Benign hematologic disorders in children : a clinical guide 9783030499808, 3030499804, 9783030499815, 3030499812, 9783030499822, 3030499820

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Benign Hematologic Disorders in Children A Clinical Guide Deepak M. Kamat Melissa Frei-Jones Editors

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Benign Hematologic Disorders in Children

Deepak M. Kamat  •  Melissa Frei-Jones Editors

Benign Hematologic Disorders in Children A Clinical Guide

Editors Deepak M. Kamat Department of Pediatrics University of Texas Health Science Center San Antonio, TX USA

Melissa Frei-Jones Department of Pediatrics Division of Hematology-Oncology The University of Texas Health Science Center San Antonio, TX USA

ISBN 978-3-030-49979-2    ISBN 978-3-030-49980-8 (eBook) https://doi.org/10.1007/978-3-030-49980-8 © Springer Nature Switzerland AG 2021 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

Preface

It is with great pleasure and immense honor we present to you Benign Hematologic Disorders in Children: A Clinical Guide. Health-care providers, including pediatricians, family physicians, and pediatric nurse practitioners, often encounter children presenting with benign hematological conditions such as anemia, thrombocytopenia, neutropenia, elevated lead levels, as well as bleeding and clotting disorders. The majority of such patients can and should be evaluated by a primary care child health provider and many conditions can successfully be managed in the outpatient setting. Some conditions need consultation and treatment by a pediatric hematologist and these patients are best co-managed with the primary care provider and the hematologist. The specialty of pediatric hematology is expanding rapidly with tremendous improvements in diagnostic procedures, preventive measures, and therapeutic modalities. Hematology involves disorders affecting the cellular and non-cellular components of blood; therefore, the content of this book addresses these disorders. For the benefit of the reader, we begin with reviewing the basic concepts and recent advances in our understanding of hematopoiesis. Next, we discuss disorders affecting red blood cells, thrombocytes, and white blood cells. This is followed by discussions on the bleeding and clotting disorders including Von willebrand disease, hemophilia, acquired disorders of coagulation, thromboembolism, and clotting disorders affecting children and adolescents. Many of us provide care to newborns in nurseries and neonatal intensive care units where we encounter babies with hematological disorders and so have included a section dealing with common hematological conditions affecting neonates including newborn screening for hemoglobin disorders. Primary bone marrow disorders are uncommon. However, because of better understanding of the pathophysiological mechanism of these disorders as well as specific and supportive care (discussed under “supportive care” section), many of these patients are leading normal lives and are taken care of by primary care providers in consultation with hematologists. To complete the disorders affecting the non-cellular component of blood, we have included complement mediated hematological disorders. Finally, even though not a part of hematology in the true

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Preface

sense but still related to blood, we have included a chapter on infantile hemangioma and common vascular malformations. We thank all the authors, who have such tremendous expertise and experience in the field of pediatric hematology, for helping us prepare this resource for the benefit of our readers. We could not have done this without them! Our sincere hope is that this book will help provide better care to children with hematological disorders all over the world. San Antonio, TX, USA San Antonio, TX, USA

Deepak M. Kamat

Melissa Frei-Jones

Contents

Part I Origin of Blood Cells 1 Hematopoiesis������������������������������������������������������������������������������������������    3 Chintan Parekh Part II Red Blood Cell Disorders 2 Nutritional Anemias: Iron Deficiency and Megaloblastic Anemia������������������������������������������������������������������������������   15 Deanna Mitchell, Jessica Foley, and Aarti Kamat 3 Lead Poisoning ����������������������������������������������������������������������������������������   31 Nicholas Newman 4 Immune and Nonimmune Hemolytic Anemia��������������������������������������   51 Christina Caruso and Satheesh Chonat 5 Sickle Cell Disease������������������������������������������������������������������������������������   65 Neethu Menon and Melissa Frei-Jones 6 Thalassemia����������������������������������������������������������������������������������������������   91 Mark Shamoun and Michael Callaghan 7 Disorders of RBC Metabolism����������������������������������������������������������������   99 Amber M. Yates 8 Red Blood Cell Membrane Defects��������������������������������������������������������  105 Amy Tang

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Contents

Part III Platelet Disorders 9 Immune Thrombocytopenia��������������������������������������������������������������������  115 Manasi Madiwale 10 Inherited and Congenital Thrombocytopenia ��������������������������������������  135 Deanna Maida 11 Platelet Disorders ������������������������������������������������������������������������������������  153 Katherine Regling and Meera Chitlur 12 Thrombocytosis in Children ������������������������������������������������������������������  175 Beverly A. Schaefer Part IV WBC Disorders 13 Neutropenia����������������������������������������������������������������������������������������������  191 Vinod K. Gidvani-Diaz 14 Granulocytosis������������������������������������������������������������������������������������������  205 Aarti Kamat and Deepak M. Kamat 15 Disorders of Granulocyte Functions������������������������������������������������������  213 Divya Seth and Pavadee Poowuttikul Part V Coagulation Disorders 16 von Willebrand Disease ��������������������������������������������������������������������������  233 Rohith Jesudas 17 Hemophilia�����������������������������������������������������������������������������������������������  247 Michael Callaghan 18 Acquired Disorders of Coagulation in Neonates and Children����������������������������������������������������������������������������  259 Daniel Gebhard and Melissa Frei-Jones 19 Thromboembolism ����������������������������������������������������������������������������������  269 Aimee Foord and Arash Mahajerin 20 Heavy Menstrual Bleeding and Bleeding Disorders in Adolescents: A Primer for the Primary Care Physician����������������������  295 Rida Abid Hasan and Ayesha Zia Part VI Neonatal Hematology 21 Newborn Screening for Hemoglobinopathies����������������������������������������  313 Melissa Frei-Jones 22 Neonatal Immune Hemolytic Anemia����������������������������������������������������  323 Alejandra Pena Hernandez

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23 Neonatal Hemostasis��������������������������������������������������������������������������������  335 Lincy Thomas 24 Disorders of Bilirubin Metabolism��������������������������������������������������������  353 Johanna M. Ascher Bartlett and Jay Shah Part VII Bone Marrow Failure Syndrome 25 Failure of Erythrocyte Production ��������������������������������������������������������  369 Anand Srinivasan 26 Inherited Bone Marrow Failure Syndromes ����������������������������������������  385 Bhakti Mehta 27 What Pediatricians Need to Know About Acquired Aplastic Anemia���������������������������������������������������������������������������������������  391 Lubna S. Mehyar Part VIII Supportive Care 28 Immunizations in the Child with Sickle Cell Disease ��������������������������  405 Emily K. Nease and Linda S. Nield 29 Transfusion Medicine for Pediatrics������������������������������������������������������  417 Bulent Ozgonenel 30 Management of Infections in Neutropenic Patients������������������������������  437 Shipra Gupta Part IX Miscellaneous 31 Pediatric Vascular Anomalies: Opportunities in Primary Care��������������������������������������������������������������������������������������������  453 Adam D. Wolfe 32 Complement Dysregulation Syndromes in Children and Adolescents������������������������������������������������������������������������  487 Chatchawin Assanasen Index������������������������������������������������������������������������������������������������������������������  503

Contributors

Johanna M. Ascher Bartlett, MD  UT Health San Antonio, San Antonio, TX, USA Chatchawin Assanasen, MD  Department of Pediatrics Division of Hematology & Oncology, UT Health San Antonio Long School of Medicine, San Antonio, TX, USA Michael  Callaghan, MD  Division of Hematology, Children’s Hospital of Michigan, Wayne State University, Detroit, MI, USA Christina  Caruso, MD  Aflac Cancer and Blood Disorders Center, Children’s Healthcare of Atlanta, Atlanta, GA, USA Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, USA Meera Chitlur, MD  Carman and Ann Adams Department of Pediatrics, Division of Hematology/Oncology, Children’s Hospital of Michigan/Central Michigan University, Detroit, MI, USA Satheesh  Chonat, MD  Aflac Cancer and Blood Disorders Center, Children’s Healthcare of Atlanta, Atlanta, GA, USA Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, USA Jessica Foley, MD  Helen DeVos Children’s Hospital, Grand Rapids, MI, USA College of Human Medicine, Michigan State University, East Lansing, MI, USA Aimee Foord, DO  Seattle Children’s Hospital, Seattle, WA, USA Melissa Frei-Jones, MD, MSCI  Pediatric Hematology-Oncology, Long School of Medicine, UT Health Science Center San Antonio, Houston, TX, USA UT Health San Antonio, Houston, TX, USA Daniel Gebhard, MD  UTHealth Long School of Medicine, San Antonio, TX, USA Vinod  K.  Gidvani-Diaz, MD, FAAP  Texas/Methodist Children’s Hospital, UT Health Joe R and Teresa Lozano Long School of Medicine, San Antonio, TX, USA xi

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Contributors

Shipra  Gupta, MD  Department of Pediatrics, Division of Pediatric Infectious Diseases, West Virginia University School of Medicine, Morgantown, WV, USA Rida  Abid  Hasan, MD  Division of Pediatric Hematology-Oncology, The University of Texas Southwestern Medical Center, Dallas, TX, USA Department(s) of Pediatrics, The University of Texas Southwestern Medical Center, Dallas, TX, USA Alejandra  Pena  Hernandez, MD  UT Health Science Center San Antonio, San Antonio, TX, USA Rohith Jesudas, MD  Bleeding and Clotting Disorders Institute, Peoria, IL, USA University of Illinois College of Medicine, Peoria, IL, USA Aarti  Kamat, MD  Helen DeVos Children’s Hospital Future fellow, Pediatric Hematology/Oncology – University of Michigan Medical Center, C.S. Mott Children’s Hospital, Ann Arbor, MI, USA Department of Pediatrics, University of Michigan, Ann Arbor, MI, USA Deepak M. Kamat, MD, PhD, FAAP  UT Health San Antonio, Department of Pediatrics, San Antonio, TX, USA Manasi Madiwale, MD  Community Medical Centers, Manteca, CA, USA Arash Mahajerin, MD  CHOC Children’s Hospital, Orange, CA, USA Deanna  Maida, MD  University of Texas Health Science Center, San Antonio, TX, USA Bhakti Mehta, MD, MPH  Amgen Inc., Thousand Oaks, CA, USA Lubna  S.  Mehyar, MD  Division of Pediatric Hematology, Oncology and Bone Marrow  Transplant, Ruby Memorial Hospital, West Virginia University, Morgantown, WV, USA Neethu  Menon, MD  UT Health McGovern School of Medicine, Houston, TX, USA Deanna Mitchell, MD  Helen DeVos Children’s Hospital, Grand Rapids, MI, USA College of Human Medicine, Michigan State University, East Lansing, MI, USA Emily  K.  Nease, MD  West Virginia University School of Medicine, Morgantown, WV, USA Nicholas Newman, DO, MS, FAAP  Department of Pediatrics and Department of Environmental & Public Health Sciences, University of Cincinnati, Pediatric Environmental Health & Lead Clinic, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA Linda S. Nield, MD  West Virginia University School of Medicine, Morgantown, WV, USA

Contributors

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Bulent  Ozgonenel, MD  Carman and Ann Adams Department of Pediatrics, Division of Hematology Oncology, Wayne State University School of Medicine, Children’s Hospital of Michigan, Detroit, MI, USA Chintan Parekh, MBBS  Children’s Hospital Los Angeles and the Keck School of Medicine at the University of Southern California, Los Angeles, CA, USA Pavadee Poowuttikul, MD  Children’s Hospital of Michigan, Division of Allergy/ Immunology, Department of Pediatrics, Central Michigan University, Detroit, MI, USA Katherine  Regling, DO  Carman and Ann Adams Department of Pediatrics, Division of Hematology/Oncology, Children’s Hospital of Michigan/Central Michigan University, Detroit, MI, USA Beverly  A.  Schaefer, MD  Roswell Park and Oishei Children’s Cancer and Blood Disorders Program, Roswell Park Comprehensive Cancer Center, Buffalo, NY, USA Department of Pediatrics, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY, USA Western New York BloodCare, Buffalo, NY, USA Divya  Seth, MD  Children’s Hospital of Michigan, Division of Allergy/ Immunology, Department of Pediatrics, Central Michigan University, Detroit, MI, USA Jay Shah, DO, MPH  UT Health San Antonio, San Antonio, TX, USA Mark Shamoun, MD  Division of Hematology, Children’s Hospital of Michigan, Central Michigan University, Mount Pleasant, MI, USA Anand  Srinivasan, MD  Jimmy Everest Section of Pediatric Hematology/ Oncology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA Amy Tang, MD  Emory University School of Medicine, Aflac Cancer and Blood Disorders Center, Children’s Healthcare of Atlanta, Atlanta, GA, USA Lincy Thomas, MD  University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA Adam D. Wolfe, MD, PhD  Baylor College of Medicine, The Children’s Hospital of San Antonio, San Antonio, TX, USA Amber M. Yates, MD  Baylor College of Medicine, Houston, TX, USA Ayesha Zia, MD  Division of Pediatric Hematology-Oncology, The University of Texas Southwestern Medical Center, Dallas, TX, USA Department(s) of Pediatrics, The University of Texas Southwestern Medical Center, Dallas, TX, USA

Part I

Origin of Blood Cells

Chapter 1

Hematopoiesis Chintan Parekh

Introduction Blood cell development (hematopoiesis) represents one of the most well-studied tissue development processes. The use of flow cytometry to define hematopoietic cell types, the investigation of hematopoietic cell differentiation pathways in cell culture and mouse models, and the characterization of molecular mechanisms through gene expression profiling and genetic manipulation experiments in human cells and mouse models have yielded a high-resolution picture of the cellular and molecular processes underlying hematopoiesis. A knowledge of the fundamentals of normal hematopoiesis is critical for understanding the pathophysiology, diagnosis, and management of blood disorders. Furthermore, the advent of next-generation sequencing-based molecular testing for diagnosis, prognostication, and management of hematological diseases in clinical practice makes a basic knowledge of the molecular mechanisms driving hematopoiesis an essential part of the clinician’s toolkit. This chapter provides an overview of the biology of normal hematopoiesis from the perspective of clinically applicable aspects of blood cell development.

Cell Development Stages during Hematopoiesis All blood cells are derived from self-renewing hematopoietic stem cells (HSC), which in postnatal life reside in the bone marrow. The presence of HSC in the bone marrow that can give rise to the entire blood cell system has been shown in both laboratory experiments in mice and in patients receiving bone marrow C. Parekh (*) Children’s Hospital Los Angeles and the Keck School of Medicine at the University of Southern California, Los Angeles, CA, USA e-mail: [email protected] © Springer Nature Switzerland AG 2021 D. M. Kamat, M. Frei-Jones (eds.), Benign Hematologic Disorders in Children, https://doi.org/10.1007/978-3-030-49980-8_1

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transplantation. HSC give rise to different blood cell lineages through differentiation into progenitor cell types that lack self-renewal capacity and are progressively more restricted in their lineage potential [1]. Progenitor cell types with distinct lineage potentials have been defined by specific cell surface antigen expression profiles using flow cytometry [2]. Lineage readouts from in vitro culture assays and the transplantation of human hematopoietic cells into immunodeficient mice (xenotransplantation) have formed the mainstay for defining the functional properties of these progenitor cell types [3]. Hematopoietic stem and progenitor cells (HSPC) are characterized by the expression of CD34. In the classical model of hematopoiesis (Fig.  1.1), the origin of common myeloid (CMP) and lymphoid progenitors (CLP) represents the bifurcation of myeloid and lymphoid lineages. Further lineage separation downstream of CMP occurs through the generation of megakaryocytic-erythroid (MEP) and granulocytic-monocytic (GMP) progenitors [1]. While the classical model is based on strict dichotomy of lineages, recent single-cell studies indicate that lineage separation may be a more continuous process where lineage biases are established in HSC and early progenitors and alternative lineage potentials are gradually extinguished to give rise to unilineage progenitors [15]. The abovementioned newer model proposes the direct origin of unilineage progenitors (e.g., megakaryocytic or erythroid progenitors) from multilineage ancestors (HSC, multipotent progenitors, CMP) rather than through an intermediate bilineage progenitor like the MEP [1]. Clinical observations of abnormalities in more than one lineage in disorders that primarily affect a single lineage are consistent with a close developmental relationship between certain lineages. For instance, iron deficiency anemia is characterized by thrombocytosis, an observation consistent with the existence of MEP [16]. HSC TPO CD34+CD38-CD90+CD45RA- HOXB3, HOXB4, HOXA9 GATA2

MPP

SPI1

CD34+CD38-CD90-CD45RATAL1 GATA1 CEBPA

LMPP

CMP

CD34+CD38+CD45RA-CD123+

MEP

GATA1 TAL1

CD34+CD38+CD45RA-CD123-

TPO Meg RUNX1 Platelets

EP EPO RBC

CD34+CD38dimCD10-CD45RA+CD62Lbright

CEBPA SPI1

GMP GMSCF CD34+CD38+CD45RA+CD123+ granulocytes GSCF

GATA2

MDP IRF8

IKZF1 CLP IL7 CD34+CD38+CD10+CD45RA+

B Dendritic cells

NOTCH1 BCL11B

PAX5 IRF8

NK

I

monocytes

Fig. 1.1  Classical model of hematopoiesis. HSC hematopoietic stem cell, MPP multipotent progenitor, LMPP lymphoid-primed multipotent progenitor, CMP common myeloid progenitor, GMP granulocytic-monocytic progenitor, MEP megakaryocytic-erythroid progenitor, MDP monocyte-­ dendritic progenitor, meg megakaryocyte. A subset of the key transcription factors [4–12] (red) and cytokines [13, 14] (blue) for HSPC cell types and lineages are depicted

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HSC are quiescent cells that divide infrequently, approximately once every 40 weeks [17], a feature that protects these long-lived cells from the accumulation of leukemogenic mutations from DNA damage due to environmental agents. In contrast, downstream progenitor cells are highly proliferative and underlie the cell number amplification required to produce the millions of blood cells that need to be generated every day. Differentiation of unilineage progenitors is associated with a loss of proliferative capacity, and terminally differentiated erythroid and myeloid progeny are nondividing cells. The rapidly dividing progenitor cells are particularly sensitive to apoptosis from DNA damage, a phenomenon that accounts for cytopenias seen after treatment with many chemotherapeutic agents. Recovery from post-­chemotherapy cytopenia is driven by the activation of quiescent HSC, which are much more resistant to such DNA damage [18].

Sites of Hematopoiesis During embryonic development in humans, hematopoiesis is first seen in the yolk sac at 3 weeks of gestational age. However, this primitive hematopoiesis is limited to the generation of red cells, macrophages, and megakaryocytes [19]. The first HSC with multilineage myeloid and lymphoid potentials appears in the aorto-­ gonad-­mesonephros and yolk sac regions at 5–6 weeks of gestational age [20]. The fetal liver is the predominant site of hematopoiesis during the second trimester [21]. The spleen forms a minor site of hematopoiesis during fetal life [22]. Liver hematopoiesis decreases in the third trimester, and the bone marrow becomes the dominant site of hematopoiesis by birth [21]. Liver hematopoiesis ceases soon after birth (usually by 5  weeks) leaving the bone marrow as the only site of physiological hematopoiesis during postnatal life [22]. While most differentiated blood cell types in postnatal life are produced in the bone marrow, T-cell production occurs in the thymus through differentiation of hematopoietic progenitors that have migrated from the bone marrow [5]. Hematopoiesis in the liver and spleen resulting in organomegaly is seen after birth (nonphysiological extramedullary hematopoiesis) in disorders with ineffective bone marrow hematopoiesis like thalassemia or as a compensatory mechanism for increased red cell destruction as in sickle cell anemia [22, 23]. While many of the underlying molecular mechanisms are shared between fetal liver and postnatal bone marrow hematopoiesis, several regulatory differences exist between the two ontogenetic phases. For instance, the switch from fetal liver to bone marrow hematopoiesis is thought to underlie the spontaneous resolution of transient myeloproliferative disorder in infants with Down syndrome, a disorder characterized by abnormal proliferation of fetal liver megakaryocytic progenitors harboring a mutation in the transcription factor gene GATA1 [24].

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Mechanisms Underlying Hematopoiesis The orderly differentiation of HSC into a myriad of blood cell types is tightly regulated by transcription factor (TF) genes in the hematopoietic cells as well as extrinsic signals from the bone marrow microenvironment including cytokines and adhesion molecule-mediated cell-to-cell interactions.

Transcription Factors Transcription factors (TF) are DNA-binding proteins that recognize specific DNA sequences to regulate the expression of a multitude of genes. TF typically recruit histone-modifying proteins and other cooperating TF to specific DNA-binding sites to form regulatory complexes that promote or inhibit the expression of target genes [25–27]. The differentiation and self-renewal of HSC are regulated by the orderly and stage-specific expression of TF, which in turn drive the expression of stem cell, progenitor cell, or lineage-specific gene networks [4]. While some TF such as the B-cell TF PAX5 are expressed in a highly lineage-­ specific fashion, TF like SPI1 (myeloid and B-cell) are shared between lineages [4]. HOXB3, HOXB4, and HOXA9 TFs are highly expressed in HSC [9]. CMP show high expression of TAL1, GATA1, and CEBPA [4]. On the other hand, generation of early lymphoid-primed progenitors is associated with downregulation of TAL1 [28]. Divergence of MEP from GMP is driven by the upregulation of TAL1 and GATA1 in MEP and that of CEBPA in GMP [4]. GATA2 regulates HSC function as well as monocyte differentiation [6]. Molecular studies have revealed germline mutations in TFs as the etiology for several hematopoietic disorders (Table 1.1). GATA2 mutations result in a monocyte immunodeficiency syndrome characterized by recurrent infections [6]. RUNX1 mutations are associated with a familial thrombocytopenia and acute myeloid leukemia/myelodysplasia predisposition syndrome [29]. Germline mutations in PAX5 Table 1.1  Hematopoietic disorders due to loss of function germline mutations in TF genes [6, 8, 10, 29–34] TF RUNX1 PAX5 IKZF1 IRF8 GATA2

Disease Familial platelet disorder with predisposition to myeloid malignancy B-ALL predisposition T,B, and myeloid combined immunodeficiency Dendritic cell and monocyte immunodeficiency Immunodeficiency syndrome with recurrent infections from monocyte deficit and pulmonary proteinosis BCL11B B+NK+T- SCID syndrome, brain anomalies, and developmental delay CEBPA AML predisposition syndrome GATA1 Diamond-Blackfan anemia

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result in a predisposition to B-cell acute lymphoblastic leukemia [30]. While most cases of Diamond-Blackfan anemia (DBA) are caused by mutations in ribosomal genes, germline GATA1 mutations that impair the synthesis of the full-length GATA1 isoform have been reported in patients with DBA who did not have known DBA ribosomal gene anomalies [33]. The variable penetrance and age of onset of clinical disease in some of these genetic disorders have important implications for screening donors when using familial donors for hematopoietic stem cell transplantation (HSCT) for bone marrow failure and immunodeficiency syndromes [35].

Cytokines Several growth factors or cytokines play a critical role in hematopoiesis. Cytokines activate specific receptors expressed on hematopoietic cells to regulate cell proliferation, survival, and differentiation. Lineage or differentiation stage-specific expression of cytokine receptors enables the regulation of specific blood cell types by a given cytokine. Cytokines are produced by local microenvironmental cells in the bone marrow (stem cell factor, SCF) as well as the liver (thrombopoietin) and kidney (erythropoietin). Thrombopoietin is required for the maintenance of self-­ renewing HSC and megakaryocytic differentiation to produce platelets. Interleukin-7 (IL-7) plays a critical role in lymphoid differentiation. G-CSF and GM-CSF are essential for myeloid differentiation into neutrophils and monocytes. Erythropoietin drives lineage commitment, survival, and differentiation of erythroid progenitors [13]. The binding of a cytokine to its receptor typically sets off a cascade of biochemical signaling events involving the phosphorylation-mediated activation of a multitude of downstream kinase proteins that ultimately lead to the transcriptional regulation of self-renewal, proliferation, cell survival, and differentiation gene networks. The JAK/STAT family of kinases, which associate with the intracellular domain of cytokine receptors, constitutes a key signal transduction pathway for several cytokines (e.g., erythropoietin, thrombopoietin) in hematopoiesis [36] (Fig. 1.2). Mutations in cytokine, cytokine receptor, or signaling transduction genes account for several very rare hematopoietic disorders (Table  1.2). While these mutations account for only an exceedingly small minority of cytopenia diseases due to defective hematopoiesis, defining these germline aberrations is essential for the elucidation of the molecular mechanisms driving human hematopoiesis, the selection of appropriate treatment, and the development of new gene therapy approaches. For instance, loss of function mutations in the thrombopoietin and erythropoietin genes causes thrombocytopenia and anemia syndromes, respectively, that are clinically similar to other inherited bone marrow failure cytopenia disorders. However, unlike most bone marrow failure syndromes resulting from intrinsic defects in hematopoietic cells, patients with germline TPO or EPO mutations do not respond to HSCT and require cytokine replacement therapy (recombinant erythropoietin or

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Fig. 1.2  Cytokine receptor signaling in hematopoietic cells. Signaling transduction downstream of activation of the thrombopoietin receptor, MPL is depicted. P phosphorylation, JAK Janus kinase, STAT signal transducer and activator of transcription proteins

TPO receptor

TPO receptor Cell membrane

TPO

P

JAK

STAT

JAK

P

P

Regulation of Gene expression

nucleus

Table 1.2  Hematopoietic disorders due to germline mutations in cytokine pathway genes [14, 37, 38] Gene MPL (thrombopoietin receptor) TPO

Type of mutation Loss of function

Disorder Amegakaryocytic thrombocytopenia; progresses to aplastic anemia

Loss of function

IL7R IL2RG CSF3R (G-CSF receptor) EPOR TPO JAK2 MPL

Loss of function Loss of function Loss of function

Amegakaryocytic thrombocytopenia; progresses to aplastic anemia T-NK+B+ SCID T-NK-B+ SCID Severe congenital neutropenia

Gain of function Gain of function Gain of function Gain of function

Hereditary erythrocytosis Hereditary thrombocytosis Hereditary thrombocytosis Hereditary thrombocytosis

thrombopoietin mimetic agents) instead. Mutations in the thrombopoietin receptor gene MPL are seen in amegakaryocytic thrombocytopenia, a disorder whose natural history is a progression to aplastic anemia. G-CSF receptor mutations have been reported in severe congenital neutropenia [14]. Mutations in the IL-7 receptor or the gamma chain component of the IL-2 receptor result in severe combined immunodeficiency due to lymphocyte defects [37].

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Cell-to-Cell Interactions HSPC reside in complex bone marrow microenvironmental niches made of osteoblast, stromal, endothelial, mesenchymal stem, and/or megakaryocyte cells [39, 40]. Several elegant studies in murine cell type-specific gene knockout models and in vitro culture systems of human hematopoietic cells have revealed the effects of specific cell populations in these niches on HSPC fate. The fate of HSC with respect to self-renewal, proliferation, and differentiation is regulated by their anatomic localization to specific niches in the bone marrow. Signals from osteoblasts in endosteal niches promote HSC self-renewal, whereas microenvironmental cues in perivascular niches promote HSC differentiation [41]. Cross talk between hematopoietic cells and the microenvironment is mediated by adhesion molecule receptors on the cell surface that interact with the extracellular matrix and adhesion molecules expressed on other cells. Specific interactions between adhesion molecule receptors and their ligands dictate the localization of HSPC to specific microenvironmental niches and induce downstream cellular proliferation, survival, self-renewal, and differentiation signaling pathways [42]. Adhesion molecule receptors expressed on HSPC, many of which overlap with those mediating the migration of leukocytes into peripheral tissues, include integrins, selectins, CXCR4, and NOTCH pathway receptors [42, 43]. CXCR4 interacts with its ligand CXCL12 expressed by microenvironmental cells including osteoblasts to promote bone marrow residence and quiescence of HSPC [44] (Fig. 1.3). Strategies to modulate adhesion molecules have been translated into therapeutic

Fig. 1.3  Bone marrow hematopoietic microenvironment. HSPC hematopoietic stem and progenitor cells, Ost osteoblasts, Str stromal cells, Endo endothelial cells, MSC mesenchymal stem cells, Meg Megakaryocytes, ECM extracellular matrix. Bidirectional arrows indicate interactions between HSPC and the microenvironment

Ost

CXCL12

ECM

MSC CXCR4 HSPC Meg CXCR4 CXCL12 stro

Endo

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applications. For instance, CXCR4 inhibitors are clinically used to mobilize HSPC from marrow niches into the blood for collection of peripheral blood stem cells for HSCT [45].

Conclusion In vitro and in vivo laboratory studies of immunophenotypically defined HSPC, and the lessons learned from patients with mutations in genes critical for hematopoiesis have resulted in the elucidation of the mechanisms underlying the generation of blood cells from HSC. Mechanistic insights about the specific transcription factors and cytokines driving the hematopoietic lineage differentiation have greatly advanced the understanding of blood disorders and led to the development of molecular diagnostic tests for hematopoietic disorders.

References 1. Cheng H, Zheng Z, Cheng T.  New paradigms on hematopoietic stem cell differentiation. Protein Cell. 2020;11(1):34–44. 2. Cimato TR, Furlage RL, Conway A, Wallace PK. Simultaneous measurement of human hematopoietic stem and progenitor cells in blood using multicolor flow cytometry. Cytometry B Clin Cytom. 2016;90(5):415–23. 3. Doulatov S, Notta F, Laurenti E, Dick JE.  Hematopoiesis: a human perspective. Cell Stem Cell. 2012;10(2):120–36. 4. Laiosa CV, Stadtfeld M, Graf T. Determinants of lymphoid-myeloid lineage diversification. Annu Rev Immunol. 2006;24(1):705–38. 5. Plum J, De Smedt M, Leclercq G, Taghon T, Kerre T, Vandekerckhove B. Human intrathymic development: a selective approach. Semin Immunopathol. 2008;30(4):411–23. 6. Collin M, Dickinson R, Bigley V. Haematopoietic and immune defects associated with GATA2 mutation. Br J Haematol. 2015;169(2):173–87. 7. Ha VL, Luong A, Li F, Casero D, Malvar J, Kim YM, et al. The T-ALL related gene BCL11B regulates the initial stages of human T-cell differentiation. Leukemia. 2017;31(11):2503–14. 8. Punwani D, Zhang Y, Yu J, Cowan MJ, Rana S, Kwan A, et al. Multisystem anomalies in severe combined immunodeficiency with mutant BCL11B. N Engl J Med. 2016;375(22):2165–76. 9. Bhatlekar S, Fields JZ, Boman BM. Role of HOX genes in stem cell differentiation and cancer. Stem Cells Int. 2018;2018:3569493. 10. Hambleton S, Salem S, Bustamante J, Bigley V, Boisson-Dupuis S, Azevedo J, et  al. IRF8 mutations and human dendritic-cell immunodeficiency. N Engl J Med. 2011;365(2):127–38. 11. Ichikawa M, Yoshimi A, Nakagawa M, Nishimoto N, Watanabe-Okochi N, Kurokawa M. A role for RUNX1 in hematopoiesis and myeloid leukemia. Int J Hematol. 2013;97(6):726–34. 12. Davis KL.  Ikaros: master of hematopoiesis, agent of leukemia. Ther Adv Hematol. 2011;2(6):359–68. 13. Metcalf D. Hematopoietic cytokines. Blood. 2008;111(2):485–91. 14. Lipton JM. Inherited thrombocytopenia and Occam’s razor. Blood. 2017;130(7):839–40. 15. Velten L, Haas SF, Raffel S, Blaszkiewicz S, Islam S, Hennig BP, et al. Human haematopoietic stem cell lineage commitment is a continuous process. Nat Cell Biol. 2017;19(4):271–81.

