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Kerry Bloom - One of the best experts on this subject based on the ideXlab platform.
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Nonrandom Distribution of Interhomolog Recombination Events Induced by Breakage of a Dicentric Chromosome in Saccharomyces cerevisiae
Genetics, 2013Co-Authors: Wei Song, Kerry Bloom, Malgorzata Gawel, Margaret Dominska, Patricia W. Greenwell, Einat Hazkani-covo, Thomas D. PetesAbstract:Dicentric Chromosomes undergo breakage in mitosis, resulting in Chromosome deletions, duplications, and translocations. In this study, we map Chromosome break sites of Dicentrics in Saccharomyces cerevisiae by a mitotic recombination assay. The assay uses a diploid strain in which one homolog has a conditional centromere in addition to a wild-type centromere, and the other homolog has only the wild-type centromere; the conditional centromere is inactive when cells are grown in galactose and is activated when the cells are switched to glucose. In addition, the two homologs are distinguishable by multiple single-nucleotide polymorphisms (SNPs). Under conditions in which the conditional centromere is activated, the functionally Dicentric Chromosome undergoes double-stranded DNA breaks (DSBs) that can be repaired by mitotic recombination with the homolog. Such recombination events often lead to loss of heterozygosity (LOH) of SNPs that are centromere distal to the crossover. Using a PCR-based assay, we determined the position of LOH in multiple independent recombination events to a resolution of ∼4 kb. This analysis shows that Dicentric Chromosomes have recombination breakpoints that are broadly distributed between the two centromeres, although there is a clustering of breakpoints within 10 kb of the conditional centromere.
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Nuclear oscillations and nuclear filament formation accompany single-strand annealing repair of a Dicentric Chromosome in Saccharomyces cerevisiae.
Journal of cell science, 2003Co-Authors: Douglas A Thrower, Jennifer Stemple, Elaine Yeh, Kerry BloomAbstract:Dicentric Chromosomes undergo breakage during mitosis as a result of the attachment of two centromeres on one sister chromatid to opposite spindle poles. Studies utilizing a conditional Dicentric Chromosome III in Saccharomyces cerevisiae have shown that Dicentric Chromosome repair occurs primarily by deletion of one centromere via a RAD52-dependent recombination pathway. We report that Dicentric Chromosome resolution requires RAD1, a gene involved in the single-strand annealing DNA repair pathway. We additionally show that single-strand annealing repair of a Dicentric Chromosome can occur in the absence of RAD52. RAD52-independent repair requires the adaptation-defective cdc5-ad allele of the yeast polo kinase and the DNA damage checkpoint gene RAD9. Dicentric Chromosome breakage in cdc5-ad rad52 mutant cells is associated with a prolonged mitotic arrest, during which nuclei undergo microtubule-dependent oscillations, accompanied by dynamic changes in nuclear morphology. We further demonstrate that the frequency of spontaneous direct repeat recombination is suppressed in yeast cells treated with benomyl, a drug that perturbs microtubules. Our findings indicate that microtubule-dependent processes facilitate recombination.
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Dicentric Chromosome stretching during anaphase reveals roles of sir2 ku in chromatin compaction in budding yeast
Molecular Biology of the Cell, 2001Co-Authors: Douglas A Thrower, Kerry BloomAbstract:We have used mitotic spindle forces to examine the role of Sir2 and Ku in chromatin compaction. Escherichia coli lac operator DNA was placed between two centromeres on a conditional Dicentric Chromosome in budding yeast cells and made visible by expression of a lac repressor– green fluorescent fusion protein. Centromeres on the same chromatid of a Dicentric Chromosome attach to opposite poles 50% of the time, resulting in Chromosome bridges during anaphase. In cells deleted for yKU70, yKU80 ,o rSIR2, a 10-kb region of the Dicentric Chromosome stretched along the spindle axis to a length of 6 m during anaphase. On spindle disassembly, stretched chromatin recoiled to the bud neck and was partitioned to mother and daughter cells after cytokinesis and cell separation. Chromatin immunoprecipitation revealed that Sir2 localizes to the lacO region in response to activation of the Dicentric Chromosome. These findings indicate that Ku and Sir proteins are required for proper chromatin compaction within regions of a Chromosome experiencing tension or DNA damage. The association of Sir2 with the affected region suggests a direct role in this process, which may include the formation of heterochromatic DNA.
