Clinical Hematology - Practical Hematology PDF

Notes in Clinical Hematology 1  Table of content 1. Hematopoiesis and Hematopoietic Growth Factors……….......3 2. Erythrocytes: Production, Function and Destruction…….…..19 3. Erythrocyte Morphology……………………….……………25 4. Erythrocytes Indices……………………………….....

Notes in Clinical Hematology 1  Table of content 1. Hematopoiesis and Hematopoietic Growth Factors……….......3 2. Erythrocytes: Production, Function and Destruction…….…..19 3. Erythrocyte Morphology……………………….……………25 4. Erythrocytes Indices………………………………...………28 5. Erythrocytes in Disease………...……………………………31 6. Leukocyte Function and Clinical Interpretation...……...……52 7. Leukocyte in disease………………………………………….61 8. Leukemia……………………………….…………………….67 9. Platelet Kinetics and Physiology………………………..…...82 10. Practical Hematology…………………………………….....91 2  Hematopoiesis and Hematopoietic Growth Factors Hematopoiesis is the process of making blood cells. The term comes from the Greek hema (blood) and poiesis (to make). There are three types of cellular elements in the blood - erythrocytes (red cells), leukocytes (white cells), and thrombocytes (platelets). Each has its own functions and differs clearly from the others. Most of the blood cells are formed in the bone marrow. Production of blood cells is highly regulated and balanced. 1. Development of the Hematopoietic System: A. Embryonic and Fetal Hematopoiesis: Hematopoiesis begins in the yolk sac during the first month of embryogenesis but gradually shifts to the liver and, to a lesser extent, the spleen. The liver is the primary site of hematopoiesis during the second trimester; however, the medullary cavities of the bones becomes the primary site of hematopoiesis after the seventh month. B. Postnatal Hematopoiesis: After birth, the bone marrow is normally the sole site of hematopoiesis (intramedullary hematopoiesis). Hematopoiesis may resume in the liver and spleen after birth in conditions associated with fibrosis of the bone marrow (extramedullary hematopoiesis). During infancy, there is active hematopoiesis in the medullary cavity of virtually every bone. With age, the hematopoietically active marrow (red marrow) is gradually replaced by inactive marrow (yellow marrow), which consists predominantly of adipose tissue. In adults, hematopoiesis is restricted to the proximal long bones and the axial skeleton (skull, vertebral bodies, ribs, sternum, and pelvis). The yellow marrow can resume active hematopoiesis under conditions of chronic hematologic stress (chronic bleeding or hemolytic anemia). 2. Bone marrow microenvironment: The medullary cavities contain vascular spaces (sinuses), 3  hematopoietic cells, and specialized stromal cells of various types. All the cells form a complex microenvironment, with numerous intricate and interdependent relationships between stromal cells and hematopoietic cells. Hematopoietic Cords: The hematopoietic cords (parenchyma) are the extravascular portions of the bone marrow and the site of blood cell production. Sinuses: The sinuses (vascular spaces) of the marrow are lined with specialized endothelial cells, which prevent the premature escape of immature cells into the peripheral blood. The basal lamina is incomplete, allowing mature cells to pass through the wall of the sinuses. Stromal Cells: The stromal cells compose the supportive tissues of the bone marrow. Some of these cells produce hematopoietic growth factors. Examples include: 1. Adventitial (reticular) cells: Modified fibroblasts that produce the reticulin framework of the bone marrow 2. Macrophages: Produce hematopoietic growth factors, store iron for hemoglobin production, and carry out phagocytosis of debris 3. Adipocytes: Store energy in the form of fat Stem Cells of Hematopoiesis: All blood cells derive from pluripotent hematopoietic stem cells. The progeny of these cells are capable of giving rise to all the different lines of mature blood cells: erythrocytes, granulocytes, monocytes, and megakaryocytes (platelets). The pluripotent stem cells are capable of self-renewal. They are rare in the bone marrow (~1 per 1,000 to 2,000 marrow cells) and cannot be recognized morphologically. The majority of pluripotent stem cells at any given time are in the resting (Go) phase of the cell cycle. 4  Expression of CD34, a marker of immature cells, is used as a marker for hematopoietic stem cells. Low numbers of hematopoietic stem cells can be found circulating in the peripheral blood (peripheral blood progenitor cells). The number of circulating progenitor cells can be greatly increased by the administration of cytotoxic chemotherapy (the number of cells increases during the recovery phase) or by the administration of high doses of hematopoietic growth factors. Circulating progenitor cells can be harvested by apheresis and have now become the primary source for bone marrow transplants in many circumstances. Pluripotent hematopoietic stem cells give rise to committed progenitor cells. This is a multistep process in which the cells become sequentially more committed to a specific lineage. These committed progenitor cells are given various names, such as colony- forming unit (CFU) or burst forming unit (BFU). The initial differentiation step is into either CFUGEMM (GEMM = granulocytes, erythrocytes, monocytes/macrophages, and megakaryocytes) or CFU- L (L = lymphoid). The CFU-GEMM gives rise to the CFU-GM (granulocyte-macrophage), BFU-E (erythroid), and CFU-Mk (megakaryocyte). Each committed progenitor cell gives rise to a thousand or more mature blood cells. Note. Myelopoiesis; the prefix "myelo"- generally refers to all aspects of bone marrow activity and is not limited to granulocytic elements alone. Consists chiefly of erythropoiesis, granulopoiesis, and thrombopoiesis. Monocytes appear to be formed in the marrow as well as elsewhere. Lymphopoiesis occurs largely in extramedullary sites such as the spleen, thymus, and lymph nodes. 3. Erythropoiesis (Erythrocyte Production): The erythron is the sum of all erythroid cells, including circulating red blood cells (RBCs) and marrow erythroid precursors. Erythroid precursors are derived from the CFU-GEMM. The earliest progenitor 5  committed exclusively to erythroid lineage is the burst-forming unit– erythroid (BFU-E); this is followed by the colony-forming unit– erythroid (CFU-E). The earliest recognizable RBC precursor is the proerythroblast, which is characterized by fine nuclear chromatin and intensely blue cytoplasm. The last nucleated RBC precursor is the orthochromatophilic erythroblast, which is characterized by well- hemoglobinized cytoplasm; the nucleus is then lost, producing the reticulocyte. 3.1 Maturation Sequence As cells develop, their morphology changes. These morphologic alterations occur gradually. As a general rule, cells are classified towards the more differentiated cell type. Immature cells are generally larger and become smaller as they mature. The nuclei of the immature cells are relatively large in relation to the amount of cytoplasm and become smaller with maturity. The chromatin of the nucleus in immature cells is delicate, fine and stippled. As the cell matures, the chromatin becomes coarse, clumped and compact. Nucleoli are found in the nucleus of immature cells. 3.2 Erythrocyte Maturation Sequence: 3.2.1 Rubriblast:  First recognizable cell in the erythrogenic series.  A large round cell which contains a large round nucleus with a thin rim of royal blue cytoplasm.  The nuclear chromatin is delicate and stippled.  The nucleus contains one to several nucleoli.  The cytoplasm is somewhat scanty and stains very basophilic. 3.2.2 Prorubricyte:  Cell is similar to the Rubriblast but smaller in size.  Nucleoli are usually absent, but remnants of the nucleoli may still be present. 6  The chromatin material is somewhat coarser.  A perinuclear clear zone may be observed.  The cytoplasm stains basophilic. 3.2.3 Rubricyte:  Cell is smaller than the Prorubricyte.  Nuclear chromatin material is coarsely clumped separated by light streaks giving the so-called cartwheel appearance.  Nucleus is round and stains very dark.  