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Tamizh Selvan G, Venkatachalam P. Potentials of cytokinesis blocked micronucleus assay in radiation triage and biological dosimetry. J Genet Eng Biotechnol 2024; 22:100409. [PMID: 39674629 PMCID: PMC11381789 DOI: 10.1016/j.jgeb.2024.100409] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2023] [Revised: 02/04/2024] [Accepted: 08/06/2024] [Indexed: 09/11/2024]
Abstract
The measurement of micronucleus (MN) in the cytokinesis-block arrested binucleated cells has been extensively used as a biomarker in many radiation biology applications in specific biodosimetry. Following radiation casualties, medical management of exposed individuals begins with triage and biological dosimetry. The cytokinesis blocked micronucleus (CBMN) assay is the alternate for the gold standard dicentric chromosome assay in radiation dose assessment. In recent years, the CBMN assay has become well-validated and emerged as a method of choice for evaluating occupational and accidental exposures scenario. It is feasible due to its cost-effective, simple, and rapid dose assessment rather than a conventional chromosome aberration assay. PubMed search tool was used with keywords of MN, biodosimetry, radiotherapy and restricted to human samples. Since Fenech and Morely developed the assay, it has undergone many technical and technological reforms as a biomarker of various applications. In this review, we have abridged recent developments of the CBMN assay in radiation triage and biodosimetry, focusing on (a) the influence of variables on dose estimation, (b) the importance of baseline frequency and reported dose-response coefficient values among different laboratories, (c) inter-laboratory comparison and (d) its limitations and means to overcome them.
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Affiliation(s)
- G Tamizh Selvan
- Central Research Laboratory, K.S. Hegde Medical Academy, NITTE (Deemed to be University), Deralakatte, Mangalore, Karnataka, India.
| | - P Venkatachalam
- Department of Human Genetics, Sri Ramachandra Institute of Higher Education and Research (Deemed to be University), Chennai, India
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Fenech M, Holland N, Zeiger E, Chang PW, Kirsch-Volders M, Bolognesi C, Stopper H, Knudsen LE, Knasmueller S, Nersesyan A, Thomas P, Dhillon V, Deo P, Franzke B, Andreassi MG, Laffon B, Wagner KH, Norppa H, da Silva J, Volpi EV, Wilkins R, Bonassi S. Objectives and achievements of the HUMN project on its 26th anniversary. MUTATION RESEARCH. REVIEWS IN MUTATION RESEARCH 2024; 794:108511. [PMID: 39233049 DOI: 10.1016/j.mrrev.2024.108511] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/20/2024] [Revised: 08/22/2024] [Accepted: 08/26/2024] [Indexed: 09/06/2024]
Abstract
Micronuclei (MN) are a nuclear abnormality that occurs when chromosome fragments or whole chromosomes are not properly segregated during mitosis and consequently are excluded from the main nuclei and wrapped within nuclear membrane to form small nuclei. This maldistribution of genetic material leads to abnormal cellular genomes which may increase risk of developmental defects, cancers, and accelerated aging. Despite the potential importance of MN as biomarkers of genotoxicity, very little was known about the optimal way to measure MN in humans, the normal ranges of values of MN in healthy humans and the prospective association of MN with developmental and degenerative diseases prior to the 1980's. In the early 1980's two important methods to measure MN in humans were developed namely, the cytokinesis-block MN (CBMN) assay using peripheral blood lymphocytes and the Buccal MN assay that measures MN in epithelial cells from the oral mucosa. These discoveries greatly increased interest to use MN assays in human studies. In 1997 the Human Micronucleus (HUMN) project was founded to initiate an international collaboration to (i) harmonise and standardise the techniques used to perform the lymphocyte CBMN assay and the Buccal MN assay; (ii) establish and collate databases of MN frequency in human populations world-wide which also captured demographic, lifestyle and environmental genotoxin exposure data and (iii) use these data to identify the most important variables affecting MN frequency and to also determine whether MN predict disease risk. In this paper we briefly describe the achievements of the HUMN project during the period from the date of its foundation on 9th September 1997 until its 26th Anniversary in 2023, which included more than 200 publications and 23 workshops world-wide.
