Abstract
The use of manual microscopy for the scoring of chromosome damage in the in vitro micronucleus assay is often associated with user subjectivity. This level of subjectivity can be reduced by using automated platforms, which have added value of faster with high-throughput and multi-endpoint capabilities. However, there is a need to assess the reproducibility and sensitivity of these automated platforms compared with the gold standard of the manual scoring. The automated flow cytometry-based MicroFlow® and image analysis-based Metafer™ were used for dose response analyses in human lymphoblastoid TK6 cells exposed to the model clastogen, methyl methanesulfonate (MMS), aneugen, carbendazim, and the weak genotoxic carcinogen, ochratoxin A (OTA). Cells were treated for 4 or 30 h, with a 26- or 0-h recovery. Flow cytometry scoring parameters and the Metafer™ image classifier were investigated, to assess any potential differences in the micronucleus (MN) dose responses. Dose response data were assessed using the benchmark dose approach with chemical and scoring system set as covariate to assess reproducibility between endpoints. A clear increase in MN frequency was observed using the MicroFlow® approach on TK6 cells treated for 30 h with MMS, carbendazim and OTA. The MicroFlow®-based MN frequencies were comparable to those derived by using the Metafer™ and manual scoring platforms. However, there was a potential overscoring of MN with the MicroFlow® due to the cell lysis step and an underscoring with the Metafer™ system based on current image classifier settings. The findings clearly demonstrate that the MicroFlow® and Metafer™ MN scoring platforms are powerful tools for automated high-throughput MN scoring and dose response analysis.
Highlights
The in vitro micronucleus assay is a robust platform for the assessment of chromosomal damage following the treatment of genotoxic agents
The findings clearly demonstrate that the MicroFlow® and MetaferTM MN scoring platforms are powerful tools for automated high-throughput MN scoring and dose response analysis
No evidence of increased cytotoxicity and cytostasis was seen from the %Relative population doubling (RPD) and the fold change in ethidium monoazide (EMA)-positive events
Summary
The in vitro micronucleus assay is a robust platform for the assessment of chromosomal damage following the treatment of genotoxic agents In this assay, a quantitative measure of the induced chromosomal damage (chromosomal breaks and chromosomal loss) is acquired by scoring micronuclei (MN) (Fenech 2000). The manual scoring procedure has been scrutinised for its subjectivity and extensive scoring time (Doherty et al 2011; Seager et al 2014) To overcome these issues, efforts have been made to automate the MN scoring platform. Efforts have been made to automate the MN scoring platform These include the use of both the semi-automated and the fully automated MN scoring approaches that are compatible with multi-endpoint MN analysis and high-through scoring (Bryce et al 2007; Varga et al 2004). Available platforms such as the Litron Laboratories automated flow cytometric platform (MicroFlow®) and the semi-automated image analysis platform (MetaferTM and PathfinderTM) are among the most widely
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
