P089 Enhanced resolution in rapid HLA typing of deceased donors: A single center experience using a new Taqman-based qPCR approach

Abstract

Aim Organ donation is a highly orchestrated process with the initial donor testing, including donor HLA typing, needing to be completed as rapidly as possible. The need for increased donor HLA typing resolution is continually growing. However, true high-resolution typing is time and resource prohibitive for deceased donors. Real-time PCR-based HLA typing assays have improved turn-around-times and are increasingly able to provide higher-resolution typing. Here we examine the performance of a new Taqman-based qPCR approach as compared to an endpoint melt curve based assay. Methods 80 DNA samples were HLA typed by first generation QTYPE (Olerup) and LinkSeq 1575 (Linkage Biosciences) for HLA-A, B, C, DRB1, DRB3/4/5, DQA1, DQB1, DPA1 and DPB1. Comparisons were made between assays for the number of common and well-documented (CWD) alleles (based on the IMTG/HLA 3.19.1 database) and the number of single antigen bead (SAB) specificities that could be resolved from the CWD typing. Results Average Roche Lightcycler 480 II run time was 42 min for the QTYPE assay and 75 min for LinkSeq. Compared to the endpoint melt curve-based LinkSeq assay, the TaqMan-based QTYPE assay defined CWD alleles at an equal or better resolution for HLA-A 13/18 loci, HLA-B 17/27 loci, HLA-C 13/15 loci, 17/17 HLA-DRB1, 3/3 HLA-DRB3/4/5 loci, 6/7 HLA-DQB1 loci, 4/6 HLA-DQA1 loci, 3/3 HLA-DPA1 and all HLA-DPB1. The number of SAB alleles that each CWD allele string was able to detect was determined using both Immucor and One Lambda SAB reagents. QTYPE resolved the multi-SAB antigens: A30, A34, A68, B13, Cw10, DR1, DR11, DR13, DR14, DR16, DRB3, DRB5, and DQ2 to a single SAB allele, while the LinkSeq assay detected CWD alleles that covered 1 + SAB alleles. A2, B44, DR4, DQ6 covered 1 + SAB in both tests, but QTYPE, on average, had better resolution for these SAB antigens. Other SAB were covered by 3 or more CWD alleles. All DPB1 were uniquely resolved by both assays except for DPB1 ∗ 04:02/105:01 in some LinkSeq tests. Conclusion The advantageous multiplexing capacity of the TaqMan based qPCR HLA typing permits greater resolution and, in general, resolves SAB alleles more consistently than the endpoint melt curve based assay. With increasing numbers of CWD HLA alleles, the TaqMan approach provides a greater potential for expansion and CWD allele discrimination. J. Lunz: Grant/Research Support; Company/Organization; Olerup. M. Wetmore: Employee; Company/Organization; Olerup. B. Passey: Employee; Company/Organization; Olerup SSP AB. D. Freedom: Grant/Research Support; Company/Organization; Olerup. G. Hill: Employee; Company/Organization; Olerup.