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Affiliation(s)
- Michael Fenech
- Health and Biomedical Innovation, UniSA Clinical and Health Sciences, University of South Australia, Adelaide 5000, Australia; Genome Health Foundation, North Brighton, SA 5048, Australia.
| | - Nina Holland
- Center for Environmental Research and Community Health (CERCH), University of California, Berkeley, Berkeley, CA, USA.
| | | | - Peter Wushou Chang
- Show Chwan Memorial Hospital, Changhwa, Taiwan; TUFTS University Medical School, Boston, USA.
| | - Micheline Kirsch-Volders
- Laboratory for Cell Genetics, Department Biology, Faculty of Sciences and Bio-engineering Sciences, Vrije Universiteit Brussel, Pleinlaan 2, Brussels 1050, Belgium.
| | - Claudia Bolognesi
- Environmental Carcinogenesis Unit, Ospedale Policlinico San Martino, Genoa, Italy.
| | - Helga Stopper
- Institute of Pharmacology and Toxicology, University of Würzburg, Würzburg 97080, Germany.
| | - Lisbeth E Knudsen
- Department of Public Health, Section of Environmental Health, University of Copenhagen, Copenhagen, Denmark.
| | - Siegfried Knasmueller
- Institute of Cancer Research, Department of Medicine I, Medical University of Vienna, Vienna, Austria.
| | - Armen Nersesyan
- Institute of Cancer Research, Department of Medicine I, Medical University of Vienna, Vienna, Austria.
| | - Philip Thomas
- CSIRO Health and Biosecurity, Adelaide 5000, Australia.
| | - Varinderpal Dhillon
- Health and Biomedical Innovation, UniSA Clinical and Health Sciences, University of South Australia, Adelaide 5000, Australia.
| | - Permal Deo
- Health and Biomedical Innovation, UniSA Clinical and Health Sciences, University of South Australia, Adelaide 5000, Australia.
| | - Bernhard Franzke
- Department of Nutritional Sciences, University of Vienna, Austria.
| | | | - Blanca Laffon
- Universidade da Coruña, Grupo DICOMOSA, CICA-Centro Interdisciplinar de Química e Bioloxía, Departamento de Psicología, Facultad de Ciencias de la Educación, and Instituto de Investigación Biomédica de A Coruña (INIBIC), Complexo Hospitalario Universitario de A Coruña (CHUAC), Sergas, A Coruña, Spain.
| | - Karl-Heinz Wagner
- Department of Nutritional Sciences, University of Vienna, Austria; Research Platform Active Ageing, University of Vienna, Austria.
| | - Hannu Norppa
- Finnish Institute of Occupational Health, Helsinki 00250, Finland.
| | - Juliana da Silva
- Laboratory of Genetic Toxicology, La Salle University (UniLaSalle), Canoas, RS 92010-000, Brazil; PPGBM, Federal University of Brazil (UFRGS), Porto Alegre 91501-970, Brazil.
| | - Emanuela V Volpi
- School of Life Sciences, University of Westminster, 115 New Cavendish Street, London W1W6UW, UK.
| | - Ruth Wilkins
- Environmental and Radiation Health Sciences Directorate, Health Canada 775 Brookfield Rd, Ottawa K1A 1C1, Canada.
| | - Stefano Bonassi
- Clinical and Molecular Epidemiology, IRCCS San Raffaele Roma, Rome 00166, Italy.
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Lee YH, Yoon HJ, Yang SS, Lee IK, Jo WS, Jeong SK, Oh SJ, Kim J, Lee Y, Seong KM. Lessons on harmonization of scoring criteria for dicentric chromosome assay in South Korea. Int J Radiat Biol 2024; 100:709-714. [PMID: 38394348 DOI: 10.1080/09553002.2024.2316603] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2023] [Accepted: 01/23/2024] [Indexed: 02/25/2024]
Abstract
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.
