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

Abstract

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.

Longitudinal multi-omic changes in the transcriptome and proteome of peripheral blood cells after a 4 Gy total body radiation dose to Rhesus macaques

“…Previous studies using the NHP model for biodosimetry have shown that the frequency of mature T-cells carrying unstable (dicentrics, micronuclei) chromosomal aberrations decreases with time after radiation exposure (16, 54, 55); re ecting their turnover (cell death and re-population) in the circulating blood. More recently, the advanced development of high-throughput CBMN (55) and dicentric assay systems (35) have accurately reconstructed dose in human and NHP blood exposed ex vivo. In addition, the CytoRADx assay platform developed by ASELL, has also been used to accurately reconstruct dose in NHPs exposed in vivo to TBI doses 0-8 Gy, 1-28 days post-irradiation (55).…”

“…More recently, the advanced development of high-throughput CBMN (55) and dicentric assay systems (35) have accurately reconstructed dose in human and NHP blood exposed ex vivo. In addition, the CytoRADx assay platform developed by ASELL, has also been used to accurately reconstruct dose in NHPs exposed in vivo to TBI doses 0-8 Gy, 1-28 days post-irradiation (55).…”

Machine learning approach for quantitative biodosimetry of partial-body or total-body radiation exposures by combining radiation-responsive biomarkers

“…Large-scale studies such as inter-laboratory comparisons reveal that the accuracy of biodosimetry for PBI scenarios still remains limited 29 , and PBI detection methods need to be tested and validated against TBI. Therefore, there is a need for development of new methods to complement existing ( e.g., cytogenetic) techniques 32 , 33 .…”

A machine learning method for improving the accuracy of radiation biodosimetry by combining data from the dicentric chromosomes and micronucleus assays

“…The DCA assay is the current “gold standard” in biodosimetry 5 . Cytogenetic biodosimetry techniques continue to be improved and developed 6 – 11 , and implementation of these assays using high-throughput automated approaches is becoming more widespread 12 – 17 . The automatic scoring technologies are advantageous because they can be employed in mass-casualty situations.…”