Phototyping: comprehensive DNA typing for HLA‐A, B, C, DRB1, DRB3, DRB4, DRB5 & DQB1 by PCR with 144 primer mixes utilizing sequence‐specific primers (PCR‐SSP)

TLDR

The authors developed a single DNA typing method using 144 sequence‑specific primer reactions to simultaneously detect all known HLA‑A, B, C, DRB1, DRB3, DRB4, DRB5, and DQB1 specificities. The integrated class I & II assay, termed Phototyping, visualizes all loci on a single photographic or electronic image using uniform reagents and PCR conditions. Phototyping achieves resolution comparable to serology, produces results in under three hours, has been applied to over 1,010 individuals, detects novel alleles, and can reduce cold ischemia times in transplantation, though about 5 % of genotypes require repeat testing due to protocol deviations or DNA quality issues.

Abstract

We have developed a single DNA typing method which uses 144 sequence-specific primer (SSP) reactions to simultaneously detect all known HLA-A, B, C, DRB1, DRB3, DRB4, DRB5 and DQB1 specificities in an allele specific or group specific manner using the same method, reagents, PCR parameters and protocols for all loci. The results from this integrated class I & II method can be visualized on a single photographic or electronic image and hence is described as "Phototyping". Phototyping has an overall resolution greater than or equivalent to good serology and results can be obtained in under 3 hours making the method suitable for genotyping potential cadaver donor peripheral blood without serological backup. This in turn produces the potential for reducing cold ischaemia times in renal transplantation as well as the application of prospective matching to cardiac and liver transplantation. The method has capacity to detect new alleles, for example, novel amplification patterns suggestive of 4 new HLA-B alleles have been detected. The Phototyping set has been used as the sole method of HLA typing for over 1010 individuals. Phototyping is not problem-free; deviations from the standard protocol, poor quality DNA and unsuitable PCR machines can result in individual PCR failures or in incorrect assignment of antigens. Approximately 5% of genotypes were repeated (either partially or fully) because of incomplete or equivocal results.