In
autoimmune hemolytic anemia autoantibodies against erythrocytes lead to increased clearance of the erythrocytes, which in turn results in a potentially fatal
hemolytic anemia. Depending on whether
IgG or
IgM antibodies are involved, response to
therapy is different. Proper identification of the isotype of the anti-erythrocyte
autoantibodies is, therefore, crucial. However, detection of
IgM autoantibodies can be challenging. We, therefore, set out to improve the detection of anti-erythrocyte
IgM. Direct detection using a flow cytometry-based approach did not yield satisfactory improvements. Next, we analyzed whether the presence of
complement C3 on a patient's erythrocytes could be used for indirect detection of anti-erythrocyte
IgM. To this end, we fractionated patients' sera by size exclusion chromatography and tested which fractions yielded
complement deposition on erythrocytes. Strikingly, we found that all patients with C3 on their erythrocytes according to standard diagnostic tests had an
IgM anti-erythrocyte component that could activate
complement, even if no such
autoantibody had been detected with any other test. This also included all tested patients with only
IgG and C3 on their erythrocytes, who would previously have been classified as having an
IgG-only mediated
autoimmune hemolytic anemia. Depleting patients' sera of either
IgG or
IgM and testing the remaining complement activation confirmed this result. In conclusion, complement activation in
autoimmune hemolytic anemia is mostly
IgM-mediated and the presence of covalent C3 on patients' erythrocytes can be taken as a footprint of the presence of anti-erythrocyte
IgM. Based on this finding, we propose a diagnostic workflow that will aid in choosing the optimal treatment strategy.