Haematology Made Easy

© 2013 Dr Erhabor & Dr Adias. All Rights Reserved.

No part of this book may be reproduced, stored in a retrieval system,

or transmitted by any means without the written permission of the author.

Published by AuthorHouse 02/06/2013

ISBN: 978-1-4772-4652-8 (sc)

978-1-4772-4651-1 (e)

Library of Congress Control Number: 2012922985

Any people depicted in stock imagery provided by Thinkstock are models,

and such images are being used for illustrative purposes only.

Certain stock imagery © Thinkstock.

Because of the dynamic nature of the Internet, any web addresses or links contained in this book may have changed since publication and may no longer be valid. The views expressed in this work are solely those of the author and do not necessarily reflect the views of the publisher, and the publisher hereby disclaims any responsibility for them.

Preface

Haematology is the science encompassing the medical study of blood and blood-producing organs. It is the study of blood, its formation and the investigation and treatment of disorders affecting the blood and the bone marrow. It is the branch of medical science concerned with diseases of the blood and blood-forming tissues. It is commonly divided into three sub-areas, according to the type and group of blood cells involved (red cells, white cells and platelets). Study of red cells disorders includes; anaemia, haemoglobinopathies. White blood cells disorders includes; leukaemia, neutropenias, myeloproliferative disorders, myelodysplastic syndromes and lymphomas while platelet and coagulation studies involves; bleeding, coagulation disorders and thrombo-embolic disorders. Common tests used in the investigation of haematological conditions include; full blood counts, blood films, bone marrow biopsies. Treatment for haematological conditions includes; medication, blood transfusion, venesection (for iron overload or polycythemia), bone marrow transplant (for leukemia and myelofibrosis), chemotherapy and radiotherapy (for leukemia). The aim of this book is to attempt to make the science of Haematology as basic and simple as possible to steer up the interest of biomedical, medical and students undertaking courses allied to medicine in the science of Haematology particularly in developing countries.

Dr Erhabor Osaro

Associate Professor of Haematology and Transfusion Medicine

Usmanu Danfodiyo University Sokoto

Acknowledgements

Our sincere thanks goes to AuthorHouse Publishers, for their assistance and contribution to the publication of this book; to our father in the Lord, Bishop David O. Oyedepo for being an inspiration in our lives through his teachings; to Pastor David Oladosu, Pastor Cyriacus Ekweme and Pastor T Davies for their spiritual oversight; to Chief Aibangbee Erhabor and Mrs Rose Erhabor; to Prof D.E. Agbonlahor, Prof O.A. Emeribe, Prof E.K. Uko, Prof O.A. Ejele, Prof C.A. Nwauche, Prof A. Ojule, Prof C. Akani, Prof S.D. Abbey, Prof R.A. Shehu, Prof L.S. Bilbis, Prof J Lori, Dr E.A.D. Alikor, Dr FI Buseri, Dr C Onwuka, Dr ZA Jeremiah, Dr A. Mainasara, Dr M.K. Dallatu, Dr H. Opurum, Dr Sonny Chinenye, Dr Mark Grey, Dr Clare Barnes, Mr David Hamer, Katie Adamson, Mr Peter Kinsella, Sharran Grey, Imran Mohammed, Cassie Thornton, Neil Fuller, Karen Farrar and Obioma Azuonwu for all their support and encouragement over the years. Our sincere thanks go to our families and friends for the encouragement while we wrote this book that will help build a culture of continuous quality improvement and create a quality consciousness among all those involved in diagnostic service delivery in the field of haematology worldwide. To the Almighty God alone be all the glory.

Contents

Chapter 1 Reference ranges in Haematology

Chapter 2 Haematopoiesis

Chapter 3 Differential Leucocyte Count (DLC)

Chapter 4 Haemostasis, platelet and blood coagulation

Chapter 5 Thrombosis, thromboembolic diseases and antithrombotic treatment

Chapter 6 Platelet-related disorders

Chapter 7 Anticoagulation and anticoagulant therapy

Chapter 8 Anaemia

Chapter 9 Iron deficiency anaemia (IDA)

Chapter 10 Haemolytic anaemia

Chapter 11 Megaloblastic (pernicious) anaemia

Chapter 12 Anaemia of chronic disease or disorders

Chapter 13 Sideroblastic anaemia

Chapter 14 Acute Haemorrhagic anaemia

Chapter 15 Aplastic anaemia and Bone marrow failure

Chapter 16 Haemochromatosis

Chapter 17 Genetic disorders of Haemoglobin (Haemoglobinopathies)

Chapter 18 Leukaemia

Chapter 19 Acute Leukaemia

Chapter 20 Hairy Cell Leukaemia

Chapter 21 Chronic leukaemia

Chapter 22 Myelopolifearative disorders

Chapter 23 Myelodysplastic syndromes

Chapter 24 Paraproteinemias (myeloma and related disorders)

Chapter 25 Lymphoma

Chapter 26 Spleen and splenomegaly syndrome

Chapter 27 Pregnancy and paediatric haematology

Chapter 28 Bone marrow and Haematological diagnosis

Chapter 29 Health and safety issues associated with working in the Haematology laboratory

