RE: Recommendations, evaluation and validation of a semi-automated, fluorescent-based scoring protocol for micronucleus testing in human cells (Mutagenesis, 29, 155-164, 2014).

We are writing in regard to the article by Seager et al. (1) that assesses and validates the Metafer slide scanning system, developed by MetaSystems (Altlussheim, Germany), for the automated scoring of micronuclei (MN) in the in vitro cytokinesis-block micronucleus assay. Specifically, we would like to highlight a further use of the Metafer system that is not widely known and may facilitate the efficient and objective measurement of cytotoxicity in cells exposed to genotoxicants. As part of Test Guideline 487 ‘The In Vitro Mammalian Cell Micronucleus Test’, the Organisation for the Economical Cooperation and Development (OECD) specifies that concurrent measures of cytotoxicity and/or cytostasis should be quantified when the cytokinesis-block method is used (2). Annex 2 of the guideline outlines calculations for cytokinesis-block proliferation index (CBPI) and the replicative index that rely on the enumeration of mononucleated, binucleated and multinucleated cells in the treated and control cultures. In their study, Seager et al. seeded satellite cultures of their cells in order to calculate the relative population doubling and relative increase in cell count as indicators of cytotoxicity/cytostasis. We would like to suggest that it would have also been possible to score the CBPI concurrently and automatically using the Metafer slide scanning system on the same slides used for scoring MN. In 2011, MetaSystems released a white paper outlining the option of using their software to count the nuclei in cells for the purposes of calculating the CBPI (3). However, the white paper contains only cursory information, and the classifier provided by MetaSystems requires modifications based on the cells of interest. With guidance from MetaSystems, we have optimised a classifier for use with a murine epithelial lung cell line, the details of which are shared below so that other investigators can also employ the platform to score both MN frequency and CBPI. Prepared slides were stained with 500 nM of propidium iodide (PI) for 5 min, then stained with 200 ng/ml of 4′-6-diamidino-2-phenylindole (DAPI) solution for 5 min and cover slips affixed with VectaShield® mounting medium (either with or without DAPI). It should be noted that this dual staining protocol addresses one of the troublesome issues noted by Seager et al. That is, when manually verifying scored slides, it can be difficult to identify whether a MN belongs to one cell or another. PI effectively defines cell boundaries by staining the cytoplasm and thus facilitates the assignment of MNs to particular cells (Figure 1). Slides were scanned on the Metafer4 platform (v3.8.6) with a Carl Zeiss Axio Imager M1 microscope, equipped with a CoolCube 1 charge-coupled device camera and Märzhäuser motorized stage that scans eight slides unattended (MetaSystems). Slides were first scored with the MNScore function to obtain the number of MN in binucleated cells and subsequently with the MetaCyte function to detect the number of nuclei in each cell for calculation of CBPI. When optimising the MetaCyte classifier, we found that first modifying the Integration Time, and specifically the Minimum Integration Time, resulted in the most improvements in cell image capture. Pressing ‘c’ on the keyboard while in the gallery allowed the captured cell contours to be verified. In addition to Integration Time, the Saturation Area (which is typically higher for the red channel), the Object Threshold (estimated using the Classify Fields Function), the Camera Gain Factor (inversely proportional to integration time and