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

Abstract

The cytokinesis-block micronucleus (CBMN) assay is a well-established technique that can be employed in triage radiation biodosimetry to estimate whole body doses of radiation to potentially exposed individuals through quantitation of the frequency of micronuclei (MN) in binucleated lymphocyte cells (BNCs). The assay has been partially automated using traditional microscope-based methods and most recently has been modified for application on the ImageStream X (IS X ) imaging flow cytometer. This modification has allowed for a similar number of BNCs to be automatically scored as compared to traditional microscopy in a much shorter time period. However, the MN frequency measured was much lower than both manual and automated slide-based methods of performing the assay. This work describes the optimized analysis template which implements newly developed functions in the IDEAS V R data analysis software for the IS X that enhances specificity for BNCs and increases the frequency of scored MN. A new dose response calibration curve is presented in which the average rate of MN per BNC is of similar magnitude to those presented in the literature using automated CBMN slide scoring methods. In addition, dose estimates were generated for nine irradiated, blinded samples and were found to be within 60.5 Gy of the delivered dose. Results demonstrate that the improved identification accuracy for MN and BNCs in the IS X -based version of the CBMN assay will translate to increased accuracy when estimating unknown radiation doses received by exposed individuals following large-scale radiological or nuclear emergencies.

Automation of the in vitro micronucleus assay using the Imagestream® imaging flow cytometer

“…The ISX-based version of the CBMN assay was shown to automatically score more BN cells in a fraction of the time compared with manual microscopy, therefore enhancing the statistical robustness and speed of the assay. A dose response calibration curve to evaluate the frequency of MN per BN cell was created from 0 to 4 Gy and was similar in magnitude to other calibration curves published in the literature (43). Finally, through the use of blinded samples it was verified that the method could generate dose estimates to within 60.5 Gy of the true dose, sufficient for triage radiation biodosimetry (44).…”

“…For all experiment samples, 20,000 events were collected at 603 magnification using a data acquisition template created in the INSPIRE (MilliporeSigma, Seattle, WA) software that controls the ISX, described below. In previous studies, DRAQ5 was used as the nuclear stain (40)(41)(42)(43)(44). In this work, Hoechst 33342 was selected as the nuclear stain due to the very sharp gradient it creates at the edge of the main nuclei and minimal cytoplasmic background staining, especially in the presence of RNase.…”

“…In IDEAS, positive and negative truth populations can be hand-tagged and saved as separate populations. These populations can then be used to set appropriate region boundaries on all bivariate plots (43). Using the merged files, truth populations consisting of 50 positive and 50 negative hand-tagged cells were created for all cell types of interest (MONO, BN, and POLY cells).…”

“…In previous publications on this work, a data analysis template was developed in IDEAS to identify candidate BN cell events (40,41,43). The analysis in this work, including masking and gating strategy, is similar but has been slightly modified since the sample preparation methods have been improved; KCl was used to swell the cells and create better separation between the main nuclei, and Hoechst was used as the DNA stain rather than DRAQ5.…”

“…Recently, an imaging flow cytometry method to perform the CBMN assay specifically for use in triage radiation biodosimetry was developed (40)(41)(42)(43)(44)(45). Results from this method demonstrated that cellular images of both the cytoplasm and DNA content could be captured at higher throughput than other automated methods and stored in a data file, eliminating the need to create microscope slides (40).…”