Development of a simple, fast, and accurate HLA-DNA typing method in the organ transplantation

从公平和效率的角度来看，器官移植中供受者的HLA分型是重要的。本研究的目的是建立一种简便、快速、准确的HLA-DNA分型系统-荧光杂交保护试验（HPA）技术，用于器官移植。以基因组DNA为靶点，采用转录介导扩增（Transcription-Mediated Amplification，TMA）方法扩增RNA，并采用序列特异性寡核苷酸（Sequence Specific Oligonucleotide，SSO）方法中的HPA检测RNA。用24种探针检测了28个HLA纯合细胞系和158名正常人的21个HLA-DRB 1等位基因或等位基因组。99.4%的样本可分型，且与血清学分型完全一致。DR 1、DR 7、DR 9、DR 11、DR 12、DR 13、DR 1403和HR 5通过一一对应与血清学完全匹配。DR 2、DR 4、DR 8和DR 14的分型用TMA-HPA进行，提取DNA需要1小时，扩增需要4 ~ 5小时，检测需要1小时，共需要6 ~ 7小时。我们进一步对琦玉县97例肾移植等待名单中的患者进行了HLA-DRB 1等位基因分型，获得了可靠的结果，这表明血清学和DNA分型之间的数据转换是可能的。建议对不可靠的DR血清学分型进行TMA-HPA再分型，以保证器官移植的公平性和有效性。

HLA typing of both donor and recipients in organ transplantation is important from the stand point of equity and efficiency. In this research, we have aimed at developing a simple, fast, and accurate HLA-DNA typing system, a chemiluminescence-based Hydridization Protection Assay (HPA) technology, to be used for the organ transplantation. RNA was amplified by a method of Transcription-Mediated Amplification (TMA) targeting at the genomic DNA and was detected by HPA which is one type of sequence-specific oligonucleotide (SSO) methods. Twenty-four probes were used to detect 21 HLA-DRB1 alleles or allele groups in 28 HLA-homozygous cell lines and 158 normal volunteers. The results were typable in 99.4% of the samples and completely concordant with the serological typing. DR1, DR7, DR9, DR11, DR12, DR13, DR1403, and HR5 were perfectly matched with the serology by one-to-one correspondence. DR2, DR4, DR8, and DR14 were typed in more detail by TMA-HPA.Operating time required for extraction of DNA was one hour, for amplification 4 to 5 hours, for detection one hour, a total of 6 to 7 hours. We further typed HLA-DRB1 alleles of 97 patients in the waiting list for renal transplantation in Saitama prefecture and obtained reliable results, which indicated the data conversion in both ways between serology and DNA typing are possible. Retyping of unreliable serologic DR typings by TMA-HPA was recommended to assure the equity and efficiency in organ transplantation.