#2402 Reproducibility of memory B-cell and HLA antibody detection in patients on kidney transplant waiting list

Abstract

Abstract Background and Aims Detection of HLA antibodies (HLA-Abs) impacts on the access and outcomes of kidney transplantation (KT). Currently, evaluation of HLA-Abs in serum is performed using single antigen bead assays (SAB). The analysis of circulating HLA-specific memory B cells (mBc), capable of differentiating to HLA-Abs secreting cells, was conceived with the aim of complementing the study of the HLA-Abs found in serum, but there is debate regarding the representativeness and reproducibility of the test. Thus, we studied HLA-Abs generated by mBc from patients on the waiting list (WL) at two different time points without sensitizing events in between and compared them with HLA-Abs detected in serum. Method We selected 7 patients on the WL for a first KT with a cPRA of 3%-100%: 4 had pregnancies and 2 had blood transfusions as known sensitizing events. We collected blood and serum samples at 2 time points separated by an average of 3.5 months (T1, T2). We cultured polyclonally activated PBMCs and isolated and concentrated IgG from the supernatant (SN) to study HLA-Abs. We tested SN and sera with SAB assays. Beads with MFI >750 were considered positive. Results SAB serum study (Fig. 1a). Eighty-two percent of the specificities found at T1 were also detected at T2, and 97% of those identified at T2 were also observed at T1. Thus, there was good concordance between T1 and T2 samples (κ = 0.869, Cohen) and the MFI the specificities correlated (p < 0.0001, Spearman). SAB SN study (Fig. 1b). Forty-one percent of the positive specificities detected at T1 were also identified at T2, and 59% of the positive specificities found at T2 had been seen at T1. Thus, there was a moderate concordance between T1 and T2 samples (κ = 0.454, Cohen) and the MFI of the specificities still correlated (p < 0.0001, Spearman). Only 42% and 35% of the positivities found in serum were also detected in the SN at T1 and T2, respectively. However, we found 9 positivities unique to the SN T1 (100% HLA-ABC) in 2 patients with cPRA>80% and we also identified a total of 20 positivities unique to the SN at T2 (45% HLA-ABC, 55% HLA-DPDQDR) in 3 patients with cPRA>80%. Conclusion The study of the degree of sensitization with SAB serum analysis remains stable between samples along time, while the mBc analysis shows less reproducibility. We identified a higher number of HLA-Ab in the study of serum compared with the study of the SN of mBc; however, we found some HLA-Abs unique to the SN. We suggest that, probably due to the small number of circulating mBC, increasing the number of samples tested for mBc improves the representativeness of the test.