hematology and oncology
Autoimmune Hemolytic Anemia
Diagnosis of Autoimmune Hemolytic Anemia
Peripheral smear, reticulocyte count, lactic dehydrogenase (LDH) Direct antiglobulin test Autoimmune hemolytic anemia should be suspected in any patient with a hemolytic anemia (as suggested by the presence of anemia and reticulocytosis). The peripheral smear usually shows microspherocytes (see photo) and a high reticulocyte count with few or no schistocytes, indicating extravascular hemolysis. Laboratory tests typically indicate hemolysis (eg, elevated LDH and indirect bilirubin). A high mean corpuscular volume (MCV) may occur due to extreme reticulocytosis. Hemolytic anemia in the setting of a low reticulocyte count is rare but can occur and is suggestive of severe disease. Autoimmune hemolytic anemia is diagnosed by detection of autoantibodies with the direct antiglobulin (direct Coombs) test (see figure). Antiglobulin serum is added to washed RBCs from the patient; agglutination indicates the presence of immunoglobulin or complement (C) bound to the RBCs. In warm antibody hemolytic anemia, IgG is nearly always present, and C3 (C3b and C3d) may be present as well. In cold antibody disease, C3 is present while IgG is usually absent. The test is ≥ 98% sensitive for autoimmune hemolytic anemia; false-negative results can occur if antibody density is very low or, rarely, if the autoantibodies are IgA. In most cases of warm antibody hemolytic anemia, the antibody is an IgG identified only as a panagglutinin, meaning the antigen specificity of the antibody can not be determined. In cold antibody disease, the antibody is usually an IgM directed against the I/i carbohydrate on the RBC surface. Antibody titers can usually be determined but do not always correlate with disease activity. The direct antiglobulin (direct Coombs) test may be positive in the absence of autoimmune hemolytic anemia, and thus should be ordered only in the proper clinical setting. A false-positive direct antiglobulin test may result from the presence of clinically insignificant antibodies. The indirect antiglobulin (indirect Coombs) test is a complementary test that consists of mixing the patient’s plasma with normal RBCs to determine whether such antibodies are free in the plasma (see figure). A positive indirect antiglobulin test and a negative direct test generally indicate an alloantibody caused by pregnancy, prior transfusions, or lectin cross-reactivity rather than immune hemolysis. Even identification of a warm antibody does not define hemolysis, because 1/10,000 healthy blood donors has a positive test result. Once autoimmune hemolytic anemia has been identified by the antiglobulin test, testing should differentiate between warm antibody hemolytic anemia and cold agglutinin disease as well as the mechanism responsible for warm antibody hemolytic anemia. This determination can often be made by observing the pattern of the direct antiglobulin reaction. Three patterns are possible: The reaction is positive with anti-IgG and negative with anti-C3. This pattern is common in idiopathic AIHA and in the drug-associated ormethyldopa-type of AIHA, usually warm antibody hemolytic anemia. The reaction is positive with anti-IgG and anti-C3. This pattern is common in patients with Systemic Lupus Erythematosus (SLE) and idiopathic AIHA, usually warm antibody hemolytic anemia, and is rare in drug-associated cases. The reaction is positive with anti-C3 but negative with anti-IgG. This pattern occurs in cold agglutinin disease (where the antibody is most commonly an IgM). It can also occur in warm antibody hemolytic anemia when the IgG antibody is of low affinity, in some drug-associated cases, and in PCH. Other studies can suggest the cause of AIHA but are not definitive. In cold agglutinin disease with unwarmed blood, RBCs clump on the peripheral smear, and automated cell counts often reveal an increased mean corpuscular volume and spuriously low hemoglobin due to such clumping; hand warming of the tube and recounting results in values significantly closer to normal. Warm antibody hemolytic anemia can often be differentiated from cold agglutinin disease by the temperature at which the direct antiglobulin test is positive; a test that is positive at temperatures≥37°C indicates warm antibody hemolytic anemia, whereas a test that is positive at lower temperatures indicates cold agglutinin disease. If paroxysmal cold hemoglobinuria (PCH) is suspected, the Donath-Landsteiner test, which is specific for PCH, should be done. In this test, the patient's serum is incubated with normal RBCs at 4°C for 30 minutes to allow for fixation of complement and then warmed to body temperature. Hemolysis of the RBCs during this test is indicative of PCH. Because the PCH antibody fixes complement at low temperatures, the direct antiglobulin (direct Coombs) test is positive for C3 and negative for IgG. However, the antibody in PCH is an IgG against the P antigen. 