Contents

Cover

Companion Website

Title Page

Copyright

Preface to the first edition

Preface to the ninth edition

Chapter 1: An introduction to haematopoiesis

Where is blood formed?

Haemopoietic stem cells

Differentiating blood cells

Myelopoiesis

Lymphopoiesis

Summary

Chapter 2: Anaemia: General principles

Anaemia

Symptoms and signs of anaemia

The normal control of red cell production

Morphological classification of anaemias

Microcytic anaemia: Iron handling and iron deficiency anaemia

Normocytic anaemia

Macrocytic anaemia

Polycythaemia (erythrocytosis)

Chapter 3: Haemolytic anaemias

Laboratory evidence of haemolysis

Clinical features of haemolysis

Classifying haemolytic anaemias and establishing a diagnosis

Congenital haemolytic anaemias

Acquired haemolytic anaemias

Chapter 4: Disorders of globin synthesis

Normal structure and function of haemoglobin

Thalassaemia

Structural haemoglobin variants

Chapter 5: Conditions associated with white cell abnormalities

Leucopenia

Leucocytosis

Lymphocytosis and lymphopenia

Flow cytometry: A technique for identification of cells in suspension

Infectious mononucleosis

Leucoerythroblastic reaction

Chapter 6: Structure and function of lymphoid tissue

Lymph node structure

Immunoglobulin structure and gene rearrangement

Natural killer cells

Cellular origin of lymphomas

Chapter 7: Lymphomas: General principles

Lymphomas versus leukaemias

Lymphomas

Chapter 8: Classification of lymphoma

Chapter 9: Neoplastic disorders of lymphoid cells

Hodgkin lymphoma

Non-Hodgkin lymphomas

Chapter 10: Myeloma and other paraproteinaemias

Multiple myeloma

Other paraproteinaemias and related disorders

Chapter 11: Neoplastic disorders of myeloid cells

Acute myeloid leukaemia

The myelodysplastic syndromes

Myeloproliferative disorders

Chapter 12: Bone marrow transplantation

Allogeneic bone marrow transplantation

Graft-versus-leukaemia/lymphoma effect

Mini-allograft or RIC transplant

Autologous bone marrow transplantation (high-dose therapy)

Chapter 13: Aplastic anaemia and pure red cell aplasia

Aplastic anaemia

Pure red cell aplasia

Chapter 14: Haemostasis, abnormal bleeding and anticoagulant therapy

Normal haemostasis

Classification of haemostatic defects

Platelets

Thrombocytopenic and non-thrombocytopenic purpura

Causes of thrombocytopenia

Abnormalities of platelet function

Platelet transfusions

Normal coagulation mechanism

The fibrinolytic mechanism

Tests for clotting defects

Congenital coagulation disorders

Acquired coagulation disorders

Anticoagulant drugs

Investigation of a patient with abnormal bleeding

Natural anticoagulant mechanisms and the prothrombotic state (thrombophilia)

Chapter 15: Blood groups and blood transfusion

Blood groups

Haemolytic disease of the newborn

Blood donation and blood components

Management of transfusion reactions

Massive transfusion

Chapter 16: Further reading

General reference books

Chapter 1 An introduction to haematopoiesis

Chapter 2 Anaemia: General principles

Chapter 3 Haemolytic anaemias

Chapter 4 Disorders of globin synthesis

Chapters 5 to 13 Malignant haematology, aplastic anaemia and pure red cell aplasia

Chapter 14 Haemostasis, abnormal bleeding and anticoagulant therapy

Chapter 15 Blood groups and blood transfusion

Index

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Library of Congress Cataloging-in-Publication Data

Lecture notes. Haematology / Nevin C. Hughes-Jones ... [et al.]. — 9th ed.
p. ; cm.
Haematology
Rev. ed. of: Lecture notes. Haematology / N.C. Hughes-Jones, S.N. Wickramasinghe, C.S.R. Hatton. 8th ed. 2009.
Includes bibliographical references and index.
ISBN 978-0-470-67359-1 (pbk. : alk. paper)
I. Hughes-Jones, N. C. (Nevin Campbell) II. Title: Haematology.
[DNLM: 1. Hematologic Diseases. 2. Blood Physiological Processes. WH 120]
616.1'5—dc23

2012031978

A catalogue record for this book is available from the British Library.

Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic books.

