Validation of raw data measurements in the comet assay

General guidance recently proposed for the comet assay concluded that "the method should be adjusted scientifically at each laboratory to obtain valid and reproducible results". However, the comet widely used metrics, Tail DNA and Tail moment, are actually based on a ratio of fluorescence signals, a relative and semi-quantitative measurement, and are quite difficult to validate according to classical criteria. As the validation of analytical methods increasingly becomes an absolute requirement in many fields, this paper investigates a scheme to study the variability of raw data measurements for computer-assisted comet measurement, including the between-operators reproducibility. In the overall analysis process, we show that the image acquisition step gives the highest variability, notably for the Tail length parameter that negatively influences the Olive tail moment. However, when the operator interacts with the system to correct obviously mistaken measurements, the reproducibility is sensibly improved. For the metrics Tail DNA and Olive tail moment, the total variability in measurements for a panel of comets quantified by different operators in real conditions is about 4%. The proposed validation scheme allows to assess the measurement process and to verify if there are any major difference between trained operators, an essential requirement for long-term investigations.