Introduction to Hematology   What is Hematology? Hematology is the science or study of blood and its diseases.  In fact the word hematology is derived from the Greek words:                           -           Haima meaning blood                         -           Logos meaning science or study   What do we study in this course?   1.         The normal physiology of blood, i.e. what are the different components of blood, what are the different blood cells, how are they formed, how are they destroyed, what is their normal % in the blood.   2.         The diseases of blood; i.e. the abnormalities that can take place in the blood and reflect themselves as blood diseases. Under this, we are going to study two broad categories of blood diseases including the anemias and the leukemias or blood cancers.   In addition, the hematology laboratory covers the study of hemostasis and thrombosis, dealing with the control of blood loss and the problems related to hemorrhage and clotting.   How is the study of blood performed? (What are the tools used?)   *          By making use of instruments to determine certain parameters including cell counts and others. *          By taking blood and by examining it under the microscope.   In other words, the study of blood could not be feasible before the days of microscopy. At that time, only the gross appearance of blood could be studied and little was understood about its composition and biological functions.   During the seventeenth century, Leeuwenkoek and others studied blood with the aid of primitive microscopes and the science of Hematology was born.       Then the study of cells under the microscope was greatly enhanced during the 19th century, when Paul Ehrlich developed staining techniques to better differentiate the various normal and abnormal cells present in human blood. In the last 70 years however, the science of clinical hematology has grown enormously and has become a broad science, linked with other sciences, including, Oncology (study of malignant changes in cells), Genetics, Immunology…..   Blood is that fluid that circulates in certain specialized vessels of the body called blood vessels.   The blood vessels are made of three major types:   1.         Arteries: are blood vessels that carry oxygenated blood from the heart to the different body organs.   2.         Veins: are blood vessels that carry the blood from the body organs back to the heart.   3.         Capillaries: are very small blood vessels between veins and arteries whose walls are so thin that they permit the exchange of fluid and gases between blood and tissues.   The blood may be thought of as a transportation system.  As it circulates throughout the body, oxygen is transported from the lungs to the tissues, products of digestion are absorbed in the intestine and carried to the various tissues of the body, and substances produced in various organs are transferred to other tissues for use.  Cellular elements of the blood may also be transported to fight infection or aid in blood coagulation. At the same time, waste products from the tissues are picked up by the blood to be excreted through the skin, kidneys and lungs. Composition of blood   The total blood volume in an adult is 5 to 6 liters, or 7 to 8% of the body weight. Blood is composed of two major constituents:                         A liquid part called plasma             A solid (cellular) part   In a normal person, approximately 55-60% of the blood is plasma of which around 90% is water. The remaining 10% is composed of: proteins, namely (albumin, globulin, and fibrinogen), carbohydrates, lipids, vitamins, hormones, enzymes, plus some inorganic salts, (Na, K, Ca, Mg, Cl, bicarbonate and phosphate).       When coagulation is prevented by the use of anticoagulants, the liquid portion of the blood is termed plasma and contains the protein fibrinogen. Several anticoagulants are available for various purposes in the clinical laboratory, but the most commonly used for hematologic procedures are EDTA (chelates Ca++), Citrate (chelates Ca++), and Heparin (acts by neutralizing thrombin, thus preventing the formation of fibrin from fibrinogen).                                          EDTA tubes   (violet stopper)                     Tubes with sodium citrate (blue stopper)                                                                                                                                                                  Tubes with heparin (green stopper)                                                                                                                                                              Simple tubes                       (red stopper)                                                If a blood specimen is allowed to clot, the liquid portion released from the clot is called serum and does not contain any fibrinogen due to the fact that the fibrinogen was utilized to form the fibrin threads of the blood clot.   