Positive virtual crossmatch with negative flow crossmatch results in two cases

Pre-transplant (Tx) presence of HLA antibodies (HLA-Ab) especially donor specific antibodies (DSA) has been correlated with post-Tx rejection. While crossmatch (XM) is the specific method to identify DSA, logistical reasons prevent performing a prospective XM in all transplants. In such cases DSA as identified by solid-phase assay (SPA) are being used to perform a virtual crossmatch (VXM). We present two cases, a heart-lung transplant and a kidney transplant, for which testing detected a presumptive DSA with discordant results: a negative flow cytometric crossmatch (FXM) and a positive VXM using SPA. The subsequent investigation determined the antibody, in both cases, was presumably directed against an epitope of a HLA-B*44 antigen found on the single antigen beads (SAB) used in the SPA but not against the native form on the donor lymphocytes used in the FXM. Manufacturing of SAB beads results in denaturation of epitopes, majority of which are removed from the final product, but residual amount is present on the final product. Denaturation of majority of antigen epitopes on single antigen beads did not remove the activity of the recipient's antibodies but it did diminish the activity of positive control serum. This indicates denaturation of some of the HLA-B*44 antigen during manufacturing of the SAB may have lead to the reactivity. Antibody mediated rejection does not appear to be associated with the titer of this antibody to denatured antigen in the first case and so clinical relevance of such antibodies is unclear. Subsequently a second case of discordant FXM and VXM was identified in a potential kidney transplant patient who went on to an uneventful transplant. In this case, lymphocytes from the donor were positively shown to express HLA-B*44:02 using known anti- HLA-B*44:02 control serum. Platelets identified as HLA-B*44:02 could adsorb the anti-HLA-B*44:02 from the control serum activity but not from that of the recipient's anti- HLA-B 44 antibody adding evidence that this antibody should best be classified as a false positive finding. The presence of such an antibody if misidentified may result in unnecessary therapy being instituted or the inappropriate denial of an organ for transplantation.

► Anti-HLA specificities detected by solid phase assays are used to perform virtual crossmatch (VXM) ► VXM is used as a substitute for actual crossmatch. ► We present two cases with positive VXM and negative flow cytometric crossmatch. ► Denatured antigens may explain discordant results. ► Antibodies to denatured antigens may be clinically irrelevant.