A CASE ORIENTED APPROACH Alan H. Rebar, DVM, PhD, DACVP

RED CELL RESPONSES IN DISEASE: A CASE ORIENTED APPROACH Alan H. Rebar, DVM, PhD, DACVP CLINICAL PATHOLOGY Introduction Anemia is one of the most common disease syndromes in domestic animals and may be either a primary process or secondary to another underlying problem. By definition, anemia is reduction in oxygen-carrying capacity of the blood and is characterized by decreases in hematocrit, red blood cell count, and hemoglobin. All anemic animals have some degree of pallor, but the presence or absence and type of other clinical signs is highly variable and is dependent on the severity of the anemia, the rapidity of onset, the state of activity of the patient, and the actual cause of disease. Here we review the approach to the diagnosis and differentiation of the causes of anemia. Only after the cause of anemia has been defined can appropriate therapy be instituted and an accurate prognosis of outcome be made. Pathophysiology of Anemia The effects of anemia are all related to reduced tissue oxygenation. Tissue hypoxia results when oxygen pressure in the microcirculatory system is inadequate to provide cells distant from the capillaries with enough oxygen for metabolism. Tissue hypoxia activates a series of physiologic compensatory mechanisms, which serve to maintain tissue oxygen levels as near normal as possible. The compensatory mechanisms that are activated are outlined below. Increased Oxygen Delivery (Reduced Red Cell Oxygen Affinity) One of the most rapid physiologic adjustments to the development of anemia (tissue hypoxia) is a shift of the oxygen-hemoglobin dissociation curve to the right. Such a shift reduces the affinity of hemoglobin for oxygen, so that a higher proportion of oxygen carried by the hemoglobin may be released to the tissues. Two mechanisms are involved in shifting the oxygen-hemoglobin dissociation curve to the right. First, whenever tissue hypoxia occurs, hypoxic cells produce more and more lactic acid via anaerobic glycolysis. The net result is a lowering of microenvironmental ph (increased acidity), and such acidity favors deoxygenation. This is known as the Bohr effect. The second mechanism is probably even more influential. Whenever anemia develops, increased levels of 2, 3 diphosphoglycerate (2, 3 DPG) are formed in red cells. The reason for increased 2, 3 DPG formation is unclear, but whatever the cause, the 2, 3 DPG produced binds to reduced hemoglobin and stabilizes it in its low oxygen affinity state. When hemoglobin is maintained in this configuration, oxygen is more readily released to the tissues. Redistribution of Blood Flow Another rapid compensatory response to anemia is the shunting of blood away from tissues with low oxygen demand (e.g., skin and kidney) to tissues with high oxygen demand (e.g., brain). Redistribution is accomplished by selective vasconstriction. The effectiveness of this mechanism is transitory and of limited value. Increased Cardiac Output Shifts in both hemoglobin dissociation curves and regional blood flow can occur quickly when tissue hypoxia develops. In cases of mild anemia, these adaptations may be adequate. However, more severe anemias require more effective compensatory changes. One such compensatory response is increased cardiac output. Increased cardiac output serves to increase tissue oxygenation by increasing the continuous supply of welloxygenated blood to hypoxic tissues. Unfortunately, increased cardiac output cannot be maintained indefinitely without deleterious effects. If left unchecked, increased cardiac output can result in cardiac failure, with all of its well-known attendant clinical signs. Expanded Red Cell Production The most appropriate compensatory response that can be made to anemia is expanded red cell production by the bone marrow such that the PCV is returned to normal. Tissue hypoxia causes the release of the erythropoietic hormone erythropoietin, most likely by the kidney. Erythropoietin is carried by the blood to the bone marrow, where it exerts its primary stimulatory effect on the unipotential red blood cell stem cell, thereby increasing red cell mass. 89

While this mechanism is the most appropriate physiologic response to anemia, it is also the slowest. Whereas tissue hypoxia has been known to cause increased levels of circulating erythropoietin within 4 6 hours, increases in circulating red cell numbers will not be seen until 4 to 6 days later. During this lag period, marrow red cell production is being expanded, but the new red cells are not yet differentiated enough for release. Clinical Signs of Anemia All of the above compensatory adjustments serve to allow the animal to cope with the problem of reduced oxygencarrying capacity of the blood (anemia). If successful, anemia may occur and resolve without clinical signs. When unsuccessful, anemia becomes a clinical syndrome. The principal presenting clinical sign of anemia is pallor. Pallor is primarily the result of reduced circulating red cell mass; however, shunting of blood away from superficial tissues is also contributory. As mentioned earlier, the other clinical features of a given anemia depend on a variety of factors such as rapidity of onset, degree of anemia, and underlying cause. As a general rule, rapidly developing (acute) anemias pose