G0-PCC-FISH derived multi-parametric biodosimetry methodology for accidental high dose and partial body exposures.

High dose radiation exposures are rare. However, medical management of such incidents is crucial due to mortality and tissue injury risks. Rapid radiation biodosimetry of high dose accidental exposures is highly challenging, considering that they usually involve non uniform fields leading to partial body exposures. The gold standard, dicentric assay and other conventional methods have limited application in such scenarios. As an alternative, we propose Premature Chromosome Condensation combined with Fluorescent In-situ Hybridization (G0-PCC-FISH) as a promising tool for partial body exposure biodosimetry. In the present study, partial body exposures were simulated ex-vivo by mixing of uniformly exposed blood with unexposed blood in varying proportions. After G0-PCC-FISH, Dolphin's approach with background correction was used to provide partial body exposure dose estimates and these were compared with those obtained from conventional dicentric assay and G0-PCC-Fragment assay (conventional G0-PCC). Dispersion analysis of aberrations from partial body exposures was carried out and compared with that of whole-body exposures. The latter was inferred from a multi-donor, wide dose range calibration curve, a-priori established for whole-body exposures. With the dispersion analysis, novel multi-parametric methodology for discerning the partial body exposure from whole body exposure and accurate dose estimation has been formulated and elucidated with the help of an example. Dose and proportion dependent reduction in sensitivity and dose estimation accuracy was observed for Dicentric assay, but not in the two PCC methods. G0-PCC-FISH was found to be most accurate for the dose estimation. G0-PCC-FISH has potential to overcome the shortcomings of current available methods and can provide rapid, accurate dose estimation of partial body and high dose accidental exposures. Biological dose estimation can be useful to predict progression of disease manifestation and can help in pre-planning of appropriate & timely medical intervention.

Synthesis and evaluation of curcumin reduced and capped gold nanoparticles as a green diagnostic probe with therapeutic potential.

Curcumin, a compound in turmeric, shows promise for its anti-cancer properties. In this study, we successfully synthesised curcumin-reduced and capped gold nanoparticles. Most evaluations have been limited to in-vitro studies for these nanoparticles; our study takes a step further by highlighting the in-vivo assessment of these curcumin-reduced and capped gold nanoparticles (GNPCs) using non-invasive imaging (SPECT and optical) and possible therapeutic potential. The GNPCs showed an average hydrodynamic diameter of 58 nm and a PDI of 0.336. The synthesised and fully characterised GNPCs showed ex-vivo hemolysis value of ≤ 1.74 % and serum stability of ≥ 95 % over 24 h. Using in-vivo non-invasive (SPECT and optical Imaging), prolonged circulation and enhanced bioavailability of GNPCs were seen. The biodistribution studies after radiolabelling GNPCs with 99 mTc complemented the optical imaging. The SPECT images showed higher uptake of the GNPCs at the tumour site, viz the contralateral muscle and the native Curcumin, resulting in a high target-to-non-target ratio that differentiated the tumour sufficiently and enhanced the diagnostics. Other organs also accumulate radiolabeled GNPCs in systemic circulation; bio dosimetry is performed. It was found that the dose received by the different organs was safe for use, and the in-vivo toxicity studies in rats indicated negligible toxicity over 30 days. The tumour growth was also reduced in mice models treated with GNPCs compared to the control. These significant findings demonstrate that GNPC shows synergistic activity in vivo, indicating its ability as a green diagnostic probe that has the potential for therapy.

Model of age-dependent dynamics and biokinetics of T-cells as natural biodosimeters.

