ANEMIA DIAGNOSIS AND CLASSIFICATION. Murray State University Dr. Wade Northington

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1 Page 1 of 15 ANEMIA DIAGNOSIS AND CLASSIFICATION Dr. Wade Northington From: is an absolute decrease in the hematocrit, hemoglobin concentration, or the RBC count. Relative anemia may occur when the plasma volume is expanded.. is not a diagnosis, but a sign of underlying disease. The objective of the laboratory is to determine the type of anemia as an aid in discovering the cause. I. Determination of the cause: A. History 1. Drug administration. 2. Exposure to toxic chemicals or plants. 3. Family or herd occurrence. 4. Recent transfusions or colostral ingestion. 5. Age at onset. B. Physical findings 1. Related to decreased capacity to transport oxygen and the compensatory efforts of the body to increase the efficiency of the erythron and reduce the work load on the heart. a. Pale mucous membranes. b. Weakness, loss of stamina, and exercise intolerance. c. Tachycardia and polypnea. d. Hypersensitivity to cold. e. Heart murmur. f. Shock if >1/3 blood volume lost in a short period. 2. Icterus, hemoglobinuria, hemorrhage, or fever. C. Laboratory findings 1. The Hct is the easiest, most accurate method for detecting anemia. Its result should be interpreted with knowledge of the hydration status and any alteration caused by splenic contraction. 2. Hb and RBC may be used to further classify the anemia, but usually are not necessary to confirm its presence.

2 Page 2 of 15 II. Classification A. Size (MCV) and Hb Concentration (MCHC) 1. Normocytic, macrocytic, microcytic. 2. Normochromic, hypochromic. (Hyperchromia does not occur) B. Bone marrow response 1. Regenerative a. Bone marrow actively responds by increasing its production of RBC's. b. Findings: i. Polychromasia. ii. Reticulocytosis. iii. Macrocytosis (increased MCV) and iv. hypochromia associated with reticulocytosis. Hypercellular bone marrow with a low M/E ratio. c. Species that have the highest reticulocyte response are those that can mount the fastest response and rate of recovery. In decreasing order: i. Dog ii. Cat iii. Cow iv. Horse d. The presence of regeneration suggests an extramarrow cause. i. Blood loss ii. Erythrocyte destruction (hemolysis) e. Bone marrow examination would reveal erythropoietic hyperplasia. f. Regeneration is difficult to detect in the horse due to the lack of reticulocytosis. Increase in MCV and RDW may give clues. Bone marrow tap might be of benefit. 2. Non-Regenerative a. Inadequate bone marrow response because of a bone marrow disorder. b. Polychromasia and reticulocytosis are absent. c. During the first 2-3 days after onset of peracute or acute hemorrhage or hemolysis, anemia may appear non-regenerative. This is the amount of time it takes the bone marrow to mount a response. d. Bone marrow exam is indicated.

3 Page 3 of 15 C. Pathophysiologic mechanism 1. Blood loss-hemorrhagic anemia. 2. Accelerated erythrocyte destruction-hemolytic anemia. 3. Reduced or defective erythropoiesis. ANEMIA FROM BLOOD LOSS (HEMORRHAGIC ANEMIA) I. Characteristics of Acute Blood Loss A. Clinical findings 1. Evidence of hemorrhage. 2. Indirect-gastrointestinal, e.g. 3. Depends on the amount of blood lost, period of time during bleeding, and site of hemorrhage. 4. Hemorrhage from multiple sites suggests clotting abnormalities. B. Laboratory findings 1. Initially the Hct will be normal because all components are lost in similar proportions. Animal may be in hypovolemic shock. 2. Splenic contraction delivers high-hct blood (80%) to the circulation, temporarily increasing the Hct. 3. Starting 2-3 hours after onset and lasting for 2-3 days, the blood volume is restored by the addition of interstitial fluid. This causes dilution of the erythrocyte mass and the signs of anemia (reduced Hct, RBC, and Hb) become evident. Plasma proteins are also reduced. 4. Platelet numbers usually increase during the first few hours. Persistent thrombocytosis may suggest continued bleeding. 5. Neutrophilic leukocytosis commonly occurs by approximately 3 hours post-hemorrhage. 6. Signs of increased erythrocyte production (polychromasia, reticulocytosis) become evident by hours and reach a maximum approximately 7 days after the onset of hemorrhage. Erythroid hyperplasia is evident in the bone marrow and precedes the changes in the peripheral blood. 7. Plasma protein concentration begins to increase in 2-3 days and returns to normal before the Hct, RBC, and Hb. 8. The hemogram returns to normal in 1-2 weeks in the dog. 9. Thrombocytopenia and subsequent hemorrhage may occur with primary bone marrow failure; the anemia in these cases is non-regenerative.

