Diagnosis of Hemolytic Anemias
Urs Giger United States
The normal life span of erythrocytes averages approximately 100–120 days in dogs and 70–78 days in cats. Accelerated erythrocyte destruction is the major mechanism in hemolytic disorders and plays a minor role in many other common anemias.
Erythrocyte destruction may take place either extra- or intravascularly. Extravascular hemolysis is the predominant form and assumed to be the mode of destruction of senescent erythrocytes in healthy animals. Extravascular destruction refers to erythrophagocytosis by macrophages of the spleen, liver, and bone marrow.
Beside the general signs of anemia such as pallor and weakness, characteristic signs of hemolysis are jaundice and pigmenturia. Jaundice is first appreciated on mucous membranes (gingiva and sclera) when the serum bilirubin level exceeds 2 mg/dl, whereas the skin becomes icteric only at higher bilirubin concentrations. Milder and chronic forms of hemolysis may not be associated with jaundice. Pigmenturia caused by hemolysis may be due to hyperbilirubinuria and hemoglobinuria. Hyperbilirubinuria persists in dogs with hemolytic disorders, whereas in cats that appears not to be a constant finding. Hemoglobinuria and hemoglobinemia are hallmark features of intravascular hemolysis and often indicate a more severe disorder. Thus, hemolytic anemias are regenerative and macrocytic-hypochromic, although in the very early stages and in some complicated acquired forms the erythroid response may be poor. Excessive destruction of erythrocytes may occur either because of an intrinsic defect in the cell itself or because of the action of extrinsic factors on normal erythrocytes. Intrinsic defects are generally inherited and the extrinsic ones are acquired.
Inherited Erythrocyte Defects
Several hereditary erythrocyte defects have been described in dogs and cats and much new information has emerged over the past decade. The mode of inheritance is autosomal recessive for all described erythrocyte defects, with the exception of feline porphyria, which is inherited as a dominant trait. They represent a large heterogeneous group of disorders and most of them occur relatively rarely. However, through inbreeding practices (popular sire, line breeding) certain erythrocyte defects have become common in certain breeds. Unless the affected breeds are closely related, the disease is likely caused by different mutations of the same gene. Most erythrocyte defects cause hemolytic disorders that lead from mild compensated to life-threatening hemolytic anemia. Accurate laboratory tests are now available for many erythrocyte defects to detect affected as well as carrier animals. Hereditary erythrocyte disorders have been classified into three groups:
1. Heme defects (porphyria) and hemoglobinopathies.
2. Membrane abnormalities including stomatocytosis and increased osmotic fragility.
3. Erythroenzymopathies such as Phosphofructokinase (PFK) and Pyruvate Kinase (PK) deficiency.
Immune-Mediated Hemolytic Anemia
Immune-mediated hemolytic anemia (IMHA) arises when an immune response targets directly or indirectly erythrocytes and hemolytic anemia ensues, and results from a breakdown in immune self-tolerance. Immune destruction of erythrocytes is initiated by the binding of IgG or IgM antibodies and complement to the surface of erythrocytes. In primary IMHA, no inciting cause can be identified, thus the synonyms idiopathic IMHA or autoimmune hemolytic anemia. In contrast, secondary IMHA is associated with an underlying condition or triggered by an agent. In addition, alloimmune hemolytic anemias such as feline neonatal isoerythrolysis and hemolytic transfusion reactions are caused by anti-erythrocytic alloantibodies. IMHA is the most common reason for hemolytic anemias in dogs. A genetic predisposition is suggested in American Cocker Spaniels that may represent up to 40% of all dogs. In other canine breeds, predisposition is less well documented and may vary geographically. As with other immune disorders, female dogs appear slightly predisposed, even when spayed. In recent studies of IMHA, an underlying disease process or trigger could be identified including drugs and infectious, neoplastic as well as other immune disorders. In babesiosis and hemobartonellosis, hemolysis appears exaggerated by immune processes. Many chronic infections including abscesses, discospondylitis, pyometra, and pyelonephritis can induce secondary immune disorders including IMHA. A temporal association between vaccination and onset of IMHA has also been suggested. As this correlation was associated with modified and killed vaccines against common infectious diseases from different manufacturers, it appears likely that vaccines may trigger or enhance a smoldering immune process rather than be the underlying cause. The higher rate of IMHA during the warmer months from May through August reported in some studies, but not others, may also suggest an infectious cause including tick-born disorders. This seasonality may vary geographically. The association of IMHA with other immune disorders, including hypothyroidism and immune-mediated thrombocytopenia (ITP), lends support to the hypothesis of a general immune disturbance. If IMHA and ITP occur concurrently, this is known as Evans’ syndrome.
