P099 Comparison of ssop versus ngs for typing of HLA-A, B, C, DRB1, DRB3/B4/B5, DQA1, DQB1, DPA1, DPB1: Toward single pass high resolution hla typing in support of solid organ and hematopoietic cell transplant programs

Abstract

Aim Many laboratories use a 2-tier HLA typing process: a first pass intermediate resolution and then high resolution, when clinically necessary. SSP or qPCR provide rapid typing for deceased donor workup; SSOP is widely employed for higher volumes; and Sanger sequencing has been the gold standard for high resolution. However, SSOP and Sanger SBT often yield ambiguous results and resolution is expensive and time consuming. Next generation sequencing offers a single pass technology to achieve unambiguous HLA allele assignments. We compared commercial SSOP and NGS systems with respect to accuracy, turnaround time, effort and resolution level. Methods Five laboratories submitted coded, blinded samples, previously typed by SSOP at each institute, to a separate lab for NGS of HLA-A, B, C at exons 1–7, DRB1, DRB3/4/5, DQA1, DQB1, DPB1 at exons 1–4, and DPA1 at exons 2–4. For up to 24 samples by SSOP or up to 48 samples by NGS, benchwork is completed by one technologist on day 1 with results available for reporting on day 2. Results At the SSOP resolution level, results were concordant except for 11 SSOP assignments in 8 specimens due to false probe reactions at DRB1, DRB5, DQA1, DQB1, and DPB1. NGS identified 21 novel sequences: one with a multi-exon deletion and 20 with SNP polymorphisms in HLA-C, DPA1, DPB1, DQA1, DRB1, DRB3, DRB4, and DRB5 alleles. Across the highly polymorphic HLA-A, B, C, DRB1 loci, only 1% of intermediate resolution, IR, SSOP results were unambiguous. Even higher resolution, HR, SSOP by 1 lab gave only 17% specific allele assignments. In contrast, over 99% of NGS results were specific unambiguous genotypes. Across all loci, NGS typing was specific except for certain discrete diploid ambiguities, also found in SSOP. Conclusions This study provides a compelling rationale for implementing NGS for single pass HLA analysis with accurate, 2 day turnaround, which includes the high resolution, unambiguous genotyping critical for unrelated donor HCT and that may be required for sensitized patients with allele specific antibodies. A.G. Smith: 2. Consultant; Company/Organization; Scisco Genetics Inc. S.E. Pereira: 4. Scientific/Medical Advisor; Company/Organization; Scisco Genetics Inc. C. Pyo: 4. Scientific/Medical Advisor; Company/Organization; Scisco Genetics Inc. W. Nelson: 5. Employee; Company/Organization; Scisco Genetics Inc. M. Bettinotti: 3. Speaker’s Bureau; Company/Organization; One Lambda ThermoFisher. M.Z. Askar: 3. Speaker’s Bureau; Company/Organization; Immucor. 4. Scientific/Medical Advisor; Company/Organization; Illumina. D.E. Geraghty: 6. Stock Shareholder; Company/Organization; Scisco Genetics Inc. Download : Download high-res image (283KB) Download : Download full-size image