birth and hematologic findings similar to those in α0-thalassemia heterozygotes in adult life. In general, the –α4.2 deletion is associated with a more severe phenotype than is the –α3.7 deletion.7

DIFFERENTIAL DIAGNOSIS The clinical and hematologic findings in homozygous β-thalassemia and hemoglobin H disease are so characteristic that the diagnosis usually is not difficult. Figure 48–20 shows a simple flowchart for laboratory investigations of a suspected case. In early childhood, distinguishing the thalassemias from the congenital sideroblastic anemias may be difficult, but the marrow appearances in the latter are quite characteristic. Because of the high

1 Hgb Constant Spring

2

3

4

5

6 Origin

Hgb A2

Hgb Bart’s

+

Figure 48–19.  Hemoglobin (Hgb) Constant Spring. Starch gel electrophoresis of 1,2, normal adult; 3,4, compound heterozygotes for hemoglobin Constant Spring and α0-thalassemia with hemoglobin H disease; 5, normal adult; and 6, compound heterozygote for α0-thalassemia and hemoglobin Constant Spring.

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LESS-COMMON FORMS OF THALASSEMIA (δβ)0-Thalassemia

The homozygous state for δβ-thalassemia is clinically milder than Cooley anemia and is one form of thalassemia intermedia.220–222 Only hemoglobin F is present; hemoglobins A and A2 are not produced. Heterozygous δβ-thalassemia is hematologically similar to β-thalassemia minor.7 The fetal hemoglobin level is higher (range: 5 to 20 percent), and the hemoglobin A2 value is normal or slightly reduced. As in βthalassemia, the fetal hemoglobin is heterogeneously distributed among the red cells, thus distinguishing this disorder from HPFH (Fig. 48–21). Heterozygosity for both β-thalassemia and δβ-thalassemia results is a condition clinically similar to but milder than Cooley anemia. The hemoglobin consists largely of hemoglobin F, with a small amount of hemoglobin A2. This finding is seen because the associated βthalassemia gene has usually been the β0 variety. δβ-Thalassemia has also been observed in individuals heterozygous for hemoglobin S or C.7

(δβ)+-Thalassemia and Hemoglobin Lepore Disorders

The hemoglobin Lepore disorders have been described in the homozygous state and in the heterozygous state, either alone or in association with β- or δβ-thalassemia, hemoglobin S, or hemoglobin C.7,9,223 In the homozygous state, approximately 20 percent of the hemoglobin is of the Lepore type and 80 percent is fetal hemoglobin. Hemoglobins A and A2 are absent. The clinical picture is variable. Some cases are identical to transfusion-dependent homozygous β-thalassemia; others are associated with the clinical picture of thalassemia intermedia. In the heterozygous state, the findings are similar to those of β-thalassemia minor. The hemoglobin consists of approximately 10 percent hemoglobin Lepore, with a reduced level of hemoglobin A2 and a slight but consistent increase in fetal hemoglobin level. The Lepore hemoglobins have been found sporadically in most racial groups. In the majority of cases, chemical analysis has shown that these hemoglobins are identical to hemoglobin Lepore Washington-Boston. Hemoglobin Lepore Hollandia and Lepore Baltimore have been observed in only a few patients.7,223

HEREDITARY PERSISTENCE OF FETAL HEMOGLOBIN The current knowledge about the molecular pathology of HPFH was described earlier in “Etiology and Pathogenesis.” Table  48–4 summarizes the currently accepted classification and nomenclature of this complex group of conditions. The different forms of HPFH are of very little clinical importance except that they may interact with thalassemia or the structural hemoglobin variants.

Hgb A

Hgb H

hemoglobin F levels encountered in juvenile chronic myelogenous leukemia, this disorder may superficially resemble β-thalassemia. However, the finding of primitive cells in the marrow, the absence of elevated hemoglobin A2 levels on hemoglobin electrophoresis, the decrease in carbonic anhydrase in juvenile chronic myelogenous leukemia, and characteristic in vitro responses of myeloid progenitors in vitro to granulocyte-monocyte colony-stimulating factor (Chap. 87) readily differentiate this disorder from β-thalassemia.

(δβ)0 Hereditary Persistence of Fetal Hemoglobin

Homozygotes for (δβ)0 HPFH have 100 percent hemoglobin F. Their blood shows mild thalassemic changes, with reduced MCH and MCV values very similar to those observed in heterozygous β-thalassemia. Similarly, they have imbalanced globin chain production, with ratios in the range of those observed in β-thalassemia heterozygotes.224 Heterozygotes have approximately 20 to 30 percent hemoglobin F, slightly

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Figure 48–20.  Flowchart showing an approach to diagnosis of the thalassemia syndromes. Hgb, hemoglobin; MCH, mean cell hemoglobin; MCV, mean corpuscular volume; RBC, red blood cell count.

reduced hemoglobin A2 values, and completely normal blood pictures. Thus, this condition appears to be an extremely well-compensated form of δβ-thalassemia in which the output of γ chains almost but not entirely compensates for the complete absence of β and δ chains. The different molecular forms of this condition show no difference in phenotype except in the proportion of Gγ chains. The African forms of (δβ)0 HPFH have been found in association with hemoglobins S and C or with β-thalassemia (Chap. 49). These compound heterozygous states are associated with little clinical disability.7

Nondeletion Types of Hereditary Persistence of Fetal Hemoglobin

Many nondeletion forms of HPFH associated with point mutations upstream from the γ-globin genes have been described (see Table  48–4). G γ β+ HPFH has been found in the heterozygous and compound heterozygous states with β-globin chain variants in African populations. No associated clinical or hematologic findings have been reported. Compound heterozygotes for Gγ β+ HPFH and hemoglobins S or C produce 45 percent of the abnormal hemoglobin, approximately 30 percent hemoglobin A, and approximately 20 percent hemoglobin F containing only Gγ chains.225,226 The most common form of nondeletion HPFH is Aγ β+ HPFH, which is found in Greeks.227–229 In the homozygous state, no clinical or hematologic abnormalities are noted. The hemoglobin findings are characterized by approximately 25 percent fetal hemoglobin and reduced hemoglobin A2 levels of approximately 0.8 percent.230 Heterozygotes, who also are hematologically normal, have 10 to 15 percent hemoglobin F, almost all of the Aγ variety. Compound heterozygotes with β-thalassemia have high hemoglobin F levels and a clinical picture that is only slightly more severe than the β-thalassemia trait. In the British form of Aγ β+ HPFH231 heterozygotes have approximately 5 to 12 percent hemoglobin F, whereas homozygotes have approximately 20 percent. No associated hematologic abnormalities are seen, although surprisingly in this form of nondeletion HPFH the hemoglobin F seems to be unevenly distributed among the red cells. A heterogeneous group of conditions is associated with persistent production of small amounts of hemoglobin F in adult life. They are

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categorized under the general heading of heterocellular HPFH. Their clinical importance is that, when they are coinherited with different forms of β-thalassemia, they may lead to greater output of hemoglobin F and, hence, to a milder phenotype. This type of interaction should be suspected when one parent of a patient with β-thalassemia intermedia has an unusually high level of hemoglobin F for the β-thalassemia trait. Similarly, unaffected lateral relatives or other family members with slightly elevated hemoglobin F levels may be found.

β-THALASSEMIA ASSOCIATED WITH β-CHAIN STRUCTURAL HEMOGLOBIN VARIANTS The most clinically important associations of β-thalassemia with β structural hemoglobin variants are sickle cell thalassemia, hemoglobin C thalassemia, and hemoglobin E thalassemia (Chap. 49). In addition, many interactions of β-thalassemia with rare structural variants have been reported.7,9,10 Sickle cell thalassemia7,232,233 occurs in parts of Africa and in the Mediterranean, particularly Greece and Italy. It also has been observed in the Middle East and parts of India. The clinical consequences of carrying one gene for hemoglobin S and one gene for β-thalassemia depend entirely on the type of β-thalassemia mutation. The interaction between the sickle cell gene and β0-thalassemia is characterized by a clinical disorder that is very similar to sickle cell anemia. Similarly, the interaction of the sickle cell gene with the more severe forms of β+-thalassemia associated with marked reduction in β-globin synthesis yields a similar clinical phenotype. On the other hand, the interaction of the sickle cell gene with very mild forms of β+-thalassemia may be quite innocuous.233 The latter disorder is characterized by mild anemia associated with splenomegaly and a hemoglobin composition of approximately 60 to 70 percent hemoglobin S, 25 percent hemoglobin A, and an elevated level of hemoglobin A2. In all these interactions, one parent shows the sickle cell trait, and the other parent shows the β-thalassemia trait. Hemoglobin C thalassemia is a mild hemolytic disorder associated with splenomegaly.7,9,10 Again, the hemoglobin pattern varies depending on whether the thalassemia gene is the β+ or β0 type. This relatively innocuous condition has been recorded mainly in North Africa, but

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A

B

Asia, is one of the most important hemoglobinopathies in the world population.7,9,10,234–240 As mentioned earlier in “Etiology and Pathogenesis,” hemoglobin E is synthesized at a reduced rate and hence produces the clinical phenotype of a mild form of β-thalassemia. Hence, when hemoglobin E is inherited with β-thalassemia—and most often this is a β0- or severe β+-thalassemia mutation in Southeast Asia and India—a marked deficit of β-chain production results, with the clinical picture of severe β-thalassemia. Hemoglobin E thalassemia shows a remarkable variability in clinical expression,234–238 ranging from a mild form of thalassemia intermedia to a transfusion-dependent condition clinically indistinguishable from homozygous β-thalassemia. The reasons for this variability of expression are not understood, although some of the factors involved are identical to those that modify other forms of β-thalassemia.239,240 In more-severe cases of hemoglobin E thalassemia, severe anemia with growth retardation, leg ulcers, bone deformity, marked tendency to infection, iron loading, and variable splenomegaly and hypersplenism are seen. Large tumor masses composed of extramedullary erythropoietic tissue may cause a variety of compression syndromes, including a clinical picture that closely mimics a cerebral tumor. Another curious picture that seems to be restricted to splenectomized patients is an obliterative occlusion of the pulmonary vasculature that is believed to result from an extremely high platelet count.241 The clinical course and complications in transfusion-dependent patients are similar to those observed in homozygous β-thalassemia. In the milder forms, the main complications are progressive hypersplenism, organ damage as a result of progressive iron loading from an increased rate of absorption, extramedullary erythropoietic tumor masses, bone disease, and infection. The blood picture shows a typical thalassemic pattern. The hemoglobin consists of E, F, and A2. Usually no hemoglobin A is present because the β0-thalassemias are particularly common in the parts of the world where hemoglobin E is found. Newer studies emphasize the complex interactions between genetic factors,239,240 differences in adaptation to anemia, particularly in early life (see “Pathophysiology” above), and the environment, notably proneness to malarial infection, that underlie the widely differing and unstable phenotypes of patients with hemoglobin E β-thalassemia.238,239

β-THALASSEMIA WITH NORMAL HEMOGLOBIN A2 LEVEL

C

Figure 48–21.  Acid elution preparations of blood films from (A)

δβ-thalassemia, (B) hereditary persistence of fetal hemoglobin, and (C) artificial mixture of fetal and adult red cells. The dark cells contain hemoglobin F. Hemoglobin F is resistant to acid elution. it also is found in West Africa. It is characterized by a mild hemolytic anemia and splenomegaly with a blood picture showing the numerous target cells characteristic of all the hemoglobin C disorders. Hemoglobin E thalassemia, which occurs at a high frequency in the eastern half of the Indian subcontinent and throughout Southeast

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Rare forms of β-thalassemia are seen in which heterozygotes have normal hemoglobin A2 levels. Their main clinical importance is that they can be confused with the more severe forms of α-thalassemia in the heterozygous state and therefore may cause difficulties in genetic counseling and prenatal diagnosis. Based on hematologic studies, two main classes of “normal hemoglobin A2 β-thalassemia”—sometimes called types 1 and 2—are seen.242 Type 1 is the “silent” form of β-thalassemia. Type 2 is heterogeneous, with many cases representing the compound heterozygous state for β-thalassemia and δ-thalassemia. “Silent” β-thalassemia7,243 is characterized by no hematologic changes in heterozygotes. Several mild forms of β-thalassemia that underlie this phenotype are described (see Refs. 44 and 45). Although this condition can be partly identified by demonstrating a mild degree of globin-chain imbalance, with α-to-β synthesis ratios of approximately 1.5:1, it can only be diagnosed with certainty by DNA analysis. Compound heterozygotes for this condition and β0-thalassemia have a mild form of β-thalassemia intermedia. Normal hemoglobin A2 β-thalassemia type 2 in heterozygotes is indistinguishable from typical β-thalassemia with elevated hemoglobin A2 levels.242 The homozygous state has not been described. The compound heterozygous state for this gene and for β-thalassemia with

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raised hemoglobin A2 levels is characterized by a clinical picture of severe transfusion-dependent β-thalassemia. Family data obtained in Italy and Sardinia suggest this condition represents the compound heterozygous state for both β-thalassemia and δ-thalassemia.244,245 Most of the δ-thalassemias have been observed trans to β-thalassemia. However, the form of δ-thalassemia resulting from loss of an A in codon 59 occurs on the same chromosome as the hemoglobin Knossos mutation, which is associated with a mild form of β-thalassemia.246 This finding explains the normal level of hemoglobin A2 associated with this condition, which is the most common form of normal hemoglobin A2 β-thalassemia in the Mediterranean region. Several other conditions, mentioned earlier in this chapter in “Etiology and Pathogenesis,” are associated with a phenotype that is indistinguishable from normal A2 β-thalassemia. These conditions include the heterozygous states for the Corfu form of δβ-thalassemia and εγδβ-thalassemia.

OTHER UNUSUAL FORMS OF β-THALASSEMIA The clinical features of the dominant β-thalassemias resemble the features of thalassemia intermedia.7 Moderate anemia and splenomegaly are seen, with a blood picture showing thalassemic red cell changes. The marrow shows erythroid hyperplasia with well-marked inclusion bodies in the red cell precursors. The latter may be seen in the blood after splenectomy. Hemoglobin analysis shows hemoglobins A and A2 are present, and the hemoglobin F level is not usually elevated much higher than that seen in β-thalassemia trait. Hemoglobin A2 levels are always raised. Other unusual varieties of β-thalassemia include those categorized by unusually high hemoglobin F or A2 levels. Most of these conditions result from deletions involving the β-globin gene and its promoter region. For example, the so-called Dutch247 form of β-thalassemia is associated with unusually high hemoglobin F levels in heterozygotes and high hemoglobin A2 levels. Several other conditions of this type, which result from different-size deletions, have been reported (see Ref. 7).

δ0-THALASSEMIA δ0-Thalassemia causes a complete absence of hemoglobin A2 in homozygotes and a reduced hemoglobin A2 level in heterozygotes.248 It is of no clinical significance except for its effect of reducing hemoglobin A2 levels in β-thalassemia heterozygotes.

εγδβ-THALASSEMIA This heterogeneous condition has been observed only in the heterozygous state in a few families.7,108,109 It is characterized by neonatal hemolysis and, in adult life, by the hematologic picture of heterozygous β-thalassemia with normal hemoglobin A2 levels.

α-THALASSEMIA IN ASSOCIATION WITH α- AND β-CHAIN HEMOGLOBIN VARIANTS Several α-globin structural variants are caused by single amino acid substitutions at α-chain loci on chromosomes that carry only a single α-chain gene. Individuals who inherit variants of this type and an α0thalassemia determinant have a form of hemoglobin H disease in which the hemoglobin consists of the α-chain variant hemoglobin and hemoglobin H. Well-documented examples include hemoglobin QH disease (– –/–αQ),249,250 hemoglobin G Philadelphia H disease (– –/–αG),251,252 and hemoglobin Hasharon H disease (– –/–αHash).253 Many examples of the coexistence of the homozygous or heterozygous states for β-chain hemoglobin variants and different α-thalassemia determinants have

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been reported.7,9,10 Particularly well-characterized disorders include the various interactions of α0- and α+-thalassemia with hemoglobin E7,234 and hemoglobin S (Chap. 49).254,255 Carriers for these hemoglobin variants who also have the α0- or α+-thalassemia traits have thalassemic red cell indices and unusually low levels of the abnormal hemoglobin. Individuals with sickle cell anemia who have α-thalassemia show thalassemic red cell changes, more persistent splenomegaly, and lower hemoglobin F values than do patients without the thalassemia genes.

THERAPY, COURSE, AND PROGNOSIS The only forms of treatment available for thalassemic children are regular blood transfusions, iron chelation therapy in an attempt to prevent iron overload, judicious use of splenectomy in cases complicated by hypersplenism, and a good standard of general pediatric care.7,9,256 Marrow transplantation has an important role in selected cases (Chap. 23).

TRANSFUSION Children with β-thalassemia who are maintained at a hemoglobin level of 9.5 to 14.0 g/dL grow and develop normally. They do not develop the distressing skeletal complications of thalassemia.7,256 Maintaining a lower hemoglobin level than this range without any deleterious effects on development and with the added advantage of reducing the level of iron loading may be possible. This regimen maintains a mean pretransfusion level that does not exceed 9.5 g/dL.257 A transfusion program should not be started too early, and it should be initiated only when the hemoglobin level is too low to be compatible with normal development. If transfusion is started too soon, thalassemia intermedia may be missed, and the child may be transfused unnecessarily. Usually blood transfusions are given every 4 weeks on an outpatient basis. To avoid transfusion reactions, washed, filtered, or frozen red cells should be used so that the majority of the white cells and plasma-protein components are removed (Chap. 138).

IRON CHELATION Every child who is maintained on a high-transfusion regimen ultimately develops iron overload and dies of siderosis of the myocardium. Therefore, such children must be started on a program of iron chelation within the first 2 to 3 years of life.256 Deferoxamine (desferrioxamine) was the first chelating agent of proven long-term value for treatment of thalassemia. It is best administered by an 8- to 12-hour overnight pump-driven infusion in the subcutaneous tissues of the anterior abdominal wall.258,259 Chelation therapy should commence by the time the serum ferritin level reaches approximately 1000 mcg/dL. In practice, this level usually is seen after the 12th to 15th transfusion. To prevent toxicity, infants must not be overchelated when the iron burden is still low. The initial dose usually is 20 mg/kg 5 nights per week, with 100 mg of oral vitamin C (200 mg in older children and adults) on the day of infusion, after the infusion has been initiated.259 Some evidence and widespread opinion indicate ascorbate precipitates myocardiopathy in these patients if it is given before deferoxamine infusion is started.260,261 In patients who are heavily iron loaded, particularly those patients with cardiac or endocrine complications, the body iron stores can be effectively lowered by continuous intravenous infusion of deferoxamine at a dose of up to 50 mg/kg body weight. The procedure usually entails insertion of an intravenous delivery system. Extensive experience with the use of deferoxamine and its toxic effects has been reported.189 No serious complications occur other than local erythema and painful subcutaneous nodules at the site of infusions and extremely rare severe allergic reactions. These reactions can

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be controlled, at least in part, by including 5 to 10 mg hydrocortisone in the infusion. Probably of greatest concern is neurosensory toxicity, which has been documented in up to 30 percent of cases. Toxicity causes high-frequency hearing loss that may become symptomatic.262,263 In a few cases, the toxicity did not respond to discontinuation of the drug, and permanent hearing loss resulted. Ocular toxicity has been reported.262 Symptoms include visual failure, night and color blindness, and field loss. Reversal of symptoms after discontinuation of the drug has been reported. Deferoxamine may cause bone changes and growth retardation, sometimes associated with bone pain. Body measurements characteristically show a reduced crown-pubis–to–pubis-heel ratio.264 These changes may be associated with radiologic abnormalities of the vertebral column. These complications can be prevented by exercising extreme care in monitoring patients receiving long-term deferoxamine therapy. Young children or individuals from whom most of the iron has been removed by chelation are at particularly high risk. Formal audiometry and ophthalmologic examinations at 6-month intervals are recommended. Because of the practical difficulties of a nightly subcutaneous infusion of deferoxamine there has been an intensive search for effective oral chelating drugs. Two of these agents are currently available, deferiprone (Ferriprox, L1) and deferasirox. The extensive literature on these agents has been reviewed.265–267 Deferiprone is administered at a dosage of 75 mg/kg in three daily doses. Unfortunately there have been limited numbers of long-term trials comparing its efficacy with deferoxamine, but overall it appears to be less effective than deferoxamine at maintaining safe body iron levels. Its administration is accompanied by a number of complications, the most important of which is neutropenia and, in some cases, agranulocytosis with some fatalities. Hence it is recommended that patients receiving this agent have a weekly white cell count. It also causes arthritis which varies in severity and between different ethnic groups. However, by virtue of its membrane-crossing capacity it has been suggested that it may be more effective in removing cardiac iron (Chap. 43). Unfortunately, to date, all the studies that suggest that it may reduce the frequency of cardiac complications in transfusion-dependent thalassemics have been retrospective and there are no long-term controlled data available. It is currently suggested that it should be used in combination with desferrioxamine, particularly for its cardiac-iron sparing effect; again, long-term prospective data are required. The initial studies of deferasirox were promising266 and suggested that this agent in doses of 5 or 10 mg/kg per day, or higher in those who are heavily iron-loaded, was as effective as desferrioxamine in containing adequate hepatic iron levels. Preliminary clinical studies also showed that this agent may be effective for removing excess cardiac iron. Recent followup data have confirmed these early observations.267 The most frequent adverse reactions to deferasirox included gastrointestinal disturbances, transient rashes, and a nonprogressive increase in serum creatinine. It is still too early to be sure about the overall effectiveness of this agent, however, or to assess its long-term safety. Because of the extremely well-documented data showing longterm survival of patients adequately treated with deferoxamine,268–270 this agent is still recommended as a first-line choice for management of transfusion-dependent thalassemia. However, particularly in view of problems of compliance and the promising trial results of deferasirox, this drug is also being used increasingly as a first line form of treatment. Further long-term follow up data regarding its efficacy are still required however. Careful monitoring of the degree of iron accumulation during chelation therapy is absolutely vital. The simplest approach, particularly in countries where most sophisticated technology is not available, is a regular estimation of the serum ferritin level, which should be

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maintained at less than 1500 mcg/L. The value of hepatic iron concentration assessment was discussed earlier in “Abnormal Iron Metabolism.” Newer noninvasive approaches to assessing body iron burden have been developed. There is now strong evidence that, with adequate calibration, the measurement and mapping of liver iron concentrations using magnetic resonance imaging (MRI) is an extremely effective approach for the regular assessment of the effectiveness of chelation therapy.271 Similarly, there have been advances in the noninvasive estimation of myocardial iron using T2* MRI. Evidence obtained using this approach suggests that there may be a variable correlation between hepatic and cardiac iron concentrations.272 Clearly functional cardiologic studies should be combined with assessment of cardiac iron levels, particularly the ejection fraction, pulmonary artery pressure, and other parameters of cardiac activity. The true value of these new approaches to assessing myocardial iron levels and function still require further study by prospective controlled trials. Increasing evidence indicates children maintained at a high hemoglobin level do not develop hypersplenism.7 However, enlargement of the spleen with increased transfusion requirements occurs commonly in patients maintained at a lower hemoglobin level. Splenectomy should be performed if transfusion requirements increase dramatically or pain develops because of the size of the spleen. Because of the risk of overwhelming pneumococcal infections, splenectomy should not be performed in children younger than age 5 years. Patients should receive a pneumococcal vaccine prior to the procedure. They then should be placed on prophylactic oral penicillin after the operation. Haemophilus influenzae type B and meningococcal vaccines also are recommended. Children with severe thalassemia are still prone to other infections. Presentation with abdominal pain, diarrhea, and vomiting should always suggest an infection with a member of the Yersinia class of bacteria. Empirical treatment should start immediately with either an aminoglycoside or a cotrimoxazole. Transfusion-transmitted virus infection is common in some populations. All chronically transfused patients should be tested annually for hepatitis C, hepatitis B, and HIV. Patients with serologic evidence of chronic active hepatitis should be considered for treatment. As mentioned earlier in “Abnormal Iron Metabolism,” subtle endocrine deficiencies are increasingly recognized, particularly those associated with growth retardation and hypogonadism. These patients require expert endocrinologic assessment and replacement therapy when appropriate.

STEM CELL TRANSPLANTATION By 1997, more than 1000 marrow transplants had been performed at three centers in Italy.273–276 Based on this experience and on later data,7 the prognosis evidently depended on the adequacy of iron chelation up to the time of transplantation. Hence, patients were divided into three classes: class I patients had a history of adequate iron chelation and neither liver fibrosis nor hepatomegaly; class II patients had one or two of these characteristics; and class III patients had all three characteristics. Among children in class I who had undergone transplantation early in the course of the disease, disease-free survival was assessed at 90 to 93 percent at 5 years, with a 4 percent risk of mortality related to the procedure. For class II patients, the intermediate-risk group, the survival and disease-free survival rates were 86 percent and 82 percent, respectively. For class III, the high-risk group, the survival and diseasefree survival rates were 62 percent and 51 percent, respectively. Apart from the immediate complications of severe infection in the posttransplantation period, most of the problems were related to development of acute or chronic graft-versus-host disease. The overall frequency of mild to severe grades ranges from 27 to 30 percent.277 Modification of

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preparative drug regimens has reduced the frequency of drug toxicity. The occurrence of mixed chimerism may be a risk factor for graft-versus-host disease. No case of hematologic malignancy has been observed in the longest followup of patients between 15 and 20 years after transplantation. Recent experience has fully confirmed these pioneering studies278 and suggests that patients without matched donors could benefit from haploidentical mother-to-child transplantation. The current status of blood stem cell therapy is discussed further in Chap. 23.

GENERAL CARE Management of thalassemia requires a high standard of general pediatric care. Infection should be treated early. If the diet is deficient in folate, supplements should be given. Supplementation probably is unnecessary in children maintained on a high-transfusion regimen. Particular attention should be paid to the ear, nose, and throat because of chronic sinus infection and middle-ear diseases resulting from bone deformity of the skull. Similarly, regular dental surveillance is essential because poorly transfused thalassemic children have a variety of deformities of the maxilla and poorly developed teeth. In the later stages of the illness, when iron loading becomes the major feature, endocrine replacement therapy may be necessary. Symptomatic treatment for metabolic bone disease and cardiac failure also may be needed.

THERAPIES OF SPECIAL TYPES OF THALASSEMIA Hemoglobin H disease usually requires no specific therapy, although splenectomy may be of value in cases associated with severe anemia and splenomegaly.7,9,10 Because splenectomy may be followed by a higher incidence of thromboembolic disease than occurs in splenectomized children with β-thalassemia,7 the spleen should be removed only in cases of extreme anemia and splenomegaly. Oxidant drugs should not be given to patients with hemoglobin H disease. The management of symptomatic sickle cell thalassemia follows the lines described for sickle cell anemia (Chap. 49). Thalassemia intermedia presents a particularly complex therapeutic problem. Whether a child with a steady-state hemoglobin level of 6 to 7 g/dL should be transfused is difficult to determine with certainty. Probably the best compromise is to watch such children very closely during the first years of life. If they grow and develop normally and no signs of bone changes are evident, they should be maintained without transfusion. If, however, their early growth pattern is retarded or their activity is limited because of their anemia, they should be placed on a regular transfusion regimen. If hypersplenism plays a role in their anemia as the children grow older, splenectomy should be performed. Because many of these patients have significant iron loading from the gastrointestinal tract, regular estimations of serum iron and ferritin should be obtained and chelation therapy instituted when appropriate.

EXPERIMENTAL APPROACHES TO TREATMENT Two main experimental approaches are being pursued in the search for more effective therapy of the thalassemias: (1) reactivation or augmentation of fetal hemoglobin production and (2) somatic gene therapy. The main rationale for employing agents that have been used in attempts to increase hemoglobin F production is based on the observation that patients recovering from cytotoxic drug therapy or during other periods of erythroid expansion may reactivate hemoglobin F synthesis. In addition, the observation that butyrate analogues might have a stimulating effect on hemoglobin F production has led to a number of studies of their potential for management of thalassemia. A number of clinical trials have been performed.279–282 Agents that have been used

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include various cytotoxic drugs, erythropoietin, and several different butyrate analogues. Overall, these agents, used alone or in combination, have produced some small effects on fetal hemoglobin production, but the results of these trials have been disappointing. Some notable exceptions were seen, however, particularly several cases of homozygosity or compound heterozygosity for hemoglobin Lepore in which use of either a combination of sodium phenylbutyrate and hydroxyurea or hydroxyurea alone produced a spectacular rise in hemoglobin F production. In the case of two homozygotes for hemoglobin Lepore, the necessity for further transfusion was eliminated.283 This finding raises the intriguing possibility that certain mutations, possibly deletions of the β-globin gene cluster, are more susceptible to this type of approach. Recent progress in searching for genetic targets for modifying fetal hemoglobin synthesis has been reviewed recently.42 The other experimental approach involves somatic gene therapy. Currently, the therapy is mainly directed at gene transfer into potential hematopoietic stem cells using retroviral vectors.284 Other approaches also are being taken, including attempts at the restoration of normal splicing in cases of splicing mutations285 and use of trans-splicing ribozymes to correct β-globin gene transcripts.286 However, studies using murine models with recombinant lentiviral vectors suggest that sustained, high-level globin gene expression may be possible, at least in this experimental system.287,288 There continues to be slow progress toward somatic-cell gene therapy as applied to the hemoglobin disorders,289 with at least one apparent success and future plans for several clinical trials.

PROGNOSIS The prognosis for patients with severe forms of β-thalassemia who are adequately treated by transfusion and chelation has improved dramatically over the years. Three large studies investigated the influence of effective long-term desferrioxamine use on the development of cardiac disease.268–270 In one study, patients who had maintained sustained reduction of body iron, as estimated by a serum ferritin level less than 2500 mcg/L over 12 years of followup, had an estimated cardiac diseasefree survival rate of 91 percent. This finding is in contrast to patients in whom most determinations of serum ferritin level exceeded this value, in whom the estimated cardiac disease-free survival rate was less than 20 percent. In a second study, the relationship between survival and totalbody iron burden was measured directly using hepatic storage iron values. Patients who had maintained hepatic iron concentrations of at least 15 mg of iron per gram of liver, dry weight, had a 32 percent probability of survival to age 25 years. No cardiac disease developed in patients who maintained hepatic iron levels below this threshold. These and other studies provide unequivocal evidence that adequate transfusion and chelation are associated with longevity and good quality of life. On the other hand, poor compliance or unavailability of chelating agents still are associated with a poor prospect of survival much beyond the second decade.

PREVENTION In parts of the world where the incidence of thalassemia is high, the disease places an immense economic burden on society. For example, if all the thalassemic children born in Cyprus were treated by regular blood transfusions and iron-chelating therapy, it was estimated that within 15 years the total medical budget of the island would be required to treat this single disease.290 Clearly, this approach was not feasible, so considerable effort was directed toward developing programs for prevention of the different forms of thalassemia. The goal of prevention can be achieved in two ways. The first is prospective genetic counseling, that is, screening total populations while

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the children still are at school and warning carriers about the potential risks of marriage to another carrier. Few data are available about the value of programs of this type; a pilot study in Greece was unsuccessful.291 Because it is believed this approach will not be successful in many populations, considerable effort has been directed toward developing prenatal diagnosis programs. Prenatal diagnosis for prevention of thalassemia entails screening mothers at the first prenatal visit, screening the father in cases in which the mother is a thalassemia carrier, and offering the couple the possibility of prenatal diagnosis and termination of pregnancy if both mother and father are carriers of a gene for a severe form of thalassemia. Currently, these programs are devoted mainly to prenatal diagnosis of the severe transfusion-dependent forms of homozygous β+ or β0-thalassemia. Considerable experience has also been gained in prenatal diagnosis of mothers at risk for having a fetus with the hemoglobin Bart hydrops syndrome, considering the distress caused by a long and difficult pregnancy and the obstetric problems resulting from the birth of a hydropic infant with a massive placenta. The first efforts at prenatal detection of β-thalassemia used fetal blood sampling and globin-chain synthesis analysis carried out at approximately week 18 of pregnancy. Despite the technical difficulties involved, the method was applied successfully in many countries and resulted in a reduced birth rate of infants with β-thalassemia.292 The technique is associated with a low maternal morbidity rate, a fetal mortality rate of approximately 3 to 4 percent, and an error rate of 1 to 2 percent. Its main disadvantage is that it must be carried out relatively late in pregnancy. For this reason, efforts turned to first trimester prenatal diagnosis. DNA technology has enabled diagnosis of important hemoglobin disorders in utero by fetal DNA analysis. Although analysis can be carried out on DNA derived from amniotic fluid, the approach has drawbacks because, again, it must be done relatively late in pregnancy, and often amniotic fluid cells must be grown in culture to obtain a sufficient amount of DNA.293 However, DNA can be obtained as early as week 9 of pregnancy by chorionic villus sampling. Although the safety of this technique remains to be fully evaluated and limb reduction deformities may occur when the procedure is carried out very early in pregnancy (9 or 10 weeks), chorionic villus sampling has become the major method for prenatal diagnosis of the thalassemias based on subsequent experience with the technique.7,293–297 Remarkable advances in DNA technology have provided a variety of methods for the direct identification of mutations in fetal DNA77 Even in families with extremely rare mutations, rapid DNA sequencing technology allows a diagnosis to be made very rapidly. The error rate using these different approaches varies, mainly depending on the experience of the particular laboratory; low rates, less than 1 percent, are reported from most centers. Potential sources of error include maternal contamination of fetal DNA and nonpaternity. The application of this new technology has caused a major reduction in the birth rate of infants with thalassemia throughout the Mediterranean region and the Middle East, and in parts of the Indian subcontinent and Southeast Asia. Several approaches continue to be explored in an attempt to avoid the use of invasive procedures like chorion villous sampling. A variety of methods are being used to harvest fetal DNA from fetal cells in maternal blood or from maternal plasma298,299 and there are increasing numbers of attempts at preimplantation diagnosis of thalassemias.300,301 There is every expectation that some of these approaches will reach the clinic in the near future.302

THALASSEMIA AS A GLOBAL HEALTH PROBLEM The remarkable advances in the diagnosis, prevention, and treatment of the thalassemias described in this chapter are only relevant to the richer

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countries of the world. In many developing countries in which there is a very high frequency of thalassemia, there are very limited facilities for their diagnosis and management. Because many of these countries are going through the epidemiologic transition, which involves improvements in nutrition, cleaner water supplies, and better public health services, babies with serious forms of thalassemia who previously would have died of infection or profound anemia are now surviving to present for treatment. Approaches to the better control and management of the thalassemias in developing countries have been reviewed.303,304 They include the development of partnerships between centers in the developed and developing countries for training workers in this field, and, once these partnerships are developed, for the further evolution of partnerships between those developing countries where there is knowledge and expertise of the field with those where no knowledge or facilities exist. Without organizations along these lines, the thalassemias will continue to cause the premature death of hundreds of thousands of infants worldwide.

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Cao A, Galanello R, Rosatelli MC: Prenatal diagnosis and screening of the haemoglobinopathies. Clin Haematol 11:215, 1998. 297. Modell B, Petrou M, Layton M, et al: Audit of prenatal diagnosis for haemoglobin disorders in the United Kingdom: The first 20 years. BMJ 315:779, 1997. 298. Cheung MC, Goldberg JD, Kan YW: Prenatal diagnosis of sickle cell anemia and thalassemia by analysis of fetal cells in maternal blood. Nat Genet 14:264, 1996. 299. Hung EC, Chiu RW, Lo YM: Detection of circulating fetal nucleic acids: A review of methods and applications. J Clin Pathol 62:308, 2009. 300. Kuliev A, Rechitsky S, Verlinsky O, et al: Preimplantation diagnosis of thalassemias. J Assist Reprod Genet 15:219, 1998. 301. Kuliev A, Rechitsky S, Verlinsky O, et al: Birth of healthy children after preimplantation diagnosis of thalassemia. J Assist Reprod Genet 16:201, 1999. 302. Cao A, Kan YW: The prevention of thalassemia. Cold Spring Harb Perspect Med 3:a011775, 2013. 303. Weatherall DJ, Akinyanju O, Fucharoen S, et al: Inherited disorders of hemoglobin, in Disease Control Priorities in Developing Countries, 2nd ed, edited by Jamison DT, Breman JG, Measham AR, Alleyne G, Claeson M, Evans DB, Jha P, Mills A, Musgrove P, p 663. Oxford University Press and the World Bank, New York, 2006. 304. A database of human hemoglobin variants and thalassemias. http://globin.bx.psu.edu/ hbvar/

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CHAPTER 49

DISORDERS OF HEMOGLOBIN STRUCTURE: SICKLE CELL ANEMIA AND RELATED ABNORMALITIES

Kavita Natrajan and Abdullah Kutlar

SUMMARY Hemoglobinopathies are the most common inherited red cell disorders worldwide. Among these disorders, sickle cell syndromes and thalassemias constitute a major public health problem. A glutamic acid to valine substitution at the sixth amino acid of the β-globin chain of human hemoglobin (HbA) results in formation of sickle hemoglobin (HbS). Sickle cell disease results from homozygosity for this mutation, or from a compound heterozygosity for sickle hemoglobin and β-thalassemia or another β-globin variant such as HbC, HbD, HbE, or HbOArab. The sickle mutation renders the hemoglobin molecule insoluble upon deoxygenation; thus red blood cells containing deoxy HbS polymer are rigid and have impaired rheologic properties. The downstream effects of the sickling process include: membrane changes leading to potassium loss and cellular dehydration, interaction of sickle hemoglobin with microvascular endothelium, neutrophils, and monocytes, hemolysis, nitric oxide depletion, release of inflammatory proteins and activation of coagulation. These processes lead to a hemolytic anemia, an inflammatory state, painful vasoocclusive episodes, and damage to multiple organ systems with a resultant shortened life expectancy.

There is considerable heterogeneity in the severity of the disease; the best known modifier of the disease is an elevated level of fetal hemoglobin (HbF), which exerts a potent antisickling effect. Concomitant α-thalassemia is also a modifier, which leads to a decrease in hemolysis. There is an interest in nonglobin genetic modifiers of sickle cell disease. Over the past 3 decades, advances in supportive care and implementation of disease-modifying therapies, such as anti–γ to β-globin switching therapies, which result in increased HbF and less HbS synthesis, and have led to an increase in life expectancy. Hydroxyurea has emerged as an effective disease-modifying agent that has been approved by the FDA for use in adults with sickle cell disease. Although its main mechanism of action is to enhance HbF production, other effects such as a decrease in neutrophils, platelets, and decreased expression of adhesion molecules contribute to its efficacy. Novel antiswitching agents, most notably, DNA methyltransferase 1 inhibitors (5′-azacytidine and decitabine) and histone deacetylase inhibitors (butyrate derivatives and others) are now in clinical trials. Evolving therapies include antiadhesive therapies to prevent interaction of sickle cells with microvascular endothelium, antiinflammatory approaches, and modulation of hemoglobin–oxygen affinity to prevent sickling. To date, the only curative therapy remains allogeneic hematopoietic stem cell transplantation.   Sickle trait, the heterozygous state for sickle hemoglobin, affects approximately 8 percent of Americans of African descent, and with rare exceptions is asymptomatic. HbC is associated with target cells and spherocytes in the blood film and splenomegaly. HbD disease is essentially asymptomatic. HbE is very common in Southeast Asia, and because of large population movements from this area, it has become a prevalent hemoglobinopathy in other regions of the world. HbE is a thalassemic variant and its coinheritance with β0-thalassemia mutations can result in severe transfusion-dependent thalassemia major. Unstable hemoglobin variants appear as rare, sporadic cases and are characterized by a Heinz body hemolytic anemia. Variants that alter the oxygen affinity of the Hb molecule lead to erythrocytosis (high oxygen affinity variants) or anemia (low oxygen affinity variants) and are rare causes of these syndromes.

 HE STRUCTURE AND FUNCTION OF T NORMAL HEMOGLOBIN Acronyms and Abbreviations: ACS, acute chest syndrome; ADMA, asymmetric dimethylarginine; AHSCT, allogeneic hematopoietic stem cell transplantation; BMP, bone morphogenic protein; 2,3-BPG, 2,3-bisphosphoglycerate; CO2, carbon dioxide; CSSCD, Cooperative Study of Sickle Cell Disease; eNOS, endothelial nitric oxide synthase; Hb, hemoglobin; HbAS, sickle cell trait; HbF, fetal hemoglobin; HbS, sickle hemoglobin; HbSC, sickle cell–HbC disease; HIF, hypoxia-inducible factor; HLA human leukocyte antigen; HPLC, high-performance liquid chromatography; IL, interleukin; iNKT cells, invariant natural killer T cells; K+, potassium; LDH, lactate dehydrogenase; MCHC, mean cell hemoglobin concentration; MCV, mean corpuscular volume; MPs, microparticles; MRI, magnetic resonance imaging; NO, nitric oxide; NT-pro-BNP, N-terminal pro–brain natriuretic peptide; O2, oxygen; P50, point at which hemoglobin is one-half saturated with oxygen; PCV7, pneumococcal polyvalent conjugate 7; PH, pulmonary hypertension; PIGF, placenta growth factor; PO2, partial pressure of oxygen; R state, relaxed oxy; SCD, sickle cell disease; SCT, stem cell transplantation; sPLA2, secretory phospholipase A2; STOP, Stroke Prevention Trial in Sickle Cell Disease; T state, tense, deoxy; TCD, transcranial Doppler; TF, tissue factor; TGF-β, transforming growth factor-beta; TNF-α, tumor necrosis factor-alpha; UDP, uridine diphosphate; UGT1A1, UDP glucuronosyltransferase 1 family; VOE, vasoocclusive episode; VTE, venous thromboembolism.

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The red protein hemoglobin (Hb) serves to transport oxygen from the lungs to the tissues and carbon dioxide (CO2) from the tissues to the lungs. Hb also binds the physiologically important nitric oxide (NO) molecule. The protein has evolved to perform its gas transport functions in a highly efficient manner. The oxygen affinity of Hb permits nearly complete saturation with oxygen in the lungs, as well as efficient oxygen unloading in the tissues because of its sigmoid oxygen dissociation curve. This curve results from the fact that Hb is a four-subunit, allosteric molecule; its conformation, and hence the oxygen affinity, changes as each successive molecule of oxygen is bound. Hb also plays an important role in acid–base balance: deoxyhemoglobin binds protons and oxyhemoglobin releases protons. Regulation of the oxygen dissociation curve to meet the needs of the body is remarkable. Hypoxic tissues become acidotic acutely, and the protons released produce a shift in the oxygen dissociation curve that enables more oxygen to be delivered to the tissue. However, longer-term acidosis or alkalosis (as occurs at high altitudes) is counteracted by modulation of red cell 2,3-bisphosphoglycerate (2,3-BPG), serving to decrease hemoglobin–oxygen affinity (Chap. 47). Normal mammalian Hbs contain two pairs of related polypeptide chains: one chain of each pair is α or α-like and the other is non-α (β, γ,

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or δ). The α-chains of all human Hbs encountered after early embryogenesis are the same. The non-α chains include the β-chain of normal adult Hb (α2β2), the γ-chain of fetal Hb (α2γ2), and the δ-chain of the minor adult Hb (HbA2 [α2δ2]), which accounts for 2.5 percent of the Hb of normal adults. Chapter 48 discusses the regulation of production of the globin chains. Certain residues in the amino acid sequence of each polypeptide chain appear to be critical to stability and function. Such residues are usually the same (invariant) in α or β chains. The NH2-terminal valines of the β chains are important in 2,3-BPG interactions. The C-terminal residues are important in the salt bridges that characterize the unliganded

molecules. Areas of contact between chains and between heme and globin tend to contain invariant residues. The non-α (β, γ, δ, or ε) chains are all 146 amino acids in length. The γ-chain of fetal Hb (HbF) differs from the β-chain by 39 residues. The γ genes are duplicated: one codes for glycine (Gγ) and the other for alanine (Aγ)7 at residue 136, giving rise to two kinds of γ chains. In addition, a common polymorphism, the substitution of threonine for isoleucine, is frequently found at residue 75 of the Aγ-chain. Approximately 75 percent of the amino acids in α and β chains are in a helical arrangement. All Hbs studied have a similar helical content (Fig. 49–1A). Eight helical areas, lettered A to H, occur in the β chains.

Figure 49–1.  A. Representation of the structure of β chains. Arrows indicate sites of substitutions in a number of unstable hemoglobins. B. The hemoglobin molecule, as deduced from x-ray diffraction studies, shown from above. The molecule is composed of four subunits: two identical α chains (light blocks) and two identical β chains (dark blocks). 2,3-BPG binds to the two β chains in the deoxyhemoglobin molecule. C. Schematic of rotation of α2β2 dimer relative to α1β1 in quaternary structure change from deoxyhemoglobin (solid lines) to carboxyhemoglobin (dashed lines). (Modified from Baldwin J, Chothia C: Haemoglobin: The structural changes related to ligand binding and its allosteric mechanism. J Mol Biol 129(2):175–220, 1979.)

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CH2 CH H 3C

CH

HC N

2

E10 (61) Val

E11 (62)

Leu FG3 (91) 4

N

HOOC · CH2

C H

Lys 1

Cδ

CH

H 3C CH2

CH2

Ce His F8 (87) Ne

Fe

protoporphyrin IX). B. Heme group and its environment in the unliganded α-chain. Only selected side chains are shown; the heme 4-propionate is omitted. (Reproduced from Gelin BR, Lee AW, Karplus M: Hemoglobin tertiary structural change on ligand binding. J Mol Biol 25;171(4):489–559, 1983.)

Leu H19 Leu F7 (136) (86)

N

N

Figure 49–2.  A. Structure of heme (ferro-

Leu F4 (83)

CH3

H C

Val FG5 (93)

CH3

3

761

His E7 (58) Leu G8 (101) Phe CD4 (46)

CH2

Tyr C7 (42)

CH2 · COOH

y x z

A

B

Hb nomenclature specifies that amino acids within helices are designated by the amino acid number and the helix letter, whereas amino acids between helices bear the number of the amino acid and the letters of the two helices. Thus, residue EF3 is the third residue of the segment connecting the E and F helices, whereas residue F8 is the eighth residue of the F helix. Alignment according to helical designation makes homology evident: Residue F8 is the proximal heme linked histidine, and the histidine on the distal side of the heme is E7. Figure 49–1B show the tertiary structure of the α and β chains. The prosthetic group of Hb is heme (ferroprotoporphyrin IX); Fig. 49–2A shows its structure. The heme group is located in a crevice between the E and F helices in each chain (Fig. 49–2B). The highly polar propionate side chains of the heme are on the surface of the molecule and are ionized at physiologic pH. The rest of the heme is inside the molecule, surrounded by nonpolar residues except for two histidines. The iron atom is linked by a coordinate bond to the imidazole nitrogen (N) of histidine F8. The E7 distal histidine, on the other side of the heme plane, is not bonded to the iron atom, but is very close to the ligand-binding site. The sigmoid oxygen dissociation curve is a function of the change of the conformation of the molecule from the liganded to the unliganded state (Table 49–1). In the deoxy state, the Hb tetramer is held together by intersubunit salt bonds (Fig. 49–3) and intersubunit hydrophobic contacts (see Fig. 49–1B), in addition to a certain number of hydrogen bonds. In deoxyhemoglobin, 2,3-BPG is situated in the central cavity between the two β chains (see Fig. 49–1B). The change in conformation of the Hb molecule is brought about by a complex, coordinated series of changes in the structure of the molecule as heme binds oxygen. The oxygen dissociation curve can be linearized by a transformation known as the Hill plot:

where K is an empiric overall constant without physicochemical basis. The slope n is taken as a convenient measure of cooperativity. Values of n in noninteracting Hbs that exhibit hyperbolic, not sigmoid, oxygen dissociation curves (e.g., myoglobin) are approximately 1. In a normal tetrameric Hb with four oxygen-reactive sites, the maximum value for n is 4.0; however, n values of 2.7 to 3.0 are found in normal Hb. The point at which the Hb is one-half saturated with oxygen (P50) is the usual measurement of oxygen affinity. It depends upon pH (the Bohr effect), temperature, and 2,3-BPG concentration. In common practice, P50 is standardized at 37°C and pH 7.20. P50 of freshly drawn blood is approximately 26.7 torr under standard conditions, but the partial pressure of oxygen (PO2) of Hb from which 2,3-BPG has been removed is only approximately 13 torr. Although fetal and newborn red cells have 2,3-BPG levels similar to those of adults, their oxygen dissociation curve is left shifted (increased oxygen affinity) with a P50 of approximately 23 torr because HbF does not react as strongly with 2,3-BPG as does HbA.

log[y/(1 – y)] = log K+n log PO2

TABLE 49–1.  Nomenclature of Hemoglobin Quaternary Structures Liganded (Oxygen Bound)

Unliganded (Reduced)

Oxy

Deoxy

R-state

T-state

Relaxed

Tense

High affinity

Low affinity

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Figure 49–3.  Salt bridges in deoxyhemoglobin (* = ionizable group less protonated at pH 9.0 than at pH 7.0). These groups account for 60% of the alkaline Bohr effect. The remainder is due to αH5 His. (Data from Perutz MF, Wilkinson AJ, Paoli M, et al: The stereochemical mechanism of the cooperative effects in hemoglobin revisited, Annu Rev Biophys Biomol Struct 1998;27:1-34.)

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 OMENCLATURE OF ABNORMAL N HEMOGLOBINS Following the molecular characterization of HbS by Ingram and colleagues in 1956, there has been a rapid and exponential increase in the number of variant or “abnormal” Hbs.1 This number now exceeds 1000. A detailed description of variant Hbs, their chemical and functional properties, and population distribution can be found on the Globin Gene Server website (http://globin.cse.psu.edu/). Initially, newly described variants were designated by letters of the alphabet (e.g., HbC, HbD, HbE, HbJ). When the letters of the alphabet were exhausted, the practice of naming the variant Hbs after the geographic location where they were first described was adapted (e.g., HbKoln, HbZurich). Variants with electrophoretic or functional properties similar to previously described abnormal Hbs were designated with the letter and the geographic location (e.g., HbDPunjab, HbESaskatoon, HbMHyde Park). Some alphabetic designations were also used to indicate electrophoretic properties of certain variants; for example, there are a number of HbDs (DPunjab, DIran, DIbadan). All of these variants share the electrophoretic properties of HbS-like mobility on alkaline (cellulose acetate) electrophoresis, whereas they move with HbA at acidic pH (citrate agar electrophoresis). Similarly, HbEs have HbC-like mobility on alkaline electrophoresis and move with HbA on citrate agar electrophoresis. The vast majority of Hb variants arise as a result of single nucleotide mutations, leading to an amino acid change in either α-, β-, δ-, or γ-globin subunits of the Hb tetramer resulting in variants of HbA (α or β), HbA2 (δ), or HbF (γ). Other mechanisms include small deletions or insertions, elongated chains, and fusions (for a detailed description of Hb variants and associated clinical syndromes, see “Other Abnormal Hemoglobins” below. The coinheritance of HbS with some other variant Hbs or β-thalassemia mutations results in a number of sickling syndromes. In the United States, the most common sickling disorder is homozygous HbS (HbSS, sickle cell anemia), which is now commonly referred to as sickle cell disease (SCD). This is followed by sickle cell-HbC disease (HbSC), sickle cell–β+-thalassemia (HbS–β+-thalassemia), and sickle cell–β0-thalassemia (HbS–β0-thalassemia). Other rarer forms include HbSDPunjab, HbSOArab, HbSLepore, and HbSE diseases. Coinheritance of a large number of β-chain variants with HbS does not result in a symptomatic sickling disorder; rather, they are clinically and hematologically indistinguishable from sickle cell trait (HbAS). HbC is found in 17 to 28 percent of West Africans, particularly east of the Niger River in the vicinity of North Ghana. The selective factors that account for this high prevalence are unknown at present, but HbC probably confers some resistance to infection with malaria. The prevalence of HbC among Americans of African descent is 2 to 3 percent. Sporadic cases also have been reported in other populations, including Italians and Afrikaners. HbDPunjab, which is now recognized to be identical with HbDLos Angeles because both have the structure α2β2121 Glu→Gln, also interacts with HbS in forming aggregates in the deoxy conformation. HbD has been found in many parts of the world, including Africa, northern Europe, and India. HbE is so prevalent that it may be the most common abnormal Hb or second in prevalence only to HbS. HbE is found principally in Burma, Thailand, Laos, Cambodia, Malaysia, and Indonesia. In some areas, HbE is found with a carrier rate of 30 percent. On the other hand, it is not prevalent among Chinese. Studies of restriction length polymorphisms in the β-globin cluster indicate the HbE mutation has arisen several times independently. It, too, probably confers some resistance to infection with malaria.

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SICKLE CELL DISEASE DEFINITION AND HISTORY The first case of SCD, reported in 1910, was that of a dental student from Grenada, Walter Clement-Noel, studying in Chicago. Dr. James Herrick and his intern, Dr. Ernest Irons, were in charge of Mr. Noel’s care between 1904 and 1907, during which time he had several bouts of fever and cough and a history of leg ulcers, jaundice, and exercise intolerance. Herrick and Irons made astute clinical observations and prepared blood films and photomicrographs of nucleated red blood cells and of red cells having a “slender sickle shape” (Fig. 49–4).2 During the next decade, two more cases of this unusual anemia were reported. In 1915, Cook and Meyer raised the question of a genetic basis for the disorder based on the family history of the third reported case. In 1917, Victor Emmel used in vitro culture to show that sickle red cells represented a physical alteration of morphologically normally appearing red cells and were not released from the marrow as sickle cells.3 He also demonstrated that morphologically normal red cells of the father of a patient became sickle shaped after in vitro culture. Vernon Mason, who reported the fourth case in 1922, coined the term sickle cell anemia after observing the similarities between all the cases reported up to that time. In 1923, Sydenstricker and Huck noted “latent-sicklers” among relatives of the diagnosed patients, confirming and expanding on Emmel’s finding. In 1927, Hahn and Gillespie showed that sickling was related to low oxygen tension and low pH. In 1933, Diggs distinguished the difference of symptomatic cases called sickle cell anemia, from asymptomatic cases that were termed sickle cell trait, and he found that approximately 8 percent of Americans of African descent had the sickle cell trait.4

Figure 49–4.  Peculiar elongated and sickle-shaped red cells from the

first report of sickle cell anemia with depiction of sickle cells. (Reproduced with permission from Herrick JB: Peculiar elongated and sickle-shaped red corpuscles in a case of severe anemia. Arch Intern Med 6:517, 1910.)

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Irving Sherman, while a medical student at Johns Hopkins, showed that sickled red cells were birefringent under a polarizing microscope and that this finding was reversible with oxygenation of the cells. This observation ultimately led Linus Pauling to study sickle Hb after being advised of this property of sickle cell by William Castle, a noted research hematologist. Indeed, in 1949, Pauling and his colleagues demonstrated electrophoretic differences between Hbs from normal, sickle cell trait, and sickle cell anemia subjects and hypothesized that there must be chemical differences, thus establishing sickle cell anemia as the first molecular disease described. In the late 1950s, Hunt and Ingram sequenced the globin peptide and linked the abnormality to a change in the amino acid composition of the β-globin chain (replacement of glutamic acid by valine at residue 6). In 1977, Marotta and coworkers showed that the corresponding change in codon 6 of the β-globin gene was GAG→GTG. The discovery of a variant fragment in HbS versus HbA during restriction endonuclease mapping of amniotic fluid cells by Y. W. Kan paved the way for antenatal diagnosis of SCD and opened the way for modern genetics using recombinant DNA technology.5 The history of sickle cell anemia serves as an inspiring reminder of the power of clinical and laboratory observations, and in an era of mechanistic basic science research, serves to highlight the importance of bedside to bench and bench to bedside research integration.6–9

EPIDEMIOLOGY The observation that sickle cell trait may have a survival advantage against some environmental factors was first suggested by Dr. Alan Raper in East Africa in 1949. Drs. Mackey and Vivarelli suggested that the environmental influence might be malaria. It was subsequently noted that blood from sickle cell trait persons contained less malarial parasites and that the sickle trait conferred some protection against malaria in early childhood. Data suggest that sickle trait is maximally protective against severe malaria as opposed to asymptomatic parasitemia or mild disease.10 The mechanism of such a protection has been the matter of much debate. Plausible mechanisms include selective sickling of parasitized red blood cells, resulting in more effective removal by the monocyte-macrophage system, and inhibitory effect on parasite growth by increased red cell potassium loss, decreased red cell pH, and increased endothelial adherence of parasitized sickle red cells. Thus, the prevalence of sickle cell anemia closely mirrors the worldwide distribution of falciparum malaria; however, as a result of migration of peoples to the industrialized Western countries, SCD has become more prevalent in areas where malaria is not endemic. The World Health Organization estimated in 2006 that 5 percent of the world population carries a gene for a hemoglobinopathy. Sickle cell anemia is highly prevalent in sub-Saharan and equatorial Africa with lesser but significant prevalence in the Middle East, India, and the Mediterranean region. Incidence of SCD in sub-Saharan African countries ranges between 1 and 2 percent, which translates into approximately 500,000 cases per year. In the Jamaican cohort study, newborn screening in 100,000 consecutive vaginal deliveries resulted in the finding of sickle cell trait in 10 percent of newborns.11 In the United States, the Centers for Disease Control and Prevention estimates that sickle cell anemia is present in 1 in 500 livebirths among Americans of African descent; 1 in 12 American of African descent have the trait, and approximately 100,000 Americans largely of African descent live with the disease. In Americans of Hispanic descent, the rate of SCD is 1 in 36,000 livebirths. Accurate population statistics of SCD are difficult to obtain in the United States because of a lack of standardized data collection and central reporting.12 As of 2002, in the United States, more than one billion dollars are spent per year on hospitalizations for SCD.13 Data from a single

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state Medicaid program estimated a lifetime cost of care of $500,000 per patient with SCD. In this patient population, cost increased with increasing age, including cost of non-SCD health issues. The majority of the costs were for inpatient healthcare utilization.14 Previously, speculation existed as to whether the sickle mutation arose once and gained worldwide distribution or whether the mutation had arisen independently in different regions of the world. The nonrandom association of restriction endonuclease polymorphisms in the β-globin cluster define the β-globin haplotype. The β-globin gene cluster yields five distinct haplotypes associated with sickle cell mutations (Chap. 9).15–17 Four of the five patterns occur in Africa and are designated as the Senegal, Benin, Bantu, and Cameroon haplotypes, whereas the fifth arose on the Indian subcontinent.18 These findings indicate that the sickle mutation arose independently at five different times.

PATHOPHYSIOLOGY The sine qua non of sickle cell anemia is a Glu→Val substitution in the sixth amino acid of the β-globin gene. However, the pathophysiologic processes that result in the clinical phenotype extend beyond the red cell (Fig. 49–5). There is marked clinical heterogeneity from one patient to another and in the same patient over time. The heterogeneity for the same genotypic abnormality therefore implies that a multitude of other factors must contribute to the pathology of sickle cell anemia. The pathology is now far removed from the simplistic theory of hypoxia-induced microvascular occlusion. Sickle cell anemia is a chronic inflammatory state punctuated by acute increase in inflammation wherein the endothelium, neutrophils and monocytes, platelets, coagulation pathways, several plasma proteins, adhesion molecules, and derangements in NO metabolism interact in concert with the abnormality in Hb polymerization described several decades ago (Fig. 49–6). Abnormal adenosine signaling and activation of invariant natural killer T (iNKT) cells have been implicated in disease pathophysiology. Added to that are the complex differences in tissue-specific vascular beds and differences in various parts of the vasculature in the same organ. Also, variation in several genes other than the β-globin gene that modify the milieu in which organ damage occurs may play a role. The pathophysiology of sickle cell anemia is described in separate sections; however, because no single, dominant pathway explains the multitude of manifestations, no single therapeutic modality serves to abrogate all of the pathology. Most experiments are in isolation in animal models or relatively simplistic experimental conditions with few in vivo studies in humans and thus do not replicate the complexity of this disorder.

Hemoglobin Polymerization

Aggregation of deoxy HbS molecules into polymers occurs when aggregates reach a thermodynamically critical size. This process is termed homogenous nucleation, and the smallest aggregate formed that favors polymer growth is called the critical nucleus.19–24 Addition of subsequent deoxy HbS molecules to already formed polymers is termed heterogenous nucleation, which results in polymer branching. Polymer growth is, therefore, an exponential process wherein there is a delay time between presence of deoxy HbS molecules and polymer formation. This delay time is inversely proportional to the concentration of HbS molecules. Polymer formation alters the rheologic properties of the red cell. The quaternary structure of oxy HbS cannot maintain axial and lateral hydrophobic contacts unlike that in the deoxygenated state, thus explaining the unsickling phenomenon upon reoxygenation.25–28 The sickling process that is initially reversible with oxygenation of deoxy HbS eventually leads to the formation of sickle-shaped red cells that

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K+ loss RBC dehydration

Hb Polymerization Sickled RBCs

Vasoocclusion Ischemia reperfusion injury

Membrane damage Lipid peroxidation ROS, XO

PS exposure Activation of coagulation TF Thrombin Protein C & S Platelet activation

Adhesion to WBCs, endothelium

Hemolysis NO scavenging Endothetial dysfunction

Increased inflammation NFκ B Activation Adhesive proteins Inflammatory cytokines Activation of WBCs, platelets

Figure 49–5.  Schema summarizing the pathophysiology of sickle cell anemia. K+, potassium; NO, nitric oxide; PS, phosphatidylserine; RBC, red blood cell; ROS, reactive oxygen species; TF, tissue factor; WBC, white blood cell; XO, xanthine oxidase.

fail to return to their normal discoid shape with oxygenation because of membrane damage imparted by repeated cycles of sickling and unsickling in the circulation. These cells are then termed irreversibly sickled cells. The rate and extent of polymerization is dependent on several factors, including intracellular Hb concentration, presence of Hbs other than HbS, blood oxygen saturation, pH, temperature, and 2,3-BPG levels.29 Microvascular occlusion by sickle red cells containing polymers is favored by prolonged transit times through the microcirculation, rapid deoxygenation and increased numbers of dense sickle red cells that contain polymers even at oxygen saturation levels found in the arterial circulation.29–32 Arguments against HbS polymerization as the major determinant of sickle cell pathophysiology include lack of clinically significant events despite constant sickling of red cells, the association of neutrophilia with vasoocclusive episodes (VOEs), and clinical features that imply macrovascular rather than microvascular perturbation, for example, large-vessel stroke.33

Cellular Dehydration

Membrane injury in HbSS red cells results in impaired cation homeostasis with decreased ability to maintain intracellular potassium concentrations. The calcium-activated potassium (K+) channel (Gardos channel), potassium-chloride cotransport channel, and a sickling-induced nonselective cation leak pathway have been implicated in sickle red cell dehydration. The net result is loss of intracellular potassium and water resulting in cellular dehydration.34–39 This change effectively increases the red cell Hb concentration, favoring sickling.

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Hemolysis and Nitric Oxide Scavenging

NO is a key component of the vascular endothelium that has vasodilatory, antiinflammatory, and antiplatelet properties.40 NO is a soluble gas synthesized from L-arginine by endothelial nitric oxide synthase (eNOS).41 Red cell L-arginase released as a consequence of sickle red cell hemolysis converts arginine to ornithine, thereby limiting L-arginine availability for NO synthesis. Decreased NO production because of elevated levels of endogenous nitric oxide synthase (NOS) inhibitors, especially asymmetric dimethylarginine (ADMA) and reduced L-arginine, have been documented in SCD especially during VOE.42–46 Reduced plasma arginine levels and elevated ADMA levels also result in NOS coupling causing production of reactive oxygen species rather than NO.47,48 Chronic hemolysis with release of plasma free Hb results in scavenging of NO with consequent endothelial dysfunction, which may favor sickle cell adherence.49,50

Abnormal Cell Adhesiveness

Seminal work by several groups showed that sickle red cells adhere to stimulated endothelium unlike their normal counterparts.51,52 Newly released red cells, called reticulocytes, express high levels of adhesion molecules, integrin α4β1, and CD36, and are more adherent than dense sickle red cells.53,54 Increased endothelial reticulocyte adhesion as compared to dense red cell adhesion is thought to be secondary to deformable red cells adhering to the endothelium behind which the dense red cells are trapped, leading to microvascular occlusion.29 Other molecules involved in sickle red cell-endothelium interactions include vascular

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adhesion molecules are upregulated, including VCAM, selectins, integrins, the acute phase reactants C-reactive protein, secretory phospholipase A2 (sPLA2), and coagulation factors are activated.64–76 Placenta growth factor (PIGF) released from erythrocytes activates monocytes to produce inflammatory cytokines and upregulates endothelin-1 signaling via the endothelin B receptor. Endothelin-1 is a potent vasoconstrictor and upregulation is associated with adverse outcomes in SCD. Placental growth factor has independently been shown to be correlated with disease severity as well.77,78 Hemin has been demonstrated to activate PIGF in mice via the erythroid Kruppel-like factor; consequently, PIGF may play an important role in the pathophysiology of iron overload as well.79 It is an open question whether inflammation is caused by abnormally adhesive red cells to the vascular endothelium or whether inflammation causes abnormal red cell adhesiveness. It is likely both occur, given that red cell adhesiveness incites endothelial activity, and infection-induced inflammation precipitates clinically significant vascular events in patients. The vascular beds in sickle cell anemia display changes akin to atherosclerotic vascular disease: large vessel intimal hyperplasia and smooth muscle proliferation.80,81 However, the characteristic lipid laden plaques of atherosclerotic vascular disease are not present.64

Ischemia–Reperfusion Injury

Akin to other disease states, such as myocardial infarction, resolution of vasoocclusion results in reperfusion injury characterized by increased oxygen free radical formation via activation of xanthine oxidase, generation of oxidant stress, lipid peroxidation, upregulation of cellular adhesion molecules, and nuclear factor-κB, a key player in the inflammatory process.64,82,83 iNKT cells propagate the inflammatory cascade in ischemia reperfusion injury and are increased and activated in patients with SCD. Agonists to adenosine 2A receptor (A2AR) on iNKT cells downregulate their activation and attenuate inflammation in mouse models of SCD.84

Figure 49–6.  Electron micrograph of negatively stained fiber of HgS and the structure deduced by three-dimensional image reconstruction. The reconstructed fiber is presented as ball models, with each ball representing a HgS tetramer. The models are presented as the outer sheath (left), the inner core (center), and a combination of both inner and outer filaments (right). (Reproduced with permission from the University of Texas Medical Branch.) cell adhesion molecule (VCAM)-1, integrin αVβ3, P-selectin, P-selectin glycoprotein ligand (PSGL)-1, E-selectin, Lutheran blood group antigen, and thrombospondin.55–60 The site of adhesion is purported to be the postcapillary venule at which site sickle red cells appear to interact with white cells adherent to the endothelium rather than engaging the endothelium directly.31 Neutrophilia is an adverse prognostic factor in sickle cell anemia. Because of their larger size, adherent leukocytes cause a greater decrease in vessel caliber than red cells. Diapedesis occurs in postcapillary venules, a site of vasoocclusion in sickle cell anemia.31,61–63 Neutrophil integrin αMβ2 microdomains capture sickle red cells causing vascular occlusion in sickle cell mouse models. Monocytes are also highly activated in sickle cell anemia, and they promote increased endothelial activation by increased production of tumor necrosis factor (TNF)-α and interleukin (IL)-1β.60 Expression of leukocyte adhesion molecules, L-selectin, and integrin αMβ2, are associated with a severe clinical phenotype.61,64

Inflammation and Chronic Vasculopathy

Sickle cell anemia is characterized by chronic leukocytosis, abnormal activation of neutrophils and monocytes, and an increase in several proinflammatory mediators including TNF-α, IL-6, and IL-1β. Several

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Activation of the Coagulation System

The initiator of coagulation, tissue factor (TF), is elevated in patients with sickle cell anemia.40,74,85–87 Microparticles (MPs) expressing TF derived from monocytes, macrophages, neutrophils and endothelial cells have been described in SCD.58,68,74,88 Conflicting results exist in the literature on the presence and contribution of TF bearing MPs. There is a lack of correlation between TF bearing MPs and procoagulant activity in SCD. Erythrocyte and platelet MPs are TF-negative and are the major component of MPs in SCD. Activation of the intrinsic pathway of coagulation by TF-negative, red cell, and platelet MPs through a phosphatidylserine-dependent mechanism appears to be the major contributor of MP-dependent coagulation activation in SCD. Perivascular TF interaction with plasma coagulation factors made possible by increased vascular permeability and phosphatidylserine exposure on the surface of red cells secondary to repeated cycles of sickling provide an impetus for the coagulation process.89 Heightened thrombin generation, platelet activation, and decreased protein C and S levels favor a procoagulant state.69,90,91 Increased plasma levels of D-dimers, thrombin–antithrombin complexes, prothrombin fragment 1.2, and plasmin–antiplasmin complexes are indicative of increased thrombinmediated coagulation with subsequent fibrinolysis.92 Plasma from sickle cell patients contains increased ultralarge von Willebrand factor multimers as a result of increased endothelial cell secretion and impaired cleavage by ADAMTS13 (a disintegrin and metalloprotease with a thrombospondin type 1 motif member 13).93

Adenosine Signaling

Cellular stress leads to the degradation of adenine nucleotides, resulting in the generation of adenosine. Adenosine homeostasis is maintained

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by two enzymes: adenosine kinase, which phosphorylates adenosine to adenosine monophosphate and adenosine deaminase, which converts adenosine to inosine. Adenosine signals through four different receptors that have differing functions. Signaling via the A2AR expressed on most leukocyte and platelets results in an antiinflammatory effect; however, signaling via the A2BR was shown to cause priapism in SCD mice via hypoxia-inducible factor (HIF)-1–mediated decrease of phosphodiesterase 5. Signaling via A2BR also leads to increased 2,3-BPG in red cells causing decreased oxygen binding affinity of Hb, which promotes sickling. Pegylated adenosine deaminase treatment of sickle mice resulted in decreased hemolysis and hypoxia reoxygenation injury.94,95

SICKLE CELL TRAIT Inheritance of only one HbS allele is termed sickle cell trait (HbAS). An estimated 300 million people carry the trait worldwide.96 The percentage of HbA is always higher (~60 percent) than HbS (~40 percent) in sickle cell trait. HbAS is considered a generally asymptomatic state with HbA in the cell preventing sickling except in the most unusual circumstances. HbAS cells sickle at O2 tension of approximately 15 torr.97 Plasma myeloperoxidase and red cell sickling have been reported to increase during exercise with fluid restriction in HbAS subjects.98 Plasma levels of VCAM-1 are higher in HbAS subjects and remain elevated following exercise compared to normal controls or HbAS with concomitant α-thalassemia, which is suggestive of subtle microcirculatory dysfunction in this population.99 Skeletal muscle capillary structures are different in HbAS subjects compared to controls. There is a 30-fold increased risk of sudden death in black army recruits with HbAS.100 Although controversial, in 2009 the National Collegiate Athletic Association recommended mandatory testing for HbAS for all its student athletes.101 Renal abnormalities are among the most common manifestations of HbAS. Anoxia, hyperosmolarity, and low pH of the renal medulla predisposes to sickling. Microscopic or gross hematuria from renal papillary necrosis is usually painless. Renal neoplasm or stones should be excluded in those with persistent gross hematuria. Isosthenuria may be seen in and may contribute to exercise induced rhabdomyolysis and sudden death.102 Renal medullary carcinoma is a rare but serious complication of HbAS. Risk of urinary tract infection is higher in females with HbAS, especially during pregnancy. End-stage renal disease occurs at an earlier age for HbAS patients with polycystic kidney disease and HbAS may contribute to erythropoietin resistance.103 Splenic infarction occurs under extreme environmental conditions in persons with HbAS; most resolve spontaneously.104,105 Caution and immediate intervention is also warranted in those HbAS individuals who develop traumatic hyphema.106 The risk of venous thromboembolism is increased twofold in HbAS subjects compared to those without the trait. The risk appears to be greater for pulmonary embolism than for deep vein thrombosis.101,105 HbAS patients do not have increased perioperative morbidity or mortality. The life span of patients with HbAS is normal.107

LABORATORY FEATURES Sickle cell anemia is characterized by a laboratory profile of evidence of hemolytic anemia with increases in lactate dehydrogenase (LDH), indirect bilirubin, reticulocyte count, and a decrease in serum haptoglobin. Anemia is usually normochromic, normocytic with a steady-state Hb level between 5 and 11 g/dL.1,108 The red cell density is increased with a normal mean cell Hb concentration (MCHC).109 Serum erythropoietin level is decreased relative to the degree of anemia.110 Elevated neutrophil and platelet levels are observed even in asymptomatic patients reflective of persistent low-grade inflammation.111–113

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Plasma tocopherol and zinc levels are low.114–116 Serum ferritin is increased, especially in iron overloaded patients. Elevated brain natriuretic peptide is seen in patients with pulmonary hypertension (PH) and congestive heart failure. Morphologically, classic sickle red cells are seen on blood film examination, and the marrow shows erythroid hyperplasia. Sickle cell anemia can be accurately diagnosed with high-performance liquid chromatography (HPLC) and isoelectric focusing.117 Rapid methods, such as solubility testing and sickling of red cells using sodium metabisulfite, are less-reliable tests.118 Polymerase chain reaction is the method of choice for prenatal diagnosis.119 No HbA is found in patients with HbSS, HbSC, or HbSβ0 diseases. Varying amounts of HbA (depending on the severity of the β-thalassemia mutation) are found in HbS–β+-thalassemia subjects.

COURSE AND PROGNOSIS Mortality from SCD in the United States has declined since 1968, coinciding with the introduction of pneumococcal polyvalent conjugate 7 (PVC7) vaccine. Comparison of mortality rates between 1979 to 1998 and 1999 to 2009 showed a 61 percent decrease in infants, 67 percent in children ages 1 to 4 years, and 35 percent decrease in children ages 5 to 19 years. Transition from pediatric to adult medical care showed an increased mortality trend with similar rises in rates during the decades of comparison.120 Average life expectancy of patients with HbSS disease in the United States is 42 and 48 years for males and females, respectively.121 In Jamaica, the population has a median survival of 53 years and 58 years for men and women, respectively, with 44 percent of individuals born prior to 1943 still living as of 2009.122 As the sickle cell population ages, causes of death change from an infectious etiology to those related to end-organ damage, such as renal failure.

CLINICAL FEATURES AND MANAGEMENT The reader is referred to the National Institutes of Health, National Heart, Lung and Blood Institute’s guidelines from 2002 for an extensive review on the topic; revised guidelines were released in the fall of 2014 at http://www.nhlbi.nih.gov/health-pro/guidelines/sickle-cell-disease-guidelines/.123 General approaches to SCD management and pain management are described separately (Table 49–2).

Sickle Cell Crises

The typical course for a sickle cell patient is that of periods of relatively normal functioning despite chronic anemia and ongoing vasoocclusion, punctuated by periods of increased pain, and serial changes in various laboratory parameters that is termed “a sickle cell crisis.” Crises have typically been classified as VOEs, aplastic crises, sequestration crises, and hyperhemolytic crises. Vasoocclusive Crises  The hallmark of SCD is the VOE. It is the most common clinical manifestation but occurs with varying frequency in different individuals. It results from increasing vasoocclusion causing tissue hypoxia, which manifests as pain. Vasoocclusion may affect any tissue, but patients typically have pain in the chest, lower back, and extremities. Abdominal pain may mimic acute abdomen from other causes. Different patients display different patterns of painful sites during a VOE, but each patient’s recurrences usually mimic the same pattern of pain. Fever is often present, even in the absence of infection. Episodes may be precipitated by dehydration, infection, and cold weather although in about most cases no precipitating factor is found.124 Figure 49–7 illustrates the phases of VOEs.125 Crises requiring readmission within 1 week occur in approximately 20 percent of patients after hospital discharge.125

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TABLE 49–2.  Pathophysiologic Mechanisms and Potential Therapeutic Targets in Sickle Cell Disease Pathophysiology/ Complication

Therapeutic Interventions

Sickle hemoglobin (HbS) polymerization

Fetal hemoglobin (HbF) induction

Cellular dehydration

Gardos channel inhibition Potassium-chloride cotransport channel inhibition

Adhesion to endothelium Red blood cell

Antiselectin Antiintegrin

White blood cell

Antiselectin Intravenous immunoglobulin Hydroxyurea (HU)

Inflammation

Nuclear factor-κB inhibition Immunomodulatory drugs HU Statins

Nitric oxide (NO) scavenging

NO donor (NO, HU, tetrahydrobiopterin) Phosphodiesterase 5 inhibition Modulation of hemolysis

Coagulation

Tissue factor inhibition Antiplatelet therapy Anticoagulation

Hyposplenism/infection

Penicillin prophylaxis

Ischemia–reperfusion

Xanthine oxidase inhibition Myeloperoxidase inhibition

Iron overload

Chelation

The characterization of crisis phases has implications for clinical research, especially in pain management, wherein interventions early in the course of a crisis could result in better outcomes for patients. Aplastic Crises  Aplastic crises in sickle cell anemia result when there is a marked reduction in red cell production in the face of ongoing hemolysis, causing an acute, severe drop in Hb level. The characteristic laboratory finding is a reticulocyte count less than 1 percent. The most common causative agent is parvovirus B19, which attaches to the P antigen receptor on erythroid progenitor cells, causing a temporary arrest in red cell production (Chap. 36). Recurrent aplastic crises by parvovirus B19 are rare because of the development of protective antibodies. Other rare complications associated with parvovirus B19 include acute splenic and/or hepatic sequestration, acute chest syndrome, marrow necrosis, and renal dysfunction. Patients usually recover within 2 weeks; however, those with severe symptomatic anemia need red cell transfusion. Siblings of SCD patients with parvovirus infections should be monitored closely for aplastic crisis given high secondary attack rates (>50 percent). Patients need to be isolated from pregnant individuals given increased risk of hydrops fetalis with parvovirus B19 infection.126 Sequestration Crises This type of crisis is characterized by sudden, massive pooling of red cells, typically in the spleen and less

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commonly in the liver.127 Splenic sequestration is typically seen in children (younger than 5 years of age) prior to autoinfarction of the spleen, but can be seen in adults with HbSC disease or HbS–β-thalassemia with persisting splenomegaly.128–130 A minor sequestration episode is usually accompanied by a Hb of more than 7 g/dL, and a major episode usually is one in which the Hb is less than 7 g/dL or the Hb has decreased by 3 g/dL from baseline.131 Acute splenic and hepatic sequestration crises can present with rapidly enlarging spleen or liver, pain, hypoxemia, and hypovolemic shock. Treatment consists of red cell transfusion. Transfusion carries the risk of hyperviscosity when the sequestration crisis resolves and the sequestered red cells are returned to the general circulation. Splenic sequestration crisis has a high rate of recurrence, especially in children. Splenectomy to prevent recurrence is debated in very young children. Some report chronic red cell exchange transfusion as a means of delaying splenectomy until the child is older while others did not see any benefit to this treatment. Patients younger than 2 years of age can be placed on chronic transfusion until they are older, at which time splenectomy should be considered. Splenectomy is recommended after the first episode of life-threatening splenic sequestration crisis or chronic hypersplenism. Partial splenectomy and emergency splenectomy during a crisis is not recommended. Parental education is important for early recognition of the problem so they can seek medical care promptly.126 Hyperhemolytic Crisis The term hyperhemolytic crisis is used to describe the occurrence of episodes of accelerated rates of hemolysis characterized by decreased blood Hb, increasing reticulocytes, and other markers of hemolysis (hyperbilirubinemia, increased LDH). Hyperhemolysis can occur during resolution of a VOE, at which time irreversibly sickled and dense red cells are rapidly destroyed, as well as from an acute or delayed hemolytic transfusion reactions.126,132

Pain Control

Patients with SCD have acute pain, chronic pain, or both. As a symptom, pain is often underrated in its intensity and undertreated by caregivers, especially inexperienced physicians. Patients are often perceived as drug-seekers or drug addicts, when in fact less than 10 percent of patients are addicted, a number comparable to other disease states. Unsatisfactory relief of pain drives patients to behaviors that appear to healthcare givers as signs of addiction—a state termed pseudoaddiction. A study comparing sickle cell anemia patients who use the emergency department frequently or infrequently found significant impairment in quality of life and increased markers of disease severity in those who use the emergency department frequently, dispelling the myth that frequent emergency department use indicates narcotic-addicted individuals when, in fact, they may have more severe disease.3,133–137 The landmark Pain in Sickle Cell Epidemiology Study revealed that adult SCD patients have pain at home approximately 55 percent of the time, which contrasts sharply to pain studies in children, who report at-home pain approximately 9 percent of the time.138,139 Acute pain is managed with opioids, nonsteroidal antiinflammatory drugs, acetaminophen, or a combination of these medications. Immediate pain assessment and frequent reassessment with appropriate application of medications until pain relief is obtained is important. For adults and children weighing more than 50 kg, morphine can be started at a dose of 0.1 to 0.15 mg/kg. The hydromorphone dose should be 0.015 to 0.020 mg/kg intravenously. These are recommended doses for opioid-naïve patients and are at the lower end of the dosing range. 123,140,141 The use of meperidine has declined because of neurologic side effects, especially in patients with renal failure, who are at risk for the serotonin syndrome in conjunction with use of other medications.142–144 However, the use of morphine is not benign and concerns of increased association of acute chest syndrome, dysphoria, and neuroexcitatory

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Changes during the progression of the painful crisis Prodromal phase

Initial phase

Established phase

Resolving phase

Dense RBC ISC RDW HDW

10 8 6

Temperature WBC count

Reticulocytes

CRP SAA

LDH CPK

(Steady state values)

RBC DI Fibrinogen Orosomucoid ESR Platelets Plasma viscosity

RDW HOW Platelets

4

Arbitrary values relativfe to steady state

Categorical pain scale

Numbness Problems with Joint effusion Problems with parasthesia er personnel signs of hospital personnel aches anxiety, fear inflammation depression

Dense RBC ISC

Hb

RBC DI

2 I 0

–2

II –1

1

2

III 3

4

5

Crisis day

IV 6

7

8

9

10 Ballas 1995, 1992 Akinola et al, 1992 Beyer et al, 1999 Jacob et al, 2005

Figure 49–7.  A typical profile of the events that develop during the evolution of a severe sickle cell painful crisis in an adult in the absence of overt infection or other complications. Such events are usually treated in the hospital with an average stay of 9 to 11 days. Pain becomes most severe by day 3 of the crisis and starts decreasing by day 6 or 7. The Roman numerals refer to the phase of the crisis: I indicates prodromal phase; II, initial phase; III, established phase; and IV, resolving phase. Dots on the x-axis indicate the time when changes became apparent; and dots on the y-axis, the relative value of change compared with the steady state indicated by the horizontal dashed line. Arrows indicate the time when certain clinical signs and symptoms may become apparent. Values shown are those reported at least twice by different investigators; values that were anecdotal, unconfirmed, or that were not reported to occur on a specific day of the crisis are not shown. CPK, creatinine phosphokinase; CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; HDW, hemoglobin distribution width; ISC, irreversibly sickled cells; LDH, lactate dehydrogenase; RBC DI, red cell deformability index; RDW, red cell distribution width; SAA, serum amyloid A. (Reproduced with permission from SK Ballas, K Gupta, P Adams-Graves: Sickle cell pain: A critical reappraisal. Blood 120(18):3647–3656, 2012.) side effects have been raised.125 Prior use of opioid therapy should be taken into consideration when deciding initial opioid doses as patients may be tolerant and require higher doses. Caution should be exercised with nonsteroidal antiinflammatory drugs and acetaminophen if there is renal or hepatic dysfunction. Patients with acute pain are better managed in a setting dedicated to sickle cell patients.145 A multidisciplinary approach is needed for pain management, especially if chronic pain is present.146,147 Opioid side effects should be anticipated and managed. Antidepressants, anticonvulsants, and clonidine can be used for neuropathic pain. Occasionally, severe, unrelenting pain may require red cell transfusion to decrease sickle Hb below 30 percent in the blood.148 There is a paucity of data regarding optimal management of pain in SCD. A randomized trial of optimizing patient controlled analgesia strategy was closed because of poor accrual.149 A trial looking at NO inhalation for treatment of VOE did not show improvement in pain.150

Pulmonary Manifestations

Acute Chest Syndrome  The acute chest syndrome (ACS) is a constellation of signs and symptoms in patients with SCD that includes a new infiltrate on chest radiograph defined by alveolar consolidation

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but not atelectasis, chest pain, fever, tachypnea, wheezing, or cough, and hypoxia (Fig. 49–8).151 However, respiratory findings on clinical examination in the absence of radiographic findings should trigger high suspicion for ACS and warrants close monitoring. ACS is the leading cause of mortality in patients with SCD.121 Etiology varies depending on age, with viral and bacterial infections dominating in the pediatric age group and fat embolization resulting from marrow necrosis during VOE dominating in adults.152,153 Important pathogens include Chlamydia pneumoniae, Mycoplasma pneumoniae, Streptococcus pneumoniae, Staphylococcus aureus, parvovirus B19, respiratory syncytial virus, and influenza. Regardless of the triggering factor, the pathogenesis of ACS involves increased intrapulmonary sickling, intrapulmonary inflammation with increased microvascular permeability, and alveolar consolidation. ACS can rapidly evolve with bilateral infiltrates and consolidation leading to acute respiratory failure requiring intubation and ventilatory assistance. Independent risk factors for respiratory failure are age older than 20 years, platelet count less than 20 × 109/L, multilobar lung involvement, and a history of cardiac disease.152 Thrombocytopenia is an independent predictor of neurologic complications during hospitalization

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Hydroxyurea should be offered to all patients with any of the risk factors for increased mortality described above.162 Asthma, Abnormal Pulmonary Function Tests, and Airway Hyperreactivity.  Asthma is a common comorbidity with higher-than-average prevalence in patients with SCD and is associated with increased risk of ACS, VOE, stroke, and mortality. Airway hyperreactivity as evidenced by a positive bronchodilator response on pulmonary function testing, irrespective of baseline function, and in response to cold air or methacholine challenge, is seen in approximately two-thirds of SCD patients. Inflammation, hypoxemia, and increased oxidative stress associated with asthma may contribute to the vasculopathy of SCD.163 Pulmonary function tests collected as part of the Cooperative Study of Sickle Cell Disease (CSSCD) revealed abnormalities in 90 percent of the 310 patients, with the majority having restrictive lung disease. Asthma treatment follows general treatment guidelines as in the non-SCD populations.164,165

Cardiac Manifestations

Figure 49–8.  Anteroposterior view of chest radiograph depicting

bilateral, patchy, lung infiltrates in a 30-year-old female with sickle cell disease and evolving acute chest syndrome.

for ACS, which was seen in 22 percent of adult patients in the National Acute Chest Syndrome study.154 The treatment of ACS includes oxygenation, incentive spirometry, adequate pain control to avoid chest splinting, antimicrobial therapy that always covers atypical bacteria and influenza when indicated, avoidance of overhydration, use of bronchodilators, and red cell transfusion to decrease intrapulmonary sickling.152,155–160 The use of glucocorticoids may attenuate the course of ACS; however, its use is not well established and readmission rates for VOE after ACS resolution are increased.153 sPLA2 has been recognized as a predictor of ACS; however, a clinical trial investigating early transfusion based on sPLA2 elevation closed because of poor accrual. Hydroxyurea therapy should be offered to all patients with a history of ACS because it reduces the incidence by 50 percent in adults and 73 percent in children.161 Pulmonary Hypertension  PH, defined by a resting mean pulmonary arterial pressure of 25 torr or higher on right-heart catheterization, is seen in 6 to 11 percent of SCD patients. An elevated tricuspid regurgitant velocity of 2.5 m/s has a positive predictive value of 25 percent for PH in SCD and is seen in one-third of these patients. PH, as defined by right-heart catheterization, elevated tricuspid regurgitant jet velocity of 2.5 m/s or higher, and a serum N-terminal pro–brain natriuretic peptide (NT-pro-BNP) level of 160 pg/mL or higher, confers an increased mortality risk.162 Abnormalities in NO metabolism, hemolysis, and inflammation contribute to the pathophysiology of PH.162 Parenchymal lung disease from repeated episodes of ACS and thromboembolism are other causal factors. Clinical symptoms of PH include fatigue, dizziness, and dyspnea on exertion, chest pain, and syncope. These may be unrecognized as being related to PH, as PH is often undiagnosed in patients with SCD. PH should be treated following guidelines set for the treatment of primary PH unrelated to SCD. Two trials looking at bosentan (endothelin receptor antagonist) in SCD patients closed because of sponsor withdrawal. A trial of sildenafil was halted early because of increased incidence of VOE. Patients who have venous thromboembolism in the setting of PH should be considered for indefinite anticoagulation.

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Anemia in SCD results in an elevated cardiac output secondary to an increased stroke volume with minimal increase in heart rate.166,167 Clinical manifestations of a hyperdynamic circulation include a forceful precordial apical impulse, systolic and diastolic flow murmurs, and tachycardia that may increase during periods of increased hemodynamic stress. Diastolic left ventricular dysfunction may begin in early childhood and is an independent risk factor for death, with even greater risk of mortality in those having PH. Left ventricular hypertrophy is common and progressive with age; left ventricular dysfunction is a late event. Myocardial infarction is an underrecognized problem in SCD. Epicardial coronary artery disease is rare; microvascular ischemia is likely causative. Sudden cardiac death has been reported in 40 percent of patients in an autopsy series.168–170 Previously sudden cardiac death was ascribed to narcotic overdose; currently, it is thought to be secondary to cardiopulmonary causes in the majority of cases. QTc prolongation, atrial and ventricular arrhythmias, nonspecific ST-T wave changes are common in SCD patients. Patients presenting with chest pain should have a thorough evaluation to rule out cardiac disease. Cardiac magnetic resonance may be a good modality to image microvascular flow and quantitate cardiac iron overload.171,172 Blood pressure in patients with SCD is significantly lower than age-, sex-, and race-matched controls, partly secondary to anemia.173 Relative hypertension is associated with end-organ damage. Diuretics may be used, keeping in mind that SCD patients have obligate hyposthenuria and are prone to dehydration, which can precipitate a VOE.

Central Nervous System

Originally thought to be a small vessel disease, stroke in SCD is a macrovascular phenomenon with devastating consequences that affects approximately 11 percent of patients younger than 20 years of age.174,175 Risk is highest in the first decade of life followed by a second smaller peak after age 29 years. Ischemic stroke is most common in children and older adults, whereas hemorrhagic stroke predominates in the third decade of life.175 Recurrent stroke is most common in the first 2 years following the primary event.176 Silent infarcts, defined as an increased T2 signal abnormality on magnetic resonance imaging (MRI), begins in infancy and has a cumulative incidence of 37 percent by age 14 years. They occur in watershed areas of the brain, are not predicted by abnormal transcranial Doppler (TCD) velocity, and may progress despite chronic transfusion.177–180 There is evidence of neurocognitive decline in asymptomatic adults despite having normal brain imaging that is attributed to anemia and hypoxemia.154 Cerebral blood flow is significantly increased in SCD because of chronic anemia and hypoxemia, but does not increase further in

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response to increased hypoxic stress, thereby predisposing to ischemia.181,182 Stenosis of large vessels, especially of the circle of Willis, without the classic atherosclerotic plaque occurs in conjunction with a multitude of other factors, including chronic hemolysis, deranged NO metabolism and impaired vascular autoregulation, and can lead to stroke.182 Rare causes of cerebral vascular disease include fat embolization and venous sinus thrombosis. Moyamoya type fragile collaterals have been reported in more than one-fifth of patients with prior stroke, possibly leading to hemorrhagic stroke in later life.183–188 Risk factors for ischemic stroke include transient ischemic attack, recent or recurrent ACS, nocturnal hypoxemia, silent infarcts, hypertension, elevated lactic dehydrogenase, and leukocytosis, whereas anemia, neutrophilia, the use of glucocorticoids, and recent transfusion are independent risk factors for hemorrhagic stroke, especially in children.175,189–195 Sickle cell genotypes other than HbSS carry a lower risk, as do patients with HbS–α-thalassemia.175,196,197 The best predictor of stroke risk, however, is an increased blood flow velocity in major intracranial arteries on TCD ultrasonography.197 Blood flow velocities less than 170 cm/s are considered normal. Velocities between 170 and 200 cm/s are termed conditional, and velocities of greater than 200 cm/s are considered high and are associated with a 10-fold increase in ischemic stroke in children 2 to 16 years of age. There is an increased frequency of stroke among siblings of patients with SCD than would be expected by chance alone, raising the possibility of other modifier genes contributing to stroke risk.183 The TNF (–308) G/A promoter polymorphism is associated with increased large-vessel stroke risk as is the IL-4–receptor gene 503 S/P variant, although it did not reach statistical significance. The clinical features of stroke in SCD encompass the classic findings of stroke in other disorders, including, but not limited to, hemiparesis, seizures, coma, paresthesias, headaches, and cranial nerve palsies. Neurocognitive deficits in IQ, memory, language, and executive function have been demonstrated.154,198 Imaging approaches for acute stroke are the same as those for non -SCD patients and includes MRI and magnetic resonance angiography. Prevention of Primary Stroke Based on the results from the Stroke Prevention in Sickle Cell Disease (STOP) Study, it is recommended that asymptomatic children with HbSS disease older than two years of age should be screened for stroke risk using TCD.197 Those with high TCD velocities should be offered a chronic red cell transfusion program for primary stroke prevention. Repeat TCD screenings should be done every 3 to 12 months even in patients who have normal or conditional baseline velocities, because they can evolve into a higher-risk category. Despite obstacles to TCD screening, clinical practice changes based on the STOP study translated into declining stroke rates since 1991.199,200 Prevention of Secondary Stroke  Patients with SCD who present with a stroke and are not on chronic transfusion should be placed on a transfusion program to prevent secondary strokes. Exchange transfusion may be preferable to periodic red cell transfusion, not only to avoid iron overload, but also to further reduce stroke risk. In a retrospective study, children who received periodic transfusion had a fivefold higher relative risk of a recurrent stroke compared to those on an exchange transfusion regimen.201 Despite chronic transfusions, patients may have a recurrent stroke, especially in patients with HbS greater than 30 percent.202 Hydroxyurea was shown to decrease high and conditional TCD velocities in more than 90 percent of patients studied.203 However, a randomized trial comparing transfusions with iron chelation to hydroxyurea with phlebotomy showed a 10 percent stroke rate in the hydroxyurea arm, thus establishing transfusion as the preferred preventive strategy.204 Anticoagulation therapy has not been studied in patients with SCD and, therefore, no recommendations can be made. Treatment guidelines

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for intracranial hemorrhage are as those for non-SCD–related intracranial hemorrhage; role of transfusion is less clear in SCD especially when cause of intracranial hemorrhage is unclear. Patients with moyamoya disease who have a particularly poor outcome may benefit from revascularization using encephaloduroarteriosyangiosis.205,206

Genitourinary Systems

Renal Failure  Sickling of HbSS erythrocytes in the hypoxic, acidic, and hypertonic environment of the renal medulla, oxidative stress, increase in prostaglandins and endothelin-1 in the kidney, and abnormalities of the renin angiotensin system contribute to the pathophysiology of renal disease in SCD.207 The incidence of renal failure varies between 4 and 20 percent.208–211 Dehydration is the most common cause of acute renal failure in SCD. Isosthenuria is highly prevalent in SCD, may increase the risk of dehydration, and is irreversible.212 Glomerular hypertrophy, focal and segmental glomerular sclerosis, and hemosiderin deposition in proximal renal tubular epithelium have been described; however, no single lesion is pathognomonic of sickle cell nephropathy. Cystatin C is an accurate marker of glomerular filtration and therefore is preferable to serum creatinine in estimating renal function.213,214 Glomerular hyperfiltration, microalbuminuria, and macroalbuminuria occur sequentially in SCD patients starting in infancy and increasing in frequency with age.122,161,215 Incidence of microalbuminuria is greater than 60 percent in those over age 35 years.213 End-stage renal disease requiring dialysis carries a poor prognosis and is associated with a median survival of 4 years.216 Angiotensin-converting enzyme inhibitors decrease proteinuria and hyperfiltration in SCD; however, large-scale studies are needed to characterize the magnitude of the benefit. Treatment of renal disease follows principles used for non-SCD kidney pathology and includes effective blood pressure control, avoidance of nephrotoxic agents, and treatment of urinary tract infection. A relative decrease in serum erythropoietin levels, proportionate to the degree of anemia is observed; however, erythropoietin treatment, with its resultant increase in Hb may cause an increase in VOEs because of an increase in blood viscosity.213 Renal tubular acidosis type IV, secondary to decreased potassium and hydrogen ion in the distal tubule can cause disproportionate acidosis and hyperkalemia in patients with declining renal function.213 Hematuria is discussed in the section on sickle cell trait. Priapism  Priapism is prevalent in at least 35 percent of male patients with SCD with devastating psychological consequences; true prevalence may be higher as it is often underreported.217–219 The mean age of episodes is 15 years and two-thirds of patients have “stuttering priapism” a term used for episodes that last less than 3 hours.220 Derangements in NO metabolism and adenosine signaling are thought to be the major contributors to priapism in SCD.94 Greater than 95 percent of priapism is the “low-flow” type resulting from ischemia, is painful, and is a medical emergency.221 Aspiration of the corpus cavernosa followed by epinephrine injections, exchange transfusion, and α and β agonists have all been used, but data regarding efficacy are sparse. α-Agonists, etilefrine 50 mg, and ephedrine 15 to 30 mg per day, seem to reduce the incidence of stuttering priapism.222 Hormonal therapies, including antiandrogens and luteinizing hormone-releasing hormone, reduce nocturnal erections but are associated with loss of libido.221 Transfusion therapy has resulted in neurologic sequelae termed “the ASPEN syndrome” (Association of Sickle Cell Disease, Priapism, Exchange Transfusion) and is thought to be secondary to hyperviscosity; care, therefore, must be taken not to increase the hematocrit beyond 30 percent. 223 In recalcitrant cases, a shunt is performed but results in permanent impotence.222 A penile prosthesis is used to ameliorate sexual dysfunction. Nocturnal Enuresis  Nocturnal enuresis is prevalent in 25 to 33 percent of the pediatric sickle cell population, which is higher compared

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to that of age-matched controls.224–226 It tends to decrease with age but is still prevalent in adults. Social and environmental factors, decreased functional bladder capacity, and decreased arousal during sleep appear to be contributing factors.

Musculoskeletal System

VOE is commonly manifested by marrow infarction causing musculoskeletal pain, swelling at involved sites, fever, and leukocytosis. Marrow hypercellularity is thought to predispose to this phenomenon by causing a decrease in local blood flow and oxygenation. Dactylitis  Dactylitis is a painful swelling of digits of the hands and feet (“hand-foot syndrome”). It occurs early in infancy as hematopoietic marrow is still present in these bones at this age. Most episodes resolve within in 2 weeks.227–230 Epiphyseal infarction can result in joint pain and swelling mimicking septic arthritis. Use of hydroxyurea in the BABY HUG trial resulted in significant reduction of rate of dactylitis.161 Osteomyelitis, Septic Arthritis, and Bone Infarction  Impaired cellular and humoral immunity together with infarction of bone contribute to this complication with an estimated prevalence of 12 percent. Atypical serotypes of Salmonella, S. aureus, and Gram-negative bacilli are the principal infectious offenders. No single lab or imaging test reliably differentiates osteomyelitis from infarction.227,229,231–235 Culture results may be nondiagnostic as patients usually receive antibiotics on presentation with fever; therefore, the presence of leukocytes in bone and joint aspirates should evoke a high suspicion for osteomyelitis.126 Septic arthritis tends to occur in joints involved with avascular necrosis, also seen following hip arthroplasty. Multiple joints may be involved. An elevated C-reactive protein should raise suspicion for septic arthritis and prompt intervention with appropriate antibiotics as needed to prevent joint deterioration and collapse.227 Vertebral body infarctions with subsequent collapse causes the classic “fish mouth” appearance of vertebrae on radiographs of the spine. Osteopenia and Osteoporosis  Osteopenia and osteoporosis are prevalent (30 to 80 percent) in patients with sickle cell anemia, with a predilection for the lumbar spine. Presence of avascular necrosis with local bone remodeling may lead to false-negative results on a bone mineral density test at the femoral neck.126 Fractures of the long bones are commonly underdiagnosed and self-reported rates of fractures in young adults with SCD are high. Etiology of osteoporosis is multifactorial with hypogonadism, hypothyroidism, nutritional deficiencies, and iron overload interfering with osteoblast function being the major causes.126,236–238 More than 50 percent of patients are vitamin D deficient with the majority (>80 percent) having less-than-optimal levels. High doses of vitamin D supplementation have resulted in improvement in chronic pain and higher levels of physical activity.239 Avascular Necrosis Vasoocclusion resulting in infarction of articular surfaces of long bone occurs most commonly in the femur followed by the humerus. It was previously thought to occur with increased frequency in HbSC disease as opposed to patients with HbSS. However, with increased longevity of HbSS patients, its prevalence is greater in patients with HbSS.240–242 As per the CSSCD estimates, 50 percent of patients by age 33 years will have avascular necrosis of the femoral head (Fig. 49–9). The presence of concurrent deletional αthalassemia (–α3·7) and a history of frequent VOEs are classic risk factors for avascular necrosis. Other risk factors include male gender, higher Hb concentration, low fetal Hb, and vitamin D deficiency.126,243,244 Polymorphisms in BMP6, ANNEXIN A2, and KLOTHO genes are associated with avascular necrosis.245 Patients present with chronic joint pain with progressive decrease in range of motion of affected joints. Multiple joints are commonly involved.246 The vast majority of untreated patients will progress to femoral head collapse within 5 years.247,248

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Figure 49–9.  Avascular necrosis of the right hip in a 31-year-old female with sickle cell disease depicting a patchy lucency and sclerosis and irregular contour of the femoral head and loss of the joint space.

Avascular necrosis has been treated with a number of modalities including core decompression, osteotomy, bone grafting, surface arthroplasty, and joint replacement. Two randomized trials in avascular necrosis compared core decompression and physical therapy versus physical therapy alone and did not show a difference in outcome between the two arms; however, followup was short, a significant number of stage III hip joints were included in one study, and sample size was limited.249 In our experience, core decompression is a useful option in early stage avascular necrosis. Several studies associate total hip replacement in SCD with a higher rate of orthopedic and medical complications. However, other studies show a lower rate of orthopedic complications. Structural bone diseases in SCD make joint replacement challenging.250–252 Hydroxyurea and chronic transfusion therapy have not been shown to reduce the risk of avascular necrosis.243

Leg Ulcers

Leg ulcers occur in 2 to 40 percent of cases with SCD and varies geographically with the highest rate being reported in Jamaica.1,253 In the United States, leg ulcers are seen in 4 to 6 percent of patients with SCD and are most common in patients older than 10 years of age.254 They occur on the lower extremities, especially on the malleoli, and cause chronic pain and disability. Venous stasis is a predisposing factor while coinheritance of α-thalassemia appears to have a protective effect. The relationship between hydroxyurea use and increased occurrence of leg ulcers is controversial.255 Polymorphisms in KLOTHO, TEK, and several other genes in the transforming growth factor (TGF)-β and bone morphogenic protein (BMP) pathways are associated with leg ulcers.245 Once established, ulcers are recalcitrant and significantly impair quality of life.256 Treatment of leg ulcers is largely empiric. Leg elevation, bed rest when practical and feasible, wet-to-dry dressings, gentle debridement, Unna boots, and treatment of infection and topical or systemic antibiotics are commonly used. The peptide encoding integrin-interaction site of many extracellular matrix proteins (RGD peptide) enhanced healing of the ulcers in preliminary studies, but, unfortunately, it never came to

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clinical practice because of nonmedical reasons.257 Increases in HbF and transfusions occasionally hasten healing of leg ulcers.258

Hepatobiliary Complications

Chronic liver abnormalities in SCD are frequent and of different etiologies that include vasoocclusion, transfusional iron overload, pigmented gallstones with bile duct obstruction, acute or chronic cholecystitis, viral hepatitis, and cholestasis.259,260 Common clinical manifestations include right upper quadrant pain, fever, hepatomegaly, nausea, and vomiting. Bilirubin levels from chronic hemolysis are usually not above 6 mg/dL, with a majority of it being the indirect fraction.261 Because some degree of aspartate transaminase elevation is seen with hemolysis, alanine transaminase elevation is a more accurate marker of liver injury. Vasoocclusion involving the hepatic sinusoids was seen in 39 percent of patients in one study, while previous reports of vasoocclusion involving the liver, termed acute sickle hepatic crisis, has been reported in 10 percent of patients. The differing prevalence is the result of varying criteria used to include biochemical and clinical abnormalities.262 Acute hepatic sequestration crisis characterized by a rapidly enlarging, tender liver and hypovolemia is akin to splenic sequestration but much rarer. It requires prompt treatment with red cell transfusion. Severe intrahepatic cholestasis with serum bilirubin levels as high as 100 mg/dL is a catastrophic situation needing exchange transfusion for resolution; synthetic liver function is lost as characterized by low serum albumin and coagulation protein abnormalities; renal impairment may occur. A more benign form of cholestasis has been described, which resolves with conservative measure.263–268 Chronic hemolysis results in an increased burden on the heme catabolic pathway leading to increased unconjugated bilirubin and formation of pigmented gallstones. The incidence of gallstones increases with age, with a reported prevalence of 50 percent at 22 years of age.269–271 The number of uridine diphosphate (UDP) glucuronosyltransferase 1 family (UGT1A1) promoter (TA) repeats (the polymorphism associated with Gilbert syndrome) is strongly associated with increased incidence of gallstones and bilirubin levels while coinherited α-thalassemia (Chap. 48) decreases bilirubin levels in patients with SCD.272 Laparoscopic cholecystectomy is recommended in symptomatic patients with cholelithiasis. The treatment of asymptomatic patients with positive findings on abdominal ultrasonography is more controversial. In the Jamaican cohort study, only 7 percent of patients with positive ultrasonograms had symptoms suggestive of biliary tract disease and needed a cholecystectomy. However, patients in the United States appear to be more symptomatic, with the majority of gallbladders removed after only a positive ultrasonogram have pathologic evidence of cholecystitis.269 Asymptomatic patients with negative screening ultrasonograms should be observed; however, timing and frequency of screening has not been standardized.

Ophthalmic Complications

The microvasculature of the retina with relative hypoxemia facilitates “sickling” akin to several other vascular beds. Microcirculatory obstruction occurs followed by neovascularization and arteriovenous aneurysms. Hemorrhage, scarring, and retinal detachment leading to blindness are the sequelae. Changes occur at the periphery, thereby sparing central vision at earlier stages. The term sickle cell retinopathy encompasses nonproliferative and proliferative changes. Nonproliferative changes include “salmon-patch” hemorrhages, peripheral retinal lesions termed “black sunbursts,” and iridescent spots, whereas neovascularization is characteristic of proliferative changes, giving a pattern of vascular lesions resembling a marine invertebrate and is termed as “sea fans.”273 Increased levels of plasma and intraocular vascular endothelial growth factor have been documented in proliferative sickle cell

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retinopathy, as have angiopoietin-1 and -2 and von Willebrand factor. Pigment epithelium derived factor, an angiogenesis inhibitor, is increased as well, especially in nonviable “sea fans.”274–276 Proliferative sickle cell retinopathy may differ from other proliferative retinopathies in that spontaneous regression of neovascularization can occur in up to 60 percent of cases.277,278 The Jamaican cohort study reported an annual incidence of 0.5 cases per 100 HbSS subjects versus 2.5 cases per 100 HbSC subjects. Prevalence was greater in HbSC subjects as well, with a 43 percent rate in the third decade versus 14 percent for those with HbSS. However, there was a 32 percent incidence of spontaneous regression. Irreversible visual loss occurred only in 2 percent of HbSC subjects up to 26 years of age observed at time of the study.277 Central retinal artery occlusion is rare in HbSS disease.279 Conjunctival vascularity is decreased in SCD patients compared to controls with further decreased vascularity and decreased conjunctival red cell velocities during vasoocclusion.280–283 An orbital compression syndrome characterized by fever, headache, orbital swelling, and visual impairment secondary to optic nerve dysfunction can occur in SCD. Orbital marrow infarction is a common cause.284 All patients with sickle hemoglobinopathies should have a yearly ophthalmology examination beginning in childhood. The examination should be carried out by an ophthalmologist and should include slitlamp examination of the anterior chamber and detailed retinal visualization including a fluorescein angiography in addition to visual acuity. The evaluation and treatment of proliferative sickle retinopathy is complicated by the fact that spontaneous regression may occur. Laser photocoagulation remains the most commonly performed procedure for this finding. Traumatic hyphema needs urgent optical referral because increased sickle red cells can cause obstruction of outflow channels, resulting in acute glaucoma. This vascular obstruction may cause decrease in retinal and optic nerve perfusion causing further visual problems. Unresolved vitreous hemorrhage and retinal detachment may need surgical intervention. Exchange transfusion to keep HbA at more than 50 percent is recommended. Central retinal artery occlusion needs urgent exchange transfusion and an ophthalmology consultation.277,285–287 Orbital compression syndrome is treated with glucocorticoids with the addition of antibiotics if concomitant infection cannot be ruled out.126

Splenic Complications

Functional asplenia defined as impaired mononuclear phagocyte system functions in the spleen occurs in 86 percent of infants with SCD.288 It is defined by the presence of Howell-Jolly bodies and absence of 99mTc (99m-technetium) splenic uptake, even in the presence of a palpable spleen. Slow blood flow in the red pulp of the spleen sets the stage for increased red cell sickling. Repeated splenic infarctions lead to “autosplenectomy.” As a consequence, patients are prone to microbial infections, especially with encapsulated microorganisms such as S. pneumoniae, Haemophilus influenzae, and Neisseria meningitidis. Hypertransfusion early in childhood, prior to age 7 years, may lead to reversal of functional asplenia. Marrow transplantation and hydroxyurea have resulted in reversal of functional asplenia in some older subjects. Splenic sequestration occurs in young children.289–293

Management during Pregnancy

Differing morbidity and mortality rates have been reported in pregnant women with SCD, some of which is attributed to geographic location and access to healthcare. Although the CSSCD data showed low rates of pregnancy loss and mortality, other studies have shown an increased mortality of 10 to 100 orders of magnitude greater as compared to non -SCD patients.285–290 Preterm delivery occurs in 30 to 50 percent of SCD

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patients and two-thirds will have infants with birth weights less than the 50th percentile.294,295 There is an increased frequency of VOEs reported during pregnancy. In a study looking at pregnancy outcomes in SCD patients compared to non-SCD patients with comorbidities, patients with SCD displayed a significantly increased incidence of venous thromboembolism (VTE), nonhemorrhagic obstetric shock (defined as pulmonary thromboembolism, amniotic fluid embolism, acute uterine inversion, and sepsis), and infection, despite being significantly younger.296,297 Other studies have shown similar findings, especially the fivefold increased risk of VTE in this population.295,297,298 Given increased risk of preeclampsia and eclampsia, patients should have close monitoring of blood pressure and proteinuria after 20 weeks of gestation. Fetal nonstress and umbilical artery Doppler studies should be undertaken after 28 weeks to identify patients who might benefit from early delivery. Studies examining prophylactic red cell transfusions in pregnancy have shown mixed results. Patients should be transfused to a Hb concentration of less than 6 g/dL, because abnormal fetal oxygenation and death have been reported below this Hb level in non-SCD populations. Otherwise, patients should be transfused based on guidelines for the nonpregnant patient with SCD.294 Based on data from animal models and small reports of spontaneous abortion or fetal death, the use of hydroxyurea is not recommended during pregnancy and breastfeeding.299,300 Hydroxyurea may decrease spermatogenesis and therefore male patients may need to stop the drug temporarily when their partners are trying to conceive. Narcotics administered for relief of pain have not been shown to cause fetal harm, but babies of mothers exposed to narcotics during pregnancy should be monitored for the neonatal abstinence syndrome.294 Despite increased concern for VTE, given insufficient data, contraception advice is similar as for women without SCD.301

Management of and Prevention of Infection

Patients with SCD are predisposed to infections for a variety of reasons, including functional asplenia and defective neutrophil responses.302–306 The magnitude of this problem was highlighted in 1971 in a landmark paper by E. Barrett-Connor.306 Functional asplenia results in susceptibility to encapsulated microorganisms, particularly to S. pneumoniae, especially in children younger than 5 years of age. The CSSCD data reported an eight-per-100-patient-years rate of invasive bacterial infection in children younger than 3 years of age.307 Given the high incidence of infection, especially in childhood, infection prevention and rapid diagnosis of established infections is of paramount importance.308,309 The pneumococcal vaccine (PCV7) can be administered in infancy with effective immunologic response prior to 2 years of age; the American Academy of Pediatrics recommends it be administered at ages 2, 4, 6, 8, and 12 to 15 months. The PCV7 vaccine decreases invasive pneumococcal disease by as much as 80 to 90 percent.310 The pneumococcal polysaccharide vaccine, PPV23, covers more serotypes but is not immunogenic prior to 24 months and response lasts for 3 years. The first dose is recommended at 24 months with additional doses 3 to 5 years later.309,311–314 Nonvaccine covered strains of S. pneumoniae are emerging as important pathogens; therefore, prompt referral of patients with suspected infection to a healthcare facility is important.315 Oral penicillin prophylaxis is recommended at a dose of 125 mg twice a day for children between 0 and 3 years of age and at 250 mg twice a day for children between 3 and 5 years of age.316 Penicillin prophylaxis beyond 5 years is recommended only for patients with recurrent pneumococcal infections or who have had surgical splenectomy. Patients allergic to penicillin are offered erythromycin. The meningococcal vaccine covers most invasive isolates of N. meningitidis and is recommended by the American Academy of Pediatrics.317 Standard pediatric immunizations protecting against

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H. influenzae and hepatitis B virus should be given. Influenza virus vaccine should be given annually because viral respiratory infection favors invasive bacterial infection. Parents and caregivers of children should be educated to recognize infections and to seek medical attention early. Diagnosis of established infections varies by site and offending agent. For invasive pneumococcal disease, ceftriaxone remains the drug of choice despite concerns of immune-mediated hemolysis. Infections seen classically in SCD patients include salmonella osteomyelitis and penumonia caused by atypical bacteria like Chlamydia and M. pneumoniae, which need to be treated with the appropriate antibiotics. The spectrum of infectious complications in adults may be different. One study reported data on blood infections in adults.302 Pneumococcal infections were rare. S. aureus was the predominant organism. Patients with S. aureus had a predilection for bone-joint infection. Those with indwelling venous catheters and a severe disease course appeared to have a high risk for bloodstream infections. Although the sickle trait confers resistance to malaria, protection is not complete and severe disease and deaths from malaria have been reported in SCD patients. Malaria chemoprophylaxis is recommended for all patients living in or traveling to endemic regions.318,319

Management during Anesthesia and Surgery

Patients with SCD should have careful monitoring of Hb concentration, hydration, oxygen, and metabolic studies in the perioperative period. Acute chest syndrome and VOE occur with higher frequency in the perioperative period. Increased age is associated with increased complications.320–322 Transfusion to keep Hb levels approximately 10 g/dL is recommended. Although a prior randomized trial showed no benefit in decreasing SCD-related complications between patients transfused aggressively to a mean HbS of less than 30 percent versus those transfused to a total Hb of 10 g/dL with mean HbS percent of 59, more recent data show reduction in clinically important events, especially serious complications, in the preoperative transfused group prior to low and moderate risk surgery.202,323 Care should be taken to avoid transfusion-induced hyperviscosity.

MODIFIERS OF DISEASE SEVERITY Some patients have a mild course with few problems related to SCD, and survive into the sixth or seventh decade. In contrast, some patients have a difficult course with multiple complications, frequent hospitalizations, severe organ damage, and a significantly shortened life expectancy.324,325 Inheritance of α-thalassemia trait and a high HbF are two factors that ameliorate many complications of SCD. Genome-wide association studies revealed three major loci associated with HbF levels: The β-globin locus on chromosome 11, an intergenic region between HBSIL and MYB genes on chromosome 6, and the BCL11 gene on chromosome 2.326 Repression of BCL11A results in increased γ-globin gene expression and, consequently, in increased HbF. The exact mechanism of how BCL11A silences γ-globin expression is unclear; its expression seems to be controlled by an erythroid specific transcription factor, KLF1 with decreased expression of BCL11A upon knockdown of KLF1 gene transcript.326,327 Inheritance of α-thalassemia and HbF level do not account for all of the clinical diversity of SCD. The completion of the human genome project has provided the impetus to study polymorphisms in candidate genes as potential modifiers of disease severity. Association of polymorphisms in candidate genes and different features of SCD such as stroke,328–330 ACS,331 bilirubin levels and cholelithiasis,332–335 avascular necrosis,245 priapism,336 and leg ulcers,253 as well as HbF levels,337–342 and HbF response to hydroxyurea,343 have been studied in different groups of patients.

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Polymorphisms in the TGF-β–BMP pathway, a ubiquitous signaling pathway that is involved in many cellular processes and pathways, have emerged as recurrent findings in many of these studies. Some of the associations have functional consequences; the association of bilirubin levels in polymorphisms in the UGT1A1 gene promoter is such an example. The 7TA repeat in the promoter leads to a decreased activity of this enzyme and hence a decrease in glucuronidation of bilirubin. Thus, the association of this polymorphism with higher bilirubin levels can be understood. On the other hand, the mechanisms by which polymorphisms in the ubiquitous TGF-β–BMP pathway are associated with various complications of SCD are unknown, and thus a causal relationship cannot yet be established. Functional studies of these variants and genomewide association studies are expected to provide a better insight into genetic modulation of the phenotype of SCD.

GENERAL MANAGEMENT OF SICKLE CELL DISEASE Pharmacotherapeutics to Increase Fetal Hemoglobin Levels

The observation that HbF results in ameliorating the phenotype of SCD led to research focused on HbF modulation as a therapy for SCD. The γ-chains of HbF are excluded from the deoxy HbS polymer; thus the presence of HbF in sickle red cells exerts a potent antisickling effect. This effect has also been supported by clinical observations; the manifestations of SCD do not become apparent in the first few months of life until the switch from γ-chain production to β-chain production is almost complete in the postnatal period. Additionally, the phenotypes of some compound heterozygous states with HbS and other inherited globin disorders that lead to increased expression of HbF in the adult life (δβ-thalassemias, hereditary persistence of HbF) are very mild (Chap. 47). In fact, compound heterozygotes for HbS and deletional hereditary persistence of HbF, in which there is continued high levels of HbF expression (30 to 35 percent) uniformly distributed in all red cells (pancellular), are clinically asymptomatic and hematologically normal. In the late 1970s, further evidence in support of the ameliorating effect of high HbF came from the observation of Saudi Arabian sickle cell anemia patients who had few, if any, symptoms of SCD, had mild anemia, and were not diagnosed until adult age.344 These individuals had HbF levels in the 20 to 25 percent range as opposed to the African patients or American patients of African descent, the majority of whom had HbF levels of approximately 5 percent. Similar patients were reported from India, and this genetic propensity for high HbF production in SCD patients was linked to a unique β-globin gene cluster haplotype (Saudi Arabian–Indian) that is distinct from those found in Africa. These observations paved the way for intense investigations on the cellular and molecular mechanisms of the fetal to adult (γ to β) switch during the perinatal period and the search for “antiswitching” agents, agents that would facilitate retaining elevated HbF levels. The observation that there is a transient increase in HbF production during recovery from marrow aplasia or suppression provided the rationale for the use of myelosuppressive agents as antiswitching therapy (Table 49–3). Antiswitching indicates a mechanism to prevent the switch from γ-globin chains to β-globin chains. Hydroxyurea  Although many myelosuppressive agents have been studied in primates and some have been used in a small number of patients, only one of these, hydroxyurea, has been used, starting in the early 1980s, in large-scale clinical trials. This is largely attributable to its excellent oral bioavailability, relatively short half-life (important from the standpoint of rapid reversibility of toxicity), no evidence that its use leads to an increase in cancer prevalence, and few side effects. Hydroxyurea is the only FDA-approved agent for the treatment of SCD. It is a ribonucleotide reductase inhibitor and is S-phase specific

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TABLE 49–3.  Antiswitching Therapies Drug

Mechanism

Hydroxyurea

Myelosuppression Antiinflammatory Nitric oxide donor Increased cyclic guanosine monophosphate

Decitabine

DNA methyltransferase 1 inhibition, i.e., hypomethylation

5′-Azacitidine

DNA methyltransferase 1 inhibition, i.e., hypomethylation

Butyrate derivatives

Histone deacetylase inhibition

Histone deacetylase inhibitors

Histone deacetylase inhibition

Immunomodulatory drugs

P38 mitogen-activated protein kinase pathway

in the cell cycle. The mechanism by which hydroxyurea increases HbF synthesis is not fully understood; it has been postulated that the myelosuppressive effect leads to the recruitment of early erythroid progenitors that have retained their fetal (γ) globin synthesis capability, giving rise to the production of red cells with a higher HbF content. Some studies show that hydroxyurea acts as a NO donor and increases HbF synthesis via the cyclic guanosine monophosphate (cGMP) pathway.345 Others suggest it works by reducing the neutrophil count, thereby reducing the contributions of neutrophils to the abnormal vascular adhesion of sickle red cells. It has several other actions that explain its efficacy in SCD other than increasing HbF. These include decrease in platelets and reticulocytes, improvement in red cell hydration, and a decrease in red cell adhesiveness to the vascular endothelium (Fig. 49–10).346–348 In the landmark Multicenter Study of Hydroxyurea, hydroxyurea was shown to decrease frequency of painful crises, ACS, hospitalizations, and blood transfusions. Followup showed a 40 percent decrease in mortality in patients randomized to the drug.160,349 Hydroxyurea is recommended in patients with three or more VOEs or history of ACS. It can be started at a dose of 15 mg/kg given as a single daily dose and escalated by 5 mg/kg per day every 8 weeks until toxicity or a maximum dose of 35 mg/kg is reached. Maximum tolerated dose is defined as the dose that targets an absolute neutrophil count of 2 to 4 × 109/L and absolute reticulocyte count 100 to 200 × 109/L.350,351 Periodic monitoring of blood cell counts and serum chemistries, especially in the first year of treatment is important. Maximal effect on HbF may not be seen until 6 to 12 months of therapy is completed. The dose should be decreased in renal failure. Although not proven to have teratogenic or leukemogenic potential in SCD patients, it is recommended that it not be used in pregnant or breastfeeding patients. Concerns about detrimental effect on spermatogenesis have also been raised based on studies in mice.352–355 Patients receiving hydroxyurea who die while on treatment are likely to be older when therapy is initiated, more anemic, likely to have Bantu or Cameron β-globin gene haplotypes, and have impaired renal function.324 Several studies have now been published on the use of hydroxyurea in infants and children. Therapy can begin between 6 and 9 months of age, is safe and well tolerated with improved growth rates, preserves organ function, and the additional benefits as seen in adults.161,351,356,357 Other Fetal Hemoglobin-Inducing Agents  Although significant advances have been made in understanding the basic mechanism(s) of the perinatal switch from γ- to β-globin synthesis, this knowledge is

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B

Figure 49–10.  Blood film from SCD patients: effect of hydroxyurea therapy. A. Blood film before therapy. Note frequent sickled cells. B. Marked decrease in sickle cells with therapy. (Reproduced with permission from Dr. Scott Drury and Dr. Elizabeth Manaloor, Department of Pathology, Medical College of Georgia.)

far from complete. Certain epigenetic mechanisms (histone deacetylation and DNA methylation) are involved in the silencing of the γ-globin genes postnatally. This has led to the use of agents that target the two common epigenetic silencing mechanisms: histone deacetylase inhibitors and DNA methyltransferase 1 inhibitors. The histone deacetylase inhibitors that have been most widely used in early phase small clinical trials in SCD and in some patients with β-thalassemia are butyrate derivatives (arginine butyrate, sodium phenyl butyrate, isobutyramide). Arginine butyrate has to be administered by IV infusion; earlier studies suggested that continuous daily infusions of arginine butyrate were not very effective in leading to a sustained increase in HbF.258 Later, it was shown that daily continuous infusion induced tachyphylaxis and hence the failure to cause a sustained HbF response. An intermittent schedule of administration (4 days, given every 4 weeks) was efficacious in increasing HbF.358 Although orally administered sodium phenyl butyrate was effective in increasing HbF, the daily doses required for maintaining a HbF response required the administration of a large number of tablets and was impractical.359 A phase II trial studying the efficacy of oral 2,2-dimethylbutyrate sodium salt (HQK1001) did not show significant increase in HbF and was associated with a trend for increased VOE.360 The two DNA methyltransferase inhibitors with antiswitching activity are 5′-azacytidine and decitabine. Both of these agents are myelosuppressive when used in higher doses; however, at low doses, they are potent inhibitors of DNA methyltransferase 1 and have been shown to increase HbF synthesis in baboons and in patients with SCD.361–368 Unlike 5′-azacytidine, which incorporates into both DNA and RNA, decitabine incorporates only in DNA and is believed to have a better genotoxicity profile. It has been effective in increasing HbF and ameliorating the disease severity in patients with SCD who have been refractory to hydroxyurea.363 Immunomodulatory agents (thalidomide and derivatives) increase HbF synthesis in erythroid colonies from SCD patients.369 Pomalidomide augments HbF in sickle cell mice.370 Data from use in sickle cell patients is awaited. The finding that the KLF-1–BCL11a axis is the major factor in the switch from β- to γ-globin has made these factors attractive targets for therapy; however, to date, no effective means of targeting these transcription factors has been developed.

Allogeneic Hematopoietic Stem Cell Transplantation

Because SCD is an inherited defect in the hematopoietic stem cell, stem cell transplantation (SCT) is an attractive option to permanently cure the disease rather than managing its sequelae piecemeal. However, the

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tremendous phenotypic variability that characterizes the disorder combined with lack of an accurate predictive model to foretell which patients are likely to have a catastrophic disease course, make selecting patients for allogeneic hematopoietic stem cell transplantation (AHSCT) challenging. AHSCT should be done in patients who are likely to have a severe disease course, but should be instituted early, prior to end-organ damage. The risk-to-benefit ratio of the morbidity and mortality associated with AHSCT has to be weighed against the disease severity of a nonmalignant hematologic disorder. AHSCT is an underused treatment modality in SCD even in eligible patients secondary to lack of donor availability and socioeconomic factors.371 Human leukocyte antigen (HLA)–matched sibling donor transplant with myeloablative conditioning represents the most common transplant type in SCD. Cerebrovascular disease, recurrent ACS, and frequent VOEs despite adequate hydroxyurea therapy are the most common indications for SCT. Data from approximately 1200 patients worldwide show an overall survival of 95 percent; early or late allograft failure resulting in disease recurrence occurs in 10 to 15 percent of patients.371,372 The most common myeloablative regimen used is busulfan, cyclophosphamide, and antithymocyte globulin; the addition of antithymocyte globulin resulted in a significant reduction in allograft rejection. Transplant-related mortality ranges between 2 and 8 percent.372 Acute graft-versus-host disease occurs in approximately 10 to 15 percent of patients, whereas chronic graft-versus-host disease has been reported in 12 to 20 percent of patients. Most series have used cyclosporine alone or in combination with methotrexate for graft-versus-host disease prophylaxis (Chap. 21). Risk of increased incidence of neurologic complications following transplantation has been ameliorated with the use of prophylactic anticonvulsants, strict control of arterial hypertension, correction of hypomagnesemia, and maintenance of Hb greater than 10 g/dL and platelets greater than 50 × 109/L. Long-term toxicity still remains a concern, especially in relation to growth, reproduction, and secondary malignancies. Followup data on AHSCT in children between 1991 and 2000 show significant gonadal toxicity and infertility, especially in females.373 AHSCT in adults is problematic given toxicity of the conditioning regimen. In an attempt to address this issue, reduced-intensity conditioning has been used but has resulted in an increased rate of graft failure. A small cohort of patients who received blood stem cells from HLA-matched siblings and used low-dose total-body radiation plus alemtuzumab as the conditioning regimen followed by sirolimus for graft-versus-host disease prophylaxis had stable engraftment at 30 months of followup.374

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Cord blood and HLA haploidentical transplantation have been used in a small number of patients with SCD, but graft failure remains a significant issue.371,375,376

Transfusion

Red cell transfusions are used frequently in SCD on an acute or chronic basis. The rationale for transfusion in SCD is twofold. Besides increasing Hb concentration, thereby increasing the oxygen-carrying capacity of the blood, transfusion also decreases the percentage of circulating HbS-containing red cells. Hb level alone should not constitute an indication to transfusion as patients adapt to their level, making it important to know the patient’s baseline Hb concentration. It is also important to calculate whether the reticulocyte count, a measure of marrow red cell production, is adequate or not. Indications for red cell transfusion include symptomatic anemia, ACS, stroke, aplastic and sequestration crises, other major organ damage secondary to vasoocclusion, and occurrence of unrelenting priapism. Transfusion is also required prior to major surgery or surgery involving critical organs. The best-established indication for chronic transfusion is stroke and an abnormal TCD velocity. Patients with other chronic or recurrent events are sometimes placed on chronic transfusion as well. Inappropriate indications for transfusion include chronic steady-state anemia, uncomplicated VOE, pregnancy, minor surgeries, infection, and avascular necrosis.377 Simple red cell or exchange transfusion can be used.378 Simple transfusion is easier to perform and is generally associated with fewer complications. Exchange transfusion, however, has the advantage of not raising total Hb, and thereby blood viscosity, while decreasing percentage of circulating sickle cells because sickle cell patients transport less oxygen to their tissues beyond a hematocrit of 30 percent as a result of increased blood viscosity.379–381 Exchange transfusion has also the advantage of not causing iron overload. Alloimmunization occurs in 20 to 50 percent of transfused SCD patients.382–384 In the United States, the majority of blood donors are of European descent, and the majority of SCD patients are of African descent (Chaps. 136 and 138). This results in blood group antigenic disparity, and antibodies to E, C, K, Jkb, S, and Fyb antigens are common. Age at first transfusion, total number of transfusions, transfusion in the context of inflammation, and influence of immunoregulatory genes may affect the rate and extent of alloimmunization.384 Extended antigen phenotyping (Kell, Duffy, Kidd, Lewis, Lutheran, P, and M&S) in addition to the usual ABO and D antigens (Chaps. 136 and 138) and leukodepletion of blood products are recommended.378,382,384,385 Delayed hemolytic transfusion reaction complicates 4 to 11 percent386 of transfusions in SCD and may present as a painful crises. It typically occurs a week after transfusion and is caused by alloantibodies to non-ABO antigens. It can cause the Hb to fall lower than the prior pretransfusion Hb and can be associated with a depressed reticulocyte count and autoantibodies. Alloantibody mediated hemolysis will present as a rapid decrease in the percent of HbA as opposed to HbS. A failure to demonstrate a new alloantibody posttransfusion should not exclude the diagnosis of delayed hemolytic transfusion reaction (Chaps. 136 and 138). Patients should be transfused only if symptomatic under such circumstances as further transfusion can exacerbate the problem.377,384 Iron overload and its attendant complications and infection transmission are the other major complications of transfusion.

