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. 2016 Jul;89(7):653-62.
doi: 10.1002/cyto.a.22887. Epub 2016 Jun 6.

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

M A Rodrigues  1   2 , C E Probst  1 , L A Beaton-Green  2 , R C Wilkins  2
Affiliations

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

M A Rodrigues et al. Cytometry A. 2016 Jul.

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(®) 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 ±0.5 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. © 2016 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of ISAC.

Keywords: ImageStreamX; automated MN analysis; biodosimetry; cytokinesis-block micronucleus assay; imaging flow cytometry; population triage.

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Figures

Figure 1
Figure 1
(A) The various steps used to create the optimized BNC mask illustrating improvement over the default mask (third row) through sequential combination of the Levelset (fourth row), Watershed (fifth row) and Range functions. The final BNC mask (sixth row) as well as the new component masks to highlight individual main nuclei (seventh and eighth rows) are shown. (B) The steps used to create the optimized MN mask. The spot identification function (second row) and dilated Levelset function (third row) are used to create the first spot mask (fourth row) while the spot identification function (fifth row) and the dilated morphology function (sixth row) are used to create the second spot mask (seventh row). Both spot masks are combined such that MN masked by either function are scored (eighth row).
Figure 2
Figure 2
Various cell images illustrating the improvement of the optimized BNC and MN masks over the original masks. (A) A BNC with zero MN. (B) A BNC with one MN that was enveloped by the original BNC mask. (C, D) BNCs with two and three MN, respectively, showing that the original BNC mask enveloped one of the MN. (E, F) BNCs with MN that were unmasked by the original MN mask now being correctly identified by the optimized MN mask.
Figure 3
Figure 3
(A) Bivariate plot of DRAQ5 Gradient RMS versus BF Gradient RMS for the selection of focused cells. (B) Bivariate plot of BF ASPECT RATIO versus BF AREA for elimination of small debris and multicellular aggregates. (C) Histogram of DRAQ5 INTENSITY used to eliminate events with insufficient nuclear stain. (D) Bivariate plot of DRAQ5 AREA versus BF CONTRAST for elimination of apoptotic cells. (E) A SPOT COUNT histogram following application of the BNC mask which separated cells containing two separate masked nuclear spots from all other events. (F) Bivariate plot of ASPECT RATIO INTENSITY components allowing for circularity comparison of the two main nuclei. (G) Bivariate plot of COMPONENT INTENSITY RATIO versus COMPONENT AREA RATIO allowing for selection of events which contained two main nuclei that were similar in both area and intensity. (H) Bivariate plot of ASPECT RATIO INTENSITY versus SHAPE RATIO that allowed for removal of events that contained two distinct but overlapping nuclei. (I) Bivariate plot of DRAQ5 SYMMETRY versus the ratio of BNC AREA to Levelset AREA for selection of cells with two well‐separated, distinct nuclei. The tagged BNC and non‐BNC populations are represented by the yellow squares/bins and blue squares/bins, respectively. Representative images captured by the ISX are shown in each panel.
Figure 4
Figure 4
Histogram of the SPOT COUNT applied to the MN mask using final BNC population from a sample irradiated with 2 Gy. Representative images captured by the ISX are shown in each panel.
Figure 5
Figure 5
(A) The rate of MN per BNC versus dose for six donors (three male, three female). The average rate of MN per BNC at each dose point is also plotted, the error bars represent the standard error of the mean and the dashed lines represent the 95% confidence intervals. (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. 18 (circles) including error bars which represent the standard error of the mean. The rate of MN per BNC from Schunck et al. 7 (solid line), Willems et al. 25 (dashed line), De Sanctis et al. 26 (dashed‐dotted line), and Thierens et al. 27 (dotted line) are also plotted using the equations provided in those publications. (C, D) Estimated dose (Gy) versus delivered dose (Gy) determined by the ISX‐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. The dashed line represents a slope of one, where the estimated dose would equal the delivered dose and the dotted lines represent ±0.5 Gy from the ideal slope. The error bars represent the standard error on the estimated dose.
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