P089 How accurate is RT-PCR typing for HLA-DPB1 alleles?

Abstract

Aim RT-PCR is a rapid method to determine HLA antigens and many laboratories utilize this assay for deceased donor typing. However, HLA-DPB1 allele level results are typically reported to UNOS without confirmation by high-resolution typing methods. Our aim was to compare the accuracy of RT-PCR to sequence based testing (SBT) in order to asses the frequency of incorrect reporting. Methods HLA-DPB1 typing on 79 deceased donors previously typed using RT-PCR reagents (Linkage Bioscience) was confirmed using SBT methods. DPB1 typing results obtained by the different methods were compared. All discrepant results were verified by LabType SSO. Results In 99% (78/79) of the cases, RT-PCR produced the same allele pairing results obtained by SBT; in the remaining case, discrepant results were obtained between RT-PCR and SBT. LabType SSO determined that the SBT result was inaccurate. The manufacturer suggested that the erroneous SBT result was most likely due to the lack of exon 3 sequence information for the allele in question. In 43% (34/79) of the cases, RT-PCR detected an ambiguous combination that included multiple common and well-documented (CWD) alleles. In 21 of the ambiguous combinations, the alternative allele was in the same G group as the reported allele; in 13 cases, the alternative allele was in a different G group and three were reported incorrectly. In the remaining non-ambiguous cases, one incorrect tying was chosen based on frequencies of the alleles, however, the CWD allele was later ruled out by SBT (Fig. 1). Download : Download high-res image (88KB) Download : Download full-size image Conclusions In general, RT-PCR methods are an efficient and accurate way to determine HLA-DPB1 alleles prior to organ allocation. However, in some situations, higher resolution methods such as SBT or SSO may be required to resolve common ambiguities and ensure that accurate typing results are reported. A procedure for revising reported alleles in the UNOS database may need to be adopted to help quantify the true prevalence of HLA-DPB1 alleles in the population.