Atlas Of Peripheral Blood : The Primary Diagnostic Tool 9780781777803, 0781777801

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Atlas of Peripheral Blood: The Primary Diagnostic Tool

Atlas of Peripheral Blood: The Primary Diagnostic Tool Irma Pereira, MT (ASCP), SH Clinical Hematology Specialist Clinical Laboratories Stanford University Stanford, California

Tracy I. George, MD Assistant Professor of Pathology Director, Clinical Hematology Laboratory Stanford University Stanford, California

Daniel A. Arber, MD Professor and Vice Chair of Pathology Medical Director, Anatomic Pathology and Clinical Laboratory Services Department of Pathology Stanford University Stanford, California

Senior Executive Editor: Jonathan W. Pine, Jr. Product Manager: Marian Bellus Vendor Manager: Alicia Jackson Senior Manufacturing Manager: Benjamin Rivera Senior Marketing Manager: Angela Panetta Designer: Teresa Mallon Production Service: SPi Global Copyright © 2012 Wolters Kluwer Health | Lippincott Williams & Wilkins Two Commerce Square 2001 Market Street Philadelphia, PA 19103 All rights reserved. This book is protected by copyright. No part of this book may be reproduced in any form or by any means, or utilized by any information storage and retrieval system without written permission from the copyright owner, except for brief quotations embodied in critical articles and reviews. Printed in the People’s Republic of China. Library of Congress Cataloging-in-Publication Data Atlas of peripheral blood : the primary diagnostic tool / [edited by] Irma Pereira, Tracy I. George, Daniel A. Arber. p. ; cm. Includes bibliographical references and index. ISBN 978-0-7817-7780-3 1. Blood—Diseases—Diagnosis—Atlases. 2. Cytodiagnosis—Atlases. I. Pereira, Irma. II. George, Tracy I. III. Arber, Daniel A., 1961– [DNLM: 1. Hematologic Diseases—diagnosis—Atlases. 2. Blood Cell Count—methods—Atlases. 3. Cytodiagnosis—methods—Atlases. WH 17] RC636.A87 2012 616.1'307582—dc23 2011026335 Care has been taken to confirm the accuracy of the information presented and to describe generally accepted practices. However, the authors, editors, and publisher are not responsible for errors or omissions or for any consequences from application of the information in this book and make no warranty, expressed or implied, with respect to the currency, completeness, or accuracy of the contents of the publication. Application of this information in a particular situation remains the professional responsibility of the practitioner; the clinical treatments described and recommended may not be considered absolute and universal recommendations. The authors, editors, and publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accordance with the current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any change in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new or infrequently employed drug. Some drugs and medical devices presented in this publication have Food and Drug Administration (FDA) clearance for limited use in restricted research settings. It is the responsibility of the health care provider to ascertain the FDA status of each drug or device planned for use in his or her clinical practice. Visit Lippincott Williams & Wilkins on the Internet at: LWW.COM. Lippincott Williams & Wilkins customer service representatives are available from 8:30 am to 6 pm, EST. 9 8 7 6 5 4 3 2 1

I would, first of all, like to dedicate this book to Dr. William Creger, Professor Emeritus of Hematology, Stanford University School of Medicine, for his continued faith and constant encouragement and mentoring for over 25 years. I would also like to thank Drs. Daniel Arber and Tracy George for their faith in my ability to teach pathology residents and fellows in the fine art of peripheral smear diagnosis. Their undying trust allowed the publication of this Atlas by supplying their very positive attitudes, their time-consuming written text, the equipment, and the time for me to succeed in this endeavor. To all the attending physicians and house staff of Stanford University Hospital, you have my undying gratitude for your confidence in me. Each new case was a learning experience for me and constantly kept me on my toes. A final thank you goes to my parents who diligently pushed me to be the best I could be in any profession I selected. They supported me with the highest degree of love and attention to insure that I was a success. I am sorry they are no longer with me to see the fulfillment of my dreams; they would have been so proud. Irma T. Pereira I dedicate this book to my husband, Chris, for his constant support, and to my many teachers, colleagues, and students. Tracy I. George I dedicate this book to my wife, Carol, for her continuous support. Daniel A. Arber

Contributors Daniel A. Arber, MD Professor and Vice Chair of Pathology Medical Director, Anatomic Pathology and Clinical Laboratory Services Department of Pathology Stanford University Stanford, California Marnelli Bautista-Quach, MD Department of Pathology Loma Linda University School of Medicine Loma Linda, California John S. Cupp, MD Staff Pathologist Department of Pathology Hoag Memorial Hospital Presbyterian Newport Beach, California Aharon G. Freud, MD, PhD Department of Pathology Stanford University Stanford, California Tracy I. George, MD Assistant Professor of Pathology Director, Clinical Hematology Laboratory Stanford University Stanford, California

Jason Kurzer, MD, PhD Instructor of Pathololgy Stanford University Stanford, California Jay Patel, MD Clinical Assistant Professor of Pathology & Laboratory Medicine University of Calgary Calgary, Alberta, Canada Irma Pereira, MT (ASCP), SH Clinical Hematology Specialist Clinical Laboratories Stanford University Stanford, California Brent Tan, MD, PhD Clinical Assistant Professor Department of Pathology Stanford University Medical Center Stanford, California James Ziai, MD Fellow, Molecular Genetic Pathology Yale University School of Medicine New Haven, Connecticut

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Preface Atlas of Peripheral Blood: The Primary Diagnostic Tool illustrates common and uncommon disease presentations in the blood. While the diagnosis of most disease is complex, often including tissue biopsies, immunophenotyping studies, or molecular genetic testing, review of the peripheral blood smear is often the first clue to the diagnostic path needed. The increasing knowledge demands for those practicing medicine today leave less time than ever for the appreciation of key morphologic features in the diagnosis of disease. Despite this, morphology remains the starting point of many diagnostic workups. This atlas is designed as a diagnostic aid for anyone who reviews peripheral blood smears, and we hope it will be useful to clinical laboratory scientists, pathologists, hematologists, residents, fellows, and students of all types as they begin the evaluation of new patients and as they monitor existing patients. Our goal is to illustrate cases as they

actually occur. To do this, we have chosen to show cases that have typical and atypical features, and in most cases, more than one example of a disease type is given in the book and through additional online images. This work reflects the decades of experience of Irma Pereira as the person on the front line in reviewing abnormal blood smears at Stanford Hospital & Clinics. We have correlated Irma’s vast experience with modern classification systems, including the 2008 World Health Organization Classification of Hematopoietic and Lymphoid Neoplasms. This approach has allowed us to merge the art of morphology with the science of current medical practice. Irma Pereira, MT (ASCP), SH Tracy I. George, MD Daniel A. Arber, MD

ix

Acknowledgments We thank Bertil Glader, MD, PhD, and the Stanford RBC Special Studies Laboratory for their help with this atlas. Irma Pereira, MT (ASCP), SH Tracy I. George, MD Daniel A. Arber, MD

xi

Contents Contributors vii Preface ix Acknowledgments

1

2

xi

Introduction

1

8

Method of Preparing Manual Blood Smears 1 Staining 1 Approach to Interpretation 1 Features of Normal Blood Smears 2

Common Red Blood Cell Changes/ Nomenclature Laboratory Red Blood Cell Measurements RBC Shape and Size Changes 8 RBC Inclusions 15 RBC Size 19

3

6

Anemia Due to Abnormal or Impaired Hemoglobin Synthesis

Anemia Due to Abnormal or Impaired DNA Synthesis

22

9 36

Megaloblastic Anemia 36 Myelodysplasia 36 Congenital Dyserythropoietic Anemia 38 Reticulocytosis 38 Liver Disease 40 Alcoholism and Alcoholic Liver Disease 41

5

Hemolytic Anemias

6

Reactive Changes in Granulocytes

7

Systemic Disorders with Granulocyte and Monocyte Inclusions 67

Granulocyte Maturation The Neutrophil 57

Introduction 67 Alder-Reilly Anomaly

67

10

Myeloproliferative Neoplasms and Myeloid and Lymphoid Neoplasms with Eosinophilia

72

Myelodysplastic Syndromes

90

Myelodysplastic/Myeloproliferative Neoplasms

97

Monocyte Maturation 97 Chronic Myelomonocytic Leukemia 97 Atypical Chronic Myeloid Leukemia, BCR-ABL1 Negative 98 Juvenile Myelomonocytic Leukemia 99 Myelodysplastic/Myeloproliferative Neoplasms, Unclassifiable 101

43

Extracorpuscular 43 Intracorpuscular 44 Southeast Asian Ovalocytosis 53 Hereditary Spherocytosis Hereditary Elliptocytosis Syndromes Acanthocytosis

70

Chronic Myelogenous Leukemia, BCR-ABL1 Positive (CML) 74 Polycythemia Vera (PV) 78 Primary Myelofibrosis (PMF) 78 Essential Thrombocythemia (ET) 80 Chronic Neutrophilic Leukemia (CNL) 82 Chronic Eosinophilic Leukemia, Not Otherwise Specified (CEL) 83 Mastocytosis 84 Myeloproliferative Neoplasm, Unclassifiable (MPN,U) 85 Myeloid and lymphoid neoplasms with Eosinophilia and Abnormalities of PDGFRA, PDGFRB, FGFR1

6

Defects in Iron Metabolism 22 Defects in Porphyrin Synthesis 23 Defects in Globin Chains Synthesis 25

4

Morquio Syndrome 67 Hunter Syndrome 69 Hurler Syndrome 70 Chediak-Higashi Syndrome May-Hegglin Anomaly 70

11

Acute Myeloid Leukemia

12

Acute Leukemias of Ambiguous Lineage

57

57

103

Ancillary Studies in Acute Myeloid Leukemia 103 Acute Myeloid Leukemia with Recurrent Genetic Abnormalities 104 Acute Myeloid Leukemia with Myelodysplasia-Related Changes 110 Therapy-Related Myeloid Neoplasms 112 Acute Myeloid Leukemia, Not Otherwise Specified 114 Myeloid Proliferations Related to Down Syndrome 121