a greater immediate risk to the patient and are associated with more severe clinical signs at a higher hematocrit then gradually developing (chronic) anemias. The reason for this is obvious; in rapid-onset anemias the compensatory mechanisms are less effective than in gradually developing anemias. Consequently, the clinical signs of acute anemias are primarily those associated with sudden hypoxia and/or sudden reductions in red cell mass. Common signs include collapse, hyperventilation, hypovolemic shock, and evidence of acute renal shutdown. In cases of acute hemolysis, hepatomegaly, splenomegaly, and jaundice can also occur. In contrast, in gradually developing anemias signs are more subtle. Hematocrits may drop to 10% or less before the owner becomes suspicious of a disease problem. The presenting sign is often increasing lethargy or inactivity. Other common signs are tachycardia and the clinical features of heart failure. Often the animal will be clinically normal until excited or stressed, when rapid breathing, respiratory distress, cyanosis, and even life-threatening collapse may all develop. Because of the severity of signs that may be precipitated by stress, these patients must be handled with great care. Diagnosis and Differentiation of the Causes of Anemia Once anemia has been recognized either as a result of laboratory evidence of reduced red cell mass or because of the presence of clinical signs, the clinician must seek to define the underlying cause, that is, to classify the anemia in a way that is useful in determining treatment. Numerous classification schemes have been developed historically. The classification scheme of greatest clinic application is known as the pathophysiologic classification scheme and attempts to divide anemias into two major subgroups on the basis of bone marrow responsiveness. A responsive anemia is one in which the bone marrow responds appropriately with increased red cell production and the release of normal young red cells into circulation. There are two major categories of responsive anemias: blood loss anemias and hemolytic anemias. Nonresponsive anemias are those in which the marrow does not respond appropriately and young red cells are not released in adequate numbers into the circulation. There are also two major categories of nonresponsive anemias: those in which marrow red cell production simply is not increased or is reduced (hypoproliferative anemias) and those in which there is marrow red cell hyperplasia but the red cells produced are abnormal and not released into circulation (the maturation defect anemias). Each of these categories will be discussed in greater detail later. Responsive versus Nonresponsive Anemia In order to classify an anemia as responsive or nonresponsive, the clinician must rely on the CBC red cell data, in particular the findings on the peripheral blood film. In all species other than the horse, the sine qua non of responsive anemias is the presence of increased numbers of young non-nucleated red blood cells in circulation. On the routine Wright s or Wright s-giemsa stained peripheral blood film, young red cells are larger than normal and are bluish because their cytoplasm, unlike that of adult RBCs, still contains ribonucleic acid and lacks a full complement of hemoglobin. Because of their tinctorial properties, these young red blood cells are called polychromatophils, and when many are present the blood smear is said to exhibit increased polychromasia. 90

Similarly, because polychromatophils are larger than adult cells, when large numbers of polychromatophils are present on the blood film there is obvious variability in red cell size or anisocytosis. Even in highly responsive anemias, there is no polychromasia in horses. Responsiveness must be assessed by bone marrow evaluation. The absolute number or percentage of immature red cells in circulation can be determined by doing a reticulocyte count. A reticulocyte smear is made by mixing a small amount of EDTA-anticoagulated blood with an equal volume of a vital stain such as new methylene blue. The mixture is allowed to incubate for approximately thirty minutes and then an air-dried smear is made. Vital stains precipitate the RNA in the cytoplasm of young red cells as a royal blue reticulum, hence the name reticulocyte. In general, the reticulocytes correlate with the polychromatophils on Wright s stained smears, but the reticulocyte smear makes the young RBCs easier to recognize, and a count based on this kind of preparation is therefore more accurate. A reticulocyte count is indicated whenever anemia is present and there is a suggestion of increased polychromasia on the routine blood film. A reticulocyte count is determined by counting the number of reticulocytes/1,000 RBCs and dividing by 10 to convert to a percentage. In dogs, pigs, and ruminants all cells containing reticulum are counted. However, in cats three forms of reticulocytes are seen. Those cells with only scattered focal precipitates of reticulum are known as punctate reticulocytes. Those cells with an abundant network of precipitated reticulum (comparable to what is seen in dogs) are known as aggregate reticulocytes. Cells with intermediate amounts of reticulum are known as intermediate reticulocytes. In cats, only aggregate reticulocytes are included in the