Standardization of the COMET-assay: Development of a procedure for improved automated scoring in a system for higher throughput analysis of the COMET-assay

The COMET-assay is a widespread method for determination of genetic damage on the DNA level. Mostly, it is conducted on a lower level of automation including a semi-automated Comet scoring procedure. However, with the increasing knowledge about the reliability and relevance of the COMET-Assay for early genotoxicity screening of drug candidates and for regulatory toxicology testing, there is an urgent need for fully automated versions leading to (a) higher throughput methods and to (b) a better standardization of the assay. The latter one is addressed by the development presented here. The semi-automated image analysis commonly applied to the COMET-assay requires a manual selection of Comets to be scored in a key step of the procedure. For that reason, the results in such a system even in a blind study design strongly depend on the subjectivity of the individual technician. This limits the possibilities of standardization and objectivity of the results considerably. Using a system for higher throughput analysis of the Comet-Assay (Ritter et al., Tox. Lett. 172 (2007) S91–S92), a set of criteria based on image analysis was developed carrying out the selection step in an automated way. The procedure was investigated with test substances and the effects of the certain criteria were evaluated. The results prove that the analysis strategy applied generated a fully automated scoring procedure which reliably selects Comets for scoring and excludes artefacts according to easily definable and visualizable rules. Therefore, it offers a comprehensive way to obtain greater objectivity and standardization in the Comet scoring