The dicentric chromosome assay (DCA) is considered the ‘gold standard’ for radiation biodosimetry [IAEA-2001], [Blakely et al. 2009], [IAEA-2011], and is an important tool for radiation dose assessment in radiation accidents. A dicentric chromosome is an aberrant chromosome bearing two or more centromeres derived from the mis-repair of two broken chromosomes [IAEA-2011]. The prevalence of these chromosomes decreases as time passes from the irradiation event, due to the limited lifespan of lymphocytes hosting them, which is typically a few months. This fact is used as a marker of recent exposure to ionizing radiation [Beaton-Green et. al. 2016], [Pinto et. al. 2010].
The DCA is challenging to use for dose determination in an emergency response setting because it is labor-intensive and time-consuming. The growth of a primary lymphocyte culture for 48h in vitro is required for the DCA, and manual scoring of dicentric chromosomes (DCs) requires at least an additional 24–48 h, resulting in an overall processing time of 72–96h for dose estimation. This makes the DCA impractical for many applications. [Ryan et. al. 2019].
To accelerate the speed of DCA analysis for dose estimation, automated tools were developed. The automated tools included harvesters [Subramanian 2020], metaphase finders [Weber et al. 1992][Ryan et al. 2019] and dicentric scoring software packages [Ryan et al. 2019]. Recently, automated dicentric chromosome aberration scoring software modules have gained wide-spread use [Romm et al. 2–13][ Gruel et al. 2013][Shirley et al. 2017][Oestreicher et al. 2018][Li et al. 2019]. The dose that was obtained with automatic dicentric scoring was found to be close to the dose obtained with manual dicentric scoring of 500 metaphases [Vaurijoux et al. 2009].
The device termed metaphase finder consists of an automated microscope, an auto-focus system, a motorized X–Y stage, a camera, and a computer. It performs image analysis of the microscopic images of glass slides and displays the positions of metaphase cells. Chromosome image quality is critical for automated dicentric chromosome (DC) detection and much effort has been invested in the development of improved image segmentation methods for selecting high quality metaphase chromosome spreads for radiation biodosimetry [Ryan et al. 2019]. There are several commercial implementations of metaphase finders [Finnon et al. 1986][Castleman 1992][Balajee et al. 2018].
The speed of metaphase finders has improved with time [Weber et al. 1992]. These systems are usually expensive and inconvenient for use with diverse morphologies of chromosome preparations (e.g. high-resolution, or condensed/double-stranded chromosomes). They are also difficult to deploy in the field [Furukawa 2019]. [Roy et al. 2007] found that using a metaphase finder increased the speed of scoring by a factor of 2. However, it was hoped that automatic scoring of a sufficient number of captured images would reduce the time needed for processing 500 metaphases from one full day, by a trained scorer, to a matter of a few minutes. Since then, progress has been made, but to enhance the accuracy of the metaphase finder systems it is likely that a deep learning approach is needed [Furukawa 2019]. It is possible to conclude, then, that metaphase analysis systems that are faster, cheaper, and more compact than what is available today, are needed [Furukawa 2019].
Enrichment of metaphase cells may be a good alternative to the use of metaphase finder systems. A single slide containing a metaphase-enriched sample may hold by itself the number of metaphase cells required to be scored at triage, or in other events where hundreds to thousands of metaphase spreads must be analyzed. In the past, as well as more recently, efforts were made to utilize this method for purposes other than biodosimetry [Wolff et al. 1972][Nagaraj et al. 2018] using differences in cell mass and volume between metaphase cells and interphase cells. It was found that (i) as cells approach metaphase their volume decreases and (ii) towards the end of cytokinesis but prior to abscission, the combined volume of the two daughter cells equals that of their mother cell during prophase [Boucrot E., et al. 2008].
Cells in mitosis were found using flow cytometry, and discriminated from G1, S, and G2 cells by analysis of a nuclear or cell suspension prepared with nonionic detergents, fixed with formaldehyde, and stained with mithramycin, propidium iodide, or ethidium bromide. The fluorescence of these fluorochromes and formaldehyde is quenched to a higher extent by interphase nuclei than by mitotic nuclei [Larsen et al. 1986].
Mitotic nuclei were found to have increased mithramycin fluorescence and decreased light scatter in comparison to those of G2 nuclei. A high correlation was found between microscope counts of mitotic figures in smear preparations of the initial cell suspension and the flow-cytometrically estimated fractions of nuclei with increased mithramycin fluorescence.