Chapter 30 Immunodeficiency disorders and AIDS

Chapter 31 Haemoparasites

About the authors

Chapter 1

Reference ranges in Haematology

Reference ranges for blood tests are sets of values used to interpret a set of diagnostic test results from blood samples. A reference range is usually defined as the set of values in which 95 percent of the normal population falls within. It is determined by collecting data from vast numbers of laboratory tests result from a large number of people in a population. Reference ranges are usually given as what are normal values found in the population. Several factors can potentially affect haematological values of an individual; timing of sample collection, containers in which they are collected, mode of transportatation, storage prior to testing, posture (mobile or confined to the bed), age, gender, environment (altitude), stress level, occupation, genetic constitution and diet. It is vital that these factors are taking into consideration in establishing reference ranges for a population. It is difficult to prove that subjects that are defined as healthy may not have infections, parasitic infection and nutritional deficiency or that they are not in a high risk group (alcoholics and smokers). For this reason, it is more feasible to talk of reference values and limits rather than normal ranges. With reference values, variables are defined when establishing the values for the reference population. This allows an individual’s result to be interpreted against a comparable normal. Reference ranges are prediction intervals between which 95% of values of a reference group fall into. This means that in such a population 2.5% of the time a sample value will be less than the lower limit of this interval, and 2.5% of the time it will be larger than the upper limit of this interval. Reference values can be arrived at by testing a number of sujects who are assumed to be representative of the population. There should be no varaition in sample collection and data should be analysed for different variables (smokers, alcohol consumption, mobile, pregnancy and ambulant subjects).

The first step in determining a given reference range is to define the population to which the reference range will apply, for example, healthy females between 20 and 30 years old. A large number of individuals from this category would be tested for a specific laboratory test. The results would be averaged and a range (plus or minus 2 standard deviations of the average) of normal values would be established. The term reference range is preferred over normal range because the reference population can be clearly defined. Rather than implying that the test results are being compared with some ill-defined concept of normal, the reference range means the results are being considered in the most relevant context. When you examine test results from different populations, you quickly discover that what is normal for one group is not necessarily normal for another group. For example, pregnancy changes many aspects of the body’s chemistry, so pregnant women have their own set of reference ranges.

Haematological values (Mean ± 2SD) based on gender

Haematological values in infancy to adolescent life (mean ± 2SD)

Bibliography

1. The procedures and vocabulary referring to reference intervals: CLSI (Committee for Laboratory Standards Institute) and IFCC (International Federation of Clinical Chemistry) CLSI - Defining, Establishing, and Verifying Reference Intervals in the Laboratory; Approved guideline - Third Edition. Document C28-A3 (ISBN 1- 56238-682-4) Wayne, PA, USA, 2008.

2. International Committee for Standardization in Haematology.The theory of reference valus.Clinical and Laboratory Haematology. Haematology 1981; 3: 369-373.

3. Balloch AJ, Cauchi MN. Reference ranges for haematological parameters in pregnancy derived from patient populations. Clinical and Laboratory Haematology 1993; 15:7-14.

4. International Committee for Standardization in Haematology. Standardization of blood specimen collection procedures for reference values. Clinical and Laboratory Haematology 1982; 4:83-86.

5. Richardson JA, Twedt D, Swaim W, et al. Diurnal change of blood counts analytes in normal subjects.American Journal of Clinical Pathology 1996; 106: 723-727.

6. International Council for Standardization in Haematology: Expert panel on Cytometry. Recommendation of the International Council for Standardization in Haematology on reporting differential leucocyte counts.Clinical and Laboratory Haematology 1995; 17: 113.

7. Appel IM, Grimminck B, Geerts J, Stiger R, Cnossen MH, Beishuizen A. Age dependency of coagulation parameters during childhood and puberty. J Thromb Haemost 2012 Aug 21. Doi: 10.1111/j.1538-7836.2012.04905.x. [Epub ahead of print]

8. Dosoo DK, Kayan K, Adu-Gyasi D, Kwara E, at al. Haematological and biochemical reference values for healthy adults in the middle belt of Ghana. PLoS One 2012; 7(4):e36308. Doi: 10.1371/journal.pone. 0036308. Epub 2012 Apr 27.

Chapter 2

Haematopoiesis

Haematopoiesis: Human blood is composed of plasma with the following cellular elements: red cells (erythrocytes), white cells (leucocytes), and platelets (thrombocytes). Haemopoiesis is the process of producing cellular constituents of the blood. It occurs throughout foetal and adult life to replace cells that are removed from the circulation. In adults, haemopoiesis occurs in bone marrow and lymphatic tissue. Haematopoietic Stem Cells (HSCs) are found in the bone marrow of adults, which can be seen in femurs, hips, ribs, sternums and other bones.

Haematopoietic Stem and Progenitor Cells: Studies in non-human mammalian models (mice) have helped to provide substantial information on haematopoiesis. Haematopoiesis begins with the pluripotential stem cells (haematopoietic stem). These cells are multipotent stem cells that give rise to all of the blood cell types from the myeloid (monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes, platelets, and dendritic cells) and lymphoid lineages (T-cells, B-cells, NK-cells). The pluripotential stem cell gives rise to the multipotential granulocyte, erythrocyte, monocyte, megakaryocyte colony-forming unit (CFU-GEMM), and lymphoid progenitor. The CFU-GEMM is a colony-forming unit that generates myeloid cells. The CFU-GEMM also gives rise to colony-forming unit granulocyte and monocyte (CFU-GM) cells. Lymphopoiesis occurs in foetal yolk sacs and the liver. The pluriotential stem cells give rise to the multi-lymphoid progenitor (MLP) which differentiates into lymphocytes by first becoming a lymphoblast (which then divides several times to become a prolymphocyte with specific cell-surface markers unique to either a T cell or B cell). The progenitor has the ability to differentiate into natural killer cells (NK) and dendritic cells.