Iron Overload

Iron overload (Chap. 43) in SCD is similar to other chronically transfused populations.169,387,388 The multicenter study of the iron overload research group showed that transfused sickle cell patients had increased

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morbidity and mortality when compared to transfused thalassemic patients and nontransfused SCD patients.389 Diagnosing significant iron overload accurately and early can be difficult. Serum ferritin is an easy, widely employed method, but is unreliable in SCD as it is an acute phase reactant. Its measurement can result in over- or underestimation and is poorly correlated to liver iron content.390 A serum ferritin value of greater than 1000 mg/mL in the steadystate has been used as an indication of iron overload. Liver iron content is the current accepted standard and a value of 7.7 mg/g dry weight is used as indication for treatment.391 However, noninvasive methods of assessment of iron overload, like superconducting quantum interference device (SQUID) or MRI T2* (Chap. 43), are becoming standard. Transfusion of 120 mL of red blood cells/kg of body weight can also be used as a chelation trigger.382 Iron chelation (Chap. 43) was typically carried out with desferrioxamine at a dose of 25 to 40 mg/kg per day given over 8 hours subcutaneously.392 Desferrioxamine can reverse cardiac iron overload. A once-daily oral iron chelator, deferasirox, is now approved and available for use in the United States. It is a tridentate ligand that binds iron with a high affinity in a 2:1 ratio. It has a half-life of 8 to 16 hours and is metabolized by glucuronidation and excreted in the feces. In an open-label phase II trial of deferasirox versus desferrioxamine in a 2:1 randomization, safety and tolerability were established. Nausea and vomiting, abdominal pain, rash, reversible increase in liver function tests, and stable increases in serum creatinine were reported. Rare cases of anaphylaxis occurring mostly in the first month of starting treatment have also been reported. Postmarketing reports suggest an increased incidence of renal failure, and caution is to be exercised in a patient population where renal insufficiency may not be readily appreciated prior to starting treatment. Postmarketing experience has also reported cases of fatal hepatotoxicity and agranulocytosis. Auditory and ophthalmic side effects occur in less than 1 percent of patients; however, annual eye and auditory examinations are recommended for deferasirox as they are for desferrioxamine. The recommended daily dose is 20 mg/kg body weight; this dose may be adjusted every 3 to 5 months in increments of 5 to 10 mg/kg if the therapeutic goal is not achieved, although the total dose should not exceed 40 mg/kg. Safety in combination with other iron chelators has not been established.393 Deferiprone is not available in the United States but has been used in other parts of the world. It is orally administered and is considered a better chelator of cardiac iron because of its ability to cross cell membranes.394 Although iron chelation in SCD follows the general guidelines of iron chelation in other iron overloaded populations, rigorous studies of its effects on morbidity and mortality in SCD are lacking.394,395

Evolving Therapies

Given the complex pathophysiology of SCD, numerous therapies targeting different pathways have been tried to ameliorate disease manifestations. Many drugs have failed to show efficacy, especially in phase II/ III trials, because of failure to choose appropriate end points or because they were too narrowly focused. Table 49–4 is a comprehensive list of trials and their outcomes. A few of the novel and promising studies are with immunomodulatory agents (thalidomide/pomalidomide), E- and P-selectin inhibitors, iNKT agonists, and Aes-103, and all are in trials as of this writing.

OTHER ABNORMAL HEMOGLOBINS The number of Hb variants discovered to the time of writing this chapter totals 1187. The vast majority of these variants are benign, without any significant clinical or hematologic problems, but are of interest to geneticists and biochemists (http://globin.cse.psu.edu). Most of the

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TABLE 49–4.  Novel Therapies for Sickle Cell Disease Mechanism of Action

Pathway T argeted in SCD

GMI1070

E-selectin inhibitor

Aes-103

Allosteric modifier of Hb

Regadenoson

Drug

Trial Phase/Type

Number Enrolled

Abnormal cell adhesiveness

I

RBC sickling, membrane stabilization under shear stress

Outcomes

Ref.

15

Decrease in coagulation, leukocyte, and endothelial cell activation

412

I/IIa

18

Decrease in pain and markers of RBC sickling

iNKT A2A receptor Inflammation agonist

I

27

Safety demonstrated; iNKT cells inhibited

413,414

Omega-3 fatty acid

Reduction in oxidative injury

Abnormal cell adhesiveness

RCT

140

Decreased VOE, anemia, and blood transfusion in supplemented group

415

Arginine

Increased NO production

NO signaling

RCT

38

Decreased parenteral opioids use and pain scores

416

Magnesium sulfate

Increased cellular hydration

Cellular dehydration

RCT

106

No difference on LOS, pain scores, or analgesia use

417

Prasugrel

P2Y12 ADP recep- Platelet activation tor antagonist

II

62

Pain rate and intensity decreased in intervention; platelet activation biomarkers decreased

418

Eptifibatide

Platelet αIIbβ3 inhibitor

Platelet activation

RCT

13

Safe but no difference in VOE resolution

419

Senicapoc

Gardos channel inhibitor

Cellular dehydration

III

144

Increased hemoglobin and hematocrit and decreased erythrocytes and reticulocytes

420

Poloxamer 188

Amphipathic copolymer

Tissue oxygenation

III

255

Safe and well tolerated and demonstrated crisis resolution in a percentage of patients (greater in children than adults)

421

TRF-1101

P-selectin inhibitor

Abnormal cell adhesiveness

II

5

Safe and increased microvascular blood flow

422

ADP, adenosine diphosphate; Hb, hemoglobin; iNKT, invariant natural killer T cell; LOS, length of stay; NO, nitric oxide; RBC, red blood cell; RCT, randomized controlled trial; SCD, sickle cell disease; VOE, vasoocclusive episode.

Hb variants are missense mutations in the globin genes (α, β, γ, or δ) resulting from single nucleotide substitutions. Other uncommon mechanisms include deletion or insertion of one or more nucleotides altering the reading frame and fusion of globin genes with deletion of intergenic DNA sequences (γβ fusion in HbKenya and δβ fusion in HbLepore), mutations of the termination codon leading to the production of elongated globin chains. Hb variants that significantly alter the structure, stability, synthesis, or function of the molecule have hematologic and/or clinical consequences. These can be classified in certain categories (Table 49–5). HbS and HbC are two examples of mutations on the surface of the Hb molecule that alter both the charge and the physical/chemical properties of the molecule with polymer formation in the case of deoxyhemoglobin S and crystallization in HbC with profound effects on the function, morphology, rheology, and life span of the red cells. Several mechanisms account for the pathogenesis of unstable Hb variants. The common mechanism involves the precipitation of the unstable Hb molecule within the red cell with attachment to the inner layer of the red cell membrane (“Heinz body” formation); red cells containing

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membrane-attached Heinz bodies (see Chap. 31, Fig. 31–11) have impaired deformability and filterability leading to their premature destruction (congenital Heinz body hemolytic anemia). Mutations in certain residues alter the oxygen affinity of the Hb molecule; a stabilization of the R (relaxed, oxy) state will result in high O2 affinity variants and erythrocytosis. Conversely, a stabilization of the T (tense, deoxy) configuration will result in a variant with low O2 affinity with enhanced unloading of O2 to the tissues with resultant cyanosis and anemia in certain cases (because of the suppression of the O2 sensing pathway) (Chaps. 32 and 50). Mutations of the heme binding site, particularly those affecting the conserved proximal (F8) and distal (E7) histidine residues, lead to the oxidation of the iron atom in heme from ferrous (Fe2+) to ferric (Fe3+) state with resultant methemoglobinemia (M Hbs) and cyanosis (Chap. 50). A group of mutations alter both the structure and the synthetic rate of the globin chain leading to a “thalassemic” phenotype (Chap. 48). These include fusion Hbs (e.g., HbLepore, where the 5′ δ-globin sequences are fused to 3′ β-globin sequences with deletion of the intergenic DNA; this puts the δβ-fusion gene under the transcriptional control of the inefficient δ-globin promoter with low expression

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TABLE 49–5.  Clinically Significant Hemoglobin Variants I. Altered physical/chemical properties A. HbS (deoxyhemoglobin S polymerization): sickle syndromes B. HbC (crystallization): hemolytic anemia; microcytosis II. Unstable hemoglobin variants: A. Congenital Heinz body hemolytic anemia (N = 135) III. Variants with altered oxygen affinity A. High-affinity variants: erythrocytosis (N = 92) B. Low-affinity variants: anemia, cyanosis IV. M hemoglobins A. Methemoglobinemia, cyanosis (N = 9) V. Variants causing a thalassemic phenotype (N = 50) A. β-Thalassemia 1. HbLepore (δβ) fusion (N = 3) 2. Aberrant RNA processing (HbE, HbKnossos, HbMalay) 3. Hyperunstable globins (HbGeneva, HbWestdale, etc.) B. α-Thalassemia 1. Chain termination mutants (HbConstant Spring) 2. Hyperunstable variants (HbQuong Sze) Data from Bunn HF, Forget BG: Hemoglobin: Molecular, Genetic, and Clinical Aspects. Philadelphia, PA: WB Saunders; 1986.

of the fusion globin (hence the thalassemic phenotype), mutations that cause both a missense mutation and create an aberrant splice site (such as HbE, HbKnossos, and HbMalay), and “hyperunstable” globins where the nascent globin chains are highly unstable, undergo rapid proteolytic degradation, and result in a reduction in the affected globin. Except for the commonly occurring variants (HbS, HbC, HbE, and HbDLos Angeles), very few abnormal Hbs have been observed in the homozygous state. Variant Hbs are usually found in the heterozygous state. Although γ-chain variants are expressed in fetal life and their level gradually decreases as the γ-globin to β-globin (fetal to adult) switch progresses during the postnatal period, β- and α-chain variants are expressed throughout life. δ-Globin variants are expressed at very low levels and can be detected only after the switch to adult globin synthesis is complete. Because α-globin chains are present in all of the Hbs expressed after the embryonic stage (HbF-α2γ2; HbA-α2β2, and HbA2α2δ2), α-chain variants are associated with the production of variant HbF (α2xγ2) and HbA2 (α2xδ2) as well. In heterozygous states, β-chain variants constitute 40 to 50 percent of the Hb in red cells; it should, however, be kept in mind that certain factors affect the amount of variant β chains in carriers. These factors include the stability of the variant, the surface charge of the variant β-chain, and the presence of concomitant α- or β-thalassemia (Chap. 48). The more unstable the variant, the lower the quantity. Surface charge of the variant also plays a role in determining the quantity in red cells; this is because the formation of the αβ-dimers (α1β1 and α2β2 contacts) is the critical first step in Hb tetramer formation, and this step is primarily driven by electrostatic interactions between α and β chains. The α-globin chains have a relatively positive surface charge, they interact more readily with relatively negatively charged β-globin variants to form αβ dimers. This is reflected in the higher percentage of negatively charged β-globin variants such as HbNBaltimore (β95Lys→Glu), which is found in approximately 50 percent in heterozygotes compared to β-globin variants with a positive surface charge, HbS (β6Glu→Val) or HbC (β6Glu→Lys) whose quantity in the

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heterozygote is 40 to 45 percent. In the presence of α-thalassemia, negatively charged β-globin variants compete more favorably for the available α-chains; this phenomenon is reflected in even lower percentages of HbS and HbC in heterozygous carriers of these variants in the presence of common deletional forms of α-thalassemia (HbS of 30 to 35 percent in individuals with heterozygous α+-thalassemia, –α/αα; and 25 to 30 percent in homozygous α+-thalassemia, –α/–α).396,397 Conversely, the amount of a β-globin variant will increase if there is a β-thalassemia allele in trans; the percentage of the variant will be inversely proportional to the output of the β-thalassemia allele; thus, the higher the variant the lower the output of the β+-thalassemia allele. In the case of a β0-thalassemia allele in trans, the variant will amount to greater than 90 percent or more of the Hb in red cells, with HbA2 and HbF constituting the remainder. The quantity of α-globin variants is also variable, depending on the α-globin gene involved, and the presence of concomitant α- or β-thalassemia. Because there are normally four α-globin loci (αα/αα) and the upstream 5′ α-globin gene (α2) is expressed at a higher level, some of the variation in the level of α-globin variants depends on which α-globin gene carries the mutation; α2-globin mutations are usually present at 20 to 25 percent of the total Hb, whereas 3′ downstream α1-globin variants are expressed at a lower level (15 to 20 percent). Concomitant α-thalassemia results in a higher level of expression of α-globin variants. Observations on the different levels of expression of the common α-globin variant, HbGPhiladelphia (α68Asn→Lys), is a case in point.398 Although this variant is found in approximately 25 percent of Northern Italians, its percentages in Americans of African descent can be either 33 or approximately 50 percent. This is clearly related to the different genotypes found in these two distinct populations: In northern Italy and Sardinia, the genotype is αGα/αα, with an expression level of approximately 30 percent, whereas in Americans of African descent, the HbGPhiladelphia mutation is commonly found on a hybrid α2α1 gene associated with the common 3.7 kb α+-thalassemia deletion (–αG/αα) with approximately 33 percent expression. When there is an α+-thalassemia deletion in trans (–αG/–α genotype), as expected, the level of HbGPhiladelwill be approximately 50 percent. Coinheritance of α-chain variants phia with β-thalassemia results in the increase of the α-chain variant.

HEMOGLOBIN C DISEASE Definition and History

HbC was the second Hb variant described after HbS.399 Homozygous HbC was described by Spaet and colleagues400 and Ranney and colleagues.401 HbC trait is found in 2 percent of Americans of African descent, and approximately one in 6000 have homozygous HbC.402 Coinheritance of HbC with HbS results in HbSC disease, which is the second most common form of SCD in the United States. There are also rare cases of HbC-β+-thalassemia and HbC–β0-thalassemia. HbC is thought to have originated in Central West Africa and in parts of West Africa, where the prevalence of HbC can reach 12.5 percent. The HbC gene was found on three distinct β-globin cluster haplotypes, termed CI, CII, and CIII; the most common is CI, accounting for 70 percent or more of HbC studied.403

Etiology and Pathogenesis

HbC is the result of a GAG→AAG transition in codon 6 of the β-globin gene, which changes the amino acid residue at this position from glutamic acid to lysine (Glu→Lys). The resultant positively charged Hb variant can easily be distinguished from HbA and HbS by electrophoresis and chromatography, including high-performance liquid chromatography. HbC does not differ from HbA in its solubility; however, purified solutions of HbC form tetragonal crystals in high-molarity phosphate buffer. The Hb in red cells from homozygous HbC individuals also

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A

B

C

D

E

F

G

H

I

779

Figure 49–11.  Blood cell morphology in patients with structural hemoglobinopathies. A. Blood film. Hemoglobin (Hb) SS disease with characteristic sickle-shaped cells and extreme elliptocytes with dense central Hb staining. Both shapes are characteristic of sickled cells. Occasional target cells. B. Phase-contrast microscopy of wet preparation. Note the three sickled cells with terminal fine-pointed projections as a result of tactoid formation and occasional target cells. C. HbSC disease. Blood film. Note the high frequency of target cells characteristic of HbC and the small dense, irregular, contracted cells reflective of their content of HbS. D. HbCC disease. Blood film. Characteristic combination of numerous target cells and a population of dense (hyperchromatic) microspherocytes. Of the nonspherocytic cells, virtually all are target cells. E. HbCC disease postsplenectomy. Blood film. Note the rod-like inclusions in two cells as a result of HbC paracrystallization. These cells are virtually all removed in patients with spleens. F. HbCC disease postsplenectomy. Phase-contrast microscopy of wet preparation. Note the HbC crystalline rod in a cell. G. HbDD disease. Blood film. Note Frequent target cells admixed with population of small spherocytes, poikilocytes, and tiny red cell fragments. H. HbEE disease. Blood film. Hypochromia, anisocytosis, and target cells. I. HbE thalassemia. Blood film. Marked anisocytosis (primarily microcytes) and poikilocytosis. Hypochromia. (Reproduced with permission from Lichtman’s Atlas of Hematology, www.accessmedicine.com.)

form crystals when incubated with hypertonic saline; HbC crystals are also observed in vivo, particularly in the red cells of splenectomized HbCC patients (Fig. 49–11F). Crystal-containing HbCC red cells have impaired deformability and filterability. HbCC red cells have a propensity for potassium (K+) loss, which is followed by water loss; unlike in sickle red cells, this K+ leak does not appear to be mediated through either the potassium chloride cotransport or the calcium ion activated K+ efflux (Gardos) channel; it is thought to be a volume-stimulated K+ efflux.402 The consequence of this K+ loss is dehydrated, often spherocytic, red cells with increased MCHC, and decreased osmotic fragility. These changes result in impaired rheologic properties of HbCC red cells; their life span is reduced to 40 days.

Clinical Features

Mild to moderate splenomegaly is a common feature of homozygous HbC. Like many other chronic hemolytic states, cholelithiasis may be present. HbCC individuals do not suffer from vasoocclusion or episodic pain. Occasionally, abdominal pain may be present and can be a

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result of splenomegaly and/or cholelithiasis. Pregnancy does not pose an increased risk to women with HbCC. Life expectancy of HbCC individuals is comparable to non-HbC Americans of African descent. In a recent single-institution study, splenomegaly and cholelithiasis occurred in approximately 2.5 percent of patients younger than 8 years of age, but it was far more common (71 percent) in individuals older than 8 years of age.404

Laboratory Features

HbCC individuals have a mild to moderate hemolytic anemia. Hb is usually in the 10 to 11 g/dL range. There is associated reticulocytosis usually in the 3 to 4 percent range. There usually is mild microcytosis (mean corpuscular volume [MCV] 70 to 75 fL) and, often, an elevated MCHC. The blood film is characteristic, showing an abundance of target cells, microspherocytes, and HbC red cell crystals, especially in splenectomized patients (see Fig. 49–11F). Indirect bilirubin may be mildly elevated. White cell and platelet counts are normal in the absence of hypersplenism.

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Differential Diagnosis

Differential diagnosis is usually achieved by Hb electrophoresis. HbC moves to a cathodic position, comigrating with HbA2, HbE, and HbOArab on alkaline pH (cellulose acetate) electrophoresis. The distinction from these Hbs can be made by electrophoresis on citrate agar in acid pH where HbE and HbA2 comigrate with HbA; HbOArab has a HbS-like mobility, and HbC has a unique migration pattern. Alternatively, newer diagnostic methods can be used; these include isoelectric focusing, where HbC can readily be distinguished from other Hbs with similar mobility on cellulose acetate electrophoresis. In cation exchange HPLC and capillary electrophoresis, HbC has a distinct elution pattern and can be distinguished from HbE and HbOArab; these latter methods also have the advantage of separating and quantifying HbA2 in HbC homozygotes and in HbC trait. This confers the advantage of readily differentiating between HbCC and rare cases of HbC– β0–thalassemia (where HbA2 is significantly higher, ~5 percent).

Therapy

The vast majority of HbCC individuals do not require any therapeutic intervention. Cholecystectomy may be required in individuals who have symptomatic gallstones. Few patients with HbCC develop hypersplenism with a reduction in white cell and platelet counts, and occasionally worsening of anemia. In such instances, splenectomy should be considered. Another indication for splenectomy is pain associated with an enlarged spleen. It is important to apply the usual precautions in patients considered for splenectomy (appropriate vaccinations, prophylactic antibiotic use, and delaying splenectomy in young children). Folic acid supplementation, as usually done in many chronic hemolytic states, is of no proven value.

HEMOGLOBIN DISEASE Definition and History

HbE (β26Glu→Lys) was the fourth abnormal Hb described.405 It is most commonly found in Southeast Asia; in some areas (in the border between Thailand, Laos, and Cambodia, the so-called HbE triangle) the reported gene frequency may reach as high as 0.50.406 This high frequency is thought to be from a protective effect against malaria. HbE is also found in other malaria-endemic areas such as Bangladesh, India, and Madagascar. HbE now has a wide distribution as a result of the large population movements from Southeast and South Asia to Western Europe and North America, and may now be the most common Hb variant worldwide.

Etiology and Pathogenesis

The GAG→AAG mutation in codon 26 of the β-globin gene not only leads to a missense mutation (Glu→Lys) at this position, but also activates a cryptic donor splice site at the boundary of exon 1 and intron 1 by increasing the sequence similarity of this site to a consensus splice sequence. The resultant aberrant splicing through this alternate site leads to a decrease in the correctly spliced messenger RNA and hence a β+-thalassemic phenotype. This is reflected in the fact that heterozygotes for HbE have 25 to 30 percent of the variant; in the presence of concomitant α-thalassemia, this quantity decreases even further. The coinheritance of HbE with a host of other globin mutants (α-thalassemia, β-thalassemia, other Hb variants), which are also common in the populations where HbE is prevalent, results in a wide spectrum of hemoglobinopathies with varying degrees of severity (HbE disorders or HbE syndromes). The most significant of these is HbE–β-thalassemia syndromes. HbE has also been reported in combination with HbS (HbSE disease).

Clinical Features

Individuals with homozygous HbE are asymptomatic. Most patients do not have hepatosplenomegaly or jaundice. They are usually diagnosed

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during screening programs or family studies of individuals with severe HbE disorders, or on routine evaluation of a blood film with significant microcytosis without anemia. HbE–β-thalassemia is a rather heterogeneous group of disorders varying from a mild thalassemia intermedia like phenotype to severe transfusion dependent thalassemia major (Chap. 48). Part of this heterogeneity results from the type of coinherited β-thalassemia mutation. Patients who are compound heterozygotes for HbE and one of the mild β+-thalassemia mutations (such as the mild promoter mutation, –28A→G) have a mild to moderate anemia, whereas patients with compound heterozygosity for HbE and one of the more severe β+-thalassemia mutations (such as IVS I nucleotide 5 or IVS II nucleotide 654 mutations) do have a more severe phenotype with severe anemia and transfusion dependency. There is also a large heterogeneity among patients with HbE–β0-thalassemia; these patients do not produce any HbA and have only HbE and varying amounts of HbF. Known factors that influence the phenotype include the ability to produce HbF and the presence of concomitant α-thalassemia. Individuals who have the propensity to synthesize significant amounts of HbF (such as those who have the Xmn I C→T mutation in the Gγ-globin promoter) are able to ameliorate the globin chain imbalance and thus have a milder phenotype. Concomitant α-thalassemia also mitigates the course of the disease by decreasing globin chain imbalance. In some cases, there may be nonglobin modifiers that impact on the phenotype. Patients with severe forms of HbE–β0-thalassemia have clinical features very similar to β-thalassemia major; they develop complications such as hypersplenism, iron overload, increased susceptibility to infections, thromboembolic complications, and heart failure, and have a shortened life expectancy.406 Splenectomized HbE–β-thalassemia patients have more pronounced intravascular hemolysis, markers of endothelia cell activation, and activation of coagulation with increased levels of cell free Hb, sE-selectin, sP-selectin, high-sensitivity C-reactive protein, and thrombin–antithrombin complex compared to nonsplenectomized patients.407

Laboratory Features

HbE-trait individuals have a borderline microcytosis (MCV in the lower 80s) but are not anemic. Homozygotes for HbE are usually only borderline or mildly anemic (Hb 11 to 13 g/dL), but they are microcytic (MCV ~70 fL). Blood film shows target cells, hypochromia, and microcytosis (see Fig. 49–11H). Osmotic fragility of the red cells is decreased. Hb electrophoresis shows greater than 90 percent HbE and 5 to 10 percent HbF. Certain chromatography techniques that can separate HbE from HbA2 reveal elevated levels of HbA2. Patients with mild forms of HbE–β+-thalassemia (Chap. 48) have Hb levels in the 9.0 to 9.5 g/dL range, whereas those with severe HbE–β+-thalassemia are more severely anemic (Hb 6.5 to 8.0 g/dL). Individuals with HbE–β0-thalassemia have varying degrees of anemia, depending on their ability to produce HbF; these patients have HbE in the 40 to 60 percent range, with the remainder being HbF. Patients with higher HbF values are less anemic.

Therapy

HbE homozygotes do not require any therapy. Patients with severe HbE–β0-thalassemia are similar to thalassemia intermedia or major; most of the latter patients should be on a chronic transfusion regimen aiming at Hb levels of approximately 10 g/dL; iron chelation should be a part of standard therapy. Splenectomy should be considered when hypersplenism develops. Patients with a thalassemia intermedia-like phenotype may require sporadic transfusions. Hydroxyurea can increase HbF levels and decrease ineffective erythropoiesis in HbE–β-thalassemia.408 AHSCT (including umbilical cord blood–derived stem cells in one patient) has also been used in HbE–β-thalassemia.

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Chapter 49: Disorders of Hemoglobin Structure: Sickle Cell Anemia and Related Abnormalities

Course and Prognosis

The prognosis is dependent upon the clinical phenotype. Patients with milder phenotypes tend to do well. Severe HbE–β-thalassemia patients require chronic red cell transfusion and iron-chelation therapy; this places a great burden on the economies of countries where this disease is prevalent. AHSCT, although potentially curative, will not be available for the vast majority of these patients. Prenatal diagnosis and neonatal screening should be an important part of the strategies to decrease the disease burden and improve care. Long-term use of hydroxyurea and other novel HbF-inducing agents as modifiers of disease (histone deacetylase inhibitors and DNA methyltransferase 1 inhibitors) can be an important addition to therapy.

HEMOGLOBIN D DISEASE HbD was the third Hb variant identified.409 The substitution in HbD is a glutamic acid to glutamine at the 121st amino acid of the β-globin chain (β121Glu→Gln). HbD has an S-like mobility on alkaline electrophoresis, but comigrates with HbA on acid pH. Subsequently, a number of other Hb variants with the same electrophoretic properties were discovered and named HbD (HbDIbadan, HbDGainesville, etc.). The most common HbD is HbDLos Angeles (β121Glu→Gln), the originally discovered HbD, which is identical to HbDPunjab. It is most commonly found in Punjab, India where 2 to 3 percent of the population have the HbD gene. Subsequently, it has also been found in a number of other populations including Europeans of Mediterranean region, and Americans of African descent.410 HbD heterozygotes are asymptomatic, are not anemic, and have normal red cell indices. Homozygotes for HbDLos Angeles are asymptomatic and are hematologically normal with normal red cell indices. Blood films may show target cells (see Fig. 49–11G). Osmotic fragility may be decreased. Compound heterozygotes for HbDLos Angeles and a β0-thalassemia mutation have mild microcytic anemia and show minimal hemolysis. Coinheritance of HbDLos Angeles with HbS results in a severe SCD phenotype not different from homozygous HbS. HbDLos Angeles should be distinguished from HbS. This can be done by a combination of routine alkaline and acid Hb electrophoretic methods. Techniques such as isoelectric focusing, HPLC, and capillary electrophoresis readily provide this distinction. Such methods allow accurate diagnosis of SCD from compound heterozygosity for HbS and HbDLos Angeles.

UNSTABLE HEMOGLOBINS Unstable Hbs form an important group of clinically significant Hb variants. Several different mechanisms lead to the generation of unstable variants, which result in a congenital hemolytic anemia with inclusion bodies in red cells (Heinz bodies), hence the term congenital Heinz body hemolytic anemia.

Definition and History

Cathie reported a 10-month-old child with hemolytic anemia, jaundice, and splenomegaly in 1952.411 Splenectomy did not result in improvement. The patient’s red cells had large Heinz bodies (Chap. 31). Similar cases were reported from around the world, and the observation that these cases were characterized by the precipitation of their hemolysate upon exposure to heat, suggested a Hb abnormality as the cause. Subsequently, nearly all of similar cases were found to have a variant Hb, and Cathie’s case was found to have Hb-Bristol (β67Val→Asp). To date, 146 unstable variants have been reported; the vast majority is sporadic cases reported only once. Few have been observed repeatedly in different populations.

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Etiology and Pathogenesis

Several different mechanisms lead to the instability of the globin molecule with precipitation in the red cell leading to hemolysis. These are summarized below. Substitutions Near the Heme Pocket  Heme is inserted into a hydrophobic pocket in each globin molecule where it is in contact with a number of invariant nonpolar amino acid residues (see Fig. 49–2). Substitution of these invariant nonpolar residues will decrease the stability of heme-globin association and ultimately lead to the instability of the globin moiety. HbZurich (β63His→Arg), HbKoln (β98Val→Met), and HbHammersmith (β42Phe→Ser) are examples of this group. Disruption of Secondary Structure (α-Helix) The secondary structure of globin chains is 75 percent in the conformation of an α helix (see Fig. 49–1). Proline residues cannot participate in an α helical conformation. Thus, the substitution of a proline residue for any other amino acid except for the first three residues of an α helix will disrupt the secondary structure and lead to the disruption and precipitation of the mutant globin chain. Mutations in α1β1 Interface  The first step in the assembly of the Hb tetramer is the formation of an αβ dimer. This structure is stabilized by a secondary structure that exposes the charged amino acids (glutamic acid, aspartic acid, lysine, and arginine) on the surface of the molecule in contact with water and stabilizes the interior of the molecule (α1β1 interface) with hydrophobic interactions. Substitution of a charged (polar) residue for a nonpolar amino acid involved in α1β1 contact will disrupt and destabilize this dimer formation and lead to the precipitation of the Hb molecule. Amino Acid Deletions  Deletion of one or more amino acid residues is expected to disrupt the secondary structure of the globin chains and may lead to instability of the mutant chain. Mutant globins with deletion of one or more residues have been reported. Examples of this type include HbLeiden (β6 or β7Glu→0), HbGun Hill (β91-95→0), and HbFrei(β23Val→0). burg Elongated Globin Chains  Some variants result from either a mutation in the termination codon or a frameshift leading to the synthesis of longer than normal globin chains. These variants tend to be unstable because of the presence of a nonfunctional fragment. Examples include HbCranston and HbTak. Whatever the underlying mechanism may be, unstable Hb variants precipitate within developing red cell precursors forming hemichromes (intermediate substances in Hb denaturation) and ultimately aggregates that attach to the inner layer of red cell membrane (Heinz bodies). Heinz bodies can be visualized with supravital stains, such as brilliant cresyl blue. Red cells with Heinz bodies have impaired rheologic properties (deformability and filterability) and are trapped in the splenic circulation (Chaps. 6, 34, and 56) with pitting of the membrane attached bodies. Hemolysis ultimately ensues. The degree of hemolysis is proportionate to the quantity and the instability of the variant.

Clinical Features

Patients with unstable Hb variants have varying degrees of hemolytic anemia. This can range from a compensated, asymptomatic hemolytic state to severe, life-threatening hemolysis. Generally, hemolytic anemia is mild to moderate and does not require therapeutic intervention. Typically, hemolysis is exacerbated by increased oxidant stress such as infections and the use of oxidant drugs. Patients may have jaundice and splenomegaly. As is the case with other chronic hemolytic states, gallstones may develop. Hypersplenism can be a problem in some cases. Many unstable Hb variants that are associated with mild, compensated hemolysis are diagnosed fortuitously or during population screening for hemoglobinopathies. Unstable variants are inherited in a mendelian pattern; they usually manifest in the heterozygous state. There are

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instances of de novo mutations without evidence of the variant in parents of an affected individual. Many of the 146 known unstable variants are found in a single individual or in a limited number of instances. However, some unstable variants like HbKoln (β98Val→Met) and HbZurich (β67His→Arg) have been found in many populations around the world.

Laboratory Features

Patients with unstable Hb variants may have varying degrees of anemia. Generally, the anemia is mild and does not require therapeutic intervention. However, exacerbation of anemia during exposure to oxidant stress (such as infections and the use of oxidant drugs) is a common feature. Features of a hemolytic state (reticulocytosis, indirect hyperbilirubinemia, elevated LDH, decreased or undetectable haptoglobin) are present. Red cell morphology shows polychromasia, anisocytosis, poikilocytosis, and occasionally basophilic stippling. A typical feature of this disorder is the presence of Heinz bodies, best visualized with supravital staining with brilliant cresyl blue as membrane attached inclusion bodies in red cells. Hb electrophoresis reveals the presence of an additional abnormal Hb band. The quantity of the variant Hb is variable and inversely proportional to the degree of instability of the abnormal Hb (e.g., the more unstable the variant, the less the quantity). More accurate quantification can be achieved with cation exchange or reversed phase HPLC. The presence of an unstable variant in the hemolysate can be demonstrated by simple tests of stability. The most commonly used tests are heat denaturation and isopropanol precipitation. The heat denaturation test is more cumbersome and time consuming and is seldom used in practice. The isopropanol precipitation test is a simple screening test for unstable variants and involves the incubation of the hemolysate with a 17 percent solution of isopropanol; hemolysates containing unstable Hb variants will form a precipitate, whereas a normal hemolysate will remain clear.

HEMOGLOBINS WITH ALTERED OXYGEN AFFINITY M Hbs result from mutations around the heme pocket that disrupt the hydrophobic nature of this structure with resultant oxidation of the iron in the heme moiety from ferrous (Fe2+) to ferric (Fe3+) state and cause methemoglobinemia (Chap. 50). Mutations in certain critical areas of the globin molecule alter the affinity of the globin for oxygen. In general, mutations that stabilize the molecule in the T state lead to low O2-affinity variants, which can clinically manifest as cyanosis or mild anemia. Mutations that stabilize the R state or destabilize the T state result in high O2-affinity variants. These variants will cause secondary polycythemia (Chap. 57). The mutations that affect the ligand binding affinity of the Hb molecule are mostly in the α1β2 interface. Rarely, mutations in the α1β1 interface lead to altered O2 affinity. Another mechanism in the generation of high O2 affinity mutants involves mutations that alter the binding of 2,3-BPG.

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375. Ruggeri A, Eapen M, Scaravadou A, et al: Umbilical cord blood transplantation for children with thalassemia and sickle cell disease. Biol Blood Marrow Transplant 17(9):1375–1382, 2011. 376. Bolanos-Meade J, Fuchs EJ, Luznik L, et al: HLA-haploidentical bone marrow transplantation with posttransplant cyclophosphamide expands the donor pool for patients with sickle cell disease. Blood 120(22):4285–4291, 2012. 377. Smith-Whitley K, Thompson AA: Indications and complications of transfusions in sickle cell disease. Pediatr Blood Cancer 59(2):358–364, 2012. 378. Telen MJ: Principles and problems of transfusion in sickle cell disease. Semin Hematol 38(4):315–323, 2001. 379. Chien S, Usami S, Bertles JF: Abnormal rheology of oxygenated blood in sickle cell anemia. J Clin Invest 49(4):623–634, 1970. 380. Morris CL, Gruppo RA, Shukla R, et al: Influence of plasma and red cell factors on the rheologic properties of oxygenated sickle blood during clinical steady state. J Lab Clin Med 118(4):332–342, 1991. 381. Schmalzer EA, Lee JO, Brown AK, et al: Viscosity of mixtures of sickle and normal red cells at varying hematocrit levels. Implications for transfusion. Transfusion 27(3):228– 233, 1987. 382. Vichinsky EP: Current issues with blood transfusions in sickle cell disease. Semin Hematol 38(1 Suppl 1):14–22, 2001. 383. Rosse WF, Gallagher D, Kinney TR, et al: Transfusion and alloimmunization in sickle cell disease. The Cooperative Study of Sickle Cell Disease. Blood 76(7):1431–1437, 1990. 384. Yazdanbakhsh K, Ware RE and Noizat-Pirenne F. Red blood cell alloimmunization in sickle cell disease: Pathophysiology, risk factors, and transfusion management. Blood 120(3):528–537, 2012. 385. Wahl S, Quirolo KC: Current issues in blood transfusion for sickle cell disease. Curr Opin Pediatr 21(1):15–21, 2009. 386. Talano JA, Hillery CA, Gottschall JL, et al: Delayed hemolytic transfusion reaction/ hyperhemolysis syndrome in children with sickle cell disease. Pediatrics 111(6 Pt 1):e661–e665, 2003. 387. Ballas SK: Iron overload is a determinant of morbidity and mortality in adult patients with sickle cell disease. Semin Hematol 38(1 Suppl 1):30–36, 2001. 388. Vichinsky E, Butensky E, Fung E, et al: Comparison of organ dysfunction in transfused patients with SCD or beta thalassemia. Am J Hematol 80(1):70–74, 2005. 389. Fung EB, Harmatz P, Milet M, et al: Morbidity and mortality in chronically transfused subjects with thalassemia and sickle cell disease: A report from the multi-center study of iron overload. Am J Hematol 82(4):255–265, 2007. 390. Brittenham GM, Cohen AR, McLaren CE, et al: Hepatic iron stores and plasma ferritin concentration in patients with sickle cell anemia and thalassemia major. Am J Hematol 42(1):81–85, 1993. 391. National Institutes of Health, Division of Blood Diseases and Resources: The Management of Sickle Cell Disease, ed 4. NIH Publication No. 02–2117. NIH, Bethesda, MD, 2002. Available at: http://www.nhlbi.nih.gov/health/prof/blood/sickle/sc_mngt.pdf 392. Silliman CC, Peterson VM, Mellman DL, et al: Iron chelation by deferoxamine in sickle cell patients with severe transfusion-induced hemosiderosis: A randomized, double-blind study of the dose-response relationship. J Lab Clin Med 122(1):48–54, 1993. 393. Vichinsky E, Onyekwere O, Porter J, et al: A randomised comparison of deferasirox versus deferoxamine for the treatment of transfusional iron overload in sickle cell disease. Br J Haematol 136(3):501–508, 2007. 394. Lucania G, Vitrano A, Filosa A, et al: Chelation treatment in sickle-cell-anaemia: Much ado about nothing? Br J Haematol 154(5):545–555, 2011. 395. Brittenham GM: Iron-chelating therapy for transfusional iron overload. N Engl J Med 364(2):146–156, 2011. 396. Steinberg MH, Adams JG 3rd, Dreiling BJ: Alpha thalassaemia in adults with sickle-cell trait. Br J Haematol 30(1):31–37, 1975. 397. Wong SC, Ali MA, Boyadjian SE: Sickle cell traits in Canada. Trimodal distribution of Hb S as a result of interaction with alpha-thalassaemia gene. Acta Haematol 65(3):157– 163, 1981. 398. Sciarratta GV, Sansone G, Ivaldi G, et al: Alternate organization of alpha G-Philadelphia globin genes among U.S. black and Italian Caucasian heterozygotes. Hemoglobin 8(6):537–547, 1984.

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399. Itano HA, Neel JV: A new inherited abnormality of human hemoglobin. Proc Natl Acad Sci U S A 36(11):613–617, 1950. 400. Spaet TH, Alway RH, Ward G: Homozygous type c hemoglobin. Pediatrics 12(5):483– 490, 1953. 401. Ranney HM, Larson DL, McCormack GH Jr: Some clinical, biochemical and genetic observations on hemoglobin C. J Clin Invest 32(12):1277–1284, 1953. 402. Nagel RL, Steinberg MH: Hb SC disease and Hb C disorders, in Disorders of Hemoglobin: Genetics, Pathophysiology, and Clinical Management, edited by Steinberg MH, Forget BG, Higgs DR, Nagel RL, pp 756–785. Cambridge University Press, Cambridge, 2001.. 403. Boehm CD, Dowling CE, Antonarakis SE, et al: Evidence supporting a single origin of the beta(C)-globin gene in blacks. Am J Hum Genet 37(4):771–777, 1985. 404. Cook CM, Smeltzer MP, Mortier NA, et al: The clinical and laboratory spectrum of Hb C [beta6(A3)Glu—>Lys, GAG>AAG] disease. Hemoglobin 37(1):16–25, 2013. 405. Itano HA, Bergren WR, Sturgeon P: Identification of fourth abnormal human hemoglobin. J Am Chem Soc 76:2278, 1954. 406. Fucharoen S: Hb E disorders, in Disorders of Hemoglobin: Genetics, Pathophysiology, and Clinical Management, edited by Steinberg MH, Forget BG, Higgs DR, Nagel RL, pp 1139–1154. Cambridge University Press, Cambridge, 2001. 407. Atichartakarn V, Chuncharunee S, Archararit N, et al: Intravascular hemolysis, vascular endothelial cell activation and thrombophilia in splenectomized patients with hemoglobin E/beta-thalassemia disease. Acta Haematol 132(1):100–107, 2014. 408. Fucharoen S, Siritanaratkul N, Winichagoon P, et al: Hydroxyurea increases hemoglobin F levels and improves the effectiveness of erythropoiesis in beta-thalassemia/hemoglobin E disease. Blood 87(3):887–892, 1996. 409. Itano HA: A third abnormal hemoglobin associated with hereditary hemolytic anemia. Proc Natl Acad Sci U S A 37(12):775–784, 1951. 410. Huisman THJ, Carver MFH, Efremov GD: A Syllabus of Human Hemoglobin Variants. The Sickle Cell Anemia Foundation, Augusta, GA, 1998. 411. Cathie IAB: Apparent idiopathic Heinz body anaemia. Great Ormond St J 3:343, 1952. 412. Wun T, Styles L, DeCastro L, et al: Phase 1 study of the E-selectin inhibitor GMI 1070 in patients with sickle cell anemia. PLoS One 9(7):e101301, 2014. 413. Nathan DG, Field J, Lin G, et al: Sickle cell disease (SCD), iNKT cells, and regadenoson infusion. Trans Am Clin Climatol Assoc 123:312–317; discussion 317–318, 2012. 414. Field JJ, Lin G, Okam MM, et al: Sickle cell vaso-occlusion causes activation of iNKT cells that is decreased by the adenosine A2A receptor agonist regadenoson. Blood 121(17):3329–3334, 2013. 415. Daak AA, Ghebremeskel K, Hassan Z, et al: Effect of omega-3 (n-3) fatty acid supplementation in patients with sickle cell anemia: Randomized, double-blind, placebo-controlled trial. Am J Clin Nutr 97(1):37–44, 2013. 416. Morris CR, Kuypers FA, Lavrisha L, et al: A randomized, placebo-controlled trial of arginine therapy for the treatment of children with sickle cell disease hospitalized with vaso-occlusive pain episodes. Haematologica 98(9):1375–1382, 2013. 417. Goldman RD, Mounstephen W, Kirby-Allen M, et al: Intravenous magnesium sulfate for vaso-occlusive episodes in sickle cell disease. Pediatrics 132(6):e1634–e1641, 2013. 418. Wun T, Soulieres D, Frelinger AL, et al: A double-blind, randomized, multicenter phase 2 study of prasugrel versus placebo in adult patients with sickle cell disease. J Hematol Oncol 6:17, 2013. 419. Desai PC, Brittain JE, Jones SK, et al: A pilot study of eptifibatide for treatment of acute pain episodes in sickle cell disease. Thromb Res 132(3):341–345, 2013. 420. Ataga KI, Reid M, Ballas SK, et al: Improvements in haemolysis and indicators of erythrocyte survival do not correlate with acute vaso-occlusive crises in patients with sickle cell disease: A phase III randomized, placebo-controlled, double-blind study of the Gardos channel blocker senicapoc (ICA-17043). Br J Haematol 153(1):92–104, 2011. 421. Orringer EP, Casella JF, Ataga KI, et al: Purified poloxamer 188 for treatment of acute vaso-occlusive crisis of sickle cell disease: A randomized controlled trial. JAMA 286(17):2099–2106, 2001. 422. Kutlar A, Ataga KI, McMahon L, et al: A potent oral P-selectin blocking agent improves microcirculatory blood flow and a marker of endothelial cell injury in patients with sickle cell disease. Am J Hematol 87(5):536–539, 2012.

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METHEMOGLOBINEMIA AND OTHER DYSHEMOGLOBINEMIAS Archana M. Agarwal and Josef  T. Prchal

SUMMARY Normal hemoglobin can be oxidized to methemoglobin. Methemoglobinemia occurs because of either increased production of oxidized hemoglobin from exposure to environmental agents or diminished reduction of oxidized hemoglobin because of underlying germline mutations. Cyanosis is virtually invariant in patients with methemoglobinemia. Hemoglobin can also bind carbon monoxide and nitric oxide, resulting in the formation of carboxyhemoglobin and nitrosohemoglobin. Sulfhemoglobinemia occurs because of increased production secondary to occupational exposure to sulphur compounds or exposure to oxidant medications. These modified hemoglobins are known as dyshemoglobins. Depending upon the severity and individual predisposition, presence of dyshemoglobins can result in varying degree of clinical manifestations. Prompt diagnosis is the key to effective and timely treatment.

METHEMOGLOBINEMIA DEFINITION AND HISTORY A bluish discoloration of the skin and mucous membrane, designated cyanosis, has been recognized since antiquity as a manifestation of lung or heart disease; however, in methemoglobinemia and sulfhemoglobinemia, it has a different molecular basis than in hemoglobin oxygen desaturation. Cyanosis resulting from drug administration has also been recognized since before 1890.1 Toxic methemoglobinemia occurs when various drugs or toxic substances either oxidize hemoglobin (Hb) directly in the circulation or facilitate its oxidation by molecular oxygen. In 1912, Sloss and Wybauw2 reported a case of a patient with idiopathic methemoglobinemia. Later Hitzenberger3 suggested that a hereditary form of methemoglobinemia might exist and, subsequently,

Acronyms and Abbreviations: AOP2, antioxidant protein 2; 2,3-BPG, 2,3bisphosphoglycerate; cGMP, cyclic guanosine monophosphate; CO, carbon monoxide; COHb, carboxyhemoglobin; GSH, reduced glutathione; N2O3, dinitrogen trioxide; NADH, nicotinamide adenine dinucleotide (reduced form); NADPH, reduced nicotinamide adenine dinucleotide phosphate; NO, nitric oxide; NOS, nitric oxide synthase; P50, the partial pressure of oxygen at which 50 percent of the blood hemoglobin is saturated with oxygen; RBC, red blood cells; SNO-Hb, S-nitrosohemoglobin; SpCO, arterial carboxyhemoglobin concentration; SpMet, arterial methemoglobin concentration; SpO2, arterial oxygen saturation.

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numerous such cases were reported.4 In 1948, Hörlein and Weber5 described a family in which eight members over four generations had cyanosis. The absorption spectrum of methemoglobin was abnormal and they demonstrated that the defect must reside in the globin portion of the molecule. Subsequently, Singer6 proposed that such abnormal hemoglobins be given the designation hemoglobin M. The cause of another form of methemoglobinemia that occurs independently of drug administration and without the existence of any abnormality of the globin portion of hemoglobin was first explained by Gibson,7 who clearly pointed to the site of the enzyme defect, nicotinamide adenine dinucleotide (reduced form) (NADH) diaphorase, also designated as methemoglobin reductase, and now known as cytochrome b5 reductase. More than 50 years after Gibson’s insightful studies, the genetic disorder that he had predicted was verified at the DNA level.8 The existence of abnormal hemoglobins that cause cyanosis through quite another mechanism was first recognized in 1968 with the description of hemoglobin Kansas.9 Here the cyanosis resulted not from methemoglobin, as occurs in hemoglobin M, but rather from an abnormally low oxygen affinity of the mutant hemoglobin. Thus, at normal oxygen tensions, a large amount of deoxygenated hemoglobin is present in the blood of affected patients.

EPIDEMIOLOGY Methemoglobinemia occurring as a result of cytochrome b5 reductase deficiency is more common among Native Americans, both in Alaska and in the continental United States, and among the Evenk people of Yakutia of Russian Siberia than in other ethnic groups.10–12 Methemoglobinemia resulting from hemoglobin M is inherited and sporadic. The occurrence of methemoglobinemia due to toxic chemicals is acquired, transient, and is also sporadic.

ETIOLOGY AND PATHOGENESIS Methemoglobinemia decreases the oxygen-carrying capacity of blood because the oxidized iron cannot reversibly bind oxygen. Moreover, when one or more iron atoms have been oxidized, the conformation of hemoglobin is changed so as to increase the oxygen affinity of the remaining ferrous heme groups. In this way methemoglobinemia exerts a dual effect in impairing the supply of oxygen to tissues.13

Toxic Methemoglobinemia

Hemoglobin is continuously oxidized in vivo from the ferrous to the ferric state. The rate of such oxidation is accelerated by many drugs and toxic chemicals, including sulfonamides, lidocaine and other aniline derivatives, and nitrites. A vast number of chemical substances may cause methemoglobinemia.14–16 Table 50–1 lists some of the agents that are responsible for clinically significant methemoglobinemia in clinical practice. The most common offenders include benzocaine and lidocaine.17–19 In some cases, the patients have been unaware that they have been ingesting one of the drugs known to produce methemoglobinemia; dapsone is apparently used in some “street drugs.”20,21 Nitrates and the nitrites contaminating water supplies or used as preservatives in foods are also common offending agents.22–30

Cytochrome b5 Reductase Deficiency

Cytochrome b5 reductase, also known as NADH diaphorase, catalyzes a step in the major pathway for methemoglobin reduction. This enzyme reduces cytochrome b5, using NADH as a hydrogen donor. The reduced cytochrome b5 reduces, in turn, methemoglobin to hemoglobin. A

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TABLE 50–1.  Some Drugs That Cause Methemoglobinemia Phenazopyridine (Pyridium)163–165 Sulfamethoxazole166 Dapsone20,167,168 Aniline88,89 Paraquat/monolinuron169–171 Nitrate22–24,81 Nitroglycerin163,172 Amyl nitrite173 Isobutyl nitrite

174

Sodium nitrite23,82 Benzocaine175–177 Prilocaine178–180 Methylene blue87 Chloramine171,181

steady-state methemoglobin level is achieved when the rate of methemoglobin formation equals the rate of methemoglobin reduction either through the cytochrome b5 reductase or through a relatively minor auxiliary mechanism such as direct chemical reduction by ascorbate and reduced glutathione. A reduced nicotinamide adenine dinucleotide phosphate (NADPH)-linked enzyme, NADPH diaphorase, does not play a role in methemoglobin reduction except when a linking dye such as methylene blue is supplied (see “Therapy, Course, and Prognosis” below). A marked diminution in the activity of cytochrome b5 reductase will result in the accumulation of the brown pigment in circulating erythrocytes. A balance to methemoglobin formation is antioxidant protein 2 (AOP2), which is present in high concentrations in human and mouse red cells (Chap. 47). This member of the peroxiredoxin protein family binds to hemoglobin and prevents both spontaneous and oxidant-induced methemoglobin formation.31 Mutations of this gene or its acquired deficiency are theoretical candidates responsible for congenital and acquired methemoglobinemia. Cyanosis resulting from abnormal hemoglobins (both hemoglobin M and low-oxygen affinity hemoglobins) is inherited as an autosomal dominant disorder. In contrast, hereditary methemoglobinemia resulting from cytochrome b5 reductase deficiency is inherited in an autosomal recessive fashion. Many mutations of cytochrome b5 reductase that cause methemoglobinemia have been identified at the nucleotide level,8 and the functional effect of some of these have been deduced from the structure of the enzyme.32,33 Although most of the mutants have been found in persons of European descent, five unique mutations were found in Chinese,34 at least three in Thais,35 two in Americans of African descent,36 and one in an Asian Indian.37 In addition, a common polymorphism (allele frequency = 0.023) has been identified in Americans of African descent; it does not appear to impair the activity of the enzyme.38 Most of the patients with cytochrome b5 reductase deficiency merely have methemoglobinemia and the enzyme deficiency is limited to the red cells, and these have been classified as having type I disease. In type II cytochrome b5 reductase deficiency, which represents 10 to 15 percent of cases of enzyme deficient congenital methemoglobinemia, cytochrome b5 reductase is decreased in all cells. In addition to cyanosis, severe developmental abnormalities can occur; most affected infants die

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in the first year of life.39,40 Patients with this form of disease are afflicted, in addition to methemoglobinemia, with a progressive encephalopathy and mental retardation. The finding that fatty acid elongation is defective in the platelets and leukocytes of such patients41 provides a clue to the type of defect that could occur in the central nervous system, where fatty acid elongation plays an important role in myelination. Rare patients with deficiency of cytochrome b5 reductase in nonerythroid cells do not suffer any neurologic disorder, and it has been suggested that they be designated as having type III disease42; however, existence of such an entity has been challenged and type III disease likely does not exist.43

Heterozygosity for Cytochrome b5 Reductase Deficiency

Heterozygotes for cytochrome b5 reductase deficiency are not usually clinically methemoglobinemic or cyanotic. However, under the stress of administration of drugs that normally induce only slight, clinically unimportant, methemoglobinemia, such persons have been reported to become severely cyanotic because of methemoglobinemia.44 Although in this report the affected patients were Ashkenazi Jews, the prevalence of cytochrome b5 reductase deficiency in 500 unselected Jewish subjects was found to be low.45 In addition, predisposition to acute toxic methemoglobinemia in heterozygous subjects for cytochrome b5 reductase deficiency seems to be quite uncommon.43 Animal models of cytochrome b5 reductase deficiency have been described in dogs, cats, and horses.46,47

Infant Susceptibility

A combination of both increased hemoglobin oxidation and decreased methemoglobin reduction also may occur. Because the activity of cytochrome b5 reductase is normally low in newborn infants,48 they are particularly susceptible to the development of methemoglobinemia. Thus, serious degrees of methemoglobinemia have been observed in infants as a result of toxic materials, such as aniline dyes used on diapers,49 and the ingestion of nitrate-contaminated water24,30 and even of beets.50 Bacterial action in the intestinal tract may reduce nitrates to nitrites, which, in turn, cause methemoglobinemia. In rural areas, fatal methemoglobinuria in infants caused by drinking water from wells contaminated with nitrates still occurs.51 Inhaled nitric oxide (NO) is approved for treatment of infants with pulmonary hypertension because of its vasodilatory effect on pulmonary vessels. During the binding and release of NO from hemoglobin, methemoglobin is formed at a higher rate. In one study of 81 premature and 82 term infants, methemoglobin was above 5 percent in preterm infants and between 2.5 and 5 percent in 16 infants.52 Methemoglobinemia occurring in acidotic infants with diarrhea is a syndrome that may have a fatal outcome.53 Such infants have normal red cell cytochrome b5 reductase activity, and the mechanism by which methemoglobinemia occurs is unknown. However, the syndrome seems most common when soy formula is being fed54 and breastfeeding appears to protect against this.51

Cytochrome b5 Deficiency

Rarely, the defect leading to methemoglobinemia may not be in the cytochrome b5 reductase that transfers hydrogen to the cytochrome b5, but rather to a deficiency in the cytochrome b5 itself.55

Hemoglobin M

The molecular mechanisms by which hemoglobin binds oxygen and releases it are discussed in Chap. 49. Heme is held in a hydrophobic “heme pocket” between the E and F α-helices of each of the four globin chains. The iron atom in the heme forms four bonds with the pyrrole nitrogen atoms of the porphyrin ring and a fifth covalent bond with the imidazole nitrogen of a histidine residue in the nearby F α-helix (Fig. 50–1).56

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A

Fe

B

A

Fe

A

O2

B

B

Figure 50–1. Diagrammatic representation of the heme group inserted into the heme pocket. A, Proximal histidine; B, distal histidine. A. In the deoxygenated form the larger ferrous atom lies out of the place of the porphyrin ring. B. In the oxygenated form the now smaller “ferric-like” atom can slip into the plane of the porphyrin ring. As a result, the proximal histidine, and helix F into which it is incorporated, are displaced. (Reproduced with permission from Lehmann H, Huntsman RG: Man’s Haemoglobins. Philadelphia PA: Lippincott Williams & Wilkins; 1974.)

This histidine, residue 87 in the α chain and 92 in the β chain, is designated as the proximal histidine. On the opposite side of the porphyrin ring the iron atom lies adjacent to another histidine residue to which, however, it is not covalently bonded. This distal histidine occupies position 58 in the α chain and position 63 in the β chain. Under normal circumstances oxygen is occasionally discharged from the heme pocket as a superoxide anion, removing an electron from the iron and leaving it in the ferric state. The enzymatic machinery of the red cell efficiently reduces the iron to the divalent form, converting the methemoglobin to hemoglobin (Chap. 47). In most of the hemoglobins M, tyrosine has been substituted for either the proximal or the distal histidine. Tyrosine can form an iron– phenolate complex that resists reduction to the divalent state by the normal metabolic systems of the erythrocyte. Four hemoglobins M are a consequence of substitution of tyrosine for histidine in the proximal and distal sites of the α and β chains. As Table 50–2 shows, these four hemoglobins M have been designated by the geographic names of their discovery, Boston, Saskatoon, Iwate, and Hyde Park. Analogous His→Tyr substitutions in the γ chain of fetal hemoglobin have also been documented and have been designated hemoglobin FMOsaka57 and FMFort Ripley.58 Another hemoglobin M, HbMMilwaukee, is formed by substitution of glutamic acid for valine in the 67th residue of the β chain. The glutamic acid side chain points toward the heme group and its γ-carboxyl group interacts with the iron atom, stabilizing it in the ferric state. It is rare for methemoglobinemia to occur as a result of hemoglobinopathies other than hemoglobins M, but HbChile (β28 Leu→Met) is such a hemoglobin. Producing hemolysis only with drug administration, this unstable hemoglobin is characterized clinically by chronic methemoglobinemia.59

TABLE 50–2.  Properties of Hemoglobins M Hemoglobin

Amino Acid Substitution

Oxygen Dissociation and Other Properties

HbMBoston

α58 (E7)His→Tyr

HbMSaskatoon

β63 (E7)His→Tyr

Clinical Effect

Reference

Very low O2 affinity, almost nonexistent heme–heme interaction, no Bohr effect

Cyanosis resulting from formation of methemoglobin

182

Increased O2 affinity, reduced hemeheme interaction, normal Bohr effect, slightly unstable

Cyanosis resulting from methemoglobin formation, mild hemolytic anemia exacerbated by ingestion of sulfonamides

182,183

HbMIwate α87 (F8)His→Tyr (HbMKankakee, HbMOldenburg, HbMSendai)

Low O2 affinity, negligible hemeheme interaction, no Bohr effect

Cyanosis resulting from formation of methemoglobin

182,184

HbMHyde Park

β92 (F8)His→Tyr

Increased O2 affinity, reduced heme interaction, normal Bohr effect, slightly unstable

Cyanosis resulting from formation of methemoglobin, mild hemolytic anemia

79

Hb M(hyde park)(HbMilwaukee 2)

 

 

 

 

HbMAkita

 

 

 

 

HbMMilwaukee

β67 (E11)Val →Glu

Low O2 affinity, reduced heme-heme interaction, normal Bohr effect, slightly unstable

Cyanosis resulting from methemoglobin formation

185

HbFMOsaka

G

γ63His→Tyr

Low O2 affinity, increased Bohr effect, methemoglobinemia

Cyanosis at birth

57

HbFMFort Ripley

G

γ92His→Tyr

Slightly increased O2 affinity

Cyanosis at birth

186

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CLINICAL FEATURES Drug Ingestion

Methemoglobinemia may be chronic or acute and acquired or congenital. Acquired severe acute methemoglobinemia, usually the consequence of drug ingestion or toxic exposure, can produce symptoms of anemia, since methemoglobin lacks the capacity to transport oxygen. Symptoms may include shortness of breath, palpitations, and vascular collapse. Chemicals that induce methemoglobinemia are often also capable of causing hemolysis, and a combination of hemolytic anemia and methemoglobinemia may occur. Chronic methemoglobinemia, whether a result of exposure to drugs or toxins or of hereditary causes, is usually asymptomatic. Cyanosis, even if present, may not be discernable in persons with very dark skin coloration.60 In instances when the methemoglobin levels are chronically very high (>20 percent of the total pigment), mild erythrocytosis may be noted (Chap. 57).

M Hemoglobins

Patients with hemoglobin M also manifest cyanosis. In the case of α-globin variants, the dusky color of the infants will be noted at birth, but the clinical manifestations of β-globin variants become apparent only after β chains have largely replaced the fetal γ chains at 6 to 9 months of age. In spite of the impaired hemoglobin function, no cardiopulmonary symptoms are observed and there is no clubbing. In the case of HbMSaskatoon and HbMHyde Park, hemolytic anemia with jaundice may be present. The hemolytic state may be exacerbated by administration of sulfonamides.61

Cytochrome b5 Reductase Deficiency

Hereditary methemoglobinemia resulting from cytochrome b5 reductase deficiency may, as noted above, be associated with mental retardation, failure to thrive and early death. In one case, skeletal anomalies were documented as well.62

LABORATORY FEATURES Toxic Methemoglobinemia

In toxic methemoglobinemia, an elevated level of methemoglobin is found, but the activity of cytochrome b5 reductase is normal. Methemoglobin levels are best measured using the change of absorbance of methemoglobin at 630 nm that occurs when cyanide is added, converting the methemoglobin to cyanmethemoglobin, a principle used in the Evelyn–Malloy method.63,64 Errors in diagnosis are frequently made when automated instruments designed to estimate levels of reduced hemoglobin, oxygenated hemoglobin, methemoglobin, and carboxyhemoglobin (COHb) are used. Most automated instruments do not properly make this distinction.65,66 The clinical incidence of methemoglobinemia can be overestimated by cooximeter measurements compared to the more specific Evelyn–Malloy method.67 Evelyn-Malloy method involves direct spectrophotometric analysis and should be used when methemoglobinemia is suspected. This is achieved by lysing the blood in a slightly acid buffer and measuring the optical density at 630 nm before and after adding a small amount of neutralized cyanide. The absorption of methemoglobin at this wavelength disappears when it is converted to cyanmethemoglobin. Although this method was described in 1938,63 it remains the most accurate technique for the estimation of methemoglobin in the blood. Details of its performance can be found in an earlier edition of this text68 and elsewhere.61 An eight-wavelength pulse oximeter, Masimo Rad-57 (Rainbow-SET Rad-57 Pulse CO-Oximeter, Masimo Inc, Irvine, CA), has been approved by the FDA for the measurement of both COHb and

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methemoglobin. The Rad-57 uses eight wavelengths of light instead of the usual two and is thereby able to measure more than two species of human hemoglobin.69 In addition to the usual SpO2 value, the Rad-57 displays SpCO and SpMet, which are the pulse oximeter’s estimates of COHb and methemoglobin concentrations, respectively. In a study on healthy human volunteers in whom controlled levels of methemoglobin and COHb were induced, the Rad-57 measured COHb with an uncertainty of ±2 percent within the range of 0 to 15 percent and measured methemoglobin with an uncertainty of 0.5 percent within the range of 0 to 12 percent,69 the usefulness of this instrument has been verified also by other studies.70,71

Cytochrome b5 Reductase Deficiency

In hereditary methemoglobinemia resulting from cytochrome b5 reductase deficiency, between 8 and 40 percent of the hemoglobin is in the form of methemoglobin. The blood may have a chocolate-brown color and cyanosis is present. Cytochrome b5 reductase activity is best measured using ferricyanide as a receptor, measuring the rate of oxidation of NADH.72,73 The residual level of enzyme activity is usually less than 20 percent of normal in patients with methemoglobinemia resulting from deficiency of this enzyme. An immunoassay has been described,74 but such an assay would not detect mutants in which enzyme molecules with impaired catalytic activity are present. For unknown reasons, glutathione reductase activity (Chap. 47) is usually also diminished.75

Cytochrome b5 Deficiency

Cytochrome b5 assays may be useful if cytochrome b5 reductase activity is normal, and the presence of hemoglobin M is ruled out.76

M Hemoglobins

Optical Spectrum Figure 50-2 illustrates the spectrum of normal methemoglobin A at pH 7.0.77 Hemoglobins M may be differentiated from methemoglobin formed from hemoglobin A by its absorption spectrum in the range of 450 to 750 nm. Because only some 20 to 35 percent of the total hemoglobin will ordinarily be the hemoglobin M, the mixed spectra of methemoglobin A and the hemoglobin M may be difficult to interpret. Therefore, it is preferable to perform these spectral studies on purified hemoglobin M isolated by electrophoretic or chromatographic means.56 Electrophoresis  All hemoglobin M samples should be converted to methemoglobin so that any difference found in electrophoresis will be the result of the amino acid substitution and not the different charge of the iron atom. Electrophoresis at pH 7.1 is most useful for separation of hemoglobins M because the imidazole groups of histidine have a net positive charge at this pH, while at higher pH levels the histidines and the substituting tyrosines are both neutral. Other Biochemical Methods  The hemoglobins M differ in their reactivity to cyanide and to azide ions.78 This property may help to identify the subunit affected, as the iron-phenolate bonds are stronger in the α-chain variants than in the β-chain variants. However, definitive identification of the variant requires peptide or DNA analysis. Hemoglobins that cause cyanosis because of a diminished oxygen affinity may be detected by determining the oxygen dissociation curve of blood, being certain that the 2,3-bisphosphoglycerate (2,3-BPG) level is normal, or by estimating the oxygen dissociation curve of hemoglobin, which has been stripped of 2,3-BPG by extensive dialysis against an appropriate buffer. Many of the hemoglobins with decreased oxygen affinity are unstable (Chap. 49) and will precipitate in the isopropanol stability test.78 In many laboratories, it may be easier to analyze the coding sequence of the globin chains at the DNA level than to attempt to determine the properties of the hemoglobin.79

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blue per kilogram body weight has produced acute hemolysis even in patients with normal glucose-6-phosphate dehydrogenase levels.89 The response to treatment is so rapid, with marked lowering or normalization of methemoglobin levels within an hour or two, that no other treatment is usually needed, but the patient should be observed carefully because continued absorption of a toxic substance from the gastrointestinal tract may cause recurrence of the methemoglobinemia. In patients who are in shock, blood transfusion may be helpful. Cimetidine, used as a selective inhibitor of N-hydroxylation, may decrease the methemoglobinemia produced by dapsone in patients with dermatitis herpetiformis.90

0.8

D

Optical density

0.6

C 0.4

Hereditary Methemoglobinemia B

0.2 A

0

793

500

600

700

Wavelength (mm)

Figure 50–2.  Absorption spectra at pH 7.0. A, Methemoglobin A; B, methemoglobin MBoston; C, methemoglobin MSaskatoon; D, methemoglobin A fluoride complex. For purposes of comparison, all the optical densities have been made equal to 0.61 at 500 nm. (Reproduced with permission from Gerald PS, George P: Second spectroscopically abnormal methemoglobin associated with hereditary cyanosis. Science 1959 Feb 13;129(3346):393-394.)

TREATMENT AND COURSE Toxic Methemoglobinemia

Acute toxic methemoglobinemia may represent a serious medical emergency. Because of the loss of oxygen-carrying capacity of the blood and the left shift in the oxygen dissociation curve that occurs when methemoglobin is present in high concentrations,80 acute methemoglobinemia may be life-threatening when the level of the pigment exceeds one-third of the total circulating hemoglobin. Levels of methemoglobin exceeding 50 percent of the total pigment may be associated with vascular collapse, coma, and death,81,82 but recovery was documented in one patient with a level as high as 81.5 percent of the total pigment.83 Methylene blue84 is an effective treatment for patients with methemoglobinemia because NADPH formed in the hexose monophosphate pathway can rapidly reduce this dye to leukomethylene blue in a reaction catalyzed by NADPH diaphorase (Chap. 47). Leukomethylene blue, in turn, nonenzymatically reduces methemoglobin to hemoglobin.85 An exception to the efficacy of this treatment exists in those patients who are glucose-6-phosphate dehydrogenase deficient (Chap. 47). In these subjects, methylene blue would not only fail to give the desired effect on methemoglobin levels, but might compound the patient’s difficulty by inducing an acute hemolytic episode86 or increasing the level of methemoglobin.87 In patients with acute toxic methemoglobinemia who are symptomatic or whose methemoglobin level is rising rapidly, the intravenous administration of 1 or 2 mg methylene blue per kilogram body weight over a period of 5 minutes is the preferred treatment because of its very rapid action.88 Use of excessive amounts of methylene blue should be avoided; the administration of repeated doses of 2 mg methylene

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The course of hereditary methemoglobinemia is generally benign (although not in type II cytochrome b5 reductase deficiency), but patients with this disorder should be shielded from exposure to aniline derivatives, nitrites, and other agents that may, even in normal persons, induce methemoglobinemia. Hereditary methemoglobinemia resulting from cytochrome b5 reductase deficiency is readily treated by the administration of ascorbic acid, 300 to 600 mg orally daily divided into three or four doses. Although intravenously administered methylene blue is very effective in correcting this type of methemoglobinemia, it is not suitable for the long-term therapy that needs to be given if the state is to be treated at all. Riboflavin administration seems to benefit some patients91 but not others.92 The iron phenolate complex that exists in the hemoglobins M prevents the reduction of ferric to ferrous iron. For this reason, the methemoglobinemia does not respond to administration of ascorbic acid or of methylene blue. No effective treatment exists for the cyanosis that is present in patients with abnormal hemoglobins with reduced oxygen affinity.

SULFHEMOGLOBIN DEFINITION AND HISTORY Sulfhemoglobinemia refers to the presence in the blood of hemoglobin derivatives that are defined by their characteristic absorption of light at 620 nm, even in the presence of cyanide. Sulfhemoglobin derives its name from the fact that it can be produced in vitro from the action of hydrogen sulfide on hemoglobin93 and that the feeding of dogs with elemental sulfur has been associated with sulfhemoglobinemia.94

ETIOLOGY AND PATHOGENESIS Sulfhemoglobin may contain one excess sulfur atom. The sulfur atom appears to be bound to a β-pyrrole carbon atom at the periphery of the porphyrin ring.95–97 Sulfhemoglobinemia has been associated with the ingestion of various drugs, particularly sulfonamides, phenacetin, acetanilid, and phenazopyridine.65,98 It also occurs independently of drug use, and has been thought to be related to chronic constipation or to purging.99 Some patients with sulfhemoglobinemia or a past history of this disorder appear to have increased levels of red blood cell reduced glutathione (GSH).100 The reason for this and its relationship to sulfhemoglobinemia are not clearly understood, but it may be of significance that some of the types of drugs that are associated with sulfhemoglobinemia cause an elevation of red cell GSH levels, probably by activating the enzyme glutathione synthase101 or by increasing intracellular glutamate levels.102 Evidence for the occurrence of hereditary sulfhemoglobinemia is not convincing,103 and it is likely that the single family reported represents a hemoglobin M hemoglobinopathy.

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CLINICAL FEATURES Sulfhemoglobinemia is characterized by cyanosis. Drugs that cause sulfhemoglobinemia often have the capacity to produce accelerated red cell destruction as well. Thus, mild hemolysis is sometimes observed in patients with sulfhemoglobinemia.

LABORATORY FEATURES Sulfhemoglobin is detected in the lysate of blood treated with ferricyanide, cyanide, and ammonia by comparing the optical density at 620 nm with that at 540 nm.63,64

TREATMENT AND COURSE Sulfhemoglobinemia is almost always a benign disorder. Unlike methemoglobin, sulfhemoglobin does not produce a left shift in the oxygen dissociation curve; instead, it decreases the affinity of hemoglobin for oxygen.98 The disorder tends to recur in the same persons after exposure to drugs but does not generally appear to affect their overall health. Unlike methemoglobin, sulfhemoglobin cannot be converted to hemoglobin. Thus, once sulfhemoglobinemia occurs, it will persist until the erythrocytes carrying the abnormal pigment reach the end of their life span.

LOW-OXYGEN-AFFINITY HEMOGLOBINS: A CAUSE OF CYANOSIS ETIOLOGY AND PATHOGENESIS In some hemoglobin variants, the deoxy conformation of the hemoglobin molecule is favored because the angle of the heme is altered from that found normally in deoxyhemoglobin. Such changes occur in HbHammersmith, HbBucuresti, HbTorino, and HbPeterborough. In other instances, the quaternary conformation is changed by mutations involving the α1β2 contact (HbKansas, HbTitusville, and HbYoshizuka). Table 50–3 summarizes the properties of abnormal hemoglobins associated with low oxygen affinity.

CLINICAL FEATURES In response to the improved tissue oxygen supply brought about by a right-shifted oxygen dissociation curve, the “oxygen sensor” of the body decreases the output of erythropoietin.104 As a result, the steady-state level of hemoglobin is diminished; mild anemia and cyanosis are characteristics of patients with hemoglobins with a decreased oxygen affinity.

LABORATORY FEATURES The affinity of hemoglobin with oxygen is expressed as P50, which is the partial pressure of oxygen at which 50 percent of the blood hemoglobin is saturated with oxygen. The venous P50 can be measured directly using a cooximeter, which is no longer easily available in either routine

or reference laboratories. A mathematical formula has been developed that can be used to calculate P50 reliably from a venous blood sample.105 Calculating P50 using this formula requires the following venous gas parameters: partial pressure of oxygen (venous), venous pH, and venous oxygen saturation, and uses an antilog mathematical function that many clinicians find difficult to use for calculation. An electronic version (in Microsoft Excel) of this mathematical formula is available for rapid calculation of P50 from venous blood gases.106 The P50 of a healthy person with normal hemoglobin is 26 ± 1.3 torr. An abnormally low P50 reflects an increased affinity of hemoglobin for oxygen and vice versa, and is especially useful for detecting those high affinity hemoglobin mutants associated with polycythemia (Chaps. 49 and 57).

DIFFERENTIAL DIAGNOSIS Cyanosis resulting from methemoglobinemia or sulfhemoglobinemia should be differentiated from cyanosis resulting from cardiac or pulmonary disease, particularly when right-to-left shunting is present. In the latter instances, the arterial oxygen tension will be low, whereas in methemoglobinemia and sulfhemoglobinemia it should be normal. One should be certain, however, that the oxygen tension was measured directly and not deduced from the percent saturation of hemoglobin. Blood from a patient with cyanosis because of arterial oxygen desaturation promptly becomes bright red upon being shaken with air. In addition, these causes of cyanosis are readily differentiated by carrying out quantitative blood methemoglobin and sulfhemoglobin levels. Because of the potential lethal nature of high levels of methemoglobin and because prompt treatment may be life-saving, a high index of suspicion is important. A patient with cyanosis whose arterial blood is brown with an SpO2 that is found to be normal on blood gas examination is likely to have methemoglobinemia. One should not rely on the readings of a standard pulse oximeter, as false readings may be obtained in the presence of methemoglobin. Rapid examination of a blood sample using an automatic analyzer, such as a cooximeter, is the first step in confirming the diagnosis. Treatment should not be delayed, but, as pointed out in “Laboratory Features” above, direct spectrophotometric analysis should be carried out on the pretreatment sample as soon as possible to distinguish between methemoglobinemia and sulfhemoglobinemia. A family history, as well as any information as to whether it is acquired or congenital, is helpful in differentiating hereditary methemoglobinemia as a result of cytochrome b5 reductase deficiency from hemoglobin M disease. The former has a recessive mode of inheritance, the latter a dominant mode. Thus, cyanosis in successive generations suggests the presence of hemoglobin M; normal parents but possibly affected siblings implies the presence of cytochrome b5 reductase. Consanguinity is more common in cytochrome b5 reductase deficiency. In cytochrome b5 reductase deficiency, incubation of the blood with small amounts of methylene blue will result in rapid reduction of the methemoglobin; in hemoglobin M disease, such reduction does not take place. The absorption spectra of methemoglobin and its derivatives

TABLE 50–3.  Some Abnormal Hemoglobins Associated with Low Oxygen Affinity Amino Acid Substitution

Oxygen Dissociation and Other Properties

HbSeattle

β70 (E14) Ala→Asp

HbKansas

β102 (G4) Asn→Thr

Hemoglobin

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Clinical Effect

Reference

Decreased O2 affinity normal heme–heme interaction

Mild chronic anemia associated with reduced urinary erythropoietin; physiologic adaptation to more efficient oxygen release to tissues

104

Very low O2 affinity, low heme– heme interaction, dissociates into dimers in ligand form

Cyanosis resulting from deoxyhemoglobin, mild anemia

187

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are normal in cytochrome b5 reductase deficiency; they are abnormal in hemoglobin M disease. In the case of toxic methemoglobinemia, cyanosis is generally of relatively recent origin, and a history of exposure to drug or toxin may usually be obtained; in hereditary methemoglobinemia, a history of lifelong cyanosis may usually be elicited.

OTHER DYSHEMOGLOBINS CARBON MONOXIDE AND CARBOXYHEMOGLOBIN Carbon monoxide (CO) is a toxic, odorless, colorless, and tasteless gas. It can be unknowingly inhaled to dangerous levels with serious clinical implications when present in high concentration in the atmosphere.107

Epidemiology

Acute CO intoxication is one of the most common causes of morbidity from poisoning in the United States. In the United States, CO poisoning results in approximately 50,000 emergency department visits per year,108,109 and approximately 500 accidental deaths as a result of CO poisoning occur annually, with the number of intentional CO-related deaths being five to 10 times higher.110,111 Primary sources of CO are home appliances, and the majority of exposures occur during the fall and winter months and during weather-related disasters.112,113 During warmer months, boating activities are another source of exposure.114 The death rate is highest among the elderly and can be attributed to delayed diagnosis because symptoms often resemble those of associated comorbidities.115,116 The exhaust produced by the typical home-use 5.5-kW generator contains as much CO as that of six idling automobiles.117 Chronic CO intoxication is commonly caused by cigarette smoking, which can increase the COHb level up to 15 percent. Houses with defective heating exhaust systems and vehicles that leak CO into the passenger compartment, either because of mechanical failure or driving with the rear hatch-door open, are the second most common cause of chronic CO exposure. Occupations that involve a high risk for CO intoxication include garage work with improper ventilation, toll booth attendants, tunnel workers, fire fighters, and workers exposed to paint remover, aerosol propellant, or organic solvents containing dichloromethane.118

Etiology and Pathogenesis

CO binds with high affinity to heme and with lesser affinities to myoglobin and cytochromes at the iron core, a site it shares with O2.119 At equilibrium in physiologic conditions, CO affinity for hemoglobin is approximately 240 times greater than that of O2. This very high equilibrium constant is the result of reaction kinetics. Contrary to popular belief, CO reacts more slowly than O2 with the heme of hemoglobin. Once CO is bound to heme, its “off ” rate is only 0.015 mol/L per second in contrast to 35 mol/L per second for O2.119 This extraordinarily slow-release process produces a very high affinity constant of CO for heme and a life-threatening danger for individuals exposed to high levels of CO. Once two molecules of CO are bound to hemoglobin, the hemoglobin switches to the relaxed (R) state, which increases the affinity of hemoglobin for oxygen. As a consequence of this phenomenon, called the Darling–Roughton effect,80 the hemoglobin O2 affinity increases in parallel with increasing CO levels, making tissue delivery of oxygen more difficult. In the absence of environmental CO, the blood of adults contains approximately 1 to 2 percent COHb. This represents approximately 80 percent of the total body CO, the remainder probably sequestered in myoglobin and other heme binding proteins. This CO is endogenously produced,120 originating from the degradation of heme by the

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rate-limiting heme oxygenase–cytochrome P450 complex, which produces CO and biliverdin. Caloric restriction, dehydration, infancy, and the genetic variations reported in Japanese and Native Americans generate higher endogenous levels of CO. Hemolytic anemia (Chap. 33), hematomas, and infection tend to increase CO production up to threefold. Fetuses and newborns have double the normal adult levels of COHb. Drugs such as diphenylhydantoin and phenobarbital, by inducing the cytochrome P450 complex, increase CO production. Normal adult level of COHb is less than 2 percent. Hemolysis can produce COHb levels of more than 2 percent. Levels more than 3 percent must have an exogenous origin, except for rare conditions as occur in carriers of abnormal hemoglobins such as HbZurich. The affinity of HbZurich for CO is approximately 65 times that of normal hemoglobin.121 Pregnant women and fetuses are particularly at risk122 because they already have higher levels of COHb. CO readily crosses the placenta, and the half-life of CO in the fetus is as much as five times longer than it is in the mother.123 The O2 affinity of fetal hemoglobin (HbF) is shifted to the left124,125 owing to its lack of 2,3-BPG binding, making the Darling– Roughton effect particularly pernicious. This is one of the reasons why cigarette smoking during pregnancy is hazardous to the fetus.

Clinical and Laboratory Features

CO poisoning is a clinical diagnosis that is confirmed by laboratory testing. Signs and symptoms consistent with CO poisoning in certain circumstances should raise the suspicion of CO intoxication. A higher index of suspicion should attend the simultaneous presentation of multiple patients from the same family or housing complex. The eight-wavelength pulse oximeter, Masimo Rad-57 (see paragraphs on “Laboratory features” of methemoglobinemia.) has been reported to be accurate in measuring COHb concentration in normal healthy volunteers,69 as well as in emergency room patients.126 Acute intoxication with CO rapidly affects the central and peripheral nervous systems and cardiopulmonary functions. Cerebral edema is common, as is impairment of the peripheral nervous system. CO induces increased capillary permeability in the lungs, resulting in acute pulmonary edema. Cardiac arrhythmias, generalized hypoxemia, and respiratory failure are the common causes of CO-related death. In survivors, considerable neuropsychological deficits might remain. In a prospective longitudinal study, approximately 45 percent of patients with CO poisoning had cognitive sequelae 6 weeks after poisoning.127,128 Acute CO intoxication in children129 sometimes has unique symptomatology resembling gastroenteritis. Surviving children are more likely to have severe sequelae such as leukoencephalopathy and severe myocardial ischemia.130 Chronic intoxication in adults might result in irritability, nausea, lethargy, headaches, and sometimes a flu-like condition. Higher COHb levels produce somnolence, palpitations, cardiomegaly, and hypertension, and could contribute to atherosclerosis. Chronic CO poisoning can produce erythrocytosis, the magnitude of which varies with the level of COHb. By increasing red cell production, chronic CO poisoning can mask the mild anemia of acquired or congenital hemolytic disorders.

Therapy, Course, and Prognosis

The most important step in the treatment for CO poisoning is prompt removal of patients from the source of CO, followed by administering 100 percent supplemental O2 via a tight-fitting mask. The serum elimination half-life of CO is 5 hours when breathing room air and 30 minutes with O2 therapy (100 percent O2 at 3 atmospheres).123 For mild to moderate cases of CO poisoning, which more often happen with chronic intoxication, removing the patient from the source of environmental CO is usually curative. If the COHb level is high, breathing 100 percent O2 will increase the rate of CO removal.

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NO NO

b93Cys-SH

Hgb:Fe"

Hgb:Fe"

Figure 50–3.  S-nitrosohemoglobin (SNO-Hb)

b93Cys-SH

and hypoxic vasodilation. (Reproduced with permission from Parker C: SNO-HB a Snow Job? The Hematologist: ASH News and Reports. 6:12;2009.)

O2

Fe":Hgb

O2 b93Cys-SNO R–State

Hgb:Fe" RBC

GSNO GSH

b93Cys-SNO T–State

Cys-SH

AE-1

Cys-SNO

AE-1

NOx (RSNO)

Vascular endothelial cells and smooth muscle cells

Vasodilation

In severe cases of CO poisoning, which more often occur with acute intoxication, after identification and removal of the source of CO, 100 percent O2 should be administered, with cardiac monitoring. Endotracheal intubation should be done in any patient with impaired mental status, and other interventions should be dictated by the symptomatology. Because of conflicting evidence, there is no absolute indication for the use of hyperbaric O2 treatment for patients with CO poisoning. Hyperbaric O2 might be indicated in patients who have obvious neurologic abnormalities, cardiac dysfunction, persistent symptoms despite normobaric O2, or metabolic acidosis.131 Hyperbaric O2 has complications of its own, such as bronchial irritation and pulmonary edema, and should be reserved for exceptional cases of CO intoxication. Locations of hyperbaric chambers throughout the world and in the United States can be found at the Undersea and Hyperbaric Medical Society website (http://www.uhms.org) under “chamber directory.” Pregnant women exposed to CO are at particularly high risk. CO poisoning is especially dangerous to the fetus because CO readily crosses the placenta and the half-life of CO in the fetus is as much as five times longer than it is in the mother. For these reasons, treatment with hyperbaric O2 should be carried out during pregnancy when the COHb levels exceed 15 percent. In a limited number of studies done on pregnant patients, hyperbaric O2 does not seem to adversely affect the fetus.132,133

NITRIC OXIDE AND NITRIC OXIDE HEMOGLOBINS Physiology and Chemistry

NO, a soluble gas, is continuously synthesized in endothelial cells by isoforms of the NO synthase (NOS) enzyme. A functional NOS transfers electrons from NADPH to its heme center, where l-arginine is oxidized to l-citrulline and NO.134 Vasodilation is caused by diffusion of NO into the smooth muscle cells, wherein NO binds avidly to the heme of soluble guanylyl cyclase, producing cyclic guanosine monophosphate

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(cGMP), which activates cGMP-dependent protein kinases and ultimately produces smooth muscle relaxation.134 Blood NO levels are set by the balance between the production of NO by NOS and the binding or scavenging of NO by the heme groups of erythrocyte hemoglobin. The half-life of NO in whole blood is extremely short, estimated to be 1.8 milliseconds.135 The short half-life of NO greatly limits its diffusional distance in blood and only maintains NO as a paracrine vasoregulator.136,137 This does not explain how hemoglobin is capable of transducing NO bioactivity far from its location of formation. Interaction of the red blood cells with NO is a complex phenomenon (Fig. 50–3). Two models have been proposed: (1) The first model, an S-nitrosohemoglobin (SNO-Hb)–dependent mechanism, proposes that NO binds to heme when the hemoglobin is in the T state (deoxygenated). In the oxygenated state, NO gets transferred from heme to a cysteine residue on the globin portion of hemoglobin, forming SNO-Hb.138,139 Nitric oxide is transported by red blood cells from the lungs to hypoxic tissues in a protected form as SNO-Hb, and is delivered in the hypoxic microvasculature at the same time as oxygen, coupling hemoglobin deoxygenation to vasodilation. (2) The second model is of deoxyhemoglobin-mediated nitrite reduction to NO.140 In the blood, deoxygenated hemoglobin functions as the predominant nitrite reductase.141 Deoxygenated hemoglobin reacts with nitrite to form NO and methemoglobin and causes vasodilation along the physiological oxygen gradient. Although this reaction is experimentally associated with NO generation, kinetic analysis suggests that NO should not be able to escape inactivation in the erythrocyte.142 This inactivation or scavenging of NO is avoided by the formation of an intermediate species, that is, dinitrogen trioxide (N2O3). Products of the nitrite–hemoglobin reaction generate N2O3 via a novel reaction of NO and nitrite-bound methemoglobin.143 N2O3 diffuses out of the red cell, later forms NO, and affects vasodilation and/or forms nitrosothiols (Fig. 50–4). According to this paradigm, nitrite, previously thought to be an inert end product of endogenous NO metabolism, is the main stable NO reservoir in blood and tissues.144,145 Nitrite is formed during normoxic conditions

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NO + NO2+ DeoxyHb + NO2–

MetHb + NO

N2O3 N2O3 Hb ––NO NO2+2+ SNO Hgb SNO + NO

MetHb + NO2– MetHb – NO2– Hb – NO2+ + NO

N2O3

MetHb – NO2– Hb + NO2+ Hb + N2O3

N2O3

797

Figure 50–4. Hemoglobin deoxygenation (purple)

occurs in capillaries. Nitrite reacts with deoxyHb that is oxidized to MetHb and NO. The NO binds to hemes of deoxyHb, and also undergoes dioxygenation to form nitrate and MetHb from oxyHb. MetHb binds nitrite to form an adduct with some Fe(II)-NO2, that is, Hb-NO. This species reacts quickly with NO to form N2O3, which can diffuse out of the red cell forming NO and effecting vasodilation and/or forming nitrosothiols (SNOs). (Reproduced with permission from Basu S, et al: Catalytic generation of N203 by the concerted nitrite reductase and anhydrase activity of hemoglobin, Nature Chemical Biology 2007 Dec;3(12):785-794.)

NO + NO2+

and then is reduced to NO and N2O3 along the physiologic oxygen and pH gradient by the heme globins.143 Cell free hemoglobin and red cell microparticles formed during hemolytic conditions and long storage of red blood cells (RBCs) lead to NO scavenging 1000 times faster than regular RBCs and to insufficient NO bioavailability.146 Stored RBCs are also stored in acidic solution that also leads to a decrease in SNO-Hb levels. This has been further substantiated by the fact that renitrosylated RBCs lead to improved oxygen delivery in animal models.147 This could explain the morbidity and mortality associated with stored RBCs. Moreover, underlying recipient endothelial dysfunction, for example, obesity or hypertension, can also induce increased RBC membrane damage in transfused blood, leading to increased microparticle formation and increased NO scavenging.148,149

Pathophysiology and Potential Therapeutic Applications

Nitric oxide was long considered highly toxic. Exogenous administration of NO by inhalation activates cytosolic guanylate cyclase, increasing intracellular levels of cGMP, and resulting in relaxation of the smooth muscles in the pulmonary arteries. Based on this observation, inhaled NO (iNO) has been used to manage the acute pulmonary hypertension seen in adult respiratory distress syndrome, sickle cell disease, and primary or secondary pulmonary hypertension. Even though NO lowers the pulmonary artery pressure and improves oxygenation in acute respiratory distress syndrome in both adults and children, it has not consistently resulted in an improvement in mortality. At present, prolonged administration of iNO is not considered as first-line therapy for pulmonary artery hypertension and instead is used only for vasoreactivity testing in these patients.150 iNO has beneficial effects in animal models, as well as in preliminary human trials of acute vasoocclusive crisis and chest syndrome associated with sickle cell disease.151–153 Some animal data suggest beneficial effects of iNO therapy in the setting of ischemia–reperfusion injury (lung, heart, and intestine).154 However, iNO is also associated with multiple side effects, such as methemoglobinemia,155 left-heart failure,156 renal insufficiency,157 and a “rebound” increase in pulmonary artery pressure upon discontinuation of iNO that may result in cardiovascular collapse.158 Direct repletion of S-nitrosothiol in the lung and blood has the potential to avoid toxicities related to iNO. In a porcine model of acute lung injury, inhaled ethyl nitrite, but not iNO, efficiently repleted lung SNO-Hb, lowered pulmonary vascular resistance, improved oxygenation dose-dependently, and had a protective effect against a decline in cardiac output.159 In humans, newborns with persistent pulmonary hypertension showed improved oxygenation and hemodynamics following ethyl

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nitrite inhalation.160 Use of cell free hemoglobin is associated with vasoconstriction and subsequent development of hypertension. Increased vascular resistance and vasoconstriction has been shown to be mediated mainly by the scavenging of NO because of the high affinity of free hemoglobin for NO.161,162

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22. Johnson CJ, Kross BC: Continuing importance of nitrate contamination of groundwater and wells in rural areas. Am J Ind Med 18(4):449, 1990. 23. Chan TY: Food-borne nitrates and nitrites as a cause of methemoglobinemia. Southeast Asian J Trop Med Public Health 27(1):189, 1996. 24. Knobeloch L, Proctor M: Eight blue babies. WMJ 100(8):43, 2001. 25. Askew GL, Finelli L, Genese CA, et al: Boilerbaisse: An outbreak of methemoglobinemia in New Jersey in 1992. Pediatrics 94(3):381, 1994. 26. Bakshi SP, Fahey JL, Pierce LE: Brief recording: Sausage cyanosis—Acquired methemoglobinemic nitrite poisoning. N Engl J Med 277(20):1072, 1967. 27. Bradberry SM, Whittington RM, Parry DA, et al: Fatal methemoglobinemia due to inhalation of isobutyl nitrite. J Toxicol Clin Toxicol 32(2):179, 1994. 28. Bradberry SM, Gazzard B, Vale JA: Methemoglobinemia caused by the accidental contamination of drinking water with sodium nitrite. J Toxicol Clin Toxicol 32(2):173, 1994. 29. Harris JC, Rumack BH, Peterson RG, et al: Methemoglobinemia resulting from absorption of nitrates. JAMA 242(26):2869, 1979. 30. Lukens JN: Landmark perspective: The legacy of well-water methemoglobinemia. JAMA 257(20):2793, 1987. 31. Stuhlmeier KM, Kao JJ, Wallbrandt P, et al: Antioxidant protein 2 prevents methemoglobin formation in erythrocyte hemolysates. Eur J Biochem 270:334, 2003. 32. Bewley M, Marohnic C, Barber M: The structure and biochemistry of NADHdependent cytochrome b5 reductase are now consistent. Biochemistry 40:13574, 2001. 33. Yamada M, Tamada T, Takeda K, et al: Elucidations of the catalytic cycle of NADHcytochrome b5 reductase by X-ray crystallography: New insights into regulation of efficient electron transfer. J Mol Biol 425(22):4295, 2013. 34. Wang Y, Wu Y, Zheng P, et al: A novel mutation in the NADH-cytochrome b5 reductase gene of a Chinese patient with recessive congenital methemoglobinemia. Blood 95:3250, 2000. 35. Shotelersuk V, Tosukhowong P, Chotivitayatarakorn P, et al: A Thai boy with hereditary enzymopenic methemoglobinemia type II. J Med Assoc Thai 83:1380, 2000. 36. Jenkins MM, Prchal JT: A novel mutation found in the 3′ domain of NADHcytochrome B5 reductase in an African-American family with type I congenital methemoglobinemia. Blood 87(7):2993, 1996. 37. Nussenzveig RH, Lingam HB, Gaikwad A, et al: A novel mutation of the cytochrome-b5 reductase gene in an Indian patient: The molecular basis of type I methemoglobinemia. Haematologica 91(11):1542, 2006. 38. Jenkins M, Prchal J: A high frequency polymorphism of NADH-cytochrome b5 reductase in African-Americans. Hum Genet 99:248, 1997. 39. Ewenczyk C, Leroux A, Roubergue A, et al: Recessive hereditary methaemoglobinaemia, type II: Delineation of the clinical spectrum. Brain 131(Pt 3):760, 2008. 40. 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Moore MR, Conrad ME, Bradley EL Jr, et al: Studies of nicotinamide adenine dinucleotide methemoglobin reductase activity in a Jewish population. Am J Hematol 12(1):13, 1982. 46. Fine DM, Eyster GE, Anderson LK, et al: Cyanosis and congenital methemoglobinemia in a puppy. J Am Anim Hosp Assoc 35(1):33, 1999. 47. Harvey JW, Ling GV, Kaneko JJ: Methemoglobin reductase deficiency in a dog. J Am Vet Med Assoc 164(10):1030, 1974. 48. Lo SC, Agar NS: NADH-methemoglobin reductase activity in the erythrocytes of newborn and adult mammals. Experientia 42(11–12):1264, 1986. 49. Graubarth J, Bloom CJ, Coleman FC, Solomon, HN. Dye poisoning in the nursery: A review of seventeen cases. JAMA 128:1155, 1945. 50. Sanchez-Echaniz J, Benito-Fernandez J, Mintegui-Raso S: Methemoglobinemia and consumption of vegetables in infants. Pediatrics 107(5):1024, 2001. 51. Hanukoglu A, Danon PN: Endogenous methemoglobinemia associated with diarrheal disease in infancy. J Pediatr Gastroenterol Nutr 23(1):1, 1996. 52. Hamon I, Gauthier-Moulinier H, Grelet-Dessioux E, et al: Methaemoglobinaemia risk factors with inhaled nitric oxide therapy in newborn infants. Acta Paediatr 99(10):1467, 2010. 53. Bricker T, Jefferson, LS, Mintz, AA: Methemoglobinemia in infants with enteritis. J Pediatr 102(1):161, 1983. 54. Murray KF, Christie DL: Dietary protein intolerance in infants with transient methemoglobinemia and diarrhea. J Pediatr 122(1):90, 1993. 55. Hegesh E, Hegesh J, Kaftory A: Congenital methemoglobinemia with a deficiency of cytochrome b5. N Engl J Med 314:757, 1986. 56. Lehmann H, Huntsman RG: Man’s Haemoglobins. Lippincott, Philadelphia, 1974. 57. Hayashi A, Fujita T, Fujimura M, et al: A new abnormal fetal hemoglobin, Hb FM-Osaka (alpha 2 gamma 2 63His replaced by Tyr). Hemoglobin 4(3–4):447, 1980. 58. Priest JR, Watterson J, Jones RT, et al: Mutant fetal hemoglobin causing cyanosis in a newborn. Pediatrics 83(5):734, 1989.

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59. Hojas-Bernal R, McNab-Martin P, Fairbanks VF, et al: Hb Chile [beta28(B10)Leu— >Met]: An unstable hemoglobin associated with chronic methemoglobinemia and sulfonamide or methylene blue-induced hemolytic anemia. Hemoglobin 23(2):125, 1999. 60. Prchal JT, Borgese N, Moore MR, et al: Congenital methemoglobinemia due to methemoglobin reductase deficiency in two unrelated American black families. Am J Med 89(4):516, 1990. 61. Wild B, Bain BJ: Investigation of abnormal haemoglobins and thalassaemia, in Dacie and Lewis Practical Haematology, edited by Lewis S, Bain B, Bates I, p 295. Churchill Livingstone, Philadelphia, 2006. 62. Yawata Y, Ding L, Tanishima K, et al: New variant of cytochrome b5 reductase deficiency (b5RKurashiki) in red cells, platelets, lymphocytes, and cultured fibroblasts with congenital methemoglobinemia, mental and neurological retardation, and skeletal anomalies. Am J Hematol 40(4):299, 1992. 63. Evelyn K, Malloy H: Microdetermination of oxyhemoglobin, methemoglobin, and sulfhemoglobin in a single sample of blood. J Biol Chem 126:655, 1938. 64. Beutler E: Carboxyhemoglobin, methemoglobin, and sulfhemoglobin determinations, in Williams Hematology, edited by Beutler E, Lichtman MA, Coller BS, Kipps TJ, p L50. McGraw-Hill, New York, 1995. 65. Halvorsen SM, Dull WL: Phenazopyridine-induced sulfhemoglobinemia: Inadvertent rechallenge. Am J Med 91(3):315, 1991. 66. Watcha MF, Connor MT, Hing AV: Pulse oximetry in methemoglobinemia. Am J Dis Child 143(7):845, 1989. 67. Molthrop D, Wheeler R, Hall K, et al: Evaluation of the methemoglobinemia associated with sulofenur. Invest New Drugs 12:99, 1994. 68. Beutler E, Gelbart T: Carboxyhemoglobin, methemoglobin, and sulf-hemoglobin determinations, in Williams Hematology, 4th ed, edited by Williams WJ, Beutler E, Erslev AJ, Lichtman MA, p 1732. McGraw-Hill, New York, 1990. 69. Barker SJ, Curry J, Redford D, et al: Measurement of carboxyhemoglobin and methemoglobin by pulse oximetry: A human volunteer study. Anesthesiology 105:892, 2006. 70. Annabi EH, Barker SJ: Severe methemoglobinemia detected by pulse oximetry. Anesth Analg 108(3):898, 2009. 71. Hampson NB: Noninvasive pulse CO-oximetry expedites evaluation and management of patients with carbon monoxide poisoning. Am J Emerg Med 30(9):2021, 2012. 72. Beutler E: Red Cell Metabolism: A Manual of Biochemical Methods. Grune & Stratton, New York, 1984. 73. Board P: NADH-ferricyanide reductase, a convenient approach to the evaluation of NADH-methaemoglobin reductase in human erythrocytes. Clin Chim Acta 109:233, 1981. 74. Lan FH, Tang YC, Huang CH, et al: Antibody-based spot test for NADH-cytochrome b5 reductase activity for the laboratory diagnosis of congenital methemoglobinemia. Clin Chim Acta 273(1):13, 1998. 75. Das Gupta A, Vaidya MS, Bapat JP, et al: Associated red cell enzyme deficiencies and their significance in a case of congenital enzymopenic methemoglobinemia. Acta Haematol 64(5):285, 1980. 76. Kaftory A, Hegesh E: Improved determination of cytochrome b5 in human erythrocytes. Clin Chem 30(8):1344, 1984. 77. Gerald PS, George P: Second spectroscopically abnormal methemoglobin associated with hereditary cyanosis. Science 129(3346):393, 1959. 78. Carrell RW, Kay R: A simple method for the detection of unstable haemoglobins. Br J Haematol 23(5):615, 1972. 79. Hutt PJ, Pisciotta AV, Fairbanks VF, et al: DNA sequence analysis proves Hb MMilwaukee-2 is due to beta-globin gene codon 92 (CAC—>TAC), the presumed mutation of Hb M-Hyde Park and Hb M-Akita. Hemoglobin 22(1):1, 1998. 80. Darling R, Roughton F: The effect of methemoglobin on the equilibrium between oxygen and hemoglobin. Am J Physiol 137:56, 1942. 81. Johnson CJ, Bonrud PA, Dosch TL, et al: Fatal outcome of methemoglobinemia in an infant. JAMA 257(20):2796, 1987. 82. Ellis M, Hiss Y, Shenkman L: Fatal methemoglobinemia caused by inadvertent contamination of a laxative solution with sodium nitrite. Isr J Med Sci 28(5):289, 1992. 83. Caudill L, Walbridge J, Kuhn G: Methemoglobinemia as a cause of coma. Ann Emerg Med 19(6):677, 1990. 84. Clifton J 2nd, Leikin JB: Methylene blue. Am J Ther 10(4):289, 2003. 85. Beutler E, Baluda MC: Methemoglobin reduction: Studies of the interaction between cell populations and of the role of methylene blue. Blood 22:323, 1963. 86. Rosen PJ, Johnson C, McGehee WG, et al: Failure of methylene blue treatment in toxic methemoglobinemia: Associations with glucose-6-phosphate dehydrogenase deficiency. Ann Intern Med 75:83, 1971. 87. Bilgin H, Ozcan B, Bilgin T: Methemoglobinemia induced by methylene blue perturbation during laparoscopy. Acta Anaesthesiol Scand 42(5):594, 1998. 88. Kearney TE, Manoguerra AS, Dunford JV Jr: Chemically induced methemoglobinemia from aniline poisoning. West J Med 140(2):282, 1984. 89. Harvey J, Keitt A: Studies of the efficacy and potential hazards of methylene blue therapy in aniline-induced methemoglobinemia. Br J Haematol 54:29, 1983. 90. Coleman MD, Rhodes LE, Scott AK, et al: The use of cimetidine to reduce dapsonedependent methaemoglobinaemia in dermatitis herpetiformis patients. Br J Clin Pharmacol 34:244, 1992. 91. Kaplan J, Chirouze M: Therapy of recessive congenital methaemoglobinemia by oral riboflavin. Lancet 2:1043, 1978. 92. Beutler E: Important recent advances in the field of red cell metabolism: Practical implications, in Erythrocytes, Thrombocytes, Leukocytes, edited by Gerlach E, Moser K, Deutsch E, Wilmanns W, p 123. George Thieme Verlag, Stuttgart, 1973.

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93. Lemberg R, Legge, JW: Hematin Compounds and Bile Pigments. Inter-science Publishers, New York, 1949. 94. Harrop GJ, Waterfield RL: Sulphemoglobinemia. JAMA 95:647, 1930. 95. Nichol A, Hendry I, Movell DB, et al: Mechanism of formation of sulfhemoglobin. Biochim Biophys Acta 156:97, 1968. 96. Berzofsky JA, Peisach J, Horecker BL: Sulfheme proteins. IV. The stoichiometry of sulfur incorporation and the isolation of sulfhemin, the prosthetic group of sulfmyoglobin. J Biol Chem 247(12):3783, 1972. 97. Berzofsky JA, Peisach J, Blumberg WE: Sulfheme proteins. II. The reversible oxygenation of ferrous sulfmyoglobin. J Biol Chem 246: 7366–7372, 1971. 98. Park CM, Nagel RL: Sulfhemoglobinemia. Clinical and molecular aspects. N Engl J Med 310(24):1579, 1984. 99. Discombe G: Sulphaemoglobinaemia and glutathione. Lancet 2:371, 1960. 100. McCutcheon A: Sulphaemoglobinaemia and glutathione. Lancet 2:290, 1960. 101. Paniker NV, Beutler E: The effect of methylene blue and diaminodiphenysulfone on red cell reduced glutathione synthesis. J Lab Clin Med 80(4):481, 1972. 102. Smith JE, Mahaffey E, Lee M: Effect of methylene blue on glutamate and reduced glutathione of rabbit erythrocytes. Biochem J 168(3):587, 1977. 103. Pandey J, Chellani H, Garg M, et al: Congenital sulfhemoglobin and transient methemoglobinemia secondary to diarrhoea. Indian J Pathol Microbiol 39(3):217, 1996. 104. Stamatoyannopoulos G, Parer JT, Finch CA: Physiologic implications of a hemoglobin with decreased oxygen affinity (hemoglobin Seattle). N Engl J Med 281(17):916, 1969. 105. Lichtman MA, Murphy MS, Adamson JW: Detection of mutant hemoglobins with altered affinity for oxygen. A simplified technique. Ann Intern Med 84(5):517, 1976. 106. Agarwal N, Mojica-Henshaw MP, Simmons ED, et al: Familial polycythemia caused by a novel mutation in the beta globin gene: Essential role of P50 in evaluation of familial polycythemia. Int J Med Sci 4(4):232, 2007. 107. Vreman HJ, Mahoney JJ, Stevenson, DK. Carbon monoxide and carboxyhemoglobin. Adv Pediatr 42:303–325, 1995. 108. Hampson NB, Weaver LK: Carbon monoxide poisoning: A new incidence for an old disease. Undersea Hyperb Med 34(3):163, 2007. 109. Weaver LK: Carbon monoxide poisoning. Crit Care Clin 15:297, 1999. 110. Centers for Disease Control and Prevention: Epidemiologic assessment of the impact of four hurricanes—Florida, 2004. MMWR Morb Mortal Wkly Rep 54(28):693, 2005. 111. Ernst A, Zibrak JD: Carbon monoxide poisoning. N Engl J Med 339:1603, 1998. 112. Chen BC, Shawn LK, Connors NJ, et al: Carbon monoxide exposures in New York City following hurricane Sandy in 2012. Clin Toxicol (Phila) 51(9):879, 2013. 113. Centers for Disease Control and Prevention: Carbon monoxide exposures after hurricane Ike—Texas, September 2008. MMWR Morb Mortal Wkly Rep 58(31):845, 2009. 114. Centers for Disease Control and Prevention: Unintentional non–fire-related carbon monoxide exposures—United States, 2001–2003. MMWR Morb Mortal Wkly Rep 54(2):36, 2005. 115. Mott JA, Wolfe MI, Alverson CJ, et al: National vehicle emissions policies and practices and declining US carbon monoxide-related mortality. JAMA 288:988, 2002. 116. Harper A, Croft-Baker J: Carbon monoxide poisoning: Undetected by both patients and their doctors. Age Ageing 33(2):105, 2004. 117. U.S. Environmental Protection Agency: Emission facts: Idling vehicle Emissions. Publication EPA420-F-98-014. USEPA, Washington, DC, 1998. Available online at: http:// www.epa.gov/oms/consumer/f98014.pdf 118. Stewart RD, Fisher TN, Hosko MJ, et al: Carboxyhemoglobin elevation after exposure to dichloromethane. Science 176:295, 1972. 119. Antonini E, Brunori M: Hemoglobin and myoglobin in their reactions with ligands. Amsterdam: North-Holland, 1971. 120. Sjostrand T: Endogenous formation of carbon monoxide in man. Nature 164(4170):580, 1949. 121. Giacometti GM, Brunori M, Antonini E, et al: The reaction of hemoglobin Zurich with oxygen and carbon monoxide. J Biol Chem 255(13):6160, 1980. 122. Balster RL, Ekelund LG, Grover RF: Evaluation of subpopulations potentially at risk to carbon monoxide exposure, in Air Quality Criteria for Carbon Monoxide edited by the U.S. EPA, p 12-1. EPA No. 600/8-90/045F. U.S. Environmental Protection Agency, Environmental Criteria and Assessment Office, Research Triangle Park, NC, 1991. 123. Hampson NB, Dunford RG, Kramer CC, et al: Selection criteria utilized for hyperbaric oxygen treatment of carbon monoxide poisoning. J Emerg Med 13:227, 1995. 124. Benesch RE, Maeda N, Benesch R: 2,3-Diphosphoglycerate and the relative affinity of adult and fetal hemoglobin for oxygen and carbon dioxide. Biochim Biophys Acta 257:178, 1972. 125. Engel RR, Rodkey FL, O’Neal JD, et al: Relative affinity of human fetal hemoglobin for CO and O2. Blood 33:37, 1969. 126. Suner S, Partridge R, Sucov A, et al: Non-invasive screening for carbon monoxide toxicity in the emergency department is valuable. Ann Emerg Med 49(5):719, 2007. 127. Weaver LK: Clinical practice. Carbon monoxide poisoning. N Engl J Med 360(12):1217, 2009. 128. Jasper BW, Hopkins RO, Duker HV, et al: Affective outcome following carbon monoxide poisoning: A prospective longitudinal study. Cogn Behav Neurol 18(2):127, 2005. 129. Gemelli F, Cattani R: Carbon monoxide poisoning in childhood. Br Med J 291:1197, 1985. 130. Lacey DJ: Neurologic sequelae of acute carbon monoxide intoxication. Am J Dis Child 135(2):145, 1981.

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131. Buckley NA, Juurlink DN, Isbister G, et al: Hyperbaric oxygen for carbon monoxide poisoning. Cochrane Database Syst Rev (4):CD002041, 2011. 132. Elkharrat D, Raphael JC, Korach JM, et al: Acute carbon monoxide intoxication and hyperbaric oxygen in pregnancy. Intensive Care Med 17:289, 1991. 133. Koren G, Shara, T, Pastuszak A, et al: A multicenter, prospective study of fetal outcome following accidental carbon monoxide poisoning in pregnancy. Reprod Toxicol 5:397, 1991. 134. Ignarro LJ: Nitric oxide. A novel signal transduction mechanism for transcellular communication. Hypertension 16(5):477, 1990. 135. Liu X, Miller MJ, Joshi MS, et al: Diffusion-limited reaction of free nitric oxide with erythrocytes. J. Biol Chem 273:18709, 1998. 136. Azarov I, Huang KT, Basu S, Gladwin MT, et al: Nitric oxide scavenging by red blood cells as a function of hematocrit and oxygenation. J Biol Chem 280:39024, 2005. 137. Kim-Shapiro DB, Schechter AN, Gladwin MT: Unraveling the reactions of nitric oxide, nitrite, and hemoglobin in physiology and therapeutics. Arterioscler Thromb Vasc Biol 26:697, 2006. 138. Stamler JS, Jia L, Eu JP, et al: Blood flow regulation by S-nitrosohemoglobin in the physiological oxygen gradient. Science 276:2034, 1997. 139. Stamler JS, Singel DJ, Piantadosi CA: SNO-hemoglobin and hypoxic vasodilation. Nat Med 14(10):1009, 2008. 140. Vitturi DA, Teng X, Toledo JC, et al: Regulation of nitrite transport in red blood cells by hemoglobin oxygen fractional saturation. Am J Physiol Heart Circ Physiol 296(5):H1398, 2009. 141. Gladwin MT, Kim-Shapiro DB. The functional nitrite reductase activity of the hemeglobins. Blood 112(7):2636, 2008. 142. Gladwin MT, Schechter AN, Kim-Shapiro DB, et al: The emerging biology of the nitrite anion. Nat Chem Biol 1(6):308, 2005. 143. Basu S, Grubina R, Huang J, et al: Catalytic generation of N2O3 by the concerted nitrite reductase and anhydrase activity of hemoglobin. Nat Chem Biol 3(12):785, 2007. 144. Lauer T, Preik M, Rassaf T, et al: Plasma nitrite rather than nitrate reflects regional endothelial nitric oxide synthase activity but lacks intrinsic vasodilator action. Proc Natl Acad Sci U S A 98(22):12814, 2001. 145. Shiva S, Wang X, Ringwood LA, et al: Ceruloplasmin is a NO oxidase and nitrite synthase that determines endocrine NO homeostasis. Nat Chem Biol 2(9):486, 2006. 146. Liu C, Zhao W, Christ GJ, et al: Nitric oxide scavenging by red cell microparticles. Free Radic Biol Med 65:1164, 2013. 147. Reynolds JD, Bennett KM, Cina AJ, et al: S-nitrosylation therapy to improve oxygen delivery of banked blood. Proc Natl Acad Sci U S A 110(28):11529, 2013. 148. Kanias T, Gladwin MT: Nitric oxide, hemolysis, and the red blood cell storage lesion: Interactions between transfusion, donor, and recipient. Transfusion 52(7):1388, 2012. 149. Kahn MJ, Maley JH, Lasker GF, et al: Updated role of nitric oxide in disorders of erythrocyte function. Cardiovasc Hematol Disord Drug Targets 13(1):83, 2013. 150. Badesch DB, Abman SH, Ahearn GS, et al: Medical therapy for pulmonary arterial hypertension: ACCP evidence-based clinical practice guidelines. Chest 126(1 Suppl):35S, 2004. 151. Martinez-Ruiz R, Montero-Huerta P, Hromi J, et al: Inhaled nitric oxide improves survival rates during hypoxia in a sickle cell (SAD) mouse model. Anesthesiology 94:1113, 2001. 152. Weiner DL, Hibberd PL, Betit P, et al: Preliminary assessment of inhaled nitric oxide for acute vaso-occlusive crisis in pediatric patients with sickle cell disease. JAMA 289:1136, 2003. 153. Sullivan KJ, Goodwin SR, Evangelist J, et al: Nitric oxide successfully used to treat acute chest syndrome of sickle cell disease in a young adolescent. Crit Care Med 27:2563, 1999. 154. McMahon TJ, Doctor A: Extrapulmonary effects of inhaled nitric oxide: Role of reversible S-nitrosylation of erythrocytic hemoglobin. Proc Am Thorac Soc 3(2):153, 2006. 155. Young JD, Dyar O, Xiong L, et al: Methaemoglobin production in normal adults inhaling low concentrations of nitric oxide. Intensive Care Med 20:581, 1994. 156. Loh E, Stamler JS, Hare JM, et al: Cardiovascular effects of inhaled nitric oxide in patients with left ventricular dysfunction. Circulation 90:2780, 1994. 157. Lundin S, Mang H, Smithies M, et al: Inhalation of nitric oxide in acute lung injury: Results of a European multicentre study. The European Study Group of Inhaled Nitric Oxide. Intensive Care Med 25:911, 1999. 158. Christenson J, Lavoie A, O’Connor M, et al: The incidence and pathogenesis of cardiopulmonary deterioration after abrupt withdrawal of inhaled nitric oxide. Am J Respir Crit Care Med 161:1443, 2000. 159. Moya MP, Gow AJ, McMahon TJ, et al: S-nitrosothiol repletion by an inhaled gas regulates pulmonary function. Proc Natl Acad Sci U S A 98:5792, 2001. 160. Moya MP, Gow AJ, Califf RM, et al: Inhaled ethyl nitrite gas for persistent pulmonary hypertension of the newborn. Lancet 360:141, 2002. 161. Gulati A, Sen AP, Sharma AC, et al: Role of ET and NO in resuscitative effect of diaspirin cross-linked hemoglobin after hemorrhage in rat. Am J Physiol 273:H827, 1997. 162. Gibson JB, Maxwell RA, Schweitzer JB, et al: Resuscitation from severe hemorrhagic shock after traumatic brain injury using saline, shed blood, or a blood substitute. Shock 17:234, 2002. 163. Paris PM, Kaplan RM, Stewart RD, et al: Methemoglobin levels following sublingual nitroglycerin in human volunteers. Ann Emerg Med 15(2):171, 1986. 164. Gavish D, Knobler H, Gottehrer N, et al: Methemoglobinemia, muscle damage and renal failure complicating phenazopyridine overdose. Isr J Med Sci 22(1):45, 1986.

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165. Christensen CM, Farrar HC, Kearns GL: Protracted methemoglobinemia after phenazopyridine overdose in an infant. J Clin Pharmacol 36(2):112, 1996. 166. Damergis JA, Stoker JM, Abadie JL: Methemoglobinemia after sulfametoxazole and trimethoprim. JAMA 249(5):590, 1983. 167. Falkenhahn M, Kannan S, O’Kane M: Unexplained acute severe methaemoglobinaemia in a young adult. Br J Anaesth 86(2):278, 2001. 168. Wagner A, Marosi C, Binder M, et al: Fatal poisoning due to dapsone in a patient with grossly elevated methaemoglobin levels. Br J Dermatol 133(5):816, 1995. 169. Ng LL, Nai KR, Polak A: Paraquat ingestion with methaemoglobinaemia treated with methylene blue. Br Med J (Clin Res Ed) 284(6327):1445, 1982. 170. Proudfoot AT: Methaemoglobinaemia due to monolinuron—Not paraquat. Br Med J (Clin Res Ed) 285(6344):812, 1982. 171. de Torres JP, Strom JA, Jaber BL, et al: Hemodialysis-associated methemoglobinemia in acute renal failure. Am J Kidney Dis 39(6):1307, 2002. 172. Gibson GR, Hunter JB, Raabe DS Jr, et al: Methemoglobinemia produced by high-dose intravenous nitroglycerin. Ann Intern Med 96(5):615, 1982. 173. Forsyth RJ, Moulden A: Methaemoglobinaemia after ingestion of amyl nitrite. Arch Dis Child 66(1):152, 1991. 174. Guss DA, Normann SA, Manoguerra AS: Clinically significant methemoglobinemia from inhalation of isobutyl nitrite. Am J Emerg Med 3(1):46, 1985. 175. Kuschner WG, Chitkara RK, Canfield J Jr, et al: Benzocaine-associated methemoglobinemia following bronchoscopy in a healthy research participant. Respir Care 45(8):953, 2000. 176. Abdallah HY, Shah SA: Methemoglobinemia induced by topical benzocaine: A warning for the endoscopist. Endoscopy 34(9):730, 2002.

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177. Novaro G, Aronow H, Militello M, et al: Benzocaine-induced methemoglobinemia: Experience from a high-volume transesophageal echocardiography laboratory. J Am Soc Echocardiogr 16:170, 2003. 178. Nilsson A, Engberg G, Henneberg S, et al: Inverse relationship between age-dependent erythrocyte activity of methaemoglobin reductase and prilocaine-induced methaemoglobinaemia during infancy. Br J Anaesth 64(1):72, 1990. 179. Duncan PG, Kobrinsky N: Prilocaine-induced methemoglobinemia in a newborn infant. Anesthesiology 59(1):75, 1983. 180. Lloyd CJ: Chemically induced methaemoglobinaemia in a neonate. Br J Oral Maxillofac Surg 30(1):63, 1992. 181. Davidovits M, Barak A, Cleper R, et al: Methaemoglobinaemia and haemolysis associated with hydrogen peroxide in a paediatric haemodialysis centre: A warning note. Nephrol Dial Transplant 18(11):2354, 2003. 182. Gerald PS, Efron ML: Chemical studies of several varieties of Hb M. Proc Natl Acad Sci U S A 47:1758, 1961. 183. Stavem P, Stromme J, Lorkin PA, et al: Haemoglobin M Saskatoon with slight constant haemolysis, markedly increased by sulphonamides. Scand J Haematol 9(6):566, 1972. 184. Hayashi N, Motokawa Y, Kikuchi G: Studies on relationships between structure and function of hemoglobin M-Iwate. J Biol Chem 241(1):79, 1966. 185. Horst J, Schafer R, Kleihauer E, et al: Analysis of the Hb M Milwaukee mutation at the DNA level. Br J Haematol 54(4):643, 1983. 186. Hain RD, Chitayat D, Cooper R, et al: Hb FM-Fort Ripley: Confirmation of autosomal dominant inheritance and diagnosis by PCR and direct nucleotide sequencing. Hum Mutat 3(3):239, 1994. 187. Reissmann KR, Ruth WE, Nomura T: A human hemoglobin with lowered oxygen affinity and impaired heme-heme interactions. J Clin Invest 40:1826, 1961.

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CHAPTER 51

FRAGMENTATION HEMOLYTIC ANEMIA

Kelty R. Baker and Joel Moake

SUMMARY Erythrocyte fragmentation and hemolysis occur when red cells are forced at high shear stress through partial vascular occlusions or over abnormal vascular surfaces. “Split” red cells, or schistocytes, are prominent on blood films under these conditions, and considerable quantities of lactate dehydrogenase are released into the blood from traumatized red cells. In the high-flow (high-shear) microvascular (arteriolar/capillary) or arterial circulation, partial vascular obstructions are caused by platelet aggregates in the systemic microvasculature during episodes of thrombotic thrombocytopenic purpura by platelet-fibrin thrombi in the renal microvasculature in the hemolytic uremic syndrome; and by malfunction of a cardiac prosthetic valve in valve-related hemolysis. Less-extensive red cell fragmentation, hemolysis, and schistocytosis occur under conditions of more moderate vascular occlusion or endothelial surface abnormalities, sometimes under conditions of lower shear stress. These latter entities include excessive platelet aggregation, fibrin polymer formation, and secondary fibrinolysis in the arterial or venous microcirculation (disseminated intravascular coagulation); in the placental vasculature in preeclampsia/eclampsia and the syndrome of hemolysis, elevated liver enzymes and low platelets (HELLP) in march hemoglobinuria; and in giant cavernous hemangiomas (the Kasabach-Merritt phenomenon).

PREECLAMPSIA/ECLAMPSIA AND HELLP SYNDROME DEFINITION AND HISTORY A life-threatening condition of pregnancy denoted by eclampsia, hemolysis, and thrombocytopenia was first noted in the German literature by Stahnke in 1922.1 Subsequently, Pritchard and coworkers described three cases in English and suggested that an immunologic process might account for both the preeclampsia or eclampsia and the hematologic abnormalities.2

Acronyms and Abbreviations:  ADAMTS13, a disintegrin and metalloproteinase with thrombospondin domain 13; ALT, alanine transaminase; aPTT, activated partial thromboplastin time; AST, aspartic acid transaminase; AT, antithrombin; DIC, disseminated intravascular coagulation; HELLP, hemolysis, elevated liver enzymes, and low platelet count; LDH, lactate dehydrogenase; MAHA, microangiopathic hemolytic anemia; NO, nitrous oxide; PGF, placental growth factor; PGI2, prostaglandin I2; PT, prothrombin time; PTT, partial thromboplastin time; sEng, soluble endoglin; sFlt-1, soluble form of fms-like tyrosine kinase 1; sVEGFR-1, soluble vascular endothelial growth factor receptor-1; TGF-β, transforming growth factor-β; TTP, thrombotic thrombocytopenic purpura; VEGF, vascular endothelial growth factor; VWF, von Willebrand factor.

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Although initially known as edema-proteinuria-hypertension gestosis type B,3 a catchier phrase, HELLP syndrome (H for hemolysis, EL for elevated liver function tests, and LP for low platelet counts), was later applied by Louis Weinstein in 1982.4

EPIDEMIOLOGY HELLP syndrome occurs in approximately 0.5 percent of pregnancies overall,5 in 4 to 12 percent of those complicated by preeclampsia (hypertension + proteinuria), and in 30 to 50 percent of those complicated by eclampsia (hypertension + proteinuria + seizures); however, approximately 15 percent of patients ultimately diagnosed with HELLP syndrome present with neither hypertension nor proteinuria.6 Twothirds of HELLP patients are diagnosed antepartum, usually between 27 and 37 weeks. The remaining one-third are diagnosed in the postpartum period, from a few to 48 hours following delivery (occasionally as long as 6 days).7,8 Risk factors for HELLP syndrome include European ancestry, multiparity, older maternal age (older than age 34 years), and a personal or familial history of the disorder.5 Although the presence of homozygosity for the 677 (C→T) polymorphism of the methylenetetrahydrofolate reductase gene may be a modest risk factor for the development of preeclampsia, this weak association does not exist for HELLP syndrome.9 Whether or not the factor V Leiden or prothrombin 20210 gene mutations are risk factors for HELLP syndrome remains controversial.10–12

ETIOLOGY AND PATHOGENESIS A developing embryo must acquire a supply of maternal blood to survive. During a normal pregnancy, the first wave of trophoblastic invasion into the decidua occurs at 10 to 12 days. This is followed by a second wave at 16 to 22 weeks, when these specialized placental epithelial cells replace the endothelium of the uterine spiral arteries and intercalate within the muscular tunica, increasing the vessels’ diameters and decreasing their resistance. As a result, the spiral arteries are remodeled into unique hybrid vessels composed of fetal and maternal cells, and the vasculature is converted into a high-flow–low-resistance system resistant to vasoconstrictors circulating in the maternal blood.13 In a preeclamptic pregnancy, the second wave fails to penetrate adequately the spiral arteries of the uterus, perhaps as a result of reduced placental expression of syncytin and subsequent altered cell fusion processes during placentogenesis.14 The resultant poorly perfused, hypoxic placenta then releases the extracellular domain (soluble) form of fmslike tyrosine kinase 1 (sFLT-1), also known as soluble vascular endothelial growth factor receptor-1 (sVEGF receptor-1, or sVEGFR-1). sVEGFR-1 functions as an antiangiogenic protein because it binds to vascular endothelial growth factor (VEGF) and placental growth factor (PGF), and prevents their interaction with endothelial cell receptors. The result is glomerular endothelial cell and placental dysfunction.15–17 Direct and indirect sequelae include increased vascular tone, hypertension, proteinuria, enhanced platelet activation and aggregation, and decreased levels of the vasodilators prostaglandin I2 (PGI2) and nitrous oxide (NO).5,17 Concurrent activation of the coagulation cascade results in platelet-fibrin deposition in the capillaries, multiorgan microvascular injury, microangiopathic hemolytic anemia, elevated liver enzymes because of hepatic necrosis, and thrombocytopenia because of peripheral consumption of platelets.5 Another antiangiogenic molecule, a soluble form of endoglin (sEng), also increases in patient serum during early and severe preeclampsia.18 Endoglin is part of the transforming growth factor-β (TGF-β) complex, and is expressed on vascular endothelial cells and syncytiotrophoblasts. The shed extracellular domain of endoglin, sEng, is

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capable of binding to and inactivating the proangiogenic growth factors, TGF-β1 and TGF-β3. The presence of elevated serum levels of both sFLT-1 (sVEGFR-1) and sEng may be associated with the progression of preeclampsia to HELLP.17,18

CLINICAL FEATURES Ninety percent of patients with HELLP syndrome present with malaise and right upper quadrant or epigastric pain. Between 45 and 86 percent have nausea or vomiting, 55 to 67 percent have edema, 31 to 50 percent have headache, and a smaller percentage complain of visual changes. Fever is not typically seen. Although hypertension is found in 85 percent of affected patients, 15 percent of those with HELLP syndrome do not develop either hypertension or proteinuria.6

LABORATORY FEATURES There is no consensus regarding the laboratory criteria necessary to diagnose HELLP syndrome, so clinical judgment in conjunction with judicious interpretation of a variety of laboratory tests constitute the diagnostic standard. In 54 to 86 percent of patients, the blood film has schistocytes, helmet cells, and burr cells consistent with microangiopathic hemolytic anemia. Reticulocytosis can be present. Low haptoglobin levels are both sensitive (83 percent) and specific (96 percent) for confirming the presence of hemolysis, and return to normal within 24 to 30 hours postpartum.6 Lactate dehydrogenase (LDH) levels are usually above normal. The ratio of LDH-5 (an isoenzyme found specifically in the liver) to total LDH is elevated in proportion to the severity of HELLP. The high LDH seen in HELLP is most likely the result, principally, of liver damage rather than hemolysis. Serum levels of aspartic acid transaminase (AST) and alanine transaminase (ALT) can be more than 100 times normal, whereas alkaline phosphatase values are typically only about twice normal and total bilirubin ranges between 1.2 and 5.0 mg/dL. Liver enzymes usually return to normal within 3 to 5 days postpartum.6 The degree of thrombocytopenia has been used in a classification system to predict maternal morbidity and mortality, the rapidity of postpartum recovery, the risk of disease recurrence, and perinatal outcome. This Mississippi triple-class system places those patients with platelet counts less than 50 × 109/L in class 1 (approximately 13 percent incidence of bleeding); those with platelet counts between 50 and 100 × 109/L in class 2 (approximately 8 percent incidence of bleeding); and those with a platelet count greater than 100 × 109/L in class 3 (no increased bleeding risk). Patients with class 1 HELLP syndrome suffer the highest incidence of perinatal morbidity and mortality, and have the most protracted recovery periods postpartum.19 There is a direct correlation between the extent of thrombocytopenia and measurements of liver function,20 but the same cannot be said for the severity of associated hepatic histopathologic changes.21 If a marrow aspiration and biopsy are performed, abundant megakaryocytes are found consistent with a consumptive thrombocytopenia causing reduction of the normal platelet life span of approximately 10 days to 3 to 5 days.19 The platelet count nadir occurs 23 to 29 hours postpartum, with subsequent normalization within 6 to 11 days.7 The prothrombin time (PT) and activated partial thromboplastin time (aPTT) are usually within normal limits, although one report cited a prolonged aPTT in 50 percent of patients.22 Although low fibrinogen levels are inconsistently found, other measures of increased coagulation and secondary fibrinolysis may be present. These include decreased protein C and antithrombin III (AT III) levels, and increased D-dimer and thrombin-AT III values. von Willebrand factor (VWF) antigen levels increase in proportion to the severity of the disease, reflecting the extent

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of endothelial damage; however, no unusually large VWF multimers are present in plasma23 and ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin domains-13) levels are within a broad normal range (ADAMTS13 normally declines moderately during pregnancy).24,25 This is in contrast to the severe deficiency of ADAMTS13 in familial and autoantibody-mediated types of thrombotic thrombocytopenic purpura (TTP).26 Unlike TTP, the thrombi found in organs involved in the HELLP syndrome contain increased amounts of fibrin and low levels of VWF.23 In patients with severe liver involvement, hepatic ultrasonography shows large, irregular, well-demarcated (or “geographical”) areas of increased echogenicity.27 Liver biopsy shows periportal or focal necrosis, platelet-fibrin deposits in the sinusoids, and vascular microthrombi. As the disease progresses, large areas of necrosis can coalesce and dissect into the liver capsule. This produces a subcapsular hematoma and the risk of hepatic rupture.5

DIFFERENTIAL DIAGNOSIS Other complications of pregnancy that can be confused with HELLP include TTP28 and the hemolytic uremic syndrome, sepsis, disseminated intravascular coagulation (DIC), connective tissue disease, antiphospholipid antibody syndrome, and acute fatty liver of pregnancy. This latter entity is also seen in the last trimester or postpartum and presents with thrombocytopenia and right upper quadrant pain, but the levels of AST and ALT only rise to 1 to 5 times normal and the PT and partial thromboplastin time (PTT) are both prolonged. Oil-red-O staining of liver biopsies demonstrates fat in the cytoplasm of centrilobular hepatocytes, and routine stains show inflammation and patchy hepatocellular necrosis as a result of the HELLP syndrome. Because it causes right upper quadrant pain and nausea, HELLP has also been misdiagnosed as viral hepatitis, biliary colic, esophageal reflux, cholecystitis, and gastric cancer. Conversely, other conditions misdiagnosed as HELLP syndrome include cardiomyopathy, dissecting aortic aneurysm, acute cocaine intoxication, essential hypertension and renal disease, and alcoholic liver disease.19

THERAPY Supportive care of HELLP includes intravenous administration of magnesium sulfate to control hypertension and prevent eclamptic seizures, management of fluids and electrolytes, judicious transfusion of blood products, stimulation of fetal lung maturation with beclomethasone, and delivery of the fetus as soon as possible.19 Indications for delivery include a severe disease presentation, maternal DIC, fetal distress, and a gestational age greater than 32 weeks with evidence of lung maturity.6 Cesarean section under general anesthesia is used in 60 to 97 percent of cases, but vaginal delivery after induction can be attempted if the fetus is older than 32 weeks of age and the mother’s cervical anatomy is favorable. Postpartum curettage is helpful in lowering the mean arterial pressure and increasing the urine output and platelet count. Transfusion therapy with packed red cells, platelets, or fresh-frozen plasma is indicated in cases complicated by severe anemia or bleeding because of coagulopathy. Although previously thought to be beneficial based upon the results of observational studies and small randomized trials, the use of dexamethasone has fallen out of favor after large randomized trials found that it didn’t reduce the duration of hospitalization, amount of blood products transfused, maternal complications, or time to normalization of laboratory abnormalities.29 Plasma exchange cannot arrest or reverse HELLP syndrome when used antepartum, but may minimize hemorrhage and morbidity when

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Chapter 51: Fragmentation Hemolytic Anemia

used peripartum. It can also be tried postpartum in the 5 percent of patients who fail to improve within 72 to 96 hours of delivery. These women are more likely to be younger than 20 years of age or nulliparous.7 Whether or not plasma exchange can effectively lower circulating levels of sVEGF and/or sEng is not known. Liver transplantation may be necessary in occasional patients with HELLP complicated by large hematomas or total hepatic necrosis. It is not yet known if replacement with some (possibly modified) form of VEGF and/or TGF-β may have future therapeutic use in preeclampsia or HELLP. A single case report describes the successful use of eculizumab to prolong by 17 days a pregnancy affected by severe HELLP, without associated maternal or fetal morbidity or mortality.30

COURSE AND PROGNOSIS Most patients stabilize within 24 to 48 hours following delivery; however, maternal death still occurs in 3 to 5 percent even with best supportive care. Mortality rates as high as 25 percent were reported prior to 1980. Events leading to maternal death include cerebral hemorrhage, cardiopulmonary arrest, DIC, adult respiratory distress syndrome, and hypoxic ischemic encephalopathy.5 Other complications include infection, placenta abruptio, postpartum hemorrhage, intraabdominal bleeding, and subcapsular liver hematomas with resultant rupture (a fatal event in 50 percent of those in whom it occurs).6 The latter patients complain of right-sided shoulder pain and are found to be in shock with ascites or pleural effusions. The hematoma is usually present in the anterior superior portion of the right lobe of the liver.5 If the liver remains intact when discovered, abdominal palpation, seizures, and emesis should be avoided or prevented. Emergency surgery is required for hepatic artery embolization or ligation, hepatic lobectomy, or even liver transplantation in patients with total hepatic necrosis.5,19 Renal complications of HELLP include acute renal failure, hyponatremia, and nephrogenic diabetes insipidus as a result of impaired hepatic metabolism of vasopressinase and resultant “resistance to vasopressin” (antidiuretic hormone). Pulmonary complications of HELLP include of pleural effusions, pulmonary edema, and adult respiratory distress syndrome. Neurologic sequelae of HELLP not mentioned above include retinal detachment, postictal cortical blindness, and hypoglycemic coma.31 Fetal morbidity and mortality are between 9 and 24 percent.6 Complications arise as a result of prematurity, placental abruption, and intrauterine asphyxia. Intrauterine growth retardation is seen in 39 percent of infants. One-third of all babies born to mothers with HELLP have thrombocytopenia, but intraventricular hemorrhage is seen in only 4 percent of thrombocytopenic infants.32 HELLP syndrome complicates 2 to 5 percent of all pregnancies,5 and can recur in as many as 27 percent of those affected during subsequent pregnancies.33 Other hypertensive disorders of pregnancy (preeclampsia or pregnancy-induced hypertension) are also relatively common in future pregnancies (27 percent of second and subsequent pregnancies).34 Women who recover from preeclampsia/HELLP may also be more likely to develop subsequent hypertension and cardiovascular disorders, possibly because of some persistent abnormal balance between proangiogenic and antiangiogenic factors.17

DISSEMINATED MALIGNANCY

EPIDEMIOLOGY Cancer-associated microangiopathic hemolytic anemia (MAHA) has been described in a wide variety of malignancies (Table 51–1). MAHA is more likely to be associated with metastatic malignant disease than with localized cancers or benign tumors.36 Approximately 80 percent of the tumors are mucinous adenocarcinomas of either the stomach (55 percent), breast (13 percent), or lung (10 percent). The median age at diagnosis is 50 years, with a slight male predominance.37

ETIOLOGY AND PATHOGENESIS MAHA as a result of malignancy can be caused by either of two distinct mechanisms: (1) DIC with intravascular occlusions (often partial) of small vessels by platelet-fibrin thrombi; or (2) intravascular tumor emboli.35,38 In the first mechanism,1 intravascular activation of coagulation may occur from excessive exposure of tissue factor on phagocytes, activated endothelial cells, or tumor cells. Alternatively, a protease in the mucin secreted by adenocarcinomas may directly activate factor X.39 Subsequent activation of coagulation factors, thrombin generation, fibrin polymer deposition, and platelet aggregation result in the formation of intravascular platelet-fibrin thrombi, and the shearing of red cells attempting to maneuver past the partial platelet-fibrin occlusions in the high-flow microvasculature. Also, circulating carcinoma mucins may interact with leukocyte L-selectin and platelet P-selectin, causing the rapid generation of platelet-rich microthrombi.40 In the second mechanism,2 intravascular tumor emboli partially occlude small vessels, mechanically or chemically disrupt the endothelium and promote platelet adherence to exposed subendothelium, coagulation activation and fibrin polymer formation, intimal hyperplasia, and vascular hypertrophy.35,37,38

TABLE 51–1.  Cancers Associated with Microangiopathic Hemolytic Anemia Gastric (55%)37,40 Breast (13%)129 Lung (10%)35 Other Adenocarcinomas   Unknown primary38  Prostate35  Colon38  Gallbladder  Pancreas  Ovary Other Malignancies  Hemangiopericytoma36  Hepatoma  Melanoma   Small cell cancer of the lung130   Testicular cancer

DEFINITION AND HISTORY

  Squamous cell cancer of the oropharynx

The association between widespread malignancy and hemolytic anemia associated with pathologic changes in small blood vessels was first noted by Brain and colleagues in 1962.35

 Thymoma

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 Erythroleukemia131

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LABORATORY FEATURES Patients with cancer-associated DIC/MAHA present with moderate-tosevere anemia. The blood film reveals schistocytes (accounting for approximately 5 to 21 percent of the red cells), burr cells, microspherocytes, reticulocytes/polychromasia, and nucleated red cells.38 Although the reticulocyte count can be high, it is an unreliable measure of hemolysis because extensive replacement of the marrow by metastatic tumor (Chap. 45) may prevent the reticulocytosis expected with MAHA. Other indicators of hemolysis that could be more reliable include increased levels of serum unconjugated bilirubin and LDH, the presence of plasma hemoglobin, and elevated urine urobilinogen and hemoglobinuria (as αβ dimers).37 Absent or low levels of haptoglobin may also be found; however, haptoglobin is an acute-phase reactant that may be increased in malignancy.38 The direct antiglobulin test is negative.37,41 Additional findings in MAHA include thrombocytopenia, with mean platelet counts of approximately 50 × 109/L (range: 3 to 225 × 109/L),37 caused by a shortened platelet life span without demonstrable sequestration of platelets in the liver or spleen. Some patients with malignant tumors, however, may have preexisting thrombocytosis, and so superimposed MAHA may reduce the platelet count only toward “normal” values.38 A normal-to-high white cell count with immature myeloid precursors may also be seen.37,38,41 Leukoerythroblastosis caused by marrow invasion (Chap. 45), along with MAHA, is highly suggestive of metastatic malignancy.38 Marrow aspiration and biopsy will demonstrate erythroid hyperplasia, normal-to-high numbers of megakaryocytes, and (in 55 percent of patients) cancer cells.41 Additional laboratory evidence of DIC has been reported in approximately 50 percent of patients with MAHA secondary to malignancy. Findings include reduced levels of fibrinogen (mean: 177 g/dL; range: 8 to 490 mg/dL), increased levels of D-dimers (or fibrin degradation products), and prolonged prothrombin and thrombin times.37 In the early phase of DIC, aPTTs may be shortened (e.g., to >5%

Haptoglobin

Decreased

Absent

Absent

LDH

500 U/L

>>500 U/L

LDH, lactate dehydrogenase. Data from Eyster E, Rothchild J, Mychajliw O: Chronic intravascular hemolysis after aortic valve replacement, Circulation 1971 Oct;44(4):657-665.