The other part of blood which was called the cellular part (formed elements) constitutes 40-45% of the blood and consists of three major components: Red blood cells, leukocytes or white blood cells, and platelets.   1.         Red blood cells: the most numerous cells in the blood are non-nucleated, deeply pigmented elements that are concerned primarily with tissue respiration, transport of O2 to the tissues and CO2 from the tissues.               The scientific name of them is erythrocytes                                                                 red     cells   When seen under the microscope, they have a reddish color which is due to the presence of a pigment called hemoglobin (Hb) that normally makes them look red and makes the whole blood look red.   2.         White blood cells or leukocytes: in the living state, the WBC(s) have a whitish grayish color, therefore we call them WBC(s) and the scientific name of them is leukocytes. Leukocytes are a heterogeneous group of nucleated cells, the major function of which is to protect the host from the external environment.   Five distinct types constitute normal blood: Neutrophils, lymphocytes, monocytes, eosinophils and basophils. Each of these cells has a characteristic morphologic appearance and more importantly, each serves a specific physiologic role.        3.         Platelets: these are small structures, they are not true cells and in fact, they are cytoplasmic fragments of giant cells in the body, called megakaryocytes (cells with a giant nucleus).   The main function of platelets is in the process of sealing an injured blood vessel or in other words, in blood clotting, and when they do so, they form a clot or a thrombus, this is why, their scientific name is thrombocytes.   Sources of blood for Hematological tests   Blood specimens may be obtained for hematologic tests either by venipuncture or by skin puncture, but venous blood is preferred to skin puncture for most hematologic tests. However there are several situations where skin puncture should be used to obtain blood specimen. Infants, particularly newborns, have a much smaller total blood volume than adults; drawing blood by routine venipuncture on a daily basis can quickly result in hospital – induced anemia. Skin puncture is also a much safer means of blood collection. For adults, skin puncture may be required because of obesity, burns, or extremely small or severely damaged veins or when IV fluid is flowing into the only accessible veins. Skin puncture is also used to save the veins of patients receiving chemotherapy and in the elderly when possible. Skin puncture can be obtained from an ear lobe or finger of an adult or from the heel of an infant.   Blood withdrawn by this procedure is collected in special microtubes.                                    How do we study blood?   In the study of blood, our interest is mainly concerning the formed elements of blood, namely the blood cells.           How to examine the blood cells?   In their living state, the great majority of blood cells are not colored and therefore, it is not possible to study them by means of the ordinary microscope. In order to study cells, they have to be colored artificially, and this process is called staining and here we use blood stains.     There are actually three different types of blood stains:   1-Vital or Supravital stains:   Some blood stains can be used on living cells and they are known as vital or supravital stains but these will be used infrequently and the more usual stains in Hematology are the regular stains.   2-Regular stains:   Regular stains are used on killed or dead cells. For that purpose, the cells have first to be killed very rapidly and preserved (to prevent degeneration) and this process is called fixing. So for proper fixing, the death of the cell has to occur very fast because we cannot allow changes to occur in the cell before it dies.   For that purpose, we use special agents known as fixatives. What are the known fixatives used in Hematology?               1.         Methanol (most commonly used fixative in Hematology)             2.         Ethanol             3.         Formalin             4.         Glacial Acetic Acid   3- Special Stains:         Special stains are used to stain specific structures in the cell.     What do we use for coloring cells?   We use a group of stains known as the Romanowsky stains and these are called after the man who first described them. The Romanowsky stains are many in type but they all consist mainly of:               A basic dye which is methylene blue (blue);                                          and             An acidic dye which is eosin (red),                                           plus A variety of intermediate stains (methylene azures: oxidation products of methylene blue) that give different shades to different structures. So these are polychrome stains because they produce multiple colors when applied to cells.   