Symposium on Clinical Laboratory Medicine
The Differential Diagnosis of Anemia Gene P. Searcy, D.V.M.*
When anemia is discovered the veterinarian should pursue this sign of disease in an orderly stepwise manner. This will involve the laboratory, but most progress and self-satisfaction will be achieved from an intimate combination of clinical findings and laboratory data. The scheme which follows will aid in understanding the pathogenesis and often the etiology of anemia. By adhering to such a scheme, the clinician may discover underlying diseases, manage the case more intelligently, and offer a more accurate prognosis. A pathophysiological approach to anemia (Table 1) is based on _.hether the decrease in erythrocyte numbers is associated with effective erythropoiesis (i.e., red cell loss due to hemorrhage or hemolysis) or impaired erythropoiesis. Clinical findings usually differ between anemias due to blood loss and impaired red cell production. Animals presented with acute onset of weakness and depression have usually experienced erythrocyte loss. Dogs and cats which have remained bright and alert for several weeks despite deereased exercise tolerance have probably suffered marrow failure. Packed cell volume or hemoglobin determination estimates erythro~"le numbers best. If one has access to an electronic cell counter, red cell counts may be obtained. However, errors are possible with these instruments, especially when counting feline erythrocytes which are significantly smaller than those of man. Evaluation of a properly prepared, well stained blood smear ith a good binocular microscope is an essential component of hematology. Scanning the smear at low magnification allows examination of the feather edge, the distribution of erythrocytes, platelets, and leukocytes, and estimation of the leukocyte count. Examination of erythrocytes with the oil immersion objective permits an assessment of erythropoietic rate which in turn characterizes the pathogenesis of the anemia. Hemorrhage or hemolysis causes a compensatory shift of immature red cells from marrow to peripheral blood *Professor of Veterinary Pathology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatechewan, Canada Veterinary Clinics of North America-Vol. 6, Nov. 4, November 1976
567
568
GENE
P.
SEARCY
Table 1. Pathophysiological Classification of Anemia ANEMIA ASSOCIATED WITH EFFECTIVE ERYTHROPOIESIS
Hemorrhage blood sucking parasites gastrointestinal lesions urinary tract lesions neoplasms with bleeding into body cavity coagulation disorders Hemolysis Acquired hemolytic anemia infectious toxigenic autoimmune Congenital hemolytic anemia hemoglobinopathies membrane defects glycolytic defects ANEMIA AssociATED WITH IMPAIRED OR INsUFFICIENT ERYTHROPOIESIS
Nutritional Deficiencies rron Anemias of Chronic Disorders chronic infection liver disease kidney disease Bone Marrow Destruction irradiation chemical agents Myeloproliferative Disorders granulocytic leukemia plasma cell myeloma erythremic myelosis erythroleukemia myelofibrosis lymphosarcoma
within approximately three days. These cells are macrocytic and polychromatophilic with Wright's or Wright-Giemsa stains. Nucleated erythrocytes (metarubricytes) also indicate an enhanced erythropoietic rate when accompanied by polychromasia and anisocytosis. Reticulocytes result from precipitation of cytoplasmic RNA in erythrocytes by stains such as new methylene blue. Hemolysis results in a greater degree of reticulocytosis than does hemorrhage because the marrow has preference for iron derived from red cell destruction. Blood containing a significant number of immature erythrocytes has an elevated mean corpuscular volume (MeV) and a decreased mean corpuscular-hemogloblin concentration (McHc). The former results from the presence ofmacrocytes, the latter from incomplete hemoglobin synthesis in reticulocytes. Laber et al. 3 have noted that cats are erythropoietically less responsive than dogs. Feline reticulocyte morphology differs from that of the dog. New methylene blue staining reveals large numbers of erythrocytes
DIFFERENTIAL DIAGNOSIS OF ANEMIA
569
,,-ith punctate basophilia in some cats in contrast to the reticular network characteristic of canine reticulocytes. The latter are also found in cat blood and perhaps should be referred to as "shift reticulocytes," since it can be safely assumed that they represent recent enhanced erythropoietic activity in contrast to the punctate cell which may be released in response to a variety of circumstances. Immature erythrocytes appear in peripheral blood in response to red cell loss within approximately 72 hours. If hemorrhage or hemolysis has occurred within that period of time, marrow examination may be necessary to evaluate erythropoiesis. Neutrophilia and thrombocytosis often accompany anemias due to hemorrhage or hemolysis. These changes occur during the regenerative phase of the anemia after reticulocytes appear.
