Aim: Multiplexed flow cytometry-based single antigen bead (SAB) immunoassays are the current standard for detection and identification of anti-HLA alloantibodies. While highly sensitive and specific, well-recognized limitations of SAB including non-linearity, saturation, reagent variability, and potential for assay inhibition limit quantitative determination of alloantibody concentrations using the assay output of Mean Fluorescence Intensity (MFI). This limitation is a barrier to accurate assessment of potential clinical impact of anti-HLA antibodies. We hypothesized that anti-HLA antibody quantification by titration, as compared to MFI, will be a more informative measure of antibody quantity.
Method: We examined the utility of SAB titration in 19 serum samples from adult patients listed for kidney transplantation with known high levels of individual anti-HLA antibodies (MFI > 8,000 and/or present at 1:10 serum dilution). SAB testing was performed using LABScreen Single Antigen Class I and Class II reagents on a FLEXMAP 3D analyzer with each serum sample was tested by a serial 4-fold dilution (neat, 1:4, 1:16, 1:64, 1:256). Antibodies detected at > 1000 MFI at any dilution (n = 625) were included in the analysis.
Results: Serum dilution notably resulted in non-proportional decreases in MFI- e.g. the first 4-fold dilution resulted in an average MFI change of -7.2%, range -100% to +5,280% (excluding 2 cases of antigen excess prozone there were undetectable in neat serum). MFI increased after dilution in 135 antibodies. Conversely, 26 antibodies became undetectable after 4-fold dilution (initial MFI values 1179 – 10,617). Several antibodies were persistently detectable at 16- (n = 236, 37.8%), 64- (n = 160, 25.6%), and 256-fold dilutions (n = 70, 11.2%).
Conclusion: These results illustrate the limitations in reliance on MFI for evaluation of anti-HLA antibody concentrations. The results here indicate that serum dilution and titration by SAB is a more robust measure of anti-HLA antibody concentration. The ability to accurately evaluate anti-HLA antibody concentrations has significant clinical implications for assessing pre-transplant immunologic risk, evaluating potential responsiveness to desensitization, and assessing responses to treatment. Current work aims to correlate dilution and titration of donor specific antibodies with biopsy proven acute rejection.
