Virtual crossmatch in the Luminex-Single Antigen beads era

High sensitivity of Luminex Single Antigen Beads (SA) assay has greatly improved HLA antibody characterization. The ability of SA in detecting low level of antibodies makes it hard to predict a crossmatch result in donor selection in order to perform a virtual crossmatch (v-XM). We retrospectively analyzed the correlation between v-XM and actual crossmatches (a-XM) performed within Jan 2007-Oct 2010. In carrying out v-XM we considered unacceptable all the donor HLA Class I/II molecules against which the patients had antibodies with normalized MFI >5000. Donors were genomically typed for HLA-A, B, DR and DQB1, so we didn't consider anti-HLA-C and -DP donor-specific antibodies (DSA). On the bases of a negative v-XM, we performed 263 XMs on sera from sensitized patients by both cytotoxic and flow-cytometric methods (CDC XM and FC XM). In 70 a-XMs the patient sera contained weak DSA (MFI < 5000). Ninety-one percent of the CDC/FC a-XM gave concordant negative results with v-XM; no DSA were detected by SA in all these sera. Four percent of the CDC/FC a-XM gave positive results; all the sera contained low level of Class I/II DSA (MFI range: 2200-4800). Five percent of the a-XM gave positive results only by FC; all these sera contained low level of Class I/II DSA (MFI range: 2100-4800). No positive CDC XM and/or FC XM results were found in the absence of DSA. The accuracy of our v-XM in predicting the outcome of a-XM was driven by a high sensitivity (100% for both CDCXM and FCXM) and specificity (94.8% for CDCXM, 80.7% for FCXM). However, 46 a-XMs were negative by both methods, even if DSA were present; in 4 cases we only found anti-DPB1 (MFI > 13000) or anti-DQA1 (MFI > 9500) DSA. In 25 of the 46 DSA positive/FCXM negative cases, patients were transplanted and no antibody-mediated rejection was observed during the follow up. In conclusion, v-XM protocol was highly useful in predicting donor-recipient immunologic compatibility and showed the importance in evaluating levels of antibodies considered permissible for transplantation. Prediction based on SA assay takes into account the amount of antigen on beads and may not be representative of the amount found on cells. The good outcome of transplant carried out in patients with low levels of DSA and negative XM underlines the importance to set a cut-off value for discarding weak and likely irrelevant DSA to perform a clinically useful v-XM.