Cytokinesis-block Micronucleus Assay by Manual and Automated Scoring

Abstract

The cytokinesis-block micronucleus assay in peripheral blood lymphocytes is one of the best standardized and validated techniques for individual radiation dose assessment. This method has been proposed as an alternative to the dicentric chromosome assay, which is considered the "gold standard" in biological dosimetry because it requires less time and cytogenetic expertise. Nevertheless, for application as a biodosimetry tool in large-scale nuclear or radiological accidents, the manually performed cytokinesis-block micronucleus assay needs further strategies (e.g., the automation of micronucleus scoring) to speed up the analysis. An essential prerequisite for radiation dose assessment is to establish a dose-effect curve. In this study, blood samples of one healthy subject were irradiated with seven increasing doses of x-ray (240 kVp, 1 Gy min⁻¹) ranging from 0.25-4.0 Gy to generate calibration curves based on manual as well as on automated scoring mode. The quality of the calibration curves was evaluated by determination of the dose prediction accuracy after the analysis of 10 blood samples from the same donor exposed to unknown radiation doses. The micronucleus frequencies in binucleated cells were scored manually as well as automatically and were used to assess the absorbed radiation doses with reference to the respective calibration curve. The accuracy of the dose assessment based on manual and automatic scoring mode was compared.

Optimized automated data analysis for the cytokinesis‐block micronucleus assay using imaging flow cytometry for high throughput radiation biodosimetry

“…Figure B shows the average calibration curve for the rate of MN per BNC from both Figure A and our previous work . Also plotted in Figure B are calibration curves from Schunck et al , Willems et al , De Sanctis et al , and Thierens et al . These curves were plotted using the quadratic equations provided by the authors and demonstrate the variability between calibration curves using automated slide‐scoring based versions of the CBMN assay.…”

“…(B) The average rate of MN per BNC from A (squares) and the average rate of MN per BNC from our previous work in Rodrigues et al (circles) including error bars which represent the standard error of the mean. The rate of MN per BNC from Schunck et al (solid line), Willems et al (dashed line), De Sanctis et al (dashed‐dotted line), and Thierens et al (dotted line) are also plotted using the equations provided in those publications. (C, D) Estimated dose (Gy) versus delivered dose (Gy) determined by the IS X ‐CBMN method for nine blinded samples using the optimized (circles) and original (squares) IDEAS ® analysis templates following 40 min ( C ) and 5 min ( D ) of data collection.…”

Automation of the Cytokinesis-Block Micronucleus Assay Using Imaging Flow Cytometry for High Throughput Radiation Biodosimetry

“…Despite this, their method was able to generate dose estimations to within 0.2 Gy of the delivered dose when scoring blinded samples and they have estimated that their method could allow for the processing of approximately 60 samples in a 12 h period. In 2014, De Amicis et al [62] and De Sanctis [63] used a similar automated slide-scoring method to generate calibration curves and also reported that the number of MN scored at 3 Gy is lower by a factor of 2, similar to the observations of Willems et al, [37] Varga et al [39] and Schunk et al [61] . Still, dose estimations were generated to within 0.3 Gy (or less) of the delivered dose for a number of blinded samples between 0-4 Gy after 8 min of slide scoring time [62,63].…”

“…Willems et al [37] were able to generate dose estimates to within 0.2 Gy of the delivered dose using automated slide-scoring however, only a single dose point (1 Gy) was examined. Additionally, in recent publications by De Amicis et al [62] and De Sanctis [63] dose estimates were generated to within 0.3 Gy (or less) of the delivered dose using automated slide-scoring for a number of doses between 0-4 Gy. The results of the work presented here demonstrates that the ISX-CBMN method, performed using standard culture time and volume conditions, is also able to estimate blinded doses between 0-4 Gy to within 0.3 Gy of the delivered dose and is comparable to results presented in other works using automated slide-scoring versions of the CBMN assay.…”

“…Although the frequency of MN scored is lower than traditional versions of the assay, this has been noted in other automated methods and is likely a consequence of the stricter scoring parameters applied in image analysis or the inability to observe MN which may be hidden behind the main nuclei in a BNC [39,[61][62][63]. While the same IDEAS® software template used to generate the dose response calibration curves would be employed to provide dose estimations in radiation biodosimetry, the lower frequency of MN would impact sensitivity and accuracy.…”