P025 : PRONASE TREATMENT OF SINGLE ANTIGEN BEADS (SAB) REVEALS A COMPLEX BIOLOGY FOR EXPLAINING DENATURED HLA ANTIGENS

Aim Microparticle-based, solid phase assays are the preferred method to detect and identify HLA antibodies. However, there are many reports of antibody reactivities, which seem to be directed against “denatured” antigen on SABs and, furthermore, appear to be clinically irrelevant. In our laboratory, such reactivities are identified by negative FlowPRA™ and positive SAB results. The goal of this study was to determine whether pre-treatment of SABs with pronase, which is known to reduce non-specific binding in the flow crossmatch (FCXM), could eliminate denatured epitopes on SABs and reduce the false-positive reactions. Methods A query of our database identified all patients who had negative FlowPRA™ and positive SAB results over the previous 12 months. Sera representing the most commonly occurring “denatured” specificities were then tested using SABs pre-treated with pronase (1:32 in RPMI media) and incubated at 37 °C for 15 min. The beads were then washed with 20% RPMI in FCS and then with bead wash buffer. The SAB assay was then performed as per manufacturer instructions. Results There were 426 total patients that met the inclusion criteria; 287 had Class I specificities, 81 had Class II specificities, and 29 had both. The most commonly identified false positive specificities for each locus were B82 (9%, ∼5000MFI), A80 (7%, ∼3500MFI), Cw1, 12, 15 (2%, ∼11,000MFI), DQ7, 8, 9 (5%, ∼6000MFI), DR18 (13%, ∼3500MFI), and DP1 (23%, ∼3500MFI). While the A80 and DQ7, 8, 9 reactivities were insensitive to pronase treatment, the B82, Cw1, 12, 15, DR18, and DP1 reactivities were completely abrogated. Conclusions Several studies have demonstrated false-positive reactivity of SABs due to denatured epitopes, which have subsequently been shown to be clinically irrelevant and do not result in a positive FCXM. Our results demonstrate that some, but not all, denatured epitopes are removed by pronase pre-treatment of the SABs. Because pronase has a broad specificity, the precise mechanism by which it reduces certain denatured epitopes but not others is unclear. These findings indicate that the concept of denatured epitopes is not a singular entity and may reflect a complex etiology that differs for each allele.