Clinical Signs of IMHA
IMHA may present at any age, but is most commonly encountered in young adult to middle-aged dogs. The clinical history is generally brief and vague. An underlying condition may be identified. An episode of vomiting or diarrhea may precede the typical signs of anemia (lethargy, weakness, exercise intolerance, pallor) and hemolysis (pigmenturia, icterus). Some animals may be febrile, presumably due to erythrocyte lysis or an underlying disease process. Others develop dyspnea indicating pulmonary problems either as the underlying disease or as a thromboembolic complication of IMHA. Physical examination may also reveal mild splenomegaly and, less commonly, mild hepatomegaly and lymphadenopathy, which again suggest a secondary cause of IMHA. Furthermore, signs attributable to their underlying disease may predominate, whereas chronic or recurrent signs of IMHA suggest a primary form.
Routine Laboratory Test Results
The anemia can be mild to life threatening and the hematocrit may precipitously drop after presentation due to active hemolysis. Although a regenerative macrocytic-hypochromic anemia would be expected, as many as one-third of all cases of IMHA are non-regenerative on presentation. The disease course may have been too acute, not yet allowing time for a regenerative response to mount. Alternatively, antibodies may be directed against erythroid precursors or the IMHA disease process may change the microenvironment of the bone marrow and thereby impair erythropoiesis. Evidence of ineffective erythropoiesis and erythrophagocytosis may be found on cytologic examination of a bone marrow aspirate. Autoagglutination of erythrocytes and spherocytosis are typical findings on blood smears.
Beside erythroid abnormalities, a leukocytosis is often present and can exceed 100,000/μl mostly due to a mature neutrophilia. Because high white blood cell counts are not generally encountered with anemia, this likely reflects a unique inflammatory and cytokine response specific for IMHA, but concomitant infection and steroid-induced leukocytosis should also be considered. Thus, hyperplasia of erythroid and myeloid cells may be present in the bone marrow. Furthermore, thrombocytopenia due to a concomitant ITP (Evans’ syndrome) or DIC may occur.
Serum analysis reveals generally hyperbilirubinemia; a serum bilirubin concentration of above 10 mg/dl has been associated with a grave prognosis. However, serum bilirubin values may only be slightly increased in chronic cases, presumably due to a highly efficient and accelerated bilirubin metabolism. Thus, high serum bilirubin values also may indicate a concomitant hepatopathy. In fact, dogs with IMHA often have increased serum liver enzymes even before steroid therapy. Hyperbilirubinuria is expected as with any other hemolytic anemia; in cats, any degree of bilirubinuria is considered important, whereas larger amounts of bilirubin are generally present in urine of dogs. There may also be evidence of a bacterial cystitis, which may indicate an underlying infectious disease or may occur secondarily due to immunoderegulation or immunosuppressive therapy.
Various imaging studies may be indicated to reveal underlying disease processes, such as neoplasia, and complications of IMHA. Evidence of thromboemboli may be detected on chest radiographs and abdominal ultrasound as well as at the site of catheters.