The multipotent CFU-GEMM gives rise to the erythroid progenitor cell, known as the erythrocyte burst-forming cell (BFU-E). The BFU-E is not a pluripotent stem cell, it is a monopotent cell only capable of proliferating to give rise to erythrocytes. BFU-E gives rise to the erythrocyte colony-forming cell (CFC-E). The CFC-E is the erythroid progenitor cell that eventually matures into an erythrocyte after several cell divisions (pronormoblast, basophilic normoblast, polychromatic normoblast, orthochromatic normoblast, reticulocytes, and erythrocytes). The CFU-E is a stage in erythroid development between the BFU-E stage and the pro-erythroblast stage. The multipotent CFU-GEMM also gives rise to the CFU-Meg (the megakaryocyte progenitor cell that gives rise to platelets). The CFU-GM (the granulocyte and monocyte progenitor cells, which give rise to monocytes and neutrophils), CFU-Ba (the basophil progenitor cells that have the potential to give rise to basophils), and the CFU-Eo (the eosinophil progenitor cells that differentiate into eosinophils).

Figure: Stages involved in Haematopoiesis

Blood-Forming Units and Functions

Sites of Blood Formation: In developing embryos, blood formation occurs in the blood islands of the yolk sac. As development progresses, blood formation occurs in the spleen, liver, and lymph nodes. When bone marrow develops, it eventually assumes the task of forming most of the blood cells. However, maturation, activation, and some proliferation of lymphoid cells occur in secondary lymphoid organs (spleen, thymus, and lymph nodes). In children, haematopoiesis occurs in the marrow of the long bones, such as the femur and tibia. In adults, it occurs mainly in the pelvis, cranium, vertebrae, and sternum. Cell culture techniques indicate that all mature blood cells derive from a primitive haemopoietic stem cell precursor by ordered cell division and differentiation. All cellular elements of blood originate from the HSCs that reside in the medulla of the bone (bone marrow) and have the unique ability to give rise to all of the different mature blood cell types. Haemopoiesis can be subdivided according to the type of cell being formed – erythroid (RBCS), myeloid (neutrophils, eosinophils, basophils, macrophages, and monocytes), lymphoid (Band T lymphocytes), and megakaryocytic (platelets).

Erythroid: The red cells play an important role in oxygen and carbon dioxide transport to and from tissues. In the early foetus, erythropoiesis takes place in the mesodermal cells of the yolk sac (functional nucleated reticulocytes and erythrocytes, which contain haemoglobin with α and β chains) and are released into the blood. By the third or fourth month, erythropoiesis moves to the spleen and liver. After seven months, erythropoiesis occurs in the bone marrow. Erythropoiesis also occurs outside the bone marrow, within the spleen or liver (non-nucleated red cells containing haemoglobin with α and β chains). This process is termed extramedullary erythropoiesis. At birth (in a full-term infant, the marrow is the primary site of red cell, platelet, and granulocyte production (neutrophils, eosinophils, and basophils) whereas monocytes and lymphocyte production takes place principally in the spleen, lymphnodes, and thymus (with minimal production in the liver and bone marrows). The bone marrow of essentially all the bones produces RBCs until age five. The tibia and femur cease to be the major sites of haematopoiesis in adult life, and haematopoiesis becomes confined to the vertebrae, sternum, pelvis, ribs, and cranial bones throughout life. If the adult is chronically stressed, the sites of erythropoiesis can reoccur in the liver and spleen. There are a number of factors controlling erythropoiesis. Erythropoietin is the key hormone that regulates red cell production. The site of erythropoiesis varies with regard to the developmental stage.

Table: Variation in Site of Haematopoiesis Based on Stage of Development

Lymphoid: Lymphocytes are the cornerstones of the adaptive immune system. Lymphocytes (T and B) play a role in the maintenance of cell-mediated and antibody (humoral)-mediated immunity. They are derived from common lymphoid progenitors. The lymphoid lineage is primarily composed of T-cells and B-cells. The Common Lymphoid Progenitor (CLP) of humans differentiates into lymphocytes by first becoming a lymphoblast. It then divides further to become a prolymphocyte with specific cell-surface markers (either a T-cell or B-cell). The progenitor can also differentiate into natural killer cells (NK) and dendritic cells. Unlike dentritic cells, T-cells, B-cells and NK cells are unique to the lymphocyte family. Dendritic cells arise from lymphoid or myeloid lineages, and they may have different roles (antigen-presenting cells) and locations, such as the skin (Langerhans cells) and the inner lining of the nose, lungs, stomach, and intestines.

Myeloid: The myeloid linage includes granulocytes, megakaryocytes, and macrophages and it is derived from common myeloid progenitors. It is involved in such diverse roles as innate immunity, adaptive immunity, and blood clotting. This process is called myelopoiesis. Granulopoiesis (granulocytopoiesis) is haematopoiesis of granulocytes. Granulocytes play a role in phagocytosis and inflammation. Granulopoiesis occurs primarily within bone marrow, and it involves the following stages: pluripotential hemopoietic stem cell to myeloblast, promyelocyte, eosinonophilic/neutrophilic/basophilic myelocytes, metamyelocyte, band cell (stab cell), and the transformation into granulocytes (eosinophils/neutrophils and basophil).

