P016 : PRONASE TREATMENT OF T LYMPHOCYTES DECREASES CDC CROSSMATCH TESTING TIME BY 50% AND MAINTAINS DSA COMPLEMENT ACTIVATING SPECIFICITY

Aim Crossmatching (XM) via AHG-CDC and Flow cytometry (FC-XM) has enhanced sensitivity for detecting donor specific antibodies (DSA) but limits IgG functional characterization. We propose Pronase treated lymphocytes enhance Amos-CDC XM sensitivity by decreasing CD55 complement (C ′ ) inhibition without loss of functional specificity. Method Five well characterized sets of T-cells/sera combinations were chosen to give borderline (≈60–120 MCS) and strong (>300 MCS) FC-XM results. Antibody specificities were determined using LabScreen® SAB. T-cells were isolated using HLA Cell Prep I Dynabeads® and pronase treated (optimized for CDC testing, 0.5 mg/mL for 10 min at 37 °C). Pronase treated Amos-CDC (PT-Amos-CDC) XM was modified for C ′ optimization (reduced incubation to 20 min). Standard Amos-CDC, AHG-CDC and FC-XM were performed in parallel. Results A total 15 XM were performed. Cumulative Class I DSA strength ranged from 5451 to 35,162 normalized MFI. Of those, 9 were positive with all methods, 1 was positive with all methods except Amos-CDC, 3 were only positive by FC-XM, 1 was positive by PT-Amos-CDC and FC-XM, and 1 was only positive by PT-Amos-CDC. Conclusion PT-Amos-CDC T-cell XM markedly reduces testing time by 50% and maintains DSA specific C ′ activation that is not assessable via AHG-CDC and FC-XM methods. This provides valuable reduction in ischemia time. Pronase and C ′ steps require optimization to enhance antibody mediated C ′ activation without inducing non-specific C ′ sensitivity. Although PT-Amos-CDC did not appear as sensitive as AHG-CDC and FC-XM, some cases are better potentiated than others and appears to be more sensitive than the standard Amos-CDC. Further studies will be useful in characterizing these antibodies (e.g., C1q-SAB & IgG subclassing) and the potential use of human C’ in the assay. PT-Amos-CDC provides a rapid cell based in vitro assay that we believe will correlate with in vivo DSA C ′ fixing potential. This makes the assay valuable not only to reduce ischemia time but also in assessing risk of hyperacute and accelerated acute rejection. Additional studies will be needed to determine if the assay can be adapted for B-cell XM as well.