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Dicentric Chromosome stretching during anaphase reveals roles of Sir2/Ku in chromatin compaction in budding yeast.
Molecular biology of the cell, 2001Co-Authors: Douglas A Thrower, Kerry BloomAbstract:We have used mitotic spindle forces to examine the role of Sir2 and Ku in chromatin compaction. Escherichia coli lac operator DNA was placed between two centromeres on a conditional Dicentric Chromosome in budding yeast cells and made visible by expression of a lac repressor-green fluorescent fusion protein. Centromeres on the same chromatid of a Dicentric Chromosome attach to opposite poles approximately 50% of the time, resulting in Chromosome bridges during anaphase. In cells deleted for yKU70, yKU80, or SIR2, a 10-kb region of the Dicentric Chromosome stretched along the spindle axis to a length of 6 microm during anaphase. On spindle disassembly, stretched chromatin recoiled to the bud neck and was partitioned to mother and daughter cells after cytokinesis and cell separation. Chromatin immunoprecipitation revealed that Sir2 localizes to the lacO region in response to activation of the Dicentric Chromosome. These findings indicate that Ku and Sir proteins are required for proper chromatin compaction within regions of a Chromosome experiencing tension or DNA damage. The association of Sir2 with the affected region suggests a direct role in this process, which may include the formation of heterochromatic DNA.
Douglas A Thrower - One of the best experts on this subject based on the ideXlab platform.
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Nuclear oscillations and nuclear filament formation accompany single-strand annealing repair of a Dicentric Chromosome in Saccharomyces cerevisiae.
Journal of cell science, 2003Co-Authors: Douglas A Thrower, Jennifer Stemple, Elaine Yeh, Kerry BloomAbstract:Dicentric Chromosomes undergo breakage during mitosis as a result of the attachment of two centromeres on one sister chromatid to opposite spindle poles. Studies utilizing a conditional Dicentric Chromosome III in Saccharomyces cerevisiae have shown that Dicentric Chromosome repair occurs primarily by deletion of one centromere via a RAD52-dependent recombination pathway. We report that Dicentric Chromosome resolution requires RAD1, a gene involved in the single-strand annealing DNA repair pathway. We additionally show that single-strand annealing repair of a Dicentric Chromosome can occur in the absence of RAD52. RAD52-independent repair requires the adaptation-defective cdc5-ad allele of the yeast polo kinase and the DNA damage checkpoint gene RAD9. Dicentric Chromosome breakage in cdc5-ad rad52 mutant cells is associated with a prolonged mitotic arrest, during which nuclei undergo microtubule-dependent oscillations, accompanied by dynamic changes in nuclear morphology. We further demonstrate that the frequency of spontaneous direct repeat recombination is suppressed in yeast cells treated with benomyl, a drug that perturbs microtubules. Our findings indicate that microtubule-dependent processes facilitate recombination.
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Dicentric Chromosome stretching during anaphase reveals roles of sir2 ku in chromatin compaction in budding yeast
Molecular Biology of the Cell, 2001Co-Authors: Douglas A Thrower, Kerry BloomAbstract:We have used mitotic spindle forces to examine the role of Sir2 and Ku in chromatin compaction. Escherichia coli lac operator DNA was placed between two centromeres on a conditional Dicentric Chromosome in budding yeast cells and made visible by expression of a lac repressor– green fluorescent fusion protein. Centromeres on the same chromatid of a Dicentric Chromosome attach to opposite poles 50% of the time, resulting in Chromosome bridges during anaphase. In cells deleted for yKU70, yKU80 ,o rSIR2, a 10-kb region of the Dicentric Chromosome stretched along the spindle axis to a length of 6 m during anaphase. On spindle disassembly, stretched chromatin recoiled to the bud neck and was partitioned to mother and daughter cells after cytokinesis and cell separation. Chromatin immunoprecipitation revealed that Sir2 localizes to the lacO region in response to activation of the Dicentric Chromosome. These findings indicate that Ku and Sir proteins are required for proper chromatin compaction within regions of a Chromosome experiencing tension or DNA damage. The association of Sir2 with the affected region suggests a direct role in this process, which may include the formation of heterochromatic DNA.
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Dicentric Chromosome stretching during anaphase reveals roles of Sir2/Ku in chromatin compaction in budding yeast.
