Hematology Lecture Notes PDF

LECTURE NOTES For Medical Laboratory Students Hematology Yared Alemu, Alemayehu Atomsa, Zewdneh Sahlemariam Jimma University In collaboration with the Ethiopia Publ...

LECTURE NOTES For Medical Laboratory Students Hematology Yared Alemu, Alemayehu Atomsa, Zewdneh Sahlemariam Jimma University In collaboration with the Ethiopia Public Health Training Initiative, The Carter Center, the Ethiopia Ministry of Health, and the Ethiopia Ministry of Education 2006  Funded under USAID Cooperative Agreement No. 663-A-00-00-0358-00. Produced in collaboration with the Ethiopia Public Health Training Initiative, The Carter Center, the Ethiopia Ministry of Health, and the Ethiopia Ministry of Education. Important Guidelines for Printing and Photocopying Limited permission is granted free of charge to print or photocopy all pages of this publication for educational, not-for-profit use by health care workers, students or faculty. All copies must retain all author credits and copyright notices included in the original document. Under no circumstances is it permissible to sell or distribute on a commercial basis, or to claim authorship of, copies of material reproduced from this publication. ©2006 by Yared Alemu, Alemayehu Atomsa, Zewdneh Sahlemariam All rights reserved. Except as expressly provided above, no part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or by any information storage and retrieval system, without written permission of the author or authors. This material is intended for educational use only by practicing health care workers or students and faculty in a health care field. PREFACE The lack of sufficient reference materials and uniformity in course syllabi has always been a problem in higher institutions in Ethiopia that are engaged in training health professionals including laboratory technologists. Hence, the authors hope that this lecture note would be immensely useful in solving this existing problem at significant level. The lecture note is intended for use by laboratory technologist both during their training and in their work places. There are twenty two chapters each beginning with specific learning objectives in which succeeding by a background of the topic in discussion. There are study questions at the end of each chapter for the reader to evaluate his understanding of the contents. In addition, important terms are defined in the glossary section at the end of the text. ACKNOWLEDGEMENT It is with sincere gratitude and pleasure that we acknowledge The Carter Center for the collaboration in preparation of this lecture note. Special thanks are due to Mohammed Awole, Serkadis Debalke, Ibrahim Ali, Misganaw B/sellasie, Abiye Shume, Shewalem Shifa and Simon G/tsadik for their assistance in reviewing and critiquing this material. For her sustained devotion and extra effort, I express my deep gratitude and sincere appreciation to Zenaye Hailemariam, who has been most supportive with scrupulous attention and dedication in helping me throughout the preparation of this lecture note (Y.A).  Table of Contents Preface.....................................................................i Acknowledgement....................................................ii Table of Contents......................................................iii Introduction...............................................................v 1. Blood.....................................................................1 2. Blood Collection....................................................42 3. Anticoagulants......................................................61 4. Preparation of Blood Smears...............................67 5. Staining of Blood Smears.....................................77 6. Hemocytometry....................................................89 7. Differential Leucocyte Count.................................122 8. Reticulocyte Count................................................136 9. Hemoglobin...........................................................146 10. Packed Cell Volume...........................................176 11. Red Cell Indices..................................................188 12. Erythrocyte Sedimentation Rate.........................197 13. Osmotic Fragility of the Red Cell........................209 14. Bone marrow smear examination.......................215 15. Lupus Erythematosus Cell..................................226 16. Red cell Morphology Study.................................232 17. Anemia................................................................244 18. Hematological Malignancies...............................311 19. Leucocyte cytochemistry....................................339 20. Hemostasis.........................................................357 21. Body fluid analysis..............................................434 22. Automation in Hematology..................................466 Glossary...................................................................477 References...............................................................567  INTRODUCTION The word hematology comes from the Greek haima (means blood) and logos (means discourse); therefore, the study of hematology is the science, or study, of blood. Hematology encompasses the study of blood cells and coagulation. Included in its concerns are analyses of the concentration, structure, and function of cells in blood; their precursors in the bone marrow; chemical constituents of plasma or serum intimately linked with blood cell structure and function; and function of platelets and proteins involved in blood coagulation. The study of blood has a very long history. Mankind probably has always been interested in the blood, since primitive man realized that loss of blood, if sufficiently great, was associated with death. And in Biblical references, “to shed blood” was a term used in the sense of “to kill”. Before the days of microscopy only the gross appearance of the blood could be studied. Clotted blood, when viewed in a glass vessel, was seen to form distinct layers and these layers were perceived to constitute the substance of the human body. Health and disease were thought to be the result of proper mixture or imbalance respectively of these layers. Microscopic examination of the blood by Leeuwenhoek and others in the seventeenth century and subsequent improvements in their rudimentary apparatus provided the means whereby theory and dogma would gradually be replaced by scientific understanding. Currently, with the advancement of technology in the field, there are automated and molecular biological techniques enable electronic manipulation of cells and detection of genetic mutations underlying the altered structure and function of cells and proteins that result in hematologic disease. Hematology CHAPTER ONE BLOOD Learning Objectives At the end of this chapter, the student shall be able to: Explain the composition of blood Describe the function of blood Describe the formation of blood cells. Explain the regulatory mechanisms in hemopoiesis Indicate the sites of hemopoiesis in infancy, childhood and adulthood.1 Composition blood 1 Hematology Blood is a circulating tissue composed of fluid plasma and cells. It is composed of different kinds of cells (occasionally called corpuscles); these formed elements of the blood constitute about 45% of whole blood. The other 55% is blood plasma, a fluid that is the blood's liquid medium, appearing yellow in color. The normal pH of human arterial blood is approximately 7.40 (normal range is 7.35-7.45), a weak alkaline solution. Blood is about 7% of the human body weight, so the average adult has a blood volume of about 5 liters, of which 2.7-3 liters is plasma. The combined surface area of all the red cells in the human body would be roughly 2000 times as great as the body's exterior surface. Blood plasma When the formed elements are removed from blood, a straw-colored liquid called plasma is left. Plasma is about 91.5% water and 8.5% solutes, most of which by weight (7%) are proteins.. Some of the proteins in plasma are also found elsewhere in the body, but those confined to blood are called plasma proteins. These proteins play a role in maintaining proper blood osmotic pressure, which is important in total body fluid balance. Most plasma proteins are synthesized by the liver, 2 Hematology including the albumins (54% of plasma proteins), globulins (38%), and fibrinogen (7%). Other solutes in plasma include waste products, such as urea, uric acid, creatinine, ammonia, and bilirubin; nutrients; vitamins; regulatory substances such as enzymes and hormones; gasses; and electrolytes. Formed elements The formed elements of the blood are broadly classified as red blood cells (erythrocytes), white blood cells (leucocytes) and platelets (thrombocytes) and their numbers remain remarkably constant for each individual in health. I. Red Blood Cells They are the most numerous cells in the blood. In adults, they are formed in the in the marrow of the bones that form the axial skeleton. Mature red cells are non- nucleated and are shaped like flattened, bilaterally indented spheres, a shape often referred to as ”biconcave disc” with a diameter 7.0-8.0µm and thickness of 1.7-2.4µm. In stained smears, only the flattened surfaces are observed; hence the appearance is circular with an area of central pallor corresponding to 3 Hematology the indented regions. They are primarily involved in tissue respiration. The red cells contain the pigment hemoglobin which has the ability to combine reversibly with 02. In the lungs, the hemoglobin in the red cell combines with 02 and releases it to the tissues of the body (where oxygen tension is low) during its circulation. Carbondioxide, a waste product of metabolism, is then absorbed from the tissues by the red cells and is transported to the lungs to be exhaled. The red cell normally survives in the blood stream for approximately 120 days after which time it is removed by the phagocytic cells of the reticuloendothelial system, broken down and some of its constituents re utilized for the formation of new cells. II. White Blood Cells They are a heterogeneous group of nucleated cells that are responsible for the body’s defenses and are transported by the blood to the various tissues where they exert their physiologic role, e.g. phagocytosis. WBCs are present in normal blood in smaller number than the red blood cells (5.0-10.0 × 103/µl in adults). Their production is in the bone marrow and lymphoid tissues (lymph nodes, lymph nodules and spleen). 4 Hematology There are five distinct cell types each with a characteristic morphologic appearance and specific physiologic role. These are: Polymorphonuclear leucocytes/granulocytes o Neutrophils o Eosinophils o Basophiles Mononuclear leucocytes oLymphocytes oMonocytes Fig. 1.1 Leucocytes 5 Hematology Polymorphonuclear Leucocytes Polymorphonuclear Leucocytes have a single nucleus with a number of lobes. They Contain small granules in their cytoplasm, and hence the name granulocytes. There are three types according to their staining reactions. Neutrophils Their size ranges from 10-12µm in diameter. They are capable of amoeboid movement. There are 2-5 lobes to their nucleus that stain purple violet. The cytoplasm stains light pink with pinkish dust like granules. Normal range: 2.0-7.5 x 103/µl. Their number increases in acute bacterial infections. Eosinophils Eosinophils have the same size as neutrophils or may be a bit larger (12-14µm).There are two lobes to their nucleus in a "spectacle" arrangement. Their nucleus stains a little paler than that of neutrophils. Eosinophils cytoplasm contains many, large, round/oval orange pink granules. They are involved in allergic reactions and in combating helminthic infections. Normal range: 40-400/ µl. Increase in their number (eosinophilia) is associated with allergic reactions and helminthiasis. 