122

Acute Undifferentiated Leukemia 122 Mixed Phenotype Acute Leukemia 123

xiii

xiv

Contents

13

Reactive Changes in Lymphocytes

14

Precursor Lymphoid Malignancies

15

Infectious Mononucleosis 128 Other Viral Infections 129 Pertussis 130 Large Granular Lymphocytes 131

Mature Small B-cell Neoplasms Involving the Blood

139

Mature T- and NK-cell Neoplasms

18

Platelets

19

Blood Changes in Infectious Disease

158

T-Cell Prolymphocytic Leukemia 158 T-Cell Large Granular Lymphocytic Leukemia 160 Sézary Syndrome 161 Adult T-Cell Leukemia/Lymphoma 162 Chronic Lymphoproliferative Disorders of NK Cells 162 Aggressive NK-Cell Leukemia 164 Other T-Cell Lymphomas 164 Blastic Plasmacytoid Dendritic Cell Neoplasm 166

168

Pure Platelet Disorders 168 Platelet Abnormalities in Other Disorders 169

173

Protists 173 Malaria Babesia Trypanosomes Bacteria 178 Fungi 180

145

Mature Intermediate to Large B-cell Neoplasms 155 Burkitt Lymphoma/Leukemia 155 Large B-Cell Lymphoma 156

17

132

B-Lymphoblastic Leukemia/Lymphoma with Recurrent Genetic Abnormalities 135 B-Lymphoblastic Leukemia/Lymphoma, Not Otherwise Specified 136 T-Lymphoblastic Leukemia/Lymphoma 136

Chronic Lymphocytic Leukemia 139 B-Prolymphocytic Leukemia 143 Lymphoplasmacytic Lymphoma 144 Multiple Myeloma/Plasma Cell Leukemia Splenic Marginal Zone Lymphoma 147 Extranodal and Nodal Marginal Zone Lymphomas 148 Hairy Cell Leukemia 149 Hairy Cell Leukemia Variant 150 Follicular Lymphoma 150 Mantle Cell Lymphoma 152

16

127

20 Index

Nonhematopoietic Tumors in the Blood

189

185

1

Introduction The peripheral blood slide review is a comprehensive qualitative examination of the blood film to detect clinically significant abnormalities in leukocyte, erythrocyte, and platelet morphology. A slide review is an appropriate diagnostic test in a patient with a suspected primary hematologic disorder or with unexplained leukocytosis, leukopenia, anemia, polycythemia, thrombocytopenia, or thrombocytosis. In addition, slide examination is useful to confirm “flagged” results from the automated hematology instrument.

METHOD OF PREPARING MANUAL BLOOD SMEARS Peripheral blood smears are made from anticoagulated blood or from a fresh drop of blood from the syringe or a finger-stick puncture. A drop of blood is placed at one end of a glass slide. The drop should be sufficiently large to produce a blood film of at least 2.5 cm. A second “pusher” slide is held at a 30-degree angle to the smear slide. This slide is drawn back until it touches the drop of blood. The blood spreads behind the pusher to nearly the full width of the pusher slide. The pusher slide then is advanced smoothly and quickly to produce a blood smear at least 2.5 cm long that ends approximately 2 cm from the end of the slide. Moving the pusher slide forward too slowly accentuates poor leukocyte distribution by pushing larger cells, such as monocytes and granulocytes, to the end and sides of the smear. When the hematocrit is higher than normal, the angle between the slides must be lowered so that the smear is not too short and thick. Conversely, when the hematocrit is lower than normal, the slide must be raised to ensure a smear with proper length and thickness. Properly prepared smears (see Fig. 1.1) should be of a wedge shape, with only a slightly rounded end, rather than bullet shaped. Properly prepared smears have a normal gradation from thick to thin. The edges of the

smear should not touch the slide edges. The slide edge is an area where small numbers of large-size malignant cells deposit, so this area should be available for scanning.

STAINING Peripheral blood smears are air-dried and stained with Romanowsky-type stains such as Wright-Giemsa. This labels DNA and RNA a deep basophilic color, thus staining nuclei blue and cytoplasm typically pink depending on cytoplasmic granule composition. The area between the cells should be clean and free of precipitated stain. A satisfactorily stained slide has the following appearance: Red blood cells (RBCs): Salmon pink Neutrophils: Dark purple chromatin, pink cytoplasm, lilac granules Eosinophils: Dark purple chromatin, pale blue cytoplasm, coral granules Basophils: Dark purple chromatin, dark blue/black granules Lymphocytes: Dark purple chromatin, sky-blue cytoplasm Monocytes: Medium blue chromatin, gray-blue cytoplasm Platelets: Violet to purple granules

APPROACH TO INTERPRETATION Examination of a properly prepared smear should be in the area of the smear called the “feathered” edge as indicated in Figure 1.1; in this area the red cells are barely overlapping or just touching each other. Good practice for slide review requires assessment (qualitative and quantitative) of leukocytes, erythrocytes, and

1

2

Atlas of Peripheral Blood

Figure 1.1 The peripheral blood smear. A properly prepared peripheral blood smear should be evenly distributed without blood touching the ends or edges of the slide. The “feathered edge” located toward the right is where the morphologic review should begin following a systematic pattern as indicated by the lines.

platelets. An initial slide evaluation is performed using a 10× lens, before switching to a higher-powered oil objective. RBCs should be assessed for significant polychromatophilia and poikilocytes, with correlation with the mean corpuscular volume (MCV) and RBC indices. Correlation with the automated platelet count should be ascertained. White blood cells (WBCs) should be assessed for number (increased, decreased, normal), leukocyte composition, inclusions, and presence of abnormal cells. Platelets should be assessed for number, clumping, satellitism, and morphology. Manual platelet estimate. Assuming that no fibrin and clumped platelets are present, count the number of platelets in a field of approximately 100 to 200 RBCs using a 100× objective. Perform the count in 10 fields. Take the average number of platelets per 10 fields and multiply by an appropriate factor for a specific microscope as listed below. After multiplication, compare this number with the automated platelet count. They should agree within approximately 20%. If not, a manual platelet count should be performed. Model of Microscope

Multiplying Factor

BX45, BX40 BH2, Laborlux S

15 20

FEATURES OF NORMAL BLOOD SMEARS In normal adults, RBCs are normocytic and normochromic with rare to occasional poikilocytes and ≤1% polychromatophilic RBCs. Normal platelets are 1.5 to 3 mm in size with fine purple-red granules, either aggregated at the center of the cell or dispersed throughout the cytoplasm. Occasional giant platelets may be seen. In a normal blood smear, the distribution of leukocyte subsets seen on the slide agrees with the automated differential counts. No

significant leukocyte abnormalities are seen. This includes no immature granulocytes, blasts, nucleated RBCs, lymphoma cells, plasma cells, prolymphocytes, microorganisms, abnormal leukocyte inclusions, or >20% bands or >5% reactive lymphocytes (Figs. 1.2 and 1.3). In healthy term infants, abnormal RBC morphology is significantly more frequent than in adults (Fig. 1.4). Immaturity of the reticuloendothelial system (e.g., the spleen) is theorized to be the cause. Abnormal shapes can include stomatocytes, echinocytes, and keratocytes, as described in more detail in Chapter 2. Spherocytes are observed in the first days of life in healthy infants. However, large populations of spherocytes, if not artifactual, are pathologic. Furthermore, spherocytes detected in infants outside of the immediate newborn period are worthy of investigation. Occasional nucleated RBCs are present in the first days of life in the peripheral blood of healthy newborns (Table 1.1). The numbers of these elements are higher in premature infants (Fig. 1.5). Premature infants may have respiratory distress due to lung immaturity and the nucleated RBCs are a marker for hypoxia. Higher erythroblast counts in cord blood are associated with acidemia and hypoxia; pregnancies in which fetal distress prompts emergency Cesarean section have higher nucleated RBC counts compared with lower counts in scheduled (elective) Cesarean section or vaginal delivery. Increased polychromatophilic red cells are a more frequent finding in neonatal erythrocytes, reflecting the higher reticulocyte count in the early days of life. Absolute reticulocyte counts are normally several times higher in neonates in the first week or two of life compared with older children and adults. After this time, elevated reticulocytes should be investigated. Platelet counts and morphology in healthy term infants, premature infants, and even in extremely premature (100 fL in an adult. They do not show significant polychromasia. Macrocytes may be oval or

Figure 2.4 Crenated cells. Crenated cells are a common artifact of aged blood, elevated pH, contact with glass, or exposure to moisture. If the majority of the RBCs on the blood film look like burr cells, then artifactual crenation is likely, and the presence of burr cells should not be reported.

round. Oval macrocytes are seen in diseases with dyserythropoiesis typically (Table 2.2; Fig. 2.7). Microcytes: Microcytes are RBCs smaller in size than normal erythrocytes, 110 fL) Liver disease (110 fL) Myelodysplastic syndrome (110 fL) Alcoholic liver disease (≥110 fL) Aplastic anemia (100–110 fL)

Sideroblastic anemia Ovalocytes/Elliptocytes: Elliptocytes vary from red cells with oval shapes (i.e., ovalocytes) to elongated rodlike cells seen in hereditary elliptocytosis syndromes (Fig. 2.9). Heriditary elliptocytosis Hereditary pyropoikilocytosis

Congenital dyserythropoietic anemia, types I and III (>115 fL)

Hypothyroidism (50% of patients)

seen with iron deficiency, hemoglobin E syndromes, and thalassemia (Fig. 2.8). Microcytes that lack central pallor are termed microspherocytes and found in congenital spherocytosis or autoimmune hemolysis. Microcytes are commonly seen in Iron deficiency Thalassemia

Figure 2.7 Oval Macrocytes. The large size of these red cells can be seen in comparison to the nucleus of the lymphocyte. Macrocytes are larger in size and also demonstrate central pallor. The majority of the macrocytes in this field are oval in shape. The presence of oval macrocytes may indicate megaloblastic anemias or myelodysplastic syndrome.

