P237 Specific pattern of non-specific ANTI-HLA antibody reactivity?

Abstract

Solid phase assays (SPAs) have significantly improved our ability to assess the presence of anti-HLA antibody (Abs) that would be deleterious to the allograft post-transplant. SPAs have also demonstrated increased specificity and sensitivity for alloantibody detection. However, several studies have identified intrinsic and extrinsic factors that negatively impact the reliability of SPAs. Thus, substances in sera that inhibit/interfere with the Abs binding have been identified. In addition, presence of denatured HLA antigens and/or inconsistent antigen density on latex beads has been noted. These factors coupled with the increased sensitivity of the SPAs could lead to false positive/false negative results. In the present study, using single antigen beads (SAB), we demonstrate the detection of a highly specific pattern of HLA Class II alloantibodies (MFI > 1,000) against a small subset of DRB1, DRB3 and DP antigens. What is remarkable about this pattern is that a near identical profile of alloantibodies was detected in at least 6 patients (5 pre- and 1 post-transplant) and was conserved over time. Thus, Abs against the following antigens (ranked from highest MFI to lowest MFI; DR52 (03:01) – DR14:02 – DP10 – DR16:01 – DR8 - DR14:54 – DR9 – DR16:02 – DR14:01 – DP13 – DP19 – DR13) were identified in all the patients. An epitope that would help explain this pattern of reactivity could not be identified. To confirm this pattern, the sera were tested using SAB from another vendor. This pattern was completely absent, suggesting that the observed reactivity was non-specific. However, pretreatment of sera with a variety of products known to reduce interfering substances did not alter the reactivity pattern suggesting that it was “real”. Testing of sera for the presence of HLA Class I and II Abs using screening beads (each coated with multiple HLA antigens) confirmed the observations made with SAB further suggesting that the observed reactivity was “real”. Finally, surrogate flow crossmatches with donor cells expressing one or more corresponding antigens were largely T- and B-cell negative suggesting that the observed reactivity was most likely non-specific (surreal?). This study underscores the complexity involved in interpreting and then assigning presence/absence of anti-HLA alloantibodies using the highly sensitive SPAs.