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Affiliation(s)
- Yang Hee Lee
- Laboratory of Biological Dosimetry, National Radiation Emergency Medical Center (NREMC), Korea Institute of Radiological & Medical Sciences (KIRAMS), Seoul, Republic of Korea
| | - Hyo Jin Yoon
- Laboratory of Biological Dosimetry, National Radiation Emergency Medical Center (NREMC), Korea Institute of Radiological & Medical Sciences (KIRAMS), Seoul, Republic of Korea
| | - Su San Yang
- Laboratory of Biological Dosimetry, National Radiation Emergency Medical Center (NREMC), Korea Institute of Radiological & Medical Sciences (KIRAMS), Seoul, Republic of Korea
| | - In Kyung Lee
- Korea Hydro & Nuclear Power Co, Radiation Health Institute, Seoul, Republic of Korea
| | - Wol Soon Jo
- Research Center, Dong Nam Institute of Radiological and Medical Sciences (DIRAMS), Busan, Republic of Korea
| | - Soo Kyung Jeong
- Research Center, Dong Nam Institute of Radiological and Medical Sciences (DIRAMS), Busan, Republic of Korea
| | - Su Jung Oh
- Research Center, Dong Nam Institute of Radiological and Medical Sciences (DIRAMS), Busan, Republic of Korea
| | - Jiin Kim
- Department of Medical Sciences, Graduate School, Soonchunhyang University, Asan, Republic of Korea
| | - Younghyun Lee
- Department of Medical Sciences, Graduate School, Soonchunhyang University, Asan, Republic of Korea
- Department of Biomedical Laboratory Science, College of Medical Sciences, Soonchunhyang University, Asan, Republic of Korea
| | - Ki Moon Seong
- Laboratory of Biological Dosimetry, National Radiation Emergency Medical Center (NREMC), Korea Institute of Radiological & Medical Sciences (KIRAMS), Seoul, Republic of Korea
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Wilkins RC, Beaton-Green LA. Development of high-throughput systems for biodosimetry. RADIATION PROTECTION DOSIMETRY 2023; 199:1477-1484. [PMID: 37721060 PMCID: PMC10720693 DOI: 10.1093/rpd/ncad060] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/29/2022] [Revised: 01/24/2023] [Accepted: 02/08/2023] [Indexed: 09/19/2023]
Abstract
Biomarkers for ionising radiation exposure have great utility in scenarios where there has been a potential exposure and physical dosimetry is missing or in dispute, such as for occupational and accidental exposures. Biomarkers that respond as a function of dose are particularly useful as biodosemeters to determine the dose of radiation to which an individual has been exposed. These dose measurements can also be used in medical scenarios to track doses from medical exposures and even have the potential to identify an individual's response to radiation exposure that could help tailor treatments. The measurement of biomarkers of exposure in medicine and for accidents, where a larger number of samples would be required, is limited by the throughput of analysis (i.e. the number of samples that could be processed and analysed), particularly for microscope-based methods, which tend to be labour-intensive. Rapid analysis in an emergency scenario, such as a large-scale accident, would provide dose estimates to medical practitioners, allowing timely administration of the appropriate medical countermeasures to help mitigate the effects of radiation exposure. In order to improve sample throughput for biomarker analysis, much effort has been devoted to automating the process from sample preparation through automated image analysis. This paper will focus mainly on biological endpoints traditionally analysed by microscopy, specifically dicentric chromosomes, micronuclei and gamma-H2AX. These endpoints provide examples where sample throughput has been improved through automated image acquisition, analysis of images acquired by microscopy, as well as methods that have been developed for analysis using imaging flow cytometry.
