From Accurate Assessment of Anti-HLA Antibody MFI to Complement-Binding Assays

Single antigen bead (SAB) and complement-binding assays are commonly used to monitor immunization in transplant patients. Like all new diagnostic assays, some considerations have to be appreciated to avoid a biased utilization. By truly decreasing antibody concentration, SAB monitoring in sensitized patients experiencing apheresis offers a good opportunity to explore analytical interference. We explored analytical artifacts by analyzing the role of prozone and saturation effects through a concrete example of a single patient who experienced immunoadsorption. We then assessed, on a larger cohort, the link between an accurate assessment of mean fluorescence intensity (MFI) and the C1q and C3d binding assays. Finally, we compared MFI with the two main available SAB assays. After immunoadsorption, the MFI of some antibodies unexpectedly rose. We showed that this increase was due, in part, to both a prozone effect and a saturation of the beads. Dithiothreitol treatment appeared to be an efficient way to avoid a prozone effect. The analysis of dilution profile was an interesting tool to detect a saturation effect. The comparison of the two main SABs revealed a systematic difference of 3000 MFI. MFI was a strong predictor of C1q and C3d positivity. Complement-positive antibodies had a higher MFI (p<0.01). Despite the great contribution of SAB assays in anti-HLA antibody assessment, the knowledge of analytical interference is necessary to avoid any misleading conclusions. With regard to the interference between MFI and complement-binding assays, their place in risk stratification has to be clarified.