HLA Antibodies Detected by Luminex-Single Antigen Beads and Virtual Crossmatch in Renal Transplantation

Abstract

Introduction: The most important immunologic consideration for kidney transplantation is the presence of preformed donor-specific anti-HLA antibodies (DSA). Solid-phase assays, such as Luminex-Single Antigen Beads (LSA), allow an accurate detection and characterization of pre-existing DSA in transplant candidates. But the ability of LSA in detecting low level of antibodies makes hard to correctly predict crossmatch results in donor selection, that is to perform a virtual-crossmatch (v-XM). In this study was to retrospectively analyze the accuracy of our v-XM protocol in predicting the results of actual-crossmatch (a-XM) in renal transplantation. We also investigated correlation between a-XM negative results and strength/specificity of pre-formed DSA. Methods: We retrospectively analyzed the correlation between v-XMs and a-XMs performed within January 2007 and June 2011 at Regional Transplant Center of Lazio (Italy). In carrying out v-XM, we considered as “unacceptable mismatches” the donor HLA class I and class II molecules against which the patients showed LSA-detected DSA with normalized MFI ≥5000. The LSA-DSA showing MFI lower than the established cutoff were defined as “acceptable DSA” for v-XM. All donors were routinely typed for HLA-A, -B, -DR and -DQB1 molecules by SSP methods. Consequently, in performing v-XM, we didn’t consider anti-HLA-C, -DQA and -DP DSA. On the basis of a negative v-XM, we performed 328 a-XMs between sera from 194 sensitized renal transplant candidates (%PRA class I = 47±39, %PRA class II = 59±32) and T/B lymphocytes from 219 cadaver donors using both cytotoxic-crossmatch (CDCXM) and flow-cytometry-crossmatch (FCXM). Results: The v-XM results predicted by LSA-DSA showed good correlation with both CDC and FC aXMs (96% and 90% respectively; Sensitivity = 100%). The high sensitivity of v-XM was related to the lack of false-negative DSA results. The limited specificity with both the XM techniques (CDCXM = 78%, FCXM = 81%) was due to the presence of “acceptable DSA” and/or anti-DQA/-DP DSA in sera used to perform 86 a-XMs. Ten of these a-XMs gave positive results by both CDC and FC assays, 20 gave positive results by FCXM only and 56 were negative by both the assays. During the study period, 104 (54%) of the 194 sensitized patients received a kidney transplant. Twenty-eight of the 104 (27%) transplanted patients had “acceptable” DSA and/or anti-DQA/-DP DSA. Late antibody mediate rejection due to preformed DSA was observed in 2 patients only; anti-DP DSA at “unacceptable” levels (MFI>5000) were present in these patients. Conclusion: Our v-XM protocol showed high sensitivity in predicting donor-recipient immunologic compatibility. Data presented here demonstrate the importance to evaluate DSA strength for implementing v-XM results in selection of recipient for kidney transplantation. Nevertheless prediction based on LSA assay feels the amount of antigen on beads and may not be representative of the amount found on cells. The findings of anti-DPB/-DQA DSA highlight the importance to define all the donor HLA molecules to perform an accurate v-XM. Finally, the good outcome of transplants carried out in patients with low levels of DSA and negative XM underlines the importance to discard discarding weak and clinically irrelevant DSA in performing v-XM.