Cytoplasm stains very basophilic in the early rubricyte stage, but the blue color is diluted out by the pink color of hemoglobin as it matures toward the next stages.  This cell can be further subdivided according to the amount of hemoglobin in the cytoplasm into basophilic, polychromatophilic and normochromic rubricyte.  The cell is the last cell in the erythrocytic series capable of cell division under normal conditions. Cell division stops when a critical hemoglobin concentration is reached. A deficiency of hemoglobin in the cell can result in extra divisions and smaller cells. (e.x. Iron deficiency results in microcytes) 3.2.4 Metarubricyte:  The nucleus is small, pyknotic, and appears as a dark blue homogeneous mass without any distinct chromatin structure. The cytoplasm stains similarly to the mature erythrocyte.  The cell is not found in the peripheral blood of normal healthy conditions. When observed in peripheral blood, it denotes a response to an anemic condition of at least 72-96 hours duration. 3.2.5 Reticulocyte or polychromatophilic cell:  This cell is larger than the mature erythrocyte and is non-nucleated.  Reticulocytes contain ribonucleic acid (RNA) for 4 days; normally, the first 3 days are spent in the marrow and fourth in the blood. 7  However, under intense stimulation by erythropoietin, reticulocytes may be released into the blood early where they may contain RNA for 2.0 to 2.5 days (shift reticulocytes).  Cell may contain a small round nuclear remnant called a Howell- Jolly body.  Cytoplasm stains slightly basophilic with Wright's stain. However, when stained with a supravital-stain such as new methylene blue or brilliant cresyl blue, precipitated ribrosomal RNA (reticulum) can be demonstrated within the cell.  The cell does not participate in normal rouleaux formation or pathologic agglutination; is more resistant to crenation and lysis; is less susceptible to mechanical trauma; has a great excess of membrane in relation to its contents, but is able to synthesize hemoglobin. 3.2.6 Erythrocyte:  These are the mature non-nucleated red blood cells.  Cell stains buff or reddish color. 4. Granulopoiesis (Granulocytes Production): Granulocytes develop in the bone marrow from undifferentiated stem cells. Granulocytes are readily differentiated from the nucleated erythrocytes by their fine, reticulated chromatin structure and bluish cytoplasm. 4.1 Maturation Sequence: Maturation of the granulocytic series of cells is characterized by the development of granules. Granules are initially formed in the progranulocytes and are called "primary or azurophil" granules. These granules are peroxidase positive and develop from fusion of small proteinaceous material by the Golgi complex. As the maturation sequence continues, the primary or azurophil granules lose their acid mucopolysaccharide and, therefore, will not stain with 8  Wright's stain. However, the myelocyte stage is characterized by the appearance of "specific" or secondary granules that persist throughout the maturation process. Cells which have granules with an affinity for blue or basic dye are identified as basophils; cells that are stained reddish-orange with the acid dye eosin are called eosinophils; and the cells with granules which do not stain intensely with either dye are called neutrophils. 4.2 Granulocyte Maturation Sequence: Several distinct developmental stages of granulocytes can be recognized morphologically. The stages are: 4.2.1 Myeloblast:  The chromatin material of the nucleus is finely stippled or has a light ground glass appearance.  The cytoplasm is somewhat scanty, basophilic, and does not contain granules. 4.2.2 Progranulocyte:  The nuclear chromatin material is coarser and slightly more clumped than that of a myeloblast.  Remnants of the nucleoli may still be present.  The cytoplasm is less basophilic than the myeloblast and contains darkly stained non-specific granules called "primary or azurophilic granules." These granules are peroxidase positive. 4.2.3 Myelocyte -- Basophil, eosinophil, neutrophil:  The nucleus of the myelocyte remains somewhat round to oval and the chromatin material is more closely clumped.  This cell contains "secondary" or specific granules that are identified by their staining properties as neutrophils, eosinophils, and basophils. These granules are peroxidase negative.  Therefore, the myelocyte and all subsequent cells of the granulocytic series should be characterized as neutrophils, eosinophils and 9  basophils.  These granules vary greatly in shape, size and concentration in domestic species.  As maturation of the granulocytes continue, the "specific granules" will increase and the "azurophilic granules" will not take the Wright's stain.  The myelocyte stage is the last stage of cell division and the first cell capable of phagocytosis. 4.2.5 Metamyelocyte -- Basophil, eosinophil, neutrophil:  This cell closely resembles the myelocyte.  The nucleus is indented and often resembles a kidney bean.  Nucleoli are not present and the nuclear chromatin material is coarser and clumped.  Cytoplasmic granules are also present. 4.2.6 Band Cell -- Basophil, eosinophil, neutrophil:  This cell has a horseshoe shaped nucleus.  The opposite sides of the nucleus are more or less parallel.  This cell may be differentiated from the metamyelocyte by the nuclear shape and the tendency for the nuclear sides to become parallel.  The nuclear chromatin material is markedly clumped. 4.2.7 Segmented Cell -- Basophil eosinophil, neutrophil:  The nucleus may be mono-lobed with clumped chromatin material, or may consist of several lobes separated by constrictions or by filaments. The cytoplasm stains very faintly. 4.3 Leukocyte Lysosomes: The term lysosome is used to describe intracellular membranous sacs containing acid hydrolytic enzymes. When leukocytes phagocytize, there is a release of lysosomal contents. Primary granules (azurophil granules) contain acid phosphatase, acid hydrolytic enzymes, basic 10  protein and one-third of the lysozyme. Secondary granules (specific granules) contain alkaline phosphatase, lactoferrin and two-thirds of the lysozyme. 5. Agranulopoiesis (Agranulocytes Production): The agranulocytic series is comprised of leukocytes devoid of specific granulation. These cells generally originate in the lymphatic system, but like the granulocytic series, they may be produced elsewhere in the body. This series includes the lymphocytic and monocytic groups. 5.1 Lymphocytic Maturation Sequence: The lymphocytic series refers to the development of the lymphocytes. Lymphocyte maturation begins in the bone marrow; B cells complete initial development in the marrow and then circulate to peripheral lymphoid tissues (lymph node, spleen, and mucosal surfaces) to await antigen exposure and final maturation into plasma cells. T-cell maturation also begins in the bone marrow; T-cell precursors then travel to the thymus (initially the cortex of the thymus, progressing down into the medulla of the thymus), where they complete maturation before being released into the blood to travel to tissues. Differentiation into T helper and T suppressor subsets occurs in the thymus. Lymphocytes are the most undifferentiated of all blood cells normally found in the peripheral blood. The nucleus does not become segmented and specific granules do not develop. Therefore, lymphocytes are "peroxidase negative". The lymphocyte cytoplasm does not develop past the blue stage. The stages in lymphocytic development are: 5.1.1 Lymphoblast:  Cell is similar to other blast cells. It is round or oval, very large, with a large round to oval reddish-purple nucleus.  The nuclear chromatin material is fine and well distributed but perhaps more coarse than in myeloblasts. 11  The nucleus contains one or two nucleoli.  The cytoplasm is bluish and nongranular and forms a thin rim around the nucleus. 5.1.2 Prolymphocyte:  The nucleus is round or oval in shape but smaller than the lymphoblast.  The nuclear chromatin is coarse and slightly clumped.  Nucleoli or remnants of nucleoli may be present.  There is an abundant amount of light blue cytoplasm around the nucleus. Also, there may be a few azurophilic granules in the cytoplasm. 