Immune-mediated hemolytic anemia () results from cross-reacting antibodies that induce enhanced red blood cell removal. Autoimmune (primary immune-mediated hemolytic anemia) hemolysis results from loss of self-tolerance and is relatively rare in horses. Most commonly, hemolysis results from adherence of cross-reacting antibodies to erythrocyte surface antigens (secondary immune-mediated hemolytic anemia). The presence of these molecules on red blood cells causes intravascular destruction by complement activation (IgM-mediated) or — most commonly — extravascular removal by macrophages. It is important for the reader to consider that any infectious agent — especially equine infectious anemia, Babesia organisms, and Anaplasma phagocytophila; exogenous substances such as penicillin and phenylbutazone; or neoplasia — may cause alterations in epitopes of the erythrocyte membrane or neoantigens that contribute to enhanced removal by immune mechanisms. Therefore identification of the inciting cause is important for complete resolution of the hemolytic crisis.
Several possibilities may explain the onset of cellular destruction. The basic mechanisms involve a change in the red blood cell or an alteration in immunologic control of self-recognition. For example, a change in the red blood cell membrane may form a novel antigen that evokes an immune response. Drugs, neoplasia, or infection may induce changes in red cell antigens. Infectious agents that express similar antigens as host red blood cell antigens result in pathogen-induced immune-mediated hemolysis, termed molecular mimicry. An example of molecular mimicry is human infection with Epstein-Barr virus. Genetic predispositions may cause a failure of self-tolerance. Failure of autoregulation has been suggested to result from reduced suppressor lymphocyte control. Finally, failure of appropriate erythropoiesis may result from precursor erythrocytes being targeted by the immune response in a manner similar to that of circulating red blood cells. The goal of the clinician should be to focus on identifying any potential inciting causes because this will allow for appropriate case management with the best prognosis for efficient and complete disease resolution.
Immune-Mediated Hemolytic Anemia: Clinical Signs and Diagnosis
Horses with immune-mediated hemolytic anemia most commonly present with signs of extravascular hemolysis, but intravascular hemolysis is possible, especially when IgM antibodies or complement is involved. Spherocytes may be present on cytology of peripheral blood smears. Diagnosis based on autoagglutination will suggest surface-bound antibody. Dilution of the sample with saline (1:1) will indicate whether true agglutination is present. If erythrocytes still agglutinate after dilution, they can be considered positive for surface-bound antibody molecules. Tests that are used for the diagnosis when autoagglutination is absent are the direct and indirect Coombs’ test; the direct test is more sensitive. The Coombs’ reagent is polyclonal sera directed against equine IgG, IgM, IgA, and C3 and is used in serial dilutions. The endpoint of the Coombs’ test is agglutination, but it can also be used to test for antibody- or complement-mediated lysis. Direct Coombs’ test may yield a false-negative result if an incomplete set of reagents is used, if blood is not tested at both 4° C and 37° C, or if severe hemolysis has resulted in removal of the majority of antibody-coated RBCs from circulation. As previously described, a new direct immunofluorescence assay that uses class-specific antibodies to equine IgM, IgG, and IgA and flow cytometry has an increased sensitivity to detect red cell antibodies for the diagnosis of immune-mediated hemolytic anemia.
Treatment of Immune-Mediated Hemolytic Anemia
Therapy will be determined based on the level of anemia. In severe cases, whole blood transfusion may be indicated. Specific guidelines are given under Blood and Blood Component Therapy (see: “Blood and Blood Component Therapy”). Current drug administration should be discontinued. If, based on confirmed sepsis, antimicrobial therapy is required, drug therapy should be continued with a molecularly dissimilar drug. After blood samples for diagnostic tests (i.e., Coombs’ test or direct immunofluorescence assay) are obtained, immunosuppressive therapy may be considered. Most affected horses require immunosuppression with corticosteroids. Because immunosuppression carries the risk of potentiating infectious agents, underlying infectious disease conditions such as equine infectious anemia should be ruled out. Glucocorticoid therapy benefits the patient in the short term by reducing the function of macrophages to recognize antibodies complexed to red blood cells and in the long term by altering antibody production by B-lymphocytes. Dexamethasone used at 0.05 to 0.2 mg/kg IV q24h has been reported to have the greatest efficacy in treating immune-mediated hemolytic anemia in horses. The packed cell volume should be monitored carefully during the course of steroid therapy, and if the patient does not respond quickly, the frequency of administration may be increased to twice daily. In some instances, it may take up to a week for the full effect of steroid therapy to be reflected by a rise in packed cell volume. Once the packed cell volume is stable at greater than 20%, the steroid therapy should be carefully tapered by 0.01 mg/kg/day, while the horse is closely monitored for recurrence of hemolytic crisis. The major adverse reactions to long-term administration of corticosteroid in horses are laminitis, tendon laxity or weakness, and immunosuppression that leads to secondary infections. Therefore the goal is to reduce to the lowest effective dose as soon as possible. Alternate day therapy should be administered for the last week of therapy. Some individuals may require therapy for several weeks until disease resolution occurs. Although only a single equine case report has been published, the use of azathioprine (5 mg/kg PO q24h) and cyclophosphamide (300 mg/m2 body surface area) was successful in managing a case of refractory immune-mediated hemolytic anemia.