Cover image: iStock © Activ Design

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Preface to the first edition

These lecture notes are designed to supply the basic knowledge of both the clinical and laboratory aspects of haematological diseases and blood transfusion. The content is broadly similar to that of the course given to medical students by the Department of Haematology at St. Mary's Hospital Medical School. References have been cited so that those who need to extend their knowledge in any particular field can do so. Most of the journals and books that are mentioned are those commonly found in every library.

At the end of each chapter I have supplied learning objectives in studying each disease. There are two main purposes in these objectives. First, they facilitate the learning process, since the process of acquisition, retention, and recall of data is greatly helped if the facts and concepts are centred around a particular objective. Secondly, many objectives are closely related to the practical problems encountered in the diagnosis and treatment of patients. For instance, the following objectives: “to understand the method of differentiation of megaloblastic anaemia due to vitamin B12 deficiency from that due to folate deficiency” and “to understand the basis for the differentiation of leukaemia into acute and chronic forms based on the clinical picture and on the peripheral blood findings” are practical problems encountered frequently in the haematology laboratory. A point of more immediate interest to the undergraduate is that examiners setting either multiple choice or essay questions will be searching for the same knowledge that is required in answering the objectives.

I should like to thank Prof. P.L. Mollison, Dr. P. Barkhan, Dr. I. Chanarin, Dr. G.J. Jenkins, and Dr. M.S. Rose for their criticism and helpful suggestions during the preparation of the manuscript and Mrs. Inge Barnett for typing the several drafts and final typescript.

N.C. Hughes-Jones

Preface to the ninth edition

The science and practice of haematology continue to advance at an extraordinary rate. At the same time, the volume of data that medical students are required to assimilate across all disciplines continues to expand. Therefore, our aim in revising the text of Lecture Notes in Haematology was to provide a comprehensive overview of this diverse subject in a way that promotes understanding of pathophysiological concepts, while highlighting the most up-to-date aspects of clinical practice. Further, to enhance active learning, we have also provided a companion website with self-test multiple-choice questions and explanations.

Dr Sunil Wickramasinghe was actively involved in Lecture Notes from its inception in the 1970s until the eighth edition. His tragically premature death in 2009 deprives us of a senior author and highly valued colleague. His contribution has been greatly missed.

We have been fortunate in having the help of many of our clinical colleagues for this edition: special thanks are due to Dr Karthik Ramasamy and Dr Adam Mead, both of Oxford University Hospitals, for kindly reviewing the chapters on myeloma and myeloid malignancies respectively. As in previous editions, we also express our thanks to Professor Kevin Gatter of the Nuffield Department of Clinical and Laboratory Sciences, University of Oxford, for generously providing many of the photomicrographs used in this text.

As ever, we are grateful to readers who have taken the time to give us valuable feedback on earlier editions. We hope that the ninth edition of Haematology Lecture Notes provides a useful introduction to this fascinating area of medicine.

Chris S. R. Hatton
Nevin C. Hughes-Jones
Deborah Hay
David Keeling

1

An introduction to haematopoiesis

Learning objectives

To understand the process of formation of blood cells

To understand the concept of a stem cell

To appreciate the process of lineage specification of blood cells

To recognize the different types of mature blood cell

To understand the normal role of each mature cell type in the blood

Where is blood formed?

Blood is normally formed early in the process of embryogenesis, with haemopoietic stem cells originating in the para-aortic mesoderm of the embryo. Primitive red blood cells, platelet precursors and macrophages are initially formed in the vasculature of the extra-embryonic yolk sac, before the principal site of haemopoiesis shifts to the fetal liver at around five to eight weeks' gestation. The liver remains the main source of blood in the fetus until shortly before birth, although the bone marrow starts to develop haemopoietic activity from as early as 10 weeks' gestation.

After birth, the marrow is the sole site of haemopoiesis in healthy individuals. During the first few years of life, nearly all the marrow cavities contain red haemopoietic marrow, but this recedes such that by adulthood haemopoiesis is limited to marrow in the vertebrae, pelvis, sternum and the proximal ends of the femora and humeri, with minor contributions from the skull bones, ribs and scapulae.

Although the sites of haemopoiesis in the adult are therefore relatively limited, other sites retain their capacity to produce blood cells if needed. In conditions in which there is an increased haemopoietic drive (such as chronic haemolytic anaemias and chronic myeloproliferative disorders), haemopoietic tissue will expand and may extend into marrow cavities that do not normally support haemopoiesis in the adult. Foci of haemopoietic tissue may also appear in the adult liver and spleen (known as extramedullary haemopoiesis).