Circulating T-lymphocytes are used as "natural biodosimeters" for estimating radiation doses, since the frequency of chromosomal aberrations induced in them is proportional to the accumulated dose. Moreover, stable chromosomal aberrations (translocations) are detected years and decades after exposure. Internal incorporation of radionuclides often leads to non-uniform exposure, which resulted in difficulties in the application of retrospective biodosimetry using T-lymphocytes. Some properties of T-lymphocytes complicate retrospective biodosimetry in this case: (1) the thymic production of T-cells depends significantly on age, the maximum is observed in early childhood; (2) the "lymphocyte-dosimeter" accumulates changes (translocations) while circulating through the body. The objective of this paper is to describe the technical characteristics of the model of age dynamics and T-cell biokinetics and approaches to assessing the dose to circulating lymphocytes under various exposure scenarios. The model allows to quantify the fractions of T-lymphocytes that were formed before and after exposure. The model takes into account the time fractions that circulating lymphocytes spend in various lymphoid organs. Age-related thymic involution was also considered. The model predicts that after internal exposure to 90Sr, the doses to T-lymphocytes can differ significantly from the doses to the bone marrow and other tissues. For uniform external γ-exposure, and for internal exposure due to non-bone -seeking radionuclides (for example, 144Ce), predicted doses to T-lymphocytes are very close to bone marrow doses. The model allows to quantify the correction factors for FISH-based doses to obtain doses to organs and tissues.

Radiation-induced gastrointestinal and cutaneous injuries: understanding models, pathologies, assessments, and clinically accepted practices.

PURPOSE: A U. S. and European joint effort fostering the development of medical countermeasures (MCMs) operable in case of radiological or nuclear emergencies. METHODS: Based on the joint engagement between the U.S. National Institute of Allergy and Infectious Diseases (NIAID) and the French Institut de Radioprotection et de Sûreté Nucléaire (IRSN), a Statement of Intent to Collaborate was signed in 2014 and a series of working group meeting were established. In December 2022, the NIAID and IRSN hosted a five-day, U.S./European meeting titled 'Radiation-Induced Cutaneous and Gastrointestinal Injuries: Advances in Understanding Pathologies, Assessment, and Clinically Accepted Practices' in Paris, France. The goals of the meeting were to bring together U.S. and European investigators to explore new research avenues for the medical management of skin and gastrointestinal injuries, including specific diagnostics for each organ system, animal models, and promising medical countermeasures (MCMs) to mitigate radiation damage. There was also an emphasis on exploring additional areas of medicine and response to understand best practices from other emergency scenarios, which could be leveraged to improve radiation preparedness, and the importance of accurate dosimetry in preclinical work. RESULTS: Subsequent to the workshop, seven collaborative projects, funded by both organizations, were established on topics ranging from MCMs and predictive biomarkers, and using physical methods to assess cutaneous radiation injuries, to mechanistic studies to understand radiation-induced damage in multiple organ systems. The importance of accurate dosimetry in preclinical works was highlighted and two recently published U.S./European commentaries that focus on the need for dosimetry standardization in the reported literature had their origins in this meeting. This commentary summarizes the workshop and open discussions among academic investigators, industry researchers, and U.S. and IRSN program representatives. CONCLUSIONS: Given the substantive progress made due to these interactions, both groups plan to expand out these meetings by incorporating high-level investigators from across the globe, while endeavoring to maintain the informal setting that was conducive to in-depth scientific discussion and enhanced the state of the science in radiation research.

Extending culture time to improve Mitotic Index for cytogenetic dosimetry.