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16. Xavier-Ferrucio J, Scanlon V, Li X, Zhang P-X, Lozovatsky L, Ayala-Lopez N, et al. Low iron promotes megakaryocytic commitment of megakaryocytic-erythroid progenitors in humans and mice. Blood. 2019;134(18):1547–57. 17. Catlin SN, Busque L, Gale RE, Guttorp P, Abkowitz JL. The replication rate of human hematopoietic stem cells in vivo. Blood. 2011;117(17):4460–6. 18. Shao L, Wang Y, Chang J, Luo Y, Meng A, Zhou D. Hematopoietic stem cell senescence and cancer therapy-induced long-term bone marrow injury. Transl Cancer Res. 2013;2(5):397–411. 19. Julien E, El Omar R, Tavian M. Origin of the hematopoietic system in the human embryo. FEBS Lett. 2016;590(22):3987–4001. 20. Ivanovs A, Rybtsov S, Welch L, Anderson RA, Turner ML, Medvinsky A.  Highly potent human hematopoietic stem cells first emerge in the intraembryonic aorta-gonad-mesonephros region. J Exp Med. 2011;208(12):2417–27. 21. Holt PG, Jones CA. The development of the immune system during pregnancy and early life. Allergy. 2000;55(8):688–97. 22. Yamamoto K, Miwa Y, Abe-Suzuki S, Abe S, Kirimura S, Onishi I, et  al. Extramedullary hematopoiesis: elucidating the function of the hematopoietic stem cell niche (Review). Mol Med Rep. 2016;13(1):587–91. 23. Bobylev D, Zhang R, Haverich A, Krueger M. Extramedullary haematopoiesis presented as intrathoracic tumour in a patient with alpha-thalassaemia. J Cardiothorac Surg. 2013;8:120. 24. Li Z, Godinho FJ, Klusmann J-H, Garriga-Canut M, Yu C, Orkin SH. Developmental stage-­ selective effect of somatically mutated leukemogenic transcription factor GATA1. Nat Genet. 2005;37(6):613–9. 25. Vaquerizas JM, Kummerfeld SK, Teichmann SA, Luscombe NM. A census of human transcription factors: function, expression and evolution. Nat Rev Genet. 2009;10(4):252–63. 26. Martinez GJ, Immunology RA. Cooperative transcription factor complexes in control. Science. 2012;338(6109):891–2. 27. Luo RX, Dean DC. Chromatin remodeling and transcriptional regulation. J Natl Cancer Inst. 1999;91(15):1288–94. 28. Kohn LA, Hao Q-L, Sasidharan R, Parekh C, Ge S, Zhu Y, et al. Lymphoid priming in human bone marrow begins before expression of CD10 with upregulation of L-selectin. Nat Immunol. 2012;13(10):963–71. 29. Kanagal-Shamanna R, Loghavi S, DiNardo CD, Medeiros LJ, Garcia-Manero G, Jabbour E, et al. Bone marrow pathologic abnormalities in familial platelet disorder with propensity for myeloid malignancy and germline RUNX1 mutation. Haematologica. 2017;102(10):1661–70. 30. Shah S, Schrader KA, Waanders E, Timms AE, Vijai J, Miething C, et al. A recurrent germline PAX5 mutation confers susceptibility to pre-B cell acute lymphoblastic leukemia. Nat Genet. 2013;45(10):1226–31. 31. Boutboul D, Kuehn HS, Van de Wyngaert Z, Niemela JE, Callebaut I, Stoddard J, et  al. Dominant-negative IKZF1 mutations cause a T, B, and myeloid cell combined immunodeficiency. J Clin Invest. 2018;128(7):3071–87. 32. Tawana K, Rio-Machin A, Preudhomme C, Fitzgibbon J.  Familial CEBPA-mutated acute myeloid leukemia. Semin Hematol. 2017;54(2):87–93. 33. Sankaran VG, Ghazvinian R, Do R, Thiru P, Vergilio J-A, Beggs AH, et al. Exome sequencing identifies GATA1 mutations resulting in Diamond-Blackfan anemia. J Clin Invest. 2012;122(7):2439–43. 34. Parrella S, Aspesi A, Quarello P, Garelli E, Pavesi E, Carando A, et  al. Loss of GATA-1 full length as a cause of Diamond–Blackfan Anemia Phenotype. Pediatr Blood Cancer. 2014;61(7):1319–21. 35. Owen CJ, Toze CL, Koochin A, Forrest DL, Smith CA, Stevens JM, et al. Five new pedigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy. Blood. 2008;112(12):4639–45. 36. Jatiani SS, Baker SJ, Silverman LR, Reddy EP.  Jak/STAT pathways in cytokine signaling and myeloproliferative disorders: approaches for targeted therapies. Genes Cancer. 2010;1(10):979–93.

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37. Tasher D, Dalal I. The genetic basis of severe combined immunodeficiency and its variants. Appl Clin Genet. 2012;5:67–80. 38. Braunstein EM, Moliterno AR. Back to biology: new insights on inheritance in myeloproliferative disorders. Curr Hematol Malig Rep. 2014;9(4):311–8. 39. Hoffman CM, Calvi LM. Minireview: complexity of hematopoietic stem cell regulation in the bone marrow microenvironment. Mol Endocrinol. 2014;28(10):1592–601. 40. Asada N, Takeishi S, Frenette PS. Complexity of bone marrow hematopoietic stem cell niche. Int J Hematol. 2017;106(1):45–54. 41. Zhang P, Zhang C, Li J, Han J, Liu X, Yang H. The physical microenvironment of hematopoietic stem cells and its emerging roles in engineering applications. Stem Cell Res Ther. 2019;10(1):327. 42. Levesque J-P, Winkler IG. Cell adhesion molecules in normal and malignant hematopoiesis: from bench to bedside. Current Stem Cell Rep. 2016;2(4):356–67. 43. Corselli M, Chin CJ, Parekh C, Sahaghian A, Wang W, Ge S, et al. Perivascular support of human hematopoietic stem/progenitor cells. Blood. 2013;121(15):2891–901. 44. Karpova D, Bonig H. Concise review: CXCR4/CXCL12 signaling in immature hematopoiesis–lessons from pharmacological and genetic models. Stem Cells. 2015 Aug;33(8):2391–9. 45. Domingues MJ, Nilsson SK, Cao B.  New agents in HSC mobilization. Int J Hematol. 2017;105(2):141–52.

Part II

Red Blood Cell Disorders

Chapter 2

Nutritional Anemias: Iron Deficiency and Megaloblastic Anemia Deanna Mitchell, Jessica Foley, and Aarti Kamat

Iron Deficiency Anemia Iron deficiency is the most common cause of anemia in infancy and childhood. Iron deficiency anemia has important health ramifications, as it has been associated with abnormal neurodevelopment [1]. Dietary modifications can prevent iron deficiency. If iron deficiency develops, simple interventions can help prevent cognitive detriments. Given the common prevalence of this disease and its impact on neurodevelopment, it is imperative that primary care physicians learn to recognize, diagnose, and treat this condition.

Prevalence Iron deficiency remains the number one cause of anemia worldwide, affecting more than 2 billion people, with a considerable effect on the lives of young children, especially in low-income and developing countries [2]. The prevalence of iron deficiency anemia is lower in the USA in comparison to developing countries; however, it remains a common problem, with a prevalence of 1.6–7.4% among children less than 5 years of age. Iron deficiency affects at least 2.4 million children in the USA [3].

D. Mitchell (*) · J. Foley Helen DeVos Children’s Hospital, Grand Rapids, MI, USA College of Human Medicine, Michigan State University, East Lansing, MI, USA e-mail: [email protected]; [email protected] A. Kamat Helen DeVos Children’s Hospital Future fellow, Pediatric Hematology/Oncology – University of Michigan Medical Center, C.S. Mott Children’s Hospital, Ann Arbor, MI, USA e-mail: [email protected] © Springer Nature Switzerland AG 2021 D. M. Kamat, M. Frei-Jones (eds.), Benign Hematologic Disorders in Children, https://doi.org/10.1007/978-3-030-49980-8_2

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In the USA, iron deficiency is more common among children living at or below the poverty level. Hispanic/Latino and Asian immigrant children have significantly higher rates of iron deficiency (17%) compared to Caucasian children 6% [4]. Prevalence of iron deficiency differs depending on body mass with obese toddlers having a higher prevalence compared to normal weight toddlers (20% vs. 7%) [3]. Children not in daycare and overweight have higher odds of iron deficiency. Other factors associated with iron deficiency include premature birth and low birth weight. The rate of iron deficiency decreases after 2 years until adolescence when 8–10% of teenage girls may develop iron deficiency and 3% will be diagnosed with iron deficiency anemia [5].

Pathophysiology Iron is critical for many body functions including energy production, respiration, DNA synthesis, and cell proliferation. Erythropoiesis, or red blood cell production, is dependent upon iron. Nearly 75% of iron is bound to heme proteins, hemoglobin, and myoglobin and is involved in oxygen transport and storage. Thirty percent of iron is otherwise bound in storage proteins: ferritin and hemosiderin of small portions are bound in critical enzyme systems, including catalase and cytochromes [6]. Total body iron increases dramatically in the first year of life which allows nearly 80% of iron to be used for hemoglobin production and iron stores. A newborn infant’s body contains 0.3–0.5 grams of iron, whereas the total iron content of an adult is 4–5 grams. An average of 0.8 mg of iron must be absorbed each day during the first 15 years of life. Iron metabolism is a relatively closed system, in which iron is continuously recycled to meet the demands of the body but particularly for red blood cell production. After full growth is achieved, the total body iron remains fixed within narrow limits. When red blood cells die, they are removed from circulation by macrophages, and iron is extracted from hemoglobin and made available to the plasma protein, transferrin, for transport to the marrow [6]. As children grow and body iron content increases, less iron is needed from the diet to meet their needs. Approximately 5% of the iron requirement for erythropoiesis comes from dietary intake in an adult, while 30% of the iron requirement for erythropoiesis must come from the diet in children [1]. Iron loss from the body is minimal and usually due to exfoliation of mucous membranes and skin. No known specific mechanisms for iron excretion through the liver or kidneys exist. Iron balance is predominantly achieved through modulation in the absorption of iron in the intestine. The average adult diet contains 10–15 mg of iron daily. Approximately 8–15 mg of iron daily is needed for optimal nutrition [6]. Dietary iron absorption in the intestine varies from 5% to 20% depending upon the physiological need. For example, intestinal absorption may increase fourfold in the setting of blood loss, pregnancy, sports activity, and hemolytic anemia [1]. Iron is absorbed mainly in the small intestine, mostly in the duodenum and first part of the jejunum. Iron absorption is regulated by the peptide hormone hepcidin, which regulates how much iron is taken up in the intestine and transported to the

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plasma. Hepcidin is synthesized primarily in the liver. Increased hepcidin levels decrease the intestinal absorption of iron by binding to and inducing the degradation of ferroportin, a transmembrane protein located on the surface of intestinal enterocytes, which facilitates the absorption of iron from the intestine. Hepcidin is regulated by the iron demand of the body. When the body is iron deficient, hepcidin concentrations are low, and thus ferroportin is available to absorb intestinal iron. Serum hepcidin expression, and subsequent intestinal iron absorption, is affected by iron stores in the form of transferrin and ferritin, erythropoiesis, bioavailability of dietary iron, and inflammatory states [7].

Iron Deficiency and Neurodevelopment Iron deficiency has been strongly linked to long-term neurological complications that affect cognitive, social, and behavioral development in infants and young children [8]. Iron deficiency, even without anemia, can adversely impact the social-­ emotional behavior of infants and influence their relationship with their caregiver. One study noted that infants with iron deficiency demonstrated increased shyness, decreased soothability, and decreased engagement [9]. Iron deficiency has its greatest impact when it occurs during fetal growth or in the first few years of life when neural systems are developing [8]. Even after iron supplementation, the cognitive and social impairments can persist in children that were formerly iron deficient [8]. One study demonstrated children who were iron deficient as infants had slower reaction times and worse inhibitory control 8–9 years after iron therapy [10]. Adolescent girls are at risk of iron deficiency and iron deficiency anemia. In a survey of adolescents in the USA, those who were iron deficient were found to have lower math scores [11]. Another study looked at iron supplementation in adolescent girls who were not anemic but had serum ferritin ≤12 micrograms/L, corresponding to iron deficiency. The girls who received iron supplementation were noted to have an improved ferritin and performed significantly better on tests of verbal learning and memory [12]. Blood loss from menstruation is expected in adolescent girls; however the resulting iron deficiency is underdiagnosed resulting in a lack of recognition of the cognitive, social, and behavioral consequences of iron deficiency in this population. Iron deficiency during adolescence occurs at a time when education is imperative to achieve a successful adulthood, and thus girls with heavy menses may suffer neurologic sequelae that will diminish their academic potential. Iron stores should be optimized prior to childbearing years [5].

Iron Deficiency and Thrombosis and Stroke in Children The association of thrombosis and iron deficiency in both children and adults has been described [13]. Thrombotic complications of iron deficiency anemia have been attributed to the secondary thrombocytosis which occurs in one third of patients.

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However, cerebral venous sinus thrombosis associated with iron deficiency has been reported without the presence of thrombocytosis [14]. Hartfield et al. described a case series of six children who developed an ischemic stroke or venous thrombosis associated with iron deficiency [15]. Iron may contribute to a hypercoagulable condition by affecting blood flow patterns within vessels due to reduced deformability and increased viscosity of microcytic red blood cells [15]. Hypoxic injury is seen particularly in the areas of the brain supplied by end arteries, such as the basal ganglia, thalamus, and hypothalamus [16]. Maguire et al. additionally described [15] previously healthy pediatric patients between 12 and 38 months of age who developed stroke and found iron deficiency anemia in 53% (8/15) of the patients. For children who were previously healthy, those who went on to develop stroke were ten times more likely to have iron deficiency than those who remained healthy and did not develop stroke. Children with iron-deficiency anemia accounted for more than half of all stroke cases in children without an underlying medical illness. This suggests that iron deficiency may be a risk factor for stroke in children [17].

Etiology of Iron Deficiency The causes of iron deficiency in children include one or more of the following: inadequate reserves at birth, inadequate intake of iron in the diet, reduced intestinal absorption of iron, or chronic blood loss. Many intrauterine conditions may cause decreased iron reserves at birth, including prematurity, twin gestation, intrauterine fetus-fetus and fetus-maternal transfusions, exchange transfusion at birth, severe iron deficiency anemia in the mother, and/or early clamping of the umbilical cord. Eighty percent of the iron present in the newborn term infant is accumulated during the third trimester of pregnancy. Therefore, infants who are born prematurely have lower iron stores. Other maternal conditions, including anemia, diabetes, conditions that cause intrauterine growth restriction, and maternal hypertension can contribute to low fetal iron stores in preterm and term babies. Additionally, premature infants require frequent lab monitoring during hospitalization which can result in iatrogenic anemia and may cause iron deficiency. The use of recombinant human erythropoietin in preterm infants in an attempt to prevent transfusion has been associated with further depletion of iron stores. Therefore, premature infants in particular require iron supplementation; this should be given at a dose of of 2–4 mg/kg/day to pretrerm and low-birth-weight infants before 6 months of age [1]. Healthy, term infants have iron stores of approximately 75 mg/kg and a mean hemoglobin of 15–17 g/dl. Term infants generally have sufficient iron stores for the first 4–6  months. During this time they are either receiving breast milk or iron-­ supplemented formula. All standard infant formulas contain a minimum of 6.7 mg/l of iron. However after 4–6  months, an infant’s diet should include iron-fortified foods such as cereals or iron-supplemented formula. Inadequate iron in infants less than 12 months of age is most commonly due to either breastfeeding without the

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initiation of iron supplementation or iron-rich foods by 6 months of age [1]. Occasionally, infants have non-fortified cow’s milk introduced prior to 12 months of age. Cow’s milk does not contain appropriate concentrations of iron for infants. Both human milk and cow’s milk have low concentrations of iron; however, the bioavailability of iron is much greater from human milk. Male et al. demonstrated in 488 healthy infants in 11 European countries that 7.2% were iron deficient at 12 months and 2.3% had iron deficiency anemia. Toddlers who were fed cow’s milk had a progressively increasing risk of iron deficiency rising to 39% [18]. Additionally, infants can also experience microscopic blood loss secondary to cow’s milk protein-­ induced colitis. This chronic blood loss contributes to the severe iron deficiency seen in excessive cow’s milk ingestion. Young children may develop a severe form of this syndrome and have significant protein-losing enteropathy with hypoalbuminemia and edema as well as iron deficiency anemia [19]. In contrast to heme iron, the absorption of nonheme iron is inhibited by casein and calcium in milk. Casein and calcium are present in much higher concentrations in cow’s milk compared to human milk [20]. Iron deficiency due to inadequate dietary intake is not just limited to children. In developing countries, iron deficiency anemia is most often secondary to insufficient iron in the diet. However, loss of blood from intestinal parasites also contributes to inadequate iron stores [2]. The most common cause of iron deficiency in adults in high-income countries is secondary to pathologic conditions such as gastrointestinal bleeding or secondary to a strict vegetarian diet. Vegetarians are at risk of iron deficiency for several reasons. A cereal-based diet decreases the iron bioavailability because phytates in grains form a complex with iron which is then poorly absorbable. Additionally, intestinal absorption of iron depends upon the form of iron ingested. Heme iron found in meat, fish, and poultry is readily absorbed with a higher bioavailability compared to nonheme iron which is found in some plants. The most readily absorbed iron is found in red meat, such as beef. Thus vegetarians should actively manage their dietary intake of iron or consider supplementation [21]. Gastrointestinal disorders including celiac disease, inflammatory bowel disease (Crohn disease), giardiasis, or other malabsorption conditions involving the duodenum can result in inadequate absorption of iron and anemia. Premature infants who suffered from necrotizing enterocolitis and have short gut syndrome frequently absorb inadequate iron [22]. Patients who have undergone bariatric surgery also are at risk for lack of iron absorption and subsequent anemia [23]. Inflammatory bowel disease, juvenile polyposis syndrome, cow’s milk protein-induced colitis, and chronic aspirin can result in increased gastrointestinal blood loss and chronic iron deficiency anemia [24]. Additional causes of iron deficiency that should be considered in a differential diagnosis include hemolysis. In rare forms of intravascular hemolysis, like paroxysmal nocturnal hemoglobinuria, iron is lost in the urine, and iron deficiency contributes to the existing hemolytic anemia. Occasionally iron deficiency is multifactorial, such as in runners and endurance athletes. Here iron deficiency may be partially due to hemolysis, blood loss, and mild inflammation [25]. There are patients with homozygous mutations in TMPRSS6, encoding the hepcidin inhibitor matriptase-2, a rare

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genetic form of anemia known as iron-refractory iron deficiency anemia (IRIDA). Patients with IRIDA have high serum hepcidin levels and therefore poor or no response to oral iron [26].

Screening Screening for anemia with hemoglobin evaluation is recommended at 1 year of age by the American Academy of Pediatrics (AAP). In addition to obtaining a hemoglobin, screening for risk factors for iron deficiency such as prematurity or low birth weight, exclusive breastfeeding without supplementation of iron or introduction of iron-containing foods, or excessive cow’s milk ingestion should be obtained [1]. If an infant has a hemoglobin concentration of less than 11 g/dL or has significant risk factors for iron deficiency, a serum ferritin and CRP (see diagnosis section) should be obtained [1]. If after 1 year of age, a child consumes greater than 24 ounces of cow’s milk or has fewer than two servings per day of iron-containing foods (meats, iron-fortified cereals), we would recommend obtaining a serum hemoglobin for further screening. Frequently milk is not introduced until 1 year of age, and the consumption increases during the 2nd and 3rd year of life. If screening is limited to only the 1-year well-child appointment, children who become iron deficient from excessive cow’s milk intake would be missed. A good diet history can be just as effective as laboratory screening for microcytic anemia. A study evaluating 205 healthy, African-American children living in a low-income setting found that a brief diet history identified children at risk for microcytic anemia 97% of the time [27]. Screening should not just be limited to young children. Several studies have indicated that cognitive impairments are present in adolescents who are iron deficient [11, 12]. Risk factors for iron deficiency in the adolescent population include heavy menses, or other blood loss, such as GI blood loss. Low body weight and malnutrition are also a risk factor for iron deficiency in this population. This may be due to inadequate intake from a vegetarian or vegan diet [21]. Children who are overweight have also been shown to have an increased prevalence of iron deficiency [28]. Additionally, athletes are at risk of iron deficiency and anemia [29]. It is imperative that the general pediatrician screen adolescent patients for these risk factors at the yearly health and wellness evaluation. A CBC and ferritin should then be obtained [28–30].

Clinical Symptoms and Signs of Iron Deficiency Anemia The classic presentation of iron deficiency anemia in the USA is the presence of microcytic, hypochromic anemia found on screening laboratory during a well-child exam in an asymptomatic young child. Children with more severe anemia may present with pallor, lethargy, poor feeding, irritability, and tachypnea. Children with

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developmental delay should be screened for iron deficiency. Febrile seizures have been associated with iron deficiency in a number of publications [31]. Exercise intolerance, decreased sports performance, and fatigue can also be symptoms of iron deficiency anemia in the older child. Iron supplementation has been reported to improve athletic performance in patients with iron deficiency anemia [25]. Pica, an intense craving for nonfood items, is a common manifestation of iron deficiency. Examples of pica can include children eating ice, clay, dirt, paper, carpet, starch, and soap. It is possible that a patient may develop concomitant lead poisoning due to iron deficiency-induced pica due to consumption of environmentally contaminated materials such as dried and lead-based paint chips [32]. Pica generally resolves rapidly with replacement of iron. Restless legs syndrome has been associated with iron deficiency in adults and children [33]. Kotagal and Silber demonstrated that serum ferritin levels in children with restless leg syndrome were low in the majority of patients (33% below the 5th percentile and 75% below the median) [34].

Diagnosis and Treatment Laboratory Testing Anemia in a pediatric patient is not always attributable to iron deficiency. Many other causes of anemia in childhood exist including anemia secondary to decreased production such as an infiltrative bone marrow process like leukemia or secondary to increased destruction such as a hemolytic anemia. Further studies may be necessary if the patient does not have risk factors or lacks the classic findings of microcytic anemia. Laboratory findings in classic iron deficiency anemia include microcytosis, anemia, elevated red blood cell distribution width (RDW), low red blood cell count, and occasionally thrombocytosis. Patients with microcytic anemia with both a low red blood count (RBC) and low MCV are likely to have iron deficiency, whereas patients with thalassemia will have a normal RBC count and a low MCV. William Mentzer described a Mentzer index: quotient of the MCV divided by the RBC count. If the quotient is less than 13, then iron deficiency anemia is more likely, whereas an index greater than 13 suggests thalassemia [35]. Peripheral smear evaluation demonstrates hypochromia, anisocytosis, and microcytosis. Reticulocyte count will be low if measured but is not required for the diagnosis of iron deficiency anemia. Classically in general pediatrics in a child with known risk factors and microcytic anemia, iron studies are not necessary. Iron studies may be required for unclear diagnoses. In the event specific testing for iron stores is needed; the three tests that provide the most information regarding iron status include serum ferritin, reticulocyte hemoglobin concentration, and serum transferrin receptor 1 concentration [1]. Serum ferritin is widely used for determination of iron stores, with a value less than

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12 μg/L indicating decreased iron stores. However, serum ferritin is an acute phase reactant and increases in the presence of inflammatory states such infection and chronic inflammation or in the presence of malignancy or liver disease. A clinician must utilize a good history and physical exam to assess for the presence of inflammation, and further iron testing is helpful in distinguishing between iron deficiency anemia and anemia of chronic disease where iron utilization is compromised by inflammation. Obtaining a C-reactive protein can be of assistance when interpreting a serum ferritin [1]. Obtaining a serum ferritin for children is a reliable screening test to assess long-term iron stores in the absence of chronic inflammation. The reticulocyte hemoglobin and serum transferrin receptor 1 tests are more accurate than the ferritin, as they are not acute phase reactants, and should be considered in the setting of inflammation [1]. Thus, for a child with microcytic anemia with a chronic disease, we would suggest ordering a serum ferritin and CRP, in addition to a good physical exam, to confirm diagnosis. In a clinically stable child with risk factors for iron deficiency and classic laboratory findings of microcytic anemia with elevated RDW, diagnosis can be confirmed by using a therapeutic challenge by initiating iron supplementation and monitoring response. Reticulocytosis develops within 1 week, and an increase of 1 gm/dL of hemoglobin after 1 month of therapeutic supplementation indicates the presence of iron deficiency anemia. If a therapeutic challenge is performed, the clinician should ensure an adequate dose of iron therapy, that there is no concern for abnormal intestinal iron absorption, and close follow-up [1].

Oral Iron Therapy Once a diagnosis of iron deficiency anemia is made, iron therapy should be initiated. Oral iron is usually effective, readily available, and inexpensive. The recommended dose is 3–6 mg/kg/day of elemental iron. Ferrous sulfate at 3 mg/kg/day has been shown to yield a greater increase in hemoglobin concentration than iron polysaccharide complex [36]. The milligrams of ferrous sulfate or iron polysaccharide complex should be converted to elemental iron concentration to avoid under dosing. Iron is best absorbed enterally when it is given on an empty stomach with orange juice and not cow’s milk which interferes with iron absorption [37]. Ascorbic acid has been shown to enhance the absorption of nonheme iron [38]. Therefore, it is often recommended that iron be taken with juice or a vitamin C supplement. Additionally, iron should be given as a once-daily dosing. When iron is given in divided doses each day, this has been shown to increase serum hepcidin levels and therefore reduces enteral absorption [39]. Thus far, studies assessing better absorption with alternate day iron dosing have been performed in iron-deficient young women [38]. We have concerns about increasing noncompliance in pediatric patients with alternate day dosing. It is recommended to treat with therapeutic iron for at least 12 weeks [36]. However, we would recommend that to adequately replete iron

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stores, the 12  weeks should start once a patient’s hemoglobin has normalized. Appropriate response to iron supplementation will show a rise of greater than 1 g/dl of hemoglobin within 2–4  weeks depending upon the degree of anemia. Poor response should be evaluated for compliance or absorption and stools tested for chronic blood loss or malabsorption. An iron absorption test can be performed in clinics to assess the serum iron response to a dose of oral iron. This is a simple test performed by obtaining an iron level when a patient has not eaten anything overnight. A dose of around 4–6  mg/kg of elemental iron is given by mouth. One to 2 hours later, a repeat serum iron level is obtained. If iron has been absorbed, then we expect to see a rise in this level from the previous one. There are very rare mutations that interfere with iron transport, resulting in iron deficiency anemia, such as iron-refractory iron deficiency anemia (IRIDA). In instances such as these, iron absorptions would be impaired. More commonly absorption is compromised due to underlying disease such as Crohn disease, jejunal feeding, or resection of the duodenum. Adverse effects of iron therapy are minimal when a dose of 3 mg/kg/day of iron was used, with constipation being the most commonly reported when compared to placebo [40]. Other adverse effects include abdominal pain and diarrhea. We would recommend liberal use of a stool softener such as lactulose or polyethylene glycol over discontinuation or dose modification of the iron supplement if constipation develops. The palatability of oral iron preparations and staining of clothing are sometimes a deterrent to adequate compliance. In young children found to have iron deficiency anemia, dietary changes are recommended including transitioning from the bottle and stopping or significantly decreasing cow’s milk ingestion. In all children older than 12 months, cow’s milk should be limited to less than 20 oz per day. Lists of iron-rich foods, including meat, Cream of Wheat, prune juice, spinach, raisins, red beans, and iron-fortified cereals, are available. However, increased dietary intake of iron-rich foods alone is inadequate to treat iron deficiency anemia.

Intravenous (IV) Iron Therapy IV iron is usually considered second-line to oral therapy in the majority of patients. Children with underlying blood loss from gastrointestinal disease or dysfunctional uterine bleeding or inadequate absorption often benefit from IV iron therapy. A number of IV iron forms exist with newer ones offering a safer toxicity profile [26]. Adverse effects may include rash, palpitations, dizziness, myalgias, and chest discomfort. Minor infusion reactions occur in less than 1% of patients and generally resolve by stopping the infusion. More serious anaphylactic reactions are rare. If a patient meets criteria for IV iron, we recommend a referral to pediatric hematology for treatment in a monitored infusion clinic.

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Vitamin B12 and Folic Acid Deficiency Nutritional deficiencies in folic acid and vitamin B12 (also known as cobalamin) can cause impaired DNA synthesis and a form of macrocytic anemia, called megaloblastic anemia. Hematopoietic precursor cells divide rapidly and are therefore susceptible to abnormal DNA synthesis caused by vitamin B12 and folate deficiencies [41]. Both folate and cobalamin are necessary in DNA synthesis, RNA synthesis, and cell duplication [50]. Megaloblastic changes in red blood cell (RBC) precursor cells are noted when DNA is unable to be appropriately synthesized. The cytoplasm in RBC precursors in the bone marrow continues to mature, while nuclear duplication slows, resulting in nuclear-cytoplasmic dyssynchrony [42]. The resulting RBCs are therefore large (macrocytic) and typically oval in shape. In addition to RBC precursors, other hematopoietic cells are affected by B12 and folate deficiencies. Patients can develop thrombocytopenia and neutropenia, with hypersegmented (greater than 5 lobes) neutrophils [43]. The resultant hypercellular bone marrow with dysplastic features can be mistaken for leukemia [42]. While folate and vitamin B12 deficiencies are the most common causes of megaloblastic anemia, certain medications that impair DNA synthesis can also cause megaloblastic anemia [44]. Rarely, megaloblastic anemia can be caused by congenital disorders resulting in folate and vitamin B12 deficiencies [45].

Folate Deficiency Dietary folate deficiency is becoming increasingly rare in developed countries where folate is supplemented in most common food products like grains and cereals. In the USA, folate deficiency is generally seen in the presence of bowel resection, chronic diarrhea, or chronic hemolytic anemia. The incidence of folate deficiency in an epidemiology study in northern India was 6% in breastfed babies 6–30 months of age and 33% in non-breastfed children of the same age [46].

Pathophysiology Dietary folates found in plant-based foods and fortified grains are absorbed in the jejunum. Then folate must be reduced to tetrahydrofolate to be biologically active and participate in DNA synthesis. Folate serves as a one-carbon donor and acceptor in many biological pathways, including the production of both purines and pyrimidines, which are fundamental for DNA synthesis. The major role of folate in DNA synthesis is to provide methyl groups which are added to other molecules. A decrease in folate intake can result in folate deficiency in a matter of weeks to months [47].

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Etiology Humans cannot synthesize folate themselves; thus we must obtain folate from sources such as dark green vegetables, fruits, legumes, and animal organs. Boiling or heating these foods can reduce their folate content [48]. Most grains and cereals are now fortified with folate. Individuals with a balanced diet should not become deficient, unless they have increased folate requirements such as in pregnancy, accelerated growth, and chronic hemolysis. Folate deficiency during pregnancy has been associated with neural tube defects in the developing fetus [49]. Those with poor diets, such as in chronic alcoholism or anorexia, and malnourished children are at higher risk for folate deficiency [48]. Folic acid deficiency is seen in infants fed goat’s milk, which has less folate than cow’s milk and supplemented formula [50]. Folic acid deficiency can also occur as a result of poor absorption, as seen in rare congenital disorders such as hereditary folate malabsorption and functional methionine synthesis deficiency or malabsorptive processes like inflammatory bowel disease (IBD), celiac disease, jejunal resection, and chronic diarrhea [51]. Finally, medications such as methotrexate and trimethoprim inhibit the enzymes that convert folate to tetrahydrofolate. Certain anticonvulsants (phenytoin, valproate, and carbamazepine) interfere with folate absorption [52]. Folate requirements are increased in the setting of chronic hemolytic anemias (sickle cell disease, hereditary spherocytosis), hemodialysis, and exfoliative skin disease [52]. These disorders are associated with increased cell turnover and increased DNA synthesis. Folic acid supplementation at a dose of 1  mg daily is helpful to prevent folate deficiency.

