Automated genetic instability studies in Oncology or Environmental Toxicology, and DNA damage evaluation in genetic toxicology and Genome Health follow-up
Drastic gain in accuracy and expert/technician time using high-content automated ScreenTox platform that can be used for large scale analysis of 5 major in vitro/in vivo assays such as Comet, Micro Nucleus, H2AX, CAS and SCE.
This allows DNA damage assessment tests for large scale monitoring of aging population or detection of pre-cancer stage, as well as tissue repair capacity for patients under chemotherapy or radiation therapy.
Automated Software modules for Comet assay, Micro Nucleus test, CAS, H2AX phosphorylation or SCE slides’ scanning and analysis may be performed on the same Pathfinder™ system.
Micro nucleus test
Analysis of micronuclei (MN) is a standard approach for assessment of chromosomal damage in exposed populations to study the genotoxic impacts of environment gene-interactions that increased steadily over the past two decades, as well as the reliable, sensitive and early assessment of DNA damage in oncology genetic instability, and in vitro/in vivo genetic toxicology. In particular, MN frequencies in cytokinesis-blocked lymphocytes (CBMN assay) is a reliable method as an early predictive biomarker for cancer risk.
To increase scoring accuracy and reliability, while saving time by 10-20 folds for various cell types (human lymphocytes, buccal cells, L51178Y, V79, CHL, CHO, TK6, HepG2…) and staining techniques, IMSTAR implemented automation of MN analysis by robust imaging system based on solid, specific image analysis algorithms, with distributed access, to be used in regulatory in vitro micronucleus studies compliant with GLP (users rights, blind scoring, audit trial) & OECD [C(97) 186] Guide Lines.
The software algorithms were designed for automated scoring of MN test in standardized preparations with detection of all cells, automated scoring of mono-, bi- and poly-nucleated cells, detection of micronuclei within these cells with high sensitivity, specificity and reliability. Automated Mitotic Index evaluation is included.
The detected micro nucleated cells can be “Reviewed and Validated” by the operator/expert using a very flexible interface.
The Customized Software Protocols (CSPs) are embedded in the Auto-MN software, specific to each cell type and staining (Giemsa, Feulgen, Acridin Orange, DAPI/CMO…), on regular or micro-well slides.
Algorithms also available for recognition of other cytogenetic events e.g. nucleo-plasmic bridges, nuclear buds, necrotic, apoptotic cells, could allow automation of a comprehensive Cytome analysis system.
Comet assay or single-cell gel electrophoresis is a well-established genotoxicity test and a simple method to detect a broad spectrum of DNA damage with high sensitivity, widely used in regulatory, mechanistic and biomonitoring studies at large scale.
For example, 110 slides are scanned by Pathfinder™ ScreenTox Auto-COMET in one day, comprising
Unattended processing for comets detection and scoring overnight by the Fully Automated system. Automated comets detection and % tail DNA evaluation of 100 – 700 randomly selected cells/slide.
Differences in % tail DNA of 100 – 700 cells scored by fully automated system analysed by one-way ANOVA analysis of variance (Dunnet’s test) to compare 3 concentrations vs corresponding control group.The influence of the concentrations and the number of cells scored on the CVs was investigated by two-way ANOVA analysis (Illustration 2).
The Auto-Comet module performs fully automated detection of thousands of cells, for reliable and accurate assessment of comets parameters (DNA% Tail, Comet Moment, Olive Tail Moment…). This approach provides also an operator-independent evaluation of DNA damage and repair in high volume data, recognized as the reference system by blue chip customers and Centers of Disease Control.
Customized Software Protocols (CSPs) are embedded in Auto-COMET software specific to: human lymphocytes (HL), HepG2, TK6, CHO, V79, Caco2, HEK, melanocytes, keratinocytes and fibroblast, for in vitro / in vivo methods.
Customized preparation techniques SOP published (Ref. 1– Azqueta et al. Mutagenesis (2011) vol. 26 no. 3 pp. 393–399; 2– Sharma A. et al. Mutation Research 749 (2012) 70-75; 3– Mutagenesis Jackson P. et al. Mutagenesis (2013) vol. 28 no.6 pp. 699-707) .
Chromosome Aberration Scoring (CAS) and Sister Chromatid Exchange (SCE)
These two modules can be used in conjunction with Metafind and Karyo.
After efficient automated metaphase finder and chromosomes’ detection, the interface offers to the user interactive tools for chromosome’ aberrations recognition, classification and numbering.
The list of aberrations can be customized to users’ needs, then stored in the database together with reports and all samples’ images and data.
Moreover, 2D-Profilometry allows automated recognition and accurate counting for Sister Chromatid Exchange test.
Protein Level Expression of H2AX
This module allows automated high content, high throughput screening of cell samples in any format (e.g. smear on pattern-slides or cell-on-chip) for quantitative evaluation of histone phosphorylation in response to radiation.
Automated molecular cytogenetics to address the needs for high-throughput quantitative studies.
Comprehensive solutions include fast and highly specific metaphase finder, very high resolution image capture, comprehensive report and large scale cross-studies.
Automated Karyotyping imaging and analysis integrating users’ requirements and interface features for optimized tools for assisted interpretation.
All operations in one batch for up to 160 slides. 3D detection of abnormalities, rearrangements, fusion, deletion, on metaphase and interphase cells (G-Band, FISH, M-FISH).
Karyotyping module performs automated chromosome detection, separation and high performance classification, with editing tools for overlapping chromosomes. Profilometry allows the recognition of multiple rearrangements.
CGH-2D profiling for automated assessment of DNA gain and loss ratio for all chromosomes, as well as genetic instability and probabilities for a given locus.
FISH module: Using FISH probes allows detection of fine chromosome abnormalities or specific gene locus position.
M-FISH: This software module allows the fully automated procedure for slide’s scanning for metaphase finding, and capture in 6 fluorescence spectral channels and fluorescence level quantification and classification of labelled chromosomes according to 24-color (combination of 6 channels).
Automated scanning of a whole sample, with detection of intra-nuclear FISH signals, in cell spreads or culture, using FISH & multi-FISH software modules.
Adapted for all FISH studies in onco-hematology and onco-genetics, requiring quantification of amplification, deletion, co-localization.