Interlaboratory evaluation of a multiplexed high information content in vitro genotoxicity assay

Abstract

We previously described a multiplexed in vitro genotoxicity assay based on flow cytometric analysis of detergent-liberated nuclei that are simultaneously stained with propidium iodide and labeled with fluorescent antibodies against p53, γH2AX, and phospho-histone H3. Inclusion of a known number of microspheres provides absolute nuclei counts. The work described herein was undertaken to evaluate the interlaboratory transferability of this assay, commercially known as MultiFlow™ DNA Damage Kit— p53, γH2AX, Phospho-histone H3. For these experiments seven laboratories studied reference chemicals from a group of 84 representing clastogens, aneugens, and non-genotoxicants. TK6 cells were exposed to chemicals in 96-well plates over a range of concentrations for 24 hrs. At 4 and 24 hrs cell aliquots were added to the MultiFlow reagent mix and following a brief incubation period flow cytometric analysis occurred, in most cases directly from a 96-well plate via a robotic walk-away data acquisition system. Multiplexed response data were evaluated using two analysis approaches, one based on global evaluation factors (i.e., cutoff values derived from all inter-laboratory data), and a second based on multinomial logistic regression that considers multiple biomarkers simultaneously. Both data analysis strategies were devised to categorize chemicals as predominately exhibiting a clastogenic, aneugenic, or non-genotoxic mode of action (MoA). Based on the aggregate 231 experiments that were performed, assay sensitivity, specificity, and concordance in relation to a priori MoA grouping were ≥ 92%. These results are encouraging as they suggest that two distinct data analysis strategies can rapidly and reliably predict new chemicals’ predominant genotoxic MoA based on data from an efficient and transferable multiplexed in vitro assay.

Predictions of genotoxic potential, mode of action, molecular targets, and potency via a tiered multiflow® assay data analysis strategy

“…Data analyses described herein were restricted to those concentrations that did not exceed the MultiFlow assay's cytotoxicity limit, that is, the top concentration of each chemical had to exhibit ≤80% reduction to RNC at the 24 hr time point. This has been described previously by Bryce et al (, ). The present report differs slightly, such that in addition to the 80% maximum cytotoxicity limit noted above, only two concentrations within the cytotoxicity range 70–80% were permitted.…”

“…Chemicals selected by Litron scientists were tested using the same experimental design described previously (Bryce et al , ). Briefly, treatments occurred in U‐bottom 96 well plates, with 198 μL TK6 cell suspension (2 × 10 5 /mL) combined with 2 μL of DMSO‐solubilized test chemical per well.…”

“…TK6 cells were prepared for analysis using reagents and instructions included in the MultiFlow® DNA Damage Kit—p53, γH2AX, Phospho‐Histone H3 (Litron Laboratories). Components and preparation of the MultiFlow working solution have been described in detail previously (Bryce et al , ). At the 4 and 24 hr sampling times, cells were resuspended with pipetting, then 25 μL were removed from each well and added to a new 96‐well plate containing 50 μL/well of pre‐aliquoted working MultiFlow reagent solution.…”

“…Representative bivariate graphs, gating logic, and position of regions were described in detail in earlier reports (Bernacki et al ; Bryce et al , ). Briefly, two biomarker measurements, γH2AX and p53, were based on the shift in median channel fluorescence intensity relative to same‐plate solvent controls.…”

“…Our laboratories have pursued the development and validation of a multiplexed flow cytometric assay that combines information from several biomarkers relevant to DNA damage response pathways and aneuploidy induction (Bryce et al , , , ; Bernacki et al ). This so‐called MultiFlow® DNA Damage Assay is formatted as an add‐and‐read test that efficiently prepares cells in microtiter plates for flow cytometric analysis.…”