reticulocyte count. Anemic animals with an increased absolute reticulocyte count (determined by multiplying the reticulocyte percentage times the total RBC count) are said to have responsive anemias, either from blood loss or hemolysis. Anemic animals without increased reticulocyte counts have nonresponsive anemias. Generally, hemolytic anemias are more responsive than blood loss anemias; it has been suggested that absolute reticulocyte counts of greater than three times the upper end of the reference range are suggestive of hemolysis. While the sine qua non of responsive anemias is reticulocytosis, there are other peripheral blood features of bone marrow responsiveness. On the peripheral blood film, increased numbers of red cells with Howell-Jolly bodies (nuclear remnants) and increased numbers of nucleated red cells may also be seen. However, it is important to realize that the number of nucleated red cells in circulation should be limited and in all cases should be significantly less than the number of reticulocytes. A peripheral blood film with large numbers of nucleated red cells and limited numbers of polychromatophils (reticulocytes) is not indicative of a responsive anemia; rather, such a finding is termed an inappropriate nucleated red cell response and indicates bone marrow stromal damage. Inappropriate nucleated red cell responses will be discussed in detail later (see cytoplasmic maturation defect anemias). The morphologic findings on the peripheral blood film also may affect the other red cell data in the CBC, in particular, the red cell indices. The red cell indices include mean cell volume (MCV), mean cell hemoglobin (MCH), and mean cell hemoglobin concentration (MCHC). The clinically important indices are MCV and MCHC. Normal values for these indices have been established for each species. The normal MCHC for all species is 31 36%; normal MCV for dogs is 60 75 fl and for cats, 40 55 fl. Increases in MCV indicate the presence of large cells (macrocytosis); decreases in MCV indicate the presence of small cells (microcytosis). Decreases in MCHC indicate reductions in hemoglobin concentration within individual cells (hypochromasia); elevations in MCHC are not biologically possible and indicate either laboratory error or in vivo or in vitro hemolysis. If the CBC has been done by a reference laboratory, the MCV and MCHC will almost always be routinely reported. If CBC data have been generated in house, then MCV and MCHC can be computed from the hematocrit, hemoglobin, and red cell count. It is important to realize that MCV and MCHC represent mean values; therefore, large numbers of cells must be abnormal before MCV and MCHC are affected. Because responsive anemias have increased numbers of large young cells without a full complement of hemoglobin, they are often macrocytic hypochromic anemias as reflected by elevations in MCV and decreases in MCHC. In contrast, the majority of the nonresponsive anemias, because they lack reticulocytosis, are normocytic (normal MCV) and normochromic (normal MCHC). Exceptions are found in the occasional nonresponsive maturation defect anemia. Nuclear maturation-defect anemias may be macrocytic and normochromic. Cytoplasmic maturation-defect 91

anemias may be microcytic (small cell size) and hypochromic in character. These features will be addressed in greater detail when the specific causes and the pathophysiology of the maturation defect anemias are considered. To this point, discussion of the diagnosis of anemia has focused on the differentiation between responsive and nonresponsive anemias. It is important to realize that this separation is at least somewhat arbitrary and that whether an anemia is classified as responsive or nonresponsive is in part dependent on when in the clinical course the anemia is observed. In addition, some anemias will appear responsive based on peripheral blood morphology but will be nonresponsive when judged by the ultimate criterion: eventual return of circulating red cells mass to normal. The remainder of this review addresses the subcategories of responsive and nonresponsive anemias. The Responsive Anemias Blood Loss Blood loss anemia is best defined as an anemia where red cell lifespan is normal but red cells are lost from the body. In response, erythropoietin levels increase, thereby stimulating increased marrow red cell production. However, even under the best conditions, the first new reticulocytes do not appear in the circulation until approximately 72 hours after hemorrhage. The reticulocyte response reaches its full expression from 4 to 7 days following the hemorrhagic episode. If only a single hemorrhagic episode has occurred and marrow response is adequate, hematocrit should have returned to normal within two weeks. The Hemolytic Anemias (See notes in these proceedings entitled: How I Diagnose and Differentiate Hemolytic Anemias ) The Nonresponsive Anemias Nonresponsive Anemias with Hypoproliferative Erythroid Marrows Hypoproliferative Anemia with Granulocytic Hyperplasia: Anemia of Inflammatory Disease The most common anemia of animals is the anemia of inflammatory disease. Unfortunately, the morphologic features of this anemia are quite bland. The anemia is mildly to moderately severe (hematocrits ranging from 20 35% in most species) and normocytic normochromic. This anemia