title: HLA class I sequence-based typing using DNA recovered from frozen plasma.
abstract: 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.
authors.affiliation: Faculty of Health Sciences, Simon Fraser University, Burnaby, British Columbia, Canada.
authors.forename: Laura A
authors.initials: LA
authors.lastname: Cotton
authors.forename: Manal
authors.initials: M
authors.lastname: Abdur Rahman
authors.forename: Carmond
authors.initials: C
authors.lastname: Ng
authors.forename: Anh Q
authors.initials: AQ
authors.lastname: Le
authors.forename: M-J
authors.initials: MJ
authors.lastname: Milloy
authors.forename: Theresa
authors.initials: T
authors.lastname: Mo
authors.forename: Zabrina L
authors.initials: ZL
authors.lastname: Brumme
mesh_terms: D000293:Adolescent; D000328:Adult; D000483:Alleles; D001483:Base Sequence; D002648:Child; D015925:Cryopreservation; D004247:DNA / Q000097:blood* / Q000235:genetics / Q000302:isolation & purification*; D005805:Genes, MHC Class I / Q000235:genetics* / Q000276:immunology; D006650:Histocompatibility Testing / Q000379:methods*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000276:immunology; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*; D055815:Young Adult
chemical_list: D004247:DNA