Haemopoietic stem cells

The process of haemopoiesis involves both the specification of individual blood cell lineages and cellular proliferation to maintain adequate circulating numbers of cells throughout life. This is accomplished using the unique properties of haemopoietic stem cells.

Long-term haemopoietic stem cells (HSCs) in the bone marrow are capable of both self-renewal and differentiation into the progenitors of individual blood cell lineages. The progenitor cells of individual lineages then undergo many rounds of division and further differentiation in order to yield populations of mature blood cells. This process can be represented as a hierarchy of cells, with HSCs giving rise to populations of precursor cells, which in turn give rise to cells increasingly committed to producing a single type of mature blood cell (Figure 1.1). Thus, the immediate progeny of HSCs are the multipotent progenitor cells, which have limited self-renewal capacity but retain the ability to differentiate into all blood cell lineages. Although there is still debate about exactly how lineage restricted subsequent precursors are, the concept of sequential and irreversible differentiation is widely accepted. In Figure 1.1, the HSC is seen giving rise to two major lineages: the lymphoid lineage, in which a common lymphoid progenitor gives rise to B and T cells; and a myeloid lineage, with a common myeloid progenitor giving rise to red cells, granulocytes and platelets. The division of haemopoiesis into myeloid and lymphoid compartments is fundamental to an understanding of haematological disease.

The process of haemopoiesis outlined above has several advantages. First, it permits the massive expansion of cell numbers needed to maintain an adequate population of mature blood cells. It also means that the production of each type of mature blood cell can be controlled individually, tailoring production to specific physiological requirements. Finally it requires relatively little proliferative activity on the part of the long-term HSCs themselves, thereby minimizing the risk of developing mutations in these crucial cells during DNA replication and cell division.

HSCs were first detected and defined functionally through experiments in which a subset of cells from the bone marrow were shown to produce blood cells of all lineages when transplanted into lethally irradiated mice, which have no haemopoietic potential of their own. Subsequent work has used cell surface markers and flow cytometric techniques (see Chapter 5) to define this population: positivity for the cell surface marker CD34 combined with negativity for CD38 describes a population of cells that is also capable of regenerating all cell lineages from the bone marrow. The cell surface marker CD34 is also used to isolate cells with multipotency and self-renewal capacity for stem cell transplantation.

Differentiating blood cells

Precisely how the ultimate lineage choice of differentiating progenitor cells is determined remains a subject of research. It has been argued that factors intrinsic to the HSC itself, such as stochastic fluctuations in transcription factors, may direct lineage specification. However, it is also known that proper regulation of HSCs and progenitor cells requires their interaction with extrinsic factors, such as non-haemopoietic cells in the bone marrow niche (e.g. endothelial cells and osteoblastic progenitors). HSCs and progenitor cells are not randomly distributed in the marrow, but exist in ordered proximity relative to mesenchymal cells, endothelial cells and the vasculature. Signalling from these non-haemopoietic cells, plus physiochemical cues such as hypoxia and blood flow, are therefore likely to influence the transcriptional activity and fate of HSCs.

Myelopoiesis

Signalling through myeloid growth factors such as granulocyte-macrophage colony stimulating factor (GM-CSF) is essential for the survival and proliferation of myeloid cells. The specification of the myeloid lineage is also known to require the interaction of a series of specific transcription factors, including C/EBPα, Core Binding Factor and c-Myb. As well as being essential for the normal formation of myeloid cells, it is becoming clear that an appreciation of these factors and others like them is critical for an understanding of myeloid diseases such as acute myeloid leukaemia (see Chapter 11).

The separation of the erythroid and megakaryocytic components of myelopoiesis requires the action of transcription factors GATA1, NF-E2 and SCL, and signalling through the growth factors thrombopoietin and erythropoietin.

Granulocytes and their function

Morphologically, myeloblasts are the earliest recognizable granulocytic cells. They are large cells, with open nuclear chromatin (Figure 1.2(a)). The successive stages through which a myeloblast matures into circulating neutrophil granulocytes are termed promyelocytes (Figure 1.2(b)), neutrophil myelocytes (Figure 1.2(c)), neutrophil metamyelocytes and neutrophil band cells. Cell division occurs in myeloblasts, promyelocytes and myelocytes, but not normally in metamyelocytes and band cells.

The maturation process of the neutrophil lineage is characterized by a reduction in size of the cell, along with the acquisition of granules containing agents essential for their microbicidal function. The nucleus also gradually begins to adopt its characteristic segmented shape (Figure 1.3).