should be suspected whenever there is a decreased hematocrit in association with an inflammatory leukogram. Bone marrow smears will be characterized generally by erythroid hypoplasia; granulocytic hyperplasia; and increases in marrow iron, macrophages, and plasma cells. Hepatic and renal disease are commonly associated with hypoproliferative non-regenerative anemias. They often represent a special case of the anemias of inflammatory disease. The pathogenesis of these anemias is often quite complex, and the morphologic appearance in the peripheral blood is therefore somewhat variable. Anemias with Selective Erythroid Hypoplasia Anemias of this nature are relatively uncommon in animals as well as in humans. The anemias seen are generally mild to moderate, although they may be quite severe. Leukograms and platelet counts remain normal. The specific causes of selective erythroid marrow depression are often obscure; however, three major mechanisms are involved: 1) reduced oxygen utilization by peripheral tissues, 2) reduced stimulation of the red cell marrow as a result of reduced erythropoietin production, and 3) selective destruction of the red cell precursors by any of a variety of toxic mechanisms. Anemias with Generalized Marrow Hypoplasia Generalized marrow hypoplasia causes severe hypoproliferative non-regenerative anemias as well as leukopenias and thrombocytopenias in the peripheral blood (aplastic anemia). Generalized marrow hypoplasia is caused by two mechanisms: 1) myelophthisis (replacement of normal marrow by abnormal elements) and 2) toxicity to the marrow affecting all cell lines. 92

1) Myelophthisic Anemias Myelophthisic anemias may be caused by both neoplastic and non-neoplastic diseases. The major neoplastic causes are the hematopoietic and lymphoid neoplasms. Principal among these are lymphosarcoma, granulocytic leukemia, erythremic myelosis, erythroleukemia, plasma cell tumors, and monocytic leukemia. These neoplasms may totally fill the marrow with malignant cells, thus eliminating space for normal marrow cell development. On rare occasions, disseminated neoplasms of other origins may also invade and replace the marrow, resulting in myelophthisis. The principal non-neoplastic disease causing myelophthisic anemia is myelofibrosis. Myelofibrosis is filling of normal marrow space by non-neoplastic fibrous connective tissue. The underlying causes of myelofibrosis have not been clearly defined but are probably multiple. Myelofibrosis is the predictable endpoint of severe marrow stromal damage and in this setting represents scarring. Myelofibrosis of this nature has been documented following exposure of the bone marrow to such things as ionizing radiation, a variety of toxic chemicals and drugs, and toxic levels of estrogen. Estrogen toxicity with marrow effects is most commonly observed in dogs and ferrets. Myelofibrosis possibly also can occur as a primary disease. Such a pathogenesis has been postulated in some cases of myelofibrosis associated with feline leukemia virus infection in cats. Regardless of the cause, peripheral blood findings in myelophthisic anemias are similar. The anemia is generally quite severe, and there is accompanying leukopenia. Platelet numbers are variable but may even be increased. There is generally marked poikilocytosis with schizocytes and the presence of dacryocytes (tear-drop-shaped erythrocytes). Diagnosis can only be confirmed by bone marrow evaluation, and when myelophthisis is suspected, core biopsies and histopathology are recommended. In neoplastic myelophthisis, aspirates are generally very cellular and contain a monotonous population of neoplastic cells. However, if the marrow is densely packed by neoplastic cells, dry taps (acellular aspirates) may be obtained. Similarly, most cases of myelofibrosis yield dry marrow taps on aspiration. It is these acellular aspirates that necessitate core biopsy evaluation. 2) Anemias Associated with General Marrow Cytotoxicity Generalized marrow cytotoxicity can be caused by a variety of both infectious and noninfectious agents. As mentioned above, marrow cytotoxicity is often an early stage in the continuum ending in myelofibrosis. Infectious causes of generalized marrow cytotoxicity include feline leukemia viral disease in cats and ehrlichiosis in dogs. Noninfectious causes include numerous drugs such as cancer chemotherapeutic agents like adriamycin, ionizing radiation, chemicals such as benzene and estrogen, or diethylstilbestrol. Clinically, generalized marrow cytotoxicity causes progressive pancytopenia. Presenting signs reflect the severity of the various cell deficits at the time of diagnosis. When thrombocytopenia is the most severe problem, bleeding is generally the presenting problem. If granulocytopenia is extreme, secondary infection is often the major clinical problem. When anemia is very marked, pale mucus membranes, lethargy, anorexia, pronounced respiratory patterns, etc., bring the animal to the veterinarian s attention. Acute stem cell destruction is more commonly associated with platelet or white cell signs; more gradual destruction of marrow elements leads to features of anemia. Diagnosis of generalized marrow toxicity depends on marrow aspiration and in some cases marrow core biopsy. Marrow findings, like clinical signs, vary with the stage of