5.1.3 Lymphocyte:  This is the mature cell of the lymphocytic series and the only cell form found in the peripheral blood in health.  Lymphocytes vary greatly in size and may be classified as small, medium or large. However, size does not determine age of these cells.  The cells are easily distorted and often appear in irregular shapes in stained preparations. The nuclear chromatin is condensed to form large, discrete almost solid clumps, with thickening of the nuclear membrane. Nucleoli are absent. Non-specific granules may be observed in the cytoplasm of these cells. 5.2 Monocytic Maturation Sequence: The monocytic series refer to the stages of development of the monocyte. Monocytes are derived from the CFU-GEMM. The maturation sequence is monoblast, promonocyte, and monocyte. After migration to tissues, they are called as macrophages. Monocytes function as phagocytic cells, antigen presenting cells, and also produce a variety of cytokines. One of the most important sites of origin is the spleen. The stages in the monocytic development are: 5.2.1 Monoblast 12  The cell is large with a round or oval nucleus. A nucleolus is present. The nuclear chromatin material is fine and well-distributed. There is a thin rim of clear blue cytoplasm around the nucleus. There are no granules present in the cytoplasm. 5.2.2 Promonocyte:  The cell is somewhat smaller than the monoblast with the nucleus being irregularly-shaped. The nuclear chromatin material is fine and spongy. There may be a nucleolus or a remnant of the nucleolus present. The cytoplasm is grayish blue and may contain non-specific granules. 5.2.3 Monocyte:  The cell is larger than a neutrophil in the thin portions of a smear.  The shape of monocytes is variable. The nuclei are usually round or kidney-shaped, but may be deeply indented or have two or more lobes connected by narrow bands. Blunt pseudopods and digestive vacuoles may be present. Monocytes are most difficult to identify and to differentiate from other cells. They are frequently mistaken for immature neutrophils and large lymphocytes. The three most characteristic features of the monocytes and the most helpful in diagnosis are the dull grayish-blue color of the cytoplasm, blunt pseudopods and the brain-like convolution of the nucleus. 6. Thrombopoiesis (Platelet or Thrombocyte Production): Cells of the megakaryocytic system are peculiar in that the nucleus undergoes multiple mitotic divisions without cytoplasmic separation, thus producing giant polyploid cells. The multiple nuclei usually remain attached to each other and are often superimposed giving a lobular appearance. The cytoplasm undergoes maturation changes characterized by the development of granules and membranes, culminating in platelet differentiation and liberation. The stages in thrombocyte development are: 13 6.1 Megakaryoblast:  The cell is large, irregularly shaped with a single or several round or oval nuclei and with a blue, nongranular cytoplasm. Nucleoli are usually present. 6.2 Promegakaryocyte:  This cell differs from the megakaryoblast in that there are bluish granules in the cytoplasm adjacent to the nucleus. The nucleus in this second stage of maturation has usually divided one or more times and the cell has increased in size. 6.3 Megakaryocyte:  The cell is very large with relatively large amounts of cytoplasm, and multiple nuclei. The cytoplasm contains numerous small, uniformly distributed granules that are reddish-blue in color. Megakaryocytes have cytoplasmic projections extending through the walls of sinuses into the lumen; fragments of cytoplasm break off into the sinus as platelets. 6.4 Thrombocyte (Platelet):  Platelets are fragments of the cytoplasm of megakaryocytes. They vary in size and shape from a barely visible structure to masses larger than red cells or leukocytes. The cytoplasm stains a light blue and contains variable numbers of small blue granules (azurophilic). 7. Hematopoietic Growth Factors: Hematopoietic growth factors are a heterogeneous group of cytokines that stimulate progenitor cells of the hematopoietic system and induce proliferation and maturation. For the most part, they are glycoproteins that are synthesized and elaborated by a variety of cells in the local milieus of the bone marrow (with the exception of erythropoietin). They bind to specific membrane receptors on the surface of the various cells of the hematopoietic system. These hormones play a critical role in the regulation of all hematopoietic 14  cells in health and disease. Major Hematopoietic Growth Factors: 1. Erythropoietin, which is synthesized by the peritubular cells of the kidney in response to hypoxemia, is always present in minute amounts in human urine. Approximately 10% of endogenous erythropoietin is secreted by the liver. This is responsible for the low- level erythroid activity in a nephric persons. 2. Interleukin-3 (IL-3) is produced by T-lymphocytes, and this factor is not lineage specific. This factor appears to stimulate production and renewal of the pluripotent stem cell compartment and is capable of stimulating pluripotent hematopoietic stem cells to differentiate into all the myeloid cell lines and perhaps lymphocytes. 3. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is synthesized and secreted by a variety of cells in the bone marrow microenvironment: stromal cells, fibroblasts, T cells, and endothelial cells. This factor stimulates growth of progenitors for granulocytes, monocytes, and erythrocytes, and often causes eosinophilia as well. It activates granulocytes and monocytes and macrophages and enhances phagocytosis and other functions of these cells. 4. Granulocyte colony-stimulating factor (G-CSF) is a potent, low- molecular-weight glycoprotein that stimulates proliferation and maturation of granulocyte precursors. Stromal cells, monocytes and macrophages, and endothelial cells produce this factor. 5. Macrophage colony-stimulating factor (M-CSF) is secreted by stromal cells, macrophages, and fibroblasts. A heavily glycosylated glycoprotein, M-CSF exists in a dimer. It is a potent stimulator of macrophage function and activation, resulting in stimulation and elaboration of other cytokines. 6. Interleukin-2 (IL-2) or T-cell growth factor (TCGF) is synthesized and secreted by activated T cells, primarily helper-T cells (CD 4 and 15  T cells). Primarily, IL-2 leads to the clonal expansion of antigen- specific T cells and the induction of the expression of IL-2 receptors (CD25) on the surface membrane of T cells. This growth factor activates T-cell cytotoxic responses and induces non-MHC restricted cytotoxic lymphocytes. To a lesser degree, it stimulates proliferation of NK cells and B cells. 7. Interleukin-4 (IL-4) and B-cell stimulating factor (BSF-1), a potent growth factor, is derived from activated T cells and mast cells. The main effect of IL-4 is the induction of proliferation and differentiation of B cells and expression of MHC class II antigens on resting B cells. IL-4 also can act on T cells, monocytes and macrophages, mast cells, fibroblasts, and endothelial cells. It may be an important factor in the modulation of host immunity and inflammatory responses in vitro. Other Cytokines that Affect Hematopoiesis 1. Interleukin-5 (IL-5) or B-cell stimulating factor (BSF-2). The major source of these growth factors is T cells. This potent eosinophil differentiation and activation factor causes a rise in peripheral blood eosinophils. IL-5 stimulates B-cell differentiation and antibody production, and it can induce IL-2 receptor expression and release of soluble Il-2 receptor proteins. 2. Interleukin-6 (IL-6) is an important, multifunctional glycoprotein. It is produced by lymphoid and nonlymphoid cells and plays a major role in the mediation of inflammation and immune response. This growth factor promotes the production of acute-phase proteins and appears to have a stimulating effect on hematopoietic stem cells. It enhances differentiation of B cells and antibody secretion. Apparently, IL-6 is an important growth factor for myeloma cells, and it has been shown to have antiviral activity. This growth factor is also a costimulant of IL-2 production and IL-2 receptor expression 16  of T cells. 