P011 : MAKING THE MOST OF HLA SINGLE ANTIGEN ANTIBODY TESTING

Abstract

Aim Solid-phase single antigen (SA) HLA antibody (Ab) testing is utilized to determine virtual cross matches (VXM) and to inform the degree of donor/recipient (in) compatibility, thus assisting in choosing induction, maintenance, and therapeutic approaches post transplant. Serum samples from highly sensitized patients (pts) are prone to inhibition “Prozone” effects, which complicate accurate assessment of antibody strength. EDTA pre-treatment or C1q-binding Ab (C1q) assays, as well as titration studies, were proposed to alleviate this problem. Methods Serum from 55 highly sensitized pts (77 SA bead assays; 7600 data points) were tested. MFI values of SA-IgG and C1q assays, as well as titration data, were compared. SA-IgG data was stratified into Strong, Strong Moderate, Weak Moderate, Weak and Negative (>10,000; 5001–10,000; 2501–5000; 1000–2500, and 10,000; 5001–10,000; 1001–5000; 200–1000; and Results “Prozone” effect, as revealed by titration studies, was observed in 47/55 pts (56/77 tests; 73%), with most samples reaching their highest MFI values at a titer of 1:16. Importantly, in many cases, discordant antibody strengths were observed between neat MFI values and titrations. Specifically, 34/55 pts (40/77 tests) exhibited Weak–Weak Moderate MFI values while antibody titers were high (1:256–>1:1024). Conversely, 14/55 pts (18/77 tests) exhibited Moderate Strong–Strong MFI values while antibody strengths by titration were low (1:1–1:8; 17 additional tests had antibody titers of 1:16). Positive C1q test results correlated with SA-IgG assays with titers of 1:32–64 and higher. Conclusion “Prozone” effect is frequently observed in SA-IgG testing for highly sensitized pts. While EDTA pretreatment of sera or C1q testing can alleviate this issue, titration studies provide additional granular information regarding the strength (affinity/avidity) of the antibody which can assist in planning for desensitization or treatment of AMR. In our hands, use of titration studies lowered the assay-to-assay variability known to affect luminex based multiplex assays and, as we have previously reported, increase the accuracy of VXM prediction.