ANEMIAS DUE TO ERYTHROCYTE LOSS After determining that the anemia is of the "erythrocyte loss" type, the next step is differentiation of hemorrhage and hemolysis. This requires thorough clinical and laboratory patient evaluation. Aspiration and examination of body fluids may reveal hemorrhage into body ca,·ities. Urine and feces should be examined for the presence of blood, keeping in mind that meat diets may result in a positive fecal occult blood test. Hemolysis may result in icterus, hemoglobinemia, and hemoglobinuria. Hemoglobin in both plasma and urine indicates intravascular hemolysis, whereas hyperbilirubinemia alone suggests reticulohistiocytic erythrocyte destruction. Icterus may only accompany hemolytic crises in dogs and cats as they are capable of rapid conjugation and excretion of bilirubin. Hemorrhagic Anemias
Parasites. Ectoparasites and endoparasites are capable of causing significant anemia and death in kittens and pups, for example, flea infestations and canine hookworms. Parasitic anemias, when chronic, become complicated by secondary iron deficiency which severely compromises compensatory erythropoiesis. Gastrointestinal Lesions. Ulceration of gastric or intestinal mucosa may result in hemorrhagic anemia; however, the incidence in dogs and cats is low. Urinary Tract Lesions. Hemorrhage may result from acute inflammatory conditions or urolithiasis, but anemia is rare. Occasionally chronic hematuria is encountered in dogs in which the etiology and pathogenesis are obscure. Spontaneous remission usually occurs in a matter of weeks.
570
GENE
P.
SEARCY
Neoplasms and Hemorrhagic Anemia. Neoplasms may become eroded and bleed, especially when located on surfaces subject to trauma. Vascular neoplasms are prone to hemorrhage. The best example, splenic hemangiosarcomas, occur in older dogs and may be characterized by periodic episodes of hemorrhagic anemia. The presence of anemia with reticulocytosis and hemoperitoneum suggests a splenic hemangiosarcoma. Coagulation Disorders. For a discussion of anemia resulting from coagulation disorders please refer to the article on "The Diagnosis of Hemostatic Disorders" in this symposium. Acquired Hemolytic Anemias
Hemolysis Caused by Infectious Agents. Feline infectious anemia is usually an acute hemolytic disease with all of the anticipated clinical findings. Blood film evaluation reveals polychromasia, anisocytosis, and Hemobartonella felis organisms. The question which must always be asked is whether the anemia is solely the result of hemobartonellosis or is the result of some other disease with organisms present as opportunists. Bone marrow injury is often the initial reason for anemia in secondary hemobartonellosis. Characteristic features of secondary hemobartonellosis are: ( l) organisms are usually not as numerous as in primary hemobartonellosis; (2) reticulocytosis is usually not consistent with the degree of anemia; (3) neutrophilia does not occur, as would be anticipated in response to hemolysis; in fact neutropenia is common; (4) the anemia may be severe with the packed cell volume perhaps ranging from 6 to 12 per cent; cats which suffer a hemolytic crisis will usually succumb; (5) abnormal nucleated cells may be visible, consistent with a myeloproliferative disorder. When any or all of these features suggest that Hemobartonella organisms may not be the sole cause of the anemia, marrow examination is indicated. If doubt remains, the cat should be treated for Hemobartonella and the response to therapy may provide further insight into the pathogenesis of the disease. Hemobartonella canis should be considered as a possible significant sequela to splenectomy, and postoperative search for the organism is indicated. Toxigenic Hemolytic Anemia. The presence of Heinz bodies on a blood film aids in the diagnosis of toxigenic hemolytic anemias. Heinz bodies are precipitated globin resulting from oxidative stresses which have overloaded the cell's protective mechanisms. The reader is referred elsewhere for a detailed description of Heinz bodies in Wright, Wright-Giemsa, or new methylene blue stained blood smears. 8 The principal cause of Heinz body hemolysis in cats has been exposure to methylene blue in urinary antiseptics. 8 Recent reports of onion toxicity in dogs indicate the necessity of keeping this in mind when confronted by Heinz-body hemolytic anemia. The dog described by
571
DIFFERENTIAL DIAGNOSIS OF ANEMIA
Spice 10 ingested dehydrated onions in the form of an onion souffle, and thus acquired a significant amount of the toxic factor N-propyl disulfide. Therapy should be supportive to allow excretion or degradation of the toxin. Autoimmune Hemolytic Anemia. Antibody-mediated hemolysis is common in dogs. It, and thrombocytopenia, are the two most prominent signs of systemic lupus erythematosus, a syndrome which also encompasses glomerulitis, synovitis, and thyroiditis. Since these latter diseases are less overt, it is not clear as to how often they accompany hemolysis or thrombocytopenia. A review of 10 cases of autoimmune hemolysis from our clinic revealed one instance of concurrent thrombocytopenia. In addition to elucidating the clinical findings and pathology of this disease, Lewis and coworkers 4 have demonstrated a virus in a colony of dogs with a high incidence of systemic lupus erythematosus. Erythrocyte destruction results from attachment of antibody to the membrane with loss of that portion by intravascular fragmentation or phagocytosis. The normal component of hemoglobin is then enclosed by a decreased amount of membrane, and disc-sphere transformation results. The spheroidal erythrocyte, because of impaired deformability, is then susceptible to retention and eventual destruction within the reticulohistiocytic system. This phenomenon is unlikely to result in hemo-
