A Comparative Evaluation of Human Leukocyte Antigen Typing by PCR–SSP and Next-generation Sequencing

Original researchwere enrolled.HIV/HBsAg/HCV-positive patients were excluded.Consent from all patients/donors was obtained. Sample CollectionBlood samples/buccal swabs (in case of blood transfusion in the last 15 days or as suggested by the clinician) of patients/donors routinely referred for HLA typing test were collected.Blood samples were collected in ethylenediaminetetraacetic acid (EDTA) vacuettes, while buccal swabs were collected in sterile transport cotton swabs. Isolation of Total Deoxyribonucleic AcidDeoxyribonucleic acid (DNA) was isolated using commercially available kit following manufacturer's protocol. IntroductIonHuman leukocyte antigens (HLA) are critical components of the immune system, encoded by genes in the major histocompatibility complex (MHC) located on the short arm of chromosome 6p21.3. [1]][3][4] These glycoproteins are essential for antigen presentation, enabling the immune system to differentiate between self and nonself.The expediency of HLA genotyping has been well established in both solid organ and hematopoietic stem cell transplantation (HSCT), where it ensures compatibility between donors and recipients. 5,6Accurate HLA typing is vital for reducing the risk of allograft rejection and graft-vs-host disease, thereby improving transplant outcomes. 7ntil recently, at GCRI, the HLA typing test for HLA-A, HLA-B, and HLA-DRB1 genes was performed using the polymerase chain reaction with sequence-specific primers (PCR-SSP) method.However, advances in next-generation sequencing (NGS) are shedding new light on this area, offering fresh insights into HLA genetics and its clinical applications, with the potential to improve transplantation strategies and patient matching. 8Moreover, the identification of allele-specific antibodies proposes benefits of using high-resolution genotyping because it can more readily ascertain donors for highly sensitized transplant candidates. 9Hence, effective management of highly sensitized transplant recipients necessitates high-resolution HLA genotyping to enhance identification of compatible donors and improve match rates.Thus, the aim of this study was to perform a broad-coverage, ultra-highresolution HLA typing in a single assay using NGS and correlate the results with those from the PCR-SSP technique.