Diagnosis In a patient who presents with macrocytic anemia, serum and erythrocyte levels of folic acid are adequate to diagnose folic acid deficiency; serum levels reflect recent intake, whereas erythrocyte levels are indicative of more chronic folic acid levels 62). Normal serum levels range from 5 to 20 ng/ml, and normal erythrocyte concentration is 150–600 ng/ml [52]. If patients have developed a megaloblastic anemia, RBCs will have an MCV >100 fl, and there are megaloblastic changes on peripheral blood smear and bone marrow. Reticulocyte count is typically low [43].

Treatment Folate supplementation at 1 mg/day should be started in individuals who are at risk for folic acid deficiency. Folic acid supplementation, either parentally or enterally, should be initiated in patients with folic acid deficiency. Treatment is typically

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0.5–1 mg/day for 3–4 weeks [52]. A small 0.1 mg dose for 1 week, with evaluation for hematologic response afterward, can be trialed if the diagnosis is unclear [43]. Once hematologic response is established, maintenance dosing can be continued at the recommended dietary allowance (0.2 mg) [52].

Vitamin B12 Deficiency Prevalence The prevalence of vitamin B12 deficiency increases with age with approximately 6% of people over 60 in the USA affected. Individuals with a vegan or vegetarian diet are at increased risk of vitamin B12 deficiency. Breastfed babies of vegan mothers are also at risk [53].

Pathophysiology Vitamin B12 is released from food by enzymes and acid in the stomach and then complexed with intrinsic factor, which is produced by gastric parietal cells in the stomach [52]. This complex is necessary for absorption in the ileum. Vitamin B12 is then broken down into adenosylcobalamin or methylcobalamin, which are used in DNA and RNA synthesis by serving as cofactors in the conversion of homocysteine to methionine and methyl-malonyl-CoA to succinyl-CoA. Deficiency of B12 can result in folate becoming trapped in the 5-methyl-THF form [41].

Etiology Like folate deficiency, the most common cause of vitamin B12 deficiency is inadequate dietary intake. The main dietary sources of vitamin B12 include meat, dairy, and eggs; therefore, veganism is a common cause of vitamin B12 deficiency [48]. Deficiency usually takes 1–2 years to develop but can be up to 5 years, due to large stores in the liver [42]. Exclusively breastfed infants of mothers who are vitamin B 12 deficient can develop deficiencies themselves [42]. Signs of deficiency in these babies can appear between 6 and 18 months of life and include macrocytic anemia and loss of motor milestones. Pernicious anemia is an autoimmune disorder that interferes with the formation of the B12-intrinsic factor complex due to the presence of anti-intrinsic factor or anti-parietal cell antibodies [52]. Pernicious anemia is a common cause of vitamin B12 deficiency when B12 intake is adequate, as it causes decreased absorption of

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vitamin B12. Pernicious anemia is rare in children. Other gastric pathologies, such as H. pylori infection and bariatric surgeries, decrease the absorption of vitamin B12 by disrupting the release of intrinsic factors [43]. Since B12 is absorbed in the ileum, disorders such as necrotizing enterocolitis which results in bowel resection and subsequent short gut syndrome, celiac disease, IBD, and pancreatic insufficiency can decrease absorption and cause deficiency [42]. Medications including metformin and antacids can also decrease the absorption of vitamin B12 [42]. Finally, rare genetic syndromes that cause decreased vitamin B12 absorption include hereditary intrinsic factor deficiency, Imerslund-Grasbeck (juvenile megaloblastic anemia) syndrome, and inborn errors of cobalamin metabolism [54–56]. Abuse of nitrous oxide can result in severe vitamin B12 deficiency [52].

Signs and Symptoms All blood elements can show megaloblastic changes; however, erythrocytes show the most marked abnormalities in size and shape, with large oval macrocytes and prominent anisopoikilocytosis. Common laboratory findings also include macrocytic anemia with MCV 100–150 fl and neutrophils with hypersegmentation of their nuclei with greater than 5 lobes [52]. Leucopenia and thrombocytopenia may also be present. The manifestations of B12 deficiency are not restricted to anemia but often present as neurologic complications [57]. Neuronal effects for vitamin B12 deficiency are partially due to reduced methylation of neuronal lipids and proteins, including myelin basic protein. Myelin basic protein comprises approximately one third of myelin, and demyelination in the setting of vitamin B12 deficiency may explain many of the neurologic manifestations, including paresthesias, numbness, gait abnormalities, cognitive impairment, and neural tube defects in fetuses [58]. The classic neurologic finding in vitamin B12 deficiency is subacute combined degeneration of the dorsal and lateral columns of the spinal cord due to demyelination, associated with ataxia, progressive weakness, and paresthesias which may progress to significant paraplegia [42]. Other non-hematologic manifestations include osteopenia and vascular occlusive disease, due to accumulation of homocysteine [52].

Diagnosis Serum vitamin B12 levels can be used to evaluate for vitamin B12 deficiency, though there is a high occurrence of both false-positive and false-negative results [42]. Serum levels of methylmalonic acid and homocysteine are typically elevated and can be helpful in making the diagnosis [52]. Detection of anti-parietal or anti-­ intrinsic factor antibodies can be used to identify pernicious anemia.

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Treatment Treatment in children is typically monthly intramuscular (IM) vitamin B12 or daily high-dose (1000-2000ug) oral supplement [59]. High-dose oral replacement has been found to be effective in cases of intestinal malabsorption. Duration of therapy depends upon the etiology. Patients with pernicious anemia need indefinite IV replacement therapy to bypass the need for intrinsic factor [42].

Bibliography 1. Baker RD, Greer FR, Committee on Nutrition American Academy of Pediatrics. Diagnosis and prevention of iron deficiency and iron-deficiency anemia in infants and young children (0–3 years of age). Pediatrics. 2010;126(5):1040–50. 2. Camaschella C. Iron-deficiency anemia. N Engl J Med. 2015;373(5):485–6. 3. Brotanek JM, Gosz J, Weitzman M, Flores G. Iron deficiency in early childhood in the United States: risk factors and racial/ethnic disparities. Pediatrics. 2007;120(3):568–75. 4. Brotanek JM, Halterman JS, Auinger P, Flores G, Weitzman M.  Iron deficiency, prolonged bottle-feeding, and racial/ethnic disparities in young children. Arch Pediatr Adolesc Med. 2005;159(11):1038–42. 5. Beard JL. Iron requirements in adolescent females. J Nutr. 2000;130(2S Suppl):440S–2S. 6. Lonnerdal B. Development of iron homeostasis in infants and young children. Am J Clin Nutr. 2017;106(Suppl 6):1575S–80S. 7. Anderson GJ, Frazer DM.  Current understanding of iron homeostasis. Am J Clin Nutr. 2017;106(Suppl 6):1559S–66S. 8. Doom JR, Georgieff MK. Striking while the iron is hot: understanding the biological and neurodevelopmental effects of iron deficiency to optimize intervention in early childhood. Curr Pediatr Rep. 2014;2(4):291–8. 9. Lozoff B, Clark KM, Jing Y, Armony-Sivan R, Angelilli ML, Jacobson SW. Dose-response relationships between iron deficiency with or without anemia and infant social-emotional behavior. J Pediatr. 2008;152(5):696–702. 10. Algarin C, Nelson CA, Peirano P, Westerlund A, Reyes S, Lozoff B. Iron-deficiency anemia in infancy and poorer cognitive inhibitory control at age 10 years. Dev Med Child Neurol. 2013;55(5):453–8. 11. Halterman JS, Kaczorowski JM, Aligne CA, Auinger P, Szilagyi PG.  Iron deficiency and cognitive achievement among school-aged children and adolescents in the United States. Pediatrics. 2001;107(6):1381–6. 12. Bruner AB, Joffe A, Duggan AK, Casella JF, Brandt J.  Randomised study of cognitive effects of iron supplementation in non-anaemic iron-deficient adolescent girls. Lancet. 1996;348(9033):992–6. 13. Franchini M, Targher G, Montagnana M, Lippi G.  Iron and thrombosis. Ann Hematol. 2008;87(3):167–73. 14. Kinoshita Y, Taniura S, Shishido H, Nojima T, Kamitani H, Watanebe T.  Cerebral venous sinus thrombosis associated with iron deficiency: two case reports. Neurol Med Chir (Tokyo). 2006;46(12):589–93. 15. Hartfield DS, Lowry NJ, Keene DL, Yager JY. Iron deficiency: a cause of stroke in infants and children. Pediatr Neurol. 1997;16(1):50–3. 16. Balci K, Utku U, Asil T, Buyukkoyuncu N. Deep cerebral vein thrombosis associated with iron deficiency anaemia in adults. J Clin Neurosci. 2007;14(2):181–4.

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17. Maguire JL, deVeber G, Parkin PC. Association between iron-deficiency anemia and stroke in young children. Pediatrics. 2007;120(5):1053–7. 18. Male C, Persson LA, Freeman V, Guerra A, van’t Hof MA, Haschke F, et al. Prevalence of iron deficiency in 12-mo-old infants from 11 European areas and influence of dietary factors on iron status (Euro-Growth study). Acta Paediatr. 2001;90(5):492–8. 19. Salstrom JL, Kent M, Liang X, Wang M. Toddlers with anasarca and severe anemia: a lesson in preventive medicine. Curr Opin Pediatr. 2012;24(1):129–33. 20. Ziegler EE. Consumption of cow’s milk as a cause of iron deficiency in infants and toddlers. Nutr Rev. 2011;69(Suppl 1):S37–42. 21. Pawlak R, Bell K. Iron status of vegetarian children: a review of literature. Ann Nutr Metab. 2017;70(2):88–99. 22. de Vizia B, Poggi V, Conenna R, Fiorillo A, Scippa L.  Iron absorption and iron defi ciency in infants and children with gastrointestinal diseases. J Pediatr Gastroenterol Nutr. 1992;14(1):21–6. 23. Parrott J, Frank L, Rabena R, Craggs-Dino L, Isom KA, Greiman L.  American society for metabolic and bariatric surgery integrated health nutritional guidelines for the Surgical Weight Loss Patient 2016 Update: Micronutrients. Surg Obes Relat Dis. 2017;13(5):727–41. 24. Camaschella C. Iron deficiency. Blood. 2019;133(1):30–9. 25. Rubeor A, Goojha C, Manning J, White J. Does iron supplementation improve performance in iron-deficient nonanemic athletes? Sports Health. 2018;10(5):400–5. 26. Girelli D, Ugolini S, Busti F, Marchi G, Castagna A. Modern iron replacement therapy: clinical and pathophysiological insights. Int J Hematol. 2018;107(1):16–30. 27. Boutry M, Needlman R.  Use of diet history in the screening of iron deficiency. Pediatrics. 1996;98(6 Pt 1):1138–42. 28. Nead KG, Halterman JS, Kaczorowski JM, Auinger P, Weitzman M. Overweight children and adolescents: a risk group for iron deficiency. Pediatrics. 2004;114(1):104–8. 29. Merkel D, Huerta M, Grotto I, Blum D, Tal O, Rachmilewitz E, et al. Prevalence of iron deficiency and anemia among strenuously trained adolescents. J Adolesc Health. 2005;37(3):220–3. 30. Johnson S, Lang A, Sturm M, O’Brien SH. Iron deficiency without anemia: a common yet under-recognized diagnosis in young women with heavy menstrual bleeding. J Pediatr Adolesc Gynecol. 2016;29(6):628–31. 31. Kumari PL, Nair MK, Nair SM, Kailas L, Geetha S. Iron deficiency as a risk factor for simple febrile seizures–a case control study. Indian Pediatr. 2012;49(1):17–9. 32. Wright RO, Shannon MW, Wright RJ, Hu H. Association between iron deficiency and low-­ level lead poisoning in an urban primary care clinic. Am J Public Health. 1999;89(7):1049–53. 33. Maheswaran M, Kushida CA. Restless legs syndrome in children. MedGenMed. 2006;8(2):79. 34. Kotagal S, Silber MH. Childhood-onset restless legs syndrome. Ann Neurol. 2004;56(6):803–7. 35. Mentzer WC Jr. Differentiation of iron deficiency from thalassaemia trait. Lancet. 1973;1(7808):882. 36. Powers JM, Buchanan GR, Adix L, Zhang S, Gao A, McCavit TL. Effect of low-dose ferrous sulfate vs iron polysaccharide complex on hemoglobin concentration in young children with nutritional iron-deficiency anemia: a randomized clinical trial. JAMA. 2017;317(22):2297–304. 37. Abrams SA, O’Brien KO, Wen J, Liang LK, Stuff JE. Absorption by 1-year-old children of an iron supplement given with cow’s milk or juice. Pediatr Res. 1996;39(1):171–5. 38. Lynch SR, Cook JD. Interaction of vitamin C and iron. Ann N Y Acad Sci. 1980;355:32–44. 39. Stoffel NU, Cercamondi CI, Brittenham G, Zeder C, Geurts-Moespot AJ, Swinkels DW, et al. Iron absorption from oral iron supplements given on consecutive versus alternate days and as single morning doses versus twice-daily split dosing in iron-depleted women: two open-label, randomised controlled trials. Lancet Haematol. 2017;4(11):e524–e33. 40. Reeves JD, Yip R. Lack of adverse side effects of oral ferrous sulfate therapy in 1-year-old infants. Pediatrics. 1985;75(2):352–5. 41. Tefferi A, Pruthi RK.  The biochemical basis of cobalamin deficiency. Mayo Clin Proc. 1994;69(2):181–6.

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4 2. Stabler SP. Clinical practice. Vitamin B12 deficiency. N Engl J Med. 2013;368(2):149–60. 43. Green R, Datta Mitra A. Megaloblastic anemias: nutritional and other causes. Med Clin North Am. 2017;101(2):297–317. 44. Quadros EV. Advances in the understanding of cobalamin assimilation and metabolism. Br J Haematol. 2010;148(2):195–204. 45. Whitehead VM. Acquired and inherited disorders of cobalamin and folate in children. Br J Haematol. 2006;134(2):125–36. 46. Taneja S, Bhandari N, Strand TA, Sommerfelt H, Refsum H, Ueland PM, et al. Cobalamin and folate status in infants and young children in a low-to-middle income community in India. Am J Clin Nutr. 2007;86(5):1302–9. 47. Lubran MM.  The biochemistry of folic acid and vitamin B 12. Ann Clin Lab Sci. 1971;1(3):236–44. 48. Diab L, Krebs NF. Vitamin excess and deficiency. Pediatr Rev. 2018;39(4):161–79. 49. Imbard A, Benoist JF, Blom HJ. Neural tube defects, folic acid and methylation. Int J Environ Res Public Health. 2013;10(9):4352–89. 50. Collins RA. Goat’s milk anemia in retrospect. Am J Clin Nutr. 1962;11:169–70. 51. Kronn D, Goldman ID. Hereditary folate malabsorption. In: Adam MP, Ardinger HH, Pagon RA, et al., editors. Seattle (WA): University of Washington, Seattle; 1993–2020. 52. Green R. Vitamin B12 deficiency from the perspective of a practicing hematologist. Blood. 2017;129(19):2603–11. 53. Allen LH. How common is vitamin B-12 deficiency? Am J Clin Nutr. 2009;89(2):693S–6S. 54. Lam JR, Schneider JL, Zhao W, Corley DA. Proton pump inhibitor and histamine 2 receptor antagonist use and vitamin B12 deficiency. JAMA. 2013;310(22):2435–42. 55. Ahmed MA, Muntingh G, Rheeder P.  Vitamin B12 deficiency in metformin-treated type-2 diabetes patients, prevalence and association with peripheral neuropathy. BMC Pharmacol Toxicol. 2016;17(1):44. 56. Kristiansen M, Aminoff M, Jacobsen C, de La Chapelle A, Krahe R, Verroust PJ, et al. Cubilin P1297L mutation associated with hereditary megaloblastic anemia 1 causes impaired recognition of intrinsic factor-vitamin B(12) by cubilin. Blood. 2000;96(2):405–9. 57. Lindenbaum J, Healton EB, Savage DG, Brust JC, Garrett TJ, Podell ER, et al. Neuropsychiatric disorders caused by cobalamin deficiency in the absence of anemia or macrocytosis. N Engl J Med. 1988;318(26):1720–8. 58. Scott JM. Folate and vitamin B12. Proc Nutr Soc. 1999;58(2):441–8. 59. Devalia V, Hamilton MS, Molloy AM, British Committee for Standards in Haematology. Guidelines for the diagnosis and treatment of cobalamin and folate disorders. Br J Haematol. 2014;166(4):496–513.

Chapter 3

Lead Poisoning Nicholas Newman

Introduction Lead is a naturally occurring metal that is malleable and corrosion resistant. It is found in the earth’s crust, and although it is not particularly abundant, it is easily accessible and has been used by humans since at least 3500 BCE [1]. Atmospheric levels of lead have increased by orders of magnitude since prehistoric times, and the modern day human body burden of lead is approximately 1000 times the level found in prehistoric specimens [2]. The toxicity of lead has been documented for thousands of years with one of the earliest descriptions of acute lead poisoning from the Greek physician, Nicander, in the second century BCE [3]. Childhood lead poisoning from house paint was painstakingly described by Dr. John Lockhart Gibson, an ophthalmologist from Brisbane, Australia, in 1904 [4]. Over the following century, it became clear that even relatively low blood lead levels had adverse effects on children’s neurodevelopment, and the Centers for Disease Control and Prevention (CDC) lowered the acceptable blood lead level from 60 mcg/dL in 1960 to 5 mcg/ dL in 2012 and may lower it further in the future as there is no safe level of lead [5, 6].

Epidemiology of Lead Exposure in Children The epidemiological triad is one framework to examine any exposure, whether it be infectious (contaminated water), electromagnetic (radiation exposure), airborne (traffic-related air pollution), or chemical (volatile organic compounds). We will use N. Newman (*) Department of Pediatrics and Department of Environmental & Public Health Sciences, University of Cincinnati, Pediatric Environmental Health & Lead Clinic, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA e-mail: [email protected] © Springer Nature Switzerland AG 2021 D. M. Kamat, M. Frei-Jones (eds.), Benign Hematologic Disorders in Children, https://doi.org/10.1007/978-3-030-49980-8_3

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Fig. 3.1 Modified epidemiological triad

Host

Vector

Agent

Environment

this framework to examine lead poisoning in children as it examines the relationship of host, agent (+/− vector), and environment interact to cause disease; see Fig. 3.1.

Host Factors The adage that “children are not little adults” is appropriate in the case of lead exposure. Children less than 6 years old consume >2.5 times the amount of water and 2–3 times the amount of food per kilogram body weight as compared to adults [7]. When exposed to lead, children less than 2 years old absorb >3 times the amount of lead than adults do [7]. For the same amount of lead present in the environment, a child absorbs a much higher amount than an adult. Young children also tend to have more frequent oral behaviors and pica. This is another risk factor for increased lead exposure. Similarly, minute ventilation in children is much higher than adults, so for any given airborne contaminant, children will be exposed to higher levels per body weight than adults. In addition, children are closer to the ground than adults and are in closer contact to re-entrained dust from the ground. This in part explains why very young children have elevated blood lead levels in response to increased soil and air lead levels [8]. Inorganic lead absorption primarily occurs through the oral and inhalational routes. Inorganic lead is found in paint and dusts; organic lead is predominantly found in leaded gasoline in the form of tetraethyl lead. Airborne particles 2.5 μm diameter tend to be swallowed and absorbed through the gastrointestinal tract [9]. Although most lead absorption appears to take place in the duodenum, the exact mechanisms for lead absorption are not known. Several mechanisms of lead absorption in the gastrointestinal tract have been proposed: active transport, diffusion, or paracellular mechanisms [9]. An active transport mechanism for lead absorption,

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divalent metal transporter 1 (DMT1), is an H+-coupled, saturable transport that also transports nonheme iron [10]. A DMT1 polymorphism has been identified in humans that are associated with increased blood lead levels [11]. The half-life for lead in blood is approximately 30 days and in bone, 10–30 years. Therefore, prolonged exposure to lead in childhood may result in a significant storage of lead in the bone that leeches out over time. Dietary factors can influence blood lead levels. Children who had not consumed breakfast had higher blood lead levels and lower zinc levels than their breakfast-­ eating counterparts [12]. Additionally, low iron levels (as measured by percent transferrin saturation) in children have been associated with increased blood lead levels [13]. Dietary calcium also impacts lead absorption, but the relationship is more complex, and there is limited evidence in children [14]. Finally, since an important target organ for lead toxicity is the brain, children are uniquely vulnerable to it because their brains are rapidly growing and developing. These neurodevelopmental processes: neuronal cell proliferation, migration of neurons, synapse formation, and myelination occur over different periods of time in different parts of the brain depending on the age of the child. Therefore, the impact of lead differs based on when during neurodevelopment the exposure takes place.

Chemical Factors Regarding Lead Lead (Pb) exists in three oxidation states Pb(0), Pb(II), and Pb(IV). In the environment, Pb(II) is the most prevalent form, most commonly as PbS (Galena), PbSO4 (anglesite), PbCO3 (cerussite or white lead), and Pb3O4 (minium) [9]. Lead carbonate (PbCO3) has been used in lead-based paints. Metallic lead is not bioavailable Pb(0), but Pb(II) is because it is divalent and readily absorbed. The +4 state is typically found only under strongly oxidizing conditions or in organic lead compounds (tetraethyl lead) such as gasoline.

Environmental Sources of Lead Currently, the primary use of lead is in lead storage batteries for automobiles [15]. Due to its desirable physical properties, lead has had many uses over the millennia such as a stabilizer for rubber and plastics as well as a pigment for paints. Lead use continues in certain cosmetics (kohl, surma, sindoor) as well as contamination of spices and other food products. Traditional medicines, particularly from Mexico, India, and Vietnam, have also been found to contain lead [16]. One of the oldest uses of lead is for plumbing components (plumbus is the Latin word for lead). Although lead in plumbing components has been eliminated in many countries, legacy infrastructure will be a source of lead exposure for some time to come. Important historical and current uses for lead are listed in Table 3.1.

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Table 3.1  Current and historical uses of lead Storage batteries Electronics Cosmetics Art paints and supplies Gasoline additive Plumbing solder Combustion of coal, oil

Paint Rubber materials Munitions Pottery glaze Stabilizer for plastics Water service lines Toys painted with lead paint

Metallurgy Electrical solder Home remedies Crayons Plumbing fixtures Pesticides

In the USA, the most common sources of lead exposure in children are deteriorating lead-based paint, in addition to lead-contaminated dust and soil [17]. Lead-­ based house paint containing lead chromate (“chrome yellow”), lead oxide (“red lead”), or lead carbonate (“white lead”) was initially banned by European governments by the League of Nations in 1922 and in the USA in 1978 [6]. Regulatory efforts have been effective in reducing lead paint levels in most of the developed world. Developing countries may not have such regulations, and sale of lead-­ containing house paints continues to be a hazard for children in India and parts of Asia [18]. Overall, lead paint and lead-containing dusts are the major contributors to childhood lead exposure. In the USA, approximately 40% of lead exposure is from dust lead, 20% from lead in drinking water, and between 10% and 20% each from soil ingestion, renovation, and soil lead [19–21]. A study of >300,000 children in Detroit, MI (USA), with blood lead levels obtained between 2001 and 2009 estimated that for every 0.0069  mcg/m3 increase in air lead level resulted in a 10% increase in blood lead levels in children 10 mcg/dL, when compared to children with blood lead levels 1.3  mcg/dL blood lead level) represented 598,000 excess cases of ADHD in the USA [39]. Following the landmark study that demonstrated an association between high dentine lead levels and poor school performance in urban, American children who had not had clinical lead poisoning [40], there have been concerns about the academic achievement of children exposed to lead. A follow-up study of a subset of this cohort demonstrated the effects of lead were persistent into late adolescence and young adulthood with a significant risk for reading problems and dropping out of high school [41]. The persistence of the effect of early life lead exposure on academic achievement has been demonstrated elsewhere. Population-based studies on school-aged children demonstrate problems with kindergarten reading readiness as well as performance on standardized achievement tests. A study of approximately 5000 children enrolling in kindergarten in Providence, Rhode Island (USA), public schools compared the scores of a reading readiness assessment test (Phonological Awareness Literacy Screening-Kindergarten) in children with blood lead levels 10 mcg/dL. The investigators found that compared with the low exposure group, children with blood lead levels 5–9 mcg/dL were 21% more likely to fail to achieve the national benchmark for reading, whereas children with blood lead levels >10 mcg/dL were 56% more likely to fail the reading benchmark [42]. A study of children in the Detroit, Michigan (USA), public schools from 2008 to 2010 examined the association between early life lead levels and standardized test scores for mathematics, science, and reading for grades 3, 5, and 8. After adjustment for covariates, they found that blood lead levels >1  mcg/dL were

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associated with increased odds of scoring “less than proficient” and that the decrease in test scores was highest at the lower lead levels [43]. Longitudinal cohort studies have also examined the long-term impact of childhood lead exposure as the cohort transition to adulthood. In a prenatal cohort recruited from Cincinnati, Ohio (USA), between 1979 and 1985 (Cincinnati Lead Study), investigators found an association between prenatal lead exposure (as measured by maternal blood lead levels) and juvenile delinquency and antisocial behavior in adolescents [44]. Investigators continued following this cohort into adulthood and found a significant association between prenatal and early childhood lead exposure and adult criminal behavior, especially arrests for violent crimes [45]. Another longitudinal cohort from New Zealand examined blood lead levels at age 11 and reported actual criminal behavior through age 45 years and found only a weak association between lead exposure and criminality [46]. Although the effect size of the relationship between lead exposure and criminality is small (R2 = 0.06–0.10), it is similar in effect size to other predictors of adult criminality such as family size, socioeconomic status, or being separated from one’s parents [47]. Compared to these other factors, lead exposure can be identified, and the means to prevent it are readily known [48]. Neuroimaging studies have explored the potential neuroanatomical and functional changes in the brain that would explain the findings from epidemiological studies. Cecil and colleagues performed the first prospective cohort study of childhood lead exposure that included brain imaging using participants in the Cincinnati Lead Study. In these young adults whose childhood lead history was prospectively obtained, they found dose-dependent decreases in gray matter volume in ventrolateral prefrontal cortex, anterior cingulate cortex, postcentral gyri, inferior parietal lobule, and cerebellum [49]. The decrease in prefrontal cortex volumes was particularly notable in males. This same group also reported effects on brain activation patterns during a verb generation task using functional MRI [50]. Although there is not a single unifying mechanism to explain lead’s toxicity, lead appears to mimic calcium in biological systems. There are multiple cellular functions in the neurons that are potential targets for lead: calmodulin, synaptotagmin, neuronal calcium sensor-1, N-methyl-D-aspartate receptor as well as G-protein-­ coupled receptors [51]. The discovery that picomolar levels of lead are equivalent to micromolar concentrations of calcium in activating brain protein kinase C suggests a possible mechanism for lead’s enhanced effect on IQ at very low levels [52]. Lead also interacts with dopamine receptors in the brain, and this may contribute to defects in executive function [53]. In addition, lead exposure has been shown to result in mitochondrial dysfunction [54].

Hematological Toxicity Lead inhibits delta-aminolevulinic acid (ALA) dehydratase, coproporphyrinogen oxidase, and ferrochelatase [55]. The partial blockage of these enzymes results in elevated urinary delta-aminolevulinic acid, urinary coproporphyrin, and erythrocyte

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(zinc) protoporphyrin. No threshold for ALA dehydratase inhibition by lead appears to exist [56]. Carriers of the ALA dehydratase 2 allele have higher blood lead levels [57]. Although anemia due to lead poisoning is not commonly seen, lead interferes with heme synthesis at blood lead levels of 25 mcg/dL or higher and after 50–70 days of exposure [19]. Chronic lead exposure may result in a basophilic stippling of red blood cells. These granules of intracytoplasmic ribosomes are visible when a blood smear is stained with Wright’s stain [58]. Acute, high-level lead exposure is associated with hemolytic anemia, and chronic lead exposure is associated with hypochromic and either normocytic or microcytic anemia [9]. Iron deficiency and iron deficiency anemia have long been associated with lead exposure [59]. Since both iron and lead absorption are in part mediated by DMT1 in the intestine, iron deficiency results in upregulation of DMT1 on the intestinal wall, and lead is absorbed in higher quantities as well.

Renal Toxicity Chronic, high-level lead exposure is a known risk factor for chronic kidney disease [60]. Lead nephropathy has been well described and is characterized by chronic interstitial nephritis [9]. Even relatively low blood lead levels are associated with decreases in glomerular filtration rate (GFR) as determined by a cystatin C-based estimating equation. Using data from 769 adolescents (aged 12–20 years) from the NHANES 1988 to 1994, participants in the highest quartile for lead levels (≥3 mcg/ dL) had 6.6  ml/min/1.73  m2 lower estimated GFR as compared with the lowest quartile (15 mcg/dL. Due to its radio-opacity, lead objects such as toys, paint chips, or other items may be easily visualized. Some clinicians may opt to use laxatives to speed up the passage of these objects. Long bone radiographs to look for “lead lines” (metaphyseal bands) are no longer routinely recommended [58].

Improving Child Resilience in Lead Poisoning Since lead exposure increases the risk for cognitive, behavioral, and school problems in children, reducing other risk factors for these adverse neurodevelopmental outcomes should be addressed. Limited evidence suggests the effects of lead on IQ may be partially reversible [67]. To improve child outcomes in lead-poisoned children, recommendations have been made regarding optimizing micronutrient intake (iron, zinc, calcium), limiting fat intake, as well as providing educational interventions. These will be summarized and reviewed briefly below.

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The relationship between iron deficiency and increased lead levels is clearly established [59, 68–71]. There is limited evidence regarding low zinc and blood lead levels [12, 72, 73]. A randomized, placebo-controlled trial of iron and zinc supplementation of 517 school-aged children in Mexico had no effect on blood lead levels but did improve iron and zinc status in deficient children [74]. Since iron deficiency is associated with neurodevelopmental deficits similar to lead exposure [75, 76], preventing iron deficiency may therefore be neuroprotective. This is quite different than using micronutrient supplements to “fight” lead poisoning as is commonly stated in public health recommendations. Similarly, although calcium supplementation of lead exposed children is questioned [14], maintaining an adequate intake of calcium may be protective against increased blood lead levels [77]. Recommendations regarding limiting fat intake were extrapolated from animal studies and may not be applicable in young children [78]. Critics of nutritional interventions caution that time and resources spent in this arena may distract from other interventions that will ultimately eliminate the source of lead exposure [79]. Once again, this evidence reinforces the primary prevention approach to lead exposure. In 2015, the CDC published Educational Interventions for Children Affected by Lead. In this document, an expert panel provided a set of recommendations to improve neurodevelopmental outcomes in children exposed to lead. Although there is a lack of evidence to recommend a specific intervention for children with lead exposure, the panel concluded that a stimulating home environment moderated the effect of lead on children’s behavior and cognition. Early childhood education programs including high-quality preschool and prekindergarten programs are recommended for children exposed to lead. Additionally more holistic programs such as Head Start that include education, health, nutrition, and social service support may also be beneficial [80]. The group also emphasized the need for additional research in the field to better determine which interventions would be most effective in children with lead exposure.