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DIFFERENTIAL DIAGNOSIS Factors that can promote valve-associated hemolysis or worsen the resultant anemia include iron deficiency (Chap. 43), because anemia increases cardiac output and shear stress and iron-poor red cells are more fragile than normal; folate deficiency (Chap. 41) arising from increased erythropoiesis; anemia of chronic disease because of endocarditis; anticoagulant-induced gastrointestinal hemorrhage (Chaps. 37, 133, and 134); and increased cardiac output as a consequence of strenuous physical exertion.82

THERAPY Appropriate therapy for hemolytic anemia arising from valvular dysfunction consists of iron and folate replacement (if deficient) and surgical repair or replacement of the malfunctioning prosthesis (if indicated).87 Poor surgical candidates with perivalvular leaks may benefit from percutaneous closure with an Amplatzer occluder device.88 Adjunctive measures to be tried include β-blockade to slow the velocity of the circulation,89 erythropoietin therapy to stimulate erythropoiesis further,90 and pentoxifylline therapy to increase the deformability of red cells.91 Although some authors have not found the use of pentoxifylline to be beneficial,92 several case reports have described amelioration of valve-related hemolysis and resultant decreased need for red cell transfusion in patients receiving pentoxifylline.93–95 A prospective study of 40 individuals with double (mitral and aortic) valve replacements randomized patients to receive either no treatment or pentoxifylline 400 mg orally three times daily for 120 days. The group who received pentoxifylline had significantly higher hemoglobin and haptoglobin levels, and significantly lower LDH, total and indirect bilirubin, and corrected reticulocyte levels, after 4 months of treatment. Of the nine patients with severe hemolysis (LDH >1500 U/L), six individuals had amelioration or complete resolution of their disease, while three patients’ hemolysis persisted unchecked, suggesting that pentoxifylline therapy is beneficial in more than 60 percent of those with valve-related hemolysis.96 Between 15 and 30 percent of patients will develop black pigment gallstones following valve surgery, the majority occurring within 6 months of the procedure. Whether this is a result of acute hemolysis associated with use of the heart–lung machine97 or chronic hemolysis because of the valve replacement itself 98,99 is uncertain; however, therapy with ursodeoxycholic acid 600 mg daily beginning 1 week before surgery significantly decreases the incidence of gallstone formation from approximately 29 percent in those who were left untreated to approximately 8 percent (P 25% are immature or atypical mast cells b. Detection of a point mutation in KIT at codon 816 in marrow, blood, or other extracutaneous organ c. Mast cells in marrow, blood, or other extracutaneous organs that coexpress CD117 with CD2 and/or CD25 d. Serum total tryptase persistently >20 ng/mL (if there is an associated myeloid disorder, this criterion is not valid) The diagnosis of systemic mastocytosis can be made if one major and one minor criterion are present or if three minor criteria are met.

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Marrow involvement is much less common in children. In a study of 17 children with cutaneous or disseminated mast cell disease, small focal mast cell lesions were observed in marrow biopsies in 10 individuals, and increased mast cells in marrow aspirate films were noted in 5.193 The focal lesions found in children usually are small and perivascular. Progression of marrow involvement in systemic mast cell disease is variable. Some adults with indolent disease appear to have stable, or even decreasing, marrow involvement over time.189 In contrast, a progressive increase in focal mast cell lesions is more commonly observed in patients with more aggressive patterns of disease.

CLINICAL PRESENTATION Even though individuals may differ in the specific pathogenesis of their disease, all patients within a given category of mastocytosis (see Table  63–5) tend to exhibit similar clinical features. Manifestations of the disease largely reflect the local and systemic consequences of mediator release from tissue mast cells. Effects caused by disruption of normal structures by local collections of mast cells also may be seen.

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At presentation, patients with mastocytosis may complain of vague and nonspecific constitutional symptoms, such as fatigue, weakness, flushing, and musculoskeletal pain. Some patients experience fever and/or weight loss.152,186 A subset of patients may present with recurrent episodes of unexplained anaphylaxis.198 However, most patients with mastocytosis and a hematologic disorder are diagnosed based on marrow biopsy findings, during the investigation of their hematologic disease.187,189 Patients with aggressive disease often present with unexplained lymphadenopathy and splenomegaly and/or hepatomegaly. Gastrointestinal disease and associated symptoms are commonly associated with systemic mastocytosis, either at presentation or as the disease progresses.174,199 Findings include nausea, vomiting, abdominal pain, and diarrhea. Peptic ulcer disease, which is thought to reflect, at least in part, the promotion of gastric acid secretion by elevated histamine levels, occurs in up to 50 percent of patients with systemic disease.199 With progressive disease, patients may develop mild malabsorption.199 If systemic involvement is advanced at the time of diagnosis, patients may exhibit lymphadenopathy, hepatomegaly, and splenomegaly during the initial evaluation.152 Because osteoporosis may accompany systemic disease, pathologic fractures may occur.

LABORATORY FEATURES When systemic mastocytosis is suspected in patients based on a combination of: reports of symptoms consistent with mediator release, identification of classical skin lesions showing a 10-fold or greater increase in mast cell numbers, an elevation in serum tryptase of greater than 20 ng/mL200 and documentation of organomegaly, an appropriate next step is to perform a marrow biopsy and aspirate.152,176,201 Additional studies, including a gastrointestinal evaluation involving radiographic studies of the upper gastrointestinal tract and small intestines, computed tomographic scan of the abdomen, and endoscopy, also may be justified. Plasma and/or urinary histamine levels may be increased in systemic mastocytosis.186 However, the isolated findings of increased levels of histamine or histamine metabolites may reflect a number of other situations, including anaphylaxis. Furthermore, the accuracy of laboratory measurement of histamine depends on the assay used. Urine histamine levels may be falsely elevated as a result of bacterial contamination, pharmacologic agents and their metabolites excreted in the urine, or diets rich in histamine or histamine precursors. Similarly, serum tryptase may be elevated after anaphylaxis. Thus, no single laboratory test showing an elevation in a mast cell mediator is diagnostic of mastocytosis. Rather, the demonstration of such mediators in blood or urine should prompt the clinician to investigate further for the presence of mastocytosis. There are patients who have symptoms of mediator release but no mastocytosis in the skin or organomegaly. Some of these patients may have experienced venom-induced anaphylaxis. These patients may also exhibit elevations in tryptase but below the 20 ng/mL which is used as a minor diagnostic criterion. In such situations, reports have suggested the detection of the D816V mutation in blood using a highly sensitive allele-specific quantitative polymerase chain reaction (qPCR) may be useful as a diagnostic parameter.201 At the time of writing of this chapter, this test is not widely available and it is being used largely in referral centers.

DIFFERENTIAL DIAGNOSIS The differential diagnosis of systemic mastocytosis includes allergic diseases; hereditary or acquired angioneurotic edema; idiopathic flushing, urticaria, and anaphylaxis; carcinoid tumor; and idiopathic capillary leak

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syndrome. When episodic hypertension is a major finding, pheochromocytoma should be considered. Significant unexplained gastroduodenal ulcer disease requires that Zollinger-Ellison gastrinoma syndrome be ruled out. Helicobacter pylori infection should be considered in all patients with ulcer disease, including patients with mastocytosis. Some diseases have hematologic findings that overlap with those of systemic mastocytosis. These disorders include tryptase-positive AML, CML with accumulation of tryptase-positive cells, primary myelofibrosis with mast cell accumulation, and acute or chronic basophilic leukemia. A somatic mutation in KIT at codon 816 (most commonly Asp816Val) is associated especially with adult-onset systemic mastocytosis. Demonstration of a codon 816 gain-of-function mutation, where the most sensitive approach is to look for its presence in sorted marrow-derived mast cells, is a minor criterion in the diagnosis of mastocytosis (see Table  63–6).

THERAPY Mastocytosis currently has no cure.202 In addition, no evidence indicates that symptomatic therapy significantly alters the course of the underlying disease.

Avoiding Triggers

Management of mastocytosis includes instructing the patient on the avoidance of factors that may trigger symptoms (presumably by direct or indirect activation of mast cell mediator production). Such factors can include temperature extremes, physical exertion, or, in some unusual cases, ingestion of ethanol, nonsteroidal antiinflammatory drugs, or opiate analgesics.174

Epinephrine and H1 or H2 Antihistamines

Anaphylaxis may follow insect stings, even in the absence of evidence of allergic sensitivity.198 Epinephrine-filled syringes and instructions on their use can be given to patients considered at risk for such a reaction. Patients with mast cell disease and a history of anaphylaxis should be advised to carry epinephrine-filled syringes, instructed on their use, and taught to self-medicate, if necessary. These patients also may benefit from the concurrent use of H1 and H2 antihistamines prophylactically. Patients may experience severe reactions to iodinated contrast materials. Thus, consideration should be given to premedicating mastocytosis patients with H1 and H2 antihistamines and prednisone. Nonsedative H1 antihistamines decrease skin irritability and pruritus.186,202 Pruritus may be relieved by approaches that maintain skin hydration. H2 antihistamines, including ranitidine and famotidine, are used to treat the gastritis and peptic ulcer disease associated with mastocytosis.186,202–204 H2 antihistamines may be titrated based on symptom control or to a particular level of gastric secretion. Proton pump inhibitors are useful for management of gastric hypersecretion.199,204

OTHER DRUG THERAPY Disodium Cromoglycate; Ketotifen

Oral administration of disodium cromoglycate may be useful for treatment of gastrointestinal cramping and diarrhea.186,202,205 The agent has been beneficial in cutaneous mast cell disease in children and infants.186 Other symptoms, including headache, have improved with administration of cromolyn sodium. Ketotifen reportedly has been effective in relieving pruritus and wheal formation in cutaneous mastocytosis.206 By contrast, one pediatric study found ketotifen was no more effective than hydroxyzine.207

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Chapter 63: Basophils, Mast Cells, and Related Disorders

Bisphosphonates

Osteoporosis in patients with mastocytosis may be unrecognized and thus undertreated, especially in patients with milder forms of disease. It is thus important to utilize dual-energy x-ray absorptiometry (DEXA) scanning in the evaluation of those with mastocytosis. Recommended approaches for the treatment of osteoporosis include calcium supplementation, consideration of estrogen replacement in postmenopausal women, and use of bisphosphonates.204

Nonsteroidal Antiinflammatory Agents

Nonsteroidal antiinflammatory agents have been useful in some patients whose primary manifestations are recurrent episodes of flushing, syncope, or both.202,204 It should be noted that these agents may exacerbate ulcer disease. Patients with a history of aspirin sensitivity should not be placed on this therapy unless they first undergo desensitization.

Glucocorticoids; Methoxypsoralen

Cutaneous lesions have been treated with either glucocorticoids208 or 8-methoxypsoralen plus ultraviolet A (PUVA),209 largely to reduce pruritus or for cosmetic improvement. No evidence indicates such approaches alter the progression of systemic disease. Relapses 3 to 6 months after cessation of PUVA therapy are common. Patients may experience a decrease in the intensity of lesions after exposure to natural sunlight. Repeated or extensive application of glucocorticoids may result in cutaneous atrophy or adrenocortical suppression.208 Systemic glucocorticoids are used to decrease significant malabsorption and ascites210 in patients with advanced disease. In adults, oral prednisone (40 to 60 mg/day) usually results in decreased symptoms over a 2- to 3-week period. After initial improvement, steroids usually can be tapered to an alternate-day regimen. However, with time, the ascites frequently recurs. Such patients reportedly can benefit from a portacaval shunt.

Interferon-α Cladribine

Patients with more advanced categories of systemic mastocytosis may be candidates for approaches directed at reducing the mast cell burden. None of these approaches has resulted in cure of the disease. For severe disease, some limited success has been reported for interferon α (IFN-α) and it is often considered to be a first-line drug of choice along with cladribine.204,211,212 It is presumed to act by restricting the proliferation of hematopoietic progenitor cells. Studies with IFN-α, often in combination with glucocorticoids, have reported variable success, with unchanged or modest reductions in marrow infiltration with mast cells, and in tryptase levels. Many patients do report symptomatic benefits. Resolutions of ascites and increased bone remineralization also have been reported. Use of IFN-α is often limited by side effects such as fever, fatigue, and cytopenias. Its use is not routinely recommended for patients with indolent systemic disease, unless there is concomitant severe osteoporosis. Cladribine (2-chlorodeoxyadenosine), a nucleoside analogue, does not require cells in active cell cycle to exert its cytotoxic activity and may be beneficial in slowly progressing neoplastic processes. The drug has myelosuppressive and immunosuppressive properties and thus cannot be recommended for patients with indolent disease.204

Hematopoietic Stem Cell Transplantation

Allogeneic stem cell transplantation (SCT) has been employed as a treatment option for patients with advanced categories of mastocytosis associated with poor survival. SCT has been used to treat a hematologic disorder associated with mastocytosis in relatively few cases.213–216 Although these studies reported favorable responses of the associated hematologic disorders, complete remission of the mast cell disease was

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reported in only one study, which used non–T-cell-depleted blood SCT in a patient with an associated myeloproliferative neoplasm.215 The value of allogeneic SCT in mastocytosis may result from the immunotherapeutic effects of the donor marrow rather than the myeloablative conditioning regimen.213 One study using nonmyeloablative blood SCT for treatment of advanced systemic mastocytosis in three patients with advanced mastocytosis reported no effect on mastocytosis progression despite the induction of a graft-versus-mast-cell response.216 Perhaps performing targeted therapy directed at the mast cell compartment before transplantation would improve outcome.

Tyrosine Kinase Inhibitors

The availability of low-molecular-weight inhibitors of tyrosine kinases suggested the mutated KIT tyrosine kinases in mastocytosis as a therapeutic target. Imatinib mesylate (Gleevec; Novartis, Basel, Switzerland) currently is the only such drug available. It has a specific inhibition profile that includes ABL1, KIT, and PDGFR (platelet-derived growth factor receptor) tyrosine kinases.217,218 Although the drug inhibits wild-type KIT and KIT bearing juxtamembrane-activating mutations similar to those found in gastrointestinal stromal tumors, it does not inhibit KIT bearing the codon 816 mutations associated with most common forms of systemic mastocytosis.219,220 This finding is attributed to a conformational change in KIT bearing the codon 816 mutation, which interferes with the association of the drug with the ATP-binding domains of the receptor. Consistent with these observations, imatinib mesylate showed a strong in vitro cytotoxic effect on mast cells bearing wild-type KIT. Mast cells bearing a codon 816 mutation isolated from marrow of patients with mastocytosis were fairly resistant to the drug.221 These studies suggest imatinib mesylate is unlikely to be an effective therapy for patients who carry codon 816 mutations. However, the drug appears to be of value when there is an imatinib-sensitive mutation or in KIT816-unmutated patients. For example, a patient with an unusual form of systemic mastocytosis associated with a KIT mutation (Phe522Cys) affecting the transmembrane region of the receptor responded to treatment with imatinib.182 Accordingly, a careful mutational analysis of a sample enriched for lesional mast cells appears to be essential in patients with mastocytosis before contemplating imatinib therapy. Other tyrosine kinase inhibitors that decrease the activity of KIT with codon 816 mutations including midostaurin (PKC412) and dasatinib are in clinical trials.222,223 Studies to date suggest that midostaurin may produce significant decreases in mast cell burden in some patients.222 Some patients with a variant of chronic eosinophilic leukemia (clonal hypereosinophilic syndrome) and FIPIL1-PDGFRA fusions exhibit elevated serum tryptase levels, increased numbers of mast cells in the marrow, some of which can appear atypical and spindle shaped, tissue fibrosis, and, like other patients who have the FIPIL1-PDGFRA fusion gene, are responsive to imatinib mesylate.224,225 Such cases are classified within the World Health Organization category of myeloid and lymphoid neoplasms with eosinophilia and abnormalities of PDGFRA, PDGFRB, or FGFR1.

Monoclonal Mast Cell Activation Syndrome

Monoclonal mast cell activation syndrome (MMAS) is a term adopted by a consensus conference to be applied to patients who are found to have one or two minor diagnostic criteria for mastocytosis but lack the full diagnostic criteria for systemic disease.226 Patients with such findings have been identified within groups of patients diagnosed with idiopathic anaphylaxis and patients with anaphylaxis to stinging insects. It is possible that these studies are identifying patients with a progressive clonal mast cell disorder that may one day meet the diagnostic criteria for systemic mastocytosis. For now, such patients are treated symptomatically and for anaphylaxis. Followup at yearly intervals is

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recommended to determine if there is evidence of an expanding mast cell compartment.

Mast Cell Activation Syndrome

The term mast cell activation syndrome (MCAS) is sometimes applied as a diagnosis for patients with episodic allergic-like signs and symptoms, including flushing, urticaria, diarrhea, and wheezing, involving two or more organ systems; and where an extensive medical evaluation has failed to identify an etiology.227,228 The assumption is that individuals to whom this diagnosis is applied are having episodes caused by release of mediators associated with hyperreactivity of mast cells that then activate spontaneously. Diagnostic criteria have been proposed to separate this proposed entity from other causes of such clinical findings. These criteria include response to antimediator therapy and an elevation in a marker of mast cell activation, such as serum tryptase, with an episode.227 Primary (clonal) and other clinical disorders associated with mast cell activation, as well as other conditions associated with vasoactive mediator release, must be eliminated as possible causes of the clinical findings, including allergic diseases, mast cell activation associated with chronic inflammatory or neoplastic disorders and chronic autoimmune urticaria. Once the diagnostic criteria are met, therapy is symptomatic. Patients must be followed regularly in the event that one of the diagnoses eliminated during the initial evaluation reaches the level of diagnosis.

Splenectomy

Splenectomy has been performed on patients with severe aggressive mastocytosis in an attempt to improve their limiting cytopenias.229 Based on comparisons to historical controls, splenectomy increased survival by an average of 12 months. Patients who had undergone splenectomy appeared to be better able to tolerate chemotherapy. Splenectomy is of no value in the management of indolent mast cell disease.

COURSE AND PROGNOSIS The prognosis of adult patients with mast cell disorders is related to the disease category. The vast majority of patients who present with UP and indolent systemic mastocytosis (ISM) have a chronic protracted course that responds to symptomatic medical management. A normal life span is expected. Few of these patients progress to more severe forms of the disease; some patients may even experience a diminution in the severity of skin lesions in later years, while their marrow findings remain unchanged.230 However, elevated serum lactate dehydrogenase levels, a late age of onset, and, in patients with SM-AHNMD, presence of a significant hematologic abnormality (such as a myeloproliferative or myelodysplastic disorder or, more rarely, overt leukemia) are indicators of a poor prognosis and shortened survival.189 The prognosis for patients with SM-AHNMD depends on the course of the associated hematologic disorder.

Mast Cell Leukemia

MCL is relatively rare and prognosis is poor.195,222 A major differential diagnosis to MCL is myelomastocytic leukemia (MML).231 Patients with MCL may have fever, anorexia, weight loss, fatigue, severe abdominal cramping, nausea, vomiting, diarrhea, flushing, hypotension, pruritus, or bone pain. Peptic ulcer and gastrointestinal bleeding, hepatomegaly, splenomegaly, and lymph node enlargement are frequent findings. Anemia is a constant feature, and thrombocytopenia is nearly always present. The total leukocyte count varies from 10,000 to 150,000/μL (10 to 150 × 109/L), and mast cells compose 10 to 90 percent of leukocytes. Marrow biopsy shows a striking increase in mast cells, sometimes up to 90 percent of marrow cells, although the leukemic mast cells often are hypogranular or agranular.195,231

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Mast Cell Sarcoma

This is an exceedingly rare tumor, characterized by nodules at various cutaneous and mucosal sites.164,176

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137. Arber DA, Brunning RD, Orazi A, et al: Acute myeloid leukaemia, not otherwise specified, in WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues, edited by Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J, Vardiman JW. p 130. IARC Press, Lyon, 2008. 138. Staal-Viliare A, Latger-Cannard V, Rault JP, et al: A case of de novo acute basophilic leukaemia: Diagnostic criteria and review of the literature. Ann Biol Clin (Paris) 64:361– 365, 2006. 139. Staal-Viliare A, Latger-Cannard V, Didion J, et al: CD203c+/CD117–, an useful phenotype profile for acute basophilic leukaemia diagnosis in cases of undifferentiated blasts. Leuk Lymphoma 48:439–441, 2007. 140. Dastugue N, Duchayne E, Kuhlein E, et al: Acute basophilic leukaemia and translocation t(X;6)(p11;q23). Br J Haematol 98:170–176, 1997. 141. Quelen C, Lippert E, Struski S, et al: Identification of a transforming MYB-GATA1 fusion gene in acute basophilic leukemia: A new entity in male infants. Blood 117:5719– 5722, 2011. 142. Pearson MG, Vardiman JW, Le Beau MM, et al: Increased numbers of marrow basophils may be associated with a t(6;9) in ANLL. Am J Hematol 18:393–403, 1985. 143. Horsman DE, Kalousek DK: Acute myelomonocytic leukemia (AML-M4) and translocation t(6;9)(p23);q34): Two additional patients with prominent myelodysplasia. Am J Hematol 26:77–82, 1987. 144. Matsuura Y, Sato N, Kimura F, et al: An increase in basophils in a case of acute myelomonocytic leukaemia associated with marrow eosinophilia and inversion of chromosome 16. Eur J Haematol 39:457–461, 1987. 145. Hoyle CF, Sherrington P, Hayhoe FG: Translocation (3;6)(q21;p21) in acute myeloid leukemia with abnormal thrombopoiesis and basophilia. Cancer Genet Cytogenet 30:261–267, 1988. 146. Nacheva EP, Grace CD, Brazma D, et al: Does BCR/ABL1 positive acute myeloid leukaemia exist? Br J Haematol 161:541–550, 2013. 147. Alsabeh R, Brynes RK, Slovak ML, et al: Acute myeloid leukemia with t(6;9) (p23;q34): Association with myelodysplasia, basophilia, and initial CD34 negative immunophenotype. Am J Clin Pathol 107:430–437, 1997. 148. Slovak ML, Gundacker H, Bloomfield CD, et al: A retrospective study of 69 patients with t(6;9)(p23;q34) AML emphasizes the need for a prospective, multicenter initiative for rare “poor prognosis” myeloid malignancies. Leukemia 20:1295–1297, 2006. 149. Oyarzo MP, Lin P, Glassman A, et al: Acute myeloid leukemia with t(6;9)(p23;q34) is associated with dysplasia and a high frequency of flt3 gene mutations. Am J Clin Pathol 122:348–358, 2004. 150. Le Beau MM, Larson RA, Bitter MA, et al: Association of an inversion of chromosome 16 with abnormal marrow eosinophils in acute myelomonocytic leukemia. A unique cytogenetic-clinicopathological association. N Engl J Med 309:630–636, 1983. 151. Lewis RA, Goetzl EJ, Wasserman SI, et al: The release of four mediators of immediate hypersensitivity from human leukemic basophils. J Immunol 114:87–92, 1975. 152. Travis WD, Li CY, Bergstralh EJ, et al: Systemic mast cell disease. Analysis of 58 cases and literature review. Medicine (Baltimore) 67:345–368, 1988. 153. Tsai M, Shih LS, Newlands GF, et al: The rat c-kit ligand, stem cell factor, induces the development of connective tissue-type and mucosal mast cells in vivo. Analysis by anatomical distribution, histochemistry, and protease phenotype. J Exp Med 174:125–131, 1991. 154. Irani AA, Garriga MM, Metcalfe DD, et al: Mast cells in cutaneous mastocytosis: Accumulation of the MCTC type. Clin Exp Allergy 20:53–58, 1990. 155. Schwartz LB, Metcalfe DD, Miller JS, et al: Tryptase levels as an indicator of mast-cell activation in systemic anaphylaxis and mastocytosis. N Engl J Med 316:1622–1626, 1987. 156. Weidner N, Horan RF, Austen KF: Mast-cell phenotype in indolent forms of mastocytosis. Ultrastructural features, fluorescence detection of avidin binding, and immunofluorescent determination of chymase, tryptase, and carboxypeptidase. Am J Pathol 140:847–857, 1992. 157. Weidner N, Austen KF: Heterogeneity of mast-cells at multiple body sites-fluorescent determination of avidin binding and immunofluorescent determination of chymase, tryptase, and carboxypeptidase content. Pathol Res Pract 189:156–162, 1993. 158. Lavker RM, Schechter NM: Cutaneous mast cell depletion results from topical corticosteroid usage. J Immunol 135:2368–2373, 1985. 159. Irani AM, Craig SS, DeBlois G, et al: Deficiency of the tryptase-positive, chymasenegative mast cell type in gastrointestinal mucosa of patients with defective T lymphocyte function. J Immunol 138:4381–4386, 1987. 160. Garriga MM, Friedman MM, Metcalfe DD: A survey of the number and distribution of mast cells in the skin of patients with mast cell disorders. J Allergy Clin Immunol 82:425–432, 1988. 161. Malone DG, Irani AM, Schwartz LB, et al: Mast cell numbers and histamine levels in synovial fluids from patients with diverse arthritides. Arthritis Rheum 29:956–963, 1986. 162. Malone DG, Wilder RL, Saavedra-Delgado AM, et al: Mast cell numbers in rheumatoid synovial tissues. Correlations with quantitative measures of lymphocytic infiltration and modulation by antiinflammatory therapy. Arthritis Rheum 30:130–137, 1987. 163. Frame B, Nixon RK: Bone-marrow mast cells in osteoporosis of aging. N Engl J Med 279:626–630, 1968. 164. Lennert K, Parwaresch MR: Mast cells and mast cell neoplasia: A review. Histopathology 3:349–365, 1979. 165. Barrett KE, Neva FA, Gam AA, et al: The immune response to nematode parasites: Modulation of mast cell numbers and function during Strongyloides stercoralis infections in nonhuman primates. Am J Trop Med Hyg 38:574–581, 1988.

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166. Bowers HM Jr, Mahapatro RC, Kennedy JW: Numbers of mast cells in the axillary lymph nodes of breast cancer patients. Cancer 43:568–573, 1979. 167. Yoo D, Lessin LS, Jensen WN: Bone-marrow mast cells in lymphoproliferative disorders. Ann Intern Med 88:753–757, 1978. 168. Yoo D, Lessin LS: Bone marrow mast cell content in preleukemic syndrome. Am J Med 73:539–542, 1982. 169. Fohlmeister I, Reber T, Fischer R: Bone marrow mast cell reaction in preleukaemic myelodysplasia and in aplastic anaemia. Virchows Arch A Pathol Anat Histopathol 405:503–509, 1985. 170. Unna PG: Beitrage zur anatomic und pathogenese der urticaria simplex und pigmentosa. Mscch Prakt Dermatol 6:EH1, 1887. 171. Nettleship E, Tay W, Med J: Rare forms of urticaria. Br Med J 2:323–330, 1869. 172. Sangster A: An anomalous mottled rash, accompanied by pruritus, factious urticaria and pigmentation, “urticaria pigmentosa (?).” Trans Clin Soc Lond 11:161, 1878. 173. Ellis JM: Urticaria pigmentosa; a report of a case with autopsy. Arch Pathol 48:426–435, 1949. 174. Carter MC, Metcalfe DD, Komarow HD: Mastocytosis. Immunol Allergy Clin North Am 34:181–196, 2014. 175. Soter NA: Mastocytosis and the skin. Hematol Oncol Clin North Am 14:537–555, vi, 2000. 176. Horny HP, Metcalfe DD, Bennett JM, et al: Mastocytosis, in WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues edited by Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J, Vardiman JW. p 54. IARC Press, Lyon, 2008. 177. Furitsu T, Tsujimura T, Tono T, et al: Identification of mutations in the coding sequence of the proto-oncogene c-kit in a human mast cell leukemia cell line causing ligandindependent activation of c-kit product. J Clin Invest 92:1736–1744, 1993. 178. Nagata H, Worobec AS, Oh CK, et al: Identification of a point mutation in the catalytic domain of the protooncogene c-kit in peripheral blood mononuclear cells of patients who have mastocytosis with an associated hematologic disorder. Proc Natl Acad Sci U S A 92:10560–10564, 1995. 179. Longley BJ, Tyrrell L, Lu SZ, et al: Somatic c-KIT activating mutation in urticaria pigmentosa and aggressive mastocytosis: Establishment of clonality in a human mast cell neoplasm. Nat Genet 12:312–314, 1996. 180. Nagata H, Okada T, Worobec AS, et al: C-kit mutation in a population of patients with mastocytosis. Int Arch Allergy Immunol 113:184–186, 1997. 181. Valent P: Mastocytosis: A paradigmatic example of a rare disease with complex biology and pathology. Am J Cancer Res 3:159–172, 2013. 182. Akin C, Fumo G, Yavuz AS, et al: A novel form of mastocytosis associated with a transmembrane c-kit mutation and response to imatinib. Blood 103:3222–3225, 2004. 183. Lahortiga I, Akin C, Cools J, et al: Activity of imatinib in systemic mastocytosis with chronic basophilic leukemia and a PRKG2-PDGFRB fusion. Haematologica 93:49–56, 2008. 184. Hirota S, Isozaki K, Moriyama Y, et al: Gain-of-function mutations of c-kit in human gastrointestinal stromal tumors. Science 279:577–580, 1998. 185. Schwaab J, Schnittger S, Sotlar K, et al: Comprehensive mutational profiling in advanced systemic mastocytosis. Blood 122:2460–2466, 2013. 186. Castells M, Metcalfe DD, Escribano L: Diagnosis and treatment of cutaneous mastocytosis in children: Practical recommendations. Am J Clin Dermatol 12:259–270, 2011. 187. Travis WD, Li CY: Pathology of the lymph node and spleen in systemic mast cell disease. Mod Pathol 1:4–14, 1988. 188. Mican JM, Di Bisceglie AM, Fong TL, et al: Hepatic involvement in mastocytosis: Clinicopathologic correlations in 41 cases. Hepatology 22:1163–1170, 1995. 189. Lawrence JB, Friedman BS, Travis WD, et al: Hematologic manifestations of systemic mast cell disease: A prospective study of laboratory and morphologic features and their relation to prognosis. Am J Med 91:612–624, 1991. 190. Horny HP, Ruck MT, Kaiserling E: Spleen findings in generalized mastocytosis. A clinicopathologic study. Cancer 70:459–468, 1992. 191. Horny HP, Parwaresch MR, Lennert K: Bone marrow findings in systemic mastocytosis. Hum Pathol 16:808–814, 1985. 192. Ridell B, Olafsson JH, Roupe G, et al: The bone marrow in urticaria pigmentosa and systemic mastocytosis. Cell composition and mast cell density in relation to urinary excretion of tele-methylimidazoleacetic acid. Arch Dermatol 122:422–427, 1986. 193. Kettelhut BV, Parker RI, Travis WD, et al: Hematopathology of the bone marrow in pediatric cutaneous mastocytosis. A study of 17 patients. Am J Clin Pathol 91:558–562, 1989. 194. Parker RI: Hematologic aspects of systemic mastocytosis. Hematol Oncol Clin North Am 14:557–568, 2000. 195. Valent P, Sotlar K, Sperr WR, et al: Refined diagnostic criteria and classification of mast cell leukemia (MCL) and myelomastocytic leukemia (MML): A consensus proposal. Ann Oncol 25:1691–1700, 2014. 196. Yang F, Tran TA, Carlson JA, et al: Paraffin section immunophenotype of cutaneous and extracutaneous mast cell disease: Comparison to other hematopoietic neoplasms. Am J Surg Pathol 24:703–709, 2000. 197. Escribano L, Diaz-Agustin B, Lopez A, et al: Immunophenotypic analysis of mast cells in mastocytosis: When and how to do it. Proposals of the Spanish Network on Mastocytosis (REMA). Cytometry B Clin Cytom 58:1–8, 2004. 198. Brockow K, Jofer C, Behrendt H, et al: Anaphylaxis in patients with mastocytosis: A study on history, clinical features and risk factors in 120 patients. Allergy 63:226–232, 2008. 199. Cherner JA, Jensen RT, Dubois A, et al: Gastrointestinal dysfunction in systemic mastocytosis. A prospective study. Gastroenterology 95:657–667, 1988.

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200. Akin C, Soto D, Brittain E, et al: Tryptase haplotype in mastocytosis: Relationship to disease variant and diagnostic utility of total tryptase levels. Clin Immunol 123:268–271, 2007. 201. Kristensen T, Vestergaard H, Bindslev-Jensen C, et al: Sensitive KIT D816V mutation analysis of blood as a diagnostic test in mastocytosis. Am J Hematol 89:493–498, 2014. 202. Siebenhaar F, Akin C, Bindslev-Jensen C, et al: Treatment strategies in mastocytosis. Immunol Allergy Clin North Am 34:433–447, 2014. 203. Frieri M, Alling DW, Metcalfe DD: Comparison of the therapeutic efficacy of cromolyn sodium with that of combined chlorpheniramine and cimetidine in systemic mastocytosis. Results of a double-blind clinical trial. Am J Med 78:9–14, 1985. 204. Robyn J, Metcalfe DD: Systemic mastocytosis. Adv Immunol 89:169–243, 2006. 205. Soter NA, Austen KF, Wasserman SI: Oral disodium cromoglycate in the treatment of systemic mastocytosis. N Engl J Med 301:465–469, 1979. 206. Czarnetzki BM: A double-blind cross-over study of the effect of ketotifen in urticaria pigmentosa. Dermatologica 166:44–47, 1983. 207. Kettelhut BV, Berkebile C, Bradley D, et al: A double-blind, placebo-controlled, crossover trial of ketotifen versus hydroxyzine in the treatment of pediatric mastocytosis. J Allergy Clin Immunol 83:866–870, 1989. 208. Barton J, Lavker RM, Schechter NM, et al: Treatment of urticaria pigmentosa with corticosteroids. Arch Dermatol 121:1516–1523, 1985. 209. Kolde G, Frosch PJ, Czarnetzki BM: Response of cutaneous mast cells to PUVA in patients with urticaria pigmentosa: Histomorphometric, ultrastructural, and biochemical investigations. J Invest Dermatol 83:175–178, 1984. 210. Reisberg IR, Oyakawa S: Mastocytosis with malabsorption, myelofibrosis, and massive ascites. Am J Gastroenterol 82:54–60, 1987. 211. Kluin-Nelemans HC, Jansen JH, Breukelman H, et al: Response to interferon alfa-2b in a patient with systemic mastocytosis. N Engl J Med 326:619–623, 1992. 212. Lim KH, Pardanani A, Tefferi A: KIT and mastocytosis. Acta Haematol 119:194–198, 2008. 213. Gromke T, Elmaagacli AH, Ditschkowski M, et al: Delayed graft-versus-mast-cell effect on systemic mastocytosis with associated clonal haematological non-mast cell lineage disease after allogeneic transplantation. Bone Marrow Transplant 48:732–733, 2013. 214. Fodinger M, Fritsch G, Winkler K, et al: Origin of human mast cells: Development from transplanted hematopoietic stem cells after allogeneic bone marrow transplantation. Blood 84:2954–2959, 1994. 215. Przepiorka D, Giralt S, Khouri I, et al: Allogeneic marrow transplantation for myeloproliferative disorders other than chronic myelogenous leukemia: Review of forty cases. Am J Hematol 57:24–28, 1998. 216. Nakamura R, Chakrabarti S, Akin C, et al: A pilot study of nonmyeloablative allogeneic hematopoietic stem cell transplant for advanced systemic mastocytosis. Bone Marrow Transplant 37:353–358, 2006. 217. Buchdunger E, Cioffi CL, Law N, et al: Abl protein-tyrosine kinase inhibitor STI571 inhibits in vitro signal transduction mediated by c-kit and platelet-derived growth factor receptors. J Pharmacol Exp Ther 295:139–145, 2000. 218. Druker BJ, Tamura S, Buchdunger E, et al: Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nat Med 2:561–566, 1996. 219. Ma Y, Zeng S, Metcalfe DD, et al: The c-KIT mutation causing human mastocytosis is resistant to STI571 and other KIT kinase inhibitors; kinases with enzymatic site mutations show different inhibitor sensitivity profiles than wild-type kinases and those with regulatory-type mutations. Blood 99:1741–1744, 2002. 220. Zermati Y, De Sepulveda P, Feger F, et al: Effect of tyrosine kinase inhibitor STI571 on the kinase activity of wild-type and various mutated c-kit receptors found in mast cell neoplasms. Oncogene 22:660–664, 2003. 221. Akin C, Brockow K, D’Ambrosio C, et al: Effects of tyrosine kinase inhibitor STI571 on human mast cells bearing wild-type or mutated c-kit. Exp Hematol 31:686–692, 2003. 222. Pardanani A: Systemic mastocytosis in adults: 2013 update on diagnosis, risk stratification, and management. Am J Hematol 88:612–624, 2013. 223. Ustun C, DeRemer DL, Akin C: Tyrosine kinase inhibitors in the treatment of systemic mastocytosis. Leuk Res 35:1143–1152, 2011. 224. Klion AD, Noel P, Akin C, et al: Elevated serum tryptase levels identify a subset of patients with a myeloproliferative variant of idiopathic hypereosinophilic syndrome associated with tissue fibrosis, poor prognosis, and imatinib responsiveness. Blood 101:4660–4666, 2003. 225. Maric I, Robyn J, Metcalfe DD, et al: KIT D816V-associated systemic mastocytosis with eosinophilia and FIP1L1/PDGFRA-associated chronic eosinophilic leukemia are distinct entities. J Allergy Clin Immunol 120:680–687, 2007. 226. Valent P, Akin C, Escribano L, et al: Standards and standardization in mastocytosis: Consensus statements on diagnostics, treatment recommendations and response criteria. Eur J Clin Invest 37:435–453, 2007. 227. Akin C, Valent P, Metcalfe DD: Mast cell activation syndrome: Proposed diagnostic criteria. J Allergy Clin Immunol 126:1099–104 e4, 2010. 228. Valent P, Akin C, Arock M, et al: Definitions, criteria and global classification of mast cell disorders with special reference to mast cell activation syndromes: A consensus proposal. Int Arch Allergy Immunol 157:215–225, 2012. 229. Friedman B, Darling G, Norton J, et al: Splenectomy in the management of systemic mast cell disease. Surgery 107:94–100, 1990. 230. Brockow K, Scott LM, Worobec AS, et al: Regression of urticaria pigmentosa in adult patients with systemic mastocytosis: Correlation with clinical patterns of disease. Arch Dermatol 138:785–790, 2002. 231. Valentini CG, Rondoni M, Pogliani EM, et al: Mast cell leukemia: A report of ten cases. Ann Hematol 87:505–508, 2008.

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CHAPTER 64

CLASSIFICATION AND CLINICAL MANIFESTATIONS OF NEUTROPHIL DISORDERS

Marshall A. Lichtman

SUMMARY Neutrophil disorders can be grouped into deficiencies, or neutropenia, excesses, or neutrophilia, and qualitative abnormalities. Neutropenia can have the severe consequence of predisposing to infection, whereas neutrophilia usually is a manifestation of an underlying inflammatory or neoplastic disease: the neutrophilia, per se, having no specific consequences. Qualitative disorders of neutrophils may lead to infection as a result of defective cell translocation to an inflammatory site or defective microbial killing. Neutropenia may reflect an inherited disease that is evident in childhood (such as congenital [hereditary] severe neutropenia), but more often it is acquired. A common cause of neutropenia is the adverse effect of a drug. Some cases of neutropenia have no evident cause. The health consequence of neutropenia is a function of the mechanism of the neutropenia, the abruptness and severity of the decrease in the blood neutrophil count, and the duration of the decrease. Neutrophils have also been identified as mediators of vascular or tissue injury. Table 64–1 provides a comprehensive categorization of quantitative and qualitative neutrophil disorders.

CLASSIFICATION Table 64–1 lists disorders that result from a primary deficiency in neutrophil numbers or function. Neutropenia or neutrophilia also occurs as part of a disorder that affects multiple blood cell lineages, as in infiltrative diseases of the marrow, or intrinsic disorders of multipotential marrow hematopoietic cells, or removal of several blood cell types in the circulation. These diseases are not included in this classification and are discussed in other chapters of this text. This classification and chapter considers disorders in which the neutrophil either is the only cell type affected or the dominant cell type affected. A pathophysiologic classification of neutrophil disorders has proved elusive. Techniques for measuring mechanisms of (1) impaired production resulting from hypoplasia or exaggerated apoptosis of marrow precursors (ineffective neutropoiesis) or (2) accelerated destruction of neutrophils are more difficult and complex than the techniques used to measure decreases in red cells or platelet concentrations. The low concentration of blood neutrophils, accentuated in neutropenic states, makes radioactive-labeling techniques for studying the kinetics of autologous cells in neutropenic subjects difficult, if not impossible.

The two compartments of neutrophils in the blood (cells marginated along vascular beds as distinct from cells circulating and counted in the blood neutrophil count [Chap. 65]), the random disappearance of neutrophils from the circulation, the short circulation time of neutrophils, the absence of practical techniques for measuring the size of the tissue neutrophil compartment, and the disappearance of neutrophils by apoptosis or excretion from the tissue compartment also make multicompartmental kinetic analysis difficult. Also, neutropenic disorders are uncommon, and few laboratories are able, or prepared, to undertake the studies necessary to define the mechanisms of their development in sporadic cases. Therefore, efforts to understand the pathophysiology and kinetics of neutropenia have been of more limited success than that of red cells or platelets. Hence, the classification of neutrophil disorders is partly pathophysiologic and partly descriptive (see Table  64–1). Classification, although imperfect, does provide a language for communication and a basis for rectification as knowledge of the cause and mechanism of each entity advances. The classification is self-explanatory except in two areas. First, certain childhood (congenital or hereditary) syndromes listed under decreased neutrophilic granulopoiesis could have been listed under chronic hypoplastic neutropenia or chronic idiopathic neutropenia; however, they seem to hold a special interest. Their unique status and their pathogenesis have become further clarified as the mutations linked to each are identified. Three childhood syndromes that are associated with neutropenia are omitted because the neutropenia is part of a more global suppression of hematopoiesis: Pearson syndrome,1,2 Fanconi anemia,3,4 and dyskeratosis congenita (Chap. 35).5,6 A second area requiring explanation is the chronic idiopathic neutropenias. This group includes (1) cases with normocellular marrows but an inadequate compensatory increase in granulopoiesis for the degree of neutropenia and (2) cases with hyperplastic granulopoiesis that apparently is ineffective as a result of apoptosis of marrow neutrophils and late precursors. Unlike hypoplastic neutropenia in which the granulocyte precursors are markedly reduced or absent, precursors are present in the marrow in the idiopathic neutropenias, but the extent of effective granulopoiesis probably is low. A variety of mutations have been discovered that are causal for inherited or sporadic neutropenia syndromes. For example, mutation of the serine protease neutrophil elastase 2 gene (ELANE) is found in 70 percent of cases of the autosomal dominant form of severe congenital neutropenia and in most cases of cyclic neutropenia.7 Kostmann syndrome is the autosomal recessive form of severe congenital neutropenia and is caused by mutations in the HAX1 gene.8 Some cases of severe congenital neutropenia have been related to mutations in GPI1, G6PC3, and others.9–11 There is evidence that these mutations result in apoptotic loss of marrow neutrophil precursors as a result of downregulation of the BCL-2 family of antiapoptotic proteins, the upregulation of the proapoptotic FAS receptor, or other apoptosis-enhancing pathways, described more fully in Chap. 65. A comprehensive listing of the genetic mutations found in monogenic congenital neutropenia and the extra hematopoietic manifestations of those disorders can be found in a publication of the Service d’Hémato Oncologie Pédiatrique Registre des neutropénies.12 Qualitative disorders of neutrophils affect their ability to enter the circulation, to leave the circulation, enter inflammatory exudates, or to ingest or kill microorganisms. Chapter 66 describes these abnormalities in more detail.

CLINICAL MANIFESTATIONS Acronyms and Abbreviations:  CD, cluster of differentiation; G-CSF, granulo-

cyte colony-stimulating factor; HLA-DR, human leukocyte antigen-D related.

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The clinical manifestations of decreased concentrations or abnormal function of neutrophils principally result from infection. The combined deficit of neutrophils and monocytes characteristic of aplastic anemia,

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Part VII: Neutrophils, Eosinophils, Basophils, and Mast Cells

TABLE 64–1.  Classification of Neutrophil Disorders I.  Quantitative Disorders of Neutrophils A. Neutropenia12,13 1. Decreased neutrophilic granulopoiesis a. Congenital severe neutropenias (Kostmann syndrome and related disorders)14,15, b. Reticular dysgenesis (congenital aleukocytosis)16,17 c. Neutropenia and exocrine pancreas dysfunction (Shwachman-Diamond syndrome)13,18 d. Neutropenia and immunoglobulin abnormality (e.g., hyperimmunoglobulin M syndrome)19–21 e. Neutropenia and disordered cellular immunity (cartilage hair hypoplasia)22,23 f. Mental retardation, anomalies, and neutropenia (Cohen syndrome)24,25 g. X-linked cardioskeletal myopathy and neutropenia (Barth syndrome)26,27 h. Myelokathexis28,29 i. Warts, hypogammaglobulinemia, infection, myelokathexis (WHIM) syndrome30,31 j. Neonatal neutropenia and maternal hypertension32,33 k.  Griscelli syndrome34 l.  Glycogen storage disease 1b35 m. Hermansky-Pudlak syndrome 236,37 n.  Wiskott-Aldrich syndrome38 o. Chronic hypoplastic neutropenia (1) Drug-induced39–42 (2) Cyclic43,44 (3) Branched-chain aminoacidemia45 p.  Acute hypoplastic neutropenia (1) Drug-induced39,46,47 (2) Infectious48 q.  Chronic idiopathic neutropenia (1) Benign (a) Familial49 (b) Sporadic50 (2) Symptomatic51–53

2.  Accelerated neutrophil destruction II.  Qualitative Disorders of Neutrophils a. Alloimmune neonatal A.  Defective adhesion of neutrophils neutropenia54–56 1. Leukocyte adhesion deficiency104,105 57–59 b.  Autoimmune neutropenia 2. Drug-induced106 (1) Idiopathic59 B. Defective locomotion and chemotaxis (2) Drug-induced59,60 1. Actin polymerization (3)  Felty syndrome61–63 abnormalities107–110 (4) Systemic lupus 2.  Neonatal neutrophils111 erythematosus64,65 3.  Interleukin-2 administration112 (5) Other autoimmune 4.  Cardiopulmonary bypass101 diseases66–71 C.  Defective microbial killing (6) Complement activation1. Chronic granulomatous induced neutropenia72 disease113,114 71,73–75 (7)  Pure white cell aplasia 2.  RAC-2 deficiency115,116 3.  Maldistribution of neutrophils 3. Myeloperoxidase deficiency117,118 a. Pseudoneutropenia76–78 4. Hyperimmunoglobulin E (Job) B. Neutrophilia syndrome119,120 1.  Increased neutrophilic granulopoiesis 5. Glucose-6-phosphate dehydrogenase deficiency121,122 a.  Hereditary neutrophilia79 b.  Trisomy 13 or 1880 6.  Extensive burns123,124 81 c.  Chronic idiopathic neutrophilia 7. Glycogen storage disease Ib125,126 82 (1) Asplenia 8.  Ethanol toxicity127,128 d. Neutrophilia or neutrophilic leu9.  End-stage renal disease129 kemoid reactions 10.  Diabetes mellitus130 (1) Inflammation83,84 D. Abnormal structure of the nucleus or of (2) Infection83–85 an organelle (3) Acute hemolysis or acute 1.  Hereditary macropolycytes131 hemorrhage83 2.  Hereditary hypersegmentation135 (4) Cancer, including granulocyte 3. Specific granule deficiency136–138 colony-stimulating factor 4.  Pelger-Huët anomaly139,140 (G-CSF)-secreting tumors86–89 5.  Alder-Reilly anomaly141 (5) Drugs (e.g., glucocorticoids, 6.  May-Hegglin anomaly142–144 lithium, granulocyte- or granulocyte-monocyte col7.  Chédiak-Higashi disease145,146 ony-stimulating factor, tumor III. Neutrophil-Induced Vascular or Tissue necrosis factor-α)83,90–94 Damage147–149 (6) Ethylene glycol exposure83 A.  Pulmonary disease150–155 (7) Exercise95,96 B.  Transfusion-related lung injury156,157 e.  Sweet syndrome97,98 C.  Renal disease158,159 f.  Cigarette smoking99,100 D.  Arterial occlusion160,161 g.  Cardiopulmonary bypass101 E.  Venous occlusion162 2. Decreased neutrophil circulatory F.  Myocardial infarction157–163,167 egress G.  Ventricular function164–168 a.  Drugs (e.g., glucocorticoids)102 H. Stroke157,169 3.  Maldistribution of neutrophils I. Neoplasia170–172 a. Pseudoneutrophilia103 J.  Sickle cell vasoocclusive crisis157,173

RAC-2, RAS-Related C3 botulinum toxin substrate 2.

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CHAPTER 64: Classification and Clinical Manifestations of Neutrophil Disorders

hairy cell leukemia, and cytotoxic therapy leads to susceptibility to a broader spectrum of infectious agents. Increased concentrations of normal neutrophils per se are usually not associated with clinical manifestations; although, increased concentrations of leukemic neutrophil precursors can produce clinical manifestations of microcirculatory leukostasis (Chap. 83). Neutrophils also play a role in deleterious vascular or tissue effects, as noted in the last entries in Table  64–1 (see “Neutrophilia” below).

NEUTROPENIA The lower limit of the normal neutrophil count is approximately 1800/μL (1.8 × 109/L) in subjects of European descent and 1400/μL (1.4 × 109/L) in subjects of African descent.174–177 An additional small proportion (~5 percent) of persons of African descent have neutrophil counts between 1000/μL (1.0 × 109/L) and 1400 (1.4 × 109/L) without evidence of associated abnormalities and this finding also may represent “ethnic neutropenia.” These findings have not been explained by exaggerated margination of neutrophils.176 Neutropenia is especially striking in Yemenite Jews, another ethnic group with very low “normal” neutrophil counts,178 and has been reported in West Africans, Caribbean inhabitants of African descent, Ethiopians, and some Arab groups.176,177 Persons of African descent do not have the increase of neutrophil count seen in Europeans who smoke or are administered glucocorticoids; however, they have an appropriate increase of neutrophils in response to infection. Americans of Mexican descent have a slightly elevated neutrophil count.176 A decrement in neutrophil concentration to 1000/μL (1.0 × 109/L) usually poses little threat in the individual with an intact immune system. If the neutrophil count drops farther, the risk of infection may increase, if the decrease reflects a decrease in flux rate into the tissues. Subjects who are chronically neutropenic, as a result of severe marrow cell production abnormalities, with counts less than 500 neutrophils/μL (0.5 × 109/L) may be at heightened risk for developing recurrent infections.179 The relationship of frequency or type of infection to neutrophil concentration is imperfect. The cause of the neutropenia, the coincidence of monocytopenia or lymphopenia, concurrent use of alcohol or glucocorticoids, exposure to nosocomial infections, and other factors influence the likelihood of infection. A breakdown in the barrier function of the skin or circumstances such as indwelling catheters, also, increase the risk of infection in severely neutropenic subjects. Lower neutrophil counts in African (Malawian) mothers infected with HIV were associated with an increased risk of HIV in their newborns.180 Infections in neutropenic subjects who are not otherwise compromised usually result from Gram-positive cocci and usually are superficial, involving skin, oropharynx, bronchi, anal canal, or vagina. However, any site can become infected and Gram-negative organisms, viruses, or opportunistic organisms can be involved. A decrease in neutrophil count can occur abruptly or gradually (Chap. 65). One type of drug-induced neutropenia is distinguished by the rapidity of onset. Abrupt-onset neutropenia more likely is severe and leads to symptoms. If the neutrophil count approaches zero (agranulocytosis), high fever; chills; necrotizing, painful oral ulcers (agranulocytic angina), and prostration may occur, presumably as a result of sepsis.181 As the disease progresses, headache, stupor, and rash may develop. In the preantibiotic era, persistent agranulocytosis had a fatality rate approaching 100 percent. Even with bactericidal, broad-spectrum antibiotics, severe, sustained neutropenia or agranulocytosis is a serious illness with a high fatality rate. Pus formation decreases in patients with severe neutropenia.182 The failure to suppurate can mislead the clinician and delay identification of the infection site because minimal physical or radiographic findings

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develop. For example, lack of pneumonic consolidation is characteristic of pneumonia in granulocytopenic subjects. An exudate, swelling, heat, and regional adenopathy are much less prevalent in granulocytopenic patients. Fever is common, and local pain, tenderness, and erythema nearly always are present despite a marked reduction in neutrophils.181 The mechanism of neutropenia and the severity of the deficiency of cells play roles in clinical manifestations. Chronic idiopathic (benign) neutropenia is associated with apparent normal granulopoiesis in the marrow and is asymptomatic even when the neutropenia has been present for prolonged periods, sometimes in the face of neutrophil counts approaching zero for prolonged periods.50 Presumably the delivery of neutrophils from marrow to tissues is sufficient to prevent infection despite the low blood pool size. Monocyte counts are normal, which may aid in host defenses because monocytes are effective phagocytes. Chronic idiopathic (symptomatic) neutropenia often is associated with pyoderma and otitis media in children. The former usually is caused by Staphylococcus aureus, Escherichia coli, and Pseudomonas species, and the latter usually results from infection by pneumococci or Pseudomonas aeruginosa. Unexplained chronic gingivitis may be a manifestation of chronic neutropenia. Pneumonia, lung abscesses, stomatitis, hepatic abscesses, or infections in other sites can occur. Chronic cyclic neutropenia is characterized by periodic oscillations in the number of neutrophils, with the nadir occurring at approximately 3-week intervals.43 During a period of neutropenia, patients develop malaise; fever; buccal, labial, or lingual ulcers; and cervical adenopathy. Furuncles, carbuncles, cellulitis, infected cuts with lymphangitis, chronic gingivitis, and abscesses of the axilla or groin may occur. Although severe infections may be fatal, life-threatening complications are uncommon. The cycling involves other hematopoietic cells as well, but the neutropenia is the most consequential functionally (Chap. 65). Some individuals have neutropenia because a larger fraction of their blood neutrophils is in the marginal rather than the circulating pool. The total blood neutrophil pool is normal, and infections do not result from this atypical distribution of neutrophils. This alteration has been called pseudoneutropenia.76–78

NEUTROPHILIA An increased neutrophil count can accompany virtually any cause of inflammation, especially inflammation caused by bacterial or fungal organisms, and a variety of cancers, especially if metastatic. Certain drugs, such as glucocorticoids or hematopoietic growth factors and minocycline, can induce neutrophilia, as can ethylene glycol intoxication (see Table 64–1). Acute hemolysis or acute hemorrhage may also result in neutrophilia. A notable cause of neutrophilia is cancers that elaborate granulocyte-colony stimulating factor (G-CSF). Numerous cancers are associated with neutrophilia and, in many cases, elaboration of very high concentrations of G-CSF has been documented. In these cases, neutrophil counts exceeding 100,000 μL (100 × 109/L) are common. Neutrophilia exceeding 50,000 neutrophils/μL (50 × 109/L) has been designated a “leukemoid reaction” and reflects an underlying inflammatory (e.g., pancreatitis), infectious (e.g., pneumococcal pneumonia), or neoplastic (e.g., carcinoma of the lung) cause. A leukemoid reaction can mimic rare types of chronic myelogenous or chronic neutrophilic leukemia. The leukemoid reaction classically (1) is composed largely of mature neutrophils with a low proportion of bands and myelocytes, (2) has increased leukocyte alkaline phosphatase reaction in neutrophils, (3) has increased granulopoiesis with normal maturation and morphology of cells in the marrow, (4) has normal cytogenetics of marrow cells, (5) has polyclonal-derived cells in women in whom such studies can be conducted (using the human androgen receptor gene assay), and (6) has cytometric analysis of neutrophils indicating a cluster

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of differentiation (CD) 13 and CD15 phenotype with absent expression of human leukocyte antigen-D related (HLA-DR) and CD34.

QUALITATIVE NEUTROPHIL ABNORMALITIES Neutrophil function depends on the ability of neutrophils to exit the marrow, adhere to vascular endothelium, move, respond to chemotactic gradients, ingest microorganisms, and kill ingested pathogens. Loss of any of these functions can predispose to infection (Chap. 66). Defects in each step of the neutrophil’s participation in the inflammatory response have been identified. Defects in adhesion molecules, cytoplasmic contractile proteins, granule synthesis or contents, or intracellular enzymes may underlie a movement, ingestion, or killing defect. These defects may be inherited or acquired. Chronic granulomatous disease113,114 and Chédiak-Higashi disease145,146 are two examples of inherited defects. Among the acquired disorders are those extrinsic to the cell, as in the movement, chemotactic, or phagocytic defects of diabetes mellitus, the effects of alcohol abuse, or glucocorticoid excess. Acquired intrinsic disorders usually are manifestations of clonal hematopoietic (myeloid) disorders such as acute myelogenous leukemia (Chap. 85). Severe defects in bacterial killing, as occur in chronic granulomatous disease, result in S. aureus, Klebsiella-Aerobacter, E. coli, and other catalase-positive bacterial infections. Suppurative lymphadenitis, pneumonia, dermatitis, hepatic abscesses, osteomyelitis, and stomatitis occur, and chronic granulomatous reactions in these sites give the disease its name. Fatality rates have been high. Functional disorders may be severe, as in chronic granulomatous disease. Mild functional disorders predispose to infections that occur infrequently and respond readily to antibiotics. Severe functional disorders result in suppurative lesions because neutrophil influx into inflammatory foci is not impaired, whereas agranulocytosis is associated with nonsuppurative lesions.

NEUTROPHIL-INDUCED VASCULAR OR TISSUE DAMAGE An overabundance of neutrophils does not result in specific clinical manifestations. Neutrophils, however, can transiently occlude capillaries, as determined by supravital microscopy, and such occlusions may reduce local blood flow transiently and contribute to the development of ischemia. Impairment of reperfusion of the coronary microcirculation has been thought to be dependent, in part, on neutrophil plugging of myocardial capillaries, but these effects can occur at normal neutrophil concentrations. An elevated neutrophil count is a feature of sickle cell disease and is a prognostic variable, increasing the likelihood of vasoocclusive events. Neutrophil adhesion to the vascular wall is an intrinsic part of the vasoocclusive events and the salutary effect of hydroxyurea is related to the decrease in neutrophil concentration that accompanies its use.157,173 In patients with ischemic vascular disease, an increased neutrophil count is associated with an increased probability of acute thrombotic episodes and the severity of chronic atherosclerosis.183 Neutrophil products may contribute to the pathogenesis of inflammatory skin, bowel, synovial, glomerular, and bronchial and interstitial pulmonary diseases (see Table 6 4–1). Diabetic retinopathy has been ascribed in part to the effects of hyperadhesive neutrophils on retinal capillaries.157 Neutrophils may act as mediators of tissue injury in stroke and myocardial infarction.157 Highly reactive oxygen products of neutrophils may be mutagens that increase the risk of neoplasia. This action may explain, for example, the development of carcinoma of the bowel in patients with chronic ulcerative colitis and the relationship between elevated leukocyte count and the occurrence of lung cancer, independent

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of the effect of cigarette usage. The oxidants, especially hypochlorous acid and chloramines, released by the neutrophil are extremely short lived and may play a role in tissue injury by inactivating several protease inhibitors in tissue fluids, permitting proteases, especially elastase, collagenase, and gelatinase, to cause tissue injury. Thrombogenesis also has been ascribed to leukocyte products.

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Lesma E, Riva E, Giovannini M, et al: Amelioration of neutrophil membrane function underlies granulocyte-colony stimulating factor action in glycogen storage disease 1b. Int J Immunopathol Pharmacol 18:297, 2005. 126. Kim SY, Jun HS, Mead PA, et al: Neutrophil stress and apoptosis underlie myeloid dysfunction in glycogen storage disease type Ib. Blood 111:5704, 2008. 127. Tamura DY, Moore EE, Patrick DA, et al: Clinically relevant concentrations of ethanol attenuate primed neutrophil bacteriocidal activity. J Trauma 44:320, 1998. 128. Breitmeier D, Becker N, Weilbach C, et al: Ethanol-induced malfunction of neutrophils respiratory burst on patients suffering from alcohol dependence. Alcohol Clin Exp Res 32:1708, 2008. 129. Porter CJ, Burden RP, Morgan AG, et al: Impaired bacterial killing and hydrogen peroxide production by polymorphonuclear neutrophils in end-stage renal failure. Nephron 77:479, 1997. 130. Hopps E, Camera A, Caimi G: [Polimorphonuclear leukocytes and diabetes mellitus] [in Italian]. Minerva Med 99:197, 2008. 131. Davidson WM, Milner RDG, Lawlor SD: Giant neutrophil leukocytes: An inherited anomaly. Br J Haematol 6:339, 1960. 135. Undritz VE: Eine neue Sippe mit Erblich—Konstitutioneller Hochsegmentierung der Neutrophilenkerne. Schweiz Med Wochenschr 94:1365, 1964. 136. Uzel G, Holland SM: White blood cell defects: Molecular discoveries and clinical management. Curr Allergy Asthma Rep 2:385, 2002. 137. Lekstrom-Himes JA, Dorman SE, Kopar P, et al: Neutrophil-specific granule deficiency results from a novel mutation with loss of function of the transcription factor CCAAT/ enhancer binding protein. J Exp Med 189:1847, 1999.

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138. Gombart AF, Koeffler HP: Neutrophil specific granule deficiency and mutations in the gene encoding transcription factor C/EBP (epsilon). Curr Opin Hematol 9:36, 2002. 139. Hoffmann K, Dreger CK, Olins AL, et al: Mutations in the gene encoding the laminin B receptor produce an altered nuclear morphology in granulocytes (Pelger-Hüet anomaly). Nat Genet 31:410, 2002. 140. Worman HJ, Bonne G: “Laminopathies”: A wide spectrum of human diseases. Exp Cell Res 313:2121, 2007. 141. Brunning RD: Morphologic alterations in nucleated blood and marrow cells in genetic disorders. Hum Pathol 1:99, 1970. 142. Oski FA, Naiman JL, Allen DM, Diamond LK: Leukocytic inclusions—Döhle bodies-associated with platelet abnormality (the May-Hegglin anomaly): Report of a family and review of the literature. Blood 20:657, 1962. 143. Pecci A, Panza E, Pujol-Moix N, et al: Position of nonmuscle myosin heavy chain IIA (NMMHC-IIA) mutations predicts the natural history of MYH9-related disease. Hum Mutat 29:409, 2008. 144. Seri M, Pecci A, Di Bari F, et al: MYH9-related disease: May-Hegglin anomaly, Sebastian syndrome, Fechtner syndrome, and Epstein syndrome are not distinct entities but represent a variable expression of a single illness. Medicine (Baltimore) 82:203, 2003. 145. Westbroek W, Adams D, Huizing M, et al: Cellular defects in Chediak-Higashi syndrome correlate with the molecular genotype and clinical phenotype. J Invest Dermatol 127:2674, 2007. 146. Lazarchick J, McRae B: Chediak-Higashi syndrome. Blood 105:4162, 2005. 147. Schmid-Schönbein GN: Leukocyte kinetics in the microcirculation. Biorheology 24:139, 1987. 148. Smedly LA, Tonnesen MG, Sandhaus RA, et al: Neutrophil-mediated injury to endothelial cells: Enhancement by endotoxin and essential role of neutrophil elastase. J Clin Invest 77:1233, 1986. 149. Weiss SJ: Tissue destruction by neutrophils. N Engl J Med 320:365, 1989. 150. Swank DW, Moore SB: Roles of the neutrophil and other mediators in adult respiratory distress syndrome. Mayo Clin Proc 64:1118, 1989. 151. MacNee W, Wiggs B, Balzberg AS, Hogg JC: The effect of cigarette smoking on neutrophil kinetics in human lungs. N Engl J Med 321:924, 1989. 152. Martin TR, Pistorese BP, Hudson LD, Maunder RJ: The function of lung and blood neutrophils in patients with the adult respiratory distress syndrome. Implication for the pathogenesis of lung infections. Am Rev Respir Dis 144:254, 1991. 153. Godek JE: Adverse effects of neutrophils on the lung. Am J Med 92(Suppl 6A):27S, 1992. 154. Palmgren MS, deShazo RO, Cater RM, et al: Mechanisms of neutrophil damage to human alveolar extracellular matrix: The role of serine and metalloproteases. J Allergy Clin Immunol 89:905, 1992. 155. Weiss ST, Segal MR, Sparrow D, Wager C: Relation of FEV1 and peripheral blood leukocyte count to total mortality. Am J Epidemiol 142:493, 1995. 156. Fung YL, Goodison KA, Wong JK, Minchinton RM: Investigating transfusion-related acute lung injury (TRALI). Intern Med J 33:286, 2003. 157. Segel GB, Halterman MW, Lichtman MA. The paradox of the neutrophil’s role in tissue injury. J Leukoc Biol 89:359, 2011. 158. Boventre JV, Colvin RB: Adhesion molecules in renal disease. Curr Opin Nephrol Hypertens 5:254, 1996. 159. Kitching AR, Holdsworth SR, Hickey MJ: Targeting leukocytes in immune glomerular diseases. Curr Med Chem 15:448, 2008. 160. Chibber R, Ben-Mahmud BM, Chibber S, Kohner EM: Leukocytes in diabetic retinopathy. Curr Diabetes Rev 3:3, 2007. 161. Fadlon E, Vordermeier S, Pearson TC, et al: Blood polymorphonuclear leukocytes from the majority of sickle cell patients in the crisis phase of the disease show adhesion to vascular endothelium and increased expression of CD64. Blood 91:266, 1998. 162. Schaub RG, Yamashita A, Simmons CA, et al: Leukocyte-mediated large vein injury and thrombosis: Pharmacologic intervention with lipoxygenase inhibitors, in Leukocyte Emigration and Its Sequelae, edited by Morat HZ, p 62. Karger, Basel, 1987. 163. Ranjadayalan K, Umachandran V, Daviews SW, et al: Thrombolytic treatment in acute myocardial infarction: Neutrophil activation, peripheral leucocyte responses, and myocardial injury. Br Heart J 66:10, 1991. 164. Welbourn CRB, Goldman G, Paterson IS, et al: Pathophysiology of ischaemia reperfusion injury: Central role of the neutrophil. Br J Surg 78:651, 1991. 165. Kassirer M, Zeltser D, Gluzman B, et al: The appearance of L-selectin (low) polymorphonuclear leukocytes in the circulating pool of peripheral blood during myocardial infarction correlates with neutrophilia and the size of the infarct. Clin Cardiol 22:721, 1999. 166. Takahashi T, Hiasa Y, Ohara Y, et al: Relationship of admission neutrophil count to microvascular injury, left ventricular dilation, and long-term outcome in patients treated with primary angioplasty for acute myocardial infarction. Circ J 72:867, 2008. 167. Takahashi T, Hiasa Y, Ohara Y, et al: Relation between neutrophil counts on admission, microvascular injury, and left ventricular functional recovery in patients with an anterior wall first acute myocardial infarction treated with primary coronary angioplasty. Am J Cardiol 100:35, 2007. 168. Kyne L, Hausdorff JM, Knight E, et al: Neutrophilia and congestive heart failure after acute myocardial infarction. Am Heart J 139:32, 2000. 169. Buck BH, Liebeskind DS, Saver JL, et al: Early neutrophilia is associated with volume of ischemic tissue in acute stroke. Stroke 39:355, 2008. 170. Trush MA, Seed JL, Kensler TW: Oxidant-dependent metabolic activation of polycyclic aromatic hydrocarbons by phorbol ester-stimulated human polymorphonuclear leukocytes: Possible link between inflammation and cancer. Proc Natl Acad Sci U S A 82:5194, 1985.

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171. Weitzman SA, Weitburg AB, Clark EP, Stossel TP: Phagocytes as carcinogens: Malignant transformation produced by human neutrophil. Science 227:1231, 1985. 172. Phillips AN, Neaton JD, Cook DG, et al: The leukocyte count and risk of lung cancer. Cancer 69:680, 1992. 173. Segel GB, Simon W, Lichtman MA. Should we still be focused on red cell hemoglobin F as the principal explanation for the salutary effect of hydroxyurea in sickle cell disease? Pediatr Blood Cancer 57:8, 2011. 174. Reed WW, Diehl LF: Leukopenia, neutropenia, and reduced hemoglobin levels in healthy American Blacks. Arch Intern Med 151:501, 1991. 175. Beutler E, West C: Hematologic differences between African-Americans and whites: The roles of iron deficiency and alpha-thalassemia on hemoglobin levels and mean corpuscular volume. Blood 106:740, 2005. 176. Hsieh MM, Everhart JE, Byrd-Holt DD, et al: Prevalence of neutropenia in the U.S. population: Age, sex, smoking status, and ethnic differences. Ann Intern Med 146:486, 2007.

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177. Grann VR, Bowman N, Joseph C, et al: Neutropenia in six ethnic groups from the Caribbean and the U.S. Cancer 113:854, 2008. 178. Berliner S, Shapira I, Toker S, et al: Benign hereditary leukopenia-neutropenia does not result from lack of low grade inflammation. A new look in the era of microinflammation. Blood Cells Mol Dis 34:135, 2005. 179. Bodey GP, Buckley M, Sathe YS: Quantitative relationships between circulating leukocytes and infection in patients with acute leukemia. Ann Intern Med 64:328, 1966. 180. Kourtis AP, Hudgens, MG, Kayira D, for the BAN study team. Neutrophil count in African mothers and newborns and HIV transmission. N Engl J Med 367:23, 2012. 181. Sickles EA, Green WH, Wiernick PH: Clinical presentation of infection in granulocytopenic patients. Arch Intern Med 135:715, 1975. 182. Dale DC, Wolff SM: Skin window studies of the acute inflammatory responses of neutropenic patients. Blood 38:138, 1971. 183. Coller B: Leukocytosis and ischemic vascular disease morbidity and mortality. Is it time to intervene? Arterioscler Thromb Vasc Biol 25:658, 2005.

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CHAPTER 65

NEUTROPENIA AND NEUTROPHILIA

David C. Dale and Karl Welte

SUMMARY Neutropenia designates a blood absolute neutrophil count that is less than 2 SD below the mean of a normal population. Neutropenia can be inherited or acquired. It usually results from decreased production of neutrophil precursor cells in the marrow. Neutropenia also can result from a shift of neutrophils from the circulating into the marginated cell pools in the circulation. Less commonly, neutropenia results from accelerated destruction of neutrophils or increased egress of neutrophil from the circulation into the tissues. When neutropenia is the sole or dominant abnormality, the condition is called “selective” or isolated” neutropenia, such as severe congenital neutropenia, chronic idiopathic neutropenia, or drug-induced neutropenia. Neutropenia can occur in other inherited or acquired marrow failure syndromes, such as severe aplastic anemia or Fanconi anemia, in which the condition is a bicytopenia or pancytopenia. In some diseases, several cell lineages are mildly affected but the reduction in neutrophil is the most severe, such as Felty syndrome. Neutropenia may be an indicator of an underlying systemic disease, such as early vitamin B12 or transcobalamin deficiency. Neutropenia, particularly severe neutropenia (neutrophil counts 20 mg). In time, however, the responses wane, requiring higher doses of corticosteroids. Standard doses of a p75:Fc fusion protein, etanercept, administrated subcutaneously twice weekly decreases the frequency, duration, and severity of attacks; thus, etanercept may provide a safer, more effective alternative then corticosteroids in controlling the disease. Therapy Colchicine treatment is effective in FMF and may prevent the development of amyloidosis.351 Prophylactic colchicine, 0.6 mg orally, two to three times a day, prevents or substantially reduces the acute attacks of FMF in most patients. Some patients can abort attack with intermittent doses of colchicine beginning at the onset of attacks (0.6 mg orally every hour for 4 hours, then every 2 hours for four doses, and then every 12 hours for 2 days). In general, patients who

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benefit from intermittent colchicine therapy are those who experience a recognizable prodrome before developing fever and clear-cut acute symptoms. Course and Prognosis The prognosis for normal longevity for patients has been excellent since the recognition that colchicine is an effective treatment of this disease. Most patients can be maintained almost entirely symptom-free. However, if amyloidosis develops, it may be followed by the nephrotic syndrome or uremia. Unless the patient receives a renal transplant, the likelihood of eventual death from renal failure is high.

Other Disorders of Neutrophil Motility

The directed migration of neutrophils from the circulation to an inflammatory site is a consequence of chemotaxis and leads to the accumulation of an exudate. For normal chemotaxis to occur, a complex series of events must be coordinated. Chemotactic factors must be generated in sufficient quantities to establish a chemotactic gradient. The neutrophils must have receptors for the chemotactic agents and mechanisms for discerning the direction of the chemotactic gradient. Depressed neutrophil chemotaxis has been observed in a wide variety of clinical conditions (see Table  66–2).360 These can be stratified as follows: (1) defects in the generation of chemotactic signals; (2) intrinsic defects of the neutrophil; and (3) direct inhibitors of neutrophil motility in response to chemotactic factors. Older patients with chemotactic disorders may be infected by a variety of microorganisms, including fungi and Gram-positive or Gram-negative bacteria. S. aureus is the most frequent bacterial offender. Typically, the skin, gingival mucosa, and regional lymph nodes are involved. Respiratory tract infections are frequent, but sepsis is rare. Delayed or inappropriate signs and symptoms of inflammation are common. Although the cells move slowly in Boyden chambers or other chemotactic assays, they do accumulate in sufficient numbers in inflammatory sites to produce pus. However, detection of patients with neutrophils that have profound defects in chemotaxis usually is accomplished through other phagocytic assays. Patients with the hereditary deficiency of complement factors C3, C5, or properidin exhibit an increased incidence of bacterial infections because they are unable to form the chemotactic peptide C5a.361 The degree to which defective chemotaxis plays a role in C3 deficiency is unclear because opsonization and ingestion rates also are abnormal in these disorders. Frequently, chemotactic disorders are associated with other impaired neutrophil functions. For instance, both glycogen storage disease type 1b362 and Shwachman-Diamond syndrome363 are chemotactic disorders frequently associated with an absolute neutrophil count below 0.5 × 109/L. Following restoration of a normal neutrophil count with G-CSF, the patients no longer are predisposed to recurrent bacterial infections in spite of a persistent chemotactic defect. Thus, a chemotactic defect observed in vitro does not correlate invariably with decreased resistance to bacterial infections in vivo. Among the impaired defense mechanisms of the neonate is neutrophil adherence and chemotaxis, as demonstrated by the in vitro response of neonatal neutrophils to a variety of chemotactic factors.322 The impaired motility of the neonatal neutrophils in part arises from the diminished ability to mobilize neutrophil β2 integrins following neutrophil activation.364 Additionally, the neonatal neutrophil may have a qualitative defect in β2-integrin function, resulting in impaired neutrophil transendothelial migration for up to 1 month after birth. At the other end of the spectrum, neutrophils from elderly loose focus during chemotaxis while their motility is unimpaired. This is caused by increased activity of PI3-K and results in less efficient bacterial killing and enhanced release of tissue destructive proteases. Inhibition of PI3K activity reverts this condition in vitro.365

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Drugs and Extrinsic Agents That Impair Neutrophil Motility

Although many pharmacologic agents can influence neutrophil function, few drugs used in clinical medicine affect neutrophil behavior in vivo. Ethanol, an inhibitor of PLD, in concentrations that occur in human blood can inhibit neutrophil locomotion and ingestion.366 Glucocorticoids, especially at high and sustained doses, inhibit neutrophil locomotion, ingestion, and degranulation.367 Administration of glucocorticoids on alternate days does not interfere with neutrophil movement.368 Epinephrine does not have a direct effect on neutrophil adhesion but cyclic adenosine monophosphate (cAMP), which is released from endothelial cells following exposure to epinephrine, can depress neutrophil adherence.369 Similarly, elevated cAMP levels following epinephrine administration may impair neutrophil adherence, leading to diminished neutrophil margination and apparent neutrophilia. Immune complexes, as seen in patients with rheumatoid arthritis or other autoimmune diseases, also can inhibit neutrophil movement by binding to neutrophil Fc receptors.

Hyperimmunoglobulin E Syndrome

Definition and History Autosomal dominant hyperimmunoglobulin E syndrome (HIES) is a disorder characterized by markedly elevated serum IgE levels, chronic dermatitis, and serious recurrent bacterial infections.370 The skin infections in these patients are remarkable for their absence of surrounding erythema, leading to the formation of “cold abscesses.” The neutrophils and monocytes from patients with this syndrome exhibit a variable, but at times profound, chemotactic defect that appears extrinsic to the neutrophil (see Table  66–2).371 The syndrome was originally described in 1966 in two red-headed, fair-skinned females who had “cold abscesses” and hyperextensible joints, which led to the appellation “Job’s syndrome.”370 Subsequently Buckley and coworkers documented the association of levels of immunoglobulin E with undue susceptibility to infection.372 Epidemiology Reports of more than 200 cases have been documented.372,373 HIES occurs in persons from diverse ethnic backgrounds and does not seem to be more common in any specific population. Etiology and Pathogenesis Both males and females have been affected, as well as members of succeeding generations, indicating that the disorder is autosomal dominant with an incomplete penetrance form of inheritance.370 STAT3 mutations cause most, if not all cases of autosomal dominant HIES. All mutations have been missense mutations or in-frame deletions, leading to the formation of full-length mutant STAT3 protein, which exerts a dominant negative effect. STAT3 is a major transduction protein affecting pathways involving wound healing angiogenesis, immunity, and cancer. The more rare autosomal recessive form is caused by mutations in dedicator of cytokinesis 8 (DOCK8), a guanine nucleotide exchange factor.374 The mechanism of the immune deficiencies in HIES remains clouded. Several reports with limited numbers of patients have conflicted results as to whether a chemotactic defect exists and whether there is a T-helper 1/T-helper 2 cytokine imbalance. Clinical Features HIES may begin as early as day 1 after birth.372 The syndrome is characterized by chronic eczematoid rashes, which are typically papular and pruritic. The rash generally involves the face and extensor surfaces of arms and legs; skin lesions are frequently sharply demarcated and usually lack surrounding erythema. By 5 years of age all patients have had a history of recurrent skin abscess formation with recurrent pneumonias, along with chronic otitis media and sinusitis. Patients may also develop septic arthritis, cellulitis, or osteomyelitis. The major offending pathogen is generally S. aureus. Other pathogens commonly infecting patients are C. albicans, H. influenzae, and pneumococci. Other associated features include coarse facial features,

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Chapter 66: Disorders of Neutrophil Function

including a prominent forehead, deep set eyes, a broad nasal bridge, a wide fleshly nasal tip, mild prognathism facial asymmetry, and hemihypertrophy.370 There is a high incidence of scoliosis, hyperextensible joints, and delayed shedding of the primary teeth.370 Occasionally, unexplained osteopenia presents, which is often complicated by recurrent bone fractures. Additionally, there is an increased risk of both Hodgkin and non-Hodgkin lymphoma. Laboratory Features Blood and sputum eosinophilia have been a consistent finding in all patients.370 Patient serum IgE levels range from three to 80 times the upper limit of normal. The serum IgE usually rises above 2000 IU/mL and often is elevated at birth. Upon reaching adulthood the IgE may decline over years, despite the clinical abnormalities of STAT3 deficiency. Usually patients have normal concentrations of IgG, IgA, and IgM, and may have elevated levels of IgD. Patients often have abnormally low anamnestic antibody response and poor antibody and cell-mediated responses to neoantigens. At times the neutrophils and monocytes of patients have a profound chemotactic defect. Differential Diagnosis Autosomal recessive-HIES (AR-HIES) is a distinct clinical entity manifested by elevated IgE ligands, and recurrent skin and cutaneous viral infections and mutations in DOCK8.370,375 Fatal sepsis occurs in AR-HIES from both Gram-positive and Gram-negative bacteria. Patients with AR-HIES have more symptomatic neurologic disease than STAT3 deficiency. Autoimmune hemolytic anemia may occur, but neutrophil chemotaxis is normal. The genetic mutation underlying AR-HIES remain unclear. Therapy remains supportive. Therapy No known therapy is curative, and management decisions are based on the clinical findings. Prophylactic trimethoprimsulfamethoxazole is effective in reducing infections with S. aureus.370 Type and route of antibiotic therapy are dictated by the results of the Gram stain and culture in patients with acute bacterial infections. Incision and drainage are essential for the management of abscesses, including superinfected pneumatoceles. Eczematoid dermatitis can be controlled with topical glucocorticoids to reduce inflammation and antihistamines to control pruritus. Intravenous immunoglobulin may decrease the number of infections for some patients. Attention needs to be paid to the scoliosis, fractures and degenerative joints by orthopedists. Retention of primary teeth requires dental expertise. Course and Prognosis If the hyperimmunoglobulin E is recognized early in life and the patient is maintained on chronic anti-Staphylococcal antibiotic therapy, the prognosis remains good. Many such patients have reached maturity, indicating that the syndrome is compatible with prolonged survival. Conversely, if the diagnosis is delayed and the patient develops infected giant pneumatoceles, secondary fungal infections may occur, leading to a morbid state.

DEFECTS IN MICROBICIDAL ACTIVITY Chronic Granulomatous Disease

Definition and History CGD is a genetic disorder affecting the function of neutrophils and monocytes. These phagocytic cells are able to ingest, but not kill, catalase-positive microorganisms because of an inability to generate antimicrobial oxygen metabolites (see Table  66–2). It is caused by mutations involving one of several genes encoding a component of the NADPH oxidase.376 In 1957, two pediatric groups caring for six male infants reported a clinical disorder of chronic suppurative lymphadenitis and recurrent fevers leading to premature deaths in the children.377,378 In the same time period, three observations assisted in providing the framework to understand the defect in the phagocytes of patients with CGD. Scientists described first that a striking increase in oxygen consumption

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was found upon particle ingestion by phagocytes, which was not related to mitochondrial oxygen metabolism.379 Next, it was found that the process of phagocytosis was accompanied by the formation of large quantities of H2O2 in the cell.380 Subsequently, it was reported that homogenates of phagocytes consume oxygen when incubated with pyridine nucleotides.381 These observations indicated that an oxidase enzyme or enzymes in the phagocytes were activated during phagocytosis to convert molecular oxygen into H2O2. It was then established that phagocytes from patients with CGD could ingest, but could not kill, the catalase-positive organisms.381 Building on previous studies that a neutrophil oxidase mediates the increase in oxygen consumption, a pyridine-dependent oxidase was found to be deficient in neutrophils of patients with CGD, which led to their inability to reduce the dye nitroblue tetrazolium (NBT) during phagocytosis of particles.382 Collectively, these studies laid the groundwork for subsequent studies to unravel the biochemical and genetic defects in CGD. Epidemiology The incidence of CGD in the United States is 1 per 200,000 livebirths, based on data from the National Institutes of Allergy and Infectious Disease Registry.383 Data from the Registry indicates that 86 percent of patients are male and 14 percent female; 80 percent are classified as white, 11 percent as black patients, and 3 percent Asians or mixed-race patients. Of the 340 patients in the Registry with adequate information for determination genetic transmission, 70 percent had the X-linked recessive form of the disease. Etiology and Pathogenesis Several laboratory tests are used to classify forms of CGD and aid in understanding its pathogenesis (Table 66–4). The diagnosis of CGD is based on a compatible clinical history and demonstration of a defective respiratory burst. Several methods detect the production of reactive oxidants. The NBT method relies on the intracellular reduction of NBT by superoxide anion to a blue formazan precipitate that can be seen microscopically.376 More sensitive methods rely on the reaction of oxidants with specific chemiluminescent and fluorescent probes. The patients with CGD may have heterogeneous array of regular symptoms and severity, depending on which subunit is defective and on the nature of the genetic mutation. Nicotinamide Adenine Dinucleotide Phosphate-Oxidase Function Engulfment of microbes by phagocytic cells is associated with a burst of oxygen consumption that is important for microbicidal killing and digestion. The respiratory burst is accompanied, not by mitochondrial respiration, but by a unique electron transport chain called the NADPH oxidase. Prior to stimulation, the components of the oxidase are physically separated into two major subcellular locations (Fig. 66–6). The membrane-bound portion of the NADPH oxidase contains a heterodimeric cytochrome b558 composed of a large, heavily glycosylated subunit with a Mr of 91 kDa, known as a gp91phox (91-kDa glycoprotein of the phagocyte oxidase), and a 22-kDa protein known as p22phox.376,384 Eighty to 90 percent of the cytochrome b558 is found in specific and gelatinase granules and secretory vesicles of the neutrophil and following neutrophil activation translocates to the plasma membrane.66,318 The heavy chain of cytochrome b contains sites for heme binding, flavin adenine dinucleotide (FAD) groups, and NADPH binding.385–388 The three-dimensional structure of cytochrome b558 indicates that the carboxyl-terminal half of the peptide contains sequences for flavin and NADPH binding.389 The amino half of the molecule is hydrophobic and contains the histidines that coordinate heme binding.390 The p22phox also contains a site for heme binding.385 The synthesis of the p22phox peptide is absolutely required for stability of gp91phox and for oxidase activity in the membrane.376 The p22phox also contains proline-rich regions that display consensus protein–protein interactions that provide a binding site for p47phox.391 Three other proteins vital to the function

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TABLE 66–4.  Diagnostic Classification of Chronic Granulomatous Disease Affected Component

Inheritance

Subtype

Membrane-Bound Cytochrome b558* Cytosol p47phox*

Cytosol p67phox*

gp91phox

X

X910

Not detectable

Normal

Normal

 

 

X91

Normal quantity, but nonfunctional

Normal

Normal

 

 

X91

Defective gp91 , which is poorly functional or expressed in a small fraction of phagocytes

Normal

Normal

p22phox

A

A220

Not detectable

Normal

Normal

 

 

A22

Normal quantity, but nonfunctional

Normal

Normal

p47

A

A47

Normal quantity

Not detectable

Normal

p67phox

 

A670

Normal

Normal

Not detectable

phox

+

phox

–

+ 0

*Detected by spectral analysis or immunoblotting. In this nomenclature, the first letter represents the mode of inheritance (-linked [X] or autosomal recessive [A]). The number indicates the phox component, which is genetically affected. The superscript symbols indicate whether the level of protein of the affected component is undetectable (0), diminished (–), or normal (+) as measured by immunoblot or spectral analysis.

Cytoplasm Fungus

Bacteria HOCl

Plasma membrane

MPO

Figure 66–6.  Possible mechanisms for the production of

Cl– Catalase or GSH H2O + O2 H2O2

OH– Fe2+

Fe3+

SOD

O2–

Phagosome O2

gp91phox FAD p22phox NADPH Rac2 p67 phox p40 phox p47phox P

FAD NADPH

P P

Rac2

Rac2

p67 phox p47 phox

phox p22 phox gp91

p40 phox Secretory vesicle, or specific granule, or gelatinase granule

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superoxide anion in neutrophils. Oxygen is reduced to superoxide (O2–) by an nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. The oxidase is a composite of (1) a 47-kDa cytosolic protein (p47); (2) a 67-kDa cytosolic protein (p67); (3) a 40-kDa cytosolic protein (p40); (4) a low-molecular-weight cytosolic G-protein, Rac2; and (5) a membrane-bound cytochrome b558. Cytochrome b consists of a 22-kDa protein subunit (p22) and a 91-kDa glycoprotein subunit (gp91), both of which contain heme. The gp91 subunit is a flavin adenine dinucleotide (FAD)-dependent flavoprotein that contains the NADPH binding site and ultimately shuttles electrons to molecular oxygen, forming O2–, and (6) the cytosol components translocate to the membrane and may serve to alter the tertiary structure of cytochrome b, to permit the flow of electrons from NADPH to O2. The p47 subunit (p47) is phosphorylated upon activation of the neutrophil. The p40phox component stabilizes the preactivation complex of p67phox. The unstable superoxide anion (O2–) is converted to hydrogen peroxide (H2O2), either spontaneously or by the enzyme superoxide dismutase (SOD). H2O2 in the presence of myeloperoxidase (MPO) converts H2O2 to hypochlorous acid (HOCl). Both H2O2 and O2– can be transformed into hydroxyl radical (OH–). H2O2 can be reduced to H2O and O2 by the enzyme catalase or by glutathione (GSH), a product of the hexose-monophosphate shunt. These reactive oxygen species are responsible for microbial killing. Normal oxidative function of the NADPH complex requires fully functional individual components.

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of this oxidase system reside in the cytosol of the resting phagocyte. Upon stimulation, translocation of p47phox takes place. Phosphorylated p47phox together with two other cytoplasmic components of the oxidase, p67phox, and a low-molecular-weight guanosine triphosphate Rac-2, translocate to the membrane, where they interact with cytoplasmic domains of the transmembrane cytochrome b558 to form the active oxidase.391,392 Both p47phox and p67phox contain SH3 (Src homology 3) domains that may participate in intramolecular and intermolecular binding with consensus proline-rich regions in p47phox.392 Phosphorylation, which occurs on serines in the cationic C-terminal region of p47phox, serves to disrupt this intermolecular interaction, making the SH3 regions available for binding to p22phox. Another cytoplasmic component with homology to p47phox has been identified as p40phox. p40phox, like p47phox, contains a PX domain, a motif that supports the binding to phosphoinositides on the cytosolic side of membranes.393 The p40phox component stabilizes the cytoplasmic complexes of p67phox and p47phox on phagosomes. Its binding of phosphatidylinositol 3 phosphate also potentiates superoxide production upon neutrophil activation.394 Cytochrome b558 spans the membrane, permitting NADPH to be oxidized at the cytoplasmic surface and oxygen to be reduced to form O2– on the outer surface of the plasma membrane or on the inner surface of the phagosomal membrane.395 Genetic Alterations Affecting Cytochrome b The most frequent form of CGD occurs in 70 percent of patients and is caused by mutations in the gp91phox gene, termed CYBB, which is located on chromosome Xp21.1.376,396 These mutations lead to the X-linked form of the disease. Large interstitial deletions causing other X-linked disorders such as retinitis pigmentosa, Duchenne muscular dystrophy, McLeod hemolytic anemia, and ornithine transcarbamylase deficiency, have been reported in a few patients with X-linked CGD.383,397–399 Mutation analysis of the gene encoding gp91 and a large group of X-linked CGD kindreds has documented many distinct defects, including point mutations, inversions, deletions, or insertions that disrupt the reading frame and nonsense mutations that create a premature stop codon.396 Some splice-site defects have also been identified. In this situation, short deletions in gp91phox mRNA are caused by point mutations that produce partial or complete exon skipping during mRNA splicing.400 This abnormality is a common cause of X-linked CGD. In the remaining patients, point mutations have been identified that generate either premature stop codons or amino acid substitutions that apparently disrupt protein stability or function and lead to a complete lack of detectable cytochrome b558 protein in phagocytic cells in most patients with X-linked CGD. In some situations, low levels of functional cytochrome b are present, whereas in others, normal levels of dysfunctional cytochrome b558 occur.401 In the latter situation there is some clustering of defects in regions of known function, such as the NADPH- or flavin-binding consensus regions.402 Approximately 10 to 15 percent of X-linked CGD arises from new germline mutations.403 A similar array of mutations has been identified in the 5 percent of CGD patients who have abnormalities in the p22phox gene, termed CYBA, which is located on chromosome 16q24.376,402,404 In this autosomal disorder, mutations in the p22phox gene result in deletions, frameshifts, and/or missense mutations. Patients with a defective p22phox gene do not express the other cytoplasmic unit polypeptide. In one patient, p22phox peptide was associated with normal amounts of cytochrome b with normal heme spectrum, but p47phox translocation membrane did not occur and there was no oxidase activation because the mutation affected a proline-rich region thought to mediate binding to one of the SH3 domains of p47phox. In gp91phox-deficient patients, p22phox mRNA is present, but it is not translated, which is consistent with the notion that either cytochrome subunit polypeptide is dependent upon the stable

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expression of the other subunit.376 Genetic Alterations Affecting Cytosolic Proteins Two other proteins have been identified as being vital to the function of the NADPH-oxidase system. Their absence results in the syndrome of CGD.405 These proteins have molecular masses of 47 kDa and 67 kDa, respectively, and are located in the cytosol of resting cells. Defects in the genes for p47phox, termed NCF1, which is found on chromosome 7q11, are responsible for the majority of all cases of autosomal recessive CGD, whereas inherited defects for the gene for neutrophil p67phox, termed NCF2, account for a small subgroup of autosomal recessive CGD.376 The function of p47phox and p67phox in regulating the respiratory burst oxidase is thought to involve activation of the electron transport function of cytochrome b558. The mutation analysis in patients with p47phox-deficient forms of CGD reveals an unusual pattern, in that more than 90 percent of mutant alleles have guanine-thymine dinucleotide deletion at the start of exon 2, resulting in frameshift and premature stop.402,406 The truncated protein is unstable in that it cannot be detected immunologically. The majority of patients appear to be homozygous for this mutation without any history of consanguinity. The p47phox gene occurs in an area of chromosome 7 that has a high degree of evolutionary duplication in normal individuals because a pseudogene highly homologous to the normal p47phox gene exists in the normal genome in this region of duplication. The pseudogene contains the same GT deletion associated with most cases of p47phox CGD. This implies that recombination of the normal gene and pseudogene with conversion of the normal gene to partial pseudotype sequence in that region may be responsible for the high relative rate of this specific mutation in diverse racial groups, which proved to be the case.407 A second rare form of CGD is caused by mutations in the gene for the p67phox cytosolic component.401 The p67phox gene, which has been mapped to the long arm of chromosome 1, spans 37 kb and contains 16 exons. The mutations identified in p67phox-deficiency CGD have included missense mutations and spliced junction mutations affecting mRNA processing, which led to nondetectable p67phox protein by immunologic means.402 Mutation of NCF4, the gene encoding p40phox, was reported in a child with granulomatous colitis. One allele had a frameshift mutation with a premature stop codon. The other had a missense mutation predicting an R105Q substitution in the PX domain which is responsible for binding to phosphatidylinositol 3 phosphate. The functional defect was inability to assemble the NADPH oxidase in the membrane of phagosomes but not on the plasma mambrane.408 Predisposition to Infection Mutations in the gene for cytochrome b558 or the cytosolic factors involved in activating the cytochrome are associated with the CGD phenotype. Figure 66–7 shows schematically the manner in which the metabolic deficiency of the CGD neutrophil predisposes the host to infection. Normal neutrophils accumulate H2O2 and other oxygen metabolites in the phagosomes containing ingested microorganisms. MPO is delivered to the phagosome by degranulation and in this setting H2O2 acts as a substrate for MPO to oxidize halide to HOCl and chloramines, which kill the microbes. The quantity of H2O2 produced by the normal neutrophils is sufficient to exceed the capacity of catalase, a H2O2-catabolizing enzyme produced by many aerobic microorganisms, including S. aureus, most Gram-negative enteric bacteria, C. albicans, and Aspergillus spp. In contrast, H2O2 is not produced by CGD neutrophils, and any generated by the microbes themselves may be destroyed by their own catalase. Thus, catalase-positive microbes can multiply inside CGD neutrophils, where they are protected from most circulating antibiotics, and can be transported to

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Part VII: Neutrophils, Eosinophils, Basophils, and Mast Cells

H2O2

catalase

O2 + H2O

H2O2 Strep.

E. coli

H O 2

O 2 H2 O 2 H2

E. coli

H2O2

E. coli

H2O2

2

H2O2

H2O2

H 2O 2

Strep. 2O 2

H

2

H 2O 2

H O 2

2

H O 2

Normal

CGD

Figure 66–7.  The pathogenesis of chronic granulomatous disease (CGD). The manner in which the metabolic deficiency of the CGD neutrophil

predisposes the host to infection is shown schematically. Normal neutrophils accumulate hydrogen peroxide (H2O2) in the phagosome containing ingested Escherichia coli. Myeloperoxidase is delivered to the phagosome by degranulation, as indicated by the closed circles, and in this setting, H2O2 acts as a substrate for myeloperoxidase to oxidize halide to hypochlorous acid and chloramines, which kill the microbes. The quantity of H2O2 produced by the normal neutrophils is sufficient to exceed the capacity of catalase, a H2O2-catabolizing enzyme of many aerobic microorganisms, including most Gram-negative enteric bacteria, Staphylococcus aureus, Candida albicans, and Aspergillus spp. When organisms such as E. coli gain entry into the CGD neutrophils, they are not exposed to H2O2 because the neutrophils do not produce it, and the H2O2 generated by microbes themselves is destroyed by their own catalase. When CGD neutrophils ingest streptococci (Strep.) or pneumococci, these organisms generate enough H2O2 to result in a microbicidal effect. On the other hand, as indicated in the middle figure, catalase-positive microbes, such as E. coli, can survive within the phagosome of the CGD neutrophil. distant sites and released to establish new foci of infection.405 Activation of the oxidase also has a pronounced effect on the pH within the phagocytic vacuole. It is controversial whether activation of the respiratory burst is associated with an alkaline phase, but the pH of the phagocytic vacuole becomes more acidic in CGD patients than in normal patients.161,409 The alkaline phase may be important for the antimicrobial and digestive functions of the neutral hydrolases released from the cytoplasmic granules into the vacuole upon phagocytosis. In CGD, the phagocytic vacuoles remain acidic and the bacteria are not digested properly.410 The impairment in the respiratory burst by CGD neutrophils leads to delayed neutrophil apoptosis and subsequent impaired clearance of degenerating neutrophils by CGD macrophages, which, in turn, predisposes the host to enhanced inflammation.411 CGD neutrophils are incapable of generating NETs and cannot trap microorganisms by this mechanism.412 The CGD macrophage is unable to clear CGD neutrophils because of a deficiency of intrinsic IL-4 production, which occurs because of defective phosphatidylserine exposure on CGD neutrophils, that is a necessary requirement to engage CGD macrophage phosphatidylserine membrane receptors and subsequent macrophage activation.411 In hematoxylin-and-eosin-stained sections from patients, macrophages eventually may contain a golden pigment, which reflects the abnormal accumulation of ingested material and also contributes to the diffuse granulomata that give CGD its descriptive name.413 On the other hand, when CGD neutrophils ingest pneumococci or streptococci, these organisms generate enough H2O2 to result in a microbicidal effect. Clinical Features Although the clinical presentation is variable, several clinical features suggest the diagnosis of CGD.376 Any patient with recurrent lymphadenitis should be considered to have CGD. Additionally, patients with bacterial hepatic abscesses, osteomyelitis at multiple sites or in the small bones of the hands and feet, a family history of recurrent infections, or unusual catalase-positive microbial infections all require clinical evaluation for this disorder. Table 66–5 lists the most

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common clinical infections that afflict CGD patients and Table 66–6 cites their prevalence. Among the various infections, only perirectal abscess, suppurative adenitis, and bacteremia/fungemia differ significantly in prevalence in the X-linked recessive and autosomal recessive CGD patients.383 Each of these conditions was twice as common in the X-linked form. The onset of clinical signs and symptoms may occur from early infancy to young adulthood. Although the majority of patients with CGD (76 percent) are diagnosed before the age of 5 years, approximately 10 percent are not diagnosed until the second decade of life, and on rare occasions, not until the third decade or later.383 The organisms infecting CGD patients have changed considerably from those initially reported between 1957 and 1976. Staphylococcus caused most of the infections in the initial cases; Klebsiella and E. coli were then the next most common pathogens. Now Aspergillus is the prominent organism causing pneumonia and is the leading cause of death in patients.376 Invasive aspergillosis can occur in the first few months of life in healthy infants as well as in those with CGD. Although aspergillosis is the most common infecting fungus in CGD, Candida and several other fungal strains have been invasive in this disorder. Burkholderia cepacia is another leading cause of death in patients with CGD. Serratia marcescens is the third leading organism that commonly infects patients with CGD. Infections are characterized by microabscesses and granuloma formation. The presence of pigmented histiocytes is helpful in establishing the diagnosis. Patients may suffer from the consequences of chronic infections including the anemia of chronic disease, lymphadenopathy, hepatosplenomegaly, chronic purulent dermatitis, restrictive lung disease, gingivitis, hydronephrosis, and gastroenteric narrowing.383 Patients with CGD are also at risk for developing colitis and chorioretinitis, and discoid lupus erythematosus.383 Several mothers of patients in whom X-linked inheritance was established had an illness resembling systemic lupus erythematosus.383 Both X-linked and autosomal recessive patients with CGD also have a

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TABLE 66–5.  Common Infecting Organisms Isolated from Chronic Granulomatous Disease Patients Infection Type

Organism

X-Linked Recessive (%)

Autosomal Recessive (%)

Pneumonia

Aspergillus spp.

41

29

 

Staphylococcus spp.

11

13

 

Burkholderia cepacia

7

11

 

Nocardia spp.

6

13

 

Serratia spp.

4

5

Abscess

 

 

 

Subcutaneous

Staphylococcus spp.

28

21

 

Serratia spp.

19

9

 

Aspergillus spp.

7

0

Liver

Staphylococcus spp.

52

52

 

Serratia spp.

6

4

 

Candida spp.

12

0

Lung

Aspergillus spp.

27

18

Perirectal

Staphylococcus spp.

9

15

Brain

Aspergillus spp.

75

25

Suppurative adenitis

Staphylococcus spp.

29

12

 

Serratia spp.

9

15

 

Candida spp.

7

4

Osteomyelitis

Serratia spp.

32

12

 

Aspergillus spp.

25

18

Bacteremia/fungemia

Salmonella spp.

20

13

 

Burkholderia cepacia

13

0

 

Candida spp.

9

25

 

Staphylococcus spp.

11

0

Data from Segal BH, Leto TL, Gallin JI, et al: Genetic, biochemical, and clinical features of chronic granulomatous disease, Medicine (Baltimore) 2000 May;79(3):170–200.

TABLE 66–6.  Prevalence of Infectious Complication of Chronic Granulomatous Disease Patients Infection Type

X-Linked Recessive (%)

Autosomal Recessive (%)

Pneumonia

80

77

Abscess (all)

68

70

 Subcutaneous

43

42

 Liver

26

33

 Lung

16

14

 Brain

3

5

 Perirectal

17

7

Suppurative adenitis

59

32

Osteomyelitis

27

21

Bacteremia/fungemia

21

10

Cellulitis

7

5

Data from Segal BH, Leto TL, Gallin JI, et al: Genetic, biochemical, and clinical features of chronic granulomatous disease, Medicine (Baltimore) 2000 May;79(3):170–200.

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similar disorder.414 It may be that these mothers’ and patients’ cells are unable to clear immune complexes sufficiently, which is a characteristic feature of CGD cells in vitro.415 Variant alleles of MBL and FcγRIIA especially in combination are associated with rheumatologic disorders in patients with CGD.416 Laboratory Findings The defect in the respiratory burst is best determined by measuring superoxide or H2O2 production in response to both soluble and particulate stimuli.417 A test that is being employed is the use of flow cytometry using dihydrorhodamine-123 fluorescence.418 Dihydrorhodamine-123 fluorescence detects oxidant production because it increases fluorescence upon oxidation.418 In most cases there is no detectable superoxide or H2O2 generation with either type of stimulus. In the variant form of CGD, however, superoxide may be produced at rates between 0.5 and 10 percent of control.419 An alternative method for measuring respiratory burst activity is the NBT test. This assay is performed by microscopically assessing the ability of individual cells to reduce NBT to purple formazan crystals following stimulation. Commonly there is no NBT reduction with most forms of CGD. In some of the variant forms, however, a high percentage of cells may contain some formazan, a finding indicative of a greatly diminished respiratory burst in most of the neutrophils. This test also permits detection of the carrier state in X-linked CGD when as few as 5 to 10 percent of the cells are NBT-negative.420

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Part VII: Neutrophils, Eosinophils, Basophils, and Mast Cells

Most sophisticated procedures can identify the molecular defect. Cytochrome b content can be measured in extracts of detergent-disrupted neutrophils by a spectrophotometric assay.420 Once the diagnosis of CGD is made, the genotype can be determined. A mosaic population of oxidation that has positive and negative neutrophils in a male patient’s mother and sister strongly suggests X-linked CGD. Lack of a mosaic pattern among female relatives does not rule out the X-linked mode of inheritance because the defect can arise spontaneously. Prenatal diagnosis of CGD is established by analysis of DNA from amniocytes or chorionic villus samples. Differential Diagnosis Leukocytes from patients with CGD have normal glucose-6-phosphate dehydrogenase (G6PD) activity. However, a few individuals with apparent CGD have been described who have neutrophils that lack or are almost lacking in G6PD activity.421,422 The erythrocytes of these patients also lack the enzyme, and the patients have chronic hemolysis. In the cases of severe neutrophil G6PD deficiency, an attenuated respiratory burst progressively decreases as a result of the depletion of intracellular NADPH, the primary substrate for the respiratory burst oxidase. CGD and G6PD deficiency can be distinguished from each other by the hemolytic anemia seen in the latter disorder and by the fact that erythrocyte G6PD activity is normal in CGD and markedly reduced in G6PD deficiency.401 A variety of studies indicate that the small GTPase Rac-2 plays an essential role in activity of the NADPH and the actin cytoskeleton in human neutrophils.383 A toddler has been described as presenting with a perirectal abscess at 5 weeks of age. This patient subsequently had necrosis of the periumbilical skin and fascia, and his surgical wounds did not heal properly. Functionally his neutrophils had multiple defective components; for example, adhesion to ligands for sLex, chemotaxis, release of primary azurophil granules upon stimulation with chemotactic peptide, and failure to undergo the respiratory burst using the same stimulus.423,424 Molecular analysis identified the asparagine for aspartic acid mutation at amino acid 57 of one allele of the Rac-2 gene.423,424 Mutant Rac-2 did not bind GTP and it inhibited and behaved as a dominant negative to impair Rac-2–mediated activation of the respiratory burst.424 Fortunately, the youngster was successfully transplanted with marrow from a HLA-identical older brother.424 Therapy, Course, and Prognosis Allogeneic hematopoietic stem cell transplantation is the only recognized curative treatment for CGD. Reduced intensity conditioning stem cell transplantation from HLAmatched donors performed in 56 patients with intractable infections and severe inflammation carried a 2-year overall survival of 96 percent.425 However, vigorous supportive care along with the use of recombinant IFN continues to be the foundation of treatment.376 Cultures must be obtained as soon as infection is suspected, as unusual organisms are commonly the source of infection and may grow promptly in vitro. Most abscesses require surgical drainage for therapeutic and diagnostic purposes, and prolonged use of antibiotics is often required. If fever occurs, it is advisable to obtain certain studies that aid in the management of septic episodes. These include roentgenograms of the chest and skeleton and a computed tomography (CT) scan of the liver because of the frequency of pneumonia, osteomyelitis, and liver abscesses.383 Arrangements should be made for prompt medical attention at the first signs of infection. With early intervention, many lesions can be managed by conservative medical means. For example, enlarging lymph nodes often regress when treated with local heat and orally administered antistaphylococcal antibiotics. It is particularly important to obtain a microbiologic diagnosis, and fine-needle aspiration may be helpful in this regard. In general, antibiotic therapy for the offending organisms is indicated and purulent masses should be drained. The cause of fever and prostration cannot always be established, and empiric

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treatment with broad-spectrum parenteral antibiotics is required. Often it is necessary to treat with antibiotics for a prolonged time until the initially elevated sedimentation rate approaches normal values. Aspergillus spp. infection requires treatment with amphotericin B or, in refractory cases, with granulocyte transfusions.376 Glucocorticoids also may be useful in the treatment of patients with antral and urethral obstruction. The risk of Aspergillus infection can be reduced by avoiding marijuana smoke and decaying plant material, such as mulch and hay, both of which contain numerous fungal spores.426 Long-term oral prophylaxis with trimethoprim-sulfamethoxazole (5 mg/kg per day of trimethoprim) is an accepted practice in the management of patients with CGD.376 Patients have prolonged infection-free periods, which result from the prevention of infections caused by S. aureus, without increasing the incidence of fungal infections. The use of itraconazole prophylactically has reduced the development of fungal infections.427,428 IFN-γ (50 mcg/m2, three times per week, subcutaneously) can reduce the number of serious bacterial and fungal infections.427,429 IFN-γ–enhanced neutrophil function in vitro has not been correlated with improvement in the activity of the neutrophil respiratory burst in patients totally lacking the ability to generate superoxide. On the other hand, its use increases the neutrophil expression of the high-affinity Fcγ receptor 1, as well as monocyte expression of FcγRI, FcγRII, FcγRIII, CD11/CD18, and HLA-DR.430 The IFN-γ protective effect in patients with CGD may involve improved microbial clearance, as suggested by the enhanced phagocytic activity by neutrophils of opsonized S. aureus. In rare, X-linked CGD patients able to generate some superoxide, IFN-γ programs granulocyte cells to increase their expression of cytochrome b, which results in normal superoxide generation.431 With the use of current prophylactic treatments, the mortality in CGD has been reduced to two patient deaths per year per 100 patients followed.376 CGD patients with mutations that result in 5 to 10 percent of normal-functioning amounts of NADPH have a mild phenotype and better clinical prognosis than do patients with complete absence of any NADPH-oxidase activity.432 Similarly, female carriers of X-linked CGD who have only 3 to 5 percent oxidase-normal neutrophils rarely get serious infections suggestive of the CGD clinical phenotype.433 Thus, even low levels or partial correction by gene therapy of CGD is likely to provide clinical benefits. In support of that hypothesis, mouse models of X-linked and p47phox-deficient CGD have been developed by gene targeting.434,435 Studies in the gp91phox- and the p47phox-deficient mouse models of CGD show that retrovirus-mediated gene-therapy-targeting of marrow progenitor cells ex vivo can result in the correction of defects in oxidant production in vivo in blood neutrophils after radiation conditioning and transplantation of marrow stem cells.436,437 Protection from infection challenge occurred even when the oxidase-corrected cells comprised less than 10 percent of circulating neutrophils. These promising results suggest that somatic gene therapy can be employed to correct defective phagocyte oxidase function in selected patients with CGD. In a phase I clinical trial, gene therapy for p47phox-deficiency CGD, five adult patients received intravenous infusions of autologous blood stem cells that were ex vivo transduced using a retrovirus encoding normal p47phox.438 Although conditioning therapy was not given prior to the stem cell infusion, functionally corrected neutrophils were detectable in blood for several months.339 In another study, long-term high-level clinical beneficial correction in ex vivo gene therapy of X-linked CGD occurred in two adult patients.439 Nonablative busulfan conditioning was used to augment gene therapy correction. There needs to be caution regarding the long-term stability and safety of gene therapy. For instance, there are concerns about gene insertion rendering patients vulnerable to developing an hematological malignancy.

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Chapter 66: Disorders of Neutrophil Function

Myeloperoxidase Deficiency

The functional and immunochemical absence of the enzyme MPO from granules of neutrophils and monocytes, but not eosinophils, is inherited as an autosomal recessive trait, with a prevalence of 1:2000.440 MPO, an enzyme that catalyzes the production of HOCl in the phagosome. In MPO deficiency, the microbicidal activity of the neutrophils is reduced early after ingestion of microorganisms (see Table  66–2). However, normal microbicidal activity is observed in approximately 1 hour after a variety of organisms are ingested.440 Thus, the MPO-deficient neutrophil uses an MPO-independent system for killing bacteria that is slower than the MPO–H2O2–halide system, but that is eventually effective in eliminating bacteria. MPO-deficient neutrophils accumulate more H2O2 than do normal neutrophils; the higher peroxide concentration improves the bactericidal activity of the affected neutrophils. In contrast to the retardation of bactericidal activity, candidacidal activity in MPO-deficient neutrophils is absent.440 The most significant clinical manifestation in a few patients with diabetes mellitus and MPO deficiency has been severe infection with C. albicans. Because this is such a common disorder of phagocytes, it is important to note that the vast majority of patients with this genetic disorder have not been unusually susceptible to pyogenic infections and do not require therapy. The complementary DNA encoding human MPO has been cloned and the gene structure, including promoter and regulatory elements, delineated.440 The gene consists of 12 exons and 11 introns and is located on the long arm of chromosome 17, and its expression is finely coordinated with expression of genes encoding other lysosomal proteins. Expression of genes for human neutrophil elastase and MPO is very similar; it is low in myeloblasts, peaks during the promyelocyte stage, and eventually drops to low levels in myelocytes. MPO is a symmetric molecule composed of four peptides, where each half consists of a heavy- and a light-chain heterodimer.440 Each heavy- and light-chain heterodimer starts as a single peptide that is cleaved during the posttranslational process to yield the heavy and light chains that form half of the mature molecules. The two halves of the molecule are associated by a disulfide linkage between heavy-subunit residues at their residue C319. The primary translation product of the gene is a single-chain peptide of 80 kDa that undergoes cotranslational glycosylation at several asparagine residues, followed by a series of modifications of these oligosaccharides. The apopromyeloperoxidase exists for a prolonged time in the endoplasmic reticulum, where it associates reversibly with several endoplasmic reticulum–resident proteins known as molecular chaperones.440 Subsequent to heme insertion, the enzymatically active promyeloperoxidase undergoes proteolytic cleavage of the pro region. Then, in a prelysosomal compartment, the single peptide is cleaved into the heavy and light subunits, which remain linked. During final sorting within the azurophil lysosome compartment, there is dimerization of half-molecules to form the mature MPO. Most patients with MPO deficiency have a missense mutation in the gene that results in replacement of arginine 569 with tryptophan.440 The mutation results in a precursor that associates with molecular chaperones, but does not incorporate heme, resulting in a maturational arrest during processing at the stage of an inactive enzymatic apopromyeloperoxidase. Other patients are heterozygotes with one allele bearing the common mutation and the other being normal, resulting in a partial deficiency.441 To date, four genotypes have been reported to cause inherited MPO deficiency, each of which results in missense mutations. In the genotype Y173C, a missense mutation results in replacement of a tyrosine at codon 173 with a cysteine residue resulting in the mutant precursor being retained in the endoplasmic reticulum by virtue of its prolonged interaction with the chaperone calnexin, and eventually

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undergoing degradation in a proteasome.440 In this way, the quality control system operating in the endoplasmic reticulum retrieves misfolded MPO precursors from the biosynthetic pathway and creates the biochemical phenotype of MPO deficiency. In another patient, a missense mutation resulted in an intact MPO molecule that acquired heme but failed to undergo proteolytic processing to a mature molecule. Acquired disorders are associated with MPO deficiency. Reported states include lead intoxication, ceroid lipofuscinosis, myelodysplastic syndromes, and acute myelogenous leukemia.442 One-half of untreated patients with acute myelogenous leukemia and 20 percent of patients with CML may have MPO deficiency.442

Deficiencies of Glutathione Reductase and Glutathione Synthetase

Neutrophils contain enzymes capable of inactivating potentially damaging reduced oxygen byproducts. Disposal of superoxide anion is accomplished through superoxide dismutase, a soluble enzyme that converts superoxide to a H2O2. H2O2 is detoxified by catalase and by the glutathione peroxidase–glutathione reductase system, which converts H2O2 to water and oxygen.443 In addition to the soluble enzymes, cellular vitamin E serves as an antioxidant to prevent damage to the surface of activated neutrophils when releasing H2O2.443 Single cases of profound deficiencies in glutathione reductase444 and glutathione synthetase443 have been associated with impaired neutrophil bactericidal activity (see Table  66–2). Both deficiencies are associated with hemolysis under conditions of oxidative stress (Chap. 48). Glutathione synthetase deficiency also has been associated with intermittent neutropenia during times of mild infection. Vitamin E has been employed to ameliorate the hemolysis and improve neutrophil function in a patient with glutathione synthetase deficiency.445 Like patients with MPO-deficient neutrophils, the patients with glutathione reductase deficiency and glutathione synthetase deficiency are not unusually susceptible to bacterial infections.

DIAGNOSTIC APPROACH TO THE PATIENT WITH SUSPECTED NEUTROPHIL DYSFUNCTION An increased susceptibility to pyogenic infections must be viewed in light of a number of factors: (1) adequacy of host defense; (2) the microbes to which the host is exposed; and (3) the conditions of the exposure. It is not always easy to establish a diagnosis of a specific neutrophil dysfunction on clinical grounds alone. Patients with recurrent pyogenic infections often yield no clues as to why they are afflicted, and patients with established deficiency of a defense mechanism may have an unimpressive clinical history. On the other hand, patients may be suspected of having a neutrophil dysfunction if they have a history of frequent bacterial or severe infections. Recurrent pulmonary infections, hepatic abscesses, and perirectal abscesses also should alert the clinician to consider further diagnostic evaluation of neutrophil function. For example, the identification of unusual catalase-positive bacteria and fungi, such as B. cepacia, S. marcescens, Nocardia, and Aspergillus, could be indicative of CGD. Because many of the tests of neutrophil function are bioassays with great variability, the results of the tests must be interpreted in light of the patient’s clinical condition. For instance, isolated chemotactic defects usually do not explain the propensity for a patient to have recurrent severe infections. Furthermore, variation in bioassays is often intensified by inflammation or infection. Figure 66–8 is an algorithm for evaluation of the patient with recurrent infection.

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Part VII: Neutrophils, Eosinophils, Basophils, and Mast Cells

Consider neutrophil G6PD deficiency Chédiak-Higashi syndrome Consider specific granule deficiency

Hemolytic anemia

Howell-Jolly bodies

1. Initial evaluation

• History, physical exam • Family history • Leukocyte, platelet, reticulocyte, and Abnormal granules differential counts Pelger-Hüet anomaly • Bacterial cultures Abnormal granules partial albinism

Thrombocytopenia, eczema Neutrophil counts 2000 IU/mL; AD hyper-IgE

If normal Chronic granulomatous disease

Complement deficiency, humoral defects

Absent O2– Abnormal NBT and DHR tests Only abnormal chemotaxis

–

3. Phagocyte evaluation • NBT test • DHR assay • Chemotaxis assays - Rebuck skin window - In vitro assay with patient control sera

If normal

Myeloperoxidase deficiency

Opsonin defect

4. Further phagocyte evaluation

Abbreviated O2 production GSH pathway

Chemotaxis Abnormal response to activated control serum Absent CD11/CD18 by decreased ingestion

Decreased ingestion with control serum • Myeloperoxidase stain • Flow cytometry to measure CD11/CD18 Decreased sLex surface glycoproteins expression by flow on neutrophils Decreased ingestion • Quantitative ingestion Diminished adhesion with patient’s serum assays (patient and to selectin ligand control sera as opsonins) • Flow cytometry to measure Failure to generate selectin on neutrophils – • Rolling on L-selectin ligand O2 when challenged with unopsonized zymosam Myeloperoxidase absent

Neutrophil G6PD deficiency LAD-1 and LAD-2 Chédiak-Higashi syndrome specific granule deficiency Rac-2 deficiency

LAD-1 Neutrophil actin dysfunction LAD-2

Rac-2 deficiency

LAD-3

Figure 66–8.  Algorithm for the workup of patients with recurrent infections. AD, autosomal dominant; CBC, complete blood count; CVID, common variable immunodeficiency; DHR, delayed hypersensitivity reaction; G6PD, glucose-6-phosphate dehydrogenase; GSH, glutathione; Ig, immunoglobulin, LAD, leukocyte adhesion deficiency; NBT, nitroblue tetrazolium; sLex, sialyl Lewis X.

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Chapter 66: Disorders of Neutrophil Function

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333. Aebi M, Helenius A, Schenk B, et al: Carbohydrate-deficient glycoprotein syndromes become congenital disorders of glycosylation: An updated nomenclature for CDG. First International Workshop on CDGS. Glycoconj J 16:669, 1999. 334. Hidalgo A, Ma S, Peired AJ, et al: Insights into leukocyte adhesion deficiency type 2 from a novel mutation in the GDP-fucose transporter gene. Blood 101:1705, 2003. 335. Kuijpers TW, van BR, Kamerbeek N, et al: Natural history and early diagnosis of LAD-1/variant syndrome. Blood 109:3529, 2007. 336. Kuijpers TW, van de V, Weterman MA, et al: LAD-1/variant syndrome is caused by mutations in FERMT3. Blood 113:4740, 2009. 337. Fischer A, Lisowska-Grospierre B, Anderson DC, et al: Leukocyte adhesion deficiency: Molecular basis and functional consequences. Immunodefic Rev 1:39, 1988. 338. Bauer TR Jr, Hickstein DD: Gene therapy for leukocyte adhesion deficiency. Curr Opin Mol Ther 2:383, 2000. 339. Malech HL, Hickstein DD: Genetics, biology and clinical management of myeloid cell primary immune deficiencies: Chronic granulomatous disease and leukocyte adhesion deficiency. Curr Opin Hematol 14:29, 2007. 340. Boxer LA, Hedley-Whyte ET, Stossel TP: Neutrophil action dysfunction and abnormal neutrophil behavior. N Engl J Med 291:1093, 1974. 341. Southwick FS, Dabiri GA, Stosse TP: Neutrophil actin dysfunction is a genetic disorder associated with partial impairment of neutrophil actin assembly in three family members. J Clin Invest 82:1525, 1988. 342. Malech HL, Gallin JI: Current concepts: Immunology neutrophils in human diseases. N Engl J Med 317:687, 1987. 343. Southwick FS, Howard TH, Holbrook T, et al: The relationship between CR3 deficiency and neutrophil actin assembly. Blood 73:1973, 1989. 344. Coates TD, Torkildson JC, Torres M, et al: An inherited defect of neutrophil motility and microfilamentous cytoskeleton associated with abnormalities in 47-kD and 89-kD proteins. Blood 78:1338, 1991. 345. Howard T, Li Y, Torres M, et al: The 47-kD protein increased in neutrophil actin dysfunction with 47-and 89-kD protein abnormalities is lymphocyte-specific protein. Blood 83:231, 1994. 346. Howard TH, Hartwig J, Cunningham C: Lymphocyte-specific protein 1 expression in eukaryotic cells reproduces the morphologic and motile abnormality of NAD 47/89 neutrophils. Blood 91:4786, 1998. 347. Camitta BM, Quesenberry PJ, Parkman R, et al: Bone marrow transplantation for an infant with neutrophil dysfunction. Exp Hematol 5:109, 1977. 348. Samuels J, Aksentijevich I, Torosyan Y, et al: Familial Mediterranean fever at the millennium. Clinical spectrum, ancient mutations, and a survey of 100 American referrals to the National Institutes of Health. Medicine (Baltimore) 77:268, 1998. 349. Siegal S: Benign paroxysmal peritonitis. Gastroenterology 12:234, 1949. 350. Drenth JP, van der Meer JW: Hereditary periodic fever. N Engl J Med 345:1748, 2001. 351. Ben-Chetrit E, Levy M: Familial Mediterranean fever. Lancet 351:659, 1998. 352. Ancient missense mutations in a new member of the RoRet gene family are likely to cause familial Mediterranean fever. The International FMF Consortium. Cell 90:797, 1997. 353. Centola M, Wood G, Frucht DM, et al: The gene for familial Mediterranean fever, MEFV, is expressed in early leukocyte development and is regulated in response to inflammatory mediators. Blood 95:3223, 2000. 354. Ryan JG, Kastner DL: Fevers, genes, and innate immunity. Curr Top Microbiol Immunol 321:169, 2008. 355. Hull KM, Shoham N, Chae JJ, et al: The expanding spectrum of systemic autoinflammatory disorders and their rheumatic manifestations. Curr Opin Rheumatol 15:61, 2003. 356. Richards N, Schaner P, Diaz A, et al: Interaction between pyrin and the apoptotic speck protein (ASC) modulates ASC-induced apoptosis. J Biol Chem 276:39320, 2001. 357. Touitou I: The spectrum of Familial Mediterranean Fever (FMF) mutations. Eur J Hum Genet 9:473, 2001. 358. Schaner P, Richards N, Wadhwa A, et al: Episodic evolution of pyrin in primates: Human mutations recapitulate ancestral amino acid states. Nat Genet 27:318, 2001. 359. Williamson LM, Hull D, Mehta R, et al: Familial Hibernian fever. Q J Med 51:469, 1982. 360. Lakshman R, Finn A: Neutrophil disorders and their management. J Clin Pathol 54:7, 2001. 361. Perlmutter DH, Colten HR: Molecular basis of complement deficiencies. Immunodefic Rev 1:105, 1989. 362. Kannourakis G: Glycogen storage disease. Semin Hematol 39:103, 2002. 363. Smith OP: Shwachman-Diamond syndrome. Semin Hematol 39:95, 2002. 364. Jones DH, Schmalstieg FC, Dempsey K, et al: Subcellular distribution and mobilization of MAC-1 (CD11b/CD18) in neonatal neutrophils. Blood 75:488, 1990. 365. Sapey E, Greenwood H, Walton G, et al: Phosphoinositide 3-kinase inhibition restores neutrophil accuracy in the elderly: Toward targeted treatments for immunosenescence. Blood 123:239, 2014. 366. Brayton RG, Stokes PE, Schwartz MS, et al: Effect of alcohol and various diseases on leukocyte mobilization, phagocytosis and intracellular bacterial killing. N Engl J Med 282:123, 1970. 367. Oseas RS, Allen J, Yang HH, et al: Mechanism of dexamethasone inhibition of chemotactic factor induced granulocyte aggregation. Blood 59:265, 1982. 368. Dale DC, Fauci AS, Wolff SM: Alternate-day prednisone. Leukocyte kinetics and susceptibility to infections. N Engl J Med 291:1154, 1974. 369. Boxer LA, Allen JM, Baehner RL: Diminished polymorphonuclear leukocyte adherence. Function dependent on release of cyclic AMP by endothelial cells after stimulation of beta-receptors by epinephrine. J Clin Invest 66:268, 1980.