Flow sorting (FACS) demonstrated that the mitotic nuclei were confined to the peak of increased mithramycin fluorescence and decreased light scatter [Larsen et al. 1986]. Another procedure for sorting and counting of mitotic cells was developed using flow-cytometric measurement of a mitosis-specific antigen [Anderson et al., 1998] and then discriminating on the antigen, which was fluorescence-tagged prior to application. This method may be used in conjunction with the measurement of cellular DNA content and of bromodeoxyuridine incorporation into cellular DNA to assign cells to the G1/G0, S, G2, or M phase of the cell cycle. Other protocols for analyzing cell cycle status using flow-cytometry that have been published include:
(1) Simultaneous analysis of a proliferation-specific marker (Ki-67) and cellular DNA content, which discriminates resting/quiescent cell populations (G0 cell) and quantifies cell cycle distribution (G1, S or G2/M, respectively) [Kim et al., 2016],
(2) Differential staining of DNA and RNA through co-staining of Hoechst-33342 and Pyronin Y, which is also useful to identify G0 cells from G1 cells [Kim et al., 2016],
(3) Following the phosphorylation of the histone H3 at Ser 10, Ser28 and Thr11. This process is tightly correlated with chromosome condensation during both mitosis and meiosis [Hendzel et al., 1997][Goto et al., 1999][Preuss et al. 2003], and the phosphorylated H3 (Ser 10) is considered as a characteristic marker of mitosis [Juan et al.,1998],
(4) Successful sorting of mammalian cells undergoing cytokinesis by selective flow-cytometric methods [Gasnereau et al., 2007]. Cultures of HeLa cells were arrested in prometaphase by nocodazole, collected by mitotic shake-off and released for 90 min into fresh medium to enrich for cells undergoing cytokinesis. After ethanol fixation and DNA staining, cells were sorted based on DNA content and DNA fluorescence signal height. This research showed that a cell population was transiently accumulated when synchronized cells exit mitosis before their entry into G1. Two properties were evident of these samples: (1) this population was highly enriched in cells undergoing cytokinesis, and (2) it was successfully sorted and analyzed by immunofluorescence and western blotting. This method of cell synchronization and sorting provided a simple means to isolate and biochemically analyze cells in cytokinesis, a period of the cell cycle that has been difficult to study by cell fractionation.
(5) Staining of cells to detect Gamma-H2AX with a fluorescent antibody (Gamma-H2AX-AF488 53BP1) and then counting them with flow cytometry (Cheyene et. Al. 2022).
There are several other biochemical methods for analyzing the cell cycle status, cellular proliferation and expression of the genes of interest. For example, several methods quoted in [Darzynkiewicz et al. 1977][Nusse et al. 1989][Nusse et al. 1990][Roti et al. 1982][Zucker 1988][Larsen et al. 1986][Di Vinci et al. 1993][Gong et al. 1995][Landberg et al. 1990]. These depend upon the changing properties of chromatin as it condenses into chromosomes, some of which are detected in whole fixed cells while others are detected in nuclei obtained from cells lysed in presence of nonionic detergents.
These depend on newly developed fluorophores that sometimes rely on antibodies to label the different entities within the cell, as it progresses through the mitotic cycle. These new methods and fluorophores also enable sorting using flow cytometry.
In this study, we developed a specific method to treat fixed cells as done by the classic DCA protocol [IAEA-2011] and used immunofluorescent staining to detect an antigen that is specific to cells in mitosis (especially in metaphase). The cells were already somewhat enriched in metaphase due to synchronizing the cells by using methotrexate and due to the use of colcemid) [Juan et al.,1998, Fox, M. H., 1987]. We then selected the mitotic cells by flow cytometric sorting. In addition, as compared to other earlier mentioned methods which uses flow sorting (FACS) capabilities, our method is potentially simpler than the other methods, and from the non-productive methods that use differences in weight and volume of cells to enrich the metaphase fraction (we assayed the enrichment method based on the weight of the cells. These data are not shown).
The goal of our enrichment method was to significantly reduce the number of slides per patient required for the DCA. We aim to perform the DCA analysis on a single slide that will contain predominantly metaphase-cells. The slide will then be analyzed by a dicentric scoring program that runs as a part of an automated chain including a fluorescent microscope and motorized stage. This will considerably shorten the processing time and will help speed up the Dicentric Chromosome Assay (DCA).