11-P

Abstract

Aim We have observed an increase in the number recipients for whom DQ only antibodies are detected. In many cases the antibody is broadly reactive with all antigens except their own, even in patients who report no sensitizing events. We were concerned that these were non-specific reactions and sought to eliminate potential interference. We compared 4 methods of IgM reduction: heat inactivation (HI), DTT, hypotonic dialysis (HD), and spin columns (SC). Methods 10 sera treated with DTT, HI, HD, SC and tested using GenProbe LSA class II. Untreated sera tested using the GP Class II phenotype beads and the One Lambda C1q assay. Hypotonic Dialysis by Slide-A-Lyzer G2 Dialysis 10K. Spin columns were PALL Nanosep 300K. Results All of the sera tested reacted with both phenotype and recombinant beads suggesting that the epitope is not denatured antigen. None of the IgM reducing treatments had significant impact on the DQ specificities detected by the GP LSA beads. The HD and SC both yielded an overall decrease in peak and mean raw MFI of 20-40%, but in general specificities were unchanged. In two cases these treatments removed the lowest antigens from the positive list and changed the epitope identified by the analysis software. In 5 cases the sensitizing DQ antigen was known (DSA from transplant or pregnancy) and included in the reactive epitope. 4/10 patients tested had no known sensitizing event to account for the presence of alloantibody. All 4 were also negative by C1q. In one case a multiparous woman demonstrated reactivity against her own DR52 allele under all conditions, except the C1q assay. Conclusions In this series IgM or other large molecule are not interfering factors. Sensitization to HLA-DQ is well documented and is associated with graft loss to AMR. Both the phenotype and single antigen beads can detect antibody that is unlikely HLA specific. In these cases further testing using antigen targeted xming should be used to confirm HLA specificity of the antibody.