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370. Freeman AF, Holland SM: The hyper-IgE syndromes. Immunol Allergy Clin North Am 28:277, 2008. 371. Engelich G, Wright DG, Hartshorn KL: Acquired disorders of phagocyte function complicating medical and surgical illnesses. Clin Infect Dis 33:2040, 2001. 372. Buckley RH: The hyper-IgE syndrome. Clin Rev Allergy Immunol 20:139, 2001. 373. Grimbacher B, Holland SM, Gallin JI, et al: Hyper-IgE syndrome with recurrent infections—An autosomal dominant multisystem disorder. N Engl J Med 340:692, 1999. 374. Zhang Q, Davis JC, Lamborn IT, et al: Combined immunodeficiency associated with DOCK8 mutations. N Engl J Med 361:2046, 2009. 375. Yong PF, Freeman AF, Engelhardt KR, et al: An update on the hyper-IgE syndromes. Arthritis Res Ther 14:228, 2012. 376. Segal BH, Leto TL, Gallin JI, et al: Genetic, biochemical, and clinical features of chronic granulomatous disease. Medicine (Baltimore) 79:170, 2000. 377. Berendes H, Bridges RA, Good RA: A fatal granulomatosus of childhood: The clinical study of a new syndrome. Minn Med 40:309, 1957. 378. Landing BH, Shirkey HS: A syndrome of recurrent infection and infiltration of viscera by pigmented lipid histiocytes. Pediatrics 20:431, 1957. 379. Sbarra AJ, Karnovsky ML: The biochemical basis of phagocytosis. I. Metabolic changes during the ingestion of particles by polymorphonuclear leukocytes. J Biol Chem 234:1355, 1959. 380. Iyer GYN, Islam MF, Quastel JH: Biochemical aspects of phagocytosis. Nature 192:535, 1961. 381. Iyer GY, Quastel JH: NADPH and NADH oxidation by guinea pig polymorphonuclear leucocytes. Can J Biochem Physiol 41:427, 1963. 382. Baehner RL, Nathan DG: Quantitative nitroblue tetrazolium test in chronic granulomatous disease. N Engl J Med 278:971, 1968. 383. Winkelstein JA, Marino MC, Johnston RB Jr, et al: Chronic granulomatous disease. Report on a national registry of 368 patients. Medicine (Baltimore) 79:155, 2000. 384. Parkos CA, Allen RA, Cochrane CG, et al: Purified cytochrome b from human granulocyte plasma membrane is comprised of two polypeptides with relative molecular weights of 91,000 and 22,000. J Clin Invest 80:732, 1987. 385. Quinn MT, Mullen ML, Jesaitis AJ: Human neutrophil cytochrome b contains multiple hemes. Evidence for heme associated with both subunits. J Biol Chem 267:7303, 1992. 386. Rotrosen D, Yeung CL, Leto TL, et al: Cytochrome b558: The flavin-binding component of the phagocyte NADPH oxidase. Science 256:1459, 1992. 387. Segal AW, West I, Wientjes F, et al: Cytochrome b-245 is a flavocytochrome containing FAD and the NADPH-binding site of the microbicidal oxidase of phagocytes. Biochem J 284:781, 1992. 388. Sumimoto H, Sakamoto N, Nozaki M, et al: Cytochrome b558, a component of the phagocyte NADPH oxidase, is a flavoprotein. Biochem Biophys Res Commun 186:1368, 1992. 389. Zhen L, Yu L, Dinauer MC: Probing the role of the carboxyl terminus of the gp91phox subunit of neutrophil flavocytochrome b558 using site-directed mutagenesis. J Biol Chem 273:6575, 1998. 390. Shatwell KP, Dancis A, Cross AR, et al: The FRE1 ferric reductase of Saccharomyces cerevisiae is a cytochrome b similar to that of NADPH oxidase. J Biol Chem 271:14240, 1996. 391. Deleo FR, Quinn MT: Assembly of the phagocyte NADPH oxidase: Molecular interaction of oxidase proteins. J Leukoc Biol 60:677, 1996. 392. Segal AW: The NADPH oxidase and chronic granulomatous disease. Mol Med Today 2:129, 1996. 393. Kanai F, Liu H, Field SJ, et al: The PX domains of p47phox and p40phox bind to lipid products of PI(3)K. Nat Cell Biol 3:675, 2001. 394. Chen J, He R, Minshall RD, et al: Characterization of a mutation in the Phox homology domain of the NADPH oxidase component p40phox identifies a mechanism for negative regulation of superoxide production. J Biol Chem 282:30273, 2007. 395. Cross AR, Jones OT: Enzymic mechanisms of superoxide production. Biochim Biophys Acta 1057:281, 1991. 396. Heyworth PG, Curnutte JT, Rae J, et al: Hematologically important mutations: X-linked chronic granulomatous disease (second update). Blood Cells Mol Dis 27:16, 2001. 397. Francke U, Ochs HD, de MB, et al: Minor Xp21 chromosome deletion in a male associated with expression of Duchenne muscular dystrophy, chronic granulomatous disease, retinitis pigmentosa, and McLeod syndrome. Am J Hum Genet 37:250, 1985. 398. Royer-Pokora B, Kunkel LM, Monaco AP, et al: Cloning the gene for an inherited human disorder—chronic granulomatous disease—on the basis of its chromosomal location. Nature 322:32, 1986. 399. Frey D, Machler M, Seger R, et al: Gene deletion in a patient with chronic granulomatous disease and McLeod syndrome: Fine mapping of the Xk gene locus. Blood 71:252, 1988. 400. de Boer M., Bolscher BG, Dinauer MC, et al: Splice site mutations are a common cause of X-linked chronic granulomatous disease. Blood 80:1553, 1992. 401. Curnutte JT, Orkin S, Dinauer MC: Genetic disorders of phagocyte function, in The Molecular Basis of Blood Diseases, 2nd ed, edited by Stammatoyannopoulos G, p 493. WB Saunders, Philadelphia, 1994. 402. Roos D, de BM, Kuribayashi F, et al: Mutations in the X-linked and autosomal recessive forms of chronic granulomatous disease. Blood 87:1663, 1996. 403. Rae J, Newburger PE, Dinauer MC, et al: X-Linked chronic granulomatous disease: Mutations in the CYBB gene encoding the gp91-phox component of respiratory-burst oxidase. Am J Hum Genet 62:1320, 1998.

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404. Dinauer MC, Pierce EA, Bruns GA, et al: Human neutrophil cytochrome b light chain (p22-phox). Gene structure, chromosomal location, and mutations in cytochrome-negative autosomal recessive chronic granulomatous disease. J Clin Invest 86:1729, 1990. 405. Segal AW: Biochemistry and molecular biology of chronic granulomatous disease. J Inherit Metab Dis 15:683, 1992. 406. Casimir CM, Bu-Ghanim HN, Rodaway AR, et al: Autosomal recessive chronic granulomatous disease caused by deletion at a dinucleotide repeat. Proc Natl Acad Sci U S A 88:2753, 1991. 407. Roos D: X-CGDbase: A database of X-CGD-causing mutations. Immunol Today 17:517, 1996. 408. Matute JD, Arias AA, Wright NA, et al: A new genetic subgroup of chronic granulomatous disease with autosomal recessive mutations in p40 phox and selective defects in neutrophil NADPH oxidase activity. Blood 114:3309, 2009. 409. Jankowski A, Scott CC, Grinstein S: Determinants of the phagosomal pH in neutrophils. J Biol Chem 277:6059, 2002. 410. Reeves EP, Lu H, Jacobs HL, et al: Killing activity of neutrophils is mediated through activation of proteases by K+ flux. Nature 416:291, 2002. 411. Fernandez-Boyanapalli RF, Frasch SC, McPhillips K, et al: Impaired apoptotic cell clearance in CGD due to altered macrophage programming is reversed by phosphatidylserine-dependent production of IL-4. Blood 113:2047, 2009. 412. Bianchi M, Hakkim A, Brinkmann V, et al: Restoration of NET formation by gene therapy in CGD controls aspergillosis. Blood 114:2619, 2009. 413. Johnston RB Jr, Baehner RL: Chronic granulomatous disease: Correlation between pathogenesis and clinical findings. Pediatrics 48:730, 1971. 414. Johnston RB Jr: Clinical aspects of chronic granulomatous disease. Curr Opin Hematol 8:17, 2001. 415. Petty HR, Francis JW, Boxer LA: Deficiency in immune complex uptake by chronic granulomatous disease neutrophils. J Cell Sci 90:425, 1988. 416. Foster CB, Lehrnbecher T, Mol F, et al: Host defense molecule polymorphisms influence the risk for immune-mediated complications in chronic granulomatous disease. J Clin Invest 102:2146, 1998. 417. Wolach B, Scharf Y, Gavrieli R, et al: Unusual late presentation of X-linked chronic granulomatous disease in an adult female with a somatic mosaic for a novel mutation in CYBB. Blood 105:61, 2005. 418. Crockard AD, Thompson JM, Boyd NA, et al: Diagnosis and carrier detection of chronic granulomatous disease in five families by flow cytometry. Int Arch Allergy Immunol 114:144, 1997. 419. Newburger PE, Luscinskas FW, Ryan T, et al: Variant chronic granulomatous disease: Modulation of the neutrophil defect by severe infection. Blood 68:914, 1986. 420. Curnutte JT: Chronic granulomatous disease: The solving of a clinical riddle at the molecular level. Clin Immunol Immunopathol 67:S2, 1993. 421. Cooper MR, DeChatelet LR, McCall CE, et al: Complete deficiency of leukocyte glucose-6-phosphate dehydrogenase with defective bactericidal activity. J Clin Invest 51:769, 1972. 422. Vives Corrons JL, Feliu E, Pujades MA, et al: Severe-glucose-6-phosphate dehydrogenase (G6PD) deficiency associated with chronic hemolytic anemia, granulocyte dysfunction, and increased susceptibility to infections: Description of a new molecular variant (G6PD Barcelona). Blood 59:428, 1982. 423. Ambruso DR, Knall C, Abell AN, et al: Human neutrophil immunodeficiency syndrome is associated with an inhibitory Rac2 mutation. Proc Natl Acad Sci U S A 97:4654, 2000. 424. Williams DA, Tao W, Yang F, et al: Dominant negative mutation of the hematopoieticspecific Rho GTPase, Rac2, is associated with a human phagocyte immunodeficiency. Blood 96:1646, 2000.

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425. Gungor T, Teira P, Slatter M, et al: Reduced-intensity conditioning and HLA-matched haemopoietic stem-cell transplantation in patients with chronic granulomatous disease: A prospective multicentre study. Lancet 383:436, 2014. 426. Chusid MJ, Gelfand JA, Nutter C, et al: Letter: Pulmonary aspergillosis, inhalation of contaminated marijuana smoke, chronic granulomatous disease. Ann Intern Med 82:682, 1975. 427. Seger RA: Modern management of chronic granulomatous disease. Br J Haematol 140:255, 2008. 428. Gallin JI, Alling DW, Malech HL, et al: Itraconazole to prevent fungal infections in chronic granulomatous disease. N Engl J Med 348:2416, 2003. 429. A controlled trial of interferon gamma to prevent infection in chronic granulomatous disease. The International Chronic Granulomatous Disease Cooperative Study Group. N Engl J Med 324:509, 1991. 430. Schiff DE, Rae J, Martin TR, et al: Increased phagocyte Fc gammaRI expression and improved Fc gamma-receptor-mediated phagocytosis after in vivo recombinant human interferon-gamma treatment of normal human subjects. Blood 90:3187, 1997. 431. Woodman RC, Erickson RW, Rae J, et al: Prolonged recombinant interferon-gamma therapy in chronic granulomatous disease: Evidence against enhanced neutrophil oxidase activity. Blood 79:1558, 1992. 432. Kuhns DB, Alvord WG, Heller T, et al: Residual NADPH oxidase and survival in chronic granulomatous disease. N Engl J Med 363:2600, 2010. 433. Malech HL, Bauer TR Jr, Hickstein DD: Prospects for gene therapy of neutrophil defects. Semin Hematol 34:355, 1997. 434. Pollock JD, Williams DA, Gifford MA, et al: Mouse model of X-linked chronic granulomatous disease, an inherited defect in phagocyte superoxide production. Nat Genet 9:202, 1995. 435. Jackson SH, Gallin JI, Holland SM: The p47phox mouse knock-out model of chronic granulomatous disease. J Exp Med 182:751, 1995. 436. Mardiney M, III, Jackson SH, Spratt SK, et al: Enhanced host defense after gene transfer in the murine p47phox-deficient model of chronic granulomatous disease. Blood 89:2268, 1997. 437. Bjorgvinsdottir H, Ding C, Pech N, et al: Retroviral-mediated gene transfer of gp91phox into bone marrow cells rescues defect in host defense against Aspergillus fumigatus in murine X-linked chronic granulomatous disease. Blood 89:41, 1997. 438. Malech HL, Maples PB, Whiting-Theobald N, et al: Prolonged production of NADPH oxidase-corrected granulocytes after gene therapy of chronic granulomatous disease. Proc Natl Acad Sci U S A 94:12133, 1997. 439. Ott MG, Schmidt M, Schwarzwaelder K, et al: Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertional activation of MDS1-EVI1, PRDM16 or SETBP1. Nat Med 12:401, 2006. 440. Hansson M, Olsson I, Nauseef WM: Biosynthesis, processing, and sorting of human myeloperoxidase. Arch Biochem Biophys 445:214, 2006. 441. Nauseef WM: Insights into myeloperoxidase biosynthesis from its inherited deficiency. J Mol Med (Berl) 76:661, 1998. 442. Nauseef WM: Myeloperoxidase deficiency. Hematol Pathol 4:165, 1990. 443. Boxer LA: The role of antioxidants in modulating neutrophil functional responses. Adv Exp Med Biol 262:19, 1990. 444. Roos D, Weening RS, Voetman AA, et al: Protection of phagocytic leukocytes by endogenous glutathione: Studies in a family with glutathione reductase deficiency. Blood 53:851, 1979. 445. Boxer LA, Oliver JM, Spielberg SP, et al: Protection of granulocytes by vitamin E in glutathione synthetase deficiency. N Engl J Med 301:901, 1979.

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Part VIII  Monocytes and Macrophages 67. Structure, Receptors, and Functions of Monocytes and Macrophages . . . . . . . . . . 1045 68. Production, Distribution, and Activation of Monocytes and Macrophages . . . . . . . 1075 69. Classification and Clinical Manifestations of Disorders of Monocytes and Macrophages . . . . . . . . . . . . . . . . . . . . . . . . . 1089

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70. Monocytosis and Monocytopenia . . . . . . 1095 71. Inflammatory and Malignant Histiocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . 1101 72. Gaucher Disease and Related Lysosomal Storage Diseases . . . . . . . . . . . 1121

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CHAPTER 67

STRUCTURE, RECEPTORS, AND FUNCTIONS OF MONOCYTES AND MACROPHAGES

Steven D. Douglas and Anne G. Douglas

SUMMARY The monocyte is a spherical cell with prominent surface ruffles and blebs when examined by scanning electron microscopy. As the monocyte enters the tissue and differentiates into a macrophage, the cell volume and number of cytoplasmic granules increase. Cell shape varies, depending on the tissue in which the macrophage resides (e.g., lung, liver, spleen, brain). A characteristic feature of macrophages is their prominent electron-dense membrane-bound lysosomes, which can be seen fusing with phagosomes to form secondary lysosomes. The latter contain ingested cellular and noncellular material in different stages of degradation. A broad range of surface receptors for many ligands, including the Fc portion of immunoglobulin, complement proteins, cytokines, chemokines, lipoproteins, and others, are on the cell surface. Macrophages differ in appearance, biochemistry, and function based on the environment in which they mature from monocytes. These differences are exemplified by the diversity among dendritic cells of lymph nodes, histiocytes of connective tissue, osteoclasts of bone, Kupffer cells of liver, microglia of the central nervous system, and macrophages of the serosal surfaces, each fashioned to meet the local needs of the mononuclear phagocyte system, which plays a role in inflammation and host defense against microbes. Modern cell biologic methods refined our knowledge of surface receptors, endocytosis, and lysosomal

Acronyms and Abbreviations:  APC, antigen-presenting cell; CD, cluster of differentiation; CR, complement receptor; CSF, colony-stimulating factor; DC, dendritic cell; EGF, epidermal growth factor; EGF-TM7, epidermal growth factor–seven transmembrane; EMR2, epidermal growth factor–like module containing mucin-like hormone receptor–like 2; FcR, Fc receptor; GM-CSF, granulocyte-monocyte colony-stimulating factor; GPCR, G-protein–coupled receptor; HLA, human leukocyte antigen; IBD, inflammatory bowel disease; IFN, interferon; Ig, immunoglobulin; IL, interleukin; IMP, intramembrane particle; IRAK, interleukin receptor-associated kinase; LFA, lymphocyte function–associated antigen; LPS, lipopolysaccharide; m-φ, macrophage; MARCO, macrophage receptor with collagenous structure; M-CSF, macrophage colony-stimulating factor; MHC, major histocompatibility complex; MPO, myeloperoxidase; NF, nuclear factor; NLR, NOD-like receptor; NOD, nucleotide-binding oligomerization domain; PI3K, phosphatidylinositol 3-kinase; PS, phosphatidylserine; SR, scavenger receptor; TGF, transforming growth factor; TLR, toll-like receptor.

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degradation, with emphasis on membrane flow and secretion. These pioneering studies culminated in the discovery of dendritic cells as potent, specialized antigen-presenting cells. Subsequent development of monoclonal antibodies and molecular cloning of surface proteins and cytokines, followed by microarray analysis and genomics, provided the sensitive and specific tools to analyze macrophage functions in vitro and in vivo. These studies have brought insights into macrophage cytotoxic and antimicrobial activities and, to a lesser extent, their trophic, homeostatic functions in the body. Macrophages play a major role in innate as well as adaptive immunity.

MONONUCLEAR PHAGOCYTE SYSTEM OVERVIEW Modern study of mammalian phagocytes began with Metchnikoff in the 19th century. An understanding of the ontogeny, kinetics, and function of phagocytic cells in animals led to the concept of the mononuclear phagocyte system.1,2 Kinetic studies indicate that marrow monoblasts and monocytes develop from the common myeloid progenitor, a derivative of the hematopoietic stem cell, and that tissue macrophages develop from monocytes that have migrated from the blood pool in response to chemotactic stimuli (Table 67-1 and Chap. 18). Tissue macrophages share many functional characteristics, such as phagocytic and microbial killing capabilities and adherence to glass or plastic surfaces in vitro. Vascular endothelium, reticular cells, and dendritic cells of lymphoid germinal centers usually are not included in the mononuclear phagocyte system, although the now obsolete term reticuloendothelial system3 denoted those cells as playing some complementary part with mononuclear phagocytes. In addition to developing the multitude of types of tissue macrophages, monocytes can differentiate into myeloid-derived dendritic cells.4,5

STRUCTURE The blood monocyte is a medium to large motile cell that can marginate along vessel walls and has a propensity for adherence to surfaces. Monocytes respond to inflammation and chemotactic stimuli by active diapedesis across vessel walls into inflammatory foci, where they can mature into macrophages, with greater phagocytic capacity and increased content of hydrolytic enzymes. Free macrophages also are present in mammary glands, alveolar spaces, pleura, peritoneum, and synovia. The somewhat less-motile fixed-tissue macrophages are found in different tissues and serous cavities. The functions of mononuclear phagocytes include phagocytosis, killing, and digestion of microorganisms, particulate material, or tissue debris; secretion of chemical mediators and regulators of the inflammatory response; interaction (as dendritic cells) with antigen and lymphocytes in the generation of the immune response; cytotoxicity, such as killing of some tumor cells; and other functions specific for macrophages of particular tissues. The development of techniques to isolate monocytes from blood of adult human subjects led to the discovery that monocytes are heterogeneous with regard to cell volumes. Isolation of purified monocytes by adherence to glass substrates or to gelatin-coated flasks or by centrifugal elutriation reveals distinct populations of monocytes.1,2 In addition to the usual 12- to 15-μm diameter (when measured on a dried blood film) monocyte, so-called regular monocytes, a somewhat smaller cell that is less active than its larger, more mature counterpart has been identified. This cell is referred to as a small immature monocyte, but its functional significance is not clear.

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Part VIII: Monocytes and Macrophages

TABLE 67–1.  Distribution of Mononuclear Phagocytes

from the progranulocyte.10,11 Peroxidase is present throughout the cell secretory apparatus in all cisternae of the rough-surfaced endoplasmic reticulum, the Golgi complex, associated vesicles, and all immature and mature granules. Cytochemical reaction products for acid phosphatase and arylsulfatase also are deposited throughout the secretory apparatus of the promonocyte.

Marrow

Tissues

Monoblasts

Liver (Kupffer cells)

Promonocytes

Lung (alveolar macrophages)

Monocytes

Connective tissue (histiocytes)

Macrophages

Spleen (red pulp macrophages)

Blood

Lymph nodes

MORPHOLOGY OF MONOCYTES

Thymus

Light Microscopy

Monocytes Body cavities Pleural macrophages

Bone (osteoclasts) Synovium (type A cells)

Peritoneal macrophages

Mucosa-associated lymphoid tissue

Inflammatory tissues

Gastrointestinal tract

Epithelioid cells

Genitourinary tract

Exudate macrophages

Endocrine organs

Multinucleate giant cells

Central nervous system (microglia) Skin (histiocyte/dendritic cells)

Data from Lewis, C, McGee, JD: The Macrophage, 2nd ed., Oxford University Press, New York, NY, 1992; Gordon S, Fraser I, Nath D. et al: Macrophages in tissues and in vitro. Curr Opin Immunol 4:25-32, 1992; Lasser A: The mononuclear phagocytic system: A review. Hum Pathol 14:108-26, 1983.

The morphology of monocytes has been investigated by light and phasecontrast optics,12 scanning and transmission electron microscopy, and freeze-fracture and freeze-etch procedures.13 On the stained blood film the monocyte has a diameter of 12 to 15 μm (Fig. 67–1). The monocyte nucleus occupies approximately half the area of the cell and usually is eccentrically placed. The nucleus most often is reniform, but may be round or irregular. It contains a characteristic chromatin net with fine strands bridging small chromatin clumps. Chromatin aggregates are arranged along the internal side of the nuclear membrane. The cytoplasm is spread out, stains grayish-blue with Wright stain, and contains a variable number of fine, pink-purple granules, which at times are sufficiently numerous to give the entire cytoplasm a pink hue. Clear cytoplasmic vacuoles and a variable number of larger azurophilic granulations often are encountered in these cells.

Phase Microscopy

Monocytes continuously emigrate from the blood into tissue, with a half-life in the blood of approximately 1 day in mice.6 Nondividing monocytes can be induced to differentiate into dendritic like cells in vitro. However, this process requires culture of the cells for 7 to 10 days with exogenous cytokines, typically interleukin (IL)-4 and granulocyte-monocyte colony-stimulating factor (GM-CSF).7 The major lineage regulator of nearly all macrophages is monocyte/macrophage colony-stimulating factor (M-CSF; also termed CSF-1) and its receptor (M-CSF R). The M-CSF R is a class III transmembrane tyrosine kinase receptor, which is expressed on most mononuclear phagocytes.8 In the presence of endothelial cells grown on an extracellular matrix, monocytes differentiate along two distinct pathways: toward dendritic cells or macrophages. Monocytes that migrate across endothelium in an abluminal to luminal direction differentiate into dendritic cells. In contrast, monocytes that remain in the subendothelial matrix differentiate into macrophages.

MORPHOLOGY OF MONOCYTE PRECURSORS Monoblasts and promonocytes are the precursors of monocytes, bearing finely dispersed nuclear chromatin and nucleoli when observed in the stained film of the marrow. The monoblast is a very-low-prevalence marrow cell, indistinguishable by light microscopy from the myeloblast. Promonocytes are 12 to 18 μm in diameter (as measured on dried blood films) and have characteristic deeply indented, irregularly shaped nuclei with condensed chromatin, and numerous cytoplasmic microfilaments. In animal studies, a small percentage of marrow cells are phagocytic, synthesize DNA, adhere to glass surfaces, and contain nonspecific esterases.9 These cells have been referred to as promonocytes and are considered as intermediate between monoblasts and the monocytes of the blood.9 Cytochemical studies identify the promonocyte in normal human marrow. Promonocytes have deeply indented and irregularly shaped nuclei and bundled and scattered single filaments in the cytoplasm. These morphologic features distinguish the promonocyte

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The monocyte nucleus has a distinct chromatin pattern on a cloudy background when examined by phase-contrast microscopy. The cytoplasm is clear gray. Mitochondria are extremely fine and occasionally form a small, juxtanuclear rosette surrounding the centrosome. The phase-dense cytoplasmic granules, varying in number, are generally at the limit of resolution of light microscopy and appear as fine intracytoplasmic dust. Monocytes contain several types of cytoplasmic vacuoles. The reniform nucleus with a juxtanuclear depression filled by a centrosome and its active undulating movement similar to that of other leukocytes are characteristic of the monocyte. The locomotion of the monocyte has the same pattern of undulating cytoplasmic veils seen in macrophages. The monocyte generally assumes a triangular shape as it moves, with one point trailing behind and the other two points advancing before the cell. Blood monocytes undergo adherence and cytoplasmic spreading following attachment to glass surfaces.14 The extent of spreading increases in the presence of antigen–antibody complexes, certain divalent metals, and proteolytic enzymes.14,15 The spread form of the monocyte reveals that the nucleus and granules are located centrally and the abundant hyaloplasm is in the periphery of the cell, terminating in a fringed border that displays undulating movement. The small monocyte may be difficult to distinguish from the large lymphocyte when examined by phase-contrast microscopy. A striking feature on phase-contrast microscopy is the ruffled plasma membrane that forms prominent phase-dense folds at the cell surface and edges. Some cells have a dense thickening at the edge of the cytoplasm, with microextensions on the thickened edge.

Scanning Electron Microscopy

The monocyte surface has very prominent ruffles and small surface blebs.16,17 Extensive ruffling on the monocyte plasma membrane is of functional significance. The monocyte is both motile and phagocytic, and these functions require physical contact with particles or cell surfaces. Reduction in the radius of curvature of the cell surface

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D

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Figure 67–1.  Blood films. This composite shows four examples of normal monocytes with different nuclear configurations. A. In this case, the nucleus is contorted on itself and the nuclear-to-cytoplasmic ratio is a bit higher than the average case. B. Another contorted nucleus with a lower nuclear-to-cytoplasmic ratio. Scattered vacuoles are common in monocytes collected in ethylenediaminetetraacetic acid (EDTA)-anticoagulated blood before film preparation. C. Characteristic reniform nuclear shape. D. Circular nuclear shape. Azurophilic granules are evident in the cytoplasm of monocytes. (Reproduced with permission from Lichtman’s Atlas of Hematology, www.accessmedicine.com.)

by formation of ruffles or microvilli may reduce repulsive forces when surface negative-charge groups on the cell approach and contact a negatively charged substratum or cell. In addition, redundancy of the cell membrane may provide reserve membrane required for locomotion and phagocytosis.

Transmission Electron Microscopy

The nucleus of the monocyte contains one or two small nucleoli surrounded by nucleolar-associated chromatin (Fig. 67–2).18 The cytoplasm contains a relatively small quantity of endoplasmic reticulum and a variable quantity of ribosomes and polysomes. The mitochondria are numerous, small, and elongated. The Golgi complex is well developed and is situated about the centrosome within the nuclear indentation. Centrioles and filamentous centriolar satellites are often visualized in this region. Microtubules are numerous, and microfibrils are found in bundles surrounding the nucleus. In cultured macrophages, collections of microfilaments are present underneath the plasma membrane near sites of cell attachment either to a substratum or to phagocytosable particles.19 The cell surface is characterized by numerous microvilli and vesicles of micropinocytosis. The cytoplasmic granules resemble the small granules found in the granulocytic series, measuring approximately 0.05 to 0.2 μm in diameter. They are dense and homogeneous and are surrounded by a limiting membrane. These granules, as with the lysosomal granules of other leukocytes, are packaged by the Golgi apparatus after their enzymatic content has been produced by the ribosomal complex of the cell.10,11 These cytoplasmic granules contain acid phosphatase and arylsulfatase and, therefore, are primary lysosomes. After endocytosis, lysosomes fuse with the phagosome, forming secondary lysosomes.

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Some monocyte granules stain positive for peroxidase, whereas others are peroxidase negative.10,11

Freeze-Fracture Microscopy

In this technique, a cell suspension is frozen, placed in a high-vacuum chamber, and struck with a blunt edge, thus producing a fracture that propagates through the frozen specimen. The utility of the procedure comes from the remarkable finding that when the fracture encounters a cell, the fracture tends to propagate along the interior of the plasma membrane and thus split the lipid bilayer into its two constituent layers. After fracture, the specimen is coated with platinum, which is electron dense when viewed with transmission electron microscopy. All cell types examined thus far by the freeze-fracture technique reveal intramembrane particles (IMPs) as the predominant topographic feature of the interior of the bilayer. Studies of the erythrocyte show that at least some particles contain intercalated membrane proteins, and this is assumed to be the case for nucleated cells as well. The distribution of IMPs is dramatically altered in a number of cell systems by physiologic stimuli, for example, hormonal stimulation. Profound changes in the distribution of IMPs on mononuclear phagocytes occur following binding of antibody-coated erythrocytes.13 Because redistribution of IMPs also occurs in some nonphagocyte Fc receptor (FcR)–bearing cells13 and after exposure to aggregated immunoglobulin (Ig) G, this alteration in IMPs presumably reflects interaction with FcR. Freeze-etch electron micrographs of the monocyte show nuclear pores traversing both lamellae of the nuclear membrane and contours of cytoplasmic lysosomes and mitochondria (Fig. 67–3).

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Figure 67–2.  Transmission electron micrograph of a monocyte. The eccentric reniform nucleus has a thinly dispersed chromatin pattern. The Golgi complex (G) is in a juxtanuclear position. Small electron-dense granules can be seen evolving in the Golgi complex. Small amounts of rough endoplasmic reticulum (er) and polyribosomes (r) are present, particularly about the cell periphery. Mitochondria (m) are concentrated in the region of the Golgi apparatus; they also are scattered in the cell periphery. Lysosomes (L) are small, electron-dense granules surrounded by a limiting membrane. The irregular ruffled cell margin is apparent with numerous microprojections (×24,000).

HISTOCHEMISTRY OF MONOCYTES Table 67–2 compares the hydrolytic enzyme contents of monocytes, neutrophils, and lymphocytes. Monocytes also give a weak but positive periodic acid–Schiff reaction (for polysaccharides) and Sudan black B reaction (for lipids). Nonspecific esterase20–22 is frequently used as a marker for monocytes. Monocyte esterases are inhibited by sodium fluoride, whereas the esterases of the granulocytic series are not. The nonspecific esterase reaction is positive in promyelocytes and myelocytes; therefore, analysis of fluoride inhibition is necessary to distinguish marrow monocytes from early myelocytes. Monocyte granules, although heterogeneous in size (0.3 to 0.6 μm), are not separable into populations by routine electron microscopic criteria. Identification of monocyte granule populations has depended on subcellular localization of monocyte enzymes by electron microscopic cytochemistry.10 Human marrow promonocytes and blood monocytes contain granules that comprise two functionally distinct populations.10,11 One population contains the enzymes acid phosphatase, arylsulfatase, and peroxidase. These granules are modified primary lysosomes and are analogous to the azurophil

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granules of the neutrophil. The monocyte azurophil granule population is heterogeneous in cytochemical reactivity for peroxidase, acid phosphatase, and arylsulfatase.23,24 Moreover, primary granules that are morphologically identical with other vesicles can be identified as lysosomes cytochemically. The other population of monocyte granules lacks alkaline phosphatase23 and is not strictly analogous to the specific granules of neutrophils.

MORPHOLOGY OF MACROPHAGES Macrophage characteristics are heralded by a significant increase in cell size, increase in the number of cytoplasmic granules, increase in the heterogeneity of cell size and shape, and increase in the number of cytoplasmic clear vacuoles in comparison to monocytes.

Light and Phase-Contrast Microscopy

In vitro culture of monocytes purified from adult human blood has provided an opportunity to observe the maturation of these cells into mature macrophages. The macrophages of the pulmonary alveoli,

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Figure 67–3.  Freeze-etch electron micrograph of a

monocyte. Fracture plane displays the large nucleus (N), with multiple nuclear pores (np) and the two lamellae of the fractured nuclear membrane (nm) evident in some regions. Membrane and cleaved surfaces of mitochondria (m) and lysosomal granules (L) can be identified in the cytoplasm.

peritoneal and pleural cavities, and inflammatory exudates are hypermature cells that have undergone in vivo stimulation and maturation. This process results in enhanced bactericidal activity1,2 because of augmentation of lysosome number and acid hydrolase content. Macrophages display attributes of morphologic specialization specific to their location and function. The fixed macrophages of the spleen (littoral

TABLE 67–2.  Cytochemical Reactions of Leukocyte Enzymes Chemical

Monocytes Neutrophils Lymphocytes

Acid phosphatase

++

+

+

β-Glucuronidase

++

+

0 to +

+

+

0

N-Acetylglucosaminidase

++

++

0

Lysozyme*

++

++

0

Naphthylamidase

++

+

0 to +

α-Naphthylbutyrate esterase†

++

0 to +

0

0 to +

++

0

Peroxidase

+

++

0

Alkaline phosphatase

0

0 to +

0

Sulfatase

Naphthol AS-D chloroacetate esterase

*Most lysozyme produced by mononuclear phagocytes is secreted rather than stored intracellularly. † α-Naphthylacetate and α-naphthylbutyrate esterase activities may appear in human T lymphocytes under certain conditions.

Data from Braunsteiner H, Schmalzl F: Cytochemistry of monocytes and macrophages. In Mononuclear Phagocytes, edited by R van Furth, p 62. Blackwell, Oxford, England, 1970; Li CY, Lam KW, Yam LT: Esterases in human leukocytes. J Histochem Cytochem 21:1-12, 1973.

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cells) are involved in the sequestration and destruction of effete or abnormal red cells and exhibit stages of erythrophagocytosis and intracytoplasmic aggregates of ferritin (Chap. 6). The macrophages of the marrow, the “nurse cells” of the erythroblastic island, play a similar role in erythrophagocytosis and iron storage and transfer (Chaps. 5 and 31). Hepatic macrophages (Kupffer cells), found in liver sinusoids, also phagocytize red cells and other cellular elements and are important sites of iron storage. Macrophages of the pulmonary alveoli, the lamina propria of the gastrointestinal tract, and the peritoneal and pleural fluids reflect in their morphology a specific function of phagocytosis of microorganisms, cells, and cellular and noncellular debris, characteristic of the specific organ location. Most macrophages are 25 to 50 μm in diameter on Wright or hematoxylin-and-eosin–stained films (Fig. 67–4). They have an eccentrically placed reniform or fusiform nucleus with one or two distinct nucleoli and finely dispersed, loosely stranded nuclear chromatin that tend to clump in the nuclear interior and along the internal aspect of the nuclear membrane (Fig. 67–5A). A juxtanuclear clear zone (Golgi complex) is well defined when the Wright stain is used. The cytoplasm shows fine granules and multiple pink-purple, large azurophil granules. The cytoplasmic borders are irregularly serrated. Cytoplasmic vacuoles are present near the cell periphery, reflecting the active pinocytosis in these cells. The surface antigen CD68, also known as macrosialin, is commonly used as a macrophage marker. Figure 67–5B shows an immunohistochemistry micrograph of a macrophage in a lymph node. The cytoplasm of the macrophage is intensely positive for CD68, while the surrounding lymphocytes are negative. On phase-contrast microscopy, living macrophages are large cells with a propensity to adhere to and spread on glass surfaces. Thus, the cell organelles are concentrated within the central portion of the cell and clear veils of hyaloplasm spread about the cell, with intense ruffling of the membrane borders. Vesicles and contractile vacuoles are seen about the cell periphery and in the cell interior. The juxtanuclear clear zone bearing the centrosome and the Golgi complex is particularly dynamic and displays an undulating motion.

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A

B

C

D

Figure 67–4.  Marrow films. Macrophages. These cells characteristically have a circular, sometimes centrally placed and sometimes eccentrically

placed nucleus dwarfed by a very large expanse of cytoplasm. A. Activated macrophage, full of cytoplasmic vacuoles and some residual ingested cellular debris. B. Macrophage stained with Prussian blue showing cytoplasmic iron granules. C. Macrophage with erythrophagocytosis. Note pale red cells (partially dehemoglobinized) undergoing hemolysis and destruction. The highly vacuolated cytoplasm is presumably the site of red cell degradation. D. Macrophage in a patient with cystinosis engorged with cystine crystals. (Reproduced with permission from Lichtman’s Atlas of Hematology, www.accessmedicine.com.)

Electron Microscopy

Scanning electron micrographs of macrophages adherent to glass surface show membrane ruffling and pseudopodia (Fig. 67–6). Transmission electron microscopy of monocyte-derived macrophages show a variable degree of differentiation, nuclear “maturity,” ribosomes,

mitochondria, and lysosome content, and the nucleus varies in shape from horseshoe to fusiform (Fig. 67–7). Clear spaces between membrane-fixed chromatin aggregates mark the sites of nuclear pores that are relatively abundant on freeze-etch electron micrographs of macrophages and monocytes (see Fig. 67–3). Polyribosomes and scant smooth

P

L Mf

Mf

A

B

Figure 67–5.  Micrographs of macrophages (Mf ). A. Hematoxylin-and-eosin stain of cytology smear (×400) showing a macrophage, a plasma cell (P), and a lymphocyte (L). B. Immunohistochemistry stain for the macrophage marker CD68 of a lymph node (×400). Numerous lymphocytes with blue nuclei surround a macrophage with brown-red cytoplasm. (Used with permission of Dr. Madalina Tuluc, Thomas Jefferson University Hospital, P­ hiladelphia, PA.)

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A

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B

Figure 67–6.  Scanning electron micrograph of cultured macrophages on coverglasses coated with (A) bovine serum albumin (BSA) or (B) with immune complexes (BSA–anti-BSA). The macrophage develops prominent peripheral membrane ruffling and numerous microadhesion points to the surface coated with immune complexes.

is related to the phagocytic activity of the cell and its rate of pinocytosis. The number and size of mitochondria vary with the phagocytic and hence metabolic activity of the cell. Mitochondria tend to be grouped about the region of the Golgi complex, although several usually are seen dispersed about the cell periphery, presumably supplying energy for the active endocytic processes occurring there.

and rough endoplasmic reticulum are seen about the cell periphery. A well-developed Golgi complex is in a juxtanuclear location. It often is multicentric and contains a concentration of vesicles, some with dense inclusions that mark them as early lysosomes. A relatively constant feature of cells engaged in endocytosis is the large number of microvilli at the cell surface. The degree of development of this surface adaptation

N

G

Figure 67–7.  Electron micrograph of monocyte-derived macrophage cultured in vitro for 9 days. G, Golgi zone; N, nucleus. Arrow on right indicates endoplasmic reticulum; arrow on left indicates mitochondria; open arrow indicates lysosomes (×7600).

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The most constant and characteristic ultrastructural features of macrophages are the electron-dense membrane-bound lysosomes that often can be seen fusing with phagosomes to form secondary lysosomes. Within the secondary lysosomes, ingested cellular, bacterial, and noncellular material can be seen in various stages of degradation, often recognizable as degenerating mitochondria or nuclear material. These secondary lysosomes also contain partially degraded material from the late stages of the endocytic process, often appearing as multilamellar lipid bodies. Microtubules and microfilaments are prominent in macrophages. Actin- and myosin-like proteins have been isolated from monocytes and partially characterized. Resting macrophages have irregular cell borders and pseudopodia pushed out in all directions. Their cytoplasm has rough endoplasmic reticulum and Golgi complex in the perinuclear area. Lipid globules, primary lysosomes, and mitochondria are characteristically prominent. Activated monocytes/macrophages are motile cells that extend a leading pseudopod as they move forward.25

RECEPTORS MEMBRANE RECEPTORS AND OTHER SURFACE PROTEINS OF MONOCYTES AND MACROPHAGES Monocyte/macrophage cells have surface receptors that have been characterized by their binding to specific monoclonal antibodies. These receptors (Fig. 67–8) are markers for origin, growth,

differentiation,26 activation, recognition, migration, and function of the monocyte/macrophage. Monocytes have been classified into distinct subtypes based on surface expression of CD14 and CD16, molecules that form part of the lipopolysaccharide (LPS) toll-like receptor (TLR) and one of the immunoglobulin FcRs, respectively. These include CD14+-bright/CD16– monocytes, CD14+-dim/CD16+ monocytes, and CD14-dim/CD16+ monocytes. Monocyte heterogeneity was initially divided into the CD14+-bright/CD16-negative cells, which comprise 90 to 95 percent of total circulating monocytes (classical monocyte)27—CD14-bright or dim refer to the fluorescence magnitude of staining using a specific CD14 monoclonal antibody. The minor subset is CD14-dim, CD16-positive, and less phagocytic than the classical monocyte. The classical monocyte produces reactive oxygen species (ROS) and cytokines in response to TLR engagement. The minor subset selectively secretes tumor necrosis factor (TNF)-α, IL-13, and CCL2 in response to viruses and immune complexes containing nucleic acids via TLR-7, TLR-8, MyD88-MEK (myeloid differentiation factor 88–MAPK kinase), and AHD.28 This minor subset, CD14-dim,29 is competent in (SR [scavenger receptor]) function of vascular, intraluminal debris and uptake of immune complexes.30 In addition, their phenotype is related to the ability to produce and secrete select cytokines.31 Macrophages are proficient at endocytosis (both fluid phase and receptor-mediated) and are highly professional phagocytes of particulates of all origin, organic (cellular, microbial) as well as inorganic foreign materials.32 In contrast, when dendritic cells (DCs) mature into antigen-presenting cells (APCs), they have reduced uptake capacity

CD31

LFA1

C2 C2 C2 C2

CR3

C2 C2

EMR2 CCR2

CX3CR1

CD36 β2 β1

v v

α2 α1

c c

β

ITAM

α

CD14 CD86 M-CSFR (CD115) MHC11 L-selection CD4

CD33 CD16 (FC receptor) GM-CSF-R

N-terminal repeats with β-propeller structure I-like domain I-domain Immunoglobulin domain Short concensus repeats Lectin-like domain EGF-like domain Leucine-rich repeats Membrane-spanning domain Hemopoeitin domain

Figure 67–8.  Schematic of selected molecules of varied structure and functions of monocyte receptors and surface antigens. (Used with permission of S. Seif, GraphisMedica, 2014.)

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expression on activated monocytes and macrophages. CD64 allows for receptor-mediated endocytosis of IgG–antigen complexes for presentation to T cells, can trigger the release of cytokines and ROS, and can play a role in granulocyte-mediated antibody-dependent cytotoxicity. The second IgG receptor, FcRII (CD32), is a widely distributed receptor present on many cell types, including monocytes, platelets, neutrophils, B cells, some T cells, and some capillary endothelium. This receptor can bind complexed IgG rather than monomeric IgG. This FcR regulates B-cell function when coengaged with the B-cell receptor for antigen, namely, surface Ig. It also can induce mediator release from myeloid cells and phagocytosis of Ig-coated particles in vitro. Finally, this FcR also can target antigen into presenting pathways. The third IgG receptor, FcRIII (CD16), is expressed by neutrophils, natural killer cells, and tissue macrophages.40 This receptor can bind Ig in immune complexes and Ig bound to cell-surface membranes. It is the main FcR responsible for antibody-dependent cellular cytotoxicity. All three FcRs specifically bind the human IgG subclasses IgG1 and IgG3 (Chap. 75). The interaction of FcR on

and induce an adaptive immune response or tolerance. Immature DCs display active macropinocytosis and capture exogenous materials for cross-presentation.33 It is convenient to classify plasma membrane uptake receptors as opsonic and nonopsonic TLRs and non–TLR-dependent. The latter category includes a range of SRs34,35 and a family of lectin-like, carbohydrate recognition molecules.36,37 Given the complex ligands presented on the surface of microorganisms and damaged host cells, or generated within the vacuolar system after uptake, these receptors frequently cooperate with one another.

Fc Receptors

FcRs for IgG are expressed on the surface of mononuclear cells, macrophages, granulocytes, and platelets.38,39 FcRs are divided into three distinct classes: FcRI, FcRII, and FcRIII (Fig. 67–9). These receptors have broad ranges of expression on different cells. The first IgG receptor, FcRI (CD64), is a receptor found on monocytes, macrophages, and activated neutrophils. This receptor binds monomeric IgG through the Fc portion of the molecule. This Ig receptor has increased

Complement regulators

Lectin-like domain GPI Ig-like domain ITAM

CD55 CD59

ITIM

huCRIg(S)

Fc

huCRIg(L)

CR4

CR3

γ–γ

α x β2

RI

Fc

γ I

γ–

µR

γ– γ

Fc

αR

I

a III

γR

α Fc α/ α

ζ ζ– –γ γ F Fc cγR γR IIb Fc II γR a I

γ ζ–

Fcξ

α

CR1 RII Fcξ

b

III

γR

α m β2

I-like domain I-domain N-terminal repeats with β-propeller structure Cysteine-rich domain Immunoglobulin domain Short concensus repeats/ complement control protein repeats GPI anchor

Figure 67–9.  Human Fc receptors and complement. Myeloid cells express a range of classical Fc receptors that initiate a variety of cellular

responses, including phagocytosis, antibody-dependent cell-mediated toxicity, antigen presentation, respiratory burst, and release of inflammatory mediators. Immunoglobulin (Ig) subclasses are bound by extracellular domains; signaling via cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM) or immunoreceptor tyrosine-based inhibition motif (ITIM) is mediated by associated membrane-spanning polypeptides. Activation and inhibitory receptors are usually coexpressed on the cell surface and function in concert, determining the magnitude of effector cell responses. Complement receptors (CRs) and membrane regulators are expressed by m-ф-CR. CR1 is broadly expressed by nucleated cells, acting as a “sink” for activated complement. CR3 (CD11b/CD18), a phagocytic receptor for C3bi-coated particles, and CR4 (CD11c/CD18) are β2 integrins, which, together with lymphocyte function-associated antigen (LFA)-1 (CD11a/CD18), mediate adhesion of myeloid cells to endothelium and extracellular matrix and migration. Human CR immunoglobin (huCRIg) are long (L) and short (S) forms of the complement-binding receptor on Kupffer cells that mediate uptake of opsonized bacteria. CD55 and CD59 are glycosylphosphatidylinositol (GPI)-anchored regulators of complement activation. (Used with permission of S. Seif, GraphisMedica, 2014.)

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macrophages with immune complexes results in cell “activation,” with an increase in phagocytosis, superoxide production, and prostaglandin and leukotriene release.

Complement Receptors

Activation of the complement system results in liberation of numerous ligands that bind to specific receptors on mononuclear phagocytes. Four receptors that bind fragments of the complement component C3 have been identified (see Fig. 67–9).41 Complement receptor (CR) 1 (or CD35) binds dimeric C3bi and is found on both monocytes and macrophages. CR3 (or CD11b) binds the complement fragment C3b. CR3 is a heterodimeric glycoprotein that is composed of two noncovalently linked polypeptides. The α chain of the polypeptide has an Mr of 185,000, and the β subunit has an Mr of 95,000. This receptor and the leukocyte antigens lymphocyte function–associated antigen (CD11a) and alpha-X integrin chain (CD11c) compose a family of heterodimers that share a common β subunit (CD18).42 This family is designated the leukocyte integrin (β2) subfamily.43 These heterodimers are involved in cell–cell interactions, including leukocyte trafficking into the tissues, binding of opsonized particles and plasma proteins, and attachment to various substrates. They also may modulate intercellular adhesion. Elimination of the integrin β2 subunit causes leukocyte adhesion deficiency.44 The classical opsonins, which promote the uptake of particles, are antibody, IgG complexed with antigens, and complement, activated by the classical pathway (antibody-dependent IgM or IgG) or recognized directly via the lectin-carbohydrate–stimulated alternative pathway. Fc and CRs are heterogeneous in structure, expression, and function, activating or inhibiting macrophage responses,45,46 as illustrated in Fig.  67–9. Other opsonins include fibronectin and milk-fat globulin.47 Through their expression of various opsonic receptors, monocytes, macrophages, and DCs perform versatile roles in innate and adaptive immunity,48 in antigen clearance and destruction, in autoimmunity, and in pathogenesis of a range of inflammatory and infectious disorders. Genetic polymorphisms influence the expression and functions of FcRs in homeostasis and disease. Although prominent in host protection, invading microorganisms may be able to exploit, even subvert these receptors to facilitate their entry and survival.49 Opsonic receptors play an important role in clearance of hematopoietic cells, for example, antibody-coated platelets, giving rise to thrombocytopenia, and in therapeutic antibody treatment, for example, to facilitate engraftment. Antibody engineering has provided novel therapeutic agents to minimize undesirable consequences, such as cell activation. The initiation or avoidance of complement activation in particular controls an important effector pathway in tissue injury and repair.

Toll-Like Receptors

The family of TLRs, identified on macrophages in mammals, is a pattern-recognition receptor that bind structurally conserved molecules derived from microorganisms, including endotoxins (LPS) and viral nucleic acids. TLRs are now considered key molecules responsible for alerting the immune system to the presence of microbial infections. For example, TLR4 is part of a recognition couple for LPS. Pathogen recognition by TLRs activates the innate immune system through the signaling pathway and provokes inflammatory responses, such as cytokine production.50 These are shown schematically in Fig. 67–10 to illustrate their diverse structures and signaling pathways. The discovery of TLR has transformed the study of innate immunity, inflammation, and adjuvant actions on APC.51–53 Receptor structures, heterogeneity of expression, microbial and endogenous ligands, and signaling have been defined, and knowledge of their regulation

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has begun to offer agents to manipulate TLR signaling in humans. The discovery of inborn errors, such as the interleukin receptor–associated kinase (IRAK)-4 deficiency,54 and the role of toll-interleukin receptor adaptor protein (TIRAP) function in Plasmodium falciparum infection,55 for example, have illustrated their role in human disease. Several concepts have emerged. From the original studies on LPS recognition and signaling by the multiprotein complex formed by CD14, LPS binding protein, and MD2, and the clarification of the distinct adaptor pathways (MyD88 [myeloid differentiation factor 88], TIRAP/MAL [MyD88 adaptor-like], TRIF [TIR domain-containing adaptor inducing interferon (IFN)-β], and TRAM [TRIF-related adaptor molecule]), the recognition and sensing of TLR ligands have become clear. The tertiary structure of TLR4 has been reported.56 TLRs are expressed either on the plasma membrane of myeloid and other cells, or within the vacuole, especially in the case of TLRs 3, 7, and 9, which are implicated in viral nucleic acid recognition. Crosstalk among nuclear factor (NF) κB, IFN, and mitogen-activated protein kinase (MAPK) kinase pathways has also become apparent.57 TLRs collaborate with other recognition receptors,58 such as dectin-1. Furthermore, a role has been proposed for TLR signaling in nontranscriptional activities, such as the kinetics of phagosome maturation in macrophages.59

Non–Toll-Like, Nonopsonic Receptors

The study of lectins and SRs has lagged behind that of the above receptors, but is gaining ground, documenting receptor expression and ligands, mainly in mouse models of inflammation and infection.35,60,61 These receptors are present on macrophages and DCs, and variably on monocytes and neutrophils. They are implicated in the recognition and uptake of microbial and host ligands, and vary in their ability to activate host defense functions. Figure 67–11 and Table 67–3 illustrate the functional attributes of these receptor systems. The mannose receptor is mainly involved in endocytosis, with a predominant intracellular localization.62,63 The multilectin mannose receptor displays dual functions, contributing to the clearance of mannoseterminal lysosomal hydrolases and of neutrophil granule glycoproteins such as MPO, as well as of hormones (e.g., thyroglobulin) and exocrine secretion products (e.g., amylase). It plays a role in the capture and transport of mannose-terminal glycoproteins to targets in spleen (marginal metallophilic macrophages) and in lymph nodes (subcapsular sinus macrophages) that express sulfated receptors for its cysteine-rich domain. The outcome of such targeting is either silent disposal or, if combined with TLR stimulation, induction of an immune response.64 In common with several other nonopsonic receptors, it can play dual, even opposing actions in host protection or in pathogenesis, as shown by ongoing studies in mice. Dectin-1 is a lectin-like receptor that is widely expressed on myeloid cells, with a single immunoreceptor tyrosine-based activation motif (ITAM)–like motif in its cytoplasmic tail.65 It recognizes β glucans, abundant in fungal walls, including bioactive zymosan particles, and has been implicated in innate resistance to fungal infection. Dectin-1 activates syk and caspase activation and recruitment domain (CARD)-9, regulating various effector pathways such as TNF-α, leukotriene production, and T-helper (Th) 17 cell activation, with heterogeneity in responses by macrophages and DCs. Dectin-1 collaborates with TLR 2/6 in the response to zymosan. Other lectins expressed by macrophages include sialic acid recognition molecules, Siglec-1 (sialoadhesin),66 an extended Ig superfamily plasma membrane protein implicated in cell– cell interactions (Chap. 68 discusses a possible role in the hematopoietic system). SRs are a diverse family of structurally unrelated, promiscuous receptors, with a predilection for polyanionic ligands, expressed by

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Figure 67–10.  The main toll-like receptor (TLR) signaling pathways and adaptor molecules. The pathways that are activated by the different receptors are multiple and complex. For example, TLR signaling involves not only nuclear factor-κB (NF-κB) activation, but also mitogen-activated protein kinases, phosphatidylinositol 3-kinase, and several other pathways that markedly affect the overall biologic response to the activation of TLRs. Dectin-1 (a β-glucan receptor) is shown as an example of various signaling-competent cell-surface pattern-recognition receptors. ASC, apoptosis-associated speck-like protein containing a caspase activation and recruitment domain; CARD, caspase activation and recruitment domain; ds, double-stranded; type I IFN, type I interferon; IFN, interferon; IκB, inhibitor of NF-κB; IL, interleukin; IPAF, interleukin-1β–converting enzyme-protease activating factor; IRF, IFN-regulatory factor; LPS, lipopolysaccharide; MDA5, melanoma differentiation-associated gene 5; MyD88, myeloid differentiation primary response gene 88; NACHT, domain present in NAIP, CIITA, HET-E, and TP-1; NALP, NACHT leucine-rich repeat and pyrin-domain-containing protein; NOD, nucleotide-binding oligomerization domain; RICK, receptor-interacting serine/threonine kinase; RIG-I, retinoic acid-inducible gene I; ss, single-stranded; TBK1, TANK-binding kinase 1; TIRAP, toll/IL-1R (TIR) domain-containing adaptor protein; TRAM, TRIF-related adaptor molecule; TRIF, TIR domain-containing adaptor protein inducing IFN-β; SYK, spleen tyrosine kinase. See text for further details. (Reproduced with permission from Trinchieri G, Sher A: Cooperation of Toll-like receptor signals in innate immune defence. Nat Rev Immunol 2007 Mar;7(3):179-190.)

diverse microorganisms, apoptotic cells, and modified host lipoproteins.34 SR-A I/II and MARCO (macrophage receptor with collagenous structure) (class A SR) are collagenous transmembrane receptors that mediate endocytosis, phagocytosis, and cell adhesion. SR-A I/II is upregulated by M-CSF and MARCO by TLR and MyD88-dependent microbial ligands, triggers of innate immune activation.67 A number of naturally occurring ligands for SR-A have been identified, including apolipoprotein A1 and Neisserial outer-surface proteins,68 as well as previously described lipid A, lipoteichoic acid, and modified (acetylated) low-density lipoproteins, among others. After initial interest primarily in its role in atherogenesis (Chap. 134), attention has also focused on innate immune functions in bacterial infection. Class B SRs, such as CD36 and SR-BI, have distinct structures and have been implicated in mycobacterial recognition as well as in the uptake and exchange of lipids.69,70 CD36, together with thrombospondin,

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plays a role in apoptotic cell uptake47 and has been implicated in macrophage fusion. Other SRs, expressed on a variety of cells as well as macrophages, have similar roles in clearance.

Human Leukocyte Antigen Class II Receptors

Monocytes and macrophages serve an important function as APCs. They bear the class II glycoproteins of the major histocompatibility gene complex, human leukocyte antigen (HLA)-DR, HLA-DP, and HLA-DQ. Expression of major histocompatibility complex (MHC) class II antigens on macrophages from different tissues varies widely. Splenic macrophages contain a high percentage of HLA-DR–positive cells (50 percent), whereas peritoneal macrophages have relatively few (10 to 20 percent).71 The proportion of Ia-positive alveolar macrophages is only approximately 5 percent.72 Lymphokines, primarily IFN-γ, can induce macrophages to express higher levels of MHC class II antigens,

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Part VIII: Monocytes and Macrophages

N E

C

E

C C

E E E

EGF-TM7 Scavenger receptor AI

N

Toll-like receptor

N N N

C C C

N N N

C

MARCO N

C

CD68

CD200R

CD36

C

S | S

S | S

S | S

S | S

C

N C

CD200 Mannose receptor

N

C S | S

C

TREM1/DAP12

Dectin1

Siglec1 N

N

Figure 67–11.  Macrophage nonopsonic and regulatory receptors. See text for further details. EGF-TM7, epidermal growth factor-seven transmembrane; MARCO, macrophage receptor with collagenous structure; Siglec, sialic acid-binding immunoglobulin-like lectin.

whereas prostaglandin E, α-fetoprotein, and glucocorticoids downregulate HLA-DR antigen expression on macrophages.

CD11

CD11 defines a family of three accessory adhesion surface glycoproteins: CD11a, CD11b, and CD11c. These proteins are distinct α subunits for three heterodimeric surface glycoproteins, each sharing a common β subunit, designated CD18. The α subunits have different isoelectric points, molecular weights, and cell distribution (Chap. 15).73 Whereas CD11a is expressed on all leukocytes, CD11b and CD11c are expressed predominantly on monocytes and macrophages, a minor subset of B lymphocytes, and most polymorphonuclear leukocytes. CD11b is expressed on more than 95 percent of fresh human monocytes and macrophages but declines rapidly on cells maintained in vitro. Antibodies specific for CD11b, such as OKM1 or Mo1, may block this CR’s ability to bind to CD3bi.74 Accordingly, these antibodies strongly inhibit CR-mediated rosetting of erythrocyte–IgM antibody–complement complexes.

CD14 and CD16

The CD14 molecule is one of the most characteristic surface antigens of the monocyte lineage. It is a polypeptide of 356 amino acids that is anchored to the plasma membrane by a phosphoinositol linkage.75 It is expressed strongly on the surface of monocytes and weakly on the surface of granulocytes and most tissue macrophages. It can be detected on some nonmyeloid cells (e.g., hepatocytes and some epithelial cells). CD14 functions as a receptor for endotoxin (LPS). LPS binds to a serum

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protein, LPS-binding protein, which facilitates the binding of LPS to CD14. The coreceptor MD2 and TLR4 also are vital in this process. When LPS binds to CD14/MD-2/TLR4 expressed by monocytes or neutrophils, the cells become activated and release cytokines such as TNF and upregulate cell surface molecules, including adhesion molecules. In vitro, soluble CD14 binds to LPS, and the complex stimulates cells that do not express CD14 to secrete cytokines and coregulate adhesion molecules.76 A subset of human blood monocytes that express low levels of CD14 molecules and high levels of the Fcγ receptor III (FcγRIII) CD16 has been identified.77,78 These CD14+CD16+ monocytes resemble alveolar but not peripheral macrophages. CD14+CD16+ monocytes represent 5 to 10 percent of blood monocytes in normal individuals and can be dramatically expanded in pathologic conditions, such as sepsis, HIV infection, and cancer. CD16+ monocytes produce high levels of proinflammatory cytokines.

CD4

T lymphocytes express several surface receptors. The surface antigen CD4 is expressed primarily in T-helper lymphocytes (Chap. 76). CD4 and its corresponding messenger RNA have been demonstrated on monocytes, macrophages, and the monocyte-like cell line U-937.79,80 Although CD4 is present at low concentrations in blood monocytes, the proportion of cells that display this plasma membrane determinant ranges from less than 5 percent to 90 percent. The CD4 molecule is involved in induction of T-lymphocyte helper functions (T4) and T proliferative responses to antigen stimulation; however, its role

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TABLE 67–3.  Ligands for Selected Nonopsonic, Non–Toll-Like Receptors Class

Receptor

Microbial Ligands

Endogenous Ligands

Function

Scavenger receptors

SR-A I/II

Gram+/– bacteria

Apoptotic cells

Phagocytosis

Lipoteichoic acid

Modified low- and high-density lipoproteins (LDL, HDL, apolipoprotein A1, apolipoprotein E)

Endocytosis

MARCO

Lipid A

AGE-modified proteins

Foam cell formation

Neisserial surface proteins

β-Amyloid

Adhesion

Gram+/– bacteria

Marginal zone B lymphocytes

Adhesion

Trehalose dimycolate

Uteroglobin-related protein

Phagocytosis

Neisserial surface proteins CD36

Innate activation

Diacylated lipopeptide from Gram+ bacteria

Apoptotic cells (with thrombospondin and vitronectin receptor)

Plasmodium falciparumparasitized erythrocytes

HDL

Uptake, exchange of lipids, adhesion

Outer rod segments Lectins

Dectin-1

β-Glucan

T lymphocytes (noncarbohydrate)

Fungal uptake and immunomodulation

DC-SIGN

Mannosyl/fucosyl glycoconjugates viruses (e.g., HIV-1, Dengue)

ICAM 2/3

Adhesion

T lymphocytes

Endocytosis

Mannose receptor

Mannosyl/fucosyl

Lysosomal hydrolases

Endocytosis

C-type lectin domains

Glycoconjugates on bacteria, viruses, fungi, parasites

Thyroglobulin

Adhesion

Cysteine-rich domain

Ribonuclease B

Antigen targeting

Fibronectin type II domain

Amylase

Adhesion

Sulfated carbohydrates in marginal zone (spleen) and subcapsular sinus (lymph node) Collagens AGE, advanced glycation end product; DC-SIGN, dendritic cell–specific intercellular adhesion molecule-3–grabbing nonintegrin; ICAM, intercellular adhesion molecule; MARCO, macrophage receptor with collagenous structure; SR, scavenger receptor. Data from Fogelman AM, Van Lenten BJ, Warden C, et al: Macrophage lipoprotein receptors. J Cell Sci (Suppl 9):135-49, 1988; Adams DO, Hamilton TA: Phagocytic cells: Cytotoxic activities of macrophages. In Inflammation: Basic Principles and Clinical Correlates 2 edition, edited by J.I. Gallin & R. Snyderman, p. 471. Raven Press, New York, NY, 1992; Werb, Z. & Goldstein, I.: Phagocytic cells: Chemotactic and effector functions of macrophages and granulocytes, 7th ed., in Basic and Clinical Immunology, edited by D. Stites & A. Terr, p. 96. Appleton and Lange, Norwalk, CT, 1991; Papadimitriou, J.M. & Ashman, R.B.: Macrophages: current views on their differentiation, structure, and function. Ultrastruct Pathol 13:343-72, 1989; Gordon, S., Perry, V.H., Rabinowitz, S., Chung, L.P. & Rosen, H.: Plasma membrane receptors of the mononuclear phagocyte system. J Cell Sci Suppl 9:1-26, 1988; Law, S.K.: C3 receptors on macrophages. J Cell Sci Suppl 9:67-97, 1988; Hume, D.A. et al.: The mononuclear phagocyte system revisited. J Leukoc Biol 72:621–7, 2002.

in the function of monocyte/macrophages has not been determined. An important aspect of the monocyte/macrophage phenotype is the presence of CD4 molecules on the surface of monocytes that can act as receptors for HIV type 1 (HIV-1). HIV-1 uses the CD4 receptors as an entry pathway for infection of monocyte/macrophages.79,80

Chemokine Receptors

Chemokines mediate their activities by binding to target cell surface chemokine receptors that belong to a large family of G-protein–coupled,

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seven transmembrane domain receptors. Human monocytes/macrophages express several chemokine receptors (Table 67–4). The chemokine receptor CCR5 has been implicated in HIV-1 infection of monocytes/macrophages.81–85 CCR5 is a major coreceptor on monocytes/macrophages for M-tropic HIV-1 infection. At least one copy of a 32-nucleotide deletion within the CCR5 gene (CCR5Δ32) has been found in approximately 4 to 16 percent of individuals, depending on their background; when in the homozygous state, individuals are highly protected against acquisition of HIV.86,87

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TABLE 67–4.  Surface Receptors of Monocytes and Macrophages Fc Receptors

Transferrin and Lactoferrin Receptors

IgG2a, IgG2b/IgG1, IgG3, IgA, IgE

Lipoprotein lipid receptors

Complement receptors

Anionic low-density lipoproteins

C3b, C3bi, C5a, C1q

PGE2, LTB4, LTC4, PAG

LPS receptors

Apolipoproteins B and E (chylomicron remnants, VLDL)

CD14 Cytokine receptors

Receptors for coagulants and anticoagulants

MIF, MAF, LIF, CF, MFF, TNF-α, IL-1, IL-2, IL-3, IL-4, IL-10, IL-18, INF-α, INF-β, INF-γ, GM-CSF, M-CSF/CSF-1

Fibrinogen/fibrin Coagulation factor VII

Chemokine receptors

α1-Antithrombin

CCR1, CCR2A, CCR2B, CCR3, CXCR4, CCR5

Heparin

Macrophage growth factor receptors

Integrins (CD11b, CD18)

M-CSF, GM-CSF

Fibronectin receptors

Receptors for peptides and small molecules

Laminin receptors

Neurokinin-1

Mannosyl, fucosyl, galactosyl residue

H1, H2,5-HT

α2-Macroglobulin-proteinase complex receptors

1,2,5-Dihydroxy vitamin D3

Toll-like receptors

N-formylated peptides

TLR2, TLR4, TLR5, TLR9

Enkephalins/endorphins

Others

Substance P

Cholinergic agonists

Hemokinin-1

α1-Adrenergic agonists

Arg-vasopressin

β2-Adrenergic agonists

Hormone receptors Insulin Glucocorticoids Angiotensin C, complement; GM, granulocyte macrophage; H1, histamine; 5-HT, 5-hydroxytryptamine; Ig, immunoglobulin; IL, interleukin; INF, interferon; LIF, leukocyte migration inhibition factor; LT, leukotriene; MAF, macrophage-activating factor; MFF, macrophage fusion factor; MIF, macrophage inhibitory factor; PAG, platelet-activating factor; PG, prostaglandin; TNF, tumor necrosis factor; VLDL, very-low-density lipoprotein. Data from Lewis C, McGee JD: The Macrophage, 2nd ed. Oxford University Press, New York, 1992; Fogelman AM, Van Lenten BJ, Warden C, et al: Macrophage lipoprotein receptors. J Cell Sci Suppl 9:135–149, 1988; Adams DO, Hamilton TA: Phagocytic cells: Cytotoxic activities of macrophages, in Inflammation: Basic Principles and Clinical Correlates, 2nd ed., edited by Gallin JI, Snyderman R, p 471. Raven Press, New York, 1992; Werb Z, Goldstein I: Phagocytic cells: Chemotactic and effector functions of macrophages and granulocytes, in Basic and Clinical Immunology, 7th ed., edited by Stites D, Terr A, p 96. Appleton and Lange, Norwalk, CT, 1991; Papadimitriou JM, Ashman RB: Macrophages: Current views on their differentiation, structure, and function. Ultrastruct Pathol 13:343–372, 1989; Gordon S, Perry VH, Rabinowitz S, et al: Plasma membrane receptors of the mononuclear phagocyte system. J Cell Sci Suppl 9:1–26, 1988; Law SK: C3 receptors on macrophages. J Cell Sci Suppl 9:67–97, 1988. Hume DA, Ross IL, Himes SR, et al: The mononuclear phagocyte system revisited. J Leukoc Biol 72:621–627, 2002.

To illustrate the dynamic interaction of macrophages and virus, a video showing an HIV-1 infected human macrophage sensing its environment was captured from a spinning disk confocal microscope using a 100× objective by Raphael Gaudin (see http://www.cellimagelibrary .org/images/41568#.VAR6eNcDfRo.email).

FUNCTION Monocytes respond to activating signals, for example, chemokines, through chemokine receptors, setting in motion a series of adhesion and migration events associated with diapedesis.88 They play a direct role in

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sepsis and in more poorly defined changes associated with intravascular coagulation and platelet activation. Their phagocytic potential is mainly expressed after adherence to the vascular endothelium. Monocytes are relatively resistant to virus infection, compared with more differentiated macrophages. These cells selectively adhere to lipid- and platelet-activated endothelium, a precursor to atherogenesis.89 Although metabolic, microbial, or environmental stimuli are normally required to induce monocyte activation, once activated monocytes express a greater potential for cytotoxicity and antimicrobial functions than resident tissue macrophages. Figure  67–11 schematically shows select surface receptors related to monocyte function. These include chemokine recognition, adhesion,

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and immunoregulatory molecules. Receptors involved in microbial recognition and innate immunity (e.g., cluster of differentiation [CD]14),90 phagocytosis (e.g., FcR, CR), secretory, and killing mechanisms are described, as are cytokine production and responses. Intracellular granule contents of monocytes include myeloperoxidase (MPO) and lysozyme, although these are less studied than in neutrophils.

MOTILITY OF MONOCYTES AND MACROPHAGES An effective monocyte response to infection is predicated upon the ability to migrate and accumulate at sites of inflammation and infection. Monocytes are capable of both random and directed movement. Random migration is nondirected movement that occurs in the absence of attracting substances. Directed movement, as a result of chemotaxis, refers to monocyte migration that occurs in response to soluble factors or stimuli and that is mediated by different types of receptors on phagocyte cell surfaces. A number of different methods have been used to study macrophage movement both in vivo91 and in vitro.92 Monocytes and macrophages are unusual among hematopoietic cells in that they are motile (ameboid type), migratory, yet capable of sessile, “fixed” life in tissues as resident and more newly recruited cells. Although not as motile as neutrophils, and more difficult to study in physiologically relevant assays in vitro, they display lineage-specific, as well as shared, yet distinct properties with DCs, which can be considered as more motile, less-adherent cells specialized for antigen capture and delivery to naïve and primed lymphocytes.93 They also share receptors and cytoskeletal properties with fibroblasts. Apart from diapedesis in response to endothelial and extravascular signals, monocytes and their progeny display polarization and specialized adhesion structures, most evident in the tight seal of osteoclasts to bone surfaces, so as to localize secretion of powerful catabolic products. Adhesion is a defining event in the differentiation of monocytes, profoundly influencing the organization of the cell, its plasma membrane, cytoplasm, and nuclear transcription machinery, as well as regulating posttranslational modification of the proteome. Monocytes express diverse integrins, implicated in outside-in as well as inside-out signaling.94 Particularly important are the β2-integrin heterodimers, restricted to myeloid cells, as opposed to β1 and β3 integrins shared with mesenchymal and other cells. The β2 integrins, lymphocyte function– associated antigen (LFA)-1 (CD11a/CD18), CR3 (CD11b/CD18), and CD11c/CD18, have been of great value in studies of monocyte/macrophage adhesion. Inhibitory and stimulatory monoclonal antibodies have been generated, and rare inborn errors of metabolism, such as the leukocyte adhesion deficiency syndrome, caused by a genetic deficiency of the common β2 chain, result in defective myeloid cell recruitment to inflammatory stimuli. The well-known sequence paradigm of rolling (mediated by L-selectin), more stable adhesion (mediated by β2 integrins), and diapedesis has been extensively studied in neutrophils (Chap. 19), and is thought to be similar for monocyte recruitment in response to chemokines, as described in Chap. 68. Monocyte-specific and constitutive migration through different tissue compartments (marrow, blood, tissues) are still poorly understood. An unresolved question is whether circulating monocytes are already “bar coded” for entry to special tissues, such as the CNS, or whether cells enter tissues stochastically from blood. The control of monocyte motility in relation to chemotaxis continues to be studied.95 In particular, the energetics and role of mitochondria in aerobic and hypoxic conditions deserve further study. Mitochondria are prominent in DCs and play a wider role than anticipated in innate

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resistance to viral infection and in cytosolic stress. Several well-known G-protein–coupled receptors (GPCRs), including the array of selective, shared, even redundant chemokine receptors, β-adrenergic receptors, and others contribute to the regulation of directed migration and other cellular functions (Table 67–5).96,97 In addition, a newly defined family of GPCR with large extracellular domains, includes myeloid-restricted members of the epidermal growth factor–seven transmembrane (EGF-TM7) subfamily with multiple EGF (epidermal growth factor) repeats. EMR2 (epidermal growth factor–like module containing mucin-like hormone receptor–like 2) and CD97, structurally related to the F4/80 antigen marker discussed in Chap. 68, likely support additional important monocyte functions.97 Their ligands include complement regulatory molecules (CD55, associated with paroxysmal nocturnal hemoglobinuria; Chap. 40) and chondroitin sulphate B, a matrix component. EMR2 expression on myeloid cells is upregulated by septic shock, its ligation on neutrophils potentiates a range of cellular responses. The roles of phosphoinositide metabolism, diacylglycerol generation, calcium fluxes, and phosphorylation/dephosphorylation in regulating actin assembly have been studied in human and mouse cells, using mainly neutrophils as a prototype.95 Genetic models of value for macrophage studies include src kinase knockout animals and the Wiskott-Aldrich syndrome. Small guanosine triphosphatases (GTPases; rac, rho, cdc42) have been implicated in diverse myeloid functions, including cell spreading and membrane ruffling. Specialized adhesion structures that deserve further study in macrophages include focal adhesion, podocyte formation (particularly prominent in osteoclasts) and possible participation in tight junctions; hemiconnexons have been reported in macrophages in marrow stroma. CR3 contributes to divalent cation-dependent adhesion of monocytes and macrophages to artificial, serum-coated substrates, such as bacteriologic plastic and the class A SR and MARCO (see “Non–Toll-Like, Nonopsonic Receptors” above), which mediate divalent cation-independent adhesion to serum-coated tissue culture plastic in vitro. However, the basis of the remarkable, even unique, protease-resistant adhesion of macrophages to foreign materials remains mysterious. Improved imaging studies, combined with genetic manipulations, will bring further insights into the regulation of monocyte/macrophage adhesion and migration in vivo.

INTERACTION WITH COAGULATION CASCADE Monocytes and resident macrophages line the sinusoids of liver (Kupffer cells) and spleen and readily recognize activated platelets, binding them for clearance and destruction. In addition, monocytes produce potent procoagulants, such as tissue factor, initiating a clotting cascade which, if dysregulated, can lead to diffuse intravascular coagulation during septic shock. Following injury and inflammation, monocytes/macrophages produce urokinase, to generate plasmin, in concert with endothelial cell-derived tissue plasminogen activator.98 Macrophage production of urokinase is regulated by phagocytic and other stimuli, and the active enzyme can bind to receptors (urokinase plasminogen activator receptor) on the cell surface in a complex interaction with protease–antiprotease complexes, thus localizing fibrinolysis, which is important in wound repair. The nature and source of the lipid tissue factor produced by monocytes is not well characterized. The cells also produce a complex mix of lipid metabolites, consisting of labile prostaglandins, leukotrienes, and thromboxanes, by utilization of arachidonate-derived precursors and substrates for phospholipase and cyclooxygenase-processing enzymes, among others.

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TABLE 67–5.  Selected G-Protein–Coupled Receptors Implicated in Functions of Monocytes and Macrophages Chemotaxis

Adhesion/Cell–Cell Contact

Activation and Resolution of Inflammation

Alternative Activation

Survival

Chemokine receptors

EGF-TM7 receptors

BAI-1

Purinergic receptors GPR86, GPR105, P2Y8, P2Y11, and P2Y12

Sphingosine-1-phosphate receptors

C5a receptor

Sphingosine-1-phosphate receptors Formyl peptide receptors

Chemokine receptors

Leukotriene B4 receptor Formyl peptide receptors Platelet-activating factor receptor

CX3CR1

Chemokine receptors C5a receptor EMR2

EMR2

Protease-activated receptors

Neuropeptide Y receptor

Platelet-activating factor receptor Leukotriene B4 receptor Neurokinin receptors Neuropeptide Y receptor Vasoactive intestine peptide receptor Prostaglandin receptors Resolvin

BAI-1, brain-specific angiogenesis inhibitor 1; EGF-TM7, epidermal growth factor–seven transmembrane; EMR2, epidermal growth factor–like module containing mucin-like hormone receptor–like 2. Data from Lattin, J.E. et al.: Expression analysis of G Protein-Coupled Receptors in mouse macrophages. Immunome Res 4:5, 2008; Yona, S., Lin, H.H., Siu, W.O., Gordon, S. & Stacey, M.: Adhesion-GPCRs: emerging roles for novel receptors. Trends Biochem Sci 33:491-500, 2008; Lattin, J. et al.: G-protein-coupled receptor expression, function, and signaling in macrophages. J Leukoc Biol 82:16–32, 2007.

RECOGNITION AND CLEARANCE OVERVIEW Resident macrophages of the liver and marrow, as well as in lung and other nonhematopoietic tissues, play a major role in the recognition, phagocytosis, and endocytosis of foreign particles and macromolecules, as well as of modified host components. Clearance can be silent, even suppressing inflammation, mediated by transforming growth factor (TGF)-β generation, as observed after the uptake of apoptotic cells by macrophages.99 Production of hematopoietic cells is balanced by their programmed senescence and increased destruction, which can be enhanced in response to microbial and other toxic substances. Macrophages initiate and perpetuate inflammation, both acute and chronic, as a result of their biosynthetic and secretory responses to injurious particles. Uptake and vacuole formation sequester the membrane-enclosed contents for digestion and possible antigen processing and presentation, a specialized property of DCs after their further differentiation from active endocytic to APCs.33 Specialized studies show that blood-derived monocytes have unique functions. For example, in the human disorder multiple sclerosis and the model experimental autoimmune encephalitis, monocyte-derived macrophages initiate demyelination at nodes of Ranvier; whereas, microglia derived from yolk-sac progenitors during embryogenesis are relatively inert at disease onset.31 To illustrate the role of macrophages in the recognition and clearance of foreign substances, images of macrophage spreading

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and engulfment of erythrocytes can be visualized by scanning electron microscopy, and the sequence of engulfment by phase-contrast optics (see video talk on macrophage phagocytosis at http://hstalks. com/?t=BL1473311). In addition, interest has grown explosively in cytosolic recognition systems, designed to protect the cell from various infectious and lytic agents.100–102 The process of autophagy shares aspects with both membrane-bound and cytoplasmic organelle injury, and has become of great current interest because of its contribution to pathogenesis of infectious, malignant, and inflammatory syndromes.103

APOPTOSIS Macrophages take up large numbers of naturally dying cells, hematopoietic and others, through a complex mechanism involving multiple, often redundant nonopsonic receptors.47,99 A possible role for complement has also been proposed. Figure 67–12 illustrates receptors and ligands that have been implicated. Apart from the SRs already discussed, they include receptors for opsonins and for milk-fat globulin, as well as for the vitronectin receptor. Phosphatidylserine (PS) expressed on the outer leaflet of apoptotic cells, contributes to apoptotic cell recognition, but its role is probably more complex as apparently healthy cells can express patches of PS on their surface and PS recognition plays a role in CD36-dependent macrophage–macrophage fusion.104 The recognition

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Figure 67–12.  Phagocytic receptors for apoptotic cell phagocytosis. Macrophages and immature myeloid dendritic cells (DCs) are the main immune cells involved in the clearance of apoptotic cells. They express broadly similar multiple receptors that can bind directly or via opsonic-soluble proteins, for example, mannose-binding lectins (MBLs) to ligands. Phosphatidylserine (PS) becomes exposed on the outer surface of the apoptotic cell and a receptor for this ligand has been long sought. A new receptor (TIM4, and related TIM1) was discovered on resident mф, with specificity for PS. Other mф populations utilize MFGE8 (a milk-fat globulin protein secreted by mф) as an opsonin. Discrimination of non-self and altered self may involve combinations of different phagocyte receptors. Apoptotic cell uptake results in an antiinflammatory response by mф (e.g., release of transforming growth factor [TGF]-β and prostaglandin E2), but has also been implicated in cross-presentation by DCs. For further details see Ref. 47. (Reproduced with permission of Savill J, Dransfield I, Gregory C, et al: A blast from the past: clearance of apoptotic cells regulates immune responses. Nat Rev Immunol 2002 Dec;2(12):965-975.)

mechanisms for uptake of necrotic cells and enucleated erythroblast nuclei by macrophages are not clear (Chap. 15).

ENDOCYTOSIS, PHAGOCYTOSIS, AND KILLING Apart from the above ligands, macrophages express receptors for endocytosis of growth factors, cytokines, peptides, and lipids. Macrophages express a functional folate receptor that is induced during activation and can be used to target drugs or tracers to macrophages in situ.105 Hemoglobin–haptoglobin complexes are internalized by CD163, a glucocorticoid-regulated receptor with a remarkable SRcysteine extracellular domain structure.106 CD163 is also upregulated by substance P.107 The cell biology of endocytosis and of phagocytosis is illustrated in Figs. 67–13 and 67–14. Apart from size and resultant involvement of the cytoskeleton, they have much in common; vesicle/phagosome formation, falling pH and initial digestion, fusion with secretory vesicles derived from the Golgi, and maturation to form secondary lysosomes/

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phagolysosomes with a more acidic pH, and further digestion.108,109 Apart from selective fusion with intracellular vesicles, there is extensive membrane flow, recycling, and fusion. Small GTPases play an important role in the control of membrane traffic.110 Early estimates revealed that a substantial fraction of surface membrane is internalized constitutively by endocytosis. Studies that used opsonic receptors to examine the uptake mechanism of antibody-coated erythrocytes via opsonic receptors gave rise to the zipper hypothesis: local segmental engagement of FcR, and circumferential flow of macrophage pseudopodia around the particle, followed by fusion at the tip, closure, and ingestion. Subsequent studies by several groups documented the role of phosphatidylinositol 3-kinase (PI3K) and phosphoinositides in the initial fusion and subsequent associations between the actin cytoskeleton and cellular membranes.111 Latex has provided a useful test particle to isolate latex-containing phagolysosomes by flotation. Proteomic analyses112 demonstrated the protein composition of phagosomes and drew attention to functional constituents in the phagolysosomal membrane.

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Phagocytosis

Antigen presentation

Endocytosis

Actin polymerization Pseudopod formation/ membrane invagination Membrane recruitment Membrane closure

Early endosome

Rab4, Rab5, other fusion proteins Partial degradation

Phagosome maturation by interaction with endocytic pathway Late endosome Rab7, Rab9 Cathepsin

Phagolysosome

Lysosome

LAMP1 LAMP2 LAMP3

Figure 67–13.  Phagocytosis and endocytosis pathways. Particulates are taken up by actin-dependent sequential maturation processes, involving

membrane fusion and fission, which intersect with the endocytic pathway at several stages. Cytosolic small guanosine triphosphatases (rabs) determine organelle-specific interactions. Membrane is recycled to the plasma membrane, with processed antigen. Progressive acidification and delivery of lysosomal hydrolases result in terminal degradation. Compartment membranes express marker proteins such as lysosomal-associated membrane protein (LAMP)-1; the pan-macrophage CD68 antigen is associated with late endosomes and lysosomes.

These observations have provided the basis for numerous investigations regarding the interactions of diverse microorganisms with the vacuolar system, which are often necessary for pathogen survival and establishment of intracellular infection (Fig. 67–15). Organisms can inhibit acidification and fusion (Mycobacterium),101,113 multiply within secondary lysosomes (Leishmania),114 escape free into the cytosol (Listeria),115 or translocate their genomes into the cytoplasm by fusion (enveloped viruses); other organisms induce variations on this theme; for example, Brucella seeks out the endoplasmic reticulum after entry and Legionella can enter macrophages by inducing a phagosome membrane of unusual composition.116 Nonpathogenic organisms or pathogens taken up via opsonic receptors or after IFN-γ activation undergo a different fate, with killing and destruction. The zipper mechanism, with tight apposition of membrane to the particle’s surface ligands, does not apply to all forms of ingestion. For example, complement opsonized particles seem to sink into the cytoplasm, and other phagosomes can be spacious. A number of key methods of visualization109 illustrate the dynamic nature of phagocytosis. Figure  67–14 illustrates some of the signaling pathways that control the cytoskeleton. Macrophages are rich in lysosomal digestive enzymes,33 activated by a falling pH of approximately 6.5 within the mature vacuole. Unless captured as peptides by MHC molecules, a feature of antigen processing by DCs, macromolecular substrates can be degraded to their constituent amino acids, sugars, or nucleic acid bases. Early studies117 probed the permeability of the lysosomal vacuolar membrane. If the

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content cannot be fully degraded because of its nature (e.g., sucrose), overload (e.g., lipid), or owing to a genetic deficiency in a catabolic enzyme (lysosomal storage diseases), it accumulates within residual lysosomes, altering macrophage gene expression and secretory output, thus mediating chronic inflammation or metabolic forms of modified inflammation, such as atherosclerosis, foam cell formation and Gaucher disease. Figure 67–16A illustrates the uptake of senescent erythrocytes, the breakdown of heme and storage of Fe2+.118 Figure 67–16B shows how phagocytosis by DCs can bring about processing and cross-presentation of exogenous antigens.119 By comparison (Fig. 67–16C), autophagy is the envelopment of damaged intracellular organelles and cytoplasm by cytoplasmic membrane, and sequestration within a digestive vacuole, resembling heterophagy (Chap. 15).116 Its biochemical and cellular basis has become of interest because of its apparent relevance to cancer, infections such as tuberculosis and Legionnaire disease, and inflammatory syndromes such as inflammatory bowel disease (IBD). Although the phagocytic mechanism has been investigated in depth, we do not understand fully how the process of internalization is controlled. For example, ingestion can be thwarted by attempts to ingest too large a particle or foreign surface, or by close apposition of plasma membrane to noninternalizable immune complexes. This results in redirecting secretory vesicles to the surface, reminiscent of osteoclast adhesion. In other circumstances, as in response to foreign bodies, and especially mycobacteria, and in the presence of

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Figure 67–14.  A model for FcγR-mediated phagocytosis. A. Signaling upstream and downstream of Rho guanosine triphosphatases during FcγR-mediated phagocytosis. Immunoglobulin (Ig) G bound to antigen on the particle binds to FcγRI receptors at the surface of the mф and induces their aggregation (shown in red). This activates a Src family tyrosine kinase (probably Lyn). Lyn phosphorylates the receptor γ chain (phosphotyrosine residues in the γ chains are depicted as red diamonds) and Syk. Syk is activated and recruited to the phosphotyrosine residues of the γ chain through its two SH2 (Src homology 2) domains. Cdc42 activation by an unknown guanine–nucleotide exchange factor (GEF) allows the recruitment of WASP (Wiskott-Aldrich syndrome protein). In turn, WASP activates the Arp2/3 complex that triggers actin polymerization to generate the protrusive force for pseudopod extension (red arrowheads). Activation of a Rac1 GEF, possibly Vav, by tyrosine phosphorylation in conjunction with PI3 kinase products (PIP3) promotes GDP/GTP (guanosine diphosphate/guanosine triphosphate) exchange on Rac1. GTP-bound Rac1 interacts with and activates the serine/threonine kinase Pak1, which may induce the actinomyosin contractility involved in phagosome closure. B. In the next step, FcγRI is rapidly down-modulated and returned to an inactive state (shown in blue), resulting in actin filament disassembly. According to this model, actin assembly proceeds as a wave at the distal rim of the pseudopodia, while actin depolymerization occurs rearward. Polyphosphoinositide phosphatases such as the SH2 domain-containing SHIP, which selectively hydrolyze PIP3, may contribute to down-modulation. Modulation of FcγRI activation may also involve tyrosine phosphatases such as SHP-1, which associates with FcγRIIb, a member of the FcγR family that may be coligated with FcγRI. In addition, PEST family phosphotyrosine phosphatases (PTPases) may contribute to dephosphorylation by interacting with PSPIP, a cytoskeletal protein that interacts with WASP. GAPs may also contribute to down-modulation by returning Cdc42/Rac1 to the inactive, GDP-bound state. Eventually, cytoskeletal proteins are shed from the ingestion site to leave the phagosome free in the cytosol (not shown here). (Reproduced with permission from Chimini G, Chavrier P: Function of Rho family proteins in actin dynamics during phagocytosis and engulfment. Nat Cell Biol 2000 Oct;2(10):E191-E196.)

the Th2 cytokines IL-4 and/or IL-13, individual macrophages can fuse to form giant cells, with a common cytoplasm and multinucleation. Several fusogenic surface molecules have been identified and DNAX-activating protein (DAP) 12 expression and signaling is important in generating a fusogenic differentiation phenotype in macrophages.120

INFLAMMASOME The recognition of the multiprotein inflammasome complex101 has stimulated intense interest in the recognition by cytosolic proteins of foreign nucleic acid, uric acid-induced injury, and breakdown products of microbial walls, for example, muramyl dipeptide. More complex peptidoglycan structures can also be recognized

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by surface receptors in Drosophila. Several reviews chart the rapid growth in our knowledge of inflammasome function in health and disease.100,102,121,122 Figure 67–17 illustrates selected nucleotide-binding oligomerization domain (NOD)-like and related receptors (NLRs) with nucleotide oligomerization and other characteristic domains. Mutations in NLR have been implicated in IBD, in periodic familial Mediterranean fever, and in a range of autohyperinflammatory syndromes.123 More specifically, NOD-2 has been implicated in Crohn disease.124,125 Excessive caspase activation and IL-1β release can be countered therapeutically with IL-1 receptor antagonists. Figure 67–18 illustrates the role of inflammasome activation in intracellular infection. Antiviral production of IFN-α and -β involves retinoidinducible gene (RIG)-I–like helicases, indicating a role for mitochondria in cytosolic sensing.

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Lysosomes

EEA1 Rab5

Rab5 Rab7

Mycobacterium

Rab7 Lamps

Listeria

Legionella

Francisella

Bacteria

Influenza

Leishmania

Candida

Virus

Parasites

Fungi

Figure 67–15.  Selected pathogens evade distinct phagocytic mechanisms. Pathogens have developed several mechanisms to enter and survive inside macrophages. Legionella pneumophila resides and multiplies in a vacuole studded with ribosomes as a result of interaction with the rough endoplasmic reticulum. The organism secretes effector molecules via its type IV secretion system into the cell, which inhibit phagosome/lysosome fusion. The Francisella tularensis phagosome acquires the early endosome markers EEA1 and Rab5 and then matures into a late endosome defined by the presence of the markers Lamp1, Lamp2, and Rab7. The late endosome does not acidify and the phagosomal membrane is disrupted, releasing the bacteria into the cytosol. The Mycobacterium tuberculosis phagosome acquires the early endosome marker Rab5 but excludes the late endosomal Lamps and Rab7. This organism also produces molecules that block fusion with the lysosome and resides and replicates in this early endosome. Acidification of the Listeria monocytogenes phagosome is essential for the perforation of the phagosomal membrane and escape of the bacteria into the cytosol. Here they mobilize the actin polymerization machinery to move within the cell and then from cell to cell. Candida albicans undergoes a conversion from a unicellular form to a multicellular hyphal form, which allows this fungus to escape the macrophage. The Leishmania mexicana phagosome develops into an acidic phagolysosome containing Rab7 where the parasite is able to survive and replicate. Viruses such as the influenza virus are able to inhibit the activation of antiviral mechanisms, such as the activation of IFN regulatory function proteins that induce IFN production upon viral infection, and enter the nucleus. Cytomegalovirus (not shown) incapacitates a range of major histocompatibility complex-antigen presenting pathways. (Used with permission of S. Seif, GraphisMedica, 2014.)

GENE EXPRESSION, SYNTHESIS, AND SECRETION The development of microarray technology has had a dramatic impact on the analysis of macrophage gene expression in response to a wide range of stimuli, including microbial ligands, cytokines, and immunomodulators. Macrophages are able to express a large number of genes and are extremely versatile in their responses to environmental cues. It has been possible to discern signatures of particular agonists, for example, IFN-α and -β and IL-4, but many caveats remain in the interpretation of such data. Heterogeneity of cellular origin, differentiation stage, and populations from diverse origins, as well as substantial species differences, make it difficult to compare results within and among experiments. Validation of more quantitative messenger RNA analysis of protein synthesis and modification is difficult, although proteomic analysis is gaining ground. The study of macrophage chromatin organization in relation to gene expression is in its infancy. There is extensive crosstalk between the secretory and endocytic pathways.126 Table 67–6 is a selected list of secretory products. This includes lysozyme, a major myelomonocytic product that is

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constitutively expressed in vitro, but upregulated in granulomata in vivo. The secretion pathway of lysozyme in monocytes and macrophages has not been defined. The well-known pro- and antiinflammatory cytokines are better characterized, both in terms of regulation and the secretion pathway.109 The response to IL-6 and TNF-α secretion in model systems shows a more complex pathway than previously recognized.127,128 In addition to these and other important growth and differentiation factors that regulate angiogenesis, for example, macrophages are able to produce and secrete enzymes and proenzymes for a range of activities, as well as their inhibitors, for example, proteinases and antiproteinases. Although the amounts of complement proteins produced, for example, are relatively small, they can be significantly concentrated in a local microenvironment. In addition, macrophages can produce a range of antimicrobial peptides and lytic agents, but their most important killing mechanisms depend on oxygen129 and nitrogen metabolites,109,114 which are illustrated in Figs. 67–19 and 67–20. Regulation of the nicotinamide adenine dinucleotide phosphate oxidase and of inducible nitric oxide synthase has been studied extensively in mice and humans through biochemical and genetic approaches. Apart from their antimicrobial activity, nitrogen metabolites contribute to signaling pathways.130 IFN-α and -β play

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Nutrient depletion Erythrocyte

Proteasome

Induction of autophagy

Heme Fe2+ HO HO Ferritin

SEC61

H

H

RAB4/ RAB11

Autophagosome

cle

y ec

R

Fusion with lysosome

TAP Fe2+

MHC class I

A

Degradation

MHC class II

B

C

Figure 67–16.  A. Macrophages have an important role in iron metabolism by processing effete erythrocytes, internalized by phagocytosis, and returning iron to the blood (through ferritin) for reuse. Dissociation of iron linked to heme on erythrocytes requires the action of heme oxygenase (HO), an enzyme present in the endoplasmic reticulum (ER). The process allowing the transfer of heme oxygenase from the ER to the phagosome lumen is so far unknown. B. Presentation of antigens from intracellular pathogens is mainly carried out by major histocompatibility complex (MHC) class II molecules loaded in phagosomes. Presentation of some pathogen antigens could also involve MHC class I molecules. Current models indicate that antigens generated by hydrolases in the phagosome lumen could use SEC61 for translocation to the cytoplasm. After processing by the proteasome, antigens could be translocated to the phagosome lumen through the transporter for antigen processing (TAP) complex where loading onto MHC class I or MHC class II molecules would occur. Transport to the cell surface from the phagosome lumen could take place by using the existing membrane recycling machinery, involving the small guanosine triphosphatases Rab4 and Rab11. C. Autophagy is a conserved membrane traffic pathway that equips eukaryotic cells to capture cytoplasmic components within a double-membrane vacuole, or autophagosome, for delivery to lysosomes. Although best known as a mechanism to survive starvation, autophagy is now recognized as a mechanism to combat infection by a variety of intracellular microbes.

IPAF inflammasome

NALP1 inflammasome

NALP3 inflammasome

NALP1 Ligand-sensing domains

NALP3

IPAF

Oligomerization domain

CARDINAL

Caspase effector domains

ASC

ASC

Interaction domains

CASP1

CASP1

LRR repeats

PYD domain

NACHT domain

CARD domain

CASP5

CASP1

Caspase domain

CASP1

FIIND

Figure 67–17.  Nucleotide-binding and oligomerization domain (NOD)–leucine-rich repeat (LRR) and inflammasome structures. NOD-like receptors (NLRs) have three structural domains: The LRR domain at the C-terminus, the NACHT (domain present in NAIP, CIITA, AHD, HET-E, TP-1) domain, and the N-terminal domain that can be a pyrin domain (PYD), a caspase activation and recruitment domain (CARD), or a baculovirus inhibitor-of-apoptosis protein repeat domain (BIR). The LRR domain is considered as the ligand-sensing motif, thus involved in the interaction with pathogen-associated molecular patterns (PAMPs), in analogy to toll-like receptors (TLRs). The NACHT domain is responsible for the oligomerization and activation of NLRs. The PYD or CARD domain of NLR is the link to downstream adaptors (such as apoptosis-associated speck-like protein containing a CARD [ASC]) or effectors (such as caspase-1). The BIR domain is proposed to act as caspase inhibitor. During NACHT LRR protein (NALP) and NALP1 inflammasome activation, NALP3 or NALP1 interact through PYD–PYD homotypic interactions with ASC, resulting in its activation. Subsequently, the CARD domain of ASC interacts with the CARD domain of caspase-1 and mediates its activation. NALP1 may also activate directly the caspase-5 through its C-terminal CARD domain. In contrast, NALP3 does not simultaneously activate caspase-5, but NALP3 can recruit a second capsase-1 through the CARD domain of CARD inhibitor of nuclear factor-κB–activating ligand (CARDINAL), a component of the NALP3 inflammasome. Interleukin-1β–converting enzyme (ICE)-protease activating factor (IPAF), that can on its own sense PAMPs, possesses a CARD domain at the N-terminal and thus may directly activate caspase-1 without ASC recruitment (“IPAF inflammasome”). (Reproduced with permission of Sidiropoulos PI, Goulielmos G, Voloudakis GK, et al: Inflammasomes and rheumatic diseases: evolving concepts. Ann Rheum Dis 2008 Oct;67(10):1382-1389.)

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Figure 67–18.  Knockout studies show that IPAF (interleukin-1β–converting enzyme-protease activating factor) is essential for the activation of caspase-1 by Salmonella typhimurium, Shigella flexneri, and Legionella pneumophila in order to induce the release of interleukin (IL)-1β, IL-18, and macrophage cell death. Sensing intracellular S. typhimurium seems to be mediated by the detection of monomeric flagellin that is secreted by the bacterial type III secretion system (and is dependent on the protein SipB from S. typhimurium) by IPAF. The type III secretion system protein IpaB is involved in sensing S. flexneri. Sensing intracellular L. pneumophila seems to be mediated by the detection of monomeric flagellin that is secreted by the type IV secretion system by NAIP5 (neuronal apoptosis inhibitor protein 5), which, in conjunction with IPAF, induces caspase-1 activation and restricts the growth of these pathogens in macrophages. Although a specific NLR (nucleotide-binding oligomerization domain-like receptor) protein that detects cytosolic Francisella tularensis has not yet been identified, the adaptor molecule ASC (apoptosis-associated speck-like protein containing a CARD) seems to be essential for counteracting infections with F. tularensis. CARD, caspase activation and recruitment domain; LRR, leucine-rich repeat; NACHT, domain present in NAIP, CIITA, HET-E, and TP-1; PYD, pyrin domain. (Reproduced with permission of Mariathasan S, Monack DM: Inflammasome adaptors and sensors: intracellular regulators of infection and inflammation. Nat Rev Immunol 2007 Jan;7(1):31-40.) an important role in macrophage antiviral activities131 and perhaps in the cellular response to bacteria.132 These cytokines also contribute significantly to immune and inflammatory pathways, as well as cancer immunoediting133 and autoimmunity.134 Macrophages may be able to produce IFN-γ, for example, under particular circumstances, but in vivo most of the cytokine derives from other sources. IFN-γ has a major impact on macrophage function (the initial name of IFN-γ was macrophage activating factor), including priming of biosynthetic and functional responses associated with cytotoxicity and inflammation in cell-mediated immunity (Fig. 67–21).135 Table 67–7 summarizes the markers and functions associated with various forms of macrophage activation and deactivation, as described in Chap. 68.136 Intracellular GTPases have been implicated in cell activation by IFN-γ, for example, and in relation to IBD.121,124,125 Similarly, the Th2 cytokines IL-4 and IL-13 induce characteristic changes in macrophage phenotype, which are associated with an alternative activation pathway. The cellular biology of alternatively activated macrophages is modified

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extensively (Fig. 67–22).137 Macrophages also express a range of inhibitory proteins, such as members of the suppressor of cytokine signaling family, that suppress cytokine production, in addition to IL-10138 and TGF-β. Lipid metabolites, mainly derived from arachidonate and other lipid precursors, provide another potent source of inflammatory and immunomodulatory products.139 The suppressive functions of monocytes and macrophages in chronic infections and experimental tumors require further study, including the development of new phenotypic markers in mice and humans.

CELLULAR INTERACTIONS In addition to cytokine and other soluble afferent and efferent responses, macrophages are able to directly interact among themselves, with all other cell types in the body, both viable and injured, as well as with all kinds of microorganisms. Their interactions are reciprocal and regulated, contributing to homeostasis and to pathogenesis, both

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TABLE 67–6.  Selected Secretion Products of Macrophages Proteins

Product

Comment

Enzymes

Lysozyme

Bulk product

Urokinase-type plasminogen activator

Regulated by inflammation

Collagenase

Regulated by inflammation

Elastase

Regulated by inflammation

Metalloproteinases

Also inhibitors

Complement

All components and regulators

Arginase

Alternative activation

Angiotensin-converting enzyme

Induced glucocorticoids, granulomas

Chitotriosidase

Gaucher disease, lysosomal storage

Acid hydrolases

All classes (mainly intracellular)

Inhibitors

TIMP Chemokines

Many C-C, C-X-C, CX3C; e.g., MCP, RANTES, IL-8

Initiates acute and chronic recruitment of myeloid and lymphoid cells

Cytokines

IL-1β, TNF-α

Pro- and antiinflammatory

IL-6, IL-10, IL-12, IL-17, IL-18, IL-23

Also antagonists, e.g., IL-1Ra

Type I IFN

Autocrine and paracrine amplification

Apolipoproteins

Apolipoprotein E

Local source, marrow origin after adoptive transfer

Growth/differentiation factors

TGF-β

Also other family members (activins), myeloid growth and differentiation

M-CSF GM-CSF FGF

Fibrosis

PDGF

Repair

VEGF

Angiogenesis

Opsonins

Fibronectin, pentraxin (PTX3)

Also uncharacterized receptor on Mф

Soluble receptors

Mannose receptor

Soluble mannose receptor

Cationic peptides

Defensins

Subpopulations and species variation

Lipids

Procoagulant

Initiation clotting

Arachidonate metabolites:

Pro- and antiinflammatory mediators

Prostaglandins Leukotrienes Thromboxanes Resolvins Metabolites

Reactive oxygen intermediates Reactive nitrogen intermediates Haem breakdown (bile pigments) Iron, B12-binding protein Vitamin D metabolites

FGF, fibroblast growth factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; IFN, interferon; IL, interleukin; MCP, monocyte chemotactic protein; M-CSF, macrophage colony-stimulating factor; PDGF, platelet-derived growth factor; RANTES, regulated on activation, normal T-cell expressed, presumed secreted; TGF, transforming growth factor; TIMP, tissue inhibitor of metalloproteinase; TNF, tumor necrosis factor; VEGF, vascular endothelial growth factor. Reproduced with permission from Firestein GS and Kelley WN: Kelley's Textbook of Rheumatology, 8th edition. Philadelphia, PA: Saunders/ Elsevier; 2008.

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NADPH oxidase complex

Bacterium 2O2 p22

p47 p40

+

NADPH 2

–.

Cl–

O2

2O 2

H2O2

gp91 p67

2e–

Glucose + NADP+ G6PD

HOCl +

MPO H

NADPH + O2 Cytochrome b558

Rac

2H+

Pentose-P + NADPH NADP+ + O2

–

2O 2 + 2H+ +

NADPH + 2H

Superoxide dismutase

Proton channel

+

– 2O2 + H2O2

A

H2O2 + Cl– Myeloperoxidase OCL– + H2O NADPH oxidase complex

Bacterium

p22

p47

+ NADPH 2

2O–2 + 2H+

2O2

p40

Superoxide dismutase –.

2O 2

2OH

–

pH

2H2O2 Catalase

gp91 p67

2e–

Rac

Hypertonic

+

NADP + 2H

+

pH

K+

H2O2 + 1O2– .OH + OH– + 1O2

OCl– + H2O 1O

2

+ Cl– + H2O

H2O2 + 1O2 H2O + O2

C

Potassium channel

B

Figure 67–19.  The respiratory burst in a phagocyte is triggered when a bacterium is phagocytosed. During the phagocytosis of bacteria by macrophages and neutrophils, the phagosome membrane pinches off and the microbe is endocytosed along with a small volume of extracellular fluid. The mechanisms discussed here are based on studies in neutrophils and are still controversial.112 Electrons are removed from nicotinamide adenine dinucleotide phosphate (NADPH) in the cytoplasm and transferred through the gp91phox component (which includes flavin adenine dinucleotide and two hemes) across the membrane, where they reduce extracellular (or intraphagosomal) O2 to O2–. Protons left behind in the cell are extruded through voltage-gated proton channels (red). Some of the reactive oxygen species (ROS) derived from O2– are indicated. Spontaneous or superoxide dismutase–catalyzed disproportionation of O2– produces hydrogen peroxide (H2O2), which may be converted to HOCl (hypochlorous acid, or household bleach) by myeloperoxidase (MPO). A. Traditional view of the respiratory burst with charge compensation by proton channels. A perfect match of one proton per electron results in no change in membrane potential, intracellular pH (pHi), or external pH (pHo) and little change in ionic strength. Because proton channels are separate molecules and for the most part operate independently of NADPH oxidase, perfect 1:1 stoichiometry is not obligatory. The large depolarization that occurs during the respiratory burst in intact neutrophils and eosinophils is likely the most important factor that causes proton channels to open, although both pHi and pHo tend to change in a direction that causes proton channels to open. That depolarization occurs demonstrates unequivocally that proton efflux initially lags behind electron efflux. B. If any fraction of the total charge compensation were mediated by K+ efflux, pHi would fall, pHo (or phagosomal pH) would increase, and the osmolality of the phagosomal contents would increase. In this model, the elevated pH and osmolality of the phagosomal contents are crucial to activating proteolytic enzymes that actually kill bacteria, as opposed to ROS, which are said to be inert. C. Respiratory burst reactions. During phagocytosis glucose is metabolized via the pentose monophosphate shunt and NADPH is formed. Cytochrome b588, which was part of the specific granule, combines with the plasma membrane NADPH oxidase and activates it. The activated NADPH oxidase uses oxygen to oxidize the NADPH. The result is the production of superoxide anion. Some of the superoxide anion is converted to H2O2 and singlet oxygen by superoxide dismutase. In addition, superoxide anion can react with H2O2 resulting in the formation of hydroxyl radical and more singlet oxygen. The result of all of these reactions is the production of the toxic oxygen compounds superoxide anion (O2–), H2O2, singlet oxygen (1O2) and hydroxyl radical (OH•). As the azurophilic granules fuse with the phagosome, myeloperoxidase is released into the phagolysosome. Myeloperoxidase uses H2O2 and halide ions (usually Cl–) to produce hypochlorite, a highly toxic substance. Some of the hypochlorite can spontaneously break down to yield singlet oxygen. The result of these reactions is the production of toxic hypochlorite (Ocl–) and singlet oxygen (1O2). (A and B, modified with permission from Decoursey TE: Voltage-gated proton channels and other proton transfer pathways, Physiol Rev 2003 Apr;83(2):475-579.)

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Figure 67–20.  The role of nitrogen metabolism in mф func-

IFN-γ TNF IL-1 Nitric oxide L-OH

arginine Citruline

NOS2

NOS2 –

L-Arginine

Ornithine aminodecarboxylase

Arginase

Polyamines

tion. Interferon-γ (IFN-γ) enhances the activity of nitric oxide synthase 2 (NOS2) to generate nitric oxide, and inhibits arginase. Interleukin (IL)-4 and IL-13 promote arginase-dependent formation of L-ornithine and, ultimately, fibroblast proliferation and collagen production. GM-CSF, granulocyte-macrophage colony-stimulating factor; TNF, tumor necrosis factor. (Adapted with permission from Hesse M1, Modolell M, La Flamme AC, et al: Differential regulation of nitric oxide synthase-2 and arginase-1 by type 1/type 2 cytokines in vivo: granulomatous pathology is shaped by the pattern of L-arginine metabolism. J Immunol 2001 Dec 1;167(11):6533-6544.)

L-Ornithine

Proline Ornithine aminotransferase Urea IL-4/IL-13 IL-10 GM-CSF

Cell proliferation Collagen production

Cytokine receptor

Figure 67–21.  Signaling pathways induced by type I and type II interferon (IFN). The type I IFNs (IFN-α and IFN-β) bind a receptor that consists of

the subunits IFN-α receptor (IFN-αR)-1 and IFN-αR2, which are constitutively associated with tyrosine kinase 2 (TYK2) and Janus kinase (JAK) 1, respectively. Type I IFN-induced JAK-STAT (signal transducer and activator of transcription) signaling is propagated similarly to IFN-γ–induced JAK-STAT signaling (below). Activated TYK2 and JAK1 phosphorylate STAT1 or STAT2. Type I IFN-induced signaling then induces homodimerization of STAT1 and heterodimerization of STAT1 and STAT2. STAT1 and STAT2 associate with the cytosolic transcription factor IFN-regulatory factor 9 (IRF9), forming a trimeric complex known as IFN-stimulated gene factor 3 (ISGF3). On entering the nucleus, ISGF3 binds IFN-stimulated response elements (ISREs). Studies of gene-targeted mice have shown that JAK1, STAT1, STAT2, and IRF9 are required for signaling through the type I IFN receptor. TYK2 is required for optimal type I IFN-induced signaling. IFN-γ signaling: IFN-γ induces reorganization of the IFN-γR subunits, IFN-γR1 and IFN-γR2, activating the Janus kinases JAK1 and JAK2, which are constitutively associated with each subunit, respectively. The JAKs phosphorylate a crucial tyrosine residue of IFNγR1, forming a STAT1-binding site; they then tyrosine phosphorylate receptor-bound STAT1, which homodimerizes through Src homology 2 (SH2) domain–phosphotyrosine interactions and is fully activated by serine phosphorylation. STAT1 homodimers enter the nucleus and bind promoters at IFN-γ–activated sites (GASs) and induce gene transcription in conjunction with coactivators, such as CBP (cyclic adenosine monophosphate-responsive–element-binding protein [CREB]), p300, and minichromosome maintenance-deficient 5 (MCM5). IFN-γ–mediated signaling is controlled by several mechanisms: by dephosphorylation of IFN-γR1, JAK1, and STAT1 (mediated by SH2 domain-containing protein tyrosine phosphatase 2 [SHP2]); by inhibition of the JAKs (mediated by suppressor of cytokine signaling 1 [SOCS1]); by proteasomal degradation of the JAKs; and by inhibition of STAT1 (mediated by protein inhibitor of activated STAT1 [PIAS1]). (Reproduced with permission from Platanias LC: Mechanisms of type-I- and type-II-interferonmediated signalling. Nat Rev Immunol 2005 May;5(5):375-386.)

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TABLE 67–7.  Immunomodulation of Macrophage Phenotype Stimulus

Category

Markers

Function

Microbial (bacterial)

Innate activation

Induction of MARCO

Enhanced phagocytosis

Costimulatory molecules

Antigen presentation

CD200

Inhibition (CD200R)

Induction MHC II

Cell-mediated immunity/delayed-type hypersensitivity

IFN-γ

Classical activation

Potentiation innate markers - TNF-α

Proinflammatory

- iNOS induction

Antimicrobial (NO) signaling

- NADPH, respiratory burst

Host defense, inflammation

LGP47 induction

Association with phagosome/intracellular pathogen killing

Downregulation of MR

Unknown

Modulation of FcR expression IL-4/IL-13

Alternative activation

Proteasomal composition

Antigen presentation

Enhanced MR

Endocytosis

Induction arginase

Humoral immunity

Induction YM1, FIZZ1 (mouse)

Th2-responses, allergy, antiparasitic

Induction CCL17 (MDC) and CCL22 (TARC)

Immunity, repair/fibrosis

Fusion, giant cell formation Upregulation

CD23 (FcRε)

Immune complexes

Modified activation

Selective IL-12 downregulation, IL-10 induction

IL-10

Deactivation

Downregulation MHC II

TGF-β

Deactivation

Downregulation of proinflammatory NO and ROI

Glucocorticoids

Deactivation

CD163 induction, monocyte recruitment downregulated, ACE induction, Stabilin induction

Antiinflammatory Homeostatic clearance of hemoglobin/ haptoglobin complexes

IFN, interferon; IL, interleukin; iNOS, inducible nitric oxide synthase; MARCO, macrophage receptor with collagenous structure; MDC, macrophage-derived chemokine; MHC, major histocompatibility complex; MR, mannose receptor; NADPH, nicotinamide adenine dinucleotide phosphate; NO, nitric oxide; ROI, reactive oxygen intermediate; TARC, thymus and activation-regulated chemokine; TGF, transforming growth factor; TNF, tumor necrosis factor.

Activated Macrophage “Ruffles” Cell membrane

Lysosomes

Mitochondrion Nucleus

Figure 67–22.  Schematic cross-section of “activated” macrophage, showing ruffling of cell membrane and cellular organelles (also see Fig. 67–15). (Used with permission of S. Seif, GraphisMedica, 2014.)

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Glut-1 CXCR4

Mitogens • IL-6 • HGF • PDGF •FGF2 • VEGF • MIF

Invasion and metastasis • TF • MMP-7 • uPA/R • MIF

FIFs-1 HIFs-2

Immunosuppression • IL-10 • PGE2 phagocytosis, Ag presentation (via inhibition of CD80)

Tie-2

Angiogenesis • VEGF • FGFS 1 & 2 • PDGF • IL-8 • TF • COX-2 • MMP-7 • Pleiotrophin • Angiotrophin-1 • Leptin • Fibronectin • Magic roundabout

acutely and following persistent injury, to chronic inflammation. Storage of poorly degraded materials in lysosomes, for example, results in sustained production of degradation products, whereas massive, acute responses have a profound impact on the systemic circulation, endocrine and nervous systems, and on metabolic pathways. Short-range interactions include giant cell formation during granulomatous inflammation, and also contact-dependent immunoregulation by surface molecules such as CD200/CD200R and SIRPα/CD47.140 Matrix and other surface interactions regulate the induction or suppression of adaptive immune responses, as well as of other functions. The availability of oxygen plays an important role in macrophage interactions with a range of other cells, both normally and in a range of pathologies inducing inflammation, repair, and malignancy (Fig. 67–23).

RELEVANCE TO HEMATOPOIETIC FUNCTIONS AND DISORDERS In addition to their essential role in host defense (innate and acquired immunity), inflammation, and repair, macrophages contribute to hematopoiesis, as well as to the turnover of hematopoietic cells and their products. Macrophages can be induced to take up folate, sense and respond to oxygen levels, and promote vascular growth, regulating the integrity of the hematopoietic microenvironment. However, they also play a central effector role in pathogenesis. Their surface expression and secretion of TNF-α, other proinflammatory cytokines, enzymes, and metabolites contribute to vascular injury and increased permeability of the microvasculature, as well as to local and systemic catabolic effects associated with chronic inflammation. In this regard, anti–TNF-α therapy is of considerable value in selected inflammatory conditions and has been extended to the treatment of cancer and rheumatologic conditions.141–144 Stromal and other resident macrophage populations provide a niche for acute and persistent infections in marrow and elsewhere, and these macrophages also contribute to trophic support of hematopoietic malignancies, such as multiple myeloma. The macrophage, therefore, provides an important target cell for selective therapeutic intervention, without undue enhancement of vulnerability to infection. Additional molecular targets are needed, based on more detailed

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Figure 67–23.  Hypoxia induces marked changes in the phenotype of macrophages. Macrophages upregulate hypoxia-inducible transcription factor (HIF)-1 and HIF-2 in hypoxia, which translocate to the nucleus to induce the expression of a wide array of target genes. Several important cell-surface receptors are upregulated in hypoxia, including the glucose receptor GLUT-1 (for increased glucose uptake as the cell switches to anaerobic glycolysis to make ATP in the absence of oxygen), the chemokine stromal cell-derived factor-1 (SDF-1) receptor CXCR4, and the angiopoietin receptor Tie-2. Hypoxia also stimulates the expression of a wide array of other protumor cytokines, enzymes, and receptors, grouped here according to their known function in tumors. Downregulation of a factor or tumor-associated macrophage function is indicated by an arrow. Ag, antigen; COX, cyclooxygenase; FGF, fibroblast growth factor; HGF, hepatocyte growth factor; MIF, macrophage migration inhibitory factor; MMP, matrix metalloproteinase; PDGF, platelet-derived growth factor; PGE2, prostaglandin E2; TF, tissue factor; uPA/R, urokinase-type plasminogen activator receptor; VEGF, vascular endothelial growth factor. (Modified with permission from Lewis CE, Hughes R: Inflammation and breast cancer. Microenvironmental factors regulating macrophage function in breast tumours: hypoxia and angiopoietin-2. Breast Cancer Res 2007;9(3):209.)

analysis of macrophage functions within their native hematopoietic tissue environment. A deeper understanding of macrophage physiologic functions and of their role in a broad range of diseases should lead to the development of fresh insights into the pathogenesis and management of hematologic disorders.

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CHAPTER 68

PRODUCTION, DISTRIBUTION, AND ACTIVATION OF MONOCYTES AND MACROPHAGES

Steven D. Douglas and Anne G. Douglas

SUMMARY Monocytes and macrophages play an important role in human biology, both as a component of the hematopoietic system and within the stroma and tissue microenvironment where they contribute trophic and clearance functions. They constitute a widely dispersed cellular system throughout the body, interacting with host cells and foreign invaders through their versatile biosynthetic and secretory responses, to maintain physiologic homeostasis. They are specialized migratory or sessile phagocytes, present within the circulation and extravascular tissue compartment, contributing to diverse pathologic processes directly and through their production of bioactive products. Because of their extensive heterogeneity and plasticity, the centrality of monocytes and their progeny has not always been recognized by hematologists. The origin, life span, and functions of the monocyte are the focus of this chapter, including their relevance to health and disease in humans, based on current understanding of their properties. The relationship of monocytes and macrophages to dendritic cells, and monocyte-derived cells with a specialized immunologic role in T-lymphocyte activation, are described. Together, macrophages and dendritic cells are major antigen-presenting cells, contributing to host defense, innate and acquired immunity, and inflammation, as well as noninfectious disease processes, both within and outside the lymphohematopoietic organs.

METHODS OF MONOCYTE AND MACROPHAGE STUDY There has been a resurgence of interest in the in situ analysis of macrophages.1 Genetic/ribonucleic acid interference manipulation, more recently with macrophage-specific/restricted promoters, has been Acronyms and Abbreviations: CR, complement receptor; DC, dendritic cell; DCSIGN, dendritic cell–specific intercellular adhesion molecule-3–grabbing nonintegrin; EMR, epidermal growth factor module-containing mucin-like hormone receptor; FACS, fluorescence-activated cell sorting; FcR, Fc receptor; GM-CSF, granulocytemacrophage colony-stimulating factor; IFN-γ, interferon-γ; IL, interleukin; LPS, lipopolysaccharide; M-CSF, macrophage colony-stimulating factor; MARCO, macrophage receptor with a collagenous structure; MR, mannose receptor; PRR, pattern recognition receptor; Sn, sialoadhesin; SR-A, scavenger receptor A; TGF, transforming growth factor; TLR, toll-like receptor; TNF-α, tumor necrosis factor-α.

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used to knock down macrophage genes or messenger RNA, and to mark cells with fluorescent labels such as green fluorescent protein. Of particular value in tracing their origins and distribution has been the use of fractalkine receptor-transgenics,2 and myeloid-specific lysozyme-Cre for targeted ablation.3 Random chemical mutagenesis has been spectacularly successful in validating known, and discovering novel, gene targets that affect macrophage functions.4,5 A wider range of experimental models (Drosophila, zebra fish) have facilitated interspecies comparisons of macrophage migration and phagocytosis in vivo.6,7 The analysis of microRNA expression8 and functions is still in its infancy and is likely to generate important insights into monocyte/macrophage gene expression in health and disease. Combined with improved imaging methods (fluorescent, nuclear magnetic resonance imaging-based, 2-photon microscopy), new insights have been obtained regarding the dynamic behavior of macrophages and dendritic cells (DCs) in vivo.9 There has been progress in provoking embryonic and induced pluripotent stem cell differentiation into macrophages and DCs in vitro, opening the possibility of introducing mutations into human genes, to complement the naturally occurring material derived from human inborn errors and resultant genetic diseases.10 Although individual-labeled cells can be followed in accessible tissues or ex vivo, the resolution, isolation, and characterization of important embedded macrophage populations are limiting. Methods of isolation from solid organs, for example, brain and even liver and gut, are prone to artifact, and macrophages are profoundly affected by removal from their natural tissue environment. Many of the genetic manipulations introduced by transgenesis are leaky and not uniform, not surprising in the light of macrophage heterogeneity. Although the fate of recently recruited cells from blood into tissues can be tracked more easily, the slowly turning over resident populations are less easily accessed, resulting in bias. Finally, there are intrinsic difficulties with human experimentation in vivo. Induced skin blisters, for example, make it possible to collect fluid and cells from sites of inflammation.11 However, the low frequency of monocytes compared with neutrophils limits the use of ex vivo indium-labeled cells for transfer studies in vivo.

PRODUCTION DEVELOPMENT OF MONOCYTES AND MACROPHAGES Macrophages and related amoeboid phagocytic cells, ancient in the evolution of multicellular organisms, are the main leukocytes responsible for innate immunity and tissue remodeling, as documented by Metchnikoff in his pioneering studies on invertebrates,12 and confirmed by contemporary studies on Drosophila melanogaster.7 In mammals, much of our knowledge of macrophage ontogeny derives from studies in the mouse. After origins from an aortic mesonephric site, the best understood phases of macrophage development occur during midfetal development, in the yolk sac, followed by fetal liver, spleen, and marrow, before and after birth.13 The association of macrophages with definitive erythropoiesis is a striking feature of fetal liver hematopoiesis from approximately day 12 of mouse development; macrophages then, for the first time, become intimately associated with nucleated erythroblasts, reaching a peak of hematopoietic cluster formation at day 14. The role of stromal macrophages in hematopoiesis within the adult is illustrated and discussed further in this chapter. The association of macrophages with erythroblasts is mediated by surface adhesion molecules,14 including a poorly characterized divalent cation-dependent receptor and the sialic acid-binding molecule sialoadhesin (Siglec1).15 The potential trophic functions of stromal

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macrophages in marrow erythropoietic islands is poorly understood, as is the considerable role of macrophages in iron and heme metabolism. Macrophages interact with cells in numerous ways; however, during erythropoiesis a special phagocytic process allows for the removal of pyknotic erythroid nuclei during the final stages of erythropoiesis. The mechanism of recognition of membrane-bound erythroid nuclei is not clear, nor its relationship to the uptake of apoptotic cells elsewhere during development. The production of granulocytes from progenitors also involves macrophage–myeloblast clusters and similar adhesion receptors. Once fetal liver hematopoiesis declines before and after birth, the macrophages in the liver adopt the features of resident Kupffer cells. The stromal macrophages associate with developing blood cells within islands of clustered cells, a feature of hematopoiesis throughout life.16 During fetal life, monocytes and macrophages are distributed through the developing vasculature, providing amoeboid, phagocytic cells implicated in tissue remodeling, for example, sculpting of digits,17 and growth of the central nervous system.18 Blood monocytes seed resident tissue macrophage populations throughout the organism, and these cells proliferate more readily in the fetus than in later life; the adhesion molecules, chemotactic signals, and receptors involved during this constitutive phase of distribution are poorly defined, but it is independent of the β2-integrin CD11b/CD18, which plays a role in myelomonocytic cell recruitment induced by inflammation in the adult.19 The appearance of macrophages during development has been correlated with fibrous scar formation after injury.17 In sum, macrophages play a major role during

development, both in hematopoiesis and in extravascular tissues, and much remains to be learned regarding their properties in the fetus.

Growth, Differentiation, and Turnover

Figure 68–1 gives an overview of differentiation of monocytic cells in the adult.20 The origins of monocytes from multipotential (progenitors colonyforming units, spleen [CFU-S]) and committed hematopoietic precursors (colony-forming units, culture [CFU-C]) and the role of lineage-restricted growth factors such as monocyte/macrophage colony-stimulating factor (M-CSF; also termed CSF-1) and granulocyte-macrophage (GM)-CSF have been studied extensively, but new details are still emerging. Both transcription factor c-Myb and receptor FLT3, M-CSF–dependent myeloid lineages (monocyte and dendritic) occur. These cell types may have different responses to tissue damage and infection.20 Monocytes share precursors with other hematopoietic cells and are closely related to granulocytes. Monocytic precursors are the source of adult tissue macrophages, as well as of myeloid DCs and osteoclasts. Their relationship to B lymphocytes and to plasmacytoid DCs is still unclear, as plasmacytoid DCs express a range of myeloid as well as lymphoid markers. There is a considerable body of knowledge about the specific growth factors and their receptors, and growing knowledge of the nature and role of transcription factors involved in monocyte/macrophage differentiation.21 Genetic and cellular abnormalities in growth and differentiation pathways underlie myeloid leukemogenesis, though rarely giving rise to monocytic leukemia.

Figure 68–1.  Differentiation of the macrophage/dendritic cell (DC) progenitor and origin of macrophage and DC subsets. CDP, common dendritic cell precursor; CMP, common myeloid progenitor; GMP, granulocyte/macrophage progenitor; HSC, hematopoietic stem cell; HSPCs, hematopoietic stem and progenitor cells; MDP, macrophage/DC progenitor; pDC, plasmacytoid dendritic cell. For further details see Ref. 20. (Used with permission of S. Seif, GraphisMedica, 2014.)

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Chapter 68: Production, Distribution, and Activation of Monocytes and Macrophages

MATURATION AND DIFFERENTIATION OF MONOCYTES AND MACROPHAGES The classic studies of Lewis and Lewis22 in 1926, Maximow23 in 1932, and Ebert and Florey24 in 1939, showed that monocytes transform into macrophages and multinucleated giant cells in vitro. Macrophages can be produced from monocytes or hematopoietic progenitor cells culture in cytokines, such as GM-CSF or M-CSF. The alterations of ultrastructure during transformation into macrophages, epithelioid cells, and giant cells have been described using purified populations of monocytes and in vitro culture techniques.25 As the monocyte matures into the macrophage, the cell enlarges in size, and the lysosomal content and the amount of hydrolytic enzymes within the lysosomes (e.g., phosphatases, esterases, β-glucuronidase, lysozyme, arylsulfatase) increase. At the time, the size and number of mitochondria increase, their energy metabolism increases concomitantly. Production of lactate also increases. The Golgi complex, which packages lysosomes, increases in size and vesicle complexity (Chap. 67). Several stimuli induce formation of multinucleated giant cells from monocytes.26

Growth Factors

M-CSF and GM-CSF are the major growth factors implicated in monocyte and macrophage differentiation. Other cytokines, such as interleukin (IL)-3 and IL-4, result in minimal monocyte proliferative

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expansion, and their genetic elimination has no effect on the lineage. M-CSF promotes survival as well as growth and differentiation of macrophages, exclusively, acting through a specific receptor (CSF-1R), encoded by the protooncogene c-FMS, which has been extensively used as a lineage marker for fluorescence-activated cell sorting (FACS) analysis (CD115) and transgenesis.27,28 The role of M-CSF has been reviewed29 and its role in macrophage and osteoclast development is illustrated in Fig. 68–2. The naturally occurring mouse mutant, op/op, gives rise to M-CSF deficiency and osteopetrosis, with marked or partial deficiency in monocyte and selected tissue macrophage populations; DC numbers are unaffected.30 Unlike PU.1 deficiency, the op/op mouse is viable, though its reproductive ability is impaired, because M-CSF also plays an important role in the reproductive system. Uterine epithelium is a rich source of M-CSF, inducing monocyte-macrophage recruitment, growth and differentiation, and upregulating scavenger receptor (SR) expression, cell adhesion, and endocytosis of modified low-density lipoproteins and other polyanionic ligands. M-CSF is produced in a soluble and membrane-bound forms, is present in plasma, and has been implicated in atherosclerosis and tumor-dependent recruitment of monocytes and macrophages. The size of the growth burst induced by M-CSF depends on the stage of differentiation of the target cell, decreasing markedly as the precursors mature into monocytes and macrophages. Adhesion and inflammatory stimuli enhance the response to growth factors and can result in macrophage proliferation at peripheral sites, for example, in granulomata.

Figure 68–2.  Regulation of macrophage and osteoclast development by macrophage colony-stimulating factor (M-CSF). Circulating M-CSF, produced by endothelial cells in blood vessels, together with locally produced M-CSF regulates the survival, proliferation, and differentiation of mononuclear phagocytes and osteoclasts. The cytokine synergizes with other hematopoietic growth factors (HGFs) to generate mononuclear progenitor cells from multipotent progenitors, and with receptor activator of nuclear factor-κB ligand (RANKL) to generate osteoclasts from mononuclear phagocytes. Brown arrows indicate cell differentiation steps; blue arrows indicate cytokine regulation. (Used with permission of S. Seif, GraphisMedica, 2014.)

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GM-CSF has a broader myeloid target profile. It is produced by many cells, including macrophages themselves, especially after inflammatory stimuli such as lipopolysaccharide (LPS), and it enhances production of monocytes and macrophages with a different morphology to that induced by M-CSF. GM-CSF is required for myeloid DC differentiation in vitro and has been widely used, alone and in combination with cytokines such as IL-4 or transforming growth factor (TGF)-β, to produce DC from mouse marrow or from human monocytes in cell culture.31,32 Its targeted deletion in mice or genetic loss of function mutants of its specific receptor chain in humans results in pulmonary alveolar proteinosis, associated with defective alveolar macrophage metabolism of pulmonary surfactant.33

Survival, Differentiation, and Turnover Overview

Once the cells have acquired the characteristics of mature monocytes/ macrophages, they display considerable heterogeneity in morphology and phenotypic plasticity. In general, their proliferative potential is limited, and their life span can vary from less than 1 day to many months, depending on their microenvironment, infections, and other stimuli. Although terminally differentiated, macrophages remain extremely active in messenger RNA and protein synthesis, with complex, often characteristic profiles of gene expression, depending on innate and acquired immune stimuli and cellular interactions. Tissue macrophages are relatively resistant to apoptosis, compared with neutrophils, but this feature changes during infection. Their active membrane turnover and endocytosis make them susceptible to toxic agents, making them targets for clearance by surviving macrophages. Sublethal injury and infection can also induce autophagy, increasingly recognized as an important component of inflammatory and infectious diseases. The remarkable ability of macrophages to undergo homotypic cell–cell fusion results in giant cell formation. This is a feature of osteoclast differentiation, depending on M-CSF and the tumor necrosis factor (TNF) family member receptor activator of nuclear factor-κB ligand (RANKL), which act on monocytic precursors to yield catabolic cells able to excavate and remodel living bone. Local adhesion and ruffling of their plasma membrane are associated with focal, polarized release of H+ and hydrolytic enzymes by monocyte-derived osteoclasts. The attempted uptake of non- or poorly degradable foreign materials induces “foreign-body giant cells,” with distinct properties; macrophagederived giant cells are also characteristic of granulomatous diseases such as tuberculosis (Langerhans giant cells; Fig. 68–3) and parasitic infections (e.g., schistosomiasis). Mycobacterial and ill-defined host lipids are able to induce giant cell formation in vitro. The mechanism of fusion involves cellular differentiation to induce a fusogenic phenotype and

A

Figure 68–3.  Microscopic image of Langhans giant cells, tuberculosis

induced. (Reproduced with permission from Y Rosen, Atlas of Granulomatous Diseases, at http://granuloma.homestead.com.)

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surface glycoprotein interactions with a selected substratum; T-helper type 2 (Th2) cytokines, such as IL-4 and IL-13, act through a common receptor chain and signaling pathway to enhance macrophage fusion.34 DNA synthesis, a feature of high-turnover granulomata associated with infection, can result in abortive cell division and cell death. These macrophage-derived giant cells are distinct from syncytia induced by fusogenic virus infection, especially paramyxoviruses and retroviruses such as the human immunodeficiency virus.