Table 2. Autoimmune Hemolytic Anemia in a Dog Feb. 23, 1973 *Mar. 9, 1973 Mar. 15, 1973 Apr. 14, 1973 tDec. 31, 1973 Hgb (g/dl) PC\'(%) RBC (X 10 6/ftl)
"cv (fl)
"CHC (g/dl) Reticulocytes (%) .\nisocytosis Spherocytes Leptocytes Polvchromasia Ho;,·ell-J oily bodies Platelets!ftl WBC/ftl '\eutrophils Band neutrophils \letamyelocytes Eosinophils b·mphocytes \lonocytes Disintegrated cells "etarubricytes Protein (g/dl) L:E cells Coombs' test
6.5 21.5 2.1 102.4 30.2 13
5.1 12.5 1.7 73.5 40.8 40
8.5 26.5 3.2 82.8 32.8 8.0
0
0 0
++++ ++
++++ ++
++++ +
++++ +
0
Increased 19,200 10,176 1,536 192 192 2,880 960 0 3,264 6.6 negative positive
Increased 47,700 34,254 6,228 1,038 0 519 5,709 0 4,152 7.9
++ ++
0 Increased 18,500 15,170 370 0 0 370 1,480 0 1,110 7.6
14.6 42 6.5 64.6 34.8 0 0 0 0 0 0 Normal 7,800 5,070 0 0 390 2,028 234 78 0 7.3
3.3 7.0 0.9 77.8 47.1 16
++++ +++ + ++++ +
404,000 30,700 19,034 3,991 921 0 2,456 2,149 0 2,149 10.0
*:VIarch 7, 1973-15 mg prednisone administered twice daily. edication was discontinued on March 20. +Hemoglobinemia and ghost erythrocytes evident. Dog died on examination table.
572
GENE
P.
SEARCY
globinemia; however, it does occur in some dogs (Table 2) and probably reflects membrane lysis due to complement involvement in the antigenantibody reaction. Theories explaining antibody formation to autologous antigens have been reviewed by Scott et al. 9 If further research confirms involvement of an infectious agent, a clearer understanding of antibody synthesis will be possible. Most dogs are presented with a hemolytic crisis of two to four days' duration and they exhibit weakness, depression, tachycardia, and perhaps fever. Icterus and occasionally hemoglobinuria are preserk Occasionally erythrocyte agglutination will be visible in freshly draWn blood within the EDT A tube or within a drop of blood. Hematology usually reveals anemia, reticulocytosis, spherocytosis, and neutrophilia. The total leukocyte count may be as high as 60,000 cells/1-.d. If the dog is presented within eight hours of the onset of hemolysis, hematology shows only anemia and spherocytosis. A positive Coombs' test confirms the presence of globulin on the erythrocyte membrane. If the test is negative, but all clinical and laboratory findings support a diagnosis of autoimmune hemolysis, therapy should be initiated. In our experience, Coombs' tests may be negative following steroid therapy. Commercial rabbit anti-dog globulin sera are available; however, complement must be inactivated and nonspecific agglutinins removed by absorption with normal canine erythrocytes. Corticosteroids are indicated because they stabilize cell membranes, which probably helps to decrease hemolytic rate. They also impair antibody synthesis and stimulate erythropoiesis. Prednisolone is recommended, 1.5 mg/kg twice daily. Response to therapy should be carefully monitored because the dosage may have to be increased for some dogs. When the packed cell volume approaches 30 per cent, the dosage should be decreased over a period of weeks until discontinued. Relapses occur and owners should be warned of this possibility. The dog described in Table 2 died of a hemolytic crisis approximately eight months after cessation of therapy. Although autoimmune hemolytic anemia has been described in cats, the incidence is lower than in dogs. The disease is more difficult to diagnose in cats because spherocytes are difficult to recognize. In addition, some commercial rabbit anti-cat sera cannot be purified to a point where normal feline erythrocytes will not agglutinate. Congenital Hemolytic Anemias Congenital hemolytic anemias may be broadly classified as hemoglobinopathies, membrane defects, and glycolytic defects. To date, one enzymopathy resulting in impaired erythrocyte glucose metabolism has been described in animals. 7 Glucose, the principal substrate for energy metabolism, is transported across the erythrocyte membrane as required. Approximately 90 per cent is degraded in the EmbdenMeyerhoff pathway to yield 2 moles of A TP per mole of glucose con-
DIFFERENTIAL DIAGNOSIS OF ANEMIA
573
sumed. Among others, A TP is responsible for maintenance of cation equilibrium, deformability, and membrane integrity. When there is malfunction of an enzyme involved in glycolysis, A TP generation is impaired and premature red cell destruction occurs. Deficiency of pyruvate kinase resulting in impaired red cell glycolysis is the etiology of congenital hemolytic anemia in Basenji and beagle dogs. Affected dogs are usually presented to veterinarians at six to twelve months of age because of reduced exercise tolerance and reduced growth rate. Clinical examination reveals pale mucous membranes and splenomegaly. Hematology reveals anemia with packed cell volume ,-alues ranging from 15 to 30 per cent and an intense reticulocytosis of up to 50 per cent. As dogs age, the anemia progressively worsens and reticulocytes decrease. The impairment of erythropoiesis results from progressive myelofibrosis and sclerosis. The pathogenesis of these alterations in bone marrow is not clear, but they are probably responsible for the death of affected dogs 3 to 5 years of age.