Molecular biology of the cell, 2001Co-Authors: Douglas A Thrower, Kerry BloomAbstract:We have used mitotic spindle forces to examine the role of Sir2 and Ku in chromatin compaction. Escherichia coli lac operator DNA was placed between two centromeres on a conditional Dicentric Chromosome in budding yeast cells and made visible by expression of a lac repressor-green fluorescent fusion protein. Centromeres on the same chromatid of a Dicentric Chromosome attach to opposite poles approximately 50% of the time, resulting in Chromosome bridges during anaphase. In cells deleted for yKU70, yKU80, or SIR2, a 10-kb region of the Dicentric Chromosome stretched along the spindle axis to a length of 6 microm during anaphase. On spindle disassembly, stretched chromatin recoiled to the bud neck and was partitioned to mother and daughter cells after cytokinesis and cell separation. Chromatin immunoprecipitation revealed that Sir2 localizes to the lacO region in response to activation of the Dicentric Chromosome. These findings indicate that Ku and Sir proteins are required for proper chromatin compaction within regions of a Chromosome experiencing tension or DNA damage. The association of Sir2 with the affected region suggests a direct role in this process, which may include the formation of heterochromatic DNA.
Mitsuaki A Yoshida - One of the best experts on this subject based on the ideXlab platform.
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Analysis of Chromosome translocation frequency after a single CT scan in adults
Journal of radiation research, 2016Co-Authors: Yu Abe, Mitsuaki A Yoshida, Tomisato Miura, Risa Ujiie, Yumiko Kurosu, Nagisa Kato, Atsushi Katafuchi, Naohiro Tsuyama, Fumihiko Kawamura, Takashi OhbaAbstract:We recently reported an increase in Dicentric Chromosome (DIC) formation after a single computed tomography (CT) scan (5.78-60.27 mSv: mean 24.24 mSv) and we recommended analysis of 2000 metaphase cells stained with Giemsa and centromere-FISH for Dicentric Chromosome assay (DCA) in cases of low-dose radiation exposure. In the present study, we analyzed the frequency of Chromosome translocations using stored Carnoy's-fixed lymphocyte specimens from the previous study; these specimens were from 12 patients who were subject to Chromosome painting of Chromosomes 1, 2 and 4. Chromosomes 1, 2 and 4 were analyzed in ∼5000 cells, which is equivalent to the whole-genome analysis of almost 2000 cells. The frequency of Chromosome translocation was higher than the number of DICs formed, both before and after CT scanning. The frequency of Chromosome translocations tended to be higher, but not significantly higher, in patients with a treatment history compared with patients without such a history. However, in contrast to the results for DIC formation, the frequency of translocations detected before and after the CT scan did not differ significantly. Therefore, analysis of Chromosome translocation may not be a suitable assay for detecting Chromosome aberrations in cases of low-dose radiation exposure from a CT scan. A significant increase in the frequency of Chromosome translocations was not likely to be detected due to the high baseline before the CT scan; the high and variable frequency of translocations was probably due to multiple confounding factors in adults.
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Increase in Dicentric Chromosome formation after a single CT scan in adults.
Scientific reports, 2015Co-Authors: Yu Abe, Mitsuaki A Yoshida, Tomisato Miura, Risa Ujiie, Yumiko Kurosu, Nagisa Kato, Atsushi Katafuchi, Naohiro Tsuyama, Takashi Ohba, Tomoko InamasuAbstract:Excess risk of leukemia and brain tumors after CT scans in children has been reported. We performed Dicentric Chromosome assay (DCAs) before and after CT scan to assess effects of low-dose ionizing radiation on Chromosomes. Peripheral blood (PB) lymphocytes were collected from 10 patients before and after a CT scan. DCA was performed by analyzing either 1,000 or 2,000 metaphases using both Giemsa staining and centromere-fluorescence in situ hybridization (Centromere-FISH). The increment of DIC formation was compared with effective radiation dose calculated using the computational dosimetry system, WAZA-ARI and dose length product (DLP) in a CT scan. Dicentric Chromosome (DIC) formation increased significantly after a single CT scan, and increased DIC formation was found in all patients. A good correlation between the increment of DIC formation determined by analysis of 2,000 metaphases using Giemsa staining and those by 2,000 metaphases using Centromere-FISH was observed. However, no correlation was observed between the increment of DIC formation and the effective radiation dose. Therefore, these results suggest that Chromosome cleavage may be induced by one CT scan, and we recommend 2,000 or more metaphases be analyzed in Giemsa staining or Centromere-FISH for DCAs in cases of low-dose radiation exposure.