6 Hematology Basophils Their size ranges from 10-12µm in diameter. Basophiles have a kidney shaped nucleus frequently obscured by a mass of large deep purple/blue staining granules. Their cytoplasmic granules contain heparin and histamine that are released at the site of inflammation. Normal range: 20-200/µl. Basophilia is rare except in cases of chronic myeloid leukemia. Mononuclear Leucocytes Lymphocytes There are two varieties:  Small Lymphocytes Their size ranges from 7-10µm in diameter. Small lymphocytes have round, deep-purple staining nucleus which occupies most of the cell. There is only a rim of pale blue staining cytoplasm. They are the predominant forms found in the blood.  Large Lymphocytes Their size ranges from 12-14µm in diameter. 7 Hematology Large lymphocytes have a little paler nucleus than small lymphocytes that is usually eccentrically placed in the cell. They have more plentiful cytoplasm that stains pale blue and may contain a few reddish granules. The average number of lymphocytes in the peripheral blood is 2500/µl. Lymphocytosis is seen in viral infections especially in children. Monocytes Monocytes are the largest white cells measuring 14-18µm in diameter. They have a centrally placed, large and ‘horseshoe’ shaped nucleus that stains pale violet. Their cytoplasm stains pale grayish blue and contains reddish blue dust-like granules and a few clear vacuoles. They are capable of ingesting bacteria and particulate matter and act as "scavenger cells" at the site of infection. Normal range: 700-1500/µl. Monocytosis is seen in bacterial infections. (e.g. tuberculosis) and protozoan infections. III. Platelets These are small, non nucleated, round/oval cells/cell fragments that stain pale blue and contain many pink granules. Their size ranges 1-4µm in diameter. They 8 Hematology are produced in the bone marrow by fragmentation of cells called megakaryocytes which are large and multinucleated cells. Their primary function is preventing blood loss from hemorrhage. When blood vessels are injured, platelets rapidly adhere to the damaged vessel and with one another to form a platelet plug. During this process, the soluble blood coagulation factors are activated to produce a mesh of insoluble fibrin around the clumped platelets. This assists and strengthens the platelet plug and produces a blood clot which prevents further blood loss. Normal range: 150-400 x 103 /µl. 1.2 Function of blood Blood has important transport, regulatory, and protective functions in the body.  Transportation Blood transport oxygen form the lungs to the cells of the body and carbon dioxide from the cells to the lungs. It also carries nutrients from the gastrointestinal tract to the cells, heat and waste products away from cells and hormones form endocrine glands to other body cells. 9 Hematology  Regulation Blood regulates pH through buffers. It also adjusts body temperature through the heat-absorbing and coolant properties of its water content and its variable rate of flow through the skin, where excess heat can be lost to the environment. Blood osmotic pressure also influences the water content of cells, principally through dissolved ions and proteins.  Protection The clotting mechanism protects against blood loss, and certain phagocytic white blood cells or specialized plasma proteins such as antibodies, interferon, and complement protect against foreign microbes and toxins. 1.3 Formation of blood cells Hemopoiesis/hematopoiesis refers to the formation and development of all types of blood cells from their parental precursors. In postnatal life in humans, erythrocytes, granulocytes, monocytes, and platelets are normally produced only in the bone marrow. Lymphocytes are produced in the secondary lymphoid organs, as well as in the bone marrow and thymus gland. There has been much debate over the years as to the nature of hemopoiesis. Although many questions 10 Hematology remain unanswered, a hypothetical scheme of hemopoiesis based on a monophyletic theory is accepted by many hematologists. According to this theory, the main blood cell groups including the red blood cells, white blood cells and platelets are derived from a pluripotent stem cell. This stem cell is the first in a sequence of regular and orderly steps of cell growth and maturation. The pluripotent stem cells may mature along morphologically and functionally diverse lines depending on the conditioning stimuli and mediators (colony-stimulating factors, erythropoietin, interleukin, etc.) and may either: Produce other stem cells and self-regenerate maintaining their original numbers, or Mature into two main directions: stem cells may become committed to the lymphoid cell line for lymphopoiesis, or toward the development of a multipotent stem cell capable of granulopoiesis, erythropoiesis and thrombopoiesis. During fetal life, hemopoiesis is first established in the yolk sac mesenchyme and later transfers to the liver and spleen. The splenic and hepatic contribution is gradually 11 Hematology taken over by the bone marrow which begins at four months and replaces the liver at term. From infancy to adulthood there is progressive change of productive marrow to occupy the central skeleton, especially the sternum, the ribs, vertebrae, sacrum, pelvic bones and the proximal portions of the long bones (humeri and femurs). Hemopoiesis occurs in a microenvironment in the bone marrow in the presence of fat cells, fibroblasts and macrophages on a bed of endothelial cells. An extracellular matrix of fibronectin, collagen and laminin combine with these cells to provide a setting in which stem cells can grow and divide. In the bone marrow, hemopoiesis occurs in the extravascular part of the red marrow which consists of a fine supporting reticulin framework interspersed with vascular channels and developing marrow cells. A single layer of endothelial cells separates the extravascular marrow compartment from the intravascular compartment. When the hemopoietic marrow cells are mature and ready to circulate in the peripheral blood, the cells leave the marrow parenchyma by passing through fine "windows" in the endothelial cells and emerge into the venous sinuses joining the peripheral circulation. 12 Hematology Fig. 1.2a Hematopoiesis 13 Hematology Fig. 1.2b Hematopoiesis Hematopoietic Regulatory Factors In general it can be stated that hemopoiesis is maintained in a steady state in which production of mature cells equals cell loss. Increased demands for cells as a consequence of disease or physiologic 14 Hematology change are met by increased cell production. Several hematopoietic growth factors stimulate differentiation along particular paths and proliferation of certain progenitor cells. Erythropoietin (EPO), a hormone produced mainly by the kidneys and in small amounts by the liver, stimulates proliferation of erythrocytes precursors, and thrombopoietin stimulates formation of thrombocytes (platelets). In addition, there are several different cytokines that regulate hematopoiesis of different blood cell types. Cytokines are small glycoproteins produce by red bone marrow cells, leucocytes, macrophages, and fibroblasts. They act locally as autocrines or paracrines that maintain normal cell functions and stimulate proliferation. Two important families of cytokines that stimulate blood cell formation are called colony stimulating factors (CSFs) and the interleukins. The classes of hematopoietic growth factors and their functions are described in Table 1.1. Table 1.1 Hematopoietic growth factors 15 Hematology Factor Function Stem Cell Growth FactorStimulates pluripotent hematopoietic (Steel factor) stem cells (hemocytoblasts) Interleukin-3 (multi-CSF*) Stimulates pluripotent hematopoietic stem cells and progenitors of eosinophils, neutrophils, basophils, monocytes, and platelets Granulocyte-MacrophageStimulates development of erythrocytes, CSF (GM-CSF) platelets, granulocytes (eosinophils, neutrophils, and basophiles,), and monocytes. Macrophage CSF (M-CSF)Stimulates development of monocytes and macrophages Granulocyte CSF (G-CSF) Stimulates development of neutrophils Interleukin-5 Stimulates development of eosinophils Interleukin-7 Stimulates development of B lymphocytes *CSF=Colony stimulating factor Extramedullary Hemopoiesis Organs that were capable of sustaining hemopoiesis in fetal life always retain this ability should the demand arise, e.g., in hemolytic anemias where there is an increased blood loss and an increased demand for red 16 Hematology blood cells. Also fatty marrow that starts to replace red marrow during childhood and which consists of 50% of fatty space of marrow of the central skeleton and proximal ends of the long bones in adults can revert to hemopoiesis as the need arises. Formation of apparently normal blood cells outside the confines of the bone marrow mainly in the liver and spleen in post fetal life is known as Extramedullary Hemopoiesis. I. Formation of Red blood cells (Erythropoiesis) 17 Hematology Erythropoiesis is the formation of erythrocytes from committed progenitor cells through a process of mitotic growth and maturation. The first recognizable erythyroid cell in the bone marrow is the proerythroblast or pronormoblast, which on Wright or Giemsa stain is a large cell with basophilic cytoplasm and an immature nuclear chromatin pattern. Subsequent cell divisions give rise to basophilic, polychromatophilic, and finally orthochromatophilic normoblasts, which are no longer capable of mitosis. During this maturation process a progressive loss of cytoplasmic RNA occurs as the product of protein synthesis, hemoglobin, accumulates within the cell; as a result the color of the cytoplasm evolves from blue to gray to pink. At the same time the nuclear chromatin pattern becomes more compact tan clumped until, at the level of the orthochromatophilic normoblast, there remains only a small dense nucleus, which is finally ejected from the cell. The resulting anucleate erythrocyte still contains some RNA and is recognizable as a reticulocyte when the RNA is precipitated and stained with dyes such as new methylene blue. 18 Hematology Normally, reticulocytes remain within the bone marrow for approximately 2 days as they continue to accumulate hemoglobin and lose some of their RNA. The reticulocyte then enters the peripheral blood, were, after about one more day, it loses its residual RNA and some of its excessive plasma membrane and becomes indistinguishable form adult erythrocytes. Under normal conditions the transit time from the pronormoblast to the reticulocyte entering the peripheral blood is about 5 days. Morphology of the red cells and their precursors A. Pronormoblast (Rubriblast) Pronormoblast is the earliest morphologically recognizable red cell precursor. Size: 20-25µm in diameter. Nucleus: large, round to oval and contains 0-2 light bluish, indistinct nucleoli. The chromatin forms a delicate network giving the nucleus a reticular appearance. Cytoplasm: there is a narrow (about 2µm) rim of dark blue cytoplasm. There may be