P240 Unmasking prozone, detecting the hidden donor specific antibody

Abstract

Given evidence that donor specific antibodies (DSA) to Human Leukocyte Antigens (HLA) adversely affect kidney transplant outcome, sensitive techniques for detecting HLA antibodies have been developed. Single-antigen (SA) Luminex-based tests, in particular, are highly sensitive and are used to define ‘unacceptable’ donor antigens in a virtual crossmatch. Although it has largely revolutionized HLA antibody detection, the Luminex SA bead assay however does occasionally present with some problematic results such non-specific reactivity and interfering substances which can make interpretation challenging. One phenomenon that labs encounter is the inhibition or ‘prozone’ effect in cases when antibody is in excess. A consequence of this is that the antibody specificities with the highest concentration can appear to have weak or absent reactivity to specific SA beads. This is believed to be caused by complement components competitively displacing the anti-IgG detecting antibody because it has been shown to be reversed with complement inhibitors such as EDTA, heat inactivation, serum dilution and C1-inhibitors. However, this inhibition effect occurs only with some patient serums and not across all the antigens. We investigated under which circumstances this occurred and whether some antigens were more likely to be affected. Post-transplant serum was tested with and without EDTA by Single Antigen Bead assay. In our center we found the inhibition effect predominantly in highly sensitized patients that had previously received grafts. Class II antigens, in particular HLA-DQ, was found to be inhibited by the presence of antibody excess, and reversed with EDTA treatment. Interestingly, the inhibitory effect was often seen with beads that specifically expressed donor specific alpha and beta chain antigen combinations, whereas beads carrying only the beta chain antigen of the donor but a different alpha chain were not affected. This indirectly suggests that the number of epitopes targeted on each HLA molecule, rather than just antibody titre, may influence the ‘prozone’ phenomenon.