HETEROGENEITY Monocytes are defined as the population of differentiated cells present in the circulation, with classical morphologic features (Chap. 67), and include the less-well defined precursors able to give rise to myeloid DCs and osteoclasts. Because of their ready availability from human blood and the sensitive methods now available to analyze their phenotype ex vivo (FACS, microarray, immunochemistry, and cytochemistry), human monocytes have been more amenable to study, whereas in the mouse, analysis of precursor–product relationship and tissue distribution have provided new insights into the fate and heterogeneity of the circulating population. The number of monocytes in the circulation depends on constitutive, steady-state production and delivery from marrow, possibly from marginated pools in spleen, as well as adhesion and diapedesis in response to unknown stimuli and enhanced recruitment in response to peripheral stimuli such as infection and inflammation. M-CSF and glucocorticoids affect their level and phenotype, as do metabolic stimuli; Chap. 70 describes clinical conditions that give rise to monocytosis. The biochemical properties and functions of monocytes are described in Chap. 67. They are relatively radioresistant once entering the circulation, where they persist for 12 to 48 hours as motile cells, with an ability to engulf particles and to adhere transiently or more stably to arterial as well as microvascular endothelium, thus modulating their phagocytic ability. Depending on interactions with the vessel wall and local differentiation, monocytes are able to crawl along and patrol the intravascular surface utilizing CD11a, a β2-integrin–dependent property.20 Mature macrophages lining the endothelium can also detach and recirculate, for example, filled with lipid stores as foam cells in atherosclerosis, and circulate heavily laden with erythroid breakdown products in malaria. The presence of significant numbers of immune cells and molecules in adipose tissue suggest vibrant interactions between the immune and metabolic systems. In obesity, the inflammatory infiltrate and activation state of macrophages in adipose tissue may contribute to insulin resistance. The cellular localization and inflammatory potentials of macrophages,35 as well as the ratio of macrophages to adipocytes,36 differ in obese and lean mice. In lean mice, macrophages in the adipose tissue have the alternate or M2 phenotype (ARG1+CD206+CD301+), are uniformly distributed, and serve a protective function as they are less inflammatory and promote insulin sensitivity by producing IL-10; however, in obese mice, macrophages distribute around necrotic adipocytes, and induce inflammation and insulin resistance.35,37 CC-chemokine receptor 2 (CCR2) and its ligand (CCL2) are critical for macrophage recruitment to adipose tissue.38 Metabolic disease can be viewed as maladaptive consequence of inflammation-induced insulin resistance, which may beneficially conserve energy resources for the immune system combatting infection for brief periods.39–43 The precursors of myeloid DC and osteoclasts may represent a subpopulation of monocytes, whose further differentiation depends on cytokines and local factors in the vessel wall, marrow, and other tissues. Ex vivo substantial numbers of monocytes give rise to myeloid DC after treatment with GM-CSF and IL-4.32 Monocytes that differentiate into macrophages do not recirculate for the most part, but persist for varying times as “resident” tissue cells that turn over locally, especially in lymph

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Figure 68–4.  Heterogeneity of monocytes in blood and the contribution of different subsets to resident and inflammatory macrophages DCs in tissues. For further details see Ref. 34. (Used with permission of S. Seif, GraphisMedica, 2014.) nodes. It is not known if the constitutive exit from blood is a stochastic process or specific to particular tissues. Phenotypic heterogeneity of monocyte populations has become a topic of intense interest, thanks to the availability of surface antigens/ receptors such as CD14, CD16 (human), and Ly6C (mouse), and analysis of chemokine/receptor expression, especially fractalkine receptor (CX3CR) and CCR2.34 Figure 68–4 illustrates the subsets and tissue progeny established by the use of genetically manipulated mice and Table 68–1 compares expression of markers to characterize monocyte subsets in mouse and human blood. The relationship of monocyte precursor subsets that give rise to inflammatory tissue macrophages and DCs is better defined than is that of those that give rise to resident cells, which turn over more slowly. Current studies aim to elucidate the subset origin of other recruited populations, for example, in atherosclerosis, normal CNS, and tumors, and in response to metabolic, traumatic, or degenerative injury. Conceptually, it is still not clear how stable these apparently distinct subsets are or whether they represent part of a continuous phenotypic spectrum, arising by modulation of subpopulations rather than irreversible, true differentiation. Separation and microarray analysis of freshly isolated monocytes will yield further information regarding this question, providing novel markers and diagnostic signatures. Removal from an in vivo environment, as well as in vitro artifacts, can profoundly alter the phenotype and function of monocytes in such studies. Imaging and in situ analysis may enable single-cell direct studies of their fate. Monocyte/macrophages have a major role in the development and progression of cardiovascular disease.44,45 In acute myocardial infarction

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macrophages with an M1 proinflammatory profile migrate to the cardiac tissue and are involved in cardiac remodeling (Chap. 134).46 In atherogenesis, there is recruitment of monocytes into the vascular wall at sites of turbulent flow. Once within the subendothelial tissue, the monocytes differentiate into macrophages and engulf oxidized low-density lipoprotein accumulated in arteries, leading to foam cell formation, atheroma development, and the secretion of profibrotic agents by adjacent vascular smooth muscle cells, resulting in a fibrous cap formation. Thus, vascular wall macrophages are key factors in initiating the atherosclerotic lesion. Moreover, macrophages activate the coagulation cascade (Chap. 67) inducing thrombus formation and vascular occlusion.

Resident Macrophage Populations in Adult Tissues Overview

It is important to describe first the nature of those macrophages present throughout the body as resident populations, in the absence of overt inflammation, before considering the altered monocyte-derived macrophages recruited to local sites by infectious or sterile inflammatory (e.g., metabolic) stimuli. The properties of such elicited macrophages are well established and are described in Chap. 67. However the functions of resident macrophages, especially in different organs, are still mysterious and are considered in outline here, with further details in Chap. 67. The use of differentiation antigens such as F4/80 and cd68 (mouse) and CD68 (human) has made it possible to define resident macrophage populations in mouse tissues,47 and to compare their anatomic relationships in the two species (Table 68–2). F4/80 (EMR1), a member of a family of epidermal growth factor-7 transmembrane (EGF-TM7)

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TABLE 68–1.  Selected Markers of Different Monocyte Subsets in Mouse and Human Blood Antigen

Human CD14hi CD16–

Human CD14+ CD16+

Mouse CCR2+ CX3CR1low

Mouse CCR2– CX3CR1hi

CHEMOKINE RECEPTORS CCR1

+

–

ND

ND

CCR2

+

–

+

–

CCR4

+

–

ND

ND

CCR5

–

+

ND

ND

CCR7

+

–

ND

ND

CXCR1

+

–

ND

ND

CXCR2

+

–

ND

ND

CXCR4

+

++

ND

ND

CX3CR1

+

++

+

++

TABLE 68–2.  Selected Markers of Mononuclear Phagocytes and Related Cells Cell Type

Antigen Markers

Other Properties

Monocytes/ macrophages  

F4/80 (mouse) EMR2 (human) CD68 CR3 (CD11b) Sialoadhesin (Siglec-1) Scavenger receptors (SR-A, MARCO) Mannose receptor M-CSF receptor

Opsonic phagocytosis; lysozyme secretion; abundant acid hydrolases      

Myeloid dendritic cells

MHC II Costimulatory molecules CD11c CD8α+/– DEC205 DC-SIGN DC-LAMP

Activation of naïve CD4 T lymphocytes

Plasmacytoid dendritic cells

CD123 B220 Lectin-like receptors (Siglec-H)

Type I interferon production; in vitro growth by flt-3 ligand

Osteoclasts

CD68 TRAP Calcitonin receptor αvβ3

Vacuolar H+ ATPase; proteinase K; resorption of living bone

OTHER RECEPTORS CD4

+

+

ND

ND

CD11a

ND

ND

+

++

CD11b

++

++

++

++

CD11c

++

+++

–

+

CD14

+++

+

ND

ND

CD31

+++

+++

++

+

CD32

+++

+

ND

ND

CD33

+++

+

ND

ND

CD43

ND

ND

–

+

CD49b

ND

ND

+

–

CD62L

++

–

+

–

CD86

+

++

ND

ND

CD115

++

++

++

++

CD116

++

++

++

++

F4/80

ND

ND

+

+

Ly6C

ND

ND

+

–

7/4

ND

ND

+

–

MHC class II

+

++

–

–

MHC, major histocompatibility complex. Adapted with permssion from Gordon S. & Taylor PR: Monocyte and macrophage heterogeneity. Nat Rev Immunol 5(12):953–964, 2005. plasma membrane molecules, is broadly present and almost exclusive to macrophages (Fig. 68–5A to C).48,49 It is related to G-protein– coupled chemokine receptors in structure, but has a large epidermal growth factor (EGF) domain extracellular extension, thought to be involved in adhesion to extracellular matrix. The human members of this family are more broadly present on myeloid cells; EGF modulecontaining mucin-like hormone receptor 2 (EMR2) is a useful tissue marker for human macrophages, although it is also present in neutrophils and immature DCs (Fig. 68–5A). Additional macrophage antigen markers useful for immunocytochemical and FACS analysis include Siglec1 (Fig. 68–5D), a sialic acid-binding lectin, the β2 integrins

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ATPase, adenosine triphosphatase; DC, dendritic cell; DC-LAMP, dendritic cell lysosomal-associated membrane protein; DC-SIGN, dendritic cell–specific intercellular adhesion molecule-3–grabbing nonintegrin; EMR, epidermal growth factor module-containing mucin-like hormone receptor; M-CSF, macrophage colony-stimulating factor; MARCO, macrophage receptor with collagenous structure; MHC, major histocompatibility complex; TRAP, tartrate-resistant acid phosphatase. note: Marker expression is variable, depending on cell localization, maturation, and activation. Some markers are also present on other myeloid cells, e.g., polymorphonuclear cells, and selected endothelial cells. CD11b/CD18 (Mac1, CR3), and CD11c, present on DCs and selected, especially alveolar, macrophages.50 Receptor antigen markers include SR-A,51 a broadly expressed macrophage receptor additionally found on sinusoidal endothelium, whereas MARCO (macrophage receptor with collagenous structure), a related collagenous SR, is more restricted in expression.52 Additional markers include lectins such as the macrophage mannose/fucose receptor (MR; Fig. 68–5E).53 CD163, a receptor for hemoglobin–haptoglobin complexes, is induced by glucocorticoids,54 IL-10,54 and substance P.55 Complement receptors (CRs) and Fc receptors (FcRs) are described in Chap. 67. The resident macrophages in tissues constitute a major dispersed organ system, responsive to endogenous and exogenous stimuli; they are highly active in uptake of particles and soluble ligands, providing not only sentinels for defense at portals of entry, but also mediating the

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Tonsil (cryostat)

EMR2

A

B

D

C

E

Figure 68–5.  Immunocytochemical detection of macrophages in human (A) and mouse (B to E) lymphohematopoietic tissues. A. Tonsil.

EMR2-positive macrophages are scattered throughout follicles and interfollicular areas. B. Liver. Kupffer cells are F4/80+, unlike sinusoidal endothelium and hepatocytes. C to E. Spleen. C. Red pulp macrophages express F4/80, unlike marginal zone cells. Macrophages in T-cell area are F4/80–, except for periarteriolar processes. D. Marginal metallophilic macrophages express sialoadhesin (Siglec1) strongly; red pulp macrophages are weakly positive. E. A subset of marginal metallophils binds a chimeric protein probe of the cysteine-rich domain of the MR-human Fc. For details see Ref. 91. (A, used with permission from of T. Marafioti. B to E, reproduced with permission from Taylor PR, Zamze S, Stillion RJ, et al: Development of a specific system for targeting protein to metallophilic macrophages. Proc Natl Acad Sci USA 101(7):1963–1968, 2004.)

clearance of damaged or dying cells and modulating the properties of viable neighboring cells. In sum, these cells provide a homeostatic, trophic function that is often overlooked in considering their role in cytotoxicity and antimicrobial host defense. The properties of macrophages in hematolymphoid organs and other tissues, with special relevance to hematologic aspects, are discussed in detail.

DISTRIBUTION HEMATOPOIETIC ORGANS Marrow

It is often overlooked that mature macrophages are important constituents of the hematopoietic stroma,56 along with fibroblastic mesenchymal cells, osteoblasts, and endothelial cells, contributing to hematopoiesis beyond their own differentiation (Figs. 68–6A to E and 68–7). Stromal macrophages in hematopoietic island clusters associate with developing erythroid and other granulocytic cells through nonphagocytic, cell–cell adhesion receptors, such as sialoadhesin and a divalent cationdependent receptor, as described for fetal liver. The potential trophic functions provided by stromal macrophages are ill-defined but include surface-expressed and secreted growth factors and cytokines. Stromal macrophages are actively endocytic and clear erythroid nuclei and apoptotic hematopoietic cells as required, rapidly degrading them for possible reutilization of iron and other nutrients. Stromal macrophages also interact with less-differentiated hematopoietic precursors through release of potent secretory products, such as IL-1, and with lymphocytic populations, including plasma cells, through IL-6. They are targets for infectious agents, for example, mycobacteria, lentiviruses, and retroviruses, and serve as reservoirs in many chronic infections, while

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expressing a reduced killing capacity, as demonstrated for other resident macrophage populations. Several monocyte and macrophage populations coexist in the marrow compartment; a network of stromal macrophages, clustered in hematopoietic islands, the developing monocytes, as well as osteoclasts and isolated macrophages in apposition to bone surfaces. Mature macrophages in human marrow contain prominent inclusions in storage disorders, such as Gaucher disease and hemosiderosis. Hemophagocytosis, a consequence of perforin deficiency in some patients, and seen in genetic syndromes and postviral infection, is a striking manifestation of excessive macrophage cytopathic activity in the marrow.57,58 Uptake of opsonized platelets by macrophage FcR and CRs in stromal and other resident tissue macrophages are important features of thrombocytopenic syndromes. The hematopoietic stem cell lineage which gives rise to monocytemacrophages and myeloid DCs also leads to production of the osteoclast lineage.59,60 Following interaction via Stat4 and RANKL (regulator of activation of nuclear factor-κB), a member of the superfamily of TNF, cells undergo differentiation, fusion, attachment to bone as osteoclasts and then function in bone remodeling.61 A common marrow progenitor cell that gives rise to both monocytes and DCs has been defined,20 including both classical DCs and the plasmacytoid DCs.62,63 This common marrow progenitor cell circulates in the blood and seeds lymphatic tissues.62,63 These short-lived, migratory cells modify T-cell responses and, unlike Langerhans cells, are replaced by bloodborne precursors.41,42 The central activity of immature classical DCs is phagocytosis, while that of mature classical DCs is cytokine production.62,63 Dendritic cells that occur in lymphoid and nonlymphoid organs have a major role in processing and presenting antigens, leading to

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unique T cell function. The “classical” DC develops from a precursor which is dependent on the growth factor receptor.62–64

Spleen

A

B

C

D

From the macrophage point of view, the spleen is the most complex organ in the body.65,66 Our knowledge is based mainly on the mouse and we know there is considerable species variation,67 as well as constitutive hematopoiesis in mouse spleen. Subpopulations observed in the mouse by marker and genetic knockout experiments include (1) macrophages in the red pulp, white pulp, and in the marginal zone, itself M-CSF dependent,30 and (2) heterogeneous, more phagocytic “metallophilic” macrophages in the outer marginal zone. Characteristic phenotypic markers are available to identify macrophages in mouse spleen (see Fig. 68–5C to E). The F4/80 antigen and the mannose receptor (MR) are restricted to mature macrophages in the red pulp, whereas CD68 is a marker for all macrophages, as well as DCs, although the mainly intracellular expression of CD68 is less prominent in DCs. There are several well-characterized markers for mouse metallophilic macrophages, including sialoadhesin (Sn), a poorly characterized protein recognized by the MOMA-1 monoclonal antibody, and ligands for MR cysteine-rich domain-Fc chimeric proteins (see Fig. 68–5E). The splenic marginal zone macrophage population develops postnatally,68 in parallel with antipolysaccharide responses to encapsulated bacteria. Functions of splenic marginal zone macrophages include clearance of senescent erythrocytes and neutrophils (red pulp), targeting of circulating antigens and pathogens (marginal zone), interferon (IFN) production, induction of secondary adaptive immune responses, regulation of hematopoiesis, and iron storage. Markers for the outer marginal zone macrophages include MARCO and SIGNR1, a mouse homologue of dendritic cell–specific intercellular adhesion molecule-3–grabbing nonintegrin (DC-SIGN). The spleen is also a site for storage and rapid deployment of monocytes, which participate in wound healing and play a role regulation of inflammation.69

Lymph Nodes

E

F

Figure 68–6.  Stromal macrophages in human marrow associate with

developing hematopoietic cells in islands/clusters. Immunocytochemical staining with antimacrophage monoclonal antibody Y1/82A of marrow section reveals a network of arborizing stromal macrophages uniformly distributed throughout the marrow interstitium (alkaline phosphatase–antialkaline phosphatase [APAAP] stain; hematoxylin counterstain). B. Marrow cells depleted of red cells and other single cells are enriched for cell clusters, most of which are erythroid clusters with a central stromal macrophage (arrows; Giemsa). C. Isolated erythroid cluster with intermediate and late normoblasts surrounding a central stromal macrophage (Giemsa). D. Isolated mixed cluster with both myeloid and erythroid cells attached to a central stromal macrophage. A dividing cell (arrow) is seen (Giemsa). E. Isolated erythroid clusters from a pathologic marrow sample show intense staining for hemosiderin of stromal macrophages with cellular processes extending between attached erythroblasts (Perl acid ferrocyanide reaction; counterstain neutral red). F. Immunocytochemical stain with antibody Y1/82A of isolated erythroid cluster. Both the stromal macrophage cell body and processes (arrows) between attached erythroblasts are visible (APAAP stain; hematoxylin counterstain). Bar = 50 μm. (Reproduced with permission from Lee, S.H. et al.: Isolation and immunocytochemical characterization of human bone marrow stromal macrophages in hemopoietic clusters. J Exp Med 168(3):1193–1198, 1988.)

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Lymph node macrophages are also heterogeneous, with distinctive Sn subcapsular cells, corresponding to marginal metallophils in their marker expression, and F4/80+ macrophages in germinal follicles and in the hilus. Macrophages in T-lymphocyte–rich areas are F4/80– or dim, as in T-cell areas in spleen, but express CD68. It is thought that antigen enters lymph nodes via afferent lymphatics and two photon experiments have defined the possible transfer of viral and other antigens and immune complexes to B lymphocytes after capture by the subcapsular sinus macrophages.70 Their contributions to the initiation of adaptive immune responses, compared with DCs, are unclear. Tingible body macrophages arise from the clearance of apoptotic B cells in germinal centers, as in the spleen.

Nonlymphohematopoietic Organs

In bulk, the gastrointestinal tract represents the largest accumulation of F4/80+ macrophages in the body, extending throughout the upper and lower gut. The small intestine is essentially sterile, and the abundant F4/80+ resident macrophages in the lamina propria express a distinct phenotype, ascribed to TGF-β production by adjacent cells.56,71–73 The liver contains an abundant population of sinusoidal F4/80+ Kupffer cells, which share some properties (FcR, MR, SR-A) with sinusoidal endothelium, which lacks F4/80. The skin has F4/80+ epidermal Langerhans cells and F4/80+ dermal macrophages, which can migrate to draining lymph nodes and differentiate into antigen-presenting DCs.74,75 The lung has a distinctive F4/80– or dim alveolar macrophage population, as well as interstitial F4/80+ macrophages. Alveolar macrophages are CD11c+ and express a range of nonopsonic phagocytic

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E G G

E

M

E

M

A

B

M

Eos

C

Figure 68–7.  Sialoadhesin (Siglec1) (arrowheads) is clustered at sites of stromal macrophage adhesion to developing cells (B, granulocytes; C, eosinophils) but diffusely present in association with erythroblasts (A). For details see Ref. 93. (Reproduced with permission from Crocker PR, Werb Z, Gordon S, et al: Ultrastructural localization of a macrophage-restricted sialic acid binding hemagglutinin, SER, in macrophage-hematopoietic cell clusters. Blood 76(6):1131–1138, 1990.) receptors (MR, SR-A), as well as FcR, but lack CR3. These cells also contain particle debris, cigarette smoke residue, and abundant lysozyme, because of exposure to irritants and uptake of carbon and dust particles, as well as of mucosal secretions in the airway. The central nervous system contains an extensive network of F4/80+ CR3+ microglia, derived from monocytes during development, when they remove apoptotic neurons.18 They differentiate into characteristic membrane-rich arborized forms within the neuropil and persist throughout adult life. Their function is obscure but may involve homeostasis and catabolism of neurotransmitters. In addition, there are perivascular F4/80+ macrophages (also MR+ SR-A+) and other F4/80+ populations in the meningeal space and choroid plexus. The endocrine, exocrine, reproductive, and urinary tracts all contain macrophage populations at sites of phagocytosis (ovary, testes) and hormonal metabolism (adrenal, thyroid, for example).71 Precise characterization of these cell types using monoclonal antibodies with specificity for human cell types in tissue requires further analysis.

ACTIVATION STATE RECRUITMENT OF MONOCYTES IN RESPONSE TO INFLAMMATION AND TUMORS The stimuli that give rise to induced recruitment of monocytes, with or without accompanying myeloid and/or lymphoid cells, and the mechanisms involved are better understood than those of constitutive tissue localization. Bacterial infections induce enhanced myelomonocytic cell recruitment and follow the stages established for neutrophils, transient arrest, and rolling on the microvascular endothelium, mediated by Lselectin, and initiated by chemotactic stimuli acting via G-proteincoupled chemokine receptors (Fig. 68–8). The β2 integrins CD11a/ CD18 and CD11b/CD18 mediate more stable adhesion. This is followed by diapedesis and interactions with CD31. Receptors implicated in subsequent extravascular migration are less defined but may include the fractalkine receptor, and intravascularly, β1- and β2-integrin, CD44, and

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EMR2. The evidence for an important role of L-selectin and β2 integrins in human phagocyte recruitment to inflammatory stimuli comes from human inborn error syndromes, mouse genetic experiments, and antibody inhibition. The role of the common β2-integrin chain (CD18) and definition of leukocyte adhesion deficiency syndrome provided a powerful paradigm for further experimental study of CD11/CD18.76,77 Cellular signaling gives rise to dynamic changes in migration/adhesion and cytoskeletal reorganization outlined further in Chap. 67. Studies of tumor-associated macrophages (TAMs) in mouse model systems, particularly mammary cancer, have identified a population of unique macrophages. The TAMs develop from marrow-derived macrophages that are inflammatory phenotypes and are recruited to the tumor.78,79 Mononuclear cell recruitment without that of other myeloid cells is a feature of viral infection and modified forms of inflammation observed in metabolic diseases, atherosclerosis, storage disorders, autoimmunity, and tumors. Different chemokine receptors and cell adhesion molecules account, in part, for more selective monocytic recruitment, although some are shared. The phenotypic heterogeneity in monocyte subsets is characterized by quantitative differences in expression of plasma membrane molecules resulting in differential recruitment of subsets in response to different stimuli. Once in the tissues, their subsequent fate also varies markedly, depending on the local environment, where newly recruited monocytes respond to tissue-specific factors. A striking example is that observed in the neutrophil, where monocytes can differentiate over a few days into highly arborized, activated microglia, resembling locally reactivated resident microglia.18 Thus, it becomes progressively more difficult to distinguish newly recruited from initially resident cells through marker analysis. Direct observation by fluorescent imaging in vivo may define precursor–product relationships more clearly. Similar issues arise in other organs, for example, lung, liver, gut, and even in skin, where static observations can be misleading. There are also common features of recruited cells irrespective of the local tissue environment, including the expression of CD11b/CD18 and monocytic adhesion molecules, and metabolic markers, such as the ability to undergo a respiratory burst and an increased proliferative potential and

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Firm adhesion Rolling CD62L shedding

Increase in CD11b Extravasation

CD54

CD99

CD62P

CD29

CD62L CD11b CD11a

Jam

CD31/PECAM

CD49d

PSGL1 Chemokines (e.g., CCR2/MCP-1) Chemokine receptor (e.g., CCL2)

N-glycans

Figure 68–8.  Recruitment. Stages of monocyte adherence to endothelium and diapedesis, induced by inflammatory stimuli. The model is mainly based on the recruitment of neutrophils, with which it shares many features, although monocyte-specific chemokines, receptors, and adhesion ligands exist, especially in constitutive and noninfectious, metabolic forms of inflammation. PECAM, platelet endothelial cell adhesion molecule.

high cell turnover rate. These monocytic markers tend to decline upon further macrophage differentiation, and in the case of myeloperoxidase may not be renewed after degranulation.

heterogeneity continues to grow in complexity, with the description of Th17, FoxP3+, and other regulatory T cells. Further details are given in Chap. 67.

HETEROGENEITY OF MACROPHAGES IN TISSUES: IMMUNOMODULATION

INNATE ACTIVATION

Characterization of the macrophages found in tissues has yielded insights into their versatility in response to microbial constituents and cytokines produced by lymphoid, other immune and nonimmune cells. Adhesion to extracellular matrix, metabolites, vascular, and hormonal changes all influence the macrophage phenotype. This variety of stimuli can selectively activate or deactivate macrophage gene and protein expression, regulating their function. Figure 68–9 illustrates some of the stereotypic signature phenotypes, and Chap. 67 further describes the innate recognition mechanisms and functional responses. Broadly considered, it is convenient to distinguish several clusters of activation properties; innate, classical, and alternative activation, and deactivation. The definition of activation has a long and confusing history, has mainly been based on limited models of analysis, typically peritoneal macrophages in vivo and in vitro, and on studies with macrophage-like cell lines. The advent of microarrays and of proteomics and systems biology has generated increasingly detailed information. It makes sense to schematize the interactions of monocytes and macrophages with microorganisms, microbial products, and Th1/Th2 lymphocytes, although this is subject to revision as CD4 T-lymphocyte

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For this discussion, innate activation is defined as direct microbial stimulation by intact bacteria or their constituents, such as LPS acting via toll-like receptor (TLR) sensors, in the absence of the major Th1/2 cytokines. For example, ethanol-killed Neisseria meningitidis, a potent immunomodulator with adjuvant-like properties, stimulates the expression of two useful markers on macrophages: MARCO and CD200. Expression of MARCO, a class A SR, is remarkably specific for macrophages (and DCs) and is regulated developmentally on the outer marginal zone macrophages, but it is inducible on most macrophage populations by TLR and myeloid differentiation factor 88 (MyD88)-dependent bacterial stimuli. It is a phagocytic and adhesion receptor providing an adaptive, enhanced ability to take up Neisseria and other bacteria, after innate activation. CD200, an immunoglobulin (Ig) superfamily member, is widely expressed on many cells, but not on resident macrophages, and part of an immunoregulatory receptor pair with CD200 R, is also induced on macrophages by innate stimuli. Lectins, such as Dectin-1, control innate activation of macrophages by β glucans in fungal walls,80 in collaboration with TLR pathways, as discussed in Chap. 67. TLR-independent innate activation by viruses, parasites, and other pathogen-associated stimuli requires further study.

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Figure 68–9.  Immunomodulation of macrophage phenotype by cytokines, microbial constituents, and glucocorticosteroids. CCL, chemokines;

CR, complement receptor; DC-SIGN, dendritic cell–specific intercellular adhesion molecule-3–grabbing nonintegrin; EMR, epidermal growth factor module-containing mucin-like hormone receptor; FPR1, formyl-peptide receptor 1; FPRL-1, formyl-peptide receptor-like 1; GC, glucocorticoids; IL, interleukin; INFγ, interferon-γ; LPS, lipopolysaccharide; MARCO, macrophage receptor with a collagenous structure; MRC-1, mannose receptor C-type 1; NO, nitric oxide; PECAM, platelet endothelial cell adhesion molecule-1; ROS, reactive oxygen species; SR-A, scavenger receptor A; TLR, toll-like receptor; TNF-α, tumor necrosis factor-α. Further details on innate modulation of phenotype by microbial products are given in Chap. 67. (Adapted with permission from Yona, S. & Gordon, S.: Inflammation: Glucocorticoids turn the monocyte switch. Immunol Cell Biol 85(2):81–82, 2007.)

CYTOKINE-INDUCED PRIMING AND ACTIVATION: CLASSICAL AND ALTERNATIVE ACTIVATION Investigations in normal humans, pathologic states, and animal models have led to the characterization of different states of macrophage polarization (Fig. 68–10).81 The terms macrophage “activation and “polarization” require well-defined nomenclature and experimental guidelines. It has been recommended that the term “activation” be used to refer to perturbation of macrophages with exogenous agents the same way that many authors use “polarization.”82 Studies of in vitro, ex vivo, and in vivo cells from animals and humans have led to descriptions of complex cellular structural, biochemical, and functional activation states of macrophages. Consequently, it is essential to describe the properties of the macrophage its activation/polarization state. There are various nomenclatures and Fig. 68–10 indicates the major features of the M1– M2 dichotomy. This dichotomy, however, is more of a continuum and there are transitional states. The most useful approach is to describe the spectrum of activation states by cell isolation technique, membrane receptors, cytokines, chemokines, and metabolic markers, and when possible, genetic modifications producing shifts in activation phenotype. This approach is essential for deciphering the role of monocyte– macrophages in disease pathogenesis. Macrophage polarization usually is driven by unique pattern recognition receptors (PRRs). Classical activation of macrophages or M1 in general83 have a proinflammatory profile. This pathway is triggered by IFN-γ followed by a microbial stimulus, LPS. The M1 macrophage is characterized by high antigen presentation and production of IL-12, IL-23, nitric oxide, and proinflammatory cytokines, including IL-1, TNF-α, IL-6, and CXCL-1, -2, -3, -5, -8, -9, and -10. The pathway to the

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alternative M2 macrophage is mediated by IL-4, IL-10, and IL-13. These cells demonstrate enhanced expression of Dectin-1, MR C1 (CD206), CD163, CCR2, CXCR1, CXCR2, and DC-SIGN.84 M2 produce high levels of IL-10 and low-levels of IL-12.85 IFN-γ, produced mainly by natural killer cells and activated Th1 CD4+ and CD8+ cytotoxic lymphocytes, induces a set of macrophage biosynthetic and effector responses, known as classical activation, because of its well-established role in enhanced macrophage functions in cell-mediated immunity, inflammation, and host defense, particularly against intracellular pathogens. Full activation of effector functions, such as the respiratory burst and generation of oxidative nitrogen metabolites, depends on a two-stage mechanism of priming by the cytokine, via specific IFN-γ receptors, followed by a local stimulus, LPS, or other TLR ligands. Although essential for host defense, including against opportunistic pathogens such as found in patients with the acquired immunodeficiency syndrome, classical activation is responsible for tissue injury and its consequences in inflammatory bowel disease, tuberculosis, and rheumatoid arthritis, although additional immunopathogenic agents, such as immune complexes, also contribute. Biochemical and cellular aspects of classical activation are described in Chap. 67. The Th2 cytokines IL-4 and IL-13, acting via a common receptor chain as well as distinct receptors, induce a characteristic signature of altered gene expression in macrophages known as alternative activation.86,87 Such primed macrophages can be induced to respond further to local, TLR-dependent phagocytic stimuli to secrete enhanced levels of proinflammatory cytokines, analogous to classically activated macrophages. Alternative activation is associated with allergy and parasitic infection, and has been implicated in humoral immunity, control of Th1-dependent inflammation, and host defense to extracellular pathogens, such as helminths. It can promote repair or, if excessive, fibrosis.

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Figure 68–10.  Schema of states of monocyte-macrophage activation. AM, alternative M2A; CM, classical (M) activation; DM, M2C deactivated; NPM, nonpolarized. Membrane markers expression in yellow. Flow cytometry and cytokines enzyme-linked immunosorbent assay (ELISA) in blue. IFN-γ, interferon-γ; IL, interleukin; LPS, lipopolysaccharide; NK, natural killer; Th1/Th2, T-helper type 1/2; TREG, T regulatory cell. (Used with permission of S. Seif, GraphisMedica, 2014.) Other forms of alternative activation have been described after stimulation of macrophages by immune complexes, acting via FcR. A major G-protein–coupled receptor that is important in macrophage activation is the Neurokinin-1 receptor.88 This receptor, which has a full-length form and a truncated splice variant, is important in macrophage signaling and calcium fluxes.89,90 An important caveat is that there are substantial species differences in the marker changes depending on the differentiation and prior activation state of the macrophages. IL-10 is a major deactivating cytokine for macrophages, produced by the macrophages themselves, as well as by Th2 lymphocytes and other sources. Acting through its own receptor, it counteracts IFN-γ, and can potentiate IL-4–induced actions. Other antiinflammatory regulators of macrophages activation include glucocorticoids and prostaglandin E2. Although less-well defined, the overall gene and protein expression profiles of macrophages are also markedly influenced by the extracellular matrix, hormones, and other immunomodulators, so that modified forms of inflammation are associated with macrophages present in lipid-rich environments, tumors, and metabolic diseases. Finally, cell–cell interactions, as well as intracellular regulatory networks, profoundly influence the functions of macrophages, and are described in Chap. 67.

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Dev Immunol 2:7–17, 1992. 69. Swirski FK, Nahrendorf M, Etzrodt M, et al: Identification of splenic reservoir monocytes and their deployment to inflammatory sites. Science 325:612–616, 2009. 70. Martinez-Pomares L, Gordon S: Antigen presentation the macrophage way. Cell 131:641–643, 2007. 71. Hume DA, Halpin D, Charlton H, Gordon S: The mononuclear phagocyte system of the mouse defined by immunohistochemical localization of antigen F4/80: Macrophages of endocrine organs. Proc Natl Acad Sci U S A 81:4174–4177, 1984. 72. Smythies LE, Maheshwari A, Clements R, et al: Mucosal IL-8 and TGF-beta recruit blood monocytes: Evidence for cross-talk between the lamina propria stroma and myeloid cells. J Leukoc Biol 80:492–499, 2006. 73. Smythies LE, Sellers M, Clements RH, et al: Human intestinal macrophages display profound inflammatory anergy despite avid phagocytic and bacteriocidal activity. J Clin Invest 115:66–75, 2005. 74. 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77. Luo BH, Carman CV, Springer TA: Structural basis of integrin regulation and signaling. Annu Rev Immunol 25:619–647, 2007. 78. Franklin RA, Liao W, Sarkar A, et al: The cellular and molecular origin of tumorassociated macrophages. Science 344:921–925, 2014. 79. Gomez Perdiguero E, Geissmann F: Cancer immunology. Identifying the infiltrators. Science 344:801–802, 2014. 80. Brown GD: Dectin-1: A signalling non-TLR pattern-recognition receptor. Nat Rev Immunol 6:33–43, 2006. 81. Wynn TA, Chawla A, Pollard JW: Macrophage biology in development, homeostasis and disease. Nature 496:445–455, 2013. 82. Murray PJ, Allen JE, Biswas SK, et al: Macrophage activation and polarization: Nomenclature and experimental guidelines. Immunity 41:14–20, 2014. 83. Herbein G, Varin A: The macrophage in HIV-1 infection: From activation to deactivation? Retrovirology 7:33, 2010. 84. Labonte AC, Tosello-Trampont AC, Hahn YS: The role of macrophage polarization in infectious and inflammatory diseases. Mol Cells 37:275–285, 2014. 85. Cassetta L, Cassol E, Poli G: Macrophage polarization in health and disease. ScientificWorldJournal 11:2391–2402, 2011. 86. Gordon S: Alternative activation of macrophages. Nat Rev Immunol 3:23–35, 2003.

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87. Martinez FO, Helming L, Gordon S: Alternative activation of macrophages: An immunologic functional perspective. Annu Rev Immunol 27:451–483, 2009. 88. Douglas SD, Leeman SE: Neurokinin-1 receptor: Functional significance in the immune system in reference to selected infections and inflammation. Ann N Y Acad Sci 1217:83–95, 2011. 89. Lai JP, Ho WZ, Kilpatrick LE, et al: Full-length and truncated neurokinin-1 receptor expression and function during monocyte/macrophage differentiation. Proc Natl Acad Sci U S A 103:7771–7776, 2006. 90. Lai JP, Lai S, Tuluc F, et al: Differences in the length of the carboxyl terminus mediate functional properties of neurokinin-1 receptor. Proc Natl Acad Sci U S A 105: 12605–12610, 2008. 91. Taylor PR, Zamze S, Stillion RJ, et al: Development of a specific system for targeting protein to metallophilic macrophages. Proc Natl Acad Sci U S A 101:1963–1968, 2004. 92. Lee SH, Crocker PR, Westaby S, et al: Isolation and immunocytochemical characterization of human bone marrow stromal macrophages in hemopoietic clusters. J Exp Med 168:1193–1198, 1988. 93. Crocker PR, Werb Z, Gordon S, Bainton DF: Ultrastructural localization of a macrophage-restricted sialic acid binding hemagglutinin, SER, in macrophagehematopoietic cell clusters. Blood 76:1131–1138, 1990.

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CLASSIFICATION AND CLINICAL MANIFESTATIONS OF DISORDERS OF MONOCYTES AND MACROPHAGES

Marshall A. Lichtman

SUMMARY Disorders that exclusively result in abnormalities of monocytes, macrophages, or dendritic cells are uncommon and usually are referred to, pathologically, as histiocytosis. These disorders can be inherited, such as familial hemophagocytic lymphohistiocytosis; inflammatory, such as infectious hemophagocytic lymphohistiocytic syndrome; or clonal (neoplastic), such as Langerhans cell histiocytosis. They can result from an inherited enzyme insufficiency in macrophages that lead to exaggerated storage of macromolecules, such as in Gaucher disease. Monocytes are critical sources for proinflammatory and inflammatory cytokines and, when inappropriately activated, can result in the lymphohistiocytic hemophagocytic syndrome with fever, intravascular coagulation, and organ pathology. A variety of hematopoietic neoplasms may have a phenotype characterized by a large proportion of monocytes. Idiopathic (clonal) monocytosis is a rare manifestation of a myelodysplastic syndrome. Some cases of myelogenous leukemia have progenitor cells that mature preferentially into leukemic monocytes, including acute monoblastic or monocytic leukemia, chronic myelomonocytic leukemia, and juvenile myelomonocytic leukemia. Two acquired diseases, hairy cell leukemia and aplastic anemia, result in a severe depression of blood monocytes (along with other blood cell types). Mutations in GATA2 are associated with severe monocytopenia and mycobacterial infections (the MonoMAC syndrome). Inherited disorders affecting white cells, such as chronic granulomatous disease and Chédiak-Higashi syndrome, result in impaired monocyte function. Monocyte dysfunction may accompany a variety of severe illnesses, such as sepsis, trauma, and cancer. Monocytes also contribute to a variety of diseases, such as Crohn disease and rheumatoid arthritis, by virtue of their being a principal source of tumor necrosis factor. Monocytes play a pathogenetic role in other complex, acquired disorders, such as thrombosis and atherogenesis. Table 69–1 catalogues the qualitative and quantitative abnormalities of monocytes, macrophages, and dendritic cells.

Acronyms and Abbreviations: CD, cluster of differentiation; GM-CSF,

granulocyte-macrophage colony-stimulating factor; HLA-DR, human leukocyte antigen-D related; IL, interleukin; MonoMAC, monocytopenia and mycobacterial infections syndrome; TNF, tumor necrosis factor.

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CLASSIFICATION Classification of monocytic disorders is difficult because few abnormalities result solely in a disturbance of monocytes or macrophages. However, the presence of monocytopenia, monocytosis, histiocytosis, or qualitative disorders of monocytes may be an important diagnostic feature or contribute to the functional abnormality in the patient. The terms histiocyte and macrophage are synonymous. The latter term is customary when discussing the biology of the cells of the mononuclear phagocyte system, which is the total pool of marrow, blood, and tissue monocytes and macrophages, formerly referred to as the reticuloendothelial system. In disease nosology, the terms histiocyte and histiocytosis continue to be used for diseases that principally involve cells derived from blood monocytes, that is, macrophages and monocytederived dendritic cells. The physician should consider the absolute monocyte count and not the percent of cells that are monocytes when evaluating the differential blood cell count before concluding that there is an inappropriate content of blood monocytes (Chap. 70). Table 69–1 lists a classification of monocyte and macrophage disorders of relevance to hematologists.

MONOCYTOPENIA Table  69–1 contains several important causes of monocytopenia. Two notable examples of disorders accompanied by severe monocytopenia are aplastic anemia and hairy cell leukemia. Pancytopenia is usual in both conditions, but the predisposition to serious infection is heightened by the deficiency in monocyte production. In hairy cell leukemia, the severe monocytopenia represents an important diagnostic clue because of its constancy. A syndrome of profound monocytopenia, often amonocytosis, associated with susceptibility to mycobacterial avian complex, fungal, and disseminated papilloma virus infections, and subsequent development of myelodysplasia or acute myelogenous leukemia in some cases, was first described in 2010 (see Table  69–1). It is accompanied by blood B-cell lymphopenia and decreased circulating and tissue dendritic cells, but not by hypogammaglobulinemia or a deficiency of tissue macrophages or skin Langerhans (dendritic) cells. It is the result of mutations of GATA2 that impair transcription of its mRNA and is usually inherited as an autosomal recessive or can occur sporadically. The mutations of GATA2 were found in germline and hematopoietic tissues, adding it to the familial leukemia genes, as well as to a slow onset (sometimes decades), complex immunodeficiency state.

MONOCYTOSIS AND HISTIOCYTOSIS Table 69–1 contains a comprehensive list of causes of monocytosis. Monocytosis is often the manifestation of an inflammatory or a neoplastic disease. Certain hematopoietic tumors, especially acute monocytic and chronic myelomonocytic leukemia, have as their principal manifestation a predominance of monocytic cells in marrow and blood. Occasionally, chronic monocytosis can precede the onset of acute myelogenous leukemia, representing an uncommon manifestation of the myelodysplastic syndromes. Dendritic cell variants of acute myelogenous leukemia have also been discovered since the advent of immunophenotyping and genotyping of acute leukemias. The precise derivation of these myeloid dendritic cells is uncertain (i.e., granulocytic or monocytic). In some cases of monocytic leukemia, the malignant clone does not appear to include progenitors of red cells and platelets. Such cases are not likely to be the result of a mutation of a multipotential hematopoietic cell. This type of progenitor cell monocytic leukemia and other histiocytic or dendritic cell tumors support the concept that primitive

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TABLE 69–1.  Disorders of Monocytes and Macrophages I. Monocytopenia A. Aplastic anemia1 B. Hairy cell leukemia2 C. MonoMAC syndrome3–7 D. Glucocorticoid therapy8,9 II. Monocytosis A. Benign (1)  Reactive monocytosis10 (2) Exercise-induced11 B. Clonal monocytosis Indolent (1)  Chronic idiopathic monocytosis12 (2)  Oligoblastic myelogenous leukemia (myelodysplasia)13 Progressive (1)  Acute monocytic leukemia14–16 (2)  Dendritic cell leukemia17–19 (3)  Progenitor cell monocytic leukemia20 (4)  Chronic myelomonocytic leukemia21,22 (5)  Juvenile myelomonocytic leukemia23 III. Macrophage Deficiency A. Osteopetrosis (isolated osteoclast deficiency)24,25 IV. Inflammatory Histiocytosis (Chap. 71) A.  Primary hemophagocytic lymphohistiocytosis26–28 (1) Familial (2) Sporadic B.  Other inherited syndromes with hemophagocytosis lymphohistiocytosis: Chédiak-Higashi, X-linked lymphoproliferative, Gracelli29 C.  Infectious hemophagocytic histiocytosis30,31 D.  Tumor-associated hemophagocytic histiocytosis31,32 E. Drug-associated hemophagocytic histiocytosis33 F. Disease-associated hemophagocytic histiocytosis29–32 G. Juvenile rheumatoid arthritis (macrophage activation syndrome)33,34 H. Sinus histiocytosis with massive lymphadenopathy35,36 V. Storage Histiocytosis (Chap. 72) A. Gaucher disease37 B. Niemann-Pick disease38 C. Gangliosidosis39 D. Sea-blue histiocytosis syndrome40

cells, committed to the monocyte-macrophage lineage, can undergo malignant transformation (Chaps. 83 and 88). Several uncommon types of histiocytosis are serious systemic diseases that may masquerade as malignant disease. However, in such cases the cytopathologic changes in monocytes or macrophages do not constitute a malignant transformation and are not monoclonal. Familial and sporadic hemophagocytic lymphohistiocytosis, infection-induced hemophagocytic syndromes, and sinus histiocytosis with massive lymphadenopathy are among such disorders (Chap. 71). Infectious hemophagocytic histiocytosis caused by Epstein-Barr virus may be a

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VI.  Clonal (Neoplastic) Histiocytosis (Chap. 71) A. Langerhans cell histiocytosis41,42 (1) Localized (2) Systemic B. Tumors or sarcomas of histiocytes and dendritic cells43 (1)  Histiocytic sarcoma (2)  Langerhans cell sarcoma (3)  Interdigitating dendritic cell sarcoma (4)  Follicular dendritic cell sarcoma VII.  Monocyte and Macrophage Dysfunction44–46 A. α1-Proteinase inhibitor deficiency47,48 B. Chédiak-Higashi syndrome49 C. Chronic granulomatous disease50,51 D. Chronic lymphocytic leukemia52,53 E. Disseminated mucocutaneous candidiasis54,55 F. Glucocorticoid therapy56,57 G. Kawasaki disease58,59 H. Malakoplakia60 I. Mycobacteriosis syndrome61–63 J. Leprosy64 K. Posttraumatic65,66 L. Septic shock-induced67–69 M. Critically ill subjects70 N. Solid tumors71,72 O. Tobacco smoking73,74 P. Marijuana smoking or cocaine inhalation75,76 Q. Whipple disease77,78 R. Human interleukin (IL)-10 effects; Epstein-Barr virus IL-10–like gene product (vIL-10)79,80 VIII. Atherogenesis81–85 IX. Thrombogenesis85–88 X. Obesity89 XI. Aging90–92

hybrid disease because of the association with an underlying monoclonal or oligoclonal proliferation of virus-infected lymphocytes. The striking activation of macrophages and the resulting cytokine elaboration and organ pathology seen in some patients with juvenile rheumatoid arthritis, referred to as the “macrophage-activation syndrome,” is closely related to other types of hemophagocytic syndromes (Chap. 71). Pediatric rheumatologists refer to the hemophagocytic syndrome in patients with juvenile rheumatoid arthritis as the “macrophage activation syndrome,” but the clinical expression is closely analogous to other acquired hemophagocytic lymphohistiocytic syndromes. In these

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hemophagocytic syndromes, it is currently thought that the inherited or acquired inability of natural killer cells and cytotoxic T lymphocytes to modulate and, eventually, abrogate the immune response is responsible for the pathologic events of cytokine storm, fever, intravascular coagulation, organ dysfunction, and intense hemophagocytosis. Tumors of histiocytes (or dendritic cells) are rare, but can be classified into several groups with a combination of morphologic and immunophenotypic markers (Chap. 71).

Q  UALITATIVE DISORDERS OF MONOCYTES Inherited abnormalities can result in dysfunctional macrophages (see Table  69–1). In these situations the abnormality is usually shared by other leukocytes, as in chronic granulomatous disease, which results from a defect in oxygen-dependent microbial killing. In ChédiakHigashi disease, defective macrophages result from an abnormality in their cell and granule membranes (Chap. 66). An indomethacinsensitive monocyte-killing defect in children is associated with a predisposition to atypical mycobacterial disease. Also, inherited or enzyme deficiencies in macrophages can result in accumulation of undegraded macromolecules, leading to various types of storage diseases. A classic example is Gaucher disease, a disorder that results from an inherited deficiency of the enzyme glucocerebrosidase, in which tissue damage results from the engorgement of macrophages with the enzyme substrate. Recombinant glucocerebrosidase, which enters macrophage lysosomes by endocytosis, can ameliorate this macrophagic disease (Chap. 72). Acquired functional abnormalities of monocytes occur in a variety of diseases and circumstances (see “VII. Monocyte and Macrophage Dysfunction” in Table  69–1). Monocyte dysfunction occurs after severe trauma, sepsis, in other critically ill patients, and in patients with metastatic cancer. Monocyte production of interleukin (IL)-12 or maturation to dendritic cells also can be impaired in cases of severe trauma, critical illness, or metastatic cancer. Some factors, such as IL-10, impair monocyte functions. A viral IL-10–like molecule encoded by the Epstein-Barr virus BCRF1 gene also might play a role in the pathogenesis of that virus infection, and may act, in part, by inhibiting monocyte function. Tobacco smoking and marijuana smoking can result in impairment of alveolar macrophage function. In several diseases, including chronic lymphocytic leukemia, Kawasaki disease, Whipple disease, and malakoplakia, specific abnormalities of monocyte function play a significant role in the immune impairment in each disorder.

CLINICAL MANIFESTATIONS OF MONOCYTE DISORDERS MONOCYTOPENIA OR MONOCYTE DYSFUNCTION Isolated monocytopenia in the absence of any other blood cell deficiency or immune deficiency has not been reported. The manifestations of such a clinical state (pure amonocytosis) must be inferred. Neutrophils, endothelial cells, and other cell types can substitute, in part, for some monocyte functions. Monocytes have antibacterial, antiviral, antifungal, and antiparasitic capabilities. They are effective phagocytes that are involved in the ingestion and inactivation of microbes, such as mycobacteria, Listeria, Brucella, trypanosomes, and other granuloma-producing organisms. Thus, their deficiency

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or functional abnormality predisposes to such infections. In association with the amonocytosis of the MonoMAC (monocytopenia and mycobacterial infections) syndrome, opportunistic infections with mycobacteria, fungi, and viral organisms are characteristic. Macrophages can serve as a reservoir for the human immunodeficiency virus and are the principal locus for the virus in the brain and in neural tissue. Deficiency in a specific subset of macrophages, the osteoclasts, results in osteopetrosis, an imbalance in bone metabolism that favors accretion. Osteoclasts normally play a key role in the closely regulated process of bone resorption and accretion, mediating the former process. Monocyte derivatives are, thereby, involved in the development of osteoporosis and other metabolic bone diseases in which the balance tips toward resorption. Bisphosphonates can inhibit osteoclast action by interfering with its function of bone resorption and by inhibiting the mevalonate pathway to geranylgeranyl diphosphate, which prevents the transformation of monocytes to osteoclasts. Thus, the deleterious clinical manifestations of macrophages are being subdued by making the monocyte a target of therapy, in this case the prevention and amelioration of postmenopausal osteoporosis, tumor-induced bone lysis, and Paget disease, as well as of others. Macrophages and their derivatives, monocyte-derived dendritic cells, process and present antigens and play a role in immune regulation. In complex systems, such as that of antibody production, abnormal macrophages might lead to defects in humoral immunity. Activated monocytes secrete more than 50 chemical mediators or monokines, which, among other things, play a vital role in cellular immunity and inflammation. In effect, they are a critical endocrine (hormone-elaborating) apparatus. The absence of monocytes from the inflammatory response and the failure to elaborate, or the inappropriate elaboration, of monokines such as IL-1, α1-proteinase inhibitor, prostaglandins, leukotrienes, plasminogen activator, elastase, tumor necrosis factor (TNF), IL-6, IL-12, and other cytokines, may cause or contribute to disease manifestations. A deficiency or impairment of monocytes has the potential of influencing several functions and systems, because monocytes are such important sources of inflammatory cytokines (Chap. 67). In contrast, the unregulated activation of monocytes can lead to deleterious cytokine elaboration. Central to this process is TNF. The monocyte is a major source of TNF, which is a principal proinflammatory cytokine, triggering the elaboration of IL-1, IL-6, and others. Monocyte-derived TNF is also the primary chemical inducer of granuloma formation. The appreciation of its latter roles resulted in therapy to sequester TNF by antibody neutralization or receptor blockade and has resulted in substantial therapeutic effects in adult and juvenile rheumatoid arthritis, psoriasis, psoriatic arthritis, and Crohn disease. The side effects of such therapy confirm the key role of TNF in suppression of intracellular pathogens, such as Mycobacterium tuberculosis (potentiation of microbial diseases by TNF sequestrants), and in the role of the monocyte in modulating demyelinization (exacerbation of multiple sclerosis in patients treated with anti-TNF). The therapeutic administration of granulocyte-monocyte colony-stimulating factor (GM-CSF) also activates monocytes to elaborate cytokines, and this effect is being used to augment cancer vaccine therapy. Monocytopenia and decreased monocyte entry into inflammatory sites occur after glucocorticoid administration. This may explain why patients treated with glucocorticoids are predisposed to infections in which monocytes play a protective role, such as those resulting from fungal, mycobacterial, and other opportunistic organisms. Dysfunctional monocytes, incapable of killing ingested microorganisms, are present in chronic granulomatous disease (Chap. 66), as well as in hematopoietic stem cell diseases, such as monocytic variants of acute myelogenous leukemia.

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TISSUE EFFECTS OF MONOCYTOSIS Benign monocytosis is not associated with specific clinical manifestations. All forms of myelogenous leukemia with a predominance of monocytes are associated with a predisposition to troublesome tissue infiltrates, especially in the skin, gingiva, lymph nodes, meninges, and anal canal. The higher the monocyte count and the higher the proportion of leukemic monocytes, the more prevalent is tissue infiltration. In some cases, the tissue infiltration of leukemic monocytes can produce symptoms: lung dysfunction, laryngeal obstruction, and intracranial vessel rupture, as well as others. Release of procoagulants leading to intravascular coagulation also occurs in myelogenous leukemia with a high proportion of monocytes. The hyperleukocytic syndrome can occur in acute monocytic leukemia with markedly elevated white cell counts (Chaps. 83 and 88).

EFFECTS OF HISTIOCYTOSIS Hemophagocytic lymphohistiocytosis usually refers to the accumulation of activated macrophages (histiocytes) in tissue sites. The cells become intensely cytophagocytic; ingestion of red cells and occasionally of leukocytes, platelets, erythroblasts in marrow, or cells in other tissue sites is an important feature of these inflammatory histiocytoses (Chap. 71). Because morphology has been misleading, the diagnosis of histiocytosis requires identification of specific cell markers. A histiocytosis may be inflammatory (polyclonal) or neoplastic (clonal). Because tissue macrophages can take on highly specialized phenotypes and localize in different tissues, histiocytosis is further defined by whether they carry markers of these cell types (e.g., Langerhans cells, interdigitating dendritic cells; Chap. 71).

THROMBOATHEROGENESIS The complex interrelationships among monocytes, atherogenesis, and coagulation are discussed in several other chapters in the text (Chaps. 115 and 134). Monocytes may play a central role in the pathologic aspects of both processes, as a repository for tissue factor, inflammatory cytokines, and a key element in the inflammatory precursor lesions of atheroma formation (Table  69–1, sections VIII and IX).

BLOOD DENDRITIC CELLS Dendritic cells and macrophages belong to a family of antigen-presenting cells and in the laboratory can be generated from a common precursor. So-called monocyte-derived dendritic cells are easily produced in the culture vessel by the appropriate cytokines. Indeed, the use of GM-CSF as an adjuvant in cancer vaccines may relate in part to the cytokine’s ability to activate monocytes and foster conversion to dendritic (antigen-presenting) cells in vivo (Chaps. 26 and 27). Dendritic cells can be defined by phenotype into two principal types—myeloid and lymphoid (plasmacytoid) dendritic cells—of which there are likely subtypes. Monocyte-derived dendritic cells are a subset of the myeloid type (Chap. 20). Flow cytometry using cluster of differentiation (CD) markers and antidendritic cell surface antibodies have permitted the enumeration of myeloid (human leukocyte antigen-D related [HLA-DR]+, CD11c+, CD123–) and lymphocytic-plasmacytoid (HLA-DR+, CD11c–, CD123+, CD303+) dendritic cells in human blood in normal subjects and subjects with disease. Their centrality in the immune response as premier antigen-presenting cells may result in nonspecific alterations in their blood concentration or function in many generalized or localized inflammatory, infectious, and neoplastic diseases. Plasmacytoid dendritic cells may be decreased in numbers with aging, further impairing

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the immune response of older individuals (Chap. 9). Dendritic cells are also profoundly decreased in patients with hairy cell leukemia and are dysfunctional in patients with chronic lymphocytic leukemia.

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lymphocyte pattern in severely injured male trauma patients. Crit Care Med 31:1722, 2003. 66. De AK, Laudanski K, Miller-Graziano CL: Failure of monocytes of trauma patients to convert to immature dendritic cells is related to preferential macrophage-colony-stimulating factor-driven macrophage differentiation. J Immunol 170:6355, 2003. 67. Venet F, Tissot S, Debard AL, et al: Decreased monocyte human leukocyte antigen-DR expression after severe burn injury: Correlation with severity and secondary septic shock. Crit Care Med 35:1910, 2007. 68. Pachot A, Cazalis MA, Venet F, et al: Decreased expression of the fractalkine receptor CX3CR1 on circulating monocytes as new feature of sepsis-induced immunosuppression. J Immunol 180:6421, 2008. 69. Tsujimoto H, Ono S, Efron PA, et al: Role of Toll-like receptors in the development of sepsis. Shock 29:315, 2008. 70. Albaiceta GM, Pedreira PR, García-Prieto E, Taboada F: Therapeutic implications of immunoparalysis in critically ill patients. Inflamm Allergy Drug Targets 6:191, 2007. 71. Sica A, Schioppa T, Mantovani A, Allavena P: Tumour-associated macrophages are a distinct M2 polarised population promoting tumour progression: Potential targets of anti-cancer therapy. Eur J Cancer 42:717, 2006. 72. Allavena P, Sica A, Solinas G, et al: The inflammatory micro-environment in tumor progression: The role of tumor-associated macrophages. Crit Rev Oncol Hematol 66:1, 2008. 73. Ryder MI, Saghizadeh M, Ding Y, et al: Effects of tobacco smoke on secretion of interleukin 1-beta, tumor necrosis factor-alpha, and transforming growth-beta from peripheral blood mononuclear cells. Oral Microbiol Immunol 17:331, 2002. 74. Chen H, Cowan MJ, Hasday JD, et al: Tobacco smoking inhibits expression of proinflammatory cytokines and activation of IL-1R-associated kinase, p38, and NF-kappaB in alveolar macrophages stimulated with TLR2 and TLR4 agonists. J Immunol 179:6097, 2007. 75. Shay AH, Choi R, Whittaker K, et al: Impairment of antimicrobial activity and nitric acid production by alveolar macrophages from smokers of marijuana and cocaine. J Infect Dis 187:700, 2003. 76. Klein TW, Cabral GA: Cannabinoid-induced immune suppression and modulation of antigen-presenting cells. J Neuroimmune Pharmacol 1:50, 2006. 77. Marth T, Neurath M, Cuccherini BA, Strober W: Defects of monocyte interleukin 12 production an humoral immunity in Whipple’s disease. Gastroenterology 113:442, 1997. 78. Desnues B, Ihrig M, Raoult D, Mege JL: Whipple’s disease: A macrophage disease. Clin Vaccine Immunol 13:170, 2006. 79. Moore KW, de Waal Maleyt R, Coffman RL, O’Garra A: Interleukin-10 and the interleukin 10 receptor. Annu Rev Immunol 19:683, 2001. 80. Dobrovolskaia MA, Vogel SN: Toll receptors, CD14, and macrophage activation and deactivation by LPS. Microbes Infect 4:903, 2002. 81. Tousoulis D, Davies G, Stefanadis C, et al: Inflammatory and thrombotic mechanisms in coronary atherosclerosis. Heart 89:993, 2003. 82. Oliveira RT, Mamoni RL, Souza JR, et al: Differential expression of cytokines, chemokines and chemokine receptors in patients with coronary artery disease. Int J Cardiol 24:17, 2009. 83. Murphy AJ, Woollard KJ, Hoang A, et al: High-density lipoprotein reduces the human monocyte inflammatory response. Arterioscler Thromb Vasc Biol 28:2071, 2008. 84. Jawie J: New insights into immunological aspects of atherosclerosis. Pol Arch Med Wewn 118:127, 2008. 85. Brambilla M, Camera M, Colnago D, et al: Tissue factor in patients with acute coronary syndromes: Expression in platelets, leukocytes, and platelet-leukocyte aggregates. Arterioscler Thromb Vasc Biol 28:947, 2008. 86. Martin J, Collot-Teixeira S, McGregor L, McGregor JL: The dialogue between endothelial cells and monocytes/macrophages in vascular syndromes. Curr Pharm Des 13:1751, 2007. 87. Napoleone E, di Santo A, Peri G, et al: The long pentraxin PTX3 up-regulates tissue factor in activated monocytes: Another link between inflammation and clotting activation. J Leukoc Biol 76:203, 2004. 88. Key NS: Platelet tissue factor: How did it get there and is it important? Semin Hematol 45(Suppl 1):S16, 2008. 89. Weisberg SP, McCann D, Desai M, et al: Obesity is associated with macrophage accumulation in adipose tissue. J Clin Invest 112:1796, 2003. 90. Giannelli S, Taddeo A, Presicce P, et al: A six-color flow cytometric assay for the analysis of peripheral blood dendritic cells. Cytometry B Clin Cytom 74:349, 2008. 91. Koga Y, Matsuzaki A, Suminoe A, et al: Expression of cytokine-associated genes in dendritic cells (DCs): Comparison between adult peripheral blood- and umbilical cord blood-derived DCs by cDNA microarray. Immunol Lett 116:55, 2008. 92. Pérez-Cabezas B, Naranjo-Gómez M, Fernández MA, et al: Reduced numbers of plasmacytoid dendritic cells in aged blood donors. Exp Gerontol 42:1033, 2007.

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CHAPTER 70

MONOCYTOSIS AND MONOCYTOPENIA

Marshall A. Lichtman

SUMMARY The blood monocyte is in transit between the marrow and tissues where it transforms (matures) into a macrophage. In tissues, the monocyte develops a phenotype characteristic of the specific tissue of residence (e.g., Kupffer cells of liver, microglia of brain, osteoclasts of bone). Because the monocyte participates in virtually all inflammatory and immune reactions, its concentration in the blood may be increased in many such conditions, including autoimmune diseases, gastrointestinal disorders, sarcoidosis, and several viral and bacterial infections. Monocytosis, an increase in the blood absolute monocyte count to more than 800/μL (0.8 × 109/L), may occur in some patients with cancer and several unrelated conditions, such as postsplenectomy states, inflammatory bowel disease, and some chronic infections (e.g., bacterial endocarditis, tuberculosis, and brucellosis). The inconsistency and unpredictability in the blood monocyte concentration among patients with the same disease is a function of its relatively small blood pool size, the damping effect of a large tissue pool, its relatively long life span, the number and complexity of effectors in the relevant cytokine network that can influence the response, and the ability to expand macrophage numbers by local mitosis in tissues. The most striking increase in blood monocyte concentration occurs with hematopoietic malignancies, especially clonal monocytosis, and monocytic or myelomonocytic leukemia. Depression, myocardial infarction, parturition, thermal injuries, and marathon competition are closely associated with monocytosis. Table 70–1 is a comprehensive list of causes of monocytosis. Monocytopenia is notable in patients with aplastic anemia or hairy cell leukemia as a feature of pancytopenia. Although other cytopenias accompany the monocytopenia, the latter contributes significantly to the predisposition to infection and in hairy cell leukemia is an aid to diagnosis because of its constancy. The MonoMAC syndrome, the result of GATA2 mutations, is associated with extreme monocytopenia and amonocytosis.

The blood monocyte is a cell in transit from marrow to tissues.1 There are two major populations of blood monocytes based on physical properties: a smaller population thought to represent a less-mature stage, has a higher buoyant density, a smaller cell volume, lacks Fc receptors,

Acronyms and Abbreviations: CD, cluster of differentiation; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-monocyte colony-stimulating factor; IL, interleukin; LPS, lipopolysaccharide; M-CSF, monocyte/macrophage colony-stimulating factor; MDS, myelodysplastic syndrome; MonoMAC, monocytopenia and Mycobacterium avium complex; NK, natural killer.

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and has greater tumoricidal activity; the larger population represents a more-mature stage, has a lower buoyant density, has a larger cell volume, displays Fc receptors, expresses more peroxidase activity, secretes larger amounts of interleukin (IL)-1, presents antigen, and mediates antibody-dependent cell-mediated cytotoxicity more efficiently. The larger population, classical monocytes that are highly phagocytic and proinflammatory, composes approximately 90 percent of blood monocytes, and strongly expresses CD14 (lipopolysaccharide receptor) but does not express CD16 (FcγRIII), designated the CD14++CD16− subset. These monocytes carry chemokine receptors CCR2hiCX3CR1lo. Of the remaining monocyte population, approximately 5 percent exhibit strong expression of CD14 and modest expression of CD16, the CD14++CD16+ “intermediate” subset, which expresses the chemokine receptors CCR2midCX3CR1hiCCR5mid, are proinflammatory and less phagocytic, and the “nonclassical” subset, which exhibits strong expression of CD16, the CD14+CD16++ subset, which expresses the chemokines CCRloCX3CR1hi, the so-called patrolling subset.2 The latter subset contains dendritic cell precursors.3 The major subsets can each be further stratified based on the expression of CD64 (FcγRI) (Chaps. 67 and 68).4 In tissues the monocyte is capable of transformation, under the influence of local environmental factors, into a macrophage. The monocyte plays an important role in acute and chronic inflammatory reactions, including granulomatous inflammation; immunologic reactions, including those involved in delayed hypersensitivity; tissue repair and reorganization; atheroma and thrombus formation; and the reaction to neoplasia and allografts. Because of the key role of monocytes in a variety of pathophysiologic reactions, a modest elevation in blood monocyte count can occur in many disparate conditions. In addition, in circumstances in which large increases in the number of macrophages are required in tissue sites, the demand may be met by local proliferation of macrophages and not be reflected either in an increased transit of monocytes through the blood compartment from marrow to tissue or in an increased concentration of blood monocytes.5 Occasionally, T-cell clones release only macrophage/monocyte colony-stimulating factor (M-CSF), which can stimulate the growth of macrophage colonies, providing a model for local control of macrophage proliferation.6

NORMAL BLOOD MONOCYTE CONCENTRATION In the first 2 weeks of life, the average absolute blood monocyte count is approximately 1000/μL (1 × 109/L; Chap. 7). There is a gradual decline in the normal monocyte count to a mean of 400/μL (0.4 × 109/L) in adulthood, at which time monocytes constitute 1 to 9 percent (mean: 4 percent) of blood leukocytes (Chap. 2). Monocytosis is present when the absolute count exceeds 800/μL (0.8 × 109/L) in adults. Men tend to have slightly higher monocyte counts than women.7 Increments in the number of blood monocytes correlate directly with increases in the total blood monocyte pool and the monocyte turnover rate.8 The blood monocyte count cycles with a periodicity of 5 days.9 Older persons have a decrease in the proportion of CD14++CD16− to CD14+CD16+ monocytes as compared to younger persons, although the functional significance of this difference has not been established.10

DISORDERS ASSOCIATED WITH MONOCYTOSIS Table  70–1 outlines the diseases reported to be associated with monocytosis. In one review, hematologic disorders represented more than 50 percent, collagen vascular diseases approximately 10 percent, and malignant disease approximately 8 percent of cases of monocytosis.11

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TABLE 70–1.  Disorders Associated with Monocytosis I.  Hematologic Disorders A.  Myeloid neoplasms 1. Myelodysplastic syndromes12–16 2.  Primary myelofibrosis17 3.  Acute monocytic leukemia18,19 4.  Acute myelomonocytic leukemia20 5.  Acute monocytic leukemia with histiocytic features21 6.  Acute myeloid dendritic cell leukemia22–24 7.  Chronic myelomonocytic leukemia25–27 8. Juvenile myelomonocytic leukemia28 9.  Chronic myelogenous leukemia (m-BCR–positive type) 29,30 10.  Polycythemia vera11 11.  Primary myelofibrosis17 B.  Chronic neutropenias31–36 C.  Drug-induced neutropenia37–39 D.  Postagranulocytic recovery40,41 E.  Lymphocytic neoplasms 1. Lymphoma43 2.  Hodgkin lymphoma44,45 3. Myeloma46,47 4. Macroglobulinemia48 5.  T-cell lymphoma49,50 6.  Chronic lymphocytic leukemia51 F.  Drug-induced pseudolymphoma52 G.  Immune hemolytic anemia11 H.  Idiopathic thrombocytopenic purpura11 I.  Postsplenectomy state53,54 II.  Inflammatory and Immune Disorders A.  Connective tissue diseases 1. Rheumatoid arthritis55 2.  Systemic lupus erythematosus56 3.  Temporal arteritis11 4. Myositis11 5.  Polyarteritis nodosa11 6. Sarcoidosis57,58

HEMATOLOGIC DISORDERS Approximately 25 percent of patients with a myelodysplastic syndrome have an increase in the absolute monocyte count.12–16 Occasional patients with a myelodysplastic syndrome may develop an absolute monocyte count as high as 30,000/μL (30 × 109/L). Chronic monocytosis may be the principal feature of a clonal myeloid disease and precede by years the development of acute myelogenous leukemia. Patients with myelodysplasia and monocytosis have a high propensity to evolve into acute or chronic myelomonocytic leukemia. Monocytosis, as a feature of primary myelofibrosis, may be a harbinger of rapid progression.17 The number of promonocytes and monocytes in blood and marrow may be increased in patients with acute myelogenous leukemia of the monocytic18,19 or myelomonocytic type.20 Acute myelogenous leukemic cells with a histiocytic (macrophagic)21 or dendritic cell phenotype have been described.22–24 Patients with chronic myelomonocytic leukemia

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B. Infections 1. Mycobacterial infections59–62 2.  Subacute bacterial endocarditis63–65 3. Brucellosis66 4.  Dengue hemorrhagic fever67 5.  Resolution phase of acute bacterial infections68 6. Syphilis69,70 7.  Cytomegalovirus infection71 8.  Varicella-zoster virus72 9. Influenza73 III.  Gastrointestinal Disorders A.  Alcoholic liver disease74 B.  Inflammatory bowel disease75 C. Sprue11 IV.  Nonhematopoietic Malignancies76–79 V.  Exogenous Cytokine Administration80–86 VI.  Myocardial Infarction87–90 VII.  Cardiac Bypass Surgery91 VIII.  Miscellaneous Conditions A.  Tetrachloroethane poisoning92 B. Parturition93,94 C.  Glucocorticoid administration95–98 D. Depression99–101 E.  Thermal injury102,103 F.  Marathon running104,105 G. Holoprosencephaly106 H.  Kawasaki disease107 I.  Wiskott-Aldrich syndrome108 J. Hemodialysis109

have, by definition, an increased absolute number of monocytes in the blood (≥1.0 × 109/L). The monocytosis may be more striking in some cases.25–27 Juvenile myelomonocytic leukemia, also, is defined in part by the increased number of monocytes in the blood and marrow.28 In some cases of acute monocytic leukemia, the monocytes are immature and have features of monoblasts or promonocytes, but in some cases they are indistinguishable by light microscopy from normal blood monocytes. Some automated instruments are dependent on the α-naphthol acetate esterase reaction to detect the proportion of monocytes in white cell differential counts. These instruments may underestimate leukemic monocytes counts, especially in cases of chronic myelomonocytic leukemia, because the leukemic monocytes have a decreased activity of the enzyme.25 An uncommon variant of Ph-positive chronic myelogenous leukemia (CML), expressing a p190 BCR-ABL transcript, is associated with a striking monocytosis in approximately 50 percent of cases.29,30

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Chapter 70: Monocytosis and Monocytopenia

Monocytosis occurs in a number of neutropenic states: cyclic neutropenia,31 chronic granulocytopenia of childhood,32 familial benign chronic neutropenia,33 infantile genetic agranulocytosis34,35 and chronic hypoplastic neutropenia.36 In human cyclic neutropenia, monocyte oscillation is reciprocal to the neutrophil cycle; the peak monocytosis, which often exceeds 2000/μL (2.0 × 109/L), occurs at the end of the neutropenic period. Monocytes often stay above 500/μL (0.5 × 109/L) throughout the cycle. In the variety of other neutropenias mentioned, monocytopoiesis often is preserved in the face of neutropenia. Transient elevations of the monocyte count have been reported in the acute phases of drug-induced agranulocytosis.37–39 Monocytosis characteristically appears later in the recovery phase of agranulocytosis and may be a harbinger of recovery.37,40,41 Some observers dispute the validity of the latter observation.42 Monocytosis can occur with lymphomas and can increase with exacerbation of disease activity.43 Monocytosis has been noted in approximately 25 percent of cases of Hodgkin lymphoma, although it does not correlate with prognosis.43,44 In contrast, one treatise on the disease reports the hematologic values of patients with Hodgkin lymphoma at the time of diagnosis; only 4 of 100 have nominal increases in absolute blood monocyte counts.45 A statistically significant increase in blood monocyte concentration has been reported in myeloma and has been correlated with the presence of λ light-chain-containing monoclonal immunoglobulin.46,47 Rare cases of M-CSF secreting lymphoid tumors have been associated with monocytosis.48,49 Monocytosis at diagnosis has been correlated with decreased survival in several lymphoma types and chronic lymphocytic leukemia.50,51 Pseudolymphoma syndrome, induced by drugs such as carbamazepine, phenytoin, phenobarbital, and valproic acid, is associated with monocytosis.52

SPLENECTOMY Monocytosis is a common feature in individuals who have had splenectomy.53,54

INFLAMMATORY AND IMMUNE DISORDERS Connective tissue diseases, including rheumatoid arthritis,55 systemic lupus erythematosus, temporal arteritis, myositis, and periarteritis nodosa, may be associated with monocytosis, although monocytosis is not common in these diseases.11 The usual alterations of the white cell count in systemic lupus erythematosus, for example, are neutropenia and lymphopenia, but 10 percent of patients have a mild monocytosis.56 An elevation of the blood monocyte count occurs in sarcoidosis57 and is inversely related to a reduction in circulating T lymphocytes.58 Infectious diseases are an uncommon cause of monocytosis. Only a few instances of infection were noted in a comprehensive review of causes of monocytosis, including tonsillitis, dental infection, recurrent liver abscesses, candidiasis, and one instance of tuberculous peritonitis.11 Tuberculosis was once a leading cause of monocytosis, because of the role of monocytes in granuloma (tubercle) formation. Neither the monocyte count nor the ratio of monocytes to lymphocytes correlates with the stage or activity of tuberculosis.59–61 Mycobacterium fortuitum infection, usually in the setting of AIDS, also is associated with monocytosis.62 Monocytosis is found in 15 to 20 percent of patients with subacute bacterial endocarditis,63,64 but is not correlated with the presence of blood macrophages, which may be present in this disease.65 A number of infections formerly thought to be associated with monocytosis are not, when examined systematically. These include rickettsial diseases, leishmaniasis, typhoid fever, malaria, and disseminated candidiasis, brucellosis,66 and dengue hemorrhagic fever.67

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A monocytosis in the resolution phase of acute infections has been noted,68 and monocytosis occurs in cases of neonatal, primary, and secondary syphilis.69,70 Certain viruses, especially cytomegalovirus varicella-zoster virus, and influenza virus induce an increase in blood monocytes.71–73

GASTROINTESTINAL DISEASES Sprue, ulcerative colitis, regional enteritis, and alcoholic liver disease are associated with monocytosis.11,74,75

NONHEMATOPOIETIC MALIGNANCIES Sixty percent of patients with nonhematologic malignancy exhibit a monocytosis that is independent of the presence or absence of metastatic disease.76 An inverse relationship of monocyte count (elevated) and T-lymphocyte concentration (decreased) has also been noted in patients with malignant disease.77 Reports of hematologic values in metastatic colon cancer and soft-tissue sarcoma have emphasized the frequency of monocytosis in patients with cancer.78,79 Consequently, if unexplained monocytosis persists, malignancy should be considered.

EXOGENOUS CYTOKINE ADMINISTRATION The administration of granulocyte-macrophage colony-stimulating factor (GM-CSF),80 IL-10,81 or granulocyte colony-stimulating factor (G-CSF)82,83 may result in mild increases in blood monocyte counts. Administration of M-CSF84,85 results in an invariable increase in blood monocytes. Doses of 40 to 120 mcg/kg per day result in the peak increase, which may reach three- to fourfold baseline, in approximately 8 days. Administration of human macrophage inflammatory protein-1α to patients or normal volunteers is associated with a brief monocytopenia followed by a monocytosis that is proportional to the dose administered.86

MYOCARDIAL INFARCTION Monocytosis occurs after myocardial infarction, reaching a peak on day 3. A correlation exists between serum creatine kinase activity and monocyte count, suggesting a relationship between extent of infarction and monocytosis.87 After myocardial infarction, persistent monocytosis is correlated with pump failure.88–90 Monocytosis is a frequent finding after cardiopulmonary bypass surgery.91 In the latter circumstance, CD14 (lipopolysaccharide [LPS] receptor) is markedly decreased on the monocyte surface and plasma-soluble CD14 is increased, changes compatible with monocyte activation.

MISCELLANEOUS CONDITIONS Other disorders associated with monocytosis include tetrachloroethane poisoning.92 Monocytosis is a frequent finding at the time of parturition.93,94 An increase in blood monocytes occurs in healthy volunteers95,96 and in patients with myelodysplastic syndrome (MDS)97,98 who are given moderately high, therapeutic-level doses of glucocorticoids. Psychiatric depression is associated with a conjoint increase in neutrophils and monocytes.99–101 The monocytosis in depressive and anxiety disorders is associated with high plasma levels of β endorphins and dysfunctional (hypophagocytic) monocytes.101 Thermal injury is accompanied by monocytosis.102,103 Competitive marathon runners have a monocytosis associated with elevated plasma levels of several cytokines, including M-CSF.104,105 An increase in blood monocytes accompanies several rare syndromes: holoprosencephaly,106 Kawasaki disease,107 and Wiskott-Aldrich.108

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BLOOD MONOCYTE SUBSET COUNTS IN DISEASE Differential monocyte subset responses (CD14++CD16− vs. CD14+CD16+) without deviation of total monocyte counts outside the normal range have been observed in older subjects and those with sepsis, AIDS, allergic disorders, dermatitides, hemodialysis, and atherosclerosis.4,10,91,109 These monocytic subset variations usually are not measured in clinical laboratories and, as yet, have little diagnostic or prognostic importance.

DISORDERS ASSOCIATED WITH MONOCYTOPENIA Table 70–2 lists the disorders associated with monocytopenia. Although monocytopenia may occur in any hematopoietic multipotential cell disease associated with pancytopenia (e.g., acute myelogenous leukemia), a decrease in monocytes is notable and constant in aplastic anemia110 and hairy cell leukemia,111 in which monocytopenia can be a helpful diagnostic clue and also a contributor to the predisposition to infection, which is an important, morbid feature of the disease. Monocytopenia occurs in a small proportion of patients with chronic lymphocytic leukemia and these patients may have a higher frequency of infections, especially by viruses.112 Severe thermal injuries also can result in monocytopenia.113 Cyclic neutropenia is also notable for intermittent periods of monocytopenia.114 Rare cases of conjoint severe neutropenia and monocytopenia occur.115 Transient monocytopenia is a feature of hemodialysis, but monocyte counts return to normal within hours after the procedure ends.109 In contrast, to reports of monocytosis noted above in “Inflammatory and Immune Disorders,” automated blood cell counts in large numbers of subjects find that a decreased absolute monocyte count is frequent in patients with rheumatoid arthritis116 or systemic lupus erythematosus,117 and in those with human immunodeficiency virus infection.118 One has to presume that these contrasting results relate to stage or activity of disease at the time of measurement. In 2010, a disease was described in which extreme monocytopenia, and sometimes amonocytosis, was the most striking abnormality in the blood counts.119 It has been named the MonoMAC syndrome because of the monocytopenia (mono) and the frequency of Mycobacterium avium complex (MAC) opportunistic infections, although persistent fungal and viral infections (especially papillomavirus), also occur. Marked decreases in blood B-cell, natural killer (NK)-cell, and dendritic cell counts are characteristic.120,121 The disease is the result of mutations in the GATA2 gene that decrease transcription of the gene message.122 It may present as an atypical type of MDS with a hypocellular marrow, but with striking dysmorphic megakaryocytes and micromegakaryocytes,

TABLE 70–2.  Disorders Associated with Monocytopenia I.  Any cause of severe leukopenia A.  Aplastic anemia110 B.  Hairy cell leukemia111 C. Other myeloid or lymphoid malignancies resulting in suppression of monocytopoiesis II. MonoMAC syndrome120–123 and Emberger syndrome124,125 (GATA2 gene mutations) III. Miscellaneous conditions (see section “Disorders Associated with Monocytopenia”)

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or with acute myelogenous leukemia.123 Because of the GATA2 gene product’s role in the development of the vascular and lymphatic systems, some cases may have the triad of lymphedema, monosomy 7, and myelodysplasia or acute myelogenous leukemia, designated the Emberger syndrome.124–127 Hematopoietic stem cell transplantation has been successful in restoring normal immunohematopoiesis in some of the patients so treated.128 Glucocorticoid hormones produce a monocytopenia, transiently, approximately 6 hours after administration to human volunteers129,130 or to patients.95 Administration of interferon-α and tumor necrosis factor-α may also cause monocytopenia.131 Monocytopenia may follow radiotherapy.132

BLOOD DENDRITIC CELL COUNTS Blood dendritic cells are composed of two phenotypic subtypes: myeloid-derived (HLA-DR+CD11c+CD123+) and lymphoid-plasmacytoidderived (HLA-DR+CD11c−CD123+). The total blood dendritic cell count can be measured by flow cytometry.133–135 Dendritic cells make up approximately 0.6 percent of blood cells (range: 0.15 to 1.30 percent) and represent 14 × 106 cells/L (range: 3 to 30 × 106 cells/L). Approximately one-third of these cells are a lymphoid-plasmacytoid–derived type and two-thirds are a myeloid-derived type.135–137 Fluctuations in blood dendritic cells are often independent of changes in total blood monocyte count. Blood dendritic cell counts decrease with aging138 and increase with surgical stress137 (and presumably other stressful reactions) in relation to plasma cortisol levels.

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18. Haferlach T, Schoch C, Schnittger S, et al: Distinct genetic patterns can be identified in acute monoblastic leukaemia (FAB AML M5a and M5b): A study of 124 patients. Br J Haematol 118:426, 2002. 19. Villeneuve P, Kim DT, Xu W, et al: The morphological subcategories of acute monocytic leukemia (M5a and M5b) share similar immunophenotypic and cytogenetic features and clinical outcomes. Leuk Res 32:269, 2008. 20. Sun X, Zhang W, Ramdas L, et al: Comparative analysis of genes regulated in acute myelomonocytic leukemia with and without inv(16)(p13q22) using microarray techniques, real-time PCR, immunohistochemistry, and flow cytometry immunophenotyping. Mod Pathol 20:811, 2007. 21. Laurencet FM, Chapius B, Roux-Lombard P, et al: Malignant histiocytosis in the leukaemic stage: A new entity (M5c-AML) in the FAB classification? Leukemia 8:502, 1994. 22. Ferran M, Gallardo F, Ferrer AM, et al: Acute myeloid dendritic cell leukaemia with specific cutaneous involvement: A diagnostic challenge. Br J Dermatol 158:1129, 2008. 23. Santiago-Schwartz F, Coppock DL, Hindenberg AA, Kern J: Identification of a malignant counterpart of the monocytic-dendritic cell progenitor in an acute myeloid leukemia. Blood 84:3054, 1994. 24. Lichtman MA, Segel GB: Uncommon phenotypes of acute myelogenous leukemia: Basophilic, mast cell, eosinophilic, and myeloid dendritic cell subtypes: A review. Blood Cells Mol Dis 35:370, 2005. 25. Frew ME, Donaldson K: Monocyte analysis in chronic myelomonocytic leukaemia. Br J Biomed Sci 54:244, 1997. 26. Onida F, Kantarjian HM, Smith TL, et al: Prognostic scoring factors and scoring systems in chronic myelomonocytic leukemia: A retrospective analysis of 213 patients. Blood 99:840, 2002. 27. Xu Y, McKenna RW, Karandikar NJ, et al: Flow cytometric analysis of monocytes as a tool for distinguishing chronic myelomonocytic leukemia from reactive monocytosis. Am J Clin Pathol 124:799, 2005. 28. Kratz CP, Niemeyer CM: Juvenile myelomonocytic leukemia. Hematology 1:100, 2005. 29. Ohsaka A, Shiina S, Kobayashi M, et al: Philadelphia chromosome-positive chronic myeloid leukemia expressing p190(BCR-ABL). Intern Med 41:1092, 2002. 30. Hur M, Song HM, Kang SH, et al: Lymphoid predominance and the absence of basophilia and splenomegaly are frequent in m-bcr-positive chronic myelogenous leukemia. Ann Hematol 81:219, 2002. 31. Wright D, Dale DC, Fauci AS, Wolff SM: Human cyclic neutropenia: Clinical review and long-term follow-up of patients. Medicine (Baltimore) 60:1, 1981. 32. Zuelzer WW, Bajoghli M: Chronic granulocytopenia in childhood. Blood 23:359, 1964. 33. Cutting HO, Lang JE: Familial benign chronic neutropenia. Ann Intern Med 61:876, 1964. 34. Krill CE, Mauer AM: Congenital agranulocytosis. J Pediatr 68:361, 1966. 35. Lang JE, Cutting HO: Infantile genetic agranulocytosis. Pediatrics 35:596, 1965. 36. Spaet TH, Dameshek W: Chronic hypoplastic neutropenia. Am J Med 13:35, 1952. 37. Robinson RL, Burk MS, Raman S: Fever, delirium, autonomic instability, and monocytosis associated with olanzapine. J Postgrad Med 49:96, 2003. 38. Graf M, Tarlov A: Agranulocytosis with monohistiocytosis associated with ampicillin therapy. Ann Intern Med 69:91, 1968. 39. Thöne J, Kessler E: Monocytosis subsequent to ziprasidone treatment: A possible side effect. Prim Care Companion J Clin Psychiatry 9:465, 2007. 40. Reznikoff P: The etiologic importance of fatigue and the prognostic significance of monocytosis in neutropenia (agranulocytosis). Am J Clin Pathol 6:205, 1936. 41. Rosenthal N, Abel HA: The significance of the monocytes in agranulocytosis (leukopenic infectious agranulocytosis). Am J Clin Pathol 6:205, 1936. 42. Pretty HM, Gosselin G, Colprian G, Long LA: Agranulocytosis: A report of 30 cases. Can Med Assoc J 93:1058, 1965. 43. Rosenberg SA, Diamond HD, Jaslowitz B, Craver LF: Lymphosarcoma: A review of 1269 cases. Medicine (Baltimore) 40:31, 1961. 44. Ultmann JE: Clinical features and diagnosis of Hodgkin’s disease. Cancer 9:297, 1966. 45. Kaplan HS: Hodgkin’s Disease, 2nd ed, Table 4.1, pp 127–128. Harvard University Press, Cambridge, MA, 1980. 46. Sewell RL: Lymphocyte abnormalities in myeloma. Br J Haematol 36:545, 1977. 47. Blom J, Nielsen H, Larsen SO, et al: A study of certain functional parameters of monocytes from patients with multiple myeloma: Comparison with monocytes from healthy individuals. Scand J Haematol 33:425, 1984. 48. Nakajima H, Mori S, Takeuchi T, et al: Monocytosis and high serum macrophage colony-stimulating factor in Waldenström’s macroglobulinemia. Blood 86:2863, 1995. 49. Tokioka T, Shimamoto Y, Motoyoshi K, Yamaguchi M: Clinical significance of monocytosis and human monocytic colony stimulating factor in patients with adult T-Cell leukaemia/lymphoma. Haematologia (Budap) 26:1, 1994. 50. Bari A, Tadmor T, Sacchi S, et al: Monocytosis has adverse prognostic significance and impacts survival in patients with T-cell lymphomas. Leuk Res 37:619, 2013. 51. Mazumdar R, Evans P, Culpin R, et al: The automated monocyte count is independently predictive of overall survival from diagnosis in chronic lymphocytic leukaemia and of survival following first-line chemotherapy. Leuk Res 37:614, 2013. 52. Choi TS, Doh KS, Kim SH, et al: Clinicopathological and genotypic aspects of anticonvulsant-induced pseudolymphoma syndrome. Br J Dermatol 148:730, 2003. 53. Durig M, Landmann RMA, Harder F: Lymphocyte subsets in human peripheral blood after splenectomy and autotransplantation of splenic tissue. J Lab Clin Med 104:110, 1984. 54. Lanng Nielson J, Romer FK, Ellegaard J: Serum angiotensin-converting enzyme and blood monocytes in splenectomized individuals. Acta Haematol 67:132, 1982.

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55. Buchan GS, Palmer DG, Gibbins BL: The response of human peripheral blood mononuclear phagocytes to rheumatoid arthritis. J Leukoc Biol 37:221, 1985. 56. Budman DR, Steinberg AD: Hematologic aspects of systemic lupus erythematosus. Current concepts. Ann Intern Med 86:220, 1977. 57. Goodwin JS, DeHaratius R, Israel H, et al: Suppressor cell function in sarcoidosis. Ann Intern Med 90:169, 1979. 58. Daniele RP, Dauber JH, Rossman MD: Immunologic abnormalities in sarcoidosis. Ann Intern Med 92:406, 1980. 59. Stobie W, England NJ, McMenemy WH: The interpretation of haemograms in pulmonary tuberculosis. Am Rev Tuberc 46:1, 1942. 60. Flinn JW: A study of the differential blood count in 1000 cases of active pulmonary tuberculosis. Ann Intern Med 2:622, 1929. 61. Singh KJ, Ahluwalia G, Sharma SK, et al: Significance of haematological reactions in patients with tuberculosis. J Assoc Physicians India 49:788, 2001. 62. Smith MB, Schnadig VJ, Boyars MC, Woods GL: Clinical and pathological features of Mycobacterium fortuitum infections: An emerging pathogen in patients with AIDS. Am J Clin Pathol 116:225, 2001. 63. Daland GA, Gottlieb L, Wallerstein RO, et al: Hematologic observations in bacterial endocarditis. J Lab Clin Med 48:827, 1956. 64. Myhre EB, Braconier JH, Sjögren U: Automated cytochemical differential leukocyte count in patients hospitalized with acute bacterial infections. Scand J Infect Dis 17:201, 1985. 65. Hill RW, Bayrd ED: Phagocytic reticuloendothelial cells in subacute bacterial endocarditis with negative cultures. Ann Intern Med 52:310, 1960. 66. Tsolia M, Drakonaki S, Messaritaki A, et al: Clinical features, complications and treatment outcome of childhood brucellosis in central Greece. J Infect 44:257, 2002. 67. Khan E, Siddiqui J, Shakoor S, et al: Dengue outbreak in Karachi, Pakistan, 2006: Experience at a tertiary care center. Trans R Soc Trop Med Hyg 101:1114, 2007. 68. Hickling RA: The monocytes in pneumonia: A clinical and hematologic study. Arch Intern Med 40:594, 1927. 69. Rosahn PD, Pearce L: The blood cytology in untreated and treated syphilis. Am J Med Sci 187:88, 1934. 70. Karyalcin G, Khanijou A, Kim KY, et al: Monocytosis in congenital syphilis. Am J Dis Child 131:782, 1977. 71. Klemola E: Cytomegalovirus infection in previously healthy adults. Ann Intern Med 79:267, 1973. 72. Tsukahara T, Yogushi A, Horiuchi Y: Significance of monocytosis in varicella herpes zoster. J Dermatol 19:94, 1992. 73. McClain MT, Park LP, Nicholson B, et al: Longitudinal analysis of leukocyte differentials in peripheral blood of patients with acute respiratory viral infections. J Clin Virol 58:689, 2013. 74. McKeever UM, O’Mahoney C, Lawlor E, et al: Monocytosis: A feature of alcoholic liver disease. Lancet 2:1492, 1983. 75. Mees AS, Berney J, Jewell DP: Monocytes in inflammatory bowel disease: Absolute monocyte counts. J Clin Pathol 33:917, 1980. 76. Barrett O Jr: Monocytosis in malignant disease. Ann Intern Med 73:991, 1970. 77. Wood GW, Neff JE, Stephens R: Relationship between monocytosis and T-lymphocyte function in human cancer. J Natl Cancer Inst 63:587, 1979. 78. Melichar B, Touskova M, Vesely P: Effect of irinotecan on the phenotype of peripheral blood leukocyte populations in patients with metastatic colorectal cancer. Hepatogastroenterology 49:967, 2002. 79. Ruka W, Rutkowski p, Kaminska J, et al: Alterations of routine blood tests in adult patients with soft tissue sarcomas: Relationships to cytokine serum levels and prognostic significance. Ann Oncol 12:1423, 2001. 80. Schmitz LL, McClure JS, Litz CE, et al: Morphologic and quantitative changes in blood and marrow cells following growth factor therapy. Am J Clin Pathol 101:67, 1994. 81. Chernoff AE, Granowitz EV, Shapiro L, et al: A randomized controlled trial of IL-10 in humans. J Immunol 154:5492, 1995. 82. Ranaghan L, Drake M, Humphreys MW, Morris TC: Leukaemoid monocytosis in M4 AML following chemotherapy: G-CSF. Clin Lab Haematol 20:49, 1998. 83. Liu CZ, Persad R, Inghirami G, et al: Transient atypical monocytosis mimic acute myelomonocytic leukemia in post-chemotherapy patients receiving G-CSF: Report of two cases. Clin Lab Haematol 26:359, 2004. 84. Weiner LM, Li W, Holmes M, et al: Phase I trial of recombinant macrophage colonystimulating factor and recombinant gamma-interferon: Toxicity, monocytosis, and clinical effects. Cancer Res 54:4084, 1994. 85. Minasian LM, Yao TJ, Steffens TA, et al: A phase I study of anti-GD3 ganglioside monoclonal antibody R24 and recombinant human macrophage-colony stimulating factor in patients with metastatic melanoma. Cancer 75:2251, 1995. 86. Marshall E, Howell AH, Powles R, et al: Clinical effects of human macrophage inflammatory protein-1 alpha MIP-1 alpha (LD78) administration in humans. Eur J Cancer 34:1023, 1998. 87. Meisel SR, Panzner H, Schecter M, et al: Peripheral monocytosis following myocardial infarction. Cardiology 90:52, 1998. 88. Maekawa Y, Anzai T, Yoshikawa T, et al: Prognostic significance of peripheral monocytosis after reperfusion acute myocardial infarction: Possible role for left ventricular remodeling. J Am Coll Cardiol 16:241, 2002. 89. Gibson WJ, Gibson CM: The association of impaired myocardial perfusion and monocytosis with late recovery of left ventricular function following primary percutaneous coronary intervention. Eur Heart J 27:2487, 2006.

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90. Hong YJ, Jeong MH, Ahn Y, et al: Relationship between peripheral monocytosis and nonrecovery of left ventricular function in patients with left ventricular dysfunction complicated with acute myocardial infarction. Circ J 71:1219, 2007. 91. Fingerle-Rowson G, Auers J, Kreuzer E, et al: Down-regulation of surface monocyte lipopolysaccharide-receptor CD14 in patients on cardiopulmonary bypass undergoing aorta-coronary bypass operation. J Thorac Cardiovasc Surg 115:1172, 1998. 92. Minot GR, Smith LW: The blood in tetrachloroethane poisoning. Arch Intern Med 28:687, 1921. 93. Siegal I, Gleichner N: Peripheral white blood cells alterations in early labor. Diagn Gynecol Obstet 3:123, 1981. 94. Buchan GS, Gibbins BL, Griffin JFT: The influence of parturition on peripheral blood mononuclear phagocyte subpopulation in pregnant women. J Leukoc Biol 37:231, 1985. 95. Rinehard JJ, Sagone AL, Balcerzak SP, et al: Effects of corticosteroid therapy on human monocyte function. N Engl J Med 292:236, 1975. 96. Shoenfeld Y, Gurewich Y, Gallant LA, et al: Prednisone-induced leukocytosis. Am J Med 71:773, 1981. 97. Morales M, Wilkes J, Lowder JN: Monocytic leukemoid reaction, glucocorticoid therapy, and myelodysplastic syndrome. Cleve Clin J Med 6:571, 1990. 98. Barker S, Scott M, Chan GT. Corticosteroids and monocytosis. N Z Med J 125:76, 2012. 99. Maes M, VanDerPlanken M, Stevens WJ, et al: Leukocytosis, monocytosis and neutrophilia: Hallmarks of severe depression. J Psychiatr Res 26:125, 1992. 100. Maes M, Lambrechts J, Suy E, et al: Absolute number and percentage of circulating natural killer, non-MHC-restricted T cytotoxic, and phagocytic cells in unipolar depression. Neuropsychobiology 29:157, 1994. 101. Castilla-Cortazar I, Castilla A, Gurpegui M: Opioid peptides and immunodysfunction in a patient with major depression and anxiety disorders. J Physiol Biochem 54:203, 1998. 102. Santangelo S, Gamelli RL, Shankar R: Myeloid commitment shifts toward monocytopoiesis after thermal injury and sepsis. Ann Surg 233:97, 2001. 103. Lovell R, Madden L, McNaughton LR, Carroll S: Effects of active and passive hyperthermia on heat shock protein 70 (HSP70). Amino Acids 34:203, 2008. 104. Kratz A, Lewandrowski KB, Siegel AJ, et al: Effect of marathon running on hematologic and biochemical laboratory parameters, including cardiac markers. Am J Clin Pathol 118:856, 2002. 105. Suzuki K, Nakaji S, Yamadi M, et al: Impact of a competitive marathon race on systemic cytokine and neutrophil responses. Med Sci Sports Exerc 35:348, 2003. 106. Jubinsky PT, Shanske AL, Pixley FJ, et al: A syndrome of holoprosencephaly, recurrent infections, and monocytosis. Am J Med Genet A 140:2742, 2006. 107. Kuo HC, Wang CL, Liang CD, et al: Persistent monocytosis after intravenous immunoglobulin therapy correlated with the development of coronary artery lesions in patients with Kawasaki disease. J Microbiol Immunol Infect 40:395, 2007. 108. Watanabe N, Yoshimi A, Kamachi Y, et al: Wiskott-Aldrich syndrome is an important differential diagnosis in male infants with juvenile myelomonocytic leukemia like features. J Pediatr Hematol Oncol 29:836, 2007. 109. Nockher WA, Wiemer J, Scherberich JE: Hemodialysis monocytopenia: Differential sequestration kinetics of CD14+CD16+ and CD14++ blood monocyte subsets. Clin Exp Immunol 123:49, 2001. 110. Twormey JJ, Douglas CC, Sharkey O Jr: The monocytopenia of aplastic anemia. Blood 41:187, 1973. 111. den Ottolander GJ, van der Burgh FJ, Lopes Cardozo P, et al: The Hemalog D automated differential counter in the diagnosis of hairy cell leukemia. Leuk Res 7:309, 1983. 112. DeRossi G, Mauro FR, Ialongo P, et al: Monocytopenia and infections in chronic lymphocytic leukemia (CLL). Eur J Haematol 46:119, 1991. 113. Peterson V, Hensbrough J, Buerk C, et al: Regulation of granulopoiesis following severe thermal injury. J Trauma 23:19, 1983. 114. Adams WH, Liu YK: Periodic neutropenia and monocytopenia. Am J Hematol 13:73, 1982.

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115. Marinone G, Roncoli B, Marinone MG Jr: Pure white cell aplasia. Semin Hematol 28:298, 1991. 116. Isenberg DA, Martin P, Hajirousou V, et al: Haematological reassessment of rheumatoid arthritis using an automated method. Br J Rheumatol 25:152, 1986. 117. Isenberg DA, Patterson KG, Todd-Pokropek A, et al: Haematological aspects of systemic lupus erythematosus: A reappraisal using automated methods. Acta Haematol 67:242, 1982. 118. Treacy M, Lai L, Costello C, et al: Peripheral blood and bone marrow abnormalities in patients with HIV related disease. Br J Haematol 65:289, 1987. 119. Vinh DC, Patel SY, Uzel G, et al: Autosomal dominant and sporadic monocytopenia with susceptibility to mycobacteria, fungi, papillomaviruses, and myelodysplasia. Blood 115:1519, 2010. 120. Camargo JF, Lobo SA, Hsu AP, et al: MonoMAC syndrome in a patient with a GATA2 mutation: Case report and review of the literature. Clin Infect Dis 57:697, 2013. 121. Hsu AP, Sampaio EP, Khan J, Calvo KR, et al: Mutations in GATA2 are associated with the autosomal dominant and sporadic monocytopenia and mycobacterial infection (MonoMAC) syndrome. Blood 118:2653, 2011. 122. Hsu AP, Johnson KD, Falcone EL, et al: GATA2 haploinsufficiency caused by mutations in a conserved intronic element leads to MonoMAC syndrome. Blood 121:3830, 2013. 123. Calvo KR, Vinh DC, Maric I, et al: Myelodysplasia in autosomal dominant and sporadic monocytopenia immunodeficiency syndrome: Diagnostic features and clinical implications. Haematologica 96:1221, 2011. 124. Ostergaard P, Simpson MA, Connell FC et al: Mutations in GATA2 cause primary lymphedema associated with a predisposition to acute myeloid leukemia (Emberger syndrome). Nat Genet 43:929, 2011. 125. Kazenwadel J, Secker GA, Liu YJ, et al: Loss-of-function germline GATA2 mutations in patients with MDS/AML or MonoMAC syndrome and primary lymphedema reveal a key role for GATA2 in the lymphatic vasculature. Blood 119:1283, 2012. 126. Spinner MA, Sanchez LA, Hsu AP, et al: GATA2 deficiency: A protean disorder of hematopoiesis, lymphatics, and immunity. Blood 123:809, 2014. 127. Dickinson RE, Milne P, Jardine L, et al: The evolution of cellular deficiency in GATA2 mutation. Blood 123:863, 2014. 128. Cuellar-Rodriguez J, Gea-Banacloche J, Freeman AF, et al: Successful allogeneic hematopoietic stem cell transplantation for GATA2 deficiency. Blood 118:3715, 2011. 129. Steer JH, Vuong Q, Joyce DA: Suppression of human monocyte tumor necrosis factor-alpha release by glucocorticoid therapy: Relationship to systemic monocytopenia and cortisol suppression. Br J Clin Pharmacol 43:383, 1997. 130. Fauci AS, Dale DC: Monocytopenia after prednisone. N Engl J Med 292:928, 1975. 131. Aulitzky WE, Tilg H, Vogel W, et al: Acute hematologic effects of interferon alpha, interferon gamma, tumor necrosis factor alpha and interleukin 2. Ann Hematol 62:25, 1991. 132. Rotman M, Ansley H, Rogow L, et al: Monocytosis: A new observation during radiotherapy. Int J Radiat Oncol Biol Phys 2:117, 1977. 133. Fearnley DB, Whyte LF, Carnoutosis SA, et al: The monitoring of human blood dendritic cell numbers. Blood 93:728, 1999. 134. Szabolcs P, Park K-D, Reese M, et al: Absolute values of dendritic cell subsets in bone marrow, cord blood, and peripheral blood enumerated by a novel method. Stem Cells 21:269, 2003. 135. Giannelli S, Taddeo A, Presicce P, et al: A six-color flow cytometric assay for the analysis of peripheral blood dendritic cells. Cytometry B Clin Cytom 74:349, 2008. 136. Koga Y, Matsuzaki A, Suminoe A, et al: Expression of cytokine-associated genes in dendritic cells (DCs): Comparison between adult peripheral blood- and umbilical cord blood-derived DCs by cDNA microarray. Immunol Lett 116:55, 2008. 137. Ho CSK, López JA, Vuckovic S, et al: Surgical and physical stress increases circulatory blood dendritic cell counts independently of monocyte counts. Blood 98:140, 2001. 138. Pérez-Cabezas B, Naranjo-Gómez M, Fernández MA, et al: Reduced numbers of plasmacytoid dendritic cells in aged blood donors. Exp Gerontol 42:1033, 2007.

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CHAPTER 71

INFLAMMATORY AND MALIGNANT HISTIOCYTOSIS

Kenneth L. McClain and Carl E. Allen

SUMMARY Diseases of the histiocyte (i.e., macrophage or dendritic cell) lineage can be divided into four groups based upon the final maturation steps from their myeloid progenitor cells: (1) Langerhans cell histiocytosis (LCH), (2) malignant histiocytoses or dendritic cell sarcomas, (3) juvenile xanthogranuloma/ Erdheim-Chester disease, Rosai-Dorfman disease, and (4) hemophagocytic lymphohistiocytosis syndromes. Storage diseases of macrophages are discussed in Chap. 72. The distinction among these diseases is based upon clinical characteristics and histopathologic staining for unique surface markers. LCH may present at birth or in adulthood with skin rash, bone pain, draining ears, oral ulcers, gingivitis, pulmonary dysfunction, chronic diarrhea, diabetes insipidus, and marrow or liver failure. Therapy for LCH in children has been studied in clinical trials by the Histiocyte Society. Treatment for adults is based primarily on case series. Although relapses are not typically rapidly fatal, they are associated with a higher risk of endocrine and central nervous system complications. The diagnostic criteria for the malignant histiocytosis have been clarified by cell-surface marker studies. Treatment options and prognosis vary widely. Erdheim-Chester disease and juvenile xanthogranuloma are phenotypically similar, but are treated differently. Erdheim-Chester disease is found almost exclusively in adults and juvenile xanthogranuloma occurs primarily in children. Rosai-Dorfman disease presents with massive cervical lymphadenopathy in most patients, but may also involve other parts of the body. There are several treatment options for Rosai-Dorfman disease, Erdheim-Chester disease, and juvenile xanthogranuloma, but no clinical trials of specific drugs have been published. Hemophagocytic lymphohistiocytosis (HLH) is characterized by pathologic inflammation and may present with infections, hepatitis, meningitis, or autoimmune diseases. Without therapy, HLH is almost universally fatal. Most patients who receive prompt diagnosis and treatment with immune suppression therapy survive.

Acronyms and Abbreviations: AHSCT, allogeneic hematopoietic stem cell

transplantation; ALL, acute lymphoblastic leukemia; ATG, antithymocyte globulin; CD, cluster designation; CT, computed tomography; DC, dendritic cell; DI, diabetes insipidus; DLCO, diffusing capacity in lung for carbon dioxide; ECD, Erdheim-Chester disease; FEV1, forced expiratory volume in 1 second; HLA-DR, human leukocyte antigen-D related; HLH, hemophagocytic lymphohistiocytosis; IFN, interferon; IL, interleukin; JXG, juvenile xanthogranuloma; LC, Langerhans cell; LCH, Langerhans cell histiocytosis; M-CSF, macrophage colony-stimulating factor; MRI, magnetic resonance imaging; NK, natural killer; PET, positron emission tomography; RDD, Rosai-Dorfman disease.

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CLASSIFICATION OF THE HISTIOCYTOSES The general description of cells in the monocyte-macrophage system (mononuclear phagocyte system) has been largely clarified (Chaps. 67 to 69), although some ambiguity remains. Nomenclature committees remain loyal to the term “histiocyte,” a designation assigned in the 19th century to tissue macrophages, although the “histiocytosis” umbrella includes functional and neoplastic disorders of a broad range of cells in the monocyte, macrophage, and dendritic cell (DC) lineages. The distinctions among diseases in this category are determined by (1) clinical findings, (2) histopathology, (3) immunocytology to define the antigens on the surface of the pathologic cells, and (4) cytogenetic or genetic features (Table 71–1). The histiocytic disorders have been classified based upon whether they are (1) DC related, (2) monocyte-macrophage related, or (3) malignancies of macrophages or DCs (Table 71–2).1,2 Evolving understanding of myelomonocytic differentiation, as well as cellular origins of histiocytic disorders, will likely necessitate revision of these classifications in the near future. The pathologic cells in Langerhans cell histiocytosis (LCH) lesions have phenotypic similarity to epidermal Langerhans cells (LCs), which has led to the hypothesis that LCH is derived from aberrant activation and/or neoplastic transformation of the epidermal LCs.3 LCH originates from aberrant proliferation and differentiation of myelomonocytic precursors.4,5 Regardless of ontogeny, LCH lesions are characterized by pathologic DCs with phenotypic similarity to epidermal LCs, including positive staining with anti-CD207 (antilangerin) and the presence of Birbeck granules identified by electron microscopy.6,7 Birbeck granules are racket-shaped inclusions that are thought to be involved in antigen processing. Cells staining with anti-CD207 and/or anti-CD1a are required for the diagnosis of LCH. Other antigens, such as S100 or HLA-DR (human leukocyte antigen-D related) are not specific for LCH. The histiocytes in Erdheim-Chester disease (ECD) and juvenile xanthogranuloma (JXG) have phenotypic similarity to the dermal–interstitial dendrocyte that stains with antibodies to CD68, fascin, and factor XIIIa. However, the DCs in these disorders also express surface CD163 that is characteristic of macrophages. Malignant histiocytosis (or “histiocytic sarcoma”) has evolved as a diagnosis of exclusion involving malignant histiocytes that lack markers for anaplastic large cell lymphoma or other hematologic malignancies. Malignant histiocytosis represents a spectrum of malignancies that presumably derive from DCs of different lineages at different stages of differentiation. Human DCs are defined as hematopoietic cells expressing high levels of major histocompatibility complex II (MCHII) and CD11c while lacking other specific lineage markers. Under normal conditions, these cells reside in tissue or circulate in the blood. Once stimulated by antigen, they migrate to lymphoid tissue and interact with effector or suppressor T cells. Malignant histiocytoses are variable and share surface markers with DC subsets: follicular DC “sarcoma” (CD21+, CD35+), interdigitating DC “sarcoma” (CD14+), and Langerhans cell “sarcoma” (CD1a+). The monocyte-macrophage disorders include Rosai-Dorfman disease (RDD) and hemophagocytic lymphohistiocytosis (HLH). RDD, also known as sinus histiocytosis with massive lymphadenopathy, has the telltale histopathologic finding of intact lymphocytes in the cytoplasm of macrophages (emperipolesis), a feature that must be present to diagnose this disorder. HLH is distinct among the diseases discussed in this chapter in that the macrophages are nonneoplastic, otherwise normal histiocytes, characterized by a pathologic reaction to aberrant stimuli.

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Part VIII: Monocytes and Macrophages

TABLE 71–1.  Differentiating Characteristics of Histiocytes* Histologic Features

LCH

Malignant Histiocytosis

ECD/JXG

HLH

RDD

HLH-DR

++

+

−

+

+

CD1a

++

+/−

−

−

−

CD14

−

+/−

++

++

++

CD68

+/−

+/−

++

++

++

CD163

−

−

+

++

++

CD207 (Langerin)

+++

+/−

−

−

−

Factor XIIIa

−

−

++

−

−

Fascin

−

+/−

++

+/−

+

Birbeck granules

+

+/−

−

−

−

Hemophagocytosis

+/−

−

−

+/−

−

Emperipolesis

−

−

−

−

+

CD, cluster of differentiation; ECD, Erdheim-Chester disease; HLH, hemophagocytic lymphohistiocytosis; JXG, Juvenile Xanthogranuloma; LCH, Langerhans cell histiocytosis; RDD, Rosai-Dorfman disease. Data from Jaffe R: The diagnostic histopathology of Langerhans cell histiocytosis, in Histiocytic Disorders of Children and Adults. Basic Science Clinical Features, and Therapy, edited by Weitzman S, Egeler RM, pp 14–39. Cambridge University Press, Cambridge, UK, 2005; Chikwava K, Jaffe R: Langerin (CD207) staining in normal pediatric tissues, reactive lymph nodes, and childhood histiocytic disorders. Pediatr Dev Pathol 7:607–614, 2004; and Lau SK, Chu PG, Weiss LM: Immunohistochemical expression of Langerin in Langerhans cell histiocytosis and non-Langerhans cell histiocytic disorders. Am J Surg Pathol 32:615–619, 2008.

TABLE 71–2.  Classification of Histiocytic Disorders 1.  Disorders of varying biologic behavior, lacking cytologic atypia a. Dendritic-cell-related Langerhans cell histiocytosis Juvenile xanthogranuloma Erdheim-Chester disease b.  Monocyte-macrophage related Hemophagocytic lymphohistiocytosis Familial and/or with identified dysfunctional gene mutation Secondary hemophagocytic syndromes Infection-associated Malignancy-associated Autoimmune-associated Other Sinus histiocytosis with massive lymphadenopathy (Rosai-Dorfman disease) Solitary histiocytoma of macrophage phenotype 2.  Malignant disorders Dendritic cell related Histiocytic sarcoma Monocyte-macrophage related Leukemias: monocytic M5A and M5B, myelomonocytic M4, chronic myelomonocytic leukemia Data from Jaffe R: The diagnostic histopathology of Langerhans cell histiocytosis, in Histiocytic Disorders of Children and Adults. Basic Science Clinical Features, and Therapy, edited by Weitzman S, Egeler RM, pp 14–39. Cambridge University Press, Cambridge, UK, 2005 and Favara BE, Feller AC, Pauli M et al: Contemporary classification of histiocytic disorders. The WHO Committee On Histiocytic/Reticulum Cell Proliferations. Reclassification Working Group of the Histiocyte Society. Med Pediatr Oncol 29:157–166, 1997.

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L  ANGERHANS CELL HISTIOCYTOSIS HISTORY LCH has a complicated history that underlies the current clinicopathologic approach to the disease. What has come to be identified as LCH was first described in case reports and series in the early 1900s.8 By the 1950s, patterns of clinical presentations had been categorized as Hand-Schüller-Christian (multifocal eosinophilic granulomas) and Letterer-Siwe (disseminated disease including marrow, spleen and liver). However, these apparently disparate entities were found to share the same histopathology: histiocytes with abundant cytoplasm and reniform nuclei among an inflammatory infiltrate that could include lymphocytes, eosinophils and macrophages. Lichtenstein hypothesized that these clinical disorders must be linked by a common etiology, and proposed the designation “Histiocytosis X,” with “X” indicating incomplete knowledge of pathogenesis and cell of origin. Two decades later, Birbeck granules, which had previously been identified only in epidermal LCs, were identified in DCs of LCH lesions by electron microscopy. Nezelof and colleagues therefore extended Lichtenstein’s hypothesis that this spectrum of disorders arises from the epidermal Langerhans cell.3 Histiocytosis X has since been regarded as “Langerhans cell histiocytosis.”

EPIDEMIOLOGY AND INHERITANCE The incidence of LCH is 2 to 10 cases per 1 million children younger than age 15 years.9–11 A survey of LCH patients in France revealed an incidence of 4 to 6 per 1 million in children younger than age 15 years. The male-to-female ratio is close to 1 and the median age of presentation is 30 months, although patients may present with the disease from birth through the ninth decade. Identical and fraternal twins with early onset of LCH have been described. There are occasional reports of affected nontwin siblings and multiple cases in one family, although it is not clear if this is significantly greater than one would expect by chance.12

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Chapter 71: Inflammatory and Malignant Histiocytosis

The relatively high rate of high-risk multisystem LCH in identical twins compared to presumed fraternal twins may be explained by shared precursor cells as well as the possibility of shared genes. An increased frequency of family members with thyroid disease,13 family members with other cancers,14 in vitro fertilization,15 and parental exposure to metal16 have also been reported as potential associations. Although inheritance of penetrant mendelian “LCH genes” in the majority of cases seems unlikely, it remains possible that there are inherited genes associated with increased risk of developing LCH.

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Molecular Pathology

and cytokine and chemokine receptors have been hypothesized to play roles in LCH pathogenesis, creating a local “cytokine storm” as well as increased circulating proinflammatory cytokines, including tumor necrosis factor α, soluble interleukin (IL)-2 receptor α, RANKL (receptor activator of nuclear factor-κB ligand), osteoprotegerin, and osteopontin.4,30,31 Although MAPK (mitogen-activated protein kinase) pathway activation in maturing DCs may drive differentiation of LCH DCs, inflammation likely plays a role in the clinical manifestations and possibly also in tumor maintenance: A unique phenomenon in LCH is that disruption of solitary LCH lesions often results in spontaneous resolution, even without clean margins.

Cell of Origin

LCH usually presents with a skin rash or painful bone lesion. Systemic symptoms of fever, weight loss, diarrhea, edema, dyspnea, polydipsia, and polyuria may also occur. In LCH, involvement of specific organs at the time of diagnosis determines the designation “high-risk” or “low-risk.” Organs that indicate high-risk of progression include liver, spleen, and marrow. Organs that indicate low-risk of progression include skin, bone, lung, lymph nodes, and pituitary gland. Patients may present with disease in one site or organ (single site or single system) or in multiple sites or organs (multisystem). Treatment decisions for patients are based on whether or not organs that indicate high-risk or low-risk of progression are involved, and if LCH presents as a single site or as a multisystem disease. Patients can have LCH of the skin, bone, lymph nodes, and pituitary in any combination and still be considered to have a low-risk of progression.

The focus of studies and reviews on LCH over the past decades has been on either an immune or a neoplastic disorder. The competing models have been (1) an inappropriate activation of an otherwise normal epidermal LC or (2) a neoplastic transformation of the epidermal LC. Twenty years ago, CD1a+ cells from LCH lesions were described as clonal, based on non–random X inactivation.17,18 Subsequently, somatic activating mutations in the BRAF oncogene were reported in 57 percent of LCH histopathologic specimens,19 with subsequent studies validating the recurrent BRAFV600E mutation at high frequency.5,20–22 BRAF is the central kinase of the RAS/RAF/MEK/ERK pathway, which is essential to numerous cell functions and is frequently mutated in cancer cells.23 Significance of BRAFV600E as a driver mutation in LCH is supported by early reports of clinical responses to BRAF inhibition in adults with combined LCH and ECD.24 Other recurrent somatic mutations in LCH may be uncovered. The cell of origin of LCH has been assumed to be the epidermal LC based on phenotypic similarities discussed above. However, the transcriptome of CD207+ cells from LCH lesions is more consistent with an immature myeloid DC phenotype than with the transcriptome of epidermal LCs.4 Furthermore, DC maturation may be heterogeneous within lesions, with variable CD1a+/CD207− populations.25,26 Immunohistochemical staining antibodies specific for BRAFV600E revealed that the mutations are not limited to CD207+ cells within LCH lesions, but also are found in CD207-negative subpopulations.21 Using the BRAFV600E mutation as a “bar code,” cells that carry the mutation were identified in circulating myelomonocytic precursors in blood and in hematopoietic stem cells in marrow aspirates of patients with clinical high-risk LCH, but not in patients with single-lesion low-risk LCH. The functional significance of this observation was supported by the ability of forced expression of BRAFV600E in myelomonocytic precursors (CD11c+ cells) to induce a disseminated LCH-like phenotype in mice.5 We therefore hypothesize that the state of differentiation of the cell in which LCH arises determines the clinical manifestations of the disease; pathologic ERK (extracellular signal-regulated kinase) activation in stem cell or early myelomonocytic precursor resulted in disseminated high-risk disease whereas ERK activation in tissue-restricted precursor resulted in localized disease. These observations define LCH as a myeloid neoplasm.

CLINICAL FEATURES

Single-Site Disease Presentation

In this situation the disease presents with involvement of one site, which can be skin, oral mucosa, bone, lymph nodes, pituitary, or thymus. Skin Lesions simulating seborrheic dermatitis of the scalp may be mistaken for prolonged “cradle cap” in infants. The lesions may be localized to intertriginous areas or may be diffuse (Fig. 71–1). The most common skin flexures affected are the groin, the perianal area, back of the ears, the neck, the armpits, and, in women, the crease below the breasts. Infants may also present with brown to purplish papules over any part of their body. This latter manifestation may be self-limited as

Inflammation and Langerhans Cell Histiocytosis

Although ERK hyperactivation may drive differentiation and proliferation of myelomonocytic precursors in LCH, the mechanisms that drive inflammation in the LCH lesions are not currently understood. The LCH DCs make up a median of 8 percent of the cells within lesions.5 Like physiologically activated DCs, they express high levels of T-cell costimulatory molecules and proinflammatory cytokines.4,27,28 The LCH lesion’s inflammatory infiltrate includes lymphocytes, macrophages, and eosinophils in variable proportions, with enrichment of regulatory CD4+CD25+ T cells (T regs).29 Dozens of cytokines, chemokines,

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Figure 71–1.  Photographs demonstrate variability in clinical presentations of Langerhans cell histiocytosis skin lesions.

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the lesions often disappear during the first year of life with no therapy. However, these patients should be evaluated for other sites of disease which may coexist with skin lesions as multisystem LCH. Some reports describe multisystem LCH arising after presenting with lesions limited to the skin,32,33 although infants with skin lesions were not observed to develop disseminated disease in a relatively large institutional series.34 In a report of 61 neonatal LCH cases, nearly 60 percent had multisystem disease and 72 percent had high-risk organ involvement.35 The overall survival was poorer in neonates with high-risk organ involvement compared to infants and children with the same extent of disease. Response to therapy at 12 weeks was more important than patient age in determining outcome. A review of 71 children with skin involvement revealed that those without other organ systems involved had nearly a 90 percent progression-free survival after initial therapy or observation. Often dermatologists or general practitioners who see a patient with skin LCH do not perform a complete evaluation searching for LCH in other sites. Thus, we found that 40% of patients referred for apparent “skin only” LCH did have other sites of disease when a complete evaluation was done.34 Isolated skin involvement is rarely observed in children older than 18 months of age.34 Children and adults may develop red papular lesions in the scalp, skin of the groin, abdomen, back, or chest that resemble the diffuse rash of Candida infection. Seborrhea-like involvement of the scalp may be mistaken for a severe case of dandruff in older individuals. Ulcerative lesions behind the ears, involving the scalp, skin of the genitalia, or perianal region are often misdiagnosed as bacterial or fungal infections. Oral Mucosa Presenting symptoms include gingival hypertrophy, ulcers of the soft or hard palate, buccal mucosa, or on the tongue and lips. Lesions of the oral mucosa may precede evidence of LCH elsewhere.36 Bone The most frequent site of LCH in children is a lytic lesion of the skull which may be asymptomatic or painful.37 LCH can occur in any bone. The most frequently involved sites are skull, femur, ribs, vertebrae, and humerus. Spine lesions are most often located in the cervical vertebrae and are frequently associated with other bone lesions. Proptosis from a LCH mass in the orbit mimics rhabdomyosarcoma, neuroblastoma, and benign fatty tumors of the eye. Some skull lesions are not only lytic but may have an accompanying mass that impinges on the dura. Whether or not this affects risk of progression or not is unknown. Lesions of the facial bones (orbit, mastoid), or anterior or middle cranial fossae (e.g., temporal, sphenoid, ethmoid, or zygomatic bone) comprise the “CNS-risk” sites. These patients have a threefold increased risk for developing diabetes insipidus (DI) and an increased risk of other CNS disease (see “Central Nervous System and Endocrine System” below). Lymph Nodes and Thymus Cervical nodes are the ones most frequently involved and may be soft or hard-matted masses with accompanying lymphedema. An enlarged thymus or mediastinal node involvement can mimic lymphoma or an infectious process and may cause asthma-like symptoms. Biopsy of the node or mass with histologic examination and microbial cultures is helpful even in patients with known LCH as lymphadenopathy may represent LCH, coexisting neoplastic disease, or infection.38 Pituitary Gland The posterior pituitary gland can be affected in LCH patients causing central DI (see “Endocrine System” below). Anterior pituitary involvement may result in impaired growth and sexual maturation.

Multisystem Disease

In multisystem LCH, the disease presents in multiple organs or body systems, including liver and spleen, marrow (high-risk sites) or bones, lungs, skin, lymph nodes endocrine system, gastrointestinal system (low-risk sites), and CNS (intermediate-risk site depending on extent).

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Liver and Spleen Patients with liver and spleen involvement have a significantly increased risk of death from LCH. Hence, these are considered “high-risk organs.”39 Hepatic infiltration by LCH lesions can be accompanied by dysfunction, leading to hypoalbuminemia with ascites, hyperbilirubinemia, and clotting factor deficiencies. Sonographic imaging, computed tomography (CT), or magnetic resonance imaging (MRI) of the liver may show hypoechoic or low-signal intensity along the portal veins or biliary tracts when the liver is involved.40 One of the most serious complications of hepatic LCH is cholestasis and progressive sclerosing cholangitis.41 The median age of children with hepatic LCH is 23 months. Patients generally present with hepatomegaly with or without splenomegaly, elevated alkaline phosphatase, liver transaminases, and γ-glutamyl transpeptidase. Biopsies may or may not show CD207+ DCs.42 A classic histologic feature is the collection of lymphocytes around bile ducts. Seventy-five percent of children with sclerosing cholangitis do not respond to chemotherapy and ultimately require liver transplantation.41 Massive splenomegaly may lead to consumptive cytopenias and respiratory compromise. Splenectomy may ameliorate severe thrombocytopenia, but the effect is generally not sustained with progressive hepatomegaly and inflammation in the setting of uncontrolled disseminated LCH. Lung The lungs were once considered a high-risk organ; however, review of a large series of patients shows that the treatment outcome for patients with lung and bone involvement is not statistically different from those with only bone LCH.43 The lungs are less frequently involved in children (13 percent) than in adults (60 percent), in whom smoking is a key etiologic factor.44 In young children with diffuse disease, therapy can halt tissue destruction and normal repair mechanisms may restore some lung parenchyma and function. “Spontaneous” pneumothorax can be the first sign of LCH in the lung. Patients also present with cough, tachypnea, or dyspnea. Ultimately, widespread fibrosis and destruction of lung tissue leads to severe pulmonary insufficiency. Declining diffusion capacity may also herald the onset of pulmonary hypertension.45 Chest radiographs may show a nonspecific interstitial infiltrate. A high-resolution CT image of the chest is needed to visualize the cystic and nodular pattern of LCH that leads to the destruction of lung tissue. Marrow Involvement of the marrow is considered an indicator of high risk. Most patients with marrow involvement are young children who also have diffuse disease in the liver, spleen, lymph nodes, and skin with significant thrombocytopenia or neutropenia, though some may also have scattered marrow involvement with more mild cytopenias.46,47 An institutional study found patients with high-risk LCH with the BRAFV600E mutation to have 0.2 to 2.1 percent of cells from the marrow aspirate carry the mutation, with only four of seven cases being reported as having abnormal histology.5 LCH patients sometimes present with hemophagocytosis in the marrow.48 The presence of CD1a+/CD207+ in the marrow or at other sites identifies hemophagocytic syndrome as secondary to LCH rather than from primary HLH (discussed in the ­section “Hemophagocytic Lymphohistiocytosis” below). Endocrine System DI is the most frequent endocrine manifestation of LCH. Patients may present with an apparent “idiopathic” DI and sometimes with an enlarged pituitary gland or stalk before other LCH lesions are identified. Approximately half of these patients will have other lesions diagnostic of LCH within a year of identifying the DI.49 A review of patients with DI and enlarged pituitary glands found the three most likely diagnoses were germinoma, LCH, and lymphoma.50 DI followed the initial LCH diagnosis at other sites by a mean of 1 year and growth hormone deficiency occurred on the average 5 years later. Historically, the 10-year risk of pituitary involvement has been reported as 24 percent.51 In one series, this incidence of DI did not decrease

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in chemotherapy-treated patients (see “Central Nervous System and ­ iabetes Insipidus” below). In another study the incidence of DI D decreased from 40 to 20 percent after 6 months of treatment with vinblastine and prednisone for patients at risk for CNS involvement.52 However, after a year of this treatment, the incidence of DI was decreased to 12 percent.39 Craniofacial Lesions Patients with multisystem disease and craniofacial involvement at the time of diagnosis, particularly of the ear, eye, and oral region, carried a significantly increased risk of developing DI (relative risk: 4.6).53 This risk increased when the disease remained active for a longer period of time or reactivated. The risk for development of DI in this population was 20 percent at 15 years after diagnosis. Up to 56 percent of DI patients will develop anterior pituitary hormone deficiencies (growth, thyroid, or gonad-stimulating hormones) within 10 years of the onset of DI.54 Gastrointestinal System A few patients with diarrhea, hematochezia, perianal fistulas, or malabsorption have been reported.55,56 Diagnosing gastrointestinal lesions in LCH is difficult because of the patchy involvement. Endoscopic evaluation may reveal CD1a+/CD207+ cells in the intestinal mucosa, though LCH involvement may be patchy and require multiple biopsies to detect. Central Nervous System and Diabetes Insipidus DI (considered both an endocrine and a CNS manifestation of LCH) can present as an early or late condition. DI caused by damage to the posterior pituitary is the most frequent initial sign (and early manifestation) of LCH in the CNS. Pituitary biopsies are rarely done and only if the stalk is larger than 6.5 mm or there is a hypothalamic mass. The pituitary enlargement may spontaneously decrease or respond to chemotherapy. 57 However, a review of 22 patients with pituitary enlargement of 6.5 mm or greater revealed that despite regression of the mass with therapy, all had anterior pituitary deficiencies as well as MRI evidence of the CNS neurodegenerative syndrome (see “Other Chronic Central Nervous System Disease Manifestations” below) and 17 (77 percent) developed clinical signs of neurodegeneration.58 Most often the diagnosis of LCH is established by biopsy of skin, bone, or lymph node of a patient who also has the pituitary abnormalities. Other Chronic Central Nervous System Disease Manifestations LCH patients may develop mass lesions of the choroid plexus, or gray or white matter.59 These lesions may contain CD1a+ DCs as well as CD8+ lymphocytes.60 A chronic CNS problem that develops in 1 to 4 percent of LCH patients is the “LCH CNS neurodegenerative syndrome” manifested by dysarthria, ataxia, dysmetria, and, sometimes, behavior changes.61,62 The brain MRI in these patients shows hyperintensity of the dentate nucleus and white matter of the cerebellum on fluid-attenuated inversion recovery (FLAIR) and T2-weighted images or hyperintense lesions of the basal ganglia on T1-weighted images (Fig. 71–2). Atrophy of the cerebellum also may be seen.59 The radiologic findings may precede the onset of symptoms by many years or can be found coincidently. Among 83 LCH patients who had at least two MRI studies of the brain for evaluation of craniofacial lesions, DI, other endocrine deficiencies, or neuropsychological symptoms, 57 percent had radiologic neurodegenerative changes at a median time of 34 months after diagnosis. Of these patients, one-quarter had clinical neurologic deficits develop 3 to 15 years after LCH diagnosis.62

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Figure 71–2.  Radiologic evidence of Langerhans cells histiocytosis and CNS neurodegenerative syndrome. T2-weighted magnetic resonance image of the brain of a patient with Langerhans cells histiocytosis showing hyperintense changes of the cerebellar white matter.

LCH, but these associations are variable.63 When the liver is involved hypoalbuminemia, elevated liver enzymes, and elevated bilirubin may be observed. Intestinal involvement may also cause hypoalbuminemia. Lytic lesions of the bone are identified by plain films, CT imaging, MRI, bone scan, or positron emission tomography (PET) scan. PET scans are useful for detecting lesions not found by bone scan or plain films and comparison PET scans are particularly good for providing evidence of healing after 6 to 12 weeks of therapy.64 An institutional series found the presence of the somatic mutation in LCH lesions to correlate with

LABORATORY FEATURES LCH is defined by characteristic inflammatory lesions including histiocytes expressing CD1a and CD207 (Fig. 71–3).1 Patients with highrisk disease may present with anemia and thrombocytopenia caused by marrow involvement and/or inflammation.46,47 An elevated sedimentation rate and thrombocytosis has been reported to correlate with active

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Figure 71–3.  Biopsy of a bone lesion in a patient with Langerhans cell histiocytosis. Langerhans cells cytoplasm and membrane stain positively for CD207 (immunoperoxidase stain with hematoxylin and eosin [H&E] counterstain).

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higher risk of initial treatment failure, and circulating cells with the BRAFV600E mutation to correlate with active disease, though these observations remain to be validated in prospective trials.5

DIFFERENTIAL DIAGNOSIS It is not unusual for infants with LCH skin lesions to have symptoms for longer than 1 year prior to having a diagnostic biopsy.34 The varied cutaneous presentations of LCH may mimic a fungal diaper rash, seborrheic scalp rash or cradle cap, congenital viral infections, neuroblastoma, contact dermatitis, or psoriasis (see Fig. 71–1). Women or men with genital lesions may be thought to have a sexually transmitted disease or other infection. Oral lesions mimic other ulcerative conditions, gingival infections, or dental caries. Copious white or green discharge from the ears resembles otitis externa. Lytic bone lesions are often thought to be evidence of a malignancy such as neuroblastoma, rhabdomyosarcoma, or Ewing sarcoma. Collapsed vertebrae from LCH may mimic tuberculosis bone disease, trauma, or osteomyelitis. The interstitial infiltrates found in LCH patients with pulmonary involvement may resemble a viral pneumonia. An enlarged thymus or mediastinal lymph nodes can cause respiratory distress and wheezing similar to asthma. Enlarged lymph nodes from LCH mimic any infiltrative condition such as lymphomas, other histiocytic diseases, infections, or immune-related conditions. Likewise, hepatosplenomegaly of LCH patients can result from the same conditions. Chronic diarrhea in LCH patients may initially be considered to be an infectious or inflammatory bowel disease. Isolated DI with enlargement of the pituitary may suggest a germinoma, lymphoma, or hypophysitis. LCH should be strongly considered when symptoms of other more common conditions do not respond to therapy. Occasionally infiltrates of LC are found in various malignancies and as such represent an attempt of the immune system to respond to that disease.65–67 Similarly LCH may be found in the thymus of patients with myasthenia gravis.68

TREATMENT Pediatric Patients

The current optimal treatment of childhood and adults LCH patients, as with other rare conditions, is on clinical trials. The U.S. National Cancer Institute website (http://www.cancer.gov/cancertopics/pdq/treatment/ lchistio/HealthProfessional) and the Histiocytosis Association (http:// www.histio.org; 1–856–589–6606) also may be useful resources. Patients with only skin LCH, some single-bone lesions (non–CNSrisk), and isolated DI have not been studied in Histiocyte Society clinical trials, but are discussed in this section. Skin-Limited Lesions Skin-limited lesions may require therapy if they are symptomatic. One approach is topical glucocorticoids,33 although rarely effective. Other approaches with reported efficacy include oral methotrexate (20 mg/m2 weekly)69 or oral thalidomide (50 to 200 mg daily).70 Topical application of nitrogen mustard may be effective for cutaneous LCH that is resistant to oral therapies, but is contraindicated for large areas of skin.71 Psoralen plus long-wave ultraviolet A radiation (PUVA) has been used as well.72 The approach is limited by the severity and distribution of the skin involvement. Single Skull Lesions of the Frontal, Parietal, or Occipital Regions, or Single Lesions of Any Other Bone Curettage or curettage plus injection of methylprednisolone may be used.73 Vertebral or Femoral Bone Lesions at Risk for Collapse Isolated radiation therapy is indicated for patients with single bone lesions of a vertebrae or the femoral neck, which are at risk of collapse.74,75 When instability of the cervical vertebrae and neurologic symptoms are present, bracing or spinal fusion may be needed.76 Certain skull lesions, not in the CNS-risk region, could also be considered for radiation therapy.