Megakaryocytic: Megakaryocytopoiesis is haematopoiesis of megakaryocytes. Platelets play a role in haemostasis (coagulation). Thrombopoiesis refers to the process of thrombocyte production. Thromobocytes are fragments of the cytoplasm from megakaryocytes. A single megakaryocyte can give rise to thousands of thrombocytes. Thrombopoietin stimulates megakaryopoiesis (megakaryocyte maturation and differentiation). Thrombopoietin binds to its receptor, c-mpl, which occurs in megakaryocyte progenitor cells. This binding signals the growth and subsequent maturation of megakaryocytes, the stability of their membranes, the formation of platelet granules, and the fragmentation into mature platelets.

Growth Factors and Cytokines Involved in Haematopoiesis: Red and white blood cell production is a highly regulated process in healthy humans, depending on need. The proliferation and self-renewal of cellular elements of blood depends on stem cell factor (SCF). Glycoprotein growth factors regulate the proliferation and maturation of these cells as they enter the blood stream from the bone marrow. Three other factors that stimulate the production of granulocytes are called colony-stimulating factors (CSFs), and they are as follows:

• Granulocyte-Macrophage CSF (GM-CSF)

• Granulocyte CSF (G-CSF)

• Macrophage CSF (M-CSF)

Cytokines: Cytokines (interleukins) are signaling proteins synthesized by helper CD4+ T lymphocytes, monocytes, macrophages, and endothelial cells. They promote the development and differentiation of haematopoietic cells. Interleukin-1 alpha and Interleukin-1 beta (IL-1α and IL-1β) are involved in the regulation of immune responses, inflammatory reactions, and haematopoiesis. Interleukin-2 (IL-2) regulates the growth and differentiation of T-cells and certain B-cells. Interleukin-3 (IL-3) controls blood cell production by controlling the production, differentiation, and function of granulocytes and macrophages. Interleukin-4 (IL-4) is produced by specialized CD4 T cells, and it plays an active role in the proliferation of B-cells and class switch. Interleukin-5 (IL-5) and eosinophil differentiation factor (EDF) play a key role in eosinophil production (eosinophilpoiesis). Interleukin-6 (IL-6) is involved in the final differentiation of B-cells. Interleukin-7 (IL-7) acts as a growth factor for early lymphoid cells of both B- and T-cell lineages. Interleukin-11 (IL-11) is a megakaryocytopoiesis (platelet production) stimulator. Interleukin-12 (IL-12) is involved in the differentiation into cytotoxic T-cells.

Cytokines that determine which types of blood cell are formed include Stem Cell Factor (SCF), Thrombopoietin (Tpo), Interleukin (IL), Granulocyte Macrophage-colony stimulating factor (GM-CSF), Erythropoietin (Epo), Macrophage-colony stimulating factor (M-CSF), Granulocyte-colony stimulating factor (G-CSF), Stromal cell-derived factor-1 FMS-like tyrosine kinase 3 ligand (FLT-3 Ligand), Tumour necrosis factor-alpha (TNF- α), and Tumour necrosis factor-beta (TGF-β). Proliferation of these cells depends upon stem cell factor whereas glycoprotein growth factors regulate the proliferation and maturation of the cells that enter the blood from the marrow (causing cells in one or more committed cell lines to proliferate and mature). Erythropoietin is required for a myeloid progenitor cell to become an erythrocyte. Thrombopoietin makes myeloid progenitor cells differentiate to megakaryocytes (platelet-forming cells).

Erythropoiesis: Erythropoiesis is the process by which red blood cells (erythrocytes) are produced. The major stimulation that initiates red cell production is suboptimal levels of oxygen in circulation (hypoxia). This stimulation results in the secretion by the kidney of the glycoprotein hormone erythropoietin. Erythropoietin, in turn, initiates the proliferation and differentiation of red cell precursors resulting in the increased production of red cells (erythropoiesis) in the red bone marrow (predominantly from seven months to adult life) and other haematopoietic tissues such as the mesodermal cells of the yolk sac (early fetal life), liver and spleen (in the first three or four months of life). Certain diseases that compromise the ability of the red marrow to produce red cells (to meet the needs of the body) can result in red cell production in other sites such as the liver and spleen (extramedullary) to compensate for the resulting anaemia. Erythropoiesis occurs in the bone marrow and is present in almost all bones from seven months until about five years of life (predominantly in the tibia and femur). When a person reaches adulthood, the predominant site shifts to other bones, which remain the main sites essentially throughout life. Those sites are as follows: vertebrae, ribs, cranial bones and sternum. Several developmental changes occur as red cells mature from primitive pluripotential stem cells to mature erythrocytes. The primitive progenitors undergo a series of differentiations from the pluripotential stem cells (pluripotent), CFU-GEMM (multipotent), BFU-E (monopotent), pronormoblast (proerythroblast), basophilic normoblast (erythroblast), polychromatic normoblast (intermediate normoblast), orthochromatic normoblast (non-nucleated late normoblast), and reticulocytes. The ultimate result is a mature red cell. The maturation time of a pronormoblast to a mature RBC is about seven days. It takes an average of five to seven days and twelve to fifteen days, respectively, for the BFU-E to mature into a CFU-E and a normoblast. Stages in the Red Cell Development Circle include: Pluripotent hematopoietic stem cell (a haemocytoblast), CFU-GEMM (a multipotent stem cell), BFU-E (a monopotent stem cell), Pronormoblast (a proerythroblast), Basophilic normoblast (an erythroblast), Polychromatophilic normoblast/intermediate normoblast, Orthochromatic normoblast (a non-nucleated, late normoblast), Reticulocyte and Mature erythrocyte.

