Automation of the Cytokinesis-Block Micronucleus Cytome Assay by Laser Scanning Cytometry and its Potential Application in Radiation Biodosimetry

Abstract

Here we describe the adaptation of laser scanning cytometry (LSC) to measure micronuclei (MN) automatically in lymphocytes. MN frequencies were determined in irradiated human lymphocytes using the cytokinesis-block technique, and the results from LSC were compared with visual scoring results obtained from slides of cells stained using Fast Green and 4',6-diamidino-2-phenylindole (DAPI). This fluorescent approach allowed clear identification of binucleated cells and detection of MN. The dose responses measured visually and by LSC showed similar trends and correlated positively (r = 0.9689; P < 0.0001). High-content analysis was developed to further automatically score MN within mono-, tri- and tetra-nucleated cells and to determine the nuclear division index and nuclear circularity values. The high-throughput nature of LSC can provide unique advantages in future DNA damage diagnostics in experimental and epidemiological studies. Importantly, it allows for co-detection of other biomarkers of interest within a single lymphocyte, and further development of this capability is anticipated.

Automation of the in vitro micronucleus assay using the Imagestream® imaging flow cytometer

“…LSC systems allow for the identification and automated enumeration of MN as well as mononucleated (MONO), BN, and POLY cells. While LSC can differentiate MN from other DNA positive objects through the use of multiple stains, many systems are still hindered by the need to create high quality microscope slides with adequate cell separation (24)(25)(26). Finally, conventional flow cytometry methods have been developed to perform the in vitro MN assay.…”

Validation of the Cytokinesis-block Micronucleus Assay Using Imaging Flow Cytometry for High Throughput Radiation Biodosimetry

“…Most of these studies involve the use of the MNScore software module integrated into the metaphase finder of the Metafer system (MetaSystems, Baden-Württemberg, Germany) (Schunck et al 2004;Varga et al 2004;Willems et al 2010;Thierens et al 2014). Recently, François et al (2014) adapted the CBMN assay for use with laser scanning cytometry (LSC) and were able to identify MN within mono-, bi-, tri-, and tetra-nucleated cells. The percentage of BNCs with MN identified with the LSC method compared well to manual slide scoring between 0-4 Gy.…”

The Use of Image Processing and Analysis in Automated Biological Dosimetry

“…The implementation of image processing and analysis for CBMN assay began three decades ago. The CBMN assay is one of the preferred techniques for assessing chromosomal damage induced by mutagens, such as radiation exposure, in human populations [18]. Several automated CBMN assay systems have previously been tested (i.e., Metafer Metasystem, PathFinder, CellScan, imaging flow cytometry) to determine whether these systems can be used during large-scale radiological events [18].…”