3. Interleukin-7 (IL-7) is produced by bone marrow stromal cells. Many other tissues express IL-7 (e.g., the spleen, fetal and adult thymus tissue). The receptor molecule for IL-7 appears to be a member of the hematopoietic receptor superfamily. IL-7 is an important growth and differentiating factor for T cells, and it may be an important viability factor for immature and nonproliferating thymocytes. This factor, which can stimulate proliferation and differentiation of human T cells, may act synergistically with other lymphokines (IL-2 and IL- 6) to stimulate the development of cytolytic T lymphocytes and lymphokine-activated killer cells from CD8 + T cells. Evidence suggests that IL-7 acts synergistically with stem cell factor in vivo to stimulate B-cell lineage development. 4. Interleukin-8 (IL-8) is a nonglycosylated protein with a molecular weight of approximately 8 kDa. It is produced by a wide variety of cells, and its elaboration can be induced by other proinflammatory molecules or stimuli (e.g., IL-1, TNF, lipopolysaccharides, infectious agents). A potent chemotactic activating factor for neutrophils, IL-8 has a wide range of other proinflammatory effects. It causes degranulation of neutrophil specific granules in the presence of cytochalasin B and induces the expression of adhesion molecules by neutrophils. In addition to being chemotactic for neutrophils and enhancing neutrophil adherents, IL-8 has been reported to be chemotactic for T lymphocytes and eosinophils. 5. Interleukin-9 (IL-9) is synthesized and secreted by T helper cells. This factor acts synergistically with IL-4 to potentiate antibody production by B cells. It also stimulates erythroid colony formation and maturation of megakaryocytes in vitro. Synergism between IL-9 and Epo is apparent. 6. Interleukin-10 (IL-10) is secreted by T cells and B cells and is 17  encoded by a gene on chromosome 1. This factor is a potent immunosuppressant of macrophage function because of its inhibitory effects on accessory function and antigen-presenting capacity of monocytes and macrophages. In addition, IL-10 can downregulate MHC class II antigen expression on macrophages, and it also inhibits numerous cytokines, including IL-1, TNF, and IL-6. 7. Interleukin-11 (IL-11) acts as an inflammatory mediator by stimulating the synthesis of hepatic acute phase reactants. This factor increases the number, size, and ploidy values of megakaryocyte colonies in vitro. IL-11 has been shown to have synergistic effects on the growth factor activity of IL-3 and IL-4 on early hematopoietic progenitors. It acts synergistically with thrombopoietin to induce megakaryocytic proliferation and maturation resulting in a substantial increase in platelets. 8. Interleukin-12 (IL-12) is produced by macrophages and B cells. This factor stimulates the production of interferon-gamma (IFN-γ) from T cells and NK cells and participates in the differentiation of helper T cells. In addition, it enhances the expansion of human helper T cells in vitro and augments cell-mediated response to infection by stimulating IL-2 and IFN-γ production. 18  Erythrocytes: Production, Function and Destruction Essential Material for RBC Production: 1. Proteins Needed for the production of globin in hemoglobin. Abnormal erythropoiesis may occur in a protein deficient diet. 2. Minerals a. Iron is an integral part of the hemoglobin molecule and is essential for Hb synthesis. b. Copper is a factor for the enzyme ALA dehydrase which is required in the synthesis of heme. c. Cobalt is essential for ruminants to synthesis B12 in the rumen. Excess Cobalt results in polycythemia (produces tissue anoxia). 3. Vitamins Essential vitamins for erythropoiesis are in the B series. Deficiencies of the following may lead to development of anemia: 1. Riboflavin (B2) 2. Pyridoxine (B6) 3. Niacin or nicotinic acid 4. Folic acid 5. Thiamine 6. B12 Erythrocytes Structure and Function: 1. Structure: a. The red cell is composed of water (55-65%) hemoglobin (30-36%) and a matrix of organic and inorganic materials. b. The red cell membrane is composed of a Triple-layered structure consisting of a lipid layer located between two protein layers. The red cell membrane is flexible but essentially nonelastic. The membrane has a negative surface charge that decreases during aging in vivo and also when red cells are exposed to antibodies. 19 c. Mammalian erythrocytes are anuclear while all other vertebrates have nucleated red cells. 2. Function: The primary function of the erythrocyte is to serve as a carrier of Hb and Hb functions as a carrier of oxygen. 3. Hemoglobin Synthesis: The hemoglobin molecule is a tetramer composed of four globin chains, each of which contains a heme ring. It has a molecular weight of approximately 64,000-68,000 and can pass an intact glomerular membrane. 3. 1. Heme a. Composed of protoporphyrin ring plus ferrous iron (Fe++) b. The properties of Hb are dependent upon maintaining iron in the reduced state (Fe++). c. The basic structure consists of four pyrrol rings linked together by four methane bridges. d. Found also in myoglobin, cytochromes, catalyze, and peroxides. e. The initial reaction in the heme synthesis pathway is the combination of glycine and succinyl coenzyme A (CoA) to form δ-aminolevulinic acid (ALA), which is catalyzed by the enzyme aminolevulinic acid synthetase (ALA synthetase). Pyridoxal 5’-phosphate (derived from pyridoxine, or vitamin B6), is an essential cofactor in the reaction. The final step in the pathway is insertion of the ferrous iron atom into protoporphyrin IX, catalyzed by the enzyme ferrochelatase. Defects in various enzymes in the heme synthesis pathway cause the porphyrias; other defects in the heme synthesis pathway result in the sideroblastic anemias. f. Lead reduces heme synthesis by inhibiting (ALA) synthesis, ALA dehydrase and heme synthetase. g. In the biogenesis of heme, copper is necessary for the enzyme ALA 20  dehydrase and B6 is necessary for the activation of glycine. 3. 2. Globin: a. Consist of two α-chains and two non-α chains (either β, δ, or γ); in the major adult hemoglobin, Hgb A, the tetramer consists of two α-β dimers. Each globin chain can carry one molecule of oxygen, so a hemoglobin tetramer can carry four oxygen molecules. b. Synthesized in the cytoplasm Excess globin synthesis stimulates heme synthesis and further inhibits globin synthesis. Globin synthesis is stimulated by excess heme which inhibits the formation of more heme. 4. Hemoglobin Types: 1. Differences in the amino acid sequence of the globin moiety are responsible for the different types of Hb. 2. Embryonic, fetal and adult types of Hb have been found. 3. More than a single Hb component exists (Hemoglobin polymorphism). The type of Hb synthesized may be influenced by environmental factors. Three Hbs are found (Hb A; Hb B; Hb C). 5. Hemoglobin Breakdown Hb may be released and degraded intravascularly or extravascularly. 5.1 Three pathways following intravascular hemolysis: a. Free Hb in the plasma is first bound to haptoglobin, an alpha 2 mucoprotein formed in the liver, and the complex is then removed rapidly and degraded mainly by the liver. b. Excess Hb in the circulation is converted to methemoglobin, which in turn dissociates, liberating hematin. Hematin is bound first to hemopexin, a glycoprotein present in the plasma, and the hematin- hemopexin complex is removed from the plasma by the hepatocytes. c. When more free Hb is present than is required to saturate all the 21  plasma haptoglobin, it is filtered by the renal glomeruli and absorbed in the proximal tubular epithelium where it is broken down to bilirubin. 1) This bilirubin is rapidly transported to the liver for excretion into the bile. 2) When the renal threshold for Hb is exceeded, Hb spills into the urine and the condition is referred to as hemoglobinuria. 