PURPOSE: To analyze the effects of extending lymphocyte cultivation time on the Mitotic Index, frequency of first-division cells, and dose estimation after irradiating blood samples with different doses of radiation. MATERIALS AND METHODS: Blood samples from two healthy male volunteers were separately irradiated with three doses (3, 5, and 6 Gy) using a 60Co gamma source (average dose rate: 1.48 kGy.h-1) and cultivated in vitro for conventional (48 h) and extended (56, 68, and 72 h) amounts of time. Colcemid (0.01 µg.mL-1) was added at the beginning of the culture period. Cells were fixed, stained with fluorescence plus Giemsa (FPG), and analyzed under a light microscope. The effects of prolonged culture duration on the Mitotic Index (MI), frequency of first-division cells (M1 cells), and the First-Division Mitotic Index (FDMI) were investigated. The estimation of delivered doses was conducted using a conventional 48h-culture calibration curve. RESULTS: Overall, cells presented higher MI (up to 12-fold) with the extension of culture, while higher radiation doses led to lower MI values (up to 80% reduction at 48 h). Cells irradiated with higher doses (5 and 6 Gy) had the most significant increase (5- to 12-fold) of MI as the cultivation was prolonged. The frequency of M1 cells decreased with the prolongation of culture for all doses (up to 75% reduction), while irradiated cells presented higher frequencies of M1 cells than non-irradiated ones. FDMI increased for all irradiated cultures but most markedly in those irradiated with higher doses (up to 10-fold). The conventional 48h-culture calibration curve proved adequate for assessing the delivered dose based on dicentric frequency following a 72-hour culture. CONCLUSION: Compared to the conventional 48-hour protocol, extending the culture length to 72 hours significantly increased the Mitotic Index and the number of first-division metaphases of irradiated lymphocytes, providing slides with a better scorable metaphase density. Extending the culture time to 72 hours, combined with FPG staining to score exclusively first-division metaphases, improved the counting of dicentric chromosomes. The methodology presented and discussed in this study can be a powerful tool for dicentric-based biodosimetry, especially when exposure to high radiation doses is involved.

LBDNet interlaboratory comparison for the dicentric chromosome assay by digitized image analysis applying weighted robust statistical methods.

PURPOSE: This interlaboratory comparison was conducted to evaluate the performance of the Latin-American Biodosimetry Network (LBDNet) in analyzing digitized images for scoring dicentric chromosomes from in vitro irradiated blood samples. The exercise also assessed the use of weighted robust algorithms to compensate the uneven expertise among the participating laboratories. METHODS: Three sets of coded images obtained through the dicentric chromosome assay from blood samples irradiated at 1.5 Gy (sample A) and 4 Gy (sample B), as well as a non-irradiated whole blood sample (sample C), were shared among LBDNet laboratories. The images were captured using the Metafer4 platform coupled with the AutoCapt module. The laboratories were requested to perform triage scoring, conventional scoring, and dose estimation. The dose estimation was carried out using either their laboratory calibration curve or a common calibration curve. A comparative statistical analysis was conducted using a weighted robust Hampel algorithm and z score to compensate for uneven expertise in dicentric analysis and dose assessment among all laboratories. RESULTS: Out of twelve laboratories, one had unsatisfactory estimated doses at 0 Gy, and two had unsatisfactory estimated doses at 1.5 Gy when using their own calibration curve and triage scoring mode. However, all doses were satisfactory at 4 Gy. Six laboratories had estimated doses within 95% uncertainty limits at 0 Gy, seven at 1.5 Gy, and four at 4 Gy. While the mean dose for sample C was significantly biased using robust algorithms, applying weights to compensate for the laboratory's analysis expertise reduced the bias by half. The bias from delivered doses was only notable for sample C. Using the common calibration curve for dose estimation reduced the standard deviation (s*) estimated by robust methods for all three samples. CONCLUSIONS: The results underscore the significance of performing interlaboratory comparison exercises that involve digitized and electronically transmitted images, even when analyzing non-irradiated samples. In situations where the participating laboratories possess different levels of proficiency, it may prove essential to employ weighted robust algorithms to achieve precise outcomes.

Local environment in yeast-based impedance biodosimeters strongly influences the measurable dose.

This work investigates the feasibility of yeast-based impedance measurements for retrospective dosimetry applications. The local environment around yeast cells in a previously developed film-badge was modeled using Geant4. A greater dose response was observed when yeast cells were surrounded by an aluminum-polymer structure, which acted as a conversion layer. Bench-top experiments were conducted using a jar-based dosimeter design that directly combined a finely-ground aluminum conversion medium with yeast powder. It was shown when irradiated in the presence of aluminum grains, yeast cells yielded a higher impedance signal, thereby indicating greater radiation-induced damage. Finally, in separate irradiation experiments, lead and aluminum sheets were placed behind yeast samples and the dosimeters were irradiated to 1 Gy. A 2-fold increase in the impedance signal was shown when samples were positioned in close contact with the lead sheet compared to the aluminum sheet. In all experiments, it was shown that the local environment significantly influences radiative energy deposition in yeast cells.