4 Page 4 of 15 C. Causes 1. Trauma 2. Surgery 3. GI ulcers 4. Hemostasis defects a. DIC b. Sweet clover c. Warfarin d. Bracken fern e. Factor X deficiency in pups II. Characteristics of Chronic Blood Loss A. Clinical findings 1. develops slowly and hypovolemia does not occur. 2. Hct can reach low values before clinical signs of anemia become obvious because slow onset allows for physiologic adaptation. B. Laboratory findings 1. Regenerative response, but less intense than acute blood loss. 2. Hypoproteinemia is usually not noted. 3. Persistent thrombocytosis may be evident. 4. Body stores of iron may become depleted and an iron-lack anemia develops. C. Causes 1. Parasitism a. Ancylostomiasis b. Strongylosis-equines c. Hemonchosis-ruminants d. Coccidiosis e. Fleas, ticks, lice 2. GI ulcers, tumors 3. Hematuria 4. Vascular neoplasms 5. Hemophilia 6. Thrombocytopenia 7. Vitamin K deficiency

5 Page 5 of 15 III. Differential features between anemias caused by external and internal hemorrhage A. External blood loss prevents reutilization of certain components (iron, plasma, and protein). These may be reabsorbed with internal hemorrhage. B. In internal hemorrhage, some erythrocytes are reabsorbed into lymphatics, particularly when hemorrhage is into body cavities, and the remainder are lysed or phagocytized, and iron and plasma proteins are reutilized. Therefore, the anemia may not be severe and recovery may be faster. ANEMIA FROM ACCELERATED ERYTHROCYTE DESTRUCTION (HEMOLYTIC ANEMIA) I. Characteristics of hemolytic anemia A. Clinical findings 1. Clinical signs of hemorrhage are absent. 2. Clinical signs, in relation to severity of anemia, may be dramatic in acute cases. 3. Icterus may be seen in acute and severe cases. 4. Hemoglobinuria and red plasma is seen if significant intravascular hemolysis occurs. B. Laboratory findings 1. Reticulocyte counts are higher in hemolytic anemias than external hemorrhagic anemias because iron from destroyed erythrocytes is more readily available for erythropoiesis than is stored iron, which has to be used when blood is lost externally. 2. Plasma protein concentration is normal or increased. 3. Neutrophilic leukocytosis and monocytosis may occur. 4. Evidence of Hb degradation (hyperbilirubinemia, hemoglobinuria). 5. Abnormal erythrocyte morphology (Heinz bodies, erythrocytic parasites, spherocytes, or poikilocytes).

6 Page 6 of 15 II. Differentiation of the Causes of Hemolytic s A. Extravascular hemolysis (occurs outside the circulating blood stream)-erythrocytes are sequestered in spleen or liver where they are phagocytized or lysed and their hemoglobin catabolized at the site. 1. Mechanisms a. Autoimmune Mediated--Antibody and/or C 3 mediated i. Antibody (IgG or IgM) reacts with cell membrane antigen. ii. C 3 is fixed into the erythrocyte membrane by the antigen/antibody reaction. iii. Macrophages have receptors for both the antibody and C 3 that facilitate attachment and complete or partial phagocytosis. In partial phagocytosis, spherocytes are formed. iv. Immune-mediated anemias may be caused by 1. Unknown-idiopathic. Autoimmune hemolytic anemia. 2. Infectious agents (FeLV, EIA, Ehrlichia, Hemobartonella) a. Alter the cell membrane exposing antigens to which the host produces antibody. b. Form immune complexes that adsorb to the cell and fix complement. c. Cross-reacting antibody may be formed in response to infection. 3. Drugs. Penicillin adsorbs to erythrocytes and acts as a hapten in the production of antibody. 4. Alterations in the immune system. a. Disturbances in T-cell lymphocytes may disrupt immune regulation. b. Coombs' positive anemia has been observed in some lymphoid malignancies. v. With Coombs' testing, warm-active IgG alone, IgG plus C 3, C 3 alone, and, rarely, cold-active IgM may be found on erythrocytes. vi. Warm-active Igm usually fixes complement to C 9 and intravascular hemolysis occurs.

