title: Validation of RNA-based molecular clonotype analysis for virus-specific CD8+ T-cells in formaldehyde-fixed specimens isolated from peripheral blood.
abstract: Recent advances in the field of molecular clonotype analysis have enabled detailed repertoire characterization of viably isolated antigen-specific T cell populations directly ex vivo. However, in the absence of a biologically contained FACS facility, peripheral blood mononuclear cell (PBMC) preparations derived from patients infected with agents such as HIV must be formaldehyde fixed to inactivate the pathogen; this procedure adversely affects nucleic acid template quality. Here, we developed and validated a method to amplify and sequence mRNA species derived from formaldehyde fixed PBMC specimens. Antigen-specific CD8+ cytotoxic T-lymphocyte populations were identified with standard fluorochrome-conjugated peptide-major histocompatibility complex class I tetramers refolded around synthetic peptides representing immunodominant epitopes from HIV p24 Gag (KRWII[M/L]GLNK/HLA B*2705) and CMV pp65 (NLVPMVATV/HLA A*0201 and TPRVTGGGAM/HLA B*0702), and acquired in separate laboratories with or without fixation. In the presence of proteinase K pre-treatment, the observed antigen-specific CD8+ T-cell repertoire determined by molecular clonotype analysis was statistically no different whether derived from fixed or unfixed PBMC. However, oligo-dT recovery methods were not suitable for use with fixed tissue as significant skewing of clonotypic representation was observed. Thus, we have developed a reliable RNA-based method for molecular clonotype analysis that is compatible with formaldehyde fixation and therefore suitable for use with primary human samples isolated by FACS outside the context of a biological safety level 3 containment facility.
authors: Centre for Immunology, St Vincent's Hospital, Sydney, NSW, Australia. d.vanbockel@cfi.unsw.edu.au
authors: David
authors: D
authors: van Bockel
authors: David A
authors: DA
authors: Price
authors: Tedi E
authors: TE
authors: Asher
authors: Vanessa
authors: V
authors: Venturi
authors: Kazuo
authors: K
authors: Suzuki
authors: Kristina
authors: K
authors: Warton
authors: Miles P
authors: MP
authors: Davenport
authors: David A
authors: DA
authors: Cooper
authors: Daniel C
authors: DC
authors: Douek
authors: Anthony D
authors: AD
authors: Kelleher
mesh_terms: D000818:Animals; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology* / Q000378:metabolism* / Q000821:virology; D002469:Cell Separation; D002478:Cells, Cultured; D002999:Clone Cells; D022801:Complementarity Determining Regions / Q000235:genetics; D005404:Fixatives; D005434:Flow Cytometry; D005557:Formaldehyde; D019674:Genes, T-Cell Receptor beta; D015497:HIV-1 / Q000276:immunology / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D016130:Immunophenotyping; D051379:Mice; D012313:RNA / Q000032:analysis* / Q000097:blood / Q000378:metabolism; D038301:Virus Inactivation
chemical_list: D022801:Complementarity Determining Regions; D005404:Fixatives; D005557:Formaldehyde; D012313:RNA
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