ANEMIA AS A CONSEQUENCE OF IMPAIRED ERYTHROPOIESIS Anemias of this type have several features in common. Since inadequate erythropoiesis is the primary cause of reduced red cell numbers, bone marrow examination is indicated. Some diseases which impair erythropoiesis also decrease red cell life span. These anemias develop slowly and, because of various compensatory mechanisms, clinical findings do not accurately reflect the severity of the reduction in red cell numbers. A high percentage of feline anemias fall within this classification. Nutritional Deficiencies Primary iron deficiency is rare in cats and dogs. Secondary iron deficiency will develop as a result of chronic hemorrhage, for example, flea and hookworm infestations. Hematology will reveal hypochromic ervthrocytes, fewer immature cells than expected with the degree of anemia, and the McHc will be reduced. The MeV may be marginally reduced, but microcytosis only occurs in severe deficiencies. Vitamin B12 deficiency is poorly documented in dogs and cats. :\legaloblastic anemia has occurred in dogs treated with anticonvulsant drugs for a prolonged period. 5 Studies in man indicate that drugs such as diphenylhydantoin, primidone, and phenobarbital impair folic acid metabolism. 6 Anemias of Chronic Disorders Impaired erythropoiesis is frequently the result of chronic infections, liver disease, renal disease, and neoplasia of organs other than the
574
Figure l. (B). (500 X.)
GE)IE
P.
SEARCY
Canine blood smears illustrating leptocytes (A) and normal erythrocytes
hemopoietic system. These diseases probably share several pathogenetic mechanisms. The significance of anemia accompanying chronic disorders is often greater than is appreciated because of their insidious onset and the wide range of accepted normal values for dogs and cats. For example, a packed cell volume of 28 per cent in a cat with a chronic infection may represent a reduction of 10 per cent for that cat. Investigation of a nonregenerative anemia frequently reveals chronic liver or renal disease. Leptocytes are common in anemias of chronic disorders , however the pathogenesis ofleptocyte development remains unclear. Morphologically, they are characterized by peripheral distribution of hemoglobin and a large distinct area of central pallor. Target cells and folded cells are also termed leptocytes (Fig. 1). Chronic Infections. Dogs and cats with chronic infections are often moderately anemic. This appears to be true of both localized and diffuse infections. For example, cats with feline infectious peritonitis may have packed cell volume values as low as 15 per cent. Cartwright and coworkers/ as well as Van Snick et al., 11 have studied the pathogenesis of anemia in humans with chronic disorders, and their findings probably apply to cats and dogs, especially when many of their conclusions were based on animal experimentation. At least three factors are implicated: ( 1) shortened red cell survival, (2) impaired marrow response to anemia, and (3) impaired flow of iron from reticulohistiocytic cells to the bone marrow.
575
DIFFERENTIAL DIAGNOSIS OF ANEMIA
Shortened red cell survival of modest degree has been demonstrated in humans with infections. The same applies to normal erythrocytes administered to these patients. Studies in rats have shown that anemia accompanying inflammation fails to trigger the mechanism which stimulates production of erythropoietin. It has been firmly established that inflammation impairs plasma transferrin iron transport to the marrow. As a consequence, plasma iron levels decrease and iron accumulates as ferritin in inflamed tissues and the reticulohistiocytic system. Lactoferrin, an iron-binding protein similar to transferrin, is produced by neutrophils. When neutrophils release lactoferrin during an inflammatory state, iron moves from transferrin to lactoferrin and is sequestered in the rcticulohistiocytic system. Since iron needed for hemoglobin synthesis originates primarily from destroyed erythrocytes rather than from iron stores, iron sequestered in the reticulohistiocytic system is not mobilized sufficiently to support erythropoiesis and anemia occurs. Reducing the inflammatory process will resolve the anemia. Administration of iron is of questionable value since it will probably be sequestered in the reticulohistiocytic system. Liver Disease. Nonresponsive anemia accompanies chronic liver disease in some dogs. The pathogenesis of this anemia is obscure and
Table 3. Anemia and Liver Disease in a Dog Hgb (g/dl) PCV (%) RBC (x 10 6/~-tl)
:-.Icv (fl)
:-.ICHC (g/dl) Reticulocvtes (%) Leptocyt~s Platelets \\"BCI~-tl
'\eutrophils Eosinophils LYmphocytes \Ionocytes Protein (g/dl)
10.2 27.5 4.5 61
37
The Differential Diagnosis of Anemia Gene P. Searcy, D.V.M.*