Life cycle of Erythrocytes: Pluripotential stem cell = CFU-GEMM (multipotent cell) = BFU-E (monopotent) = Pronormoblast = Basophillic normoblast = Polychromatic normoblast = Orthochromatic normoblast = Reticulocyte = Mature Erythrocytes.

The mature cell is released from the bone marrow into peripheral circulation at the reticulocyte stage. Under normal circumstances, reticulocytes constitute about 1 per cent of circulating red blood cells. The reticulocyte matures after one to two days in circulation into a mature, functional, and fully haemoglobinized red blood cell. Iron, vitamin B12, and folate play a major role in the red cell maturation process. Deficiency of iron results in a red cell with a cytoplasm that is sub-optimally haemoglobinized (iron deficiency anaemia) whereas a vitamin B12 or folate deficiency can result in maturation challenges resulting in anaemia (due to the marrow producing abnormally large red blood cells that cannot function properly). This condition is known as pernicious anaemia. A common cause of vitamin B12 and folate deficiency is not eating enough food that contains vitamin B12. A vegetarian or vegan diet can cause a deficiency because vitamin B12 is commonly found in foods of animal origin (meat, fish, eggs, and milk). Also, a deficiency can arise if the small intestine cannot absorb vitamin B12 produced after digestion (due to a deficiency of intrinsic factor produced by the stomach, which absorbs vitamin B12 from food). Intrinsic factor deficiency can result from the following: development of antibodies against the cells that produce intrinsic factor, carcinoma of the stomach and ulcers, diseases of the small intestine, post-bowel surgery, and fish tapeworm infestation.

Stages in Normoblastic Differentiation

Proerythroblast: The proerythroblast is the earliest of four stages in the development of the normoblast. Proerythroblasts arise from the CFU-E (colony-forming unit erythroid) cells, and they give rise to basophilic erythroblasts. A pronormoblast is typically 14–20 µm in size with a round, centrally located nucleus about the size or a myeloblast. The nuclear chromatin is coarser, more reticular, and more condensed compared to a myeloblast. The pronormoblast will have multiple prominent nucleoli.

Figure: Proerythroblast

Basophilic Normoblast or Early Normoblast: The basophilic normoblast is slightly smaller in size than the pronormoblast (12–17 µm diameter). The chromatin is more condensed than that of the pronormoblast. They have closed nucleoli (the absence of nucleoli is the major distinguishing feature between a basophilic normoblast and a pronormoblast). The ribosome content of cytoplasm increases, and the cytoplasm becomes more basophilic. This is a sign of the beginning of haemoglobin synthesis (globin chain synthesis). They have a grainy and reticular textured chromatin.

Figure: Basophilic Normoblast

Polychromatophilic Normoblast/Intermediate Normoblast

• The polychromatophilic normoblast is slightly smaller in size than the basophilic normoblast (12–15 µm diameter).

• The nuclear chromatin condenses even further (it becomes more clumped) with no nucleoli.

• The nucleus becomes smaller (7–9 µm diameter).

• Haemoglobin production in the cytoplasm of a polychromatophilic normoblast is more evident when one observes the colour shift from deep basophilic to grey.

Figure: Polychromatic Normoblast

Orthochromatic Normoblast (Non-Nucleated Late Normoblast): The orthochromatic normoblast is slightly smaller in size than the polychromatophilic normoblast (8–12 µm diameter). Cytoplasm tends towards full haemoglobinization. The nuclear chromatin condenses further, and the nucleus shrinks and tends to become more peripheral and (eventually) extruded as the cell matures to become a reticulocyte.

Figure: Orthochromatic Normoblast

Reticulocyte: Reticulocytes are young anucleate (non-nucleated) erythrocytes. An orthochromatic normoblast becomes a reticulocyte once the nucleus is extruded. Reticulocytes are larger than mature red cells. They are about twice the size of normal, mature red cells. They appear as polychromatophlic cells on Wright’s stained blood film (cytoplasm is stained greyish). The cytoplasm contains a fine reticulum with cytoplasmic organelles (ribosomes, Golgi bodies, and mitochondria). They are demonstrable using supravital stains (new methylene or brilliant cresyl blue) that show a basophilic reticulum under vital staining. They appear as a reticular (mesh-like) network of residual rRNA that has not yet been extruded from the cell.

Figure: Reticulocytes

Figure: Reticulocytes Stained with a Supravital Stain

Significant Changes That Occur as a Red Cell Matures: The following characteristics can be seen changing in the erythrocytes when they are maturing:

• There is significant reduction in the cell size.

• The nuclear size decreases. The nucleus is initially large with open chromatin. However, as the cell matures, the nuclear chromatin condenses, becomes smaller, and is eventually extruded at the orthochromatic normoblast stage (before becoming a reticulocyte).

• The cytoplasmic RNA and DNA increases, and the cell becomes essentially basophilic with a bluish cytoplasm.

• Staining reaction of the cytoplasm changes characteristically from blue to salmon pink due to a decline in the amount of cytoplasmic RNA and DNA and optimum haemoglobinization.

