GenEvolutioN is pleased to share new findings on the use of telomere and centromere FISH staining (fluorescence in situ hybridization) to enhance the detection of chromosomal aberrations in a dicentric chromosome assay (DCA). Conducted as part of the RENEB 2021 inter-laboratory comparison, this approach significantly improves accuracy in biological dosimetry, particularly for applications in radiation emergency medicine and large-scale genotoxic screenings.
1. Context and Objectives
In a radiological or nuclear emergency, rapid and accurate dose estimation is vital for triage and immediate medical decision-making. Conventional DCA methods often rely on uniform staining techniques (e.g., Giemsa or DAPI), which can underestimate certain aberrations. By contrast, telomere–centromere FISH staining provides distinct fluorescent markers at chromosome ends and centromere regions, improving the detection of dicentrics that may otherwise go unnoticed.
Key Goals:
•Enhance Detection: Reduce the underestimation of dicentric chromosomes using FISH staining.
•Evaluate Feasibility: Assess ease of use and reliability under emergency conditions (triage mode).
• Compare Endpoints: Investigate two cytogenetic assays—dicentric chromosome assay (DCA) and cytokinesis-block micronucleus assay (CBMN)—to classify exposure levels accurately.
2. Methods: RENEB Inter-Laboratory Comparison
Study Design
• Three Blinded Samples exposed to X-rays at 0.0, 1.2, and 3.5 Gy.
• Whole Blood from a Single Donor (male, age 32).
•Multiple Laboratories: 14 labs for the CBMN assay, 33 labs for the DCA.
•Each lab reported dose estimates in a short timeframe, simulating emergency scenario constraints.
Telomere–Centromere FISH Staining
•Telomere (red) and Centromere (green) Probes: Clearly highlight chromosome ends and centromeres.
•Improved Aberration Identification: Distinguish genuinely dicentric chromosomes from closely adjacent centromeres that appear as a single entity under uniform staining.
Endpoints
1. Dicentric Chromosome Assay (DCA)
•150 metaphases scored per sample in semi-automated mode.
•High-level expertise required for Giemsa, more straightforward with FISH staining.
2. Cytokinesis-Block Micronucleus Assay (CBMN)
•1000 binucleated (BN) cells scored in semi-automated mode.
•FISH can also be applied to differentiate clastogenic (telomere-positive, centromere-negative) from aneugenic (centromere-positive) micronuclei.
3. Key Findings
•Reduced Undercounting of Dicentrics: Telomere–centromere FISH minimizes false negatives by revealing closely spaced centromeres as truly separate.
•Accurate Dose Estimation: Both DCA and CBMN effectively classified the blinded samples into correct triage categories (low, medium, high).
•Faster Triage Mode: Participating labs successfully reported results quickly, supporting emergency medical management.
•Ease of Interpretation: FISH signals provide a clearer, more direct indication of chromosome breakage or mis-segregation, lowering the required skill threshold compared to uniform stains.
4. Implications for Radiation Emergency Medicine
1.High-Throughput: This approach scales well for processing large cohorts, crucial during mass exposure events.
2.Improved Reliability: Early identification of significantly exposed individuals leads to timely clinical interventions.
3.Versatile Applications: Beyond radiation emergencies, telomere–centromere FISH can enhance genotoxic agent detection in chemical or pharmaceutical industries.
GenEvolutioN’s participation in the RENEB 2021 inter-laboratory exercise demonstrates how telomere–centromere FISH staining revolutionizes dicentric chromosome assays. By reducing underestimation of dicentrics and streamlining detection, laboratories gain a robust, accurate method for biological dosimetry, essential for both emergency response and routine toxicological assessments.
