Aim: In the current study, flow cytometry crossmatch (FCXM) using urine culture derived cells (UCDC) versus FCXM with donor lymphocytes was evaluated. The detection of donor specific antibody (DSA) by FCXM using UCDC from recipients suffering from antibody mediated rejection with documented DSA by single antigen bead (SAB) assay was retrospectively performed. The application of UCDC for delineating the specificity of HLA antibodies was also investigated, especially with patients’ samples with difficult assignment.
Methods: UCDC were prepared from fresh urine samples collected from kidney recipients with known donor HLA typing. Degree of donor derived cell enrichment in these UCDC were confirmed by Short Tandem Repeat (STR) analysis. Antigen expression levels of HLA were compared between donor lymphocytes and UCDC using flow cytometry. FCXM was performed using UCDC against patients’ samples in those patients with suspicion of DSA in order to confirm the presence of DSA.
Results: STR analysis confirmed that 100% donor derived cells were successfully obtained from UCDC derived from 50 kidney transplant recipients. Comparable mean channel shift (MCS) was observed in FCXM with UCDC and donor lymphocytes (P = 0.345), demonstrating similar expression of HLA antigens on these two cells by evaluation of anti-HLA-ABC and anti-HLA-DR, -DP and -DQ after interferon-gamma stimulation. Positive FCXM results were also obtained with patient sera with suspected anti-HLA Class I or Class II antibodies targeting the donor mismatched HLA antigens expressed in UCDC (Figure 1).
Conclusion: Our results suggest that UCDC is a feasible alternative for detecting DSA using FCXM. As UCDC express the exact HLA antigens of the donors, they are unlimited sources for revitalizing of donor cells, making retrospective donor-specific crossmatch feasible to allow accurate DSA interpretation in particular when the donor mismatched antigens are not covered in the panel of commercial SAB.