Role of Erythropoietin: Erythropoietin is a glycoprotein that is synthesized in the kidney, often in response to hypoxia (resulting from low oxygen levels). Erythropoietin regulates the process of erythropoiesis. In normal circumstance (absence of disease), there should be a proportional relationship between red cell production and destruction. However, disease states in which the numbers of red cells present are inadequate the body in a bid to ensure optimum oxygen delivery to the tissues trigger erythropoietin production in the kidney and liver. The peptide hormone hepcidin (produced by the liver) may also play a role in the regulation of erythropoiesis and haemoglobin production. Hepcidin controls iron absorption in the gastrointestinal tract and iron release from reticuloendothelial tissue. Iron from macrophages in the bone marrow is a major component of heme, a major part of the haemoglobin molecule that enhances the functionality of the red cells. Synthetically produced erythropoietin (recombinant DNA technology, epoetin alpha) is available and used in many clinical settings.

Use of synthetically produced erythropoietin in clinical settings:

• To treat anaemia in patients with anaemia related to kidney dysfunction associated with low erythropoietin production.

• To treat patients with kidney disease who require regular dialysis to treat and prevent anaemia.

• To treat anaemia associated with HIV medication (Zidovudine or AZT).

• To treat anaemia related to bone marrow suppression or failure, resulting from radiation or chemotherapy treatment for cancer.

• To treat anaemia associated with haematological and non-haematological carcinoma.

Function of Erythropoietin

• Increases the synthesis of haemoglobin in red cell precursors.

• Releases marrow reticulocytes (polychromatophilic cells) into the peripheral blood as a means of compensating for chronic anaemia.

• Reduces maturation time of red cells and their precursors.

• Proliferates the number of erythroid-committed stem cells and red cell precursors.

Increased erythropoietin levels in anaemic patients may be due to a decrease in bone marrow function (bone marrow failure). The presence of anaemia associated with low or normal erythropoietin levels is indicative of kidney disease – this is often due to the fact that the kidney is producing suboptimal levels of the hormone. The absence of anaemia associated with high erythropoietin levels is indicative of an increased synthesis of erythropoietin by the kidneys or other tissues in the body. The presence of excess RBC production and associated low or normal erythropoietin levels is indicative of polycythaemia.

Table: Causes of Increased and Reduced Levels of Erythropoietin in Plasma and Urine

Other Agents That Facilitates Erythropoiesis: Chronic anaemia can occur in many situations such as chemotherapy treatment, aplastic anaemia, pernicious anaemia, bone marrow malignancies, various vitamin or mineral deficiencies (B12, iron, or folate), disease states (anaemia due to chronic disease) and other causes of anaemia resulting from increased destruction and bleeding. In such situations, the demand for red cell production increases. Apart from the hormone erythropoietin, other agents facilitate erythropoiesis include; Vitamin B12 and folate, Iron, manganese, and cobalt, Hormones such as androgens and thyroxine and Amino acids

Defeactive or Extramedullary Haematopoiesis (EMH): In adult life, haematopoiesis occurs principally in the extravascular part of the red marrow. In some cases, the liver, thymus, lymph nodes, and spleen may resume their haematopoietic function. Other less common sites include adipose tissues, kidneys, endosteums (medullary cavities of long bones), adrenal glands, intrathoracic regions, and broad ligaments. This is called extramedullary haematopoiesis. EMH is often a remedial response to haematopoiesis failure in the bone marrow. EMH may cause an enlargement in the responsible organs (hepatomegaly, splenomegaly). EMH is usually associated with pathologic processes. Examples of diseases associated with extramedullary Haemopoiesis include; Myelofibrosis with myeloid metaplasia, Aplastic anaemia and other chronic anaemia, Foetal development, Pernicious anaemia, Myeloscelerosis, Secondary carcinoma of the bone, Bone marrow failure, Myeloproliferative disorders, Haemoglobinopathies (SCD and Thalassaemia), Bone marrow infiltration and Haemolytic disease of the foetus and newborn (HDFN).

Myelopoiesis: The myeloid progenitor CFU-GEMM can differentiate in the bone marrow into granulocytes and macrophages (mature monocytes). Granulocytes are also called polymorphonuclear leucocytes because of their characteristically shaped nuclei and cytoplasmic granules. Granulocyte includes; neutrophils, eosinophils, and basophils. A granulocyte differentiates into a distinct cell type by a process called granulopoiesis. In this process, it first transforms from a common myeloblast (myeloid progenitor) to a common promyelocyte. This promyelocyte gives rise to a unique myelocyte that can either be eosinophilic, basophilic, or neutrophilic (based on the staining of the cytoplasmic granules). The myelocyte then differentiates further into a metamyelocyte and then a band cell before becoming a mature neutrophil, eosinophil, or basophil.

Figure: Stages of Development in Myelopoiesis

Stages of Development in the Myeloid Series

Myeloblast: Most primitive precursor in the granulocytic series, which matures into the promyelocyte. Its size ranges from 10–20 µm, with no granules. The myeloblast nucleus is large and round or oval, and it has an open, unclumped nuclear chromatin. The nucleus contains several nucleoli. The cell has an insignificant basophilic cytoplasm.

Figure: Myeloblast

It is difficult to differentiate between a myeloblast, a monoblast, and a lymphoblast in a blood film. Use of special stains that detect the presence of enzymes such as esterase and peroxidase in the blast cytoplasm facilitates the classification in acute leukaemia. The major differentiating features from a blood smear are as follows:

• In lymphoblasts, the nuclear chromatin is denser and coarser, and it shows more aggregation compared to myeloblasts.

