Report from the HLA class II typing by PCR-SSP Multicentre Study

Eur J Immunogenet. 1997 Jun;24(3):191-199. doi: 10.1111/j.1365-2370.1997.00261.x.

Abstract

Results from 360 HLA-DR and -DQ 'low-resolution' typings with polymerase chain reaction sequence-specific primers (PCR-SSP), performed by nine laboratories, were analysed for their overall utility in routinely defining the HLA-DR1-DR18, DR51-DR53 and DQ1-DQ9 specificities in less than 2.5 h. Thirty EDTA blood samples and 10 DNA samples were distributed and analysed by each laboratory. DNA was extracted using a rapid bromide salt extraction protocol. Complete HLA-DR and -DQ typings were performed, three by three, on pre-aliquoted 96-tube PCR trays. When compared with reference typing, 351/360 (98%) correct DR typings were obtained, whereas 320/360 (89%) of the DQ phenotypes were correctly assigned. The time for three complete HLA-DR and -DQ 'low-resolution' typings, including DNA extraction, ranged from 2.0 h to 2.3 h. Unfortunately, an unusually high level of PCR amplification failures was observed (3%), probably due to diffusion and a significant volume loss from some of the pre-aliquoted primer mixes. Consequently, only 52% of the typings were without any amplification failure, and 0-2 amplification failures where found in 88% of the PCR-SSP typings performed. The number of HLA-DR-DQ retypings needed was 7 and 8%, respectively, reflecting the low number of typings where allelic identification was directly affected by the relatively high level of amplification failures in this study. Thus, a 91-98% success rate of correctly identified HLA-DR and -DQ alleles could be maintained, even under suboptimal typing conditions.

Publication types

  • Comparative Study
  • Multicenter Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alleles
  • DNA / isolation & purification
  • DNA Primers
  • Electrophoresis, Agar Gel
  • Genes, MHC Class II / genetics*
  • HLA-DQ Antigens / analysis*
  • HLA-DQ Antigens / genetics
  • HLA-DR Antigens / analysis*
  • HLA-DR Antigens / genetics
  • Histocompatibility Testing / methods*
  • Humans
  • Polymerase Chain Reaction
  • Polymorphism, Restriction Fragment Length
  • Sensitivity and Specificity
  • Specimen Handling

Substances

  • DNA Primers
  • HLA-DQ Antigens
  • HLA-DR Antigens
  • DNA