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An automated platform for cytogenetic biodosimetry, the "Rapid Automated Biodosimetry Tool II (RABiT-II)," adapts the dicentric chromosome assay (DCA) for high-throughput mass-screening of the population after a large-scale radiological event. To validate this test, the U.S. Federal Drug Administration (FDA) recommends demonstrating that the high-throughput biodosimetric assay in question correctly reports the dose in an in vivo model. Here we describe the use of rhesus macaques (Macaca mulatta) to augment human studies and validate the accuracy of the high-throughput version of the DCA. To perform analysis, we developed the 17/22-mer peptide nucleic acid (PNA) probes that bind to the rhesus macaque's centromeres. To our knowledge, these are the first custom PNA probes with high specificity that can be used for chromosome analysis in M. mulatta. The accuracy of fully-automated chromosome analysis was improved by optimizing a low-temperature telomere PNA FISH staining in multiwell plates and adding the telomere detection feature to our custom chromosome detection software, FluorQuantDic V4. The dicentric frequencies estimated from in vitro irradiated rhesus macaque samples were compared to human blood samples of individuals subjected to the same ex vivo irradiation conditions. The results of the RABiT-II DCA analysis suggest that, in the lymphocyte system, the dose responses to gamma radiation in the rhesus macaques were similar to those in humans, with small but statistically significant differences between these two model systems.

We report the development of system for packaging critical components of the traditional collection kit to make an integrated fingerstick blood collector for self-collecting blood samples of 100 μl or more for radiation countermeasures. A miniaturized vacuum tube system (VacuStor system) has been developed to facilitate liquid reagent storage, simple operation and reduced sample contamination. Vacuum shelf life of the VacuStor tube has been analyzed by the ideal gas law and gas permeation theory, and multiple ways to extend vacuum shelf life beyond one year have been demonstrated, including low temperature storage, Parylene barrier coating and container vacuum bag sealing. Self-collection was also demonstrated by healthy donors without any previous fingerstick collection experience. The collected blood samples showed similar behavior in terms of gene expression and cytogenetic biodosimetry assays comparing to the traditionally collected samples. The integrated collector may alleviate the sample collection bottleneck for radiation countermeasures following a large-scale nuclear event, and may be useful in other applications with its self-collection and liquid reagent sample preprocessing capabilities.

We developed a fully-automated dicentric chromosome assay (DCA) in multiwell plates. All operations, from sample loading to chromosome scoring, are performed, without human intervention, by the second-generation Rapid Automated Biodosimetry Tool II (RABiT-II) robotic system, a plate imager and custom software, FluorQuantDic. The system requires small volumes of blood (30 µl per individual) to determine radiation dose received as a result of a radiation accident or terrorist attack. To visualize dicentrics in multiwell plates, we implemented a non-classical protocol for centromere FISH staining at 37°C. The RABiT-II performs rapid analysis of chromosomes after extracting them from metaphase cells. With the use of multiwell plates, many samples can be screened at the same time. Thus, the RABiT-II DCA provides an advantage during triage when risk-based stratification and medical management are required for a large population exposed to unknown levels of ionizing radiation.

The cytokinesis-block micronucleus (CBMN) assay is considered to be the most suitable biodosimetry method for automation. Previously, we automated this assay on a commercial robotic biotech high-throughput system (RABiT-II) adopting both a traditional and an accelerated micronucleus protocol, using centrifugation steps for both lymphocyte harvesting and washing, after whole blood culturing. Here we describe further development of our accelerated CBMN assay protocol for use on high-throughput/high content screening (HTS/HCS) robotic systems without a centrifuge. This opens the way for implementation of the CBMN assay on a wider range of commercial automated HTS/HCS systems and thus increases the potential capacity for dose estimates following a mass-casualty radiological event.

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 × 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.

In this work, we describe a fully automated cytokinesis-block micronucleus (CBMN) assay with a significantly shortened time to result, motivated by the need for rapid high-throughput biodosimetric estimation of radiation doses from small-volume human blood samples. The Rapid Automated Biodosimetry Tool (RABiT-II) currently consists of two commercial automated systems: a PerkinElmer cell::explorer Workstation and a GE Healthcare IN Cell Analyzer 2000 Imager. Blood samples (30 μl) from eight healthy volunteers were gamma-ray irradiated ex vivo with 0 (control), 0.5, 1.5, 2.5, 3.5 or 4.5 Gy and processed with full automation in 96-well plates on the RABiT-II system. The total cell culture time was 54 h and total assay time was 3 days. DAPI-stained fixed samples were imaged on an IN Cell Analyzer 2000 with fully-automated image analysis using the GE Healthcare IN Cell Developer Toolbox version 1.9. A CBMN dose-response calibration curve was established, after which the capability of the system to predict known doses was assessed. Various radiation doses for irradiated samples from two donors were estimated within 20% of the true dose (±0.5 Gy below 2 Gy) in 97% of the samples, with the doses in some 5 Gy irradiated samples being underestimated by up to 25%. In summary, the findings from this work demonstrate that the accelerated CBMN assay can be automated in a high-throughput format, using commercial biotech robotic systems, in 96-well plates, providing a rapid and reliable bioassay for radiation exposure.

Biodosimetry is an essential tool for providing timely assessments of radiation exposure. For a large mass-casualty event involving exposure to ionizing radiation, it is of utmost importance to rapidly provide dose information for medical treatment. The well-established cytokinesis-block micronucleus (CBMN) assay is a validated method for biodosimetry. However, the need for an accelerated sample processing is required for the CBMN assay to be a suitable population triage tool. We report here on the development of a high-throughput and miniaturized version of the CMBN assay for accelerated sample processing.

The RABiT (Rapid Automated Biodosimetry Tool) is a dedicated Robotic platform for the automation of cytogenetics-based biodosimetry assays. The RABiT was developed to fulfill the critical requirement for triage following a mass radiological or nuclear event. Starting from well-characterized and accepted assays we developed a custom robotic platform to automate them. We present here a brief historical overview of the RABiT program at Columbia University from its inception in 2005 until the RABiT was dismantled at the end of 2015. The main focus of this paper is to demonstrate how the biological assays drove development of the custom robotic systems and in turn new advances in commercial robotic platforms inspired small modifications in the assays to allow replacing customized robotics with 'off the shelf' systems. Currently, a second-generation, RABiT II, system at Columbia University, consisting of a PerkinElmer cell::explorer, was programmed to perform the RABiT assays and is undergoing testing and optimization studies.

Here the general concept of the combined use of plates and tubes in racks compatible with the American National Standards Institute/the Society for Laboratory Automation and Screening microplate formats as the next generation platforms for increasing the throughput of biodosimetry assays was described. These platforms can be used at different stages of biodosimetry assays starting from blood collection into microtubes organised in standardised racks and ending with the cytogenetic analysis of samples in standardised multiwell and multichannel plates. Robotically friendly platforms can be used for different biodosimetry assays in minimally equipped laboratories and on cost-effective automated universal biotech systems.