Diagnostic Laboratory Test Results
A diagnosis of IMHA must demonstrate accelerated immune destruction of erythrocytes. Thus, beside documenting a hemolytic anemia, a search after antibodies or complement or both directed against erythrocytes is required, i.e., one or more of the following three hallmarks has to be present to reach a definitive diagnosis of IMHA:
1. Marked spherocytosis.
2. True autoagglutination.
3. Positive direct Coombs’ test.
Spherocytes are spherical erythrocytes that appear microcytic with no central pallor. They result from either partial phagocytosis or lysis and are rigid and extremely fragile in the erythrocyte osmotic fragility test. Because spherocytes have lost some membrane, they do not have any reserves to expand in hypotonic solution. Large numbers of spherocytes are present in approximately two-thirds of dogs with IMHA, but small numbers may also be seen with hypophosphatemia, zinc intoxication, and microangiopathic hemolysis.
Anti-erythrocytic IgM and, in large quantities, IgG antibodies may cause direct autoagglutination. Autoagglutination may be visible to the naked eye when blood (at low hematocrit) is in an EDTA tube or placed on a glass slide (macroscopic agglutination) or may become apparent as small clumps of erythrocytes on a stained blood smear or in saline wet mount (microscopic agglutination). Autoagglutination has to be distinguished from rouleaux formation where erythrocytes stack up on top of each other. For yet unexplained reasons, canine erythrocytes have a tendency to unspecifically agglutinate in the presence of plasma at colder temperatures. Mixing one drop of blood with one drop of saline may not break up this unspecific form of agglutination. It is, therefore, important to determine whether the agglutination persists after “saline washing” which has been termed true autoagglutination. This is accomplished by adding three times physiologic saline solution to a tube of blood after repeated centrifugation and removal of supernatant including the plasma.
Direct Coombs’ Test
The direct Coombs’ test, also known as direct antiglobulin test, is used to detect antibodies and/or complement on the erythrocyte surface when the anti-erythrocyte antibody strength or concentration is too low (subagglutinating titer) to cause spontaneous autoagglutination. The so-called “incomplete” antibodies on erythrocytes, together with species-specific antiglobulins against IgG, IgM and C3b (Coombs reagents), allow antibody bridging and thereby agglutination and/or lysis of coated erythrocytes. Separate IgG, IgM and C3b, as well as polyvalent Coombs’ reagents, are available for dogs and cats. They are added at varied concentrations after washing the patient’s erythrocytes free of plasma. The mixture is generally incubated at 37°C, and then centrifuged, and supernatant and pellet are analyzed for hemolysis and agglutination, respectively. Performance of the direct Coombs’ test at colder temperatures (4°C and 20°C) is rarely indicated, because cold agglutinins and hemolysins are rarely strong enough and rarely active at near normal body temperatures (30°C) to cause disease. Cold agglutinins and hemolysins of clinical importance are generally IgM-antibodies at very high titer with thermal amplitude that reaches 30°C. Because the same erythrocyte washing procedure is used in the direct Coombs’ test as for the true autoagglutination test and the end point of the Coombs’ reaction is agglutination and lysis of erythrocytes, true autoagglutination precludes the performance of a direct Coombs’ test.
Positive direct Coombs’ test results are reported as +1 to +4 or in the form of dilutions of the Coombs’ reagent that causes agglutination and/or lysis. The strength of the Coombs’ reaction does not necessarily predict the severity of hemolysis. In order to reach a definitive diagnosis of IMHA, the direct Coombs’ test should be positive, however, this does not discriminate between primary and secondary IMHA. Dogs with negative Coombs’ test results should be reevaluated for other causes of hemolytic anemia. However, a small proportion of dogs may have IMHA, despite a negative Coombs’ test result. False-negative Coombs’ test results may occur because of insufficient quantities of bound antibodies and many technical reasons (inappropriate reagents or dilutions). Negative results are also seen in animals in which the disease is in remission, however, a few days of immunosuppressive therapy will likely not reverse the test results. In fact, treated animals may still have positive Coombs’ test results long after the hemolytic anemia resolved. False-positive Coombs’ test results occur only rarely, e.g., after an incompatible transfusion or because of technical problems.
