HLA class I sequence-based typing using DNA recovered from frozen plasma

J Immunol Methods. 2012 Aug 31;382(1-2):40-7. doi: 10.1016/j.jim.2012.05.003. Epub 2012 May 11.


We describe a rapid, reliable and cost-effective method for intermediate-to-high-resolution sequence-based HLA class I typing using frozen plasma as a source of genomic DNA. The plasma samples investigated had a median age of 8.5 years. Total nucleic acids were isolated from matched frozen PBMC (~2.5 million) and plasma (500 μl) samples from a panel of 25 individuals using commercial silica-based kits. Extractions yielded median [IQR] nucleic acid concentrations of 85.7 [47.0-130.0]ng/μl and 2.2 [1.7-2.6]ng/μl from PBMC and plasma, respectively. Following extraction, ~1000 base pair regions spanning exons 2 and 3 of HLA-A, -B and -C were amplified independently via nested PCR using universal, locus-specific primers and sequenced directly. Chromatogram analysis was performed using commercial DNA sequence analysis software and allele interpretation was performed using a free web-based tool. HLA-A, -B and -C amplification rates were 100% and chromatograms were of uniformly high quality with clearly distinguishable mixed bases regardless of DNA source. Concordance between PBMC and plasma-derived HLA types was 100% at the allele and protein levels. At the nucleotide level, a single partially discordant base (resulting from a failure to call both peaks in a mixed base) was observed out of >46,975 bases sequenced (>99.9% concordance). This protocol has previously been used to perform HLA class I typing from a variety of genomic DNA sources including PBMC, whole blood, granulocyte pellets and serum, from specimens up to 30 years old. This method provides comparable specificity to conventional sequence-based approaches and could be applied in situations where cell samples are unavailable or DNA quantities are limiting.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adolescent
  • Adult
  • Alleles
  • Base Sequence
  • Child
  • Cryopreservation
  • DNA / blood*
  • DNA / genetics
  • DNA / isolation & purification*
  • Genes, MHC Class I / genetics*
  • Genes, MHC Class I / immunology
  • Histocompatibility Testing / methods*
  • Humans
  • Leukocytes, Mononuclear / cytology
  • Leukocytes, Mononuclear / immunology
  • Polymerase Chain Reaction
  • Sequence Analysis, DNA / methods*
  • Young Adult


  • DNA