Southeast Asian ovalocytosis Iron deficiency anemia (e.g., pencil cells; Fig. 2.10) Myelodysplastic syndromes and megaloblastic anemia (e.g., oval macrocytes) Oxidative Changes (Bite Cells): Oxidative damage to red cells results in denatured or precipitated hemoglobin, Heinz bodies. When the spleen “bites” out the rigid Heinz body, the typical bite cell remains. Because the cell has lost surface membrane, it will attempt to seal off by first forming a “veil” over the bite. This cell then becomes a spherocyte (Figs. 2.11 and 2.12). Patients will have both bite cells and spherocytes. Patients with glucose-6-phosphate deficiency (G6PD) are sensitive to oxidant injury, from drugs, infections, or Fava beans.

12

Atlas of Peripheral Blood

Figure 2.8 Microcytes. Microcytes are RBCs 15%, and ∼30% microcytes MCV 55–65 fL, or 50%–75% microcytes MCV < 55 fL, or more than 75% microcyte

20

Atlas of Peripheral Blood

TABLE 2.5

TABLE 2.7

GRADING OF MACROCYTOSIS

GRADING OF HYPOCHROMIA

1+

Slight

2+ 3+ 4+

MCV 98–105 fL or MCV normal with RDW > 15%, and ∼10% macrocytes MCV 105–110 fL, >50% macrocytes MCV 110–140 fL, >50% macrocytes MCV > 140 fL, >50% macrocytes

Autoagglutination and Rouleaux: Aggregates or 3-dimensional clumps of RBCs are described as autoagglutination, which is typically antibody mediated (i.e., IgM antibodies also known as cold agglutinins). Rouleaux or “stacking” of three or more RBCs is due to high protein states such as multiple myeloma or polyclonal hypergammaglobulinemia. A qualitative grading system is shown in Table 2.6. RBC Hemoglobin Concentration: Normal RBCs have central pallor that occupies approximately one-third of the cell. Increased central pallor is termed hypochromia and can be qualitatively graded as shown in Table 2.7. Poikilocytosis: Some degree of poikilocytosis (variation in RBC shape) can be seen on normal adult blood smears. This poikilocytosis usually occurs from a mix of cells types. Cell types that normally occur more often are referred to as high-frequency cell types, while those that are rare in normal blood are low-frequency cell types (see Table 2.8). Based on the percentage of high- or lowfrequency cells in the blood, the degree of poikilocytosis can be graded. Such grading can be helpful to ascertain the significance of the poikilocytes in different disease states (Table 2.9). Table 2.10 highlights distinct red cell abnormalities and their associated disorders.

Central pallor >1/2 but 2/3 of cell. Pale outer rim of hemoglobin. Marked Only thin rim of pink cytoplasm remains.

TABLE 2.8 HIGH- AND LOW-FREQUENCY RBC TYPES AND CHANGES THAT CONTRIBUTE TO POIKILOCYTOSIS Frequency High

Acanthocyte Burr cell Ovalocyte Sickle cell Spherocyte Target cell Stomatocyte

Low

Autoagglutination Bizarre cell Blister cell Helmet cell Macrocyte Microcyte Oxidative change Pencil cell Polychromatophilic red cell Rouleaux Schistocyte Teardrop cell

TABLE 2.6

TABLE 2.9

GRADING OF AUTOAGGLUTINATION AND ROULEAUX

GRADING OF POIKILOCYTOSIS BASED ON PERCENTAGE OF HIGH- OR LOWFREQUENCY RBC CHANGES

1±

2±

3±

4±

Autoagglutination and 10% 20%–50% 60%–75% >75% Rouleaux Note: Percentage (%) is defined as the number of RBCs involved.

Quantifier High frequency Low frequency

1+ 2%–5% 1%–2%

2+

3+

5%–10% 10%–30% 2%–5% 5%–10%

4+ >30% >10%

Chapter 2: Common Red Blood Cell Changes/Nomenclature

21

TABLE 2.10 DISORDERS ASSOCIATED WITH VARIOUS RBC ABNORMALITIES Abnormality

Associated Disorder

Oval macrocytes with hypochromia, Pappenheimer bodies in hypochromic RBCs, or 1+ bizarre microcytes with central pallor; dysplastic nRBCs >1+ Pappenheimer bodies

Myelodysplasia, sideroblastic anemia

≥2+ Rouleaux ≥1+ Autoagglutination Moderate to marked hypochromia Microcytosis with 1+ to 2+ coarse basophilic stippling Sickle cells, hemoglobin C crystalsa ≥2+ Polychromatophilic red cells ≥1+ Teardrop-shaped RBCs 1+ MAP change (≥1+ schistocytes plus any number of helmet cells, spherocytes)b 1+ Oxidative change (≥1+ “bite cells” and/or large fragmented cells with puddled hemoglobin plus any number of spherocytes)b ≥2+ Spherocytes (except hyposplenic state) ≥2+ Acanthocytes, burr cells or target cells ≥80% Stomatocytes with even distribution on the slide confirmed by wet preparation

Sideroblastic anemia, ineffective erythropoiesis, severe hemolysis Hypergammaglobulinemia, including monoclonal gammopathy Autoimmune hemolysis, cold agglutinins Severe iron deficiency Heavy metal poisoning, thalassemia Hemoglobinopathy Hemolytic anemia, blood loss Bone marrow fibrosis (including primary myelofibrosis), megaloblastic anemia, thalassemia MAP hemolysis, respiratory distress syndrome in a premature infant, severe liver disease Oxidative hemolysis, RBC enzyme deficiencies

Autoimmune hemolysis, hereditary spherocytosis Hemoglobinopathy, liver disease, renal disease Hereditary stomatocytosis

a

See Chapter 3. See Chapter 5.

b

BIBLIOGRAPHY Bain BJ. Morphology in the diagnosis of red cell disorders. Hematology. 2005;10(Suppl 1):178–181. Glassy EF, ed. Color Atlas of Hematology. An Illustrated Field Guide Based on Proficiency Testing. Northfield, IL: College of American Pathologists; 1998.

Perkins SL. Examination of the blood and bone marrow. In: Greer JP, Foerster J, Rodgers GM, et al., eds. Wintrobe’s Clinical Hematology. 12th ed. Philadelphia, PA: Lippincott Williams & Wilkins, Inc.; 2008. Pierre RV. Red cell morphology and the peripheral blood film. Clin Lab Med. 2002;22(1):25–61.

3

Anemia Due to Abnormal or Impaired Hemoglobin Synthesis Anemias due to abnormal hemoglobin synthesis or impaired hemoglobin synthesis are characterized by a microcytic anemia. There are three broad categories of anemias of this type—anemias caused by defects in (1) iron metabolism, (2) porphyrin synthesis, and (3) globin chain synthesis.

DEFECTS IN IRON METABOLISM Perturbations in iron metabolism include iron deficiency, anemia of chronic disease, congenital abnormalities such as atransferrinemia, and acquired conditions (i.e., idiopathic pulmonary hemosiderosis).

Iron Deficiency The etiology of iron deficiency includes decreased intake, increased turnover/consumption, and loss of iron. Decreased iron intake ■

Decreased dietary oral intake

■

Malabsorption (i.e., gastrointestinal surgery) or malabsorption syndromes

Increased iron consumption ■

Pregnancy and the peripartum period

■

Intravascular hemolysis, with hemoglobinuria

■

Growth spurts in infants and children

22

Loss of iron ■

Bleeding

■

Dialysis

In pregnancy and the peripartum state, a combination of iron diversion to the fetus, blood loss at delivery, and lactation amounts to a loss of approximately 900 mg of iron. This is the equivalent of approximately 2 L of blood with regards to the iron content. Approximately 30 mg of iron alone is used monthly in lactation. Bleeding may be due to a variety of causes and typically is gastrointestinal bleeding (e.g., colon carcinoma, peptic ulcer) or menstrual blood loss. Loss of iron through dialysis occurs through two mechanisms. In the first, blood is trapped in the dialyzing equipment. In the second, a microcytic anemia can occur as a result of contaminating the dialysate with aluminum, thereby inhibiting heme synthesis. The progression of iron deficiency goes through three phases. The first phase represents iron depletion; this is the earliest stage of iron deficiency. Storage iron in reticuloendothelial cells in the liver and bone marrow is reduced or absent. This reduction of storage iron results in decreased serum ferritin levels. At this early stage of iron deficiency, serum iron, hemoglobin, and the hematocrit may be relatively normal. The RDW (red cell distribution width) may only be slightly elevated. The second phase represents iron deficient erythropoiesis. The amount of storage iron is reduced or absent with

Chapter 3: Anemia Due to Abnormal or Impaired Hemoglobin Synthesis

23

Figure 3.1 Iron deficiency. Severe iron deficiency is evident in this patient whose blood smear shows a predominance of small (microcytic) erythrocytes with markedly increased central pallor (hypochromic) resulting in a low MCHC. RBCs show a wide variation in shape (poikilocytosis) and size (anisocytosis) with frequent pencil cells resulting in an elevated RDW. Polychromatophilic red cells are reduced for the degree of anemia, indicating insufficient new RBC production.

low serum iron levels and low transferrin saturation, but without frank anemia. Depletion of iron stores occurs and hemoglobin levels fall to below 11 g/dL in woman and below 13 g/dL in men. The red blood cell (RBC) morphology is still normal, and the mean corpuscular volume (MCV) is in the low 80s fL. Microcytosis starts at 100 fL for individuals >12 years of age, but this will vary when the age is 100 × 109/L. In contrast to the aforementioned conditions, reticulocytes are a normal blood constituent and a subset of reticulocytes is visual in the peripheral blood as polychromatophilic red cells. Polychromatophilic red

TABLE 4.3 CONGENITAL DYSERYTHROPOIETIC ANEMIAS

Relative incidence/ age Inheritance pattern (gene involved) Morphology

Severity Ancillary studies

Type I

Type II

Type III

Second most common form Childhood/adolescence Autosomal recessive (CDAN1)

Most frequent form Adolescence/early adulthood Autosomal recessive (SEC23B)

Rarest form (few cases reported in select families) Autosomal dominant (CDAN3)

