Aim: Deceased donor (DD) HLA typing must be accurate and have a short turnaround time (TAT) to accommodate allocation timeline restrictions. Our laboratory has experienced an 80% increase from 5 years ago in the number of deceased donors tested, with the majority of local donors currently being DCD donors that require expedited workup.
To optimize the workflow of DD HLA typing currently performed by Luminex-based SSOP, our laboratory (1) evaluated alternative methods that can provide shorter TAT or higher resolution, and (2) selected and validated a high-resolution typing method for clinical use.
Methods: One real time PCR, and two 3rd generation nanopore-based sequencing assays were evaluated. While the real time PCR method had a short TAT (1.5h) and was able to identify common antigens, it offered low resolution and higher number of ambiguities compared to SSOP. In contrast, both nanopore-based sequencing assays offered high-resolution unambiguous results, with only slightly longer TAT (3-5h). Differences between the two sequencing methods included gene coverage, length of PCR and library preparation steps, sequencing time and software. Considering all these parameters, one of the two nanopore-based methods was chosen for validation, using a representative mix of residual clinical samples, comparing results to the 2nd generation method (NGS) currently used for clinical HLA typing. Accuracy, precision, resolution of ambiguities, allele dropouts, novel alleles, data quality and flow cell carry-over were evaluated.
Results: Analysis of 43 samples so far indicated 100% concordance with the clinical NGS for HLA-A, B, C, DRB1, DQB1, DPA1 and DPB1. Concordance for DQA1 was 95% due to NGS failure in two samples, and for DRB345 it was 93%. Low concordance in DRB345 was due to DRB4*01:01 dropouts in 5 samples by the clinical NGS, which is a known limitation of the assay. Of interest, (1) there were no dropouts for any locus when sequenced by the nanopore method, and (2) there were fewer ambiguities overall. No significant carryover was observed between washes (evaluated on 7 flow cells), allowing for cost-saving by flow cell re-use between samples.
Conclusion: Nanopore sequencing-based HLA typing is the path forward for DD testing, as it provides unambiguous allele level results in a short timeframe (3-4h). Laboratories should adapt this method because it simplifies the process and accuracy of virtual crossmatch.