When anemia is discovered the veterinarian should pursue this sign of disease in an orderly stepwise manner. This will involve the laboratory, but most progress and self-satisfaction will be achieved from an intimate combination of clinical findings and laboratory data. The scheme which follows will aid in understanding the pathogenesis and often the etiology of anemia. By adhering to such a scheme, the clinician may discover underlying diseases, manage the case more intelligently, and offer a more accurate prognosis. A pathophysiological approach to anemia (Table 1) is based on _.hether the decrease in erythrocyte numbers is associated with effective erythropoiesis (i.e., red cell loss due to hemorrhage or hemolysis) or impaired erythropoiesis. Clinical findings usually differ between anemias due to blood loss and impaired red cell production. Animals presented with acute onset of weakness and depression have usually experienced erythrocyte loss. Dogs and cats which have remained bright and alert for several weeks despite deereased exercise tolerance have probably suffered marrow failure. Packed cell volume or hemoglobin determination estimates erythro~"le numbers best. If one has access to an electronic cell counter, red cell counts may be obtained. However, errors are possible with these instruments, especially when counting feline erythrocytes which are significantly smaller than those of man. Evaluation of a properly prepared, well stained blood smear ith a good binocular microscope is an essential component of hematology. Scanning the smear at low magnification allows examination of the feather edge, the distribution of erythrocytes, platelets, and leukocytes, and estimation of the leukocyte count. Examination of erythrocytes with the oil immersion objective permits an assessment of erythropoietic rate which in turn characterizes the pathogenesis of the anemia. Hemorrhage or hemolysis causes a compensatory shift of immature red cells from marrow to peripheral blood *Professor of Veterinary Pathology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatechewan, Canada Veterinary Clinics of North America-Vol. 6, Nov. 4, November 1976
567
568
GENE
P.
SEARCY
Table 1. Pathophysiological Classification of Anemia ANEMIA ASSOCIATED WITH EFFECTIVE ERYTHROPOIESIS
Hemorrhage blood sucking parasites gastrointestinal lesions urinary tract lesions neoplasms with bleeding into body cavity coagulation disorders Hemolysis Acquired hemolytic anemia infectious toxigenic autoimmune Congenital hemolytic anemia hemoglobinopathies membrane defects glycolytic defects ANEMIA AssociATED WITH IMPAIRED OR INsUFFICIENT ERYTHROPOIESIS
Nutritional Deficiencies rron Anemias of Chronic Disorders chronic infection liver disease kidney disease Bone Marrow Destruction irradiation chemical agents Myeloproliferative Disorders granulocytic leukemia plasma cell myeloma erythremic myelosis erythroleukemia myelofibrosis lymphosarcoma
within approximately three days. These cells are macrocytic and polychromatophilic with Wright's or Wright-Giemsa stains. Nucleated erythrocytes (metarubricytes) also indicate an enhanced erythropoietic rate when accompanied by polychromasia and anisocytosis. Reticulocytes result from precipitation of cytoplasmic RNA in erythrocytes by stains such as new methylene blue. Hemolysis results in a greater degree of reticulocytosis than does hemorrhage because the marrow has preference for iron derived from red cell destruction. Blood containing a significant number of immature erythrocytes has an elevated mean corpuscular volume (MeV) and a decreased mean corpuscular-hemogloblin concentration (McHc). The former results from the presence ofmacrocytes, the latter from incomplete hemoglobin synthesis in reticulocytes. Laber et al. 3 have noted that cats are erythropoietically less responsive than dogs. Feline reticulocyte morphology differs from that of the dog. New methylene blue staining reveals large numbers of erythrocytes
DIFFERENTIAL DIAGNOSIS OF ANEMIA
569
,,-ith punctate basophilia in some cats in contrast to the reticular network characteristic of canine reticulocytes. The latter are also found in cat blood and perhaps should be referred to as "shift reticulocytes," since it can be safely assumed that they represent recent enhanced erythropoietic activity in contrast to the punctate cell which may be released in response to a variety of circumstances. Immature erythrocytes appear in peripheral blood in response to red cell loss within approximately 72 hours. If hemorrhage or hemolysis has occurred within that period of time, marrow examination may be necessary to evaluate erythropoiesis. Neutrophilia and thrombocytosis often accompany anemias due to hemorrhage or hemolysis. These changes occur during the regenerative phase of the anemia after reticulocytes appear.
