Multiwell-based G0-PCC assay for radiation biodosimetry.

In cytogenetic biodosimetry, assessing radiation exposure typically requires over 48 hours for cells to reach mitosis, significantly delaying the administration of crucial radiation countermeasures needed within the first 24 hours post-exposure. To improve medical response times, we incorporated the G0-Premature Chromosome Condensation (G0-PCC) technique with the Rapid Automated Biodosimetry Tool-II (RABiT-II), creating a faster alternative for large-scale radiation emergencies. Our findings revealed that using a lower concentration of Calyculin A (Cal A) than recommended effectively increased the yield of highly-condensed G0-PCC cells (hPCC). However, integrating recombinant CDK1/Cyclin B kinase, vital for chromosome condensation, proved challenging due to the properties of these proteins affecting interactions with cellular membranes. Interestingly, Cal A alone was capable of inducing chromosome compaction in some G0 cells even in the absence of mitotic kinases, although these chromosomes displayed atypical morphologies. This suggests that Cal A mechanism for compacting G0 chromatin may differ from condensation driven by mitotic kinases. Additionally, we observed a correlation between radiation dose and extent of hPCC chromosome fragmentation, which allowed us to automate radiation damage quantification using a Convolutional Neural Network (CNN). Our method can address the need for a same-day cytogenetic biodosimetry test in radiation emergency situations.

RABiT-III: an Automated Micronucleus Assay at a Non-Specialized Biodosimetry Facility.

Micronuclei, detected through the cytokinesis-block micronucleus assay, are valuable indicators of ionizing radiation exposure, especially in short-term lymphocyte cultures. The peripheral human blood lymphocyte assay is recognized as a prime candidate for automated biodosimetry. In a prior project at the Columbia University Center for Radiological Research, we automated this assay using the 96-well ANSI/SLAS microplate standard format and relied on established biotech robotic systems named Rapid Automated Biodosimetry Tool (RABiT). In this study, we present the application of a similar automated biotech setup at an external high-throughput facility (RABiT-III) to implement the same automated cytokinesis-block micronucleus assay. Specifically, we employed the Agilent BRAVO liquid-handling system and GE IN Cell Analyzer 6000 imaging system in conjunction with the PerkinElmer Columbus image data storage and analysis system. Notably, this analysis system features an embedded PhenoLOGIC machine learning module, simplifying the creation of cell classification algorithms for CBMN assay image analysis and enabling the generation of radiation dose-response curves. This investigation underscores the adaptability of the RABiT-II CBMN protocol to diverse RABiT-III biotech robotic platforms in non-specialized biodosimetry centers. Furthermore, it highlights the advantages of machine learning in rapidly developing algorithms crucial for the high-throughput automated analysis of RABiT-III images.

Sex and dose rate effects in automated cytogenetics.

Testing and validation of biodosimetry assays is routinely performed using conventional dose rate irradiation platforms, at a dose rate of approximately 1 Gy/min. In contrast, the exposures from an improvised nuclear device will be delivered over a large range of dose rates with a prompt irradiation component, delivered in less than 1 µs, and a protracted component delivered over hours and days. We present preliminary data from a large demographic study we have undertaken for investigation of age, sex and dose rate effects on dicentric and micronucleus yields. Our data demonstrate reduced dicentric and micronucleus yields at very high dose rates. Additionally, we have seen small differences between males and females, with males having slightly fewer micronuclei and slightly more dicentrics than females, at high doses.

Improved prediction of breast cancer risk based on phenotypic DNA damage repair capacity in peripheral blood B cells.

