Automated Triage Radiation Biodosimetry: Integrating Imaging Flow Cytometry with High-Throughput Robotics to Perform the Cytokinesis-Block Micronucleus Assay

Abstract

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 use of a centrifuge-free RABiT-II system for high-throughput micronucleus analysis

“…Moreover, our protocols for RABiT-II cell harvesting with the use of centrifuge [9,15,17] as well as described in this paper centrifuge-free RABiT-II approach were developed for keeping all reagents and plates at room temperature so that more workstations without automated heaters/coolers could be used for CBMN assay sample preparation. Thus, almost any ANSI/SLAC-format compatible robotic cell handling system meeting the minimum requirements of the centrifuge-free RABiT-II approach can be used for sample preparation with the use of the corresponding RABiT-II systems has the potential to increase the capacity for biodosimetry response during a large-scale radiological/nuclear event.…”

Development of a high-throughput γ-H2AX assay based on imaging flow cytometry

“…Fresh blood aliquots (1 mL) were dispensed into 15 mL conical bottom tubes (Santa Cruz Biotechnology, Dallas, TX) and were irradiated with γ rays (0, and 4 Gy) using a Gammacell ® 40 137 Cesium irradiator (Atomic Energy of Canada, Ltd., Chalk River, ON). The blood sample tubes were placed on their side in the middle of the chamber and irradiated with a dose rate of 0.73 Gy/min (35). The 137 Cs irradiator was calibrated annually with TLDs and homogeneity of exposure across the sample volume was verified using EBT3 Gafchromic film with less than 2% variation within the sample (Ashland Advanced Materials, Gafchromic, Bridgewater, NJ).…”

New High Throughput Approaches to Detect Partial-body and Neutron Exposures on an Individual Basis

“…partial-body photon exposures, or vs. neutron + photon mixtures. Specifically, using our highthroughput CBMN assay 5,9,46 we wish to evaluate on an individual basis: 1) the photon and the neutron doses and the fraction of neutrons in the total dose after a mixed exposure, 2) whether there was indeed a partial body exposure. Our study design (shown schematically in Fig.…”

“…Thus, there are a larger number of undamaged cells than would be expected based on Poisson statistics, coupled with more damage in those that are traversed by ionizing tracks. While these phenomena has been observed for many years and applied to the dicentric assay, essentially by analyzing the proportion of undamaged metaphases [33][34][35] , these approaches can be directly applied to high-throughput assays 5,6 .…”

“…A rapid assessment of exposure type would need to be made for a large number of individuals following a large-scale radiological event. Consequently, there is a well-recognized need for development and utilization of high-throughput assays that can discriminate between these complex irradiation scenarios like partial-body shielding and/or neutron + photon mixtures from simpler exposures such as homogeneous total-body photon exposure [4][5][6] . Such reconstruction of exposure type is important for making appropriate triage and treatment decisions in mass casualty situations.…”