Investigation and Mitigation of Sources of Lead Exposure In some jurisdictions, an elevated blood lead level in a child automatically triggers an evaluation by local public health authorities. Typically, the response from public health depends on the child’s blood lead level. In the USA, depending upon region, this could mean a full lead risk assessment for a child with a blood lead level of 5 mcg/dL or no risk assessment, regardless of the blood lead level. One easy mnemonic in obtaining an environmental health history is “ACHHOO.” This mnemonic will guide the examiner through settings and activities relevant in an environmental health history [81]:

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Activities: School, childcare, after school, sports, other caregivers, and places of worship Community: General characteristics, industry, agriculture, waste sites, water quality, and source Household: Type of dwelling, age, condition, heating/cooking fuel, and pesticide use Hobbies: Arts, crafts, fishing, and hunting Occupation: Known exposures from work of patient or parent/caregivers; occupations Oral behaviors: Pica/mouthing Although this list is not comprehensive, it can be used as a starting point with a series of open-ended questions to aid in obtaining a complete environmental health history and to identify possible lead hazards. Important sources of lead exposure in children and possible mitigation strategies are summarized in Table 3.2. All educational and clinic-based strategies are second best to a lead risk assessment and subsequent lead abatement [82]. Clinicians should encourage elimination of sources of lead exposure as often as possible. Table 3.2  Important sources of lead exposure in children and mitigation strategies Source Interior dust lead

Water lead [83]

Soil ingestion/ pica

Renovation [20]

Detailsa Mitigation strategies Typical sources: deteriorating lead-based paint, Elimination or stabilization tracking in of lead-contaminated soil of deteriorating painted surfaces Wet wiping, vacuuming of interior horizontal surfacesb Doormats Leaving shoes outside or at the door Lead service lines, lead-containing fixtures and Avoid using hot water for solder, lead plumbing consumption Water filtration Elimination of lead-­ containing components Seen in young children and older children with Substitution oral behavior developmental delays with non-lead-containing item (chew toy) Behavioral modification Residences with lead-based paint (in USA, Identify lead-based paint housing built prior to 1978) prior to renovation Follow US EPA Renovation, Repair and Painting Rulec Young children and women of childbearing age should avoid premises during renovation (continued)

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Table 3.2 (continued) Source Soil lead [84]

Detailsa Particularly in urban areas and nonurban areas with unusually high exposure

Mitigation strategies Create barrier using ground cover or other item (i.e., mulch) Identify source of lead contamination and eliminate (i.e., adjacent deteriorating housing with lead-based paint) Removal of lead-­ contaminated soil and replacement with non-contaminated soil “Leave work at work” Para-occupational Construction or demolition, plumbing, lead Use of work clothing, battery manufacture, welders or brazers, exposure proper hygiene, changing munition plant workers, workers at firing (take-home exposure) [15, 85] ranges, law enforcement/military, glassmakers, clothes before leaving work artists, pottery workers, e-waste recyclers, marine/aviation mechanics, artisanal mining operations Foods and spices Spices from a number of countries, especially Avoid use Seek sources of spices that [22, 86] East Asia and Central Asia are 3rd-party certified lead Georgian saffron, curry, fenugreek, turmeric, free hot chili powder; candies from Vietnam or Mexico (particularly tamarind), lozeena (Iraqi food flavoring) Avoid use Traditional Greta and Azarcon (Latin American remedies [86, 87] constipation remedy) Ayurveda (metals found in 30–65% of samples) Ghasard (Indian digestion aid) Ba-baw-san (Chinese colic remedy) Saoott or Cebagin (Middle Eastern teething powder) Bint al dahab (Iranian colic remedy) Santrinj (Saudi Arabian teething powder) Pay-loo-ah (Southeast Asian treatment for fever and rash) Daw tway gaw mo gah (Burmese multi-­ symptom infant remedy) Cosmetics [88] Sindoor, kohl kajal, surma, tiro, tozali, kwalli Avoid use (all commonly used in Africa, Middle East or Asia); progressive hair dyes Toys and other Painted items, some metal toys Watch for recall items announcements Avoid use Not a comprehensive list since there are many sources of lead A systematic review by the Cochrane Collaboration found limited effectiveness of household cleaning interventions to avoid childhood lead exposure [82]. There is evidence that these cleaning interventions may reduce the number of children with blood lead levels >15 mcg/dL [89] c https://www.epa.gov/lead/lead-renovation-repair-and-painting-program a

b

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Interventions Based on Lead Level In the USA, the American Academy of Pediatrics (AAP) and the CDC recommend testing publicly insured children at 1 and 2 years of age and again at 3 to 6 years of age if not previously tested [19, 90]. State and local health jurisdictions may have other requirements for lead testing in children. Clinicians should check with their local public health authorities for details. Lead testing may be performed if there is a suspicion of lead exposure based on an environmental history. The purpose of lead testing is to identify children who may need public health or clinical interventions. If primary prevention measures for lead poisoning have failed, children with lead exposure require comprehensive interventions to prevent further increases in blood lead levels, to implement changes to rapidly decrease blood lead levels, and to address other risk factors for neurodevelopmental problems. Although little evidence exists that the effects of lead poisoning are reversible, evidence does exist to suggest that the duration blood lead levels are elevated which is more important than a single peak level [91]. A modified version of the PEHSU medical management recommendations for lead exposed children is below in Table  3.3 [64]. These recommendations are meant to be general guidance and should be adapted to local laws and regulations and used as a guide but not as a replacement for clinician judgment.

Chelation Treatment for Lead Poisoning After removing a child from the source of lead exposure, children with blood lead levels ≥45 mcg/dL should be considered for chelation therapy to reduce lead levels. Chelating agents are medications that contain a partially negatively charged moiety (thiol or nitrogen-oxygen) that bind to metals and increase their solubility and allow

Table 3.3  Medical management recommendations for children with elevated blood lead levels Lead Level Recommendation 1. Review lab results with family. For reference, in the USA, the geometric 30,000/μL. Second-­ line therapies are also often used in children with refractory, chronic, or persistent ITP. It is important to ensure that a child with ITP, who is unresponsive to first-line therapies, has undergone an appropriate work-up for secondary causes as detailed previously. There are no established trials comparing the various modalities of second-­line treatments in childhood ITP.  The choice of second-line agents often depends on patient/parental preference, ease of administration, toxicity profile, physician experience, and perceived efficacy [44].

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Rituximab is an anti-CD20 monoclonal antibody that targets B-cells which are CD20+. It has been used as a second-line treatment modality in ITP and is administered intravenously as a weekly dose of 375 mg/m2 for 4 weeks. Due to iatrogenic B-cell depletion after rituximab treatment, these children may develop acquired immunoglobulin deficiency and require IVIG infusions to prevent serious illnesses from B-cell immunodeficiency. Approximately 30% of patients treated with rituximab have a durable response after B-cell reconstitution 9–12 months after treatment [45–49]. Other second-line treatments also target the immune system to decrease production of antiplatelet antibodies and include mycophenolate mofetil [50], cyclosporine [51], azathioprine [52], danazol [53], cyclophosphamide [54], interferon [55], and alemtuzumab [56] (Table  9.2). Dapsone is a non-­ immunomodulatory second-line agent with an interesting mechanism of action in ITP compared to the other medications. Dapsone functions by causing mild hemolysis and overwhelming the RES [57]. Thrombopoietin (TPO) receptor agonists, eltrombopag and romiplostim, have been in the limelight in recent years due to their increasing use in chronic ITP and

Table 9.2  Second-line therapeutic options in ITP Drug Rituximab Alemtuzumab Azathioprine

Mechanism of action Anti-CD20 antibody Targets B-cells Anti-CD52 antibody Targets both B- and T-cells Purine synthesis inhibitor

Cyclophosphamide

Alkylating agent, inhibits DNA-RNA cross-linking

Cyclosporine

Calcineurin inhibitor, affects T-cell function Calcineurin inhibitor, affects T-cell function Inosine monophosphate dehydrogenase inhibitor, affects T-cell function Androgen analog, decreases antiplatelet antibody production Hemolysis, saturating RES, anti-inflammatory effect Microtubule polymerization inhibitor Unclear, likely related to immunomodulatory effect of interferon TPO receptor agonists

Tacrolimus Mycophenolate mofetil Danazol Dapsone Vincristine Interferon-alpha

Eltrombopag, romiplostim

Adverse effects Hypogammaglobulinemia Profound immunosuppression Immunosuppression, secondary malignancy Hemorrhagic cystitis, immunosuppression, secondary malignancy Hirsutism, hypertension, renal insufficiency Hypertension, hyperglycemia Gastrointestinal intolerance

Hirsutism, acne, liver toxicity Gastrointestinal disturbances, methemoglobinemia Constipation, peripheral neuropathy, leukopenia Flu-like symptoms, leukopenia

Headache, arthralgia, nasopharyngitis, liver toxicity

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aplastic anemia in adults. Both eltrombopag and romiplostim have shown promising results with good platelet response noted in children [58–60]. Rationale for use of TPO receptor agonists in chronic ITP is that TPO levels are often inappropriately low in patients with ITP and TPO receptor agonists stimulate platelet synthesis [17, 18]. Eltrombopag was approved by the FDA in 2015 for use in chronic ITP in children older than 1 year of age [61]. Eltrombopag is administered as an initial dose of 50 mg orally once a day, with dose titration done based on response; maximal dose is 75 mg/day [58, 59, 62]. Patients of Asian ethnic backgrounds have been reported to have a higher incidence of hepatotoxicity; hence, a starting dose of 25 mg/day is recommended in this population [58, 59, 62]. In younger children aged 1–5 years, a weight-based dosing was studied by Bussell et al.; 0.7 mg/kg/day up to a maximum of 2  mg/kg based on response maybe used in this patient population [58]. Eltrombopag must be taken on an empty stomach to prevent absorption issues. Romiplostim can be initiated at a dose of 1 mcg/kg/week as a subcutaneous injection, with a maximal dose of 10 mcg/kg/week [60, 62]. Like eltrombopag, romiplostim has been FDA-approved for treatment of children 1 year old and older with a diagnosis of ITP for at least 6 months [63]. Both medications are recommended for use in patients with chronic ITP and insufficient response to corticosteroids, IVIG, or splenectomy. During the phase of initial dose titration with TPO receptor agonists, platelet counts need to be monitored weekly. When a platelet count of >50,000/μL has been achieved for 4 weeks, frequency of platelet count monitoring can be reduced to once a month. Treatment should be withheld if platelet count is >200,000/μL. It is recommended to monitor liver enzymes simultaneously while in the initial phase of optimal dose titration of eltrombopag. Therapy with TPO receptor agonists may be discontinued if platelets have not increased to >50,000/μL after 4 weeks of optimal therapy. Adverse effects of TPO receptor agonists include headache, nausea, fatigue, diarrhea, arthralgia, nasopharyngitis, and hepatotoxicity (described with eltrombopag). In addition, increased bone marrow reticulin, similar to that seen in myelofibrosis, has been reported with TPO receptor agonists especially romiplostim [64]. The bone marrow fibrotic changes are reversible in early stages and will recede once therapy with TPO receptor agonists is discontinued [64].

Splenectomy Historically, splenectomy was the second-line therapy for chronic ITP which was unresponsive to other therapies. Utilization of splenectomy as a therapeutic option in childhood ITP has decreased in recent times due to availability of more effective and less invasive second-line therapeutic modalities. Splenectomy is often avoided in children due to the risk for overwhelming postsplenectomy sepsis caused by encapsulated organisms; risk for this is greatest in children less than 5 years of age. If deemed necessary, splenectomy is often delayed and performed when the child is older than 5 years. Children should be vaccinated appropriately against encapsulated organisms including pneumococcus, Haemophilus influenzae group B, and

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Meningococcus; these should be performed a minimum of 2  weeks prior to the surgery. Splenectomy removes the major site for platelet destruction in ITP, in addition to reducing autoantibody production. Approximately 70–80% of children with ITP have remission of their thrombocytopenia after splenectomy [65–67]. Platelet count can be increased perioperatively by using IVIG, corticosteroids, or anti-D IgG. Intraoperative bleeding is rare if perioperative count of 50,000–100,000/μL is achieved. Increase in platelet count is seen immediately after the surgery in most patients. In case of persistent severe thrombocytopenia after splenectomy, the possibility of an accessory spleen being present should be considered. Evaluation for an accessory spleen can be done by a Technetium99m-labeled radionuclide scan. Longterm risks of splenectomy include risk for life-threatening sepsis, thrombocytosis, thromboembolism, and pulmonary hypertension; the latter are adverse effects reported in adults postsplenectomy [68].

Conclusion ITP is a common hematological illness encountered in young children. Older children may present with secondary ITP, usually due to concurrent rheumatologic or immunologic disorders. There has been an evolution in the knowledge related to ITP pathogenesis, from primarily being labeled as a B-cell-mediated autoimmune disease to recent knowledge about low TPO levels playing an important role in ITP. Treatment of ITP often poses a therapeutic dilemma with options ranging from observation alone to IVIG, corticosteroids, and anti-D IgG. Newer agents such as TPO receptor agonists have shown promising results in childhood ITP.

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45. Rao A, Kelly M, Musselman M, Ramadas J, Wilson D, Grossman W, Shenoy S. Safety, efficacy, and immune reconstitution after rituximab therapy in pediatric patients with chronic or refractory hematologic autoimmune cytopenias. Pediatr Blood Cancer. 2008;50(4):822–5. 46. Bennett CM, Rogers ZR, Kinnamon DD, Bussel JB, Mahoney DH, Abshire TC, Sawaf H, Moore TB, Loh ML, Glader BE, McCarthy MC.  Prospective phase 1/2 study of rituximab in childhood and adolescent chronic immune thrombocytopenic purpura. Blood. 2006;107(7):2639–42. 47. Mueller BU, Bennett CM, Feldman HA, Bussel JB, Abshire TC, Moore TB, Sawaf H, Loh ML, Rogers ZR, Glader BE, McCarthy MC. One year follow-up of children and adolescents with chronic immune thrombocytopenic purpura (ITP) treated with rituximab. Pediatr Blood Cancer. 2009;52(2):259–62. 48. Wang J, Wiley JM, Luddy R, Greenberg J, Feuerstein MA, Bussel JB.  Chronic immune thrombocytopenic purpura in children: assessment of rituximab treatment. J Pediatr. 2005;146(2):217–21. 49. Taube T, von Stackelberg A, Schulte-Overberg U, Henze G, Schmid H, Reinhard H. Chronic immune thrombocytopenic purpura in children: assessment of rituximab treatment. J Pediatr. 2006;148(3):423. 50. Provan D, Moss AJ, Newland AC, Bussel JB. Efficacy of mycophenolate mofetil as single-agent therapy for refractory immune thrombocytopenic purpura. Am J Hematol. 2006;81(1):19–25. 51. Emilia G, Messora C, Longo G, Bertesi M. Long-term salvage treatment by cyclosporin in refractory autoimmune haematological disorders. Br J Haematol. 1996;93(2):341–4. 52. Bouroncle BA, Doan CA. Refractory idiopathic thrombocytopenic purpura treated with azathioprine. N Engl J Med. 1966;275(12):630–5. 53. Schreiber AD, Chien P, Tomaski A, Cines DB. Effect of danazol in immune thrombocytopenic purpura. N Engl J Med. 1987;316(9):503–8. 54. Pizzuto J, Ambriz R. Therapeutic experience on 934 adults with idiopathic thrombocytopenic purpura: Multicentric Trial of the Cooperative Latin American group on Hemostasis and Thrombosis. Blood. 1984;64(6):1179–83. 55. Donato H, Kohan R, Picón A, Rovó A, Rapetti MC, Schvartzman G, Lavergne M, de Galvagni A, Rosso A, Rendo P. α-Interferon therapy induces improvement of platelet counts in children with chronic idiopathic thrombocytopenic purpura. J Pediatr Hematol Oncol. 2001;23(9):598–603. 56. Willis F, Marsh JC, Bevan DH, Killick SB, Lucas G, Griffiths R, Ouwehand W, Hale G, Waldmann H, Gordon-Smith EC. The effect of treatment with Campath-1H in patients with autoimmune cytopenias. Br J Haematol. 2001;114(4):891–8. 57. Damodar S, Viswabandya A, George B, Mathews V, Chandy M, Srivastava A. Dapsone for chronic idiopathic thrombocytopenic purpura in children and adults–a report on 90 patients. Eur J Haematol. 2005;75(4):328–31. 58. Bussel JB, de Miguel PG, Despotovic JM, Grainger JD, Sevilla J, Blanchette VS, Krishnamurti L, Connor P, David M, Boayue KB, Matthews DC. Eltrombopag for the treatment of children with persistent and chronic immune thrombocytopenia (PETIT): a randomised, multicentre, placebo-controlled study. Lancet Haematol. 2015;2(8):e315–25. 59. Grainger JD, Locatelli F, Chotsampancharoen T, Donyush E, Pongtanakul B, Komvilaisak P, Sosothikul D, Drelichman G, Sirachainan N, Holzhauer S, Lebedev V. Eltrombopag for children with chronic immune thrombocytopenia (PETIT2): a randomised, multicentre, placebo-­ controlled trial. Lancet. 2015;386(10004):1649–58. 60. Tarantino MD, Bussel JB, Blanchette VS, Despotovic J, Bennett C, Raj A, Williams B, Beam D, Morales J, Rose MJ, Carpenter N. Romiplostim in children with immune thrombocytopenia: a phase 3, randomised, double-blind, placebo-controlled study. Lancet. 2016;388(10039):45–54. 61. Ehrlich LA, Kwitkowski VE, Reaman G, Ko CW, Nie L, Pazdur R, Farrell AT. US Food and Drug Administration approval summary: eltrombopag for the treatment of pediatric patients with chronic immune (idiopathic) thrombocytopenia. Pediatr Blood Cancer. 2017;64(12):e26657.

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62. Kühne T.  Advances in chemical pharmacotherapy for the treatment of pediatric immune thrombocytopenia. Expert Opin Pharmacother. 2018;19(7):667–76. 63. Neunert CE, Rose MJ. Romiplostim for the management of pediatric immune thrombocytopenia: drug development and current practice. Blood Adv. 2019;3(12):1907–15. 64. Ghanima W, Geyer JT, Lee CS, Boiocchi L, Imahiyerobo AA, Orazi A, Bussel JB.  Bone marrow fibrosis in 66 patients with immune thrombocytopenia treated with thrombopoietin-­ receptor agonists: a single-center, long-term follow-up. Haematologica. 2014;99(5):937–44. 65. Aronis S, Platokouki H, Avgeri M, Pergantou H, Keramidas D. Retrospective evaluation of long-term efficacy and safety of splenectomy in chronic idiopathic thrombocytopenic purpura in children. Acta Paediatr. 2004;93(5):638–42. 66. Donato H, Picón A, Rapetti MC, Rosso A, Schvartzman G, Drozdowski C, Di Santo JJ. Splenectomy and spontaneous remission in children with chronic idiopathic thrombocytopenic purpura. Pediatr Blood Cancer. 2006;47(S5):737–9. 67. Wang T, Xu M, Ji L, Yang R. Splenectomy for chronic idiopathic thrombocytopenic purpura in children: a single center study in China. Acta Haematol. 2006;115(1–2):39–45. 68. Rørholt M, Ghanima W, Farkas DK, Nørgaard M. Risk of cardiovascular events and pulmonary hypertension following splenectomy—a Danish population-based cohort study from 1996–2012. Haematologica. 2017;102(8):1333–41.

Chapter 10

Inherited and Congenital Thrombocytopenia Deanna Maida

Introduction Although rare, congenital and inherited causes of thrombocytopenia require prompt recognition by the general practitioner followed by hematology referral in order to provide optimal and timely care. Oftentimes, thrombocytopenia is concomitant with other sequelae, as the genetic changes resulting in thrombocytopenia may affect other biologic systems. This chapter will provide the tools necessary to be able to identify findings suggestive of inherited or congenital thrombocytopenia, providing review of definitions, testing modalities, and the importance of history and physical examination. Generally, inherited platelet disorders are caused by an error in megakaryocytopoiesis (the platelet production process) or are related to decreased platelet function as a result of genetic error in the creation of the platelet itself. By becoming familiar with these diagnoses, both the primary care provider and the hematologist can, together, effectively manage and counsel patients in the outpatient setting.

The Platelet Life Cycle Platelet Birth The development, birth, role, and subsequent demise of a platelet are crucial to hemostasis. Understanding the basics of this process will help guide understanding of the pathophysiology of platelet disorders. Platelets are not only needed to achieve D. Maida (*) University of Texas Health Science Center, San Antonio, TX, USA e-mail: [email protected] © Springer Nature Switzerland AG 2021 D. M. Kamat, M. Frei-Jones (eds.), Benign Hematologic Disorders in Children, https://doi.org/10.1007/978-3-030-49980-8_10

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clinical hemostasis, but their signaling capabilities are used to promote healing, recruiting, and remodeling [1–3]. The signaling mechanism for platelet production is complex and not fully understood. Simply put, pluripotent stem cells housed within the bone marrow differentiate into megakaryocytes, a process regulated by growth factors such as cytokines (i.e., thrombopoietin (TPO), stem cell factor, erythropoietin) and interleukins (i.e., IL-3, IL-6, IL-9, IL-11) as studied in mouse and human models [4–8]. The most specific pathway involves thrombopoietin, a protein produced in the liver that is essential for megakaryocytopoiesis and platelet production signaling [5, 6, 9]. Its receptor, the myeloproliferative ligand receptor (c-Mpl), is located on the surface of the megakaryocyte, housed within the bone marrow [6]. The receptor is made up of an extracellular domain that binds TPO, a transmembrane domain, and an intracellular domain which activates the JAK2 signaling cascade and through a series of additional signaling cascades that are beyond the scope of this chapter [6]. In order to prepare itself for life outside of the megakaryocytes, the “proplatelet” must undergo structural changes in preparation for release into vascular rich areas of the bone marrow [5, 10]. The megakaryocyte has reached peak development once the proplatelet is released into its mature platelet form within the blood stream [10]. Resting platelet morphology is disc-like, with a mean platelet volume (MPV) of 6–9 femtoliters [11, 12], but may vary based on method of measurement used [13] Therefore, it is recommended to review local laboratory reference ranges. A normal platelet count ranges from 150 to 450 k per microliter at any given time, and platelets are expected to perform several tasks during their lifespan of about 10 days [11].

Platelet Anatomy and Function Platelets flow along the vessel wall, making sure the vascular integrity is intact. Crucial to this process is (1) adequate platelet number, (2) appropriate platelet receptors for adhesion, and (3) release of cellular contents, each of which has a specific function to fulfill in order to achieve maximal hemostasis. Once platelets have reached their maximal lifespan and have not been consumed, they are thought to be cleared by the liver and spleen [14, 15]. At the resting phase of its life, platelets appear disc-like, given the intricate pattern of microtubules [12]. Their outer membrane is home to key receptors called “glycoproteins” (GPs) [12]. Integral receptors include the GP1b and GPIIa/IIIb. GP1b receptor forms a complex with coagulation factors IX and V and ultimately leads to the binding of platelets to the injured endothelial wall through the binding of von Willebrand factor (vWF) [12, 16]. Platelet activation also stimulates changes that allow the GPIIb/IIIa receptor to bind fibrinogen and vWF, ultimately leading to platelet aggregation [12]. In addition to GPs, tissue factor is also found in the platelet surface, playing a role in binding of coagulation factors which ultimately leads to thrombin generation [12].

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For being an anucleate structure, platelets have a very intricate intracellular pattern of contents, which include a system of microtubules and granules. The microtubular system is integral to the function of platelets, as it will allow for conformational change not only to allow aggregation but also to activate integrins and expel intracellular signaling contents (called granules) to propagate the hemostatic response [1, 12]. There are three major types of granules: alpha, dense, and lysosomes. The exact chemicals which are released and pathways activated are too numerous and complicated to review here, but it is important to understand several key players. Alpha granules release procoagulant factors such as V, XI, XIII, and prothrombin but also release anticoagulant factors including protein S, plasminogen, and tissue factor pathway inhibitor [1, 12]. Dense granules release ADP, 5-HT, and Ca2+, among other chemicals that are responsible for cascade signaling that ultimately results in hemostatic response. Both alpha and dense granules have been found to play a role in inflammation, healing, and immune modulation [1, 2, 12].

Neonatal Platelet Variation It is worthwhile to mention that there are several differences in the neonatal vs adult platelet structure and function. Platelets have been detected in the first trimester and reach “adult” size and number by the second trimester [17]. However, much is left to be studied regarding neonatal platelet activity as it relates to adult function and when the transition from neonatal to adult is complete [17, 18].

Clinical Evaluation History The most common reasons for thrombocytopenia are typically acquired. Only rarely are causes inherited. As in every patient case, history and physical exam is crucial, as many inherited forms of thrombocytopenia can be ruled out by history alone. A comprehensive list of acquired risk factors are outside the scope of this chapter but may include prenatal risk factors, drug effects, alloimmune or autoimmune thrombocytopenia, malignancy, consumptive processes, infectious etiologies, etc. A thorough history should include questions pertaining to bleeding symptoms along with a complete review of systems to rule out secondary causes (Table 10.1). It is important to ask specific questions such as duration and frequency of bleeding as well as need for blood transfusions. To tease out prolonged bleeding and bruising from normal, age-appropriate findings, specific questions must be asked to determine severity. For example, nosebleeds lasting more than 15 minutes despite adequate intervention or bruising on fatty or soft body parts should raise alertness.

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Table 10.1  Patient history Birth history Pregnancy or delivery complications? Was mother receiving any medications? Did mother or baby experience bleeding? Did the infant have to spend any extra time in the nursery or ICU? If so, what was the indication? Are there any birth defects? Was there bruising on the infant’s head? Did the infant have a bleed in the brain? Was the (male) infant circumcised at birth? How long did it take to heal? Did the umbilical stump heal well? Was there prolonged bleeding from the heel stick for newborn screen testing? Has the child… Ever been told they are anemic or have low number of red blood cells? Required iron supplementation? Required a blood transfusion? Indication? Had any cuts that did not stop bleeding or required medical evaluation? Experienced nosebleeds? If so, how frequently? One or both nares? What intervention was used? Duration of bleed? Experienced easy bruising? Specific location? Had bloody or dark black stools? Had blood in the urine? Had excessive bleeding with tooth eruption? Experienced any purple/red pinpoint rash on the skin? Been on any medications? Has the female adolescent… Achieved menarche? Does the patient use pads or tampons? Level of absorbency? Amount soaked? Frequency of changing? Duration of menstruation? Frequency of menses? Surgical history Has the child had any surgeries, including circumcision (male) or dental procedures? Were surgeries tolerated well? Was there mention of excessive bleeding or need for blood transfusion? Family history Is there any family history of bleeding disorders? Has anyone in the family ever required a blood transfusion? Ask about maternal surgical and menstrual history as well as delivery history. Does the patient have any siblings? Are they healthy? Do they experience easy bleeding or bruising symptoms?

Bruising isolated to shins is typical for an active child. Family history should also be addressed, as there are many inherited forms of bleeding related to thrombocytopenia. There are several screening questionnaires developed to predict possibility of harboring a bleeding disorder, but many children have not achieved significant hemostatic challenges to find them very informative [19–21].

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Physical Exam The physical exam should be focused yet thorough, considering age-appropriate developmental milestones, looking for signs of thrombocytopenia or altered platelet function. This includes a complete skin exam with inspection for bruising, making note of size and topography and presence of petechiae. Mucous membranes should be evaluated for signs of bleeding in the mouth or nares. Indications of anemia secondary to blood loss +/− iron deficiency should also be evaluated (i.e., pallor to the skin, nail beds, and conjunctiva, tachycardia, and presence of heart flow murmur). Finally, an abdominal exam should focus on palpation for hepatosplenomegaly or abdominal masses.

Laboratory Evaluation Initial testing includes a screening complete blood count (CBC) to look for degree of thrombocytopenia and evidence of involvement of other cell lines produced in the bone marrow. A patient at any age should have a platelet count of at least 150 k per microliter. A range from 100 to 149 k, from 50 to 100 k, and from 0 to50 k is considered mild, moderate, and severe thrombocytopenia, respectively. Typically, patients who have quantitative defects are not symptomatic until they are in the severe range. Those who have qualitative defects have variable phenotypes, as function does not correlate with degree of thrombocytopenia, possibly due to underestimation of the platelet count due to macrothrombocytes. The mean platelet volume (MPV) is also useful to characterize thrombocytopenia, as certain diagnoses tend to favor either large or small morphologies. A peripheral smear for pathologic consultation must also be obtained to rule out pseudo-thrombocytopenia (due to platelet clumping or analyzer misreading of giant platelets as neutrophils) and to visualize the morphology of the platelet cell. Findings include confirmation of cell number, size, and at times intracellular platelet contents and the presence of abnormal findings in other cell lines. An immature platelet fraction (IPF), although not widely used in pediatric patients given the paucity of pediatric-­specific norms, can be helpful to determine whether thrombocytopenia is secondary to decreased production (i.e., low IPF) versus losses or destruction (i.e., you would expect an increased IPF to compensate for ongoing losses) [22]. The standard INR, aPTT, and thrombin times will be unaffected by platelet disorders and will therefore be normal. How to proceed with additional testing depends on each case individually in consultation with a pediatric hematologist. It is important to recognize and refer, as many of these tests are specialized and require prompt processing in a pathology lab. Several options are outlined below.

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PFA-100 A simple screening test can include a platelet function analyzer test (PFA-100), a measurement of platelet aggregation or response in the presence of agonist such as epinephrine and collagen or ADP and collagen by measuring the closure time, or time it takes for the sample to occlude [23, 24]. Whole blood volume required for this testing makes it an appealing screening test for pediatric patients [24]. This test generally requires a platelet count greater than 100,000/L and adequate hematocrit [25]. Medications affecting platelet function such as aspirin may affect the results. Results may be helpful for the diagnosis of von Willebrand disease and severe platelet disorders but is not very sensitive or specific [24].

Light Transmission Aggregometry When suspicion for an inherited platelet disorder is high, platelet aggregometry has been used among hematologists given its improved specificity over the PFA-100. Using plasma rich in platelets, light or optical transmission is used to determine platelet aggregation in the presence of several different agonists such as epinephrine, ADP, collagen, ristocetin, and arachidonic acid [26]. Several different platelet disorders can be diagnosed in this manner, as each has a uniquely distinct aggregation pattern in the presence of different agonists [27]. Testing can also be affected by many medications that interact with platelet function, and so careful history and testing preparation is warranted.

Flow Cytometry Flow cytometry is a useful way to identify cell surface receptors. Therefore, it is commonly used to diagnose qualitative platelet disorders. A patient’s fluid sample is combined with specific antibodies to cell surface receptors of interest [25]. Light scatter and fluorescence are measured as the sample passes through lasers [28]. Ultimately, the presence and quantification of cell surface receptors can be made.

Additional Testing Most recently, thromboelastography is being used to look at the entire coagulation process in real time. It allows a hematologist to follow the formation, strength, and duration of the clot, and a certain pattern may allude to a platelet function disorder. Bone

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marrow evaluation, flow cytometry, electron microscopy, and genetic sequencing are additional tests that may be ordered by the hematologist in consultation with the pathologist given a particular pattern of findings. These individual tests will not be discussed here but will be mentioned later in the chapter as it relates to a specific diagnosis.

Inherited Platelet Disorders Clinical diagnosis of inherited platelet disorders will be discussed based on whether they are primarily quantitative or qualitative.

