Automated Micronucleus Scoring Research Articles

Improving radiation dosimetry with an automated micronucleus scoring system: correction of automated scoring errors

Radiation dose estimations performed by automated counting of micronuclei (MN) have been studied for their utility for triage following large-scale radiological incidents; although speed is essential, it also is essential to estimate radiation doses as accurately as possible for long-term epidemiological follow-up. Our goal in this study was to evaluate and improve the performance of automated MN counting for biodosimetry using the cytokinesis-block micronucleus (CBMN) assay. We measured false detection rates and used them to improve the accuracy of dosimetry. The average false-positive rate for binucleated cells was 1.14%; average false-positive and -negative MN rates were 1.03% and 3.50%, respectively. Detection errors seemed to be correlated with radiation dose. Correction of errors by visual inspection of images used for automated counting, called the semi-automated and manual scoring method, increased accuracy of dose estimation. Our findings suggest that dose assessment of the automated MN scoring system can be improved by subsequent error correction, which could be useful for performing biodosimetry on large numbers of people rapidly, accurately, and efficiently.

Radiation monitoring with plant-based biotas and an automated micronucleus scoring approach

Radiation monitoring with plant-based biotas and an automated micronucleus scoring approach

A scanning and image processing system with integrated design for automated micronucleus scoring

Purpose: Our aim was to design a compact and cost-effective optical microscopic system for automated non-fluorescent micronucleus (MN) scoring whose performance can reach the accuracy of visual scoring with the help of minimal user interaction and also gives an option for fully automatic scoring with an accuracy suitable for triage purposes.Materials and methods: The concept of Radometer MN-Series (RS-MN) microscopic system designed by Radosys was to develop hardware and software layers in parallel in order to optimize the performance in automated MN scoring. A MN assay slide is automatically scanned by the RS-MN then the binucleated cells and micronuclei are automatically identified. Processing 1000 cells takes 10–60 minutes by automatic scoring (scanning plus image processing) depending on sample quality and required accuracy. The manual revision of the cell gallery takes an extra 10 minutes per sample. Dose response curves are determined for manual, automatic and semi-automatic scoring methods.Results: The combination of object-sensitive autofocusing method and the multi-layer image acquisition is able to reduce the minimum resolvable dose by 14%. The MN yields obtained from the manual, semi-automatic and automatic scoring methods are well correlating (Pearson’s correlation coefficients are between 0.977 and 0.998). In order to compare the reliability of the results of visual and automatic scoring, an extended analysis on uncertainty contributors was performed. For a dose of 1 Gy, the estimated relative uncertainty from the Poisson characteristics of MN yield is 17–19% for the manual and 20–38% for the automated scoring. Other uncertainty factors (differences in donor radiosensitivity, scorer performance, and sample preparations) can contribute to this error fall within a similar range: 3–16%. Taking into account all of the possible uncertainties, the minimum resolvable dose for the manual (0.48 Gy) is the two-thirds of that of the automatic scoring (0.61 Gy).Conclusions: The results verify that the fully automatic mode of RS-MN is suitable for triage purposes. The performance of the user interacted semi-automatic mode is comparable with the reference manual scoring. Its performance reaches up to other non-fluorescent automatic systems and offers a compact and cost-effective alternative.

Seven benzimidazole pesticides combined at sub-threshold levels induce micronuclei in vitro

Benzimidazoles act by disrupting microtubule polymerisation and are capable of inducing the formation of micronuclei. Considering the similarities in their mechanisms of action (inhibition of microtubule assembly by binding to the colchicine-binding site on tubulin monomers), combination effects according to the principles of concentration addition might occur. If so, it is to be expected that several benzimidazoles contribute to micronucleus formation even when each single one is present at or below threshold levels. This would have profound implications for risk assessment, but the idea has never been tested rigorously. To fill this gap, we analysed micronucleus frequencies for seven benzimidazoles, including the fungicide benomyl, its metabolite carbendazim, the anthelmintics albendazole, albendazole oxide, flubendazole, mebendazole and oxibendazole. Thiabendazole was also tested but was inactive. We used the cytochalasin-blocked micronucleus assay with CHO-K1 cells according to OECD guidelines, and employed an automated micronucleus scoring system based on image analysis to establish quantitative concentration–response relationships for the seven active benzimidazoles. Based on this information, we predicted additive combination effects for a mixture of the seven benzimidazoles by using the concepts of concentration addition and independent action. The observed effects of the mixture agreed very well with those predicted by concentration addition. Independent action underestimated the observed combined effects by a large margin. With a mixture that combined all benzimidazoles at their estimated threshold concentrations for micronucleus induction, micronucleus frequencies of ~15.5% were observed, correctly anticipated by concentration addition. On the basis of independent action, this mixture was expected to produce no effects. Our data provide convincing evidence that concentration addition is applicable to combinations of benzimidazoles that form micronuclei by disrupting microtubule polymerisation. They present a rationale for grouping these chemicals together for the purpose of cumulative risk assessment.