5.2 Extravascular hemolysis Extravascularly, Hb is broken down by the reticuloendothelial system (RES) into hemotoidin which unites with a plasma protein and becomes hemobilirubin (indirecting-reacting, unconjugated or free bilirubin). Hemobilirubin is then transported to the liver where it is conjugated with glucuronic acid to become cholebilirubin (direct- reacting, conjugated or bound bilirubin). 6. Erythrocytes Metabolism Erythrocytes generate energy from glucose via the Embden-Meyerhof pathway (anaerobic glycolysis), which turns glucose into lactate and generates adenosine triphosphate (ATP) for energy (controlling the active movement of Na+ out of and K+ into the cell to maintain cell shape). Approximately 10% of glucose is shunted into the hexose monophosphate shunt, which generates reducing potential for the cell in the form of NADPH, the reduced form of nicotinamide adenine dinucleotide phosphate (NADP). The first enzyme in the pathway is glucose-6-phosphate dehydrogenase. An additional accessory pathway off the Embden-Meyerhof pathway is the Rapoport-Luebering shunt, which generates 2,3- diphosphoglycerate (2,3- DPG), which is the primary physiologic regulator of the oxygen affinity of hemoglobin. Another product of the Embden-Meyerhof pathway is reducing equivalents in the form of NADH, the reduced form of nicotinamide adenine dinucleotide 22  (NAD). In the normal functional state, the iron atoms in hemoglobin are in the ferrous (Fe2+) state. The iron atom can be oxidized to the ferric (Fe3+) state with the production of methemoglobin and superoxide (O2–). Methemoglobin, in which iron atoms are in the ferric (Fe3+) state, is useless as an oxygen carrier. Unless it is reduced back to hemoglobin (Fe2+), methemoglobin may be oxidized to form hemichromes and then aggregates of denatured hemoglobin called Heinz bodies. Hemichromes and Heinz bodies can attach to and damage the cell membrane and, if present in sufficient quantity, can cause lysis of the erythrocyte. Methemoglobin is reduced back to hemoglobin by the methemoglobin reductase enzyme system, which requires NADH, generated as part of glycolysis. Superoxide (O2–) is a potent oxidizing agent and will damage the erythrocyte unless it is neutralized. Superoxide is converted to hydrogen peroxide (H2O2) by the enzyme superoxide dismutase; hydrogen peroxide is itself an oxidizing agent and must be neutralized by reduced glutathione (GSH). Oxidized glutathione (GSSG) is reduced by the enzyme glutathione reductase, which requires NADPH generated by the hexose monophosphate shunt. If methemoglobin is not reduced to hemoglobin, or GSSG reduced back to glutathione, the result is premature destruction of the erythrocyte (hemolysis). Remember: 1. The EM pathway produces NADP which is utilized for the enzymatic reduction of methemoglobin (Fe+++) and the enzyme methemoglobin reductase to the functional Hb (Fe++) capable of transporting oxygen. 2. The pentose phosphate shunt produces NADPH which is utilized for the conversion of oxidized glutathione (GSSG) to reduced glutathione (GSH) with the enzyme glutathione reductase. Reduced glutathione is required for the non-enzymatic conversion of 23  methemoglobin to functional hemoglobin. Reduced glutathione is necessary to protect erythrocytes against hemolysis by oxidant drugs. Inability to generate GSH may lead to precipitation of Hb in the form of Heinz bodies producing Heinz body anemia. 3. Deficiencies of certain enzymes of the EM pathway such as pyruvate kinase and glucose-6-phosphate dehydrogenase resulting in hematologic disorders have been reported. The RBC has a shortened life resulting in a nonspherocytic hemolytic anemia. A tremendous response to the anemia is seen by numerous metarubricytes and polychromatophilic cells. 24  Erythrocyte Morphology Normal Morphology: The morphological features of mature red blood cells of most mammals are generally very similar in that they all lack nuclei, stain reddish to reddish-orange, and generally are biconcave, discoid-shaped cells. The major differences are found in the size of the red blood cells and the degree of central pallor. The central pallor is the lighter-staining area in the middle of the cell, resulting from a close association of the membranes in this region. Definitions of Morphological Variations: 1. Anisocytosis: Variation in the size of red blood cells. The large cells are most likely immature and are reticulocytes indicating early release from the marrow. 2. Polychromasia: Variation in the staining of red cells giving a mixture of blue and red cells. The bluish-red blood cells are generally large and represent reticulocytes. The blue color is due to residual RNA (ribosomes and mitochrondria). Associated with increased erythropoietic activity. 3. Hypochromasia: Decreased staining of red blood cells and increased central pallor due to a reduction of hemoglobin (Hb) in the cell. Most common cause is iron deficiency. 4. Poikilocytosis: Variation in the shape of red blood cells. May be pear shaped, tear shaped, etc. Should not be confused with crenation. Commonly seen in chronic blood loss, iron deficiency anemia, and occasionally in diseases characterized by increased red blood cell fragility. Poikilocytes may be removed prematurely from the circulation, potentiating the anemic state. 5. Macrocytes: Larger than normal red blood cells with increased mean corpuscular volume (MCV). May exhibit polychromasia and represent reticulocytes. Can occur in B12 and folic acid deficiency anemias. 25 6. Microcytes: Small red blood cells with decreased MCV. Observed in iron and pyridoxine deficiency anemias. 7. Leptocytes (Target Cells): A thin red blood cell with increased membrane or surface area. Observed in chronic debilitating diseases and anemia. 8. Spherocytes: Red blood cells with reduced amounts of membrane for amount of volume; opposite of leptocyte. Small, dark cell with no central pallor. Observed in autoimmune and isoimmune hemolytic anemias. May be observed following transfusions. Removed prematurely from circulation. 9. Basophilic Stippling: Punctate basophilia of Wright stained red blood cells due to RNA. Occurs in responding anemias and in lead poisoning. Do not confuse with parasites. 10. Howell Jolly Bodies: Small, round, usually eccentrically located, densely staine basophilic bodies considered to be nuclear remnants in erythrocytes. They may occur in some immature erythrocytes released to peripheral blood in response to anemia. May be increased in anemias in remissions. Do not confuse with parasites. 11. Heinz Bodies: Denatured hemoglobin occurring as refractive structures formed in red blood cells as the result of the toxic effects of certain drugs or chemicals; e.g., phenothiazine toxicosis and methylene blue toxicosis. These bodies do not stain with Wright stain. Must use new methylene blue to stain the cells. 12. Er Bodies: Erythrocyte refractive bodies similar to Heinz bodies. Increased in a variety of diseases. May play a role in the development of anemia. 13. Reticulocytes: These cells are immature red blood cells, typically composing about 1% of the red blood cells. In the process of erythropoiesis (red blood cell formation), reticulocytes develop and mature in the bone marrow and then circulate for about a day in the 26  blood stream before developing into mature red blood cells. Like mature red blood cells, in mammals, reticulocytes do not have a cell nucleus. They are called reticulocytes because of a reticular (mesh- like) network of ribosomal RNA that becomes visible under a microscope with certain stains such as new methylene blue. Increased reticulocytes in the peripheral blood indicate an increase in bone marrow activity and can be taken as an indicator of effective erythropoiesis. Premature release of reticulocytes under the influence of increased levels of erythropoietin, results in cells that are very large, contain more reticulum and are termed "shift" or stimulated reticulocytes. Reticulocytes normally mature in the peripheral blood between 19 and 43 hours with an average of 31 hours. They appear in the peripheral blood in about four days in response to hemorrhage or hemolysis and reach their peak by the seventh day. 14. Nucleated RBC: Represents early release of immature nucleated erythroid cells, usually metarubricytes. They usually indicate a bone marrow response and are a sign of intense erythrogenesis when accompanied by polychromatophilic cells in the peripheral blood. 