Allele-level resolution data at primary HLA typing is the ideal for most histocompatibility testing laboratories. Many high-throughput molecular HLA typing approaches are unable to determine the phase of observed DNA sequence polymorphisms, leading to ambiguous results. The use of higher resolution methods is often restricted due to cost and time limitations. Here we report on the feasibility of using Pacific Biosciences’ Single Molecule Real-Time (SMRT) DNA sequencing technology for high-resolution and high-throughput HLA typing. Seven DNA samples were typed for HLA-A, -B and -C. The results showed that SMRT DNA sequencing technology was able to generate sequences that spanned entire HLA Class I genes that allowed for accurate allele calling. Eight novel genomic HLA class I sequences were identified, four were novel alleles, three were confirmed as genomic sequence extensions and one corrected an existing genomic reference sequence. This method has the potential to revolutionize the field of HLA typing. The clinical impact of achieving this level of resolution HLA typing data is likely to considerable, particularly in applications such as organ and blood stem cell transplantation where matching donors and recipients for their HLA is of utmost importance.

HLA matching at an allelic-level resolution for volunteer unrelated donor (VUD) hematopoietic cell transplantation (HCT) results in improved survival and fewer post-transplant complications. Limitations in typing technologies used for the hyperpolymorphic HLA genes have meant that variations outside of the antigen recognition domain (ARD) have not been previously characterized in HCT. Our aim was to explore the extent of diversity outside of the ARD and determine the impact of this diversity on transplant outcome. Eight hundred ninety-one VUD-HCT donors and their recipients transplanted for a hematologic malignancy in the United Kingdom were retrospectively HLA typed at an ultra-high resolution (UHR) for HLA-A,-B,-C,-DRB1,-DQB1, and-DPB1 using nextgeneration sequencing technology. Matching was determined at full gene level for HLA class I and at a coding DNA sequence level for HLA class II genes. The HLA matching status changed in 29.1% of pairs after UHR HLA typing. The 12/12 UHR HLA matched patients had significantly improved 5-year overall survival when compared with those believed to be 12/12 HLA matches based on their original HLA typing but were found to be mismatched after UHR HLA typing (54.8% versus 30.1%, P = .022). Survival was also significantly better in 12/12 UHR HLAmatched patients when compared with those with any degree of mismatch at this level of resolution (55.1% versus 40.1%, P = .005). This study shows that better HLA matching, found when typing is done at UHR that includes exons outside of the ARD, introns, and untranslated regions, can significantly improve outcomes for recipients of a VUD-HCT for a hematologic malignancy and should be prospectively performed at donor selection.

57 58Improving haematopoietic cell transplantation outcomes by selection of an HLA 59 matched unrelated donor is best practice, however donor selection by secondary 60 characteristics is controversial. We studied 1271 recipients with haematological 61 malignancies who underwent T cell depleted allografts and who had complete 62 data on HLA matching status for six loci (HLA-A, -B, -C, -DRB1, -DQB1, -DPB1) and 63 clinical outcome data. 5-year overall survival was 40.6%. HLA mismatching (at 64 HLA-A, -B, -C, -DRB1, -DQB1) (Relative Risk (RR) 1.22, 95% CI 1.2-1.5, p=0.033 for 65 1 mismatch and RR 1.46, 95% CI 1.1-1.9, p=0.009 for >1 mismatch) and CMV 66 mismatching (RR 1.37, 95% CI 1.2-1.6, p<0.001) were significantly associated 67 with inferior survival. Donors under 30 years were associated with a trend 68 towards better survival (RR 1.17, 95% CI 0.99-1.4, p=0.069). In a multivariate 69 model for mortality combining CMV and HLA match status, we found a RR of 1.36 70 (95% CI 1.1-1.7, p=0.003) for HLA matched/CMV mismatched, a RR of 1.