• Lymphoblasts generally have 1–2 nucleoli whereas myeloblasts have 2–5 nucleoli.

• Myeloblasts tend to have a fine chromatin and a pale nucleus.

Promyelocyte

• The promyelocytes have a diameter that is 10–20 µm and round, oval nucleoli.

• They show a more condensed nuclear chromatin and less prominent nucleoli.

• The cytoplasm contains characteristic azurophilic myeloperoxidase, acid phosphatase, and esterase enzymes containing granules (primary granules).

• Neutrophilic promyelocytes tend to be larger than the myeloblasts.

Figure: Metamyelocytes

Myelocytes

• Myelocytes have a diameter of 10–18 µm. They are slightly smaller than promyelocytes.

• They have eccentric, round-oval nuclei. The nuclear chromatin is coarse and condensed with small, non-visible nucleoli.

• Primary azurophilic granules still persist in the cytoplasm, but secondary granules begin to predominate.

• Cytoplasmic basophilia decreases and eventually disappears.

Figure: Myelocytes

Metamyelocyte: Metamyelocytes have a diameter of 10–18 µm. They are slightly smaller than myelocytes. They have characteristic kidney-shaped nuclei and relatively densely clumped nuclear chromatin with no nucleoli. The kidney shaped nucleus is a major feature that differentiates a metamyelocytes from myelocytes. The cytoplasm is faintly pink and non-basophilic. The cytoplasm becomes filled with predominantly secondary granules (although primary granules persist, and tertiary granules begin to appear in the cytoplasm).

Figure Metamyelocyte

Band Forms: It has a curved (but non-lobular or unsegmented) nucleus. The chromatin is heavily clumped, and there are secondary or specific granules (neutrophilic, eosinophilic, and basophilic predominate). There are an increased proportion of neutrophilic bands in the peripheral blood. This is often referred to as a left shift, and it may indicate the presence of infection or inflammation.

Figure: Neutrophilic band form

Mature Neutrophils, Eosinophils, and Basophils

Neutrophils: Neutrophils or polymorphonuclear neutrophils (PMNs) are white cells with a diameter of 12–15 µm.. They have 2–5 lobes. The chromatin of the segmented neutrophil is coarsely clumped. The cytoplasm is stained faint pink, and it is filled with fine pink secondary granules. The number of lobes and the extent of granulation are diagnostic. Neutrophils with more than five lobes are called hyper-segmented neutrophils (megaloblastic anaemia such as pernicious anaemia).

Figure: Neutrophils

Eosinophils: Eosinophils have a diameter of 10–15 µm. Their cytoplasm is filled with large, red-orange granules that contain eosinophil peroxidise. They are generally low in number (1–3 per cent), and they most often increase in number in individuals with allergies and parasitic infections. The nuclei are bi-lobed.

Figure: Eosinophilia

Basophils: Basophils are 10–15 µm cells with large, purplish-black, coarse granules.. The primary granules are peroxidise positive, and they contain heparin and histamine.. Basophilia, as an isolated finding, is uncommon. However, it is a common feature of myeloproliferative disorders (and particularly prominent in chronic myelogenous leukaemia).

Figure: Basophils

Monopoiesis: Monocytes are produced by the bone marrow from haematopoietic stem cell precursors called monoblasts. Monocytes differentiate and mature from monoblasts into promonocytes and then to matured monocytes. They circulate in the bloodstream for about one to three days, where they move into tissues throughout the body. They constitute between 3–8 per cent of all leucocytes in the blood. Half of them are stored as a reserve in the spleen in clusters in the red pulp’s Cords of Billroth. In the tissues, monocytes mature into different types of macrophages and help protect tissues from foreign substances. .

CFU-GM = Monoblast, promonocyte and monocyte

Monoblast: A monoblast has a diameter of about 12–20 µm. It has a large nucleus. The nuclear: cytoplasm ratio is about 4:1 or 3:1. The nucleus is round to oval and has fine nuclear chromatin. The nucleus has distinct 1–4 nucleoli.. The cytoplasm is devoid of granules (agranular), and it stains slightly

Figure: Myeloblast

Promyelocyte: The cell has an average diameter of 14–18 µm. It is an intermediate between the monoblast and monocyte, and it has a coarse chromatin structure and one or two nucleoli.. It has a large, convoluted nucleus.. The cytoplasm is stained grey-blue and may contain a few fine azurophilic granules..

Figure: Promonocyte

Monocyte: Monocytes are the largest of the white blood cells. They have an average diameter of 15–18 µm. The cytoplasm is stained grey-blue, and it may show vacuolation and fine azurophilic granules. Monocytes have abundant cytoplasm and a large, distinctive, kidney-shaped nucleus. Monocytes and their macrophage and dendritic-cell progeny serve three main functions in the immune system: phagocytosis, antigen presentation, and cytokine production.

Figure: Monocyte

Megakaryocytopoiesis: In the bone marrow, the multipotent CFU-GEMM cells differentiate by a process of mitosis into the megakaryocytic precursor called the megakaryocyte CFU (CFU-Meg) under the influence of cytokines CSF, thrombopoietin, and IL-3. The CFU-Meg then develops into the megakaryoblast. And then it develops into a megakaryocye before developing into mature platelets.