a perinuclear halo. The nuclear/cytoplasm ratio is about 8:1. B. Basophilic Normoblast 19 Hematology Size: 16-18µm in diameter. Nucleus: round or oval and smaller than in the previous stage. The chromatin forms delicate clumps so that its pattern appears to be denser and coarser than that seen in the pronormoblast. No nucleoli are seen. Cytoplasm: slightly wider ring of deep blue cytoplasm than in the pronormoblast and there is a perinuclear halo. The nuclear/cytoplasm ratio is about 6:1 C. Polychromatophilic Normoblast Size: 12-14µm in diameter Nucleus: smaller than in the previous cell, has a thick membrane, and contains coarse chromatin masses. Cytoplasm: as the nucleus is shrinking the band of cytoplasm is widening. It has a lilac (polychromatic) tint because of beginning of hemoglobinization. The nuclear cytoplasmic ratio varies from 2:1 to 4:1. D. Orthochromatic Normoblast Size: 10-12µm in diameter. Nucleus: small and central or eccentric with condensed homogeneous structure less chromatin. It is ultimately 20 Hematology lost by extrusion. Cytoplasm: a wide rim of pink cytoplasm surrounds the shrinking nucleus. The entire cell is somewhat smaller than the polychromatophilic normoblast. The nuclear / cytoplasmic ratio varies from 1:2-1:3. E. Reticulocyte After the expulsion of the nucleus a large somewhat basophilic anuclear cell remains which when stained with new methylene blue, is seen to contain a network of bluish granules. This network is responsible for the name of the cell and consists of precipitated ribosomes. As the bone marrow reticulocyte matures the network becomes smaller, finer, thinner, and finally within 3 days disappears. About 1% of reticulocytes enter the peripheral circulation. Size: 8-10µm in diameter Nucleus: the reticulocyte does not contain a nucleus. Cytoplasm: faintly basophilic (blue) F. Mature erythrocyte Size: 7-8µm in diameter 21 Hematology Cytoplasm: biconcave, orange-pink with a pale staining center occupying one-third of the cell area. Regulation of Erythropoiesis Erythropoietic activity is regulated by the hormone erythropoietin which in turn is regulated by the level of tissue oxygen. Erythropoietin is a heavily glycosylated hormone (40% carbohydrate) with a polypeptide of 165 aminoacids. Normally, 90% of the hormone is produced in the peritubular (juxtaglomerular) complex of the kidneys and 10% in the liver and elsewhere. There are no preformed stores of erythropoietin and the stimulus to the production of the hormone is the oxygen tension in the tissues (including the kidneys). When there is tissue airhypoxia due to: Low blood hemoglobin levels (e.g., anemia) Imped oxygen release from hemoglobin for some structural or metabolic defects (e.g., the hemoglobinopathies) Poor blood flow as in severe circulatory defects. Low atmospheric oxygen (e.g., high altitude) Erythropoietin production increases and this stimulates erythropoiesis by increasing the number of progenitor cells committed to erythropoiesis. 22 Hematology Erythropoietin accelerates nearly every stage of red cell production: It increases the rate at which the committed stem cells divide and differentiate It increases the rate of cell division It speeds up the incorporation of iron into the developing red cells It shortens the time cell maturation, and It hastens the entry of reticulocytes into the peripheral circulation Similarly, increased oxygen supply to the tissues due to: Increased red cell mass (e.g., polycythemia) Ability of hemoglobin to release oxygen to the tissues more readily than normal reduces the erythropoietin drive. Ineffective erythropoiesis/Intramedullary hemolysis Erythropoiesis is not entirely efficient since 10-15% of eryhtropoiesis in a normal bone marrow is ineffective, i.e., the developing erythroblasts die within the marrow without producing mature cells. Together with their hemoglobin, they are ingested by macrophages. This 23 Hematology process is substantially increased in a number of anemias. Megaloblastic Erythropoiesis Megaloblasts are pathologic cells that are not present in the normal adult bone marrow, their appearance being caused by a deficiency in vitamin B12 or folic acid or both leading to defective DNA synthesis. In megaloblastic erythropoiesis, the nucleus and cytoplasm do not mature at the same rate so that nuclear maturation lags behind cytoplasmic hemoglobinization. This nuclear lag appears to be caused by interference with DNA synthesis while RNA and protein synthesis continue at a normal rate. The end stage of megaloblastic maturation is the megalocyte which is abnormally large in size (9-12µm in diameter). II. Formation of white blood cells (Leucopoiesis) Granulopoiesis and Monocytopoiesis Neutrophils and monocytes, which evolve into macrophages when they enter the tissues, are arise form a common committed progenitor. The myeloblast is the earliest recognizable precursor in the granulocytic series that is found in the bone marrow. On division the myeloblast gives rise to promyelocyte which contain 24 Hematology abundant dark “azurophilic” primary granules that overlie both nucleus and cytoplasm. With subsequent cell divisions these primary granules become progressively diluted by the secondary, less conspicuous “neutrophilic” granules that are characteristic of the mature cells. This concomitant cell division and maturation sequence continues form promyelocytes to early myelocytes, late myelocytes, and they metamyelocytes, which are no longer capable of cell division. As the metamyelocyte matures the nucleus becomes more attenuated and the cell is then called a “band” or “stab” form. Subsequent segmentation of the nucleus gives rise to the mature neutrophil or polymorphonuclear leucocyte. The average interval from the initiation of granulopoiesis to the entry of the mature neutrophil into the circulation is 10 to 13 days. The mature neutrophil remains in the circulation for only about 10 to 14 hours before entering the tissue, where it soon dies after performing its phagocytic function. Neutrophil Granulocyte and Precursors A. Myeloblast Size and shape: the myeloblast is 20-25µm in diameter and has a round or oval shape. 25 Hematology Nucleus: large, oval or round, and eccentric. It has a thin nuclear membrane and finely dispersed, granular, purplish, pale chromatin with well-demarcated, pink, evenly distributed parachromatin: 2-5 light blue-gray nucleoli surrounded by dense chromatin are seen. Cytoplasm: the cytoplasmic mass is small in comparison to the nucleus, producing a nuclear/ cytoplasmic ratio of 7:1. It stains basophilic (bluish) and shows a small indistinct, paranuclear, lighter staining halo (golgi apparatus). The cytoplasm lacks granules. B. Promyelocyte Size and Shape: The promyelocyte is 15-20µm in diameter and round or oval in shape. Nucleus: the nucleus is still large but is beginning to shrink. It is round or oval, eccentric, possibly slightly indented, and surrounded by a thin membrane. With in the finely of granular purplish pale chromatin, 1-3 nucleoli may be faintly visible. Cytoplasm: It is pale blue; it is some what large in area than in myeloblast, so the nuclear/cytoplasmic ratio is 4:1 or 5:1. The basophilia is not quite as intense as in myeloblasts. The non-specific, peroxidase-containing 26 Hematology azurophilic granules are characteristic of the promyelocyte stage of development. C. Myelocyte Size and shape: 14-18µm in diameter and round. Nucleus: Condensed, oval, slightly indented, and eccentric. The chromatin is coarse. Nucleoli are absent. Cytoplasm: Light pink and contains neutrophilic granules (brownish) that may cover the nucleus and are coarse in the younger cells but become finer as the cell matures. The nuclear/cytopalsmic ratio is about 2:1 or 1:5:1. D. Metamyelocyte (Juvenile cell) The last cell of the granulocyte series capable of mitotic division; further stage in the development are caused by maturation and non-division. Size and shape: 12-14µm in diameter and round. Nucleus: Eccentric, condensed, and indented or kidney-shaped. The nuclear membrane is thick and heavy, and the chromatin is concentrated into irregular thick and thin areas. 27 Hematology Cytoplasm: abundant and pale or pink; it contains both specific and non-specific (few) granules that in the neutrophilic metamylocytes vary in size, whereas the basophilic and eosinophilic granules are large and equal in size. The nuclear/cytoplasmic ration is 1:l. E. Band Granulocyte (Stab Cell) The juvenile cell or the band cell are the youngest granulocytes normally found in the peripheral blood. Size: 10-12µm in diameter Nucleus: elongated, curved and usually U shaped, but it may be twisted. It is not segmented but may be slightly indented at one two points. The chromatin is continuous thick and coarse, and parachromatin is scanty. Cytoplasm: contains specific and a few non-specific granules and is pink or colorless. The nuclear/ cytoplasmic ratio is 1:2 F. Segmented granulocyte Size: 10-12µm in diameter. Nucleus: eccentric with heavy, thick chromatin masses. 28 Hematology It is divided into 2-5 lobes connected to each other by thin bridges of chromatin membrane. The ratio of segmented to band forms is of clinical significance and is normally about 10:1. Cytoplasm: abundant and slightly eosinophilic (pinkish) or colorless and contains specific granules. The neutrophilic granules are very fine in texture and do not overlay the nucleus. The nuclear/cytoplasmic ratio is 1:2. Eosinophilic Granulocyte and Precursors Eosinophils mature in the same manner as neutrophils. The eosinophlic myeloblast is not recognizable as such. In the eosinophilic promyelocyte in the Wright-Giemsa stained preparation the granule are at first bluish and later mature into orange granules, which are larger than neutrophilic granules are round or ovoid and are prominent in the eosinophilic myelocyte. Mature Eosinophil Size and shape: 11-13µm in diameter, slightly larger than a segmented polymorphonuclear granulocyte. Nucleus: usually bilobed, rarely single- or tri-lobed and 29 Hematology contains dense chromatin masses. Eosinophils with more than two nuclear lobes are seen in vitamin B12 and folic acid deficiency and in allergic disorders. Cytoplasm: densely filled with orange-pink granules so that its pale blue color can be appreciated only if the granules escape. The granules are uniform in size, large and do not cover the nucleus. Basophilic Granulocyte and Precursors The early maturation of the basophilic granulocyte is similar to that of the neutrophlic granulocyte. Mature Basophil Size: Somewhat smaller than eosiniphils, measuring 10-12µm in diameter. Nucleus: Indented giving rise to an S pattern. It is difficult to see the nucleus because it contains less chromatin and is masked by the cytoplasmic granules. Cytoplasm: Pale blue to pale pink and contains granules that often overlie the nucleus but do not fill the cytoplasm as completely as the eosinophilis granules do. 30 Hematology Monocytes and their Precursors Monoblast Since the monoblast can not be differentiated from the myeloblast on morphologic or histochemical criteria, one may assume that the myeloblast can give rise to myeloid and monocytic cells. Size: 15-25µm in diameter. Nucleus: Round or oval and at times notched and indented. The chromatin is delicate blue to purple stippling with small, regular, pink, pale or blue parachromatin areas. The nucleoli (3-5 in number) are pale blue, large and round. Cytoplasm: Relatively