Cutaneous Radiation Injuries: REAC/TS Clinical Experience.

The Radiation Emergency Assistance Center/Training Site (REAC/TS) is one of the US Department of Energy (DOE)/National Nuclear Security Administration (NNSA) Nuclear Emergency Response Team (NEST) assets and has been responding to radiological incidents since 1976. REAC/TS is in the Oak Ridge Institute for Science and Education (ORISE). A critical part of the REAC/TS mission is to provide emergency response, advice, and consultation on injuries and illnesses caused from ionizing radiation. Fortunately, radiation injuries are not frequent, but when they occur, they are more likely to be cutaneous radiation injuries (CRI) or internal contamination. In this paper, we will review selected cases from the REAC/TS experience in order to illustrate cutaneous patterns of injury and treatment options.

Lessons on harmonization of scoring criteria for dicentric chromosome assay in South Korea.

PURPOSE: Networking with other biodosimetry laboratories is necessary to assess the radiation exposure of many individuals in large-scale radiological accidents. The Korea biodosimetry network, K-BioDos, prepared harmonized scoring guidelines for dicentric chromosome assay to obtain homogeneous results within the network and investigated the efficiency of the guidelines. MATERIALS AND METHODS: Three laboratories in K-BioDos harmonized the scoring guidelines for dicentric chromosome assay. The results of scoring dicentric chromosomes using the harmonized scoring guidelines were compared with the laboratories' results using their own methods. Feedback was collected from the scorers following the three intercomparison exercises in 3 consecutive years. RESULTS: K-BioDos members showed comparable capacity to score dicentrics in the three exercises. However, the results of the K-BioDos guidelines showed no significant improvement over those of the scorers' own methods. According to the scorers, our harmonized guidelines led to more rejected metaphases and ultimately decreased the number of scorable metaphases compared with their own methods. Moreover, the scoring time was sometimes longer with the K-BioDos protocol because some scorers were not yet familiar with the guidelines, though most scorers reported that the time decreased or was unchanged. These challenges may cause low adherence to the guidelines. Most scorers expressed willingness to use the guidelines to select scorable metaphases or identify dicentrics for other biodosimetry works, whereas one did not want to use it due to the difference from their calibration curves. CONCLUSIONS: We identified potential resistance to following the harmonized guidelines and received requests for more detailed methods. Our findings suggest that the harmonized criteria should be continually updated, and education and training should be provided for all scorers. These changes could allow members within the biodosimetry network to successfully collaborate and support each other in large-scale radiological accidents.

Validation of genes for H-ARS severity prediction in leukemia patients - interspecies comparison, challenges, and promises.