Of the Romanowsky groups of stains, the following are standards:               Leishman stain             Jenner stain             May-Grun Wald stain             Wright stain             Giemsa   Modifications differ in the ratios of dye components and the manufacturing methods used to oxidize methylene blue. The above 4 stains stain best the cytoplasm and the granules and the Giemsa stain stains best the nucleus and this is why we stain the chromosomes with it.    Principle of staining with the Romanowsky type of stains:   These stains act by chemical interaction with the various substances present in the cell (granules, ribosomes, mitochondria) and this reaction depends on the pH of the structure.   Acidic substances in the cell take the basic stain (methylene blue) so they stain bluish in color and these are called basophilic because they like the basic dye. Ex(s) of these are RNA, DNA, certain cytoplasmic proteins and granules of basophils. For instance, the more RNA there are in the cell, the more bluish is the cell (ex, immature cells, plasma cells). On the other hand, all basic structures in the cell will take up the acidic stain (eosin), so they appear red in color. So, basic structures are called acidophilic or eosinophilic because they like the acidic dye: ex(s), some proteins such as Hb, some cytoplasmic constituents and granules of eosinophils. Finally, substances with intermediate or neutral pH will take intermediate colors and as such these substances are called neutrophilic.    Of these stains, we are mainly concerned with the Wright stain and Giemsa stain. Since the Wright stain gives excellent details of the cytoplasm and the granules and the Giemsa stain is excellent for nuclear detail, ideally what we do is stain first with the Wright stain and after that with Giemsa stain.   Procedure of staining with the Romanowsky stains   Manual staining methods   Rack Method (uses rods overlying a sink that hold slides in a horizontal position)   A.        Staining with the Wright stain               *          Obtain a clean slide, place at the edge a drop of blood and spread it, then   allow it to dry in the air.                 *          Place the smear on a flat surface and add to its surface a known volume of Wright stain.       Since the Wright stain powder is dissolved in absolute methanol, we do not need to fix the cells before staining. During the first two minutes, actual fixing of the cells takes place.   *          After two minutes, add an equal volume of phosphate buffer solution to each slide. The pH of the buffer will affect the quality of staining, the optimum being between pH 6.4 and 6.8. The buffer-stain solution is mixed gently by blowing on it. A greenish metallic sheen indicates the proper stain: buffer ratio.               Leave for 4 minutes during which actual staining is taking place.   *          Wash with tap water. Flush thoroughly the staining mixture from the slide until all stain is removed. Do not over wash.   B.        Stain with Giemsa   *          Add buffer on the slide.   *          Add few drops of Giemsa and leave for 2 minutes.                Ideally, we should add the drops of Giemsa on the edges of the slide and then blow in order to get an even coloration of the slide.   *          Wash with tap water. *          The slides are cleaned off from the back with gauze and the specimen is air dried. N.B:        Iif we want to stain with Giemsa stain alone, we should start by fixing the slides, because the Giemsa stain is diluted twice, so we cannot use it to fix the slides with.   Advantages and disadvantages of the rack method   The rack method is very cost effective for a small number of slides because it uses a minimum of stain solution/slide.   In addition, one or several slides may be stained simultaneously. The disadvantages of the rack method include the time involved and the inconsistency in the staining quality related to variances in the amounts of stain and buffer/slide and the timing/step.   Automated staining methods   Carousel type   This instrument sprays a measured amount of stain reagents on the smears as they are transported in a rotating carousel.    Advantages of automated stainers include the following:   1.         They are time saving. Once the slides are loaded into the instrument, the operator can walk away and staining time is less than 10’.   2.         Stain quality is consistent from smear to smear.

Blood Film Inspection   Examination of the peripheral blood smear should be considered, along with review of the results of peripheral blood counts and red blood cell indices, an essential component of the initial evaluation of all patients with hematologic disorders. The examination of blood films stained with Wright and Giemsa stain frequently provides important clues in the diagnosis of anemias and various disorders of leukocytes and platelets.     How does a mature RBC look?   An RBC has a strange shape -- a biconcave disc (the thickness at the periphery is 1½ times the center). If the red cell has this shape, it will have a greater surface area for 02 exchange since the main function of this cell is in gas exchange. The RBC has a d of around 7-7.5m and it is normally saturated with hemoglobin, so that a normal RBC cannot contain more hemoglobin than it has. This RBC appears dark at the edges and lighter in the center because it is biconcave in shape and this light area in the center is called central pallor.   