the disease and the nature of the stem cell destruction. Marrow aspirates taken within a few days of acute stem cell destruction contain evidence of marrow necrosis and/or cytotoxicity. Toxic changes in precursors include cytoplasmic basophilia and vacuolation, nuclear vacuolation, and cytonuclear dissociation in all cell lines. Numerous broken cells may be seen. Large numbers of macrophages containing phagocytized cellular debris may be present. Necrosis of marrow can be difficult to recognize in marrow aspirates but is characterized by increased amounts of basophilic background material (necrotic amorphous proteinaceous debris) and poorly staining cells with little nuclear and cytoplasmic detail. Bone marrow necrosis should be confirmed histologically with core biopsies. If the disease is more advanced when first recognized clinically, marrow aspirates often are very low in cellularity and difficult to evaluate because one is uncertain if the aspirate sample is truly representative of marrow cellularity. 93

When repeated hypocellular samples are obtained in animals with pancytopenia, aplastic anemia should be strongly suspected. However, confirmation requires core biopsy histopathology. After a diagnosis of aplastic anemia due to generalized marrow cytotoxicity has been established, an attempt to identify a specific cause should be made. A complete and detailed history of recent drug therapy and/or possible exposure to toxic material should be obtained. Infectious diseases such as feline leukemia and ehrlichiosis can be ruled out serologically. Nonresponsive Anemia with Hyperproliferative Bone Marrows (Maturation Ddefect Anemias) Nuclear Maturation Defect Anemias (Megaloblastic Anemias) Nuclear maturation defect anemias are relatively uncommon in dogs, but occur with some frequency in cats. Cattle suffering from cobalt deficiency (cobalt is an essential element for normal vitamin B-12 function) may also exhibit nuclear maturation defect anemia. The principal problem in these anemias is the presence of an acquired bone marrow defect in which precursor nuclei of all cell lines fail to mature and divide normally while cytoplasmic maturation proceeds unimpaired. The nuclear defect is a result of abnormal or reduced DNA synthesis. The bone marrow in these animals is hypercellular, and all cell lines are affected and are left shifted. In the red cell series this nuclear/cytoplasmic asynchrony and failure of division results in the formation of cells known as megaloblasts. Megaloblasts are large red cell precursors with immature, bizarre, pale staining nuclei, which contain irregular chromatin clumps. The cytoplasm of megaloblasts is clearly too hemoglobinized for the degree of nuclear maturation. Recognizing these cells in the marrow is the key to the proper diagnosis of the nuclear maturation defect anemias. While definitive diagnosis of the nuclear maturation defect anemias resides in the bone marrow, peripheral blood findings are generally suggestive that the problem is present. There is generally a mild nonresponsive anemia with normocytic normochromic to macrocytic normochromic red cell indices. Since all marrow cell lines are involved, there is also usually a mild leukopenia and thrombocytopenia. Peripheral smears generally show anisocytosis with occasional giant fully hemoglobinized red cells being present. Red cells containing bizarre and multiple nuclear fragments may also be seen. In more severe cases, occasional megaloblasts are observed on the smears. Cytoplasmic Maturation Defect Anemias The cytoplasmic maturation defect anemias are the inverse of the nuclear maturation defect anemias; in these anemias nuclear development of red cell precursors proceeds normally, but cytoplasmic development is arrested because of failure to form hemoglobin. Morphologically the result is a hypercellular red cell bone marrow with a build-up of very small metarubricytes with scant cytoplasm. In effect, red cell precursors continue to divide but are not released into circulation because they never acquire an adequate complement of hemoglobin. This evidence of increased erythropoiesis without release of reticulocytes into circulation is known as ineffective erythropoiesis. In humans, there are a large number of inherited and acquired cytoplasmic defect anemias; in dogs and cats the two of clinical importance are iron deficiency anemia and the anemia of lead poisoning. The anemia of lead poisoning merits special consideration. Lead interferes with the synthesis of hemoglobin at several points and consequently causes a true cytoplasmic maturation arrest anemia characterized by red cell marrow hyperplasia, ineffective erythropoiesis, and a normocytic normochromic nonresponsive peripheral blood red cell picture. Lead also is believed to cause marrow stromal damage, with resultant leakage of nucleated red blood cells into circulation. Consequently, lead poisoning causes an inappropriate nucleated red blood cell response in the peripheral blood the presence of large numbers of nucleated red blood cells in the absence of significant polychromasia. Nucleated red cells in the absence of polychromasia should never be interpreted as evidence of appropriate marrow responsiveness; rather, this finding is most commonly indicative (as in lead poisoning) of marrow stromal damage. 94