Reference cells in the comet assay

doi:10.1016/j.mrgentox.2019.05.020

Mutation Research/Genetic Toxicology and Environmental Mutagenesis

Volume 845, September 2019, 403064

https://doi.org/10.1016/j.mrgentox.2019.05.020 Get rights and content

Highlights

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Fish blood cells may be used as reference cells in the comet assay.
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With one-fourth of human genome size, turbot and human cells are differentiated.
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Reference cells improve standardization of protocols and inter-laboratory results.

Section snippets

Internal standards for comparing comet assay results from different laboratories

In both in vitro and human cohort studies, comet assay results obtained in different experiments and different laboratories are compared and analysed. Negative and positive controls are obviously required to demonstrate that the assay is actually working, but in addition standardization of results is needed to allow quantification and evaluation of data. In particular, when comparing and analysing results from different human cohorts, as in the hCOMET Project (CA COST Action CA15132 – The comet

Conclusions

Cells of moderately different genome size represent novel opportunities as internal reference cells. We expect that a systematic use of such references should contribute to improved standardization of comet assay data.

Conflict of interest

None.

The kind assistance of Drøbak Akvarium with its leader Klaus Bareksten is greatly appreciated.

Acknowledgements

This work was supported by the European Cooperation for Science and Technology (CA COST Action CA15132 - The comet assay as a human biomonitoring tool (hCOMET)); and by the Research Council of Norway through its Centres of Excellence funding scheme, project number 223268/F50 (CERAD, Centre for Environmental Radioactivity). Experimental contributions by Anna Safar and Kristine B. Gutzkow, and discussions with Terje Christensen are gratefully acknowledged.

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Electrophoresis in the comet assay
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The comet assay: topical issues
Mutagenesis
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M. Zainol et al.
Introducing a true internal standard for the Comet assay to minimize intra- and inter-experiment variability in measures of DNA damage and repair
Nucleic Acids Res.
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There are more references available in the full text version of this article.

Cited by (2)

Calibration of the comet assay using ionising radiation
2023, Mutation Research - Genetic Toxicology and Environmental Mutagenesis
Citation Excerpt :
Another attractive possibility would be the use of irradiated reference cells which could be taken through the comet assay after mixing with sample cells, acting as a true internal control. Two different approaches have been described for specific scoring of cells in such mixtures [25,30]. To summarise, we have investigated the causes of variation in comet assay results between laboratories.
Several trials have attempted to identify sources of inter-laboratory variability in comet assay results, aiming at achieving more equal responses. Ionising radiation induces a defined level of DNA single-strand breaks (per dose/base pairs) and is used as a reference when comparing comet results but relies on accurately determined radiation doses. In this ring test we studied the significance of dose calibrations and comet assay protocol differences, with the object of identifying causes of variability and how to deal with them. Eight participating laboratories, using either x-ray or gamma radiation units, measured dose rates using alanine pellet dosimeters that were subsequently sent to a specialised laboratory for analysis. We found substantial deviations between calibrated and nominal (uncalibrated) dose rates, with up to 46% difference comparing highest and lowest values. Three additional dosimetry systems were employed in some laboratories: thermoluminescence detectors and two aqueous chemical dosimeters. Fricke’s and Benzoic Acid dosimetry solutions gave reliable quantitative dose estimations using local equipment. Mononuclear cells from fresh human blood or mammalian cell lines were irradiated locally with calibrated (alanine) radiation doses and analysed for DNA damage using a standardised comet assay protocol and a lab-specific protocol. The dose response of eight laboratories, calculated against calibrated radiation doses, was linear with slope variance CV= 29% with the lab-specific protocol, reduced to CV= 16% with the standard protocol. Variation between laboratories indicate post-irradiation repair differences. Intra-laboratory variation was very low judging from the dose response of 8 donors (CV=4%). Electrophoresis conditions were different in the lab-specific protocols explaining some dose response variations which were reduced by systematic corrections for electrophoresis conditions. The study shows that comet assay data obtained in different laboratories can be compared quantitatively using calibrated radiation doses and that systematic corrections for electrophoresis conditions are useful.
Introduction to hCOMET special issue, ‘Comet assay in vitro’
2019, Mutation Research - Genetic Toxicology and Environmental Mutagenesis

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Letter to the EditorReference cells in the comet assay

Highlights

Section snippets

Internal standards for comparing comet assay results from different laboratories

Conclusions

Conflict of interest

Acknowledgements

Electrophoresis in the comet assay

The comet assay: topical issues

Mutagenesis

Introducing a true internal standard for the Comet assay to minimize intra- and inter-experiment variability in measures of DNA damage and repair

Nucleic Acids Res.

Letter to the Editor
Reference cells in the comet assay