Objective To evaluate the heterozygous ambiguity resolution primers (HARPs) method in resolving ambiguous genotyping results of human leukocyte antigen (HLA) genes in Chinese Hart population, and choose some appropriate HARPs primers. Methods HLA-A, HLA-B and HLA-DRB1 genes of 416 southern Chinese Han individuals were genotyped by sequence-based-typing(SBT) method and then the ambiguous genotyping samples were sequenced again by HARPs primers provided by American Atria company. Results The percentage of ambiguous genotyping samples resolved by HARPs for HLA-A, HLA-B and HLA-DRBI locus was 86.3% (132/153), 73.9% (130/176) and 38.1% (85/223) respectively. Among them, 48.5% (64/132)HLA-A, 80.0% (104/130)HLA-B and all HLA-DRB1(85/85)samples only need one primer, 47.7 % (63/132)HLA-A and 20.0% (26/130)HLA-B samples need two primers. Three to six different HARPs primers can resolve more than 90% ambiguities. Conclusion HARPs is a convenient method and could be a routine method to resolve ambiguities for HLA-A, HLA-B and HLA-DRB1 genes genotyped by SBT in Chinese Han population.

BACKGROUND: Polymerase chain reaction-sequence based typing (PCR-SBT) as a gold standard of human leucocyte antigen (HLA) typing, is widely used in clinical transplantation typing and HLA typing of donors from Chinese Marrow Donor Program. However, the high proportion of ambiguity typing is the most disadvantage of the method. Therefore, it is urgent to find an ideal solution for HLA typing. OBJECTIVE: To investigate the ambiguity distribution of HLA-A, B and DRB1 loci by SBT method in Han population in southern China and evaluate its possible solutions. DESIGN, TIME AND SETTING: An observational measurement was performed at Key Laboratory of Immunogenetics, Institute of Transfusion Medicine, Shenzhen Blood Center, from August 2007 to August 2008. PARTICIPANTS: 416 Han marrow donors from Chinese Marrow Donor Program in Shenzhen were enrolled in the experiment. The nationality and native place of the donors were determined by self telling. METHODS: HLA-A, B and DRB1 loci of all donors were genotyped by PCR-SBT, and then the ambiguity distribution of the three loci was analyzed. The typing ambiguities were resolved by high-resolution polymerase chain reaction- sequence-specific primers (PCR-SSPs) and heterozygous ambiguity resolution primers (HARPs) methods, respectively. The frequency of genotypes was calculated with the direct count method, and then, the relative probability of true alleles was calculated. MAIN OUTCOME MEASURES: Distribution of HLA-A, B and DRB1 loci in 416 donors by SBT method. Ambiguity distribution and classification of HLA-A, B and DRB1 loci by SBT method. Ability of high-resolution PCR-SSPs and HARPs methods to resolve ambiguous results. RESULTS: The ambiguity rate of the HLA-A, B and DRB1 loci among the 416 samples were 80.29%. About 80% HLA-A, 80% HLA-B and < 40% HLA-DRB1 ambiguities could be solved by HARPs method, and the other by high-resolution PCR-SSPs method. According to calculation of the allele frequency data, the relative probability of 75% true allele combination was higher than that of 98% among the 553 ambiguous combinations. CONCLUSION: HARPs and high-resolution PCR-SSPs methods have high abilities to solve HLA ambiguities both locate inside and outside the sequencing region, respectively. And they could complement each other. Application of allele frequency data had practical value to resolve ambiguities for large scale donors of HLA typing.