7 Page 7 of 15 b. Decreased erythrocyte deformability i. Changes in the erythrocyte membrane, increase in internal viscosity, or decrease in surface area/volume ratio predisposes to splenic sequestration and phagocytosis by macrophages. ii. Examples 1. Shistocytes of microangiopathic anemia 2. Spherocytes of immune-mediated anemia 3. Parasitized erythrocytes 4. Eccentrocytes or Heinz body-containing cells c. Reduced glycolysis and ATP content of the erythrocyte i. The affected cell is predisposed to removal by splenic macrophages. ii. Reduction in glycolysis occurs with normal aging. d. Increased macrophage activity i. Phagocytosis of normal erythrocytes may occur. ii. iii. Associated with conditions causing splenomegaly where there is excessive sequestration of erythrocytes and exposure to macrophages. The condition in humans has been called hypersplenism e. Intravascular causes of hemolysis do not lyse all erythrocytes; some altered cells may remain that are removed by phagocytosis. 2. Clinical and laboratory characteristics of phagocytic (extravascular) hemolysis. a. Usually chronic with insidious onset. b. A regenerative response is associated with an increase in plasma proteins. c. Hemoglobinemia and hemoglobinuria are absent. d. Hyperbilirubinemia occurs if the magnitude of the hemolysis is sufficient to exceed uptake, conjugation, and excretion by the liver. Unconjugated bilirubin

8 Page 8 of 15 usually predominates early, but later conjugated bilirubin may be prominent. e. The bone marrow response may compensate for the destruction of erythrocytes in cases of low-grade hemolysis, and the Hct is in the normal range. "Compensated hemolytic anemia." f. Neutrophilia, monocytosis, and thrombocytosis are common. g. Splenomegaly may result from increased macrophage activity and extramedullary hematopoiesis. h. Low-grade extravascular hemolysis occurs in many anemias that are primarily nonhemolytic (e.g., anemia of chronic renal disease, iron-deficiency anemia). Referred to as the "hemolytic component" of other types of anemia. 3. Aids in identification of the specific cause of extravascular hemolysis. a. History of a particular breed and/or littermates affected may suggest hereditary causes. b. Additional laboratory findings may include: i. Positive direct Coombs' test. ii. Abnormal erythrocyte morphology in a variety of anemias. Erythrocytic parasites, spherocytes, schistocytes, and keratocytes suggest the possibility of excessive erythrocyte phagocytosis. B. Intravascular hemolysis-erythrocytes are destroyed within the circulation, releasing hemoglobin into the plasma where it is either removed by the liver or excreted by the kidneys. 1. Mechanisms: The erythrocyte membrane must be significantly disrupted to allow escape of the Hb molecule into the plasma. Most of the mechanisms of intravascular hemolysis are extrinsic or extracorpuscular defects-the erythrocyte is initially normal. a. Complement-mediated lysis. i. Complement is incorporated into the erythrocyte membrane by antigen-antibody reactions occurring on the surface. If activated to C 9, a large enough membrane defect occurs for Hb to escape.

9 Page 9 of 15 ii. It occurs most commonly in immune-mediated anemias when IgM, a good complement fixer, is involved. IgG is a poor complement fixer, but can occasionally cause complement-mediated lysis. iii. This is the mechanism in most cause cases of neonatal isoerythrolysis and transfusion reactions. iv. When complement is fixed only to C 3, extravascular phagocytosis is facilitated, not intravascular lysis. b. Physical injury. i. Traumatic disruption of the membrane can occur from the shearing effect of fibrin formed intravascularly. ii. Since in most cases the fibrin is formed in small blood vessels, this type of anemia is called microangiopathic anemia. iii. Causes: 1. DIC 2. Coagulation 3. Vasculitis 4. Heartworm disease iv. Shistocytes or keratocytes are abnormally shaped erythrocytes whose membranes have been altered by trauma. Their presence suggests this mechanism as the cause of the anemia. c. Oxidative injury. i. Oxidants affect the erythrocyte in three ways: 1. Denaturation of Hb with Heinz body or eccentrocyte formation. 2. Oxidation of membrane proteins. 3. Oxidation of hemoglobin iron with the formation of methemoglobin. This interferes with oxygen transport but does not cause anemia. ii. iii. iv. Cause membrane damage sufficient for hemoglobin to escape the cell. If intravascular lysis does not occur, the cells are removed by phagocytosis. The erythrocyte is protected from daily exposure to oxidants via two pathways: 1. Reduced glutathione, which neutralizes oxidants, is produced and maintained in