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Affiliation(s)
- Ruth C Wilkins
- Consumer and Clinical Radiation Protection Bureau, Health Canada, Ottawa K1A 1C1, Canada
| | - Lindsay A Beaton-Green
- Consumer and Clinical Radiation Protection Bureau, Health Canada, Ottawa K1A 1C1, Canada
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Schunck C, Lörch T, Kowalski R, Porter M, Mahnke R, Capaccio C, Perrier J, Damer K. A standardized and automated CBMN assay for biological dosimetry: the CytoRADx™ system. RADIATION PROTECTION DOSIMETRY 2023; 199:1516-1519. [PMID: 37721072 DOI: 10.1093/rpd/ncad027] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/29/2022] [Revised: 12/06/2022] [Accepted: 01/25/2023] [Indexed: 09/19/2023]
Abstract
Major nuclear accidents can result in many casualties, and it is important to assess the absorbed radiation dose to support treatment decisions. Biological dosimetry (BD) allows retrospective determination of dose using biological markers. To achieve consistent cytogenetic assay results across labs, the current practice requires each lab to generate periodic, unique calibration curves using in vitro dose-effect experiments. Here, we present CytoRADx™, a standardized biodosimetry system that integrates automated dose calculation in a high-throughput platform without the need for lab-specific calibration curves. CytoRADx consists of an improved, standardized Cytokinesis Block Micronucleus assay combined with automated analysis utilizing an established slide scanning device. We tested CytoRADx for accuracy and reproducibility across different instruments, sites, days and operators. Our results demonstrate that CytoRADx eliminates the time-consuming, lab-specific calibration curves, allowing multiple laboratories to obtain consistent results and to distribute the testing burden in the event of a large-scale accident.
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Affiliation(s)
| | - Thomas Lörch
- MetaSystems Hard & Software GmbH, Altlussheim, Germany
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Shirley BC, Mucaki EJ, Knoll JHM, Rogan PK. Radiation exposure determination in a secure, cloud-based online environment. RADIATION PROTECTION DOSIMETRY 2023; 199:1465-1471. [PMID: 37721084 DOI: 10.1093/rpd/ncac266] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/16/2022] [Revised: 10/19/2022] [Accepted: 11/16/2022] [Indexed: 09/19/2023]
Abstract
Rapid sample processing and interpretation of estimated exposures will be critical for triaging exposed individuals after a major radiation incident. The dicentric chromosome (DC) assay assesses absorbed radiation using metaphase cells from blood. The Automated Dicentric Chromosome Identifier and Dose Estimator System (ADCI) identifies DCs and determines radiation doses. This study aimed to broaden accessibility and speed of this system, while protecting data and software integrity. ADCI Online is a secure web-streaming platform accessible worldwide from local servers. Cloud-based systems containing data and software are separated until they are linked for radiation exposure estimation. Dose estimates are identical to ADCI on dedicated computer hardware. Image processing and selection, calibration curve generation, and dose estimation of 9 test samples completed in < 2 days. ADCI Online has the capacity to alleviate analytic bottlenecks in intermediate-to-large radiation incidents. Multiple cloned software instances configured on different cloud environments accelerated dose estimation to within clinically relevant time frames.
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Affiliation(s)
- Ben C Shirley
- CytoGnomix Inc., 60 N. Centre Rd., London Ontario N5X 3X5, Canada
| | - Eliseos J Mucaki
- Dept. Biochemistry, University of Western Ontario, London Ontario N6A 3K7, Canada
| | - Joan H M Knoll
- CytoGnomix Inc., 60 N. Centre Rd., London Ontario N5X 3X5, Canada
- Dept. Pathology and Laboratory Medicine, University of Western Ontario, London Ontario N6A 3K7, Canada
| | - Peter K Rogan
- CytoGnomix Inc., 60 N. Centre Rd., London Ontario N5X 3X5, Canada
- Dept. Biochemistry, University of Western Ontario, London Ontario N6A 3K7, Canada
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Bertucci A, Wilkins RC, Lachapelle S, Turner HC, Brenner DJ, Garty G. Comparison of Isolated Lymphocyte and Whole Blood-Based CBMN Assays for Radiation Triage. Cytogenet Genome Res 2023; 163:110-120. [PMID: 37573770 PMCID: PMC10859551 DOI: 10.1159/000533488] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2023] [Accepted: 08/06/2023] [Indexed: 08/15/2023] Open
Abstract
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.