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biological dosimetry by the triage Dicentric Chromosome assay further validation of international networking
Radiation Measurements, 2011Co-Authors: Ruth C Wilkins, Horst Romm, Ursula Oestreicher, Leonora Marro, Mitsuaki A Yoshida, Y Suto, Pataje G S PrasannaAbstract:Abstract Biological dosimetry is an essential tool for estimating radiation doses received to personnel when physical dosimetry is not available or inadequate. The current preferred biodosimetry method is based on the measurement of radiation-specific Dicentric Chromosomes in exposed individuals’ peripheral blood lymphocytes. However, this method is labor-, time- and expertise-demanding. Consequently, for mass casualty applications, strategies have been developed to increase its throughput. One such strategy is to develop validated cytogenetic biodosimetry laboratory networks, both national and international. In a previous study, the Dicentric Chromosome assay (DCA) was validated in our cytogenetic biodosimetry network involving five geographically dispersed laboratories. A complementary strategy to further enhance the throughput of the DCA among inter-laboratory networks is to use a triage DCA where dose assessments are made by truncating the labor-demanding and time-consuming metaphase spread analysis to 20 – 50 metaphase spreads instead of routine 500 – 1000 metaphase spread analysis. Our laboratory network also validated this triage DCA, however, these dose estimates were made using calibration curves generated in each laboratory from the blood samples irradiated in a single laboratory. In an emergency situation, dose estimates made using pre-existing calibration curves which may vary according to radiation type and dose rate and therefore influence the assessed dose. Here, we analyze the effect of using a pre-existing calibration curve on assessed dose among our network laboratories. The dose estimates were made by analyzing 1000 metaphase spreads as well as triage quality scoring and compared to actual physical doses applied to the samples for validation. The dose estimates in the laboratory partners were in good agreement with the applied physical doses and determined to be adequate for guidance in the treatment of acute radiation syndrome.
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biological dosimetry by the triage Dicentric Chromosome assay potential implications for treatment of acute radiation syndrome in radiological mass casualties
Radiation Research, 2011Co-Authors: Horst Romm, Gordon K. Livingston, Ruth C Wilkins, Mitsuaki A Yoshida, Akio A. Awa, Mark S. Jenkins, Terry C. Pellmar, Norman C Coleman, Patricia Lillishearne, Ursula OestreicherAbstract:Biological dosimetry is an essential tool for estimating radiation dose. The Dicentric Chromosome assay (DCA) is currently the tool of choice. Because the assay is labor-intensive and time-consuming, strategies are needed to increase throughput for use in radiation mass casualty incidents. One such strategy is to truncate metaphase spread analysis for triage dose estimates by scoring 50 or fewer metaphases, compared to a routine analysis of 500 to 1000 metaphases, and to increase throughput using a large group of scorers in a biodosimetry network. Previously, the National Institutes for Allergies and Infectious Diseases (NIAID) and the Armed Forces Radiobiology Research Institute (AFRRI) sponsored a double-blinded interlaboratory comparison among five established international cytogenetic biodosimetry laboratories to determine the variability in calibration curves and in dose measurements in unknown, irradiated samples. In the present study, we further analyzed the published data from this previous study to investigate how the number of metaphase spreads influences dose prediction accuracy and how this information could be of value in the triage and management of people at risk for the acute radiation syndrome (ARS). Although, as expected, accuracy decreased with lower numbers of metaphase spreads analyzed, predicted doses by the laboratories were in good agreement and were judged to be adequate to guide diagnosis and treatment of ARS. These results demonstrate that for rapid triage, a network of cytogenetic biodosimetry laboratories can accurately assess doses even with a lower number of scored metaphases.
