title: Simplified method for the collection, storage, and comet assay analysis of DNA damage in whole blood.
abstract: Single-cell gel electrophoresis (comet assay) is one of the most common methods used to measure oxidatively damaged DNA in peripheral blood mononuclear cells (PBMC), as a biomarker of oxidative stress in vivo. However, storage, extraction, and assay workup of blood samples are associated with a risk of artifactual formation of damage. Previous reports using this approach to study DNA damage in PBMC have, for the most part, required the isolation of PBMC before immediate analysis or freezing in cryopreservative. This is very time-consuming and a significant drain on human resources. Here, we report the successful storage of whole blood in ~250 μl volumes, at -80°C, without cryopreservative, for up to 1 month without artifactual formation of DNA damage. Furthermore, this blood is amenable for direct use in both the alkaline and the enzyme-modified comet assay, without the need for prior isolation of PBMC. In contrast, storage of larger volumes (e.g., 5 ml) of whole blood leads to an increase in damage with longer term storage even at -80°C, unless a cryopreservative is present. Our "small volume" approach may be suitable for archived blood samples, facilitating analysis of biobanks when prior isolation of PBMC has not been performed.
authors.affiliation: Oxidative Stress Group, Department of Cancer Studies and Molecular Medicine, University of Leicester, Leicester, LE2 7LX, UK.
authors.forename: Kamla
authors.initials: K
authors.lastname: Al-Salmani
authors.forename: Hussein H K
authors.initials: HH
authors.lastname: Abbas
authors.forename: Sjors
authors.initials: S
authors.lastname: Schulpen
authors.forename: Mahsa
authors.initials: M
authors.lastname: Karbaschi
authors.forename: Intisar
authors.initials: I
authors.lastname: Abdalla
authors.forename: Karen J
authors.initials: KJ
authors.lastname: Bowman
authors.forename: Kwok K
authors.initials: KK
authors.lastname: So
authors.forename: Mark D
authors.initials: MD
authors.lastname: Evans
authors.forename: George D D
authors.initials: GD
authors.lastname: Jones
authors.forename: Roger W
authors.initials: RW
authors.lastname: Godschalk
authors.forename: Marcus S
authors.initials: MS
authors.lastname: Cooke
mesh_terms: D015415:Biomarkers / Q000032:analysis*; D001800:Blood Specimen Collection; D002460:Cell Line; D020552:Comet Assay / Q000379:methods / Q000639:trends; D015925:Cryopreservation; D004247:DNA / Q000378:metabolism*; D004249:DNA Damage / Q000235:genetics* / Q000276:immunology; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000378:metabolism* / Q000473:pathology; D018384:Oxidative Stress
chemical_list: D015415:Biomarkers; D004247:DNA