Disorders of Platelet Production Many quantitative disorders are secondary to genetic mutations that cause additional systemic findings. Although neonatal thrombocytopenia is quite common in the ICU setting, thrombocytopenia due to decreased production occurs in less than 5% of infants with thrombocytopenia [11]. Congenital Amegakaryocytic Thrombocytopenia Congenital amegakaryocytic thrombocytopenia (CAMT) is an exceedingly rare diagnosis defined by the lack of megakaryocytes within the bone marrow [29]. Inherited in an autosomal recessive fashion, it is caused by a mutation in the MPO gene, which encodes the thrombopoietin receptor c-Mpl [29] . Without the c-Mpl receptor, megakaryocytes cannot mature and produce platelets in response to TPO stimulation. Thus, TPO levels are often elevated [29]. Platelet counts are usually less than 50 k, and symptoms are related to bleeding. Bone marrow biopsy reveals low to absent number of megakaryocytes. Diagnosis can be made based on clinical signs and symptoms coupled with molecular testing. For reasons that are not well understood, patients will go on to develop bone marrow failure by the time they reach childhood. Additional sequelae include marrow dysplasia and leukemia. Treatment for CAMT is bone marrow transplant, preferably with a matched sibling donor, ideally before complete bone marrow failure occurs [29]. Thrombocytopenia with Absent Radii Thrombocytopenia with absent radii (TAR) is a complex, poorly understood rare genetic disorder defined by low platelet count and absent radii. Diagnosis is based on platelet counts (having severe thrombocytopenia early in life that increases with

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age to ~100 k in toddlerhood and may vary by stress and interestingly ingestion of cow’s milk), clinical features of absent radii, and molecular testing [30, 31]. Typically, absence of radii with the presence of thumbs is the most common bony abnormality, but dysplasia of the ulna, humerus, metacarpals, and shoulder girdle may exist as well as lower extremity deformities [31]. Additional manifestations include but are not limited to cardiac defects, gastrointestinal complications (particularly related to milk protein intolerance), genitourinary anomalies, and transient leukemoid reactions [31]. The inheritance pattern is autosomal recessive and complex, with genetic abnormalities at particular alleles within the RBM8A gene [31]. These genetic abnormalities result in decreased megakaryocytopoiesis and, therefore, results in decreased platelet production that is unresponsive to TPO [30]. Genetic consultation is appropriate given complete penetrance and inheritance risk [31] . Management of TAR is dependent upon disease phenotype and bleeding symptoms. Careful assessment of all possible organic systems should be evaluated, including orthopedic evaluation of all limbs, echocardiography, renal ultrasound and function, and genetic evaluation [31]. Platelet transfusions should be reserved for clinical bleeding that must be controlled. However, treatment of thrombocytopenia with platelet transfusions in the neonatal period is crucial to prevent serious morbidities. Avoidance of cow’s milk protein is suggested to reduce gastroenteritis and decrease risk for thrombocytopenic fluctuation [31] . Platelet counts will be followed over time by the hematologist. Prognosis is excellent, particularly after the newborn thrombocytopenia phase. TAR is frequently misdiagnosed, as it shares similar clinical findings to Fanconi anemia, Holt-Oram syndrome, Roberts syndrome, and VACTERL, for example, owing to radial defects [31]. With clinical and laboratory findings similar to TAR, Fanconi anemia can easily mimic findings of TAR, particularly early in the disease process when only thrombocytopenia may be present [30] . However, patients with Fanconi anemia typically also have missing thumbs among other organic findings [30, 31]. Holt-Oram, Robert syndrome, and VACTERL are not associated with thrombocytopenia. Amegakaryocytic Thrombocytopenia with Radioulnar Synostosis Similar to CAMT and TAR, amegakaryocytic thrombocytopenia with radioulnar synostosis (ATRUS) is a very rare diagnosis characterized by neonatal thrombocytopenia and bilateral radioulnar fusion [32]. Genetic loci involved have not been well described, although autosomal dominant mutations within the HOXA11 gene have been reported [32]. Initial treatment is supportive, although many children will go on to develop myelofibrosis, requiring bone marrow transplant [32].

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Wiskott-Aldrich Syndrome Wiskott-Aldrich syndrome (WAS) comprises the historical triad: immunodeficiency, eczema, and thrombocytopenia [33, 34]. A rare diagnosis with an incidence of 1–4/1,000,000, WAS is inherited in an X-linked fashion, mainly affecting males [35]. More than 300 mutations within the WAS gene have been identified [36]. Thrombocytopenia may be moderate to severe and is typically microcytic [33]. The relationship between these mutations and thrombocytopenia is less understood, but two theories include decreased platelet production and increased splenic platelet destruction secondary to abnormal internal platelet drivers [37, 38]. Clinical presentation is variable and depends on symptoms present at time of visit [35]. A scoring system has been developed to aid in diagnosis and to help distinguish from the closely related X-linked thrombocytopenia diagnosis, discussed later in the chapter [35, 38]. Without treatment, survival is poor, as patients succumb to infection and hematologic malignancy [35]. Supportive measures include infection prevention and management of thrombocytopenia, ultimately requiring a bone marrow transplant for cure [35, 38]. Gene therapy trials are a promising new endeavor, with ongoing trials [39]. X-Linked Thrombocytopenia X-linked thrombocytopenia (XLT) is a rare diagnosis resulting from a specific WAS gene mutations, often missense or splice site, causing a mildly low activity profile, and leading to a more variable phenotype compared to WAS [39]. Typically, patients have microthrombocytopenia [33, 39]. Again, the scoring system used to define WAS and XLT can be helpful but may also change over time, as patients may go on to develop autoimmunity [33, 39]. Conflicting data exists as to whether these patients are at risk for malignancy later in life [33, 39], likely owing to the variable phenotypes observed. Patients are managed with supportive care and rarely require bone marrow transplant [39]. ANKRD26-Related Thrombocytopenia ANKRD26-related thrombocytopenia (ANKRD26-RT) is thought to be one of the more common inherited forms of thrombocytopenia [40]. Inherited in an autosomal dominant fashion, the mutation in the ANKRD26 gene causes a defective protein, leading to abnormal megakaryocytopoiesis, and mild-to-severe normocytic thrombocytopenia [40]. Bleeding symptoms tend to be mild [40]. Through recent years, an increasing number of cases of myeloid malignancy and dysplasia have been reported, which is thought to have variable penetrance [40, 41]. Treatment is supportive, obtaining a bone marrow biopsy if clinically indicated [41].

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ACTN1-Related Thrombocytopenia ACTN1-related thrombocytopenia (ACTN1-RT) is comprised of mutations within the ACTN1 gene that cause altered proplatelet cytoskeleton formation and, thus, thrombocytopenia [42, 43]. Clinical bleeding symptoms are rare, and only mild thrombocytopenia is typically identified [42]. Bone Marrow Failure Syndromes Associated with Thrombocytopenia Important to mention are the bone marrow failure syndromes, as many have pathology that includes thrombocytopenia. These diagnoses include Fanconi anemia, dyskeratosis congenita, Shwachman-Diamond syndrome, and Pearson syndrome, among others. Several of the more common diagnoses will be discussed here. Fanconi Anemia Fanconi anemia is a rare autosomal recessive or X-linked recessive disorder defined by physical anomalies and progressive bone marrow failure [40]. Typical manifestations in the newborn period include but are not limited to low birth weight, cardiac anomalies, skeletal anomalies (i.e., absent or hypoplastic thumbs, radii, flat thenar eminence), triangular facies, and renal anomalies [44]. While many patients with Fanconi anemia present with isolated thrombocytopenia [45], cytopenias are uncommon at birth [44, 46]. There are only case reports of neonatal thrombocytopenia as the presenting manifestation of Fanconi anemia [47]. Typically, the hematologic manifestations of bone marrow failure typically occur in the first decade of life [46]. Patients are at risk for myelodysplastic syndrome, acute myelogenous leukemia, and solid tumors [44, 46]. Hematopoietic stem cell transplant is reserved for patients with severe bone marrow failure, keeping in mind that transplant treats only the hematologic manifestations [46]. Otherwise, thrombocytopenia is treated by way of supportive care with platelet transfusions and androgen therapy [44]. Dyskeratosis Congenita Dyskeratosis congenita is a rare diagnosis with complicated inheritance pattern that causes the well-known triad of reticular pigmentation of the skin, oral leukoplakia, and nail dystrophy [46]. Bone marrow failure typically presents in the adolescent and young adult population, with thrombocytopenia frequently an initial manifestation [48]. It is rare to see hematologic manifestations at birth, but two subtypes of disease, Hoyeraal-Hreidarsson syndrome and Revesz syndrome, can present with neonatal complications including cerebellar hypoplasia, developmental delay, immunodeficiency, enteropathy, retinopathy, and intracranial calcifications [46]. Patients have increased risk for myelodysplastic syndrome, leukemia, pulmonary fibrosis, liver disease, and squamous cell cancers [46]. Once bone marrow failure

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develops, some patients may respond to androgen therapy, and stem cell transplant is reserved for specific cases and treats only the hematologic manifestations [46]. Shwachman-Diamond Syndrome Shwachman-Diamond syndrome is a rare disease inherited in an autosomal recessive fashion, typically causing neutropenia, exocrine pancreatic insufficiency, and skeletal anomalies [46]. Additional cytopenias may occur, even progressing onto bone marrow failure [46]. Treatment is typically supportive, and bone marrow transplant is reserved for special populations [46]. Familial Platelet Disorder with Associated Myeloid Malignancy Familial platelet disorder with associated myeloid malignancy is an autosomal dominantly inherited disorder with mild-to-moderate thrombocytopenia and qualitative platelet disorders [49]. Platelets typically have a dense granule deficiency along with abnormal platelet aggregation studies [49]. The genetic mutation resides within the RUNX1 gene, and the lifetime risk for development of leukemia or myelodysplasia is ~40% [49]. Other autosomal dominantly inherited platelet disorders that are associated with malignancy include the ANKRD26-related thrombocytopenia (previously discussed) and ETV6-related thrombocytopenia [47]. Patients are at risk for development of leukemias and myelodysplasia [49]. The ETV6-­ related thrombocytopenia mutation is also inherited in an autosomal dominant fashion with mild-to-moderate thrombocytopenia with impaired platelet function that may lead to bleeding symptoms [49]. This mutation is identified in ~20% of patients with pre-B acute lymphoblastic leukemia and has also been seen in ~30%v of other hematologic cancers [49]. MYH-9-Related Disorders Formerly known individually as Epstein syndrome, May-Hegglin anomaly, Fechner syndrome, and Sebastian syndrome, these syndromes have been collectively mapped to a mutation in the MYH9 gene, which encodes myosin IIa, a protein expressed in many cells and organs such as the platelets, leukocytes, kidney, and cochlea [50, 51]. Due to the inherited or de novo mutation, the proplatelet is unable to be released from the megakaryocyte. This leads to varying degrees of macrothrombocytopenia, with correlating bleeding symptoms; however, platelet function is preserved [51]. Neutrophil inclusions (a buildup on intracellular myosin II) are also frequently seen in combination with macrothrombocytopenia on peripheral smear [51]. Patients are also at risk for development of hearing loss, nephropathy, cataracts, and transaminitis at various points throughout their life [51]. Treatment of thrombocytopenia is supportive. Careful consideration must be taken, as this group of disorders has been mistaken for immune thrombocytopenia (ITP) and has been mistakenly treated as such [50, 51].

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Pearson Syndrome Pearson syndrome is a rare mitochondrial deletion disorder, causing cytopenias and metabolic disorders [52]. Neonatal onset of thrombocytopenia occurs in ~9% of patients [52]. Treatment is mainly supportive.

Qualitative Platelet Disorders Isolated qualitative platelet disorders are exceedingly rare and limited to just several diagnoses. Qualitative disorders typically present with varying degrees of clinical bleeding, with or without thrombocytopenia. Platelet function testing becomes essential for diagnosis. Bernard-Soulier Bernard-Soulier is a rare bleeding disorder with an incidence of 1 per 1,000,000 [53]. Ultimately, there is impaired assembly of the GP1b-IX-V receptor complex on the surface of the platelet. Typically responsible for von Willebrand factor binding and platelet activation, this receptor complex is crucial to initiation of hemostasis [53]. Clinical manifestations may vary but include mucocutaneous bleeding that can present in infancy, gastrointestinal bleeding, menorrhagia, and surgical bleeding [53]. Routine laboratory tests demonstrate mild-to-severe macrothrombocytopenia, by mechanisms not well understood, although automated analyzers may misinterpret true platelet counts by underestimating platelet number due to larger platelet size [53, 54]. Screening coagulation testing includes platelet function analyzer −100, which is prolonged for both the epinephrine and ADP cartridges [54]. Platelet aggregometry will demonstrate failure to agglutinate to ristocetin despite addition of normal plasma [54]. Flow cytometry reveals decreased expression of the GPIX and the GP1bα, leading to the diagnosis [54]. Molecular analyses of the GP1BA, GP1BB, and GP9 genes have shown various mutations and inheritance patterns in multiple genes, including autosomal recessive and autosomal dominant missense mutations, and are notably more common in offspring of consanguineous unions [53]. Treatment approaches are limited and include platelet transfusions and antifibrinolytics for severe bleeding [54]. DiGeorge Syndrome DiGeorge syndrome is an autosomal dominantly inherited disorder with mutation located at 22q11.21. Clinical manifestations include cardiac, thyroid, and facial anomalies with mild macrothrombocytopenia seen on lab evaluation. This disorder is mentioned here because the genetic mutation responsible creates a dysfunctional

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GP1b-IX-V complex. Therefore, symptoms will be consistent with mild Bernard-­ Soulier, although studies defining bleeding risk are ongoing [55]. Glanzmann Thrombasthenia Glanzmann thrombasthenia is an autosomal recessive inherited defect with an incidence 1 per 1,000,000. It affects the platelet receptor GPIIb/IIIa, which causes failure of platelet aggregation [54]. There are two mechanisms that outline the pathology: (1) a quantitative defect that results in reduced number of platelet cell surface GPIIb/ IIIa receptors (more common) and (2) a qualitative defect that renders the GPIIb/IIIa cell surface receptor nonfunctional. Both cause failure of platelets to bind to fibrinogen and von Willebrand protein, leading to poor platelet aggregation [54, 56]. Patients have mild-to-severe clinical bleeding symptoms including petechiae, mucocutaneous bleeding, bruising, menorrhagia, and trauma-­ related bleeding [54]. Laboratory analysis demonstrates normal platelet count to moderate (macro)thrombocytopenia [54]. PFA-100 is abnormally prolonged for both epinephrine and ADP.  However, platelet aggregometry demonstrates impaired aggregation with every agonist (i.e., ADP, thrombin, epinephrine, arachidonic acid) except ristocetin [54, 56]. Flow cytometry is useful in diagnosing the quantitative defects, as there will be low to absent markers for the GPIIb/IIIa cell surface receptor. To diagnose the qualitative defect, other flow cytometry methods are used to demonstrate lack of platelet aggregation to activated GPIIb/IIIa [54]. Molecular analysis to date has revealed more than 400 specific mutations on 2 genes [56]. Therapy is mainly supportive and includes platelet transfusions, antifibrinolytics, and recombinant activated factor VII for severe bleeding [54, 56]. Hematopoietic stem cell transplant has been performed in select severe cases with good outcome [54, 56]. Granule Defects As mentioned earlier in this chapter, platelets contain three types of granules that are released upon platelet activation so that the recruiting and aggregation of platelets remain localized. The contents released from within the granules are then used for hemostatic signaling. Dense and alpha granule deficiencies will be discussed here and can occur alone, together, or in association with other genetic syndromes. The details of these processes are quite intricate and beyond the scope of this chapter. For now, we will focus on the core concepts. Dense granules are crucial for platelet aggregation as they are the source of ADP signaling and serotonin release for aggregation [12]. Patients tend to show variable bleeding severity [57]. Electron microscopy demonstrates absence or paucity of the granules [57]. Syndromes that can occur with dense granule defects include Hermansky-Pudlak and Chediak-Higashi [12, 57]. Hermansky-Pudlak is an autosomal recessive disorder associated with specific clinical signs including oculocutaneous albinism and bleeding diathesis due to absence of dense granules.

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Chediak-Higashi also demonstrates autosomal recessive inheritance and is characterized by oculocutaneous albinism and immunodeficiency [57, 58]. Alpha granules are responsible for the release of many cellular signaling pathways, including procoagulants and anticoagulants, growth factors, and immune modulation [12]. Gray platelet syndrome is a type of alpha granule deficiency. It is inherited in an autosomal recessive fashion, and patients have macrothrombocytopenia and variable bleeding symptoms ranging from mild to severe [58]. Patients often have myelofibrosis and splenomegaly due, in part, to extramedullary hematopoiesis [58]. Electron microscopy is useful in identification of the presence or absence of alpha granules. Pseudo-von Willebrand Disease Pseudo-von Willebrand disease (pseudo-vWD), also known as “platelet-type von Willebrand disease,” is due to a platelet-specific defect that causes increased affinity of the GP-Ib-IX platelet cell surface receptor complex to the large von Willebrand multimers [59]. This binding leads to platelet activation and subsequent agglutination and platelet consumption. Consequently, patients develop thrombocytopenia and also show decreased high-molecular-weight multimers [59]. This diagnosis should be differentiated from type 2B von Willebrand disease (2B-vWD), as treatment is different depending on the diagnosis [59]. The type 2B-vWD defect resides in the von Willebrand factor protein itself, which causes increased affinity for the GPIb-IX-V platelet receptor complex [60]. Again, the increased binding causes platelet agglutination, potentially resulting in mild thrombocytopenia and decreased large multimers. There are reported methods that are used to guide possible diagnosis, but the gold standard remains genetic testing [59]. Treatment for pseudo-vWD is supportive platelet transfusion during bleeding episodes [59]. Treatment for type 2B-vWD is von Willebrand infusion [59].

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7. Drayer AL, Boer AK, Los EL, Esselink MT, Vellenga E.  Stem cell factor synergistically enhances thrombopoietin-induced STAT5 signaling in megakaryocyte progenitors through JAK2 and src kinase. Stem Cells (Dayton, Ohio). 2005;23(2):240–51. 8. Metcalf D, Di Rago L, Mifsud S. Synergistic and inhibitory interactions in the in vitro control of murine megakaryocyte colony formation. Stem Cells (Dayton, Ohio). 2002;20(6):552–60. 9. Kuter DJ. The biology of thrombopoietin and thrombopoietin receptor agonists. Int J Hematol. 2013;98(1):10–23. 10. Thon JN, Italiano JE. Platelet formation. Semin Hematol. 2010;47(3):220–6. 11. Orkin S, Fisher D, Ginsburg D, Look AT, Lux S, Nathan D. Nathan and Oski’s hematology and oncology of infancy and childhood. 8th ed. Philadelphia: Elsevier Saunders; 2014. 12. Gremmel T, Frelinger AL, Michelson AD.  Platelet physiology. Semin Thromb Hemost. 2016;42(3):191–204. 13. Jackson SR, Carter JM.  Platelet volume: laboratory measurement and clinical application. Blood Rev. 1993;7(2):104–13. 14. Grozovsky R, Hoffmeister KM, Falet H. Novel clearance mechanisms of platelets. Curr Opin Hematol. 2010;17(6):585–9. 15. Quach ME, Chen W, Li R. Mechanisms of platelet clearance and translation to improve platelet storage. Blood. 2018;131(14):1512–21. 16. Estevez B, Du X. New concepts and mechanisms of platelet activation signaling. Physiology (Bethesda, MD). 2017;32(2):162–77. 17. Tesfamariam B.  Distinct characteristics of neonatal platelet reactivity. Pharmacol Res. 2017;123:1–9. 18. Sitaru AG, Holzhauer S, Speer CP, Singer D, Obergfell A, Walter U, Grossmann R. Neonatal platelets from cord blood and peripheral blood. Platelets. 2005;16(3–4):203–10. 19. Mittal N, Naridze R, James P, Shott S, Valentino LA. Utility of a paediatric bleeding questionnaire as a screening tool for von Willebrand disease in apparently healthy children. Haemophilia. 2015;21(6):806–11. 20. Elbatarny M, Mollah S, Grabell J, Bae S, Deforest M, Tuttle A, Hopman W, Clark DS, Mauer AC, Bowman M, et al. Normal range of bleeding scores for the ISTH-BAT: Adult and pediatric data from the merging project. Haemophilia. 2014;20(6):831–5. 21. O’Brien SH. An update on pediatric bleeding disorders: bleeding scores, benign joint hypermobility, and platelet function testing in the evaluation of the child with bleeding symptoms. Am J Hematol. 2012;87(Suppl 1):S40–4. 22. Schmoeller D, Picarelli MM, Paz Munhoz T, Poli DF, Staub HL. Mean platelet volume and immature platelet fraction in autoimmune disorders. Front Med. 2017;4:146. 23. Kundu SK, Heilmann EJ, Sio R, Garcia C, Davidson RM, Ostgaard RA.  Description of an in  vitro platelet function analyzer--PFA-100. Semin Thromb Hemost. 1995;21(Suppl 2):106–12. 24. Dovlatova N, Heptinstall S.  Platelet aggregation measured by single-platelet counting and using PFA-100 devices. Platelets. 2018;29(7):656–61. 25. Kuiper GJAJM, Houben R, Wetzels RJH, Verhezen PWM, Oerle RV, ten Cate H, Henskens YMC, Lancé MD. The use of regression analysis in determining reference intervals for low hematocrit and thrombocyte count in multiple electrode aggregometry and platelet function analyzer 100 testing of platelet function. Platelets. 2017;28(7):668–75. 26. Koltai K, Kesmarky G, Feher G, Tibold A, Toth K. Platelet aggregometry testing: molecular mechanisms, techniques and clinical implications. Int J Mol Sci. 2017;18(8):1803. https://doi. org/10.3390/ijms18081803. 27. Dawood BB, Lowe GC, Lordkipanidze M, Bem D, Daly ME, Makris M, Mumford A, Wilde JT, Watson SP.  Evaluation of participants with suspected heritable platelet function disorders including recommendation and validation of a streamlined agonist panel. Blood. 2012;120(25):5041–9. 28. McKinnon KM. Flow cytometry: an overview. Curr Protoc Immunol. 2018;120, 5.1.1–5.1.11. https://doi.org/10.1002/cpim.40.

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29. Geddis AE. Congenital amegakaryocytic thrombocytopenia and thrombocytopenia with absent radii. Hematol Oncol Clin North Am. 2009;23(2):321–31. 30. Manukjan G, Bosing H, Schmugge M, Strauss G, Schulze H. Impact of genetic variants on haematopoiesis in patients with thrombocytopenia absent radii (TAR) syndrome. Br J Haematol. 2017;179(4):606–17. 31. Toriello H. Thrombocytopenia absent radius syndrome. In: GeneReviews®. Seattle: University of Washington; 2009. 32. Geddis AE. Inherited thrombocytopenias: an approach to diagnosis and management. Int J Lab Hematol. 2013;35(1):14–25. 33. Chandra S, Bronicki L, Nagaraj CB, Zhang K. WAS-related disorders. In: Adam MP, Ardinger HH, Pagon RA, et  al., editors. GeneReviews®. Seattle: University of Washington, Seattle. GeneReviews is a registered trademark of the University of Washington, Seattle. All rights reserved; 1993. CI: Copyright © 1993–2019; GR: HHSN276201400262U/NLM NIH HHS/ United States; OTO: NLM. 34. Shekhovtsova Z, Bonfim C, Ruggeri A, Nichele S, Page K, AlSeraihy A, Barriga F, de Toledo Codina JS, Veys P, Boelens JJ, et  al. A risk factor analysis of outcomes after unrelated cord blood transplantation for children with Wiskott-Aldrich syndrome. Haematologica. 2017;102(6):1112–9. 35. Buchbinder D, Nugent DJ, Fillipovich AH.  Wiskott-Aldrich syndrome: diagnosis, current management, and emerging treatments. Appl Clin Genet. 2014;7:55–66. 36. Rivers E, Thrasher AJ. Wiskott-Aldrich syndrome protein: emerging mechanisms in immunity. Eur J Immunol. 2017;47(11):1857–66. 37. Ochs HD, Filipovich AH, Veys P, Cowan MJ, Kapoor N. Wiskott-Aldrich syndrome: Diagnosis, clinical and laboratory manifestations, and treatment. Biol Blood Marrow Transplant. 2009;15(1, Supplement):84–90. 38. Massaad MJ, Ramesh N, Geha RS. Wiskott-Aldrich syndrome: a comprehensive review. Ann N Y Acad Sci. 2013;1285:26–43. 39. Rivers E, Worth A, Thrasher AJ, Burns SO. How I manage patients with Wiskott Aldrich syndrome. Br J Haematol. 2019;185(4):647–55. 40. Balduini A, Raslova H, Di Buduo CA, Donada A, Ballmaier M, Germeshausen M, Balduini CL.  Clinic, pathogenic mechanisms and drug testing of two inherited thrombocytopenias, ANKRD26-related thrombocytopenia and MYH9-related diseases. Eur J Med Genet. 2018;61(11):715–22. 41. Perez Botero J, Dugan SN, Anderson MW.  ANKRD26-related thrombocytopenia. In: Adam MP, Ardinger HH, Pagon RA, et  al., editors. GeneReviews®. Seattle: University of Washington, Seattle. GeneReviews is a registered trademark of the University of Washington, Seattle. All rights reserved; 1993. CI: Copyright © 199–2019; GR: HHSN276201400262U/ NLM NIH HHS/United States; OTO: NLM. 42. Faleschini M, Melazzini F, Marconi C, Giangregorio T, Pippucci T, Cigalini E, Pecci A, Bottega R, Ramenghi U, Siitonen T, et al. ACTN1 mutations lead to a benign form of platelet macrocytosis not always associated with thrombocytopenia. Br J Haematol. 2018;183(2):276–88. 43. Kunishima S, Okuno Y, Yoshida K, Shiraishi Y, Sanada M, Muramatsu H, Chiba K, Tanaka H, Miyazaki K, Sakai M, et al. ACTN1 mutations cause congenital macrothrombocytopenia. Am J Hum Genet. 2013;92(3):431–8. 44. Savage SA, Walsh MF. Myelodysplastic syndrome, acute myeloid leukemia, and cancer surveillance in Fanconi anemia. Hematol Oncol Clin North Am. 2018;32(4):657–68. 45. Butturini A, Gale RP, Verlander PC, Adler-Brecher B, Gillio AP, Auerbach AD. Hematologic abnormalities in Fanconi anemia: an International Fanconi Anemia Registry study. Blood. 1994;84(5):1650–5. 46. Khincha PP, Savage SA. Neonatal manifestations of inherited bone marrow failure syndromes. Semin Fetal Neonatal Med. 2016;21(1):57–65. 47. Landmann E, Bluetters-Sawatzki R, Schindler D, Gortner L. Fanconi anemia in a neonate with pancytopenia. J Pediatr. 2004;145(1):125–7. 48. Dokal I. Dyskeratosis congenita. Hematology. 2011;2011(1):480–6.

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49. Galera P, Dulau-Florea A, Calvo KR. Inherited thrombocytopenia and platelet disorders with germline predisposition to myeloid neoplasia. Int J Lab Hematol. 2019;41(Suppl 1):131–41. https://doi.org/10.1111/ijlh.12999. 50. Fernandez-Prado R, Carriazo-Julio SM, Torra R, Ortiz A, Perez-Gomez MV. MYH9-related disease: it does exist, may be more frequent than you think and requires specific therapy. Clin Kidney J. 2019;12(4):488–93. 51. Pecci A, Ma X, Savoia A, Adelstein RS. MYH9: structure, functions and role of non-muscle myosin IIA in human disease. Gene. 2018;664:152–67. 52. Manea EM, Leverger G, Bellmann F, Stanescu PA, Mircea A, Lèbre AS, Rötig A, Munnich A. Pearson syndrome in the neonatal period: two case reports and review of the literature. J Pediatr Hematol/Oncol. 2009;31(12):947–51. 53. Boeckelmann D, Hengartner H, Greinacher A, Nowak-Gottl U, Sachs UJ, Peter K, Sandrock-­ Lang K, Zieger B. Patients with Bernard-Soulier syndrome and different severity of the bleeding phenotype. Blood Cells Mol Dis. 2017;67:69–74. 54. Grainger JD, Thachil J, Will AM.  How we treat the platelet glycoprotein defects; glanzmann thrombasthenia and bernard soulier syndrome in children and adults. Br J Haematol. 2018;182(5):621–32. 55. Lambert MP, Arulselvan A, Schott A, Markham SJ, Crowley TB, Zackai EH, McDonald-­ McGinn DM. The 22q11.2 deletion syndrome: Cancer predisposition, platelet abnormalities and cytopenias. Am J Med Genet A. 2018;176(10):2121–7. 56. Poon MC, Di Minno G, d’Oiron R, Zotz R.  New insights into the treatment of glanzmann thrombasthenia. Transfus Med Rev. 2016;30(2):92–9. 57. Ambrosio AL, Di Pietro S. Storage pool diseases illuminate platelet dense granule biogenesis. Platelets. 2017;28(2):138–46. 58. Chen CH, Lo RW, Urban D, Pluthero FG, Kahr WH. Alpha-granule biogenesis: from disease to discovery. Platelets. 2017;28(2):147–54. 59. Enayat MS, Guilliatt AM, Lester W, Wilde JT, Williams MD, Hill FG. Distinguishing between type 2B and pseudo-von Willebrand disease and its clinical importance. Br J Haematol. 2006;133(6):664–6. 60. Kroner P, Kluessendorf M, Scott J, Montgomery R. Expressed full-length von Willebrand factor containing missense mutations linked to type IIB von Willebrand disease shows enhanced binding to platelets. Blood. 1992;79:2048–55.

Chapter 11

Platelet Disorders Katherine Regling and Meera Chitlur

Introduction Platelets were first described by Max Schultze in 1865; however, it was Giulio Bizzozero who demonstrated that they were the first component of blood to adhere to damaged blood vessel walls in vivo and in vitro in 1882 [1]. Visually, platelets are small, disc-shaped, anuclear, cellular fragments that range in concentration from 150,000 to 300,000/mm3 in healthy individuals [2]. The platelets’ primary role is to maintain hemostasis by adhering to the site of vascular injury and forming a platelet plug upon which coagulation factors are assembled to generate thrombin. The platelet also secretes cytokines/chemokines and possesses many surface receptors involved in inflammatory and immune responses. Here we focus on the hemostatic effects of platelets and the disorders of hemostasis resulting from platelet dysfunction. Classification of platelet disorders is often complex but can be divided into quantitative versus qualitative abnormalities. They can also be classified into congenital (inherited) or acquired disorders (Fig. 11.1).

Clinical Features of Platelet Disorders Congenital or acquired platelet disorders can result in a bleeding diathesis of varying severity. Typically, patients manifest with mild-to-moderate mucocutaneous bleeding, but severe bleeding can be seen in inherited platelet disorders such as Glanzmann thrombasthenia (GT). Both males and females are equally affected, K. Regling (*) · M. Chitlur Carman and Ann Adams Department of Pediatrics, Division of Hematology/Oncology, Children’s Hospital of Michigan/Central Michigan University, Detroit, MI, USA e-mail: [email protected]; [email protected] © Springer Nature Switzerland AG 2021 D. M. Kamat, M. Frei-Jones (eds.), Benign Hematologic Disorders in Children, https://doi.org/10.1007/978-3-030-49980-8_11

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Valproic acid

suppression

Chemotheray-related bone marrow

Essential thrombocythemia

Production Defects

Others

Infection

Kasabach-Merritt syndrome

Thrombotic microangiopathy

Combined alpha/dense granule defects

Gray Platelet syndrome

Chediak-Higashi syndrome

Hermansky-Pudlak syndrome

Platelet Granule Defects

Thromboxane A2 deficiency

Phospholpase A2 deficiency

Drug-induced

Impaired cyclooxygenase enzyme activity

NAIT

Impaired arachidonic acid metabolism

Signal Transuction Defects

Scott syndrome

Glycoprotein VI deficiency

Others

Purpura Fulminans

Systemic Lupus Erythematosus

Henoch-Schӧnlein Purpura

Systemic Disease

Others

Platelet-type von Willebrand disease G protein-coupled receptor defects

Dipyridamole

Aspirin

Drug-Induced

Acquired Disorders

Glanzmann Thrombasthenia

Bernard-Soulier syndrome

Receptor Defects

Congenital Disorders

Normal platelet count

Lymphoproliferative/Autoimmune

ITP

Increased Destruction

Burns

Hypothermia

Hypersplenism

Sequestration

EDTA-dependent antibodies

Laboratory artifact

Spurious

Acquired Disorders

Fig. 11.1  Diagnostic approach for the most common platelet disorders

MYH9 disorders

Jacobsen syndrome

Wiskott-Aldrich Syndrome

Others

TAR syndrome

CAMT

Production Defects

Congenital Disorders

Thrombocytopenia

Positive Bleeding History

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with common symptoms including epistaxis, ecchymosis, excessive bleeding from minor cuts and abrasions, gingival bleeding, postoperative bleeding, GI bleeding, or rarely hemarthrosis. Heavy menstrual bleeding is a common presenting symptom seen in 68% of females with platelet disorders [3]. While life-threatening GI bleeding or intracranial hemorrhage (ICH) are uncommon, they can occur in both disorders of platelet number, such as immune thrombocytopenic purpura (ITP), as well as disorders of platelet function such as GT [4, 5].