ANEMIAS DUE TO ERYTHROCYTE LOSS After determining that the anemia is of the "erythrocyte loss" type, the next step is differentiation of hemorrhage and hemolysis. This requires thorough clinical and laboratory patient evaluation. Aspiration and examination of body fluids may reveal hemorrhage into body ca,·ities. Urine and feces should be examined for the presence of blood, keeping in mind that meat diets may result in a positive fecal occult blood test. Hemolysis may result in icterus, hemoglobinemia, and hemoglobinuria. Hemoglobin in both plasma and urine indicates intravascular hemolysis, whereas hyperbilirubinemia alone suggests reticulohistiocytic erythrocyte destruction. Icterus may only accompany hemolytic crises in dogs and cats as they are capable of rapid conjugation and excretion of bilirubin. Hemorrhagic Anemias
Parasites. Ectoparasites and endoparasites are capable of causing significant anemia and death in kittens and pups, for example, flea infestations and canine hookworms. Parasitic anemias, when chronic, become complicated by secondary iron deficiency which severely compromises compensatory erythropoiesis. Gastrointestinal Lesions. Ulceration of gastric or intestinal mucosa may result in hemorrhagic anemia; however, the incidence in dogs and cats is low. Urinary Tract Lesions. Hemorrhage may result from acute inflammatory conditions or urolithiasis, but anemia is rare. Occasionally chronic hematuria is encountered in dogs in which the etiology and pathogenesis are obscure. Spontaneous remission usually occurs in a matter of weeks.
570
GENE
P.
SEARCY
Neoplasms and Hemorrhagic Anemia. Neoplasms may become eroded and bleed, especially when located on surfaces subject to trauma. Vascular neoplasms are prone to hemorrhage. The best example, splenic hemangiosarcomas, occur in older dogs and may be characterized by periodic episodes of hemorrhagic anemia. The presence of anemia with reticulocytosis and hemoperitoneum suggests a splenic hemangiosarcoma. Coagulation Disorders. For a discussion of anemia resulting from coagulation disorders please refer to the article on "The Diagnosis of Hemostatic Disorders" in this symposium. Acquired Hemolytic Anemias
Hemolysis Caused by Infectious Agents. Feline infectious anemia is usually an acute hemolytic disease with all of the anticipated clinical findings. Blood film evaluation reveals polychromasia, anisocytosis, and Hemobartonella felis organisms. The question which must always be asked is whether the anemia is solely the result of hemobartonellosis or is the result of some other disease with organisms present as opportunists. Bone marrow injury is often the initial reason for anemia in secondary hemobartonellosis. Characteristic features of secondary hemobartonellosis are: ( l) organisms are usually not as numerous as in primary hemobartonellosis; (2) reticulocytosis is usually not consistent with the degree of anemia; (3) neutrophilia does not occur, as would be anticipated in response to hemolysis; in fact neutropenia is common; (4) the anemia may be severe with the packed cell volume perhaps ranging from 6 to 12 per cent; cats which suffer a hemolytic crisis will usually succumb; (5) abnormal nucleated cells may be visible, consistent with a myeloproliferative disorder. When any or all of these features suggest that Hemobartonella organisms may not be the sole cause of the anemia, marrow examination is indicated. If doubt remains, the cat should be treated for Hemobartonella and the response to therapy may provide further insight into the pathogenesis of the disease. Hemobartonella canis should be considered as a possible significant sequela to splenectomy, and postoperative search for the organism is indicated. Toxigenic Hemolytic Anemia. The presence of Heinz bodies on a blood film aids in the diagnosis of toxigenic hemolytic anemias. Heinz bodies are precipitated globin resulting from oxidative stresses which have overloaded the cell's protective mechanisms. The reader is referred elsewhere for a detailed description of Heinz bodies in Wright, Wright-Giemsa, or new methylene blue stained blood smears. 8 The principal cause of Heinz body hemolysis in cats has been exposure to methylene blue in urinary antiseptics. 8 Recent reports of onion toxicity in dogs indicate the necessity of keeping this in mind when confronted by Heinz-body hemolytic anemia. The dog described by
571
DIFFERENTIAL DIAGNOSIS OF ANEMIA
Spice 10 ingested dehydrated onions in the form of an onion souffle, and thus acquired a significant amount of the toxic factor N-propyl disulfide. Therapy should be supportive to allow excretion or degradation of the toxin. Autoimmune Hemolytic Anemia. Antibody-mediated hemolysis is common in dogs. It, and thrombocytopenia, are the two most prominent signs of systemic lupus erythematosus, a syndrome which also encompasses glomerulitis, synovitis, and thyroiditis. Since these latter diseases are less overt, it is not clear as to how often they accompany hemolysis or thrombocytopenia. A review of 10 cases of autoimmune hemolysis from our clinic revealed one instance of concurrent thrombocytopenia. In addition to elucidating the clinical findings and pathology of this disease, Lewis and coworkers 4 have demonstrated a virus in a colony of dogs with a high incidence of systemic lupus erythematosus. Erythrocyte destruction results from attachment of antibody to the membrane with loss of that portion by intravascular fragmentation or phagocytosis. The normal component of hemoglobin is then enclosed by a decreased amount of membrane, and disc-sphere transformation results. The spheroidal erythrocyte, because of impaired deformability, is then susceptible to retention and eventual destruction within the reticulohistiocytic system. This phenomenon is unlikely to result in hemo-