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Interlaboratory Comparison of the Dicentric Chromosome Assay for Radiation Biodosimetry in Mass Casualty Events
Radiation research, 2008Co-Authors: Ruth C Wilkins, Gordon K. Livingston, Horst Romm, Ursula Oestreicher, Mitsuaki A Yoshida, Tzu-cheg Kao, Akio A. Awa, Mark S. Jenkins, Terry C. Pellmar, Pataje G S PrasannaAbstract:Abstract Wilkins, R. C., Romm, H., Kao, T-C., Awa, A. A., Yoshida, M. A., Livingston, G. K., Jenkins, M. S., Oestreicher, U., Pellmar, T. C. and Prasanna, P. G. S. Interlaboratory Comparison of the Dicentric Chromosome Assay for Radiation Biodosimetry in Mass Casualty Events. Radiat. Res. 169, 551–560 (2008). This interlaboratory comparison validates the Dicentric Chromosome assay for assessing radiation dose in mass casualty accidents and identifies the advantages and limitations of an international biodosimetry network. The assay's validity and accuracy were determined among five laboratories following the International Organization for Standardization guidelines. Blood samples irradiated at the Armed Forces Radiobiology Research Institute were shipped to all laboratories, which constructed individual radiation calibration curves and assessed the dose to dose-blinded samples. Each laboratory constructed a dose–effect calibration curve for the yield of Dicentrics for 60Co γ rays in the 0 to 5-Gy range, u...
Ruth C Wilkins - One of the best experts on this subject based on the ideXlab platform.
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RADIATION DOSE ESTIMATION BY COMPLETELY AUTOMATED INTERPRETATION OF THE Dicentric Chromosome ASSAY
Radiation protection dosimetry, 2019Co-Authors: Ben C. Shirley, Ruth C Wilkins, Joan H.m. Knoll, Farrah Norton, Peter K. RoganAbstract:Accuracy of the automated Dicentric Chromosome (DC) assay relies on metaphase image selection. This study validates a software framework to find the best image selection models that mitigate inter-sample variability. Evaluation methods to determine model quality include the Poisson goodness-of-fit of DC distributions for each sample, residuals after calibration curve fitting and leave-one-out dose estimation errors. The process iteratively searches a pool of selection model candidates by modifying statistical and filter cut-offs to rank the best candidates according to their respective evaluation scores. Evaluation scores minimize the sum of squared errors relative to the actual radiation dose of the calibration samples. For one laboratory, the minimum score for the curve fit residual method was 0.0475 Gy2, compared to 1.1975 Gy2 without image selection. Application of optimal selection models using samples of unknown exposure produced estimated doses within 0.5 Gy of physical dose. Model optimization standardizes image selection among samples and provides relief from manual DC scoring, improving accuracy and consistency of dose estimation.
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Validation of the Dicentric Chromosome assay for radiation biological dosimetry in South Korea.
Journal of radiation research, 2019Co-Authors: Younghyun Lee, Ruth C Wilkins, Young Woo Jin, Seongjae JangAbstract:The Dicentric Chromosome assay (DCA) is a well-established biodosimetry test to estimate exposure to ionizing radiation. The Korea Institute of Radiological and Medical Sciences (KIRAMS) established a DCA protocol as a medical response to radiation emergencies in South Korea. To maintain its accuracy and performance, intercomparison exercises with Health Canada (HC) have been conducted; herein, we aimed to validate our capacity of DCA analysis based on those results. Blood samples irradiated at HC were shipped to KIRAMS to assess the irradiation dose to blinded samples using conventional DCA full scoring and triage-based techniques (conventional DCA scoring in triage mode and DCA QuickScan method). Actual doses fell within the 95% confidence intervals of dose estimates for 70-100% of the blinded samples in 2015-2018. All methods discriminated binary dose categories, reflecting clinical significance. This DCA can be used as a reliable radiation biodosimetry tool in preparation for radiation accidents in South Korea.
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Accurate cytogenetic biodosimetry through automated Dicentric Chromosome curation and metaphase cell selection.
