Variation of DNA damage levels in peripheral blood mononuclear cells isolated in different laboratories

Abstract

This study investigated the levels of DNA strand breaks and formamidopyrimidine DNA glycosylase (FPG) sensitive sites, as assessed by the comet assay, in peripheral blood mononuclear cells (PBMC) from healthy women from five different countries in Europe. The laboratory in each country (referred to as 'centre') collected and cryopreserved PBMC samples from three donors, using a standardised cell isolation protocol. The samples were analysed in 13 different laboratories for DNA damage, which is measured by the comet assay. The study aim was to assess variation in DNA damage in PBMC samples that were collected in the same way and processed using the same blood isolation procedure. The inter-laboratory variation was the prominent contributor to the overall variation. The inter-laboratory coefficient of variation decreased for both DNA strand breaks (from 68 to 26%) and FPG sensitive sites (from 57 to 12%) by standardisation of the primary comet assay endpoint with calibration curve samples. The level of DNA strand breaks in the samples from two of the centres (0.56-0.61 lesions/10(6) bp) was significantly higher compared with the other three centres (0.41-0.45 lesions/10(6) bp). In contrast, there was no difference between the levels of FPG sensitive sites in PBMC samples from healthy donors in the different centres (0.41-0.52 lesion/10(6) bp).

Application of the comet assay in human biomonitoring: An hCOMET perspective

“…Inter-laboratory variation in DNA damage levels has been described since the early 2000s through ring-trials involving multiple laboratories [5][6][7]. Later ring-trials have shown reductions in inter-laboratory variation in DNA damage levels on cryopreserved cells by using standardization with reference standards and standardized comet assay procedures, although some variation persists even after such means of standardization [8][9][10][11][12][13].…”

Ionizing radiation biomarkers in epidemiological studies – An update

“…The choice of tissues will also depend on the purpose of the study and what is feasible and ethical to collect from the study population. The method of blood collection and treatment of samples, as well as the factors discussed above relating to technique and protocols, impact on γH2AX quantification and can explain part of the variation seen between laboratories in γH2AX measurements [88,90,91]…”

“…Non-targeted approaches such as metabolomics, lipidomics and proteomics have provided promising preliminary results for new biomarkers of exposure to radionuclides, as demonstrated by recent studies of uranium (lowest estimated calculated dose of 0.15 mGy in the kidney), 137 Cs (lowest average cumulated dose of 4mGy) and 90 Sr (lowest average cumulated dose of 1 Gy) contamination in rats [216,217,[265][266][267]. Translocations, complex chromosomal rearrangements and micronuclei in peripheral blood lymphocytes (see above) are also potentially useful to estimate cumulated red bone marrow doses resulting from protracted mixed exposure to internal and external IR following moderate to high doses, although further work is needed for complete validation [12,56,59,60,268].…”

In vitro effects of low-level aldehyde exposures on human umbilical vein endothelial cells

“…35 Briefly, the alkaline comet assay was conducted under normal conditions (without pre-incubation with Fpg) and with a pre-incubation with Fpg. 35 Briefly, the alkaline comet assay was conducted under normal conditions (without pre-incubation with Fpg) and with a pre-incubation with Fpg.…”