10 Page 10 of 15 the reduced state by the hexosemonophosphate pathway. 2. Iron is maintained in the reduced state by methemoglobin reductase and methemoglobin accumulation is minimized. v. In most cases, the causative oxidant is drug or diet derived. It or its intermediates are directly oxidative or interfere with formation of reduced glutathione. vi. Heinz bodies or eccentrocytes suggest this mechanism. d. Osmotic lysis. i. Membrane alterations occur that allow excess water to be drawn into the normally hypertonic cell and lysis occurs. ii. Hypotonic intravenous fluids have the same effect. e. Other membrane alterations. i. Castor beans-ricin. Causes direct lysis ii. Snake venoms iii. Bacterial toxins iv. Parasites (Babesia) 2. Clinical and laboratory characteristics of intravascular hemolytic anemia. a. Most cases present as peracute or acute episodes. b. History may reveal exposure to causative drugs or plants, recent transfusion of blood, or recent ingestion of colostrum. c. A regenerative response occurs, but it may not be evident in early stages. d. Hemoglobinemia is the principal feature of intravascular hemolysis. i. Red discoloration of plasma ii. Increased MCHC e. Hemoglobinuria may occur hours following hemolysis if the concentration of free hemoglobin saturates the available haptoglobin and hemopexin and exceeds the capacity of the renal tubular epithelial cells to absorb and metabolize any hemoglobin that passes the glomerular filter.

11 Page 11 of 15 f. Hemosiderinuria occurs if there is sufficient renal tubular epithelial cell absorption and metabolism to form detectable hemosiderin. g. Hyperbilirubinemia i. Bilirubin is not formed until 8-10 hours after the onset of the hemolytic episode. ii. Hyperbilirubinemia will occur if bilirubin formation is of sufficient magnitude to exceed the capacity of the liver to remove it from iii. plasma, and conjugate and excrete it into bile. Unconjugated bilirubin is the predominant form early. Conjugated bilirubin becomes more prominent with time and occasionally may be the major form present. h. Additional laboratory findings may include schistocytes, keratocytes, Heinz bodies, eccentrocytes, erythrocytic parasites, positive Coombs' test. ANEMIA FROM REDUCED OR DEFECTIVE ERYTHROPOIESIS s caused by reduced or defective erythropoiesis are non-regenerative and characte4rized by an abnormal bone marrow that cannot maintain effective erythropoiesis. The clinic course is usually long and the onset insidious. I. General considerations. A. Mechanisms: 1. Proper function of the bone marrow in the maintenance of normal erythrocytic mass requires adequate: a. Precursor cells b. Nutrients (iron and B vitamins) c. Stimulation (erythropoietin) 2. Bone marrow failure may be primary (intramarrow disease resulting in inadequate stem and progenitor cells) o secondary from extramarrow causes (nutrient or erythropoietin lack). 3. Bone marrow failure may be selective for the erythroid series or may also affect the other cell lines. B. Bone marrow response 1. When the number of precursor cells or erythropoietic stimulation is inadequate, the erythroid marrow is hypocellular.

12 Page 12 of Maturation abnormalities which characterize the nutritional deficiencies, are associated with a Hypercellular marrow and ineffective erythropoiesis. (Usually the failure of the erythrocytes produced to be delivered to the blood). Usually abnormal cells are seen--microcytes, macrocytes. 3. All degrees of bone marrow failure can occur, from complete aplasia to a suboptimal response of the erythroid marrow following hemorrhage or hemolysis. II. Differentiation of anemias caused by reduced or defective erythropoiesis. An attempt to classify the pathogenic mechanism based on erythrocyte morphology, blood neutrophil and platelet numbers, and bone marrow cellularity. A. Normocytic, normochromic anemia; normal or increased neutrophil and platelet numbers; increased M/E ratio caused by hypocellular erythroid marrow. 1. of erythropoietin lack. a. Chronic renal disease. i. proportional to severity of the uremia. ii. Causes 1. Erythropoietin deficiency caused by destruction of the secreting peritubular cells. 2. Hemolysis caused by factors in uremic plasma. 3. Gastrointestinal hemorrhage from abnormal platelet function and vascular lesions. 4. Inhibitors of erythropoiesis in uremic plasma. b. Endocrinopathies i. Cushings ii. Hypoandrogenism iii. Hypopituitarism 2. of chronic disorders (ACD) a. Occurs in chronic infectious, inflammatory, or neoplastic disorders. b. Cytokines involved with the inflammatory process initiate the anemia. i. Inhibits erythroid progenitor cells ii. Inhibits erythropoietin production iii. Impairment of iron release from macrophages