large in amount, contains a few azurophile granules, and stains pale blue or gray. The cytoplasm filling the nucleus indentation is lighter in color than the surrounding cytoplasm. The surrounding cytoplasm may contain Auer bodies. Promonocyte The earliest monocytic cell recognizable as belonging to the monocytic series is the promonocyte, which is capable of mitotic division. Its product, the mature 31 Hematology monocyte, is only capable of maturation into a macrophage. Size: 15-20µm in diameter. Nucleus: Large, ovoid to round, convoluted, grooved, and indented. The chromatin forms a loose open network containing a few larger clumps. There may be two or more nucleoli. Cytoplasm: sparse, gray-blue, contains fine azurophilic granules. The nuclear/cytoplasmic ratio is about 7:1 Monocyte Size: 14-18µm in diameter. Nucleus: Eccentric or central, is kidney shaped and often lobulated. The chromatin network consists of fine, pale, loose, linear threads producing small areas of thickening at their junctions. No nucleolus is seen. The overall impression is that of a pale nucleus quite variable in shape. Cytoplasm: Abundant, opaque, gray-blue, and unevenly stained and may be vacuolated. Lymphopoiesis 32 Hematology The precursor of the lymphocyte is believed to be the primitive mulipotential stem cell that also gives rise to the pluirpotenital myeloid stem cell for the granulocytic, erythyroid, and megakaryocytic cell lines. Lymphoid precursor cells travel to specific sites, where they differentiate into cells capable of either expressing cell- mediated immune responses or secreting immunoglobulins. The influence for the former type of differentiation in humans is the thymus gland; the resulting cells are defined as thymus-dependent lymphocytes, or T cells. The site of the formation of lymphocytes with the potential to differentiate into antibody-producing cells has not been identified in humans, although it may be the tonsils or bone marrow. In chickens it is the bursa of Fabricius, and for this reason these bursa-dependent lymphocytes are called B cells. B cells ultimately differentiate into morphologically distinct, antibody-producing cells called plasma cells Lymphocytes and Precursors Lymphoblast Size: 15-20µm in diameter. Nucleus: Central, round or oval and the chromatin has a stippled pattern. The nuclear membrane is distinct and 33 Hematology one or two pink nucleoli are present and are usually well outlined. Cytoplasm: Non-granular and sky blue and may have a darker blue border. It forms a thin perinuclear ring. Prolymphocyte Size: 14-18µm in diameter. Nucleus: Oval but slightly indented and may show a faint nucleolus. The chromatin is slightly condensed into a mosaic pattern. Cytoplasm: there is a thin rim of basophlic, homogeneous cytoplasm that may show a few azurophilic granules and vacuoles. Lymphocytes There are two varieties and the morphologic difference lies mainly in the amount of cytoplasm, but functionally most small lymphocytes are T cells and most large lymphocytes are B cells. Small Lymphocyte Size: 7-10µm in diameter. 34 Hematology Nucleus: round or oval to kidney shaped and occupies nine tenths of the cell diameter. The chromatin is dense and clumped. A poorly defined nucleolus may be seen. Cytoplasm: It is basophilic and forms a narrow rim around the nucleus or at times a thin blue line only. Large Lymphocyte Size: 12-14µm in diameter Nucleus: the dense, oval, or slightly indented nucleus is centrally or eccentricity located. Its chromatin is dense and clumped. Cytoplasm: abundant, gray to pale blue, unevenly stained, and streaked at times. A few azurophilic granules are contained in 30-60% of the cells. These are large granular lymphocytes (LGLs). III. Formation of platelets (Thrombopoiesis) Platelets are produced in the bone marrow by fragmentation of the cytoplasm of megakaryocytes. The precursor of the megakaryocyte-the megakaryoblast- arises by a process of differentiation for the hemopoietic stem cell. The megakaryoblast produces megakaryocytes, distinctive large cell that are the 35 Hematology source of circulating platelets. Megakaryocyte development takes place in a unique manner. The nuclear DNA of megakaryoblasts and early megakaryocytes reduplicates without cell division, a process known as endomitosis. As a result, a mature megakaryocytes has a polyploidy nucleus, that is, multiple nuclei each containing a full complement of DNA and originating from the same locust within the cell. Mature megakaryocytes are 8 n to 36 n.The final stage of platelet production occurs when the mature megakaryocyte sends cytoplasmic projections into the marrow sinusoids and sheds platelets into the circulation. It takes approximately 5 days from a megakaryoblast to become a mature megakaryocyte. Each megakaryocyte produces from 1000 to 8000 platelets. The platelet normally survives form 7 to 10 days in the peripheral blood. Morphology of the Platelets and their Precursors Megakaryoblast Size: ranges from 10-30µm in diameter. The cell is smaller than its mature forms but larger than all other blast cells. 36 Hematology Nucleus: the single, large, oval or indented nucleus has a loose chromatin structure and a delicate nuclear membrane. Multi-lobulated nuclei also occur representing a polyploid stage. Several pale blue nucleoli are difficult to see. The parachromatin is pink. Cytoplasm: the cytoplasm forms a scanty, bluish, patchy, irregular ring around the nucleus. The periphery shows cytoplasmic projections and pseudopodia like structures. The immediate perinuclear zone is lighter than the periphery. Promegakaryocyte Size: ranges from 20-50µm in diameter. It is larger than the megakaryoblast and in the process of maturation it reaches the size of the stage III cell. Nucleus: large, indented and poly-lobulated. The chromatin appears to have coarse heavily stained strands and may show clumping. The total number of nucleoli is decreased and they are more difficult to see than in the blast cell. The chromatin is thin and fine. Cytoplasm: intensely basophilic, filled with increasing 37 Hematology numbers of azurophilic granules radiating from the golgi apparatus toward the periphery sparing a thin peripheral ring that remains blue in color. Granular Megakaryocyte The majority of the megakaryocytes of a bone marrow aspirate are in stage III which is characterized by progressive nuclear condensation and indentation and the beginning of platelet formation within the cytoplasm. Size: ranges from 30-100µm in diameter and is the largest cell found in the bone marrow. Cytoplasm: a large amount of polychromatic cytoplasm produces blunt, smooth, pseudopodia-like projections that contain aggregates of azurophilic granules surrounded by pale halos. These structures give rise to platelets at the periphery of the megakaryocytes. Platelets Size: varies from 1-4µm in diameter. Nucleus: no nucleus is present. In Wright - Giemsa stained films, platelets appear as 38 Hematology small, bright azure, rounded or elongated bodies with a delicately granular structure. 39 Hematology Review Questions 1. What is hemopoiesis and how is the process regulated? 2. What are the hemopoietic tissues during fetal life, in infancy, in childhood and in adulthood? 3. What are the effects of the hormone erythropoietin on red cell development and maturation 4. Explain what megaloblastic erythropoiesis is. 5. State the main functions of blood. 40 Hematology CHAPTER TWO BLOOD COLLECTION Learning objectives At the end of this chapter, the student shall be able to: List safety precautions considered in collecting blood samples List the possible source of blood samples for hematological investigation Describe the advantage of peripheral blood collection Explain the advantage and disadvantage of venous blood collection Describe the mechanism for preventing hemolysis Introduction Blood is the body fluid used most frequently for analytical purposes. Blood must be collected with care and adequate safety precautions to ensure test results are reliable, contamination of the sample is avoided and infection from blood transmissible pathogens is prevented. The proper collection and reliable 41 Hematology processing of blood specimens is a vital part of the laboratory diagnostic process in hematology as well as other laboratory disciplines. Unless an appropriately designed procedure is observed and strictly followed, reliability can not be placed on subsequent laboratory results even if the test itself is performed carefully. All material of human origin should be regarded as capable of transmitting infection. Specimens from patients suffering from, or at risk of, hepatitis or human immunodeficiency virus (HIV) infection require particular care. When collecting blood sample, the operator should wear disposable rubber gloves. The operator is also strongly advised to cover any cuts, abrasions or skin breaks on the hand with adhesive tape and wear gloves. Care must be taken when handling especially, syringes and needles as needle-stick injuries are the most commonly encountered accidents. Do not recap used needles by hand. Should a needle-stick injury occur, immediately remove gloves and vigorously squeeze the wound while flushing the bleeding with running tap water and then thoroughly scrub the wound with cotton balls soaked in 0.1% hypochlorite solution. Used disposable syringes and needles and other sharp items such as 42 Hematology lancets must be placed in puncture-resistant container for subsequent decontamination or disposal. Three general procedures for obtaining blood are (1) Skin puncture, (2) venipuncture, and (3) arterial puncture. The technique used to obtain the blood specimen is critical in order to maintain its integrity. Even so, arterial and venous blood differs in important respects. Arterial blood is essentially uniform in composition throughout the body. The composition of venous blood varies and is dependent on metabolic activity of the perfused organ or tissue. Site of collection can affect the venous composition. Venous blood is oxygen deficient relative to arterial blood, but also differs in pH, carbon dioxide concentration, and packed cell volume. Blood obtained by skin puncture is an admixture of blood from arterioles, venules, and capillaries. Increased pressure in the arterioles yields a specimen enriched in arterial blood. Skin puncture blood also contains interstitial and intracellular fluids. 2.1 Capillary blood collection Capillary blood (peripheral blood / microblood samples) 43 Hematology is frequently used when only small quantities of blood are required, e.g., for hemoglobin quantitation, for WBC and RBC counts and for blood smear preparation. It is also used when venipuncture is impractical, e.g. in infants, in cases of sever burns, in extreme obesity where locating the veins could be a problem and in patients whose arm veins are being used for intravenous medication. Sites of Puncture Adults and children: palmar surface of the tip of the ring or middle finger or free margin of the ear lobe. Infants: plantar surface of the big toe or the heel. Note: Edematous, congested and cyanotic sites should not be punctured. Cold sites should not be punctured as samples collected from cold sites give falsely high results of hemoglobin and cell counts. Site should be massaged until it is warm and pink. 44 Hematology Fig 2.1 Peripheral blood collection from adult person Fig 2.2 Skin puncture from infants 45 Hematology Materials Required Gauze pads or cotton, 70% alcohol, sterile disposable lancet Method 1. Rub the site vigorously with a gauze pad or cotton moistened with 70% alcohol to remove dirt and epithelial debris and to increase blood circulation in the area. If the heel is to be punctured, it should first be warmed by immersion in a warm water or applying a hot towel compress. Otherwise values significantly higher than those in venous blood may be obtained. 