High resolution HLA-DRB1 SSP typing for cadaveric donor transplantation

Abstract: An HLA-DRB1 typing procedure by means of sequence-specific primer (SSP) amplification was developed for 65 different DRB1 subtypes. Subtyping is achieved by the performance of two subsequent PCR assays (PCR-1 assay and PCR-2 assay) using a limited number of reactions. The PCR-1 assay 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 second 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 template. PCR reactions were carried out using unpurified primers in reaction volumes of 20 μ1 and 100 ng of chromosomal DNA. After 3 hours, the results of the PCR-1 assay were analyzed and subsequently subtyping results in the PCR-2 assay were obtained in another 1.5 hours. A total of 249 DNA samples was typed by this method. No false positive nor false negative results were obtained in DRB1 typing of 32 homozygous cell lines, 56 serologically well-defined panel cells and 125 unrelated individuals. Segregation of the amplification patterns was investigated in 36 members of 7 two-generation families. DRB1 subtyping revealed codominant Mendelian segregation for all subtypes investigated. In conclusion, LR-HR-PCR-SSP typing is a fast and reliable typing technique for routine DNA typing purposes which gives complete DRB1 subtyping within 4.5 h. Besides low-resolution DRB typing, also high-resolution DRB subtyping for prospective HLA-DR matching in cadaveric renal transplantation is possible by this method.