F1000Research, 2017Co-Authors: Jin Liu, Ruth C Wilkins, Joan H.m. Knoll, Farrah Flegal, Peter K. RoganAbstract:Accurate digital image analysis of abnormal microscopic structures relies on high quality images and on minimizing the rates of false positive (FP) and negative objects in images. Cytogenetic biodosimetry detects Dicentric Chromosomes (DCs) that arise from exposure to ionizing radiation, and determines radiation dose received based on DC frequency. Improvements in automated DC recognition increase the accuracy of dose estimates by reclassifying FP DCs as monocentric Chromosomes or Chromosome fragments. We also present image segmentation methods to rank high quality digital metaphase images and eliminate suboptimal metaphase cells. A set of Chromosome morphology segmentation methods selectively filtered out FP DCs arising primarily from sister chromatid separation, Chromosome fragmentation, and cellular debris. This reduced FPs by an average of 55% and was highly specific to these abnormal structures (≥97.7%) in three samples. Additional filters selectively removed images with incomplete, highly overlapped, or missing metaphase cells, or with poor overall Chromosome morphologies that increased FP rates. Image selection is optimized and FP DCs are minimized by combining multiple feature based segmentation filters and a novel image sorting procedure based on the known distribution of Chromosome lengths. Applying the same image segmentation filtering procedures to both calibration and test samples reduced the average dose estimation error from 0.4 Gy to
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accurate cytogenetic biodosimetry through automated Dicentric Chromosome curation and metaphase cell selection
F1000Research, 2017Co-Authors: Jin Liu, Ruth C Wilkins, Joan H.m. Knoll, Farrah Flegal, Peter K. RoganAbstract:Accurate digital image analysis of abnormal microscopic structures relies on high quality images and on minimizing the rates of false positive (FP) and negative objects in images. Cytogenetic biodosimetry detects Dicentric Chromosomes (DCs) that arise from exposure to ionizing radiation, and determines radiation dose received based on DC frequency. Improvements in automated DC recognition increase the accuracy of dose estimates by reclassifying FP DCs as monocentric Chromosomes or Chromosome fragments. We also present image segmentation methods to rank high quality digital metaphase images and eliminate suboptimal metaphase cells. A set of Chromosome morphology segmentation methods selectively filtered out FP DCs arising primarily from sister chromatid separation, Chromosome fragmentation, and cellular debris. This reduced FPs by an average of 55% and was highly specific to these abnormal structures (≥97.7%) in three samples. Additional filters selectively removed images with incomplete, highly overlapped, or missing metaphase cells, or with poor overall Chromosome morphologies that increased FP rates. Image selection is optimized and FP DCs are minimized by combining multiple feature based segmentation filters and a novel image sorting procedure based on the known distribution of Chromosome lengths. Applying the same image segmentation filtering procedures to both calibration and test samples reduced the average dose estimation error from 0.4 Gy to <0.2 Gy, obviating the need to first manually review these images. This reliable and scalable solution enables batch processing for multiple samples of unknown dose, and meets current requirements for triage radiation biodosimetry of high quality metaphase cell preparations.
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accurate cytogenetic biodosimetry through automation of Dicentric Chromosome curation and metaphase cell selection
bioRxiv, 2017Co-Authors: Jin Liu, Ruth C Wilkins, Joan H.m. Knoll, Farrah Flegal, Peter K. RoganAbstract:Software to automate digital pathology relies on image quality and the rates of false positive and negative objects in these images. Cytogenetic biodosimetry detects Dicentric Chromosomes (DCs) that arise from exposure to ionizing radiation, and determines radiation dose received from the frequency of DCs. We present image segmentation methods to rank high quality cytogenetic images and eliminate suboptimal metaphase cell data based on novel quality measures. Improvements in DC recognition increase the accuracy of dose estimates, by reducing false positive (FP) DC detection. A set of Chromosome morphology segmentation methods selectively filtered out false DCs, arising primarily from extended prometaphase Chromosomes, sister chromatid separation and Chromosome fragmentation. This reduced FPs by 55% and was highly specific to the abnormal structures (≥97.7%). Additional procedures were then developed to fully automate image review, resulting in 6 image-level filters that, when combined, selectively remove images with consistently unparsable or incorrectly segmented Chromosome morphologies. Overall, these filters can eliminate half of the FPs detected by manual image review. Optimal image selection and FP DCs are minimized by combining multiple feature based segmentation filters and a novel image sorting procedure based on the known distribution of Chromosome lengths. Consequently, the average dose estimation error was reduced from 0.4Gy to <0.2Gy with minimal manual review required. These image filtering approaches constitute a reliable and scalable solution that results in more accurate radiation dose estimates.
Adayabalam S. Balajee - One of the best experts on this subject based on the ideXlab platform.