2. After the skin has dried, make a puncture 2-3mm deep with a sterile lancet. A rapid and firm puncture should be made with control of the depth. A deep puncture is no more painful than a superficial one and makes repeated punctures unnecessary. The first drop of blood which contains tissue juices should be wiped away. The site should not be squeeze or pressed to get blood since this dilutes it with fluid from the tissues. Rather, a freely flowing blood should be taken or a moderate pressure some distance above the puncture site is allowable. 3. Stop the blood flow by applying slight pressure with 46 Hematology a gauze pad or cotton at the site. Advantages of Capillary Blood It is obtained with ease. It is the preferred specimen for making peripheral blood films since no anticoagulant is added that affect cell morphology. Disadvantages of Capillary Blood Only small amounts of blood can be obtained and repeated examinations require a new specimen. Platelet count can not be performed on capillary blood since some platelets are unavoidably lost by adherence onto the wound. Precision is poorer in capillary than venous blood because of variation in blood flow and dilution with interstitial fluid. Blood in microtubes frequently hemolyses and hemolysis interferes with most laboratory tests. 2.2. Venous Blood Collection A venous blood sample is used for most tests that require anticoagulation or larger quantities of blood, 47 Hematology plasma or serum. Sites of Puncture The veins that are generally used for venipuncture are those in the forearm, wrist or ankle. The veins in the antecubital fossa of the arm are the preferred sites for venipuncture. They are larger than those in the wrist or ankle regions and hence are easily located and palpated in most people. The three main veins in the forearm are the cephalic, the median cephalic, and the median basilic. In infants and children, venipuncture presents special problems because of the small size of the veins and difficulty controlling the patient. Puncture of the external jugular vein in the neck region and the femoral vein in the inguinal area is the procedure of choice for obtaining blood. 48 Hematology Fig 2.3 venipuncture Materials Sterile syringe and needle, vacuum tube, vacuum tube holder and two-way needle (if the vacutainer method is to be employed), tourniquet, gauze pads or cotton, 70% alcohol, test tubes with or without anticoagulant. Method 1. Assemble the necessary materials and equipment. Remove the syringe from its protective wrapper and the needle from the cap and assemble them allowing the cap to remain covering the needle 49 Hematology until use. Attach the needle so that the bevel faces in the same direction as the graduation mark on the syringe. Check to make sure the needle is sharp, the syringe moves smoothly and there is no air left in the barrel. The gauge and the length of the needle used depend on the size and depth of the vein to be punctured. The gauge number varies inversely with the diameter of the needle. The needle should not be too fine or too long; those of 19 or 21G are suitable for most adults, and 23G for children, the latter especially with a short shaft (about 15mm). The International Organization for standardization has established a standard (ISO 7864) with the following diameters for the different gauges: 19G=1.1mm; 21G=0.8mm; 23G=0.6mm. If the vacutainer method is to be used, thread the short end of the double-pointed needle into the holder and push the tube forward until the top of the stopper meets the guide mark on the holder. The point of the needle will thus be embedded in the stopper without puncturing it and loosing the vacuum in the tube. 2. Identify the patient and allow him/her to sit 50 Hematology comfortably preferably in an armchair stretching his/ her arm. 3. Prepare the arm by swabbing the antecubital fossa with a gauze pad or cotton moistened with 70% alcohol. Allow it to dry in the air or use a dry pad or cotton. The area should not be touched once cleaned. 4. Apply a tourniquet at a point about 6-8cm above the bend of the elbow making a loop in such a way that a gentle tug on the protruding ends will release it. It should be just tight enough to reduce venous blood flow in the area and enlarge the veins and make them prominent and palpable. The patient should also be instructed to grasp and open his/her fist to aid in the build up of pr essu re in the are a o f t h e p u n c t u re. Alternatively, the veins can be visualized by gently tapping the antecubital fossa or applying a warm towel compress. 5. Grasp the back of the patient’s arm at the elbow and anchor the selected vein by drawing the skin slightly taut over the vein. 6. Using the assembled syringe and needle, enter the skin first and then the vein. To insert the needle properly into the vein, the 51 Hematology index finger is placed along side the hub of the needle with the bevel facing up. The needle should be pointing in the same direction as the vein. The point of the needle is then advanced 0.5-1.0cm into the subcutaneous tissue (at an angle of 450) and is pushed forward at a lesser angle to pierce the vein wall. If the needle is properly in the vein, blood will begin to enter the syringe spontaneously. If not, the piston is gently withdrawn at a rate equal to the flow of blood. With the vacutainer system, when in the vein, the vacuum tube is pushed into the needle holder all the way so that the blood flows into the tube under vacuum. The tourniquet should be released the moment blood starts entering the syringe/vacuum tube since some hemoconcentration will develop after one minute of venous stasis. 7. Apply a ball of cotton to the puncture site and gently withdraw the needle. Instruct the patient to press on the cotton. 8. With the syringe and needle system, first cover the needle with its cap, remove it from the nozzle of the 52 Hematology syringe and gently expel the blood into a tube (with or without anticoagulant). Stopper the tube and invert gently to mix the blood with the anticoagulant. The sample should never be shaked. With the vacutainer system, remove the tube from the vacutainer holder and if the tube is with added anticoagulant, gently invert several times. Label the tubes with patient’s name, hospital number and other information required by the hospital. 9. Reinspect the venipuncture site to ascertain that the bleeding has stopped. Do not let the patient go until the bleeding stops Advantages of Venous Blood By providing sufficient amount of blood it allows various tests to be repeated in case of accident or breakage or for the all-important checking of a doubtful result. It also frequently allows the performance of additional tests that may be suggested by the results of those already ordered or that may occur to the clinician as afterthoughts. Aliquots of the specimen (plasma and serum) 53 Hematology may be frozen for future reference. It reduces the possibility of error resulting from dilution with interstitial fluid or constriction of skin vessels by cold that may occur in taking blood by skin puncture. Disadvantages of Venous Blood It is a bit a lengthy procedure that requires more preparation than the capillary method. It is technically difficult in children, obese individuals and in patients in shock. Hemolysis must be prevented because it leads to lowered red cell counts and interferes with many chemical tests. Hematoma (or blood clot formation inside or outside the veins) must be prevented. Difference between peripheral and venous Blood Venous blood and peripheral blood are not quite the same, even if the latter is free flowing, and it is likely that free flowing blood obtained by skin puncture is more arteriolar in origin. The PCV, red cell count and hemoglobin content of peripheral blood are slightly greater than in venous blood. The total leucocyte and neutrophil counts are higher by about 8% and the 54 Hematology monocyte count by 12%. Conversely, the platelet count appears to be higher by about 9% in venous than peripheral blood. This may be due to adhesion of platelets to the site of the skin puncture. Advantages of the Vacutainer Method of Venous Blood Collection It is an ideal means of collecting multiple samples with ease. The multiple sample needle used in the vacutainer method has a special adaptation that prevents blood from leaking out during exchange of tubes. The use of evacuated tube eliminates many of the factors that cause hemolysis. No preparation of anticoagulants and containers needed. One can choose among a wide range of tube size and contained anticoagulant. Because the evacuated tubes are sterile possible bacterial contamination is prevented and hence provides the ideal blood sample for microbiological analysis. 2.3. Arterial puncture Arterial blood is used to measure oxygen and carbon 55 Hematology dioxide tension, and to measure pH (arterial blood gases-ABG). These blood gas measurements are critical in assessment of oxygenation problems encountered in patients with pneumonia, pneumonitis, and pulmonary embolism. Arterial punctures are technically more difficult to perform than venous punctures. Increased pressure in the arteries makes it more difficulty to stop bleeding with the undesired development of a hematoma. Arterial selection includes radial, brachial, and femoral arteries in order of choice. Sites not to be selected are irritated, edematous, near a wound, or in an area of an arteriovenous (AV) shunt or fistula. Prevention of Hemolysis Make sure the syringe, needle and test tubes are dry and free from detergent as traces of water or detergent cause hemolysis. Use smooth, good quality sharp needles. Gentleness should be the watch word. Avoid rough handling of blood at any stage. Do not eject the blood from the syringe through the needle as this may cause mechanical destruction of the cells. Transfer the blood from the syringe by gently ejecting down the side of the tube. Mix blood with 56 Hematology anticoagulant by gentle inversion not by shaking. Tourniquet should not be too tight and should be released before blood is aspirated. If examination is to be delayed beyond 1-3 hrs, do not allow the sample to stand unsealed or at room temperature. Stopper and store in a refrigerator at 4OC. Blood should not be stored in a freezer because the red cells will hemolyse on thawing. Make sure that all solutions with which blood is to be mixed or diluted are correctly prepared and are isotonic. Hypotonic solutions will lead to hemolysis. When obtaining blood by skin puncture make sure the skin is dry before pricking and to use sharp, 2-3mm lancets that produce clean puncture wounds. The blood should be allowed to escape freely. 57 Hematology Review Questions 1. What are the sources of blood sample for hematological investigations? 2. What are the anatomical sites of collection in these sources in the different age groups? 3. What are the advantages as well as the draw backs of taking/using blood samples from each of these sources? 