Three types of multinuclearity: 10%–31% multinuclearity in marrow Giant erythroblasts present in the peripheral blood 1. Binucleate cells with nuclear Pseudo-Gaucher cells commonly bridging found Extreme marrow erythroid 2. Giant cells with two irregular Moderate-size variation and chromasia multinuclearity (up to 12 nuclei nuclei without bridging seen in one erythroblast) 3. Binucleate cells with two disparate nuclei lacking uniform size or structure Moderate to severe anemia Mild to severe anemia Mild anemia Gene mutation analysis Gene mutation analysis Gene mutation analysis Elevated serum thymidine kinase Elevated serum thymidine Hams test positive (HEMPAS kinase type: [hereditary erythroblastic multinuclearity positive for acidified serum])

Chapter 4: Anemia Due to Abnormal or Impaired DNA Synthesis

39

Figure 4.3 Congenital dyserythropoietic anemia, type I. This blood smear is from a 3-year-old child with an MCV of 104 fL (normal 75 to 87 fL). In addition to oval macrocytes, teardrop cells and a dimorphic red cell population composed of hypochromic, microcytic red cells and normochromic, normocytic red cells are seen.

cells are round in shape, not showing the dysmorphic, oval features of megaloblastic anemia, myelodysplasia, or CDA. The hallmark of reticulocytosis as a cause of macrocytosis in the peripheral blood is the presence of increased polychromatophilic red cells, which show a gray-blue appearance. This is due to the presence of very small amounts of residual ribosomal RNA, which

Figure 4.4 Reticulocytosis. As a result of various causes, including blood loss and hemolysis, reticulocytosis can be present in the patient as manifested by increased numbers of polychromatophilic red cells in the peripheral blood. Due to their large size, polychromatophilic red cells are a recognized cause of macrocytosis. In this image, these cells are apparent by their round, large-size, and gray-blue cytoplasm (polychromasia), the latter owing to the presence of residual ribosomal RNA. This image was taken from a premature infant.

sometimes becomes most obvious after air drying as fine basophilic stippling, a feature that should not be misinterpreted as the pathologic coarse basophilic stippling. Supravital stains (e.g., methylene blue) can be used to better identify reticulocytes as they are more effective at precipitating RNA than a Wright-Giemsa preparation (Fig. 4.4).

40

Atlas of Peripheral Blood

Figure 4.5 Liver cancer. Although a nucleated cell is not present in this image for reference, the red cells are macrocytic and round in shape. Occasional target cells, a feature of liver disease, are also present.

LIVER DISEASE Nonalcoholic liver disease, such as liver cancer and viral hepatitis, can produce macrocytosis, characterized by round macrocytes, and other associated red cell changes including target cells and acanthocytes. The MCV is

usually between 100 and 110 fL, and reticulocytosis is not typically seen. Membrane lipids are increased in liver disease, correlating with the membrane surface area, which is presumably responsible for the increase in MCV in these conditions. The severity of liver disease does not correlate with the degree of macrocytosis (Figs. 4.5 and 4.6).

Figure 4.6 Hepatitis. The red cells in this field are normochromic, round in shape, and slightly increased in size compared to the nucleus of a normal lymphocyte. Target cells, a feature of liver disease, due to excess plasma lipids resulting in increased membrane lipids, are also present in this image.

Chapter 4: Anemia Due to Abnormal or Impaired DNA Synthesis

41

Figure 4.7 Alcoholism. Although excess alcohol intake may be associated with folate deficiency, resulting in the changes of megaloblastic anemia, chronic alcohol intake can in itself produce macrocytosis (sometimes without an accompanying anemia), composed of round macrocytes, as seen in this image. The macrocytosis is not due to impaired DNA synthesis, but it is thought to be secondary to the direct toxic effect of acetaldehyde (by-product of ethanol metabolism) on bone marrow hematopoiesis.

ALCOHOLISM AND ALCOHOLIC LIVER DISEASE Alcohol is the most common cause of macrocytosis, with the MCV having been used in screening procedures for the detection of alcohol abuse. Macrocytosis can be seen

Figure 4.8 Alcoholic liver disease. It is thought that excess lipids that attach to red cell membranes contribute to the macrocytosis seen in liver disease, the cells of which are typically round in shape as opposed to the oval macrocytes of megaloblastic anemia and myelodysplasia. In addition, target cells are a frequent finding in alcoholic liver disease as well as other causes of liver disease.

in the absence of or in association with liver disease and is most common in alcoholic individuals without anemia; anemia is more commonly observed in alcoholic patients with liver disease. In the absence of concomitant nutritional deficiency (B12 or folate), the MCV is typically between 100 and 110 fL, and the macrocytic red cells are

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round in shape. They do not exhibit the increased poikilocytosis of megaloblastic anemia. Although round macrocytes can be seen in alcoholism (without liver disease) and alcoholic liver disease, the typical changes seen in cirrhosis (i.e., target cells and acanthocytes) are not present in the setting of chronic alcohol intake without liver disease (Figs. 4.7 and 4.8). Both conditions lack the presence of reticulocytosis. In contrast to megaloblastic anemia, which results from impaired DNA synthesis and consequent red cell dysmaturation, the macrocytosis of alcoholism is caused by the direct toxic effect of a by-product of ethanol metabolism, acetaldehyde, on bone marrow hematopoietic progenitors, which can also produce thrombocytopenia due to impaired platelet production.

BIBLIOGRAPHY Colon-Otero G, Menke D, Hook CC. A practical approach to the differential diagnosis and evaluation of the adult patient with macrocytic anemia. Med Clin North Am. 1992;76(3):581–597. Kaferle J, Strzoda CE. Evaluation of macrocytosis. Am Fam Physician. 2009;79(3):203–208. Lindenbaum J, Roman MJ. Nutritional anemia in alcoholism. Am J Clin Nutr. 1980;33(12):2727–2735. Pierre RV. Reticulocytes. Their usefulness and measurement in peripheral blood. Clin Lab Med. 2002;22(1):63–79. Scott JM, Weir DG. Drug-induced megaloblastic change. Clin Haematol. 1980;9(3):587–606. Wickramasinghe SN. Congenital dyserythropoietic anaemias: clinical features, haematological morphology and new biochemical data. Blood Reviews. 1998;12:178–200.

Hemolytic Anemias

5

Marnelli Bautista-Quach, MD

Anemias secondary to hemolysis can be classified into two main groups: (1) extracorpuscular or extrinsic agents instigating red blood cell (RBC) destruction or hemolysis and (2) intracorpuscular or intrinsic, inherited RBC membrane or enzymatic defects. While RBC production is normal, the life span of the RBC is decreased due to the abnormal environment and/or RBC membrane defect.

EXTRACORPUSCULAR Microangiopathic Hemolytic Anemia (Intravascular Hemolysis) Pathologic activation of the coagulation cascade with concomitant fibrinolytic dysregulation leads to consumptive coagulopathy or disseminated intravascular coagulation (DIC), frequently with consumption of platelets and increased circulating fibrin strands. In nonconsumptive processes, a sticky hyaline-like substance within the capillaries traps platelets leading to thrombocytopenia. In both instances, shearing of RBCs occurs as they rapidly pass through the microvasculature lined with either fibrin or hyaline-like meshwork (i.e., intravascular hemolysis), resulting in the formation of fragmented cells (e.g., schistocytes, helmet cells) and microspherocytes as illustrated in Figs. 5.1 and 5.2. Heart valve replacement or valvular disorders may also cause traumatic hemolysis due to sheering of red cells (Table 5.1).

Autoimmune Hemolytic Anemia Hemolysis occurs when the red cells, coated with immunoglobulins (IgM or IgG) or complement factor (C3), activate the complement cascade resulting in intravascular destruction, or attracts macrophages of

the reticuloendothelial system, producing extravascular hemolysis. Many of the autoimmune hemolytic anemias (AIHAs) are associated with lymphoproliferative disorders. The direct antiglobulin test or direct Coombs test (DAT) detects RBCs coated with antibodies or C3. The presence of alloantibodies secondary to exposure to foreign RBC antigens either through transfusion or pregnancy is ascertained by the indirect antiglobulin test. The antibodies could be cold or warm reacting, or inducible by drugs. 1. Cold autoagglutinins: The antibody is usually the IgM type, which has complement-binding ability, resulting in a positive DAT with C3. Large three-dimensional aggregates of agglutinated RBCs are seen in peripheral blood smears as shown in Figure 5.3. This phenomenon may cause a falsely decreased RBC count with a normal hemoglobin and high mean corpuscular volume (MCV) value on automated hematology analyzers due to the instrument counting clumps of red cells as single cells with increased diameters, and hence a falsely high MCV. Agglutination is reversed by warming the blood to 37°C. Mycoplasma pneumoniae infection or lymphoma may prompt production of cold autoagglutinins. 2. Warm autoagglutinins: This condition is mediated predominately by IgG antibodies. Increased numbers of spherocytes, nucleated RBCs, and polychromatophilic red cells are encountered in the peripheral blood smear (Fig. 5.4). The disease may be idiopathic or related to other conditions such as lymphoma or autoimmune disorders, particularly systemic lupus erythematosus. Drugs such as penicillin, 6-mercaptopurine, ribavirin, and a-methyldopa may also promote warm autoantibody production, a process known as druginduced immune hemolysis.

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The Mechanics of Microangiopathy This demonstrates the area of RBC folding over the fibrin strand Point of fold

Point of fold

RBCs = 1 Schistocyte 1 Helmet Cell

RBC = 2 Helmet Cells

= Fibrin, micro thrombi, narrow vessels, or organ attachment

RBC folded in half over fibrin

Point of fold

= Fibrin

= Fibrin

RBC folded 1st quarter

RBCs in circulation

RBCs = 1 Helmet Cell 1 Schistocyte

RBC folded at ¾

RBCs in circulation RBCs in circulation

Figure 5.1 Mechanism of microangiopathic hemolysis. Depending on where the RBC folds over the fibrin strand, different sizes of fragmented red cells will be present in the circulation. As shown in this example, these could be the small schistocyte or the larger helmet cell. This point of contact may represent a fibrin strand, microthrombi, a narrow vessel or torturous vessel, or some other physical object that sheers the RBC.