PURPOSE: In a previous baboon-study, a total of 29 genes were identified for clinical outcome prediction of the hematologic, acute, radiation, syndrome (H-ARS) severity. Among them, four genes (FDXR, DDB2, POU2AF1, WNT3) appeared promising and were validated in five leukemia patients. Within this study, we sought further in-vivo validation in a larger number of whole-body irradiated patients. MATERIAL AND METHODS: Peripheral blood was drawn from 10 leukemia patients before and up to 3 days during a fractionated (2 Gy/day) total-body irradiation (TBI) with 2-12Gy. After RNA-isolation, gene expression (GE) was evaluated on 31 genes widely used in biodosimetry and H-ARS prediction employing qRT-PCR. A customized low-density-array (LDA) allowed simultanously analyzing all genes, the 96-well format further examined the four most promising genes. Fold-changes (FC) in GE relative to pre-irradiation were calculated. RESULTS: Five patients suffering from acute-lymphoblastic-leukemia (ALL) respectively non-Hodgkin-lymphoma (NHL) revealed sufficient RNA-amounts and corresponding lymphocyte and neutrophile counts for running qRT-PCR, while acute-myeloid-leukemia (AML) and one myelofibrosis patient could not supply enough RNA. Generally, 1-2µg total RNA was isolated, whereas up to 10-fold differences in RNA-quantities (associated suppressed GE-changes) were identified among pre-exposure and exposure samples. From 31 genes, 23 were expressed in at least one of the pre-exposure samples. Relative to pre-exposure, the number of expressed genes could halve at 48 and 72h after irradiation. Using the LDA, 13 genes were validated in human samples. The four most promising genes (vid. sup.) were either undetermined or too close to pre-exposure. However, they were measured using the more sensitive 96-well format, except WNT3, which wasn´t detectable. As in previous studies, an opposite regulation in GE for FDXR in leukemia patients (up-regulated) relative to baboons (down-regulated) was reconfirmed. Radiation-induced GE-changes of DDB2 (up-regulated) and POU2AF1 (down-regulated) behaved similarly in both species. Hence, 16 out of 23 genes of two species showed GE-changes in the same direction, and up-regulated FDXR as in human studies were revalidated. CONCLUSION: Identified genes for H-ARS severity prediction, previously detected in baboons, were validated in ALL but not in AML patients. Limitations related to leukemia type, associated reduced RNA amounts, suppressed GE changes, and methodological challenges must be considered as factors negatively affecting the total number of validated genes. Based on that, we propose additional controls including blood cell counts and preferably fluorescence-based RNA quantity measurements for selecting promising samples and using a more sensitive 96-well format for candidate genes with low baseline copy numbers.

Sex differences in radiation research.

PURPOSE: The Sex Differences in Radiation Research workshop addressed the role of sex as a confounder in radiation research and its implication in real-world radiological and nuclear applications. METHODS: In April 2022, HHS-wide partners from the Radiation and Nuclear Countermeasures Program, the Office of Research on Women's Health National Institutes of Health Office of Women's Health, U.S. Food and Drug Administration, and the Radiological and Nuclear Countermeasures Branch at the Biomedical Advanced Research and Development Authority conducted a workshop to address the scientific implication and knowledge gaps in understanding sex in basic and translational research. The goals of this workshop were to examine sex differences in 1. Radiation animal models and understand how these may affect radiation medical countermeasure development; 2. Biodosimetry and/or biomarkers used to assess acute radiation syndrome, delayed effects of acute radiation exposure, and/or predict major organ morbidities; 3. medical research that lacks representation from both sexes. In addition, regulatory policies that influence inclusion of women in research, and the gaps that exist in drug development and device clearance were discussed. Finally, real-world sex differences in human health scenarios were also considered. RESULTS: This report provides an overview of the two-day workshop, and open discussion among academic investigators, industry researchers, and U.S. government representatives. CONCLUSIONS: This meeting highlighted that current study designs lack the power to determine statistical significance based on sex, and much is unknown about the underlying factors that contribute to these differences. Investigators should accommodate both sexes in all stages of research to ensure that the outcome is robust, reproducible, and accurate, and will benefit public health.

Evaluating the radioprotective effect of green barley juice on male rats.

PURPOSE: DNA damage accounts for most biological effects of ionizing radiation. Antioxidants are known for their protective effect by preventing DNA damage. This pilot study aimed to evaluate the potential radioprotective effect of Natural SOD®, a green barley juice rich in antioxidants, on DNA damage in the testes and lymphocytes of Wistar rats exposed to ionizing radiation. MATERIALS AND METHODS: Male Wistar rats (n = 15) were selected and equally divided into three groups. Rats in one of the groups were pretreated orally with Natural SOD® for 14 days, while rats in another group were sham-pretreated with saline solution. Rats in both these groups were afterwards subjected to a single dose of 6 Gy X-ray whole-body irradiation. The control group did not receive any treatment and was not irradiated. Shortly after X-ray exposure, all rats were sacrificed and testes and blood were collected. Gamma-H2AX and histopathological assessment in the testes, along with comet assay of lymphocytes were performed. RESULTS: Histopathological examination of the testes showed no significant architectural alterations. Immunofluorescent staining of γ-H2AX revealed more DNA double-strand break sites in testicular cells from sham animals compared to Natural SOD® pretreated rats. Alkaline comet assay results showed increased DNA damage in lymphocytes of irradiated rats compared to the control group with little differences between the pretreated groups. Animals pretreated with Natural SOD showed slightly reduced DNA damage compared to sham-pretreated rats. These findings suggest a potential protective effect of Natural SOD® against radiation-induced DNA damage. CONCLUSIONS: Natural SOD® exhibited a potential prophylactic radioprotective effect in rats, particularly in testes. Further investigations to determine medium and long-term effects of X-ray in animals administered Natural SOD® are needed to better estimate the radioprotective effect.

