CANTYPE( HLA ABC ALLELE-LEVEL GENOTYPING: A MULTISTEP ASSAY USING PCR AND IMMOBILIZED OLIGOPROBES
I.M. Buyse, C. Couture, S. Stephens, S. Ouellet, S. Tavoularis. Head Office, Canadian Blood Services, Ottawa, ON, Canada.
In Tissue Antigens: 50: 291-302, 1997, we reported a reverse hybridization assay for allele level-resolution DRB genotyping. Here we describe a multi-step reverse SSOP assay allowing both intermediate and allele-level A, B, and Cw genotyping, that accommodates both single-sample and batch processing. Slightly different typing strategies were developed for each of the A, B, and Cw genes to reflect the degree and nature of the polymorphism present. For each locus, a generic amplicon covering all allelic polymorphisms present in exons 2 and 3 is initially generated and hybridized to a generic panel of SSO probes (57 for A, 58 for B, 44 for C), allowing intermediate genotyping. Depending on the genotype, a second step may be needed. For the A locus, the second step involves either extended amplification and hybridization of 55 SSO probes to resolve specific groups of alleles (AXT primer pairs 1A & 1C resolve all A*02 alleles, AXT primer pairs 2A & 2C resolve A*0104N and all A*24, A*30 alleles, and primer pair 3A resolves all A*11, A*25, A*26 A*66, A*68 alleles), or sequence-specific amplification of individual alleles followed by hybridization to either generic or extended typing membranes. For the B locus, the original B generic amplicons are rehybridized to an additional panel of 39 SSO probes and the combined extended hybridization patterns analyzed. Alternatively, sequence-specific amplicons of individual alleles are hybridized to the extended panel. For Cw locus typing, a second step is necessary only to resolve particular genotypes. The assay allows 100% allele-level A, B, and Cw genotyping. A computer program assists in the analysis of hybridization patterns. All hybridization membranes work under stringency conditions identical to HLA class II. The A & B Generic assays have been used to type over 3,000 donors from the Canadian Unrelated Bone Marrow Donor Registry. The Cw Typing Kit has been validated using both proficiency samples and 10th International Workshop cell lines. These typing assays proved to be reliable and specific. Moreover, because all polymorphic areas are covered by SSO probes, the Cantype( kits extensively screen the encoded alleles and thus allow the identification of novel alleles.