General Evaluation for Platelet Dysfunction History  A comprehensive history should include personal bleeding symptoms, family history of bleeding symptoms or disorders, medication history, and surgical and dental procedure history, as a part of the evaluation for platelet dysfunction. Medication history is important because common over-the-counter medications such as nonsteroidal anti-inflammatory drugs and herbal remedies can be associated with platelet dysfunction. A detailed family history is important as some familial thrombocytopenias are associated with an increased predisposition to myeloid malignancies and as such should be appropriately managed. The timing of symptom onset is variable. Severe inherited platelet dysfunction may manifest soon after birth with umbilical stump bleeding, cephalohematoma, or bleeding from circumcision, while classical, acute ITP often presents in young children with mucosal bleeding. Bleeding Assessment and Screening  Since bleeding history is often very subjective, standardized bleeding questionnaires and bleeding scores have been developed and found to be useful to determine the extent of bleeding symptoms in patients with platelet quantitative and functional disorders [6–8]. Laboratory Work-Up  Complete blood counts, coagulation assays (prothrombin time and partial thromboplastin time), and fibrinogen should be obtained to rule out common coagulation disorders. Testing for von Willebrand disease (VWD) may be necessary as it is also associated with mucocutaneous bleeding. Automated Platelet Count and Size  Automated hematology analyzers can provide a platelet count rapidly to screen for quantitative abnormalities. The platelet count can be affected by other factors such as platelet activation during sample collection. Ethylenediaminetetraacetic acid (EDTA)-dependent in vitro platelet clumping may result in pseudothrombocytopenia that can be avoided by utilizing sodium citrate or acid citrate dextrose tubes for testing [9, 10]. Normal mean platelet volume (MPV) ranges between 5 and 11 fL in most laboratories. Small platelet size (MPV 11 fL) can been seen in disorders such as ITP and Bernard-Soulier syndrome (BSS). Large platelets may be miscounted as another cell type such as red blood cells due to size causing pseudothrombocytopenia due to misclassification.

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Light Microscopy and Peripheral Smear Review  Manual review of the peripheral blood smear (Fig. 11.1) is essential to determine platelet morphology as most automated analyzers can detect excessively large or small platelets but are unable to detect granular defects. Platelet clumping may be suggestive of pseudothrombocytopenia. Platelets that appear pale or agranular may be a sign of gray platelet syndrome (GPS), and the presence of intracytoplasmic inclusions such as Döhle bodies in neutrophils are diagnostic of myosin heavy-chain 9 (MYH9)-related disorders. Decreased platelet numbers in addition to fragmented red blood cells (RBC) are representative of microangiopathic hemolysis as seen in hemolytic uremic syndrome (HUS), thrombotic thrombocytopenic purpura (TTP), or disseminated intravascular coagulation (DIC). Electron Microscopy  Defects in platelet alpha or delta granules (storage pool disorders) can only be assessed by platelet light transmission electron microscopy where the ultrastructure is visualized by either whole-mount or thin section preparation. Examples of disorders with dense granule deficiency include Hermansky-­ Pudlak syndrome (HPS), Chediak-Higashi syndrome (CHS), WAS, and thrombocytopenia with absent radii syndrome (TAR). Abnormal cytoplasmic inclusions in leucocytes are characteristic of CHS. Disorders of α-granule deficiency are rarer and include GPS and X-linked GATA1 macrothrombocytopenia. Platelet Function Analysis  Platelet function analysis by PFA-100® (Siemens, Tarrytown, NY) was designed as a replacement for the bleeding time which is now universally considered obsolete. The PFA-100® is useful in screening for severe platelet function disorders such as GT but has poor sensitivity for mild platelet function disorders. The PFA-100® is also commonly used to measure efficacy of antiplatelet therapy like aspirin. Platelet Aggregation and Secretion Study  Platelet aggregation assays allow evaluation of platelet function using different agonists at various concentrations that target specific receptors on the cell membrane surface. Commonly used agonists include collagen, adenosine diphosphate (ADP), epinephrine, arachidonic acid (AA), ristocetin, and thrombin receptor activation peptide. The two methods currently available are light transmission aggregometry (LTA) and whole blood aggregometry (WBA). While LTA is considered the gold standard of platelet function testing [11, 12], the need for large volumes of blood makes this an extremely difficult test to perform in small children. The advantages of WBA include smaller blood volume requirement, the ability to measure granular secretion, and platelet function testing in the presence of other blood components that more closely mimics the in vivo environment [12, 13]. Flow Cytometric Assessment of Surface Receptors and Activation  Platelet flow cytometry is useful to assess hereditary platelet disorders of glycoprotein (GP) deficiencies. Markedly decreased GPIIb and GPIIIa expression levels are diagnostic for GT, while BSS is characterized by markedly decreased GPIX and GPIbα expression [12].

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Genetic Analysis  Genetic testing allows for accurate diagnosis of subtle or nonspecific platelet abnormalities. Single-gene tests and platelet disorder gene panels based on technologies such as whole-exome sequencing and next-generation sequencing are available commercially and are commonly used for evaluation of disorders such as HPS, congenital amegakaryocytic thrombocytopenia (CAMT), and WAS.

Qualitative Disorders Inherited Platelet Disorders Receptor Defects Bernard-Soulier Syndrome BSS is an autosomal recessive platelet receptor defect characterized by a prolonged bleeding time/PFA-100® closure time and macrothrombocytopenia [14]. The frequency of BSS has been reported at 1:106 with approximately 1:500 individuals being carriers for BSS. Heterozygosity for BSS is associated with DiGeorge syndrome and benign Mediterranean macrothrombocytopenia [15]. In BSS, platelets exhibit defective adhesion to the sub-endothelium due to mutations of the von Willebrand receptor, known as the GPIb-IX-V complex, on the platelet membrane. In normal individuals, the GPIb-IX-V complex binds with von Willebrand factor (VWF) and allows for further platelet activation and aggregation to occur [15]. In vitro, BSS platelets show normal platelet aggregation with ADP, adrenaline, collagen, and AA with a delayed response to thrombin and absent aggregation with ristocetin [2, 16]. Patients with BSS show adequate numbers of megakaryocytes in the bone marrow, but show evidence of dysmegakaryopoiesis, which has been hypothesized to contribute to the macrothrombocytopenia in BSS [17]. Clinically, individuals with BSS report a history of mild-to-moderate mucosal bleeding; however, they may experience significant hemorrhage after traumatic events. Females may report severe heavy menstrual bleeding. Glanzmann Thrombasthenia GT is an autosomal recessive bleeding disorder associated with severe platelet dysfunction with normal platelet counts and morphology associated with failure of the platelets to bind fibrinogen due to a deficiency of the platelet fibrinogen receptor αIIb-β3 resulting in defective aggregation [2, 18]. The genes for αIIb and B3 are located on chromosome 17. Platelet aggregometry is diagnostic and shows absence of response to ADP, epinephrine, and collagen but a normal response to ristocetin.

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Although rare worldwide, it is found at higher incidences among certain consanguineous relationships including Arab populations [19, 20], Iraqi Jews [21], French gypsies [22], and individuals from southern India [23]. Clinically, the disorder is characterized by repetitive, and sometimes severe, mucocutaneous bleeding from a young age, with epistaxis and gastrointestinal bleeding being the most common. Frequently, young children require iron supplementation due to chronic blood loss. Unprovoked intracranial and gastrointestinal bleeding can occur and contributes to the 5–10% lifelong mortality rate [2]. These patients may also experience larger hemorrhages (i.e., joint or muscle hematomas) which is more characteristic of hemophilia. Platelet-Type von Willebrand Disease (Pseudo-von Willebrand Disease) Platelet-type (pseudo-) VWD is an autosomal dominant disorder characterized by mild-to-moderate bleeding mucocutaneous bleeding. Contrary to type 2B VWD, pseudo-VWD is caused by a change in the platelet GPIb/IX complex that leads to increased affinity for normal VWF multimers resulting in platelet clumping and clearance and subsequent mild thrombocytopenia. Platelet Granule Defects Platelets contain several different types of granules, including dense granules, alpha granules, and lysosomes whose contents are essential for platelet-platelet and platelet-­endothelial interactions (Fig. 11.2). Defects in release or secretion of these granular contents are associated with a mild bleeding phenotype. Dense Granule Defects The two most common dense granule deficiency disorders are HPS and CHS. HPS is characterized by oculocutaneous albinism that is caused by mutations in the HPS-1 to HPS-9 genes which encode for proteins involved in the biogenesis of lysosome-related organelles, including the melanosomes in melanocytes and the delta granules in platelets. The disease is inherited as an autosomal recessive disorder with the highest incidence in those from Puerto Rico, although other small populations exist in Switzerland, Japan, and Ireland [24, 25]. Clinically, patients have photophobia, rotatory nystagmus, decreased visual acuity, and a mild-to-moderate bleeding diathesis. The accumulation of the ceroid-like proteins may induce progressive pulmonary fibrosis and granulomatous colitis. The pulmonary fibrosis generally begins in early adulthood and is the major cause of mortality in this population. CHS is an extremely rare autosomal disorder that is characterized by immunodeficiency, dense granule defects, and partial albinism. Patients typically have a white

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forelock or light hair secondary to abnormally large melanosomes. The white blood cells show defective chemotaxis and decreased bactericidal activity. Because of this, many patients die within the first two decades of life due to severe infections, acute bleeding, or the development of hemophagocytic lymphohistiocytosis (HLH); those who survive into adulthood often have significant neurologic dysfunction [26, 27]. HLH occurs in approximately 85% of patients with CHS [26]. The disorder is caused by mutations on the lysosomal trafficking regulator (LYST) gene on chromosome 1q42.1-42.2 [28]. Alpha-Granule Defects Patients with alpha-granule deficiencies have similar bleeding phenotypes to those with dense granule deficiency and associated macrothrombocytopenia with prolonged bleeding time. The disorder was first named GPS, as the platelets appear gray on peripheral blood smears, in 1971 by Raccuglia [29]. Platelet aggregometry shows significant impairment in response to thrombin with variable responses to other agonists including ADP, collagen, AA, and ristocetin [30]. Other Rare Platelet Disorders Other disorders of defects in platelet granules (combined alpha-granule and dense granule defects), receptors (GPVI, G protein-coupled receptor), and signal transduction [thromboxane X2 (TXA2) and cyclooxygenase] are described which are rare and often associated with mild bleeding manifestations.

Alpha Granules Vacuoles and Tubular Networks Dense Granule Glycogen

Mitochondria

Fig. 11.2  Platelet ultrastructure by electron microscopy. (Photo courtesy of Deepti M.  Warad, M.B.B.S.; Mayo Clinic, Rochester, MN 55905)

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Acquired Platelet Disorders Drug-Induced Platelet Dysfunction Many drugs have been shown to inhibit platelet function; however, in most patients, this inhibition does not give rise to clinical symptoms or require treatment but can cause bleeding in those with an underlying bleeding diathesis. The mechanism by which the platelets become dysfunctional is dependent on which medication is used. For example, aspirin inhibits production of TXA2 through the cyclooxygenase pathway [31], whereas dipyridamole is a vasodilatory medication that interferes with platelet function by increasing the intracellular concentration of cyclic adenosine monophosphate [32]. Systemic Disease Patients with severe systemic diseases that induce renal insufficiency with uremia or liver disease are at significantly increased risk of platelet dysfunction. Bleeding symptoms can include petechiae, purpura, gastrointestinal hemorrhage, and others. Henoch-Schӧnlein Purpura is a transient, systemic vasculitic disease of school-­ aged children that affects the capillaries of the skin, synovial membranes, renal mesangium, and small intestines. Generally, an acute febrile illness occurs followed by raised purpuric papules over the lower extremities and buttocks which resolves in 6 weeks. Treatment is generally symptomatic care, but glucocorticoids have been shown to improve the skin, joint, and gastrointestinal symptoms if severe. Systemic lupus erythematosus can often present with isolated thrombocytopenia which is immune-mediated. Symptoms and management are similar to ITP.

Quantitative Disorders Inherited Disorders Disorders of Platelet Production Congenital Amegakaryocytic Thrombocytopenia Congenital amegakaryocytic thrombocytopenia (CAMT) is characterized by severe thrombocytopenia, absent or near absence of megakaryocytes in the bone marrow, and an increased risk for the development of bone marrow failure. Eventual progression to pancytopenia occurs in late childhood. Many patients have severe bleeding that does not improve with age, thus necessitating the need for bone marrow transplantation. The disorder is due to mutations of the thrombopoietin (TPO) receptor (Mpl) [33].

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Thrombocytopenia with Absent Radii Syndrome TAR syndrome is a rare inherited or de novo deletion of chromosome 1q21.1 in the majority of affected patients [34]. It is generally inherited in an autosomal recessive fashion but may be autosomal dominant with variable expressivity. Affected patients have a hypomegakaryocytic thrombocytopenia and bilateral absent radii. Approximately, 50% of patients have other skeletal abnormalities of the upper and lower limbs including ulna and/or humeral hypoplasia, phocomelia, hip dysplasia, patellar subluxation, and bowed legs, 25% have renal anomalies, and 15% have cardiac anomalies [35]. Thrombocytopenia is more severe in the neonatal period and can be associated with GI or intracranial hemorrhage but gradually improves throughout the first year of life. Cow’s milk allergy is common in TAR and worsens the degree of thrombocytopenia. Bone marrow evaluation shows decreased number of megakaryocytes with an elevated serum TPO and normal Mpl levels on the hematopoietic cells, suggesting lapse in communication between the TPO receptor and the downstream signaling events [34]. Other Inherited Disorders Wiskott-Aldrich Syndrome WAS is an inherited microthrombocytopenia with eczema and a severe combined immunodeficiency. The defect is located on the X-chromosome of the WASP (Wiskott-Aldrich syndrome protein) gene at Xp11.22 [36]. The WASP protein is heavily involved in actin polymerization, regulation of the cytoskeleton, signal transduction, and apoptosis of the lymphocyte and megakaryocyte cell lines. The immunodeficiency arises from the inability to form antibodies to carbohydrate antigens, as well as defects in their B and T lymphocytes [36, 37]. The platelets are small in size and can be less than 10,000 platelets/uL. Patients can be cured with allogeneic hematopoietic stem cell transplantation (HSCT), and more recently gene therapy trials are being conducted as a curative option [38, 39]. X-linked thrombocytopenia is a WAS-related disorder due to mutations in the WAS gene that causes production of an altered protein. X-linked thrombocytopenia is not associated with immunodeficiency but often still has profound thrombocytopenia. Jacobsen Syndrome Jacobsen syndrome (JS) is a genetic disease involving partial deletions of the long arm of chromosome 11. Patients typically have physical and developmental growth delays, dysmorphic facies, and thrombocytopenia or pancytopenia. More than 80% of patients with JS are affected by Paris-Trousseau syndrome, which is

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characterized by neonatal thrombocytopenia that may resolve as the child ages, but with persistent platelet dysfunction [40, 41]. Peripheral smear shows giant platelets and giant α-granules and increased micromegakaryocytes on bone marrow evaluation [41]. Myosin Heavy-Chain 9-Related Thrombocytopenia MYH9 disorders are a group of macrothrombocytopenias associated with a normal coagulation profile and leukocytes containing large, spindle-shaped inclusions (Dӧhle bodies). The MYH9 gene is located on chromosome 22, and mutations are inherited in an autosomal dominant fashion, although up to 35% of cases are sporadic [42, 43]. These patients may also be affected by sensorineural hearing loss, nephritis, and ocular anomalies. Both bleeding symptoms and degree of thrombocytopenia are variable, with most patients having mild-to-moderate symptoms (i.e., bruising, epistaxis, and heavy menstrual bleeding).

Acquired Platelet Disorders Spurious Thrombocytopenia Laboratory artifact is a common cause of deceptive thrombocytopenia, often due to platelet activation during blood collection, platelet clumping due to agglutinated platelets, adherence of platelets to leukocytes forming platelet “satellites,” and pseudothrombocytopenia due to in  vitro agglutination by EDTA-dependent antibodies [10]. Pseudothrombocytopenia is due to EDTA-dependent IgM (sometimes IgG) antibodies. This phenomenon can be confirmed by assessing the platelet count using a different anticoagulant such as citrate, oxalate, or heparin [10]. Thrombocytopenia Caused by Sequestration Hypersplenism The spleen is a lymphatic organ which plays an important role in the filtering of RBCs and general immune function. A healthy human spleen contains less than 2% of the RBC mass and about a third of the body’s platelets [44]. Under certain disease states causing splenomegaly, it has been demonstrated that the fraction of platelets sequestered in the spleen increases in proportion to the spleen size [44]. Generally, the thrombocytopenia is mild, and usually does not require any clinical interventions.

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Hypothermia Thrombocytopenia has been shown to occur when body temperatures drop below 25° Celsius in both experimental animal and human studies [45, 46]. Thrombocytopenia is a well-recognized entity in neonates with hypoxic ischemic encephalopathy who undergo therapeutic hypothermia; this drop in platelet count is variable, and the platelets return to circulation as rewarming takes place [47]. Thrombocytopenia Secondary to Increased Platelet Destruction Immune Thrombocytopenic Purpura ITP is an acquired autoimmune disorder characterized by low circulating platelet count of 1192 × 109/L (maximum 4500 × 109/L) [35]. Approximately two thirds of children with pET have platelet counts >1000 × 109/L [39]. In the 206 patients in whom full driver mutation analysis was performed, 31% were positive for JAK2 V617F, 10% for CALR, and 2% for MPL with 57% of the cases being triple-negative. These are key biologic differences compared to aET, in whom there is a much lower frequency of triple-negative cases and higher percentages of JAK2- or CALR-positive cases [40]. The altered frequency of driver mutations likely influences clinical phenotype and propensity for thrombotic and fibrotic transformation in pET, illustrating the need for long-term observational cohort studies.

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Splenomegaly and hepatomegaly have been reported in 50% and 25% of pET, respectively [3, 35]. Similar to aET, 30% of pET have had thromboembolic or hemorrhagic complications at the time of diagnosis or later [3, 35]. Unusual thromboses involving the splanchnic veins, cerebral veins may be a presenting symptom. Bleeding tendency in ET may be due to multiple etiologies including qualitative platelet defects, [41] and acquired Von Willebrand syndrome, particularly for patients with sustained extreme thrombocytosis [42].

Natural History Compared with other MPNs, aET has a more indolent course, less severe splenomegaly and a life span that approaches that of normal individuals [43]. In aET, the major cause of morbidity and shortened survival are thrombosis and evolution to myelofibrosis with rare progression to AML, yet the incidence of these events is believed to be much lower in pET. In a cohort of 50 children with pET and HT (n = 34 and 16, respectively) with a median follow-up 128 months, 5% of patients developed a thrombotic event, 0% developed leukemia, and 14% of gestations had first trimester miscarriage (similar to rate in general population) [27]. These complications were not influenced by JAK2 mutational status or additional thrombophilic abnormalities; however patients received varied treatment approaches that may have affected risk. The optimal treatment strategy to prevent complications in pET is unknown, and the lower rates of complications and thrombosis suggest that a more conservative approach may be warranted in pET. Longer observation periods are needed to define lifetime risk for evolution to acute leukemia or myelofibrosis [27].

Workup for Patients with Thrombocytosis When reactive thrombocytosis is encountered, it is important to obtain a detailed personal and family history, exclude inflammation and iron deficiency, and obtain platelet counts over several months. See Fig. 12.1 for a diagnostic algorithm. For suspected HT, serum TPO, sequencing TPO, MPL, JAK2 genes, as well as family testing. TPO is elevated in certain hereditary causes [24, 44]. For suspected ET, bone marrow biopsy and histology are needed. The most challenging problem is to understand if a child with prolonged, but not secondary thrombocytosis really has pET [45]. Diagnostic workup requires molecular and histological studies, and histological review by an expert pathologist in pediatric MPNs is recommended.

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Platelet count >450 x109/L

Check acute phase reactants (ESR, CRP, fibrinogen)

Elevated

Normal

Reactive thrombocytosis

Repeat CBC in 3-4 weeks

Repeat CBC 3-4 weeks

Persistent elevated platelets >450

Iron studies

Iron deficiency →treat

Repeat CBC on iron repletion

Secondary causes of thrombocytosis ruled out

Negative

Monitor for possibility of MPN, consider additional genetic testing

Genetic testing for BCR-ABL, JAK2 V617F, MPLW515K/L EPO & TPO levels Obtain family history/CBC

Bone marrow aspiration and biopsy

Positive

Megakaryocyte hyperplasia, pleomorphism, hyperlobation

MPN essential vs hereditary thrombocytosis if other MPN excluded. If FH+, TPO elevated or genetic testing negative consider additional testing for CALR, MPL and THPO

Fig. 12.1 Suggested algorithm for workup of pediatric thrombocytosis. (Adapted from Kucine et al.)

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Treatment As additional long-term data emerges regarding low rates of complications in pET, a more conservative approach has been suggested by expert hematologists [1, 45]. For asymptomatic children, a “watch and wait” approach has been recommended. Patients with low-risk symptoms of headaches, hepatosplenomegaly, additional thrombophilia, or cardiac risk factors are recommended to start low-dose aspirin [1, 45]. Treatment of primary thrombocytosis is not recommended if platelet counts are less than 1500/nL, and there is no history of bleeding or thrombosis [3]. Patients with history of VTE need anticoagulation. Cytoreductive therapies should be reserved for severe cases, including patients who have failed low-risk therapy, have history of thrombosis or severe bleeding, or have persistent extreme thrombocytosis. Options for cytoreductive therapy include hydroxyurea, interferon, and anagrelide [1, 45]. Ruxolitinib is a JAK1/2 inhibitor approved for adults with PV, PMF, and ET. The phase II study showed that ruxolitinib was non-inferior to current second-line treatments for aET, significantly improved some disease-related symptoms, but rates of thrombosis, hemorrhage, or transformation were not different [46]. Ruxolitinib has been studied in children with relapsed malignancies, and graft-versus-host disease, but limited data exists regarding efficacy and in JAK2 V617F pET [47]. Thrombocytopheresis has been used to emergently reduce platelet counts in adults with acute severe thrombotic or hemorrhagic complications, but data regarding safety and utility is scarce in pediatrics [48]. Treatment for secondary thrombocytosis is not recommended, even in extreme thrombocytosis. Concern for portal and splanchnic vein thrombosis postoperatively has led to initiation of cytoreductive therapy as standard of care in TPIAT [19]; however there are no current guidelines to support routine use of anticoagulation postsplenectomy in children.

Summary Elevated platelet count in pediatrics is a common incidental finding in hospitalized and outpatient children, and the majority of cases are reactive and transient in nature. When evaluating a child with thrombocytosis, the most challenging problem for pediatricians is to understand if a child with prolonged and apparently not reactive thrombocytosis may really have ET [45]. Nonreactive thrombocytosis due to HT or pET is rare, but the classification does not fully adapt to adult criteria. Children with ET are expected to have long-life expectancy, and treatment in these children must be effective, safe, and well-tolerated in the long term.

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References 1. Kucine N, Chastain KM, Mahler MB, Bussel JB.  Primary thrombocytosis in children. Haematologica. 2014;99:620–8. https://doi.org/10.3324/haematol.2013.092684. 2. Sutor AH.  Thrombocytosis. In: Pediatric hematology. London, Edinburgh, New  York, Philadelphia, Sydney, Toronto: Churchill Livingstone; 1999. p. 455–64. 3. Dame C, Sutor AH.  Primary and secondary thrombocytosis in childhood. Br J Haematol. 2005;129:165–77. https://doi.org/10.1111/j.1365-2141.2004.05329.x. 4. Chiarello P, Magnolia M, Rubino M, et  al. Thrombocytosis in children. Minerva Pediatr. 2011;63:507–13. 5. Heath HW, Pearson HA. Thrombocytosis in pediatric outpatients. J Pediatr. 1989;114:805–7. https://doi.org/10.1016/s0022-3476(89)80141-6. 6. Kaushansky K, Lok S, Holly RD, et  al. Promotion of megakaryocyte progenitor expansion and differentiation by the c-Mpl ligand thrombopoietin. Nature. 1994;369:568–71. https://doi. org/10.1038/369568a0. 7. Zeigler FC, de Sauvage F, Widmer HR, et  al. In vitro megakaryocytopoietic and thrombopoietic activity of c-mpl ligand (TPO) on purified murine hematopoietic stem cells. Blood. 1994;84:4045–52. 8. Ghilardi N, Wiestner A, Kikuchi M, et al. Hereditary thrombocythaemia in a Japanese family is caused by a novel point mutation in the thrombopoietin gene. Br J Haematol. 1999;107:310–6. https://doi.org/10.1046/j.1365-2141.1999.01710.x. 9. Wiestner A, Schlemper RJ, van der Maas AP, Skoda RC. An activating splice donor mutation in the thrombopoietin gene causes hereditary thrombocythaemia. Nat Genet. 1998;18:49–52. https://doi.org/10.1038/ng0198-49. 10. Wolber EM, Fandrey J, Frackowski U, Jelkmann W.  Hepatic thrombopoietin mRNA is increased in acute inflammation. Thromb Haemost. 2001;86:1421–4. 11. Ishiguro A, Suzuki Y, Mito M, et  al. Elevation of serum thrombopoietin precedes thrombocytosis in acute infections. Br J Haematol. 2002;116:612–8. https://doi. org/10.1046/j.0007-1048.2001.03304.x. 12. Chan KW, Kaikov Y, Wadsworth LD. Thrombocytosis in childhood: a survey of 94 patients. Pediatrics. 1989;84:1064–7. 13. Matsubara K, Fukaya T, Nigami H, et  al. Age-dependent changes in the incidence and etiology of childhood thrombocytosis. Acta Haematol. 2004;111:132–7. https://doi. org/10.1159/000076520. 14. Ellaurie M. Thrombocytosis in pediatric HIV infection. Clin Pediatr (Phila). 2004;43:627–9. https://doi.org/10.1177/000992280404300707. 15. Xavier-Ferrucio J, Scanlon V, Li X, et al. Low iron promotes megakaryocytic commitment of megakaryocytic-erythroid progenitors in humans and mice. Blood. 2019;134:1547–57. https:// doi.org/10.1182/blood.2019002039. 16. Del Rey Hurtado de Mendoza B, Esponera CB, Izquierdo Renau M, Iglesias Platas I.  Asymptomatic late thrombocytosis is a common finding in very preterm infants even in the absence of erythropoietin treatment. J Int Med Res. 2019;47:1504–11. https://doi. org/10.1177/0300060518821033. 17. Schnell BR, Seipel K, Bacher U, et al. Rebound thrombocytosis after induction chemotherapy is a strong biomarker for favorable outcome in AML patients. HemaSphere. 2019;3:e180. https://doi.org/10.1097/HS9.0000000000000180. 18. Aster RH. Pooling of platelets in the spleen: role in the pathogenesis of “hypersplenic” thrombocytopenia. J Clin Invest. 1966;45:645–57. https://doi.org/10.1172/JCI105380. 19. Boucher AA, Luchtman-Jones L, Palumbo JS, et al. Extreme thrombocytosis after pediatric pancreatectomy with islet autotransplantation is unique compared to other Postsplenectomy states. J Pediatr Surg. 2019; https://doi.org/10.1016/j.jpedsurg.2019.09.019. 20. Gurria JP, Boucher AA, Hornung L, et al. Thrombopoietin contributes to extreme thrombocytosis after pediatric pancreatectomy with islet autotransplantation. Pancreas. 2019;48:652–5. https://doi.org/10.1097/MPA.0000000000001313.

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21. Vo QT, Thompson DF.  A review and assessment of drug-induced thrombocytosis. Ann Pharmacother. 2019;53:523–36. https://doi.org/10.1177/1060028018819450. 22. Rose SR, Petersen NJ, Gardner TJ, et  al. Etiology of thrombocytosis in a general medicine population: analysis of 801 cases with emphasis on infectious causes. J Clin Med Res. 2012;4:415–23. https://doi.org/10.4021/jocmr1125w. 23. Hasle H. Incidence of essential thrombocythaemia in children. Br J Haematol. 2000;110:751. https://doi.org/10.1046/j.1365-2141.2000.02239-7.x. 24. McMullin MF.  Diagnostic workflow for hereditary erythrocytosis and thrombocyto sis. Hematol Am Soc Hematol Educ Program. 2019;2019:391–6. https://doi.org/10.1182/ hematology.2019000047. 25. Posthuma HLA, Skoda RC, Jacob FA, et  al. Hereditary thrombocytosis not as innocent as thought? Development into acute leukemia and myelofibrosis. Blood. 2010;116:3375–6. https://doi.org/10.1182/blood-2010-06-290718. 26. Teofili L, Giona F, Torti L, et  al. Hereditary thrombocytosis caused by MPLSer505Asn is associated with a high thrombotic risk, splenomegaly and progression to bone marrow fibrosis. Haematologica. 2010;95:65–70. https://doi.org/10.3324/haematol.2009.007542. 27. Giona F, Teofili L, Moleti ML, et al. Thrombocythemia and polycythemia in patients younger than 20 years at diagnosis: clinical and biologic features, treatment, and long-term outcome. Blood. 2012;119:2219–27. https://doi.org/10.1182/blood-2011-08-371328. 28. Randi ML, Geranio G, Bertozzi I, et  al. Are all cases of paediatric essential thrombocythaemia really myeloproliferative neoplasms? Analysis of a large cohort. Br J Haematol. 2015;169:584–9. https://doi.org/10.1111/bjh.13329. 29. Boklan JL, Walsh AM, de la Maza MC, et  al. Pediatric chronic myeloid leukemia presenting with extreme thrombocytosis simulating essential Thrombocythemia. J Pediatr Hematol Oncol. 2018;40:456–7. https://doi.org/10.1097/MPH.0000000000001154. 30. Kucine N, Al-Kawaaz M, Hajje D, et al. Difficulty distinguishing essential thrombocythaemia from polycythaemia vera in children with JAK2 V617F-positive myeloproliferative neoplasms. Br J Haematol. 2019;185:136–9. https://doi.org/10.1111/bjh.15386. 31. WHO Classification of Tumours. Revised 4th Edition, Volume 2. Edited by Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J. World Health Organization classification of tumors and haemotopoietic and lymphoid tissues, IARC Publications, Lyon, France. 2017. 32. Putti MC, Pizzi M, Bertozzi I, et  al. Bone marrow histology for the diagnosis of essential thrombocythemia in children: a multicenter Italian study. Blood. 2017;129:3040–2. https://doi. org/10.1182/blood-2017-01-761767. 33. Tefferi A, Wassie EA, Guglielmelli P, et al. Type 1 versus type 2 calreticulin mutations in essential thrombocythemia: a collaborative study of 1027 patients. Am J Hematol. 2014;89:E121–4. https://doi.org/10.1002/ajh.23743 34. Randi ML, Putti MC, Scapin M, et  al. Pediatric patients with essential thrombocythe mia are mostly polyclonal and V617FJAK2 negative. Blood. 2006;108:3600–2. https://doi. org/10.1182/blood-2006-04-014746. 35. Ianotto J-C, Curto-Garcia N, Lauermanova M, et  al. Characteristics and outcomes of patients with essential thrombocythemia or polycythemia vera diagnosed before 20 years of age: a systematic review. Haematologica. 2019;104:1580–8. https://doi.org/10.3324/ haematol.2018.200832. 36. Karow A, Nienhold R, Lundberg P, et al. Mutational profile of childhood myeloproliferative neoplasms. Leukemia. 2015;29:2407–9. https://doi.org/10.1038/leu.2015.205. 37. Langabeer SE, Haslam K, McMahon C.  Distinct driver mutation profiles of childhood and adolescent essential thrombocythemia. Pediatr Blood Cancer. 2015;62:175–6. https://doi. org/10.1002/pbc.25190. 38. Langabeer SE, Haslam K, McMahon C. A prenatal origin of childhood essential thrombocythaemia. Br J Haematol. 2013;163:676–8. https://doi.org/10.1111/bjh.12533. 39. Dror Y, Blanchette VS.  Essential thrombocythaemia in children. Br J Haematol. 1999;107:691–8. https://doi.org/10.1046/j.1365-2141.1999.01545.x.