Applicability of Scoring Calyculin A-Induced Premature Chromosome Condensation Objects for Dose Assessment Including for Radiotherapy Patients.

As an extension to a previous study, a linear calibration curve covering doses from 0 to 10 Gy was constructed and evaluated in the present study using calyculin A-induced premature chromosome condensation (PCC) by scoring excess PCC objects. The main aim of this study was to assess the applicability of this PCC assay for doses below 2 Gy that are critical for triage categorization. Two separate blind tests involving a total of 6 doses were carried out; 4 out of 6 dose estimates were within the 95% confidence limits (95% CL) with the other 2 just outside. In addition, blood samples from five cancer patients undergoing external beam radiotherapy (RT) were also analyzed, and the results showed whole-body dose estimates statistically comparable to the dicentric chromosome assay (DCA) results. This is the first time that calyculin A-induced PCC was used to analyze clinical samples by scoring excess objects. Although dose estimates for the pre-RT patient samples were found to be significantly higher than the mean value for the healthy donors and were also significantly higher than those obtained using DCA, all these pre-treatment patients fell into the same category as those who may have received a low dose (<1 Gy) and do not require immediate medical care during emergency triage. Additionally, for radiological accidents with unknown exposure scenario, PCC objects and rings can be scored in parallel for the assessment of both low- and high-dose exposures. In conclusion, scoring excess objects using calyculin A-induced PCC is confirmed to be another potential biodosimetry tool in radiological emergency particularly in mass casualty scenarios, even though the data need to be interpreted with caution when cancer patients are among the casualties.

DDB2 and MDM2 genes are promising markers for radiation diagnosis and estimation of radiation dose independent of trauma and burns.

In the field of biodosimetry, the current accepted method for evaluating radiation dose fails to meet the need of rapid, large-scale screening, and most RNA marker-related studies of biodosimetry are concentrating on a single type of ray, while some other potential factors, such as trauma and burns, have not been covered. Microarray datasets that contain the data of human peripheral blood samples exposed to X-ray, neutron, and γ-ray radiation were obtained from the GEO database. Totally, 33 multi-type ray co-induced genes were obtained at first from the differentially expressed genes (DEGs) and key genes identified by weighted gene co-expression network analysis (WGCNA), and these genes were mainly enriched in DNA damage, cellular apoptosis, and p53 signaling pathway. Following transcriptome sequencing of blood samples from 11 healthy volunteers, 13 patients with severe burns, and 37 patients with severe trauma, 6635 trauma-related DEGs and 7703 burn-related DEGs were obtained. Through the exclusion method, a total of 12 radiation-specific genes independent of trauma and burns were identified. ROC curve analysis revealed that the DDB2 gene performed the best in diagnosis of all three types of ray radiation, while correlation analysis showed that the MDM2 gene was the best in assessment of radiation dose. The results of multiple-linear regression analysis indicated that such analysis could improve the accuracy in assessment of radiation dose. Moreover, the DDB2 and MDM2 genes remained effective in radiation diagnosis and assessment of radiation dose in an external dataset. In general, the study brings new insights into radiation biodosimetry.

The NIAID/RNCP Biodosimetry Program: An Overview.