Mature neutrophils have the ability to migrate to areas of inflammation (chemotaxis), where they become marginated in the vessel lumen and pass into the tissues through interaction with selectins, integrins and other cell adhesion molecules. Once primed by cytokines such as TNFα and IFNγ, neutrophils are able to phagocytose opsonized microbes, and destroy them by deploying their toxic intracellular contents. This release of reactive oxygen species (the ‘respiratory burst') provides a substrate for the enzyme myeloperoxidase (MPO), which then generates hypochlorous acid with direct cytotoxic effects. The granules of neutrophils also contain an array of antimicrobial agents, including defensins, chymotrypsin and gelatinases.

Eosinophils (a subset of granulocytes with bright pink granules on haematoxylin and eosin-stained blood films) have a similar ability to phagocytose and destroy micro-organisms, but are classically associated with the immune response to parasitic infection. They are often found in high numbers in patients with allergy and atopy. IL-5 signalling appears to be critical for their differentiation from granulocyte precursors.

Basophils are the least common of the granulocytes. They contain very prominent cytoplasmic granules on H&E staining, which have stores of histamine and heparin as well as proteolytic enzymes. They are involved in a variety of immune and inflammatory responses, but it is unusual to see a marked elevation or depression in their numbers in specific reactive conditions.

Monocytopoiesis and monocyte function

The cell classes belonging to the monocyte–macrophage lineage are, in increasing order of maturity, monoblasts, promonocytes, marrow monocytes, blood monocytes and tissue macrophages. Their synthesis is controlled in part by the activity of GM-CSF. Functionally, monocytes have a variety of immune roles: as the precursors of tissue macrophages and dendritic cells, their roles include phagocytosis, antibody presentation to other immune cells, and a contribution to the cytokine milieu. Phagocytosis of micro-organisms and cells coated with antibody (with their exposed Fc fragments) and complement occurs via binding to Fc and C3b receptors on the surface of monocytes and macrophages. Bacteria and fungi that are not antibody coated are phagocytosed after binding to mannose receptors on the phagocyte surface. As with neutrophils, the killing of phagocytosed micro-organisms by monocytes/macrophages involves superoxide dependent and O2- independent mechanisms.

Megakaryocytes and platelet function

Megakaryocytes are the cells which give rise to platelets. During megakaryocyte formation, driven by the action of the growth factor thrombopoietin (TPO), there is replication of DNA without cell division. This leads to the generation of very large mononucleate cells that are markedly polyploid. A mature megakaryocyte is illustrated in Figure 1.4. Large numbers of platelets are formed from the cytoplasm of each mature megakaryocyte; these are rapidly discharged directly into the marrow sinusoids. The residual ‘bare' megakaryocyte nucleus is then phagocytosed by macrophages.

TPO is the key regulator of normal platelet production. This protein, which is produced by the liver, binds to TPO receptors on the megakaryocyte membrane. Downstream signalling through mechanisms including the JAK/STAT pathway allows an increase in megakaryocyte ploidy, and also cytoplasmic maturation such that increased numbers of platelets are released. TPO is also able to bind to the surface of platelets themselves; thus when platelet numbers are high, TPO is sequestered on the platelet membranes, leaving less available to act on the megakaryocytes to promote further platelet production. In this way, a negative feedback loop is created, maintaining platelet numbers within stable limits.

The fundamental role of platelets is in primary haemostasis, through their interactions with von Willebrand factor and the exposed collagen of damaged endothelial surfaces (see Chapter 14).

Erythropoiesis and red cell function

The specification of the erythroid lineage requires a balanced interaction between transcription factors GATA1 and other haemopoietic transcription factors, including PU.1 and FOG1. Once committed to an erythroid fate, the expansion of erythroid precursors takes place, driven largely by signalling through the erythropoietin receptor.

The hormone erythropoietin is expressed principally in the cortical interstitial cells of the kidney, where its transcription is modulated in response to hypoxaemia. The transcription factor hypoxia inducible factor (HIF-1) is induced in cells exposed to hypoxaemic conditions enhances expression of the erythropoietin gene. Increased levels of erythropoietin are therefore available to interact with the Epo receptor on red cell progenitor membranes, activating an erythroid-specific signal transduction cascade, and leading to enhanced proliferation and terminal differentiation of erythroid cells.