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40. Rumi E, Cazzola M.  Diagnosis, risk stratification, and response evaluation in classi cal myeloproliferative neoplasms. Blood. 2017;129:680–92. https://doi.org/10.1182/ blood-2016-10-695957. 41. Polokhov DM, Ershov NM, Ignatova AA, et al. Platelet function and blood coagulation system status in childhood essential thrombocythemia. Platelets. 2019:1–11. https://doi.org/10.108 0/09537104.2019.1704710. 42. Schneider C, Stutz-Grunder E, Lüer S, et al. Fulminant essential Thrombocythemia associated with acquired Von Willebrand syndrome and bleeding episodes in a 14-year-old girl. Hamostaseologie. 2019; https://doi.org/10.1055/s-0039-1679929. 43. Finazzi G.  Ruxolitinib in ET: not all MPN are equal. Blood. 2017;130:1873–4. https://doi. org/10.1182/blood-2017-08-802165. 44. Nelson ND, Marcogliese A, Bergstrom K, et al. Thrombopoietin measurement as a key component in the evaluation of pediatric thrombocytosis. Pediatr Blood Cancer. 2016;63:1484–7. https://doi.org/10.1002/pbc.26032. 45. Randi ML, Bertozzi I, Putti MC. Contemporary management of essential thrombocythemia in children. Expert Rev Hematol. 2019;12:367–73. https://doi.org/10.1080/17474086.201 9.1602034. 46. Harrison CN, Mead AJ, Panchal A, et al. Ruxolitinib vs best available therapy for ET intolerant or resistant to hydroxycarbamide. Blood. 2017;130:1889–97. https://doi.org/10.1182/ blood-2017-05-785790. 47. Loh ML, Tasian SK, Rabin KR, et  al. A phase 1 dosing study of ruxolitinib in children with relapsed or refractory solid tumors, leukemias, or myeloproliferative neoplasms: a Children’s Oncology Group phase 1 consortium study (ADVL1011). Pediatr Blood Cancer. 2015;62:1717–24. https://doi.org/10.1002/pbc.25575. 48. Boddu P, Falchi L, Hosing C, et al. The role of thrombocytapheresis in the contemporary management of hyperthrombocytosis in myeloproliferative neoplasms: a case-based review. Leuk Res. 2017;58:14–22. https://doi.org/10.1016/j.leukres.2017.03.008.

Part IV

WBC Disorders

Chapter 13

Neutropenia Vinod K. Gidvani-Diaz

Introduction Neutrophils, also termed granulocytes or polymorphonuclear leukocytes, are derived from bone marrow stem cells. They mediate acute inflammation and play a critical role in the cellular defense against bacterial and fungal infections through generation of cytokines and their phagocytic functions. Approximately 1–1.5 × 109/ kg neutrophils are produced daily in the bone marrow, but only 2–5% enter the circulation. The lifespan of a neutrophil is 7–10 days [1].

Definition and Epidemiology Inherited and acquired neutropenia are rare disorders whose reported frequencies range from 1/100,000 for acquired autoimmune forms to 1/1,000,000 for congenital forms. The absolute neutrophil count (ANC) is calculated by multiplying the total white blood cell (WBC) count by the combined percentages of neutrophils and bands and dividing the result by 100. Neutropenia is defined by an ANC of less than 3000 for term infants to 1 week of age, less than 1000 in infants and children 1 week to 2 years, and less than 1500 after 2 years of age. For patients older than 1 year of age, neutropenia is defined as mild with an ANC between 1000 and 1500, moderate with an ANC between 500 and 1000, and severe when the ANC is below 500. In some ethnic groups, specifically individuals with African heritage and from some

V. K. Gidvani-Diaz (*) Texas/Methodist Children’s Hospital, UT Health Joe R and Teresa Lozano Long School of Medicine, San Antonio, TX, USA e-mail: [email protected] © Springer Nature Switzerland AG 2021 D. M. Kamat, M. Frei-Jones (eds.), Benign Hematologic Disorders in Children, https://doi.org/10.1007/978-3-030-49980-8_13

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Middle Eastern regions, the lower limit of neutropenia is lower, i.e., 800–1000. This is termed benign familial or benign ethnic neutropenia [4]. Neutropenia can result from decreased marrow production, increased margination or sequestration, or increased destruction. Neutropenia can be acute or chronic and be the result of congenital or acquired etiologies.

Approach to the Neutropenic Child The age at presentation of neutropenia provides clues to the likely etiology. Neonatal neutropenia is often identified in premature infants in association with maternal hypertension or preeclampsia. Severe neutropenia in newborns can also occur in the setting of sepsis. In full-term infants who appear well, consider alloimmune or isoimmune neutropenia. Neutropenia in young children is most commonly encountered in the setting of a concurrent or recent viral infection and results from bone marrow suppression. Evaluation of the neutropenic child starts with careful attention to the history and physical examination. Key symptoms include recurrent stomatitis, gingival inflammation, recurrent severe diaper dermatitis, skin and sinopulmonary infections, as well as invasive bacterial infections, the latter being prevalent in severe congenital neutropenia. A detailed fever pattern should be correlated to physical manifestations; for example, some children with cyclic neutropenia will develop mouth ulcers contemporaneous with fever development. Stooling pattern and growth history are likewise important to note in the context of bone marrow failure syndromes, such as the stooling pattern and failure to thrive seen in patients with Shwachman-Diamond or the poor growth often seen in patients with immunodeficiencies. The exam can guide the clinician toward specific bone marrow failure syndromes. Important features to document include the examination of the gingiva, skin, and extremities. Initial laboratory evaluation should always include a complete blood count with review of the peripheral blood smear. The presence of additional cytopenias such as anemia or thrombocytopenia should prompt earlier referral for hematology evaluation. Second-line testing is guided by the history and physical exam, as well as the chronicity and pattern of the neutropenia. For isolated neutropenia, prior to referral to the specialist, it is appropriate for the general pediatrician to evaluate for anti-neutrophil antibodies and to screen for immunodeficiencies with immunoglobulin levels. The further evaluation of neutropenia by the specialist may include genetic testing for specific mutations associated with inherited bone marrow failure syndromes, and bone marrow examination may be indicated to further evaluate severe persistent neutropenia with attention to myeloid maturation and performance of cytogenetics/FISH (fluorescence in situ hybridization) for myelodysplastic syndrome (MDS)/acute myelogenous leukemia (AML) done. Patients with new-onset severe neutropenia (ANC 7 days and/or a menstrual blood loss of >80 mL per cycle [11–13]. Clinical history details of changing sanitary pads more than once per hour, clots of greater than one inch in diameter, or evidence of iron deficiency anemia (IDA) predict blood loss of >80 mL with a sensitivity of 60% and specificity of 86% [14]. A more recent patient-centric definition of HMB adopted by the International Federation of Gynecology and Obstetrics (FIGO) is “any menstrual blood loss which interferes

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with a woman’s physical, social, emotional, and/or material quality of life” [2, 15]. The International Federation of Gynecology and Obstetrics also recommends against using terms such as menorrhagia, dysfunctional uterine bleeding, or hypermenorrhea. Abnormal uterine bleeding and HMB are the suggested terms to describe menstrual bleeding that is abnormal in frequency, volume, duration, and cycle regularity [16].

The Etiology of HMB The etiology of HMB is multifactorial [13]. International Federation of Gynecology and Obstetrics and ACOG, after years of consensus forming, have agreed on a nomenclature system called PALM-COEIN to distinguish between structural and nonstructural causes [2, 17]. PALM refers to the most common structural causes of HMB seen in reproductive age women, whereas COEIN refers to the common nonstructural causes and includes Coagulation, Ovulatory dysfunction, Endometrial, Iatrogenic, Not yet classified (Table 20.1) [17]. Nonstructural causes resulting in HMB are more common in adolescents, specifically anovulation from the immaturity of the hypothalamic-pituitary-ovarian axis in the 1–3 years after menarche but which can last up to 5 years [13, 18–20]. Anovulation is followed by inherited bleeding disorders that can be found in up to Table 20.1 The International Federation of Gynecology and Obstetrics PALM-COIEN classification PALM (structural)

Coagulopathy

Ovulatory Dysfunction

Endometrial Iatrogenic

Not otherwise Classified

Polyp Adenomyosis Leiomyoma Malignancy and Hyperplasia Von Willebrand disease Platelet function disorders Thrombocytopenia (ITP etc.) Factor deficiencies Other bleeding disorders Immaturity of the hypothalamic-pituitary-ovarian axis Hyperandrogenism (polycystic ovarian syndrome) Thyroid disease Anorexia, weight loss Pregnancy Sexually transmitted infections (gonorrhea, chlamydia) Medications (anticoagulation, tricyclic antidepressants, SSRIs, steroids, herbal, i.e., ginseng) Long-acting reversible contraceptives Inadequate estrogen dose Trauma Others

ITP immune thrombocytopenic purpura, SSRIs Selective serotonin release inhibitors

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35% in adolescents with HMB resulting from defects in either primary or secondary hemostasis [3]. Half of these women will have a history of HMB since menarche [3, 21–23]. For many adolescents, menses are the first hemostatic challenge that disrupts uterine blood vessels and requires intact function of the platelet, clotting factors, and fibrinolytic pathways [24]. Bleeding disorders can be a part of the etiology either alone or concurrently with other causes [3]. Among bleeding disorders affecting adolescents with HMB, the most common is von Willebrand disease (VWD) followed by platelet function disorders (PFDs), but various other disorders may be detected based on geographical location and consanguinity (Table 20.2). Von Willebrand disease is defined as either antigen or Table 20.2  Common bleeding disorders in women with heavy menstrual bleeding Disorder Von Willebrand disease

Prevalence Specific interventions 5–36% DDAVP trial (contraindicated in type 2B)

 Type 1  Type 2A, 2B, 2M, 2N

VWF concentrates in severe type 3

 Type 3 Low von Willebrand factor Platelet function defects  Platelet storage pool defects  Qualitative platelet  Dysfunction Thrombocytopenia

12–20%

 ITP  Autoimmune disorders  (Lupus etc.) Factory deficiency

8–9%

 Factor V, VII, X, XI, XIII  Hemophilia A carrier (VIII)  Hemophilia B carrier (XI) Fibrinolytic disorders  Hypofibrinogenemia  Afibrinogenemia  Dysfibrinogenemia  PAI-1 deficiency Collagen defects: generalized joint hypermobility syndromes, Ehlers-Danlos syndrome, Marfan syndrome

16% 2–44%

Antifibrinolytics DDAVP trial Antifibrinolytics

1%

ITP: steroids, IVIG, rituximab, TPO-agonists

Antifibrinolytics DDAVP trial in hemophilia A carriers Antifibrinolytics Factor concentrates Antifibrinolytics Cryoprecipitate (in deficiency state)

DDAVP trial Antifibrinolytics

DDAVP 1-deamino-8-D-arginine vasopressin, VWF von Willebrand factor, ITP immune, thrombocytopenic purpura, IVIG intravenous immunoglobulin, TPO thrombopoietin receptor, PAI plasminogen activator inhibitor

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activity levels 1 cm) bruises and petechiae may suggest thrombocytopenia or underlying bleeding disorders. Findings of obesity, acanthosis nigricans, hirsutism, and acne may

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Table 20.3  Proposed history taking questions in the evaluation of heavy menstrual bleeding in adolescents Menstrual  HMB since onset of menses  Frequency (frequent if 8 days)  Regularity (irregular if shortest to longest cycle variation is ≥8 days)  Volume    Pads per day (heavy if >6 pads/day),    Changing pads (more than every 1–2 hours)    Frequent soiling of clothes/bedsheets  Sensation of gushing or presence of clots (greater than 2 inch in diameter) Personal  History of iron deficiency or symptoms of IDA (low ferritin, headaches, dizziness, fatigue, pica)  Mucosal bleeding (epistaxis)    How often, prolonged if >10 minutes    Required medical intervention such as ER visit  Easy bruising (unexplained bruising, with minor trauma, bruising on abdomen/central areas and >2 cms)  Bleeding with hemostatic challenges either heavier than expected, prolonged, or recurrent (surgery of any type, tonsillectomy, dental procedures)  Prolonged bleeding from minor wounds (trivial cuts bleeding >10 mins)  Blood in oral cavity or GI tract without a known cause  Bleeding that required blood transfusions  Muscle or joint bleeding Family history  Known bleeding disorders including hemophilia  Frequent or prolonged epistaxis (>10 minutes)  History of iron deficiency anemia due to recurrent blood loss (after minor surgery, during menses, mucosal bleeds)  Females with HMB or anemia or postpartum hemorrhage  Blood transfusions after minor surgeries or with HMB  Collagen disorders (Ehlers-Danlos, Marfan syndrome, hypermobility syndromes, Osteogenesis imperfecta) HMB heavy menstrual bleeding, IDA iron deficiency anemia, ER emergency room, GI gastrointestinal

suggest polycystic ovarian syndrome (PCOS). Evaluation should include assessment of joint hypermobility syndromes with a Beighton score, hyperextensible skin, and/or abnormal scarring [36]. An external pelvic exam should consist of external visualization for any trauma, foreign bodies, and virilization. The utility of an invasive exam such as a bimanual or pelvic exam for HMB is debated [24, 37].

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Laboratory Evaluation Initial Evaluation Laboratory evaluation should occur in a stepwise manner for adolescents with HMB (Fig. 20.1). There are a few essential non-hematological tests to evaluate for HMB [38]. These include urine pregnancy test to rule out any pregnancy-related causes, urine for sexually transmitted diseases such as gonorrhea and chlamydia, thyroid studies including thyroid-stimulating hormone, and free T4 to evaluate for thyroid disease and free testosterone to assess for PCOS. The initial hematologic evaluation of patients with a suspected bleeding disorder should include a complete blood count and a review of peripheral blood smear for platelet and red blood cell (RBC) morphology (Table  20.4). Smear morphology may identify the presence of large platelets, which can suggest rare disorders such as Bernard-Soulier or myosin heavy chain-9 (MYH9)-related disorders. Heavy Menstrual Bleeding

Consider PALM-COEIN etiology

Consider further testing: Clotting factors: PT, PTT Fibrinogen defects: Fibrinogen, thrombin time Von Willebrand Disease: VWF antigen, Factor VIII activity, VWF: RCo, multimer analysis

Abnormal test results or normal test results with suspicion for BD

Positive history and/or exam findings of possible BD or iron deficiency

Labs: CBC Iron studies: ferritin, TIBC

Findings of Iron Deficiency: · Hemoglobin < 12 g/dL with MCV low or normal < 100 · Ferritin < 30ng/mL with elevated TIBC

Referral to multidisciplinary clinic

Start Ferrous Sulfate 325 (65mg elemental) mg Consider Hormonal Therapy

Repeat labs at 1 month. If no response to iron therapy despite adherence or side effects, consider IV iron

Fig. 20.1 Approach to heavy menstrual bleeding. PALM-COEIN Polyp; Adenomyosis; Leiomyoma; Malignancy and Hyperplasia-Coagulation; Ovulatory dysfunction; Endometrial; Iatrogenic; Not yet classified, BD bleeding disorder, CBC complete blood count, TIBC total iron-­ binding capacity, PT prothrombin time, PTT partial thromboplastin time, VWF von Willebrand factor, RCo ristocetin cofactor activity, MCV mean corpuscular volume, IV intravenous

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Table 20.4  Laboratory tests used in evaluation for bleeding disorders Lab test CBC Smear morphology for large platelets Ferritin, TIBC Prothrombin time Activated partial thromboplastin time Fibrinogen Thrombin time VWF antigen, VWF ristocetin cofactor activity, Factor VIII, multimer analysis PFA-100 Platelet aggregation testing Platelet electron microscopy Platelet flow cytometry

Factor levels (II, V, VII, VIII, IX, X, XI and XIII)

Evaluation Thrombocytopenia (ITP, etc.) Microcytic anemia (iron deficiency, thalassemia) Platelets disorders such as MYH9 (Döhle bodies), Bernard-Soulier Screen for iron deficiency Vitamin K deficiency, factor VII deficiency, liver disease Prolonged vitamin K deficiency, factor deficiencies (VIII, IX, XI, XII) Presence of fibrinogen (afibrinogenemia) Assesses for the presence and function of fibrinogen (dysfibrinogenemia) Presence of VWD or low VWF Severe bleeding disorder (Glanzmann, Bernard Soulier, severe type III VWD, etc.) Platelet function disorders Gray platelet syndrome (absence of alpha granules) Hermansky-Pudlak syndrome or Chediak Higashi (absence of dense granule) Platelet storage pool defects Defects in platelet signal transduction Hemophilia A/B carriers Factor VII deficiency Factor XI deficiency Factor XIII deficiency

CBC complete blood count, ITP immune thrombocytopenic purpura, MYH9 myosin heavy chain 9, VWF von Willebrand factor, VWD von Willebrand disease, PFA platelet function assay

Testing for Iron Deficiency Anemia The degree of anemia, hypochromia, and microcytosis will also identify if IDA is present. Anemia is found in 25% of women who present with HMB, and about 50% have evidence of iron deficiency alone [39]. We recommend further screening of iron stores with ferritin and total iron-binding capacity (TIBC). Early manifestation can be limited to just iron deficiency (serum ferritin 12  g/dL), or anemia is apparent with lower (2 days for plasmapheresis or plateletpheresis procedures) [1, 2]. A health history questionnaire is used to screen donors who may not be suitable for donations. Individuals who are taking teratogenic medications such as B. Ozgonenel (*) Carman and Ann Adams Department of Pediatrics, Division of Hematology Oncology, Wayne State University School of Medicine, Children’s Hospital of Michigan, Detroit, MI, USA e-mail: [email protected] © Springer Nature Switzerland AG 2021 D. M. Kamat, M. Frei-Jones (eds.), Benign Hematologic Disorders in Children, https://doi.org/10.1007/978-3-030-49980-8_29

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isotretinoin or finasteride; those who have a history of high-risk behaviors for exposure to human immunodeficiency virus (HIV) infection, infectious hepatitis, malaria, Chagas disease, or variant Creutzfeldt-Jakob disease (vCJD); and those for whom the donation process may not be safe (e.g., pregnant women up to 6 weeks of postpartum period) are deferred, either permanently or temporarily depending on the identified risk factor. The donor undergoes a brief physical examination and hemoglobin check. Donors who have acceptable physical examination criteria and a hemoglobin level greater than 12.5 gm/dL are accepted for donation. The blood donation occurs through one of two ways: whole blood collection or collection of a specific blood component through apheresis. When whole blood is collected, it is obtained through venipuncture from one of the antecubital veins. Appropriate disinfection of the antecubital area is essential for avoiding introduction of skin bacteria into the collected blood. A large-bore needle such as a 16-gauge needle is used for venipuncture. Donors who weigh at least 110 lb (50 kg) can donate up to 525 mL of blood, but those who weigh less should donate proportionately less. The collected whole blood is subsequently separated into components via centrifugation. Apheresis donation is accomplished through a single venipuncture. Whole blood is collected in the single-use collection system and the instrument utilizes centrifugation to separate the blood component of choice, red blood cells (RBCs), platelets, or plasma. After the target component is collected, the other blood components are returned to the donor through the same needle. The collected blood is typed for ABO and Rh antigens and screened for unusual RBC antibodies. Donor blood is also extensively tested for some infectious agents (Table 29.1). Sometimes more than one method is used to screen for a specific infection to improve the detection of the infection. If the testing is positive, the test is repeated, and if confirmed on the second test, the unit is destroyed. The donor is notified of the positive result and deferred from future donations.

 rocessing of Blood Components at the Collection P and Processing Facility Donated blood undergoes several steps in processing before the various products are ready for issue from the collection and processing facility to hospital blood banks [5]. FRACTIONATION: If whole blood is collected, individual components (i.e., RBCs, platelets, and plasma) are separated through centrifugation. The centrifuged components are collected into satellite bags that are connected to the whole blood bag through plastic tubing. This “closed” system prevents contamination during the separation process. PRESERVATION: The initial collection bag contains an anticoagulant/preservative solution, which is either CPD (citrate-phosphate-dextrose) or CPDA-1 (citratephosphate-dextrose-adenine). Citrate is the anticoagulant. Phosphate is necessary to

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Table 29.1  Infectious disease screening in donated blood and the methodology used Microorganism(s) screened Syphilis Hepatitis B virus

HIV types 1 and 2 Hepatitis C

Testing methodology Serology HbsAg (hepatitis B surface antigen) Antibody against hepatitis B core antigen NAT Anti-HIV 1/2 antibodies NAT Anti-HCV antibodies NAT Anti-HTLV antibodies

Human T-cell lymphotropic virus (HTLV) I and II West Nile virus NAT Trypanosoma cruzi (Chagas disease) Anti-T. cruzi antibody Babesia microti Anti-B. microti antibody NAT Zika virus NAT

Year introduced 1950s 1971 1986 2009 1985 1999 1990 1999 1998 2003 2007 2012 2012 2016

NAT nucleic acid testing. The last column shows the year when American Red Cross incorporated routine testing of donated blood for the infectious agent [3, 4] Universal screening of blood components for cytomegalovirus (CMV) is not routinely performed. However, some units are randomly screened for anti-CMV antibodies to maintain a source of CMV-seronegative units. (Indications for CMV-negative units will be discussed further below)

replenish 2,3-diphosphoglycerate (2,3-DPG) as decreased levels of 2,3-­DPG shift the hemoglobin-oxygen dissociation curve to the left and oxygen release from hemoglobin to the hypoxic tissues is impaired. Dextrose is necessary for RBC energy. Glucose is converted to lactate through glycolysis, producing adenosine triphosphate (ATP) for the red blood cell, and adenine is necessary for ATP production. Packed red blood cell (PRBC) units stored in CPDA-1 have a shelf life of 35 days, but additive solutions have more glucose and adenine, extending the shelf life to 42 days. The hematocrit of a CPDA-1 unit can be as high as 80%, but units prepared with an additive solution have a lower hematocrit between 55% and 65%. In situations where high post-transfusion hematocrits should be avoided, such as in patients with sickle cell disease, clinicians should consider the difference in hematocrit in their volume considerations. In general, 10 mL/kg of a unit with CPDA-1 is equivalent to 12 mL/kg of a unit prepared with additive solution. PRE-STORAGE LEUKOREDUCTION: Blood components undergo leukocyte depletion, also called leukoreduction or leukofiltration, prior to storage and further processing, thus arriving at blood banks already leukoreduced, obviating the need to use bedside leukocyte filters. Pre-storage leukoreduction of a blood component has several advantages, including minimizing the occurrence of febrile non-hemolytic transfusion reactions, minimizing HLA (human leukocyte antigen) alloimmunization and platelet refractoriness, and prevention of transmission of leukotropic viruses residing in white blood cells (WBCs), such as CMV or EBV (Epstein-Barr

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virus). In fact, leukoreduced blood components can be considered CMV-safe even if they have been obtained from a CMV-seropositive donor. Post-storage leukoreduction may not have the above advantages as WBCs can still release cytokines or leukotropic viruses into the component solution during storage, and leukocyte fragments containing HLA may pass through bedside filters. BACTERIAL CULTURE OF PLATELET UNITS: Regardless of the method of collection, platelet units are stored at room temperature (220–24 °C), which may facilitate bacterial growth in the platelet unit. In fact, bacterial sepsis in the recipient is most frequently associated with platelet transfusion. Most processing facilities have elected to culture platelet units prior to issuing them to blood banks to avoid transfusion-associated bacteremia or sepsis. Units that signal bacterial growth are discarded, and only those with negative growth at 2 days are issued to blood banks. This diminishes the already short, 7-day shelf life of platelets, and most units that reach the blood bank have only 5 days left before expiration [6]. PATHOGEN INACTIVATION OF PLATELET UNITS: This novel method is soon going to replace bacterial culturing of platelet units despite a labor-intensive process that involves injecting platelet units with amotosalen. Amotosalen-injected units are exposed to ultraviolet A rays, which leads to breakdown of psoralen-bound DNA of viruses, bacteria, and WBCs. Platelets do not have a nucleus and therefore are not affected by this process [6].

Processing of Blood Components at the Hospital Blood Bank STORAGE and INFCTION CONTROL: Table  29.2 summarizes the storage requirements and shelf lives for blood components [7, 8]. Figure 29.1 shows platelet storage at room temperature with gentle agitation. When a PRBC unit leaves the blood bank, it must be used for transfusion within 4 hours or otherwise discarded to minimize infectious risk. PRBC units specific to recipients may also be frozen in a glycerol solution and stored at freezing temperatures up to 10 years. Table 29.2  Storage requirements and shelf lives of blood components Blood component PRBC units Platelet units

FFP, cryoprecipitate

Temperature At refrigerator shelf temperature of 1–6 degrees Celsius (°C) At room temperature of 20–24 °C with continuous gentle agitation At freezing temperatures of ≤ −18 °C

°C = degrees Celsius

Shelf life 35 days – units stored in CPDA-1 42 days – units stored in additive solution 5 days if the unit was cultured at the collection facility 7 days if the unit underwent pathogen inactivation Up to a year

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Fig. 29.1  Platelets are stored at room temperature with continuous gentle agitation

Platelet units can be screened for presence of bacterial cell wall components prior to issuing to the patient. This strategy aims to reduce transfusion-transmitted bacterial infections. IRRADIATION: Cellular blood components need to be irradiated prior to transfusing to certain patients to prevent transfusion-associated graft versus host disease (TA-GVHD). Irradiation prevents the donor T lymphocytes from proliferating in the recipient and launching a cellular immune attack on the recipient’s tissues. Gamma irradiation of the unit can be accomplished with an irradiator found in most blood banks. The expiry date of the PRBC units is reduced to 28 days after an irradiation or original expiration date, whichever is shorter. Irradiation also causes an efflux of potassium into the extracellular fluid in stored units, and that’s why irradiated units should be transfused as soon as possible to avert hyperkalemia in patients at risk, such as newborns [9]. Pathogen-inactivated units do not need to be irradiated as they have already received ultraviolet A irradiation [7, 8]. Cellular blood products should be irradiated for the following transfusions [10]: • Intrauterine transfusions. • Transfusions given to newborns or infants younger than 4 months of age (some centers irradiate only for transfusions given to low-birth-weight preterm infants). • Transfusions from blood-related donors. • Transfusions following hematopoietic stem cell transplantation. • Transfusions given to individuals with congenital immunodeficiency. • Transfusions given to patients receiving chemotherapy or radiation treatment for cancer. • Transfusions given to patients receiving intense immunosuppressive treatment. • Transfusions of HLA-matched blood components. • All granulocyte transfusions. • All cellular transfusions need to be irradiated in homogenous populations where there is very little HLA variability. For example, the Japanese population is relatively homogenous, and there is very little HLA variability, and cases of ­TA-­GVHD have occurred even after transfusions from unrelated donors to immunocompetent individuals [11].

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WASHING: Washing is a process that aims to reduce the plasma component of cellular blood components such as PRBC units or platelet units. The indications for this processing include severe or multiple allergic reactions in the recipient. The goal is to reduce exposure to the inciting allergen in the plasma. The washing process itself is accomplished by infusing 1–2 liters of normal saline into the unit and then centrifuging the unit to remove the supernatant. The process unfortunately leads to loss of the cellular product as well, and up to 20% of the RBC yield and 33% of the platelet yield are also lost. This process is labor-intensive, takes time, and reduces the expiry to 24 hours for PRBCs and 4 hours for platelet units and therefore should not be ordered unless there is a strong indication for its use [7, 8].