Hemolytic anemia may develop upon exposure to several parasitic, bacterial and viral agents due to the direct action of the infecting agent or its products on erythrocytes. Few rickettsial and protozoal organisms are capable of infecting erythrocytes directly and cause severe hemolytic anemia. Other infectious agents may induce along with other major clinical signs indirectly a hemolytic component. Thus, any infection may trigger the production of humoral antibodies against host erythrocytes, and together with an activated complement and phagocytic system, the rate of erythrocyte destruction may be markedly accelerated. Furthermore, during bacterial (e.g., Leptospira, Clostridia, Streptococci, Staphylococci) septicemia, specific hemolysins can be produced and result in hemolytic anemia.
Chemical and Physical Induced Hemolytic Anemia
Many compounds including chemical agents, drugs, and food components and additives can induce oxidative damage to erythrocytes leading to a hemolytic anemia. Many of these agents are derivatives of aromatic organic compounds. In some cases, the chemical itself acts as an oxidative agent, but more often, the compound or its metabolite interacts with oxygen to form free radicals and peroxides. Extracellularly produced oxidants injure the membrane, whereas oxidants generated intracellularly attack hemoglobin as well as membrane structures. Thus, a single agent may inflict erythrocyte injury, thereby hemolysis and reduced oxygen delivery to tissue by one or all three of:
1. Oxidation of heme iron resulting in methemoglobin production.
2. Oxidative denaturation of hemoglobin leading to Heinz body formation.
3. Membrane damage causing impaired deformability and ion transport.
Severe hypophosphatemia causing hemolysis in dogs and cats has been associated with diabetes mellitus, hepatic lipidosis, and primary hyperparathyroidism as well as with enteral and parenteral hyperalimentation (starvation-refeeding syndrome) and oral administration of phosphate-binding antacids. During insulin, fluid and bicarbonate treatment of (ketoacidotic) diabetic animals, the phosphate value in plasma declines precipitously. Hypophosphatemia occurs due to intracellular phosphate shifts, enhanced renal losses, and reduced intestinal absorption of phosphate. In addition to myopathy and cardiac and neurologic dysfunction, acute hemolytic anemia, characterized by a rapid drop in PCV and mild intravascular lysis and Heinz body formation is observed in animals with hypophosphatemia. In clinical practice hemolysis occurs with phosphate values of < 2.5 mg/dl. The pathogenesis of the hypophosphatemia-induced anemia is likely related to decreased erythrocytic deformability and increased osmotic fragility as well as susceptibility to oxidative injury.
A large variety of conditions may cause physical damage to erythrocytes that leads to cell fragmentation and intra- as well as extravascular hemolysis. In case of water intoxication due to near drowning in fresh water, erythrocytes undergo hypotonic lysis similar to the swelling and lysis of erythrocytes in the in vitro osmotic fragility test. Heat stroke and severe burns can inflict thermal injury to erythrocytes. Heart valve disease as well as cardiovascular implants and intravenous catheters can induce mechanical damage to erythrocytes as much as dirofilariasis, particularly in the form of the caval syndrome. In addition, other endothelial damage caused by vasculitis, hemangiosarcoma and other tumors, various splenic diseases or torsion, and liver disease can injure erythrocytes. A hemolytic-uremic syndrome characterized by acute renal failure, platelet activation leading to thrombocytopenia and thrombosis, and microangiopathic hemolytic anemia has recently been described in dogs. Similarly, disseminated intravascular coagulation is associated with a fragmentation hemolysis. A diagnosis of microangiopathic hemolysis, which is often subclinical and only rarely causes overt intravascular hemolytic anemia, is made by identifying the triggering condition and the characterization of schistocytes (shizocytes). These erythrocyte fragments appear on blood smears as small, misshapen, often triangular or helmet-shaped structures. Schistocytes are important even in small numbers and cannot be fabricated by poor blood smear preparation.
1. Giger, Urs (2000), “Regenerative Anemias Caused by Blood Loss or Hemolysis,” Textbook of Veterinary Internal Medicine, S.J. Ettinger and E.C. Feldman, ed. Philadelphia, PA, Saunders.
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