The direct antiglobulin test (Coombs’ test) is a laboratory test for detection of autoantibodies associated with immune-mediated hemolytic anemia.
Understanding when direct antiglobulin (Coombs’) testing is necessary, and appropriate interpretation of the test results is important to an accurate diagnosis of IMHA. A direct antiglobulin test is used to detect the presence of antibody against red blood cells when the anti-erythrocyte concentration is too low to cause spontaneous autoagglutination. There is a direct antiglobulin test and an indirect antiglobulin (Coombs') test. The direct antiglobulin test detects antibodies attached to red blood cells and is the most useful test in diagnosing IMHA. The indirect antiglobulin test detects antibodies to red blood cells that are present in the patient’s serum. Since most anti-erythrocyte antibody is found attached to the RBC’s the direct antiglobulin test is a more sensitive test and results in fewer false negatives. Test results of a direct antiglobulin test are reported simply as positive or negative. The actual interpretation of this result is not so simple and should be given more attention.
Washed patient erythrocytes are incubated with the Coombs’ reagent. The Coombs' serum may be monovalent (directed against one immunoglobulin class such as IgG or complement fragment) or may polyvalent (directed against IgG, IgM, and complement). The Coombs' serum must be species-specific for an accurate result. If autoantibody to immunoglobulins and/or complement are present on the patient’s RBCs, the Coombs’ reagent antibodies bind to them and crosslinking occurs. This is visible as gross or microscopic agglutination and represents a positive test result.
Many laboratories perform dilutions of the Coombs’ reagent to decrease the chance of false negative reactions due to the presence of very large amounts of antibody in undiluted samples. The phenomenon of a false negative test result is referred to as a prozone effect.9 Dilution of the Coombs' reagent allows the correct ratio between red cell antibody and reagent to allow agglutination. Titers are not commonly reported with test results since there is no clear association between the titer and disease severity or any further diagnostic information available from the titer value.
Most studies report some degree of false negative test results with use of the direct antiglobulin test to detect agglutination. Two studies noted a sensitivity of only 60%.6,8 Other studies also noted the occurrence of negative test results in dogs with IMHA. For this reason, it is important to search for other possible causes of hemolysis. Furthermore, the possibility of IMHA should not be summarily dismissed because a negative test result.
- Wilkerson MJ (2000) Isotype-specific antibodies in horses and dogs with immune-mediated hemolytic anemia. J Vet Intern Med 14:190-196
- Klag AR, Giger U, Shofer FS (1993) Idiopathic immune-mediated hemolytic anemia in dogs: 42 cases (1986-1990). J Am Vet Med Assoc 202:783-788