Interlaboratory evaluation of a flow cytometric, high content in vitro micronucleus assay

Abstract

An international, multi-lab trial was conducted to evaluate a flow cytometry-based method for scoring micronuclei in mouse lymphoma L5178Y cells [Avlasevich et al., Environ. Molec. Mutagen. 47 (2006) [56][57][58][59][60][61][62][63][64][65][66]. A reference laboratory investigated the potential of six chemicals to induce micronuclei-the genotoxicants mitomycin C, etoposide, and vinblastine, and the non-genotoxicants sucrose, staurosporine, or dexamethasone. The latter two non-genotoxicants were selected as extreme challenges to the assay because of their potent apoptogenic activity. Three collaborating laboratories were supplied with prototype In Vitro MicroFlow ™ kits, and each was assigned one genotoxicant and one non-genotoxicant. Cells were treated continuously for 24 hrs over a range of concentrations up to 5 mg/ml, or overtly cytotoxic concentrations. Micronuclei were scored via standard microscopy and flow cytometry. In addition to enumerating micronucleus frequencies, a cytotoxicity measurement that is simultaneously acquired with the flow cytometric micronucleus scoring procedure was evaluated (Flow-NBR). With this method, latex particles served as counting beads, and facilitated relative survival measurements that exclude the presence of dead/dying cells. For comparison purposes, additional cytotoxicity endpoints were measured, including several that are based on cell number, and others that reflect compromised membrane integrity, including dye permeability and/or phospholipid distribution. Key findings for this set of compounds include the following: (1) significant discrepancies in top concentration selection were found when cytotoxicity measurements were based on different methods, with the Flow-NBR approach tending to be the most sensitive, (2) both microscopy-and flow cytometry-based scoring methods detected concentrationdependent micronucleus formation for the three genotoxic agents studied, with good agreement between the reference laboratory and the collaborating laboratories, and (3) whereas flow cytometric analyses showed no significant increases for the non-genotoxicants when top concentration selection was based on Flow-NBR, significantly elevated micronucleus frequencies were observed for concentrations that were chosen based on less-sensitive cytotoxicity assays. Collectively, these results indicate that rapid assessment of genotoxicity can be accomplished with a relatively simple Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access

Assessing of genotoxicity of 16 centralized source-waters in China by means of the SOS/umu assay and the micronucleus test: Initial identification of the potential genotoxicants by use of a GC/MS method and the QSAR Toolbox 3.0

“…The counting beads were used to calculate the cell viability which was described by Bryce et al [25], and cytotoxicity was described as percentage relative survival (% RS). The samples were gently tapped up and down to re-suspend the cells before analysis of micronuclei (MN) and nuclei.…”

Delayed Mitogenic Stimulation Decreases DNA Damage Assessed by Micronucleus Assay in Human Peripheral Blood Lymphocytes after 60CO Irradiation

“…Recent reports from various laboratories have demonstrated its potential applicability for dose assessment. CBMN assay is now being considered as an attractive tool for triage biodosimetry, because of its advantages such as simplicity of scoring, accuracy, and most importantly ease of automation using microscopy and flow cytometry (Hayashi et al 2000;Bryce et al 2008;Willems et al 2010). Moreover, now a days the protocols are standardized in laboratories present all over the world by conducting an inter lab comparison, to minimize variations, by sharing of workloads and by conducting validations between the labs (Garcia et al 1995;Fenech et al 2003;Yoshida et al 2007;Beinke et al 2011).…”

Development and Validation of a Higher Throughput Screening Approach to Genotoxicity Testing Using the GADD45a-GFP GreenScreen HC Assay

“…A higher throughput in vitro micronucleus assay using mouse lymphoma L5178Y cells has been developed in which automated scoring of micronuclei and cytotoxicity is achieved by cell-staining approaches with analysis by flow cytometer. 15 The protocol was shown to be transferable between laboratories, but the number of validation compounds and the diversity of their chemistry are currently limited. There is also a routinely used microplate format for the mammalian thymidine kinse mutation assay (MLA), although this still requires many weeks for dose setting and the selection of revertants for counting.…”