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Updated: Feb 22, 2026
An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry
Published on: May 13, 2019
Classification of in vitro genotoxicants using a novel multiplexed biomarker assay compared to the flow cytometric
Sabrina Wilde1, Miriam Dambowsky1, Claudia Hempt1
1Investigational Toxicology, Bayer AG, Berlin, Germany.
Abstract:
Regulatory in vitro genotoxicity testing exhibits shortcomings in specificity and mode of action (MoA) information. Thus, the aim of this work was to evaluate the performance of the novel MultiFlow® assay composed of mechanistic biomarkers quantified in TK6 cells after treatment (4 and 24 hr): γH2AX (DNA double strand breaks), phosphorylated H3 (mitotic cells), translocated p53 (genotoxicity), and cleaved PARP1 (apoptosis). A reference dataset of 31 compounds with well-established MoA was studied using the MicroFlow® micronucleus assay. A positive call was raised following the earlier published criteria from Litron Laboratories. In the light of our data, these evaluation criteria should probably be adjusted since only 8/11 (73%) nongenotoxicants and 18/20 (90%) genotoxicants were correctly identified. Moreover, there is a need for new in vitro tools to delineate the predominant MoA as in the MicroFlow® assay only 5/9 (56%) aneugens and 4/11 (36%) clastogens were correctly classified. In contrast, the MultiFlow® assay provides more in-depth information about the MoA and therefore reliably discriminates clastogens, aneugens, and nongenotoxicants. By using a lab-specific, practical threshold for the aforementioned biomarkers, 10/11 (91%) nongenotoxicants and 19/20 genotoxicants (95%), 9/11 (82%) clastogens, and 8/9 (89%) aneugens were correctly categorized, suggesting a clear improvement over the MicroFlow® . Furthermore, the MultiFlow markers were benchmarked against established methods to assess the validity of the data. Altogether, these findings demonstrated good agreement between the MultiFlow® assay and the benchmarking methods. Finally, p21 may improve class discrimination given the correct identification of 4/4 (100%) aneugens and 2/5 (40%) clastogens. Environ. Mol. Mutagen. 58:662-677, 2017. © 2017 Wiley Periodicals, Inc.
Insights
The novel MultiFlow assay accurately identifies genotoxicants and their mechanisms of action, outperforming older methods. This advancement improves in vitro genotoxicity testing specificity and mode of action information.
Area of Science:
- Toxicology
- Genetics
- Biomarker Discovery
Background:
- Current in vitro genotoxicity testing lacks specificity and detailed mode of action (MoA) information.
- Existing assays, like the MicroFlow micronucleus assay, show limitations in classifying genotoxicant types (clastogens vs. aneugens).
Purpose of the Study:
- To evaluate the performance of the MultiFlow assay using mechanistic biomarkers for genotoxicity testing.
- To assess the assay's ability to differentiate between clastogens, aneugens, and non-genotoxicants.
Main Methods:
- The MultiFlow assay measured biomarkers (γH2AX, p-H3, p53, cleaved PARP1) in TK6 cells post-treatment.
- A reference dataset of 31 compounds was tested using both MultiFlow and MicroFlow assays.
- Lab-specific thresholds were applied to MultiFlow biomarker data for classification.
Main Results:
- MultiFlow correctly identified 95% of genotoxicants and 91% of non-genotoxicants using lab-specific thresholds.
- The assay demonstrated improved classification of clastogens (82%) and aneugens (89%) compared to MicroFlow.
- Benchmarking confirmed good agreement between MultiFlow assay data and established methods.
Conclusions:
- The MultiFlow assay offers enhanced specificity and MoA information for in vitro genotoxicity testing.
- It reliably discriminates between clastogens, aneugens, and non-genotoxicants, surpassing the MicroFlow assay's capabilities.
- Further investigation with biomarkers like p21 may further refine genotoxicant class discrimination.
