Optimized flow cytometry to measure anti-ABO immunoglobulin G

Background: An accurate measurement of ABO antibody levels before and after ABO-incompatible organ transplantations is critical. We verified several assay steps of a previously reported protocol using flow cytometry, improved its shortcomings, and developed a more optimized protocol, which we named Flow ABO Ab, for the anti-ABO immunoglobulin G (IgG). Methods: Fixed red blood cells (RBCs) were sensitized with dithiothreitol-treated sera at 4°C. Residual hemagglutination and hemolysis were monitored on a time-dependent plot. Only singlet RBCs were analyzed, excluding RBC agglutinates, to obtain mean fluorescence intensity (MFI) ratios. Results: The sensitivity for anti-ABO IgG was compared between Flow ABO Ab and the column agglutination technique (CAT) in 34 seradiluted to have borderline reactivity of anti-ABO IgG. Flow ABO Ab yielded a positivity rate of 68% (23/34), whereas CAT yielded 50% (17/34) (P =.18). The CAT titer was highly correlated with the mean fluorescence intensity (MFI) ratio of Flow ABO Ab (r = 0.843, P<.001) in 17 undiluted sera. Using Flow ABO Ab, all group A (30 [100%]) and group B (30 [100%]) healthy individuals tested positive for anti-B and anti-A, respectively. Conclusions: Our protocol markedly improved on the previously reported protocol and provided its analytical performance to be comparable to that of CAT, suggesting its potential as an additional effective tool for the measurement of anti-ABO IgG. Further studies are needed to clarify the target MFI ratio for transplantation and whether Flow ABO Ab is preferable to CAT for a correlation with a clinical outcome.