4. How do you minimize or avoid the occurrence of hemolysis in blood samples for hematological investigations? 5. What is the difference between samples collected from these two sources in terms of hematological parameters? 58 Hematology CHAPTER THREE ANTICOAGULANTS Learning objectives At the end of this chapter, the student shall be able to: Define anticoagulants Describe the proportion, mechanism of anticoagulation and advantages of EDTA, Trisodium citrate, double oxalates and heparin anticoagulants. Prepare the different anticoagulants in the right concentration Introduction Anticoagulants are chemical substances that are added to blood to prevent coagulation. In other words, certain steps are involved in blood coagulation, but if one of the factors is removed or inactivated, the coagulation reaction will not take place. The substances responsible for this removal or inactivation are called anticoagulants. While clotted blood is desirable for certain laboratory investigations, most hematology procedures require an anticoagulated whole blood. 59 Hematology For various purposes, a number of different anticoagulants are available. EDTA and sodium citrate remove calcium which is essential for coagulation. Calcium is either precipitated as insoluble oxalate (crystals of which may be seen in oxalated blood) or bound in a non-ionized form. Heparin works in a different way; it neutralizes thrombin by inhibiting the interaction of several clotting factors in the presence of a plasma cofactor, antithrombin III. Sodium citrate or heparin can be used to render blood incoagulable before transfusion. For better long-term preservation of red cells for certain tests and for transfusion purposes, citrate is used in combination with dextrose in the form of acid-citrate-dextrose (ACD), citrate-phosphate- dextrose (CPD) or Alserver’s solution. 3.1. Ethylenediamine tetraacetic acid Ethylenediamine tetraacetic acid (EDTA) has become the standard hematology anticoagulant because of its very efficient and complete anticoagulation and its lack of effect on the size (morphology) or number of blood cells in the specimen. Its disodium or tripotassium salt are used. The anticoagulant recommended by the ICSH is the dipotassium salt. It is the preferred anticoagulant for cell counts and morphological studies. It is especially 60 Hematology the anticoagulant of choice for platelet counts and platelet function tests since it prevents platelet aggregation. It exerts its effect by tightly binding (chelating) ionic calcium thus effectively blocking coagulation. The dilithium salt of EDTA is equally effective as an anticoagulant, and its use has the advantage that the same sample of blood can be used for chemical investigation. The amount of EDTA necessary for the complete chelation of Calcium is balanced with the desire to minimize cellular damage so that standardizing bodies have recommended a concentration of 1.5±0.25mg of Na2 or K3 EDTA per 1ml of blood (e.g. 0.02ml of 10% (W/V) solution of K3EDTA is used for 1ml of blood). This concentration does not appear to adversely affect any of the erythrocyte or leucocyte parameters. 3.2 Trisodium Citrate Sodium citrate combines with calcium, thereby preventing the conversion of prothrombin to thrombin, and coagulation does not occur. 100-120 mmol/l 61 Hematology trisodium citrate (32g/l) is the anticoagulant of choice in coagulation studies. Nine volumes of blood are added to 1 volume of the sodium citrate solution and immediately well mixed with it. Sodium citrate is also the anticoagulant for the erythrocyte sedimentation rate (ESR); for this, 4 volumes of venous blood are diluted with 1 volume of the sodium citrate solution. 3.3. Balanced or double oxalate Salts of oxalic acid by virtue of their ability to bind and precipitate calcium as calcium oxalate serve as suitable anticoagulants for many hematologic investigations. Three parts of ammonium oxalate is balanced with two parts of potassium oxalate (neither salt is suitable by itself, i.e., ammonium oxalate causes cellular swelling and potassium oxalate causes erythrocyte shrinkage). It is used in the proportion of 1-2mg/ml of blood. 3.4. Heparin Heparin is an excellent natural anticoagulant extracted from mammalian liver or pancreas. It is more expensive than the artificial ones and has a temporary effect of 62 Hematology only 24 hours. Heparin prevents clotting by inactivating thrombin, thus preventing conversion of fibrinogen to fibrin. It is the best anticoagulant when absolute minimal hemolysis is required (e.g., osmotic fragility test and hematocrit determination). It is unsatisfactory for leucocyte and platelet and leucocyte counts as it causes cell clumping and also for blood film preparation since it causes a troublesome diffuse blue background in Wright-stained smears. It is used in the proportion of 0.1-0.2mg of the dry salt for 1ml of blood. 63 Hematology Review Questions 1. Define anticoagulant. 2. List the anticoagulants that are commonly used in hematology. How does each of these anticoagulants exert their functions? 3. Write the proportion of the volume of blood to the volume of each if these anticoagulants. 64 Hematology CHAPTER FOUR PREPARATION OF BLOOD SMEARS Learning objectives At the end of this chapter, the student shall be able to: Explain the purpose of preparing blood films Prepare thin blood films on slides and cover glasses Explain the spinner method of preparing blood film Identify the desirable qualities of a thin blood film Prepare thick blood films Microscopic examination of the peripheral blood is most often done by preparing, staining, and examining a thin film (smear) of blood on glass slide. A great deal of information can be obtained from the examination of a blood film. With the use of automatic counting devices that determine hemoglobin, hematocrit, red cell, white cell, and platelet counts together with MCV, MCH, MCHC, and RDW, white cell differential, and histograms, there is a tendency to place less emphasis on the routine examination of the peripheral blood film. However, these same automated results may also point 65 Hematology to the need to examine the blood film microscopically to confirm the presence of disease suggested by the results or for early detection of disease. Of course, in a laboratory without access to such automated information, the microscopic examination of the peripheral blood film is invaluable. Examination of the blood film is an important part of the hematologic evaluation and the validity or reliability of the information obtained from blood film evaluation, the differential leucocyte count in particular depends heavily on well-made and well- stained films. While blood film preparation is a disarmingly simple straight - forward procedure, there is abundant and continuing evidence that the quality of blood films in routine hematology practice leaves much room for improvement. If not made from skin puncture, films should be prepared within 1 hour of blood collection into EDTA. Adequate mixing is necessary prior to film preparation if the blood has been standing for any appreciable period of time. 4.1 Preparation of thin blood films Three methods of making films are described: the two- slide or wedge method, the coverglass method, and the 66 Hematology spinner method. Preparation of blood films on glass slides has the following advantages: Slides are not easily broken Slides are easier to label When large numbers of films are to be dealt with, slides will be found much easier to handle. Method I. Wedge method (Two-slide method) A small drop of blood is placed in the center line of a slide about 1-2cm from one end. Another slide, the spreading slide placed in front of the drop of blood at an angle of 300 to the slide and then is moved back to make contact with the drop. The drop will spread out quickly along the line of contact of the spreader with the slide. Once the blood has spread completely, the spreader is moved forward smoothly and with a moderate speed. The drop should be of such size that the film is 3-4cm in length (approx. 3/4th of the length of the slide). It is essential that the slide used as a spreader have a smooth edge and should be narrower in breadth than the slide on which the film is prepared so that the edges of the film can be readily examined. 67 Hematology Fig 4.1 preparing a glass spreader to make blood films It can be prepared in the laboratory by breaking off 2mm from both corners so that its breadth is 4mm less than the total slide breadth. If the edges of the spreader are rough, films with ragged tails will result and gross qualitative irregularity in the distribution of cells will be the rule. The bigger leucocytes (neutrophils and monocytes) will accumulate in the margins and tail while lymphocytes will predominate in the body of the film. The ideal thickness of the film is such that there is some overlap of the red cells through out much of the film’s length and separation and lack of distortion towards the tail of the film. Thickness and length of the film are affected by 68 Hematology speed of spreading and the angle at which the spreader slide is held. The faster the film is spread the thicker and shorter it will be. The bigger the angle of spreading the thicker will be the film. Once the slide is dry, the name of the patient and date or a reference number is written on the head of the film using a lead pencil or graphite. If these are not available, writing can be done by scratching with the edge of a slide. A paper label should be affixed to the slide after staining. Fig 4.2 (a) Preparation of blood film 69 Hematology Fig 4.2 (b) Good blood film II. Cover glass method 22mm × 22mm cover glasses are required. Touch a clean cover glass to the top of a small drop of blood without touching the skin and place it blood side down, cross- wise on another cover glass so that the corners will as an eight-pointed star. If the drop is not too large and if the cover glasses are perfectly clean, the blood will spread out evenly and quickly in a thin layer between the two surfaces. Cover glasses should be placed film side up on a clean paper and allowed to dry in the air. After they are stained they are mounted film side down with permount film side down on glass slides. III. Spinner method 70 Hematology Blood films that combine the advantages of easy handling of the wedge slide and uniform distribution of cells of the coverglass preparation may be made with special types of centrifuges known as spinners. The spinner slide produces a uniform blood film, in which all cells are separated (a monolayer) and randomly distributed. White cells can be easily identified at any spot in the film On a wedge smear there is a disproportion of monocytes at the tip of the feather edge, of neutrophils just in from the feather edge, and of both at the later edges of the film. This is of little practical significance, but it does result in slightly lower monocyte counts in wedge films. Desirable qualities of a thin blood film The availability of sufficient working area. Acceptable morphology within working area and minimum distortion of the distribution of the blood cells in particular the leucocytes. Gradual transition to thickness from the thick to thin areas terminating in a feather like edge. No ridges, holes or waves. Margins of the film should be smooth, continuous and accessible for oil-immersion examination. The minimum length of the film should be 3.0cm 71 Hematology (approximately 3/4th of the length of the slide. 