15. Parasites: Located within the red blood cell or on the cell surface. 27  Erythrocytes Indices Red cell indices are mean cell volume (MCV), mean cell hemoglobin (MCH), and mean cell hemoglobin concentration (MCHC). They are also called as “absolute values”. They are derived from the values of hemoglobin, packed cell volume (PCV or hematocrit), and red cell count. Red cell indices are accurately measured by automated hematology analyzers. Recently, a new parameter called red cell distribution width (RDW) has been introduced. Uses: 1. Morphological classification of anemia: Based on values of red cell indices, anemia is classified into three main types: normocytic normochromic, microcytic hypochromic, and macrocytic normochromic. Calculation of red cell indices is especially helpful in mild or moderate anemia when red cell changes are subtle and often difficult to appreciate on stained blood smear. 2. Differentiation of iron deficiency anemia from thalassemia trait: In iron deficiency, MCV, MCH, and MCHC are low, while in thalassemia trait, MCV and MCH are low and MCHC is normal. Mean Cell Volume:  The MCV, the average volume of red cells, is calculated from the Hct and the red cell count.  MCV = Hct × 1000/RBC (in millions per µL), expressed in femtoliters or cubic micrometers.  If the Hct = 0.45 and the red cell count = 5 × 1012/L, 1 L will contain 5 × 1012 red cells, which occupy a volume of 0.45 L. Causes of Increased MCV Megaloblastic anemia 28  Non-megaloblastic macrocytosis: reticulocytosis Newborns. Causes of Low MCV Microcytic hypochromic anemia MCV is normal in normocytic normochromic anemia (acute blood loss, hemolysis, aplastic anemia). Mean Cell Hemoglobin:  The MCH is the content (weight) of Hb of the average red cell; it is calculated from the Hb concentration and the red cell count.  The value is expressed in picograms. If the Hb = 15 g/dL and the red cell count is 5 × 1012/L, 1 L contains 150 g of Hb distributed in 5 × 1012cells.  MCH is decreased in microcytic hypochromic anemia, and increased in macrocytic anemia and in newborns. Mean Cell Hemoglobin Concentration:  The mean cell hemoglobin concentration (MCHC) is the average concentration of Hb in a given volume of packed red cells. It is calculated from the Hb concentration and the Hct.  MCHC is raised in hereditary spherocytosis, and is decreased in hypochromic anemia. MCHC corresponds with degree of hemoglobinization of red cells on a blood smear. If MCHC is normal, 29  red cell is normochromic, and if low, red cell is hypochromic. Red Cell Distribution Width (RDW):  Some automated analyzers measure red cell distribution width or RDW. It is a measure of degree of variation in red cell size (anisocytosis) in a blood sample. It is helpful in differential diagnosis of some anemias. Amongst microcytic anemias, RDW is low in β thalassemia trait, high in iron deficiency anemia, and normal in anemia of chronic disease 30  Erythrocytes in Disease A. Erythrocytosis and Polycythemia: Red blood cell production typically is in part regulated by the hormone erythropoietin (EPO). When oxygen sensors located in the renal cortex become hypoxic, hypoxia – inducible factors are generated and incite EPO gene transcription. The resultant EPO then stimulates erythroid precursors in the bone marrow to increase RBC numbers, thereby enhancing the oxygen carrying capacity of the blood. Erythrocytosis is defined as an increase in peripheral RBC numbers, hemoglobin concentration, and calculated hematocrit or PCV above reference intervals. In human medicine, polycythemia sometimes denotes not only erythrocytosis, but also concurrent increases in white blood cell and platelet counts. Classification based on pathogenesis: 1. Relative Erythrocytosis (Hemoconcentration) In relative erythrocytosis, the increased PCV is not accompanied by an expanded RBC mass, but rather develops from diminished plasma volume and hemoconcentration due to fluid loss (i.e. severe dehydration associated with vomiting, diarrhea, or polyuria without sufficient water intake and shock). Another cause of relative erythrocytosis is splenic contraction associated with excitement or anxiety (transient mild erythrocytosis unassociated with clinical signs). The plasma protein concentration is elevated. 2. Absolute Erythrocytosis Absolute erythrocytosis, defined as a true increase in RBC mass, can develop from primary or secondary causes. 2.1 Primary Erythrocytosis (Polycythemia Vera) The primary erythrocytosis (polycythemia vera) is a chronic myeloproliferative disorder resulting from the clonal expansion of hematopoietic progenitor cells. In humans, polycythemia vera is 31  associated with splenomegaly, leukocytosis, and thrombocytosis, and may progress to myelofibrosis or leukemia. All cellular elements of the bone marrow are affected. There is an absolute increase in the numbers of erythrocytes and total blood volume; however, the total protein remains the same. 2.2 Secondary Erythrocytosis Secondary erythrocytosis develops from excessive production of EPO. If EPO is secreted in response to systemic hypoxia, then the resultant erythrocytosis represents an appropriate compensatory response. If, on the other hand, the increased EPO secretion is not associated with systemic tissue hypoxia, then the response is inappropriate. Endocrinopathy – associated erythrocytosis is another type of secondary erythrocytosis resulting from hormonal (other than solely EPO -mediated) stimulation of erythropoiesis. Appropriate Secondary Erythrocytosis usually associated with: 1. Cardiac Disorder 2. Chronic severe pulmonary disease 3. Oxygen affinity hemoglobinopathies Inappropriate Secondary Erythrocytosis (Excess production of EPO in the absence of systemic hypoxia) usually associated with: (1) EPO - secreting tumors of the kidneys or other organs (2) Kidney disorders e.g. renal cysts, hydronephrosis, and glomerulonephritis result in regional hypoxia leading to increased EPO production. Endocrinopathy - Associated Erythrocytosis Hormones other than EPO, such as cortisol, androgen, thyroxine, and growth hormone, also may stimulate erythropoiesis either directly, or indirectly through increased production of EPO or alternate pathophysiologic mechanisms. 32 B. Anemia: 1. It is a reduction below normal in the total number of red blood cells, PCV, hemoglobin concentration or all three. It is not a diagnosis but rather a symptom of an underlying disease process. Treatment should not be directed at the anemia except as an emergency matter. 2. Clinical signs are related to the decrease in oxygen carrying capacity of the blood. a. Signs and symptoms include: ① Pale mucous membranes ② Weakness or loss of stamina ③ Tachycardia, heart murmur ④ Shock if one-third of the blood volume is lost b. Signs are less marked if onset is gradual. The animal adapts to the decreased erythron. c. Icterus, hemoglobinuria, hemorrhage, edema or fever may be observed depending on the pathophysiologic mechanism involved. Classification: 1. Classification of Anemia According to Morphology 1.1 Red Blood Cell Size The size of the red cell is determined by visual interpretation on a stained blood smear or more accurately by the mean corpuscular value. 1.1.1 Macrocytic (Increased MCV) Transitory macrocytic condition is observed in cases where the marrow is responding to increased red blood cell need and discharging more immature cells (reticulocytes and nucleated red cells), which are larger, into the peripheral blood. e.g. Blood loss or hemolysis. True macrocytic is observed in conditions where there is interference with maturation in the prorubicyte - rubricyte stage and reduced cell 33  division resulting in large cells being released into the blood. e.g. B12, folic acid like deficiency. 1.1.2 Normocytic (Normal MCV) There is interference with erythropoiesis at the stem cell level resulting in fewer cells entering the maturation process. Those that do enter the process develop normally and are released at the normal time. e.g. selective anemias of chronic infections, chronic renal disease, chronic liver disease and aplastic anemias. 