86-P: CAN LABTYPE SSO MEDIUM RESOLUTION RESOLVE DNA SEQUENCING BASED TYPING AMBIGUITIES?

Abstract

High-resolution HLA typing plays a central role in medical decisions concerning the matching of stem cell transplant recipients to unrelated donors. However, DNA sequencing based typing (SBT) may result with three or more allele combination. We undertook this study to validate whether luminex based PCR-SSO method can resolve these SBT ambiguities. HLA medium and high resolution genotyping was performed using Luminex-based sequence-specific oligonucleotide (SSO) (Thermofiser, LabtypeSSO) and Sequencing based typing (Atria Genetics HLA SBT kits) respectively. Fluorochrome-labeled DNA fragments were electrophoresed by capillary electrophoresis on an ABI 3130 Genetic Analyzer. Heterozygous Ambiguity Resolving Primers (HARPs) were used as the gold standard method to resolve ambiguity. A total of 64 DNA samples typed by SSO and SBT (15 A, 14 B, 19 C and 16 DRB1) showed HLA assignments that have more than four possible antigen/allele combinations. SSO based typing resolved 57% of ambiguities for HLA-A; 51% for HLA-B, 60% for HLA-C and 65% for HLA-DRB1 locus. On the contrary, HARPS resolved 94% of ambiguities for HLA-A and -B, 90% for HLA-C and 83% for HLA-DRB1. Overall, SSO significantly resolve ambiguities for Class I (p < 0.0001, 95% CI=1.99 - 4.38) and Class II (p < 0.0001 95% CI=2.08-4.78). HARPs analysis confirmed the SBT ambiguity resolution by SSO. Significant ambiguity resolution of SBT results can be achieved by SSO, which would significantly reduce the need of additional amplifications and financial cost involved in HARP analysis. Therefore, we recommend the combination of SSO and SBT for the generation of high resolution HLA typing and advocate the use of HARPS only in cases of unresolved HLA typing by both SSO and SBT. Also, it is important that SSO probe reaction patterns be reviewed thoroughly to assure the right allele combinations.