Visual comet scoring revisited: a guide to scoring comet assay slides and obtaining reliable results

Research output: Contribution to journalJournal articleResearchpeer-review

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

Measurement of DNA migration in the comet assay can be done by image analysis or visual scoring. The latter accounts for 20-25% of the published comet assay results. Here we assess the intra- and inter-investigator variability in visual scoring of comets. We include three training sets of comet images, which can be used as reference for researchers who wish to use visual scoring of comets. Investigators in 11 different laboratories scored the comet images using a five-class scoring system. There is inter-investigator variation in the three training sets of comets (i.e. coefficient of variation (CV) = 9.7%, 19.8% and 15.2% in training sets I-III, respectively). However, there is also a positive correlation of inter-investigator scoring in the three training sets (r = 0.60). Overall, 36% of the variation is attributed to inter-investigator variation and 64% stems from intra-investigator variation in scoring between comets (i.e. the comets in training sets I-III look slightly different and this gives rise to heterogeneity in scoring). Intra-investigator variation in scoring was also assessed by repeated analysis of the training sets by the same investigator. There was larger variation when the training sets were scored over a period of six months (CV = 5.9-9.6%) as compared to one week (CV = 1.3-6.1%). A subsequent study revealed a high inter-investigator variation when premade slides, prepared in a central laboratory, were stained and scored by investigators in different laboratories (CV = 105% and 18-20% in premade slides with comets from unexposed and hydrogen peroxide-exposed cells, respectively). The results indicate that further standardization of visual scoring is desirable. Nevertheless, the analysis demonstrates that visual scoring is a reliable way of analysing DNA migration in comets.

Original languageEnglish
Issue number5
Pages (from-to)253–263
Number of pages11
Publication statusPublished - 2023

Bibliographical note

© 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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