1.1.3 Microcytic (Reduced MCV) This is the type seen in iron deficiency anemia. A deficiency in iron results in less Hb being formed. Since the cell stops division when a certain critical hemoglobin concentration is reached, in iron deficiency there is often an extra division resulting in smaller cells. e.g. iron, copper and pyridoxine deficiency. 1.2 Hemoglobin Concentration Determined by prominence of central pallor in stained smear and more accurately by the mean corpuscular hemoglobin concentration (MCHC) and mean corpuscular hemoglobin (MCH). 1.2.1. Hyperchromic The cell cannot become supersaturated with Hb so this state does not actually exist. The MCH may be increased in cases where the MCV is elevated but not the MCHC. Elevated MCHC may indicate free hemoglobin in the plasma due to hemolysis, Heinz bodies in RBCs, or to excessive amounts of EDTA which cause shrinkage of RBCs. 1.2.2 Normochromic Normal Hb concentration is found in most anemias of animals. 1.2.3 Hypochromic Reduced Hb concentration is characteristic of iron, copper and pyridoxine deficiency anemias, and in blood loss and hemolysis where the number of immature cells greatly exceeds the number of mature 34  red blood cells. 2. Classification of Anemia According to Response 2.1 Regenerative The bone marrow is capable of responding to the anemia by increasing its production of red blood cells. The ability to respond with increased activity indicates that the site of pathologic alteration is not in the bone marrow. i.e. blood loss and hemolytic anemias. Signs of increased erythrocyte production by the bone marrow may include: a. Reticulocytosis b. Polychromasia c. Basophilic stippling d. Anisocytosis e. Nucleated red cells f. Howell-jolly bodies 2.2 Non-Regenerative The bone marrow is not able to respond to the anemic state. In fact, the inability of the marrow to produce red blood cells is the reason for the anemia. Therefore regenerative signs are absent. Red Cell morphology is normal in depression anemias and aplastic anemias. 3. Classification of Anemia by Pathophysiologic Mechanism 3.1 Blood loss anemia (Hemorrhagic Anemia) 3.1.1 Acute Blood Loss Acute blood loss - Anemia occurs when 25-40% of the circulating blood volume is lost. 1) Causes a. Trauma - ruptured liver or spleen b. Surgery c. Vascular neoplasm (hemangiosarcoma) d. Coagulation defects 35  e. Severe parasitism - hookworms, Haemonchus f. Gastrointestinal ulceration 2) External vs Internal Acute Blood Loss External blood loss results in a reduction of red blood cells, plasma proteins, and plasma volume. Iron is lost to the exterior and not available for utilization. Acute blood loss seldom results in iron deficiency since adults generally have sufficient iron stores to regenerate 2-3 times the total blood volume of red blood cells. Internal blood loss is primarily into body cavities, 2/3 of the blood is reabsorbed by the lymphatics (completed in 24-72 hours) while 1/3 is broken down by the RE system. If a large amount of blood is broken down it may simulate a hemolytic anemia (hyperbilirubinemia). 3) Response The response following acute hemorrhage depends upon the amount of blood loss, period of time during which bleeding occurs and the site of hemorrhage. A) Initially only the blood volume is reduced. The patient may be in shock due to blood loss (hypovolemia). The plasma and cells are both lost and may present as a normal hemogram. B) Thrombocyte numbers increase during the next few hours. C) Three hours later, a neutrophilic leukocytosis derived from the marginal pool occurs. D) Starting at 2-3 hours and proceeding to 48-72 hours, the blood volume is restored by the addition of fluid resulting in reduced PCV, Hb, and red blood cell numbers (anemia). Plasma protein concentration is also reduced. No regenerative signs are present at this stage. E) At 72-96 hours, the signs of increased red blood cell production are evident in the peripheral blood. i.e. polychromasia, nucleated red 36  blood cells (NRBC), reticulocytosis. May actually have an elevated MCV at this stage due to large young cells. F) Leukocytosis with left shift may accompany red cell response due to dual stimulation of bone marrow. G) Hemogram returns to normal in 1-2 weeks after a single acute episode. PCV returns to normal more rapidly than red blood count and Hb because of the large size of young cells. H) If reticulocytosis persists longer than 2-3 weeks look for continuous bleeding. 3.1.2 Chronic Blood loss Chronic blood loss - Small amount of blood lost over a long period. 1) Causes: a) Parasitism b) GI ulcers c) Vascular neoplasia d) Coagulation defects 1. Hemophilia 2. Thrombocytopenia 3. Vitamin K, prothrombin deficiency 2) Response: a) Anemia develops slowly - nonhypovolemia. b) Hematologic parameters resemble the regenerative stages of acute blood loss. c) The severity and longevity of the anemia is variable depending on the amount of blood being lost. d) With time and continuation of blood loss, regenerative signs become less obvious as the bone marrow becomes exhausted. Leptocytosis and poikilocytosis are severe. e) If the body stores of iron are depleted, an iron deficiency develops. i.e. hypochromic, microcytic. 37  f) In severe cases the marrow response may end abruptly resulting in a depression type anemia. g) Hypoproteinemia often complicates the anemia. 3.2 Anemias due to accelerated erythrocyte destruction (Hemolytic Anemia) a. Causes 1) Intrinsic - Hemolysis stems from a defect in the patient's red blood cells. a) Abnormal hemoglobins. b) Red blood cell enzymes deficiencies. c) Membrane abnormalities. 2) Extrinsic - Red blood cells damaged by some external factor a) Antibodies b) Toxins c) Parasites d) Chemicals e) Mechanical b. Site of Destruction Damaged red blood cells (by intrinsic or extrinsic means) may lyse within the circulation (intravascular hemolysis) or be phagocytized by the reticuloendothelial cells (extravascular hemolysis). In many cases, hemolysis takes place in both locations but one will usually predominate. 1) Intravascular hemolysis - more acute, seen in: a) Leptospirosis b) Clostridium perfringes Type A c) Clostridium hemolyticium (bacillary hemoglobinuria) d) Babesiosis e) Chronic copper poisoning - acute hemolytic crisis resulting from 38  stress precipitated release of copper stored in abnormal concentration in the liver. f) Phenothiazine poisoning - Heinz bodies in red blood cells. g) Onion poisoning. h) Lead poisoning - characterized by large numbers of basophilic stippled red blood cells and metarubricytes in the presence of a normal or slightly lowered PCV. Polychromia and reticulocytosis. i) Post-parturient hemoglobinuria j) Porphyria k) Isoimmune hemolytic disease l) Incompatible transfusions 2) Extravascular hemolysis a) Anaplasmosis. b) Hemobartonellosis c) Eperythrozoonosis d) Infectious anemia Virus e) PK deficiency - inherited, autosomal recessive disease, anemia evident early in life. f) Autoimmune hemolytic anemia c. Response The laboratory picture of hemolytic anemia will vary according to the clinical course and site of hemolysis 1) Peracute - Sudden onset a) Clinical course of a few to 48 hours b) Type observed in incompatible transfusions and acquired immunological disease. Usually intra-vascular type hemolysis. c) No signs of marrow response during this period, too early d) Plasma may be hemolysed (hemoglobinemia) e) If massive and haptoglobins are saturated, will have hemoglobinuria in later stages 39  f) Spontaneous agglutination may occur in immunologic types. g) Icterus only in later stages and only if liver's capacity to conjugate bilirubin is exceeded. h) Splenomegaly 2) Acute a) Onset more gradual - week or more - usually intravascular type hemolysis b) Signs of bone marrow response c) Icterus, hemoglobinemia d) Hemoglobinuria e) Agglutination of red blood cells and/or spherocytosis in immunologic types. f) Neutrophilic leukocytosis, eosinopenia and lymphopenia due to stress may occur. g) Splenomegaly 3) Chronic a) Slower onset- weeks b) Usually extravascular type hemolysis c) Anemia may not be very evident if destruction is slow enough for increased erythropoiesis to compensate d) Regenerative bone marrow e) Icterus, if marked enough to surpass liver's ability to conjugate bilirubin f) Usually no hemoglobinemia or hemoglobinuria g) Usually leukocytosis (neutrophilia) 40  3.2.1 Autoimmune Hemolytic Anemia (AIHA) 1. Definition An acquired hemolytic disease in which the life span of the RBC is shortened due to the coating of the cell by an abnormal globulin. The body forms antibodies (autoantibodies) against its own RBC's. The membrane of these RBC's is altered and the cell is removed prematurely by the RE system (extravascular hemolysis). Two main forms of AIHA are noted. 