Novel flow cytometric assay 'Flow IAT' reveals hidden IgM driving complement activation in autoimmune hemolytic anemia

Autoimmune hemolytic anemia (AIHA) is caused by antibody-driven red blood cell (RBC) destruction, which can involve complement activation. Correct detection of complement involvement in AIHA is now more relevant since the introduction of novel complement inhibitors. The Data Registry of AIHA (DRAIHA) study is a Dutch national cohort set up to investigate pathophysiology and diagnostic predictors. We developed a novel flow cytometry-based assay to assess serum-derived IgG and IgM binding and complement activation in AIHA and used this assay to analyze categorized DRAIHA samples (cold AIHA and warm AIHA with and without complement). In our novel flow cytometric indirect antiglobulin test (Flow IAT), bromelain-treated group O donor RBCs were incubated with serum from AIHA patients (n=48) or controls (n=21) in EDTA. Cells were washed and incubated with donor serum as complement source, with anti-C5 to prevent lysis. The RBCs were analyzed for IgM, IgG, C4 and C3 opsonization, using flow cytometry. IgM binding correlated strongly with C4 and C3 deposition, whereas IgG binding correlated poorly. The strong correlation between IgM and C4 remained when analyzing per AIHA subtype. The Flow IAT demonstrated enhanced sensitivity for detecting IgM antibodies compared to conventional diagnostics, while its sensitivity for IgG detection was lower. In conclusion, we developed the Flow IAT, a novel indirect serum-based assay using flow cytometry to quantify binding of circulating anti-RBC antibodies and antibody-mediated complement activation. The Flow IAT successfully identified the presence of complement-activating autoantibodies and demonstrated that IgM strongly correlated with complement activation in both cold and warm AIHA.