Comparison of sequence-specific oligonucleotide probe vs next generation sequencing for HLA-A, B, C, DRB1, DRB3/B4/B5, DQA1, DQB1, DPA1, and DPB1 typing: Toward single-pass high-resolution HLA typing in support of solid organ and hematopoietic cell transplant programs.
Many clinical laboratories supporting solid organ transplant programs use multiple HLA genotyping technologies, depending on individual laboratory needs. Sequence-specific primers and quantitative polymerase chain reaction (qPCR) serve the rapid turnaround necessary for deceased donor workup, while sequence-specific oligonucleotide probe (SSOP) technology is widely employed for higher volumes. When clinical need mandates high-resolution data, Sanger sequencing-based typing (SBT) has been the "gold standard." However, all those methods commonly yield ambiguous typing results that utilize valuable laboratory resources when resolution is required. In solid organ transplantation, high-resolution typing may provide critical information for highly sensitized patients with donor-specific anti-HLA antibodies (DSA), particularly when DSA involve HLA alleles not discriminated by SSOP typing. Arguments against routine use of SBT include assay complexity, long turnaround times (TAT), and increased costs. Here, we compare a next generation sequencing (NGS) technology with SSOP for accuracy, effort, turnaround time, and level of resolution for genotyping of 11 HLA loci among 289 specimens from five clinical laboratories. Results were concordant except for SSOP misassignments in eight specimens and 21 novel sequences uniquely identified by NGS. With few exceptions, SSOP generated ambiguous results while NGS provided unambiguous three-field allele assignments. For complete HLA genotyping of up to 24 samples by either SSOP or NGS, bench work was completed on day 1 and typing results were available on day 2. This study provides compelling evidence that, although not viable for STAT typing of deceased donors, a single-pass NGS HLA typing method has direct application for solid organ transplantation.
['Smith|Anajane G|AG|', 'Pereira|Shalini|S|', 'Jaramillo|Andrés|A|0000-0002-0910-1460', 'Stoll|Scott T|ST|', 'Khan|Faisal M|FM|', 'Berka|Noureddine|N|', 'Mostafa|Ahmed A|AA|', 'Pando|Marcelo J|MJ|', 'Usenko|Crystal Y|CY|', 'Bettinotti|Maria P|MP|', 'Pyo|Chul-Woo|CW|', 'Nelson|Wyatt C|WC|', 'Willis|Amanda|A|', 'Askar|Medhat|M|', 'Geraghty|Daniel E|DE|']
D000483:Alleles D060005:Genotyping Techniques D006680:HLA Antigens D018380:Hematopoietic Stem Cell Transplantation D059014:High-Throughput Nucleotide Sequencing D006650:Histocompatibility Testing D006801:Humans D015345:Oligonucleotide Probes D016377:Organ Transplantation
next generation sequencing solid organ transplantation HLA genotyping