Established in 2004, the Radiation and Nuclear Countermeasures Program (RNCP), within the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health has the central mission to advance medical countermeasure mitigators/therapeutics, and biomarkers and technologies to assess, triage, and inform medical management of patients experiencing acute radiation syndrome and/or the delayed effects of acute radiation exposure. The RNCP biodosimetry mission space encompasses: (1) basic research to elucidate novel approaches for rapid and accurate assessment of radiation exposure, (2) studies to support advanced development for US Food and Drug Administration (FDA) clearance of promising triage or treatment devices/approaches, (3) characterization of biomarkers and/or assays to determine degree of tissue or organ dose that can predict outcome of radiation injuries (i.e., organ failure, morbidity, and/or mortality), and (4) outreach efforts to facilitate interactions with researchers developing cutting edge biodosimetry approaches. Thus far, no biodosimetry device has been FDA cleared for use during a radiological/nuclear incident. At NIAID, advancement of radiation biomarkers and biodosimetry approaches is facilitated by a variety of funding mechanisms (grants, contracts, cooperative and interagency agreements, and Small Business Innovation Research awards), with the objective of advancing devices and assays toward clearance, as outlined in the FDA's Radiation Biodosimetry Medical Countermeasure Devices Guidance. The ultimate goal of the RNCP biodosimetry program is to develop and establish accurate and reliable biodosimetry tools that will improve radiation preparedness and ultimately save lives during a radiological or nuclear incident.

Application of the Cytokinesis-Block Micronucleus Assay for High-Dose Exposures Using Imaging Flow Cytometry.

The cytokinesis-block micronucleus assay is a well-established method to assess radiation-induced genetic damage in human cells. This assay has been adapted to imaging flow cytometry (IFC), allowing automated analysis of many cells, and eliminating the need to create microscope slides. Furthermore, to improve the efficiency of assay performance, a small-volume method previously developed was employed. Irradiated human blood samples were cultured, stained, and analyzed by IFC to produce images of the cells. Samples were run using both manual and 96-well plate automated acquisition. Multiple parameter-based image features were collected for each sample, and the results were compared to confirm that these acquisition methods are functionally identical. This paper details the multi-parametric analysis developed and the resulting calibration curves up to 10 Gy. The calibration curves were created using a quadratic random coefficient model with Poisson errors, as well as a logistic discriminant function. The curves were then validated with blinded, irradiated samples, using relative bias and relative mean square error. Overall, the accuracy of the dose estimates was adequate for triage dosimetry (within 1 Gy of the true dose) over 90% of the time for lower doses and about half the time for higher doses, with the lowest success rate between 5 and 6 Gy where the calibration curve reached its peak and there was the smallest change in MN/BNC with dose. This work describes the application of a novel multi-parametric analysis that fits the calibration curves and allows dose estimates up to 10 Gy, which were previously limited to 4 Gy. Furthermore, it demonstrates that the results from samples acquired manually and with the autosampler are functionally similar.

Effects of cyclophosphamide and mitomycin C on radiation-induced transcriptional biomarkers in human lymphoblastoid cells.

