title: Effect of blood storage conditions on DNA repair capacity measurements in peripheral blood mononuclear cells.
abstract: Due to the great number of genes involved in DNA repair and the interactions among the pathways responsible for the repair of different types of DNA damage, there is an increasing need for simple and reliable approaches to phenotypically assess DNA repair capacity (DRC). The use of peripheral blood mononuclear cells (PBMCs) in DRC assays is particularly useful for human monitoring studies. However, in such studies it is not always possible to collect and process samples on the same day as the blood is taken. We performed a genotype-phenotype correlation study on DRC on 225 healthy subjects. Due to the large number of blood samples to be processed, PBMCs were either isolated and cryopreserved on the same day of blood collection (day 1) or on the following day after 24h blood storage at room temperature (day 2-RT). Samples processed in different days showed a significant difference in the DRC evaluated as 8-oxoguanine glycosylase activity (OGG assay) in cell extracts (p<0.0001) and as benzo[a]pyrene diol epoxide (BPDE)-induced damage repair by the comet assay (p=0.05). No apparent effect of the blood storage conditions on the outcome of γ-ray induced H2AX phosphorylation assay was reported. These results prompted us to further analyze the effects of blood storage conditions by performing a validation study. Three blood samples were simultaneously taken from ten healthy donors, PBMCs were isolated and cryopreserved as follows: immediately after blood collection (day 1); on the following day, after blood storage at RT (day 2-RT); or after blood storage at 4°C (day 2-4°C). DRC was then evaluated using phenotypic assays. The γ-ray induced H2AX phosphorylation assay has been confirmed as the only assay that showed good reproducibility independently of the blood storage conditions. The measurement of OGG assay was most affected by the blood storage conditions.
authors.affiliation: Human Genetics Foundation - HuGeF, Via Nizza 52, I-10126 Turin, Italy. Electronic address: alessandra.allione@hugef-torino.org.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Allione
authors.forename: Paola
authors.initials: P
authors.lastname: Porcedda
authors.forename: Alessia
authors.initials: A
authors.lastname: Russo
authors.forename: Fulvio
authors.initials: F
authors.lastname: Ricceri
authors.forename: Valeria
authors.initials: V
authors.lastname: Simonelli
authors.forename: Anna
authors.initials: A
authors.lastname: Minoprio
authors.forename: Simonetta
authors.initials: S
authors.lastname: Guarrera
authors.forename: Barbara
authors.initials: B
authors.lastname: Pardini
authors.forename: Filomena
authors.initials: F
authors.lastname: Mazzei
authors.forename: Eugenia
authors.initials: E
authors.lastname: Dogliotti
authors.forename: Claudia
authors.initials: C
authors.lastname: Giachino
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Matullo
mesh_terms: D000293:Adolescent; D000328:Adult; D001793:Blood Preservation / Q000009:adverse effects / Q000379:methods*; D001800:Blood Specimen Collection / Q000009:adverse effects / Q000379:methods; D020552:Comet Assay; D015925:Cryopreservation; D004260:DNA Repair / Q000502:physiology*; D005260:Female; D056726:Genetic Association Studies / Q000592:standards; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D013696:Temperature; D055815:Young Adult
keywords: 8-oxoguanine glycosylase; BER; BPDE; Blood storage; Comet assay; DNA repair; DNA repair capacity; DRC; DSBR; FPG; H2AX phosphorylation; IR; NER; OGG1; OGG1 activity; PBMCs; RT; base excision repair; benzo[a]pyrene diol epoxide; double-strand break repair; formamidopyrimidine DNA glycosylase; ionizing radiation; nucleotide excision repair; peripheral blood mononuclear cells; room temperature