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optimization and validation of automated Dicentric Chromosome analysis for radiological nuclear triage applications
Mutation Research-genetic Toxicology and Environmental Mutagenesis, 2019Co-Authors: Terri L. Ryan, Maria Escalona, Carol J. Iddins, Joseph Albanese, Tammy Smith, Adayabalam S. BalajeeAbstract:Abstract Dicentric Chromosome Assay (DCA) is the most preferred cytogenetic technique for absorbed radiation dose assessment in exposed humans. However, DCA is somewhat impractical for triage application owing to its labor intensive and time consuming nature. Although lymphocyte culture for 48 h in vitro is inevitable for DCA, manual scoring of Dicentric Chromosomes (DCs) requires an additional time of 24–48 h, making the overall turnaround time of 72–96 h for dose estimation. To accelerate the speed of DC analysis for dose estimation, an automated tool was optimized and validated for triage mode of scoring. Several image training files were created to improve the specificity of automated DC analysis algorithm. Accuracy and efficiency of the automated (unsupervised) DC scoring was compared with the semi-automated scoring that involved human verification and correction of DCs (elimination of false positives and inclusion of true positives). DC scoring was performed by both automated and semi-automated modes for different doses of X-rays and γ-rays (0 Gy–5 Gy). Biodoses estimated from the frequencies of DCs detected by both automated (unsupervised) and semi-automated (supervised) scoring modes were grossly similar to the actual delivered doses in the range of 0.5 to 3 Gy of low LET radiation. We suggest that the automated DC tool can be effectively used for large scale radiological/nuclear incidents where a rapid segregation is essential for prioritizing moderately or severely exposed humans to receive appropriate medical countermeasures.
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Optimization and validation of automated Dicentric Chromosome analysis for radiological/nuclear triage applications.
Mutation research, 2019Co-Authors: Terri L. Ryan, Tammy L. Smith, Maria Escalona, Carol J. Iddins, Joseph Albanese, Adayabalam S. BalajeeAbstract:Dicentric Chromosome Assay (DCA) is the most preferred cytogenetic technique for absorbed radiation dose assessment in exposed humans. However, DCA is somewhat impractical for triage application owing to its labor intensive and time consuming nature. Although lymphocyte culture for 48 h in vitro is inevitable for DCA, manual scoring of Dicentric Chromosomes (DCs) requires an additional time of 24-48 h, making the overall turnaround time of 72-96 h for dose estimation. To accelerate the speed of DC analysis for dose estimation, an automated tool was optimized and validated for triage mode of scoring. Several image training files were created to improve the specificity of automated DC analysis algorithm. Accuracy and efficiency of the automated (unsupervised) DC scoring was compared with the semi-automated scoring that involved human verification and correction of DCs (elimination of false positives and inclusion of true positives). DC scoring was performed by both automated and semi-automated modes for different doses of X-rays and γ-rays (0 Gy-5 Gy). Biodoses estimated from the frequencies of DCs detected by both automated (unsupervised) and semi-automated (supervised) scoring modes were grossly similar to the actual delivered doses in the range of 0.5 to 3 Gy of low LET radiation. We suggest that the automated DC tool can be effectively used for large scale radiological/nuclear incidents where a rapid segregation is essential for prioritizing moderately or severely exposed humans to receive appropriate medical countermeasures.
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Extension of lymphocyte viability for radiation biodosimetry: Potential implications for radiological/nuclear mass casualty incidents.
Journal of Cellular Biochemistry, 2018Co-Authors: Tammy L. Smith, Maria Escalona, Terri L. Ryan, Jacob T. Sanders, Gordon K. Livingston, Adayabalam S. BalajeeAbstract:Dicentric Chromosome assay (DCA) is routinely used for estimating the absorbed radiation dose in exposed humans. Optimal lymphocyte viability is crucial for reliable dose estimation and most cytogenetic laboratories prefer the receipt of blood samples within 24 to 36 hours after collection. Delays in the shipment/receipt of samples can occur sometimes under certain unforeseen circumstances: (1) Adverse weather conditions, (2) distant location of blood collection sites, and (3) shipping and handling of a large number of samples after radiological/nuclear mass casualty incident(s). To circumvent some of these limitations, we evaluated the suitability of ex vivo irradiated blood samples stored in the presence of phytohemagglutinin (PHA) for 7 days at ambient temperature (22-24°C) for radiation biodosimetry. Blood samples stored in the presence of PHA for up to 7 days showed a higher mitotic index than blood samples stored without PHA. To verify the use of stored blood samples for DCA, frequencies of X-rays induced Dicentric Chromosomes were analyzed in the blood samples that were cultured either 24 hours after exposure or 7 days later after storage. Our results indicate that storage of ex vivo irradiated blood samples in the presence of PHA at ambient temperature was found optimal for DCA and that the radiation doses estimated by Dicentric Chromosome frequencies were grossly similar between the fresh and stored blood samples. Our study suggests that reliable and accurate biodosimetry results can be obtained for triage using blood samples stored for up to a week at ambient temperature in the presence of PHA.