Background: Standard Breast Cancer (BC) risk prediction models based only on epidemiologic factors generally have quite poor performance, and there have been a number of risk scores proposed to improve them, such as AI-based mammographic information, polygenic risk scores and pathogenic variants. Even with these additions BC risk prediction performance is still at best moderate. In that decreased DNA repair capacity (DRC) is a major risk factor for development of cancer, we investigated the potential to improve BC risk prediction models by including a measured phenotypic DRC assay. Methods: Using blood samples from the Breast Cancer Family Registry we assessed the performance of phenotypic markers of DRC in 46 matched pairs of individuals, one from each pair with BC (with blood drawn before BC diagnosis) and the other from controls matched by age and time since blood draw. We assessed DRC in thawed cryopreserved peripheral blood mononuclear cells (PBMCs) by measuring γ-H2AX yields (a marker for DNA double-strand breaks) at multiple times from 1 to 20 hrs after a radiation challenge. The studies were performed using surface markers to discriminate between different PBMC subtypes. Results: The parameter Fres, the residual damage signal in PBMC B cells at 20 hrs post challenge, was the strongest predictor of breast cancer with an AUC (Area Under receiver-operator Curve) of 0.89 [95% Confidence Interval: 0.84-0.93] and a BC status prediction accuracy of 0.80. To illustrate the combined use of a phenotypic predictor with standard BC predictors, we combined Fres in B cells with age at blood draw, and found that the combination resulted in significantly greater BC predictive power (AUC of 0.97 [95% CI: 0.94-0.99]), an increase of 13 percentage points over age alone. Conclusions: If replicated in larger studies, these results suggest that inclusion of a fingerstick-based phenotypic DRC blood test has the potential to markedly improve BC risk prediction.

High-throughput measurement of double strand break global repair phenotype in peripheral blood mononuclear cells after long-term cryopreservation.

Peripheral blood mononuclear cells (PBMCs) are a useful model for biochemical assays, particularly for etiological studies. We describe here a method for measuring DNA repair capacity (DRC) in archival cryogenically preserved PBMCs. To model DRC, we measured γ-H2AX repair kinetics in thawed PBMCs after irradiation with 3 Gy gamma rays. Time-dependent fluorescently labeled γ-H2AX levels were measured at five time points from 1 to 20 h, yielding an estimate of global DRC repair kinetics as well as a measure of unrepaired double strand breaks at 20 h. While γ-H2AX levels are traditionally measured by either microscopy or flow-cytometry, we developed a protocol for imaging flow cytometry (IFC) that combines the detailed information of microscopy with the statistical power of flow methods. The visual imaging component of the IFC allows for monitoring aspects such as cellular health and apoptosis as well as fluorescence localization of the γ-H2AX signal, which ensures the power and significance of this technique. Application of a machine-learning based image classification improved flow cytometry fluorescent measurements by identifying apoptotic cells unable to undergo DNA repair. We present here DRC repair parameters from 18 frozen archival PBMCs and 28 fresh blood samples collected from a demographically diverse cohort of women measured in a high-throughput IFC format. This thaw method and assay can be used alone or in conjunction with other assays to measure etiological phenotypes in cryogenic biobanks of PBMCs.

Validation of a High-Throughput Dicentric Chromosome Assay Using Complex Radiation Exposures.