Table 2. Autoimmune Hemolytic Anemia in a Dog Feb. 23, 1973 *Mar. 9, 1973 Mar. 15, 1973 Apr. 14, 1973 tDec. 31, 1973 Hgb (g/dl) PC\'(%) RBC (X 10 6/ftl)
"cv (fl)
"CHC (g/dl) Reticulocytes (%) .\nisocytosis Spherocytes Leptocytes Polvchromasia Ho;,·ell-J oily bodies Platelets!ftl WBC/ftl '\eutrophils Band neutrophils \letamyelocytes Eosinophils b·mphocytes \lonocytes Disintegrated cells "etarubricytes Protein (g/dl) L:E cells Coombs' test
6.5 21.5 2.1 102.4 30.2 13
5.1 12.5 1.7 73.5 40.8 40
8.5 26.5 3.2 82.8 32.8 8.0
0
0 0
++++ ++
++++ ++
++++ +
++++ +
0
Increased 19,200 10,176 1,536 192 192 2,880 960 0 3,264 6.6 negative positive
Increased 47,700 34,254 6,228 1,038 0 519 5,709 0 4,152 7.9
++ ++
0 Increased 18,500 15,170 370 0 0 370 1,480 0 1,110 7.6
14.6 42 6.5 64.6 34.8 0 0 0 0 0 0 Normal 7,800 5,070 0 0 390 2,028 234 78 0 7.3
3.3 7.0 0.9 77.8 47.1 16
++++ +++ + ++++ +
404,000 30,700 19,034 3,991 921 0 2,456 2,149 0 2,149 10.0
*:VIarch 7, 1973-15 mg prednisone administered twice daily. edication was discontinued on March 20. +Hemoglobinemia and ghost erythrocytes evident. Dog died on examination table.
572
GENE
P.
SEARCY
globinemia; however, it does occur in some dogs (Table 2) and probably reflects membrane lysis due to complement involvement in the antigenantibody reaction. Theories explaining antibody formation to autologous antigens have been reviewed by Scott et al. 9 If further research confirms involvement of an infectious agent, a clearer understanding of antibody synthesis will be possible. Most dogs are presented with a hemolytic crisis of two to four days' duration and they exhibit weakness, depression, tachycardia, and perhaps fever. Icterus and occasionally hemoglobinuria are preserk Occasionally erythrocyte agglutination will be visible in freshly draWn blood within the EDT A tube or within a drop of blood. Hematology usually reveals anemia, reticulocytosis, spherocytosis, and neutrophilia. The total leukocyte count may be as high as 60,000 cells/1-.d. If the dog is presented within eight hours of the onset of hemolysis, hematology shows only anemia and spherocytosis. A positive Coombs' test confirms the presence of globulin on the erythrocyte membrane. If the test is negative, but all clinical and laboratory findings support a diagnosis of autoimmune hemolysis, therapy should be initiated. In our experience, Coombs' tests may be negative following steroid therapy. Commercial rabbit anti-dog globulin sera are available; however, complement must be inactivated and nonspecific agglutinins removed by absorption with normal canine erythrocytes. Corticosteroids are indicated because they stabilize cell membranes, which probably helps to decrease hemolytic rate. They also impair antibody synthesis and stimulate erythropoiesis. Prednisolone is recommended, 1.5 mg/kg twice daily. Response to therapy should be carefully monitored because the dosage may have to be increased for some dogs. When the packed cell volume approaches 30 per cent, the dosage should be decreased over a period of weeks until discontinued. Relapses occur and owners should be warned of this possibility. The dog described in Table 2 died of a hemolytic crisis approximately eight months after cessation of therapy. Although autoimmune hemolytic anemia has been described in cats, the incidence is lower than in dogs. The disease is more difficult to diagnose in cats because spherocytes are difficult to recognize. In addition, some commercial rabbit anti-cat sera cannot be purified to a point where normal feline erythrocytes will not agglutinate. Congenital Hemolytic Anemias Congenital hemolytic anemias may be broadly classified as hemoglobinopathies, membrane defects, and glycolytic defects. To date, one enzymopathy resulting in impaired erythrocyte glucose metabolism has been described in animals. 7 Glucose, the principal substrate for energy metabolism, is transported across the erythrocyte membrane as required. Approximately 90 per cent is degraded in the EmbdenMeyerhoff pathway to yield 2 moles of A TP per mole of glucose con-
DIFFERENTIAL DIAGNOSIS OF ANEMIA
573
sumed. Among others, A TP is responsible for maintenance of cation equilibrium, deformability, and membrane integrity. When there is malfunction of an enzyme involved in glycolysis, A TP generation is impaired and premature red cell destruction occurs. Deficiency of pyruvate kinase resulting in impaired red cell glycolysis is the etiology of congenital hemolytic anemia in Basenji and beagle dogs. Affected dogs are usually presented to veterinarians at six to twelve months of age because of reduced exercise tolerance and reduced growth rate. Clinical examination reveals pale mucous membranes and splenomegaly. Hematology reveals anemia with packed cell volume ,-alues ranging from 15 to 30 per cent and an intense reticulocytosis of up to 50 per cent. As dogs age, the anemia progressively worsens and reticulocytes decrease. The impairment of erythropoiesis results from progressive myelofibrosis and sclerosis. The pathogenesis of these alterations in bone marrow is not clear, but they are probably responsible for the death of affected dogs 3 to 5 years of age.