A semi-automated micronucleus-centromere assay to assess low-dose radiation exposure in human lymphocytes

Purpose: The in vitro micronucleus (MN) assay is a reliable method to assess radiation-induced chromosomal damage in human peripheral blood lymphocytes. It is used to evaluate in vivo radiation over-exposure and to assess in vitro chromosomal radiosensitivity. A limitation of the MN assay is the relatively high and variable spontaneous MN frequency that restricts low-dose estimation to doses of about 0.3 gray (Gy). As radiation-induced MN mainly contain acentric fragments and spontaneous MN originate from lagging chromosomes, both MN types can be distinguished from each other by using fluorescence in situ hybridisation (FISH) with a pan-centromeric probe. The aim of this study was to investigate if the sensitivity, reliability and processing time of the MN assay can be enhanced by combining the automated MN assay with pan-centromere scoring.Materials and methods: Blood samples from 10 healthy donors were irradiated in vitro with low doses of gamma-rays. Dose response curves were determined for fully-automated and semi-automated MN scoring and semi-automated scoring of centromere negative MN (MNCM−).Results: A good correlation was obtained between fully-automated and semi-automated MN scoring (r2 = 0.9973) and between fully automated MN scoring and semi-automated scoring of MNCM− (r2 = 0.998). With the Wilcoxon test, a significant p value was obtained between 0 and 0.2 Gy for the fully-automated MN analysis, between 0 and 0.1 Gy for semi-automated MN analysis and between 0 and 0.05 Gy for semi-automated scoring of MNCM−.Conclusion: The semi-automated micronucleus-centromere assay combines high-speed MN analysis with a more accurate assessment in the low-dose range which makes it of special interest for large-scale radiation applications.

Automated micronucleus (MN) scoring for population triage in case of large radiation accidents.

In case of a large radiation accident, when hundreds of people may be exposed, it is very important to distinguish the severely exposed individuals, who require early medical treatment, from those less exposed. In such a situation, specific biological indicators should promptly indicate whether the whole-body dose received by the victims exceeded 1Gy, the level above which it is necessary to provide medical care. Once the exposed population has undergone such an early triage, a second step may be indicated for a more accurate assessment of the exposure. Up to now, scoring of dicentrics is the reference to estimate the dose received in case of individual overexposures. However, to obtain a reliable dose estimate, 500 metaphases should be screened for the presence of dicentrics, a very time consuming procedure, that can only be applied to small populations of exposed individuals. The aim of our study was to develop a quick population triage method based on automated micronucleus (MN) scoring. Automated MN scoring was performed using a MN software module specifically developed by Metasystems for the Metafer4 platform. Standard dose response curves were established for manual, automated and semi-automated MN scoring. To this aim, blood samples of 10 individuals were irradiated with in vitro doses of 0-0,2-0,5-1-2-3Gy Co-60 γ-rays. Whole blood cultures were set up and cells were harvested after 70h, applying rigorous culture/harvest conditions. Cells were stained with DAPI for manual and automated scoring. For both scoring methods 500 binucleated (BN) cells were counted. The reproducibility, accuracy and sensitivity of the manual versus automated scoring procedure were analyzed. A comparison between manual and automated scoring of the same slides, revealed a drop in the MN yield for the automated scoring procedure. However, the automated scores were as reproducible as the manual scores and both scores were highly correlated. Scoring of 500 BN cells allows us to detect a dose of 0,5 Gy, with 95% confidence, for both manual and automated MN scoring procedures (Mann-Whitney U test, p<0.05). The semi-automated scoring procedure, taking into account false positives and false negatives in the MN class, didn’t result in a better accuracy or reproducibility. With the automated MN scoring procedure, about 80 slides (500 BN cells) can be scored in 1 day with the Metafer4. In conclusion, our preliminary results show that automated MN scoring is a quick and reliable method, useful for a first population triage in case of large radiation accidents.

Validation of an automated image analysis micronucleus scoring system

The Loats Automated Micronucleus Scoring System was developed to assist with the evaluation of compounds for the ability to induce micronuclei in mouse bone marrow polychromatic erythrocytes (PCE). This image analysis system can identify PCE as well as normochromatic erythocytes (NCE) and calculate the PCE/NCE ratio as an index for bone marrow toxicity. Two studies were conducted to provide slides for a comparison of micronucleated PCE values collected manually to those collected by the automated system. Mitomycin C was used as a micronucleus-inducing agent to elicit a positive response and Lilly compound 303497 was used as an example of a compound negative for the induction of micronuclei. No statistically significant differences were observed between micronucleus counts obtained manually and those obtained by the automated system. The PCE/NCE ratios calculated by the automated system were also similar to those determined from the manually collected PCE and NCE counts for the vehicle and positive controls, however, differences in the ratios were observed in compound treatment groups. These differences were attributed to a larger population of transitional cells in the treated groups. These results confirm that the Loats Automated Micronucleus Scoring System is an acceptable alternative to manual evaluation of mouse bone marrow slides for the incidence of micronucleated PCE.

Automated Micronucleus Scoring Workshop

Automated Micronucleus Scoring Workshop

Automated Micronucleus Scoring Workshop November 17–18, 1988

Automated Micronucleus Scoring Workshop November 17–18, 1988 Get access Mutagenesis, Volume 3, Issue 6, November 1988, Page 532, https://doi.org/10.1093/mutage/3.6.532 Published: 01 November 1988