中华骨髓库造血干细胞捐献志愿者HLA基因分型中模棱两可结果解决方案的探讨

目的 探讨中华骨髓库造血干细胞捐献志愿者HLA基因分型中模棱两可结果的解决方案。方法 采用聚合酶链式反应(PCR)-测序分型技术(SBT)对20621名中华骨髓库供者的HLA-A、B、DRB1基因进行高分辨分型,并对其中的模棱两可分型结果进行精确分型。根据精确分型后的HLA等位基因频率计算模棱两可结果中真基因型的相...

Study on Resolution Program of Ambiguous HLA Genotypes for the Bone Marrow Donors of China Marrow Donor Program

Objective To study the resolution program of ambiguous HLA genotypes for China Marrow Donor Program(CMDP). Methods 20621 donors from CMDP were genotyped by PCR-SBT method for their HLA-A, B and DRB1 loci. The ambiguous genotyping samples were identified by using other molecular biological methods. Furthermore, the relative frequencies of true genotype for ambiguous samples were calculated by allele frequencies which from the above accurately genotyping results. Result The ratio of ambiguous genotypes was 53.88％(11 110/20 621), 66.43％ (13 697/20 621) and 70.95％(14 630/20 621) for HLA-A, B, DRB1 locus in the 20 621 donors, respectively. Among them, the ratio of ambiguous allelie-pairs for HLA-A, B, DRB1 locus was 1.34 ％ (277/20 621), 23.30％ (4 804/20 621)and 58.76％ (12 116/20 621), respectively. The ratio of ambiguous genotypes was 52. 54 ％ (10 834/20621), 56.13％ (11 574/20 621) and 59.76％ (12 323/20 621), respectively. Two alleles were found only B * 07105/07:06 and DRB1 * 08 : 03/08 : 14 allelic-pairs in 15 kinds of allelic-pair, the other 13 kinds only found one allele. Among the 11110 HLA-A, 13697 HLA-B and 14630 HLA-DRB1 ambiguity samples,The relative frequencies of 98.27%(10 918/11 110) HLA-A, 98.46%(13 486/13 697)HLA-B and 99.47％ (14 553/14 630)HLA-DRB1 samples were higher than 95%. The genotype with relative frequency which were lower than 50％ could be found in A * 01:01/11:04, A * 24:03/33:10, A * 33:01/66:01, B * 35:27/55:02, B* 38:01/78:02, B* 44:02/55: 16, B* 53:01/55:01, DRB1 * 03:02/13:01, DRB1 * 04:10/ 13:03, DRB1 * 11 : 06/13:03 and DRB1 * 12: 01/15: 04, etc. The accuracies of HLA-A, B and DRB1 genotypes by PCR-SBT method were all higher than 98％[99.07% (20 429/20 621), 98.98％ (20 410/20 621) and 99.63%(20 544/20 621) respectively], which just directly based on the relative frequency but discounted any other molecular biological methods to resolve the ambiguities. Each locus had about one percent samples with wrong genotypes. Conclusion It is a simple, rapid and cost-saving method with higher accuracy and reliability using relative frequency which calculated from allele frequencies to directly identify the ambiguous HLA genotypes in large-scale donors genotyped by PCR-SBT method, but it must be carefully used in the identity-test of patient-donor pairs before the transplantation.