Hemolytic Anemias – Focus On Immune-Mediated Hemolytic Anemia
Urs Giger, PD Dr. med. vet. MS FVH, DACVIM, DECVIM
Extravascular hemolysis is the predominant form and also 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. Macrophages destroy ingested erythrocytes and unconjugated bilirubin is released into the plasma. The albumin-bound (indirect) bilirubin is conjugated (direct bilirubin) in hepatocytes. The conjugated bilirubin is excreted, but in the presence of massive heme breakdown, conjugated bilirubin leaks back into the plasma and is excreted also in the urine. Normal serum values do not exceed 0.4 mg/dl. A rise in heme degradation will result in hyperbilirubinemia and hyperbilirubinuria.
Less commonly, erythrocytes are lysed within the systemic circulation as a consequence of membrane permeability changes or cellular fragmentation. With intravascular hemolysis, lysis can be readily recognized by the presence of hemoglobinemia and hemoglobinuria. The hemoglobinemia has to be distinguished from artifactual increases in plasma hemoglobin caused by poor blood collection and handling as well as delayed plasma separation. Plasma hemoglobin concentrations can be measured by an accurate and simple hemoglobinometer (HemoCue).The clinical features and laboratory test abnormalities of the various hemolytic disorders are similar. 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. Hemoglobinuria and hemoglobinemia are hallmark features of intravascular hemolysis and often indicate a more severe disorder. As the urine dipstick only identifies heme (labeled “blood”), hemoglobinuria has to be differentiated from hematuria (whole erythrocytes) by microscopic examination of a urine sediment. 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.
Inherited Erythrocyte Defects
Several hereditary erythrocyte defects that autosomal recessively inherited have been described in dogs. 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 hemolytic to life-threatening 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:
- Hemoglobinopathies: thus far not described in dogs
- Membrane defects: increased osmotic fragility, spherocytosis, and stomatocytosis
- Erythroenzymopathies: phosphofructokinase (PFK) and pyruvate kinase deficiency (PK)
Chemical-Induced Hemolytic Anemias
Many compounds including chemical agents (New methylene blue, naphthaline), several drugs, and food components (onions, propylene glycol) 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 and thereby hemolysis and reduced oxygen delivery to tissue by one of all three of
Therapy is supportive and directed at removing the triggering agent.
Severe hypophosphatemia causing acute hemolysis in dogs 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 clinical practice hemolysis occurs with phosphate values of <2.5 mg/dl. The pathogenesis of the hypophosphatemia-induced anemia is likely related to depletion of erythrocytic ATP and GSH which leads to decreased deformability and increased osmotic fragility as well as susceptibility to oxidative injury.
Hypophosphatemic animals need to receive oral or parenteral phosphate supplementation. Aggressive intravenous phosphate therapy is often needed in severely hypophosphatemic and anorexic or vomiting animals. An initial dosage of 0.01-0.03 mmol/kg/h Na or K phosphate appears safe and effective, but serum phosphorus and calcium concentrations should be measured every 6 hours and the dose should be adjusted and route switched to oral when appropriate. Potential complications of intravenous phosphate supplementation include hypocalcemia, acute renal failure and dystrophic soft tissue calcification.
Microangiopathic Hemolytic Anemia
A large variety of conditions may cause physical damage to erythrocytes, which leads to cell fragmentation and intra- as well as extravascular hemolysis. 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. In addition, other endothelial damage caused by vasculitis, hemangiosarcoma and other tumors, various splenic diseases or torsion, and liver disease can injure erythrocytes. Similarly, disseminated intravascular coagulation is associated with a fragmentation hemolysis. Schistocytes (fragmented erythrocytes) are important even in small numbers and cannot be fabricated by poor blood smear preparation. On the other hand, schistocytes are observed with a variety of other anemias including chronic iron deficiency states and zinc intoxication. Concomitantly, a thrombocytopenia and a coagulopathy are often present. Beside supportive care, therapy is directed at the underlying disease and control of DIC. Blood component therapy should be considered. The prognosis is guarded to poor, if the underlying disease cannot be corrected.
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 protozoal organisms are capable of infecting erythrocytes directly and cause severe hemolytic anemia (Pitbull terriers and Greyhounds with babesia infections). Other infectious agents may induce along with other major clinical signs indirectly a hemolytic component, for instance dirofilariasis. 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. Treatment against infectious agent along with supportive care and transfusions for improved oxygen carrying transport are effective.
Immune-Mediated Hemolytic Anemia
Immune-mediated hemolytic anemia (IMHA) arises when an immune response targets directly or indirectly erythrocytes and hemolytic anemia ensues. Until the anti-erythrocytic antibodies are identified and the pathogenesis is better understood, the nomenclature and classification of IMHA remains imprecise and sometimes confusing. In primary IMHA no inciting cause can be identified, thus the synonyms idiopathic IMHA or autoimmune hemolytic anemia (AIHA). In contrast, secondary IMHA is associated with an underlying condition or triggered by an agent. In addition, alloimmune hemolytic anemias, such as hemolytic transfusion reactions, are caused by anti-erythrocytic alloantibodies. Hemolysis of the newborn has not been reported in dogs.