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Skull Lesions in the Mastoid, Temporal, Orbital or Base of Skull Bones (CNS-Risk Lesions) The purpose of treating these patients with systemic therapy is to decrease the risk of developing DI. In a large series of patients with CNS-risk lesions who received little or no chemotherapy there was a 20 to 50 percent incidence of DI compared to incidence rates of 10 percent in patients treated with systemic chemotherapy.52 The current standard of care, based on the LCH-III study, is to treat patients with single or multifocal lesions in CNS risk sites for 12 months with intravenous vinblastine and oral prednisone: weekly intravenous vinblastine (6 mg/m2) for 7 weeks, with daily oral prednisone (40 mg/ m2) for 4 weeks followed by a 2-week taper. If there is a good response by 6 weeks, then vinblastine frequency is decreased to every 3 weeks. After the first 6 weeks, oral prednisone is given for 5 days at 40 mg/m2 every 3 weeks with the vinblastine intravenous. Patients with suboptimal responses by 6 weeks are given an additional 6 weeks of weekly intravenous vinblastine.39 Multiple Bone Lesions or Combinations of Skin, Lymph Node, or Pituitary Gland Involvement with or Without Bone Lesions Patients should be treated for 12 months with intravenous vinblastine and oral prednisone as outlined for the CNS-risk lesions. Both shorter (60 percent of patients) and circumferential sheathing of the aorta (>60 percent of patients), as well as retroperitoneal

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DIFFERENTIAL DIAGNOSIS Although histologically distinct, the clinical features may suggest LCH, RDD, JXG, or xanthoma disseminatum. Some clinical features overlap with sarcoidosis, amyloidosis, Paget disease, Ormond disease (idiopathic retroperitoneal fibrosis), and Whipple disease (intestinal lipodystrophy). The histologic features can be confused with Gaucher disease, Niemann-Pick disease, mucopolysaccharidosis, or malakoplakia.172

Subcutaneous IFN-α and pegylated IFN-α are considered the first-line treatments for ECD.173 Survival has been improved using doses of 3 million units, 3 times a week.174–178 When the standard dose is ineffective, increasing the IFN-α dose to greater than 18 million units per week or use of pegylated IFN-α to a dose greater than 180 mcg/wk is recommended. Treatments have been extended for as long as 3 years. Patients treated with the high-dose regimens had a stabilization of CNS disease in 64 percent and of cardiac involvement in 79 percent. Earlier published treatment results include a review of 37 patients treated with glucocorticoids, usually 1 mg/kg per day, orally, resulting in decreased exophthalmos or general symptoms in 20 patients.156 Among these patients, glucocorticoids were effective in six patients, transiently effective in four, and ineffective in eight. Of eight patients treated with a variety of chemotherapy agents and glucocorticoids, four had improvement. Radiation was ineffective for orbital masses, but transiently relieved bone pain. A series of six patients treated with oral imatinib mesylate reported two had stable disease and one an initial response before worsening. 179 Some patients have been treated effectively with intravenous cladribine.180 Anticytokine treatments with anakinra, infliximab, and tocilizumab have had varying degrees of success in a limited number of patients. Anakinra is given at 1 to 2 mg/kg per day, intravenously, and may work best for patients with bone pain and other systemic symptoms.181–183 However, it seems to be less effective than IFN-α. The same can be said for the anti–tumor necrosis factor α drugs, intravenous infliximab and intravenous etanercept. Clinical trials currently open to open to ECD patients include: • NCTT01524978 Vemurafenib: anti-BRAFV600E • NCT01727206 Tocilizumab: anti–IL-6 (phase II clinical trial) • ACTRN12613001321730: Sirolimus and prednisone (prospective trial)

COURSE AND PROGNOSIS Nearly 60 percent of ECD patients die of their disease; 36 percent die within 6 months. The mean survival duration is less than 3 years. Cardiac, pulmonary, and renal failure are the primary causes of death.

JUVENILE XANTHOGRANULOMA DEFINITION AND HISTORY JXG is a histiocytic disorder that affects the skin with multiple nodules in the head, neck, and trunk primarily in children, although adults can also be affected.184 The lesional cells are derived from dermal dendrocytes. Systemic involvement occurs in a few cases. Rudolf Virchow may have been the first to describe a child with what he called “cutaneous xanthomas” in 1871.

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EPIDEMIOLOGY

THERAPY

Children with solitary lesions have a median age of onset of 2 years with a male-to-female ratio of 1.5:1. Children with multiple lesions have a median age of onset of 5 months and have a male-to-female ratio of 12:1. No population study of JXG has been reported, so the precise incidence is unknown. However, a review of JXG from the Kiel Pediatric Tumor Registry recorded 129 (0.52 percent) cases of JXG and 800 (3.3 percent) cases of LCH among 24,600 children over a 36-year period.

Patients with a single or only a few lesions need no therapy. An excisional biopsy can be used, if desired for cosmetic reasons. For the rare patients who have systemic disease and require treatment a wide variety of chemotherapy and radiotherapy regimens have been reported.191–193 Inclusion of a vinca alkaloid and a glucocorticoid is associated with better overall response rates than single agents. A child with CNS JXG who failed to respond to vinblastine was successfully treated with cladribine.194 A series of four children with systemic and CNS JXG were successfully treated with clofarabine.84

ETIOLOGY AND PATHOGENESIS

COURSE AND PROGNOSIS

There is no known cause of JXG. Patients with JXG and neurofibromatosis types 1 and 2, as well as the triad of the aforementioned diseases with juvenile chronic myelogenous leukemia, have been reported.185–187 These and other cases have suggested an increased risk of leukemia in neurofibromatosis patients with JXG, but there is no rigorous proof for this association.188,189

CLINICAL FEATURES The majority of patients are children younger than 2 years of age who have solitary skin nodules on their head, neck, or trunk.184,190 The lesion is most often the same color as surrounding skin, but may be erythematous or yellowish. Rarely, nodules may be in the subcutaneous fat, deep soft tissue, or skeletal muscle. Organ involvement is rare, but has been reported in the soft tissue, CNS, bone, lung, liver, spleen, pancreas, adrenal, intestines, kidneys, lymph nodes, marrow, and heart.184,190,191 Systemic symptoms and signs occur only if these organ systems are involved.

LABORATORY FEATURES Immunohistochemical staining of biopsies is necessary to differentiate JXG from other histiocytic lesions. JXG classically stains with a macrophage marker such as antibodies to CD68 or Ki-M1P, factor XIIIa, fascin, vimentin, and often CD4. They are negative for S100 and antiCD1a. There are three characteristic histologic patterns: early JXG, classic JXG, and transitional JXG.190 Early JXG is characterized by small- to intermediate-size mononuclear histiocytes in sheet-like infiltrates. The cells in this category have only small quantities of lipid in the cytoplasm and Touton-type giant cells are absent. This type has relatively more mitoses than the others, but there is no cytologic atypia. Classic JXG exhibits abundant vacuolated, foamy histiocytes with Touton giant cells (lipid-laden histiocytes with multiple nuclei and a small amount of centrally oriented cytoplasm). Transitional JXG has a predominance of spindle-shaped cells resembling benign fibrous histiocytoma with foamy histiocytes and occasional giant cells.190 Biopsies also contain lymphocytes, eosinophils, and occasionally Charcot-Leyden crystals. If the marrow is involved, patients may have cytopenias. Liver infiltration may cause elevation of liver enzymes, hypoalbuminemia, and an elevated erythrocyte sedimentation rate. Pituitary involvement may lead to DI. Hypercalcemia has been reported. CNS lesions can lead to hydrocephalus, seizures, and developmental delay.

DIFFERENTIAL DIAGNOSIS LCH is the disease most often confused with JXG. Other disorders to be considered include fibrohistiocytic lesion not otherwise specified, reticulohistiocytoma, hemangioendothelioma, Spitz nevus, malignant fibrous histiocytoma, and rhabdomyosarcoma or other malignancies.

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Patients with only skin or soft-tissue involvement all survive and in a majority of cases, the lesions spontaneously disappear over time. Infants with large retroperitoneal masses, liver, marrow, or CNS involvement usually survive with chemotherapy treatment. Two of 17 patients with multisystem JXG reported in the literature died despite multiagent chemotherapy.192

S  INUS HISTIOCYTOSIS WITH MASSIVE LYMPHADENOPATHY (ROSAIDORFMAN DISEASE) DEFINITION AND HISTORY Rosai and Dorfman recognized this nonmalignant proliferation of histiocytes as a unique histopathologic entity, which is part of the differential diagnosis of massive lymphadenopathy.195 Although this disease is self-limited in some patients, others with airway obstruction, multiple bone lesions, orbital or brain tumors require therapy.84,196

EPIDEMIOLOGY RDD is found throughout the world as a disease of children and young adults (mean age: 20.6 years). Most of our knowledge about it is the result of analysis of the 423 cases in the registry developed by Rosai and Dorfman in which there was no gender, ethnic, or socioeconomic predilection. Persons of African and European descent are equally represented; people of Asian descent less so. In cases of digestive system disease, males and persons of African descent were more commonly affected.197 Intracranial disease is found in patients with a mean age of 37.5 years. There is an apparent increase in rheumatologic disorders and hemolytic anemia among these patients.198,199 Germline mutations in the nucleoside transporter SLC29A3 have been described in patients with rare familial syndromes that include lymphadenopathy characteristic of RDD.200

ETIOLOGY AND PATHOGENESIS Although associations with various herpes virus infections have been reported, these most likely represent detection of lymphocytes or macrophages harboring these viruses with no relation to etiology. A model for the key histopathologic finding, emperipolesis of lymphocytes by macrophages, has been proposed.201 These authors hypothesized that macrophage-activating cytokines could stimulate the macrophages to ingest lymphocytes. The cells in the lesions of this disorder are polyclonal.202

CLINICAL FEATURES Massive, painless bilateral cervical adenopathy is the presenting finding in 87 percent of patients. Some have fever, night sweats, malaise, and

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weight loss. A few patients have polyarthralgia, rheumatoid arthritis, glomerulonephritis, asthma, and diabetes mellitus. Painless maculopapular eruptions, sometimes reddish or bluish, or yellow xanthomatous rashes occur in 16 percent of patients. Subcutaneous nodules can be found anywhere in the body. Another 16 percent of patients have nasal cavity and paranasal sinus involvement with obstruction of the airways, epistaxis, septal displacement, and mass lesions infiltrating the sinuses. Ten percent have eyelid or orbital masses with proptosis. Unlike patients with LCH, patients with RDD uncommonly (10 percent) have osteolytic bone lesions. These have irregular borders, but may have sclerotic margins. Bilateral parotid or submandibular gland swelling may also be present. Less than 10 percent of patients have CNS, intracranial, epidural, or dural masses, as solitary or multiple lesions leading to headaches, nerve palsies, or syncope. Other organ system involvement in 1 to 3 percent of cases includes the kidney, genitourinary tract, lungs, larynx, liver, tonsil, breast, gastrointestinal tract, and heart. Up to 43 percent of patients have lymphadenopathy coupled with extranodal involvement of the skin, soft tissue, upper respiratory tract, bone, eye, or retroorbital tissue.203

LABORATORY FEATURES Patients may have a hemolytic anemia or anemia of chronic disease, elevated erythrocyte sedimentation rate, and polyclonal hyperimmunoglobulinemia. Elevation of liver enzymes and other laboratory abnormalities depend on the organs involved.204 Hepatic features include capsular and pericapsular fibrosis. The lymph node sinuses are enlarged by a proliferation of histiocytes with large round or oval vesicular nuclei and a prominent nucleolus. Mitoses are rare. The cytoplasm is pale and eosinophilic, although some may have a foamy cytoplasm. The key diagnostic finding is intact lymphocytes in macrophages (active ingestion, or emperipolesis, the penetration of a smaller cell into larger one). Because the lymphocytes are inside vacuoles, they are not degraded. Accompanying the histiocytes are numerous plasma cells. The pathologic macrophages in this disease infiltrate the sinuses of lymph nodes and are phagocytosing lymphocytes and plasma cells as well as erythrocytes. Although the histiocytes are S100-positive, they are CD1a-negative, unlike the LCs, which are positive for both markers. The macrophages express CD68, CD14, CD15, lysozyme, transferrin receptor, IL-2 receptor, and CD163.196

DIFFERENTIAL DIAGNOSIS Any other cause of lymphadenopathy, such as infections, lymphomas, leukemias, Gaucher disease, melanoma, and other malignancies, should be ruled out by a biopsy. The massive cervical lymph nodes are strikingly similar to those of patients with the autoimmune lymphoproliferative syndrome.205 Inflammatory pseudotumor and RDD have been found in the same patient suggesting a histologic continuum.206 Clinicians should be aware that the sinuses of many reactive lymph nodes contain macrophages (histiocytes) and pathologists will report that presence as “sinus histiocytes or sinus histiocytosis.” This is not evidence for RDD because in those cases the sinus histiocytes do not have lymphocytes within their cytoplasm.

THERAPY Many cases are self-limited and do not require therapy. Surgery may be useful for symptomatic treatment of local large lymph nodes. Multiorgan involvement or dysfunction, and association with immune dysfunction are poor prognostic indicators and indicate the necessity of treatment.207 Several therapies have been used, including glucocorticoids and chemotherapy, with success in some cases. Several case reports have described improvement or cure of patients with the disease

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with oral dexamethasone, oral methotrexate, oral 6-mercaptopurine, intravenous cladribine, or intravenous vinorelbine plus methotrexate.208–211 Intravenous clofarabine may be the best therapy for patients with bone and CNS involvement.84

COURSE AND PROGNOSIS Most patients will have a slow but steady decrease in the size of their lymph nodes over months to years. For those patients requiring treatment because of impingement on vital organs responses are variable. Because no clinical trials have been done, treatment has been based on anecdotal reports.

HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS DEFINITION AND HISTORY Farquhar and Claireux first described this disease in siblings in 1952.212 Although many case reports using several eponyms ensued, Henter and Elinder provided a logical organization of the diverse clinical presentations.213 HLH is an aggressive and potentially fatal syndrome that results from inappropriate prolonged activation of lymphocytes and macrophages. The name describes the characteristic (but not diagnostic) pathologic finding of macrophages engulfing all types of blood cells in marrow, lymph nodes, spleen, or liver biopsies (see Fig. 71–3). HLH is also known as autosomal recessive familial HLH, familial erythrophagocytic lymphohistiocytosis, viral-associated hemophagocytic syndrome, and infection-associated hemophagocytosis. “Primary” or “familial” HLH has been used to describe young children with HLH with known gene mutations or a family history of HLH. Older children with HLH, or children without identifiable mutations, are sometimes described as having “secondary” or “acquired” HLH with the assumption that the condition is caused by infection or other stimulus and not a result of genetic predisposition. The same mutations may be present in both situations, and there is no rapid and definitive gene-testing strategy to distinguish the two groups. In general, presentation and outcome are the same for primary and acquired HLH.214 Hypomorphic mutations in HLH-associated genes and compound heterozygous mutations have been described in patients who develop HLH at an older age or in the context of autoimmune disease.215,216 Thus, this distinction is not clinically useful in the acute setting as they both must be diagnosed promptly and treated aggressively.

EPIDEMIOLOGY The incidence of HLH in Sweden was estimated at 1.2 children per 1 million children per year, or 1 in 50,000 livebirths with equal sex distribution.213 At the Texas Children’s Hospital, HLH was diagnosed in 1 of 3000 inpatient admissions in a 2-year study.217 The incidence in adults is unknown and the outcomes may be worse than for children.218 Many adult patients with HLH also have lymphoma.219

ETIOLOGY AND PATHOGENESIS Defects in the function of natural killer (NK) cells and cytotoxic T cells have been found in HLH patients. This results in the inappropriate activation of T cells and macrophages, which produce proinflammatory cytokines, including IFN-γ, tumor necrosis factor-α, IL-6, IL-10, IL-12, and soluble IL-2 receptor-α (sCD25).220,221 In an animal model, perforin deficiency leads to inability to “prune” antigen-presenting DCs, resulting in increased activation of cytotoxic CD8+ T cells.222 The hypercytokinemia and pathologic activation of T cells and macrophages result in multiorgan dysfunction that can rapidly lead to death.

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Perforin Expression

Perforin was identified as a candidate HLH gene by gene mapping and was confirmed by poor expression of perforin in NK cells and cytotoxic T lymphocytes of HLH patients.223,224 Some HLH characteristics were reproducible in PRF1 knockout mice.225 Perforin is secreted from NK cells and cytotoxic T cells upon activation by target cells and introduces pores in the target cell membrane, allowing granzyme to enter and trigger apoptosis.226

Other Defects Causing Hemophagocytic Lymphohistiocytosis

Mutations in other genes encoding proteins involved in NK and cytotoxic T-cell–mediated killing of target cells also have been discovered in patients with HLH, including UNC13D (encodes MUNC13-4), STX11 (encodes syntaxin 11), and UNC18B (encodes STXBP2).227 Mutations in the gene that encodes RAB27a (protein that controls secretion of lytic granules) have also been identified in patients with Griscelli syndrome.228

Immune Deficiencies Associated with Hemophagocytic Lymphohistiocytosis

Patients with other immune deficiencies associated with lysosomal trafficking defects (e.g., Chédiak-Higashi syndrome, HermanskyPudlak syndrome type II) also have a high frequency of HLH.229 HLH, often associated with infection by the Epstein-Barr virus, is the most common fatal complication of X-linked lymphoproliferative disease (XLP1/SH2D1A and XLP2/XIAP).230

CLINICAL FEATURES Initial signs and symptoms of HLH mimic more common problems (e.g., fever of unknown origin or sepsis).231 Confounding diagnoses such as infection, autoimmune disease, hepatitis, multisystem organ failure, encephalitis, and malignancy do not exclude a diagnosis of HLH. Important clues include an acutely ill patient with unexplained fever, rash, or neurologic symptoms. A medical history of immune deficiency should bring HLH to mind. Family history of consanguinity, recurrent spontaneous abortions, or HLH in siblings (or symptoms suggesting undiagnosed HLH) may be suggestive of a risk for HLH. Prominent early clinical signs in one study included fever (91 percent), hepatomegaly (90 percent), splenomegaly (84 percent), neurologic signs (47 percent), rash (43 percent), and lymphadenopathy (42 percent).232 Another study found 75 percent of patients with HLH to have CNS symptoms that may mimic encephalitis.233 Patients with HLH develop liver failure with markedly elevated conjugated bilirubin, pancytopenia, coagulopathy, renal failure heralded by hyponatremia, and pulmonary failure similar to acute respiratory distress syndrome with interstitial infiltrates on chest radiography.231

TABLE 71–4.  Clinical Criteria for Diagnosis of Hemophagocytic Lymphohistiocytosis Hemophagocytic lymphohistiocytosis (HLH) diagnosis is established with at least five of the following: •  Fever •  Splenomegaly •  Cytopenias in at least two cell lines: •  Hemoglobin 2000 mcg/L may be more specific) •  Soluble cD25 (soluble interleukin-2 receptor) >2400 U/mL or •  HLH-associated gene mutations

cytometry degranulation assays that measure membrane CD107a are also effective for identifying patients with lymphocytes with impaired cytotoxic function.236 Hemophagocytosis is sometimes misunderstood as pathognomonic and necessary for the diagnosis of HLH, but biopsies fail to demonstrate hemophagocytosis in approximately one-third of patients (Fig. 71-4).237 HLH changes over time such that the cytokine stimulation resulting in hemophagocytosis may be modest early in the disease, or the marrow may progress to become aplastic with few macrophages available to engage in hemophagocytosis. Repeat marrow aspirates and biopsies, as well as lymph node or liver biopsies, may be helpful. Finding hemophagocytosis is highly suggestive of HLH, but is neither necessary nor sufficient to make the diagnosis. Cerebrospinal fluid should be tested in patients with signs of CNS abnormalities; pleocytosis, hyperproteinemia and hemophagocytosis support HLH with CNS involvement.

Diagnostic Criteria

The cumulative experiences from the first prospective international treatment protocol sponsored by the Histiocyte Society, HLH-94, as well as other observations and studies, have led to the Histiocyte Society treatment protocol HLH-2004, which includes diagnostic guidelines (Table 71–4).234 The HLH criteria are derived from retrospective analysis of patients treated on HLH-94 and describe patients with extreme pathologic inflammation and associated defects in cytotoxic immune function. Biallelic mutations in HLH-associated (or monoallelic in case of X-linked genes) are diagnostic for HLH, but generally not helpful for acute management although genetic results are becoming available more quickly. More rapid flow cytometry studies can identify absence of protein expression of PRF1, SAP (XLP1), or XIAP (XLP2).235 Flow

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Figure 71–4.  Hemophagocytosis by macrophages. Marrow aspirate treated with Wright-Giemsa stain illustrating prominent hemophagocytosis of multiple cell types by macrophages (arrows) in the marrow of a patient with hemophagocytic lymphohistiocytosis.

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LABORATORY FEATURES Ferritin

Although no one diagnostic criterion is sufficient to make the diagnosis of HLH, a highly elevated serum ferritin along with four other criteria is strongly indicative. A ferritin concentration of greater than 500 mcg/L was included in the HLH-2004 diagnostic criteria because a survey found that most children with infectious diseases had levels less than that level and those with rheumatologic diagnoses only rarely had higher levels. Ferritin concentrations greater than 500 mcg/L were 100 percent sensitive for HLH in a retrospective review over a 2-year period.217 However, at this level there is considerable overlap with other disorders. Ferritin concentrations more than 10,000 mcg/L were 90 percent sensitive and 96 percent specific for HLH with very minimal overlap with sepsis, infections, and liver failure. Analysis of ferritin values in an extended cohort suggests that 2000 mcg/L may be a more appropriate measure for diagnosis of HLH than 500 mcg/L.238 The following tests should be done on a previously healthy patient who presents with persistent fevers, hepatosplenomegaly, and cytopenia of at least two cell lines: serum ferritin, aspartate aminotransferase/ alanine aminotransferase, lactate dehydrogenase, bilirubin, coagulation studies, fibrinogen, triglycerides. A marrow biopsy and aspirate is needed, as well as a lumbar puncture for spinal fluid examination. NK cell function, perforin expression of T cells and NK cells, and sCD25 concentrations should be evaluated, usually requiring access to specialty laboratories, if there is clinical suspicion for HLH. Following daily serum ferritin levels is useful because rapidly rising ferritin is a strong indicator of HLH, and inferior outcomes are associated with slow normalization.239 It may be necessary to repeat the marrow biopsy or biopsy an enlarged liver or lymph nodes, if the first marrow biopsy fails to show hemophagocytosis and clinical suspicion of HLH is high.

DIFFERENTIAL DIAGNOSIS Patients with fever of unknown origin, moderate infections, sepsis, multiorgan dysfunction, hepatitis, anemia and thrombocytopenia, and autoimmune phenomena such as Kawasaki disease, lupus erythematosus, or rheumatoid arthritis may present with features that overlap the diagnostic criteria for HLH. These may represent alternative or concurrent diagnoses. One must consider HLH if no clear diagnosis is established of the above mentioned entities is evident and the patient is deteriorating. Identification of an underlying immune deficiency such as X-linked lymphoproliferative disease (Chap. 80), Griscelli syndrome (Chap. 80), or Chédiak-Higashi syndrome (Chap. 80) should increase the suspicion of HLH. Epstein-Barr virus, cytomegalovirus, and other herpes virus infections are the most frequent viral infections associated with HLH. A wide variety of bacterial fungal and protozoal infections may also lead to HLH.

THERAPY Before treatment with immune-modulating therapy fewer than 10 percent of patients with HLH survived.240 After case reports and case series described patients successfully treated with strategies that included aggressive immune suppression, podophyllotoxin derivatives, or a combination of immune suppression with etoposide, a prospective treatment protocol was developed that included induction therapy with oral or intravenous dexamethasone and intravenous etoposide, followed by continuous treatment with oral cyclosporine and pulses of dexamethasone and intravenous etoposide.241–243 Patients with CNS symptoms or cerebrospinal fluid lymphocytosis or pleocytosis also received intrathecal methotrexate. Patients with resistant disease, recurrent disease, or familial HLH underwent AHSCT. The overall estimated 3-year survival

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on the HLH-94 protocol was 55 percent.243 In a study of patients with Epstein-Barr virus–associated HLH, early administration of intravenous etoposide was associated with improved outcomes.244 Etoposide was recently demonstrated to have specific cytotoxicity to activated T cells, which may explain why it is effective in HLH.245 A second protocol, HLH-2004, containing minor modifications from the first included starting oral cyclosporine at the onset of induction therapy, adding glucocorticoids to intrathecal therapy in patients with CNS disease, adding etoposide to conditioning in patients who undergo AHSCT, and considering depletion of T cells in patients who receive stem cells from unrelated donors.243 At this time, we consider HLH-94 the standard of care. Data are not yet available to evaluate the benefits of early cyclosporine, and it has known risks including increasing susceptibility to posterior reversible encephalopathy syndrome (PRES).246 Intravenous antithymocyte globulin (ATG) has been used as a primary treatment of 38 cases of familial HLH.247 It was intended that all of these patients undergo AHSCT, which ultimately cured 16 of 19 cases. ATG was ineffective for patients who had been previously treated with etoposide, dexamethasone, and cyclosporine and who had relapsed while on therapy. A study that is open as of this writing, hybrid immunotherapy for HLH (HIT-HLH; clinicaltrials.gov: NCT01104025) combines strategies of early immune suppression with ATG and prolonged immune suppression with etoposide. A significant number of patients with HLH will fail to respond to initial therapy or will develop recurrent episodes of inflammation while awaiting AHSCT. Treatment failures and recurrences are associated with very high rates of mortality. Escalation of dexamethasone and etoposide is a typical first step for patients with recurrence. Additional salvage strategies that have been reported include infliximab, dalizubumab, anakinra, and other agents.231 A retrospective multiinstitutional study reported that 77 percent of patients who received alemtuzumab therapy for refractory or recurrent HLH survived to AHSCT.248 A clinical trial is currently open to test the efficacy and safety of inhibition of IFNγ in patients with HLH with recurrent inflammation (clinicaltrial.gov: NCT01818492). AHSCT may be indicated for patients with familial HLH or with gene defects, CNS disease, or who relapse either on or off HLH therapy. Long-term survival was 50 to 65 percent with myeloablative conditioning, but patients experience significant treatment-related morbidity and mortality.214,249 Institutional series have demonstrated improved survival and decreased treatment-associated complications with reduced intensity conditioning (RIC) strategies that include alemtuzumab. RIC strategies are associated with improved survival.248,250 A multicenter clinical trial (Reduced Intensity Conditioning for Children and Adults with Hemophagocytic Syndromes or Selected Primary Immune Deficiencies [RICHI]) is currently testing the safety and efficacy of RIC with “intermediate” timed alemtuzumab.251 (clinicaltrials.gov:NCT01998633) Patients with HLH are generally acutely ill, and therapies for HLH may exacerbate cytopenias and susceptibility to opportunistic infections. Patients may require multiple transfusions of red cells, platelets, and fresh frozen plasma. Prophylaxis against Pneumocystis carinii infection with sulfamethoxazole and against fungi with fluconazole is necessary. Newly diagnosed HLH patients should have human leukocyte antigen typing done and a donor search initiated in case AHSCT is required for therapy.

Macrophage Activation Syndrome

This nomenclature describes patients with symptoms and signs of HLH in the setting of juvenile rheumatoid arthritis or systemic lupus erythematosus.252 Similar to classic HLH, macrophage activation is

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characterized by proliferation of macrophages and T cells. Patients present with continuous fever, purpura, hepatosplenomegaly, mental status changes, cytopenias, coagulopathy, and hypofibrinogenemia. Laboratory findings may include defective NK cell function and low perforin expression, as seen in HLH. In the setting of pathologic inflammation driven by autoimmune disease, patients may be successfully treated with therapy targeted against the underlying autoimmune disease.253 Treatment with dexamethasone and etoposide therapy is recommended if patients fail to improve after a brief trial of therapy appropriate for rheumatologic disease.

COURSE AND PROGNOSIS Patients with HLH are often critically ill, functionally immunosuppressed, and receive toxic chemotherapy. They should be treated at institutions familiar with the complications of chemotherapy and immune suppression. Splenectomy is recommended only in the case of life-threatening respiratory compromise. Some patients have an initial good response to therapy with etoposide and dexamethasone, but then have progressive disease as evidenced by elevation of the serum ferritin, worsening coagulopathy, or need for increased respiratory, blood pressure, or renal support. Although it may seem counterintuitive to treat critically ill patients with immune suppression, patients with HLH require this approach to have a chance to survive to clear the inflammatory trigger or overcome inherited immune defects with AHSCT.

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Paulli M, Bergamaschi G, Tonon L, et al: Evidence for a polyclonal nature of the cell infiltrate in sinus histiocytosis with massive lymphadenopathy (Rosai-Dorfman disease). Br J Haematol 91:415–418, 1995. 203. Foucar E, Rosai J, Dorfman R: Sinus histiocytosis with massive lymphadenopathy (Rosai-Dorfman disease): Review of the entity. Semin Diagn Pathol 7:19–73, 1990. 204. Chow CP, Ho HK, Chan GC, et al: Congenital Rosai-Dorfman disease presenting with anemia, thrombocytopenia, and hepatomegaly. Pediatr Blood Cancer 52:415–417, 2009. 205. Price S, Shaw PA, Seitz A, et al: Natural history of autoimmune lymphoproliferative syndrome associated with FAS gene mutations. Blood 123:1989–1999, 2014. 206. Govender D, Chetty R: Inflammatory pseudotumour and Rosai-Dorfman disease of soft tissue: A histological continuum? J Clin Pathol 50:79–81, 1997. 207. Pulsoni A, Anghel G, Falcucci P, et al: Treatment of sinus histiocytosis with massive lymphadenopathy (Rosai-Dorfman disease): Report of a case and literature review. Am J Hematol 69:67–71, 2002. 208. Horneff G, Jurgens H, Hort W, et al: Sinus histiocytosis with massive lymphadenopathy (Rosai-Dorfman disease): Response to methotrexate and mercaptopurine. Med Pediatr Oncol 27:187–192, 1996. 209. Perry R, Penk J, Kapoor N, Shah A: Vinorelbine and methotrexate for the treatment of Rosai-Dorfman Disease in children. Pediatr Blood Cancer 200584–85. 210. Rodriguez-Galindo C, Helton KJ, Sanchez ND, et al: Extranodal Rosai-Dorfman disease in children. J Pediatr Hematol Oncol 26:19–24, 2004. 211. Stine KC, Westfall C: Sinus histiocytosis with massive lymphadenopathy (SHML) prednisone resistant but dexamethasone sensitive. Pediatr Blood Cancer 44:92–94, 2005.

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212. Farquhar JW, MacGregor AR, Richmond J: Familial haemophagocytic reticulosis. Br Med J 2:1561–1564, 1958. 213. Henter JI, Elinder G, Soder O, Ost A: Incidence in Sweden and clinical features of familial hemophagocytic lymphohistiocytosis. Acta Paediatr Scand 80:428–435, 1991. 214. Henter JI, Samuelsson-Horne A, Arico M, et al: Treatment of hemophagocytic lymphohistiocytosis with HLH-94 immunochemotherapy and bone marrow transplantation. Blood 100:2367–2373, 2002. 215. Zhang K, Biroschak J, Glass DN, et al: Macrophage activation syndrome in patients with systemic juvenile idiopathic arthritis is associated with MUNC13–4 polymorphisms. Arthritis Rheum 58:2892–2896, 2008. 216. Zhang K, Chandrakasan S, Chapman H, et al: Synergistic defects of different molecules in the cytotoxic pathway lead to clinical familial hemophagocytic lymphohistiocytosis. Blood 124:1331–1334, 2014. 217. Allen CE, Yu X, Kozinetz CA, McClain KL: Highly elevated ferritin levels and the diagnosis of hemophagocytic lymphohistiocytosis. Pediatr Blood Cancer 50:1227–1235, 2008. 218. Parikh SA, Kapoor P, Letendre L, et al: Prognostic factors and outcomes of adults with hemophagocytic lymphohistiocytosis. Mayo Clin Proc 89:484–492, 2014. 219. Li F, Li P, Zhang R, et al: Identification of clinical features of lymphoma-associated hemophagocytic syndrome (LAHS): An analysis of 69 patients with hemophagocytic syndrome from a single-center in central region of China. Med Oncol 31:902, 2014. 220. Henter JI, Elinder G, Soder O, et al: Hypercytokinemia in familial hemophagocytic lymphohistiocytosis. Blood 78:2918–2922, 1991. 221. Imashuku S, Hibi S, Sako M, et al: Heterogeneity of immune markers in hemophagocytic lymphohistiocytosis: Comparative study of 9 familial and 14 familial inheritance-unproved cases. J Pediatr Hematol Oncol 20:207–214, 1998. 222. Terrell CE, Jordan MB: Perforin deficiency impairs a critical immunoregulatory loop involving murine CD8(+) T cells and dendritic cells. Blood 121:5184–5191, 2013. 223. Feldmann J, Le DF, Ouachee-Chardin M, et al: Functional consequences of perforin gene mutations in 22 patients with familial haemophagocytic lymphohistiocytosis. Br J Haematol 117:965–972, 2002. 224. Kogawa K, Lee SM, Villanueva J, et al: Perforin expression in cytotoxic lymphocytes from patients with hemophagocytic lymphohistiocytosis and their family members. Blood 99:61–66, 2002. 225. Jordan MB, Hildeman D, Kappler J, Marrack P: An animal model of hemophagocytic lymphohistiocytosis (HLH): CD8+ T cells and interferon gamma are essential for the disorder. Blood 104:735–743, 2004. 226. de Saint BG, Menasche G, Fischer A: Molecular mechanisms of biogenesis and exocytosis of cytotoxic granules. Nat Rev Immunol 10:568–579, 2010. 227. zu Stadt U, Rohr J, Seifert W, et al: Familial hemophagocytic lymphohistiocytosis type 5 (FHL-5) is caused by mutations in Munc18-2 and impaired binding to syntaxin 11. Am J Human Genetics 85:482–492, 2009. 228. zur Stadt U, Beutel K, Kolberg S, et al: Mutation spectrum in children with primary hemophagocytic lymphohistiocytosis: Molecular and functional analyses of PRF1, UNC13D, STX11, and RAB27A. Hum Mutat 27:62–68, 2006. 229. Chandrakasan S, Filipovich AH: Hemophagocytic lymphohistiocytosis: Advances in pathophysiology, diagnosis, and treatment. J Pediatr 163:1253–1259, 2013. 230. Marsh RA, Bleesing JJ, Filipovich AH: Using flow cytometry to screen patients for X-linked lymphoproliferative disease due to SAP deficiency and XIAP deficiency. J Immunol Methods 362:1–9, 2010. 231. Jordan MB, Allen CE, Weitzman S, et al: How I treat hemophagocytic lymphohistiocytosis. Blood 118:4041–4052, 2011. 232. Janka GE, Belohradsky BH, Daumling S, et al: Familial lymphohistiocytosis. Haematol Blood Transfus 27:245–253, 1981. 233. Horne A, Trottestam H, Arico M, et al: Frequency and spectrum of central nervous system involvement in 193 children with haemophagocytic lymphohistiocytosis. Br J Haematol 140:327–335, 2008. 234. Henter JI, Horne A, Arico M, et al: HLH-2004: Diagnostic and therapeutic guidelines for hemophagocytic lymphohistiocytosis. Pediatr Blood Cancer 48:124–131, 2007. 235. Marsh RA, Bleesing JJ, Filipovich AH: Using flow cytometry to screen patients for X-linked lymphoproliferative disease due to SAP deficiency and XIAP deficiency. J Immunol Methods 362:1–9, 2010. 236. Bryceson YT, Pende D, Maul-Pavicic A, et al: A prospective evaluation of degranulation assays in the rapid diagnosis of familial hemophagocytic syndromes. Blood 119: 2754–2763, 2012. 237. Gupta A, Weitzman S, Abdelhaleem M: The role of hemophagocytosis in bone marrow aspirates in the diagnosis of hemophagocytic lymphohistiocytosis. Pediatr Blood Cancer 50:192–194, 2008. 238. Lehmberg K, McClain KL, Janka GE, Allen CE: Determination of an appropriate cutoff value for ferritin in the diagnosis of hemophagocytic lymphohistiocytosis. Pediatr Blood Cancer 61:2101–2103, 2014. 239. Lin TF, Ferlic-Stark LL, Allen CE, et al: Rate of decline of ferritin in patients with hemophagocytic lymphohistiocytosis as a prognostic variable for mortality. Pediatr Blood Cancer 56:154–155, 2011. 240. Janka GE, Lehmberg K: Hemophagocytic lymphohistiocytosis: Pathogenesis and treatment. Hematology Am Soc Hematol Educ Program 2013:605–611, 2013. 241. Ambruso DR, Hays T, Zwartjes WJ, et al: Successful treatment of lymphohistiocytic reticulosis with phagocytosis with epipodophyllotoxin VP 16–213. Cancer 45:2516–2520, 1980. 242. Fischer A, Virelizier JL, Arenzana-Seisdedos F, et al: Treatment of four patients with erythrophagocytic lymphohistiocytosis by a combination of epipodophyllotoxin, steroids, intrathecal methotrexate, and cranial irradiation. Pediatrics 76:263–268, 1985.

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243. Henter JI, Samuelsson-Horne A, Arico M, et al: Treatment of hemophagocytic lymphohistiocytosis with HLH-94 immunochemotherapy and bone marrow transplantation. Blood 100:2367–2373, 2002. 244. Imashuku S: Treatment of Epstein-Barr virus-related hemophagocytic lymphohistiocytosis (EBV-HLH); update 2010. J Pediatr Hematol Oncol 33:35–39, 2011. 245. Johnson TS, Terrell CE, Millen SH, et al: Etoposide selectively ablates activated T cells to control the immunoregulatory disorder hemophagocytic lymphohistiocytosis. J Immunol 192:84–91, 2014. 246. Thompson PA, Allen CE, Horton T, et al: Severe neurologic side effects in patients being treated for hemophagocytic lymphohistiocytosis. Pediatr Blood Cancer 52:621–625, 2009. 247. Mahlaoui N, Ouachee-Chardin M, de Saint BG, et al: Immunotherapy of familial hemophagocytic lymphohistiocytosis with antithymocyte globulins: A single-center retrospective report of 38 patients. Pediatrics 120:e622–e628, 2007. 248. Marsh RA, Allen CE, McClain KL, et al: Salvage therapy of refractory hemophagocytic lymphohistiocytosis with alemtuzumab. Pediatr Blood Cancer 60:101–109, 2013.

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249. Horne A, Janka G, Maarten ER, et al: Haematopoietic stem cell transplantation in haemophagocytic lymphohistiocytosis. Br J Haematol 129:622–630, 2005. 250. Cooper N, Rao K, Goulden N, et al: The use of reduced-intensity stem cell transplantation in haemophagocytic lymphohistiocytosis and Langerhans cell histiocytosis. Bone Marrow Transplant 42 Suppl 2:S47–S50, 2008. 251. Marsh RA, Kim MO, Liu C, et al: An intermediate alemtuzumab schedule reduces the incidence of mixed chimerism following reduced-intensity conditioning hematopoietic cell transplantation for hemophagocytic lymphohistiocytosis. Biol Blood Marrow Transplant 19:1625–1631, 2013. 252. Grom AA, Mellins ED: Macrophage activation syndrome: Advances towards understanding pathogenesis. Curr Opin Rheumatol 22:561–566, 2010. 253. Schulert GS, Grom AA: Macrophage activation syndrome and cytokine-directed therapies. Best Pract Res Clin Rheumatol 28:277–292, 2014. 254. Lau SK, Chu PG, Weiss LM: Immunohistochemical expression of Langerin in Langerhans cell histiocytosis and non-Langerhans cell histiocytic disorders. Am J Surg Pathol 32:615–619, 2008.

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GAUCHER DISEASE AND RELATED LYSOSOMAL STORAGE DISEASES

patients contains typical foam cells with small droplets in the cytoplasm and sea-blue histiocytes. Substrate reduction therapy was approved for patients with type C disease in 2008 in Europe; pharmacologic chaperone therapy is being attempted.   Fabry disease, Wolman/cholesteryl ester storage disease (CESD), and GM1-gangliosidoses are other lipid storage diseases characterized by hepatosplenomegaly; GM2-gangliosidosis by hepatomegaly only. CESD patients may result in anemia and have sea-blue histiocytes. They are usually not cared for by hematologists and will not be discussed in this chapter.

Ari Zimran and Deborah Elstein

SUMMARY Gaucher disease and Niemann-Pick disease are the two lipid storage disorders that are most likely to be encountered by the hematologist because both may cause hepatosplenomegaly and cytopenias.   Gaucher disease is a common autosomal recessive lipid storage disorder, with an increased prevalence among Ashkenazi Jews, in whom the estimated birth occurrence is 1 in 850. Deficiency of the enzyme β-glucocerebrosidase results in accumulation of the sphingolipid glucocerebroside in the cells of the macrophage-monocyte system. Patients with the most prevalent form, type 1, have no primary neuronopathic symptoms, whereas there is involvement of the central nervous system in type 2 and type 3. Diagnosis of Gaucher disease depends on demonstration of decreased enzymatic activity of β-glucocerebrosidase combined with identification of mutations in the β-glucocerebrosidase gene at the DNA level, usually with elevation of biomarkers, such as chitotriosidase as ancillary confirmation and means of followup. Disease manifestations include hepatosplenomegaly, thrombocytopenia, anemia, osteopenia/osteoporosis with pathologic fractures and osteonecrosis, and, less commonly, pulmonary infiltration. Many patients, especially those homozygous for the common N370S mutation, are putatively protected against neurologic involvement, albeit there is evidence of a genetic risk factor for Parkinson disease. Generally, many patients with type 1 may be asymptomatic or so mildly affected that they may not present until their fifth or sixth decade and do not require disease-specific therapy, whereas for those with more severe signs and symptoms, enzyme replacement therapy (currently three infusible enzymes) is available. Substrate reduction therapy is an oral modality but is associated with a more problematic safety profile. Pharmacological chaperones and oral enzyme are being tested.   Niemann-Pick disease is a heterogeneous group of autosomal recessive disorders. Type A and type B result from deficiency of the enzyme sphingomyelinase, whereas type C results from mutations in the NPC1 or NPC2 gene, which appears to be involved in cholesterol trafficking and resulting in accumulation of cholesterol as well as sphingomyelin. Type A is a lethal infantile form with marked progressive neurologic involvement. Type B is a later-onset form with no neurologic involvement but hepatosplenomegaly in many patients. Patients with type C disease manifest progressive neurologic involvement and hepatosplenomegaly, but may survive into adulthood. The marrow of these

Acronyms and Abbreviations: cDNA, complementary DNA; ERT, enzyme replacement therapy; MRI, magnetic resonance imaging; PC, pharmacologic chaperone; SRT, substrate reduction therapy.

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D  EFINITION OF GLYCOLIPID STORAGE DISEASES The glycolipid storage diseases are hereditary disorders in which one or more tissues become engorged with specific lipids, because of deficiencies of the lysosomal enzymes required for hydrolysis of one of the glycosidic bonds. Figure 72–1 shows the catabolic pathway of glycosphingolipids and lists the diseases that are involved in impaired degradation because of specific enzyme deficiencies. The type of lipid and its tissue distribution have a characteristic pattern in each disorder. This chapter deals mainly with Gaucher disease, in which glucocerebroside is stored. It is a common lysosomal storage disorder and also the one with the most hematologic features. The second storage disorder with some hematologic features is Niemann-Pick disease, in which the accumulated material is sphingomyelin and/or cholesterol. The remaining lysosomal diseases (Fabry disease, Wolman/cholesteryl ester storage disease, and GM1- and GM2-gangliosidoses), in which there is hepatosplenomegaly but few hematologic abnormalities, are not reviewed in this chapter.

GAUCHER DISEASE HISTORY AND DEFINITION Gaucher disease was first described by P.C.E. Gaucher in 1882, who thought that the large splenic cells of a young woman seen postmortem were evidence of a primary neoplasm.1 The term Gaucher disease appeared first in 1905, when the autosomal recessive genetic nature of the disorder was elucidated.2 In 1934, it was shown that glucocerebroside is the storage material in Gaucher disease,3 and in 1965, the primary defect was recognized as a deficiency of glucocerebrosidase resulting in an impairment of degradation of glucocerebroside.4,5 Enzymatic purification ultimately led to the cloning of the gene in 1985,6,7 unraveling of its structure, and identification of many glucocerebrosidase mutations.8 Disease-specific enzyme replacement therapy (ERT) was first introduced in 1991.9

EPIDEMIOLOGY Gaucher disease is inherited as an autosomal recessive disorder. Although panethnic, type 1 is most common among the Ashkenazi Jews, with a carriership prevalence of 1 in 17 and an expected frequency of the disease in 1 in 850 livebirths.10 Two distinct forms of Gaucher disease, type 3b and type 3c, are also relatively common in Norrbottnia in northern Sweden,11 and near the Palestinian town of Jenin, respectively.12 In the general population, the estimated frequency (based on large-scale neonatal screening projects in three countries is in the range of 1 in 50,000 to 1 in 100,000 persons.13

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Gal-GalNAc NANA

Gal-Glc-Cer [GM1] Generalized gangliosidosis

GalNAc NANA

Gal Gal-Glc-Cer [GM2]

GalNAc-Gal-Gal-Glc-Cer [Globoside]

Tay-Sachs disease

Sandhoff disease GalNAc GalNAc NANA-Gal-Glc-Cer [GM3] Gal-Gal-Glc-Cer

Figure 72–1. The catabolic pathways of selected glycosphingolipids involved in some of the glycolipid storage diseases. Solid squares depict the blocked pathways caused by specific inherited deficiencies of enzymes, which give rise to the accumulation of the respective substrates. The names of the various diseases are shown above the names of the deficient enzymes. (Reproduced with permission from Lichtman’s Atlas of Hematology, www.accessmedicine.com.)

Fabry disease Neuraminidase

NANA

Gal

Gal-Glc-Cer

Gal Glc-Cer Gaucher disease Glc Gal

SO3H2 SO3H-Gal-Cer Metachromatic leukodystrophy Arylsulfatase A

Gal-Cer

Coline-P

Ceramide

Krabbe disease

Phosphorylcholine-Cer [Sphingomyelin] Niemann-Pick disease Sphingomyelinase

Farber disease Ceramidase

Fatty acid Sphingosine

The high prevalence of at least two Gaucher mutation, N370S and 84GG (and possibly R496H14 and others), among Ashkenazi Jews, and the existence of other lysosomal diseases within this ethnic group, may reflect, in addition to a founder effect, a selective advantage. However, a selective advantage because of greater resistance to tuberculosis15 or superior intelligence16 has not been proven. Animal studies suggest that the selective advantage may be the higher circulating serum levels of glucocerebroside that have antiinflammatory and beneficial immunomodulary effects.17

ETIOLOGY AND PATHOGENESIS Enzymatic Basis

During normal growth, development, and senescence, parts of or whole cells are continually replaced. Breakdown of complex constituents of cells requires sequential enzymatic degradation. Such degradation occurs largely in secondary lysosomes, organelles formed by the fusion of primary lysosomes with phagocytic vacuoles containing ingested material. Gaucher disease is the result of a hereditary deficiency in the activity of a lysosomal enzyme, glucocerebrosidase, required for glycolipid degradation. The reduced activity of glucocerebrosidase results in accumulation of glucocerebroside in macrophages engorgement with

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glucocerebroside induces increased cell size and cytoplasmic striations, leading to the formation of “Gaucher cells” (see Fig. 72–1). Inherent in subsequent lysosomal dysfunction is a dysregulation of metabolites and the consequent lack of coordination of cellular metabolism. These changes may explain the elaboration of various cytokines and other biomarkers because of engorgement of macrophages. Accumulating evidence indicates that in addition to the glucocerebrosidase enzyme (GBA1), a second, nonlysosomal glucocerebrosidase, GBA2, a cytosolic protein that tightly associates with cellular membranes, may be integral to the pathogenesis of Gaucher disease, affecting its phenotype by potentially interacting with GBA1.18–20 In rare instances, severe forms of Gaucher disease are associated with deficiency of saposin C, a heat-stable glucocerebrosidase co-factor.21,22

GENETIC BASIS OF GAUCHER DISEASE The glucocerebrosidase gene is located on chromosome 1q21. A pseudogene, with 96 percent sequence homology, has been identified approximately 16 kb downstream from the functional gene. Nearly 300 point mutations causing Gaucher disease have been described8,23; most are point mutations, missense, nonsense, frameshift, and splice-site mutations, but there are also insertions, deletions, and recombinant alleles.

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Some mutations result from recombinant events between the functional gene and its pseudogene.8 Since 2000, approximately 20 of these mutated enzymes have undergone crystallography showing the divergence of ligands in the active site and with various degrees of glycosylation.23 Among Ashkenazi Jews, the predominant mutation is N370S which accounts for approximately 75 percent of mutant alleles among Jewish patients and approximately 30 percent of the alleles among non-Jewish patients. Homozygosity for N370S is characterized by relatively milder phenotypes (although the phenotype is very heterogenous and severe cases are seen24). N370S has heretofore been considered “protective” against the development of neuronopathic features. The second common mutation found almost exclusively among Ashkenazi Jews is one that usually causes a severe phenotype. Five or six common mutations account for approximately 97 percent of alleles among Jews, but account for less than 75 percent of alleles among non-Jews.8,25–27 Although controversial, premarital/prenatal screening for common mutations has become frequent among Ashkenazi Jews.28,29, The second most common mutation is L444P, which when homozygous accounts for most patients with the neuronopathic type 3 disease, and is the most prevalent mutation in Asians, Arabs, and Norrbottnians. Patients with the unique variant of progressive calcifications of cardiac valves, type 3c, are uniformly homozygous for a point mutation D409H.12 Despite some relationship between specific mutations and the clinical course, genotype–phenotype correlation is imperfect. Elucidation of the three-dimensional structure of the glucocerebrosidase by crystallography has also not improved prediction of disease severity based on the location of mutations in the native protein.30 The majority of the mutations cause glucocerebrosidase misfolding, which may lead to early degradation of the enzyme in the

endoplasmic reticulum.31,32 The investigation of the proteotoxic effect of the misfolded mutant enzyme in the endoplasmic reticulum has led to the development of the new therapeutic modality of pharmacologic chaperones (PCs). PCs are targeted to stabilize the mutated glucocerebrosidase and allow its appropriate trafficking from endoplasmic reticulum to Golgi and, finally, to the lysosome.

CLINICAL FEATURES Three major types of Gaucher disease are differentiated clinically based on absence (type 1) or presence of neurologic features (types 2 and 3).33 Table 72–1 summarizes key clinical, genetic, and demographic features. Although it has been suggested that there is a phenotypic continuum,34,35 it is still useful to think of Gaucher disease as three distinct forms to facilitate genetic counseling and management decisions. There is variability in disease severity of all types of Gaucher disease. Type 1 disease may be asymptomatic and be discovered in the course of population surveys of Ashkenazi Jews,28 or incidentally during evaluation of an unrelated hematologic disorder.

Fatigue

Fatigue is a common complaint, usually not invariably related to anemia, but also quite common in nonanemic patients and may be a result of elevated inflammatory cytokines.36

Organomegaly

In symptomatic patients, the spleen is typically enlarged,37 whether barely palpable or massively enlarged causing positional symptoms, such as early satiety or abdominal discomfort. Splenic infarction and subcapsular hematoma are uncommon. Hepatomegaly is usually asymptomatic, but it may cause abdominal discomfort and in splenectomized

TABLE 72–1.  Characteristics of the Three Types of Gaucher Disease  

TYPE 1

TYPE 2

TYPE 3

Subtype

Asymptomatic

Symptomatic

Neonatal

Infantile

3a

3b

3c

Common genotype

N370S/N370S or 2 mild mutations

N370S/other or 2 mild mutations

Two null or recombinant mutations

One null and one severe mutations

None

L444P/L444P

D409H/D409H

Ethnic predilection

Ashkenazi Jews Ashkenazi Jews None

None

None

Norrbottnians, Asians, Arabs

Palestinian Arabs, Japanese

Common presenting features

None

HepatospleHydrops fetalis; SNGP, strabisnomegaly, congenital mus, opisthohypersplenism, ichthyosis tonus, trismus bleeding, bone pains

SNGP; myoclonic seizures

SNGP; hepatosplenomegaly growth retardation

SNGP; cardiac valves’ calcifications

CNS involvement

None

None

Lethal

Severe

SNGP; slowly progressive neurologic deterioration

SNGP; gradual cognitive deterioration

SNGP; brachycephalus

Bone involvement

None

Mild to severe (variable)

None

None

Mild

Moderate to severe; kyphosis (gibbus)

Minimal

Lung involvement

None

None to (rarely) Severe severe

Severe

Mild to moderate

Moderate to severe

Minimal

Death during childhood

Death in mid-adulthood

Death in early adulthood

Life Expectancy Normal

Normal/ near-normal

Neonatal death Death before age 3 years

SNGP, supranuclear gaze palsy.

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A

B

Figure 72–2.  A. Histologic section of “Gaucheroma” showing hemorrhagic mass with nucleated red blood cells covered by a fibrous capsule. B. Histologic section at a higher magnification showing nucleated red blood cells admixed with numerous Gaucher cells. (Used with permission of Prof. Eliezer Rosenmann, Shaare Zedek Medical Center, Jerusalem, Israel.)

patients or others with very severe disease, liver fibrosis and later cirrhosis,38 with or without portal hypertension, may occur; hepatocellular carcinoma may evolve.39 An increased incidence of nonalcoholic fatty liver disease has been observed.40

splenectomized patients54; it has not been reported in children.55 Pulmonary function tests may reveal abnormalities, such as reduced diffusion capacity in approximately two-thirds of patients.56

Lymphadenopathy

Reduced hemoglobin levels are also primarily a result of hypersplenism and marrow replacement by Gaucher cells, but additional causes include iron deficiency, vitamin B12 deficiency, and autoimmune hemolysis.46,47

Bone involvement is usually the main cause of morbidity in symptomatic patients and can occur in any long bone.57 Patchy areas of bone demineralization and infarction are seen (Fig. 72–3A), and asymptomatic widening of the distal femur known as Erlenmeyer flask deformity is very common (Fig. 72–3B). Bone metabolism markers indicate that bone resorption predominates,58 but the overall mechanisms underlying development of bone lesions are poorly understood. Children may have delayed bone age and delayed eruption of the teeth.59 Bone pain is probably the most troublesome symptom of Gaucher disease. Bone pain may be related to the pathologic processes evident by radiography, magnetic resonance imaging (MRI), and computerized tomography, or have the character of a “crisis,” which is a self-limiting, albeit exquisitely painful event, associated with signs of acute local and/or systemic inflammation (Fig. 72–3D). Aseptic necrosis of large joints, mainly the femoral heads but also the shoulders and knees (and rarely even in smaller joints) and vertebral collapse are particularly common typically among untreated patients with genotypes resulting in more severe phenotypes (Fig. 72–3C and E).

Gaucheromas

Gynecologic Manifestations and Fertility

Lymphadenopathy has been described,41 including a severe proteinlosing form,42 which is a clinical management problem.

Hemorrhagic Events

Epistaxis, easy bruising, and hemorrhage after surgical or dental procedures and bleeding during labor are common presenting symptoms. These manifestations usually are related to thrombocytopenia as the result of hypersplenism or marrow replacement by Gaucher cells, but platelet dysfunction and decreased levels of coagulation factors have also been described and hence should be assessed prior to surgical procedure or before delivery.43–45 Coagulation factor deficiencies may result from liver disease or consumption coagulopathy.

Anemia

“Gaucheromas” (Fig. 72–2), which are possibly extraosseous in origin48 and/or may mimic a malignant process,49,50 appear idiosyncratically, but possibly after some invasive procedure such as hip surgery; these have been described to be at increased risk of hemorrhaging when manipulated.

Pulmonary Disease

Severe pulmonary disease with cyanosis and clubbing occurs in some patients with advanced liver involvement, and is usually a consequence of hepatopulmonary syndrome with or without infiltration of the lungs by Gaucher cells.51,52 Mild pulmonary hypertension may be detected by echocardiography,53 but may (rarely) be severe especially among

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Bone Disease

Gynecologic and obstetric problems are common and are mainly related to bleeding tendency,60 which may explain why females are more likely to be diagnosed. Delayed menarche and menorrhagia are common, and increased risk of recurrent abortions has been reported.61 Fertility is unaffected in males and females.

Ophthalmologic Disorders

Organs other than the spleen, liver, bones, and lungs may be affected. Many patients have pinguecula and a few a pterygium at the corneoscleral limbus.62 Additional findings include uveitis and preretinal white spots in rare cases.63

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A

B

C

D

1125

E

Figure 72–3.  Gaucher-related skeletal involvement including (A) humerus with chevron or herring-bone pattern; (B) Erlenmeyer flask deformity

of the proximal femur; (C) plain radiograph of osteonecrosis of the left hip; (D) magnetic resonance image of pelvis and thighs that was performed 2 weeks after bone crisis of the right thigh. Bone edema is seen in the upper part of the femur at the level of lesser trochanter. Chronic marrow signal changes are seen in both femurs; (E) vertebral collapse. (Used with permission of Dr. Ehud Lebel, Shaare Zedek Medical Center, Jerusalem, Israel.)

Kidney Disease

Renal manifestations are rare and limited to case reports of nephrotic syndrome and renal cell carcinoma.64 Nonetheless, many patients seem to have benign urinary hyperfiltration.65

Neurologic Findings

Neurologic symptoms constitute the hallmark of types 2 and 3 diseases.66 Particularly notable and pathognomonic are oculomotor abnormalities, especially supranuclear gaze palsy (SNGP), which is typically noted horizontally,67 but might occur in the vertical plane as well. Patients with type 2 disease develop hypertonia of the neck muscles with extreme arching of the neck (opisthotonus), bulbar signs, limb rigidity, seizures, and sometimes choreoathetoid movements. In these patients, the SNGP becomes a fixed convergent squint, often facilitating differentiating between type 2 patients, who are terminal by 2 to 3 years of age, and the severe type 3a patients, who may survive longer. Patients with type 3a disease exhibit progressive neurologic abnormalities such as myoclonus and dementia.68 Patients with type 3b disease display aggressive visceral and skeletal involvement but neurologic manifestations are largely limited to horizontal SNGP.68 Patients with type 3c disease exhibit SNGP, mild visceral involvement, and fatally progressive calcifications of mitral and tricuspid valves and of the large arteries.12,68–70 Several neurologic abnormalities have been observed in patients with type 1 disease, including peripheral neuropathy71,72 and an increased prevalence of Parkinson disease (the latter also among carriers of a single mutation).73–77 Carriers of severe mutations (e.g., null alleles) were reported to have a 13.6-fold increased risk of Parkinson disease compared to controls, whereas carriers of the more benign mutations have a 2.2-fold increased risk.78 A meta-analysis of patients with Parkinson disease has confirmed this strong association between mutations in the glucocerebrosidase gene and Parkinson disease, which is marked by an earlier age of onset and higher prevalence of cognitive changes.78,79

Predisposition to Infections

An increased tendency to infections is sometimes seen, occurring among splenectomized patients or severely affected patients, some of whom may have defective neutrophil chemotaxis.80,81 Bacterial osteomyelitis is most often iatrogenic following surgical intervention at the site of a bone crisis. In children, linear growth retardation is common regardless of disease severity,82 but a compensatory “catch-up” growth may occur by early adulthood.83

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Predisposition to Neoplasia

There is a higher prevalence of neoplastic disorders in patients with Gaucher disease.84,85 Myeloma has been established to be more prevalent.84,85 Other hematologic malignancies,86 hepatocellular carcinoma, and renal cell carcinoma, may also have increased prevalence.87 Although elevated levels of interleukin-6 in patients with Gaucher disease may link Gaucher disease and myeloma,88 there is no explanation at present for increased incidence of other types of cancer. Some malignancies may be less common.87 The impact of ERT on either an increased or decreased development of malignancies has not been determined.

LABORATORY FEATURES Blood Counts

The complete blood count in patients with Gaucher disease may be normal or may reflect the effects of hypersplenism. A normocytic, normochromic anemia is frequently present, but hemoglobin levels only rarely fall below 8 g/dL. A modest reticulocytosis is often present in anemic patients. The white cell count may be decreased to as low as 1.0 × 109/L, but milder degrees of leukopenia are more common. The differential count is normal, but splenectomized patients tend to show a lymphocytosis. A defect of leukocyte chemotaxis which may be corrected by ERT,89 and in some patients is associated with a tendency to bacterial infections80; monocyte dysfunction has also been reported.81 Thrombocytopenia is typically more prominent than anemia.46 In an anemic patient with an intact spleen and normal range platelet counts, there is probably an alternative reason for the low hemoglobin level, unrelated to Gaucher disease. Thrombocytopenia may be quite severe, even in an otherwise mildly affected patient. In splenectomized patients, anemia is more likely in the absence of thrombocytopenia; white cell count and platelet counts are usually higher than normal. Severe anisocytosis and poikilocytosis also occur in splenectomized patients, with many target cells, some nucleated red cells and Howell-Jolly bodies. During bone crises, leukocytosis, thrombocytosis, and elevated erythrocyte sedimentation are seen. Other markers of inflammation have been noted regardless of disease severity: elevated fibrinogen levels, elevated high-sensitivity C-reactive protein, and increased adhesion and aggregation of red blood cells.90,91

Other Hematologic Findings

Clotting factor abnormalities may be induced by activated macrophages41,42 or may be found when there is liver involvement. Factor IX deficiency may be a laboratory artifact related to the effect of

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accumulated lipid on platelet membranes.92 Factor XI deficiency is common among Ashkenazi Jewish patients because of its high coincidental prevalence in this ethnic group.93 Bleeding tendency may also result from defective aggregation or adhesion of platelets33 and therefore platelet function and/or thromboelastography should be tested before surgical and dental procedures and labor.44,94

Biochemical and Immunologic Findings

In most patients, liver function tests are within normal limits but in conjunction with more severe disease, splenectomy, and/or comorbidities (hepatitic B and/or C, or autoimmune diseases) abnormal liver function tests may be seen. Because of the increased prevalence of cholelithiasis,95,96 cholestatic findings may occur. Renal function tests are usually normal.64 Many patients present with polyclonal gammopathies. Monoclonal gammopathies are found in 1 to 20 percent of patients, particularly older patients.79–82 Increased levels of autoantibodies have been reported,97 and may indicate coincide with autoimmune diseases such as Hashimoto thyroiditis, rheumatoid arthritis, or immune hemolytic anemia. Biochemical abnormalities have been used as surrogate markers in Gaucher disease. In the past, increased activities of serum acid phosphatase, angiotensin-converting enzyme, serum ferritin, and other hydrolases, such as β-hexosaminidase or β-glucuronidase, were used.

A

Other biomarkers correlate better with the extent of glucocerebroside storage. The most widely used biomarker is chitotriosidase,98 which is undetectable in healthy subjects (its physiologic role is unknown), but is elevated, often several thousand-fold, in patients with Gaucher disease. Chitotriosidase measurement is useful for monitoring both untreated patients, to assess stability versus deterioration, and treated patients, to assess response to therapy. A change in chitotriosidase levels rather than absolute values is used for monitoring. In approximately 6 percent of people, it is undetectable, and for those patients, measurements of chemokine CCL18/PARC which is predominantly produced by Gaucher cells, can be used.99 A potentially more sensitive and more specific biomarker has been identified: the lyso-glucosylsphingosine (lyso-Gb1),100 which may be preferred as a more reproducible biomarker, using a more operatorfriendly assay. Serum iron levels may be low in patients because of iron deficiency related to bleeding or chronic inflammation. Deficiencies of vitamin B12101 and vitamin D102 have been described, albeit these are also very common in the general population. Serum ferritin levels are usually elevated.

Gaucher Cells

Gaucher cells, found mainly in the marrow, spleen, and liver (Fig. 72–4), have small, usually eccentrically placed nuclei and cytoplasm with

B

C

Figure 72–4.  A. “Gaucher cell” from the marrow of a patient with Gaucher disease. B. Histomicrograph of a Gaucher spleen with marked infiltration of

the red pulp by Gaucher cells. C. Liver infiltrated by Gaucher cells (the pale pink cells). (Marrow image used with permission of Prof. Chaim Hershko, Shaare Zedek Medical Center, Jerusalem, Israel; spleen and liver images used with permission of Prof. Gail Amir, Hadassah Medical Center, Jerusalem, Israel.)

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characteristic crinkles or striations. The cytoplasm is stained by the periodic acid-Schiff technique. Electron microscopy demonstrates cytoplasmic spindle- or rod-shaped, membrane-bound inclusion bodies 0.6 to 4 μm in diameter, consisting of numerous small tubules, 130 to 750 Å in diameter, that are composed of twisted multilayers in negatively stained preparations.103

DIFFERENTIAL DIAGNOSIS Diagnosis

The diagnosis of Gaucher disease should be considered in (1) any patient who presents with unexplained splenomegaly, thrombocytopenia, frequent nosebleeds, anemia, acute or chronic bone pain; (2) children with short stature for their age; and (3) nontraumatic avascular necrosis of a large joint at any age, especially if is associated with any of the above features. A definitive diagnosis requires a reduced enzymatic activity of β-glucocerebrosidase in leukocytes,104,105 cultured fibroblasts, or amniocytes obtained during prenatal diagnosis. Measurement of glucocerebrosidase levels is supplemented by mutational analysis. This is important for prognosis, particularly in children, and for detection of carriers among affected families. While rapid polymerase chain reaction-based tests are often performed for five or seven common mutations, especially among Ashkenazi Jews as a “first-pass,” it is highly recommended to perform whole-genome sequencing106 to rigorously establish the molecular diagnosis. Marrow aspiration as a means of diagnosis is only indicated when other hematologic diseases must be considered.94,105 Gaucher cells are often sparse and thorough examination under low-power may be required to find them. Cells indistinguishable by light microscopy from typical Gaucher cells may also be seen in patients with other disorders such as chronic myelogenous leukemia, Hodgkin lymphoma, myeloma, and acquired immunodeficiency syndrome. The latter patients do not lack the ability to catabolize glucocerebroside, but the great inflow of globoside into phagocytic cells exceeds their capacity to hydrolyze glucocerebroside, forming “pseudo-Gaucher cells.” Prenatal diagnosis can be established by examining cultured amniocytes obtained by amniocentesis for measurement of glucocerebrosidase activity104 or by examining amniocytes or chorionic villi DNA for known mutations.

Heterozygote Detection

Heterozygotes for Gaucher disease have neither Gaucher cells in their marrow nor stigmata of Gaucher disease (other than the increased risk of Parkinson disease). Existence of a carrier state can be demonstrated by reduced glucocerebrosidase activity to approximately 50 percent of normal values. However, regardless of methodology, enzyme activity among heterozygotes overlaps the normal range and hence definitive diagnosis of heterozygous status only can be made by mutational analysis. Currently various methodologies are being developed to allow noninvasive prenatal diagnosis of monogenic diseases like Gaucher disease; the most promising of these is molecular analysis of cell-free fetal DNA.107

THERAPY Symptomatic Treatment

Symptoms and signs related to massive enlargement of the spleen (e.g., pancytopenia, early satiety, abdominal discomfort, and growth retardation in children) can be resolved by splenectomy. However, because of the efficacy of ERT, splenectomy should only be a last resort as it often induces progressive liver and bony complications, and increases

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the risk of infection with encapsulated organisms. Partial splenectomy has not proved useful, with both regrowth of the remnant and risk of osteonecrosis. When bone lesions result in fractures or osteonecrosis (see Fig. 72–3D), orthopedic procedures may be required. Joint replacement is generally uneventful, with good functional outcome and quality of life. The success of arthroplasties is enhanced by adherence to preoperative protocols including assessment of bleeding tendency, prophylactic use of antibiotic therapy, particularly in splenectomized patients, and early post-operative ambulation.108 Deficiencies of iron, vitamin B12, or vitamin D should be corrected and calcium supplementation is recommended in patients with osteoporosis receiving bisphosphonates.109 Use of erythropoietin may be required for management of anemia because of marrow failure.110

Enzyme Replacement Therapy

The use of alglucerase,9 the first mannose-terminated, placental-derived enzyme, was approved in 1991, and the recombinant form, imiglucerase, albeit with one amino acid R495H that differs from the wild-type protein owing to a cloning artifact in the original complementary DNA (cDNA), was introduced in 1994.111 Two intravenous preparations, one with the perfect native-enzyme sequence developed in a human cell line, velaglucerase alfa,112 and the other, a carrot root cell-derived with the imiglucerase core sequence, taliglucerase alfa,113 have completed phase 3 clinical trials and are available. Phase 2 clinical trials with taliglucerase alfa are currently underway in which the same carrot cells, expressing taliglucerase alfa, are used as vehicle for oral delivery of the enzyme. The response to ERTs is most gratifying.9,111–116 Decreased spleen and liver volumes and increased hemoglobin levels and platelet counts usually occur within 6 months of therapy with biweekly doses between 15 and 60 U/kg. Platelet counts in patients with massively enlarged spleens may require longer periods to respond, but improvement continues within the first 2 years of therapy. Thereafter, patients treated with imiglucerase stabilize even while on the same dose.116 The bone response is slower and less predictable. Osteonecrosis and lytic lesions do not respond to ERT. Quantitative chemical shift imaging, a sensitive modality to show changes in the marrow, including response to ERT (Fig. 72–5),117 is a resource available in only one site worldwide and, hence, various other imaging modalities, especially MRI-based modalities, but also bone densitometry and plain radiographs, are used as needed to document skeletal status. ERT may or may not improve pathologic pulmonary findings. Because the enzyme is a large molecule, it does not cross the blood– brain barrier, and hence, does not impact neuronopathic features.118,119 All ERTs are safe, having few side effects that are usually transient.112,113,120 Hypersensitivity reactions have been reported with each type of ERT, but only rare cases of anaphylaxis. Most patients with such reactions may continue ERT with or without premedication; it is advisable to avoid the administration of glucocorticoids for this purpose because of an increased risk of osteonecrosis. For each ERT there is a different percent of patients who may develop antibodies either shortly after initiation of treatment or over time. Another side effect is weight gain with some concerns about changes in insulin resistance and the development of metabolic syndrome,121 including steatohepatitis. Because of the excellent safety profile, many patients receive therapy at home122 and many female patients are comfortable continuing with ERT during pregnancy and lactation.123,124 The effects of treatment are unaffected by switching from imiglucerase to velaglucerase alfa125 or taliglucerase alfa.126 The two major disadvantages of ERTs are the apparent lifetime dependency on intravenous infusions and the extremely high cost.

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1.0

0.0 T = 0 FF = 0.11

T=3 FF = 0.24

Guidelines and/or expert opinions usually recommend the use of relatively high doses.127,128 Yet, it is evident that for most symptomatic patients, there is no justification for doses higher than 30 to 60 U/kg per month, and for patients with asymptomatic type 1 disease, ERT should not be encouraged.129

Substrate Reduction Therapy

The possibility that decreasing the formation of glucocerebroside from ceramide and glucose, referred to as substrate reduction therapy (SRT),130 might favorably impact disease parameters was proposed in the 1970s.131 Oral miglustat (N-butyldeoxynojirimycin),130 a glucose analogue that inhibits glucocerebroside synthase, has been licensed for treatment of patients for whom ERT is not suitable or not a therapeutic option according to the two preeminent regulatory authorities’ definitions. This circumscribed approval stems from inferior efficacy of miglustat relative to ERT and a problematic safety profile including peripheral neuropathies, tremor, and memory impairment. Miglustat is effective in reducing hepatosplenomegaly in Gaucher disease when given as 100 mg, three times daily.132 Response to miglustat is dose-dependent; lower doses yield suboptimal improvement without reducing frequency of side effects.133 Miglustat has also been studied as maintenance therapy in patients previously treated with imiglucerase.133 A practical advantage was that it could be considered in type 3 patients because as a small molecule it crosses the blood–brain barrier and impacts neurologic signs. Unfortunately, a clinical trial failed to achieve the end points and, hence, there is no indication for this drug in neuronopathic Gaucher disease. Another SRT, a ceramide analogue, eliglustat tartrate,134 has been granted FDA approval. However, it has a more problematic safety profile compared to ERT (including cardiac events), the efficacy parameters.135 The robust database derived from long-term followup from phase 2 and from three different phase 3 clinical trials136 indicates it can be useful. However, unlike miglustat, it cannot cross the blood–brain barrier and should be targeted to type 1 patients only.

Pharmacologic Chaperones

A new approach to lysosomal storage diseases is “chaperone therapy.” PC therapy is based on in vitro experiments showing that some misfolded mutants of glucocerebrosidase are destroyed prior to their export from the endoplasmic reticulum to the lysosome.137,138 Under these circumstances, a reversible inhibitor stabilizes the mutant enzyme, enabling its passage to the lysosome without losing activity. Clinical trials with the first PC for Gaucher disease, isofagomine tartrate which had been shown to increase mutant enzyme activity in cells, tissues,139 and healthy volunteers during the phase 1 trial, failed in the phase 2 clinical trial when only 1 of 18 patients with type 1 showed a beneficial effect.140 Another PC, ambroxol, an expectorant that is available without prescription in many countries and has decades-long safety experience,

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T=7 FF = 0.45

Figure 72–5. Color-coded fat fraction measurements using quantitative chemical shift imaging in an adult patient with type 1 Gaucher disease. Annual measurements show increase in fat fraction with specific therapy (mean value in 1994 = 0.11; mean value in 2001 = 0.45). (Used with permission of Dr. Mario Maas, Academic Medical Center, Amsterdam, The Netherlands.)

was administered in a pilot study to adult type 1 patients141; clinical trials in type 3 patients are planned.

Organ Transplantation

Because the macrophage is a derived from hematopoietic stem cells, allogeneic hematopoietic stem cell transplantation should cure Gaucher disease. Although some enthusiasm was expressed for this approach, the short-term risks of transplantation markedly limit the number of suitable candidates. Effective ERT further limits the appropriateness of transplantation. Liver transplantation has been performed in a few patients with severe hepatic failure.142

COURSE AND PROGNOSIS Age of onset, severity of clinical manifestations, and degree of progression are partially related to genotype. Patients homozygous for the N370S mutation tend to present with symptoms and signs at an older age with relatively milder manifestations, and usually have a relatively stable disease. By contrast, compound heterozygotes for N370S and a “severe” mutation (such as N370S/84GG or N370S/L444P) usually present with the disease during childhood, and if untreated, progress continuously with both visceral and skeletal complications.108,143,144 Patients homozygous for the L444P mutation will develop neuronopathic disease with deteriorating neurologic signs and symptoms and their life span is reduced.65 Although the genotype of the patient provides a benchmark for prognosis, there is much variability in patients with the same genotype, including between siblings with the same genotype. The availability of ERT has changed the natural course of the disease allowing normal growth and development in most patients, even in those with “severe” genotypes. Nevertheless, some patients still develop skeletal complications despite ERT and there is concern regarding development of associated diseases, such as myeloma, other malignancies, or Parkinson disease.120 Prior to the availability of ERT, patients with severe type 1 or type 3, died at an early age because of liver disease, bleeding, or sepsis. With the advent of ERT, typical causes of death are malignancy, cardiovascular disease, and cerebrovascular disease.145 In type 2 disease, death usually results from neurologic complications within the first 4 years of life65; there is also a lethal neonatal variant. Total absence of glucocerebrosidase may not be compatible with life.

NIEMANN-PICK DISEASE HISTORY AND CLASSIFICATION In 1914, Niemann, a Berlin pediatrician, reported the case of an infant who died at age 18 months with a disorder that seemed atypical for Gaucher disease because of its early onset and rapid course.146 In 1927, Pick identified this as a unique disorder of rapid, progressive

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neurodegeneration in infants.147 The first adult patients identified had massive hepatosplenomegaly but no neurologic involvement. The predominant phospholipid accumulating in this disorder is sphingomyelin. In 1966, a deficiency of sphingomyelinase activity was demonstrated in a patient with Niemann-Pick disease.148 Niemann-Pick is not a single entity; it comprises a group of disorders in which sphingomyelin storage occurs. Type A and type B disease, the classic forms of the disorder, represent an infantile neuronopathic and a later-onset nonneuronopathic form, respectively.149 Type C, the most common form of Niemann-Pick disease, is a neuronopathic disorder, usually with an onset in early childhood, that results from an abnormality in cholesterol transport.150 The sphingomyelinase gene is normal in type C disease, but mutations occur in one of two genes which have been designated NPC1 and NPC2; the proteins they code may function in closely related steps of cholesterol transport. The designation type D disease was once applied to a population isolate in Nova Scotia151 but because these individuals also have an NPC1 mutation, this term is no longer used.

EPIDEMIOLOGY Niemann-Pick type A and type B diseases, also referred to as acid-sphingomyelinase deficiency, are panethnic disorders. There is a relatively high prevalence of type A disease among Ashkenazi Jews with a carrier rate of approximately 1 in 90.152 Three mutations account for 90 percent of Ashkenazi Jewish patients. Type B is common among individuals from the Maghreb region and the Arabian peninsula,153 with three and two mutations accounting for 75 percent and 85 percent of Turkish and Arabic patients, respectively. Type C disease is relatively common in a Nova Scotia isolate,151 in a Hispanic population from the Upper Rio Grande Valley in the United States,154 and in Western Europe.155 The prevalence of Niemann-Pick type C disease in European populations is estimated to be 1 in 120,000 to 1 in 150,000 Europeans.156

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PATHOLOGY AND CLINICAL MANIFESTATIONS The most characteristic histopathologic feature of the various forms of Niemann-Pick disease is the presence of foam histiocytes (Fig. 72–6), mainly in lymphoid tissues, but these may be present throughout the body. The foam cells contain largely sphingomyelin and cholesterol, the storage of cholesterol being more prominent in type C disease. Type A disease presents in infancy. During the first months of life, affected infants gain weight at a diminished rate, the abdomen enlarges, and development is delayed. The patients usually cannot sit and lose physical capabilities already achieved and become blind and deaf. Some infants have a protracted course of jaundice of unknown cause. During the second year of life, the child lies still with nearly flaccid hyporeflexic extremities; there is massive hepatosplenomegaly, mild lymphadenopathy, and often a fine xanthomatous rash. Bone lesions may occur. Type B disease usually presents in the first decade of life with hepatosplenomegaly, but may not be noted until adulthood. Neurologic manifestations are usually absent. Pulmonary infiltrates are common. The absence of a cherry-red spot in the macula and longer life expectancy differentiate type B from type A. Sea-blue histiocytes are sometimes found in the marrow, and a number of patients had been diagnosed as having sea-blue histiocytosis before a deficiency in sphingomyelinase was demonstrated.165 Patients with type C disease often have neonatal jaundice, normal early childhood, and then develop hypotonia, dementia, ataxia, dysarthria, dystonia, seizures, gelastic cataplexy, and cognitive decline.165 Hepatosplenomegaly is common.149 Presentation may be at any age, even in the seventh decade,165 but the “classic” description is of

ETIOLOGY AND PATHOGENESIS Type A and type B are autosomal recessive diseases caused by loss of function mutations in the gene for sphingomyelinase,157 which is required for cleaving the bond between ceramide and phosphorylcholine (see Fig. 72–1). Nonsense mutations seem to cause the more severe type A disease, while missense mutations are found in the milder type B disorder.157 Although sphingomyelinase is believed to be a part of an apoptosis-signaling pathway by generating ceramide from sphingomyelin,158 no relationship between disease severity and this pathway has been established. Type C disease also is an autosomal recessive disorder and is caused by mutations in either the NPC1159,160 or NPC2160 gene. The function of the proteins encoded by these genes is unknown, but was suggested to be related to intracellular cholesterol transport.160,161 The NPC1 gene encodes for a multi-pass transmembrane protein that localizes to the late endosome. The NPC2 protein is soluble. The NPC1 mutations account for more than 95 percent of cases.161 There are only a few cases of NPC2 mutations manifested in neonates by severe liver and lung involvement, and progressive neurologic involvement leading to death by 4 years of age; there is a juvenile form in which there seems to be good genotype–phenotype correlation.162 NPC1 deficiency is associated with induction of autophagy by the class III-P13K (phosphatidylinositide 3′-kinase)/beclin-1 complex.163 A naturally occurring murine model of the disease exists.164

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Figure 72–6.  Typical foam cell from the marrow of a patient with Niemann-Pick disease.

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juvenile dystonic lipidosis with neonatal icterus and hepatosplenomegaly. In infants and toddlers, hepatosplenomegaly may be the only sign. In patients with a later-onset, there are variable presentations, but psychiatric signs and symptoms (disinhibition and deteriorating executive function) predominate.165

LABORATORY FEATURES AND DIFFERENTIAL DIAGNOSIS Hemoglobin values may be normal, or mild anemia may be present. Approximately 75 percent of lymphocytes contain one to nine vacuoles with a diameter of 2 μm. Electron microscopy reveals that these vacuoles are lipid-filled lysosomes.166 The marrow contains typical foam cells whose diameter ranges between 20 and 100 μm. Small droplets are scattered throughout the cytoplasm (see Fig. 72–6). The cytoplasm of these cells stains only very faintly with the periodic acid-Schiff reagent. Phase microscopy of unstained preparations clearly reveals droplets in the cytoplasm of Niemann-Pick foam cells that distinguish them from Gaucher cells. Sea-blue histiocytes may be present in the spleen and marrow.158,164 Type A and type B disease can be distinguished from other disorders by identification of the lipid as sphingomyelin and by demonstration of sphingomyelinase deficiency in leukocytes or cultured fibroblasts.166,167 Patients with type A disease have acid sphingomyelinase activity levels less than 5 percent of normal in in vitro cultures of lymphoblasts or fibroblasts. In type B disease, acid sphingomyelinase activity levels range between 2 and 10 percent of normal levels. Monospecific antibodies against sphingomyelinase are used to differentiate between type A and type B disease.168 Heterozygotes may be detected by measurement of sphingomyelinase activity of cultured fibroblasts.169 For patients with type C disease, the presence of foam cells is the only indication of the disease. Prenatal diagnosis of the three types of the disease is possible, but is difficult in type C.170

TREATMENT There is no effective treatment for types A and B disease, but there has been an announcement of a successful phase 1b trial with ERT for type B disease and plans for continuation into a phase 2 trial.171 Splenectomy is only rarely required, because death usually occurs from other manifestations of the disease before hypersplenism becomes clinically important. Liver transplantation in type A disease corrects hepatic pathology but has little long-term benefit172; similarly, allogeneic hematopoietic stem cell transplantation does not ameliorate the neurologic deterioration in type B disease,173 nor does it affect the course of type C disease.174 Somatic cell gene therapy has been attempted in knockout type B mice175 and was effective in ameliorating visceral signs but had no effect on neurologic signs. SRT was attempted in a patient with type C disease.176 Depletion of glycosphingolipids by miglustat130 despite having no direct effect on cholesterol metabolism, corrected abnormal lipid trafficking177 and indicated that glycosphingolipid accumulation is a primary pathogenetic event in type C disease. A clinical trial using miglustat at a dose of 200 mg three times daily in an open-label 12-month trial with extension to 66 months in juvenile and adult patients with type C disease and in some children, resulted in improvement or stabilization.177 Subsequent long-term exposure in adults, and some children179 led to its commercial approval and use clinically in many countries. Miglustat improved neurologic manifestations such as ambulation, manipulation, speech, and swallowing; there are some gastrointestinal side effects.177–179

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COURSE AND PROGNOSIS The prognosis in type A Niemann-Pick disease is dire; death nearly always occurs before the third year of life. Patients with type B disease may survive into childhood or longer and there is now hope for a disease-specific ERT. Patients with type C disease usually die in the second decade of life, but some patients with mild disease have a normal life span. New hope for patients with type C emanates from the potential for identification of disease-specific oxysterols as both biomarkers180 of the disease and as harbingers of PC therapy for misfolded variants.181

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CRC Press, Boca Raton, FL, 2007. 144. Taddei TH, Kacena KA, Yang M, et al: The underrecognized progressive nature of N370S Gaucher disease and assessment of cancer risk in 403 patients. Am J Hematol 84:208, 2009. 145. Weinreb NJ, Deegan P, Kacena KA, et al: Life expectancy in Gaucher disease type 1. Am J Hematol 83:896, 2008. 146. Niemann A: Ein unbekanntes Krankheitsbild. Jahrbuch Kinderheilkunde 79:1, 1914. 147. Pick L: Uber die lipoidzellige Splenhepatomegalie Typus Niemann-Pick als Stoffwechselerkrankung. Med Klin 23:1483, 1927. 148. Brady RO, Kanfer JN, Mock MB, et al: The metabolism of sphingomyelin II. Evidence of an enzymatic deficiency in Niemann-Pick disease. Proc Natl Acad Sci U S A 55:366, 1966. 149. Schuchman EH, Desnick RJ: Niemann-Pick disease types A and B: Acid sphingomyelinase deficiencies, in The Metabolic and Molecular Bases of Inherited Disease, 7th ed, edited by Scriver CR, Beaudet AL, Sly WS, Valle D, p 2601. McGraw-Hill, New York, 1995. 150. Pentchev PG, Vanier MT, Suzuki K, et al: Niemann-Pick disease type C: A cellular cholesterol lipidosis, in The Metabolic and Molecular Bases of Inherited Disease, 7th ed, edited by Scriver CR, Beaudet AL, Sly WS, Valle D, p 2625. McGraw-Hill, New York, 1995. 151. Greer WL, Riddell DC, Murty S, et al: Linkage disequilibrium mapping of the Nova Scotia variant of Niemann-Pick disease. Clin Genet 55:248, 1999. 152. Schuchman EH, Miranda SR: Niemann-Pick disease: Mutation update, genotype/phenotype correlations, and prospects for genetic testing. Genet Test 1:13, 1997. 153. Simonaro CM, Desnick RJ, McGovern MM, et al: The demographics and distribution of type B Niemann-Pick disease: Novel mutations lead to new genotype/phenotype correlations. Am J Hum Genet 71:1413, 2002. 154. Wenger DA, Barth G, Githens JH: Nine cases of sphingomyelin lipidosis, a new variant in Spanish-American children. Juvenile variant of Niemann-Pick Disease with foamy and sea-blue histiocytes. Am J Dis Child 131:955, 1977. 155. Millat G, Marçais C, Rafi MA, et al: Niemann-Pick C1 disease: The I1061T substitution is a frequent mutant allele in patients of Western European descent and correlates with a classic juvenile phenotype. Am J Hum Genet 65:1321, 1999. 156. Patterson MC, Vanier MT, Suzuki K, et al: Niemann-Pick disease type C: A lipid trafficking disorder, in The Metabolic and Molecular Bases of Inherited Disease, 8th ed, edited by Scriver CR, Beaudet AL, Sly WS, Valle D, Childs B, Kinzler KW, Vogelstein B, p 3611. McGraw-Hill, New York, 2001. 157. Takahashi T, Suchi M, Desnick RJ, et al: Identification and expression of five mutations in the human acid sphingomyelinase gene causing types A and B Niemann-Pick disease. Molecular evidence for genetic heterogeneity in the neuronopathic and non-neuronopathic forms. J Biol Chem 267:12552, 1992.

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158. De Maria R, Rippo MR, Schuchman EH, et al: Acidic sphingomyelinase (ASM) is necessary for fas-induced GD3 ganglioside accumulation and efficient apoptosis of lymphoid cells. J Exp Med 187:897, 1998. 159. Carstea ED, Morris JA, Coleman KG, et al: Niemann-Pick C1 disease gene: Homology to mediators of cholesterol homeostasis. Science 277:228, 1997. 160. Park WD, O’Brien JF, Lundquist PA, et al: Identification of 58 novel mutations in Niemann-Pick disease type C: Correlation with biochemical phenotype and importance of PTC1-like domains in NPC1. Hum Mutat 22:313, 2003. 161. Millat G, Chikh K, Naureckiene S, et al: Niemann-Pick disease type C: Spectrum of HE1 mutations and genotype/phenotype correlations in the NPC2 group. Am J Hum Genet 69:1013, 2001. 162. Verot L, Chikh K, Freydière E, et al: Niemann-Pick C disease: Functional characterization of three NPC2 mutations and clinical and molecular update on patients with NPC2. Clin Genet 71:320, 2007. 163. Pacheco CD, Kunkel R, Lieberman AP: Autophagy in Niemann-Pick C disease is dependent upon Beclin-1 and responsive to lipid trafficking defects. Hum Mol Genet 16:1495, 2007. 164. Loftus SK, Morris JA, Carstea ED, et al: Murine model of Niemann-Pick C disease: Mutation in a cholesterol homeostasis gene. Science 277:232, 1997. 165. Golde DW, Schneider EL, Bainton EL, et al: Pathogenesis of one variant of sea-blue histiocytosis. Lab Invest 33:371, 1975. 166. Patterson MC: A riddle wrapped in a mystery: Understanding Niemann-Pick disease, type C. Neurologist 9:301, 2003. 167. Lazarus SS, Vethamany VG, Schneck L, et al: Fine structure and histochemistry of peripheral blood cells in Niemann-Pick disease. Lab Invest 17:155, 1967. 168. Brady RO: Sphingomyelin lipidoses: Niemann-Pick disease, in The Metabolic Basis of Inherited Disease, edited by JB Stanbury, JB Wyngaarden, DS Fredrickson, JL Goldstein, MS Brown, p 831. McGraw-Hill, New York, 1983. 169. Gal AE, Brady RO, Hibberg SR, et al: A practical chromogenic procedure for the detection of homozygotes and heterozygous carriers of Niemann-Pick disease. N Engl J Med 293:632, 1975.

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170. Vanier MT. Prenatal diagnosis of Niemann-Pick diseases types A, B and C. Prenat Diagn 22:630, 2002. 171. Mount Sinai International Center for Types A and B Niemann-Pick Disease: http:// www.mssm.edu/niemann-pick 172. Daloze P, Delvin EE, Glorieux FH, et al: Replacement therapy for inherited enzyme deficiency: Liver orthotopic transplantation in Niemann-Pick disease type A. Am J Med Genet 1:229, 1977. 173. Victor S, Coulter JB, Besley GT, et al: Niemann-Pick disease: Sixteen-year follow-up of allogeneic bone marrow transplantation in a type B variant. J Inherit Metab Dis 26:775, 2003. 174. Hsu YS, Hwu WL, Huang SF, et al: Niemann-Pick disease type C (a cellular cholesterol lipidosis) treated by bone marrow transplantation. Bone Marrow Transplant 24:103, 1999. 175. Miranda SR, Erlich S, Friedrich VL Jr, et al: Hematopoietic stem cell gene therapy leads to marked visceral organ improvements and a delayed onset of neurological abnormalities in the acid sphingomyelinase deficient mouse model of Niemann-Pick disease. Gene Ther 7:1768, 2000. 176. Lachmann RH, te Vruchte D, Lloyd-Evans E, et al: Treatment with miglustat reverses the lipid-trafficking defect in Niemann-Pick disease type C. Neurobiol Dis 16:654, 2004. 177. Patterson MC, Vecchio D, Prady H, et al: Miglustat for treatment of Niemann-Pick C disease: A randomised controlled study. Lancet Neurol 6:765, 2007. 178. Wraith JE, Vecchio D, Jacklin E, et al: Miglustat in adult and juvenile patients with Niemann-Pick disease type C: Long-term data from a clinical trial. Mol Genet Metab 99:351, 2010. 179. Patterson MC, Vecchio D, Jacklin E, et al: Long-term miglustat therapy in children with Niemann-Pick disease type C. J Child Neurol 25:300, 2010. 180. Jiang X, Sidhu R, Porter FD, et al: A sensitive and specific LC-MS/MS method for rapid diagnosis of Niemann-Pick C1 disease from human plasma. J Lipid Res 52:1435, 2011. 181. Ohgane K, Karaki F, Dodo K, Hashimoto Y: Discovery of oxysterol-derived pharmacological chaperones for NPC1: Implication for the existence of second sterol-binding site. Chem Biol 20:391, 2013.

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Part IX  Lymphocytes and Plasma Cells 73. The Structure of Lymphocytes and Plasma Cells . . . . . . . . . . . . . . . . . . . . . . 1137

79. Lymphocytosis and Lymphocytopenia . . . . . . . . . . . . . . . . 1199

74.  Lymphopoiesis . . . . . . . . . . . . . . . . . . . 1149

80.  Immunodeficiency Diseases . . . . . . 1211

75. Functions of B Lymphocytes and Plasma Cells in Immunoglobin Production . . . . . . . . . . . . . . . . . . . . . . . . 1159

81. Hematologic Manifestations of Acquired Immunodeficiency Syndrome . . . . . . . . . . . . . . . . . . . . . . . . 1239

76. Functions of T Lymphocytes: T-Cell Receptors for Antigen . . . . . . . . . . . . 1175

82.  Mononucleosis Syndromes . . . . . . . 1261

77.  Functions of Natural Killer Cells . . . 1189 78. Classification and Clinical Manifestations of Lymphocyte and Plasma Cell Disorders . . . . . . . . . . . . . 1195

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CHAPTER 73

THE STRUCTURE OF LYMPHOCYTES AND PLASMA CELLS

Natarajan Muthusamy and Michael A. Caligiuri*

SUMMARY Lymphocytes are a heterogeneous population of blood cells that can be distinguished from other leukocytes by their characteristic morphology and structural features. Mature lymphocytes can be divided into several functional types and subtypes based on their organs of development and function. The major classes of lymphocytes include T cells, B cells, and natural killer (NK) cells. T lymphocytes develop in the thymus (Chaps. 6, 74, and 76) and are exported to the blood and lymphoid organs. They are responsible for cell-mediated cytotoxic reactions and for delayed hypersensitivity responses (Chap. 76). T lymphocytes also produce the cytokines that regulate immune responses and provide helper activity for B cells. B lymphocytes can capture, internalize, and present antigens to T cells and are the precursors of ­immunoglobulin-secreting plasma cells (Chap. 75). NK cells account for innate immunity against infectious agents and transformed cells that have altered expression of transplantation antigens (Chap. 77). Blood T and B lymphocytes are indistinguishable by light and electron microscopy. NK cells tend to be larger cells with relatively large granules scattered in their cytoplasm. B cells can mature into plasma cells upon activation by engagement with antigen or with certain B cell mitogens. Although the different lymphocyte subpopulations appear similar by morphology they have distinct surface and intracellular protein expression patterns. These subpopulations, as defined by antigen expression, reflect different functional subsets, maturation stages, and activation stages. This chapter describes the light and transmission electron microscopic structures of lymphocytes and plasma cells and the major structural features reflected by surface antigens that are characteristic of each lymphocyte type. The chapter also provides information on biophysical and biochemical features of human lymphocytes.

Acronyms and Abbreviations: ADAM, a disintegrin and a metalloprotease; BTK, Bruton tyrosine kinase; CD, cluster of differentiation; Ig, immunoglobulin; lck, leukocyte tyrosine kinase; LGL, large granular lymphocyte; MHC, major histocompatibility complex; NK, natural killer; TCR, T-cell receptor; TdT, terminal deoxynucleotidyl transferase; TFH, follicular helper T cells; Th, T helper cells; TREG, T-regulatory cell; ZAP-70, zeta-associated protein of 70 kDa.

* This chapter was written by H. Elizabeth Broome in the 8th edition and some of the text and images have been retained.

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DEFINITION AND HISTORY Lymphocytes and plasma cells first were described in 1774 and 1875, respectively.1 Studies during the subsequent 75 years with improved histologic techniques and light microscope optics furthered understanding of the lymphoid organs and the distribution of lymphocytes.2–6 By the mid-20th century, awareness that the immune system had at least two ­components—one governing humoral immunity and one governing cellular immunity—led to early concepts of different lymphocyte subsets. Also, at the same time came the discovery that the thymus and bursa of Fabricius in birds were the source of what came to be known as T (thymic-derived) and B (bursa-derived) lymphocytes, respectively, and that the marrow was the bursa equivalent in humans (human B cells therefore could represent marrow-derived cells). This discovery coupled with descriptions of inherited absence of the thymus leading to loss of cellular immunity but retention of humoral immunity and cases of retention of cellular immunity in children who were deficient in antibody production, eventually led to our current understanding of the division of labor among what originally appeared to be a common lymphocyte pool, morphologically. The later advent of monoclonal antibodies against numerous surface antigens coupled with flow cytometry, in vitro functional assays, molecular techniques to distinguish between B cells and T cells, and experiments using inbred strains of mice brought us to our current state of knowledge of the immune response and its abnormalities. Flow cytometry identifies a multitude of lymphocyte subsets based on antigen expression patterns. These immunophenotypic subsets correlate closely with function as determined by in vitro and in vivo testing. T lymphocytes, B lymphocytes, and natural killer (NK) cells represent three major blood lymphocyte functional subsets. The marrow and thymus contain precursor cells that resemble lymphocytes but lack function without differentiation and maturation into various lymphocyte subsets. Plasma cells are terminally differentiated B lymphocytes that produce immunoglobulin and mostly reside in marrow, lymph nodes, and other lymphoid tissues. (Chap. 6).

 ICROSCOPY AND HISTOCHEMISTRY M OF NORMAL BLOOD LYMPHOCYTES LIGHT MICROSCOPY Classic studies of blood and tissues defined lymphocytes as spherical and/or ovoid cells that have diameters from 6 to 15 μm when flattened on glass slides.4 Some of these studies described two separate broad types of lymphocytes based on size: small lymphocytes with diameters of 6 to 9 μm and large lymphocytes with diameters of 9 to 15 μm. Patients with acute viral illnesses have increased numbers of circulating large, “reactive,” lymphocytes. Other illnesses, such as infection with Bordetella pertussis and autoimmune disorders, can cause blood to have increased small lymphocytes or lymphocytes with plasma cell-like morphology (Chap. 78). The mean absolute number of circulating small lymphocytes in normal adults is 2.5 × 109/L (Chap. 2).7 Children have higher lymphocyte counts that trend downward until they reach adult levels at approximately 8 to 10 years of age (Chap. 7).8 Most lymphocytes in normal blood are small with an ovoid or kidney-shaped nucleus that stains purple, has densely packed nuclear chromatin, and occupies approximately 90 percent of the cell area (Fig. 73–1A and B) by Romanowsky polychromatic stains (e.g., Giemsa or Wright) of air-dried films. A small rim of cytoplasm stains light blue. Nucleoli rarely are observed in Wright-stained films, but nucleoli in these cells may become visible in certain preparations, such as cytospin slides, or after prolonged storage in anti-coagulated blood collection tubes.

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A

B

C

E

F

G

D

H

Figure 73–1.  Wright-Giemsa stained blood films showing (A) a normal, small lymphocyte, monocyte, and segmented neutrophil; (B) normal small lymphocyte and two medium-sized lymphocytes; (C) neutrophil and two lymphocytes with morphologic features characteristic of Bordetella pertussis infection (small size, cleaved nuclei, and scant cytoplasm); (D) reactive lymphocytes; and (E) large granular lymphocyte and small lymphocyte. Wright-Giemsa stained marrow films showing (F) normal plasma cell; (G) two normal plasma cells, one nucleated red cell, and one neutrophil; and (H) two plasma cells with one containing many Russell bodies. A minority of lymphocytes in normal blood have morphology that defines them as large granular lymphocytes (LGLs).9 These LGLs are slightly larger than most lymphocytes, having an increased area of light blue or clear cytoplasm. LGL cytoplasm contains a number of coarse pink granules, usually 5 to 15 per cell, and occasional clear vacuoles. In a normal adult, approximately 5 percent but up to 10 to 15 percent of blood lymphocytes are LGLs (see Fig. 73–1E).9 The LGLs in blood are composed of NK cells and a subset of cluster of differentiation (CD) 8+ T lymphocytes, indistinguishable by their morphology.

PHASE-CONTRAST MICROSCOPY Active movement of lymphocytes is studied by phase-contrast, or ­interference-contrast, microscopy. Lymphocytes move slowly with a “hand mirror” appearance. Cytoplasmic spreading does not occur. However, during cell movement, a thickening occurs in the cytoplasmic rim, which houses most of the cell’s organelles, including the Golgi apparatus.

TRANSMISSION ELECTRON MICROSCOPY AND CYTOCHEMISTRY The blood lymphocyte measures approximately 5 μm in spherical diameter as visualized by transmission electron microscopy.10 The nucleus has an abundance of electron-dense, condensed heterochromatin, a feature characteristic of nonproliferating cells. The nucleoli are round in section, approximately 0.5 to 1.4 μm in diameter. They are composed of three distinct and concentrically arranged structural units: the central region or agranular zone; the middle, fibrillar region; and the granular zone, which contains intranucleolar chromatin. The lymphocyte’s nuclear membrane contains nuclear pores and a perinuclear space. The cytoplasmic organelles of the lymphocytes are characteristic of eukaryotic cells. Some organelles, such as the Golgi zone, are poorly developed. The cytoplasm contains free ribosomes, occasional

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ribosome clusters, and strands of rough-surfaced endoplasmic reticulum (Fig. 73–2). Centrioles, mitochondria, microtubules (diameter approximately 0.25 μm), and microfilaments (diameter approximately 0.07 μm) are present in the cytoplasm adjacent to the cell membrane. The cytoplasm also contains lysosomes, which are approximately 0.4 μm in diameter, are electron opaque, and contain classic lysosomal enzymes (e.g., acid phosphatase, β-glucuronidase, and acid ribonuclease).11 The lymphocyte plasma membrane stains with colloidal iron, a marker for membrane sialic acid. Lymphocyte cell membranes and cell coat glycoproteins are shown with other electron-dense markers, including phosphotungstic acid, lanthanum colloid, and ruthenium red. Most T lymphocytes have a localized “dot” pattern when stained for acid phosphatase, acid and neutral nonspecific esterases, β-glucuronidase, and N-acetyl-β-glucosaminidase.12 LGLs stain for acid hydrolases with a dispersed, granular reaction pattern.13 B lymphocytes either lack esterase and acid phosphatase or show minimal scattered granular staining.

SCANNING ELECTRON MICROSCOPY Scanning electron microscopy provides three-dimensional information.14 However, the resolution achieved with scanning electron microscopy, approximately 0.1 μm, is considerably less than that possible with transmission electron microscopy, generally 0.002 to 0.0039 μm. Normal blood lymphocytes, washed and collected on silver membranes and fixed in glutaraldehyde, have a spherical topography with varying numbers of stubby or finger-like microvilli (Fig. 73–3).15 In contrast, monocytes are much larger, have few microvilli, and display ruffled membranes and ridge-like profiles (Chap. 67). Lymphocyte microvilli contain parallel bundles of actin filaments that undergo continuous assembly and disassembly.16 The function of lymphocyte microvilli probably includes segregating surface receptors involved in extravasation. Two receptors involved in the initial rolling phase of extravasation, L-selectin and α4β7 integrin,17 localize to microvillar tips. In contrast, the β2 integrins that mediate stable adhesion

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Ribosome Lysosome

Euchromatin Heterochromatin

Nucleolus Nucleus

Rough-surfaced endoplasmic reticulum

Golgi Centriole Mitochondrion

A

B

Figure 73–2.  A. Transmission electron micrograph of normal human blood lymphocyte (×12,000). B. Diagrammatic representation of normal

blood lymphocyte, with organelles labeled.

and diapedesis localize to nonprotrusive regions of the cell surface. This spatial separation of surface receptors might enable a temporal segregation of adhesive function during extravasation. Lymphocytes expressing chimeric L-selectin constructs that no longer localize to microvilli do not roll on L-selectin ligands, supporting this hypothesis.18

MORPHOLOGIC CHANGES ASSOCIATED WITH ACTIVATION Lymphocyte stimulation is associated with a complex sequence of morphologic and biochemical events. Activation of B and T lymphocytes results in the transformation of the small, resting lymphocyte into proliferating large cells with abundant highly basophilic cytoplasm, irregularly condensed or smudgy chromatin, and round to slightly irregular

Figure 73–3.  Scanning electron micrograph of normal blood lym-

phocytes separated by the Ficoll-Hypaque method. Cells show varying numbers of microvilli (×5000). (Used with permission of Dr. Aaron Polliack of the Department of Hematology, Hebrew University Hadassah Medical School, Jerusalem, Israel.)

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nuclear outlines (see Fig. 73–1D). Nucleoli may be evident by light microscopy in these cells, but they usually are not prominent. Infection with B. pertussis causes an increase in blood lymphocytes with a particular activated morphology characterized by small size, scant cytoplasm, and cleaved nuclei with mature chromatin (see Fig. 73–1C). Activated lymphocytes proliferate and mature into effector lymphocytes and memory cells. Effector cells include helper T cells, cytolytic T cells, and plasma cells (see Figs. 73-1F and G, 73–4, and 73–5). In vitro, plant lectins, bacterial products, polymeric substances, and enzymes activate lymphocytes and cause mitosis. Such agents are called mitogens. Some mitogens are specific for either B or T lymphocytes, whereas other mitogens stimulate both.19 Approximately 4 hours after mitogen stimulation, lymphocytes show increased nucleolar size and an increase in the number and concentration of granules in the granular zone. These changes are followed by an increase in fibrillar zones and increased intranucleolar chromatin. Nucleolar chromatin becomes more electron lucent or dispersed. From 48 to 72 hours following the addition of phytohemagglutinin, the volume of the cytoplasm increases. In addition, the cytoplasm contains an increased number of ribosomal clusters and more rough-surfaced endoplasmic reticulum. The activated cell has increased numbers of lysosomes and a larger Golgi complex with more components.20 Under some circumstances (e.g., cultures of human lymphocytes stimulated for 7 to 10 days with pokeweed mitogen), some B cells form well-developed Golgi and plasmacytoid features.21 Similar plasmacytoid cells are observed in antigen-stimulated lymph nodes, during graft rejection in vivo, and in some in vitro systems, including the mixed lymphocyte culture. In lymph nodes, the stimulated lymphoid cells may be referred to by various pathologists as lymphoblasts, immunoblasts, centroblasts, or large lymphoid cells. Morphologic criteria for these cells overlap. Following stimulation with antigen or mitogens, the lymphocyte enters the cell cycle. The fate and function of lymphocytes that traverse the cell cycle can be divided into two pathways. Some lymphocytes undergo several mitotic cycles and then return to the G0 phase, indistinguishable in morphology from the original nonactivated cells.

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Part IX: Lymphocytes and Plasma Cells

Some of them then become memory cells, programmed to remember the stimulating antigen and thus respond more rapidly to reexposure to the original antigen. Alternatively, they become terminally differentiated effector lymphocytes, such as plasma cells or cytotoxic T cells (Chaps. 75 and 76).

 ICROSCOPY AND HISTOCHEMISTRY M OF PLASMA CELLS MORPHOLOGIC STUDIES

Figure 73–4.  Transmission electron micrograph of lymphocyte from

normal individual incubated with phytohemagglutinin20 for 3 days. The transformed cell has a large Golgi zone (G) and many ribosomal aggregates (arrows). The nucleus is euchromatic (×7500).

Plasma cells derive from small B lymphocytes after activation in the correct environment. The characteristic feature of plasma cells is abundant cytoplasmic and secretory immunoglobulin (Ig). A fully mature plasma cell lacks surface Ig expression. Each plasma cell has the same clonal rearrangement of its V(D)J (variable diversity joining) Ig genes as its predecessor B lymphocyte (Chap. 75). Several mitotic divisions may occur during cellular differentiation from the resting lymphocyte to the plasmablast to the immature plasma cell. Immature plasma cells can undergo successive waves of mitosis in the medullary cords of lymph nodes in response to antigen.22 Cell transfer experiments demonstrated that these transformed cells later mature into antibody-producing plasma cells.23 Pokeweed mitogen induces B lymphocytes to transform into plasma cells after 7 to 10 days of culture.24 These plasma cells infrequently contain large electron-dense inclusions (Russell bodies), which may measure 2 to 3 μm in diameter (see Fig. 73–1H).25 Russell bodies, cytoplasmic Ig in the endoplasmic reticulum, sometimes are dissolved during the staining procedure. They usually occur in pathologic states but may be found in plasma cells from normal lymph nodes or marrow.

LIGHT MICROSCOPY, HISTOCHEMISTRY, AND ELECTRON MICROSCOPY

Figure 73–5.  Transmission electron micrograph of plasmacytoid cell

present in culture of lymphocytes from a patient with chronic lymphocytic leukemia incubated with pokeweed mitogen for 7 days. The nucleolus (N) and rough-surfaced endoplasmic reticulum (arrows) are evident (×9000). (Reproduced with permission from Cohnen G, Douglas SD, Konig E, Brittinger G: Pokeweed mitogen response of lymphocytes in chronic lymphocytic leukemia: A fine structural study, Blood 1973 Oct;42(4):591-600)

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The mature plasma cell has a characteristic basophilic cytoplasm and an eccentric nucleus when treated with a polychrome stain. The nuclear polarity is attributable to a large paranuclear zone, which corresponds to the Golgi apparatus. The typical mature plasma cell spread on a slide usually is round or oval and has a diameter of 9 to 20 μm, with a mean cell diameter of 14 μm and a mean nuclear diameter of 8.5 μm (see Fig. 73–1F and G).26 The nuclear heterochromatin is coarse and distributed in a pattern that sometimes resembles the spokes of a wheel (cartwheel nucleus) on paraffin sections. Normal plasma cells may occasionally have two or more nuclei. Cytochemical features of plasma cells include positive staining for β-glucuronidase and mitochondrial enzyme markers. They do not stain for peroxidase or nonspecific esterase.27 Plasma cells in patients with certain diseases may have different histochemical properties. Plasma cell size and morphology may be altered substantially in myeloma and macroglobulinemia (Chaps. 107 and 109, respectively). Plasma cells with two or three nuclei are more frequent in marrows from patients with plasma cell dyscrasias. Periodic acid-Schiff stains may reveal cytoplasmic or nuclear inclusions in clonal plasma cells.28 Under some circumstances, amyloid inclusions in plasma cells have been detected by electron microscopy.29 In hemochromatosis and hemosiderosis, plasma cells may contain hemosiderin when examined by electron microscopy.30 The plasma cell is packed with a rough-surfaced endoplasmic reticulum having numerous attached ribosomes as seen by electron microscopy. A large, circumscribed Golgi zone forms a paranuclear halo when observed by light microscopy. The nucleus has dense areas of heterochromatin. The Golgi zone contains lamellae, vesicles, vacuoles, and a number of granules. Mitochondria are located between the strands of endoplasmic reticulum.31

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Chapter 73: The Structure of Lymphocytes and Plasma Cells

ANTIGENS OF HUMAN LYMPHOCYTES B LYMPHOCYTE ANTIGENS Figure 73–6 summarizes the expression of antigens on cells of the B-lymphocyte lineage, including committed progenitor B cells and pre-B cells. Chapter 74 discusses these cells and the maturation stages they represent. Figure  73–6 also lists antigens that are expressed or increased upon B-cell activation. Of the B-cell–associated antigens that are commonly used, only a few are restricted to cells of the B lineage. Of these antigens, only CD20, CD22, and Pax 5 are not found on other cell types. Pax 5, a transcription factor, is a “master regulator” of B-cell development32,33 that is expressed from the precursor stage through all B-cell maturation until it is lost at the plasma cell stage. Demonstration of monoclonal surface Ig allows diagnosis of clonal, neoplastic B cells. CD20 is the target of rituximab, a monoclonal antibody commonly used for treatment of B-cell neoplasms. CD19 is restricted mostly to B cells, but may be expressed weakly by follicular dendritic cells. CD19 is expressed by B cells at all stages of maturation, including the committed B-cell progenitor and most normal plasma cells. As such, it is the best-defined pan–B-cell surface antigen. In addition to the CD antigens and Igs, B cells express the three major histocompatibility complex (MHC) class II antigens: DR, DP, DQ. These antigens are heterodimers of heavy chains and light chains that are encoded by genes within the D complex of the human leukocyte antigen (HLA) complex (Chap. 137). MHC class I antigens are expressed on all nucleated cells.

B-1 B Cells and CD5+ B Cells

B-1 B cells have distinctive activation requirements and high levels of CD44 and interleukin (IL)-5 receptor α (IL-5Rα). They proliferate more rapidly than other B cells to stimuli such as IgM crosslinking, possibly because of having constitutively activated nuclear signal transducer and activator of transcription 3 (STAT3).34 Many, but not all, B-1 cells express CD5, a 67-kDa transmembrane glycoprotein that is more brightly expressed by T cells. These cells are designated CD5 B cells.35 B-1 B cells do not express other T-cell markers but do express all other pan–B-cell surface antigens. Various agents modulate B-cell expression of CD5.36 B-1 B cells are found in umbilical cord blood,37 adult blood, the pleura and peritoneum, and all major secondary

Committed progenitor

Early pre-B cell

Pre-B cell

1141

lymphoid organs; they are rare in the marrow.38 These cells apparently are enriched for cells that spontaneously produce polyreactive autoantibodies.39–41

Plasma Cells

Many B cell differentiation antigens are not expressed by the mature plasma cell, including CD20, Pax-5, surface Ig, and HLA class II antigens (see Fig. 73–6). Of the cells of the B lineage, plasma cells are distinctive in that they express CD138 and bright CD38.42 Clonal plasma cell neoplasms usually have antigen expression distinct from normal plasma cells including aberrant expression of CD20, CD28, CD56, and CD117. Clonal plasma cells usually aberrantly lack expression of CD19 and CD27.43

T-LYMPHOCYTE AND NATURAL KILLER CELL ANTIGENS Figure 73–7 and Table 73–1 summarize the expression of antigens on cells of the T-lymphocyte and NK lineages. All lymphocyte progenitors originate in the marrow, but T lymphocytes have their own special organ for maturation—the thymus—whereas NK cells appear to differentiate in secondary lymphoid tissue (Chap. 6).

Thymocyte

The thymus promotes the development of antigen-specific T lymphocytes and eliminates self-reactive T lymphocytes. There are three general stages of thymocyte maturation based on the surface CD4 and CD8 expression: double negative, double positive, and single positive. These stages have corresponding anatomic localization within the thymus with the least-mature double-negative cells located in the subcapsular area and the most-mature single-positive cells located in the medulla (Chap. 6). The most immature T lymphocytes in the thymus populate the subcapsular areas and express CD2, CD5, and CD7, antigens present on T lymphocytes of all stages. Capsular, “double-negative” thymocytes also express CD1a, cytoplasmic CD3, and terminal deoxynucleotidyl transferase (TdT). The majority of thymocytes are at the “double-positive” stage within the cortical area. These are the cells undergoing positive selection. Once the thymocytes achieve their “education” without dying, they mature to the single-positive stage in the medulla. These varying stages of immature T-cell maturation in the thymus have corresponding cell phenotypes in T-lymphoblastic leukemia/lymphoma.

Mature B cell

Activated B cell

Germinal center Plasma cell B cell

CD45 TdT/CD34 CD19 HLA-DR CD10 CD20/22 Pax-5 clgM CD38 slgM clgG/A/E CD138

Figure 73–6.  Clinically useful antigens expressed during B-lymphocyte maturation. The intensity of the antigen expression at each stage of B-lymphocyte maturation is depicted by gradient density of bars on the graph. c, cytoplasmic; s, surface.

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Prothymocyte

Subcapsular thymocyte

Cortical thymocyte

Medullary thymocyte

Mature T lymphocyte

CD45

Figure 73–7. Clinically useful antigens expressed during T-lymphocyte maturation. The intensity of the antigen expression at each stage of T-lymphocyte maturation is depicted by gradient density of bars on the graph.

CD34 TdT CD7∗ CD2 CD5 cCD3 CD4 CD8 CD3/TCR ∗CD7 is expressed on most, but not all, mature T lymphocytes.

Mature T Lymphocytes

Small, mature T lymphocytes are the most common lymphocytes in blood. T lymphocytes recognize antigen in the context of the MHC through binding with the T-cell receptor (TCR). Signaling from the TCR involves many membrane proteins, including CD3, a three-­subunit complex expressed by early thymocytes and mature T cells.44 It is tightly linked to the T-cell antigen receptor (Chap. 76). Most T lymphocytes have the α/β TCR on their surface, but a few percent of blood lymphocytes have the γ/δ TCR.

CD4 and CD8 Lymphocyte Subsets

Mature T cells express either CD4 or CD8, but not both. CD4, a member of the Ig supergene family, is a single-chain transmembrane glycoprotein.45 CD8 is a 34-kDa dimeric transmembrane glycoprotein.46 Most T cells express the α and β subunits of CD8. CD4 and CD8 act as coreceptors during T-cell activation by antigen. CD4 recognizes MHC II and CD8 recognizes MHC class I (Chap. 76). CD4 also is a coreceptor for the

TABLE 73–1.  Mature Natural Killer and T-Lymphocyte Subsets NK or T-Cell Subset

Antigens

CD4+ helper T cells

CD2, CD3, CD4, CD5, CD7, and TCR α/β Subsets selectively express CD10, CD25,49 CD57,99 and FoxP3100

CD8+ cytolytic T cells

CD2, CD3, CD5, CD7, CD8, and TCR α/β Subsets selectively express CD16,55 CD56, CD57, cytolytic enzymes101

NK cells

CD2, CD7, KIRs (multiple), NKp46 (NCR1) Negative for CD3, TCR (α/β or γ/δ) Subsets express either CD16-negative, dim and CD56 bright, or CD16 bright and CD56 dim54 Cytolytic enzymes

γ/δ T cells

CD2, CD3, CD7, TCR γ/δ Usually negative for CD4 Subsets express CD5, CD8, and cytolytic enzymes

CD, cluster of differentiation; KIR, killer cell immunoglobulin-like receptor; NK, natural killer; TCR, T cell receptor.

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human immunodeficiency virus47 (Chap. 81), as are CCR5 (chemokine receptor 5) and CXCR4 (chemokine-related receptor). The majority of CD8 cells are cytolytic when appropriately activated. Subsets of CD4+ T cells have helper function for activation and maturation of cytolytic cells or B cells. Other CD4 subsets have regulatory activity including the T-regulatory (TREG) cells that induce immune tolerance, and follicular T-helper (TFH) cells that promote B cell maturation and differentiation in germinal centers. TREG and TFH cells have unique phenotypes. TREG cells express the low affinity receptor for IL-2 (CD25); and the transcription factor forkhead box P3 (FoxP3).48 Follicular helper T-TFH cells express CD10 and CD57. Malignant counterparts to both subsets occur. Adult T-cell leukemia/ lymphoma expresses both CD25 and FoxP3 and is associated with marked immunosuppression.49 Angioimmunoblastic T-cell lymphoma has characteristic clonal T cells that express CD10 and CD57 just like TFH cells, and this lymphoma is associated with polyclonal hypergammaglobulinemia and the expansion and proliferation of both B cells and CD21+ follicular dendritic cells.50 T-helper-17 (Th17) cells that secrete IL-17 exhibit critical roles in mucosal defense and in autoimmune disease pathogenesis.51

Natural Killer Cells

The NK cell is defined as an effector cell that is not MHC restricted and has the capacity for spontaneous cytotoxicity toward various target cells (Chap. 77). Most NK cells have LGL morphology (see Fig. 73–1E).52 However, not all NK cells have LGL morphology, and not all LGL cells are NK cells. Many are cytolytic T lymphocytes. Cytolytic T lymphocytes and NK cells share many granule contents that can be detected by immunohistochemistry or flow cytometry. These include TIA-1, an RNA binding protein, and several granzymes, which are granule enzymes with serine protease activity. Despite their relative morphologic homogeneity, NK cells comprise several subpopulations with distinct phenotypes. Human NK cells characteristically express CD16 (FcγRIII) and CD56 but not TCRα/β or TCRγ/δ, CD3, or CD4.53,54 CD8 is found on approximately 30 to 50 percent of NK cells. CD8 on NK cells is dim by flow cytometry and is of the β-homodimer form. CD16 (FcγRIII) is a low-affinity receptor that binds to IgG, which is bound specifically to antigens present on cells targeted for destruction in antibody-dependent cell-mediated cytotoxicity.55 CD16 is expressed on all NK cells, neutrophils, and tissue macrophages. CD56 is the neural cell adhesion molecule and is seen on most NK cells in either low (“dim”) or high (“bright”) density.53,56 This 200-kDa protein is expressed at higher levels following activation.

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1143

LYMPHOCYTE SURFACE ANTIGENS

LYMPHOCYTE MEMBRANE

Lymphocyte subsets generally cannot be distinguished from one another by morphology. Most resting lymphocytes appear as small round cells with a dense nucleus and little cytoplasm. However, this homogeneous appearance is deceptive, as these cells comprise many functionally distinct subpopulations. These subsets can be distinguished through the differential expression of cell-surface proteins, each of which can be recognized by a specific monoclonal antibody. Coupled with the biochemical analyses of the surface molecules that are recognized by each of these antibodies, many lymphocyte surface antigens have been defined. Typically, it is necessary to monitor for coexpression of two or more cell-surface proteins to define a functional subset of lymphocytes. The same cell-surface protein is often expressed by more than one cell subset. For example, both helper and cytotoxic T cells express CD3, the proteins associated with the TCR for antigen (Chap. 76). Expression of both CD3 and CD4 helps to distinguish mature Th cells from cytotoxic T cells that express CD3 and CD8, and from other cells, such as dendritic cells, that express CD4 but lack expression of CD3 (Chap. 76). As noted above (see “CD4 and CD8 Lymphocyte Subsets”), TREG cells are defined by the coexpression of CD3, CD4, CD25, and cytoplasmic FoxP3.48 For these and other types of lymphocytes, it is the expression of a characteristic constellation of surface and cytoplasmic molecules, rather than the expression of any one particular marker, that generally helps to distinguish one subset of lymphocytes from another. Fluorescent probes also can be used to identify antigen-specific lymphocytes.57 Each clone of B lymphocytes expresses Ig capable of binding a particular antigen (Chap. 75). The frequencies of B cells specific for one antigen are estimated to range from 1 in 100,000 to 1 in 1,000,000 cells or less. Populations of lymphocytes enriched for B cells binding to a specific antigen can be stained using antigen coupled to probes, allowing for the detection and isolation of ­antigen-specific B cells using flow cytometry.58 Alternatively, flowbased techniques can be used to monitor for antigen-specific B cells that are activated by contact with antigen.59 T lymphocytes, however, generally recognize antigen in the form of peptides nestled into ­molecules of the MHC (Chap. 76). Thus identification and isolation of ­antigen-specific T cells require more complex probes using multimeric complexes comprised of specific peptide antigen complexed with the relevant MHC molecule.57

The lymphocyte plasma membrane is composed of equal parts of weight of protein and glycosphingolipids and 6 percent by weight of carbohydrate.63 The molar ratio of cholesterol to phospholipid is approximately 0.5.64,65 Phosphatidylcholine is the predominant phospholipid in the lymphocyte plasma membrane, but phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and sphingomyelin are also present. Approximately half the membrane fatty acids are saturated. The membrane proteins are usually glycosylated. The glycosphingolipids and protein receptors of lymphocytes often are organized in glycolipoprotein microdomains termed lipid rafts.66,67 Such lipid rafts sequester various protein receptors, coreceptors, and accessory molecules that together are involved in lymphocyte cell signaling, cytoskeletal reorganization, and/or membrane trafficking.68 As such, the surface molecules on lymphocytes are not randomly distributed.

COMPOSITION OF LYMPHOCYTES Unfortunately, few studies of the composition and biochemistry of lymphocytes have used purified lymphocyte subpopulations. Because mature helper T cells are the predominant blood lymphocyte of normal adults, many reported biochemical parameters are most relevant to this subpopulation.

ION AND WATER CONTENT The resting blood lymphocyte has a mean cell volume of 200 μm3 and contains 71 ± 1.2 percent by weight of water.60 The total lymphocyte cation content is 35 femtomoles (fmol) per cell, of which 22 to 28 fmol per cell is potassium, and 7.9 ± 3.2 fmol per cell is sodium.60 Lymphocyte membranes have both voltage-gated and calcium-activated potassium channels that regulate cell volume. Pharmacologic inhibition of these channels blocks T-cell activation. The calcium content of resting lymphocytes has been estimated at 580 to 800 pmol/106 cells.61 Cytosolic free calcium concentrations are relatively low in resting lymphocytes (approximately 0.1 μM) but increase severalfold after activation.62

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Extracellular Membrane-Associated Enzymes (Ectoenzymes)

Exposed on the exterior surface of lymphocytes are several enzymes called ectoenzymes (Table 73–2). Generally, the number of surface enzyme molecules is low compared with that of other surface molecules, such as those involved in lymphocyte adhesion. This probably reflects the fact that these molecules are catalytic and have a higher functional specific activity than do molecules involved in adhesion events, where multiple interactions over large surface areas are required. As such, it is possible that many more enzymes are present than the ones currently recognized because they are expressed at levels that are not detectable by conventional methods using monoclonal antibodies and flow cytometry. Some of the surface enzymes are involved in nucleotide metabolism (see Table  73–2). For example, CD73 is an ecto-5′-nucleotidase that catalyzes the 5′ dephosphorylation of purine and pyrimidine riboand deoxyribonucleoside monophosphates to nucleosides that can be taken up by transport systems.69 This ecto-5′-nucleotidase is attached to the plasma membrane by a glycerol phosphatidylinositol anchor. In addition, lymphocytes express CD26,70 which is a membrane protein that can associate with adenosine deaminase, the levels of which are increased after activation.71 The shedding of adenosine deaminase by stimulated cells may explain why plasma levels of this enzyme are increased in early HIV infection and in other diseases associated with immune activation.72 The ectoenzymes of nucleotide metabolism may regulate lymphocyte and granulocyte function at sites of inflammation. Activated T lymphocytes can release ATP, which, in turn, can bind to specific plasma membrane ATP receptors.73 In addition, CD38 can catalyze the transient formation of cyclic adenosine 5′-diphosphate-ribose, a new second-messenger molecule directly involved in the control of calcium homeostasis by means of receptor-mediated release of calcium from ryanodine-sensitive intracellular stores.74 The consequent increase in calcium mobilization and phospholipid breakdown can provoke activation or death, depending on the target cell. Subsequently, the dephosphorylation of ATP generates adenosine, which can interact with A2 receptors on the plasma membranes of neutrophils, monocytes, and lymphocytes.75 The engagement of A2 receptors elevates adenosine 3′,5′-cyclic phosphate levels, counteracting the effects of ATP on cell activation. The deamination of adenosine permits the cycle to begin anew. The ectodomains of several other surface antigens can possess proteolytic activity. For example, CD10 (or CALLA) also has neutral endopeptidase activity,76 and CD26 has dipeptidyl peptidase IV activity.77 These enzymes may play a role in modulating the binding of lymphocytes to other cells and to the extracellular matrix. In addition, inhibition of the

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TABLE 73–2.  Ectoenzymes Expressed by Lymphocytes Surface Molecule

Enzymatic Activity

Function

Reference

CD10

Neutral endopeptidase, EC 3.4.24.11

Metalloproteinase that may also play a role in the metabolic stability of glucagon-like peptide-1

76

CD13

Aminopeptidase N, EC 3.4.11.2

Aminopeptidase involved in trimming peptides bound to major histocompatibility complex class II molecules and cleaving macrophage inflammatory protein (MIP)-1 chemokine to alter target cell specificity. Also served as Rc for coronavirus

102

CD26

Dipeptidylpeptidase IV, EC 3.4.14.5

Serine peptidase that may be involved in T-cell signaling and T-cell activation

77

CD38

ADP ribosyl cyclase, EC 3.4.14.5

Ectoenzyme with NAD glycohydrolase, ADP ribosyl cyclase, and cyclic ADP ribose hydrolase activities

74

CD39

Ecto (Ca2+, Mg2+)-apyrase (ecto-ATPase)

Ectoenzyme with ADPase and ATPase activities that plays a role in regulat- 103 ing platelet aggregation

CD73

Ecto-5′-nucleotidase

Ecto-5′-nucleotidase that may play a role in T-cell signaling

CD143

Peptidyl-dipeptide hydroPeptidyl-dipeptide hydrolase that is involved in the metabolism of vasolase (angiotensin-converting active peptides angiotensin II and bradykinin enzyme)

104

CD156a

ADAM8 metalloprotease

Matrix metalloprotease that may play a role in leukocyte extravasation

78

CD156b

ADAM17 metalloprotease

Metalloprotease that cleaves membrane-bound tumor necrosis factor and 79 transforming growth factor-α to release the soluble cytokine

CD157

ADP ribosyl cyclase and cyclic ADP ribose hydrolase

ADP ribosyl cyclase and cyclic ADP ribose hydrolase that may play a role in lymphocyte development. Like CD38, this enzyme also is involved in the metabolism of NAD

105

CD224

γ-Glutamyltranspeptidase, EC2.3.2.2

γ-Glutamyltranspeptidase role in γ-glutamyl cycle involving the degradation and neosynthesis of glutathione

106

69

ADAM, a disintegrin and a metalloprotease; ADP, adenosine 5′-diphosphate; ADPase, adenosine 5′-diphosphatase; ATPase, adenosine 5′-triphosphate; CD, cluster of differentiation; NAD, nicotinamide adenine dinucleotide.

catalytic activity of CD26 can provoke many cellular effects, including induction of tyrosine phosphorylation and p38 mitogen-­activated protein kinase activation, as well as suppression of DNA synthesis and reduced production of various cytokines. As such, these ectoenzymes could play an important role in lymphocyte activation. Some membrane-bound proteases have a disintegrin and a metalloprotease domain, termed ADAM (a disintegrin and a metalloprotease).78 One such member of this family of proteins is the tumor necrosis factor-α converting enzyme, otherwise known as ADAM17 (CD156b).79 These enzymes cleave other surface molecules, such as tumor necrosis factor, thereby releasing the soluble active cytokine. In addition, they may play an important role in modifying the activity of cytokines or other cell-surface molecules that are present in the vicinity of the plasma membrane.

Intracellular Membrane-Associated Enzymes

Transmembrane proteins that have cytoplasmic regions with kinase or phosphatase activities are common in biology although relatively few of these are restricted to lymphocytes. Nevertheless, many cytoplasmic domains of transmembrane proteins interact directly with enzymes that are restricted or preferentially expressed by lymphocytes or lymphocyte subsets (Chaps. 75 and 76). B lymphocytes, for example, selectively express Bruton tyrosine kinase (BTK), a tyrosine kinase that plays a critical role in signal transduction via surface Ig receptors.80 Moreover, mutations that disrupt the function of such kinases can impair B-cell development, leading to dysregulated B-cell function or immune deficiency.81 On the other hand, T-cell development and function rely heavily on cytoplasmic receptor-associated tyrosine kinases, such as the

Kaushansky_chapter 73_p1135-1148.indd 1144

zeta-associated protein of 70 kDa (ZAP-70), leukocyte tyrosine kinase (lck), or fyn. ZAP-70 interacts with the ζ-chain (CD247) of the TCR for antigen,82 whereas the latter enzymes, lck and fyn, are Src family tyrosine kinases that interact with cytoplasmic domains of various accessory molecules, including CD2, CD4, CD8, CD44, CD50, and/ or CD137.83 Through such interactions, these receptor protein tyrosine kinases play important roles in signal transduction following immune recognition and/or cognate intercellular immune interactions. In addition, lymphocytes possess an important class of intracellular molecules, known collectively as adapter proteins, that have no intrinsic enzymatic activity.84 These adaptor proteins can serve as a scaffolding for the assembly of kinases and other signaling molecules following antigen-receptor ligation. One important adaptor protein expressed in B lymphocytes is B-cell linker protein (BLNK; Chap. 75).85 On the other hand, T cells use a distinct adaptor protein called linker for activation of T cells (LAT).86 These molecules couple proximal biochemical events initiated by surface-receptor ligation with more distal signaling pathways by recruiting other cytosolic proteins (Chaps. 75 and 76).

