Aim: The virtual crossmatch (VXM) relies on accurate donor HLA typing for histocompatibility assessment. In the deceased donor (DD) setting, molecular typing is most often performed by real time PCR due to its ease and speed. However, PCR typing is of low-to-intermediate resolution, which can potentially hinder VXM. Next generation sequencing (NGS) offers high-resolution typing that can permit accurate evaluation even in the presence of allele-specific antibodies. Herein, we assess the concordance between DD typing performed by PCR and NGS to determine the impact that NGS typing can have on the VXM.
Methods: Routine PCR typing (LinkSeqTM HLA-ABCDRDQDP SABR 384 Kit; One Lambda/Thermofisher) on DDs was correlated to NGS typing (CareDx) performed on select cases to resolve ambiguities. High-resolution typing was imputed from PCR results using HaploStats.org, selecting the most common haplotype for DD ethnicity. Discrepancies between methods were noted and quantified. VXMs, initially based on PCR typing, were re-executed retrospectively using the NGS-based DD typing. Impact of NGS donor typing on the VXM was assessed based on the utility NGS provided in overcoming limitations encountered with PCR results.
Results: In total, 40 DDs were typed by PCR and NGS. DDs were assessed against 54 transplant recipients in 56 VXMs. Characteristics of DDs and recipients are provided in Table 1. Concordance between PCR and NGS is provided in Table 2, along with the percentage of correct alleles identified outright on PCR. Overall concordance between the two methods was 82.2%. PCR was unable to resolve between common alleles 47.3 % of the time. Of the 56 VXMs, NGS would have impacted the VXM assessment in one case (1.7%), where DSA was definitively ruled out.
Conclusion: NGS was superior in providing a single unambiguous result. Nonetheless, even though concordance between PCR and NGS was 82.2%, PCR still appeared adequate for VXM for most cases. However, the method of imputation may have impacted concordance. Though the percentage of cases in which NGS enhanced VXM assessment was low, the overall impact could be greater if extrapolated to the total number of VXM performed in a year (e.g., 30-40 cases/year for 2000 VXM). The advent of rapid NGS typing by nanopore technology promises increased accessibility to high-resolution results for DDs, which can be used in VXM as well as molecular matching.
