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Updated: May 24, 2026
An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry
Published on: May 13, 2019
Re-evaluation of a reported increased micronucleus frequency in lymphocytes of workers occupationally exposed to
Günter Speit1, Carina Ladeira, Regina Linsenmeyer
1Institut für Humangenetik, Universität Ulm, Ulm, Germany. guenter.speit@uni-ulm.de
Abstract:
A replicate evaluation of increased micronucleus (MN) frequencies in peripheral lymphocytes of workers occupationally exposed to formaldehyde (FA) was undertaken to verify the observed effect and to determine scoring variability. May-Grünwald-Giemsa-stained slides were obtained from a previously performed cytokinesis-block micronucleus test (CBMNT) with 56 workers in anatomy and pathology laboratories and 85 controls. The first evaluation by one scorer (scorer 1) had led to a highly significant difference between workers and controls (3.96 vs 0.81 MN per 1000 cells). The slides were coded before re-evaluation and the code was broken after the complete re-evaluation of the study. A total of 1000 binucleated cells (BNC) were analysed per subject and the frequency of MN (in ‰) was determined. Slides were distributed equally and randomly between two scorers, so that the scorers had no knowledge of the exposure status. Scorer 2 (32 exposed, 36 controls) measured increased MN frequencies in exposed workers (9.88 vs 6.81). Statistical analysis with the two-sample Wilcoxon test indicated that this difference was not significant (p=0.17). Scorer 3 (20 exposed, 46 controls) obtained a similar result, but slightly higher values for the comparison of exposed and controls (19.0 vs 12.89; p=0.089). Combining the results of the two scorers (13.38 vs 10.22), a significant difference between exposed and controls (p=0.028) was obtained when the stratified Wilcoxon test with the scorers as strata was applied. Interestingly, the re-evaluation of the slides led to clearly higher MN frequencies for exposed and controls compared with the first evaluation. Bland-Altman plots indicated that the agreement between the measurements of the different scorers was very poor, as shown by mean differences of 5.9 between scorer 1 and scorer 2 and 13.0 between scorer 1 and scorer 3. Calculation of the intra-class correlation coefficient (ICC) revealed that all scorer comparisons in this study were far from acceptable for the reliability of this assay. Possible implications for the use of the CBMNT in human biomonitoring studies are discussed.
Insights
This study re-evaluated micronucleus (MN) frequencies in formaldehyde-exposed workers, finding significant differences only when combining scorer data, highlighting poor inter-scorer reliability in biomonitoring. Occupational formaldehyde exposure requires careful assessment due to potential genotoxicity.
Area of Science:
- Environmental Health
- Toxicology
- Biomonitoring
Background:
- Occupational exposure to formaldehyde (FA) has been linked to increased micronucleus (MN) frequencies in peripheral lymphocytes.
- Previous studies reported significant increases, necessitating replicate evaluation to confirm findings and assess scoring variability.
Purpose of the Study:
- To verify the observed increase in MN frequencies in workers exposed to formaldehyde.
- To determine the scoring variability associated with the cytokinesis-block micronucleus test (CBMNT) in occupational biomonitoring.
Main Methods:
- Re-evaluation of May-Grünwald-Giemsa-stained slides from 56 formaldehyde-exposed workers and 85 controls using the CBMNT.
- Slides were randomly distributed between two independent scorers unaware of exposure status.
- Analysis involved scoring 1000 binucleated cells (BNC) per subject and statistical analysis using Wilcoxon tests and Bland-Altman plots.
Main Results:
- Individual scorers showed non-significant differences in MN frequencies between exposed workers and controls (Scorer 2: p=0.17; Scorer 3: p=0.089).
- Combining data from both scorers yielded a significant difference (p=0.028).
- Bland-Altman analysis revealed very poor agreement between scorers (mean differences 5.9 and 13.0), and low intra-class correlation coefficients (ICC) indicated unreliable assay performance.
Conclusions:
- The reliability of the CBMNT assay for human biomonitoring is questionable due to significant inter-scorer variability.
- While formaldehyde exposure may increase MN frequencies, the current scoring methods present challenges for consistent and reliable detection.
- Further standardization of CBMNT scoring protocols is crucial for accurate occupational exposure assessment.