Heat-associated and Other Traumatic Anemias Exposure to extremely high temperatures can damage the RBC membrane leading to significant variability in red cell size and shape (Fig. 5.5). Splenomegaly or hypersplenism is another factor that can induce mechanical destruction of RBCs leading to normocytic normochromic anemia. Leukocytes and platelets may also decrease in number secondary to sequestration, resulting in additional cytopenias.

INTRACORPUSCULAR Oxidative Hemolytic Anemia Glucose-6-phosphate dehydrogenase (G6PD) and pyruvate kinase (PK) deficiencies are the two most common hereditary RBC enzyme disorders that can cause hemolytic anemia (Fig. 5.6). G6PD deficiency: This is the most common RBC enzyme deficiency affecting more than 400 million people. The incidence is highest in Africans, people of Mediterranean descent, and Southeast Asians. Given

Chapter 5: Hemolytic Anemias

45

Figure 5.2 A: A remarkable number of schistocytes are present in this patient with microangiopathic hemolytic anemia due to hemolytic uremic syndrome. Note the absence of platelets. B: In this patient with toxic shock syndrome, the neutrophil at bottom contains numerous toxic granulations. At top, an immature granulocyte with vacuolated cytoplasm and toxic granulation is present. Numerous schistocytes are present among the red cells and there is a striking lack of platelets.

the high incidence of G6PD deficiency in areas of the world where malaria was once endemic, this deficiency is thought to be protective against infection by malaria. The hexose monophosphate shunt pathway is essential in protecting RBCs from oxidative injury. G6PD provides reducing capacity by sustaining levels of nicotinamide adenine dinucleotide phosphate (NADPH). Glutathione reductase transforms the oxidized glutathione (GSSG) into its reduced form (GSH) with the aid of the coenzyme, NADPH, thereby maintaining a repository of GSH (Fig. 5.7).

1. Class I, II variants: In the class I variant, patients have extremely depleted enzyme levels (17% ∼5%–6% ∼10%–12%

supporting that HPP is a form of HE. HPP is seen in individuals with homozygous mutations of spectrin (i.e., mutation inherited from both parents). This leads to severe disruption of spectrin self-association. Patients with HPP are most often of African origin and frequently have complications of severe anemia including growth retardation, gallbladder disease, splenomegaly, and bone abnormalities. The peripheral blood smears typically have large numbers of fragmented red cells with spherocytes, nonspecific poikilocytes, red cell budding, and occasional elliptocytes (Fig. 5.16). The morphologic picture resembles the blood smear findings in a patient with severe thermal burns (Fig. 5.5). The fundamental difference in HPP versus HE is that the red cell membrane in HPP is usually markedly deficient in spectrin protein, in addition to having a functionally abnormal protein. This may be due, in part, to increased degradation of the abnormal spectrin protein being produced. Infants affected with HE are generally not symptomatic until 4 to 6 months of life. However, a subset of neonates, particularly those with a-spectrin abnormalities may have a “neonatal poikilocytosis syndrome.” These neonates have jaundice and a peripheral blood morphologic picture similar to HPP with moderately severe hemolytic anemia. This usually abates by 4 months to 2 years of age, followed by a common HE that is often asymptomatic.

Chapter 5: Hemolytic Anemias

Incubated Osmotic Fragility 100 90

Positive osmotic fragility

80 70 Hemolysis %

Figure 5.14 Osmotic fragility plot. The solid line depicts a left-shifted curve characteristic of hereditary spherocytosis. However, other conditions such as immune hemolytic anemias characterized by increased numbers of spherocytes may also demonstrate a positive osmotic fragility result. The osmotic fragility test measures the in vitro lysis of red cells suspended in solutions of decreasing osmolarity. The red cell is freely water permeable and thus, when red cells are placed into a hypotonic solution, water is osmotically drawn into the red cell. As the cell swells, it becomes spherical. After the critical volume of the cell is reached, the membrane begins to leak large molecules such as hemoglobin. This release of hemoglobin into the supernatant is measured spectrophotometrically. As a result of membrane loss, spherocytes have less capacity for additional swelling and thus are more fragile than normal red cells when placed into hypotonic solution.

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Normal Control

60 50 40 30 20 10 0 0.15

0.14

0.13

0.12

0.11

0.1

0.09 0.08 NaCl (M)

Control

0.07

0.06

0.05

0.04

0.03

Patient

SOUTHEAST ASIAN OVALOCYTOSIS TABLE 5.4 INHERITED RBC MEMBRANE MUTATIONS Disorder

Percentage

Hereditary elliptocytosis 65 a-Spectrin

b-Spectrin

30

Protein 4.1R

5

Southeast asian ovalocytosisb Band 3

100

Hereditary spherocytosis Ankyrin 50–60 Other proteins 30–40

Common Findings Heterozygous— asymptomatic Homozygousa—severe hemolytic anemia Heterozygous—variable clinical findings Homozygousa—usually fatal Heterozygous— asymptomatic or little hemolysis Homozygousa—severe hemolytic anemia

Heterozygous— asymptomatic/mild Homozygous—fatal in utero

SAO is a RBC membrane disorder that is commonly seen in malaria endemic areas in Melanesia, Malaysia, Philippines, Indonesia, and southern Thailand. The prevalence ranges from 5% to 25% of individuals in these areas. Similar to HE, the disease is inherited in an autosomal dominant manner, although only heterozygous forms are seen. In contrast to HE, only one mutation has been identified, which affects the band 3 protein in the RBC membrane (see Table 5.4). This mutation results in a very rigid, but mechanically stable red cell membrane. Affected individuals surprisingly experience no hemolysis or minimal red cell hemolysis despite the increased red cell membrane rigidity. This abnormality is presumed to have evolved also as a protective mechanism against invasion of malarial parasites. Peripheral blood smears show rounded ovalocytes that contain one or two transverse ridges or a single longitudinal slit. Some of these cells may have the appearance of either stomatocytes or “shoe buckles” with two areas of central pallor bisected by a hemoglobin bridge.

Variable clinical findings

a Includes compound heterozygous cases in which individuals have two different HE-related spectrin mutations (one from each parent) instead of the same spectrin mutation inherited from both parents (homozygous). b Also known as hereditary ovalocytosis.

Acanthocytosis Abetalipoproteinemia is a rare autosomal recessive disorder due to deficiencies of apolipoproteins B-48

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TABLE 5.5 CLASSIFICATION OF COMMON HE PHENOTYPES HE type

Clinical Findings

Peripheral blood smear

Typical HE Silent carrier HE with hemolysis

Asymptomatic typically Asymptomatic Sporadic hemolysis in response to infection, moderate to severe compensated hemolysis Moderate to severe transfusion-dependent hemolysis Neonatal jaundice, severe hemolysis

Marked elliptocytosis Normal red cell morphology Marked elliptocytosis, fragmented red cells, poikilocytes

Homozygous, compound heterozygous HE Hereditary pyropoikilocytosis HE with infantile poikilocytosis HE with dyserythropoiesis

Neonatal hemolysis, jaundice By 6–12 months of age, hemolysis and poikilocytosis resolve Sporadic hemolysis due to ineffective, dysplastic erythropoiesis

and B-100 or defects in the microsomal triglyceride transfer protein. It is characterized by lipid malabsorption resulting in exceedingly low levels of plasma phospholipids, cholesterol (20%. B: A closer image of basophils with large purple granules and lobated nucleus with an accompanying purple haze of degranulation on the smear. C: Dysplastic hypogranular neutrophils and a marked thrombocytopenia < 100 × 109/L are present in this patient with accelerated phase. A small blast is seen in the center, right next to a promyelocyte.

gene. The diagnosis of CML requires the presence of the t(9;22)(q34;q11) or BCR-ABL1. Typically, CML goes through three stages (Table 8.3; Figs. 8.2–8.6): The vast majority of patients present with CML in the chronic phase. In addition to elevated white blood cell (WBC) counts, patients typically present with normal to elevated numbers of platelets that may exceed 1000 × 109/L but thrombosis is unusual. Due to this excess cell proliferation, serum lactate dehydrogenase and uric acid are typically increased as well. The leukocytosis of CML consists primarily of neutrophils, immature granulocytes with an accompanying basophilia, and often eosinophilia. Nucleated red cells, giant platelets, megakaryocytic nuclei, and abnormalities such as hypogranular leukocytes, binucleate leukocytes, hypersegmented neutrophils, mitotic cells,

giant metamyelocytes, and leukocytes with basophilic and eosinophilic granules may also be seen. The differential diagnosis of CML includes myeloid leukemoid reactions (see Chapter 6). Reactive causes of basophilia are shown in Table 8.4.

TABLE 8.4 REACTIVE CAUSES OF BASOPHILIA Diabetes Drugs (i.e., estrogen, IL-3) Hyperlipidemia Hypersensitivity reactions Myxoedema Tuberculosis Ulcerative colitis

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Figure 8.4 Chronic myelogenous leukemia, myeloid blast phase. A: Promyelocytes and blasts comprise most of the leukocytes in this patient transforming to blast phase of CML. B: Three large basophilic blasts with cytoplasmic vacuoles suggesting monocytic differentiation are adjacent to a small lymphocyte. C: At right a megakaryoblast is present with cytoplasmic blebbing above a giant platelet in this patient who transformed to a blast phase characterized by megakaryocytic differentiation. At left a blast is present with agranular basophilic cytoplasm.

TABLE 8.5 NATURAL HISTORY OF POLYCYTHEMIA VERA Prepolycythemic phase

→ Polycythemic phase

Mild erythrocytosis

Increased red cell mass

Median survival

10–15 y

→ Postpolycythemic myelofibrosis Cytopenias with leukoerythroblastosis and dacrocytes, bone marrow fibrosis, splenomegaly with extramedullary hematopoiesis 2–3 y

In the prepolycythemic phase of the disease, only a mild erythrocytosis is present, but a persistent elevation in red cell mass is noted in the polycythemic phase. This corresponds to hemoglobin levels of >16.5 g/dL in women and >18.5 g/dL in men. In addition, there is typically an accompanying leukocytosis of WBC > 12 × 109/L involving neutrophils, immature granulocytes and basophils. Approximately one-half of patients will have a thrombocytosis that may exceed 1,000 × 109/L. Some patients will have hypochromic and microcytic red cells representing a concomitant iron deficiency due to excess red cell production and depletion of iron stores. In the spent phase of the disease (postpolycythemic myelofibrosis), the red cell mass has normalized or is decreased and the marrow has been replaced by fibrosis with resulting cytopenias, splenomegaly with extramedullary hematopoiesis, and a subsequent leukoerythroblastic smear with dacrocytes. At this stage of the disease, the median survival has markedly decreased with mortality due to hemorrhage and thrombosis, with one-fifth of patients transforming to myelodysplasia and/or acute myeloid leukemia.

