Evaluation of erythrocyte autoantibodies with flow cytometric phagocytosis assay

Abstract

Aims: Cellular assays such as the monocyte monolayer assay have been used to evaluate the clinical significance of antibodies to red blood cells (RBCs). Flow cytometry (FCM) has also been applied to antibody screening and diagnosing autoimmune hemolytic anemia (AIHA). Methods: RBC eluate from 131 patients with positive direct antiglobulin tests, along with three human monoclonal antibodies (two anti-D and one anti-Dib) were used to evaluate a new FCM two-color phagocytosis assay using markers that bind RBCs and monocytes. Dual-positive event percentages were compared with antibody titers and trimmed mean fluorescence intensity (MFI) from the 131 eluates and three monoclonal antibodies. Results: Percent positive by the FCM phagocytosis assay correlated well (R2=0.755) with trimmed-MFI in serial dilutions of two human IgG1 monoclonal antibodies, anti-D and anti-Dib, and 131 clinical samples with titers ranging from 2 to 2048. IgG2 antibodies showed lower phagocytosis relative to titers and MFI associated with these antibodies. Conclusion: The FCM phagocytosis assay can be used to evaluate AIHA-associated antibodies, with great potential to standardize immunohematology testing, especially in regard to establishing the clinical significance of RBC alloantibodies.