Finding the hidden IgM: a study of complement-activating autoantibodies in different autoimmune hemolytic anemia subtypes using an in vitro flow indirect antiglobulin test (flow IAT)
Autoimmune hemolytic anemia (AIHA) is characterized by autoantibody-driven red blood cell (RBC) destruction. The majority of AIHA is classified as either warm AIHA (typically IgG) or cold AIHA (typically IgM). IgM autoantibodies cause hemolysis through complement activation, IgG predominantly through phagocytosis via Fc-receptors on macrophages in the spleen. The direct antiglobulin test (DAT), a standard test for AIHA, detects antibodies and complement (C3d) on patient RBCs, but lacks sensitivity for IgM. In warm AIHA with DAT positive for both IgG and C3d, it is thought that some IgG’s may activate complement. Alternatively, IgM may be underdetected in this AIHA subtype. Correctly determining the mechanism is increasingly important as more targeted therapeutics, such as complement inhibitors and FcRn inhibitors are developed. We tested if a serum-based flow cytometry assay (flow IAT) would be useful to quantify circulating autoantibodies and their complement-activating capacity in AIHA patients.
Serum samples from AIHA patients were collected in the DRAIHA study, and subclassified as warm AIHA with complement (DAT IgG+, C+, cold agglutinin titer <64), warm AIHA without complement (DAT IgG+, C-) and cold AIHA (DAT IgG-/weak, C+). Serotyped bromelain-treated RBCs of healthy donors were incubated with serum from AIHA patients (n=51) or healthy controls (HC, n=21) in EDTA. RBCs were washed and incubated with healthy serum as a fresh complement source, with addition of anti-C5 to prevent lysis. RBCs were analyzed using flow cytometry for C3 and C4 deposition and IgG and IgM binding.
Among all AIHA subtypes, IgM binding and not IgG binding correlated strongly with C3 and C4 deposition. IgG binding was only detected in samples classified as warm AIHA, with or without complement deposition in DAT. IgM binding and complement deposition were almost exclusively detected in cold AIHA and in DAT complement positive warm AIHA, but not in warm AIHA without complement. Inhibiting C1q abrogated all complement deposition, underlining that complement activation in this set-up is classical pathway-mediated.
We successfully tested AIHA patient sera using flow IAT to quantify binding of circulating antibodies of both IgG and IgM class and antibody-mediated complement activation on donor RBCs. Regardless of AIHA subtype, the observed complement activation could be attributed to the binding of IgM, whereas IgG was not associated with complement activation. These results indicate that IgM may have a larger role in warm AIHA than previously recognized which may have clinical implications. Lastly, our assay proved suitable as a serum-based test for classical complement pathway involvement in AIHA and testing of complement inhibitors.
* & # these authors contributed equally