Chapter 8: Myeloproliferative Neoplasms and Myeloid and Lymphoid Neoplasms with Eosinophilia

Figure 8.5 Chronic myelogenous leukemia, lymphoid blast phase. A: Inapproximately 20% to 30% of cases of blast phase, the blasts are lymphoid. Rare cases present with both myeloid and lineage differentiation of blasts. In this field, two lymphoblasts at top and left are present in a background containing neutrophils, an eosinophil, and a basophil. B: Lymphoblasts with very high nuclear-to-cytoplasmic ratios, scant basophilic cytoplasm, and smooth chromatin are present at 3 o’clock, 7 o’clock, and 10 o’clock. The remaining leukocytes include neutrophils, an eosinophil, and a hypogranular promyelocyte.

Figure 8.6 An unusual case of chronic myelogenous leukemia, chronic phase, and diffuse large B-cell lymphoma circulating in the peripheral blood. A: A deeply basophilic large atypical lymphoma cell is in the center of the image with a band at left. A basophil, myelocyte, and monocyte are also present in blood conspicuous for its lack of platelets. B: The corresponding bone marrow aspirate from this patient demonstrates sheets of large lymphoma cells. Immunophenotypic studies demonstrated support for diffuse large B-cell lymphoma in the bone marrow and blood.

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TABLE 8.6

TABLE 8.7

APPARENT VERSUS TRUE CAUSES OF ERYTHROCYTOSIS

REACTIVE CAUSES OF ERYTHROCYTOSIS

Apparent: Decrease in plasma volume giving the appearance of an increased red blood cell content (hemoglobin/hematocrit) ■ Decreased plasma volume due to dehydration or chronically contracted plasma volume True: Real increase in the total intravascular red blood cell mass ■ Primary erythrocytosis (low to normal EPO level) ● Polycythemia vera ■ Secondary erythrocytosis (normal to high EPO level) ● Reactive causes EPO, erythropoietin.

POLYCYTHEMIA VERA (PV) In PV, there is an increased production of erythrocytes independent of regulators of normal erythropoiesis resulting in expansion of the red cell mass despite low or normal levels of erythropoietin (a regulator of erythropoiesis). Almost all patients carry a mutation in the Janus 2 kinase gene (JAK2) resulting in an erythroid proliferation, as well as proliferation of megakaryocytes and granulocytes. The three phases of PV are as follows (Table 8.5): Symptoms arise from the erythrocytosis that causes hyperviscosity and subsequent venous and arterial thromboses (i.e., myocardial infarctions, strokes, venous thromboses of the legs). Venous occlusion of intra-abdominal vessels such as mesenteric, portal, and hepatic veins (Budd-Chiari syndrome) is highly suggestive of PV. A majority of patients present with splenomegaly and plethora, as well as other symptoms related to erythrocytosis (i.e., headache, dizziness, paresthesias, visual disturbances, and painful feet). The latter finding may be associated with digital ischemia despite palpable pulses. Erythromelalgia (erythema, warmth, and a burning sensation in the hands and feet) may also be seen. In some patients, concomitant platelet abnormalities result in problems of hemostasis including epistaxis and bleeding of gums and gastrointestinal tract. Approximately 50% of patients will present with itching on exposure to water thought to be related to histamine release from basophils. The diagnosis of PV depends on finding a genuine increase in red cells without other explanations as shown in Tables 8.6–8.8 (Fig. 8.7). The presence of the

Congenital Mutant high oxygen affinity hemoglobin Congenital low 2,3-DPG Mutational defects in hypoxia sensing Acquired Arterial hypoxemia High altitude Cyanotic congenital heart disease Chronic lung disease, smoking; obstructive sleep apnea Renal lesions Renal tumors, cysts, diffuse parenchymal disease, hydronephrosis, renal artery stenosis, renal transplantation Endocrine lesions Adrenal tumors Hepatic lesions Hepatoma, cirrhosis, hepatitis Miscellaneous tumors Cerebellar hemangioblastoma, uterine fibroids, lung carcinoma Drugs Androgens Italicized text indicates common causes of reactive erythrocytosis.

JAK2 mutation in > 95% of patients is helpful in excluding reactive causes of polycythemia.

PRIMARY MYELOFIBROSIS (PMF) Primary myelofibrosis (PMF) is a disorder characterized by a clonal proliferation of megakaryocytes and granulocytic precursors in the bone marrow accompanied by reactive marrow fibrosis thought to be secondary to local release of fibrogenic growth factors. This replacement of hematopoietic marrow by fibrosis leads to extramedullary hematopoiesis, most commonly in spleen and liver, with subsequent organomegaly and compression of adjacent normal structures. While 30% to 40% of patients are asymptomatic at diagnosis with disease suggested by physical examination (e.g., splenomegaly) or abnormal blood tests, symptoms are usually due to anemia, hypermetabolic state, splenomegaly, or thrombocytopenia. Anemia leads to fatigue, dyspnea, weakness, and palpitations, while a hypermetabolic state can cause weight loss, fever, sweats, gout, or renal stones. The massive splenomegaly seen in primary myelofibrosis often

Chapter 8: Myeloproliferative Neoplasms and Myeloid and Lymphoid Neoplasms with Eosinophilia

79

Figure 8.7 Polycythemia vera. A: In this peripheral blood smear from a patient in the polycythemic stage of PV, there are increased numbers of erythrocytes and platelets as well as a mild leukocytosis with a neutrophilia and mild basophilia. B: Erythrocytes may be microcytic and hypochromic as shown in this image, where the erythrocytes are smaller in size than the lymphocyte nucleus. Central pallor is increased in many of the red cells from the normal one-third to one-half of the red cell diameter. C: Numerous pencil cells are present in this PV patient presenting with microcytic and hypochromic red cells due to the excess red cells production by bone marrow, that depletes iron stores. D: In the spent phase or postpolycythemic myelofibrosis phase of the disease, the blood findings resemble those found in patients with primary myelofibrosis. Notably the blood smears show a leukoerythroblastic picture and poikilocytosis with frequent dacrocytes; dacrocytes and hypochromic erythrocytes are present in this image. At this stage of the disease, the red blood cell mass decreases. E: Transformation to acute leukemia in a patient with long-standing history of PV and treatment with cytotoxic agents. The blasts showed evidence of megakaryocytic differentiation by immunophenotyping. The background is notable for numerous large and giant hypogranular platelets and hypochromic erythrocytes.

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TABLE 8.8 PRACTICAL DIAGNOSIS OF POLYCYTHEMIA VERA ■

■ ■

Elevated hemoglobin ● If Hb is normal, and PV is suspected, look for causes for a masked erythrocytosis JAK2 V617F mutation Low EPO levela

Hb, hemoglobin; PV, polycythemia vera; EPO, erythropoietin level. The JAK2 V617F mutation is present in > 95% of patients with PV. Smaller numbers of patients have a mutation in exon 12 of JAK2. a The EPO level may be low or sometimes normal in these patients.

results in abdominal discomfort and early satiety. Low platelets or abnormal platelet function can cause hemorrhage. The disease course of primary myelofibrosis starts in a prefibrotic or cellular phase terminating in a fibrotic phase (Table 8.9). The cellular phase describes the bone marrow, which is hypercellular at this stage with minimal fibrosis. Once in the fibrotic phase, the marrow has been replaced by large amounts of fibrosis with accompanying abnormalities in megakaryocytes. This megakaryocytic atypia explains the peripheral smear findings of giant and bizarre platelets, circulating megakaryocyte nuclei, and even micromegakaryocytes (Fig. 8.8). Patients in the fibrotic phase have a reduced life expectancy and may progress to myelodysplasia and/or acute leukemia. In establishing a diagnosis of myelofibrosis, criteria have been outlined by the World Health Organization (Table 8.10). These include a morphology criterion, an

exclusionary criterion, and a molecular criterion. If no molecular abnormalities are detected, then the exclusion of secondary causes of fibrosis is required. These are listed in Table 8.11.

ESSENTIAL THROMBOCYTHEMIA (ET) A clonal population of mature megakaryocytes in the bone marrow in ET produces excess numbers of platelets resulting in the characteristic thrombocytosis, often exceeding 1,000 × 109/L in this disease. The average age at diagnosis is 50 to 60 years and the majority of cases are discovered by routine blood tests in asymptomatic patients. The high platelet count can result in symptoms usually related to vessel thrombosis or abnormal vascular reactivity such as dizziness, headaches, visual disturbances, transient ischemic attacks, digital ischemia, and paresthesias. Thromboses may involve arteries or veins, and hemorrhage is less frequent. Some patients develop acquired von Willebrand disease in which von Willebrand multimers adhere to platelets and are removed from the circulation. Patients may also present with erythromelalgia, an asymmetric erythema, burning pain, and warmth of hands and feet exacerbated by heat, exercise, or dependency. Splenomegaly is usually minimal. In the blood smear, large numbers of platelets are seen, which vary considerably in size, from small to giant (Fig. 8.9). White blood cells (WBC) and red blood cells are usually normal in number and morphology. The diagnosis of ET requires a sustained platelet count of > 450 × 109/L, typical bone marrow findings of increased

TABLE 8.9 PRIMARY MYELOFIBROSIS TIME COURSE AND PERIPHERAL BLOOD SMEAR FINDINGS Cellular phase Diagnosis Median survival CBC

Blood smear morphology

10–15 y Anemia Thrombocytosis Mild leukocytosis NRBCs Dacrocytes Immature granulocytes Large platelets

→

Fibrotic phase Most patients diagnosed 3–7 y Anemia High/low platelets High/low white cells Leukoerythroblastic smear with many dacrocytes Giant and bizarre platelets Megakaryocyte nuclei Micromegakaryocytes Few blasts common

Leukoerythroblastic smear refers to the presence in the blood smear of nucleated red cells and immature granulocytes. CBC, complete blood cell count.