High-power view of a normal peripheral blood smear. Several platelets and a normal lymphocyte can also be seen. The red cells are of relatively uniform size and shape. The diameter of the normal red cell should approximate that of the nucleus of the small lymphocyte; central pallor should equal one-third of its diameter. Courtesy of Carola von Kapff, SH (ASCP). Normal human red blood cells are biconcave disks with a mean diameter of about 7.5 μm. Erythrocytes are slightly smaller than small lymphocytes. The hemoglobin of red cells is located peripherally, leaving an area of central pallor equal to approximately 30 to 45% of the diameter of the cells. Cells of normal size and hemoglobin content (color) are termed normocytic and normochromic.    Examination of peripheral blood films of normal persons reveals small numbers of poikilocytes, usually less than 2%. In the assessment of the significance of poikilocytosis, one must identify the predominant abnormal morphologic form and exclude artifactual alterations of the red cells.   I. Red Blood Cell Abnormalities Changes in RBC Size Anisocytosis: Refers to variation in the size of RBC(s). Normally, there is minimal variation in the size of RBC(s). When the variation is significant, we call it anisocytosis. Anisocytosis is not characteristic of any one disease, but it is present in a great majority of erythroid disorders and in virtually all anemias. Microcytosis When the MCV < 80fl, the RBC (s) are said to be microcytic. There are many types of anemias where the average cell is smaller than normal. These include: Fe-deficiency anemia Thalassemia Hereditary Sideroblastic Anemia Macrocytosis When the MCV > 100 fl, the RBC (s) are said to be macrocytic. Macrocytosis is encountered in many instances:             Megaloblastic anemia New born babies  Active erythropoiesis Liver disease (chronic) i.e. Alcoholism.             (Altered lipid content of the red cell membrane)     Changes in [Hb] Normochromic RBC (s) The normal RBC is saturated with Hb and as such the central pallor constitutes around one third of the cell size. This cell is known as a normochromic RBC. When the amount of hemoglobin per red cell is less than normal Þ the central pallor becomes larger and the RBC is said to be hypochromic. Hypochromic RBC(s) are seen in Fe-deficiency anemia, Thalassemia as well as Hereditary Sideroblastic Anemia. N.B.: A red blood cell without central pallor is one of two things: (a)        If it is smaller than normal, then it is a spherocyte. (b)        If the red blood cell is larger than normal and the color is slightly bluish, this is called a polychromatophilic RBC.   Changes in RBC shape Poikilocytosis: Refers to the presence of abnormally shaped RBC(s).   1.         Ovalocyte, elliptocyte or cigar shaped RBC The commonest abnormal shape is the ovalocyte, elliptocyte or cigar shaped RBC, characteristic of the following disorders:      Fe-deficiency  Thalassemia Megaloblastic anemia (Macroovalocytes[WU9] )   However note that these cells may be normally present in the blood smear in small numbers.   N.B: The cigar shaped RBC is very similar to the ovalocyte or elliptocyte with slight modifications, i.e. it is a little bit longer and thinner.   When the great majority of RBC(s) (~ 95%) are ovalocytes, elliptocytes or cigar shaped Þ we call the condition congenital ovalocytosis or elliptocytosis.  This condition is inherited as an autosomal dominant gene. In congenital elliptocytosis, we have one of two cases:               * 95% of subjects with elliptocytosis are benign and have no disease. Here we do not know what makes the RBC deformed to become elliptical but these cells are known to function normally. * Only 5% show a condition of hemolytic anemia (abnormality or defect in spectrin, a skeletal protein present in the RBC membrane).   2.         Stomatocyte Stomatocytes are erythrocytes with an elongated (mouth-like) area of central pallor. As many as 3% of RBC(s) may be stomatocytes in a normal smear. The presence of few stomatocytes in a peripheral blood smear is entirely harmless, but when they are increased in number Þ we get a case of congenital stomatocytosis. As is the case with congenital elliptocytosis, almost all cases are harmless and only ~ 5% present a hemolytic type of anemia. Stomatocytes might be seen as a non-specific finding in a variety of situations, such as liver disease (cirrhosis), alcoholism or may even be considered an artifact of preparation.   3.         Tear-drop cells (Pear shaped) Tear-drop cells may be encountered in a variety of anemias. However, when, these cells are numerous, it is very suggestive of myelofibrosis (a condition where the BM becomes full of fibrous tissue) that would lead to extramedullary hematopoiesis or blood cell production outside the bone marrow.   4.         