HIGH-RESOLUTION HLA-DRB1 SSP TYPING FOR CADAVERIC DONOR TRANSPLANTATION

An HLA-DRB1 typing procedure by means of sequence-specific primer (SSP ) amplification was developed for 65 different DRB1 subtypes. Subtypin g is achieved by the performance of two subsequent PCR assays (PCR-I a ssay and PCR-2 assay) using a limited number of reactions. The PCR-1 a ssay determined low-resolution HLA-DRB1 typing, i.e. the serologically defined specificities DR1, 2, 3, 4, 11, 12, 6, 7, 8, 9 and 10. The se cond exon of the DRB1 gene is amplified also in this PCR-1 assay. High -resolution subtyping for positively identified alleles was performed in the PCR-2 assay with the exon-2 product from PCR-1 assay as DNA tem plate. PCR reactions were carried out using unpurified primers in reac tion volumes of 20 mu l and 100 ng of chromosomal DNA. After 3 hours, the results of the PCR-1 assay were analyzed and subsequently subtypin g results in the PCR-2 assay were obtained in another 1.5 hours. A tot al of 249 DNA samples was typed by this method. No false positive nor false negative results were obtained in DRB1 typing of 32 homozygous c ell lines, 56 serologically well-defined panel cells and 125 unrelated individuals. Segregation of the amplification patterns was investigat ed in 36 members of 7 two-generation families. DRB1 subtyping revealed codominant Mendelian segregation for all subtypes investigated. In co nclusion, LR-HR-PCR-SSP typing is a fast and reliable typing technique for routine DNA typing purposes which gives complete DRB1 subtyping w ithin 4.5 h. Besides low-resolution DRB typing, also high-resolution D RB subtyping for prospective HLA-DR matching in cadaveric renal transp lantation is possible by this method.