Chapter 8: Myeloproliferative Neoplasms and Myeloid and Lymphoid Neoplasms with Eosinophilia

Figure 8.8 Primary myelofibrosis (PMF). A: A leukoerythroblastic smear is present accompanied by a marked thrombocytosis with many large and occasional giant and bizarre platelets. The leukoerythroblastosis refers to immature granulocytes accompanied by nucleated red blood cells. The typical dacrocytes seen in PMF are not observed in this patient who is status postsplenectomy. B: This image shows a large blast at left with a giant hypogranular platelet at top right and a well-granulated giant platelet at bottom. Scattered dacrocytes (tear-drop shaped erythrocytes) are present in the background. C: Dacrocytes are clearly visible with their tips pointing in different directions (spurious teardrops may be seen when all of the tips point in the same direction on a smear). At bottom left, a blunted dacrocyte with coarse basophilic stippling is noted. D: Another image of the blood from the patient shown in (A) reveals numerous large and giant platelets with many hypogranular forms and a giant and bizarre platelet at top. E: Acute myeloid leukemia arising from PMF with a large blast at center in a background of dacrocytes and scattered hypochromic erythrocytes.

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TABLE 8.10 WORLD HEALTH ORGANIZATION DIAGNOSTIC CRITERIA FOR PRIMARY MYELOFIBROSIS Major Atypical megakaryocytic hyperplasia with fibrosis in bone marrow or, if no fibrosis, a hypercellular marrow with granulocytic hyperplasia ■ Does not meet criteria for CML, MDS, PV or other myeloid neoplasm ■ JAK2 V617F mutation or other clonal marker (e.g. MPL W515K/L), or if no clonal marker, then exclusion of secondary causes of fibrosis are required Minor ■ Anemia ■ LDH increase ■ Leukoerythroblastic smear ■ Splenomegaly ■

The diagnosis of primary myelofibrosis requires all major criteria and two of the minor criteria. CML, chronic myelogenous leukemia, BCR-ABL1 positive; MDS, myelodysplastic syndrome; PV, polycythemia vera; LDH, lactate dehydrogenase.

numbers of enlarged and mature megakaryocytes, and evidence of a clonal marker. Typically one-half of patients have evidence of the JAK2 mutation. If no clonal marker is evident, then causes of a reactive thrombocytosis must be excluded (Table 8.12). ET is considered a diagnosis of exclusion and criteria for the other MPNs such as CML, PMF, and PV or other myeloid neoplasms, should not be met.

CHRONIC NEUTROPHILIC LEUKEMIA (CNL) CNL is a very rare disorder with granulocytic hypercellularity in the bone marrow resulting in a persistent blood neutrophilia. The peripheral blood shows more than 80% segmented neutrophils and bands out

Figure 8.9 Essential thrombocythemia (ET). A: A striking thrombocytosis is present with increased numbers of large platelets as well as many hypogranular forms. A giant and bizarre hypogranular platelet is just below the center of the field. B: An accompanying neutrophilia is present with the thrombocytosis. A giant hypogranular platelet is present at top, but many smaller platelets are also hypogranular. Erythrocytes are hypochromic in this patient with ET who has had many gastrointestinal bleeds.

Chapter 8: Myeloproliferative Neoplasms and Myeloid and Lymphoid Neoplasms with Eosinophilia

83

TABLE 8.11

TABLE 8.12

DIFFERENTIAL DIAGNOSIS OF PRIMARY MYELOFIBROSIS

REACTIVE CAUSES OF THROMBOCYTOSIS

Chronic MPN other than PMF Myelodysplastic syndrome Acute panmyelosis with myelofibrosis Acute myeloid leukemia Mastocytosis Malignant histiocytosis Vitamin D deficiency Connective tissue disease Autoimmune myelofibrosis Lymphomas Hairy cell leukemia Plasma cell myeloma Metastatic cancer Renal osteodystrophy Infections Gray platelet syndrome

of total white cells with 5% myeloblasts in blood or marrow. Mast cells may be difficult to morphologically distinguish from basophils, although the latter cell type has a segmented nucleus with condensed chromatin similar to a neutrophil with many coarse dense purple granules that overlay and obscure the nucleus. Immunophenotyping (e.g., flow cytometry, immunohistochemistry) can distinguish mast cells from basophils (Fig. 8.12).

MYELOPROLIFERATIVE NEOPLASM, UNCLASSIFIABLE (MPN,U) Disorders in this category fail to fall into the other diagnostic entities related above. This may be because the disease is early in its course and has not yet fully developed into a clear diagnostic category, or the disease is late in the course with end-stage findings such as advanced myelofibrosis and lacks earlier diagnostic features. Or, some diseases possess features of two or more MPNs. By definition, these patients lack the BCR-ABL1 fusion gene or abnormalities of PDGFRA, PDGFRB, or FGFR1 (Fig. 8.13). Myeloid and lymphoid neoplasms with eosinophilia and abnormalities of platelet-derived growth factor receptor alpha (PDGFRA), platelet-derived growth factor receptor beta (PDGFRB), or fibroblast growth factor receptor 1 (FGFR1) These three rare diseases all result from formation of a fusion gene that encodes an aberrant tyrosine kinase with resulting eosinophilia, but involve different fusion genes. A list of all primary bone marrow

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Figure 8.13 Myeloproliferative neoplasm, unclassifiable. A leukocytosis is present in this patient with neutrophilia and immature granulocytes. Frequent myelocytes are noted and significant dysplasia is not seen. This patient lacked BCR-ABL1 and lacked an absolute monocytosis and did not meet criteria for chronic neutrophilic leukemia. Reactive causes of a neutrophilia were also excluded.

disorders resulting in eosinophilia is presented in Table 8.14. Reactive causes of eosinophilia are far more common than myeloid and lymphoid neoplasms with eosinophilia and should be excluded (Table 8.15). Men are overwhelmingly affected by PDGFRA related neoplasms that usually present as chronic eosinophilic leukemias, typically the result of the FIP1L1-PDGFRA fusion gene, and may also involve the neutrophil and mast cell lineages. Serum tryptase may be elevated and patients present with anemia, and/or thrombocytopenia, and splenomegaly. Standard cytogenetic karyotyping studies are typically normal and the fusion gene

TABLE 8.14 PRIMARY BONE MARROW DISORDERS ASSOCIATED WITH EOSINOPHILIA ■ ■ ■ ■ ■ ■ ■

Acute myeloid leukemia or acute lymphoblastic leukemia Chronic myelogenous leukemia, BCR-ABL1 positive Myelodysplastic syndrome (MDS) MPN or MDS/MPN overlap syndromes Systemic mastocytosis Chronic eosinophilic leukemia Myeloid or lymphoid neoplasms associated with abnormalities of PDGFRA, PDGFRB or FGFR1

MDS, myelodysplastic syndrome; MPN, myeloproliferative neoplasm.

TABLE 8.15 REACTIVE CAUSES OF EOSINOPHILIA Infections ■ Parasites, bacteria, viruses, fungi, rickettsiae Allergy/hypersensitivity diseases ■ Asthma, rhinitis, drug reactions, allergic bronchopulmonary aspergillosis, allergic gastroenteritis Connective tissue diseases ■ Churg-Strauss, Wegener granulomatosis, systemic lupus erythematosus, rheumatoid arthritis, scleroderma, eosinophilic myositis Pulmonary ■ Bronchiectasis, cystic fibrosis, Löffler syndrome, eosinophilic granuloma of lung Cardiac ■ Tropical endocardial fibrosis, endomyocardial fibrosis or myocarditis Dermatologic ■ Atopic dermatitis, urticaria, eczema, dermatitis herpetiformis Malignancy ■ Hodgkin lymphoma, non-Hodgkin lymphoma, lung, breast, renal cancers Gastrointestinal ■ Eosinophilic gastroenteritis, celiac disease Metabolic ■ Adrenal insufficiency Other ■ IL-2 therapy, L-tryptophan ingestion, toxic oil ingestion, renal graft rejection

Chapter 8: Myeloproliferative Neoplasms and Myeloid and Lymphoid Neoplasms with Eosinophilia

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Figure 8.14 Myeloid neoplasm with eosinophilia and PDGFRA. This 50-yearold male presented with splenomegaly and a leukocytosis of 19.8 × 109/L with a marked eosinophilia, hemoglobin of 13.1 g/dL, and platelet count of 181 × 109/L. While cytogenetic karyotype was normal, FISH showed evidence of the FIP1L1-PDGFRA fusion gene. A: Eosinophils are markedly increased in number and contain paler eosinophilic granules than normal, that do not fill the cytoplasm of the cell. B: This eosinophil contains multiple small vacuoles as well and has a nucleus that is hypersegmented with four lobes instead of the normal two to three lobes.

must be detected using fluorescence in situ hybridization (FISH) or reverse transcriptase polymerase chain reaction (RT-PCR). Patients are extremely sensitive to the tyrosine kinase inhibitor imatinib. PDGFRBrelated neoplasms may present with features of chronic myelomonocytic leukemia with eosinophilia, atypical chronic myeloid leukemias, chronic eosinophilic leukemia, or MPN with eosinophilia. The hematologic and morphologic picture depends on the partner gene that rearranges with PDGFRB. Unlike abnormalities with PDGFRA, PDGFRB abnormalities are typically detected using standard cytogenetic karyotyping. For example, the most common rearrangement t(5;12) (q31-q33;p12) results in an ETV6-PDGFRB fusion gene. Molecular testing is recommended to confirm the result of cytogenetic studies. Middle-aged men are more affected by PDGFRB-related neoplasms and in addition to eosinophilia, patients present with splenomegaly and organ dysfunction related to eosinophilia. Serum tryptase may also be elevated. Depending on the partner gene, the hematologic picture can vary. Invariably, the white cell count is increased and may be accompanied

by anemia and thrombocytopenia. Eosinophils, neutrophils, monocytes, and precursors of neutrophils and eosinophils may all be variably increased (Fig. 8.15). Importantly, these patients are also sensitive to imatinib therapy. The last disorder, myeloid and lymphoid neoplasms with FGFR1 abnormalities, is not sensitive to imatinib therapy and currently carries a very poor prognosis; current recommended therapy is hematopoietic stem cell transplantation. FGFR1– related neoplasms are derived from a pluripotent stem cell and are also known as 8p11 stem cell leukemia/ lymphoma syndrome. The presentation can be as an MPN, AML, or T- or B-lymphoblastic lymphoma or as a mixed phenotype acute leukemia (Fig. 8.16); hence, the disease can involve blood, marrow, lymph nodes, liver, and spleen. The disease is very aggressive and typically terminates in acute leukemia within 1 to 2 years. The key to the diagnosis is the detection of the 8p11 by cytogenetic studies. Multiple different partner genes have been reported with FGFR1 (located at 8p11), with the most common being the ZNF198 located at 13q12.

