NESTED POLYMERASE CHAIN REACTION-BASED HLA CLASS-II TYPING FOR THE UNIQUE IDENTIFICATION OF FORMALIN-FIXED AND PARAFFIN-EMBEDDED TISSUE

Human leukocyte antigen (HLA) genotyping is routinely performed prior to organ transplantation using peripheral blood leukocyte-derived DNA. In addition, polymerase chain reaction (PCR)-based methods have permi tted HLA genotyping using DNA extracted from formalin-fixed and paraff in-embedded tissue, with proven applications in HLA-disease associatio n studies and surgical biopsy identification. The utility of current t echniques may be limited by the poor yield of intact DNA from such par affin biopsies. This paper describes a new nested PCR-based HLA class II genotyping method,which reliably detects HLA DRB alleles within DNA extracted from even extremely small paraffin biopsies. This method co mprises initial PCR amplification of exon II sequences of the HLA DRB1 , 3, 4, and 5 genes using generic PCR primers. Identification of the H LA DRB1 alleles and detection of the DRB3, 4, and 5 genes is then perf ormed using a series of separate individual second-round PCR reactions , each of which contains PCR primer pairs detecting a single HLA DRB a llele or group of alleles (PCR-SSP). The ability of this method to det ect 19 individual HLA DRB1 alleles or groups of alleles, covering all common DRB1 specificities, was confirmed via concordant results when c ompared with 'direct' (single amplification step) PCR-SSP analysis of one cell line-derived and nine peripheral blood-derived DNA samples, a nd with five DNA samples extracted from paraffin biopsies. The techniq ue was then successfully applied to 11 further paraffin biopsy-derived DNA samples, of which ten were untypable by 'direct' PCR-SSP analysis , from five cases in which doubt existed as to the individual origin o f the tissues.