title: Evaluation of the automated MicroFlow®and Metafer™ platforms for high-throughput micronucleus scoring and dose response analysis in human lymphoblastoid TK6 cells.
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.
authors.affiliation: Institute of Life Science, School of Medicine, Swansea University, Swansea, SA2 8PP, UK. jatin_verma12@yahoo.com.
authors.forename: Jatin R
authors.initials: JR
authors.lastname: Verma
authors.affiliation: Institute of Life Science, School of Medicine, Swansea University, Swansea, SA2 8PP, UK.
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Rees
authors.affiliation: Institute of Life Science, School of Medicine, Swansea University, Swansea, SA2 8PP, UK.
authors.forename: Eleanor C
authors.initials: EC
authors.lastname: Wilde
authors.affiliation: Institute of Life Science, School of Medicine, Swansea University, Swansea, SA2 8PP, UK.
authors.forename: Catherine A
authors.initials: CA
authors.lastname: Thornton
authors.affiliation: Institute of Life Science, School of Medicine, Swansea University, Swansea, SA2 8PP, UK.
authors.forename: Gareth J S
authors.initials: GJS
authors.lastname: Jenkins
authors.affiliation: Institute of Life Science, School of Medicine, Swansea University, Swansea, SA2 8PP, UK.
authors.forename: Shareen H
authors.initials: SH
authors.lastname: Doak
authors.affiliation: Institute of Life Science, School of Medicine, Swansea University, Swansea, SA2 8PP, UK.
authors.forename: George E
authors.initials: GE
authors.lastname: Johnson
mesh_terms: D001331:Automation; D001562:Benzimidazoles / Q000633:toxicity; D002219:Carbamates / Q000633:toxicity; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D057166:High-Throughput Screening Assays / Q000295:instrumentation / Q000379:methods; D006801:Humans; D008741:Methyl Methanesulfonate / Q000633:toxicity; D015162:Micronucleus Tests / Q000295:instrumentation* / Q000379:methods*; D009153:Mutagens / Q000633:toxicity; D009793:Ochratoxins / Q000633:toxicity; D015203:Reproducibility of Results
chemical_list: D001562:Benzimidazoles; D002219:Carbamates; D009153:Mutagens; D009793:Ochratoxins; C025589:ochratoxin A; D008741:Methyl Methanesulfonate; C006698:carbendazim
keywords: Automation; Dose response; Metafer™; MicroFlow®; Micronucleus