Immune mechanisms - Regardless of the underlying cause, IMHA 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. Under most clinical circumstances, immune destruction is an extravascular process that depends on recognition of erythrocytes opsonized with IgG or complement or both by specific receptors on reticuloendothelial cells. Macrophages with engulfed erythrocytes may be noted by cytologic examination of tissue aspirates. Alternatively, only portions of the membrane are removed by phagocytes, leaving erythrocytes with reduced surface area to volume ratio, thereby forming spherocytes.
Underlying Conditions and Predispositions - Historically, in most dogs with IMHA, no underlying condition was ever identified and, thus, considered to be primary or idiopathic IMHA. However, in more recent studies of autoimmune hemolytic anemia, an underlying disease process or trigger could be identified including drugs and infectious, neoplastic, as well as other immune disorders. In babesiosis, hemolysis is exaggerated by immune processes, and many chronic bacterial infections, including abscesses, discospondylitis, pyometra, and pyelonephritis, can induce secondary IMHA. A temporal association between vaccination and onset of IMHA has also been suggested: In a limited retrospective study, one quarter of all dogs with IMHA of unknown cause was vaccinated within one month of onset of clinical signs. 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. The seasonality may vary geographically. If IMHA and ITP occur concurrently, this is known as Evans syndrome. A genetic predisposition is suggested in some dogs by the breed predilection and familial occurrence: American Cocker spaniels 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.
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 peracute, 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 or DIC may occur.
Serum analysis reveals generally a hyperbilirubinemia, and 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. The degree of hemoglobinemia – a sign of intravascular hemolysis – can vary drastically and rapidly, and is associated with hemoglobinuria. Hyperbilirubinuria is expected as with any other hemolytic anemia, and 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:
Spherocytosis – Spherocytes are spherical erythrocytes that appear microcytic with no central pallor. They result either from partial phagocytosis or lysis and are rigid and extremely fragile in the erythrocyte osmotic fragility test. 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.
Autoagglutination – 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 blood after repeated centrifugation and removal of the 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. They are added at varied concentrations after washing the patient’s erythrocytes free of plasma and mixtures are generally incubated at 37°C. Cold agglutinins and hemolysins of clinical importance are generally IgM-antibodies at very high titers. 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 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 (titer) 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). Furthermore, the test result may be negative in the absence of an inciting agent, e.g., drug. 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, and treated animals may still have positive Coombs’ test results long after the hemolytic anemia resolved.
Therapy - Because the severity of IMHA ranges from indolent to life-threatening disease, therapy has to be tailored for each patient and depends in part on whether the IMHA is primary or secondary. Removal of the triggering agent or treatment of the underlying condition can bring the IMHA under control. Thus, in cases of secondary IMHA due to infection, antiprotozoal or antibiotics should be instituted. Because of the potential of underlying occult infection and the predisposition to infection from immunoderegulation associated with IMHA and immunosuppressive therapy, antibiotic therapy is generally indicated. Non-essential drugs, particularly those implicated to cause an immune reaction, should immediately be withdrawn. Despite these interventions, transfusion and immunosuppressive therapy are likely still required in the initial control of secondary IMHA.In case of signs of tissue hypoxia due to severe anemia and a dropping hematocrit, packed red blood cell transfusions are beneficial. The increased oxygen-carrying capacity provided by transfused cells may be sufficient to maintain the animal for the few days required for other treatment modalities to become effective. The notion that transfusions are especially hazardous in animals with IMHA has been overemphasized and is not supported by recent retrospective studies. As the anti-erythrocytic antibody in IMHA is not an alloantibody, the destruction of transfused cells is no higher than autologous erythrocytes. However, autoagglutination may hamper, even after RBC washing, accurate blood typing and crossmatching tests, thus, only DEA 1.1 negative blood should be transfused in autoagglutinating dogs. If compatible blood is not available, the recently FDA-approved bovine hemoglobin solution can be administered and provides increased oxygen-carrying capacity and plasma expansion. In contrast, oxygen inhalation therapy is of little benefit, unless the animal is suffering from pulmonary disease such as pulmonary thromboemboli. Thanks to adequate transfusion support, animals with IMHA rarely die because of anemia, but because of secondary complications such as thromboemboli and infections.