CYTOPLASMIC STRUCTURES CYTOMATRIX Beneath the lymphocyte’s plasma membrane is a fully developed cytomatrix with several different structural and mechanical proteins, including tubulin, actin, myosin, tropomyosin, α-actinin, filamin, and a spectrinlike molecule, which are important in the formation of the immunologic synapse that forms during cognate intercellular interactions.87 These are

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Chapter 73: The Structure of Lymphocytes and Plasma Cells

arranged into typical microfilaments, microtubules, and intermediate filaments. Lymphocyte activation by antigens or mitogens can lead to changes in the interaction of membrane components with the cytoskeleton, allowing for antigen processing, Ig secretion, or cell-mediated cytotoxic reactions.88

ORGANELLES In large part the composition and metabolism of long-lived blood T lymphocytes reflect their resting state. The T cells have a high nuclear-­ to-cytoplasmic ratio, few ribosomes or mitochondria, and scant endoplasmic reticulum. Glycogen stores are meager. The DNA content of the resting small lymphocyte, 8 pg per cell, is the same amount in other diploid cells. In contrast, the RNA content averages 2.5 pg per cell, yielding an RNA-to-DNA ratio of approximately 0.32.89 This value is less than in most other human cells, as a result of the small amount of ribosomal RNA in most lymphocytes. In contrast to most lymphocytes, however, plasma cells have a high RNA-to-DNA ratio. These cells are the end products of B-cell differentiation and are committed to the synthesis, assembly, and secretion of Ig. Accordingly, these cells have a well-developed rough endoplasmic reticulum and Golgi apparatus, but lack many of the surface receptors found on most lymphocytes. Mature plasma cells are probably terminally differentiated and have a low rate of DNA synthesis and abundant RNA, reflecting the plasma cell’s high-level synthesis of Ig.

Lysosomes

The few lysosomes in blood lymphocytes contain several different acid hydrolases, including acid phosphatase, β-glucuronidase, β-galactosidase, β-hexosaminidase, α-arabinosidase, α-galactosidase, α-mannosidase, α-glucosidase, and β-glucosidase.90–92 Acid hydrolase activities are generally higher in T cells than in non-T lymphocytes. Lysosomal acid esterase, assayed histochemically with α-naphthyl acetate as substrate, has a characteristic punctate appearance in mature T lymphocytes.93 Secretory lysosomes are specialized organelles that combine catabolic functions of conventional lysosomes with the capacity to be secreted upon induction.94 An example of such secretory lysosomes are the specialized cytoplasmic granules of T cells and NK cells that are responsible for the cytotoxic effector function of these cells.

Cytoplasmic Granules

In contrast to other lymphocytes, cytotoxic T lymphocytes and NK cells possess abundant cytoplasmic granules. These contain a pore-forming proteolytic enzyme, termed perforin, and a series of serine proteinases with specific proapoptotic activity, called granzymes.95 To protect against possible autolysis by granule contents, cytotoxic lymphocytes possess serine-proteinase inhibitors, termed serpins.96 As an additional safeguard, the granzymes of resting lymphocytes are stored as inactive proenzymes. Cytotoxic lymphocytes rely primarily on the perforin/granzyme system to kill their targets.97 Upon contact with its target cell, the cytotoxic lymphocyte converts the granzymes into active forms by a lysosomal cysteine protease called dipeptidyl peptidase I.98 Then perforin introduces a pore in the membrane, allowing the activated granzymes and other granule contents to pass into the cytoplasm and then the nucleus of the cell targeted for destruction.95 In vitro studies indicate that granzyme nuclear import is independent of ATP, cannot be inhibited by nonhydrolyzable guanosine triphosphate analogues, and involves binding within the nucleus, unlike conventional signal-dependent nuclear protein import. The perforin-dependent nuclear entry of granzymes precedes the nuclear events of apoptosis, such as DNA fragmentation and breakdown of the nuclear envelope (Chap. 15).

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ACKNOWLEDGMENTS This chapter was adapted from “Morphology of Lymphocytes and Plasma Cells” by H. Elizabeth Broome and “Composition and Biochemistry of Lymphocytes and Plasma Cells” by Thomas J. Kipps in the earlier edition.

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63. Crumpton MJ, Snary D: Preparation and properties of lymphocyte plasma membrane. Contemp Top Mol Immunol 3:27–56, 1974. 64. Goppelt M, Eichhorn R, Krebs G, Resch K: Lipid composition of functional domains of the lymphocyte plasma membrane. Biochim Biophys Acta 854:184–190, 1986. 65. Johnson SM, Robinson R: The composition and fluidity of normal and leukaemic or lymphomatous lymphocyte plasma membranes in mouse and man. Biochim Biophys Acta 558:282–295, 1979. 66. Jury EC, Flores-Borja F, Kabouridis PS: Lipid rafts in T cell signalling and disease. Semin Cell Dev Biol 18:608–615, 2007. 67. Gupta N, DeFranco AL: Lipid rafts and B cell signaling. Semin Cell Dev Biol 18: 616–626, 2007. 68. Landry A, Xavier R: Isolation and analysis of lipid rafts in cell-cell interactions. Methods Mol Biol 341:251–282, 2006. 69. Colgan SP, Eltzschig HK, Eckle T, Thompson LF: Physiological roles for ecto-5′-nucleotidase (CD73). Purinergic Signal 2:351–360, 2006. 70. Havre PA, Abe M, Urasaki Y, et al: The role of CD26/dipeptidyl peptidase IV in cancer. Front Biosci 13:1634–1645, 2008. 71. Kameoka J, Tanaka T, Nojima Y, et al: Direct association of adenosine deaminase with a T cell activation antigen, CD26. Science 261:466–469, 1993. 72. Ohtsuki T, Tsuda H, Morimoto C: Good or evil: CD26 and HIV infection. J Dermatol Sci 22:152–160, 2000. 73. Swennen EL, Coolen EJ, Arts IC, et al: Time-dependent effects of ATP and its degradation products on inflammatory markers in human blood ex vivo. Immunobiology 213:389–397, 2008. 74. Partida-Sanchez S, Rivero-Nava L, Shi G, Lund FE: CD38: An ecto-enzyme at the crossroads of innate and adaptive immune responses. Adv Exp Med Biol 590:171–183, 2007. 75. Kumar V, Sharma A: Adenosine: An endogenous modulator of innate immune system with therapeutic potential. Eur J Pharmacol 616:7–15, 2009. 76. Plamboeck A, Holst JJ, Carr RD, Deacon CF: Neutral endopeptidase 24.11 and dipeptidyl peptidase IV are both involved in regulating the metabolic stability of glucagon-like peptide-1 in vivo. Adv Exp Med Biol 524:303–312, 2003. 77. Ohnuma K, Takahashi N, Yamochi T, et al: Role of CD26/dipeptidyl peptidase IV in human T cell activation and function. Front Biosci 13:2299–2310, 2008. 78. Yamamoto S, Higuchi Y, Yoshiyama K, et al: ADAM family proteins in the immune system. Immunol Today 20:278–284, 1999. 79. Black RA: Tumor necrosis factor-alpha converting enzyme. Int J Biochem Cell Biol 34:1–5, 2002. 80. Lindvall JM, Blomberg KE, Väliaho J, et al: Bruton’s tyrosine kinase: Cell biology, sequence conservation, mutation spectrum, siRNA modifications, and expression profiling. Immunol Rev 203:200–215, 2005. 81. Kurosaki T, Hikida M: Tyrosine kinases and their substrates in B lymphocytes. Immunol Rev 228:132–148, 2009. 82. Au-Yeung BB, Deindl S, Hsu LY, et al: The structure, regulation, function of ZAP-70. Immunol Rev 228:41–57, 2009. 83. Salmond RJ, Filby A, Qureshi I, et al: T-cell receptor proximal signaling via the Srcfamily kinases, Lck and Fyn, influences T-cell activation, differentiation, and tolerance. Immunol Rev 228:9–22, 2009. 84. Leo A, Schraven B: Adapters in lymphocyte signalling. Curr Opin Immunol 13:307–316, 2001. 85. Tsukada S, Baba Y, Watanabe D: Btk and BLNK in B cell development. Adv Immunol 77:123–162, 2001. 86. Aguado E, Martinez-Florensa M, Aparicio P: Activation of T lymphocytes and the role of the adapter LAT. Transpl Immunol 17:23–26, 2006. 87. Rey M, Sanchez-Madrid F, Valenzuela-Fernandez A: The role of actomyosin and the microtubular network in both the immunological synapse and T cell activation. Front Biosci 12:437–447, 2007. 88. Miletic AV, Swat M, Fujikawa K, Swat W: Cytoskeletal remodeling in lymphocyte activation. Curr Opin Immunol 15:261–268, 2003. 89. Glen AC: Measurement of DNA and RNA in human peripheral blood lymphocytes. Clin Chem 13:299–313, 1967. 90. Beaumelle BD, Gibson A, Hopkins CR: Isolation and preliminary characterization of the major membrane boundaries of the endocytic pathway in lymphocytes. J Cell Biol 111:1811–1823, 1990. 91. Casey TM, Meade JL, Hewitt EW: Organelle proteomics: Identification of the exocytic machinery associated with the natural killer cell secretory lysosome. Mol Cell Proteomics 6:767–780, 2007. 92. Qu P, Du H, Wilkes DS, Yan C: Critical roles of lysosomal acid lipase in T cell development and function. Am J Pathol 174:944–956, 2009. 93. Kulenkampff J, Janossy G, Greaves MF: Acid esterase in human lymphoid cells and leukaemic blasts: A marker for T lymphocytes. Br J Haematol 36:231–240, 1977. 94. Lettau M, Schmidt H, Kabelitz D, Janssen O: Secretory lysosomes and their cargo in T and NK cells. Immunol Lett 108:10–19, 2007. 95. Chavez-Galan L, Arenas-Del Angel MC, Zenteno E, et al: Cell death mechanisms induced by cytotoxic lymphocytes. Cell Mol Immunol 6:15–25, 2009. 96. Bots M, Medema JP: Serpins in T cell immunity. J Leukoc Biol 84:1238–1247, 2008. 97. Trapani JA, Smyth MJ: Functional significance of the perforin/granzyme cell death pathway. Nat Rev Immunol 2:735–747, 2002. 98. Pham CT, Ley TJ: Dipeptidyl peptidase I is required for the processing and activation of granzymes A and B in vivo. Proc Natl Acad Sci U S A 96:8627–8632, 1999.

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99. Maeda T, Yamada H, Nagamine R, et al: Involvement of CD4+, CD57+ T cells in the disease activity of rheumatoid arthritis. Arthritis Rheum 46:379–384, 2002. 100. Fontenot JD, Gavin MA, Rudensky AY: Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat Immunol 4:330–336, 2003. 101. Chattopadhyay PK, Betts MR, Price DA, et al: The cytolytic enzymes granzyme A, granzyme B, and perforin: Expression patterns, cell distribution, and their relationship to cell maturity and bright CD57 expression. J Leukoc Biol 85:88–97, 2009. 102. Tani K, Ogushi F, Huang L, et al: CD13/aminopeptidase N, a novel chemoattractant for T lymphocytes in pulmonary sarcoidosis. Am J Respir Crit Care Med 161:1636–1642, 2000.

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103. Schulte am Esch J 2nd, Sévigny J, Kaczmarek E, et al: Structural elements and limited proteolysis of CD39 influence ATP diphosphohydrolase activity. Biochemistry 38: 2248–2258, 1999. 104. Bauvois B: Transmembrane proteases in cell growth and invasion: New contributors to angiogenesis? Oncogene 23:317–329, 2004. 105. Ortolan E, Vacca P, Capobianco A, et al: CD157, the Janus of CD38 but with a unique personality. Cell Biochem Funct 20:309–322, 2002. 106. Stark AA, Porat N, Volohonsky G, et al: The role of gamma-glutamyl transpeptidase in the biosynthesis of glutathione. Biofactors 17:139–149, 2003.

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during embryogenesis lags behind development of the myeloid and erythroid lineages. Although myeloid, erythroid, and natural killer (NK) cells can be produced from all extraembryonic and embryonic sites, B and T lymphocytes are predominantly generated from so-called definitive hematopoietic stem cells (HSCs) in the embryo proper.1

Christopher S. Seet and Gay M. Crooks

MURINE HEMATOPOIETIC DEVELOPMENT

LYMPHOPOIESIS SUMMARY Lymphopoiesis refers to the process by which the cellular components of the immune system (i.e., T cells, B cells, and natural killer cells, and certain dendritic cells) are produced during hematopoietic differentiation. This process begins with the hematopoietic stem cell and continues through progenitor stages down a series of mostly diverging lineage pathways, ultimately resulting in the remarkable diversity and flexibility of the immune system. Although the more terminal events in lymphocyte differentiation and function have been defined in detail (Chaps. 75 to 77), the earliest events during which hematopoietic stem cells undergo lymphoid lineage commitment are lesswell understood and still controversial. Although the conceptual framework for the questions of lymphoid commitment has been established largely on studies in the mouse, experimental systems now exist to better understand how such events are controlled in humans. This chapter summarizes what is known about the ontogeny of lymphoid development and the control of lymphoid differentiation, and discusses some of the persisting controversies in the field.

LYMPHOPOIESIS DURING PRENATAL DEVELOPMENT Blood is formed from a succession of sites during embryonic and fetal development, beginning outside the embryo in the yolk sac. Soon afterward, hematopoiesis begins in the embryo proper, initially in the para-aortic splanchnopleura (PAS) and aorto-gonad-mesonephros (AGM) regions, then the fetal liver, spleen, and finally the fetal marrow (Chap. 7). With each change of anatomical site, the range of hematopoiesis becomes progressively more complex and similar to that of the adult (Fig. 74–1). When assigning hematopoietic function to each developmental stage, it is important to distinguish the lineage “potential” of stem and progenitor cells that arise from certain areas (i.e., the ability to generate specific lineages in vitro from immature cells removed from a region) from the spontaneous physiologic production of lineages in each region. With this distinction in mind, the onset of lymphopoiesis

Acronyms and Abbreviations: AGM, aorto-gonad-mesonephros; BM, bone marrow; BCR, B-cell receptor; CLP, common lymphoid progenitor; CT, computed tomography; DC, dendritic cell; DN, double negative; E, days of gestation; EBF, early B-cell factor; FACS, fluorescence-activated cell sorting; FLT3, Fms-like tyrosine kinase 3; HSC, hematopoietic stem cell; Ig, immunoglobulin; IL, interleukin; JAK3, Janus kinase 3; LMPP, lymphoid-primed multipotent progenitor; LSK, linnegsca-1+c-kit+; NK, natural killer; PAS, para-aortic splanchnopleura; SCID, severe combined immunodeficiency.

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Most of the studies exploring embryonic and fetal hematopoiesis have been performed using mouse models. Although the timing of each developmental stage has been carefully mapped, it has long been a source of controversy as to whether hematopoiesis in the embryo is initiated from colonizing precursors from the extraembryonic yolk sac, or whether the embryonic sites of hematopoiesis arise independently from the yolk sac.2–6 This debate has implications for understanding the lineages generated at different sites of hematopoiesis and thus for tracing the ancestry of the lymphoid cells that are produced in the mammalian embryo. One reason for the difficulty in assigning the exact organ in which lineages are generated, is that each site of hematopoiesis is active during overlapping periods (see Fig. 74–1). In addition, once circulation has been established, it is difficult to rule out the possibility that stem cells and progenitors found in one location did not migrate from another. However studies using Ncx1–/– mice, which lack both heartbeat and circulation,7,8 are beginning to dissect the autonomous lineage potentials of these distinct embryonic hematopoietic tissues. The first wave of hematopoiesis in the mouse begins in the extraembryonic tissue of the yolk sac by 7.5 days of gestation (E7.5), before circulation is established.9,10 This initial stage of so-called primitive hematopoiesis produces mostly erythrocytes and macrophages. Although lymphocytes are not detectable at this time,10 the contribution of first-wave progenitors to downstream fetal lymphopoiesis has been suggested by the identification of a lymphomyeloid progenitor in the E9.5 yolk sac, which expresses Rag-1, one of the earliest lymphoidspecific events,11 as well as of a distinct progenitor with B-1/marginal zone B cell potential.12 Further studies have identified both thymic-repopulating and multipotent potential in the yolk sac,13,14 indicating emerging changes to our understanding of primitive hematopoiesis. The murine placenta has also been identified as an autonomous source of multipotent hematopoietic cells as early as E8.58; however, the direct contribution of either yolk sac or placental progenitors to definitive lymphoid development remains to be determined. Definitive HSCs that are capable of generating all lymphohematopoietic lineages first appear in the PAS/AGM region at E8.5 to E9.3,10 High-level, multilineage reconstituting activity typical of definitive HSC can be found in the murine AGM region by E10.5. However, although AGM cells can produce all lineages, including T and B lymphocytes in vitro, lymphocytes do not spontaneously develop in the fetus until hematopoiesis has begun in the fetal liver. Rag-1 expression, one of the earliest lymphoid-specific events, can be found in the E11 murine fetal liver.10 T-cell potential has been identified in the yolk sac and PAS as early as E8.25 to E9.5 of murine gestation13; however, T-cell differentiation in vivo begins with the colonization of the thymus around E11 by stem or progenitor cells that migrate to the thymus from the AGM, fetal liver, and, later still, the fetal marrow.15,16

HUMAN HEMATOPOIETIC DEVELOPMENT Hematopoietic cells have been identified in the human yolk sac as early as day 18 of embryonic life, at which time, like the mouse, they are almost exclusively comprised of erythrocytes and, to a lesser extent, monocytes and macrophages (see Fig. 74–1).17 Although no lymphocytes are seen in the yolk sac, yolk sac progenitors do have NK cell potential under certain in vitro conditions.17,18 The same yolk sac progenitors, however,

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Figure 74–1.  Timing of lymphohematopoiesis during prenatal development. Shown is the timeline for activity in each site of hematopoiesis in the embryo and fetus of (A) human and (B) mouse. B and T cells are first detected in vivo in fetal liver and thymus, respectively, at times shown. AGM, aorto-gonad-mesonephros; PAS, para-aortic splanchnopleura. do not possess the capacity for B- or T-cell development, even when placed in culture conditions that permit lymphoid differentiation.18 As in the mouse, definitive hematopoiesis develops first in the AGM region derived from the splanchnopleura, as evidenced by the finding that the AGM is the site where CD34+ cells with the capacity for full lymphoid and myeloid differentiation are first found in the human embryo.18,19 The AGM develops at day 27 of gestation in the human, when human HSCs are generated as clusters of two or three cells arising from the endothelium specifically on the ventral surface of the preumbilical region of the aorta. These cells are clonogenic and highly proliferative, rapidly increasing to several thousand in number and spreading further along the aorta. However, hematopoiesis exists only transiently in the AGM, disappearing entirely by day 40.17 Although lymphoid cells can be produced in culture from cells extracted from the AGM17,18 the HSC of the AGM do not produce mature cells in situ; instead their role is to migrate and colonize the fetal liver, producing the next wave of hematopoiesis. As in the mouse, recent studies have also detected multipotent progenitors with lymphoid potential in the human placenta. These reports suggest that hematopoietic stem/progenitor cells may also be generated de novo from the large vessels of the chorionic plate as early as week 5 of gestation, with multipotent HSC detectable at 15 weeks.20,21 However, the contribution of placental HSC to fetal liver or marrow colonization remains unclear. Although blood cells are first detectable in the human fetal liver as early as day 23, they exist at this time only as erythroid and myeloid cells associated with hepatic sinusoids. These erythroid cells consist of megaloblasts expressing embryonic hemoglobins (globin chains ζ and ε), and no CD34+ cells are seen in the fetal liver during this early phase. It is likely that this first stage of fetal liver hematopoiesis is secondary to colonization of more mature cells from the yolk sac. By day 30, AGM-derived

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CD34+ cells appear in the fetal liver,22 and by day 32, these cells are able to maintain long-term hematopoiesis in vitro.22 Erythroid cells in the fetal liver at this later stage of definitive hematopoiesis consist of enucleated macrocytes producing fetal hemoglobin (globin chains α and γ). In normal development, as with the yolk sac and AGM, hematopoiesis in the fetal liver is transient, disappearing by 20 weeks of gestation.1 The final wave of hematopoietic development takes place in the fetal marrow, starting around 11 weeks of gestation. The initial cells seen in the marrow are CD15+ myeloid cells and glycophorin A+ erythroid cells, and hematopoiesis is again associated with the endothelium, taking place in the medullary sinusoids before osteoblast formation.23 Eventually, CD34+ cells are found in the fetal marrow and behave functionally as true HSC, generating B, T, NK, and myeloid and erythroid lineages.1 HSC have found their final niche, and lifelong, self-renewing lymphohematopoiesis resides permanently in the marrow thereafter.

THYMIC DEVELOPMENT The human thymic microenvironment begins to develop at approximately 4 weeks’ gestation and then undergoes three developmental phases.24 The first phase occurs between 4 and 8 weeks’ gestation, with the appearance of thymic epithelium arising as a product of the third and fourth pharyngeal pouches25 and the expansion of thymic epithelial cells. The second phase occurs between 9 and 15 weeks’ gestation and is characterized by the development of subcapsular, cortical, and medullary regions.24 Thymic colonization by fetal liver-derived progenitors and lymphocyte production begins at approximately week 9.25 The ability of thymocytes to respond to the mitogen phytohemagglutinin is detectable as early as 10 weeks’ gestation,26 and alloreactive, phenotypically mature T cells can be found by 13 to 16 weeks’ gestation.27

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The third phase occurs from 16 weeks’ gestation until age 1 to 2 years and is characterized by robust intrathymic T-cell maturation (Chaps. 6 and 76). An exhaustive study of 136 human postnatal thymuses ranging from neonatal life to more than 90 years old, found that essentially all postnatal thymic growth (based on weight and volume) occurs during the first postnatal year, mostly in the first few months of life.28 From the age of 12 months, the human thymus undergoes steady involution, with a reduction of thymocytes and thymic epithelium, particularly the medulla, and a corresponding increase in fatty infiltration of the perivascular space.28 Whereas mice lose approximately 90 percent of the wet weight of the thymus during life, the increasing fatty infiltration in the perivascular space in the aging human thymus maintains the total size of the thymus in healthy people into late life.28 Radiologic studies using computed tomography (CT) scans have confirmed that total thymic size remains stable in humans throughout life although parenchymal tissue atrophies dramatically (~95 percent; Chaps. 6 and 9).29

lineage may emerge prior to the development of the adaptive immune system.12 At present, there is no clear evidence that humans have similar subpopulations of B-1 and B-2 cells during development.31

B-CELL DEVELOPMENT

Dendritic-like cells which express class II major histocompatibility (MHC) antigens are produced at all stages of embryonic and fetal hematopoiesis, being first detected in the human yolk sac and mesenchyme as early as 4 to 8 weeks, before development of the fetal marrow or thymus.31 Dendritic cells (DCs) are detectable at each site of hematopoiesis as soon as they become active, in the human fetal thymus at 11 to 14 weeks, marrow at 14 to 17 weeks, spleen at 16 weeks, and tonsils at 23 weeks.39,40 DC and macrophages are closely related and phenotypically similar (Chap. 67), both expressing MHC class II. Thus, clear discrimination of these two cell types can be difficult, particularly as many studies that provided information on human DC development were conducted before all the molecular and antibody tools for analysis of DC were available.

The hallmark characteristic of a mature B cell circulating in blood or residing in secondary lymphoid tissue is the expression of cell-surface immunoglobulin (Ig). The cell-surface Ig consists of μ, δ, γ, α, or ε heavy chains disulfide-linked to κ or λ light chains (Chap. 75). The cell-surface Ig and the associated signaling molecules Igα (CD79a) and Igβ (CD79b) are referred to as the B-cell receptor (BCR). Progenitor (pro-) B cells are defined by the absence of both cytoplasmic μ heavy chains and cellsurface BCR. Precursor (pre-) B cells are defined by the presence of cytoplasmic μ heavy chains in the absence of cell-surface BCR. This minimal definition of pro-B, pre-B, and B cells forms the basis of the current detailed model of human B-cell development.30 B-cell development can be divided into two stages: an antigen-independent stage that occurs primarily in fetal liver and fetal and adult marrow, and an antigendependent stage that occurs primarily in secondary lymphoid tissue, such as spleen and lymph node. The first B cells detectable in the human fetus are found in the fetal liver25 at approximately 8 weeks’ gestation, with the appearance of cytoplasmic IgM+ pre-B cells; by 10 to 12 weeks, surface IgM+ B cells are seen in the fetal liver31 and fetal omentum.32 B-cell and IgM production move to the fetal marrow and spleen by 17 weeks of gestation (Chaps. 6, 7, and 75).33,34 From the end of the second trimester throughout adult life, marrow is the exclusive origin of B-cell development.35 The frequency of early B-lineage cells as a percentage of the total nucleated lymphohematopoietic cell pool is higher in fetal than in adult marrow. However, the ratio between pro-B, pre-B, and immature B cells and the mitotic activity within these fractions is relatively constant.36 In murine B-cell development, two functionally and immunophenotypically distinct types of B cells, B-1 and B-2, have been described.31 Most B cells in adult mice are B-2 cells, which form part of the adaptive immune system by their ability to interact with T cells and undergo immunoglobulin heavy chain class switching. The B-1 cells make up approximately 5 percent of adult murine lymphocytes, but demonstrate a far less diverse immunoglobulin repertoire than the B-2 cells, responding to carbohydrate antigens and other T-cell–independent immunogens and forming part of the innate immune system. Murine B-1 cells are marked by their expression of CD11b, and are found in multiple sites, including the spleen, intestine, and the pleural and peritoneal cavities.37,38 The B-1 cells can be further divided into B-1a cells (which secrete immunoglobulins spontaneously) and B-1b cells (in which immunoglobulin production is induced) based on the expression of the marker CD5. The demonstration of a B-1a/marginal zone B-cell– restricted progenitor in the E9 extraembryonic yolk sac suggests the B-1

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NATURAL KILLER CELL DEVELOPMENT Functional NK cells can be detected in the human fetal liver as early as 9 to 10 weeks of gestation,26 but NK cell differentiation can be induced in vitro from progenitors derived at all stages of hematopoietic development, even those from the yolk sac.1,17,18 Thus, the onset of NK potential is not equivalent to the full lymphoid potential of definitive hematopoiesis, as NK potential can be assigned to a range of progenitor types that exist at different stages, including primitive hematopoiesis. NK cell production can be considered as providing an essential defense mechanism for the developing mammalian embryo prior to development of more complex pathways of adaptive immunity.

DENDRITIC CELL DEVELOPMENT

DIFFERENTIATION PATHWAYS FOR LYMPHOCYTE PRODUCTION The conceptual framework for how the lymphocyte lineages are generated from HSC was developed largely from studies using genetically engineered mice and murine transplant models. Although necessary and useful as a starting point, caution should be exercised in translating the results of the murine studies to human lymphopoiesis, or in assuming for any species that only one pathway to lymphopoiesis exists at all stages of ontogeny.41,42 Additionally, the conclusions about lineage relationships of isolated populations are influenced by limitations inherent in any of the in vitro or in vivo assays employed to examine differentiation potential.43 For several decades, our understanding of hematopoiesis has been built on a hierarchical schema in which all the pathways of differentiation lead away from a multipotent HSC, and progress through discrete progenitor stages that mark each branch-point of lineage commitment (Chap. 18). In the classical paradigm, the earliest differentiation “decision” made by an HSC is to enter one of two pathways, marked by either a common lymphoid progenitor (CLP) or a common myeloid progenitor (CMP), which has full myeloid and erythromegakaryocytic differentiation potential (Fig. 74–2).44 With each successive stage of differentiation, lineage-specific cell-surface markers and transcription factors are upregulated, and alternative lineage potentials are lost. Consequently, the CLP is defined as a single cell that can give rise to all lymphoid lineages (B, T, and NK), but cannot generate myeloid, erythroid, or megakaryocytic lineages. The concept of progenitor populations marking two mutually exclusive differentiation pathways, one limited to myeloid and erythromegakaryocytic potential and the second defining

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Figure 74–2.  Postnatal pathways of lymphopoiesis in mice and humans. The key immunophenotype used to isolate each population is shown in boxes on the right. In parentheses under the main immunophenotypes are other markers also associated with each population. The exact relationship between lymphoid-primed multipotential progenitors (LMPPs) and common lymphoid progenitors (CLPs), and between LMPPs and myeloid progenitors remains controversial, as does the main cell type that initiates thymopoiesis (all shown as dashed lines). In both mice and humans, dendritic cells (DCs) can be produced in vitro from all prospectively identified lymphoid progenitors as well as myeloid progenitors. A. Murine lymphoid progenitor pathways. Populations with multilineage potential include long-term hematopoietic stem cells (LT-HSCs) and multipotential progenitors (MPPs). FLT3+ LMPPs, have full lymphoid (T-, B-, and natural killer [NK] cell developmental potential) and limited myeloid (nonclonogenic, mostly monocyte) potential. In the CLP, all myeloid potential is lost and full lymphoid potential remains. B. Human lymphoid progenitor pathways. Populations with multilineage potential include HSC and LMPP (aka multilymphoid progenitor [MLP]), which have full lymphoid (T-, B-, and NK cell developmental potential) and limited myeloid (mostly monocyte) potential. The CD10+ CLP has predominantly B, and some NK and T potential and is almost devoid of myeloid potential. Alternative phenotypic markers reported for each progenitor type with similar lineage potential are shown. BM, marrow; GMP, granulocyte-macrophage progenitor; MEP, megakaryocyte-erythroid progenitor.

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lymphoid commitment, was held long before cells that satisfied the criteria of CLP were identified.45–47 In contrast, the existence of single clonogenic cells with myeloid, erythroid, and megakaryocyte lineages was shown more than three decades ago through the use of in vitro clonal assays to demonstrate so-called colony-forming unit-granulocyte erythromyeloid megakaryocyte,48 and later confirmed using markers to prospectively isolate such cells in mice49 and humans.50 The lymphocyte lineages were assumed to be closely related because of a number of associations, for example, common anatomical sites of T and B lymphopoiesis (spleen, lymph nodes; Chap. 6), similar molecular mechanisms that regulate T-cell receptor and B-cell immunoglobulin rearrangements (Chaps. 75 and 76), and severe B- and T-lymphoid defects that result from single genetic mutations in mice (Chap. 80).51 Development of flow cytometry (synonym: fluorescence-activated cell sorting [FACS]) made possible the isolation of rare hematopoietic cell populations and the subsequent interrogation of lineage potential using in vitro cultures and in vivo reconstitution studies (Chap. 18). Primitive multilymphoid progenitors with little or no clonogenic myeloid or erythroid potential have now been isolated from human tissue using flow cytometry with combinations of various cell-surface markers.52–54 However, it seems likely that lineage relationships are lessstrictly organized than once believed. Studies in mice show that the erythroid and megakaryocytic lineages can branch off at an earlier point in hematopoiesis, and that lymphoid (i.e., T, B, and NK) and myeloid (or at least monocytic) lineages can arise from the same pathway through a so-called lymphoid-primed multipotent progenitor (LMPP).55 It remains unclear which of these lineage differentiation pathways are most physiologically significant during steady-state hematopoiesis, but it is likely that more than one pathway can exist simultaneously and alternative pathways may predominate during different stages of ontogeny and from different sites of hematopoiesis.

MURINE LYMPHOID PROGENITORS In 1997, investigators working with murine marrow cells, isolated progenitors that possessed no myeloid or erythromegakaryocytic potential, but when transplanted into irradiated recipients could rapidly restore T-, B-, and NK cell lineages.56 Clonal in vitro and in vivo studies showed that all lymphoid lineages were derived from a single common progenitor, thus proving the existence of the long-assumed CLP and supporting the classical model of lymphopoiesis. This study isolated cells based in part on expression of interleukin-7 receptor alpha (IL-7Rα).56 The IL-7Rα+ CLP do not express hematopoietic markers associated with fully differentiated lineages (they are called “lineage negative” or “linneg” cells). As an indication that they are more differentiated than multilineage HSCs, expression of certain HSC-related cell-surface markers (Sca-1, Thy-1, c-kit) is downregulated.56 Thus the full immunophenotype assigned to the murine CLP is Linneg IL-7Rα+ Thy-1neg Sca-1lo c-kitlo. This contrasts the murine CLP immunophenotype with that of the murine HSC, which is found within the Linneg IL-7Rαneg Thy-1lo Sca1hi c-kithi population.56 Work with murine marrow has prompted a reexamination of when the lymphoid lineage pathways diverge from those of the myeloid and erythroid lineages (see Fig. 74–2). A population from murine marrow cells, defined largely by expression of the receptor FLT3 (Fms-like tyrosine kinase 3), has been shown to possess full lymphoid and some myeloid potential, but not erythroid or megakaryocytic potential.55,57 These linnegsca-1+c-kit+CD34+FLT3hi (synonym: LSK CD34+FLT3hi) cells are primed for lymphoid commitment in that they have downregulated genes involved in erythromegakaryocytic differentiation and upregulated lymphoid-associated genes.58 They have thus been termed LMPPs.55,58 Although they are able to generate monocytes and granulocytes in vitro, their differentiation potential is nonetheless skewed

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heavily to lymphoid cells; after transplantation into irradiated recipients, LSK CD34+FLT3hi cells rapidly reconstitute B and T lymphopoiesis. However, unlike the multipotent LSK CD34+FLT3neg cells, reconstitution of myeloid lineages in vivo from LSK CD34+FLT3hi is very limited, and importantly lacks granulocytic potential.55 Indeed, subsequent in vivo fate-mapping studies have questioned the physiologic importance of a lymphomyeloid differentiation pathway to steady-state myeloid development, both in the marrow and thymus.59,60 One confusing factor in defining lineage potential is the ability of both myeloid and lymphoid progenitor populations to differentiate into DCs, at least in vitro (Chap. 21).61–63 As in vitro-derived DC express many cell-surface markers common to myeloid antigen-presenting cells, irrespective of the lineage of origin, the extent to which identification of in vitro myeloid potential is confounded by a DC program is unclear.

HUMAN LYMPHOID PROGENITORS The CD34 cell-surface marker is expressed on human HSCs and on a variety of different types of hematopoietic progenitors, including those restricted to lymphoid development (Chap. 18).64,65 CD34 has been combined with additional cell-surface markers to identify human multilymphoid progenitors, for example, CD10,52,66 CD7,53,54,67,68 and CD45RA.52,67 Similar to the mouse CLP, in addition to the upregulation of expression of the aforementioned markers, lymphoid commitment is accompanied by downregulation of certain HSC cell-surface markers, such as c-kit and Thy-1.41 As in murine studies, no one marker used in isolation is able to define a human lymphoid progenitor.65 For example, although the expression of CD7 can be used to define a subset of CD34+linnegCD38neg cells in cord blood that are multilymphoid progenitors without myeloid or erythroid potential,53,54 CD34+linnegCD38+CD7+ cells from cord blood have full lineage (lymphoid, myeloid, and erythroid) potential. Furthermore, when comparing the progenitor populations identified in human studies with those described from murine experiments it is important to recognize that species differences exist between cellsurface markers.41 For example, IL-7Rα expression is used to define murine CLP,56 but CD34+linnegCD38negCD7+ multilymphoid progenitors in human cord blood do not express IL-7Rα,53 and CD34+linnegCD38+IL-7Rα+ cells in human cord blood have both myeloid, lymphoid and even some erythroid potential. A different ontogeny and source of hematopoietic cells will also introduce unexpected variations of progenitor immunophenotype and function.41 Whereas most murine studies have been conducted with adult murine marrow, most human studies have been performed with umbilical cord blood, a more logistically available source containing progenitors that are significantly more proliferative than marrow.69 Again using the example of the CD34+linnegCD38negCD7+ multilymphoid progenitor, although this immunophenotype can be used to identify multilymphoid progenitors in cord blood,53 the same markers cannot be used in human marrow because CD34+linnegCD38neg marrow cells do not express CD7. Two candidates for the human equivalent of the murine LMPP have been described, both of which coexpress FLT3: one in the marrow identified as CD34+linnegCD38+CD45RA+ and high expression of CD62L (Lselectin)70; and another in the marrow and cord blood characterized as a CD34+linnegCD38negThy-1lo/negCD45RA+ multilymphoid progenitor (MLP).71 The hierarchical relationship of these two populations to each other, or to the marrow CD34+linnegCD38+CD45RA+CD10+ CLP is unclear; and as in the murine system, the physiologic contributions of a lymphomyeloid developmental pathway to steady-state hematopoiesis has yet to be determined. Adding to the concept of possible independence of dendritic cell development, DC potential has been found in all the primitive human lymphoid progenitors reported.52,53,67,70–72

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THYMIC PROGENITORS It was long assumed that lymphoid commitment in the marrow precedes thymic seeding and T-cell development. However, despite the clear existence of lymphoid-committed progenitors within the marrow, the dominant cell type that migrates from the marrow and seeds the thymus to initiate thymopoiesis is still a matter of controversy. As described above, a variety of marrow-derived lymphoid-restricted progenitors and LMPPs are each able to generate T cells in vitro and in vivo. However, careful examination of the thymus has revealed primitive progenitors that have not only lymphoid, but also myeloid and erythroid potential. Such rare cells have been identified in murine thymus, where they are referred to as early thymic progenitors (ETP),73 and also in human thymus, where they have the phenotype CD34+linnegCD1anegCD7neg.74,75 The lineage potential of such cells as well as the sharing of many cell-surface markers and similar gene expression profile to HSCs, suggest strongly that HSCs or at least multipotent progenitors are able to seed the thymus directly without a preceding stage of lymphoid commitment in the marrow. Which of these alternative progenitor types are dominant in terms of their contribution to steady-state thymopoiesis is yet to be determined76; however, it is likely that early thymic progenitor lineage potential is itself dynamic, based on colonization of the murine thymus with temporally distinct waves of both lymphoidrestricted and multipotent thymic-seeding progenitors during embryonic development.77

CHALLENGES IN FUNCTIONAL CHARACTERIZATION OF LYMPHOID PROGENITORS The accurate assignment of lineage potential to immunophenotypically defined progenitors requires clonal analysis. Although clonal assays for myelo-erythromegakaryocytic progenitors have existed for more than 30 years,48 the ability to differentiate HSCs along lymphoid pathways has been relatively recent, particularly for human studies.43 In vitro assays for human lymphoid potential became available when it was observed that selected murine stromal cell lines were capable of supporting B-cell, NK cell, and DC differentiation from primitive human HSCs.78–80 T-cell differentiation systems are more complex, requiring an in vitro model that recapitulates the unique environment of the thymus. Originally this was only possible using the fetal thymic organ culture method, a system in which large numbers of murine or human progenitors are seeded into whole thymic lobes in so-called hanging drop cultures.81 A more efficient in vitro system for studying murine and human T-cell differentiation has been developed using a murine stromal monolayer that expresses the Notch ligand Delta-like 1 (“OP9-DL1 stroma”).82 However, none of the in vitro T-cell culture systems simultaneously support B-cell development, making proof of full T- and B-lymphoid potential at a clonal level technically problematic. In vivo transplantation of a single murine HSC can prove multilineage potential at a clonal level, but this also is technically difficult, especially when studying progenitor populations that are not self-renewing. In vivo studies with human cells are particularly challenging as they rely on xenogeneic transplant models with low engraftment efficiency.41,43

REGULATION OF LYMPHOPOIESIS CYTOKINES IN LYMPHOPOIESIS The many cytokine pathways that regulate lymphoid development, differentiation, and function are too numerous and complex for a full description here. However, the cytokine receptors of the common gamma (γc) chain family should be mentioned particularly because

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of their biologic importance in lymphopoiesis and their clinical relevance in primary immune deficiency disease. The γc subunit is a signaling component of six different cytokine receptors, interleukin (IL)-2,83 IL-4,84,85 IL-7,86,87 IL-9,88 IL-15,89 and IL-21,90 all of which act on different stages and pathways involved in lymphopoiesis.51,91,92 All six γcdependent receptors are unique in their activation of the Janus kinase 3 (JAK3) tyrosine kinase, a molecule that directly interacts with γc to mediate signaling.93 In addition to the γc subunit, each of these receptors are comprised of an α subunit through which specific ligands bind; IL-2R and IL-15R also share a common β subunit.51 Null mutations of γc result in severe combined immunodeficiency (SCID) syndromes in mice and humans. However differences in the specific lineages affected reveal important species differences in cytokine dependency.51 The most important of these differences is in the requirement for IL-7 signaling in human and murine B-cell development. Adult murine B-cell development has an absolute requirement for IL-7 to IL-7R interaction and subsequent downstream signaling involving the γc subunit of the IL-7R and JAK3.94 In contrast, IL-7 is not essential for human B-cell development. X-linked SCID patients with mutations in the γc cytokine-receptor subunit exhibit profound thymic hypoplasia and an absence of NK cells but normal or elevated numbers of B cells.51 SCID patients with mutations in JAK395,96 or the IL-7R97 also have normal numbers of blood B cells. Although B-cell numbers are normal, B-cell function in patients with γc-deficient SCID is not normal and patients are hypogammaglobulinemic, presumably partly as a result of the role of IL-4 in B-cell function and the absence of T-cell interactions in antibody production. These collective results indicate IL-7 is not essential for at least the numerically normal development of human B cells. NK cells are absent in patients with γc-deficient and JAK3-deficient SCID, but are normal in IL-7Rα deficiency.65,97,98 NK cells are also absent in mice deficient in IL-15,99 IL-15Rα,100 or IL-2Rβ (a subunit shared by IL-2R and IL-15R),101 demonstrating the essential role of IL-15, but not IL-7, in NK cell development. Although no null mutations for IL-15 or its receptor have been described in humans, a familial NK cell deficiency has been described in humans in which the response to IL-15 and IL-2 appears to be subnormal.102 The production of both B and NK cells in patients with IL-7Rα deficiency, shows that in humans IL-7 is not required for the earliest stages of lymphoid commitment or growth of CLPs. This point is further supported with the finding that multilymphoid CD34+CD38negCD7+ progenitors in human cord blood do not express IL-7Rα,53 and that early lymphoid progenitor subsets are preserved in the marrow of γc and JAK3-deficient patients.103 In contrast to B cells and NK cells, however, T-cell development is absolutely dependent on IL-7 in both mice and humans.91 In both species, mutations of any portion of the IL-7 signaling pathway, that is, γc, IL-7Rα, or JAK3, completely prevents T-cell development.51 IL-2, in contrast, although an important cytokine in proliferation and function of mature T cells, is not essential for thymopoiesis; mutations in IL-2,104 IL-2Rα, or IL2Rβ105 result in functional T-cell defects, but T cells are not absent.

TRANSCRIPTIONAL REGULATION IN LYMPHOPOIESIS The hierarchical differentiation pathways that lead irreversibly to the diverse array of functionally specialized mature lymphocytes are regulated by groups of genes expressed and repressed in a complex, precisely orchestrated sequence. As with cytokine regulation, our understanding of which transcriptional factors control each stage of differentiation has been developed using a combination of gene expression analyses in isolated progenitors and precursors, and an examination of the functional consequences of genetic mutations in mice and humans. The review

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in this chapter focuses on genes that regulate the earliest commitment decisions in the production of lymphoid progenitors; regulation of later differentiation stages in each lineage is discussed in Chaps. 75 to 77, respectively. The complex interplay between groups of genes involved in hematopoietic differentiation has been likened to a multidimensional network whose “regulatory space” is formed by a dynamic balance between certain transcriptional regulators.106 Expression analysis of multiple genes in defined progenitor populations demonstrates levels of promiscuity at early stages of hematopoiesis, that preclude assignment of any unique gene expression pattern to each stage.106–108 As differentiation proceeds, a more specific “genetic fingerprint” for each lineage develops.

Regulation of Early Lymphoid Commitment

Ikaros  Although no single gene has been identified as a lymphoidspecific master regulator, several transcription factors have been shown to be essential for the early stages of lymphopoiesis. The gene Ikaros, which encodes a family of DNA-binding zinc finger proteins, was identified in murine knockout studies as essential for all fetal lymphopoiesis.109,110 However, in the postnatal setting, the role of Ikaros is more complex and less specific. Adult Ikarosnull mice completely lack B cells, and although T cells are produced, their differentiation is abnormal.111 A murine study has suggested that Ikaros is not required for the initial lymphomyeloid versus myeloerythroid commitment decision, and that not only lymphoid differentiation, but also certain fate choices in the myeloerythroid pathway are affected by Ikaros.112 As the expression of two key lymphoid cytokine receptors, FLT3 and IL-7Rα, is dependent on Ikaros, and as these markers are used to isolate murine LMPP and CLP, respectively, it is still not completely clear at which exact lymphoid progenitor stage Ikaros exerts its effects.112 In addition to lymphoid progenitors, Ikaros isoforms are also expressed in HSCs, and myeloid lineages in mice112–116 and humans.116,117 Although Ikaros may act as a typical transcription factor in some settings, Ikaros also affects gene expression through its role in chromatin formation.118 Pu.1  The transcription factor PU.1 is essential for normal B- and T-lymphocyte development, but its effects are highly dose dependent. At high levels of PU.1, key myeloid regulatory genes are upregulated and macrophage differentiation is induced preferentially over lymphoid differentiation.119 Low-level expression of PU.1, however, is essential for lymphopoiesis.120,121 Mice in which PU.1 is completely absent lack B cells and have abnormal fetal thymopoiesis. However, studies with mice in which PU.1 is deleted specifically in B-lineage cells show that PU.1 is not essential for B-cell differentiation beyond the pre-B stage.122 It is likely that the critical role for PU.1 in murine lymphopoiesis lies in its upregulation of expression of the receptor for IL-7, which as mentioned above, is a key cytokine in both B and T lymphopoiesis in mice.120 E2A  E2A (encoded by TCF3) generates two basic helix-loop-helix proteins, E12 and E47, through differential splicing.123 Murine studies suggest that E2A is necessary for lymphoid priming of multipotent progenitors and that the E2A proteins prime expression of a number of lymphoid-associated genes.124 There is a dose-dependent requirement for E2A expression in the development of LMPP and CLP.124 Both Band T-lineage commitment are severely reduced in the absence of E2A, but Ikaros and PU.1 expression are normal.124–126 E2A affects B lymphopoiesis in part through upregulation of early B-cell factor (EBF)127 and T lymphopoiesis through upregulation of expression and function of the key T-cell specification factor Notch 1.128

Regulation of B-Cell Commitment

The transcription factors Ikaros, PU.1, E2A, EBF, and Pax5 are essential for normal B-cell differentiation. Mice that have functional deletions

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in any one of these genes have severely abnormal B-cell development; however, of these genes, only EBF and Pax5 are B-cell specific within the hematopoietic system. Pax5  Pax5 is expressed specifically in B-lineage–committed progenitors and is required for normal expression of the B-lineage genes CD19 and CD79a.121 Pax5–/– mice are blocked at the pro-B cell stage, but express most early B-cell–related genes.129 Although Pax5 can activate a small subset of B-lineage genes, its main function in B-cell differentiation appears to be the suppression of T-cell and myeloid transcriptional programs at the murine pro-B–cell stage, thus enforcing commitment to the B lineage.121,129,130 Consistent with this role, PU.1, E2A, and EBF function earlier than Pax5 in B lymphopoiesis, and forced expression of Pax5 does not rescue the B-cell defect seen in EBF–/– mice or PU.1–/– mice.121 Ebf  EBF (encoded by EBF1) is a helix-loop-helix zinc finger protein that activates a B-lineage transcriptional program, and induces B lymphoid in preference to myeloid development, in part by antagonizing the expression of genes encoding alternative lineages such as C/EBPα (CCAAT/enhancer binding protein), Id2, and PU.1,131 and, in part, by inducing Pax5 expression.121 Ebf1–/– lymphoid progenitor populations from mice lack the ability to generate B cells but retain the ability to generate T, NK, and myeloid cells.131 Overexpression of EBF in multipotent progenitors promotes B-cell production at the expense of myeloid differentiation.131 EBF and E2A function cooperatively in early B lymphopoiesis124; however, overexpression of EBF can rescue B-cell differentiation in E2A-deficient mice, including activation of Pax5.132 Pax5 overexpression however cannot rescue the B-cell defect in EBF–/– mice,121 demonstrating a critical, Pax5-independent role of EBF in early B-cell fate decisions.

Regulation of T-Cell Commitment

Notch  Upon arrival into the thymus, multipotent progenitors from the marrow become rapidly committed to the T- and NK-cell pathways. The most important environmental cue for T-cell commitment is delivered by the thymic epithelium in the form of the Notch ligands, Delta-like 1 (DLL1) and Delta-like 4 (DLL4).133 Binding of one of these ligands to the Notch 1 receptor expressed on the surface of thymocyte precursors causes activation of intracellular Notch and a series of transcriptional programs turn on to switch lineage fate toward the T lineage at the expense of B-cell development.133 In mice, Notch is absolutely required for T-cell differentiation and proliferation, including β selection.134 Analogous to control of early B cell differentiation by E2A, Notch signaling activates a transcriptional network which includes factors critical for lineage specification (GATA-3, TCF-1), and commitment (BCL11b).135 However, although Notch signaling is necessary for murine thymopoiesis it is not sufficient for activation of the full complement of T-cell genes.136 The ability of hematopoietic progenitors to respond to Notch signaling and commit to T-lineage fate depends on a balance between positive and negative regulators. Combinations of at least four other transcription factors are required to initiate T-cell development: PU.1, Ikaros, Runx family factors, and E2A.124,133 In addition, leukemia-lymphoma–related factor (LRF/Pokemon, encoded by Zbtb7a) must be downregulated to allow Notch signaling to induce T-cell fate decisions.137 Notch signaling also plays important roles at later stages of thymocyte differentiation.133 The effects of Notch signaling have been extensively studied in mice, but the exact stages and processes regulated by Notch appear to differ between mice and humans. For example, using in vitro studies of human T-cell development, it appears that while Notch is essential for early thymocyte proliferation, it is not required for β selection or T-cell receptor αβ differentiation.138,139 As with so much of the information described in this chapter, the most important challenge that lies ahead is

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to translate the detailed mechanistic framework developed from murine studies into careful investigations of human lymphopoiesis. GATA-3  GATA-3 is a key transcriptional factor for T-cell development, and is essential at various stages of differentiation. However, in addition to T cells, GATA-3 is also expressed in uncommitted HSCs, CLPs, and even in nonhematopoietic cells, and its effects are complex and highly dose-dependent.33,135,140 Tcf-1  TCF-1 (encoded by the TCF7 gene) is a transcription factor essential for T-cell development, and is directly activated by Notch signaling.141,142 In ETPs, TCF-1 promotes cell survival as well as activation of T-lineage specific genes, including Gata3 and Bcl11b.141,142 Induction of a T-cell specific transcriptional program by TCF-1 can occur even in the absence of Notch signaling; however, it cannot activate the essential T-lineage gene Ptcra,142 indicating that, as in B-cell development, T-cell specification occurs through both hierarchical and combinatorial transcription factor interactions. Bcl11b  BCL11B was identified as a transcription factor required for the normal generation of αβ T cells during β selection; however, upregulation of Bcl11b first occurs at the earlier CD4negCD8neg (double negative)-2 (DN2) stage, likely through transcriptional activation by TCF-1.135 In DN2 cells, Bcl11b appears to contribute minimally to the T-lineage specification program governed by Notch/E2A/GATA-3/ TCF-1 activity, but rather is required for the suppression of stem/multipotent progenitor-associated genes, which marks the loss of myeloid potential and final commitment to the T-cell lineage.143

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Kohn LA, Hao QL, Sasidharan R, et al: Lymphoid priming in human bone marrow begins before expression of CD10 with upregulation of L-selectin. Nat Immunol 13:963, 2012. 71. Doulatov S, Notta F, Eppert K, et al: Revised map of the human progenitor hierarchy shows the origin of macrophages and dendritic cells in early lymphoid development. Nat Immunol 11:585, 2010. 72. Bjorck P, Kincade PW: CD19+ pro-B cells can give rise to dendritic cells in vitro. J Immunol 161:5795, 1998. 73. Allman D, Sambandam A, Kim S, et al: Thymopoiesis independent of common lymphoid progenitors. Nat Immunol 4:168, 2003. 74. Hao QL, George AA, Zhu J, et al: Human intrathymic lineage commitment is marked by differential CD7 expression: Identification of CD7– lympho-myeloid thymic progenitors. Blood 111:1318, 2008. 75. Weerkamp F, Baert MR, Brugman MH, et al: Human thymus contains multipotent progenitors with T/B lymphoid, myeloid, and erythroid lineage potential. Blood 107: 3131, 2006. 76. Bhandoola A, Sambandam A, Allman D, et al: Early T lineage progenitors: New insights, but old questions remain. J Immunol 171:5653, 2003. 77. Ramond C, Berthault C, Burlen-Defranoux O, et al: Two waves of distinct hematopoietic progenitor cells colonize the fetal thymus. Nat Immunol 15:27, 2014. 78. Rawlings DJ, Quan S, Hao QL, et al: Differentiation of human CD34+CD38– cord blood stem cells into B cell progenitors in vitro. Exp Hematol 25:66, 1997. 79. Berardi AC, Meffre E, Pflumio F, et al: Individual CD34+CD38lowCD19–CD10– progenitor cells from human cord blood generate B lymphocytes and granulocytes. Blood 89:3554, 1997. 80. Miller JS, McCullar V, Punzel M, et al: Single adult human CD34(+)/Lin–/CD38(–) progenitors give rise to natural killer cells, B-lineage cells, dendritic cells, and myeloid cells. Blood 93:96, 1999. 81. Plum J, De Smedt M, Verhasselt B, et al: Human T lymphopoiesis. In vitro and in vivo study models. Ann N Y Acad Sci 917:724, 2000. 82. Awong G, Herer E, Surh CD, et al: Characterization in vitro and engraftment potential in vivo of human progenitor T cells generated from hematopoietic stem cells. Blood 114:972, 2009. 83. Noguchi M, Yi H, Rosenblatt HM, et al: Interleukin-2 receptor gamma chain mutation results in X-linked severe combined immunodeficiency in humans. Cell 73:147, 1993. 84. Kondo M, Takeshita T, Ishii N, et al: Sharing of the interleukin-2 (IL-2) receptor gamma chain between receptors for IL-2 and IL-4. Science 262:1874, 1993. 85. Russell SM, Keegan AD, Harada N, et al: Interleukin-2 receptor gamma chain: A functional component of the interleukin-4 receptor. Science 262:1880, 1993. 86. Noguchi M, Nakamura Y, Russell SM, et al: Interleukin-2 receptor gamma chain: A functional component of the interleukin-7 receptor. Science 262:1877, 1993. 87. Kondo M, Takeshita T, Higuchi M, et al: Functional participation of the IL-2 receptor gamma chain in IL-7 receptor complexes. Science 263:1453, 1994. 88. Kimura Y, Takeshita T, Kondo M, et al: Sharing of the IL-2 receptor gamma chain with the functional IL-9 receptor complex. Int Immunol 7:115, 1995. 89. Giri JG, Ahdieh M, Eisenman J, et al: Utilization of the beta and gamma chains of the IL-2 receptor by the novel cytokine IL-15. EMBO J 13:2822, 1994. 90. Asao H, Okuyama C, Kumaki S, et al: Cutting edge: The common gamma-chain is an indispensable subunit of the IL-21 receptor complex. J Immunol 167:1, 2001. 91. Kang J, Der SD: Cytokine functions in the formative stages of a lymphocyte’s life. Curr Opin Immunol 16:180, 2004. 92. Di Santo JP, Kuhn R, Muller W: Common cytokine receptor gamma chain (gamma c)-dependent cytokines: Understanding in vivo functions by gene targeting. Immunol Rev 148:19, 1995.

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93. Russell SM, Johnston JA, Noguchi M, et al: Interaction of IL-2R beta and gamma c chains with Jak1 and Jak3: Implications for XSCID and XCID. Science 266:1042, 1994. 94. Candeias S, Muegge K, Durum SK: IL-7 receptor and VDJ recombination: Trophic versus mechanistic actions. Immunity 6:501, 1997. 95. Macchi P, Villa A, Giliani S, et al: Mutations of Jak-3 gene in patients with autosomal severe combined immune deficiency (SCID). Nature 377:65, 1995. 96. Russell SM, Tayebi N, Nakajima H, et al: Mutation of Jak3 in a patient with SCID: Essential role of Jak3 in lymphoid development. Science 270:797, 1995. 97. Puel A, Ziegler SF, Buckley RH, Leonard WJ: Defective IL7R expression in T(–) B(+) NK(+) severe combined immunodeficiency. Nat Genet 20:394, 1998. 98. Giliani S, Mori L, de Saint Basile G, et al: Interleukin-7 receptor alpha (IL-7Ralpha) deficiency: Cellular and molecular bases. Analysis of clinical, immunological, and molecular features in 16 novel patients. Immunol Rev 203:110, 2005. 99. Kennedy MK, Glaccum M, Brown SN, et al: Reversible defects in natural killer and memory CD8 T cell lineages in interleukin 15-deficient mice. J Exp Med 191:771, 2000. 100. Lodolce JP, Boone DL, Chai S, et al: IL-15 receptor maintains lymphoid homeostasis by supporting lymphocyte homing and proliferation. Immunity 9:669, 1998. 101. Suzuki H, Kündig TM, Furlonger C, et al: Deregulated T cell activation and autoimmunity in mice lacking interleukin-2 receptor beta. Science 268:1472, 1995. 102. Eidenschenk C, Jouanguy E, Alcaïs A, et al: Familial NK cell deficiency associated with impaired IL-2– and IL-15–dependent survival of lymphocytes. J Immunol 177:8835, 2006. 103. Kohn LA, Seet CS, Scholes J, et al: Human lymphoid development in the absence of common γ-chain receptor signaling. J Immunol 192:5050, 2014. 104. Weinberg K, Parkman R: Severe combined immunodeficiency due to a specific defect in the production of interleukin-2. N Engl J Med 322:1718, 1990. 105. Gilmour KC, Fujii H, Cranston T, et al: Defective expression of the interleukin-2/interleukin-15 receptor beta subunit leads to a natural killer cell-deficient form of severe combined immunodeficiency. Blood 98:877, 2001. 106. Warren LA, Rothenberg EV: Regulatory coding of lymphoid lineage choice by hematopoietic transcription factors. Curr Opin Immunol 15:166, 2003. 107. Akashi K, He X, Chen J, et al: Transcriptional accessibility for genes of multiple tissues and hematopoietic lineages is hierarchically controlled during early hematopoiesis. Blood 101:383, 2003. 108. Miyamoto T, Iwasaki H, Reizis B, et al: Myeloid or lymphoid promiscuity as a critical step in hematopoietic lineage commitment. Dev Cell 3:137, 2002. 109. Georgopoulos K, Bigby M, Wang JH, et al: The Ikaros gene is required for the development of all lymphoid lineages. Cell 79:143, 1994. 110. Wang JH, Nichogiannopoulou A, Wu L, et al: Selective defects in the development of the fetal and adult lymphoid system in mice with an Ikaros null mutation. Immunity 5:537, 1996. 111. Georgopoulos K, Winandy S, Avitahl N: The role of the Ikaros gene in lymphocyte development and homeostasis. Annu Rev Immunol 15:155, 1997. 112. Yoshida T, Ng SY, Zuniga-Pflucker JC, Georgopoulos K: Early hematopoietic lineage restrictions directed by Ikaros. Nat Immunol 7:382, 2006. 113. Nichogiannopoulou A, Trevisan M, Neben S, et al: Defects in hemopoietic stem cell activity in Ikaros mutant mice. J Exp Med 190:1201, 1999. 114. Wu L, Nichogiannopoulou A, Shortman K, Georgopoulos K: Cell-autonomous defects in dendritic cell populations of Ikaros mutant mice point to a developmental relationship with the lymphoid lineage. Immunity 7:483, 1997. 115. Klug CA, Morrison SJ, Masek M, et al: Hematopoietic stem cells and lymphoid progenitors express different Ikaros isoforms, and Ikaros is localized to heterochromatin in immature lymphocytes. Proc Natl Acad Sci U S A 95:657, 1998. 116. Payne KJ, Huang G, Sahakian E, et al: Ikaros isoform x is selectively expressed in myeloid differentiation. J Immunol 170:3091, 2003. 117. Payne KJ, Nicolas JH, Zhu JY, et al: Cutting edge: Predominant expression of a novel Ikaros isoform in normal human hemopoiesis. J Immunol 167:1867, 2001. 118. Cobb BS, Smale ST: Ikaros-family proteins: In search of molecular functions during lymphocyte development. Curr Top Microbiol Immunol 290:29, 2005. 119. DeKoter RP, Walsh JC, Singh H: PU.1 regulates both cytokine-dependent proliferation and differentiation of granulocyte/macrophage progenitors. EMBO J 17:4456, 1998. 120. DeKoter RP, Lee HJ, Singh H: PU.1 regulates expression of the interleukin-7 receptor in lymphoid progenitors. Immunity 16:297, 2002. 121. Medina KL, Ponqubala JM, Reddy KL, et al: Assembling a gene regulatory network for specification of the B cell fate. Dev Cell 7:607, 2004. 122. Polli M, Dakic A, Light A, et al: The development of functional B lymphocytes in conditional PU.1 knock-out mice. Blood 106:2083, 2005. 123. Murre C: Helix-loop-helix proteins and lymphocyte development. Nat Immunol 6:1079, 2005. 124. Dias S, Månsson R, Gurbuxani S, et al: E2A proteins promote development of lymphoid-primed multipotent progenitors. Immunity 29:217, 2008. 125. Bain G, Engel I, Robanus Maandag EC, et al: E2A deficiency leads to abnormalities in alphabeta T-cell development and to rapid development of T-cell lymphomas. Mol Cell Biol 17: 4782, 1997. 126. Bain G, Robanus Maandag EC, te Riele HP, et al: Both E12 and E47 allow commitment to the B cell lineage. Immunity 6:145, 1997. 127. Kee BL, Murre C: Induction of early B cell factor (EBF) and multiple B lineage genes by the basic helix-loop-helix transcription factor E12. J Exp Med 188:699, 1998. 128. Ikawa T, Kawamoto H, Goldrath AW, Murre C: E proteins and Notch signaling cooperate to promote T cell lineage specification and commitment. J Exp Med 203:1329, 2006.

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129. Nutt SL, Heavey B, Rolink AG, Busslinger M: Commitment to the B-lymphoid lineage depends on the transcription factor Pax5. Nature 401:556, 1999. 130. Cobaleda C, Schebesta A, Delogu A, Busslinger M: Pax5: The guardian of B cell identity and function. Nat Immunol 8:463, 2007. 131. Pongubala JM, Northrup DL, Lancki DW, et al: Transcription factor EBF restricts alternative lineage options and promotes B cell fate commitment independently of Pax5. Nat Immunol 9:203, 2008. 132. Seet CS, Brumbaugh RL, Kee BL: Early B cell factor promotes B lymphopoiesis with reduced interleukin 7 responsiveness in the absence of E2A. J Exp Med 199:1689, 2004. 133. Rothenberg EV, Moore JE, Yui MA: Launching the T-cell-lineage developmental programme. Nat Rev Immunol 8:9, 2008. 134. Maillard I, Tu L, Sambandam A, et al: The requirement for Notch signaling at the beta-selection checkpoint in vivo is absolute and independent of the pre-T cell receptor. J Exp Med 203:2239, 2006. 135. Rothenberg EV: Transcriptional drivers of the T-cell lineage program. Curr Opin Immunol 24:132, 2012. 136. Taghon TN, David ES, Zúñiga-Pflücker JC, Rothenberg EV: Delayed, asynchronous, and reversible T-lineage specification induced by Notch/Delta signaling. Genes Dev 19:965, 2005.

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137. Maeda T, Merghoub T, Hobbs RM, et al: Regulation of B versus T lymphoid lineage fate decision by the proto-oncogene LRF. Science 316:860, 2007. 138. Taghon T, Van de Walle I, De Smet G, et al: Notch signaling is required for proliferation but not for differentiation at a well-defined beta-selection checkpoint during human T-cell development. Blood 113:3254, 2009. 139. Van de Walle I, De Smet G, De Smedt M, et al: An early decrease in Notch activation is required for human TCR-alphabeta lineage differentiation at the expense of TCRgammadelta T cells. Blood 113:2988, 2009. 140. Taghon T, Yui MA, Rothenberg EV: Mast cell lineage diversion of T lineage precursors by the essential T cell transcription factor GATA-3. Nat Immunol 8:845, 2007. 141. Germar K, Dose M, Konstantinou T, et al: T-cell factor 1 is a gatekeeper for T-cell specification in response to Notch signaling. Proc Natl Acad Sci U S A 108:20060, 2011. 142. Weber BN, Chi AW, Chavez A, et al: A critical role for TCF-1 in T-lineage specification and differentiation. Nature 476:63, 2011. 143. Li L, Leid M, Rothenberg EV: An early T cell lineage commitment checkpoint dependent on the transcription factor Bcl11b. Science 329:89, 2010.

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CHAPTER 75

FUNCTIONS OF B LYMPHOCYTES AND PLASMA CELLS IN IMMUNOGLOBULIN PRODUCTION

Thomas J. Kipps

SUMMARY Much of our immune defense against invading organisms is predicated upon the tremendous diversity of immunoglobulin molecules. Immunoglobulins are glycoproteins produced by B lymphocytes and plasma cells. These molecules can be considered receptors because the primary function of the immunoglobulin molecule is to bind antigen. A single person can synthesize 10 to 100 million different immunoglobulin molecules, each having a distinct antigen-binding specificity. The great diversity in this so-called humoral immune system allows us to generate antibodies specific for a variety of substances, including synthetic molecules not naturally present in our environment. Despite the diversity in the specificities of antibody

Acronyms and Abbreviations: ADCC, antibody-dependent cellular cytotoxicity; AID, activation-induced deaminase; BACH2, basic leucine zipper transcription factor 2; BCL-6, B-cell chronic lymphocytic leukemia/lymphoma 6; BiP, immunoglobulin-binding protein; Blimp-1, B-lymphocyte-induced maturation protein-1; BLNK, B-cell linker protein; BTK, Bruton tyrosine kinase; C, constant; CDR, complementarity determining region; CRI, cross-reactive idiotype; CSR, class switch recombination; D, diversity; DLBCL, diffuse large B-cell lymphoma; DNA-PK, DNA protein kinase; E2F1, E2F transcription factor 1; EBF1, early B-cell factor 1; ERGIC, ER-Golgi-intermediate compartment; FR, framework region; H, heavy; HMG, high-mobility group protein; Ig, immunoglobulin; IL, interleukin; IRF4, interferon regulatory factor 4; ITAM, immunoreceptor tyrosine-based activation motif; κ, immunoglobulin kappa light chain; Kde, kappa-deleting element; λ, immunoglobulin lambda light chain; L, light; MITF, microphthalmia-associated transcription factor; MYBL1 and 2, v-myb myeloblastosis viral oncogene homologues 1 and 2; NHEJ, nonhomologous DNA end-joining; PAX5, paired box gene 5; PDI, protein disulphide isomerase; PLC, phospholipase C; POU2AF1, Pou domain, class 2, associating factor 1; POU2F2, Pou domain, class 2, factor 2; PRDM1, positive regulatory domain 1-binding factor-1; RAG, recombination-­activating gene; RSS, recombination signal sequence; SCID, severe combined immunodeficiency; SHP-1, Src homology 2 domain-containing protein tyrosine phosphatase-1; SHIP-1, phosphatidylinositol-3,4,5-trisphosphate 5-phosphatase 1; TCFE2A, transcription factor E2a; UNG, uracil-DNA glycosylase; V, variable-region gene; V(D)J, exon created by a rearranged immunoglobulin heavychain variable-region gene, diversity gene segment, and joining gene segment; XBP1, X-box binding protein-1.

Kaushansky_chapter 75_p1159-1174.indd 1159

molecules, the binding of antibody to antigen initiates a limited series of biologically important effector functions, such as complement activation and/or adherence of the immune complex to receptors on leukocytes. The eventual outcome is the clearance and degradation of the foreign substance. This chapter describes the structure of immunoglobulins and outlines the mechanisms by which B cells produce molecules of such tremendous diversity with defined effector functions.

I MMUNOGLOBULIN STRUCTURE AND FUNCTION BASIC STRUCTURE All naturally occurring immunoglobulin molecules are composed of one or several basic units consisting of two identical heavy (H) chains and two identical light (L) chains (Fig. 75–1).1 The four polypeptides are held in a symmetrical, Y-shaped structure by disulfide bonds and noncovalent interactions.2–4 The internal disulfide bonds of the heavy and light chains cause the polypeptides to fold into compact globe-shaped regions called domains, each containing approximately 110 to 120 amino acid residues. Each domain is composed of β-pleated sheets that are stabilized by a conserved disulfide bond (Fig. 75–1). The light chains have two domains; the heavy chains have four or five domains. The aminoterminal domains of the heavy and light chains are designated the variable (V) regions because their primary structure varies markedly among different immunoglobulin molecules. The carboxyterminal domains are referred to as constant (C) regions because their primary structure is the same among immunoglobulins of the same class or subclass. The amino acids in the light- and heavy-chain variable regions interact to form an antigen-binding site. Each fourchain immunoglobulin basic unit has two identical binding sites. The constant-region domains of the heavy and light chains provide stability for the immunoglobulin molecule. The heavy-chain constant regions also mediate the specific effector functions of the different immunoglobulin classes (Table 75–1).

LIGHT CHAINS Immunoglobulin light chains have an approximate Mr of 23,000. They are divided into two types, κ and λ, based upon multiple amino acid sequence differences in the single constant-region domain.5 The λ chains are divided further into subclasses. The proportion of κ-to-λ chains in adult human plasma is approximately 2:1. The immunoglobulin lightchain constant region has no known effector function. Its main purpose may be to allow for proper assembly and release of an intact immunoglobulin molecule. Soon after synthesis, the antibody light-chain constant region associates with the nascent immunoglobulin heavy chain (see Fig. 75–1), releasing the latter from the immunoglobulin-binding protein (BiP). BiP is a heat shock protein that, in the absence of antibody light chain, binds the first constant-region domain of the newly synthesized heavy chain, thereby retaining the heavy-chain polypeptide in the cell’s endoplasmic reticulum.6

HEAVY CHAINS Immunoglobulin heavy chains have an Mr of 50,000 to 70,000, depending upon the number and length of the constant-region domains. The five major isotypes of heavy chains—γ, α, μ, δ, and ε—determine the five corresponding classes of immunoglobulin (Ig): IgG, IgA, IgM, IgD, and IgE. The individual immunoglobulin molecules of each isotype may contain either κ or λ light chains, but not both. Tables  75–1 and 75–2

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Figure 75–1.  Model of an immunoglobulin (Ig) G molecule. The lightchain domains VL and CL and the heavy-chain domains VH, Cγ1(or CH1), Cγ2 (or CH2), and Cγ3 (or CH3) are labeled inside the respective immunoglobulin domain. Dotted red colored lines indicate intrachain and interchain disulfide bonds. The aminoterminus (N) and carboxyl-terminus (C) of each polypeptide are indicated. The hinge region also is indicated. Digestion by pepsin cleaves the molecule at the carboxyl side of the hinge region, which generates Fc and F(ab′)2 fragments, as indicated on the right. The F(ab′)2 fragment is bivalent, as it is held together by the disulfide bridges in the hinge region. On the other hand, digestion of the molecule by papain degrades the Fc portion and generates monovalent Fab fragments, as the cleavage site for papain is on the aminoterminal side of the disulfide bridges of the hinge region. summarize the distinct physical and functional properties of the human immunoglobulin classes.

IgG

Approximately 80 percent of the immunoglobulins in adult plasma are IgG. The IgG molecule is composed of the basic 150-kDa immunoglobulin four-chain structure plus approximately 3 percent carbohydrate. Near the junction of the two arms of the Y-shaped immunoglobulin molecule, the two heavy chains interact to form a flexible “hinge” region (see Fig. 75–1). Exposed between constant-region globular domains, the hinge region is attacked readily by the proteolytic enzyme papain or pepsin. Figure   75–1 shows the cleavage sites. Digestion of IgG with papain yields three fragments. The single Fc piece contains the carboxy-terminal region of both heavy chains. The two identical F(ab) pieces contain the entire light chain and the aminoterminal portion

of the heavy chain. The Fc-regions also contain a binding epitope for the neonatal Fc receptor (FcRn), responsible for the extended half-life, placental transport, and bidirectional transport of IgG to mucosal surfaces.7 As such, IgG molecules effectively penetrate extravascular spaces and readily cross the placental barrier to provide passive immunity to the newborn. IgG is the predominant antibody produced during the secondary immune response to antigen. The average half-life of circulating IgG molecules is approximately 21 days, although the exact value varies among the IgG subclasses (Table 75–3). Within the IgG class are four major subclasses, designated IgG1, IgG2, IgG3, and IgG4.7 The most abundant subclass is IgG1, which constitutes 60 percent of the total IgG in plasma. All IgG subclasses have a similar molecular mass except for IgG3, which has a much longer hinge region than any other IgG subclass. The IgG3 hinge region is approximately four times as long as the IgG1 hinge, containing up to 62 amino acids (including 21 prolines and 11 cysteines), forming a polyproline helix with limited flexibility. Because of this, IgG3 myeloma protein may aggregate spontaneously to produce a hyperviscosity syndrome. Each subclass has a distinct heavy-chain constant region and mediates different effector functions (see Table   75–3). Whereas IgG1 and IgG3 proteins activate complement via the classic pathway, IgG2 molecules fix complement poorly and IgG4 proteins not at all. Antibody responses to soluble protein antigens and membrane proteins primarily induce IgG1, but also lower levels of the other subclasses, mostly IgG3 and IgG4. On the other hand, IgG antibody responses to bacterial capsular polysaccharide antigens typically are restricted to IgG2; IgG2 deficiency can result in the virtual absence of IgG anticarbohydrate antibodies. Viral infections generally induce IgG antibodies of the IgG1 and IgG3 subclasses, with IgG3 antibodies appearing first in the course of the infection. The IgG4 subclass typically is produced in response to allergens. Because IgG4 has relatively low affinity to activating FcγRIII, but relatively high affinity to the inhibiting FcγRII, it may serve to prevent excessive immune responses against sterile antigens, such as bee venom, which do not pose infectious threats. As such, IgG4 has been called a “blocking antibody” in the context of allergy, where it may compete with IgE for allergen binding. For antigens found on pathogens, the bound IgG can: (1) tag the pathogen for ingestion and destruction by phagocytes, a process called opsonization; (2) activate complement; and/or (3) direct antibodydependent cell-mediated cytotoxicity (ADCC). Either aggregated IgG

TABLE 75–1.  Physical Properties of Human Immunoglobulins IgG

IgA

IgM

IgD

IgE

Heavy-chain class

γ

α

μ

δ

ε

Heavy chain subclass

γ1, γ2, γ3, γ4

α1, α2

—

—

—

No. of heavy-chain domains

4

4

5

4

5

Secretory form

Monomer

Monomer, dimer

Pentamer

Monomer

Monomer

Molecular mass (Da)

150,000

160,000 (monomer) 400,000 (secretory)

900,000

184,000

188,000

Antigen-binding valency

2

2 (monomer) 4 (secretory)

10

2

2

Serum concentration (mg/mL)

8–16

1.4–4.0

0.5–2.0

0–0.4

17–450 ng/mL

Percent of total immunoglobulin

80

13

6

1

0.002

Electrophoretic mobility

γ

Fast γ to β

Slow γ

Fast γ

Fast γ

Percent carbohydrate

3

8

12

13

12

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TABLE 75–2.  Biologic Properties of Human Immunoglobulins

TABLE 75–3.  Characteristics of Major IgG Subclasses

IgG

IgA

IgM

IgD

IgE

IgG1

IgG2

IgG3

IgG4

Heavy chain subclass

Percent of body pool in intravascular space

45

42

76

75

51

γ1

γ2

γ3

γ4

Molecular mass (kDa)

146

146

170

146

Percent of intravascular pool catabolized per day

6.7

25

18

37

89

Serum concentration (mg/mL)

7

4

0.5

0.6

Normal synthetic rate (mg/kg per day)

33

24

6.7

0.4

0.02

Relative abundance (%)

60

32

4

Serum half-life (days)

Serum half-life (days)

21

5.8

10

2.8

2.3

Placental Transfer

Placental transfer

Yes

No

No

No

No

Cytophilic for mast cells and basophils

No

No

No

No

Yes

Complement fixation (C1q binding)

Binding to macrophages and other phagocytes

Yes

No

No

No

Yes

Reactivity with ­staphylococcal protein A

Yes

No

No

No

No

Antibody-dependent­ c­ ell-mediated cytotoxicity

Yes

No

No

No

No

4 21

21

21

7-21

++++

++

++/++++ *

+++

++

+

+++

–

a

FcR Binding FcγRI (CD64)

+++

–

++++

++

FcγRIIaH131 (CD32)†

+++

++

++++

++

FcγRIIaR131 (CD32)†

+++

+

++++

++

+

–

++

+

FcγRIIb/c (CD32) FcγRIIIaF158 (CD16)

++

–

++++

–

FcγRIIIaV158 (CD16)‡

+++

+

++++

–

‡

Complement fixation   Classic pathway

Yes

No

Yes

No

No

FcγRIIIb (CD16)

+++

–

++++

++

  Alternative pathway

No

Yes

No

No

No

FcγRn (at pH 30 IU/dL).31 In a large, multicenter European study of 150 type 1 VWD families, about one-third of cases historically diagnosed to have type 1 VWD were found to have abnormal multimers, and of these nearly all (95 percent) had a putative VWF gene mutation and significantly lower VWF:Ag, VWF ristocetin cofactor activity (VWF:RCo), assay of FVIII activity (FVIII:C), and VWF collagen-binding assay (VWF:CB) levels. Conversely, index cases with normal multimers had higher laboratory VWF values and fewer identifiable VWF mutations (55 percent), suggesting that the pathogenic mechanism(s) underlying this cohort of “true” type 1 VWD patients is more genetically complex.162 Given the complex biosynthesis and processing of VWF, defects at a number of other loci could also be expected to result in quantitative VWF abnormalities (reviewed in Ref. 176). This concept is supported by families with type 1 VWD in which bleeding histories and low ristocetin cofactor activities do not always cosegregate with genetic markers at the VWF locus,31,177 while one or more genetic factors outside the VWF locus may be associated with the variation in bleeding severity observed within VWD pedigrees.178,179 It is interesting to note that a spontaneous mouse model of type 1 VWD exhibits up to 20-fold reductions in plasma VWF as a result of an unusual mutation in a glycosyltransferase gene, leading to aberrant posttranslational processing of VWF and accelerated clearance from plasma.180 Similar mechanisms affecting VWF survival, perhaps combined with altered proteolysis,181–183 may

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explain the observed modifying effect of the ABO blood group glycosyltransferases on plasma VWF survival.184 Additional genetic factors have been implicated to influence VWF via altered survival, including the clearance receptors CLEC4M and LRP1 (CD91) (reviewed in Ref. 185). The biologic consequences of VWF modifiers identified in normal populations are unclear, and studies are needed to determine their significance in VWD.

Type 3 von Willebrand Disease

Patients with type 3 VWD account for 1 to 5 percent of clinically significant VWD, have very low or undetectable levels of plasma and platelet VWF:Ag and VWF:RCo, and generally present early in life with severe bleeding.186 FVIII coagulant activity is markedly reduced but usually detectable at levels of 3 to 10 percent of normal. Type 3 VWD has generally been considered an autosomal recessive disorder, but in a recent Canadian study of 100 individuals in 34 families, 48 percent of  “carriers” had a diagnosis of type 1 VWD,161 suggesting the dominant type 1 VWD pattern of inheritance is common in type 3 VWD families. Mutations associated with type 3 VWD have been reported throughout the VWF gene (http://www.vwf.group.shef.ac.uk/). Gross VWF gene deletion detectable by Southern blot26,187–190 or multiple ligation-probe amplification161,191 is the molecular mechanism for type 3 VWD in only a small subset of families. However, large deletions may confer an increased risk for the development of alloantibodies against VWF.26,189 A similar correlation between gene deletion and risk for alloantibody formation has been observed in hemophilia (Chap. 123). Comparative analysis of VWF genomic DNA and platelet VWF mRNA has identified nondeletion defects resulting in complete loss of VWF mRNA expression as a molecular mechanism in some patients with type 3 VWD.192,193 A number of nonsense and frameshift mutations that would be predicted to result in loss of VWF protein expression or in expression of a markedly truncated or disrupted protein have been identified in some type 3 VWD families.168,194–196 A frameshift mutation in exon 18 appears to be a particularly common cause of type 3 VWD in the Swedish population and has been shown to be the defect responsible for VWD in the original Åland Island pedigree.197,198 This mutation results in a stable mRNA encoding a truncated protein that is rapidly degraded in the cell.199 This mutation also appears to be common among type 3 VWD patients in Germany,200 but not in the United States.201

Type 2A von Willebrand Disease

Type 2A is the most common qualitative variant of VWD and is generally associated with autosomal dominant inheritance and selective loss of the large and intermediate VWF multimers from plasma (see Fig. 126–4). A 176-kDa proteolytic fragment present in normal individuals is markedly increased in quantity in many type 2A VWD patients. This fragment is consistent with proteolytic cleavage of the peptide bond between Tyr1605 and Met1606.98,202 Based on this observation, initial DNA sequence analysis in patients centered on VWF exon 28, in the region encoding this segment of the VWF protein, leading to the identification of the first point mutations responsible for VWD.203 Since that time, a large number of mutations have been identified, accounting for the majority of type 2A VWD patients.194 Many of these mutations are clustered within a 134-amino-acid segment of the VWF A2 domain (between Gly1505 and Glu1638; see Fig. 126–3), and the most common, Arg1597Trp, appears to account for about one-third of type 2A VWD patients.194,195,204 Type 2A VWD mutations have been grouped by two distinct molecular mechanisms. In the first subset, classified as group 1, the type 2A VWD mutation has been commonly considered a defect in intracellular transport, with retention of mutant VWF in the ER. In addition to retention or degradation of mutant VWF in the ER, type 2A mutations

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can also disrupt intracellular processing and secretion via defective multimerization and/or loss of regulated storage.205 In the second subset, or group 2, mutant VWF is normally processed and secreted in vitro, and thus loss of multimers in vivo is presumed to occur based on increased susceptibility to proteolysis in plasma98,206–209 at the Tyr1605-Met1606 site cleaved by ADAMTS13.101,210 The susceptibility of type 2A VWD mutations to proteolysis by ADAMTS13 in vitro supports accelerated proteolysis as a mechanism for the loss of high-molecular-weight VWF multimers in these patients.204 The multimer structure of platelet VWF correlates well with the underlying type 2A mechanisms. Group 1 patients show loss of large VWF multimers within platelets as a result of defective synthesis, while group 2 patients have normal VWF multimers within the protected environment of the α granule.206 These observations confirm the earlier subclassification of type 2A VWD based on platelet multimers.156 Subclassification into group 1 or 2 might be expected to predict response to DDAVP therapy, although this remains to be demonstrated. In addition to the major classes of type 2A VWD described above, a number of rare variants historically classified as types IIC to IIH, type IB, and “platelet discordant” are included in the more general type 2A category. Most of these rare variants were distinguished on the basis of subtle differences in the multimer pattern (see Fig. 126–4; multimer changes relative to the location of type 2 mutations is reviewed in Ref. 211). The IIC variant is usually inherited as an autosomal recessive trait and is associated with loss of large multimers and a prominent dimer band. Several mutations have been identified in the VWFpp of these patients,212–214 presumably interfering with multimer assembly and/or trafficking to storage granules. A mutation at the C terminus of VWF, interfering with dimer formation, was described in a patient with the IID variant.215 Most of the other reported variants of type 2A VWD are quite rare, often limited to single case reports.

Type 2B von Willebrand Disease

Type 2B VWD is usually inherited as an autosomal dominant disorder and is characterized by thrombocytopenia and loss of large VWF multimers. The plasma VWF in type 2B VWD binds to normal platelets in the presence of lower concentrations of ristocetin than does normal VWF and can aggregate platelets spontaneously. Accelerated clearance of the resulting complexes between platelets and the large, most adhesive forms of VWF accounts for the thrombocytopenia and the characteristic multimer pattern (see Fig. 126–4). The peculiar functional abnormality characteristic of type 2B VWD suggested a molecular defect within the GPIb binding domain of VWF. For this reason, initial DNA sequence analysis focused on the corresponding portion of VWF exon 28.216,217 Type 2B mutations are located within the VWF A1 domain at one surface of the described crystallographic structure.124,129 The four most common mutations are clustered within a 36-amino-acid stretch between Arg1306 and Arg1341 (see Fig. 126–3); together, these account for more than 80 percent of type 2B VWD patients.195 Functional analysis of mutant recombinant VWF218–222 confirms that these single-amino-acid substitutions are sufficient to account for increased GPIb binding and the resulting characteristic type 2B VWD phenotype. Structural studies of type 2B VWD mutations show that these residues interact with the leucine rich repeats of GPIb thought to be critical to the VWF A1–GPIb interactions under shear.223 Type 2B mutations have now been modeled extensively in mice, all of which exhibited accelerated VWF clearance, as expected.224 Type 2B VWD mice also had short-lived platelets, with evidence of macrophage-mediated platelet clearance.225 In these models, platelets were observed to be coated by type 2B VWF,225 a phenomenon that may contribute to a previously unsuspected acquired platelet function defect.226 Interestingly, mice with the same type 2B mutations exhibit variable loss

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of large multimers224 and varying degrees of thrombocytopenia,227 similar to the variation observed in human pedigrees. Individual type 2B patients can also exhibit varying multimer structure and platelet counts over time. For example, two siblings with the Arg1306Trp mutation and abnormal multimers intermittently regained normal VWF multimer distribution during periods of thrombocytopenia.228 Families have been described that exhibit enhanced VWF binding to GPIb but a normal distribution of VWF multimers. These variants, previously referred to as type I New York, type I Malmö, and type I Sydney, are now all designated as type 2B VWD. Type I New York and type I Malmö are caused by the same VWF mutation, Pro1266Leu. This mutation is located within the cluster of type 2B mutations in the VWF A1 domain and results in a similar increase in platelet GPIb binding.229

Type 2N von Willebrand Disease

As described in Chap. 123, hemophilia A results from defects in the FVIII gene and is inherited in an X-linked recessive manner. Distinct from hemophilia A, families have been reported in which the inheritance of low FVIII appeared to be autosomal, based on the occurrence of affected females or direct transmission from an affected father.230,231 Several cases of an apparent autosomal recessive decrease in FVIII were shown to be caused by decreased VWF binding of FVIII,232–234 now referred to as VWD type 2N, after the Normandy province of origin of the first patient. DNA sequence analysis has identified more than 37 distinct mutations235 associated with this disorder, most located at the VWF N terminus (see Fig. 126–3) (curated in the Scientific and Standardisation Committee of the International Society on Thrombosis and Haemostasis VWF Database, http://www.vwf.group.shef.ac.uk/). One of these mutations, Arg854Gln, appears to be particularly common, may contribute to variability in the severity of type 1 VWD in some cases,236 and may also cause a VWF secretion defect.237 Rare cases of misdiagnosis of type 2N have led to treatment with recombinant FVIII for presumed hemophilia A, with poor responses and adverse clinical outcomes.238

Type 2M von Willebrand Disease

This category was classically reserved for rare VWD variants in which a defect in VWF platelet-dependent function leads to significant bleeding but VWF multimer structure is not affected (although some have subtle multimer abnormalities). Most contemporary type 2M variants are indeed associated with absent ristocetin cofactor activity but normal platelet binding with other agonists. A total of 28 type 2M VWD mutations have been described,235 including a number of other families with normal VWF multimers and disproportionately decreased ristocetin cofactor activity,239,240 families with a combination of defects in VWF:CB and VWF–GPIb interactions of varying severities,241,242 and mutations with isolated defects in VWF:CB with normal VWF:RCo activity.235 Several families have also been described with a VWD variant (VWD Vicenza) characterized by larger-than-normal VWF multimers and classified as either type 1 or type 2M VWD.243 Genetic linkage analysis indicates that the Vicenza defect lies within the VWF gene,244 and mutations within the VWF gene have been reported to be associated with VWD Vicenza.245 The underlying molecular mechanism responsible for the VWD Vicenza phenotype remains controversial,246 although recent kinetic modeling suggests that altered VWF survival alone could account for the VWF perturbations observed in this disorder.247

CLINICAL FEATURES INHERITANCE Type 1 VWD is generally transmitted as an autosomal dominant disorder, and accounts for approximately 70 percent of clinically significant VWD. However, disease expressivity is variable, and penetrance is

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incomplete.168 Laboratory values and clinical symptoms can vary considerably, even within the same individual, and establishing a definite diagnosis of VWD is often difficult. In two large families with type 1 VWD, only 65 percent of individuals with both an affected parent and an affected descendent had significant clinical symptoms.249 For comparison, 23 percent of the unrelated spouses of the patients, who presumably did not have a bleeding disorder, were judged to have a positive bleeding history. A number of factors have long been known to modify VWF levels, including ABO blood group, secretor blood group, estrogens, thyroid hormones, age, and stress.250–252 ABO blood group is the best characterized of these factors. Genome-wide linkage has repeatedly confirmed strong linkage between the ABO locus and VWF levels (reviewed in Ref. 253). Mean VWF:Ag levels are approximately 75 percent for type O individuals and 123 percent for type AB individuals when compared to a pool of normal donor plasmas. Thus, it may be difficult to differentiate between a low-normal VWF value and mild type 1 VWD in blood group O individuals. In recent years, additional modifiers of VWF have been identified in large genetic association studies,254,255 including genes associated with VWF intracellular trafficking (STXBP5256,257) and VWF clearance (CLEC4M258). Additionally, a genome-wide association study identified a novel genetic locus on chromosome 2 contributing to variation in plasma VWF.259 The variable expressivity and incomplete penetrance of type 1 VWD and overlap in VWF levels between mild type 1 VWD and normal populations has complicated the determination of accurate incidence figures for VWD, with estimates ranging from as high as 1 percent260,261 to as low as 2 to 10 per 100,000 population.262 In general, the type 2 VWD variants, which comprise 20 to 30 percent of all VWD diagnoses,263 are more uniformly penetrant. Type 2A and type 2B VWD account for the vast majority of patients with qualitative VWF abnormalities. Types 2A, 2B, and 2M are generally autosomal dominant in inheritance, although Type 2N and other rare cases of apparent recessive inheritance have been reported. Estimates of prevalence for severe (type 3) VWD range from 0.5 to 5.3 per 1,000,000 population.264–266 Although this variant is frequently defined as autosomal recessive in inheritance, this is not a consistent finding. As described above, one or both parents of a severe VWD patient can be clinically asymptomatic and have entirely normal laboratory test results, although in many families one or both parents appear to be affected with classic type 1 VWD. Thus, in some families, severe VWD may represent the homozygous form of type 1 VWD. In this model, the apparent recessive inheritance in a subset of families could simply be the result of the incomplete penetrance of type 1 VWD. Alternatively, there may be a fundamental difference in the molecular mechanisms responsible for type 1 and type 3 VWD.168 Compound heterozygosity (the presence of more than one VWF gene mutation) can occur, and the clinical presentation in such cases can depend on the interaction between the different mutant VWF proteins. Compound heterozygosity can impact response to therapy because of a complex VWD phenotype and has implications for genetic counseling. If compound heterozygosity is deduced from the family history and/or laboratory studies or discovered during genetic testing, the most recent update to the VWD nomenclature represents both types separated by a slash (/), such as VWD type 2B/2N.151

CLINICAL SYMPTOMS Mucocutaneous bleeding is the most common symptom in patients with type 1 VWD.249 It is important to note that more than 20 percent of normal individuals may give a positive bleeding history.267 Bleeding assessment scores have evolved over many years,268 leading the International Society on Thrombosis and Haemostasis to propose a unified

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Bleeding Assessment Test269 for research purposes. Although high bleeding scores are suggestive of a bleeding diathesis and can predict future bleeding,270 no bleeding questionnaire as yet is clearly diagnostic for VWD. These observations, together with the limited sensitivity and specificity of the currently available laboratory tests (see below), makes the diagnosis of mild VWD quite difficult and probably contributes to the wide range of prevalence figures for type 1 VWD currently in the literature. A National Heart Lung and Blood Institute Expert Panel has proposed clinical guidelines for evaluating patients to determine whether laboratory testing for VWD or other bleeding disorders is warranted.150 Epistaxis occurs in approximately 60 percent of type 1 VWD patients, 40 percent have easy bruising and hematomas, 35 percent have menorrhagia, and 35 percent have gingival bleeding. Gastrointestinal bleeding occurs in approximately 10 percent of patients.150 An apparent association between hereditary hemorrhagic telangiectasia (HHT) and VWD has been reported in several families. The causative genes in HHT were identified and are located on chromosomes 9q33–34, and 12q13271 (Chap. 122), distinct from the VWF gene on chromosome 12p13. However, because inheriting VWD is likely to increase the severity of bleeding from HHT, the diagnosis is more likely to be made in patients inheriting both defects.272 Mucocutaneous bleeding is common after trauma, with approximately 50 percent of patients reporting bleeding after dental extraction, approximately 35 percent after trauma or wounds, 25 percent postpartum, and 20 percent postoperatively. Hemarthroses in patients with moderate disease are extremely rare and are generally only encountered after major trauma. The bleeding symptoms can be quite variable among patients within the same family and even in the same patient over time. An individual may experience postpartum bleeding with one pregnancy but not with others, and clinical symptoms in mildly to moderately affected type 1 individuals often ameliorate by the second or third decade of life. Aside from an infrequent type 3 patient, death from bleeding rarely occurs in VWD. Thrombocytopenia is a common feature of type 2B VWD and is not seen in any other form of VWD. Most patients only experience thrombocytopenia at times of increased VWF production or secretion, such as during physical effort, in pregnancy, in newborn infants, postoperatively, or if an infection develops. The platelet count rarely drops to levels thought to contribute to clinical bleeding.273,274 Infants with type 2B VWD may present with neonatal thrombocytopenia, which could be confused with neonatal alloimmune thrombocytopenia, neonatal sepsis, or congenital thrombocytopenia. Patients who are homozygous or compound heterozygous for type 2N VWD generally have normal levels of VWF:Ag and VWF:RCo and normal VWF platelet adhesive function. However, FVIII levels are moderately decreased, resulting in a mild to moderate hemophilia-like phenotype.146 In contrast to patients with classic hemophilia A (FVIII deficiency), these patients do not respond to infusion of purified FVIII and should be treated with VWF-containing concentrates.275 Heterozygotes for this disorder may have mildly decreased FVIII levels but are generally asymptomatic. Although type 2N VWD appears to be considerably less common than classic hemophilia A, it should be considered in the differential diagnosis of FVIII deficiency, particularly if any features suggest an autosomal pattern of inheritance. Although the FVIII level rarely drops below 5 percent, type 2N VWD mutation can be associated with FVIII levels as low as 1 percent, when co-inherited with a type 3 VWD allele.276 The latter observation further suggests that a diagnosis of type 2N VWD should also be considered in patients with marked reductions of FVIII. Patients with type 3 VWD can suffer from severe clinical bleeding and experience hemarthroses and muscle hematomas, as in severe

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hemophilia A (Chap. 123). After infusion of VWF-containing plasma fractions, some of these patients develop anti-VWF antibodies that neutralize VWF (reviewed in Ref. 277). Other heritable coagulopathies can coexist with VWF deficiency. An evaluation for other factor deficiencies or platelet disorders should be considered in patients that have a suggestive family history, a bleeding phenotype out or proportion or inconsistent with an expected VWD pattern, or a poor response to therapy. In VWD patients with combination coagulopathies, treatment of both disorders may be necessary to achieve a good clinical result.278

LABORATORY FEATURES In the initial laboratory evaluation of patients suspected by history of having VWD, the following tests are routinely performed: assay of FVIII:C, VWF:Ag, and VWF:RCo. Other tests that are commonly used include RIPA, VWF:CB, and VWF multimer analysis. Routine coagulation studies, such as prothrombin time (PT) or activated partial-thromboplastin time (aPTT), are generally not useful in the evaluation of VWD. However, the aPTT can be prolonged in subjects with VWF deficiency,279 or in patients with homozygous type 2N VWD, because of the reduction in FVIII level. The wide range of normal and the considerable overlap with the levels observed in type 1 VWD make borderline levels difficult to interpret. A variety of concurrent diseases and drugs may modify the results of individual tests. Many conditions, such as recent exercise, age, pregnancy, time of the menstrual cycle, estrogen therapy, hypo- or hyperthyroidism, diabetes, uremia, liver disease, infection, myeloproliferative neoplasms, or malignancy can affect the FVIII activity, VWF:Ag, and ristocetin cofactor activity levels. These values can be regarded as acute-phase reactants, and even minor illnesses can increase the levels in a VWD patient to normal. Appropriate processing of laboratory specimens is also critical as VWF parameters can be artifactually skewed (either high or low) by phlebotomy conditions or specimen handling (reviewed in Ref. 150). Even controlling for many of these factors, the coefficients of variation of repeated VWF:Ag and ristocetin cofactor assays in a single person are quite large,280 and can be influenced by numerous factors including diurnal variation.281 For this reason, repeated measurements are usually necessary, and the diagnosis of VWD or its exclusion should generally not be based on a single set of laboratory values. The laboratory diagnosis of type 1 VWD can be confounded by the wide range of VWF levels in “normals” and borderline laboratory results. An alternative strategy is to classify some patients for whom the diagnosis of VWD is ambiguous as “low VWF,” recognizing that these patients may have an increased risk of bleeding without labeling them as type 1 VWD.282,283 In response to this need to distinguish those patients with VWD from nonbleeding individuals with moderately low levels of VWF (30 to 50 IU/dL), a threshold of less than 30 IU/dL has been recommended.150 In clinical practice there remains wide variation in the assignment of normal VWF ranges and in the interpretation of laboratory results to make a VWD diagnoses.284–286

FACTOR VIII FVIII levels in VWD patients are generally coordinately decreased along with plasma VWF, although skewing of FVIII-to-VWF ratios can be observed.287 Levels in type 3 VWD generally range from 3 to 10 percent. In contrast, the levels in type 1 and the type 2 VWD variants (other than 2N) are variable and usually only mildly or moderately decreased. The FVIII level in type 2N VWD is more severely decreased, but rarely to less than 5 percent.

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VON WILLEBRAND FACTOR ANTIGEN Plasma VWF:Ag is usually quantitated by electroimmunoassay or an enzyme-linked immunosorbent assay (ELISA) technique. In type 1 VWD, the VWF:Ag assay usually parallels VWF:RCo, but it has lower specificity and sensitivity than the VWF:RCo assay. In patients with type 2 VWD, the VWF:Ag is variably decreased but can be normal (see Table  126–2).

RISTOCETIN COFACTOR ACTIVITY The standard measure of VWF activity, the VWF:RCo quantitates the ability of plasma VWF to agglutinate platelets via platelet membrane GPIbα in the presence of ristocetin.288 In the most common method, normal platelets washed free of plasma VWF are used either as fresh platelets or after formaldehyde fixation. This assay has long been reported to be the most sensitive and specific single test for the detection of VWD.289 Numerous alternative methods have been proposed as adjuncts or replacements of the standard platelet-based ristocetin cofactor activity assay. However, none as yet can serve as a surrogate for VWF:RCo (reviewed in Ref. 290). Ristocetin cofactor activity is generally decreased coordinately with VWF:Ag and FVIII in type 1 VWD patients. In type 2 VWD variants, ristocetin cofactor activity can be disproportionately decreased, as is usually the case in type 2A variants (and sometimes type 2B), because of the greater dependence of the ristocetin-mediated platelet– VWF interaction on the presence of larger VWF multimers, and in type 2M, because of decreased VWF-platelet interactions (see Table  126–2). Thus, the VWF:RCo-to-VWF:Ag ratio has been proposed as a means to distinguish between type 1 and type 2 VWD, with a ratio of VWF:RCo-to-VWF:Ag of less than 0.7 being indicative of a qualitative (type 2) VWF defect.151 However, in patients with very low VWF:Ag levels this ratio may not be reliable because of the limits of sensitivity of most VWF:RCo assays. The VWF:RCo assay is uninformative in the presence of the VWF Asp1472His variant, which interferes with the VWF– ristocetin interaction in vitro.155

RISTOCETIN-INDUCED PLATELET AGGLUTINATION Similar to the ristocetin cofactor assay above, the RIPA assay also measures platelet agglutination caused by ristocetin-mediated VWF binding to platelet membrane GPIbα. In the case of RIPA, ristocetin is added directly to patient platelet-rich plasma and platelet aggregation is measured. Hyperresponsiveness to RIPA results either from a type 2B VWD mutation or an intrinsic defect in the platelet (platelet-type or pseudo-VWD). In these disorders, patient platelet-rich plasma agglutinates spontaneously or at low ristocetin concentrations of only 0.2 to 0.7 mg/mL. At these concentrations, normal platelet-rich plasma does not agglutinate. Type 2B and platelet-type VWD can be distinguished by RIPA experiments performed with separated patient platelets or plasma mixed with the corresponding component from a normal individual or paraformaldehyde-fixed platelets. The RIPA is generally reduced in most other subtypes of VWD (see Table  126–2).

MULTIMER ANALYSIS Analysis of plasma VWF multimers is critical for the proper diagnosis and subclassification of VWD (see Fig. 126–4). This is generally accomplished by agarose gel electrophoresis of plasma VWF to separate VWF multimers on the basis of molecular size, with the largest multimers

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migrating more slowly than the intermediate or smaller multimers. The multimers may be visualized by autoradiography after incubation with 125I-monospecific antihuman VWF antibody or, more commonly, by nonradioactive immunologic techniques. The normal multimeric distribution is an orderly ladder of major protein bands of increasing molecular weight, going from the smallest to the largest VWF multimers (see Fig. 126–4). Each normal multimer has a fine structure consisting of one major component and two to four satellite bands.291 Type 2B and most of the type 2A variants were initially distinguished from each other on the basis of subtle variations in the satellite band pattern. In a large European multicenter type 1 VWD study, careful analysis of VWF multimers in subjects historically diagnosed as type 1 VWD, including patients diagnosed at experienced centers, found one-third of “type 1” VWD patients had subtly abnormal multimers.292 Although this previously would have required reclassification of these patients as type 2 VWD, the most recent update on the classification of VWD by the International Society on Thrombosis and Haemostasis Subcommittee on von Willebrand Factor expanded the category of type 1 VWD to permit subtle VWF multimer abnormalities.151 The authors of the European type 1 VWD study note that having samples from the index case, affected family members, and unaffected family members on one gel made qualitative defects more readily detectable, and that intermediate-resolution multimer gels were superior to low-resolution multimer gels in detecting abnormalities in this population. Use of VWF tests sensitive to VWF multimer structure have been proposed by some experts as proxies for VWF multimer testing, such the VWF:RCo-to-VWF:Ag ratio (VWF RCo:Ag ratio) or VWF:CB assay.293 In studies comparing these approaches as surrogates for VWF multimer assays, the (VWF RCo:Ag ratio) ratio was found to be less sensitive than multimer gel techniques in identifying qualitative VWF defects,292 while VWF:CB an detect some type 2M VWD patients who have normal VWF multimers.294,295 These observations support a continued important role for VWF multimer analysis in the laboratory evaluation of VWD.

ADDITIONAL LABORATORY TESTS As a result of the variable sensitivity and specificity of laboratory testing for VWD, additional diagnostic studies may be useful in the classification of VWD patients. The VWF:CB measures VWF binding to collagen (type I, type III, type VI, or mixed) by ELISA. As above, assays based upon VWF:CB can complement the VWF:RCo in detecting type 2 VWD variants,296–299 and an abnormal VWF:CB-to-VWF:Ag ratio (VWF CB:Ag ratio) is suggestive of a qualitative VWF defect.151 Abnormalities in VWF:CB can reflect loss of high-molecular-weight multimers and/ or the discrete loss of collagen binding caused by a type 2M mutation. Use of VWF:CB assays is expanding in clinical practice, with select sites including this test routinely in initial VWD diagnostic laboratory testing. Another group of tests are included in the term VWF “activity” (VWF:Act). These tests seek to assess VWF-GPIb binding capacity independent of ristocetin, usually by using antibodies to the VWF A1 domain in ELISAs. These tests are easily confused with the VWF:RCo, which has also been referred to as “VWF activity.” None of these tests measures activity; rather both VWF:RCo and VWF:Act are sensitive to VWF conformation. The VWF:Act does not always provide the same results as VWF:RCo, particularly with regards to type 2M VWD, and is not considered a substitute for the VWF:RCo (reviewed in Ref. 290). When type 2N VWD is suspected, VWF:FVIII binding capacity can be measured.234 Specific assays of FVIII binding to VWF (VWF:FVIIIB) have been developed and can be used to confirm the diagnosis of type 2N VWD.300,301 Type 2N carriers do not always exhibit a decrease in VWF:FVIIIB, but a decreased VWF:FVIIIB-to-VWF:Ag

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ratio may correlate with heterozygosity for a type 2N VWF mutation.302 Although this assay is widely used in European hemostasis laboratories, its availability in the United States is currently limited to a few specialized reference laboratories. An assay measuring the VWFpp can be used to calculate the VWFpp:antigen ratio (VWFpp:Ag ratio) to detect a subset of VWD patients with decreased VWF survival. Good correlation has been reported between subjects with significantly shortened VWF half-life after DDAVP challenge and an increased VWFpp:Ag ratio.287,303,304 This assay is currently available in a few reference laboratories. A normal platelet VWF:Ag in the setting of decreased plasma VWF laboratory parameters also suggests an accelerated clearance phenotype such as that seen in VWD type Vicenza,305 but platelet VWF:Ag testing also is not widely available in clinical laboratories. A number of other assays for VWF activity have been developed. The PFA-100 system, which measures platelet binding under high shear,306,307 is controversial in the diagnosis or monitoring of VWD. Although the PFA-100 is usually abnormal in type 2 VWD and in more severe type 1 and type 3 VWD cases, milder type 1 VWD and some type 2 VWD patients can have normal results.150 Other VWF assays can measure binding of an antibody to the GPIb binding site on VWF as a proposed screening test for VWD.308–311 Additional assays can measure platelet agglutination induced by botrocetin (which is no longer commercially available) and other snake venom proteins.312 In the National Heart Lung and Blood Institute Expert Panel guidelines (http://www. nhlbi.nih.gov/files/docs/guidelines/vwd.pdf), none of these tests are recommended for screening for VWD.150 With advances in understanding the molecular genetics of VWD, it is now possible to precisely diagnose and subclassify many variants of VWD on the basis of DNA mutations (reviewed in Ref. 313). DNA testing, particularly for type 2 VWD mutations which cluster within specific regions of the VWF gene (see Fig. 126–3), can be used to confirm the diagnosis and is available in specialized reference laboratories. The analysis of type 3 and type 1 VWD is more complex, as the currently known mutations are scattered throughout the gene235 and account only for a subset of patients. The bleeding time is mentioned here for historical purposes only, as it was used as a screening test for VWD and other abnormalities of platelet function. Bleeding time varied considerably with the experience of the operator and a variety of other factors, did not prolong with FVIII deficiency, and correlated poorty with bleeding risk. Thus, the bleeding time is no longer recommended in the evaluation of VWD.150

PRENATAL TESTING Given the mild clinical phenotype of most patients with the common variants of VWD, prenatal diagnosis for the purpose of deciding on terminating a pregnancy is rarely performed. However, type 3 VWD patients often have a profound bleeding disorder, similar to or more severe than classic hemophilia, and some families may request prenatal diagnosis. In those cases of VWD in which the precise mutation is known, DNA diagnosis can be performed rapidly and accurately by polymerase chain reaction (PCR) from amniotic fluid or chorionic villus biopsies (reviewed in Ref. 313) and would be expected to be compatible with new noninvasive prenatal testing methods.314 In those cases where the mutation is unknown, diagnosis can still be attempted by genetic linkage analysis using the large panel of known polymorphisms within the VWF gene.313 Although all cases of VWD analyzed to date appear to be linked to the VWF gene, the possibility of locus heterogeneity (i.e., a similar phenotype caused by a mutation in a gene other than VWF) should be considered.315 As with all DNA testing, if prenatal testing is

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being considered, genetic counseling should be provided before the decision to test is made as well as following the procedure.

DIFFERENTIAL DIAGNOSIS PLATELET-TYPE (PSEUDO-) VON WILLEBRAND DISEASE Platelet-type (pseudo-) VWD is a platelet defect that phenotypically mimics VWD (Chap. 120). Patients have mucocutaneous bleeding, plasma VWF often lacks the largest multimers, RIPA is enhanced at low concentrations of ristocetin, and thrombocytopenia of variable degree is often present. Molecular analysis has identified missense mutations within the GPIbα gene as the molecular basis for pseudo-VWD. These mutations are located within the segment of GPIb that encodes the VWF binding domain and appear to induce the conformational change complementary to that produced in VWF by type 2B VWD mutations (reviewed in Ref. 316). The specialized RIPA test should be performed at low ristocetin concentrations to distinguish type 2B and platelet type VWD from type 2A VWD. In this test, purified normal plasma VWF or cryoprecipitate added to platelet preparations from patients with platelet-type VWD causes platelet aggregation, distinguishing this disorder from type 2B VWD where patient platelets aggregate only at higher ristocetin concentrations. In addition, type 2B VWD plasma transfers the enhanced RIPA to normal platelets, whereas plasma from patients with platelettype VWD interacts normally with control platelets.

ACQUIRED VON WILLEBRAND SYNDROME Acquired VWD, or acquired von Willebrand syndrome (AVWS), is a relatively rare acquired bleeding disorder that usually presents as a late-onset bleeding diathesis in a patient with no prior bleeding history and a negative family history of bleeding (reviewed in Ref. 317). Decreased levels of FVIII, VWF:Ag, and VWF:RCo are common, and VWF multimers can be abnormal. AVWS is usually associated with another underlying disorder and has been reported to occur in patients with myeloproliferative neoplasms,318 amyloidosis,319 benign or malignant B-cell disorders,320 hypothyroidism,321 autoimmune disorders,322 several solid tumors (particularly Wilms tumor),323 cardiac or vascular defects (such as aortic stenosis),324 ventricular assist devices,325 or in association with several drugs, including ciprofloxacin and valproic acid.326,327 The mechanisms that cause AVWS can generally be attributed to an associated medical condition. A variety of B-cell disorders have been associated with the development of anti-VWF autoantibodies. In most cases the AVWS appears to be due to rapid clearance of VWF induced by the circulating inhibitor, although these antibodies may also interfere with VWF function. Hypothyroidism results in decreased VWF synthesis.321 In some cases of malignancy, AVWS is thought to be due to selective adsorption of VWF to the tumor cells or in myeloproliferative neoplasms, clearance/alterations of VWF by the high circulating platelet mass. AVWS associated with valvular heart disease, ventricular assist devices, or certain drugs, VWF may be lost by accelerated destruction or proteolysis under shear.325–327 Although the VWF multimers in AVWS usually exhibit a type 2A pattern with relative depletion of the large multimer forms, AVWS can manifest as a wide range of VWD phenotypes.322,328 Distinguishing AVWS from genetic VWD can be difficult, as testing for the associated autoantibodies is generally not available in the clinical setting. The diagnosis often rests on the late onset of the disease, the absence of a family history, and the identification of an associated underlying disorder.

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Management of AVWS is generally aimed at treating the underlying disorder. VWF levels and bleeding symptoms often improve with successful treatment of hypothyroidism or an associated malignancy. Refractory patients have been treated with glucocorticoids, plasma exchange, intravenous gamma globulin, rituximab, DDAVP, and VWFcontaining FVIII concentrates.317,329

THERAPY, COURSE, AND PROGNOSIS The mainstays of therapy for VWD are DDAVP, which induces secretion of both VWF and FVIII (reviewed in Ref. 330), and replacement therapy with VWF-containing plasma concentrates. The choice of treatment in any given patient depends upon the type and severity of VWD, the clinical setting, and the type of hemostatic challenge that must be met. Type 1 patients are most often treated with DDAVP alone, types 2A and 2B with a combination of DDAVP and a VWF-containing FVIII product, and type 2N and type 3 patients with VWF-containing concentrates.150 A previous history of trauma or surgery and the success of previous treatment are important parameters to include in assessing the risk of bleeding. Prophylaxis is used in anticipation of hemostatic challenges,150 such as dental extractions, and is efficacious in preventing recurrent bleeding in severe VWD patients.331 Although in general there is a correlation between normal hemostasis and correction of VWF and FVIII activity, this does not occur in all cases.

DESMOPRESSIN DDAVP is an analogue of antidiuretic hormone that acts through type 2 vasopressin receptors to induce secretion of FVIII and VWF, likely via cyclic adenosine monophosphate–mediated secretion from the WeibelPalade bodies in endothelial cells.83 When DDAVP is administered to healthy subjects, it causes sustained increases of FVIII and ristocetin cofactor activity for approximately 4 hours.332 Patients with type 1 VWD treated with DDAVP release unusually high-molecular-weight VWF multimers into the circulation for 1 to 3 hours after the infusion.332,333 Therapy with DDAVP often increases the FVIII activity, VWF:Ag, and ristocetin cofactor activity to two to five times the basal level. DDAVP has become a mainstay for the treatment of mild hemophilia and VWD334 because it is relatively inexpensive, widely available, and avoids the risks of plasma-derived products. Approximately 80 percent of type 1 VWD patients have excellent responses to DDAVP, although this figure may be substantially lower depending on the criteria for diagnosis and response.335 It is regularly used in the setting of mild to moderate bleeding and for prophylaxis of patients undergoing surgical procedures. DDAVP is administered at a dose of 0.3mcg/kg continuous intravenous infusion over 30 minutes. DDAVP is also available for subcutaneous injection (at the same 0.3mcg/kg dose) and in intranasal form (at a fixed dose of 300mcg for adults and 150mcg for children), which appears to be similar in efficacy to intravenous administration,336,337 although the response may be more variable. The response to DDAVP in any given individual with VWD is generally reproducible and predicts response to future doses as long as the follow doses are at least 2 to 4 days later. In one study, 22 type 1 VWD patients showed a departure of less than 20 percent from the mean FVIII peak level calculated from two separate infusions. In addition, the consistency of response in one patient reliably predicted the future response of that patient and other affected family members.338 In a study of 77 type 1 VWD patients, DDAVP response was associated both with VWF mutation and baseline multimeric pattern, although subtle abnormalities in VWF multimers did not preclude a patient response to DDAVP. Interestingly, patients with the same VWF mutation did not necessarily exhibit the same degree of responsiveness to DDAVP, implying the

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influence of other factors in the magnitude of DDAVP effect.339 For patients requiring repeated infusions of DDAVP, the FVIII activity and VWF responses may not be of the same magnitude as after the first infusion. Although this decay in response has considerable individual variability, after one infusion of DDAVP per day for 4 days it was found that the responses on days 2 to 4 were reduced approximately 30 percent compared to day 1.336–338,340 Therefore, in patients for whom DDAVP is potentially the treatment of choice, a test dose should be given at the planned therapeutic dose and route in advance of the first required course of treatment with measurements of before and after VWF and FVIII:C levels to ensure an adequate therapeutic response. Sampling additional time points after DDAVP infusion should be considered as some type 1 and type 2 VWD patients have a significantly shortened VWF half-life and it may be more appropriate to treat with VWF replacement therapy in clinical scenarios requiring more durable therapy to maintain hemostasis. For patients with type 1 VWD who are undergoing surgical procedures, DDAVP can be administered 1 hour before surgery and approximately every 12 hours thereafter for up to two to four doses before loss of clinically significant response. Patients should be monitored for response of FVIII and ristocetin cofactor activity and side effects, particularly hyponatremia (and then should be water restricted), when DDAVP is administered at frequent intervals. VWF-containing FVIII concentrates should be available for infusion as backup. Approximately 20 to 25 percent of patients with VWD do not respond adequately to DDAVP. Type 2 VWD patients are less likely to have a response than type 1 patients,341,335 and virtually no patients with type 3 VWD respond. The response to DDAVP of patients with type 2A VWD is variable. Although most patients respond only transiently, some patients exhibit complete hemostatic correction after DDAVP infusion.342,343 It has been hypothesized that the differences in DDAVP efficacy among type 2A patients may correspond to the type of mutation, with better responses predicted in patients with group 2 mutations. A prospective study of the biologic response to DDAVP in well-characterized VWD patients included type 2A VWD patients with both group 1 and group 2 defects. Although patients with group 2 mutations had greater improvements in VWF:RCo and shortening of bleeding times than patients with group 1 defects, neither groups could be classified as responders.341 Common side effects of DDAVP administration are mild cutaneous vasodilation resulting in a feeling of heat, facial flushing, tachycardia, tingling, and headaches. The potential for dilutional hyponatremia, especially in elderly and very young patients and with repeat dosing, requires appropriate attention to fluid restriction, as it may result in seizures. There have been isolated reports of acute arterial thrombosis associated with administration of DDAVP, but the risk appears to be very low when judged against the total number of patients treated. DDAVP is contraindicated in patients with unstable coronary artery disease because of increased risk of thrombotic events, such as myocardial infarction.344 Patients receiving DDAVP at closely spaced intervals of less than 24 to 48 hours can develop tachyphylaxis.340 Many experts consider DDAVP to be contraindicated in the treatment of type 2B VWD, as the high-molecular-weight VWF released from storage sites has an increased affinity for binding to GPIb and might be expected to induce spontaneous platelet aggregation and worsening thrombocytopenia.229 However, there are reports of DDAVP used successfully in type 2B VWD patients, with an associated shortening of bleeding times and variable thrombocytopenia.345,346 Although type 2N patients can exhibit increased FVIII:C levels after DDAVP, in some cases the FVIII:C levels rapidly decline, likely a result of the absence of stabilizing normal VWF, attenuating clinical efficacy. Type 2M patients generally do not have a satisfactory response to DDAVP.341,347

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VON WILLEBRAND FACTOR REPLACEMENT THERAPY It is important to determine the response to DDAVP for each individual so as to avoid the unnecessary use of plasma products. For type 3 VWD patients and other patients unresponsive to DDAVP, the use of selected virus-inactivated, VWF-containing FVIII concentrates is generally safe and effective.150 Humate-P, Alphanate, Wilate, and Koate are all acceptable commercial VWF-containing plasma concentrates that have been evaluated in VWD replacement therapy in clinical studies, and other VWF-containing FVIII concentrates may also be effective. A plasmafree recombinant VWF–recombinant FVIII combination (rVWF-rFVIII) shows promise in clinical trials and may become available in the near future.348 Cryoprecipitate was useful in the past, but because it is not generally treated to inactivate bloodborne pathogens, is less targeted to correcting the VWD hemostatic defect, and its administration is associated with a large volume load, it is less desirable. It is important to note that most standard FVIII concentrates and all recombinant FVIII products are not effective in VWD because they lack clinically significant quantities of VWF. Although such products can substantially increase circulating FVIII:C, the infused factor is short-lived in the circulation in the absence of stabilizing VWF.349 Only preparations that contain large quantities of VWF with well-preserved multimer structure are suitable for use in VWD patients. In practice, VWD replacement therapy dosing and timing has been largely empiric. Recommendations for therapy have been outlined based upon the degree and nature of hemorrhage and experience in clinical practice.150 The objective is to elevate FVIII:C and VWF:RCo until bleeding stops and healing is complete. In general, replacement goals of FVIII:C and VWF:RCo should be initial replacement to greater than 100 IU/dL and maintenance of greater than 50 IU/dL for 7 to 14 days for major trauma, surgery, or central nervous system hemorrhage; greater than 30 to 50 IU/dL for 3 to 5 days for minor surgery or bleeding; greater than 50 IU/dL for delivery and continued for at least 3 to 5 days in the postpartum period; greater than 30 to 50 IU/dL for 1 to 5 days for dental extractions and minor surgery; and greater than 20 to 50 IU/dL for mucous membrane bleeding or menorrhagia. Laboratory monitoring of posttreatment FVIII:C and VWF levels is important in guiding therapy and avoidance of supratherapeutic replacement doses (>200 IU/dL VWF:RCo, >250 IU/dL FVIII), which are associated with an increased risk of thrombosis.150,350,351 Although thrombosis is rare overall, VWD patients on prolonged therapy or with central access catheters appear to be at higher risk.352 In patients who have concomitant thrombocytopenia associated with or in addition to VWD, it may be necessary to transfuse platelets in addition to factor concentrates. If clinical bleeding continues, additional replacement therapy must be given and searches undertaken for other hemostatic defects. Type 3 VWD patients receiving multiple transfusions can develop antibodies directed against VWF (reviewed in Ref. 277). Continued replacement with VWF-containing concentrates is contraindicated because of the risk of anaphylaxis. A variety of approaches to the management of VWD inhibitors, similar to the treatment of FVIII inhibitors in hemophilia A (Chap. 123), have been attempted. Immunosuppression, recombinant FVIII, and recombinant factor VIIa have been reported to be useful in patients with type 3 VWD who have developed anti-VWF antibodies.

OTHER NONREPLACEMENT THERAPIES Fibrinolytic inhibitors, such as ε-aminocaproic acid or tranexamic acid, have been used effectively in some VWD patients. Antifibrinolytics are commonly used alone or in conjunction with DDAVP or a

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plasma-derived VWF replacement product in patients with gynecologic bleeding, mucous membrane bleeding, or undergoing dental procedures.150 Fibrinolytic inhibitors can be delivered systemically or topically and are generally well tolerated, but rarely can cause nausea or diarrhea and are contraindicated in patients with gross hematuria. Estrogens or oral contraceptives have been used empirically in treating menorrhagia. In addition to their effects on the ovaries and uterus, some estrogens can increase plasma VWF levels. Patients with VWD frequently normalize their levels of FVIII, VWF:Ag, and VWF:RCo during pregnancy (Chap. 8). Postpartum hemorrhage within the first few days after parturition may be related to the relatively rapid return of FVIII and VWF activities to prepregnancy levels, and postpartum hemorrhage in all forms of VWD may occur as long as 1 month postpartum. In pregnant patients with type 1 VWD, the FVIII and ristocetin cofactor activities usually rise above 50 percent. These patients usually do not require any specific therapy at the time of parturition. In contrast, individuals who have 30 percent or less FVIII or variant forms of VWD are more likely to require prophylactic therapy before delivery. In a recent study, women receiving treatment for VWD postpartum were unexpectedly found not to have corrected to target levels.353 Therefore, laboratory testing is recommended at term and should be considered in the postpartum period in patients at risk for immediate and/or delayed bleeding complications or receiving therapy. Recombinant activated factor VII (rFVIIa, or NovoSeven) has also been successfully used in VWD patients with severe hemorrhage refractory to VWF replacement therapy and in bleeding patients with anti-VWF antibodies (reviewed in Ref. 354). In the case of minor accessible bleeding, topical drugs such as fibrin sealants or topical bovine thrombin may also be considered when standard VWD therapies fail to provide adequate local hemostasis.150

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231. Graham JB, Barrow ES, Roberts HR, et al: Dominant inheritance of hemophilia A in three generations of women. Blood 46:175–188, 1975. 232. Mazurier C, Gaucher C, Jorieux S, et al: Evidence for a von Willebrand factor defect in factor VIII binding in three members of a family previously misdiagnosed mild haemophilia A and haemophilia A carriers: Consequences for therapy and genetic counselling. Br J Haematol 76:372–379, 1990. 233. Mazurier C, Diéval J, Jorieux S, et al: A new von Willebrand Factor (vWF) defect in a patient with factor VIII (FVIII) deficiency but with normal levels and multimeric patterns of both plasma and platelet vWF. Characterization of abnormal vWF/FVIII interaction. Blood 75:20–26, 1990. 234. Nishino M, Girma J-P, Rothschild C, et al: New variant of von Willebrand disease with defective binding to factor VIII. Blood 74:1591–1599, 1989. 235. Hampshire DJ, Goodeve AC: The international society on thrombosis and haematosis von Willebrand disease database: An update. Semin Thromb Hemost 37:470–479, 2011. 236. Eikenboom JC, Reitsma PH, Peerlinck KM, Briët E: Recessive inheritance of von Willebrand’s disease type I. Lancet 341:982–986, 1993. 237. Castaman G, Giacomelli SH, Jacobi P, et al: Homozygous type 2N R854W von Willebrand factor is poorly secreted and causes a severe von Willebrand disease phenotype. J Thromb Haemost 8:2011–2016, 2010. 238. Gupta M, Lillicrap D, Stain AM, Friedman KD, Carcao MD: Therapeutic consequences for misdiagnosis of type 2N von Willebrand disease. Pediatr Blood Cancer 57:1081– 1083, 2011. 239. Nichols WC, Cooney KA, Ginsburg D, Ruggeri ZM: Von Willebrand disease, in Thrombosis and Hemorrhage, edited by J Loscalzo, AI Schafer, pp 539–559. Lipincott Williams & Wilkins, Philadelphia, 2003. 240. Meyer D, Fressinaud E, Gaucher C, et al: Gene defects in 150 unrelated French cases with type 2 von Willebrand disease: From the patient to the gene. Thromb Haemost 78:451–456, 1997. 241. McKinnon TA, Nowak AA, Cutler J, et al: Characterisation of von Willebrand factor A1 domain mutants I1416N and I1416T: Correlation of clinical phenotype with flow-based platelet adhesion. J Thromb Haemost 10:1409–1416, 2012. 242. Larsen DM, Haberichter SL, Gill JC, et al: Variability in platelet- and collagen-binding defects in type 2M von Willebrand disease. Haemophilia 19:590–594, 2013. 243. Mannucci PM, Lombardi R, Castaman G, et al: von Willebrand disease “Vicenza” with larger-than-normal (supranormal) von Willebrand factor multimers. Blood 71:65–70, 1988. 244. Randi AM, Sacchi E, Castaman GC, et al: The genetic defect of type I von Willebrand disease “Vicenza” is linked to the von Willebrand factor gene. Thromb Haemost 69: 173–176, 1993. 245. Casonato A, Pontara E, Sartorello F, et al: Reduced von Willebrand factor survival in type Vicenza von Willebrand disease. Blood 99:180–184, 2002. 246. Castaman G, Rodeghiero F, Mannucci PM: The elusive pathogenesis of von Willebrand disease Vicenza. Blood 99:4243–4244, 2002. 247. Gezsi A, Budde U, Deak I, et al: Accelerated clearance alone explains ultra-large multimers in von Willebrand disease Vicenza. J Thromb Haemost 8:1273–1280, 2010. 248. Berkowitz SD, Ruggeri ZM, Zimmerman TS: Von Willebrand disease, in Coagulation and Bleeding Disorders. The Role of Factor VIII and von Willebrand Factor, edited by TS Zimmerman, ZM Ruggeri, pp 215–259. Marcel Dekker, New York, 1989. 249. Miller CH, Graham JB, Goldin LR, Elston RC: Genetics of classic von Willebrand’s disease. I. Phenotypic variation within families. Blood 54:117–145, 1979. 250. Gill JC, Endres-Brooks J, Bauer PJ, et al: The effect of ABO blood group on the diagnosis of von Willebrand disease. Blood 69:1691–1695, 1987. 251. Orstavik KH, Kornstad L, Reisner H, Berg K: Possible effect of secretor locus on plasma concentration of Factor VIII and von Willebrand factor. Blood 73:990–993, 1989. 252. O’Donnell J, Boulton FE, Manning RA, Laffan MA: Genotype at the secretor blood group locus is a determinant of plasma von Willebrand factor level. Br J Haematol 116:350–356, 2002. 253. Franchini M, Crestani S, Frattini F, et al: ABO blood group and von Willebrand factor: Biological implications. Clin Chem Lab Med 52:1273–1276, 2014. 254. Antoni G, Oudot-Mellakh T, Dimitromanolakis A, et al: Combined analysis of three genome-wide association studies on vWF and FVIII plasma levels. BMC Med Genet 12:102, 2011. 255. Smith NL, Chen MH, Dehghan A, et al: Novel associations of multiple genetic loci with plasma levels of factor VII, factor VIII, and von Willebrand factor: The CHARGE (Cohorts for Heart and Aging Research in Genome Epidemiology) Consortium. Circulation 121:1382–1392, 2010. 256. Zhu Q, Yamakuchi M, Ture S, et al: Syntaxin-binding protein STXBP5 inhibits endothelial exocytosis and promotes platelet secretion. J Clin Invest 124:4503–4516, 2014. 257. Ye S, Huang Y, Joshi S, et al: Platelet secretion and hemostasis require syntaxin-binding protein STXBP5. J Clin Invest 124:4517–4528, 2014. 258. Rydz N, Swystun LL, Notley C, et al: The C-type lectin receptor CLEC4M binds, internalizes, and clears von Willebrand factor and contributes to the variation in plasma von Willebrand factor levels. Blood 121:5228–5237, 2013. 259. Desch KC, Ozel AB, Siemieniak D, et al: Linkage analysis identifies a locus for plasma von Willebrand factor undetected by genome-wide association. Proc Natl Acad Sci U S A 110:588–593, 2013. 260. Werner EJ, Broxson EH, Tucker EL, et al: Prevalence of von Willebrand disease in children: A multiethnic study. J Pediatr 123:893–898, 1993. 261. Rodeghiero F, Castaman G, Dini E: Epidemiological investigation of the prevalence of von Willebrand’s disease. Blood 69:454–459, 1987.

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262. Sadler JE: Von Willebrand disease type 1: A diagnosis in search of a disease. Blood 101:2089–2093, 2003. 263. Lillicrap D: Von Willebrand disease: Advances in pathogenetic understanding, diagnosis, and therapy. Blood 122:3735–3740, 2013. 264. Weiss HJ, Ball AP, Mannucci PM: Incidence of severe von Willebrand’s disease. N Engl J Med 307:127, 1982. 265. Berliner SA, Seligsohn U, Zivelin A, et al: A relatively high frequency of severe (type III) von Willebrand’s disease in Israel. Br J Haematol 62:535–543, 1986. 266. Mannucci PM, Bloom AL, Larrieu MJ, et al: Atherosclerosis and von Willebrand factor. I. Prevalence of severe von Willebrand’s disease in western Europe and Israel. Br J Haematol 57:163–169, 1984. 267. Nosek-Cenkowska B, Cheang MS, Pizzi NJ, et al: Bleeding/bruising symptomatology in children with and without bleeding disorders. Thromb Haemost 65:237–241, 1991. 268. Rydz N, James PD: The evolution and value of bleeding assessment tools. J Thromb Haemost 10:2223–2229, 2012. 269. Elbatarny M, Mollah S, Grabell J, et al: Normal range of bleeding scores for the ISTHBAT: Adult and pediatric data from the merging project. Haemophilia 20:831–835, 2014. 270. Federici AB, Bucciarelli P, Castaman G, et al: The bleeding score predicts clinical outcomes and replacement therapy in adults with von Willebrand disease. Blood 123: 4037–4044, 2014. 271. van den Driesche S, Mummery CL, Westermann CJ: Hereditary hemorrhagic telangiectasia: An update on transforming growth factor beta signaling in vasculogenesis and angiogenesis. Cardiovasc Res 58:20–31, 2003. 272. Iannuzzi MC, Hidaka N, Boehnke ML, et al: Analysis of the relationship of von Willebrand disease (vWD) and hereditary hemorrhagic telangiectasia and identification of a potential type IIA vWD mutation (IIe865 to Thr). Am J Hum Genet 48:757–763, 1991. 273. Rick ME, Williams SB, Sacher RA, McKeown LP: Thrombocytopenia associated with pregnancy in a patient with type IIB von Willebrand’s disease. Blood 69:786–789, 1987. 274. Mazurier C, Parquet-Gernez A, Goudemand J, et al: Investigation of a large kindred with type IIB von Willebrand’s disease, dominant inheritance and age-dependent thrombocytopenia. Br J Haematol 69:499–505, 1988. 275. Gupta M, Lillicrap D, Stain AM, et al: Therapeutic consequences for misdiagnosis of type 2N von Willebrand disease. Pediatr Blood Cancer 57:1081–1083, 2011. 276. Schneppenheim R, Budde U, Krey S, et al: Results of a screening for von Willebrand disease type 2N in patients with suspected haemophilia A or von Willebrand disease type 1. Thromb Haemost 76:598–602, 1996. 277. James PD, Lillicrap D, Mannucci PM: Alloantibodies in von Willebrand disease. Blood 122:636–640, 2013. 278. Asatiani E, Kessler CM: Multiple congenital coagulopathies co-expressed with von Willebrand’s disease: The experience of Hemophilia Region III Treatment Centers over 25 years and review of the literature. Haemophilia 13:685–696, 2007. 279. Lippi G, Franchini M, Poli G, et al: Is the activated partial thromboplastin time suitable to screen for von Willebrand factor deficiencies? Blood Coagul Fibrinolysis 18:361–364, 2007. 280. Abildgaard CF, Suzuki Z, Harrison J, et al: Serial studies in von Willebrand’s disease: Variability versus “variants.” Blood 56:712–716, 1980. 281. Timm A, Fahrenkrug J, Jorgensen HL, et al: Diurnal variation of von Willebrand factor in plasma: The Bispebjerg study of diurnal variations. Eur J Haematol 93:48–53, 2014. 282. Sadler JE: Von Willebrand disease type 1: A diagnosis in search of a disease. Blood 101:2089–2093, 2003. 283. Sadler JE: New concepts in von Willebrand disease. Annu Rev Med 56:173–191, 2005. 284. Quiroga T, Goycoolea M, Belmont S, et al: Quantitative impact of using different criteria for the laboratory diagnosis of type 1 von Willebrand disease. J Thromb Haemost 12:1238–1243, 2014. 285. Hayward CP, Moffat KA, Plumhoff E, Van Cott EM: Approaches to investigating common bleeding disorders: An evaluation of North American coagulation laboratory practices. Am J Hematol 87 Suppl 1:S45–S50, 2012. 286. Favaloro EJ, Bonar R, Chapman K, et al: Differential sensitivity of von Willebrand factor (VWF) “activity” assays to large and small VWF molecular weight forms: A cross-laboratory study comparing ristocetin cofactor, collagen-binding and mAb-based assays. J Thromb Haemost 10:1043–1054, 2012. 287. Eikenboom J, Federici AB, Dirven RJ, et al: VWF propeptide and ratios between VWF, VWF propeptide, and FVIII in the characterization of type 1 von Willebrand disease. Blood 121:2336–2339, 2013. 288. Weiss HJ, Hoyer LW, Rickles FR, et al: Quantitative assay of a plasma factor deficient in von Willebrand’s disease that is necessary for platelet aggregation. J Clin Invest 52:2708– 2716, 1973. 289. Rodeghiero F, Castaman G, Tosetto A: Von Willebrand factor antigen is less sensitive than ristocetin cofactor for the diagnosis of type I von Willebrand disease-results based on an epidemiological investigation. Thromb Haemost 64:349–352, 1990. 290. Favaloro EJ: Diagnosis and classification of von Willebrand disease: A review of the differential utility of various functional von Willebrand factor assays. Blood Coagul Fibrinolysis 22:553–564, 2011. 291. Ruggeri ZM, Zimmerman TS: The complex multimeric composition of factor VIII/von Willebrand Factor. Blood 57:1140–1143, 1981. 292. Budde U, Schneppenheim R, Eikenboom J, et al: Detailed von Willebrand factor multimer analysis in patients with von Willebrand disease in the European study, molecular and clinical markers for the diagnosis and management of type 1 von Willebrand disease (MCMDM-1VWD). J Thromb Haemost 6:762–771, 2008.