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Affiliation(s)
- Antonella Bertucci
- Center for Radiological Research, Columbia University, New York, NY, USA
- Currently at: Loma Linda University Medical Center, Loma Linda, CA, USA
| | - Ruth C. Wilkins
- Consumer and Clinical Radiation Protection Bureau, Health Canada, Ottawa, ON, Canada
| | - Sylvie Lachapelle
- Consumer and Clinical Radiation Protection Bureau, Health Canada, Ottawa, ON, Canada
| | - Helen C. Turner
- Center for Radiological Research, Columbia University, New York, NY, USA
| | - David J. Brenner
- Center for Radiological Research, Columbia University, New York, NY, USA
| | - Guy Garty
- Center for Radiological Research, Columbia University, New York, NY, USA
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Beaton-Green LA, Mayenburg JM, Marro L, Hassan EM, Cuadros Sanchez S, Darwish R, Lachapelle S, Adam N, Burtt JJ, Van Den Hanenberg C, Rodrigues MA, Wang Q, Brenner DJ, Turner HC, Wilkins RC. Application of the Cytokinesis-Block Micronucleus Assay for High-Dose Exposures Using Imaging Flow Cytometry. Cytogenet Genome Res 2023; 163:131-142. [PMID: 37527635 DOI: 10.1159/000532124] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2023] [Accepted: 07/20/2023] [Indexed: 08/03/2023] Open
Abstract
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.
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Affiliation(s)
- Lindsay A Beaton-Green
- Environmental and Radiation Health Sciences Directorate, Health Canada, Ottawa, Ontario, Canada
| | - Jessica M Mayenburg
- Environmental and Radiation Health Sciences Directorate, Health Canada, Ottawa, Ontario, Canada
| | - Leonora Marro
- Environmental and Radiation Health Sciences Directorate, Health Canada, Ottawa, Ontario, Canada
| | - Eman M Hassan
- Environmental and Radiation Health Sciences Directorate, Health Canada, Ottawa, Ontario, Canada
| | - Sarita Cuadros Sanchez
- Environmental and Radiation Health Sciences Directorate, Health Canada, Ottawa, Ontario, Canada
| | - Riham Darwish
- Environmental and Radiation Health Sciences Directorate, Health Canada, Ottawa, Ontario, Canada
| | - Sylvie Lachapelle
- Environmental and Radiation Health Sciences Directorate, Health Canada, Ottawa, Ontario, Canada
| | - Nadine Adam
- Environmental and Radiation Health Sciences Directorate, Health Canada, Ottawa, Ontario, Canada
| | - Julie J Burtt
- Environmental and Radiation Health Sciences Directorate, Health Canada, Ottawa, Ontario, Canada
| | - Cyndi Van Den Hanenberg
- Environmental and Radiation Health Sciences Directorate, Health Canada, Ottawa, Ontario, Canada
| | | | - Qi Wang
- Center for Radiological Research, Columbia University Medical Center, New York, New York, USA
| | - David J Brenner
- Center for Radiological Research, Columbia University Medical Center, New York, New York, USA
| | - Helen C Turner
- Center for Radiological Research, Columbia University Medical Center, New York, New York, USA
| | - Ruth C Wilkins
- Environmental and Radiation Health Sciences Directorate, Health Canada, Ottawa, Ontario, Canada
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9