The main goal in the treatment of IMHA focuses on controlling the immune response by reducing phagocytosis, complement activation, and anti-erythrocytic antibody production. Glucocorticosteroids are the initial treatment of choice for IMHA. They interfere with both the expression and function of macrophage Fc receptors and thereby immediately impair the clearance of antibody-coated erythrocytes by the macrophage system. In addition, glucocorticosteroids may reduce the degree of antibody binding and complement activation on erythrocytes, and only after weeks, diminish the production of autoantibodies. Oral prednisone or prednisolone at a dose of 1-2 mg/kg twice daily is the mainstay treatment. Alternatively, oral or parenteral dexamethasone at an equipotent dose of 0.6 mg/kg daily can be used, but is likely not more beneficial. A response reflected by a stabilized or even rising hematocrit, an appropriate reticulocytosis, less autoagglutination, and fewer spherocytes, can be expected within days. As glucocorticosteroid therapy is associated with well-known side effects, the initial dose will then be tapered by reducing the amount by one-third every 7-10 days. In secondary IMHA with appropriate control of the underlying disease, the tapering can be accomplished more rapidly. Because of the potential of gastrointestinal ulceration by steroids, gastrointestinal protectance such as sucralfate may be considered. Despite an apparent recovery as judged by reaching a normal hematocrit, particularly animals with primary IMHA may continue to have a positive Coombs’ test for weeks to months and could obviously relapse. Such relapses may be controlled by the same treatment as initially used, but a more gradual tapering regime may be used, which leaves the animal on an every other day prednisone therapy for months.
Other immunosuppressive therapy is warranted when prednisone fails, only controls the disease at persistently high doses, or causes unacceptable side effects. They are generally used together with prednisone but may eventually be used independently. Cytotoxic drugs were historically first added. Cyclophosphamide, an alkalating and potent myelosuppressive agent, has been advocated in cases of fulminate IMHA. However, a recent randomized limited prospective trial comparing prednisone versus a combination of prednisone and cyclophosphamide did not find any beneficial effects of cyclophosphamide in the acute management of IMHA. Retrospective studies with cyclophosphamide and/or azathioprine, an anti-metabolite, were similarly disappointing. These drugs inhibit lymphocytes and thereby suppress the anti-erythrocyte antibody production within weeks. These agents are, therefore, likely not effective in the acute management of IMHA, but may have a place in the long-term control of refractory and relapsing cases. A reasonable regimen for dogs might include either cyclophosphamide at 2 mg/kg every other day or azathioprine at 2 mg/kg SID/EOD. The risk versus benefit ratio should be carefully considered when using these drugs.
Recently, several other immunosuppressive agents have been used on a limited bases in conjunction with prednisone, and anecdotal success has been reported in dogs and humans. As all of these agents interfere with antibody action and macrophage function, they can elicit more immediate effects. Cyclosporine, an expensive but potent immunosuppressive agent most commonly used in preventing graft rejection and graft vs. host disease in transplant patients, may be beneficial in controlling the immune response in dogs with IMHA. A dose regimen of 10 mg/kg SID may be used initially, but blood concentrations should be periodically monitored to achieve an effective but safe level. Intravenous human immunoglobulin (IVIG; two doses of 1 g/kg 24 hrs apart) may be helpful in the short-term treatment of dogs with IMHA or refractory cases. IVIG can block Fc receptors on macrophages, thereby reducing Fc-mediated phagocytosis of IgG-coated erythrocytes, interfere with complement action, and suppress antibody production.
Splenectomy may be considered in IMHA patients failing prednisone and other immunosuppressive therapy, requiring long-term high dose therapy to remain in remission, or suffering, intractable drug side effects. In addition, histologic examination of the spleen may provide evidence of an underlying disease. The response to splenectomy remains to be determined in animals. There is a slight risk of developing overwhelming infections immediately post-splenectomy and systemic bacterial infections subsequently.
Thrombemboli and DIC are unique serious complications that greatly contribute to the morbidity and mortality of patients with IMHA. Although the pathogenesis remains unknown, venipuncture, catheters, and glucocorticosteroid therapy represent predisposing conditions. Thus far, no study has documented any successful prevention and/or management protocol for these life-threatening hemostatic problems in IMHA. Predisposing factors should, whenever possible, be limited and adequate perfusion and oxygenation of tissue should be provided with fluids and transfusions. Generally, anticoagulant therapy is instituted only after there is some evidence or suspicion of thromboemboli. Heparin at a dose of 100 IU/kg sc every 6 hours or by continuous infusion is the most commonly used drug. Other complications are likely related to drug therapy.
Despite appropriate implementation of the above therapeutic strategies, the mortality rate remains high; in fact, there is an impression that the fulminate form of IMHA is more frequently encountered today. Depending on the type of practice (primary to tertiary), mortality rates from 20-75% have been reported. Negative prognostic indicators are rapid drop in PCV, high serum bilirubin levels, non-regenerative anemia, intravascular hemolysis, autoagglutination, and thromboembolic complications.
Giger, U: “Regenerative Anemias Caused by Blood Loss or Hemolysis,” Textbook of Veterinary Internal Medicine, S.J. Ettinger and E.C. Feldman, Eds. 5th Edition. Philadelphia, PA, Saunders, pp 1784-1804, 2000.
Prof. Dr. Urs Giger, Veterinary Hospital University of Pennsylvania,