Blood Components Packed Red Blood Cells Source PRBC units can be obtained via centrifugation of the whole blood or via erythrocytapheresis. PRBC units generally weigh between 250 and 350 mL [7]. Young Versus Old Blood Several changes occur in the RBCs as the PRBC unit ages. ATP and 2,3-DPG are depleted towards the end of the 2nd week of storage, and the intracellular potassium leaks into the unit storage solution. In most stable patients, this does not cause any clinical problems as the extracellular potassium in the solution is minimal and the depleted ATP and 2,3-DPG are restored within hours. In vulnerable populations such as newborns and patients with severe cardiopulmonary conditions, however, PRBC units stored less than 7–10 days are preferred to avoid hyperkalemia [10]. Pretransfusion Testing RBCs carry several carbohydrate or protein antigens that may be present or absent in different individuals. Antigens that are perceived as “non-self” and thus provoke an immune reaction in an individual are called “blood groups.” The major blood groups are the ABO and the RhD antigens. ABO antigens are carbohydrate antigens. Individuals lacking a certain ABO antigen develop antibodies “naturally” without prior exposure via transfusion. The theory is these antibodies develop during infancy after exposure to antigens carried through exposure to gut bacteria. Antibodies against RhD antigen, however, require previous exposure to the antigen. Therefore an RhD-negative individual receiving

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RhD-positive blood transfusion or an RhD-negative woman pregnant with an RhD-­ positive fetus develops antibodies against the Rh antigen. These antibodies could initiate a strong hemolytic reaction in a subsequent exposure, either via transfusion or during pregnancy. The blood bank technician types the recipient’s blood for ABO and RhD antigens and screens for auto- and alloantibodies. This screen is an indirect antiglobulin test using the Coombs reagent to identify antibodies present in the recipient’s serum. This combined testing is referred to as a “type and screen.” The technician then runs a crossmatch of the donated blood and the recipient’s sample. In major crossmatch, donor’s RBCs are tested in the presence of recipient serum. If the recipient has antibodies against the donor RBC antigens, an agglutination occurs which can be detected visually. In minor crossmatch, donor serum is tested against recipient RBCs [12]. Volume and Rate The typical transfusion volume of PRBCs for most stable patients is 10–12 mL/kg which can be given over 2 hours. Stable patients usually require one to two units depending upon body weight. Patients with active bleeding may require higher volumes of transfusion or more frequent transfusions to replenish ongoing losses. Patients receiving chronic blood transfusions such as thalassemia major or sickle cell disease may receive higher volumes of 12–15  mL/kg. Newborn infants with severe anemia are usually given higher volumes up to 15 mL/kg. Transfusion can be delivered much faster if the patient has developed acute anemia due to active bleeding or severe hemolysis. Transfusions should be given slowly to patients who have very low hemoglobin levels due to chronic anemia to avoid precipitating heart failure. Since the transfusion time cannot be extended over the abovementioned 4-hour limit, smaller aliquots of 5–6 mL/kg of body weight should be given in such cases [13]. Indications Basic Principles The hemoglobin inside RBCs carries oxygen from the lungs to capillaries where oxygen is released into the tissues. In situations with low hemoglobin levels, compensatory mechanisms, such as oxygen dissociation curve shift, may be sufficient to maintain adequate tissue oxygenation. The basic indication for PRBC transfusion is treatment of severe anemia that disrupts oxygen delivery to tissues. Many other factors, such as the patient’s age, comorbidities, acuity of the condition, and availability of other treatment options for anemia, may affect this indication [13, 14]. Both the public and the medical community have had legitimate concerns about adverse effects of transfusion. These include not only infection transmission and

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acute transfusion reactions but also HLA alloimmunization, alloantibody formation, and transfusion-related immunomodulation. Inappropriate transfusions expose patients to adverse effects, incur unnecessary costs, and waste an otherwise precious resource that could have benefited another patient in need of transfusion. Therefore, the decision to transfuse should include the following considerations of clinical transfusion: 1 . Transfuse only and only if it is absolutely necessary. 2. Avoid transfusing for arbitrary “triggers.” 3. Transfuse only as much as needed. If one unit will achieve the clinical goal, a second unit does not need to be given. PRBC transfusions should not be given as a volume expander unless the patient is actively hemorrhaging, as a substitute for iron, vitamin B12, or erythropoietin which can be administered pharmacologically, to help with wound healing or to satisfy a patient’s or physician’s desire to have a certain numerical hemoglobin value. PRBC Transfusion Indications by Disease Category Chronic anemias secondary to deficiencies of iron, folic acid, vitamin B12, or erythropoietin should be ideally managed by replacing the deficient hematopoietic substance. Such children with chronic anemia can tolerate very low levels of hemoglobin, and it is not unusual for severe anemia to be discovered during a routine blood count check. For such asymptomatic patients, transfusion could be held even for hemoglobin levels as low as 5 gm/dL. Patients with hemoglobin levels below 5 gm/dl or those who have symptoms and signs related to severe anemia such as headache, pre-syncope, syncope, or heart failure should receive a transfusion. The transfusion should be undertaken very slowly and in small volumes in such patients. Typically the volume for transfusion is 5–6  mL/kg given over 3  hours, with careful monitoring of cardiovascular status. If the etiology of the anemia is due to iron, folic acid, or vitamin B12, supplementation should be started in addition to the transfusion. If the patient is found to be deficient in erythropoietin, renal function should be evaluated and recombinant erythropoietin administration begun [13, 14]. The acute onset of severe anemia, however, will not have allowed development of compensatory mechanisms seen in chronic anemias, and patients may require transfusion at higher initial hemoglobin levels. These patients can be transfused more rapidly especially if there is a concern that the severe anemia is leading to shock. Patients with such severe acute anemia include those with active hemorrhage (gastrointestinal, vaginal, surgical, or traumatic) and those with acute hemolysis (secondary to thrombotic thrombocytopenic purpura, glucose-6-phosphate dehydrogenase deficiency, or autoimmune hemolytic anemia (AIHA)). Note that in patients with AIHA, the blood bank may require more time to investigate the involved antibody and to crossmatch units. Because rapidly evolving

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severe anemia could easily decompensate, patients with severe anemia may not be able to wait for perfectly matched blood. In such situations, even an incompatible unit may be transfused. Slow transfusion, warming the patient and the unit in the presence of cold antibodies, providing fluids, and immunosuppressive therapy such as steroids may slow the hemolysis and provide the patient with a more stable hemoglobin until the autoimmune hemolysis is controlled. Patients with hemoglobinopathies, severe red cell enzyme defects and red cell membranopathies, congenital dyserythropoietic anemia (CDA), and myelodysplastic syndrome may need to receive transfusion on a scheduled, chronic basis. In patients with ineffective erythropoiesis (i.e., thalassemia, CDA), PRBC transfusions aim to maintain hemoglobin at a higher level to suppress expanded erythropoiesis and thereby prevent medullary expansion. The aim is to achieve a pretransfusion or nadir hemoglobin of 9–10 gr/dL in such patients. Transfusions could be given at 15–18  mL/kg every 3–6  weeks based on patient characteristics. In patients with hereditary spherocytosis, the abnormal RBCs are broken down in the spleen, while severe anemia results in extramedullary hematopoiesis in the spleen and further splenomegaly, leading to a vicious cycle of hemolysis causing anemia causing splenomegaly causing more hemolysis and more severe anemia. The aim of the transfusion is to break this cycle by both elevating the hemoglobin and thus suppressing extramedullary erythropoiesis and providing red blood cells that will not be destroyed in the spleen. In patients with sickle cell disease (SCD), PRBC transfusion is given to decrease the hemoglobin S concentrations below 20–50% depending upon the indication for transfusion. Reducing the HbS% significantly reduces the sickling process. Transfusion in SCD can either be given on a chronic basis such as in patients with a history of stroke or on an acute basis, such as those presenting with acute chest syndrome. Simple transfusions may be adequate for acute anemia or increased hemolysis. However, in the setting of stroke, severe ACS or multi-organ failure exchange transfusions are preferred to more rapidly reduce the HbS percentage. Exchange transfusions can be performed using an automated instrument or manually where the patient undergoes a phlebotomy of 5 mL/kg and receives 12 ml/kg of PRBC in additive solution (10 mL/kg if the PRBC solution is prepared only with CPDA-1). Patients undergoing myelosuppressive chemotherapy or radiation treatment for pediatric cancers usually receive PRBC transfusions when their hemoglobin drops to 7–8 gm/dL. The anemia in these patients does not respond to erythropoietic substances such as iron or erythropoietin, and the use of recombinant erythropoietin in patients with pediatric cancers is controversial because of the risk of inducing tumor growth. Myelosuppression after chemotherapy or radiation therapy usually takes 2–3  weeks (longer in some cases) to recover. By the time the bone marrow has recovered, the next cycle of myelosuppressive therapy starts. Patients with critical illness with stable cardiopulmonary status can usually be transfused at hemoglobin levels as low as 7 gm/dl. Past studies have shown that transfusing at higher hemoglobin levels does not impart any clinical benefit and could even be harmful [15].

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Platelets Source Platelet units can be collected as either random donor platelets (RDP), which are obtained via centrifugation of a whole blood donation, or single donor units (SDU), which are obtained via apheresis. RDP units have a volume of ~50 mL and contain at least 55 billion platelets per unit, but the larger apheresis units have a volume of 200–250 mL and contain at least 300 billion platelets per unit. Blood processing centers in the United States have largely shifted to collecting apheresis platelets, and the platelets obtained from whole blood donations are usually discarded. If obtained, however, 4–6 RDP units are usually pooled into one random donor unit (RDU) [16, 17]. Pretransfusion Testing In general, there is no pretransfusion testing for platelets other than the recipient blood type as platelets should be plasma compatible. Many blood banks issue platelet units without regard to ABO compatibility between the donor and recipient. However, platelets do carry ABO antigens and, if transfused to a recipient with a major incompatibility, may lead to “platelet refractoriness,” meaning an unexpectedly low rise in the platelet count after transfusion. Although platelet refractoriness might have other causes, most experts recommend attempting transfusion with ABO-identical platelet units prior to embarking on a search for other causes, such anti-HLA antibodies. Platelets are also contaminated with minute amounts of RBCs, increasing the risk of Rh-sensitization if a unit obtained from an Rh-positive donor is transfused to an Rh-negative recipient. Female Rh-negative patients of reproductive age should receive Rh-negative units or anti-D injections to avoid sensitization after a transfusion of Rh-positive platelet unit. Volume and Rate Most experts do not recommend transfusing more than six random donor units or one single apheresis unit at a time. Platelet transfusions can be given over 30–60 minutes. Slower transfusion compromises post-transfusion platelet count and activity. Indication The decision to transfuse platelets depends on the patient’s clinical condition, the status of plasma phase coagulation, the platelet count, the cause of the thrombocytopenia, and the functional capacity of the patient’s own platelets. In the face of decreased production and platelet counts less than 10,000–20,000/μL, the risk of

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severe, spontaneous bleeding is increased markedly, and, in the absence of immune-­ mediated thrombocytopenia (ITP), transfusion should be considered. Under certain circumstances, especially with platelet dysfunction or when receiving anticoagulation treatment, transfusions at higher platelet counts may be necessary to prevent or treat bleeding [17–19].

Plasma-Based Components Source The two most commonly used plasma-based blood components are fresh frozen plasma (FFP) and cryoprecipitate. FFP is obtained either through plasma apheresis or via separation of the plasma component of a whole blood collection. Cryoprecipitate is manufactured from the precipitated portion of plasma thawed at 1–6 °C [16]. Pretransfusion Testing Although a pretransfusion crossmatch testing is not necessary, the plasma should be compatible with the recipient’s ABO antigens. Individuals with AB blood type can universally donate plasma to all recipients, whereas individuals with O blood type can donate only to recipients with group O. Volume and Rate Both FFP and cryoprecipitate can be given in a rapid transfusion over 30–60 minutes. FFP is given at a dose of 10–15 ml/kg. Cryoprecipitate dosing is as follows: 1 cryoprecipitate unit for every 5 kg to raise the fibrinogen by 100 mg/dL [20]. Indication The indication for FFP is replacement of plasma coagulation factors in the setting of active bleeding associated with multiple clotting factor deficiencies. In most hereditary factor deficiencies, such as factor VIII deficiency or vWD, commercially prepared concentrates contain higher concentrations of these factors and, because of viral inactivation, impose less infectious risk and are more appropriate than FFP. FFP units are the blood components most commonly transfused for inappropriate indications. Such inappropriate use may involve the treatment of prolongation of prothrombin time (PT) or activated partial thromboplastin time (aPTT) in the absence of clinical bleeding or for supplementation of volume or albumin or

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replacement of coagulation factors for which individual factor concentrates are readily available. Cryoprecipitate contains four important coagulation factors: factor VIII, von Willebrand factor, factor XIII, and fibrinogen. Specific concentrates for each of these individual factors now are available, thus decreasing the utilization of cryoprecipitate as a treatment for specific clotting factor deficiencies; however, it is still commonly used to replenish fibrinogen in states such as disseminated intravascular coagulation (DIC), as the fibrinogen solution is not easily available and is more expensive.

Granulocytes Granulocyte transfusions had fallen out of favor because of severe side effects including ARDS but are now making a comeback for severely neutropenic patients with severe sepsis. The complications and availability are still significant issues with the use of this blood component. Granulocyte donors receive granulocyte colony-­stimulating factor injections and/or steroids to increase blood granulocyte numbers prior to donation [16]. With better supportive care over the past 10 years, the need for granulocytes in neutropenic patients with severe bacterial infections has decreased. Indications still remain for severe bacterial or fungal infections unresponsive to vigorous medical therapy in either newborns or older children with bone marrow failure or patients with neutrophil dysfunction. Newer mobilization schemes using G-CSF and steroids in donors result in granulocyte collections with at least 50 billion neutrophils. This may provide a better product for patients requiring granulocyte support.

Adverse Effects of Transfusion Transfusion of blood components can be associated with either acute or delayed complications. Acute transfusion reactions manifest themselves either during the transfusion of the unit or soon after the transfusion is completed, and reactions occurring within 24 hours after the start of the transfusion are included within this category. Delayed adverse complications are usually referred to as “post-­transfusion complications” and occur within days, months, or years after the transfusion [13, 20–22]. Signs and symptoms of acute transfusion reactions vary based on type of reaction but include skin manifestations such as urticaria or angioedema; respiratory symptoms such as wheezing, tachypnea, dyspnea, or hypoxia; cardiovascular symptoms such as hypotension, tachycardia, and shock; inflammatory signs and symptoms such as fever and rigors; or pain in the back or flank. In the event of a suspected transfusion reaction, the transfusion should immediately be stopped when such signs and symptoms occur in a patient being transfused. The patient should be

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urgently evaluated, and appropriate supportive care (oxygen, intravenous fluids, medications etc.) should be provided. Patients with mild allergic reactions limited to skin (i.e., urticaria only) can resume the transfusion with the same unit once the reaction subsides and the patient is stable. Further investigation is not necessary. For all other reactions, the involved unit should be returned to the blood bank for crossmatch and a microbiologic culture, and patient specimens should be obtained for a laboratory investigation. Such investigations aim to exclude mostly a hemolytic reaction (direct Coombs test, indirect bilirubin level, complete blood counts, plasma and urine hemoglobin), but a blood culture should also be obtained if bacterial infection is suspected.

Acute transfusion reactions include the following: ACUTE HEMOLYTIC TRANSFUSION REACTION: This reaction can occur as a result of a clerical error, where incompatible RBCs are transfused because of an error in sampling, crossmatch, or patient identification. It can also occur because of previously undetected alloantibodies in the recipient. The responsible antibody may be either immunoglobulin (Ig) M or immunoglobulin G (IgG) in the case of anti-Aor anti-B-related acute intravascular hemolysis. Complement-fixing IgG antibodies can also cause acute hemolysis in the case of anti-Kidd or anti-Kell antibodies. Hemolytic antibodies cause lysis of red blood cells through complement activation. This results in hemoglobinemia and hemoglobinuria. The complement activation also triggers the kinin-bradykinin system, releases anaphylatoxins, and causes release of further inflammatory cytokines. The severe storm of physiologic changes following a hemolytic transfusion reaction can cause fever and rigors; back, flank, or chest pain; nausea and vomiting; dark urine; respiratory difficulty; and hypotension which could progress to shock. The patient may complain of feeling “impending doom,” meaning feeling like he/she is going to die. The clinical picture can be confused with an infectious process due to fever or with an allergic reaction because of wheezing and shortness of breathing. Laboratory investigations show evidence of disseminated intravascular coagulation, acute renal failure, positive direct Coombs test, indirect hyperbilirubinemia, presence of free hemoglobin in the plasma and hemoglobinuria, and presence of schistocytes and spherocytes on the smear.

 ransfusion-Related Acute Lung Injury (TRALI) T and Transfusion-Associated Circulatory Overload (TACO) TRANSFUSION-RELATED ACUTE LUNG INJURY (TRALI) AND  TRANSFUSION-ASSOCIATED CIRCULATORY OVERLOAD (TACO): TRALI is caused by anti-neutrophil or anti-HLA (human leukocyte antigen) antibodies in the donor plasma that interact with the neutrophils in the recipient’s pulmonary circulation. This interaction results in neutrophil activation, altered vascular

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permeability, and capillary leak syndrome in the pulmonary capillaries. The end result is pulmonary edema. TRALI presents with respiratory distress associated with hypoxia within 6 hours after the start of transfusion, accompanied by radiographic findings of acute pulmonary edema. Other causes of pulmonary edema should be excluded to be able to make the diagnosis of TRALI. Clinically, physicians have to distinguish between TRALI and TACO, which has a similar clinical and radiographic presentation. TACO is associated with elevations in brain-­ natriuretic peptide (BNP) in the serum and responds promptly to diuretics and fluid restriction, whereas TRALI is associated with normal BNP levels and does not respond to diuretics or fluid administration. Acute hemolytic transfusion reaction, bacterial contamination and sepsis, and allergic reactions can also cause respiratory symptoms; therefore, a full transfusion reaction investigation needs to be undertaken in the patient who develops tachypnea, dyspnea, wheezing, or hypoxia during or soon after a transfusion. Blood culture should be sent from the patient, and the suspected unit (if available) should be sent to the blood bank for crossmatch and bacterial culture. In situations where other causes of respiratory distress have been excluded, there is a high probability of TRALI. In such situations, the diagnosis can be verified by testing the donor for antibodies against HLA or neutrophil antigens and testing the recipient for HLA and neutrophil antigen specificities. If an antibody is detected in the donor along with a corresponding antigen in the recipient, the diagnosis of TRALI can be confirmed. Patients who develop TRALI require respiratory support and critical care. Fortunately the event is self-limited, and the patient recovers within a few days in most cases. In a few cases, however, the patient dies of TRALI, which in fact is the number 1 cause of transfusion-related death in the United States. The donor implicated in a case of TRALI should be deferred from donations. Interestingly, TRALI occurs mostly in units with a large plasma content such as platelet units or FFP. Multigravid women donors have higher incidence of implicating antibodies, and exclusion of such donors can decrease the incidence of TRALI. In fact, some countries now collect only male plasma, and this practice has led to a fall in the incidence of TRALI.

Allergic Reactions ALLERGIC REACTIONS: are usually triggered by plasma proteins and thus are more common with FFP or platelet transfusions. Clinically, allergic reactions can be mild (with only urticarial skin reaction); moderate (respiratory symptoms secondary to angioedema of the airway or bronchospasm); or

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severe (hypotension and shock along with respiratory ± skin manifestations). Moderate to severe reactions are called “anaphylaxis” if truly mediated by IgE and “anaphylactoid reactions” if mediated through other antibodies. For example, IgG4 antibodies cause this reaction by binding to Fc receptors on mast cells and basophils. Mild allergic reactions are the most common transfusion reactions. As described above, STOP the transfusion and assess the patient in the event of an allergic reaction. If the reaction is limited to skin manifestations only and resolves upon treatment with antihistamines, the unit may be restarted at a slower rate. Note that mild reactions usually appear late during the course of the transfusion and are not generalized. If an urticarial reaction occurred immediately after start of a transfusion, it should not be considered as a “mild” reaction. Rapidity of onset predicts the severity of the reaction. Do not restart the unit if there is perioral swelling or laryngospasm, even if the urticarial rash has cleared. The treatment for moderate to severe allergic reactions includes epinephrine and steroids. 1:1000 epinephrine solution (1 mg/mL) is administered subcutaneously at a dose of 0.2 to 0.5 mL for adults and 0.1 mL for every 10 kg of body weight for children. The dose may be repeated every 15–30 minutes as needed. Patients who have had multiple reactions can be given premedications, such as hydrocortisone and antihistamines. Patients who have had an anaphylactic reaction should be investigated for IgA deficiency or haptoglobin deficiency. If such a deficiency is discovered, patients can be given transfusions from IgA deficient or haptoglobin-­deficient donors. If no such deficiency is discovered, and the patient is going to receive a cellular unit such as PRBC or a platelet unit, then the units should be “washed,” meaning the cells are suspended in a saline solution and the plasma is removed by centrifugation.

Febrile Non-hemolytic Transfusion Reaction (FNHTR) Fever is defined as temperature >38 °C or a rise in temperature >1 °C or >2 °F in the 4 hours after the start of transfusion. Shaking chills may accompany fever. In FNHTR, the patient does not have any other signs or symptoms, and specifically there is no hypotension, and laboratory investigations exclude bacterial contamination or hemolytic transfusion reaction. The cause of this reaction is cytokine accumulation in the plasma compartment of the unit. This was a common transfusion reaction in the past, but since the introduction of pre-storage leukocyte filtration, the incidence has decreased significantly. Treatment includes antipyretics.

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 ransfusion-Associated Graft Versus Host Disease T (TA-GVHD) TRANSFUSION-ASSOCIATED GRAFT VERSUS HOST DISEASE (TA-GVHD): This is a rare complication thanks to implementation of irradiation when indicated. The graft versus host disease reaction takes place particularly in the skin, bone marrow, liver, and gut, giving rise to symptoms and signs of fever, erythematous rash that progresses to desquamation, diarrhea, and liver dysfunction or failure. The mortality is as high as 90% after TA-GVHD [10].

Transfusion Avoidance Blood transfusion is a life-saving therapeutic modality, but like all therapeutic measures, it should only be used for appropriate indications. Blood transfusion is an expensive limited resource, associated with numerous adverse effects. Some patients and families may wish to avoid transfusion because of religious or personal beliefs. Therefore, the clinician should be familiar with transfusion-sparing strategies and should employ them in an effort to avoid or at least to minimize transfusion exposure [23]. Blood conservation in children undergoing surgery can be achieved through several modalities: 1. Preoperative hemoglobin level can be increased by using erythropoiesis-­ stimulating agents and iron supplementation, either orally or parenterally. 2. Autologous blood transfusion can be given to the patient. This involves preoperative autologous blood donation, reinfusion of shed blood during surgery, and acute normovolemic hemodilution. 3. Operative blood losses can be reduced using anti-fibrinolytic agents, such as tranexamic acid. 4. Rates of postoperative transfusion can be reduced by using transfusion algorithms.

Setting Evidence-Based Clinical Guidelines Hospital committees that oversee blood component use should set evidence-based guidelines to help clinicians decide when and how much to transfuse. For example, most children who are not actively bleeding and who are stable from a cardiopulmonary point can tolerate hemoglobin levels as low as 7 gm/dL. Increasing absolute reticulocyte counts can help anticipate erythropoietic recovery and can help avoid transfusion. If the anemia is related to the deficiency of a substance that can be easily replaced (iron, vitamin B12, erythropoietin, etc.), replacement of the deficient substance is preferable to transfusion.

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The amount that will be delivered should also be decided upon judiciously. Whenever possible, the patient should be exposed to as few units as possible. Consider the two cases below: • Case #1: A 15-year-old girl who presented to the hospital with syncope and was later found to have a hemoglobin level of 7.5 gm/dL is suffering from heavy menstrual bleeding. The patient still has active bleeding, is tachycardic, and is hypotensive. The patient has been started on intravenous fluids, intravenous estrogen, and intravenous iron. The attending clinician also wants to transfuse PRBC to this patient who weighs 50 kg. The calculation for 12 mL/kg of PRBC yields 600  mL.  The two matching PRBC units available at the hospital blood bank have 300 mL and 350 mL of volume each. • Case #2: A 15-year-old girl who is receiving chemotherapy for Hodgkin lymphoma was found to have a hemoglobin of 7.2 gr/dL with an absolute reticulocyte count of 10,000/mm3. The patient is clinically stable. She will be discharged home after a PRBC transfusion and is expected to return in a week for her subsequent chemotherapy visit. The patient weighs 50  kg. The calculation for 12 mL/kg of PRBC yields 600 ml. The hospital blood bank has two units available, one with a volume of 300 mL and the other 350 mL. The case #1 absolutely needs an acute increase in her hemoglobin level as she is symptomatic with active bleeding. Although the intravenous iron will increase the hemoglobin, a clinically significant rise will not occur until at least a week after the start of the iron treatment. The initial hemoglobin of 7.5 gr/dL may be an underestimate of the patient’s actual hemoglobin, which possibly is much lower. This patient should be given both units totaling 650 mL, and the hemoglobin should be monitored closely. The case #2 is clinically stable but has a low (50%). However, as a primary hematologic defect, it is not surprising that many other organ systems may also be affected at diagnosis including the cardiovascular (43%), neurologic (48%), pulmonary (46%), and gastrointestinal (37%) systems [19–24]. Disease pathogenesis of aHUS has been clearly correlated to the underlying genetic defect. The most common mutation, complement factor H (CFH), has been identified in up to 25% of children diagnosed with aHUS and can be used to illustrate this process [21]. An initiating event, more commonly of an infectious etiology but principally related to any underlying inflammatory response, triggers one of the complement pathways. The alternative pathway’s feedback mechanism expands the end-product of the cascade by producing multiple MAC complexes rapidly destroying the surface of complement-activated endothelial cells. A chain reaction ensues as more cells become involved. As a potent inhibitor of the complement alternative pathway, mutations in CFH reduce its capability to mitigate this amplification cascade causing unregulated complement activity. TMA develops as fibrin webs produced from the complement system’s interactions with the coagulation cascade begin damaging red blood cells (RBC) through mechanical sheer forces flowing through the microenvironment. Microthrombi rapidly form reducing platelet numbers ultimately occluding the vascular space causing distal anoxia and infarction perpetuating further damage and inflammatory stimulation through complement over activation (Fig. 32.2).

2 Lead to fibrin webs and resulting TMA 3 Microthrombi causing distal necrosis / inflammation

necrosi s 4 Distal necrosis & inflammation incite ffurther h complement l activation & TMA

Fig. 32.2  Thrombotic microangiopathy (TMA) development. (1) Complement activation leads to endothelial cell damage in the local microvascular space. (2) Platelet activation and thrombin activation result in thrombocytopenia and fibrin web formation damaging RBCs (schistocytes). (3) Local microthrombi occlude the vascular space causing distal infarction and further inflammation. (4) Distal necrosis and inflammation incite further complement expanding the TMA-affected environment

1 Complement activation and endothelial damage

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Described in 1981, CFH deficiency was the first of many subsequent deficiencies and mutations identified contributing to complement dysregulation syndrome phenotype [20, 21]. Other mutations associated with aHUS have been described including singular and combined mutations of CFI, THBD, C3, CFBD, MCP, and DGKE [20, 21]. Together they still characterize only 30–40% of patients diagnosed with aHUS. Screening for genetic mutations at presentation is not recommended as the results are generally not readily available and a negative result cannot alter therapeutic decision-making. Genetic screening is recommended for patients after excluding alternative diagnoses, in patients with relapsed disease, familial history of disease, pregnancy or postpartum-disease-related complications, posttransplantation disease, and prior to organ transplantation planned for patients with aHUS [18]. Such characterization is necessary to establish prognostic risk and progression potential, procreative genetic counseling to parents and family members, as well as organ transplant preparation. Current genetic testing technologies primarily utilize next-generation sequencing analysis. Compared to other options, it remains cost-­ effective and allows for the simultaneous study of all potentially relevant genes. Use of whole exome sequencing has been reported but remains limited to research laboratories [18]. Due to diagnostic limitations, aHUS has remained a diagnosis of exclusion with an ever-widening differential of diseases presenting with TMA (MAHA, thrombocytopenia, organ damage). Complement levels have not proven to be useful as diagnostic tools in this disease and tissue biopsy has only served to confirm the presence of TMA. As noted above, STEC HUS can easily be ruled out using fecal immunological assays for Shiga toxin. Thrombotic thrombocytopenic purpura (TTP) can be ruled out through ADAMTS13 assays with >10% activity levels. Cobalamin C defects can be ruled out through the examination of the complete blood count (CBC) noting megaloblastosis, with serum elevations in homocysteine, and urine elevations in methylmalonic acid (MMA) [18, 25]. Atypical HUS with coexisting disease may need to be considered in the setting of autoimmune-related disorders such as systemic lupus erythematosus (SLE), malignancy, and hematopoietic stem cell transplantation [18]. TMA presenting during pregnancy or postpartum presents another interesting dilemma. Approximately one-third of women with hemolysis, elevated liver enzymes, and low platelet count (HELLP) syndrome have an identified complement mutation, suggesting a true underlying complement dysregulation syndrome, and up to 86% of females presenting with TMA during pregnancy have a complement mutation as well [26, 27]. Anti-CFH antibodies have also been identified contributing to aHUS presentations. Assays for anti-CFH are recommended for all patients to differentiate this disease variant [18]. Prior to the widespread use of monoclonal complement inhibition, therapeutic interventions for aHUS have relied on empiric immunosuppressive therapies and use of plasma exchange or plasma infusion (PE/PI). Other reported research trials have included double liver–kidney organ transplantation [18]. Eculizumab, a monoclonal humanized anti-C5 antibody, has transformed therapeutic management of complement-mediated disease processes. Eculizumab prevents C5 cleavage and the formation of C5a and MAC inhibiting complement activity despite upstream

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dysregulation [28]. Distal effects include reduction of the C5a proinflammatory and MAC prothrombotic contributions to the disease process. In multiple clinical trials, eculizumab has proven benefit over PE/PI by halting disease progression and in cases of early initiation, improvement in end organ dysfunction [29–32]. These results have led to federal drug administration (FDA) approval for eculizumab as the primary treatment for pediatric patients with aHUS.

Pneumococcal Hemolytic Uremic Syndrome Another rare though emerging pediatric disease presenting with TMA is that of HUS associated with Streptococcus pneumonia (spHUS). Accounting for 5–15% of all HUS cases, the epidemiology of spHUS has changed with the introduction of new vaccines [33, 34]. Prior to the use of the 7-valent pneumococcal conjugate vaccine, the predominant serotypes associated with spHUS were 14, 6B, 9 V, 19, 3, 8 and 23F [34–37]. Following the introduction of this vaccine, a relative increase in cases of spHUS associated with strains not covered by the vaccine was observed with serotypes 3, 6A, 12F, and 19A [34]. Similarly, in another study a significant rise in serotype 19A infections was observed from 1% in the pre-vaccine era to 20% in the post-vaccine era [34]. This observation suggests the etiology of spHUS may not be serotype specific but more related to the emergence of new invasive non-­ vaccine-­covered serotypes. Recent reports have also suggested that the incidence of spHUS seems to be increasing. However, it is unknown whether this reflects a true increase in number of new cases or an increasing awareness of spHUS. Globally, the epidemiology of spHUS varies as well noting that the Taiwanese population identified spHUS much more frequently than the more common STEC HUS [36–38]. Generally, children under 2 years of age have the highest incidence of spHUS with associations of pneumonia with or without effusion/empyema in 65–92% of cases [36–39]. Meningitis, the second highest incidence, was also associated with more severe cases [36–38]. Reported cases recount a typical clinical course of spHUS developing 3–13 days after pneumococcal infection [33, 35–38]. Suppurative diseases with heavy bacterial loads have also been suggested to increase the risk of developing spHUS [33, 35–38]. Compared with STEC HUS, patients with spHUS were noted to have more severe presentations and longer durations of complications. Involvement by other organ systems including the pancreas, liver, and heart have also been reported [33, 35–38]. The diagnosis of spHUS includes findings of pneumococcal infection in the setting of TMA (MAHA, thrombocytopenia, and organ injury) but can be challenging due to the similarities in clinical and laboratory findings with disseminated intravascular coagulation (DIC). Patients with DIC, however, will tend to have more pronounced coagulopathies and abnormal fibrinogen levels [33]. The Canadian Pediatric Society has defined spHUS cases by evidence of S. pneumonia infection, evidence of HUS, and the presence of thrombotic microangiopathy on biopsy [33]. Other classification schemes have been proposed, which include probable and

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possible case definitions primarily defined by the presence of a positive direct antiglobulin (DAT) test which has been shown to be very sensitive (90%) but less specific in identifying spHUS [33]. Disease pathogenesis of spHUS remains unknown. Several proposed theories, however, actively involve direct complement interaction. The most accepted theory currently involves a cryptantigen named the Thomsen–Friedenreich antigen. This normally hidden antigen is exposed by the S. pneumonia neuraminidase. Once exposed from the cell surface, it can interact with preformed IgM antibodies leading to the TMA phenotype [33]. Another theory on the pathogenesis of spHUS involves the disruption of factor H binding sites by neuraminidase resulting in complement dysregulation and overactivation [40]. Other studies have also suggested that factor H may be directly inhibited by different serotypes of pneumococcus affecting complement regulation [41]. Current treatment for spHUS remains supportive. As with STEC HUS, management includes maintaining fluid and electrolyte balance, nutrition, and the treatment of underlying infections. The American Academy of Pediatrics (AAP) specifically recommends antibiotic (vancomycin and an extended spectrum cephalosporin) use in invasive pneumococcal infections until specific antibiotic sensitivities are identified [33, 37]. Due to a concern that anti-Thomsen-Friedenreich antibodies may be present in donated blood products, washed blood products are generally recommended to be used whenever possible to theoretically prevent any disease aggravation by their potential presence [33, 37]. Similarly, transfusions of fresh frozen plasma should be avoided unless there is active bleeding. Plasmapheresis has also been advocated as a potential treatment modality for spHUS. Theoretical removal of neuraminidase or anti-Thomsen-Friedenreich antibodies with factor replacement is one of the potential benefits of this intervention [33, 37]. Data are, however, extremely limited with variable results from small case studies in this patient population. In contrast to STEC HUS, spHUS has a higher mortality rate of 2–12% of patients noting that those with meningitis are at a much higher risk [33, 35–38]. Surviving patients may also have long-term complications such as end-stage kidney disease (10–16%) [33, 35–38].

Paroxysmal Nocturnal Hemoglobinuria Like aHUS, paroxysmal nocturnal hemoglobinuria (PNH) is another disease model of complement dysregulation. PNH is an acquired hematopoietic stem cell disorder characterized by sensitivity of the stem cell progeny to destruction by complement pathways with resultant clinical complications. The prevalence of PNH has been estimated at 15.9/ million individuals [42, 43]. More commonly observed in older individuals (20–50 years), pediatric PNH (5–18 years) is extremely rare accounting for 10% of reported cases [42–44]. The protein encoded by the PIG-A gene is necessary for the synthesis of the GPI anchor essential for various cell surface proteins. Two inhibitors of complement, CD55 and CD59, also require GPI anchors for their

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Table 32.1  International PNH Interest Group classification with management recommendations Category Classical PNH

Rate of hemolysis High

Bone marrow status Normocellular to hypercellular with normal morphology

Clone size High (>50%)

PNH/BMF

Mild

Concomitant bone marrow failure syndrome

Variable (