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Port M, Barquinero JF, Endesfelder D, Moquet J, Oestreicher U, Terzoudi G, Trompier F, Vral A, Abe Y, Ainsbury L, Alkebsi L, Amundson S, Badie C, Baeyens A, Balajee A, Balázs K, Barnard S, Bassinet C, Beaton-Green L, Beinke C, Bobyk L, Brochard P, Brzoska K, Bucher M, Ciesielski B, Cuceu C, Discher M, D,Oca M, Domínguez I, Doucha-Senf S, Dumitrescu A, Duy P, Finot F, Garty G, Ghandhi S, Gregoire E, Goh V, Güçlü I, Hadjiiska L, Hargitai R, Hristova R, Ishii K, Kis E, Juniewicz M, Kriehuber R, Lacombe J, Lee Y, Lopez Riego M, Lumniczky K, Mai T, Maltar-Strmečki N, Marrale M, Martinez J, Marciniak A, Maznyk N, McKeever S, Meher P, Milanova M, Miura T, Gil OM, Montoro A, Domene MM, Mrozik A, Nakayama R, O’Brien G, Oskamp D, Ostheim P, Pajic J, Pastor N, Patrono C, Pujol-Canadell M, Rodriguez MP, Repin M, Romanyukha A, Rößler U, Sabatier L, Sakai A, Scherthan H, Schüle S, Seong K, Sevriukova O, Sholom S, Sommer S, Suto Y, Sypko T, Szatmári T, Takahashi-Sugai M, Takebayashi K, Testa A, Testard I, Tichy A, Triantopoulou S, Tsuyama N, Unverricht-Yeboah M, Valente M, Van Hoey O, Wilkins R, Wojcik A, Wojewodzka M, Younghyun L, et alPort M, Barquinero JF, Endesfelder D, Moquet J, Oestreicher U, Terzoudi G, Trompier F, Vral A, Abe Y, Ainsbury L, Alkebsi L, Amundson S, Badie C, Baeyens A, Balajee A, Balázs K, Barnard S, Bassinet C, Beaton-Green L, Beinke C, Bobyk L, Brochard P, Brzoska K, Bucher M, Ciesielski B, Cuceu C, Discher M, D,Oca M, Domínguez I, Doucha-Senf S, Dumitrescu A, Duy P, Finot F, Garty G, Ghandhi S, Gregoire E, Goh V, Güçlü I, Hadjiiska L, Hargitai R, Hristova R, Ishii K, Kis E, Juniewicz M, Kriehuber R, Lacombe J, Lee Y, Lopez Riego M, Lumniczky K, Mai T, Maltar-Strmečki N, Marrale M, Martinez J, Marciniak A, Maznyk N, McKeever S, Meher P, Milanova M, Miura T, Gil OM, Montoro A, Domene MM, Mrozik A, Nakayama R, O’Brien G, Oskamp D, Ostheim P, Pajic J, Pastor N, Patrono C, Pujol-Canadell M, Rodriguez MP, Repin M, Romanyukha A, Rößler U, Sabatier L, Sakai A, Scherthan H, Schüle S, Seong K, Sevriukova O, Sholom S, Sommer S, Suto Y, Sypko T, Szatmári T, Takahashi-Sugai M, Takebayashi K, Testa A, Testard I, Tichy A, Triantopoulou S, Tsuyama N, Unverricht-Yeboah M, Valente M, Van Hoey O, Wilkins R, Wojcik A, Wojewodzka M, Younghyun L, Zafiropoulos D, Abend M. RENEB Inter-Laboratory Comparison 2021: Inter-Assay Comparison of Eight Dosimetry Assays. Radiat Res 2023; 199:535-555. [PMID: 37310880 PMCID: PMC10508307 DOI: 10.1667/rade-22-00207.1] [Show More Authors] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2022] [Accepted: 01/10/2023] [Indexed: 06/15/2023]
Abstract
Tools for radiation exposure reconstruction are required to support the medical management of radiation victims in radiological or nuclear incidents. Different biological and physical dosimetry assays can be used for various exposure scenarios to estimate the dose of ionizing radiation a person has absorbed. Regular validation of the techniques through inter-laboratory comparisons (ILC) is essential to guarantee high quality results. In the current RENEB inter-laboratory comparison, the performance quality of established cytogenetic assays [dicentric chromosome assay (DCA), cytokinesis-block micronucleus assay (CBMN), stable chromosomal translocation assay (FISH) and premature chromosome condensation assay (PCC)] was tested in comparison to molecular biological assays [gamma-H2AX foci (gH2AX), gene expression (GE)] and physical dosimetry-based assays [electron paramagnetic resonance (EPR), optically or thermally stimulated luminescence (LUM)]. Three blinded coded samples (e.g., blood, enamel or mobiles) were exposed to 0, 1.2 or 3.5 Gy X-ray reference doses (240 kVp, 1 Gy/min). These doses roughly correspond to clinically relevant groups of unexposed to low exposed (0-1 Gy), moderately exposed (1-2 Gy, no severe acute health effects expected) and highly exposed individuals (>2 Gy, requiring early intensive medical care). In the frame of the current RENEB inter-laboratory comparison, samples were sent to 86 specialized teams in 46 organizations from 27 nations for dose estimation