Sickle cells Sickle cells are deformed RBC(s) in the shape of a sickle or crescent. To be considered as sickle cells, they must come to a point at one end. These cells are associated with HbS and are found in sickle cell anemia.   5.         Crenated or spiculated RBC(s) (Acanthocytes/Echinocytes)             Acanthocytes (Spur cells) Acanthocytes1(below) are RBC(s) with irregularly spaced projections. These cells have a decreased survival time (condition associated with hemolysis) and are predominantly found in a condition known as acanthocytosis or abetalipoproteinemia (absence of b-lipoprotein from the RBC membrane). This condition is normally inherited; however, acanthocytosis may be acquired in association with a number of conditions such as alcoholic cirrhosis and malabsorption states. In this condition, the RBC membrane undergoes distortion to give finger - like projections. At most, there would be 5 projections.               Echinocytes (burr cells) Echinocytes (burr cells) have multiple short pointed projections uniformly spaced over the cell surface. The central pallor is retained. Echinocytes are often confused with acanthocytes (spur cells). However, the projections of echinocytes are smaller, more numerous (6-10/RBC), and more regular than those of acanthocytes. Echinocytes are characteristic of end stage liver disease or kidney failure and should be differentiated from acanthocytes or spur cells.   They can also be seen as an artifact of slide preparation or prolonged specimen storage.   6.         Schistocytes Schistocytes or fragmented RBC(s) are encountered in a great majority of hemolytic anemias especially intravascular hemolytic anemias. They are also seen in conditions in which we have ineffective erythropoiesis (destruction of RBC(s) in the bone marrow).   7.         Helmet or triangular cells There is no special appearance for schistocytes but two specific forms are important in diagnosis. The fragmented RBC may appear triangular or may have the appearance of a helmet cell (like Napoleon’s hat). A triangular cell or a helmet cell, characterizes a type of anemia in which the RBC(s) are fragmented within the blood vessels, these latter containing fibrin threads. This anemia is a hemolytic anemia that occurs secondary to intravascular coagulation. 8.         Target cell The normal RBC shape is governed by a wide surface area/volume ratio for adequate exchange of gases. If you decrease the internal volume of the cell, or you increase the surface area keeping the volume constant, the RBC membrane will undergo folding and will look like a target.   Thus any condition which removes Hb from inside the cell will wrinkle the RBC. This is a mark of ¯ volume compared to the surface area. Under this, we have anemias due to ¯ in heme synthesis (namely Fe-deficiency anemia) or due to ¯ in globin synthesis (Hemoglobinopathies such as thalassemia or sickle cell anemia).   On the other hand, the second component involves an increased surface area compared to volume. The RBC membrane is rich in free cholesterol and this cholesterol exchanges freely with the free cholesterol in plasma. So the higher the free cholesterol in plasma Þ the more cholesterol is taken up by the RBC membrane Þ the bigger is the membrane Þ the RBC becomes a target cell. Free cholesterol in the body is taken up by the liver and in the liver, it is esterified (addition of molecules) and the liver would excrete esterified cholesterol. In severe liver disease, the esterification of cholesterol is depressed and the free cholesterol in the blood increases Þ formation of target cells in the peripheral blood. Target, Spur and burr cells in liver disease N.B: This may also appear in normal persons after splenectomy.   9.         Spherocyte The spherocyte is a small red blood cell with no central pallor that appears a little bit darker in color. Spherocytosis could be inherited or acquired (in association with ABO/ Rh incompatibility or autoimmune hemolytic anemias) and it is always associated with hemolysis due to the rigidity of the membrane. 10.       Polychromasia and stippling The polychromatophilic RBC is an immature non nucleated RBC which appears diffusely bluish in color when stained with a regular Romanousky stain.     This is an early or immature reticulocyte with a significant number of ribosomes and RNA still present in the cytoplasm. Because RNA is acidic in nature, it takes up methylene blue. Since the wright stain contains alcohol Þ we have fixation of the cells Þ the remaining RNA and   ribosomes diffuse in the cell to give it a diffuse grayish-bluish color. This cell is known as a reticulocyte when stained with a supravital stain. So if we do not fix the RBC(s), but subject them to a potent oxidizing agent such as Brilliant Cresyl Blue Þ we will precipitate the remaining RNA and ribosomes so that these give a deep blue precipitate instead of showing a diffuse grayish color.   N.B: All polychromatophils are reticulocytes, however not all reticulocytes are polychromatophils on a Wright's stained blood smear. Maturing reticulocytes are readily visualized in New Methylene Blue / Brilliant Cresyl Blue-stained blood smears, but they do not contain sufficient RNA to appear blue-purple in Wright's stained smears. So normally polychromasia will not be detected unless the reticulocyte count is above 2%.   