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

Journal of Immunological Methods
Laura A CottonZabrina L Brumme

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...Continue Reading

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Citations

Oct 12, 2012·Journal of Virology·Jonathan M CarlsonUNKNOWN International HIV Adaptation Collaborative
Sep 28, 2012·Journal of Virology·Denis R ChoperaUNKNOWN CAPRISA 002 Study Team
Sep 13, 2014·Retrovirology·Eric MartinZabrina L Brumme
Jul 28, 2016·Clinical Immunology : the Official Journal of the Clinical Immunology Society·Robert WinchesterOliver FitzGerald
Nov 3, 2017·Journal of Virology·Maribel Soto-NavaUNKNOWN HIV MexNet Group
Jun 27, 2014·Journal of Virology·Xiaomei T KuangMark A Brockman
Nov 13, 2015·Journal of Virology·Natalie N KinlochZabrina L Brumme
Jun 14, 2019·Journal of Virology·Rachel L MillerUNKNOWN ORCHID Study Group
Apr 19, 2018·Scientific Reports·Humberto Valenzuela-PonceUNKNOWN Mesoamerican HIV Project Group
May 30, 2019·Frontiers in Immunology·Michael TuenRalf Duerr
May 16, 2018·Frontiers in Genetics·Kim N T TonMartin A Kennedy
Sep 12, 2019·Frontiers in Immunology·Keith E Garrison, Melanie R Gubbels Bupp
Dec 4, 2015·Clinical Cancer Research : an Official Journal of the American Association for Cancer Research·Julie S NielsenBrad H Nelson

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