Validation of biodosimetry assays is routinely performed using primarily orthovoltage irradiators at a conventional dose rate of approximately 1 Gy/min. However, incidental/ accidental exposures caused by nuclear weapons can be more complex. The aim of this work was to simulate the DNA damage effects mimicking those caused by the detonation of a several kilotons improvised nuclear device (IND). For this, we modeled complex exposures to: 1. a mixed (photons + IND-neutrons) field and 2. different dose rates that may come from the blast, nuclear fallout, or ground deposition of radionuclides (ground shine). Additionally, we assessed whether myeloid cytokines affect the precision of radiation dose estimation by modulating the frequency of dicentric chromosomes. To mimic different exposure scenarios, several irradiation systems were used. In a mixed field study, human blood samples were exposed to a photon field enriched with neutrons (ranging from 10% to 37%) from a source that mimics Hiroshima's A-bomb's energy spectrum (0.2-9 MeV). Using statistical analysis, we assessed whether photons and neutrons act in an additive or synergistic way to form dicentrics. For the dose rates study, human blood was exposed to photons or electrons at dose rates ranging from low (where the dose was spread over 32 h) to extremely high (where the dose was delivered in a fraction of a microsecond). Potential effects of cytokine treatment on biodosimetry dose predictions were analyzed in irradiated blood subjected to Neupogen or Neulasta for 24 or 48 h at the concentration recommended to forestall manifestation of an acute radiation syndrome in bomb survivors. All measurements were performed using a robotic station, the Rapid Automated Biodosimetry Tool II, programmed to culture lymphocytes and score dicentrics in multiwell plates (the RABiT-II DCA). In agreement with classical concepts of radiation biology, the RABiT-II DCA calibration curves suggested that the frequency of dicentrics depends on the type of radiation and is modulated by changes in the dose rate. The resulting dose-response curves suggested an intermediate dicentric yields and additive effects of photons and IND-neutrons in the mixed field. At ultra-high dose rate (600 Gy/s), affected lymphocytes exhibited significantly fewer dicentrics (P < 0.004, t test). In contrast, we did not find the dose-response modification effects of radiomitigators on the yields of dicentrics (Bonferroni corrected P > 0.006, ANOVA test). This result suggests no bias in the dose predictions should be expected after emergency cytokine treatment initiated up to 48 h prior to blood collection for dicentric analysis.

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

A large-scale malicious or accidental radiological event can expose vast numbers of people to ionizing radiation. The dicentric chromosome (DCA) and cytokinesis-block micronucleus (CBMN) assays are well-established biodosimetry methods for estimating individual absorbed doses after radiation exposure. Here we used machine learning (ML) to test the hypothesis that combining automated DCA and CBMN assays will improve dose reconstruction accuracy, compared with using either cytogenetic assay alone. We analyzed 1349 blood sample aliquots from 155 donors of different ages (3-69 years) and sexes (49.1% males), ex vivo irradiated with 0-8 Gy at dose rates from 0.08 Gy/day to ≥ 600 Gy/s. We compared the performances of several state-of-the-art ensemble ML methods and found that random forest generated the best results, with R2 for actual vs. reconstructed doses on a testing data subset = 0.845, and mean absolute error = 0.628 Gy. The most important predictor variables were CBMN and DCA frequencies, and age. Removing CBMN or DCA data from the model significantly increased squared errors on testing data (p-values 3.4 × 10-8 and 1.1 × 10-6, respectively). These findings demonstrate the promising potential of combining CBMN and DCA assay data to reconstruct radiation doses in realistic scenarios of heterogeneous populations exposed to a mass-casualty radiological event.

Transportation container for pre-processing cytogenetic assays in radiation accidents.

We report a shipping container that enables a disruptive logistics for cytogenetic biodosimetry for radiation countermeasures through pre-processing cell culture during transportation. The container showed precise temperature control (< 0.01 °C) with uniform sample temperature (< 0.1 °C) to meet the biodosimetry assay requirements. Using an existing insulated shipping box and long shelf life alkaline batteries makes it ideal for national stockpile. Dose curve of cytogenetic biodosimetry assay using the shipping container showed clear dose response and high linear correlation with the control dose curve using a laboratory incubator (Pearson's correlation coefficient: 0.992). The container's ability of pre-processing biological samples during transportation could have a significant impact on radiation countermeasure, as well as potential impacts in other applications such as biobanking, novel molecular or cell-based assays or therapies.

The RABiT-II DCA in the Rhesus Macaque Model.

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.

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

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.

Development of an integrated fingerstick blood self-collection device for radiation countermeasures.

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.

RABiT-II-DCA: A Fully-automated Dicentric Chromosome Assay in Multiwell Plates.

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.

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

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.

RABiT-II: A Fully-Automated Micronucleus Assay System with Shortened Time to Result.

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.

RABiT-II: Implementation of a High-Throughput Micronucleus Biodosimetry Assay on Commercial Biotech Robotic Systems.