In chronic hemolytic anemias however, we may have pathologic precipitation of the ribosomes and the cell is then known as a stippled red blood cell. N.B: Polychromatic cells/ stippled RBCs would appear as reticulocytes when stained with a supravital stain.   11.       Diserythropoiesis This refers to an abnormality in the nuclear membrane resulting in bilobed or multilobulated erythroblasts.   12.       Dimorphic picture/appearance Dimorphic picture/appearance describes heterogeneity in the size of red blood cells, usually with two distinct populations.  It can be found in partially treated iron deficiency, mixed deficiency anemias  (e.g. folate/B12 and iron together), following red cell transfusion, or in cases of sideroblastic anaemia.   13.       Rouleaux Rouleaux are stacked/clumped groups of red cells caused by the presence of high levels of circulating acute-phase proteins which increase red cell 'stickiness'. They are often an indicator that a patient has a high ESR and are seen in infections, inflammations and Multiple Myeloma.   II.        RBC Inclusions   Red blood cell inclusions can arise from a variety of sources. Correct identification of these abnormalities is important since it can provide insights into metabolic, physiologic, and pathologic conditions affecting the red blood cells.   1.         Howell-Jolly bodies Howell-Jolly bodies are round, purple staining nuclear fragments seen in the RBC. They usually appear in conditions where we have chromosomal breakage. As this RBC passes through the spleen, the spleen will take out the HJB and therefore the cell will pass out without it. So, when we see HJB(s) in a peripheral blood smear, it means that the spleen is not present or if present its function is impaired.   HJB are common:                                                 1) In some hemolytic anemias (i.e. Thalassemia and SCA)                                     2) In megaloblastic anemia     2.         Cabot rings Cabot rings are purple-staining, threadlike filaments in the shape of a ring or as a figure of eight (8) appearing in the RBC. They are thought to be nuclear remnants and more specifically derived from the microtubules of the mitotic spindle. Cabot rings are seen in conditions where we have disturbed erythropoiesis and most frequently in thalassemia and megaloblastic anemia, basically in similar conditions to Howell-Jolly bodies.     3.         Basophilic stippling Basophilic stippling is the fine or coarse deep blue to purple staining inclusion that appears in immature RBC(s) stained with Wright and Giemsa. Stippling represents aggregates of ribosomes and is associated with chronic hemolytic anemias. The three disorders particularly associated with coarse basophilic stippling are lead poisoning, sideroblastic anemia, and thalassemia.   4.         HbC crystals These are elongated crystals with blunt ends that appear darkly stained in color and are associated with HbC disease.   5.         Pappenheimer bodies Pappenheimer bodies (siderotic granules) are small, irregular, dark staining granules that appear near the periphery of a RBC in a film stained with Wright and Giemsa. With Perls’ Prussian Blue stain (special stain for iron), these bodies stain positively, indicating their iron content. The spleen normally removes these inclusions without destroying the cell. However, after splenectomy, pappenheimer bodies are visible on the blood film. Normally, no more than three small iron particles are noted in developing RBC(s) in the B.M.   An erythrocyte that is positive for siderotic (iron) granules in a Prussian Blue stain is designated a siderocyte. A normoblast (nucleated erythrocyte) with siderotic granules is called a sideroblast.   With severe disturbance of Hb synthesis, pathologic sideroblasts and siderocytes are present in the B.M. and P.B. Siderotic granules may be present in sideroblastic anemia   and refractory anemias.     6.         Heinz bodies  Heinz bodies which are clumps of denatured Hb are RBC inclusions which are stained with a supravital stain. With Romanowsky stains, they appear as pale focal areas within the RBC (Peripheral hemoglobinization). The RBC(s) here are also known as bite cells.   In man, the finding of Heinz bodies in the blood is associated with G6PD deficiency, following exposure to certain drugs or oxidants.   Heinz bodies (arrow) do not appear with the regular stains.(Wright’s-Giemsa stain with 100x objective). They appear as a pale focal area. Photograph courtesy of Dr. Perry Bain.   Heinz bodies are more prominent with the use of a vital stain (New methylene blue stain with 100x objective). Photograph courtesy of Dr.Perry Bain.   7.         Hb H inclusions These are bluish green granules that appear with Supravital Stains only. They are precipitates of b globin chains and are associated with Hb H disease (a form of α thalassemia).