Aim: To distinguish between HLA specific antibodies with legitimate biological activity from false Luminex HLA antibody reactivities using an adsorption crossmatch with elution (AXE) protocol.
Methods: The AXE protocol selects lymphocytes expressing HLA antigens of interest and crossmatching them with serum of interest containing HLA donor specific antibodies (DSA). Only bone fide HLA DSA will adsorb to the HLA antigen of interest. The cells are washed and the bound antibodies are then eluted from the cells. Antibodies are then identified using the single antigen bead (SAB) Luminex assay. The results from the SAB assay are then compared to SAB results from unadsorbed sera.
Results: Sera (n=30) containing dubious HLA antibody profiles were selected and crossmatched with selected cells using the AXE protocol. The resulting eluates were tested with the SAB assay and compared to previously untreated SAB results. These results were also compared to Halifaster flow crossmatch results. Suspected false positive antibodies identified by Luminex were not adsorbed donor cells and agreed with Halifaster flow cytometry crossmatch results.
Conclusions: The AXE protocol allows labs to determine if anti-HLA antibodies seen in Luminex bind to native HLA on donor cells, indicating real biological activity. AXE can therefore define whether Luminex profiles reflect in vivo binding to HLA antigens. Given the concordance with flow crossmatch the AXE technique could improve interpretation of the flow cytometry crossmatch, although future studies are needed.