4.2. Preparation of thick blood smears Thick blood smears are widely used in the diagnosis of blood parasites particularly malaria. It gives a higher percentage of positive diagnosis in much less time since it has ten times the thickness of normal smears. Five minutes spent in examining a thick blood film is equivalent to one hour spent in traversing the whole length of a thin blood film. Method Place a small drop of blood on a clean slide and spread it with an applicator stick or the corner of another slide until small prints are just visible through the blood smear. This corresponds to a circle of approximately 2cm diameter. 72 Hematology Review Questions 1. What is a thin blood film? 2. Which technique of blood film preparation is commonly employed and how is the method of preparation? 3. What are the desirable qualities of a thin blood film? 4. What are the possible effects of using a blood sample that has been standing at room temperature for some time on blood cell morphology? 73 Hematology CHAPTER FIVE STAINING OF BLOOD SMEARS Learning objectives At the end of this chapter, the student shall be able to: Describe the general principle of staining blood films Perform then technique of staining thin blood films with Romanowsky dyes Describe the appearance of cells and cell components in Romanowsky-stained blood films Explain the principle of thick blood film preparation with Giemsa and Field’s stains Stain blood films with the panoptic stains List the problems that arise in staining and the possible remedies Introduction Ehrlich was the first to use aniline dyes at first in sequence and latter as a premixed acidic – basic stains (neutral dyes). Jenner (1880) found that the precipitate formed when eosin and methylene blue are mixed could 74 Hematology be dissolved in methyl alcohol to form a useful stain combining certain properties of both parent dye stuffs. Romanowsky (1890) found that when old (ripened and therefore "polychromed") methylene blue solution is mixed with eosin and the precipitate dissolved in methyl alcohol, a stain results that has a wider range than Jenner’s stain staining cell nuclei and platelet granules (which Jenner’s mixture failed to stain). 5.1. Principle of staining Acidic dyes such as eosin unites with the basic components of the cell (cytoplasm) and hence the cytoplasm is said to be eosinophilic (acidic). Conversely, basic stains like methylene blue are attracted to and combine with the acidic parts of the cell (nucleic acid and nucleoproteins of the nucleus) and hence these structures are called basophilic. Other structures stained by combination of the two are neutrophilic 5.2. Romanowsky stains in common use 75 Hematology Modern Romanowsky stains in common, e.g., Wright and Leishman, are basically similar to Romanowsky’s original method, the difference being the method of polychroming the methylene blue. I. Wright stain In its preparation, the methylene blue is polychromed by heating with sodium carbonate. It is purchased as a solution ready to use or as a powder. Staining Method 1. Place the air-dried smear film side up on a staining rack (two parallel glass rods kept 5cm apart). 2. Cover the smear with undiluted stain and leave for 1 minute. The methyl alcohol in the satin fixes the smear. When it is planned to use an aqueous or diluted stain, the air dried smear must first be fixed by flooding for 3-5 minutes with absolute methanol. if films are left unfixed for a day or more, it will be found that the background of dried plasma stains pale blue and this is impossible to remove without spoiling the staining of the blood cells. 3. Dilute with distilled water (approximately equal volume) until a metallic scum appears. Mix by 76 Hematology blowing. Allow this diluted stain to act for 3-5 minutes. 4. Without disturbing the slide, flood with distilled water and wash until the thinner parts of the film are pinkish red. II. Leishman Stain In its preparation, the methylene blue is polychromed by heating a 1 % solution with 0.5% sodium carbonate at 650C for 12 hours after which a further ripening is allowed to proceed for 10 days before it is mixed with an equal volume of 0.1% eosin B. Staining method The method is similar to that used in Wright’s stain except for step 3. With Leshman’s stain, dilution is effected with approximately two volume of distilled water to one volume of stain (the best guide is the appearance of a metallic scum). Microscopic appearance of cells and cell components in Romanowsky-stained blood films (Films stained with either Wright or Leishman stains are pinkish in color when viewed with the naked eye): Red cells - pink with a central pale area 77 Hematology Nuclei of leucocytes - blue to purple Cytoplasmic neutrophilic granules - tan Eosinophilic granules - red orange each distinctly discernible Basophilic granules - dark blue Cytoplasm of monocytes - faint blue gray Platelets - violet granules Malaria parasites - sky blue cytoplasm and red purple chromatin III. Giemsa stain Instead of empirically polychromed dyes, this stain employs various azure compounds (thionine and its methyl derivative) with eosin and methylene blue). This is an alcohol-based Romanowsky stain that required dilution in pH 7.1-7.2 buffered water before used. It gives the best staining of malaria parasites in thick films. It is commonly used in combination with Jenner or May – Grunwald stains it constitutes “panoptic staining". Staining of thick smears The stains used employ the principle of destroying the red cells and staining leucocytes and parasites. The method using Giemsa stain is satisfactory. 78 Hematology Method 1. Cover the air-dried smear with a 1:10 diluted Giemsa using buffered distilled water at pH 6.8 as a diluent. Do not fix the films before staining. Leave the stain to act for 15-30 minutes. Do not fix the films before staining. 2. Wash with distilled water and air dry. IV. Panoptic staining Panoptic staining consists of a combination of a Romanowsky stain with another stain, e.g. Giemsa. This improves the staining of cytoplasmic granules and other bodies like nucleoli of blast cells. Popular methods are Jenner - Giemsa and May-Grunwald - Giemsa. A. Jenner-Giemsa method 1. Dry the films in the air then fix by immersing in a jar containing methanol for 10-20 minutes. For bone marrow films leave for 20-25 minutes. 2. Transfer the films to a staining jar containing Jenner's stain freshly diluted with 4 volumes of buffered water and leave for 4 minutes. 79 Hematology 3. Transfer the slides without washing to a jar containing Giemsa stain freshly diluted with 9 volumes of buffered water pH 6.8. Allow to stain for 7-10 minutes. 4. Transfer the slides to a jar containing buffered water, pH 6.8; rapidly wash in 3 or 4 changes of water and finally allow to stand undisturbed in water for 2-5 minutes for differentiation to take place. 5. Place the slides on end to dry. B. May-Grünwald-Giemsa method 1. Dry the films in the air then fix by immersing in a jar containing methanol for 10-20 minutes. For bone marrow films leave for 20-25 minutes. 2. Transfer the films to a staining jar containing May- Grünwald's stain freshly diluted with an equal volume of buffered water and leave for 15 minutes. 3. Transfer the slides without washing to a jar containing Giemsa's stain freshly diluted with 9 volumes of buffered water pH 6.8. Allow to stain for 10-15 minutes. 4. Transfer the slides to a jar containing buffered water, pH 6.8; rapidly wash in 3 or 4 changes of water and finally allow to stand undisturbed in water for 2-5 minutes for differentiation to take place. 80 Hematology 5. Place the slides on end to dry. V. Field's stain Field’s stain was introduced to provide a quick method for staining thick films for malaria parasites. It this water-based Romanowsky stain is composed of two solutions, Field’s stain A and Field’s stand B. It is buffered to the correct pH and neither solution requires dilution when staining thick films. When staining thin films, Field’s stain B requires dilution. Compared with Giemsa working stain, Field’s stains are more stable. They stain fresh blood films, well, particularly thick films. The rapid technique is ideally suited for staining blood films from waiting outpatients and when reports are required urgently. Thin film Field’s staining technique Required Field’s stain A Field’s stain B, diluted 1 in 5 Buffered pH 7.1-7.2 water Method 1. Place the slide on a staining rack and cover the methanol-fixed thin film with approximately 0.5ml of 81 Hematology diluted Field’s stain B. 2. Add immediately an equal volume of Field’s stain A and mix with the diluted Field’s stain B. Leave to stain for 1 minute. The stain can be easily applied and mixed on the slide by using 1ml graduated plastic bulb pipettes. 3. Wash off the stain with clean water. Wipe the back of the slide clean and place it in a draining rack for the film to air-dry. Thick film Field’s staining technique Required Container of fields’ stain A Container of Field’s stain B Two containers of clean water (need not be buffered) Method 1. Holding the slide with the dried thick film facing downwards, dip the slide into Field’s stain A for 5 seconds. Drain off the excess stain by touching a corner of the slide against the side of the container. 2. Wash gently for about 5 seconds in clean water. Drain off the excess water. 3. Dip the slide into Field’s stain B for 3 seconds. Drain off the excess stain. 82 Hematology 4. Wash gently in clean water. Wipe the back of the slide clean and place it upright in a draining rack for the film to air-dry. 5.3 Problems in staining I. Excessively blue stain Causes: too thick films, prolonged staining, inadequate washing, too high alkalinity of stain or diluent Appearance: erythrocytes-blue green, nuclear chromatin-deep blue to black, granules of neutrophils-deeply stained and appear large and prominent. Correction: preparing films with ideal thickness, reducing staining time, using less stain and more diluent, prolonging washing, adjust pH of buffer or prepare a new batch of stain. II. Excessively pink stain Causes: insufficient staining, prolonged washing, too high acidity of the stain or buffer (exposure of stain or buffer to acid fumes). Appearance: erythrocytes-bright red or orange, nuclear chromatin-pale blue, granules of 83 Hematology eosinophils-sparkling brilliant red Correction: prolonging staining time, reducing washing, preparing a new batch of stain. III. Precipitate on the film Causes: unclean slides, drying during the period of staining, inadequate washing of slide at the end of the staining period Correction: use clean slides, cover the smear with generous amount of the stain, wash the slide until thinner parts of the film are pinkish 84 Hematology Review Questions 1. What is the general principle of staining blood films with Romanowsky dyes? 2. What are the various Romanowsky stains used for staining of blood films? 3. Describe the appearance of cells and cell components in Romanowsky- stained thin blood films. 4. What are the staining problems that give rise to unsatisfactory results? How do you correct these problems? 5. What is panoptic staining? What is the advantage of panoptic stains over simple Romanowsky dyes? 6. What is the principle of thick film staining? List two dyes that are commonly used in thick blood film staining? 