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Figure 8.15 Myeloid neoplasm with eosinophilia and PDGFRB. This 72-year-old male had a chronic anemia of unclear etiology with a rising WBC count. At the time of bone marrow examination, laboratory values are as follows: WBC 38.1 × 109/L, Hb 10.7 g/dL, platelet count 170 × 109/L. Cytogenetic studies showed a t(5;12)(q33;p13) and FISH studies confirmed the ETV6-PDGFRB rearrangement. A: Examination of WBCs showed a neutrophilia and eosinophilia in this patient with scattered immature granulocytes. In this field, two eosinophils containing small vacuoles surround a neutrophil. In the bottom of the image, a promyelocyte with numerous primary granules is present. B: Eosinophils again contain small vacuoles and granules do not occupy all of the cytoplasm. The neutrophil at bottom is vacuolated. C: The bone marrow biopsy is almost 100% cellular with a marked myeloid hyperplasia with accompanying marrow eosinophilia. Scattered atypical megakaryocytes are also seen. D: A reticulin stain shows marked reticulin fibrosis in the bone marrow biopsy, which explains the difficulty in obtaining a bone marrow aspirate (smear not shown).

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Figure 8.16 Myeloid and lymphoid neoplasm with eosinophilia and FGFR1. A: 46-year-old male with inguinal lymphadenopathy showing a T-lymphoblastic lymphoma. Cytocentrifuge smear of the lymph node submitted for flow cytometry shows an eosinophilia admixed with small and large lymphoblasts. B: A concurrent peripheral blood smear with WBC 33 × 109/L, RBC 6.45 × 1012/L, Hb 16.7 g/dL, MCV 77fL, PLT 158 × 109/L with absolute neutrophils 26 × 109/L, absolute monocytes 3.3 × 109/L, and absolute eosinophils 2.7 × 109/L. Blood smear highlights the eosinophilia and immature granulocytes. The bone marrow was hypercellular with a myeloid and megakaryocytic hyperplasia and eosinophilia, with cytogenetics showing a t(8;13); (p11;q12); PCR confirmed the ZNF198-FGFR1 fusion gene.

BIBLIOGRAPHY Anastasi J. The myeloproliferative neoplasms: insights into molecular pathogenesis and changes in WHO classification and criteria for diagnosis. Hematol Oncol Clin North Am. 2009;23(4):693–708. George TI. Pathology of the myeloproliferative diseases. In: Greer JP, Foerster J, Rodgers GM, et al., eds. Wintrobe’s Clinical

Hematology. 12th ed. Philadelphia, PA: Lippincott Williams & Wilkins; 2008. George TI, Horny HP. Systemic mastocytosis. Surg Path Clinic. 2010;3(4):1185–1202. Gotlib J. Eosinophilic myeloid disorders: new classification and novel therapeutic strategies. Curr Opin Hematol. 2010;17(2):117–124. Vardiman JW. Chronic myelogenous leukemia, BCR-ABL1 positive. Am J Clin Pathol. 2009;132(2):250–260.

Myelodysplastic Syndromes Myelodysplastic syndrome (MDS) represents a group of clonal neoplasms that primarily involve the peripheral blood and bone marrow. These proliferations have been termed “preleukemia” in the past and patients with MDS will often transform to acute myeloid leukemia or succumb due to bone marrow failure. While the complete diagnostic evaluation requires examination of bone marrow aspirate smears and a bone marrow biopsy, review of the peripheral blood morphology is also essential and the blood provides many valuable diagnostic clues in MDS. Patients with MDS characteristically present with one or more cytopenias and MDS should be diagnosed with extreme caution in the absence of a cytopenia, especially in the absence of anemia. In the blood, the morphologic features of MDS occur as red blood cell, white blood cell, and platelet changes as well as the presence and enumeration of blast cells. As with all diagnostic smears, the interpretation of myelodysplastic changes is dependent on high quality and well-stained smears, as staining artifact can mimic some dysplastic changes. Additionally, some reactive changes may mimic MDS and complete knowledge of the clinical history, including drug history is needed to properly interpret smears for the presence of MDS. Dysplasia of the red blood cell series (dyserythropoiesis) is most easily recognized in marrow nucleated red blood cells by identification of erythroid nuclear abnormalities that include irregular nuclear contours, nuclear budding, and nuclear to cytoplasmic asynchrony. Cytoplasmic changes of dyserythropoiesis include nuclear vacuolization, often containing periodic acid-Schiff (PAS) positivity, or the presence of cytoplasmic siderotic granules that may include ring sideroblasts. When nucleated red blood cells are present in the blood, such features may be apparent.

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9 Buffy coat preparations may concentrate the nucleated red blood cells. In cases with numerous nucleated red blood cells in the blood, an iron stain may be useful to identify increases in red blood cell iron incorporation and ring sideroblasts with iron granules ringing one-third or more of the nucleus. However, most cases of MDS do not show nucleated red blood cells in the blood and the nonnucleated red blood cell changes are less specific. Red blood cell dysplasia in MDS should be accompanied by the presence of anemia, usually in the absence of an adequate reticulocytosis or red blood cell polychromasia. Red blood cells in many cases of MDS are macrocytic and often show moderate to marked anisocytosis and poikilocytosis, including hypochromic cells, teardrop-shaped red blood cells, and cell with Pappenheimer granules. In refractory anemia with ring sideroblasts, the red cells often show a dimorphic population of cells that include a population of macrocytes and a second population of normochromic normocytic red blood cells (Figs. 9.1–9.3). Unfortunately, none of these changes are specific for MDS. As described previously (see Chapter 4), anisopoikilocytosis, macrocytosis, and even the presence of ring sideroblasts may occur in nonneoplastic conditions. Dysplasia of the white blood cell series (dysgranulopoiesis) is also characterized by nuclear and cytoplasmic abnormalities. Because the bone marrow of many MDS patients shows limited granulocyte maturation, the peripheral blood is often the best site for identification of dysgranulopoiesis. The most typical features include nuclei that have clumped nuclear chromatin of a segmented neutrophil but fail to segment fully. These neutrophils may have an unsegmented nucleus that is shaped more like a myelocyte or may have a bilobed nucleus (pseudo–Pelger-Huët anomaly). These dysplastic cells tend to be small in size and have hypogranular cytoplasm.

Chapter 9: Myelodysplastic Syndromes

Figure 9.1 Dysplastic nucleated red blood cells. A: This buffy coat preparation of peripheral blood shows a dysplastic nucleated red blood cell. The nucleus is irregular and a nuclear fragment is present in the cytoplasm. B: This peripheral blood smear shows a nucleated red blood cell with blebbing and an adjacent blast cell. C: This nucleated red blood cell shows prominent granules suggestive of a ring sideroblast.

Figure 9.2 A,B: Red blood cell changes of myelodysplasia. The red blood cells show a dimorphic population with teardrop-shaped macrocytes and smaller, hypochromic cells.

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Figure 9.3 A,B: Red blood cell changes of myelodysplasia. These examples show more poikilocytosis, including teardrop-shaped red blood cells and fragmented cells.

Some cases, however, may have pseudo Chediak-Higashi granules or even hypersegmented nuclei. As described in Chapter 6, the nuclear changes alone are not specific for MDS and are also seen in patients with the inherited Pelger-Huët anomaly or as a reactive change secondary to some therapeutic drugs. One must also differentiate pyknotic nuclei in neutrophils from a poorly preserved specimen from truly dysplastic cells as well. With poor staining, normal neutrophils may appear to be hypogranular and well-stained cells with more normal cytoplasmic granules as an internal control should be identified before cells are considered hypogranular. The combination of both nuclear hyposegmentation and hypogranular cytoplasm appears to be the most reliable for suggesting MDS (Figs. 9.4 and 9.5). While megakaryocyte dysplasia cannot be assessed on peripheral blood smears, the platelets of dysplastic megakaryocytes are usually abnormal. In most MDS types, the platelet count is low or normal, but it may be elevated in MDS associated with isolated del(5q). Hypogranular platelets are often found in MDS and these may be normal or large in size (Fig. 9.6). Identification and enumeration of blast cells in the blood are also important in MDS. Blasts in MDS are almost always myeloblasts and may show features that range from undifferentiated blasts with scant agranular cytoplasm to cells with monocytoid features to blasts with granules and Auer rods. Blasts in MDS are

not distinguishable from the blasts of most types of acute myeloid leukemia (see Chapter 11). Auer rods may occasionally be seen in more mature granulocytes as well, and these rod-like collections of myeloperoxidase granules are an abnormal finding in any cell type. Because some studies have linked the presence of Auer rods in MDS to a worse prognosis, cases with Auer rods even in the absence of an increase in blasts are classified as refractory anemia with excess blasts-2 (RAEB-2) (Fig. 9.7). Careful enumeration of blast cells in the blood is essential for the proper classification of MDS and at least 200 white blood cells should be counted to properly determine the blood blast percentage. The blast percentages in the blood vary by disease category, but clearly identifying cases with 1% or more peripheral blood blasts from those with