*Purpose: In this study we present a new diagnostic approach to HLA typing in kidney transplant recipients using cultured renal tubular cells from urine samples facilitating clinical assessment of donor specificity of de novo occurring HLA antibodies.

*Methods: Urine samples of 10 patients after allogenic kidney transplantation (KTx) were collected and a primary culture of renal tubular cells was established according to a published protocol (Zhou et al. 2012, Nature protocols). After approximately 2 – 4 weeks of tissue culture and the third passage of cells DNA extraction was performed from a 10 cm² culture dish. For this study, patients were selected for the existence of stored spleen tissue to validate the analysis. To avoid contamination with patients’ autologous renal cells, only KTx patients without residual urine excretion were included. HLA typing was performed, comparing DNA from cultured tubular cells to DNA from spleen, both derived from the donor. To further rule out potential contamination of urine samples with patient’s autologous renal cells, fluorescence in situ hybridization (FISH) of tubular cultures was performed, as well as analysis for genetic polymorphic markers by human multiplex short tandem repeats.

*Results: Most attempts to establish a culture of renal tubular cells were successful, where rarely microbial contamination or insufficient proliferation of cells required a repeat urine sampling. Relevant contamination with autologous renal tubular cells was successfully ruled out using FISH or by analyzing polymorphic markers. Comparison of HLA typing from cultured renal tubular cells to their respective splenic control samples showed a consistent match in all patient samples. In selected cases we were already able to clinically interpret these findings in the context of circulating HLA antibodies.

*Conclusions: Screening for HLA antibodies has broadly become a yearly routine after KTx. Detection of previously unidentifiable de novo HLA antibodies is common during the follow-up after KTx due to widespread use of highly sensitive single antigen solid phase tests specific for individual HLA alleles. Assessing donor specificity of these antibodies, however, is frequently limited by incomplete or unavailable donor HLA typing data and / or donor samples for re-typing.

Theoretically, a graft biopsy could deliver donor DNA for HLA typing. However, this avenue is not frequently used since it is an invasive procedure and the acquired tissue is heterogeneous with varying degrees of recipient cellular material. We suggest that this new diagnostic approach for HLA typing in KTx patients offers a non-invasive option to determine donor specificity of de novo HLA antibodies typically occurring many years after transplantation. We propose that this technique is valuable in clinical management of humoral allograft rejection.

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