HLA-I类PCR-SSP基因分型在异基因造血干细胞移植中的应用

Application of HLA class I PCR-SSP genotyping method in allogeneic hematopoietic stem cell transplantation

目的 提高异基因造血干细胞移植供、受者HLA I类配型的精确度 ,有效防止移植物抗宿主病 (GVHD)的发生。方法 采用准确、简便的DNA、RNA提取方法和HLA I类PCR SSP基因分型方法。建立HLA A33、A6 8、B40、B13PCR SSP分型方法以及HLA基因序列测序分析。结果 对 30 0例中血清学难以判断结果的 30例临床标本进行基因水平的研究发现 ,DNA及RNA提取方法可以满足此项研究对样本的要求。HLA I类PCR SSP分型方法具有良好的稳定性、可靠性和特异性 ;重复率达 10 0 %。同时 ,HLA A33、A6 8、B40、B13结果与HLA I类PCR SSP分型结果一致。HLA基因测序分析进一步肯定了HLA I类PCR SSP分型的特异性。HLA I类PCR SSP不受某些血清学影响因素的干扰。整个实验过程仅耗时 2 .5h。结论 HLA I类PCR SSP基因分型方法准确、特异、重复性好 ,可作为临床骨髓移植配型和正常人群无关供者筛选的常规方法 。

Objective To improve the accuracy of HLA class I typing methods for allogeneic hematopoietic stem cell transplant donor recipient pairs, and to effectively prevent graft vs host disease (GVHD). Methods An accurate, simple DNA extraction method and HLA class I PCR sequence specificprimer (SSP) genotyping method were employed. HLA A33, A68, B40, B13 PCR SSP and HLA gene sequencing method were developed. Results The results of 30 cases which could not be easily typed by serological methods in 300 cases indicated that DNA and RNA extraction methods could satisfy the experimental requests. The HLA class I PCR SSP method was confirmed to be reliable, stable and specific with the reproducible rate being 100%. Meanwhile, the results of HLA A33, A68, B40, B13 PCR SSP were in accordance to HLA class I PCR SSP typing results. The HLA gene sequencing results of some special cases further confirmed the specificity of HLA class I PCR SSP typing results. HLA class IPCR SSP results were not affected by some factors which might confuse the serological results and the whole experiment took only 2.5?h. ConclusionsHLA class I PCR SSP was specific and accurate, and could be used as routine typing method for HLA matching in allogeneic hematopoietic stem cell transplantation and for selecting unrelated donors, and would play important role in preventing GVHD.