25 Assessment of concordance between real-time PCR and Next-Generation Sequencing for HLA typing in deceased donors

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Abstract Accurate identification of donor-specific antibodies (DSA) plays a critical role in assessing immunological risk for solid organ transplantation, with high-resolution HLA typing being a key component of this process. Short-read next-generation sequencing (NGS) offers two-field high-resolution (2F-HR) HLA typing, but its application in deceased donors is often impractical due to time constraints. As a result, most tissue typing laboratories use faster, lower-resolution methods like real-time PCR (qPCR), though qPCR provides limited gene coverage, which may lead to ambiguous results due to the inability to fully exclude certain HLA alleles. This study aimed to evaluate the concordance between two-field HLA alleles identified through qPCR and those determined using NGS. Additionally, we examined the impact of potential allele discrepancies on DSA assignment during pre-transplant assessments. A retrospective analysis was conducted on 67 deceased donors at our center between January and September 2024. Donor HLA typing was initially performed using qPCR by the organ procurement organization’s HLA laboratory, followed by high-resolution NGS typing in our center’s laboratory. DSA detection, both pre- and post-transplant, utilized Luminex-based single antigen bead assays. The study found a 94.7% concordance between qPCR and NGS results, with the highest agreement observed for HLA-DPB1 (98.2%), HLA-DQB1 (93.4%), and HLA-DRB1 (97%). However, the lowest concordance (90.2%) was seen for HLA-DRB*3/4/5. All discordant alleles were further analyzed for their potential impact on DSA assignments in virtual crossmatch (VXM) evaluations, which assess immunologic compatibility between a recipient’s alloantibody profile and the donor’s histocompatibility antigens. The analysis demonstrated negligible effects on DSA assignments. While HLA-DPA1 and DQA1 exhibited the highest incidence of discordant allele assignments represented on the bead panel, no waitlisted recipient displayed antibodies against either the qPCR or NGS-discordant alleles. In conclusion, these results confirm that while qPCR generally aligns well with NGS for high-resolution HLA typing, certain loci present challenges in accurate allele assignment. Further, the impact of HLA typing discrepancies on DSA identification in VXM assessments was insignificant, supporting qPCR as a reliable and efficient tool for HLA typing in deceased donor transplants. Future directions of this research include expanding the study to include a larger cohort of deceased donors from multiple centers to validate the findings and assess their applicability across various populations. Additionally, the study will explore whether discordant alleles affect eplet mismatches (molecular mismatch) and influence post-transplant outcomes, such as rejection rates and graft survival.

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