Prozone phenomenon in pretransplant testing: An interesting conundrum involving solid‑phase and cell‑based assays

BACKGROUND: Human leukocyte antigen (HLA) is a major determinant in deciding upon solid organ histocompatibility. Donor‑specific anti‑HLA antibodies (Donor‑specific anti‑HLA antibodies (DSAs)) are always a contraindication for solid organ transplantation, and identification of DSA becomes very crucial before transplantation to provide long‑term graft survival. For identification of DSA, usually, either cell‑based or HLA bead‑based assay is being used in laboratories. However, both cell‑based and bead‑based assays have certain limitations. One such common limitation is “prozone effect,” which can give false‑negative results. Here, we would like to present a small pilot study to analyze the effect of the prozone phenomenon in the cell‑based and HLA bead‑based assays and its utility in histocompatibility testing. MATERIALS AND METHODS: In a series of four experiments, cell‑based assay, flow cytometric cross‑match (FCXM), and HLA bead‑based flow cytometric panel reactive antibodies (PRAs) were performed. Single‑antigen bead (SAB) testing was conducted as a first experiment on four known positives samples for anti‑HLA antibody‑antibodies. In the second experiment, these four samples were pooled together (called pooled sera in the text) and tested for FCXM and PRA. In the third experiment, known commercially available positive control sera were mixed with pooled positive sera (positive control sera + pooled sera) to prepare, what we have called “positive concoction” in the text. In the fourth experiment, the positive concoction was diluted serially (1:2, 1:4, 1:8, and 1:16) and FCXM and PRA were performed again to analyze and compare the prozone effect. RESULTS: Pooled sera did not have the expected median fluorescence intensity (MFI) values in FCXM assay, whereas the PRA was showing >90% positivity. In positive concoction, the MFI of FCXM assay was observed to be declining; however, PRA values remained almost constant. Dilutions of the pooled sera showed that MFI values of FCXM assays were increased suddenly after dilution. The highest MFI values were observed in 1:4 dilution of the sera, and then, it declined gradually, but the PRA values remained almost constant even after serial dilutions. CONCLUSION: In our experimental findings, it was clear that cell‑based assay (FCXM) was more severely affected by the prozone, whereas solid‑phase (flow PRA) assay remained resistant to prozone.