85 Hematology CHAPTER SIX HEMOCYTOMETRY Learning objectives At the end of this chapter, the student shall be able to: Discuss the general principles of manual hemocytometry List the materials that are basically required in manual hemocytometry Identify the sources of error in manual hemocytometry Mention the diluting fluid, dilution factor and areas of counting on the chamber for the RBC, WBC, platelet and eosinophil count Perform RBC, WBC, platelet and eosinophil counts Discuss the clinical significance and normal values of each of the cell counts. Introduction Visual counting of blood cells is an acceptable 86 Hematology alternative to electronic counting for white cell and platelet counts. It is not recommended for routine red cell counts because the number of cells which can be counted within a reasonable time in the routine laboratory will be too few to ensure a precise result. Yet it is still necessary for the technologist to be able to use this method effectively and to know its limitations. Any cell counting procedure includes three steps: dilution of the blood, sampling the diluted suspension into a measured volume, and counting the cells in that volume. The main principles for such examinations are: Selection of a diluting fluid that not only will dilute the cells to manageable levels but will either identify them in some fashion or destroy contaminant cellular elements. The use of a special glass counting chamber called hemocytometer that will present the cells to the observer in such a way that the number of cells per unit volume of fluid can be counted. Counting Chambers The hemocytometer is a thick glass slide with inscribed platforms of known area and precisely controlled depth under the coverslip. In the center of the upper surface 87 Hematology there are ruled areas separated by moats/channels from the rest of the slide and two raised transverse bars one of which is present on each side of the ruled area. The ruled portion may be in the center of the chamber (single chamber) or there may be an upper and lower ruled portion (double chamber). The double chamber is to be recommended since it enables duplicate counts to be made rapidly. When an optically plane cover glass is rested on the raised bars there is a predetermined gap or chamber formed between its lower surface and the ruled area (fig. 6.1). This is called the depth of the chamber and it varies with the type of the chamber. The ruled area itself is divided by microscopic lines into a pattern that varies again with the type of the chamber. The counting chamber recommended for cell counts is a metallized surface (Bright-line) double cell Improved N e u b a u e r r u l e d c h a m b e r. N o n - m e t a l l i z e d hemocytometer are less expensive, but they are not recommended. It is more difficult to count WBCs reliable using this type of chamber because the background rulings and cells are not as easily seen. Not-metallized chambers are also more difficult to fill. 88 Hematology Although there are a number of hemocytometer, it is the improved Neubauer counting chamber which is sued for most routine cell counts: I. Ordinary Neubauer counting chamber The central platform is set 0.1mm below the level of the two side ones, giving the chamber a depth of 0.1mm. The engraving covers an area of 9mm2 divided into 9 squares of 1mm2 each. The 4 corner squares are divided into 16 squares, each with an area of 1/16 of a mm2. The central ruled area of 1mm2 is divided into 16 large squares by sets of triple lines. These large squares are further subdivided into 16 small squares by single lines. The width of the triple lines dividing the large squares is the same as the width of a small square. Two adjacent sides of the ruled area are bounded by triple lines, the other two by single lines. Each side is, therefore, divided into 20 equal divisions (the width of 16 small squares and 4 sets of triple lines). Each small square is, therefore, 1/20 of 1mm squared that is 1/400 of 1mm 2. II. The Improved Neubauer Counting Chamber The depth between the lower surface of the cover glass which is on the raised bars and the ruled area is 0.1mm. 89 Hematology Each ruled area is a square of 9mm divided into nine large squares each of 1mm side. The central square of these nine is divided by engraved lines into 400 tiny squares of arranged in 25 groups of 16 by triple boundary lines. Each large square is 1mm2, each of the 25 medium squares is of 0.04mm2 area and each of the 400 tiny squares has an area of 0.0025mm2. 90 Hematology Fig. 6.1a Improved Neubauer ruled counting chamber. Fig. 6.1b: View of the improved Neubauer counting chamber III. Fuchs-Rosenthal counting chamber This chamber was originally designed for counting cells in cerebrospinal fluid, but as such a relatively large area is covered, it is preferred by some workers for counting leucocytes. The depth is 0.2mm and the ruled area consists of 16mm squares divided by triple lines. These squares are subdivided to form 16 smaller squares, each with an area of 1/16 of 1mm2 (figure 6.2). Another type of Fuchs-Rosenthal chamber is now available, 91 Hematology which has the same depth as the one described, but is ruled over 9mm2 only. Fig. 6.2: Fuchs-Rosenthal counting chamber IV. Burker ruled counting chamber Like the Neubauer counting chamber, this has a ruled area of 9mm2 and a depth of 0.1mm. To count white cells using Burker Chamber, the four large corner squares are used (4mm2) and the same calculation as describe for the Improved Neubauer ruled chamber is used. 92 Hematology (a) (b) Fig. 6.3 (a) Ruled area of the Burker counting chamber; (b) enlarged view showing actual measurements. Dilution of the Sample Dilution of sample is accomplished by using either a thomma pipette or the tube dilution method. With tubes larger volumes of blood and diluting fluid are used and the greater will be the accuracy as compared with the smaller volumes used in the thomma pipette techniques. Thomma pipettes are small calibrated diluting pipettes designed for either white cell or red cell count. Counting and Calculation The diluted cells are introduced into the counting chamber and allowed to settle. They are then counted in 93 Hematology the designated area (s). Cells lying on or touching the upper or left boundary lines are included in the count while those on the lower and right boundary lines are disregarded. Fig 6.4 Examples of white blood cells counted in a representative area. Calculation No. of cells/mm3 = N × DF ; No. of cells/l = N × DF × 106 A×d A×d Where, N = no. of cells counted in a given area DF = dilution factor A = area of counting in mm2 d = depth of the counting chamber (Volume of chamber = A × d) 94 Hematology 6.1 White blood cell count A white blood cell count (total leucocyte count – TLC) is used to investigate infections and unexplained fever and to monitor treatments which can cause leucopenia. In most situations when a total WBC count is requested it is usual to perform also a differential WBC count. EDTA anticoagulated blood or capillary blood can be used for counting white cells. Heparin or sodium citrate anticoagulated blood must not be used. Principle Whole blood is diluted 1 in 20 an acid reagent which hemolyzes the red cells (not the nucleus of nucleated red cells), leaving the whit cells to be counted. White cells are counted microscopically suing an Improved Neubauer ruled counting chamber (hemocytometer) and the number of WBCs per liter of blood calculated. When after examining a stained blood film, many nucleated red cells are present (more than 10%), the WBC count should be corrected. Diluting Fluid Turk’s solution - 2% aqueous solution of acetic acid 95 Hematology colored pale violet with gentian violet or pale blue with methylene blue. The glacial acetic acid causes erythrocyte lysis while the gentian violet lightly stains the leucocytes permitting easier enumeration. Test method Thomma White Cell Pipette The long stem is divided into 10 equal parts with “0.5” and “1” engraved on it. On the short limb just above the bulb, the mark “11” is engraved. When blood is drawn up to the 0.5 mark and diluent to the 11 mark, the sample of blood (now in the bulb) is diluted 1:20. Once the pipette accurately filled to the mark, the rubber suction (or mouth piece) is carefully removed, with the pipette held horizontally and only one finger sealing the tip. Both ends of the pipette may then be sealed with special small rubber sealing caps or with the middle finger on the tip and the thumb on the other end. The pipette is shaken mechanically or manually for 2 minutes. A bead contained in the bulb of the pipette aids in the mixing. If shaking is done manually, the shaking motions should be varied and alternated. The cover glass is placed on the chamber and a slight pressure applied to the ends of the cover glass until a 96 Hematology “rain bow” or Newton’s diffraction rings are revealed on either side. Once the diluted blood in the pipette has been thoroughly mixed, a few drops are expelled to discard the cell-free diluting fluid in the long stem of the pipette. With the index finger forming a controlled seal over the end of the pipette, which is held at an angle of 450 , the tip of the pipette is brought up to the edge of the cover glass and by gentle release of index finger pressure, fluid is allowed to run out slowly until the counting platform is covered. The fluid is drawn into the chamber by capillary attraction. Care must be taken not to overfill the chamber which will result in overflow into the channels. If blood is diluted with the tube technique (in which 20µl of blood is taken with a sahli pipette and mixed with 0.38ml of diluting fluid in a small tube). Charging is accomplished by using disposable capillary tubes or long stem Pasteur pipettes. The chamber is placed in position on the microscope stage and is allowed to stand for 2 or 3 minutes so that the cells will settle. All apparatus should be cleaned thoroughly after each use. Pipettes (thomma and sahli) should be washed well with a sequence of water and acetone (filled with 97 Hematology each fluid three or four times) and air drawn after the acetone until the inside of the pipette is thoroughly dry. Pipettes should be periodically cleaned with potassium dichromate cleaning solution or hydrogen peroxide. Hemocytometers should be washed in distilled water immediately after use and dried with gauze or tissue paper. They should be stored in such a way as to avoid breakage and scratching of the counting surface. Performance of the Count The counting chamber is surveyed with the low power objective to ascertain whether the cells are evenly distributed. Then the number of cells in four large squares is counted. Calculation If N is the number of leucocytes in four large squares, then the number of cells per mm3 is given by: No. of leucocytes/mm3 = N × DF Vol. Where N is the number of leucocytes in an area of 4mm2 DF is the dilution factor equal to 20 Vol. is the total volume on which the count is 98 Hematology done and is given by the total area of count multiplied by the depth of the chamber (0.1mm for the improved Neubauer counting chamber. Substituting these values in the above formula: No. of leucocytes/mm3 = N × 50, N ≥ 100* * 100 cells is a reasonable and practical figure for visual counts. When the leucocyte count is low (below 4.0 × 103/mm3), it is advisable for greater accuracy to use a 1:10 dilution, i.e., take blood to the “1” mark of the pipette and diluting fluid to the 11 mark. The corrected leucocyte count Nucleated red cells will be c