Figure: Developmental Development of Platelets

Megakaryoblast: The cell is slightly basophilic. It is a 20–30 µm diameter cell with a large oval or kidney-shaped non-lobulated nucleus. The nucleus contains several nucleoli. It has an insignificant non-granular cytoplasm.

Figure: A Normal Marrow with a Magakaryoblast

Promegakaryocyte: The cytoplasm is intensely basophilic. It has a diameter of 30 µm and a large oval or kidney-shaped non-lobulated nucleus. The nucleus is a dense chromatin, and it may show mild lobulation. The cytoplasm contains fine azurophilic granules.

Figure: A Normal Marrow with a Promegakaryocyte

Megakaryocyte: The cell is around 50–100µm with a single, indented nucleus. Each megakaryocyte fragments into between 1000 and 3000 platelets. The cytoplasm is basophilic (light blue), and it contains azurophilic granules. Megakaryocytes normally account for approximately 0.05–0.1 per cent of all nucleated bone marrow cells.

Figure: Two Megakaryocytes

Mature Platelets: Platelets are small, 2–4 µm-sized discoid cell fragments. The cytoplasm is stained light blue, and it contains purple-reddish granules. They are produced by the fragmentation of precursor cells called megakaryocytes. Platelets play a key role in haemostasis, and they are involved in the formation of blood clot following a break in vascular endothelium by forming a platelet plug at the site of injury. They are short-lived. They have an average life span of five to nine days. Patients with extremely low platelet counts (thrombocytopenia) are prone to bleeding. Patients with high platelet counts (thrombocytosis or thrombocythaemia) are prone to thrombosis.

Figure: Platelets

Role of Thrombopoietin in Thrombopoiesis: Like erythropoietin, thrombopoietin (TPO) is a glycoprotein hormone synthesized by the liver and the kidney. Its function is to regulate the production of thrombocytes (platelets) in the bone marrow by stimulating CFU-Meg to mature into megakaryocytes. TPO is mainly produced in the liver, and it is regulated by a negative feedback mechanism. The function of TPO is to bind to TPO receptors on the surface of megakaryocytes and platelets. In severe thrombocytopenia, the liver is stimulated to synthesize a high level of TPO, which facilitates the binding of TPO to its receptors on megakaryocytes (thus enabling the differention and maturation of CFU-Meg into mature platelets). The TPO level is kept low when there is no physiologic need for the production of platelets.

Figure: Platelet Clumping

Bibliography

1. Armitage J.O. Emerging applications for recombinant human granulocyte-macrophage colony stimulating factor. Blood 1998; 92:491-4508.

2. Kawamoto, Wada, Katsura. A revised scheme for developmental pathways of haematopoietic cells: the myeloid-based model. International Immunology 2010.

3. Palis J, Segel GB. Developmental biology of erythropoiesis. Blood Rev. 1988; 12 (2): 106–114.

4. Wickremansinghe R.G, Hoffbrand A.V. Biochemical and genetic control of apoptosis: relevance to normal haematopoiesis and haematological malignancies. Blood 1999; 93: 3587-3600.

Chapter 3

Differential Leucocyte Count (DLC)

Differential white blood cell count is an examination and enumeration of the distribution of the various leucocytes (neutrophils, lymphocytes, eosinophils monocytes, and basophils) in a stained blood smear. The different kinds of white cells are counted and reported as percentages or absolute values of the total examined. Differential white blood cell counts provide more specific information related to infections and diseases. The relative levels of the various white cell types can vary in different disease states, and they can be of diagnostic value in haematology. Differential leukocyte counts are made by microscopic examination of a blood smear, counting 100-200 leukocytes, and expressing the fraction of the specific leukocyte types as percentages of the total leukocyte count. Many public medical laboratories use automated techniques for analysis of samples due to the few laboratory staff members required and the increased speed of these automated analyzers.

Why Request a DLC? WBC differential counts have become a cornerstone in haematological diagnosis, and they are widely used for the following; screening, case finding, diagnosing, monitoring of haematologic and non-haematologic disorders, diagnosing bacterial or viral infectious disease, evaluating allergic conditions, diagnosing and monitoring malignant diseases such as leukaemia, monitoring a patient’s progress and/or response to treatment (a decline in absolute neutrophil counts in patients treated for bacterial infection or septicaemia being treated with antibiotics is often seen as a positive response). However, the manual DLC is associated with several challenges; it is a time-consuming process, it is labour intensive and expensive and the 100-cell differential count is often criticized for its statistical shortcomings because of its small sampling size. To obviate these challenges, most modern three and five-part differential analyzers are equipped with flagging algorithms that alert users to possible morphological (or other) abnormalities that require manual morphology review. The advantage of five-part over three-part systems is a potential for reduction in manual review rates. With automated analyzers, a manual DLC may be indicated in the following situations;

• Presence of abnormal or immature cells

• Severe neutropenia (< 1x 10⁹/L)

• Leucopoenia (WBC < 1 x 10⁹/L)

• Presence of a blast flag

• Presence of abnormal lymphocyte flag

• Presence of atypical lymphocyte flag

• Iron deficiency anaemia picture (presence of a high RBC count with low haemoglobin, mean cell haemoglobin (MCH) and mean cell volume (MCV)

• Presence of nexplained low platelet count (< 100 x 10⁹/L) when platelet clumping is suspected to confirm presence of platelet clumping that may affect the platelet count obtained.

• When a deficiency disease or disorder