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293. Flood VH, Gill JC, Friedman KD, et al: Collagen binding provides a sensitive screen for variant von Willebrand disease. Clin Chem 59:684–691, 2013. 294. Flood VH, Lederman CA, Wren JS, et al: Absent collagen binding in a VWF A3 domain mutant: Utility of the VWF:CB in diagnosis of VWD. J Thromb Haemost 8:1431–1433, 2010. 295. Flood VH, Gill JC, Christopherson PA, et al: Critical von Willebrand factor A1 domain residues influence type VI collagen binding. J Thromb Haemost 10:1417–1424, 2012. 296. Favaloro EJ, Dean M, Grispo L, et al: Von Willebrand’s disease: Use of collagen binding assay provides potential improvement to laboratory monitoring of desmopressin (DDAVP) therapy. Am J Hematol 45:205–211, 1994. 297. Riddell AF, Jenkins PV, Nitu-Whalley IC, et al: Use of the collagen-binding assay for von Willebrand factor in the analysis of type 2M von Willebrand disease: A comparison with the ristocetin cofactor assay. Br J Haematol 116:187–192, 2002. 298. Popov J, Zhukov O, Ruden S, et al: Performance and clinical utility of a commercial von Willebrand factor collagen binding assay for laboratory diagnosis of von Willebrand disease. Clin Chem 52:1965–1967, 2006. 299. Meiring M, Badenhorst PN, Kelderman M: Performance and utility of a cost-effective collagen-binding assay for the laboratory diagnosis of Von Willebrand disease. Clin Chem Lab Med 45:1068–1072, 2007. 300. Mazurier C, Meyer D: Factor VIII binding assay of von Willebrand factor and the diagnosis of type 2N von Willebrand disease-results of an international survey. On behalf of the Subcommittee on von Willebrand Factor of the Scientific and Standardization Committee of the ISTH. Thromb Haemost 76:270–274, 1996. 301. Zhukov O, Popov J, Ramos R, et al: Measurement of von Willebrand factor-FVIII binding activity in patients with suspected von Willebrand disease type 2N: Application of an ELISA-based assay in a reference laboratory. Haemophilia 15:788–796, 2009. 302. Casonato A, Pontara E, Sartorello F, et al: Identifying carriers of type 2N von Willebrand disease: Procedures and significance. Clin Appl Thromb Hemost 13:194–200, 2007. 303. Haberichter SL, Balistreri M, Christopherson P, et al: Assay of the von Willebrand factor (VWF) propeptide to identify patients with type 1 von Willebrand disease with decreased VWF survival. Blood 108:3344–3351, 2006. 304. Haberichter SL, Castaman G, Budde U, et al: Identification of type 1 von Willebrand disease patients with reduced von Willebrand factor survival by assay of the VWF propeptide in the European study: Molecular and clinical markers for the diagnosis and management of type 1 VWD (MCMDM-1VWD). Blood 111:4979–4985, 2008. 305. Casonato A, Pontara E, Sartorello F, et al: Identifying type Vicenza von Willebrand disease. J Lab Clin Med 147:96–102, 2006. 306. Fressinaud E, Veyradier A, Truchaud F, et al: Screening for von Willebrand disease with a new analyzer using high shear stress: A study of 60 cases. Blood 91:1325–1331, 1998. 307. Cattaneo M, Federici AB, Lecchi A, et al: Evaluation of the PFA-100 system in the diagnosis and therapeutic monitoring of patients with von Willebrand disease. Thromb Haemost 82:35–39, 1999. 308. De Vleeschauwer A, Devreese K: Comparison of a new automated von Willebrand factor activity assay with an aggregation von Willebrand ristocetin cofactor activity assay for the diagnosis of von Willebrand disease. Blood Coagul Fibrinolysis 17:353–358, 2006. 309. Salem RO, Van Cott EM: A new automated screening assay for the diagnosis of von Willebrand disease. Am J Clin Pathol 127:730–735, 2007. 310. Sucker C, Senft B, Scharf RE, Zotz RB: Determination of von Willebrand factor activity: Evaluation of the HaemosIL assay in comparison with established procedures. Clin Appl Thromb Hemost 12:305–310, 2006. 311. Pinol M, Sales M, Costa M, et al: Evaluation of a new turbidimetric assay for von Willebrand factor activity useful in the general screening of von Willebrand disease. Haematologica 92:712–713, 2007. 312. Fujimura Y, Kawasaki T, Titani K: Snake venom proteins modulating the interaction between von Willebrand factor and platelet glycoprotein Ib. Thromb Haemost 76:633– 639, 1996. 313. Keeney S, Bowen D, Cumming A, et al: The molecular analysis of von Willebrand disease: A guideline from the UK Haemophilia Centre Doctors’ Organisation Haemophilia Genetics Laboratory Network. Haemophilia 14:1099–1111, 2008. 314. Snyder MW, Simmons LE, Kitzman JO, et al: Noninvasive fetal genome sequencing: A primer. Prenat Diagn 33:547–554, 2013. 315. James PD, Lillicrap D: The molecular characterization of von Willebrand disease: Good in parts. Br J Haematol 161:166–176, 2013. 316. Othman M, Kaur H, Emsley J: Platelet-type von Willebrand disease: New insights into the molecular pathophysiology of a unique platelet defect. Semin Thromb Hemost 39:663–673, 2013. 317. Federici AB, Budde U, Castaman G, et al: Current diagnostic and therapeutic approaches to patients with acquired von Willebrand syndrome: A 2013 update. Semin Thromb Hemost 39:191–201, 2013. 318. Budde U, Schaefer G, Mueller N, et al: Acquired von Willebrand’s disease in the myeloproliferative syndrome. Blood 64:981–985, 1984. 319. Kos CA, Ward JE, Malek K, et al: Association of acquired von Willebrand syndrome with AL amyloidosis. Am J Hematol 82:363–367, 2007. 320. Mannucci PM, Lombardi R, Bader R, et al: Studies of the pathophysiology of acquired von Willebrand’s disease in seven patients with lymphoproliferative disorders or benign monoclonal gammopathies. Blood 64:614–621, 1984. 321. Rogers JS, Shane SR, Jencks FS: Factor VIII activity and thyroid function. 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322. Viallard JF, Pellegrin JL, Vergnes C, et al: Three cases of acquired von Willebrand disease associated with systemic lupus erythematosus. Br J Haematol 105:532–537, 1999. 323. Scott JP, Montgomery RR, Tubergen DG, Hays T: Acquired von Willebrand’s disease in association with Wilm’s tumor: Regression following treatment. Blood 58:665–669, 1981. 324. Warkentin TE, Moore JC, Morgan DG: Aortic stenosis and bleeding gastrointestinal angiodysplasia: Is acquired von Willebrand’s disease the link? Lancet 340:35–37, 1992. 325. Geisen U, Heilmann C, Beyersdorf F, et al: Non-surgical bleeding in patients with ventricular assist devices could be explained by acquired von Willebrand disease. Eur J Cardiothorac Surg 33:679–684, 2008. 326. Castaman G, Lattuada A, Mannucci PM, Rodeghiero F: Characterization of two cases of acquired transitory von Willebrand syndrome with ciprofloxacin: Evidence for heightened proteolysis of von Willebrand factor. Am J Hematol 49:83–86, 1995. 327. Tefferi A, Nichols WL: Acquired von Willebrand disease: Concise review of occurrence, diagnosis, pathogenesis, and treatment. Am J Med 103:536–540, 1997. 328. Kumar S, Pruthi RK, Nichols WL: Acquired von Willebrand disease. Mayo Clin Proc 77:181–187, 2002. 329. Kanakry JA, Gladstone DE: Maintaining hemostasis in acquired von Willebrand syndrome: A review of intravenous immunoglobulin and the importance of rituximab dose scheduling. Transfusion 53:1730–1735, 2013. 330. Svensson PJ, Bergqvist PB, Juul KV, Berntorp E: Desmopressin in treatment of haematological disorders and in prevention of surgical bleeding. Blood Rev 28:95–102, 2014. 331. Abshire TC, Federici AB, Alvarez MT, et al: Prophylaxis in severe forms of von Willebrand’s disease: Results from the von Willebrand Disease Prophylaxis Network (VWD PN). Haemophilia 19:76–81, 2013. 332. Mannucci PM, Ruggeri ZM, Pareti FI, Capitanio A: 1-Deamino-8-d-arginine vasopressin: A new pharmacological approach to the management of haemophilia and von Willebrands’ diseases. Lancet 1:869–872, 1977. 333. Ruggeri ZM, Mannucci PM, Lombardi R, Federici AB, Zimmerman TS: Multimeric composition of factor VIII/von Willebrand Factor following administration of DDAVP: Implications for pathophysiology and therapy of von Willebrand’s disease subtypes. Blood 59:1272–1278, 1982. 334. Mannucci PM: Desmopressin (DDAVP) in the treatment of bleeding disorders: The first 20 years. Blood 90:2515–2521, 1997. 335. Federici AB, Mazurier C, Berntorp E, et al: Biologic response to desmopressin in patients with severe type 1 and type 2 von Willebrand disease: Results of a multicenter European study. Blood 103:2032–2038, 2004. 336. Lethagen S, Harris AS, Nilsson IM: Intranasal desmopressin (DDAVP) by spray in mild hemophilia A and von Willebrand’s disease type I. Blut 60:187–191, 1990. 337. Rose EH, Aledort LM: Nasal spray desmopressin (DDAVP) for mild hemophilia A and von Willebrand disease. Ann Intern Med Intern Med 114:563–568, 1991. 338. Rodeghiero F, Castaman G, Di Bona E, Ruggeri M: Consistency of responses to repeated DDAVP infusions in patients with von Willebrand’s disease and hemophilia A. Blood 74:1997–2000, 1989.

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339. Castaman G, Lethagen S, Federici AB, et al: Response to desmopressin is influenced by the genotype and phenotype in type 1 von Willebrand disease (VWD): Results from the European Study MCMDM-1VWD. Blood 111:3531–3539, 2008. 340. Mannucci PM, Bettega D, Cattaneo M: Patterns of development of tachyphylaxis in patients with haemophilia and von Willebrand disease after repeated doses of desmopressin (DDAVP). Br J Haematol 82:87–93, 1992. 341. Federici AB, Mazurier C, Berntorp E, et al: Biologic response to desmopressin in patients with severe type 1 and type 2 von Willebrand disease: Results of a multicenter European study. Blood 103:2032–2038, 2004. 342. de la Fuente B, Kasper CK, Rickles FR, Hoyer LW: Response of patients with mild and moderate hemophilia A and von Willebrand’s disease to treatment with desmopressin. Ann Intern Med 103:6–14, 1985. 343. Gralnick HR, Williams SB, McKeown LP, et al: DDAVP in type IIa von Willebrand’s disease. Blood 67:465–468, 1986. 344. Mannucci PM: Treatment of von Willebrand’s disease. N Engl J Med 351:683–694, 2004. 345. Casonato A, Sartori MT, De Marco L, Girolami A: 1-Desamino-8-D-arginine vasopressin (DDAVP) infusion in type IIB von Willebrand’s disease: Shortening of bleeding time and induction of a variable pseudothrombocytopenia. Thromb Haemost 64: 117–120, 1990. 346. McKeown LP, Connaghan G, Wilson O, et al: 1-Desamino-8-arginine-vasopressin corrects the hemostatic defects in type 2B von Willebrand’s disease. Am J Hematol 51: 158–163, 1996. 347. Mazurier C, Gaucher C, Jorieux S, Goudemand M: Biological effect of desmopressin in eight patients with type 2N (“Normandy”) von Willebrand disease. Collaborative Group. Br J Haematol 88:849–854, 1994. 348. Mannucci PM, Kempton C, Millar C, et al: Pharmacokinetics and safety of a novel recombinant human von Willebrand factor manufactured with a plasma-free method: A prospective clinical trial. Blood 122:648–657, 2013. 349. Morfini M, Mannucci PM, Tenconi PM, et al: Pharmacokinetics of monoclonallypurified and recombinant factor VIII in patients with severe von Willebrand disease. Thromb Haemost 70:270–272, 1993. 350. Makris M, Colvin B, Gupta V, et al: Venous thrombosis following the use of intermediate purity FVIII concentrate to treat patients with von Willebrand’s disease. Thromb Haemost 88:387–388, 2002. 351. Mannucci PM, Chediak J, Hanna W, et al: Treatment of von Willebrand disease with a high-purity factor VIII/von Willebrand factor concentrate: A prospective, multicenter study. Blood 99:450–456, 2002. 352. Coppola A, Franchini M, Makris M, et al: Thrombotic adverse events to coagulation factor concentrates for treatment of patients with haemophilia and von Willebrand disease: A systematic review of prospective studies. Haemophilia 18:e173–e187, 2012. 353. James AH, Konkle BA, Kouides P, et al: Postpartum von Willebrand factor levels in women with and without von Willebrand disease and implications for prophylaxis. Haemophilia 21:81–87, 2015. 354. Sucker C, Scharf RE, Zotz RB: Use of recombinant factor VIIa in inherited and acquired von Willebrand disease. Clin Appl Thromb Hemost 15:27–31, 2009.

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CHAPTER 127

ANTIBODY-MEDIATED COAGULATION FACTOR DEFICIENCIES

Sean R. Stowell, John S. (Pete) Lollar, and Shannon L. Meeks

SUMMARY Clinically significant autoantibodies to coagulation factors deficiencies are uncommon, but can produce life-threatening bleeding and death. The most commonly targeted coagulation factor in autoimmunity is factor VIII. Acquired hemophilia A, which results from these antibodies, can either be idiopathic or associated with older age, other autoimmune disorders, malignancy, the postpartum period, and the use of drugs such as penicillin and sulfonamides. Bleeding in acquired hemophilia A is treated with factor VIII bypassing agents. The underlying autoimmune disorder frequently responds to immunosuppressive medication. Antiprothrombin antibodies usually are found in patients with lupus anticoagulant and are associated with bleeding. Antibodies of von Willebrand factor are found in patients with type 3 von Willebrand disease in response to infusion of plasma concentrates containing von Willebrand factor. Antibodies to factor V can occur as autoantibodies or as cross-reacting antibovine factor V antibodies that develop after exposure to bovine thrombin products that are contaminated with factor V. Pathogenic autoantibodies also have been described that target thrombin, factor IX, factor XI, factor XIII, protein C, protein S, and the endothelial cell protein C receptor.

DEFINITION AND HISTORY Antibodies directed against coagulation factors can develop as an acquired, autoimmune phenomenon. These “circulating anticoagulants” or “inhibitors” were recognized as early as 1906 as a cause of an acquired bleeding disorder.1 The most common coagulation factor targeted in autoimmunity is factor VIII. The key feature that distinguishes antibody-mediated from other acquired coagulation factor deficiencies, such as impaired synthesis (e.g., a result of vitamin K deficiency) or increased consumption (e.g., in disseminated intravascular coagulation), is the ability of the patient’s plasma to inhibit the coagulation of normal plasma. Inhibitors also can develop in response to replacement therapy in patients with congenital coagulation factor deficiencies as discussed in Chap. 123.

Acronyms and Abbreviations: APC, activated protein C; aPCC, activated prothrombin complex concentrate; aPTT, activated partial thromboplastin time; BU, Bethesda units; CTLA4, cytotoxic T-lymphocyte associated protein 4; DAMP, damage-associated molecular patters; EACH, European Acquired Hemophilia Registry; FEIBA, factor eight inhibitor bypasssing agent; PAPP, pathogen-asscoiated molecular patterns; rVIIa, recombinant activated factor VII.

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ACQUIRED HEMOPHILIA A DEFINITIONS AND EPIDEMIOLOGY The incidence of autoantibodies to factor VIII, which is the most commonly targeted coagulation factor in autoimmunity, is 1.4 per million people per year.2–4 The associated clinical condition is called acquired hemophilia A. Approximately 40 to 50 percent of acquired hemophilia A patients have underlying conditions, including other autoimmune disorders (e.g., rheumatoid arthritis and systemic lupus erythematosus), malignancy, pregnancy, or a history consistent with a drug reaction.5 The remaining idiopathic cases most commonly occur in elderly patients of either sex with the median age at diagnosis being in the mid-70s.

MECHANISMS OF ANTIBODY DEVELOPMENT Even though adaptive immunity provides a unique ability to recognize a nearly infinite range of antigenic determinants, mechanisms of immunologic tolerance exist that reduce the probability of autoimmunity.6 Self non-self discrimination provides the key foundation upon which immune activity can be specifically directed toward potential pathogens.6 However, self non-self discrimination alone does not possess the capacity to distinguish innocuous antigens from antigens associated with a real threat of infection.7 As a result, an elaborate network of innate immune factors also exist that recognize potential danger in the form of cellular injury or conserved determinants on pathogens themselves, often referred to as damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs), respectively.7,8 Activation of immune cell function following exposure to PAMPs or DAMPs provide the necessary signals required for an efficient immunologic response to foreign antigen.7–9 The development of anti–factor VIII antibodies following factor VIII infusion in individuals with hemophilia A provides a classic example of the deleterious outcome of alloantibody formation following exposure to alloantigen. In this scenario, the factor VIII protein is foreign to the patients; consequently, central tolerance to the factor VIII protein does not occur. In contrast, acquired hemophilia results from loss of previous tolerance to a self antigen.10–12 For alloantibody development in patients with hemophilia A, individual variability in factor VIII levels accounts for some of the divergent level of tolerance to factor VIII observed. However, some individuals with undetectable levels of factor VIII antigen fail to generate factor VIII inhibitors, regardless of factor VIII exposure. Although these individuals would not be predicted to be tolerized to factor VIII, 70 to 80 percent of patients with baseline factor VIII levels of less than 1 percent do not develop an immune response to repeated dosing and are considered tolerized.13–16 For the 20 to 30 percent of patients who develop inhibitors there are both genetic and nongenetic risk factors for inhibitor development. Patients with a positive family history of inhibitors, those who have large factor VIII gene deletions, and nonwhites have a higher risk of inhibitor development.17–20 The non– factor VIII genes—interleukin-10, tumor necrosis factor-α, and cytotoxic T-lymphocyte antigen 4–318 allele—are associated with inhibitor development.17–20 Nongenetic risk factors, such as infusing factor at the time of a “danger” signal (e.g., a surgical procedure), intense factor exposure, and prophylaxis versus no prophylaxis, also are associated with inhibitor development.16 Patients who receive factor at the time of a “danger” signal may experience sufficient tissue injury to provide the necessary immune activation through DAMPs. Furthermore, it remains possible that low grade and potentially clinically undetectable infection may provide low levels of PAMPs that could likewise stimulate anti–factor VIII antibodies following factor VIII exposure. However, while PAMPs and/or DAMPs may provide the important immune activation signals,21,22

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several studies using animal models suggest that significant factor VIII antibody development can occur in the absence of known tissue injury or DAMP exposure.23 Consistent with this, immune activation can occur in the apparent absence of DAMPs or PAMPs toward several model antigens.24 Unique B-cell populations, especially those in the spleen, can rapidly respond to bloodborne antigens in the absence of any identifiable PAMPs or tissue injury, suggesting that these cells may be uniquely poised to respond to factor VIII.25 Consistent with this, in experimental models, splenectomy can significantly inhibit factor VIII inhibitor development following factor VIII exposure,26,27 suggesting that several of these unique B-cell populations may be involved in the development of factor VIII antibodies irrespective of DAMP or PAMP exposure.25,26 Although examples of antigens inducing B-cell activation in the absence of known DAMPs or PAMPs exist, most of these antigens require crosslinking of cell-surface B-cell receptors for efficient activation and therefore reflect highly repetitive antigenic structures.28 In contrast, factor VIII represents a soluble antigen with little inherent predicted crosslinking ability. Most soluble antigen of this type actually induce tolerance following injection, likely because of the inability of soluble monovalent antigens to adequately crosslink and thereby stimulate B-cell receptors. Although factor VIII can exist in a soluble, monovalent form, it remains possible that factor VIII may form complexes with highermolecular-weight species and thus form a network of factor VIII antigens that may serve as a suitable substrate for efficient B-cell receptor crosslinking and subsequent activation. Consistent with this, induction of tolerance to factor VIII by exposure to high levels of factor VIII may partially reflect a saturation of sites for factor VIII complex formation,29 which may, in turn, result in B-cell exposure to high levels of soluble, monovalent factor VIII. However, if this occurs, studies suggest that it likely takes place independent of interactions with von Willebrand factor, the primary binding partner of factor VIII, or its own coagulant activity.30 Clearly, there is much more to learn regarding the immunologic factors responsible for factor VIII inhibitor development. In contrast to generating alloantibodies following factor VIII infusion, some patients generate autoantibodies against factor VIII, which can result in acquired factor VIII deficiency. As coagulation typically occurs at sites of inflammation and injury where DAMPs presumably are generated, tolerance to factor VIII may unfortunately be lost in these settings. Additionally, nonproteolytic and proteolytic degradation of coagulation proteins potentially could present neoepitopes. However, the fact that the development of acquired factor VIII deficiency is rare (1.4 per million population) provides a testimony to the ability of the immune system to discriminate efficiently between infectious non-self from noninfectious self.12 Essentially, nothing is known about the breakdown of tolerance in patients that develop autoantibodies to coagulation factors.

MOLECULAR PATHOLOGY Factor VIII inhibitors in congenital and acquired hemophilia nearly always consist of a polyclonal immunoglobulin (Ig) G population. Although IgG4 accounts for only 5 percent of the total IgG in normal plasma, it usually is a major, but not the sole, component of the anti– factor VIII antibody population.31 IgG4 antibodies do not fix complement, which has been cited as a reason that immune complex disease is not observed in factor VIII inhibitor patients. However, it is more likely that factor VIII simply is not present in sufficient quantity to form enough immune complex deposition to mediate tissue damage. Factor VIII contains a sequence of domains designated A1-A2B-ap-A3-C1-C2 (Chap. 123). During the activation of factor VIII by thrombin, the B and ap domains are released, producing an A1/ A2/A3-C1-C2 activated factor VIII heterotrimer.32 Anti–factor VIII

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antibodies in both congenital and acquired hemophilia A inhibitor are primarily directed to the A2 and C2 domains, although antibodies to all domains have been described.33–35 The similarity in the properties of antibodies in congenital and acquired hemophilia, which represent very different immunologic settings, suggests that intrinsic structural features in the factor VIII molecule are an important determinant driving the immune response. Epitope spreading from a single “problem” epitope, which has been implicated in some autoantibody phenomena,36 does not appear to be a property of factor VIII inhibitors because anti–C2 antibodies can occur in the absence of anti–A2 antibodies and vice versa. The only known biologic function of factor VIII is to become proteolytically activated and participate as a cofactor for factor IXa during intrinsic pathway factor X activation on phospholipid membranes. Theoretically, antibodies could inhibit factor VIII procoagulant function in several ways, including blocking the binding of factor VIIIa to factor IXa, factor X, or phospholipid, or by interfering with the proteolytic activation of factor VIII. Some anti-A2 antibodies map to a region bounded by Arg484-Ile50837 and inhibit activated factor VIII by blocking its ability to bind factor X.38 Anti-C2 antibodies bind to the NH2-terminal half of the C2 domain.39 Anti-C2 antibodies have been identified that inhibit the binding of activated factor VIII to phospholipid membranes,40 which is critical for its interaction with the platelet surfaces. However, the C2 domain also apparently contributes to the binding of factor VIII to its activators, thrombin and factor Xa.41–43 Consistent with this, anti–C2 inhibitors have been identified that block factor VIII activation.41,44 Factor VIII inhibitors also have been identified in approximately 20 percent of normal healthy donors.45 These inhibitors inhibit factor VIII activity in pooled normal plasma, but not autologous plasma, indicating that they are not autoantibodies, but rather alloantibodies directed against an unidentified polymorphism. Anti–factor VIII IgG also has been identified in all normal plasmas tested by affinity chromatography on immobilized factor VIII.46 The increased sensitivity of the method is a consequence of its ability to resolve anti–factor VIII antibodies from anti–anti–factor VIII idiotypic antibodies that also are present. Idiotypic regulation has been proposed as a mechanism for controlling autoantibody activity in vivo.47

CLINICAL FEATURES Acquired hemophilia A patients usually present with spontaneous bleeding, which often is severe and life- or limb-threatening, although large cohort studies have shown that approximately 30 percent of patients do not require hemostatic management.2,48 Patients with acquired hemophilia are more likely to have a severe bleeding diathesis than congenital hemophilia A inhibitor patients.49 Additionally, in contrast to patients with congenital hemophilia A, hemarthrosis in these patients is rare. The reasons for these differences is puzzling, especially in light of the fact that the properties of factor VIII inhibitors in the two patient populations is similar. As noted above, inhibitors can block factor VIII function in several ways. Conceivably, unidentified mechanistic differences in inhibitor action account for the difference in clinical severity. Factor VIII inhibitors sometimes resolve spontaneously. However, it is not possible to predict in which subset of patients this will occur.

LABORATORY FEATURES AND DIFFERENTIAL DIAGNOSIS The new onset of an acquired bleeding disorder should immediately lead to screening tests that include an activated partial thromboplastin time (aPTT), a prothrombin time, and a platelet count. Patients with acquired hemophilia A have a prolonged aPTT resulting from decreased

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or absent factor VIII activity in the intrinsic pathway of blood coagulation. The autoantibody inhibits the factor VIII in the plasma from normal individuals, which forms the basis of mixing study that is used to screen for inhibitors. The presence of a prolonged aPTT in a mixing study establishes the diagnosis of a circulating anticoagulant. Specific factor assays then are performed to determine whether a specific coagulation factor inhibitor or a lupus anticoagulant is present. The activity of other intrinsic pathway coagulation factors may be decreased in the presence of high titer factor VIII inhibitors. However, the levels of these factors normalize at increasing dilutions of patient plasma, whereas factor VIII activity remains decreased. Once the identity of an inhibitor has been established, its titer is determined using the Bethesda assay.50 Inhibitors frequently take minutes to hours to maximally inhibit factor VIII. Therefore, dilutions of patient plasma are preincubated with normal plasma for 2 hours at 37°C. The inhibitor titer is defined as the dilution of patient plasma that produces 50 percent inhibition of the factor VIII activity and is expressed in Bethesda units per milliliter (BU/mL). Inhibitors are classified informally as low titer or high titer when the titers are less than 5 BU/mL or greater than 5 to 10 BU/mL, respectively. The Bethesda assay has been modified by the addition of 0.1 M imidazole, pH 7.4, and by diluting test plasma into factor VIII–deficient plasma during the preincubation phase to prevent assay variation resulting from pH changes and adsorptive losses of factor VIII.51 This “Nijmegen” modification of the Bethesda assay decreases false-positive low-titer inhibitors.52 Patients with acquired hemophilia often have measurable residual factor VIII activity. This activity may cause an underestimate of the inhibitory titer. Preanalytical heat treatment has been proposed as a simple way to denature factor VIII to allow for more accurate determination of titer in both patients with acquired hemophilia A and patients with congenital hemophilia A who may have infused factor VIII.53,54 Factor VIII inhibitors are classified based on the kinetics and extent of inactivation of factor VIII in plasma.55 Type I inhibitors follow second-order kinetics and inactivate factor VIII completely, which would be expected for a simple bimolecular antigen-antibody reaction. Type II inhibitors inactivate factor VIII incompletely and display more complex kinetics of inhibition. Hemophilia A inhibitor patients and acquired hemophilia A patients tend to have type I and type II inhibitors, respectively.56 However, the borderline between type I and type II inhibitors is not always clear and the distinction is not useful clinically. Additionally in a recent observational study of patients with acquired hemophilia in the United Kingdom, factor VIII levels and inhibitor titers at presentation were not predictive of the severity of bleeding events. The median factor VIII level and inhibitory titers were nearly identical for patients with fatal bleeding events compared to those who did not require treatment for their bleeding symptoms.2

TREATMENT The severe bleeding that often is the presenting feature of this disorder requires urgent action to establish a diagnosis and initiate therapeutic measures. Ideally, this is carried out in a setting where factor VIII inhibitors can be identified and quantitated and where there is subspecialty expertise in the management of bleeding disorders. Invasive procedures should be performed only if absolutely necessary and venipuncture should be kept to a minimum given the risk of significant bleeding.57 Treatment of patients with acquired hemophilia A depends on the inhibitor titer. Although no prospective trials are available, clinical experience indicates that patients with a factor VIII inhibitor titer of less than 5 BU/mL often are treated successfully with sufficient doses of recombinant or plasma-derived factor VIII to neutralize the inhibitor. Patients with titers between 5 and 10 BU/mL also may respond to factor VIII,

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whereas those with titers greater than 10 BU/mL generally do not respond. Formulas exist to calculate the amount of factor VIII needed to treat a patient, but these are rough estimates at best. The efficacy of factor VIII concentrates was lower than that of bypassing agents in a large registry study, which was likely secondary to challenges in appropriately dosing the factor VIII concentrate.48 Desmopressin can be administered by intravenous, subcutaneous, or intranasal routes and results in an increase in plasma von Willebrand factor levels and factor VIII activity.58 Its potential use is in patients with baseline factor VIII levels greater than 5 IU/dL and minor bleeding. However, like factor VIII concentrates, response is not predictable and close monitoring of hemostatic efficacy and factor VIII levels is needed. Factor VIII bypassing agents, which drive the coagulation mechanism through the extrinsic pathway, are the mainstays of management of patients with a high titer of an inhibitor. Two agents, recombinant activated factor VII (rFVIIa; NovoSeven RT) and plasma-derived antiinhibitor coagulant complex (AICC; FEIBA VH Immuno, also called activated prothrombin complex concentrate [aPCC]) are commercially available and approved by the U.S. Food and Drug Administration for treatment of acquired hemophilia A. Although no comparative trials have been done, analysis of the European Acquired Haemophilia (EACH2) Registry showed similar hemostatic efficacy between rFVIIa and aPCC at approximately 90 percent.48 Similar hemostatic efficacy between rFVIIa and aPCC has been seen in the treatment of congenital hemophilia A with inhibitors.59 The recommended dose range of rFVIIa for the treatment of patients with acquired hemophilia is 70 to 90 mcg/kg repeated every 2 to 3 hours until hemostasis is achieved. aPCC is given at doses of 50 to 100 U/kg every 8 to 12 hours, but should not exceed 200 U/kg per day. Lower doses (50 to 75 U/kg) are used for mild bleeding, whereas higher doses (100 U/kg) are given for severe limb or life-threatening bleeding. Treatment should be continued until there are clear signs of clinical improvement. Although there are similar rates of efficacy between the two available bypassing agents, not all patients respond. Additionally, there are no widely accepted methods available for predicting response to therapy or monitoring patients on therapy. The use of thromboelastography and the thrombin generation assays as a predictor of response to therapy in congenital hemophilia A and inhibitors has been reported but large clinical studies linking clinical data to outcome are lacking, leaving clinical response as the only available monitoring option.60 The major serious adverse event associated with bypassing agents is thrombosis. The EACH2 Registry reported similar rates in patients treated with rFVIIa or aPCC.48 However, the risk of thrombosis is considered low when used for approved indications at the recommended doses. The incidence of thrombosis in patients with acquired hemophilia A treated with bypassing agents appears higher than that for patients with congenital hemophilia. This is probably because of cardiovascular risk factors in the acquired hemophilia population given their age and associated medical conditions. Escalating doses of either bypassing agent or combination of the two agents should be done with caution, especially in older patients. Factor VIII inhibitors usually cross-react poorly with porcine factor VIII.61 A commercial plasma-derived porcine factor VIII concentrate was useful in the treatment of factor VIII inhibitor patients for approximately 20 years,62 but was discontinued in 2004 because of viral contamination of the product. Porcine factor VIII has the advantage of potentially being guided by laboratory monitoring of recovery of factor VIII activity in plasma. However, the development of antiporcine factor VIII antibodies may preclude its long-term use. A phase II/III clinical trial of a recombinant porcine factor VIII product has been completed in patients with acquired hemophilia A63 and a phase II trial has been completed in congenital hemophilia inhibitor patients.64

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Although acquired inhibitors may remit spontaneously, fatal bleeding may occur up to several months after the initial diagnosis, even in patients who present with mild bleeding. Therefore, immunosuppressive therapy at the time of diagnosis to eradicate the inhibitor is recommended.57 A variety of immunosuppressive agents have been used, including cyclophosphamide, azathioprine, cyclosporine, intravenous immunoglobulin, and rituximab. Immune tolerance induction using human factor VIII similar to what is done for patients with congenital hemophilia A and inhibitors has been used successfully. Additionally, plasmapheresis and immunoadsorption of the inhibitory antibody have been used. First-line immunosuppressive regimens at many centers consist of glucocorticoids alone or glucocorticoids combined with cyclophosphamide.65 No appropriately powered randomized studies have been performed, so the information available is from a single small randomized study, case reports, national surveys, and large registry data. The single randomized trial of 31 patients comparing prednisone and cyclophosphamide showed no difference in the treatment arms. A national registry study also showed no difference with 76 percent of patients achieving complete remission in the steroid arm and 78 percent in the steroids plus cytotoxic agent arm.66 The EACH2 Registry has the largest reported experience with 331 patients and reported a higher rate of stable complete remission at 70 percent for patients treated with steroids and cyclophosphamide compared with 48 percent for steroids alone and 59 percent for rituximab containing regimens. Extensive analysis to control for potential confounding factors in this non-randomized study confirmed that stable complete remission was more likely with a steroid and cyclophosphamide than steroids alone (odds ratio of 3.25). The median time to remission was 5 weeks in patients treated with steroids alone or steroids and cyclophosphamide and 10 weeks in patients treated with rituximab. There have been no studies that have shown a difference in long-term outcomes including survival and sustained remission.57,67 The rarity of this disease, the severity of bleeding at onset, and the delay in diagnosis of these patients has all contributed to the lack of controlled trials. Given the lack of controlled trial clinical management decisions are guided from the limited data available and clinical judgment.

 CQUIRED ANTIBODIES TO OTHER A COAGULATION FACTORS ANTI–FACTOR V AND ANTITHROMBIN ANTIBODIES Thrombin and factor V inhibitors are discussed together because of their frequent coexistence in immune responses to commercial products that contain thrombin. Thrombin products have been used widely in surgical and endoscopic procedures. It has been estimated that more than 500,000 patients are treated annually with products containing thrombin.68 Thrombin is used either alone or as a component of fibrin sealants, which consist of fibrinogen and thrombin preparations that are mixed together at the wound site to form a topical fibrin clot.69 Additionally, factor XIII sometimes is added to crosslink and stabilize the clot. Fibrin sealants contain thrombin and fibrinogen derived from human plasma, whereas stand-alone thrombin products are prepared from bovine plasma. Both types of products are heavily contaminated with other plasma proteins, including factor V and prothrombin.70,71 Almost all patients exposed to bovine proteins develop a detectable immune response. In half of these patients antibovine antibodies

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cross-react with human thrombin, factor V, or prothrombin.68 Usually, these antibodies are subclinical.72 However, mild to life-threatening hemorrhage can occur, especially if the titer of anti–human factor V antibodies is high. The risk of bleeding is higher in patients who receive bovine thrombin products more than once because of the development of a secondary immune response. There have been no clinical trials comparing the safety and efficacy of fibrin sealants to stand-alone thrombin products. Because fibrin sealants are composed mainly of human proteins, they may be less immunogenic. However, anti–factor V antibodies have been reported in a patient receiving fibrin sealant.73 There currently is no stand-alone human thrombin product. It seems likely that the development of highly purified plasma-derived or recombinant products containing human thrombin in the presence or absence of human fibrinogen would decrease the incidence of antithrombin and anti–factor V antibodies.70 Autoantibodies to thrombin are rare. However, the mechanisms of action of antithrombin antibodies have been studied extensively because of the wealth of information about thrombin structure and function.74–77 In contrast, approximately half of the 105 cases of inhibitory anti–factor V antibodies reported and reviewed between 1955 and 1997 appeared to be autoantibodies not associated with the exposure to bovine thrombin products.72 β-Lactam antibiotics also are associated with anti–factor V autoantibodies and may partly explain the increased incidence with surgery. In approximately 20 percent of cases of autoantibody formation, no underlying disease was identified. Anti–factor V autoantibodies have been identified rarely in patients with autoimmune diseases, solid tumors, and monoclonal gammopathies. In addition to autoantibody formation, alloantibodies to factor V have developed in patients with severe factor V deficiency in response to replacement therapy with fresh-frozen plasma. Patients with inhibitory antibodies to factor V have prolonged prothrombin and aPTT, low factor V levels, and a normal thrombin time. The diagnosis of a factor V inhibitor is based on the specific loss of factor V coagulant activity when patient and normal plasma are mixed in a coagulation assay. The antibody titer can be defined as in the factor VIII Bethesda assay as the dilution of test plasma that produces 50 percent inhibition of factor V activity. Not all patients with factor V inhibitors have hemorrhagic manifestations. Factor V inhibitors anecdotally produce a less serious bleeding disorder than factor VIII inhibitors. The relationship between inhibitor titer and bleeding has not been studied. The reported incidence of bleeding has been higher in patients with autoantibodies to factor V compared to anti–factor V antibodies in patients receiving bovine thrombin. However, this may reflect a bias resulting from the reason the patient sought medical attention. Factor V contains an A1-A2-B-A3-C1-C2 domain structure that is homologous to factor VIII. Also, like factor VIII, the N-terminal half of the factor V C2 domain contains a phospholipid-binding site78that is necessary for normal procoagulant function79 and is targeted by factor V inhibitors.80,81

ANTIPROTHROMBIN ANTIBODIES Antiprothrombin antibodies are most commonly associated with the antiphospholipid syndrome. The antiphospholipid syndrome is caused by lupus anticoagulants, which are defined as antibodies that produce phospholipid-dependent prolongation of in vitro coagulation assays. Anionic phospholipids participate as cofactors for the lupus anticoagulant binding to protein antigens, primarily β2-glycoprotein I82 and prothrombin.83 The antibody–antigen complexes compete for the binding of coagulation factors to the phospholipid present in coagulation assays and produce the lupus anticoagulant phenomenon.

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The role of prothrombin in the generation of lupus anticoagulant activity initially was suggested from studies of a bleeding patient with severe hypoprothrombinemia. However, in the absence of hypoprothrombinemia, lupus anticoagulants do not produce a bleeding diathesis and bleeding in patients with lupus anticoagulants is uncommon.84 Antiprothrombin antibodies are associated with an increased incidence of thrombosis in these patients.85 In patients with antiprothrombin antibodies and hypoprothrombinemia, precipitating, noninhibitory antibodies are present and prothrombin antigen levels are low, indicating that the hypoprothrombinemia is the result of rapid clearance of antigen–antibody complexes.86 However, most patients with lupus anticoagulants have demonstrable antiprothrombin antibodies but do not have hypoprothrombinemia.87 Thus, antibody-mediated hypoprothrombinemia appears to represent a relatively uncommon evolution of the autoimmune response to prothrombin in patients with lupus anticoagulants.

ANTIBODIES TO COMPONENTS OF THE PROTEIN C SYSTEM An acquired inhibitor to protein C associated with a fatal thrombotic disorder has been reported,88 but evidently is rare. In contrast, there is a relatively high prevalence of pathogenic anti-protein S antibodies. Inhibitory antibodies to protein S were detected in five of 15 patients with acquired protein S deficiency.89 Anti–protein S antibodies, but not antibodies to cardiolipin, β2-glycoprotein I, prothrombin, or protein C, appear to be a risk factor for acquired activated protein C (APC) resistance, defined as APC resistance in the absence of the factor V Leiden mutation, and for deep venous thrombosis.90 Additionally, antibodies to the endothelial cell protein receptor have been identified that are associated with fetal death in patients with the antiphospholipid syndrome.91

ACQUIRED ANTIBODIES TO OTHER COAGULATION FACTORS Clinically significant antibodies to coagulation factors other than factor VIII, factor V, and prothrombin that produce acquired bleeding disorders are sufficiently rare that they merit case reports, which are only incompletely listed here. In contrast to acquired hemophilia A, acquired hemophilia B is extremely rare.92,93 Patients with antifibrinogen antibodies have been identified either who are asymptomatic with abnormal laboratory values94or who have abnormal bleeding.95 Patients with abnormal bleeding associated with acquired inhibitors to factor VII,96 factor X,97 factor XI,98 or factor XIII98–107 also have been described. The development of alloantibodies against von Willebrand factor occurs in patients with type 3 von Willebrand disease in response to treatment with plasma concentrates that contain von Willebrand factor.108 Acquired von Willebrand disease can be caused by adsorption of von Willebrand factor to tumor cells, loss of high-molecular-weight von Willebrand factor multimers at as well as by autoantibodies to von Willebrand factor.109

REFERENCES 1. Margolius A Jr, Jackson DP, Ratnoff OD: Circulating anticoagulants: A study of 40 cases and a review of the literature. Medicine (Baltimore) 40:145–202, 1961. 2. Collins PW, Hirsch S, Baglin TP, et al: Acquired hemophilia A in the United Kingdom: A 2-year national surveillance study by the United Kingdom Haemophilia Centre Doctors’ Organisation. Blood 109(5):1870–1877, 2007. 3. Borg JY, Guillet B, Le Cam-Duchez V, et al: Outcome of acquired haemophilia in France: The prospective SACHA (Surveillance des Auto antiCorps au cours de l’Hemophilie Acquise) registry. Haemophilia 19(4):564–570, 2013. 4. Knoebl P, Marco P, Baudo F, et al: Demographic and clinical data in acquired hemophilia A: Results from the European Acquired Haemophilia Registry (EACH2). J Thromb Haemost 10(4):622–631, 2012.

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5. Green D, Lechner K: A survey of 215 non-hemophilic patients with inhibitors to Factor VIII. Thromb Haemost 45(3):200–203, 1981. 6. Hogquist KA, Baldwin TA, Jameson SC: Central tolerance: Learning self-control in the thymus. Nat Rev Immunol 5(10):772–782, 2005. 7. Janeway CA Jr, Medzhitov R: Innate immune recognition. Annu Rev Immunol 20: 197–216, 2002. 8. Matzinger P: Tolerance, danger, and the extended family. Annu Rev Immunol 12: 991–1045, 1994. 9. Rubtsov AV, Swanson CL, Troy S, et al: TLR agonists promote marginal zone B cell activation and facilitate T-dependent IgM responses. J Immunol 180(6):3882–3888, 2008. 10. Lollar P: Pathogenic antibodies to coagulation factors. Part one: Factor VIII and factor IX. J Thromb Haemost 2(7):1082–1095, 2004. 11. Dunn AL, Abshire TC: Current issues in prophylactic therapy for persons with hemophilia. Acta Haematol 115(3–4):162–171, 2006. 12. Franchini M, Lippi G: Acquired factor VIII inhibitors. Blood 112(2):250–255, 2008. 13. White GC 2nd, Kempton CL, Grimsley A, et al: Cellular immune responses in hemophilia: Why do inhibitors develop in some, but not all hemophiliacs? J Thromb Haemost 3(8):1676–1681, 2005. 14. Lorenzo JI, Lopez A, Altisent C, Aznar JA: Incidence of factor VIII inhibitors in severe haemophilia: The importance of patient age. Br J Haematol 113(3):600–603, 2001. 15. Lusher JM, Arkin S, Abildgaard CF, Schwartz RS: Recombinant factor VIII for the treatment of previously untreated patients with hemophilia A. Safety, efficacy, and development of inhibitors. Kogenate Previously Untreated Patient Study Group. N Engl J Med 328(7):453–459, 1993. 16. Gouw SC, van der Bom JG, Marijke van den Berg H: Treatment-related risk factors of inhibitor development in previously untreated patients with hemophilia A: The CANAL cohort study. Blood 109(11):4648–4654, 2007. 17. Astermark J, Berntorp E, White GC, et al: The Malmo International Brother Study (MIBS): Further support for genetic predisposition to inhibitor development in hemophilia patients. Haemophilia 7(3):267–272, 2001. 18. Astermark J, Oldenburg J, Escobar M, et al: The Malmo International Brother Study (MIBS). Genetic defects and inhibitor development in siblings with severe hemophilia A [see comment]. Haematologica 90(7):924–931, 2005. 19. Goodeve A: The incidence of inhibitor development according to specific mutations— and treatment? [review] [8 refs]. Blood Coagul Fibrinolysis 14 Suppl 1:S17–S21, 2003. 20. Oldenburg J, Schroder J, Brackmann HH, et al: Environmental and genetic factors influencing inhibitor development [review] [44 refs]. Semin Hematol 41(1 Suppl 1): 82–88, 2004. 21. Hendrickson JE, Desmarets M, Deshpande SS, et al: Recipient inflammation affects the frequency and magnitude of immunization to transfused red blood cells. Transfusion 46(9):1526–1536, 2006. 22. Hendrickson JE, Chadwick TE, Roback JD, et al: Inflammation enhances consumption and presentation of transfused RBC antigens by dendritic cells. Blood 110(7): 2736–2743, 2007. 23. Meeks SL, Healey JF, Parker ET, et al: Antihuman factor VIII C2 domain antibodies in hemophilia A mice recognize a functionally complex continuous spectrum of epitopes dominated by inhibitors of factor VIII activation. Blood 110(13):4234–4242, 2007. 24. Stowell SR, Henry KL, Smith NH, et al: Alloantibodies to a paternally derived RBC KEL antigen lead to hemolytic disease of the fetus/newborn in a murine model. Blood 122(8):1494–1504, 2013. 25. Martin F, Kearney JF: Marginal-zone B cells. Nat Rev Immunol 2(5):323–335, 2002. 26. Navarrete A, Dasgupta S, Delignat S, et al: Splenic marginal zone antigen-presenting cells are critical for the primary allo-immune response to therapeutic factor VIII in hemophilia A. J Thromb Haemost 7(11):1816–1823, 2009. 27. Zhang AH, Skupsky J, Scott DW: Effect of B-cell depletion using anti-CD20 therapy on inhibitory antibody formation to human FVIII in hemophilia A mice. Blood 117(7):2223–2226, 2011. 28. Bachmann MF, Rohrer UH, Kundig TM, et al: The influence of antigen organization on B cell responsiveness. Science 262(5138):1448–1451, 1993. 29. Kempton CL, White GC 2nd: How we treat a hemophilia A patient with a factor VIII inhibitor. Blood 113(1):11–17, 2009. 30. Meeks SL, Cox CL, Healey JF, et al: A major determinant of the immunogenicity of factor VIII in a murine model is independent of its procoagulant function. Blood 120(12):2512–2520, 2012. 31. Hoyer LW, Gawryl MS, de la Fuente B: Immunochemical characterization of factor VIII inhibitors. Prog Clin Biol Res 150:73–85, 1984. 32. Lollar P, Parker CG: Subunit structure of thrombin-activated porcine factor VIII. Biochemistry 28(2):666–674, 1989. 33. Fulcher CA, de Graaf Mahoney S, Roberts JR, et al: Localization of human factor FVIII inhibitor epitopes to two polypeptide fragments. Proc Natl Acad Sci U S A 82(22): 7728–7732, 1985. 34. Prescott R, Nakai H, Saenko EL, et al: The inhibitor antibody response is more complex in hemophilia A patients than in most nonhemophiliacs with factor VIII autoantibodies. Recombinate and Kogenate Study Groups. Blood 89(10):3663–3671, 1997. 35. Scandella D, Mattingly M, de Graaf S, Fulcher CA: Localization of epitopes for human factor VIII inhibitor antibodies by immunoblotting and antibody neutralization. Blood 74(5):1618–1626, 1989. 36. James JA, Harley JB: B-cell epitope spreading in autoimmunity. Immunol Rev 164: 185–200, 1998.

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37. Healey JF, Barrow RT, Tamim HM, et al: Residues Glu2181-Val2243 contain a major determinant of the inhibitory epitope in the C2 domain of human factor VIII. Blood 92(10):3701–3709, 1998. 38. Lollar P, Parker ET, Curtis JE, et al: Inhibition of human factor VIIIa by anti-A2 subunit antibodies. J Clin Invest 93(6):2497–2504, 1994. 39. Healey JF, Lubin IM, Nakai H, et al: Residues 484–508 contain a major determinant of the inhibitory epitope in the A2 domain of human factor VIII. J Biol Chem 270(24):14505–14509, 1995. 40. Arai M, Scandella D, Hoyer LW: Molecular basis of factor VIII inhibition by human antibodies. Antibodies that bind to the factor VIII light chain prevent the interaction of factor VIII with phospholipid. J Clin Invest 83(6):1978–1984, 1989. 41. Nogami K, Shima M, Hosokawa K, et al: Factor VIII C2 domain contains the thrombin-binding site responsible for thrombin-catalyzed cleavage at Arg1689. J Biol Chem 275(33):25774–25780, 2000. 42. Nogami K, Shima M, Hosokawa K, et al: Role of factor VIII C2 domain in factor VIII binding to factor Xa. J Biol Chem 274(43):31000–31007, 1999. 43. Saenko EL, Shima M, Rajalakshmi KJ, Scandella D: A role for the C2 domain of factor VIII in binding to von Willebrand factor. J Biol Chem 269(15):11601–11605, 1994. 44. Meeks SL, Healey JF, Parker ET, et al: Nonclassical anti-C2 domain antibodies are present in patients with factor VIII inhibitors. Blood 112(4):1151–1153, 2008. 45. Algiman M, Dietrich G, Nydegger UE, et al: Natural antibodies to factor VIII (antihemophilic factor) in healthy individuals. Proc Natl Acad Sci U S A 89(9):3795–3799, 1992. 46. Gilles JG, Saint-Remy JM: Healthy subjects produce both anti-factor VIII and specific anti-idiotypic antibodies. J Clin Invest 94(4):1496–1505, 1994. 47. Guilbert B, Dighiero G, Avrameas S: Naturally occurring antibodies against nine common antigens in human sera. I. Detection, isolation and characterization. J Immunol 128(6):2779–2787, 1982. 48. Baudo F, Collins P, Huth-Kuhne A, et al: Management of bleeding in acquired hemophilia A: Results from the European Acquired Haemophilia (EACH2) Registry. Blood 120(1):39–46, 2012. 49. Ludlam CA, Morrison AE, Kessler C: Treatment of acquired hemophilia. Semin Hematol 31(2 Suppl 4):16–19, 1994. 50. Kasper CK, Aledort L, Aronson D, et al: Proceedings: A more uniform measurement of factor VIII inhibitors. Thromb Diath Haemorrh 34(2):612, 1975. 51. Verbruggen B, Novakova I, Wessels H, et al: The Nijmegen modification of the Bethesda assay for factor VIII:C inhibitors: Improved specificity and reliability. Thromb Haemost 73(2):247–251, 1995. 52. Giles AR, Verbruggen B, Rivard GE, et al: A detailed comparison of the performance of the standard versus the Nijmegen modification of the Bethesda assay in detecting factor VIII:C inhibitors in the haemophilia A population of Canada. Association of Hemophilia Centre Directors of Canada. Factor VIII/IX Subcommittee of Scientific and Standardization Committee of International Society on Thrombosis and Haemostasis. Thromb Haemost 79(4):872–875, 1998. 53. Batty P, Platton S, Bowles L, et al: Pre-analytical heat treatment and a FVIII ELISA improve factor VIII antibody detection in acquired haemophilia A. Br J Haematol 166(6):953–956, 2014. 54. Soucie JM, Miller CH, Kelly FM, et al: A study of prospective surveillance for inhibitors among persons with haemophilia in the United States. Haemophilia 20(2):230–237, 2014. 55. Biggs R, Austen DE, Denson KW, et al: The mode of action of antibodies which destroy factor VIII. II. Antibodies which give complex concentration graphs. Br J Haematol 23(2):137–155, 1972. 56. Hoyer LW, Scandella D: Factor VIII inhibitors: Structure and function in autoantibody and hemophilia A patients. Semin Hematol 31(2 Suppl 4):1–5, 1994. 57. Collins PW, Chalmers E, Hart DP, et al: Diagnosis and treatment of factor VIII and IX inhibitors in congenital haemophilia: (4th edition). UK Haemophilia Centre Doctors Organization. Br J Haematol 160(2):153–170, 2013. 58. Franchini M, Lippi G: The use of desmopressin in acquired haemophilia A: A systematic review. Blood Transfus 9(4):377–382, 2011. 59. Astermark J, Donfield SM, DiMichele DM, et al: A randomized comparison of bypassing agents in hemophilia complicated by an inhibitor: The FEIBA NovoSeven Comparative (FENOC) Study. Blood 109(2):546–551, 2007. 60. Young G, Sorensen B, Dargaud Y, et al: Thrombin generation and whole blood viscoelastic assays in the management of hemophilia: Current state of art and future perspectives. Blood 121(11):1944–1950, 2013. 61. Brettler DB, Forsberg AD, Levine PH, et al: The use of porcine factor VIII concentrate (Hyate:C) in the treatment of patients with inhibitor antibodies to factor VIII. A multicenter US experience. Arch Intern Med 149(6):1381–1385, 1989. 62. Hay CR: Porcine factor VIII: Past, present and future. Haematologica 85(10 Suppl): 21–24; discussion 24–25, 2000. 63. Kruse-Jarres R, St-Louis J, Greist A, et al: Efficacy and safety of OBI-1, an antihaemophilic factor VIII (recombinant), porcine sequence, in subjects with acquired haemophilia A. Haemophilia 21(2):162–170,2015. 64. Kempton CL, Abshire TC, Deveras RA, et al: Pharmacokinetics and safety of OBI-1, a recombinant B domain-deleted porcine factor VIII, in subjects with haemophilia A. Haemophilia 18(5):798–804, 2012. 65. Collins P, Baudo F, Huth-Kuhne A, et al: Consensus recommendations for the diagnosis and treatment of acquired hemophilia A. BMC Res Notes 3:161, 2010.

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66. Green D, Rademaker AW, Briet E: A prospective, randomized trial of prednisone and cyclophosphamide in the treatment of patients with factor VIII autoantibodies. Thromb Haemost 70(5):753–757, 1993. 67. Collins P, Baudo F, Knoebl P, et al: Immunosuppression for acquired hemophilia A: Results from the European Acquired Haemophilia Registry (EACH2). Blood 120(1): 47–55, 2012. 68. Schoenecker JG, Johnson RK, Lesher AP, et al: Exposure of mice to topical bovine thrombin induces systemic autoimmunity. Am J Pathol 159(5):1957–1969, 2001. 69. Ortel TL, Charles LA, Keller FG, et al: Topical thrombin and acquired coagulation factor inhibitors: Clinical spectrum and laboratory diagnosis. Am J Hematol 45(2): 128–135, 1994. 70. Schoenecker JG, Johnson RK, Fields RC, et al: Relative purity of thrombin-based hemostatic agents used in surgery. J Am Coll Surg 197(4):580–590, 2003. 71. Zehnder JL, Leung LL: Development of antibodies to thrombin and factor V with recurrent bleeding in a patient exposed to topical bovine thrombin. Blood 76(10): 2011–2016, 1990. 72. Knobl P, Lechner K: Acquired factor V inhibitors. Baillieres Clin Haematol 11(2): 305–318, 1998. 73. Caers J, Reekmans A, Jochmans K, et al: Factor V inhibitor after injection of human thrombin (tissucol) into a bleeding peptic ulcer. Endoscopy 35(6):542–544, 2003. 74. Arnaud E, Lafay M, Gaussem P, et al: An autoantibody directed against human thrombin anion-binding exosite in a patient with arterial thrombosis: Effects on platelets, endothelial cells, and protein C activation. Blood 84(6):1843–1850, 1994. 75. La Spada AR, Skalhegg BS, Henderson R, et al: Brief report: Fatal hemorrhage in a patient with an acquired inhibitor of human thrombin. N Engl J Med 333(8):494–497, 1995. 76. Lian F, He L, Colwell NS, et al: Anticoagulant activities of a monoclonal antibody that binds to exosite II of thrombin. Biochemistry 40(29):8508–8513, 2001. 77. Sie P, Bezeaud A, Dupouy D, et al: An acquired antithrombin autoantibody directed toward the catalytic center of the enzyme. J Clin Invest 88(1):290–296, 1991. 78. Macedo-Ribeiro S, Bode W, Huber R, et al: Crystal structures of the membrane-binding C2 domain of human coagulation factor V. Nature 402(6760):434–439, 1999. 79. Ortel TL, Devore-Carter D, Quinn-Allen M, Kane WH: Deletion analysis of recombinant human factor V. Evidence for a phosphatidylserine binding site in the second C-type domain. J Biol Chem 267(6):4189–4198, 1992. 80. Izumi T, Kim SW, Greist A, et al: Fine mapping of inhibitory anti-factor V antibodies using factor V C2 domain mutants. Identification of two antigenic epitopes involved in phospholipid binding. Thromb Haemost 85(6):1048–1054, 2001. 81. Ortel TL, Moore KD, Quinn-Allen MA, et al: Inhibitory anti-factor V antibodies bind to the factor V C2 domain and are associated with hemorrhagic manifestations. Blood 91(11):4188–4196, 1998. 82. McNeil HP, Simpson RJ, Chesterman CN, Krilis SA: Anti-phospholipid antibodies are directed against a complex antigen that includes a lipid-binding inhibitor of coagulation: Beta 2-glycoprotein I (apolipoprotein H). Proc Natl Acad Sci U S A 87(11): 4120–4124, 1990. 83. Fleck RA, Rapaport SI, Rao LV: Anti-prothrombin antibodies and the lupus anticoagulant. Blood 72(2):512–519, 1988. 84. Feinstein DI, Rapaport SI: Acquired inhibitors of blood coagulation. Prog Hemost Thromb 1:75–95, 1972. 85. Lakos G, Kiss E, Regeczy N, et al: Antiprothrombin and antiannexin V antibodies imply risk of thrombosis in patients with systemic autoimmune diseases. J Rheumatol 27(4):924–929, 2000. 86. Bajaj SP, Rapaport SI, Fierer DS, et al: A mechanism for the hypoprothrombinemia of the acquired hypoprothrombinemia-lupus anticoagulant syndrome. Blood 61(4): 684–692, 1983. 87. Edson JR, Vogt JM, Hasegawa DK: Abnormal prothrombin crossed-immunoelectrophoresis in patients with lupus inhibitors. Blood 64(4):807–816, 1984. 88. Mitchell CA, Rowell JA, Hau L, et al: A fatal thrombotic disorder associated with an acquired inhibitor of protein C. N Engl J Med 317(26):1638–1642, 1987. 89. Sorice M, Arcieri P, Griggi T, et al: Inhibition of protein S by autoantibodies in patients with acquired protein S deficiency. Thromb Haemost 75(4):555–559, 1996. 90. Nojima J, Kuratsune H, Suehisa E, et al: Acquired activated protein C resistance associated with anti-protein S antibody as a strong risk factor for DVT in non-SLE patients. Thromb Haemost 88(5):716–722, 2002. 91. Hurtado V, Montes R, Gris JC, et al: Autoantibodies against EPCR are found in antiphospholipid syndrome and are a risk factor for fetal death. Blood 104(5):1369–1374, 2004. 92. Boggio LN, Green D: Acquired hemophilia. Rev Clin Exp Hematol 5(4):389–404; quiz following 431, 2001. 93. Krishnamurthy P, Hawche C, Evans G, Winter M: A rare case of an acquired inhibitor to factor IX. Haemophilia 17(4):712–713, 2011. 94. Nawarawong W, Wyshock E, Meloni FJ, et al: The rate of fibrinopeptide B release modulates the rate of clot formation: A study with an acquired inhibitor to fibrinopeptide B release. Br J Haematol 79(2):296–301, 1991. 95. Ruiz-Arguelles A: Spontaneous reversal of acquired autoimmune dysfibrinogenemia probably due to an antiidiotypic antibody directed to an interspecies cross-reactive idiotype expressed on antifibrinogen antibodies. J Clin Invest 82(3):958–963, 1988. 96. Aguilar C, Lucia JF, Hernandez P: A case of an inhibitor autoantibody to coagulation factor VII. Haemophilia 9(1):119–120, 2003.

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Chapter 127: Antibody-mediated Coagulation Factor Deficiencies

97. Rao LV, Zivelin A, Iturbe I, Rapaport SI: Antibody-induced acute factor X deficiency: Clinical manifestations and properties of the antibody. Thromb Haemost 72(3): 363–371, 1994. 98. Goodrick MJ, Prentice AG, Copplestone JA, et al: Acquired factor XI inhibitor in chronic lymphocytic leukaemia. J Clin Pathol 45(4):352–353, 1992. 99. Ajzner E, Schlammadinger A, Kerenyi A, et al: Severe bleeding complications caused by an autoantibody against the B subunit of plasma factor XIII: A novel form of acquired factor XIII deficiency. Blood 113(3):723–725, 2009. 100. Daly HM, Carson PJ, Smith JK: Intracerebral haemorrhage due to acquired factor XIII inhibitor—Successful response to factor XIII concentrate. Blood Coagul Fibrinolysis 2(4):507–514, 1991. 101. Fukue H, Anderson K, McPhedran P, et al: A unique factor XIII inhibitor to a fibrin-binding site on factor XIIIA. Blood 79(1):65–74, 1992. 102. Krumdieck R, Shaw DR, Huang ST, et al: Hemorrhagic disorder due to an isoniazidassociated acquired factor XIII inhibitor in a patient with Waldenstrom’s macroglobulinemia. Am J Med 90(5):639–645, 1991. 103. Lopaciuk S, Bykowska K, McDonagh JM, et al: Difference between type I autoimmune inhibitors of fibrin stabilization in two patients with severe hemorrhagic disorder. J Clin Invest 61(5):1196–1203, 1978.

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104. Lorand L, Maldonado N, Fradera J, et al: Haemorrhagic syndrome of autoimmune origin with a specific inhibitor against fibrin stabilizing factor (factor XIII). Br J Haematol 23(1):17–27, 1972. 105. Lorand L, Velasco PT, Murthy SN, et al: Autoimmune antibody in a hemorrhagic patient interacts with thrombin-activated factor XIII in a unique manner. Blood 93(3):909–917, 1999. 106. Lorand L, Velasco PT, Rinne JR, et al: Autoimmune antibody (IgG Kansas) against the fibrin stabilizing factor (factor XIII) system. Proc Natl Acad Sci U S A 85(1):232–236, 1988. 107. Tosetto A, Rodeghiero F, Gatto E, et al: An acquired hemorrhagic disorder of fibrin crosslinking due to IgG antibodies to FXIII, successfully treated with FXIII replacement and cyclophosphamide. Am J Hematol 48(1):34–39, 1995. 108. James PD, Lillicrap D, Mannucci PM: Alloantibodies in von Willebrand disease. Blood 122(5):636–640, 2013. 109. Federici AB: Acquired von Willebrand syndrome: Is it an extremely rare disorder or do we see only the tip of the iceberg? J Thromb Haemost 6(4):565–568, 2008.

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2191

CHAPTER 128

HEMOSTATIC ALTERATIONS IN LIVER DISEASE AND LIVER TRANSPLANTATION

Frank W.G. Leebeek and Ton Lisman

SUMMARY In patients with acute liver failure or chronic liver disease, many changes in the hemostatic system occur. The liver is the site of synthesis of nearly all coagulation factors, both pro- and anticoagulant proteins. A reduced synthesis function of the liver will lead to reduced levels of these factors in circulation. In addition, the liver is involved in the clearance of many activated coagulation factors and protein-inhibitor complexes from the circulation, which, in turn, can lead to activation of the coagulation system if liver function is impaired. Furthermore, the liver is involved in the synthesis and clearance of pro- and antifibrinolytic proteins, which may lead to a shift in the balance of the fibrinolytic system. Also primary hemostasis might be affected in liver disease because of thrombocytopenia and impaired platelet function, which is frequently encountered in these patients. It is evident that patients with liver disease have frequent bleeding episodes, mainly in the gastrointestinal tract, such as variceal bleeding. It has been a longstanding dogma that patients with liver disease are at a high risk of bleeding caused by the above mentioned hemostatic changes. However, in recent years this cause of the bleeding tendency has been questioned because of the concomitant reductions of pro- and anticoagulant factors and pro- and antifibrinolytic factors. More recent studies using more sophisticated coagulation tests showed that thrombin generation is normal in patients with chronic liver failure and that some may even have a prothrombotic phenotype. This led to the development of a model of a rebalanced hemostatic system in these patients, which may have immediate implications for treatment. Hematologists and other clinicians taking care of patients with acute liver failure of chronic liver disease, such as cirrhosis, are still faced with the questions whether these patients need correction of the changes in hemostasis before interventions such as paracentesis, biopsies, dental care, and surgery. It was generally believed that replacement therapy with frozen plasma or prothrombin complex concentrate was indicated. However, based on these new findings, physicians should now be more restrictive in the use of hemostatic agents and blood products in these patients both in liver disease and during liver transplantation.

Acronyms and Abbreviations: ADAMTS13, a disintegrin-like and metalloprotease with thrombospondin domain 13; aPTT, activated partial thromboplastin time; DDAVP, 1-deamino-8-d-arginine vasopressin; DIC, disseminated intravascular coagulation; FFP, fresh-frozen plasma; HAT, hepatic artery thrombosis; INR, international normalized ratio; ISI, international sensitivity index; MELD, model of end-stage liver disease; PAI-1, plasminogen activator inhibitor 1; PFA, platelet function analyzer; PT, prothrombin time; PVT, portal vein thrombosis; TAFI, thrombin-activatable fibrinolysis inhibitor; t-PA, tissue-type plasminogen activator; VWF, von Willebrand factor.

Kaushansky_chapter 128_p2191-2198.indd 2191

The liver plays a central role in the hemostatic system. Liver parenchymal cells are the site of synthesis of most coagulation factors (except factor VIII), the natural inhibitors of coagulation, including protein C, protein S, and antithrombin, and essential components of the fibrinolytic system, such as plasminogen, α2-antiplasmin, and thrombin activatable fibrinolysis inhibitor (TAFI). The liver also regulates hemostasis and fibrinolysis by clearing activated coagulation factors and coagulation factor-inhibitor complexes from the circulation. In addition, changes in primary hemostasis mediated by platelets, von Willebrand factor (VWF) and ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type 1 repeats) may occur. Therefore, when acute or chronic liver dysfunction is present in patients with liver disease, complicated hemostatic derangement may occur, which can lead to bleeding, thrombosis, or neither bleeding nor thrombosis.

 EMOSTATIC ALTERATIONS IN H CHRONIC LIVER DISEASE PRIMARY HEMOSTASIS More than 75 percent of patients with chronic liver disease, especially in moderate to severe cirrhosis (Child B and C) have reduced levels of platelets (5 years), patients with stroke, and patients with thrombotic events that were associated with a provocative factor such as trauma, surgery, stasis, pregnancy, and estrogens.

THROMBOSIS The accumulated evidence from randomized controlled trials indicates that patients with APS and thrombosis should be treated with warfarin for the long-term, and maintained at a therapeutic international normalized ratio (INR) of 2.0 to 3.0.305 Patients with arterial thrombosis may require a higher anticoagulant intensity as a retrospective study showed that a higher intensity (INR >3.0) was necessary for preventing recurrences in this group of patients, but this issue is controversial.306 Two other studies reported no benefit for high-intensity warfarin but the number of patients with arterial thrombosis was not high.299,305 The issue of appropriate antithrombotic treatment of aPL-associated stroke is even more controversial. One major study concluded that there was no benefit for warfarin anticoagulation compared to aspirin therapy.307 For patients treated acutely with intravenous UFH, care must be taken to determine whether the patient has a preexisting LA that can interfere with aPTT monitoring of heparin levels. This problem can be circumvented by using an LA-insensitive aPTT reagent or be avoided by treatment, where appropriate, with a LMWH. An important practical consequence of the LA effect is that prothrombin time and INR results can be artifactually elevated in some patients with APS and LAs treated with warfarin anticoagulant therapy.308 A multicenter study reported that all but one of the commercial thromboplastins in use at nine centers provided acceptable INR values for APS patients with LA.309 New thromboplastins should be checked for their responsiveness to LA prior to their use in monitoring oral anticoagulant treatment in patients with APS. Chromogenic factor X (CFX) assays can be used as an alternative to INR for APS patients, especially in patients with a prolonged baseline prothrombin time prior to initiating warfarin therapy, those who are persistently positive for LA and those patients who continue to have recurrent venous thromboembolism

9/18/15 5:10 PM

Chapter 131: The Antiphospholipid Syndrome

(VTE).310 Therapeutic CFX values range from 20 to 40 percent; thus, a CFX of 40 percent would approximate an INR of 2.0, and a CFX of 20 percent would approximate an INR of 3.0. New oral anticoagulants (NOACs), either direct factor Xa or thrombin inhibitors, are effective for treatment of VTE.311 However, their use specifically in APS patients has not been thoroughly evaluated. In two recent case studies, NOACs have failed to prevent thrombosis in APS patients. Of six APS patients studied, five suffered recurrent VTE and one suffered a recurrent TIA after transitioning to NOACs.312,313 At the time of writing, a prospective randomized controlled trial of warfarin versus rivaroxaban named “RAPS” (Rivaroxaban in Antiphospholipid Syndrome) is ongoing in patients with thrombotic APS (study ISRCTN68222801). Until the trial is concluded (expected completion in 2015), caution should be applied in using NOACs for APS patients. Fibrinolytic treatment has been reported for patients with primary APS and extensive thrombosis of the common femoral and iliac veins extending to the lower vena cava,314 acute ischemic stroke,315 and acute myocardial infarction.316 The antimalarial drug hydroxychloroquine (HCQ) is associated with reduced risk of thrombosis in patients with APS271–273,317–319 and SLE.319–321 The potential effectiveness of this treatment has been supported by an animal model for aPL thrombosis322 and by a recent report that HCQ directly disrupts aPL IgG–β2GPI complexes,323 and also reverses the aPL antibody-mediated disruption of annexin A5 binding on phospholipid bilayers324 and on human placental syncitiotrophoblasts.325 In a longitudinal cohort study consisting of 272 patients with the APS and 152 taking HCQ (17 of 272 patients on warfarin, 203 were on prednisolone, 112 on azathioprine, 38 on aspirin) investigators found fewer thrombotic complications for patients on HCQ (odds ratio [OR] 0.17, 95% confidence interval [CI] 0.07 to 0.44; p 99.9

M

Some

Some

Most

Few

D/nc

No

Rare

Rare

78

70

M: common, usually not clinically significant

N

Some

Some

Most

Rare

D/nc

No

Rare

(Rare)

72

74

N: rare, usually not clinically significant

S

Some

Some

Some Most

V/nc

Some

Yes

Mild – Sev 55

31

s

Few

Most

Few

Most

V/nc

Rare

Yes

No – Sev

89

97

U

—

Most

—

Most

nc/nc

Rare

Yes

Mild–Sev

100

99.7

Lewis

Ii

MNSs

b

SsU: clinically significant autoantibody specificities reported

9/21/15 4:31 PM

(continued )

Part XIII: Transfusion Medicine

Kaushansky_chapter 136_p2327-2352.indd 2344

TABLE 136–5.  Summary of Selected Erythrocyte Antibodies (Continued )

Kaushansky_chapter 136_p2327-2352.indd 2345

Kell

K k

Some

Most

Few

Most

nc/D

Rare

Yes

Mild–Sev

9

2

K: very common immune antibody

—

Most

Rare

Most

nc/D

No

Yes

Mild–Sev

99.9

—

a

—

Most

Rare

Most

nc/D

No

Yes

Mild–Sev

2.3

—

Kpb

—

Most

Rare

Most

nc/D

No

Yes

Mild–Mod >99.9

100

Js

a

—

Most

Rare

Rare

nc/D

No

Yes

Mild–Sev

—

20

Js

b

—

Most

—

—

nc/D

No

Yes

Mild–Sev

>99.9

99

Fya

—

Most

Rare

Most

D/nc

Rare

Yes

Mild–Sev

66

10

Fy

b

—

Most

Rare

Most

D/nc

Rare

Yes

Mild

83

23

a

Few

Most

Rare

Most

I/nc

Yes

Yes

Mild–Mod 77

92

Jkb

Few

Most

Rare

Most

I/nc

Yes

Yes

No–Mild

72

41

Lu

a

Some

Few

Most

Few

nc(V)/D

No

No

No–Mild

7.7

—

Mild RBC destruction

Lub

Some

Some

Few

Most

nc(V)/D

No

Yes

Mild

99.9

—

Lu glycoprotein on placental tissue may adsorb maternal Lu antibodies

Xg

Xga

Some

Most

Rare

Most

D/nc

Some

No

No

64(m)

–

Xga: poor immunogen

89(f )

–

Yt

Kp

Duffy Kidd

Lutheran

Colton

a

Yt

–

Most

No

Most

D(V)/D(V) No

No– Mod

No

99.7

–

Ytb

–

Most

No

Most

D(V)/D

No

No

No

8

–

Ch

Rare

Most

–

Most

D/nc

No

No

No

96

–

Rg

Most

D/nc

No

No

No

98

–

–

Most

–

a

–

Most

Some Most

nc/nc

Some

No– Mod

Mild–Sev

99.9

–

Cob

–

Most

Some Most

nc/nc

Rare

No– Mod

Mild

10

–

Most

–

nc/V

No

No– Mild

No

>99.9

>99.9

Co

Cromer

General group

Diego

Dia

–

Most

Some Most

nc/nc

Rare

Yes

Mild–Sev

Rare

–

Dib

–

Most

No

Most

nc/nc

No

No– Mod

Mild

100

–

Doa

–

Most

No

Most

nc/D(V)

No

Yes

+DAT

67

–

Dob

–

Most

No

Most

nc/D(V)

No

Yes

+DAT

83

–

Dombrock

Most

Fya: common immune antibody Jka: associated with delayed HTR; hemolytic; disappears quickly from serum

Yt: some antibody examples clinically significant, others not Ch/Rg: associated with C4 complement, clinically insignificant antibodies

Dia: antigen found in South American Indians and Asians

Doa Dob: poor immunogens

Chapter 136: Erythrocyte Antigens and Antibodies

Ch/Rg

Jk

Autoantibodies reported

(continued )

2345

9/21/15 4:31 PM

2346

Ig Class Blood Group

Serologic Activity

Implicated in

Antigen Frequency (%)

Antibody

IgM

IgG

RT

37°C AHG

Activates ENZ/DTT Complement HTR

HDFN

Whites Blacks

Hy

–

Most

–

Most

nc(I)/D(V) No

No – Mod

+DAT

>99

– Hy– and Jo(a–): found only in blacks Gy(a–) (Donull) found in eastern Europeans and Japanese

Gya

–

Most

–

Most

nc(I)/D(V) No

No– Moderate

+DAT

>99

–

Joa

–

Most

–

Most

nc(I)/D(V) No

No– Mod

No

>99

–

Most

–

Most

D/nc

Yes

No– Mod

(+DAT)

>99.9

>99.9

Ge: located on glycophorins C and D In: located on CD44 adhesion protein

Gerbich

General group

Indian

Ina

–

Most

–

Most

D/D

No

Yes

(+DAT)

99.9

–

JMH

–

Most

–

Most

D/D

No

No

No

>99.9

>99.9

Knops

Scianna

JMH

Comments

D, destroyed; DTT, dithiothreitol; ENZ, enzyme (papain/ficin); I, increased; nc, no change; V, variable.

Knops antigens associated with CR1 (complement) receptor, clinically insignificant antibodies

Sc1: some antibodies react in serum but not plasma

JMH: carrier protein CDw108

Part XIII: Transfusion Medicine

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TABLE 136–5.  Summary of Selected Antierythrocyte Antibodies (Continued )

9/21/15 4:31 PM

Chapter 136: Erythrocyte Antigens and Antibodies

RBC membrane in vivo, at the lower temperatures in the extremities, and activate the complement cascade in the core of the body. Because such IgM antibodies dissociate from RBCs at higher temperatures, their reactivity may be detected in routine antiglobulin tests (using polyspecific antiglobulin) by virtue of the complement components that remain bound to the red cell membrane.11,16

Immunoglobulin A

IgA is the primary Ig in body secretions, where it exists predominantly as a dimer with a secretory component (Chap. 75). IgA does not cross the placenta or fix complement, but aggregated IgA can activate the alternative pathway of complement, and IgA can trigger cell-mediated events. Multimeric IgA antibodies in serum are seen as hemagglutinins in blood bank tests and most often are associated with anti-A or anti-B.

IMMUNOGLOBULIN IN THE FETUS AND NEWBORN Initially, the fetus acquires low levels of maternal IgG, probably by diffusion across the placenta. These levels rise significantly between 20 and 33 weeks’ gestation as a selective transport system matures and maternal IgG is actively transported across the placenta. Thus, almost all blood group antibodies detected in the fetus and newborn originate from the mother and disappear within the first few months of life. Actual fetal antibody production begins shortly before birth with low levels of IgM, followed by IgG and IgA several weeks after birth. Anti-A and anti-B usually are readily detected by age 2 to 6 months. Because of this late immune response in the newborn and because maternal antibody is so predominant at birth, blood bank standards permit abbreviated testing on neonates younger than 4 months.56 If available, the mother’s serum is used (and preferred) for identifying antibodies in a newborn and for crossmatching RBC components.

NATURALLY OCCURRING ANTIBODIES Naturally Occurring Antibodies in Development

An antibody is said to be naturally occurring when it is found in the serum of an individual who has not been exposed to the antigen through transfusion or pregnancy. These antibodies most likely are heteroagglutinins produced in response to substances in the environment that are similar to those on RBC antigens. Evidence supporting this concept has come from studies on the formation of anti-B in chickens.57 Chicks raised in a normal environment made anti-B within the first 30 days of life, whereas chicks raised in a germ-free environment did not make anti-B by day 60. Naturally occurring anti-A and anti-B in humans, also called isoagglutinins, can increase in titer following ingestion or inhalation of suitable bacteria.58 However, a great many antigens that likely are not present in the environment have been associated with naturally occurring antibodies, so the stimulus for naturally occurring antibodies is not clearly known.

Blood Group Associations and Presence of Naturally Occurring Antibodies

Naturally occurring alloantibodies are commonly associated with the carbohydrate antigens of the ABO, LE, and P1PK blood group systems. Anti-A and anti-B are expected in people who lack the corresponding antigens, as are antibodies specific for H, PP1Pk, or P antigens. Naturally ­occurring antibodies reactive with A1, Lea, Leb, or P1 determinants also are seen frequently. Carbohydrate antigens, especially those with repetitive epitopes, can stimulate B cells to make specific antibody without the aid of helper T cells. Such thymus-independent immune responses typically result in antigen-specific antibodies of the IgM class.

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Within other systems,16 anti-Sda, anti-Vw, and anti-Wra are found in up to 2 percent of normal people. Other, less-common antibody specificities in approximate order of descending occurrence are anti-M, -S, -N, -Ge, -K, -Lua, -Dia, and -Xga. Rh antigens are thought to reside only on RBCs, but apparent naturally occurring anti-D has been reported in 0.15 percent of Rh-negative donors and anti-E in more than 0.1 percent of Rh-positive donors when more sensitive enzyme detection methods are used. Examples of naturally occurring anti-C, anti-CW, and anti-CX also have been described.4–6 Some naturally occurring antibodies exist as autoagglutinins (e.g., anti-H and anti-I). Patients with autoimmune hemolytic anemia can produce many antibodies to low-prevalence antigens with no specific stimulus, in addition to autoantibody.5,6,16,40

Characteristics of Naturally Occurring Alloantibodies

Most naturally occurring antibodies are IgM, but some have an IgG component and a few are predominantly IgG. Some anti-A or anti-B may even be of the IgA class. Antibodies that cause direct agglutination of saline-suspended RBCs most commonly are of the IgM class. However, even IgG antibodies may cause agglutination of RBCs when they bind antigens that are present at high density on the RBC membrane, such as the ABO or MN antigens. With the exception of anti-A and anti-B, most common naturally occurring antibodies do not react at body temperature and are considered clinically insignificant. However, if they are found to react at 37°C, providing crossmatch-compatible blood for transfusion is prudent.

ANTIBODIES GENERATED IN RESPONSE TO IMMUNIZATION: IMMUNE ANTIBODIES Blood Group Associations and Occurrence of Immune Antibodies

Immune antibodies are produced following exposure to foreign RBC antigens through pregnancy or transfusion. The primary immune response is seen several weeks to several months after the first exposure to antigen. IgM usually is associated with early primary responses, but whether it is always the first antibody class made is unclear. In most individuals, IgG soon predominates. This process is characteristic of a thymus-dependent immune response, where T cells help induce B cells to undergo isotype switching from IgM to IgG. In a secondary or anamnestic response, antibody concentration starts to increase several days to several weeks following exposure, and IgG may rise to very high levels. Some IgG antibodies remain detectable for decades after a stimulus. Others, especially Kidd antibodies, can disappear after several months and are more commonly associated with delayed hemolytic transfusion reactions.5,6,16 Immune antibodies are found more commonly in individuals who have been multiply transfused than in multiparous women. This situation occurs because in pregnancy the immunizing dose of red cells often is too small to elicit a primary response and the foreign antigens are limited to those of the father.16 Anti-D used to be the most common immune antibody, but with the advent of Rh matching of donors and recipients in the late 1940s and use of RhIg prophylaxis since the 1970s, its incidence has sharply decreased. Anti-D is present in 0.27 to 0.56 percent of transfusion recipients, 0.10 to 0.20 percent of pregnant women, and 0.16 to 0.25 percent of healthy blood donors.16 In contrast, the occurrence of immune antibodies other than anti-D has increased. Specificities other than anti-D have been reported in approximately 0.6 percent of transfusion recipients, 0.14 percent of pregnant women, and 0.19 percent of healthy blood donors. Pooled data from three 5-year periods and approximately 300,000 patients

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Part XIII: Transfusion Medicine

suggest the absolute occurrence of Rh antibodies other than anti-D is 0.22 percent, other than anti-K is 0.19 percent, other than anti-Fya is 0.05 percent, and other than anti-Jka is 0.04 percent.16 The rate of alloimmunization in patients with sickle cell anemia was 18.6 percent in one survey, and 55 percent of the immunized patients made more than one antibody. The most common specificities were anti-C, anti-E, and anti-K.16

Characteristics of Immune Antibodies

Immune antibodies most often are IgG but may be IgM and sometimes are IgA. Most immune antibodies react at body temperature and are considered clinically significant, except those directed against Bg, Knops, Csa, JMH, and sometimes Yta and Lutheran antigens.

C  LINICAL SIGNIFICANCE OF ERYTHROCYTE ANTIBODIES Information about the clinical significance of alloantibodies is available at www.nybloodcenter.org.59,60

HEMOLYTIC TRANSFUSION REACTIONS Clinically significant antibodies are capable of destroying transfused RBCs. The severity of the reaction varies with antigen density and antibody characteristics. Antibodies commonly associated with intravascular hemolysis include anti-A, anti-B, anti-Jka, and anti-Jkb. ABO incompatibility is the most potent cause of immediate hemolytic reactions because A and B antigens are strongly expressed on RBCs and the antibodies so efficiently bind complement. Kidd antibodies are associated more often with delayed hemolytic reactions because they typically are difficult to detect and can disappear quickly from the circulation. IgG antiJka appears to bind complement only when traces of IgM anti-Jka are present.16 Anti-PP1Pk, anti-Vel, and anti-Lea have been associated with hemolysis, but such examples are rare. Extravascular hemolysis occurs with IgG1 and IgG3 antibodies that react at body temperature; that is, immune antibodies reactive with Rh, Kidd, Kell, Duffy, or Ss antigens. These antibodies make up the bulk of clinically significant antibodies. Antibodies not expected to cause RBC destruction are those that react only at temperatures below 37°C and IgG antibodies of the IgG2 or IgG4 subclass.16

HEMOLYTIC DISEASE OF THE FETUS AND NEWBORN HDFN is caused by blood group incompatibility between a sensitized mother and her antigen-positive fetus (Chap. 55). The antibodies most significant in HDFN are those that cross the placenta (IgG1 and IgG3), react at body temperature to cause red cell destruction, and are directed against well-developed RBC antigens. ABO incompatibility most commonly is seen, but ABO HDFN is clinically mild, presumably because the antigens are not fully expressed at birth. Antibodies directed against the D antigen can cause severe HDFN, and fetal health should be carefully monitored when anti-D titers are greater than 16. The severity of HDFN is less predictable with other blood group antibodies and can vary from mild to severe. For example, anti-K and anti-Ge3 not only causes red cell hemolysis but also may suppress erythropoiesis.4,6,

AUTOIMMUNE HEMOLYTIC ANEMIA Autoimmune hemolytic anemia is caused by the production of “warm-” or “cold-” reactive autoantibodies directed against RBC

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antigens (Chap. 54).40 Production can be triggered by disease, viral infection, or drugs; from breakdown in immune system tolerance to self-antigens; or from exposure to foreign antigens that induce antibodies that crossreact with self-RBC antigens. Autologous specificity is not always obvious because antigen expression can be depressed when autoantibody is present.40 Warm autoantibodies react best at 37°C and are primarily IgG (rarely IgM or IgA). Most are directed against the Rh protein, but Wrb, Kell, Kidd, and U blood group specificities have been reported.40 Cold-reactive autoantibodies are primarily IgM. They react best at temperatures below 25°C but can agglutinate RBCs or activate complement at or near 37°C, causing hemolysis or vascular occlusion upon exposure to cold.16 Patients with cold agglutinin disease often have C3d on their RBCs, which can provide some protection from hemolysis. Most cold-reactive autoantibodies have anti-I activity. Reactivity with i, H, Pr, P, or other antigenic specificities is much less common. The biphasic cold-reactive IgG antibody associated with paroxysmal cold hemoglobinuria (“Donath-Landsteiner” antibody) typically reacts with the high-prevalence antigen P (GLOB). It attaches to RBCs in the cold and very efficiently activates complement before it dissociates at warmer temperatures.

DISEASES ASSOCIATED WITH ANTIBODY PRODUCTION Table   136–4 lists diseases associated with specific antibody production. These antibodies cause autoimmune hemolytic anemia only if the patient carries the corresponding antigen.

SEROLOGIC DETECTION OF ERYTHROCYTE ANTIGENS AND ANTIBODIES ABO ABO grouping is the single most important test performed in the transfusion service because it is the fundamental basis for determining blood compatibility. ABO grouping is determined by testing RBCs with licensed antisera to identify the A or B antigens they carry (forward, or cell, grouping) and by testing the corresponding serum or plasma with known A and B cells to identify the antibodies present (reverse, or serum, grouping). Positive reactions are seen as hemagglutination or hemolysis, and the results of one test should confirm the results of the other. If results are discrepant or reactions are weaker than expected, the cause must be investigated before the ABO group can be interpreted with confidence. Discrepancies can be related to RBC anomalies, serum anomalies, or both, and they may be associated with disease.5,11,16 Table 136–6 lists common causes, excluding clerical and technical error. If the ABO group of a patient cannot be determined, group O blood can be used for transfusion.

Rh The D type is the next most important test performed for blood compatibility. Individuals whose RBCs type D+ are called Rh-positive, and those who type D– are called Rh-negative, provided controls are acceptable. Blood donors who type D– using standard typing sera are tested further for weak D expression using more sensitive methods, such as an indirect antiglobulin test. Donors with weak D antigen are considered

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TABLE 136–6.  Common Causes of Abo Discrepancies RED CELLS MAY APPEAR TO HAVE Weak or missing antigens

Weak subgroup of A or B antigen Excess soluble A or B antigen in plasma Disease-associated loss (leukemia) ABO nonidentical marrow transplantation ABO nonidentical red blood cell (RBC) transfusions

Extra antigens

Positive direct antiglobulin test Antibody to reagent additive or dye Rouleaux or cold agglutinin on cells Disease-associated acquisition (polyagglutination)

SERUM MAY APPEAR TO HAVE Weak or missing antibody

Age related (newborns or the very elderly) Disease-associated immunosuppression Congenital hypogammaglobulinemia ABO nonidentical marrow transplantation

Extra antibody

Alloantibodies (A1 Lea, Leb, P1 M, N) Autoantibodies (I, i, H, Pr, P) Rouleaux Antibodies to additives in reagent RBCs Passive antibody acquisition from transfusion or from passenger lymphocytes in organ transplantation

Rh-positive. Testing for weak D is optional for transfusion recipients and pregnant women.56

EXTENDED ANTIGEN PHENOTYPING Reagent antisera to detect other common antigens (e.g., CcEe, MNSs, Kk, FyaFyb, JkaJkb) are available and used when identification of the red cell phenotype is essential to antibody identification, blood compatibility, determination of zygosity, or paternity or forensic issues. Extended phenotyping is especially important to patients who are at high risk for alloimmunization from chronic blood transfusion, for example, those with sickle cell anemia or thalassemia. Ideally, an extended RBC phenotype of patients who are likely to be chronically transfused should be determined prior to initiation of transfusion therapy. Prediction of a blood group antigen can be made by testing DNA of a patient, even in the presence of transfused RBCs.27

ANTIBODY SCREEN The antibody screen, or indirect antiglobulin test, detects “atypical” or “unexpected” antibodies in the serum (i.e., other than anti-A and anti-B) using group O reagent red cells that are known to carry various combinations of antigens. The methods used must be able to detect clinically significant antibodies. Typically, serum or plasma and screening cells are incubated at 37°C with an additive to potentiate antibody– antigen reactions, then an indirect antiglobulin test is performed. Hemagglutination or hemolysis at any point is a positive reaction, indicating the presence of naturally occurring or immune alloantibody or autoantibody. The antibody screen will not detect all atypical antibodies in serum, such as antibodies to low-prevalence antigens not present on

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screening cells and antibodies that are not apparent at 37°C and in the antiglobulin phase.

DIRECT ANTIGLOBULIN TEST The direct antiglobulin test (often referred to as the direct Coombs test, a term discouraged by Robin Coombs because he said that Race and Mourant were also key to the description of the test) detects antibody or complement bound to RBCs in vivo. Red cells are washed free of serum and then mixed with an antiglobulin reagent that agglutinates RBCs coated with IgG or the C3 component of complement. Positive direct antiglobulin test results are associated with the following: (1) transfusion reactions, in which recipient alloantibody coats transfused donor RBCs or transfused donor antibody coats recipient RBCs; (2) HDFN, in which maternal antibody crosses the placenta and coats fetal RBCs; (3) autoimmune hemolytic anemias, in which autoantibody coats the patient’s own RBCs; (4) drug or drug–antibody complex interactions with RBCs that sometimes lead to hemolysis; (5) passenger lymphocyte syndrome, in which transient antibody produced by passenger lymphocytes from a transplanted organ coats recipient RBCs; and (6) hypergammaglobulinemia, in which Ig nonspecifically adsorb onto circulating RBCs. A positive direct antiglobulin test result does not always indicate decreased red cell survival. As many as 10 percent of hospital patients and 0.1 percent of blood donors have a positive direct antiglobulin test result with no clinical indication of hemolysis.11

COMPATIBILITY TESTING Compatibility testing refers to a set of donor and recipient tests that are performed prior to red cell transfusion. The collecting facility tests donors for ABO, Rh, and unexpected antibody. However, transfusing hospitals retest the ABO (and D on Rh-negative units) to verify the accuracy of the blood label.56 Routine recipient testing includes an ABO, D, and antibody screening on a blood sample collected within 3 days of the intended transfusion. Results are checked against historical records to verify ABO, D, and antibody status.56 If the recipient has a negative antibody screening test result and no history of clinically significant antibodies, a serologic immediate spin crossmatch between recipient serum and donor red cells or a “computer crossmatch” (wherein computer software compares the ABO test results of both donor and recipient) is required to confirm ABO compatibility.11 If clinically significant antibodies are detected in a recipient’s serum or previously were identified, red cell components should test negative for the corresponding antigens and be crossmatch compatible at 37°C by the antiglobulin test. The chance of finding compatible units usually reflects the antigen prevalence in the population, that is, 91 percent of units should be compatible with a patient making anti-K because 9 percent of the population is K+. This reasoning will not be valid if the local donor population varies significantly from the general population. When more than one antibody is present, the probability of finding compatible blood is the product of the prevalence (probability) of each independent antigen tested. For example, only 21 percent of units will be compatible for the recipient having both anti-K and anti-Jka: (0.91 for K–) × (0.23 for Jk[a–]) = 0.21. When multiple clinically significant antibodies or an antibody directed against a high-prevalence antigen are present, finding compatible RBC components can be extremely difficult. Such antibody producers should be encouraged to give autologous donations prior to their elective blood needs. If the patient is not a candidate for autologous donation, compatible units may be found by testing the patient’s siblings or by asking regional blood suppliers to check their rare donor inventories and files. Such procurement requires additional time.

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TABLE 136–7.  ABO-Rh Compatibility Guidelines Compatible Blood Groups Antigen on Red Cells

Antibody in Serum

Donor Red Cells

Donor Plasma

If recipient blood group is A

A

Anti-B

A, O

A, AB

B

B

Anti-A

B, O

B, AB

O

O

Anti-A, anti-B

O

O, A, B, AB

AB

A, B

None

AB, A, B, O

AB

Rhpositive

D

None

Rh-positive, Rh-negative

Rh not considered

Anti-D Rh-negative only if immunized

Rh not considered

RhNone negative

Whole blood must be identical to recipient’s blood group. Red blood cell (RBC) products must be compatible with recipient’s serum. Plasma products should be compatible with recipient’s RBCs. Platelet and cryoprecipitate products should be compatible with recipient’s RBCs, but any ABO group can be given if compatible products are not available. Repeat donor testing and crossmatching are not performed for plasma and platelet components, but the recipient’s ABO and Rh phenotypes must be known for appropriate selection of components. Table 136–7 gives general ABO-D compatibility guidelines.

ANTIBODY IDENTIFICATION All unexpected antibodies should be investigated. Those detected in serum or plasma as an ABO discrepancy, a positive antibody screening result, or an incompatible crossmatch are identified using a panel of eight to 16 different group O red cells that have been typed for antigens corresponding to clinically significant antibodies. Serum reactions with these RBCs are compared to their antigen typing to determine specificity.11 For example, an antibody that reacts with all K+ RBCs but not with K– cells most likely is anti-K. A control of autologous RBCs and serum is tested concurrently with panel RBCs. Absence of reactivity with autologous cells implies the antibody is an alloantibody, whereas a positive result suggests autoantibody or a positive direct antiglobulin test result. Once antibody specificity is identified, the patient’s RBCs are tested for the corresponding antigen. If the alloantibody is anti-K, the cells should type K–. Such antigen typing helps to confirm serum findings. When antibody is detected both on red cells (a positive direct antiglobulin test result) and in serum, only the antibody in serum is identified unless a review of the medical, pregnancy and transfusion history offers evidence that the antibodies might be different. When antibody is detected only on RBCs and in vivo hemolysis is suspected, the antibody can be eluted from the patient’s RBCs and tested against panel RBCs to identify the specificity.

REFERENCES 1. Lewis M, Anstee DJ, Bird GWG, et al: Blood group terminology 1990. ISBT working party on terminology for red cell surface antigens. Vox Sang 58:152, 1990. 2. Lögdberg L, Reid ME, Zelinsky T: Human blood group genes 2010: Chromosomal locations and cloning strategies revisited. Transfus Med Rev 25:36, 2011.

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3. Cartron JP, Bailly P, Le Van Kim C, et al: Insights into the structure and function of membrane polypeptides carrying blood group antigens. Vox Sang 74(Suppl 2):29, 1998. 4. Daniels G: Human Blood Groups, 3rd ed. Blackwell Science, Oxford, 2013. 5. Issitt PD, Anstee DJ: Applied Blood Group Serology, 4th ed. Montgomery Scientific, Durham, NC, 1998. 6. Reid ME, Lomas-Francis C, Olsson ML: Blood Group Antigen FactsBook, 3rd ed. Academic Press, San Diego, 2012. 7. Telen MJ: Erythrocyte blood group antigens: Not so simple after all. Blood 85:299, 1995. 8. Cartron JP, Colin Y: Structural and functional diversity of blood group antigens. Transfus Clin Biol 8:163, 2001. 9. Bruce LJ, Ghosh S, King MJ, et al: Absence of CD47 in protein 4.2-deficient hereditary spherocytosis in man: An interaction between the Rh complex and the band 3 complex. Blood 100:1878, 2002. 10. Reid ME, Mohandas N: Red blood cell blood group antigens: Structure and function. Semin Hematol 41:93, 2004. 11. Fung MK, Grossman BJ, Hillyer C, et al, editors: Technical Manual, 18th ed. American Association of Blood Banks, Bethesda, MD, 2014. 12. Storry JR, Castilho L, Daniels G, et al: International Society of Blood Transfusion Working Party on Red Cell Immunogenetics and Terminology: Cancun report (2012). Vox Sang 107:90, 2014. 13. Daniels GL, Anstee DJ, Cartron J-P, et al: Blood group terminology 1995. ISBT working party on terminology for red cell surface antigens. Vox Sang 69:265, 1995. 14. Garratty G, Dzik WH, Issitt PD, et al: Terminology for blood group antigens and genes: Historical origins and guidelines in the new millennium. Transfusion 40:477, 2000. 15. Reid ME, Lomas-Francis C: Blood Group Antigens & Antibodies: A Guide to Clinical Relevance & Technical Tips. Star Bright Books, New York, 2007. 16. Klein HG, Anstee DJ: Mollison’s Blood Transfusion in Clinical Medicine, 11th ed. Wiley-Blackwell, Oxford, 2006. 17. Clausen H, White T, Takio K, et al: Isolation to homogeneity and partial characterization of a histo-blood group A defined Fuca1—>2Gala1—>3-N-acetylglucosaminyltransferase from human lung tissue. J Biol Chem 265:1139, 1990. 18. Yamamoto F, Marken J, Tsuji T, et al: Cloning and characterization of DNA complementary to human UDP-GalNAc: Fuca1—>2Gala1—>3GalNAc transferase (histoblood group A transferase) mRNA. J Biol Chem 265:1146, 1990. 19. Yamamoto F, Hakomori S: Sugar-nucleotide donor specificity of histo-blood group A and B transferases is based on amino acid substitutions. J Biol Chem 265:19257, 1990. 20. Yamamoto F, Clausen H, White T, et al: Molecular genetic basis of the histo-blood group ABO system. Nature 345:229, 1990. 21. Chester MA, Olsson ML: The ABO blood group gene: A locus of considerable genetic diversity. Transfus Med Rev 15:177, 2001. 22. Olsson ML, Chester MA: Polymorphism and recombination events at the ABO locus: A major challenge for genomic ABO blood grouping strategies. Transfus Med 11:295, 2001. 23. Garratty G: Association of blood groups and disease: Do blood group antigens and antibodies have a biological role? Hist Philos Life Sci 18:321, 1996. 24. Avent ND, Reid ME: The Rh blood group system: A review. Blood 95:375, 2000. 25. Tippett P, Lomas-Francis C, Wallace M: The Rh antigen D: Partial D antigens and associated low incidence antigens. Vox Sang 70:123, 1996. 26. Huang C-H, Liu PZ, Cheng JG: Molecular biology and genetics of the Rh blood group system. Semin Hematol 37:150, 2000. 27. Reid ME: Applications of DNA-based assays in blood group antigen and antibody identification. Transfusion 43:1748, 2003. 28. Giblett ER: A critique of the theoretical hazard of inter vs. intra-racial transfusion. Transfusion 1:233, 1961. 29. Tippett P: Regulator genes affecting red cell antigens [review]. Transfus Med Rev 4:56, 1990. 30. Singleton BK, Burton NM, Green C, et al: Mutations in EKLF/KLF1 form the molecular basis of the rare blood group In(Lu) phenotype. Blood 112:2081, 2008. 31. Okubo Y, Yamaguchi H, Nagao N, et al: Heterogeneity of the phenotype Jk(a–b–) found in Japanese. Transfusion 26:237, 1986. 32. Hakomori S: Blood group ABH and Ii antigens of human erythrocytes: Chemistry, polymorphism, and their developmental change. Semin Hematol 18:39, 1981. 33. Spitalnik PF, Spitalnik SL: The P blood group system: Biochemical, serological, and clinical aspects. Transfus Med Rev 9:110, 1995. 34. Pogo AO, Chaudhuri A: The Duffy protein: A malarial and chemokine receptor. Semin Hematol 37:122, 2000. 35. Araten DJ, Swirsky D, Karadimitris A, et al: Cytogenetic and morphological abnormalities in paroxysmal nocturnal haemoglobinuria. Br J Haematol 115:360, 2001. 36. Tippett P, Ellis NA: The Xg blood group system: A review. Transfus Med Rev 12:233, 1998. 37. Cartron J-P, Rahuel C: Human erythrocyte glycophorins: Protein and gene structure analyses. Transfus Med Rev 6:63, 1992. 38. Tournamille C, Colin Y, Cartron JP, Le Van Kim C: Disruption of a GATA motif in the Duffy gene promoter abolishes erythroid gene expression in Duffy-negative individuals. Nat Genet 10:224, 1995. 39. Mourant AE, Kopec AC, Domaniewska-Sobczak K: Distribution of the Human Blood Groups and Other Polymorphisms, 2nd ed. Oxford University Press, London, 1976. 40. Petz LD, Garratty G: Acquired Immune Hemolytic Anemias, 2nd ed. Churchill Livingstone, New York, 2003. 41. Moulds JM, Moulds JJ: Blood group associations with parasites, bacteria, and viruses. Transfus Med Rev 14:302, 2000.

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42. Cartron JP: Molecular basis of red cell protein antigen deficiencies. Vox Sang 78:7, 2000. 43. Lee S, Russo D, Redman CM: The Kell blood group system: Kell and XK membrane proteins. Semin Hematol 37:113, 2000. 44. Danek A, Rubio JP, Rampoldi L, et al: McLeod neuroacanthocytosis: Genotype and phenotype. Ann Neurol 50:755, 2001. 45. Luhn K, Wild MK, Eckhardt M, et al: The gene defective in leukocyte adhesion deficiency II encodes a putative GDP-fucose transporter. Nat Genet 28:69, 2001. 46. Etzioni A, Tonetti M: Leukocyte adhesion deficiency II-from A to almost Z. Immunol Rev 178:138, 2000. 47. Yu L-C, Twu Y-C, Chang C-Y, Lin M: Molecular basis of the adult i phenotype and the gene responsible for the expression of the human blood group I antigen. Blood 98:3840, 2001. 48. Inaba N, Hiruma T, Togayachi A, et al: A novel I-branching beta-1,6-N-acetylglucosaminyltransferase involved in human blood group I antigen expression. Blood 101:2870, 2003. 49. Yu LC, Twu YC, Chou ML, et al: The molecular genetics of the human I locus and molecular background explaining the partial association of the adult i phenotype with congenital cataracts. Blood 101:2081, 2003. 50. Agre P, King LS, Yasui M, et al: Aquaporin water channels—From atomic structure to clinical medicine. J Physiol 542:3, 2002. 51. Crew VK, Burton N, Kagan A, et al: CD151, the first member of the tetraspanin (TM4) superfamily detected on erythrocytes, is essential for the correct assembly of human basement membranes in kidney and skin. Blood 104:2217, 2004.

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52. Rock JA, Shirey RS, Braine HG, et al: Plasmapheresis for the treatment of repeated early pregnancy wastage associated with anti-P. Obstet Gynecol 66:57S, 1985. 53. Udden MM, Umeda M, Hirano Y, Marcus DM: New abnormalities in the morphology, cell surface receptors, and electrolyte metabolism of In(Lu) erythrocytes. Blood 69:52, 1987. 54. Heaton DC, McLoughlin K: Jk(a–b–) red blood cells resist urea lysis. Transfusion 22:70, 1982. 55. Sands JM: Molecular mechanisms of urea transport. J Membr Biol 191:149, 2003. 56. Standards Committee of American Association of Blood Banks: Standards for Blood Banks and Transfusion Services, 29th ed. American Associations of Blood Banks, Bethesda, MD, 2014. 57. Springer GF, Horton RE, Forbes M: Origin of anti-human blood group B agglutinins in white leghorn chicks. J Exp Med 110:221, 1959. 58. Springer GF, Horton RE: Blood group isoantibody stimulation in man by feeding blood group-active bacteria. J Clin Invest 48:1280, 1969. 59. Reid ME, Øyen R, Marsh WL: Summary of the clinical significance of blood group alloantibodies. Semin Hematol 37:197, 2000. 60. Poole J, Daniels G: Blood group antibodies and their significance in transfusion medicine. Transfus Med Rev 21:58, 2007.

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CHAPTER 137

HUMAN LEUKOCYTE AND PLATELET ANTIGENS

Myra Coppage, David Stroncek, Janice McFarland, and Neil Blumberg

SUMMARY The human leukocyte antigens (HLAs) are highly polymorphic glycoproteins encoded by the major histocompatibility complex on chromosome 6. Their biologic function is presentation of antigenic peptides to T lymphocytes, and there are two major classes: class I (A, B, and C loci) and class II (DR, DQ, and DP loci). Class I antigens are present on almost all nucleated cells, whereas class II antigens are primarily expressed on B cells and other antigenpresenting cells such as dendritic cells, endothelial cells, and monocytes. These antigens play key roles in hematopoietic cell transplantation acceptance/ rejection and allosensitization to nonleukoreduced blood transfusions leading to platelet transfusion refractoriness, with lesser, but distinct roles in solidorgan transplantation. Other clinically important lineage-specific white cell antigens include those on neutrophils, which are much less polymorphic and less commonly a cause of clinical problems than the HLA system. Antibody to neutrophil antigens plays a role in autoimmune neutropenia, and reactions such as transfusion-related acute lung injury. Platelets also possess a relatively limited number of polymorphic antigens that are involved in clinical problems such as posttransfusion purpura and platelet transfusion refractoriness, and neonatal problems such as alloimmune thrombocytopenia.

 UMAN LEUKOCYTE ANTIGENS (MAJOR H HISTOCOMPATIBILITY COMPLEX) DEFINITION The human leukocyte antigens (HLAs) are highly polymorphic glycoproteins encoded by a region of genes known as the major histocompatibility complex (MHC) located on chromosome 6p21 and covering a region of approximately 7.6 Mbp.1,2 After ABO antigens, HLA antigens are the major barrier to transplantation. Their biologic function is to present antigenic peptides to T lymphocytes. The MHC codes for several groups of antigens. The best understood are the highly polymorphic, classical class I (HLA-A, HLA-B, and HLA-C) and class II (HLA-DR, HLA-DQ, and HLA-DP) antigens. Class I antigens are ubiquitous and present on most nucleated somatic cells. Class II antigens exhibit more

Acronyms and Abbreviations: CDC, complement-dependent cytotoxicity; ELISA, enzyme-linked immunosorbent assay; GP, glycoprotein; GVHD, graft-versus-host disease; HLA, human leukocyte antigen; HNA, human neutrophil antigen; HPA, human platelet antigen; MHC, major histocompatibility complex; NAIT, neonatal alloimmune thrombocytopenia; NMDP, National Marrow Donor Program; PCR, polymerase chain reaction; PRA, panel reactive antibody; PTP, posttransfusion purpura; SSO, sequence-specific oligonucleotide; SSP, sequence-specific primer; TRALI, transfusion-­ related acute lung injury; WHO, World Health Organization.

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restricted distribution, with varying levels of expression on B cells, dendritic cells, monocytes, macrophages, and endothelial cells. However, class II antigens can be induced on many cell types through activation.3 The nonclassical class Ib antigens HLA-E, HLA-F, and HLA-G, and the MHC class I chain-related antigens are much less polymorphic, their function less understood, and their tissue expression more limited. In addition the MHC region codes for a number of pseudogenes. This chapter focuses on the classic class I and II molecules because of their importance in transfusion and transplantation. The two major classes of HLA antigens are homologous. However, there are areas of high variability (polymorphism) that distinguish individual HLA molecules (alleles) and confer antigen specificity. HLA antigens are codominantly expressed so that each individual expresses two antigens at each locus (A, B, DR, etc.). As of July 2014, many thousands of HLA alleles had been characterized.5 Table 137–1 lists the number of known HLA alleles at each locus.

GENETICS OF THE MAJOR HISTOCOMPATIBILITY COMPLEX The first sequence map of the MHC encompassed approximately 3.6 Mbp on chromosome 6p21 and was divided into three regions: class I, class II, and class III genes.2 Newer analysis confirming high linkage disequilibrium and conserved synteny led to the concept of an extended MHC (xMHC), and a new map was produced in 2004.6 The xMHC occupies approximately 7.6 Mbp, and is composed of five subregions, which include the classical classes I, II, and III genes. Class II genes are the most centromeric and occupy approximately 1 Mbp of DNA. The genes are ordered sequentially beginning with HLA-DP genes followed by HLA-DM, TAP, HLA-DQ and lastly the HLA-DR genes. The class III genes occupy space between the class I and class II genes. The class III genes include genes that encode other proteins that participate in immune response such as complement, heat shock proteins, tumor necrosis factor, and other lymphocyte antigens. Telomeric are the class I genes sequentially as MICA, MICB, HLA-B, HLA-C, HLA-E, HLA-A, HLA-F, and HLA-G. Extended class I genes include histone clusters and zinc finger genes. Figure 137–1B is a representative map of the MHC.

STRUCTURE AND FUNCTION Class I Antigens

The HLA-A, -B, and -C molecules are transmembrane glycoproteins with an Mr 56,000.7 Each is a heterodimer composed of one α heavy chain (Mr 45,000) noncovalently bound to β2-microglobulin (Mr 11,000). The α heavy chain is the polymorphic glycoprotein encoded by the MHC genes. The extracellular region of the α chain consists of three domains (α1, α2, α3) based on folding and disulfide bonding (see Fig. 137–1A). Antigenicity resides in the α1 and α2 domains, the areas of highest polymorphism. These two chains form a platform composed of a single β-pleated sheet “floor” topped by two α helices with a cleft or groove between them. The structure is supported by the third, α3, domain of the heavy chain in conjunction with β2-microglobulin, which stabilizes the molecule on the cell surface. Class I HLA molecules present peptide fragments from endogenously derived proteins (e.g., viral infection, intracellular bacteria, or transformation) to CD8+ T cells. The highly polymorphic groove permits presentation of highly variable peptides of nine amino acids average length. Class I HLA-A, -B, and -C antigens are found on most nucleated somatic cells.8 Platelets express HLA-A antigens, but lack some HLA-B and most HLA-C antigens.9

Class II Antigens

The class II antigens are also transmembrane glycoproteins formed by two noncovalently bound chains.12 Both the α heavy chain (Mr 34,000)

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TABLE 137–1.  Number of Known Alleles for Each Human Leukocyte Antigen Locus as of July 2014 HLA CLASS I Gene

A

B

C

E

F

G

Alleles

2884

3590

2375

15

22

50

Gene

DRA

DRB

DQA

DQB

DPA

DPB

Alleles

7

1642

52

664

38

422

Gene

MICA

MICB

TAP1

TAP2

Alleles

100

40

12

12

HLA CLASS II

NON-HLA

HLA, human leukocyte antigen (HLA). Data from Robinson J, Waller MJ, Parham P, et al: IMGT/HLA and IMGT/ MHC: Sequence databases for the study of the major histocompatibility complex. Nucleic Acids Res 31(1):311–314, 2003. and the light β chain (Mr 29,000) are encoded in the MHC region. Class II molecules, like class I, consist of an extracellular hydrophilic NH2– terminal region, a hydrophobic transmembrane region, and an intracellular COOH– terminus region. Unlike class I antigens, the extracellular regions of each chain contain only two domains. The two domains of the α chain are designated α1 and α2, and the two domains of the β-chain are called β1 and β2. The α chain of HLA-DR is constant for all HLA-DR molecules, whereas the β chain is polymorphic and determines specificity of the molecule. Both α and β chains of HLA-DQ and -DP are polymorphic, although the β chain is more so than the α chain. In all class II antigens the β1 domain represents the most polymorphic region. The structure of HLA-DR is essentially similar to the structure of class I molecules. Class II antigens present peptides from exogenous sources, such as bacterial pathogens, to CD4+ cells. The binding groove is more open than that of class I, and peptides of longer length (11 to 18 amino

acids) are accommodated.13,14 Class II antigens have a more restricted tissue distribution, being found primarily on B lymphocytes and other antigen-presenting cells such as dendritic cells, monocytes, and macrophages. They may also be expressed on activated endothelial cells and T lymphocytes.12 The extraordinarily polymorphic nature of HLA has probably evolved because of the need to present a very large array of different antigenic peptides in host defense. Antigen processing and presentation is a tightly regulated process, especially among the professional ­antigen-presenting cells such as dendritic cells. A number of alternative mechanisms have been demonstrated in vitro, such as cross-presentation, whereby dendritic cells transfer antigen derived from endocytic sources to the class I pathway, but are poorly understood.15 One promising area of research is the ability of HLA molecules to present antigenic peptides derived from tumors. Such peptides could arise via point mutation, or reactivation of a normally silent gene that produces a peptide that can bind to HLA and induce a T-cell immune response. Several such peptides (melanoma-associated gene [MAGE] antigens) have been identified for melanoma.16

NOMENCLATURE Distinguishing polymorphic variations among HLA antigens is clinically important in stem cell transplantation. Terminology used to describe accepted HLA alleles or antigens is standardized by the World Health Organization (WHO), Nomenclature Committee for Factors of the HLA System, which issues biannual reports and monthly updates.4 In addition, an HLA dictionary defining HLA antigens, their assigned nomenclature, and serologic equivalents is published periodically.5 The nomenclature committee approved major changes to the system that were implemented in 2010.17 The revisions were designed to ­accommodate the unexpected number of new sequenced alleles. Under this system, colons are used as delimiters to separate fields. The first field signifies the allele family that often corresponds to the serological antigen. The second field denotes the alleles, assigned in order of determination. The third field is used for defining synonymous nucleotide substitutions.

Figure 137–1.  A. Schematic of the HLA-A2 molecule. The peptide groove is formed by the α helices and β-pleated sheet floor. The groove holds

processed peptide antigen. The peptide and the polymorphic α helices interact with the T-cell receptor. B. Representative diagram of the genes of the MHC on chromosome 6. (A, reproduced with permission from Bjorkman PJ, Saper MA, Samraoui B, et al: The foreign antigen binding site and T cell recognition regions of class I histocompatibility antigens. Nature 329(6139):512–518, 1987. B, adapted with permission from Campbell RD, Trowsdale J: Map of the human MHC, Immunol Today 14(7):349–352, 1993.)

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The last field defines alleles that differ by sequence polymorphisms in introns or in the 5′ or 3′ untranslated regions that flank the exons and introns. In addition, there are suffixes that are used to describe expression status. Null alleles (not expressed) are identified by the suffix “N.” Low surface expression is represented by “L.” Secreted molecules not present at the surface are assigned “S.”

INHERITANCE OF MAJOR HISTOCOMPATIBILITY COMPLEX ANTIGENS The genes of the MHC demonstrate more polymorphism than any other genetic system; that is, many alleles exist for each locus. Each individual, however, has one allele for each locus per chromosome, and therefore, encodes two HLA antigens per locus. The identification of each HLA antigen of an individual is called a phenotype. Because HLA genes are closely linked, recombination within the MHC is rare (≤1%), and a complete set of HLA genes usually is inherited from each parent as a unit. The genes inherited from each parent are referred to as a haplotype. Maternal and paternal haplotypes can be identified through family studies. Identification of both haplotypes of an individual provides the genotype. Family studies consist of typing for the HLA-A, HLA-B, HLA-C, HLA-DR, and HLA-DQ antigens to identify haplotypes and to rule out genetic recombination within the MHC. Because HLA genes are inherited together on a single chromosome, four combinations of maternal and paternal haplotypes are possible provided no recombination occurs (Fig. 137–2).

Linkage Disequilibrium

Because the MHC is so highly polymorphic, the probability that any two unrelated individuals are HLA identical is extremely low. However, the system exhibits a phenomenon known as linkage disequilibrium. That is, HLA alleles are inherited together on the same chromosome

Father

Mother

ab

cd

2355

more often than would be predicted if HLA loci were in equilibrium. At equilibrium, the frequency of an allele at one locus is independent of the frequencies of alleles at linked loci. For example, the gene frequency of HLA-A1 is 0.145 and that of HLA-B8 is 0.1 in North American whites. Given no preferential association between A1 and B8, then the haplotype frequency would be 0.0145 (0.145 × 0.1). However, population studies demonstrate that the actual frequency of the HLA-A1, B8 haplotype is 0.0726.18 The degree of linkage disequilibrium is defined as the observed frequency minus its expected frequency, 0.0581 in this example. Although particular alleles found in linkage disequilibria differ for various racial groups, all racial groups display significant disequilibria. Different races and ethnic groups can vary greatly in the frequency with which HLA antigens are found.19

HUMAN LEUKOCYTE ANTIGEN TYPING Tissue typing for HLA antigens can be performed by various methods using serologic, cellular, and molecular technologies. The most frequent procedures used in the clinical setting are serologic and molecular. Cellular assays such as the mixed lymphocyte reaction and the primed lymphocyte test were common prior to the widespread adoption of DNA methods. Compared to DNA techniques, cellular methods are labor-intensive and require the use of radioisotopes; they are mainly used in research laboratories.

Serology

The microlymphocytotoxicity complement-dependent cytotoxicity (CDC) test has been a fundamental procedure for defining HLA antigens for more than 30 years,20 although it has been supplanted by molecular typing methods. In this assay, a suspension of lymphocytes is incubated with human alloantisera or monoclonal antibody in a microtiter tray.21 Rabbit serum is added as a source of complement. Cell death is induced when antibody binds to antigen on the cell surface and the complement cascade activated. Death is visualized microscopically by the uptake of vital dye or by immunofluorescence. Panels used to determine a patient’s HLA type consist of two to four antisera that recognize the same specificity, which requires approximately 150 different reagents for class I antigens and 80 to 150 for class II antigens. Antisera are usually obtained from multiparous women, multiply transfused patients, and from patients who have rejected allografts. Monoclonal antibodies are also commercially available for many HLA specificities. Serology for class II (DR and DQ) antigens requires enrichment for B lymphocytes, which can be accomplished with antibody or immunomagnetic bead reagents.

Molecular Human Leukocyte Antigen Typing

#2 ac

Child #1 ac

Code: a b c d

#3 ad Haplotype: A1, B8, A2, B44, A3, B7, A11, B55,

Cw1, Cw2, Cw3, Cw3,

#4 bc

DR17, DR11, DR15, DR4,

#5 bd

DR52, DR52, DR51, DR53,

DQ2 DQ7 DQ6 DQ8

Figure 137–2.  Pedigree representing inheritance of HLA antigens.

Each of the four parental haplotypes is coded by a letter: a and b represent paternal haplotypes; c and d represent maternal haplotypes. Each child inherits one paternal and one maternal haplotype such that four combinations are possible.

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The development of the polymerase chain reaction (PCR)22 revolutionized the approach to HLA typing. Several DNA-based methods are commonly accepted for HLA typing. These include sequence-based typing, sequence-specific primer (SSP) amplification23 and sequencespecific oligonucleotide (SSO) probe hybridization. All of these methods involve amplification of genomic DNA from selected portions of HLA genes with oligonucleotide primer pairs. Generally exons 2 and 3 of class I and exon 2 of class II genes are amplified. These exons encode most of the polymorphisms of the classes I and II molecules. Molecular HLA typing is primarily of clinical interest in marrow/blood stem cell transplantation. The advent of “next-generation sequencing” (NGS) strategies has proven useful to high-throughput sequencing of HLA genes. NGS methods also overcome limitations of Sanger-based methods, including combination ambiguities that result from heterozygous samples in diploid genomes or between alleles where sequence varies outside the target region (i.e., exons 2 and 3). NGS methods are described as

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massively parallel allowing for many overlapping reads of the same sequence area. Multiple platforms using different chemistries are available; bioinformatics expertise is required to analyze the extensive data provided by these methods.

Detection of Antibodies to Human Leukocyte Antigen Molecules

In addition to typing for HLA antigens, most laboratories also use technology to detect antibodies to HLA antigens. This is very important for solid-organ transplantation where the presence of anti-HLA antibodies can cause irreversible rejection upon transplantation. It is of less concern for marrow/stem cell transplantation where donors and recipients are generally matched for HLA antigens. The microcytotoxicity serologic test is still in use, but solid phase assays have become standard practice as they are more sensitive than the CDC method. These assays include enzyme-linked immunosorbent assay (ELISA) and microbead-based flow assays such as FlowPRA and Luminex assays. These tests require HLA antigen, in either recombinant or native form, bound to a solid surface such as a microsphere and used to capture alloantibody in patient serum. Analysis of the reaction patterns yields information about the breadth of alloimmunization, or PRA (panel reactive antibody), and the specificity of the reactions. Prior to most solid-organ transplants, a donor-specific crossmatch is also performed to ensure that the recipient does not have anti-HLA antibodies against donor HLA antigens. Crossmatches are performed by microlymphocytotoxicity (CDC), flow cytometry, and by solid-phase (ELISA and microbead) assays. Labs are now adopting the “virtual” crossmatch using data from the sensitive microbead assays to predict crossmatch outcome. For low-risk cases this allows a transplant to proceed without waiting for a physical crossmatch and shortens cold ischemic time for an organ.24

CLINICAL APPLICATIONS The HLA antigens coded by the MHC play a central role in transplantation, regulation of immune responses, and susceptibility to a variety of diseases. The most common application, however, is the field of transplantation. In renal and stem cell transplantation, allografts from HLA-identical sibling donors have significantly greater survival than grafts from nonmatched family or unrelated donors. For solid-organ transplantation, a living donor is not always available or feasible (e.g., for heart transplantation). HLA typing for matching of kidneys and pancreas is performed at the HLA-A, HLA-B, and HLA-DR loci at low resolution (serologic or antigen level by DNA). In the early years of renal transplantation a high degree of match was sought between recipients and donors. However, as more potent immunotherapies have developed, the level and use of HLA matching has declined. HLA matching is not prospectively performed for hepatic or cardiac transplantation. Detection of alloantibody by screening techniques and the donor specific crossmatch is of prime importance for kidney and heart transplants where its existence could cause a hyperacute rejection and graft failure. The role of alloantibody is less clear in the immediate posttransplantation period, but may be detrimental to long-term survival.25 In the last several years, several “paired donor exchanges” have arisen to assist recipients who have incompatible, but willing living donors. These programs, such as the National Kidney Registry allow donor-recipient pairs to swap within or between transplantation centers. Chains of up to 30 cross-country swaps in the United States have facilitated more than 1200 transplantations since its inception. Marrow or stem cell transplantation entails problems other than allograft survival. In these therapies an immunocompetent graft is transplanted to an immunocompromised/immunoablated host.

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The graft may recognize the host tissue as foreign and mount an immune response resulting graft-versus-host disease (GVHD). With HLA-identical sibling donors, disease-free survival of greater than 80 percent can be achieved for some hematopoietic malignancies.26,27 However, fewer than 30 percent of individuals have an HLA-identical sibling. For these patients, alternative donors, such as phenotypically matched unrelated volunteers and partially matched family members, may be considered. However, the risks and incidence of graft failure and GVHD are higher than seen with HLA-identical siblings, and increase with the level of HLA disparity. HLA typing for stem cell transplants is generally performed by molecular methods. For those with a family donor, low-resolution typing may be sufficient to identify a match. However for unrelated or haploidentical family donors, high-­ resolution (allele-level) typing for HLA-A, HLA-B, HLA-C, HLA-DR, and HLA-DQ should be performed, and is required by the national registry program (National Marrow Donor Program [NMDP]).26 HLA alloantibody is becoming common, especially when incompletely matched donors are used. Patients requiring platelet transfusions may be broadly sensitized to HLA-A, and -B (i.e., have high PRA) through prior transfusions (particularly nonleukoreduced) or pregnancies. HLA antibody screening to select nonreactive donors and/or HLA donor platelet matching may enable these refractory patients to achieve improved platelet transfusion count increments. HLA typing at one or a few antigens or alleles may also be performed to support diagnosis of diseases associated with specific HLA antigens. The most common of these is the association between HLAB27 and ankylosing spondylitis28 and HLA-DQ2’s association with narcolepsy.29 HLA typing may also be performed to determine eligibility for vaccine trials that use peptides and HLA.30,31 HLA antigens also are implicated in drug hypersensitivity. For example, HLA-B*5701 is associated with hypersensitivity to the drug for treatment of the human immunodeficiency virus, abacavir.32 HLA tetramers may be used to monitor the efficacy of HLA-based peptide vaccines. Recombinant HLA molecules are loaded with the peptide vaccine and linked via a fluoresceinated streptavidin molecule. They are incubated with patient blood lymphocytes. Effector T cells specific for the peptide-HLA will be bound by the tetramer and monitored by flow cytometry.

 EUTROPHIL ANTIGENS N AND ANTIBODIES Clinically significant alloantigens expressed only or predominantly by neutrophils are known as human neutrophil antigens (HNAs).33 In this nomenclature, the antigen systems are indicated by integers, and specific antigens within each system are designated alphabetically by date of publication (Table 137–2).

THE HNA-1 ANTIGEN SYSTEM HNA-1 Antigens

The neutrophil-specific HNA-1 antigen system is made up of the four antigens alleles, HNA-1a, -1b, -1c and -1d (see Table  137–2).34 HNA-1 antigens are located on the low-affinity Fcγ receptor IIIb (FcγRIIIb), CD16, and are expressed only on neutrophils.35–38 FcγRIIIb and HNA-1 antigens are expressed on all segmented neutrophils, on approximately one-half of neutrophilic metamyelocytes, and on approximately 10 percent of neutrophilic myelocytes.39 Soluble FcγRIIIb is present in plasma and has the same HNA-1 polymorphisms found on neutrophils.40

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TABLE 137–2.  Human Neutrophil Antigens Location of Antigens

System

Alleles

Genes

HNA-1

HNA-1a, -1b, -1c, and 1d

FcγRIIIb

FCGR3B*01, FCGR3B*02, and FCGR3B*03

HNA-2

HNA-2

NB1gp

CD177

HNA-3

HNA-3a

Choline ­transporter-like protein-2 (CTL2)

SLC44A2*01

HNA-4

HNA-4a

αM integrin, C3bi-receptor (CR3)(CD11b)

ITGAM*01

HNA-5

HNA-5a

αL integrin, LFA-1(CD11a)

ITGAL*01

Molecular Biology

FcγRIIIb and the HNA-1 antigens are encoded by the FCGR3B gene located on chromosome 1q23–24. FCGR3B is highly homologous to FCGR3A, which encodes FcγRIIIa. In addition to the polymorphic FCGRB3B nucleotides only four others differ between FCGR3B and FCGR3A. The most important difference between the two genes is a C-to-T change at 733 in FCGR3B that creates a stop codon. As a result, FCGR3A has 21 more amino acids than FCGR3B and FCGR3B is a glycosylphosphatidylinositol (GPI)-anchored rather than a transmembrane glycoprotein (GP). The four HNA-1 system antigens are encoded by three alleles. The FCGR3B*01 allele differs from FCGR3B*02 by only five nucleotides in the coding region, at positions 141, 147, 227, 277, and 349.35–38 Four of the nucleotide changes result in changes in amino acid sequence between the HNA-1a and HNA-1b forms of FcγRIIIb. The glycosylation pattern of FcγRIIIb differs between the two antigens because of two nucleotide changes at bases 227 and 277. The HNA-1b form of FcγRIIIb has six N-linked glycosylation sites and the HNA-1a form has four glycosylation sites. The FCGR3B*03 allele is identical to FCGR3B*02 except for a C-to-A substitution at nucleotide 266 resulting in an alanine to aspartate change at amino acid 78 of FcγRIIIb.34 In many cases, FCGR3B*03 exists on the same chromosome with a second or duplicate FCGR3B

gene.41,42 FCGR3B*03 encodes both HNA-1b and HNA-1c. FCGR3B*02 also encodes HNA-1d which is characterized by the FcγRIIIb sequence Ala78–Asn82.43 The antigen frequencies of three of the alleles vary widely among different racial groups. Among whites, HNA-1b is the most common antigen (Table 137–3),44–46,48–50 but in Japanese and Chinese populations HNA-1a is most common.44,46,48,49,51 The frequency of the gene encoding HNA-1c antigen also varies among racial groups. HNA-1c is expressed by neutrophils in 4 to 5 percent of whites, 25 to 38 percent of African Americans, and 10 percent of Brazilians.52,53 The antigen frequency of HNA-1d should be nearly the same as that of HNA-1b, but it has not yet been investigated. Several other sequence variations in FCGR3B have been described.46 Most of these chimeric alleles have single-base substitutions involving one of the five single nucleotide polymorphisms (SNPs) that distinguish FCGR3B*1 and FCGR3B*2. FCGR3B alleles that most closely resembled FCGR3B*2 were found more often in African Americans than in whites or Japanese.46,47 Genetic deficiencies of neutrophil FcγRIIIb and HNA-1 antigens have also been reported. Among whites the incidence of individuals homozygous for FCGR3B deletion is approximately 0.1 percent.52,54,55 However, among Africans and African Americans the incidence is much higher. In one study, 3 of 126 Africans were found to be FCGR3B deficient,48 and in another study, 1 of 53 were found to be FCGR3B deficient.46

Function of HNA-1 Antigens

Polymorphisms in FcγRIIIb have some effect on neutrophil function. Neutrophils that are homozygous for HNA-1b have a lower affinity for immunoglobulin (Ig) G3 than those homozygous for HNA-1a.56 Neutrophils from those who are homozygous for HNA-1b phagocytize erythrocytes sensitized with IgG1 and IgG3 anti-Rh monoclonal antibodies and bacteria opsonized with IgG1 at a lower level than neutrophils homozygous for HNA-1a.57,58

THE HNA-2 ANTIGEN SYSTEM The HNA-2 Antigen

HNA-2 is an isoantigen without allelic variation. HNA-2 is expressed only on neutrophils, neutrophilic metamyelocytes, and myelocytes.39,59 It’s unique in that it is expressed on subpopulations of neutrophils. The mean size of the HNA-2-positive subpopulation of neutrophils is 45 to

TABLE 137–3.  Human Neutrophil Antigen Frequencies Antigen Frequencies System

Alleles

Europeans/ North Americans

Africans and African Americans

Asians

Brazilians/ Argentineans

HNA-1

HNA-1a

58%

59–67%

91%

68%

HNA-1b

88%

71–88%

54%

76%

HNA-1c

5%

25–38%