Robots to the Rescue: Automated Analysis of Cytogenetic Biomarkers for Genotoxic Risk Assessment
Cytogenetic methods play a pivotal role in human biomonitoring, serving as essential tools for evaluating the presence and extent of chromosomal damage in populations exposed to various genotoxic agents. These agents may be biological, chemical, or physical in nature, and air pollution, in particular, encompasses all three categories.
One of the most widely employed cytogenetic techniques is the cytokinesis-block micronucleus (CBMN) assay, which quantifies the frequency of micronuclei (MNi) in different cell types, including peripheral blood lymphocytes, liver cells, and epithelial cells such as those in the buccal mucosa or lungs. The CBMN assay conducted on human peripheral blood lymphocytes is among the most validated approaches for biomonitoring individuals exposed to chemical and physical genotoxic agents. An elevated MNi frequency in otherwise healthy individuals is indicative of genomic instability, a condition that heightens the risk of carcinogenesis, making this assay a valuable predictive tool in cancer risk assessment. The CBMN assay assesses several parameters indicative of both cytotoxic and genotoxic effects, which may be influenced by a range of environmental and biological factors.
Micronuclei (MNi) are small, oval-shaped chromatin bodies that separate from the main nucleus, arising from the condensation of acentric chromosome fragments or entire chromosomes that fail to be incorporated into daughter nuclei during anaphase. Their presence serves as a quantitative biomarker of structural and numerical chromosome aberrations resulting from genotoxic exposures. Apart from representing the products of biological errors, MNi can also indicate other biological effects in terms of their leakage into the cytoplasm which may trigger an immune response. It was recently shown that these nuclear structures are not only biomarkers of disease but also play an active role in tumour biology. Many consequences of MNi formation on tumour biology are dependent on the frequent and irreversible rupture of their nuclear envelopes, which results in the exposure of their DNA contents to the cytoplasm. In addition to MNi, the CBMN assay can identify nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs). NPBs are typically formed due to dicentric chromosomes, defective DNA repair mechanisms, or errors in sister chromatid separation, while NBUDs often signal gene amplification events or the elimination of excess genetic material from aneuploid cells. Despite their small size, these parameters have a significant impact on cells and their microenvironment, collectively offering a comprehensive assessment of genomic stability.
Despite its significant advantages, the CBMN assay has certain limitations, including the necessity for highly trained personnel, labour-intensive procedures, and potential observer subjectivity. To mitigate these challenges, the adoption of automated systems is becoming increasingly crucial. A particularly effective solution is the Metafer slide scanning software (MetaSystems, Germany, https://metasystems-international.com/), which enables high-throughput automated analysis across various microscopy applications. The motorised scanning stage is equipped with two motors that enable precise movement in four primary directions (left, right, forward, and backward). An essential component of this system is the 80-position slide feeder, designed with sixteen frames, each capable of holding up to five slides. For manual adjustments, a trackball allows users to navigate the stage to a specific area of interest. Additionally, an automated arm seamlessly selects the designated frame from the slide feeder and loads it onto the microscope stage, ensuring a smooth and efficient workflow. Metafer streamlines the automated scoring of MNi and other chromosomal aberrations, enabling the analysis of large cell populations with high accuracy. Additionally, the system supports image storage for retrospective analysis, further enhancing its reliability and utility in cytogenetic research.
Given these capabilities, Metafer represents a promising advancement in cytogenetic biomonitoring. Its application is particularly valuable for improving the risk assessment of populations exposed to genotoxic hazards, contributing not only to cancer prevention but also to high-throughput biodosimetry in the aftermath of large-scale radio-nuclear incidents.
As part of the EDIAQI project, Metafer is actively utilised by partner institutions to enhance automated cytogenetic analysis. At the Institute for Medical Research and Occupational Health (IMROH, https://www.imi.hr/en/) in Zagreb, Croatia, the system is applied for the automated detection of MNi in human peripheral blood lymphocytes, while at the National Institute of Biology (NIB, https://www.nib.si/) in Ljubljana, Slovenia, it is employed for the analysis of MNi in HepG2 cells. A key aspect of the EDIAQI project is the transfer of knowledge between partner institutions, fostering a deeper understanding of the technology and its application. This exchange of expertise will enhance the implementation of automated methodologies, streamline workflows, and support future validation of the method. By strengthening collaboration between laboratories, the project emphasises the growing importance of automation in biomedical research, ultimately improving efficiency and accuracy in genomic stability assessment.
Figure 1. Automated Micronucleus Assay Workflow Using the Metafer System and Zeiss Microscope
This collage illustrates various stages of the micronucleus (MN) assay and its automation using the Metafer system integrated with a Zeiss microscope. (A) MN cultures prepared for analysis. (B) Blood samples undergoing centrifugation, with the cell pellet visible at the bottom. (C) Fixed cell slides placed onto the Metafer slide-frame. (D) Metafer software identifying focus points on the slide. (E) Visual analysis of binuclear cells, identified and counted by Metafer. (F) Visualisation of binuclear cell locations on the slide. (G) Microscope objectives used for fluorescence microscopy. (H) A library of identified binuclear cells within the Metafer system. (I) DAPI-stained cells observed under the microscope, highlighting nuclei for analysis.
Note: This article has been published on behalf of Luka Kazensky & Goran Gajski, Institute for Medical Research and Occupational Health (IMROH), Zagreb, Croatia.
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