PURPOSE: Ionizing radiation (IR)-induced transcriptional changes are considered a potential biodosimetry for dose evaluation and health risk monitoring of acute or chronic radiation exposure. It is crucial to understand the impact of confounding factors on the radiation-responsive gene expressions for accurate and reproducible dose assessment. This study aims to explore the potential influence of exposures to chemotherapeutic agents such as cyclophosphamide (CP) and mitomycin C (MMC) on IR-induced transcriptional biomarkers. METHODS: The human B lymphoblastoid cells (AHH-1) were exposed to 0, 20, 50, 100, 200 and 500 µg/ml CP or 0, 0.025, 0.05, 0.1 and 1 µg/ml MMC, respectively. The appropriate concentrations of CP and MMC were added for 1 h before irradiation with 0, 2, 4 and 6 Gy of 60Co γ-rays at a dose rate of 1 Gy/min. Cell viability was evaluated by CCK-8 assay. The gene expression responses of 18 radiation-induced transcriptional biomarkers were examined at 24 h after exposures to CP and MMC, respectively. The expression levels of five crucial DNA interstrand crosslinks (ICLs) repair genes were also evaluated. The biodosimetry models were established based on the specific radiation-responsive gene combinations. RESULTS: The baseline transcriptional levels of the 18 selected genes were slightly affected by CP treatment in the absence of IR, while the transcript responses to IR could be inhibited as the concentration of CP up to 50 µg/ml. MMC treatment up-regulated the background levels in most radiation-responsive gene expressions. Of 18 genes, only the relative mRNA expression levels of CDKN1A and BBC3 were repressed after treatment with IR and MMC in combination. The relative mRNA level of RAD51 was significantly up-regulated after exposure to CP, while the expression of FANCD2, RAD51 and BLM showed an overall increase in response to MMC treatment. After irradiation, the relative mRNA expression levels of FANCD2, BRCA2 and RAD51 exhibited dose-dependent increases in IR alone and MMC treatment groups. In addition, the biodosimetry models were established using 2-4 radiation-responsive genes based on different radiation exposure scenarios. CONCLUSION: Our findings suggested that IR-induced gene expression changes were slightly affected after exposure to a relatively low concentration of CP and MMC. Gene expression combinations might improve the broad applicability of transcriptional biodosimetry across diverse radiation exposures.

Comparison of Isolated Lymphocyte and Whole Blood-Based CBMN Assays for Radiation Triage.

Following a mass-casualty nuclear/radiological event, there will be an important need for rapid and accurate estimation of absorbed dose for biological triage. The cytokinesis-block micronucleus (CBMN) assay is an established and validated cytogenetic biomarker used to assess DNA damage in irradiated peripheral blood lymphocytes. Here, we describe an intercomparison experiment between two biodosimetry laboratories, located at Columbia University (CU) and Health Canada (HC) that performed different variants of the human blood CBMN assay to reconstruct dose in human blood, with CU performing the assay on isolated lymphocytes and using semi-automated scoring whereas HC used the more conventional whole blood assay. Although the micronucleus yields varied significantly between the two assays, the predicted doses closely matched up to 4 Gy - the range from which the HC calibration curve was previously established. These results highlight the importance of a robust calibration curve(s) across a wide age range of donors that match the exposure scenario as closely as possible and that will account for differences in methodology between laboratories. We have seen that at low doses, variability in the results may be attributed to variation in the processing while at higher doses the variation is dominated by inter-individual variation in cell proliferation. This interlaboratory collaboration further highlights the usefulness of the CBMN endpoint to accurately reconstruct absorbed dose in human blood after ionizing radiation exposure.

Retrospective Evaluation of Cytogenetic Effects Induced by Internal Radioiodine Exposure: A 27-Year Follow-Up Study.

Radioiodine (131I) is widely used in the treatment of hyperthyroidism and as an effective ablative therapy for differentiated thyroid cancer. Radioiodine (131I) constitutes 90% of the currently used therapies in the field of nuclear medicine. Here, we report the cytogenetic findings of a long-term follow-up study of 27 years on a male patient who received two rounds of radioiodine treatment within a span of 26 months between 1992 and 1994 for his papillary thyroid cancer. A comprehensive cytogenetic follow-up study utilizing cytokinesis blocked micronucleus assay, dicentric chromosome assay, genome wide translocations and inversions was initiated on this patient since the first administration of radioiodine in 1992. Frequencies of micronuclei (0.006/cell) and dicentric chromosomes (0.008/cell) detected in the current study were grossly similar to that reported earlier in 2019. The mFISH analysis detected chromosome aberrations in 8.6% of the cells in the form of both unbalanced and balanced translocations. Additionally, a clonal translocation involving chromosomes 14p; 15q was observed in 2 of the 500 cells analyzed. Out of the 500 cells examined, one cell showed a complex translocation (involving chromosomes 9, 10, and 16) besides 5 other chromosome rearrangements. Collectively, our study indicates that the past radioiodine exposure results in long-lasting chromosome damage and that the persistence of translocations can be useful for both retrospective biodosimetry and for monitoring chromosome instability in the lymphocytes of radioiodine exposed individuals.