13 Page 13 of 15 c. Erythrocyte life span reduced. d. Laboratory findings include: i. Low serum iron ii. Low total iron binding capacity iii. Increased bone marrow macrophage iron iv. Mild-moderate anemia that is usually nonprogressive v. Microcytosis and hypochromia rarely occur 3. Feline leukemia virus (FeLV)_associated nonregenerative anemia a. Erythroid stem and progenitor cells are selectively killed by FeLV. b. May be macrocytic. 4. Pure red cell aplasia a. Characterized by a selective loss of erythroid precursors in the bone marrow. b. Thought to be immune mediated. 5. Unknown mechanisms a. Liver disease b. Vitamin E deficiency B. Normocytic, normochromic anemia; neutropenia and/or thrombocytopenia; M/E ratio is difficult to determine because of hypocellularity. 1. Aplastic anemia a. Disease of the multipotential stem cell or bone marrow microenvironment leading to a pancytopenia. b. Concomitant deficiency in erythropoiesis, granulopoiesis, and anemia because of the shorter life spans of the cells. c. Causes i. Drugs, chemicals, plants ii. Irradiation iii. Cytotoxic T cells or antibody iv. Infectious agents 2. Myelophthisic anemia a. The bone marrow is physically replaced by an abnormal proliferation of cells. i. Myeloproliferative disorders-leukemias ii. iii. iv. v. Metastatic cancer 3. caused by infectious agents a. Ehrlichiosis b. FeLV c. Canine Parvo Myelofibrosis Osteosclerosis Diffuse granulomatous osteomyelitis

14 Page 14 of 15 C. Microcytic, hypochromic anemia; variable neutrophil and platelet number; usually a hypercellular marrow with a variable M/E ratio. 1. Iron deficiency a. Chronic hemorrhage b. Dietary deficiency, especially in young milk-fed animals c. Ineffective erythropoiesis early; hypoplastic later d. Laboratory findings: i. Low serum iron ii. Variable iron-binding capacity iii. Microcytosis iv. Hypochromasia v. Poikilocytes vi. Hypercellular bone marrow with disproportionate number of late rubricytes and metarubricytes. Because the critical level of hemoglobin necessary to stop cell division is not reached and an extra division occurs. 2. Pyridoxine deficiency. This vitamin is a cofactor in heme synthesis and a deficiency leads to a failure to utilize iron. 3. Copper deficiency. Copper-containing ceruloplasmin is important in iron absorption and transfer between gut, macrophages, and transferrin. D. Macrocytic, normochromic anemia; variable neutrophil and platelet number; M/E ratio usually low because of hypercellular erythroid marrow. 1. Ruminants on cobalt-deficient or molybdenum-rich pastures. 2. Vitamin BB12 and folic acid deficiency. 3. Erythemic myelosis or erythroleukemia. 4. FeLV infection. 5. POLYCYTHEMIA Polycythemia is an increase in the Hct, RBC count, and Hb concentration. The opposite of anemia. I. Spurious or relative polycythemia. The total RBC mass is normal. A. Dehydration 1. Decrease in plasma volume results in a relative increase in the Hct, RBC count, Hb concentration, and plasma protein concentrations. 2. Determination of dehydration is based on physical examination and not laboratory tests. 3. Mechanisms:

15 Page 15 of 15 a. Water loss caused by vomiting, diarrhea, excessive diuresis, water deprivation, perspiration, or febrile dehydration. b. Internal fluid loss in shock via increased vascular permeability. 4. Daily fluctuations of the Hct of 2-5% in sick animals are usually the consequence of hydration changes. B. Redistribution of erythrocytes. 1. Excitement causes epinephrine release and splenic contraction delivers high-hct blood into the general circulation. 2. Common in the horse and cat. II. Absolute polycythemia. The total RBC mass is increased because of increased erythropoiesis. Plasma volume and plasma protein concentration are normal. A. Primary absolute polycythemia (polycythemia vera) is a myeloproliferative disorder of stem cells. 1. Erythropoietin levels are normal or decreased. 2. Thrombocytosis and leukocytosis occasionally accompany the erythrocytosis. B. Secondary absolute polycythemia is caused by increased Erythropoietin secretion. 1. Appropriate compensatory epo secretion occurs during chronic hypoxia. a. High altitude b. Chronic pulmonary disease c. Cardiovascular abnormalities with right to left shunting 2. Inappropriate epo secretion occurs in some cases of hydronephrosis or renal cysts, and epo-secreting tumors.

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