CytoRADx: A High-Throughput, Standardized Biodosimetry Diagnostic System Based on the Cytokinesis-Block Micronucleus Assay.

In a large-scale catastrophe, such as a nuclear detonation in a major city, it will be crucial to accurately diagnose large numbers of people to direct scarce medical resources to those in greatest need. Currently no FDA-cleared tests are available to diagnose radiation exposures, which can lead to complex, life-threatening injuries. To address this gap, we have achieved substantial advancements in radiation biodosimetry through refinement and adaptation of the cytokinesis-block micronucleus (CBMN) assay as a high throughput, quantitative diagnostic test. The classical CBMN approach, which quantifies micronuclei (MN) resulting from DNA damage, suffers from considerable time and expert labor requirements, in addition to a lack of universal methodology across laboratories. We have developed the CytoRADx™ System to address these drawbacks by implementing a standardized reagent kit, optimized assay protocol, fully automated microscopy and image analysis, and integrated dose prediction. These enhancements allow the CytoRADx System to obtain high-throughput, standardized results without specialized labor or laboratory-specific calibration curves. The CytoRADx System has been optimized for use with both humans and non-human primates (NHP) to quantify radiation dose-dependent formation of micronuclei in lymphocytes, observed using whole blood samples. Cell nuclei and resulting MN are fluorescently stained and preserved on durable microscope slides using materials provided in the kit. Up to 1,000 slides per day are subsequently scanned using the commercially based RADxScan™ Imager with customized software, which automatically quantifies the cellular features and calculates the radiation dose. Using less than 1 mL of blood, irradiated ex vivo, our system has demonstrated accurate and precise measurement of exposures from 0 to 8 Gy (90% of results within 1 Gy of delivered dose). These results were obtained from 636 human samples (24 distinct donors) and 445 NHP samples (30 distinct subjects). The system demonstrated comparable results during in vivo studies, including an investigation of 43 NHPs receiving single-dose total-body irradiation. System performance is repeatable across laboratories, operators, and instruments. Results are also statistically similar across diverse populations, considering various demographics, common medications, medical conditions, and acute injuries associated with radiological disasters. Dose calculations are stable over time as well, providing reproducible results for at least 28 days postirradiation, and for blood specimens collected and stored at room temperature for at least 72 h. The CytoRADx System provides significant advancements in the field of biodosimetry that will enable accurate diagnoses across diverse populations in large-scale emergency scenarios. In addition, our technological enhancements to the well-established CBMN assay provide a pathway for future diagnostic applications, such as toxicology and oncology.

Environmental monitoring for biological threat agents using the autonomous pathogen detection system with multiplexed polymerase chain reaction.

We have developed and field-tested a now operational civilian biodefense capability that continuously monitors the air in high-risk locations for biological threat agents. This stand-alone instrument, called the Autonomous Pathogen Detection System (APDS), collects and selectively concentrates particles from the air into liquid samples and analyzes the samples using multiplexed PCR amplification coupled with microsphere array detection. During laboratory testing, we evaluated the APDS instrument's response to Bacillus anthracis and Yersinia pestis by spiking the liquid sample stream with viable spores and cells, bead-beaten lysates, and purified DNA extracts. APDS results were also compared to a manual real-time PCR method. Field data acquired during 74 days of continuous operation at a mass-transit subway station are presented to demonstrate the specificity and reliability of the APDS. The U.S. Department of Homeland Security recently selected the APDS reported herein as the first autonomous detector component of their BioWatch antiterrorism program. This sophisticated field-deployed surveillance capability now generates actionable data in one-tenth the time of manual filter collection and analysis.

An integrated proteomic workflow for two-dimensional differential gel electrophoresis and robotic spot picking.

New technologies have advanced the field of proteomics, and a number of companies have developed innovative platforms to drive this research. However, significant challenges are often encountered when trying to integrate complementary technologies from multiple manufacturers. We have developed a software and hardware solution to integrate the Ettan two-dimensional difference gel electrophoresis (2-D DIGE) system (GE Healthcare) with the Investigator ProPic spot picking robot (Genomic Solutions). We have analyzed protein sample preparations from bacterial and mammalian sources to demonstrate a new workflow with increased throughput for gel-based proteomics.