Inter-laboratory variation in measurement of DNA damage by the alkaline comet assay in the hCOMET ring trial

Research output: Contribution to journalJournal articleResearchpeer-review

  • Amaya Azqueta
  • Miguel Collia
  • Tamara Bakuradze
  • Elke Richling
  • Ezgi Eyluel Bankoglu
  • Helga Stopper
  • Victoria Claudino Bastos
  • Sabine A S Langie
  • Sara Ristori
  • Francesca Scavone
  • Lisa Giovannelli
  • Maria Wojewódzka
  • Marcin Kruszewski
  • Vanessa Valdiglesias
  • Blanca Laffon
  • Carla Costa
  • Solange Costa
  • João Paulo Teixeira
  • Mirko Marino
  • Cristian Del Bo'
  • Patrizia Riso
  • Congying Zhang
  • Sergey Shaposhnikov
  • Andrew Collins

The comet assay is a simple and versatile method for measurement of DNA damage in eukaryotic cells. More specifically, the assay detects DNA migration from agarose gel-embedded nucleoids, which depends on assay conditions and the level of DNA damage. Certain steps in the comet assay procedure have substantial impact on the magnitude of DNA migration (e.g. electric potential and time of electrophoresis). Inter-laboratory variation in DNA migration levels occurs because there is no agreement on optimal assay conditions or suitable assay controls. The purpose of the hCOMET ring trial was to test potassium bromate (KBrO3) as a positive control for the formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay. To this end, participating laboratories used semi-standardized protocols for cell culture (i.e. cell culture, KBrO3 exposure, and cryopreservation of cells) and comet assay procedures, whereas the data acquisition was not standardized (i.e. staining of comets and image analysis). Segregation of the total variation into partial standard deviation (SD) indicates importance of cell culture procedures (SD = 10.9), comet assay procedures (SD = 12.3), staining (SD = 7.9) and image analysis (SD = 0.5) on the overall inter-laboratory variation of DNA migration (SD = 18.2). Future studies should assess sources of variation in each of these steps. On the positive side, the hCOMET ring trial demonstrates that KBrO3 is a robust positive control for the Fpg-modified comet assay. In conclusion, the hCOMET ring trial has demonstrated a high reproducibility of detecting genotoxic effects by the comet assay, but inter-laboratory variation of DNA migration levels is a concern.

Original languageEnglish
Issue number5
Pages (from-to)283–294
Number of pages12
Publication statusPublished - 2023

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© The Author(s) 2023. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail:

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