We demonstrate the use of high-throughput biodosimetry platforms based on commercial high-throughput/high-content screening robotic systems. The cytokinesis-block micronucleus (CBMN) assay, using only 20 µl whole blood from a fingerstick, was implemented on a PerkinElmer cell::explorer and General Electric IN Cell Analyzer 2000. On average 500 binucleated cells per sample were detected by our FluorQuantMN software. A calibration curve was generated in the radiation dose range up to 5.0 Gy using the data from 8 donors and 48,083 binucleated cells in total. The study described here demonstrates that high-throughput radiation biodosimetry is practical using current commercial high-throughput/high-content screening robotic systems, which can be readily programmed to perform and analyze robotics-optimized cytogenetic assays. Application to other commercial high-throughput/high-content screening systems beyond the ones used in this study is clearly practical. This approach will allow much wider access to high-throughput biodosimetric screening for large-scale radiological incidents than is currently available.

Liquid Handling Optimization in High-Throughput Biodosimetry Tool.

Due to the need of high-speed and efficient biodosimetric assays for triage and therapy in the event of radiological or nuclear attack, a robotically based automated biodosimetry tool (RABiT) has been developed over the past few years. Adapting the micronucleus assay from filter plates to V-shaped plates presented challenges in the liquid handling, namely, cell splashing out of the V-shaped well plate during the cell harvesting, poor cell distribution on the bottom of the image plate during the dispensing, and cell loss from the image plate during the aspiration in the liquid handling process. Experimental and numerical investigations were carried out to better understand the phenomena and mitigate the problems. Surface tension and contact angle among the fluids and the plate wall were accounted for in the discrete and multiphase numerical models. Experimental conditions were optimized based on the numerical results showing the relationship between nozzle speed and amount of splashed liquid, and the relationship between aspiration speed and number of escaped cells. Using these optimized parameters, numbers of micronuclei in binucleated cells showed the same dose dependence in the RABiT-prepared samples as those in the manually prepared ones. Micronucleus assay protocol was fully realized on RABiT.

Development of a High-Throughput and Miniaturized Cytokinesis-Block Micronucleus Assay for Use as a Biological Dosimetry Population Triage Tool.

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.

An automated imaging system for radiation biodosimetry.

We describe here an automated imaging system developed at the Center for High Throughput Minimally Invasive Radiation Biodosimetry. The imaging system is built around a fast, sensitive sCMOS camera and rapid switchable LED light source. It features complete automation of all the steps of the imaging process and contains built-in feedback loops to ensure proper operation. The imaging system is intended as a back end to the RABiT-a robotic platform for radiation biodosimetry. It is intended to automate image acquisition and analysis for four biodosimetry assays for which we have developed automated protocols: The Cytokinesis Blocked Micronucleus assay, the γ-H2AX assay, the Dicentric assay (using PNA or FISH probes) and the RABiT-BAND assay.

Next generation platforms for high-throughput biodosimetry.

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.

New sequence-based data on the relative DNA contents of chromosomes in the normal male and female human diploid genomes for radiation molecular cytogenetics.

BACKGROUND: The objective of this work is to obtain the correct relative DNA contents of chromosomes in the normal male and female human diploid genomes for the use at FISH analysis of radiation-induced chromosome aberrations. RESULTS: The relative DNA contents of chromosomes in the male and female human diploid genomes have been calculated from the publicly available international Human Genome Project data. New sequence-based data on the relative DNA contents of human chromosomes were compared with the data recommended by the International Atomic Energy Agency in 2001. The differences in the values of the relative DNA contents of chromosomes obtained by using different approaches for 15 human chromosomes, mainly for large chromosomes, were below 2%. For the chromosomes 13, 17, 20 and 22 the differences were above 5%. CONCLUSION: New sequence-based data on the relative DNA contents of chromosomes in the normal male and female human diploid genomes were obtained. This approach, based on the genome sequence, can be recommended for the use in radiation molecular cytogenetics.