Morphologically, the differentiation and maturation of erythroid cells are shown in Figure 1.5. Pro-erythroblasts are early erythroid progenitors in the bone marrow recognizable by their large size, their dark blue cytoplasm, their dispersed nuclear chromatin and nucleoli. As the cells mature, they become smaller with less basophilic cytoplasm (see Figure 1.5). Cell division continues until the cells reach the late polychromatic normoblast stage, when cells extrude their nucleus. At this point the cell is termed a reticulocyte (Figure 1.6) and is released from the marrow into the peripheral blood. Reticulocytes are characterized by their slightly larger size and bluish staining contrasted with mature red cells. After one to two days in circulation, reticulocytes lose their remaining ribosomes and become mature red cells.

The red cell function is to carry oxygen, bound to the haem moiety of haemoglobin, from the lungs to the peripheral tissues. The details of haemoglobin structure and function (and diseases resulting from perturbation of these) are discussed further in Chapter 4.

Lymphopoiesis

The structure and function of lymphoid tissue are the focus of Chapter 6. Lymphoid cells are thought to arise from multilymphoid progenitor cells in the fetal marrow. Although incompletely characterized, these progenitors are known to feature CD45 and CD7 cell surface markers. The transcription factor Ikaros has been shown to be critical for lymphopoiesis in mouse models; Pax5 is among several transcription factors needed for B cell development, while GATA3 and Notch signalling are essential for T cell maturation.

The development of B lymphocytes commences in the fetal liver and fetal marrow. Here, progenitor B cells develop into pre-B cells (defined by the presence of the cytoplasmic μ chain of the B-cell receptor) and then into mature B cells. During this time, the genes for the immunoglobulin light and heavy chains are rearranged, allowing the production of immunoglobulins with a wide array of antigenic specificities. Subsequent B cell maturation requires antigen exposure in the lymph nodes and other secondary lymphoid tissues, with the mature B cell having the capacity to recognize non-self antigens and produce large quantities of specific immunoglobulin.

T cells, by contrast, are formed in the thymus, where lymphocyte progenitors from the fetal liver migrate in early gestation. These earliest immature T cells express neither CD4 nor CD8 and undergo rearrangement of the T cell receptor genes to permit cell surface expression of the T cell receptor (TCR). As with the surface immunoglobulin or B cell receptor, the process of rearrangement yields a vast collection of potential T cell receptors, with the ability to recognize a wide range of different antigens. During the process of maturation, T cells acquire both CD4 and CD8 cell surface markers (double positive thymocytes) and undergo a process of positive selection to ensure that the survival only of those that are able to interact adequately with MHC molecules on antigen-presenting cells. T cells that interact with MHC Class I become CD8 positive only, while those that interact with MHC class II down-regulate their CD8 expression and become CD4 T cells. A further phase of negative selection ensures that T cells that interact very strongly with ‘self-antigens' in the thymus undergo apoptosis.

CD4+ lymphocytes are known as T ‘helper' cells, and they form the majority of the circulating T cell population. Their roles include the production of cytokines to promote an inflammatory response in presence of the appropriate antigen. Such cytokines include interferon γ (from the Th1 class of CD4+ cells) and interleukins 4, 5 and 13 (from the Th2 subset of CD4+ cells). The effects of cytokine production include activation of the monocyte/macrophage system, the promotion of granulocyte maturation and the induction of antibody synthesis by B cells.

CD8+ lymphocytes are T suppressor/cytotoxic cells, comprising approximately one quarter of the T cells in the peripheral blood. Their function is to destroy any cells expressing a peptide to which their T cell receptor can bind (e.g. virally infected cells).

A small minority of mature lymphocytes are distinct from both B and T cell lineages. These are the natural killer (NK) cells, which have a role in the innate immune system, through cell-mediated cytotoxicity.

All these stages of both B and T cell development have the morphological features of either lymphoblasts or lymphocytes. The identification of different lymphocyte precursors is therefore based not on morphology but on properties including reactivity with certain monoclonal antibodies, their immunoglobulin gene or TCR gene rearrangement status, and the presence of immunoglobulin or TCR on the surface membrane (Tables 1.1 and 1.2). In the peripheral blood, lymphocytes may be small and compact (Figure 1.7) or may appear large with azurophilic cytoplasmic granules (Figure 1.8). Such large granular lymphocytes include cytotoxic T cells and NK cells.

Table 1.1 The sequence of events during B-cell differentiation.

Table 1.2 The sequence of events during T-cell differentiation.

Summary

Table 1.3 summarizes the role of each mature cell type in the peripheral blood. It is the abnormal production, function or destruction of these cells that constitutes the study of clinical haematology, and that forms the basis of the rest of this text.

Table 1.3 The main functions of blood cells.