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Development of electronic training and telescoring tools to increase the surge capacity of Dicentric Chromosome scorers for radiological/nuclear mass casualty incidents.
Applied Radiation and Isotopes, 2018Co-Authors: Adayabalam S. Balajee, Maria Escalona, Carol J. Iddins, Igor Shuryak, Don Hanlon, Gordon K. Livingston, Nicholas DainiakAbstract:Abstract Dicentric Chromosome assay (DCA) is most frequently used for estimating the absorbed radiation dose in the peripheral blood lymphocytes of humans after occupational or incidental radiation exposure. DCA is considered to be the “gold standard” for estimating the absorbed radiation dose because the Dicentric Chromosome formation is fairly specific to ionizing radiation exposure and its baseline frequency is extremely low in non-exposed humans. However, performance of DCA for biodosimetry is labor intensive and time-consuming making its application impractical for radiological/nuclear mass casualty incidents. Realizing the critical need for rapid dose estimation particularly after radiological/nuclear disaster events, several laboratories have initiated efforts to automate some of the procedural steps involved in DCA. Although metaphase image capture and Dicentric Chromosome analysis have been automated using commercially available platforms, lack or an insufficient number of these platforms may pose a serious bottleneck when hundreds and thousands of samples need to be analyzed for rapid dose estimation. To circumvent this problem, a web-based approach for telescoring was initiated by our laboratory, which enabled the cytogeneticists around the globe to analyze and score digital images. To further increase the surge capacity of Dicentric scorers, we recently initiated a Dicentric training and scoring exercise involving a total of 50 volunteers at all academic levels without any prerequisite for experience in radiation cytogenetics. Out of the 50 volunteers enrolled thus far, only one outlier was found who overestimated the absorbed radiation dose. Our approach of training the civilians in Dicentric Chromosome analysis holds great promise for increasing the surge capacity of Dicentric Chromosome scorers for a rapid biodosimetry in the case of mass casualty scenarios.
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DEVELOPMENT OF A MINIATURIZED VERSION OF Dicentric Chromosome ASSAY TOOL FOR RADIOLOGICAL TRIAGE.
Radiation protection dosimetry, 2018Co-Authors: Adayabalam S. Balajee, Tammy L. Smith, Maria Escalona, Terri L. Ryan, Nicholas DainiakAbstract:Use of ionizing radiation (IR) in various industrial, medical and other applications can potentially increase the risk of medical, occupational or accidental human exposure. Additionally, in the event of a radiological or nuclear (R/N) incident, several tens of hundreds and thousands of people are likely to be exposed to IR. IR causes serious health effects including mortality from acute radiation syndrome and therefore it is imperative to determine the absorbed radiation dose, which will enable physicians in making an appropriate clinical 'life-saving' decision. The 'Dicentric Chromosome Assay (DCA)' is the gold standard for estimating the absorbed radiation dose but its performance is time consuming and laborious. Further, timely evaluation of Dicentric Chromosomes (DCs) for dose estimation in a large number of samples provides a bottleneck because of a limited number of trained personnel and a prolonged time for manual analysis. To circumvent some of these technical issues, we developed and optimized a miniaturized high throughput version of DCA (mini-DCA) in a 96-microtube matrix with bar-coded 1.4 ml tubes to enable the processing of a large number of samples. To increase the speed of DC analysis for radiation dose estimation, a semi-automated scoring was optimized using the Metafer DCScore algorithm. The accuracy of mini-DCA in dose estimation was verified and validated though comparison with conventional DCA performed in 15 ml conical tubes. The mini-DCA considerably reduced the sample processing time by a factor of 4 when compared to the conventional DCA. Further, the radiation doses estimated by mini-DCA using the triage mode of scoring (50 cells or 30 DCs) were similar to that of conventional DCA using 300-500 cells. The mini-DCA coupled with semi-automated DC scoring not only reduced the sample processing and analysis times by a factor of 4 but also enabled the processing of a large number of samples at once. Our mini-DCA method, once automated for high throughput robotic platforms, will be an effective radiological triage tool for mass casualty incidents.