No abstract

The cytokinesis-block micronucleus (CBMN) assay is employed in biological dosimetry to determine the dose of radiation to an exposed individual from the frequency of micronuclei (MN) in binucleated lymphocyte cells. The method has been partially automated for the use in mass casualty events, but it would be advantageous to further automate the method for increased throughput. Recently, automated image analysis has been successfully applied to the traditional, slide-scoring-based method of the CBMN assay. However, with the development of new technologies such as the imaging flow cytometer, it is now possible to adapt this microscope-based assay to an automated imaging flow cytometry method. The ImageStream(X) is an imaging flow cytometer that has adequate sensitivity to quantify radiation doses larger than 1 Gy while adding the increased throughput of traditional flow cytometry. The protocol and analysis presented in this work adapts the CBMN assay for the use on the ImageStream(X). Ex vivo-irradiated whole blood samples cultured for CBMN were analyzed on the ImageStream(X), and preliminary results indicate that binucleated cells and MN can be identified, imaged and enumerated automatically by imaging flow cytometry. Details of the method development, gating strategy and the dose response curve generated are presented and indicate that adaptation of the CBMN assay for the use with imaging flow cytometry has potential for high-throughput analysis following a mass casualty radiological event.

The cytokinesis-block micronucleus (CBMN) assay is an established technique in radiation biological dosimetry for estimating the dose to an individual by measuring the frequency of micronuclei (MN) in binucleated lymphocyte cells (BNCs). The assay has been partially automated using slide-scoring algorithms, but an automated multiparameter method without the need of the slide-making procedure would be advantageous to further increase throughput for application in mass casualty events. The development of the ImageStream X (ISX) imaging flow cytometer has made it possible to adapt the CBMN assay to an automated imaging flow cytometry (FCM) method. The protocol and analysis presented in this work tailor and expand the assay to a multiparameter biodosimetry tool. Ex vivo irradiated whole blood samples were cultured, processed, and analyzed on the ISX and BNCs, MN, and mononuclear cells were imaged, identified, and enumerated automatically and simultaneously. Details on development of the method, gating strategy, and dose response curves generated for the rate of MN per BNC, percentage of mononuclear cells as well as the replication index are presented. Results indicate that adapting the CBMN assay for use in imaging FCM has produced a rapid, robust, multiparameter analysis method with higher throughput than is currently available with standard microscopy. We conclude that the ISX-CBMN method may be an advantageous tool following a radiological event where triage biodosimetry must be performed on a large number of casualties. V C 2014 International Society for Advancement of Cytometry

The cytokinesis-block micronucleus (CBMN) assay has become a fully-validated and standardized method for radiation biodosimetry. The assay is typically performed using microscopy, which is labor intensive, time consuming and impractical after a large-scale radiological/nuclear event. Imaging flow cytometry (IFC), which combines the statistical power of traditional flow cytometry with the sensitivity and specificity of microscopy, has been recently used to perform the CBMN assay. Since this technology is capable of automated sample acquisition and multi-file analysis, we have integrated IFC into our Rapid Automated Biodosimetry Technology (RABiT-II). Assay development and optimization studies were designed to increase the yield of binucleated cells (BNCs), and improve data acquisition and analysis templates to increase the speed and accuracy of image analysis. Human peripheral blood samples were exposed ex vivo with up to 4 Gy of c rays at a dose rate of 0.73 Gy/min. After irradiation, samples were transferred to microtubes (total volume of 1 ml including blood and media) and organized into a standard 8 3 12 plate format. Sample processing methods were modified by increasing the blood-to-media ratio, adding hypotonic solution prior to cell fixation and optimizing nuclear DRAQ5 staining, leading to an increase of 81% in BNC yield. Modification of the imaging processing algorithms within IFC software also improved BNC and MN identification, and reduced the average time of image analysis by 78%. Finally, 50 ll of irradiated whole blood was cultured with 200 ll of media in 96-well plates. All sample processing steps were performed automatically using the RABiT-II cell::explorer robotic system adopting the optimized IFC-CBMN assay protocol. The results presented here detail a novel, high-throughput RABiT-IFC CBMN assay that possesses the potential to increase capacity for triage biodosimetry during a large-scale radiological/nuclear event.

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.