1. Hemolytic anemia unaccompanied by any coexisting disease. 2. Hemolytic anemia accompanied by thrombocytopenia or thrombocytopenic purpura. Approximately 1/3 of the cases are of this type. 2. Clinical Signs 1. Sudden onset, course variable, usually fatal 2. Signs referable to anemia a. Pallor b. Weakness c. Shortness of breath d. Tachycardia, heart murmur 3. Signs referable to hemolysis a. Very slight icterus in some cases b. Dark orange feces c. Dark urine d. Splenomegaly e. Hepatomegally f. Increased body temperature 4. Signs referable to thrombocytopenia a. Epistaxis b. Gingival, ocular, oral hemorrhages c. Petechial hemorrhages in the skin 41  d. Melena 3. Clinico-Pathological Findings 1. Regenerative anemia--hypochromia in some cases. 2. Neutrophilic leukocytosis with left shift, lymphopenia. 3. Spherocytosis - Not present in all cases. Small, dark RBC's with unaltered volume but reduced diameter. Should have no central pallor - removed by RE system. 4. May disappear with therapy or remission. 5. Spontaneous agglutination of RBC's may occur - usually involves spherocytes. 6. Hemolysed plasma - intravascular hemolysis is not characteristic of AIHA but fragile spherocytes may rupture intravascular or when blood is drawn. 7. Positive Coombs' test or absence of spherocytes does not rule out the disease. Often becomes negative with treatment. 3.3 Reduced or Defective Erythropoiesis Site of the problem is in the bone marrow in contrast to peripheral blood, as is the case in hemorrhagic and hemolytic anemias. a. Secondary Anemia (Anemias of chronic disorders) - Selective depression of erythropoiesis. The three disease categories associated with anemia of chronic disease are chronic infection, inflammation, and malignancy. There is a block in release of storage iron from macrophages for erythropoiesis that is mediated by inflammatory cytokines. 1) Chronic Inflammatory Disease - Chronic suppurative diseases. The anemia does not respond to hematinics and improves only when the underlying disorder improves. The anemia is mild to moderately severe. a) Normocytic, normochromic (rarely microcytic, hypochromic mild) 42 b) Non-regenerative c) Both serum iron and transferring levels are reduced. Iron binding capacity is low. Tissue iron stores are increased. There is an impairment of release of iron from the RE cell. Low serum iron seems to make the bone marrow less responsive to erythropoietin 2) Chronic renal disease a) Gradual onset b) Normocytic, normochromic anemia c) Non-regenerative anemia d) Shortened red blood cell survival time (usually proportional to degree of azotemia) - erythrophagia evident in RE system. Reduced erythropoietin production by the kidney and resulting lack of stem cell stimulation 3) Neoplastic Disease - (non-marrow infiltrating) a) Particularly in disseminated malignancies and/or necrotic neoplastic situations. b) Similar to secondary anemias associated with chronic inflammatory disease in its pathogenesis and blood picture. 43  3.4 Aplastic Anemia a. Characterized by 1. Pancytopenia in peripheral blood (granulo-cytopenia, thrombocytopenia and anemia) and decreased cellularity in bone marrow. 2. Normocytic, normochromic, non-regenerative anemia 3. Decreased cellularity in bone marrow - fat replacement. M/E ratio usually normal. 4. Damage is to the multipotential stem cell. 5. Since the life span of white blood cells and platelets are short (7-10 days), severe neutropenia and thrombocytopenia precede the anemia (RBC life span - long). 6. Overwhelming infection or uncontrollable hemorrhage is a frequent complication b. Causes 1) Irradiation - lymphocytes and bone marrow cells highly sensitive. Mature red blood cells are resistant 2) Poisoning 3) Chemicals a) Alkylating Agents, antimetabolites, and other classes of cancer chemotherapeutic drugs b) Benzene and cyclic hydrocarbons c) Insecticides d) Chloramphenicol, sulfonamides, estradiol, Anti-convulsive agents, Phenylbutazone and others. Must be administered over a long period of time. Individual idiosyncrasy is often involved. 4) Chronic blood loss over a long period of time; abrupt aplasia in terminal stages. 5) Myelophthisis anemia - physical replacement of hematopoietic marrow. 44 a) Neoplasia - Myeloproliferative disorders and other infiltrating neoplasms b) Osteodystrophia fibrosa and other bone diseases rarely severe enough. 45  3.5. Nutritional Anemias The bone marrow can usually respond but in an ineffective manner. 3.5.1 Megaloblastic Anemia Megaloblastic anemia results from deficiency of either folate or vitamin B12. Folate and vitamin B12 are essential for synthesis of deoxyribonucleic acid (DNA); in case of deficiency, therefore, there is defective synthesis of DNA in all the proliferating cells including bone marrow cells. In the deficient state there is not enough nucleic acid for cell division in the prorubricyte and rubricyte stages resulting in fewer divisions and therefore, the final cell is larger (macrocyte). A.B12 Deficiency Common in man. B12 is prepared for absorption by an intrinsic factor in the stomach, a lack of which causes pernicious anemia, a macrocytic anemia associated with leukopenia and thrombocytopenia. B12 deficiency develops in animals on cobalt deficient pasture. Cobalt is essential in the molecular structure of B12. B. Folic acid deficiency - similar to B12. Functions in nucleic acid metabolism. Results in a macrocytic anemia. Hematological findings: 1. Macrocytic anemia (mean cell volume >100 fl in adults). Elevation of mean cell volume is an early sign and precedes the onset of anemia. 2. Pancytopenia is common. 3. Blood smear shows oval macrocytosis, basophilic stippling, Howell- Jolly bodies, and hypersegmentation of neutrophils (>5% of neutrophils showing 5 or more lobes). 4. Reticulocyte count is normal or low. 5. Bone marrow shows megaloblasts, erythroid hyperplasia, and giant metamyelocytes and bands. C. Cobalt deficiency Required for B12 synthesis. 46 D. Iron deficiency  1/2 to 3/4 of the body iron is in Hb. The remainder is in myoglobin, enzymes of the cytochrome oxidase system and stored iron. Absorption of iron from the gut is controlled by need. A high pH and low HCl concentration impair absorption. Transported in blood bound to transferrin and stored as ferritin or hemosiderin. Most of the Hb iron is re-utilized. Because normal daily iron requirement is so low it would take years to deplete the body stores of iron and produce anemia. Iron deficiency in the adult means abnormal chronic blood loss. Stages of Iron Deficiency Stage 1 (Iron depletion): Depletion of storage iron (low serum ferritin), normal transport iron (serum iron, total iron binding capacity), normal hemoglobin Stage 2 (Low transport iron): Depletion of storage iron, low transport iron, normal hemoglobin Stage 3: (Low hemoglobin production): Depletion of storage iron, low transport iron, low hemoglobin Blood picture: a) Microcytic, hypochromic anemia. b) Since accumulation of Hb in the cell is one of the means of halting division. A deficiency of iron (Hb) would result in additional divisions and therefore smaller cells with reduced hemoglobin. c) Low PCV and Hb but red blood cell count may be normal. d) May see regenerative signs of anemia. e) Poikilocytosis is common f) Erythroid hyperplasia in marrow. Higher numbers of late rubricytes and metarubricytes. g) Typically, serum iron is low, TIBC is increased, and transferrin saturation is