ANEMIA AS A CONSEQUENCE OF IMPAIRED ERYTHROPOIESIS Anemias of this type have several features in common. Since inadequate erythropoiesis is the primary cause of reduced red cell numbers, bone marrow examination is indicated. Some diseases which impair erythropoiesis also decrease red cell life span. These anemias develop slowly and, because of various compensatory mechanisms, clinical findings do not accurately reflect the severity of the reduction in red cell numbers. A high percentage of feline anemias fall within this classification. Nutritional Deficiencies Primary iron deficiency is rare in cats and dogs. Secondary iron deficiency will develop as a result of chronic hemorrhage, for example, flea and hookworm infestations. Hematology will reveal hypochromic ervthrocytes, fewer immature cells than expected with the degree of anemia, and the McHc will be reduced. The MeV may be marginally reduced, but microcytosis only occurs in severe deficiencies. Vitamin B12 deficiency is poorly documented in dogs and cats. :\legaloblastic anemia has occurred in dogs treated with anticonvulsant drugs for a prolonged period. 5 Studies in man indicate that drugs such as diphenylhydantoin, primidone, and phenobarbital impair folic acid metabolism. 6 Anemias of Chronic Disorders Impaired erythropoiesis is frequently the result of chronic infections, liver disease, renal disease, and neoplasia of organs other than the
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Figure l. (B). (500 X.)
GE)IE
P.
SEARCY
Canine blood smears illustrating leptocytes (A) and normal erythrocytes
hemopoietic system. These diseases probably share several pathogenetic mechanisms. The significance of anemia accompanying chronic disorders is often greater than is appreciated because of their insidious onset and the wide range of accepted normal values for dogs and cats. For example, a packed cell volume of 28 per cent in a cat with a chronic infection may represent a reduction of 10 per cent for that cat. Investigation of a nonregenerative anemia frequently reveals chronic liver or renal disease. Leptocytes are common in anemias of chronic disorders , however the pathogenesis ofleptocyte development remains unclear. Morphologically, they are characterized by peripheral distribution of hemoglobin and a large distinct area of central pallor. Target cells and folded cells are also termed leptocytes (Fig. 1). Chronic Infections. Dogs and cats with chronic infections are often moderately anemic. This appears to be true of both localized and diffuse infections. For example, cats with feline infectious peritonitis may have packed cell volume values as low as 15 per cent. Cartwright and coworkers/ as well as Van Snick et al., 11 have studied the pathogenesis of anemia in humans with chronic disorders, and their findings probably apply to cats and dogs, especially when many of their conclusions were based on animal experimentation. At least three factors are implicated: ( 1) shortened red cell survival, (2) impaired marrow response to anemia, and (3) impaired flow of iron from reticulohistiocytic cells to the bone marrow.
575
DIFFERENTIAL DIAGNOSIS OF ANEMIA
Shortened red cell survival of modest degree has been demonstrated in humans with infections. The same applies to normal erythrocytes administered to these patients. Studies in rats have shown that anemia accompanying inflammation fails to trigger the mechanism which stimulates production of erythropoietin. It has been firmly established that inflammation impairs plasma transferrin iron transport to the marrow. As a consequence, plasma iron levels decrease and iron accumulates as ferritin in inflamed tissues and the reticulohistiocytic system. Lactoferrin, an iron-binding protein similar to transferrin, is produced by neutrophils. When neutrophils release lactoferrin during an inflammatory state, iron moves from transferrin to lactoferrin and is sequestered in the rcticulohistiocytic system. Since iron needed for hemoglobin synthesis originates primarily from destroyed erythrocytes rather than from iron stores, iron sequestered in the reticulohistiocytic system is not mobilized sufficiently to support erythropoiesis and anemia occurs. Reducing the inflammatory process will resolve the anemia. Administration of iron is of questionable value since it will probably be sequestered in the reticulohistiocytic system. Liver Disease. Nonresponsive anemia accompanies chronic liver disease in some dogs. The pathogenesis of this anemia is obscure and
Table 3. Anemia and Liver Disease in a Dog Hgb (g/dl) PCV (%) RBC (x 10 6/~-tl)
:-.Icv (fl)
:-.ICHC (g/dl) Reticulocvtes (%) Leptocyt~s Platelets \\"BCI~-tl
'\eutrophils Eosinophils LYmphocytes \Ionocytes Protein (g/dl)
10.2 27.5 4.5 61
37