and identification of three clinically relevant groups. The time for sending early crude reports and more precise reports was documented for each laboratory and assay where possible. The quality of dose estimates was analyzed with three different levels of granularity, 1. by calculating the frequency of correctly reported clinically relevant dose categories, 2. by determining the number of dose estimates within the uncertainty intervals recommended for triage dosimetry (±0.5 Gy or ±1.0 Gy for doses <2.5 Gy or >2.5 Gy), and 3. by calculating the absolute difference (AD) of estimated doses relative to the reference doses. In total, 554 dose estimates were submitted within the 6-week period given before the exercise was closed. For samples processed with the highest priority, earliest dose estimates/categories were reported within 5-10 h of receipt for GE, gH2AX, LUM, EPR, 2-3 days for DCA, CBMN and within 6-7 days for the FISH assay. For the unirradiated control sample, the categorization in the correct clinically relevant group (0-1 Gy) as well as the allocation to the triage uncertainty interval was, with the exception of a few outliers, successfully performed for all assays. For the 3.5 Gy sample the percentage of correct classifications to the clinically relevant group (≥2 Gy) was between 89-100% for all assays, with the exception of gH2AX. For the 1.2 Gy sample, an exact allocation to the clinically relevant group was more difficult and 0-50% or 0-48% of the estimates were wrongly classified into the lowest or highest dose categories, respectively. For the irradiated samples, the correct allocation to the triage uncertainty intervals varied considerably between assays for the 1.2 Gy (29-76%) and 3.5 Gy (17-100%) samples. While a systematic shift towards higher doses was observed for the cytogenetic-based assays, extreme outliers exceeding the reference doses 2-6 fold were observed for EPR, FISH and GE assays. These outliers were related to a particular material examined (tooth enamel for EPR assay, reported as kerma in enamel, but when converted into the proper quantity, i.e. to kerma in air, expected dose estimates could be recalculated in most cases), the level of experience of the teams (FISH) and methodological uncertainties (GE). This was the first RENEB ILC where everything, from blood sampling to irradiation and shipment of the samples, was organized and realized at the same institution, for several biological and physical retrospective dosimetry assays. Almost all assays appeared comparably applicable for the identification of unexposed and highly exposed individuals and the allocation of medical relevant groups, with the latter requiring medical support for the acute radiation scenario simulated in this exercise. However, extreme outliers or a systematic shift of dose estimates have been observed for some assays. Possible reasons will be discussed in the assay specific papers of this special issue. In summary, this ILC clearly demonstrates the need to conduct regular exercises to identify research needs, but also to identify technical problems and to optimize the design of future ILCs.
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Affiliation(s)
- M. Port
- Bundeswehr Institute of Radiobiology, Munich, Germany
| | | | | | - J. Moquet
- UK Health Security Agency, Radiation, Chemical and Environmental Hazards Division, Oxfordshire, United Kingdom
| | | | - G. Terzoudi
- National Centre for Scientific Research “Demokritos”, Health Physics, Radiobiology & Cytogenetics Laboratory, Agia Paraskevi, Greece
| | - F. Trompier
- Institut de Radioprotection et de Surete Nucleaire, Fontenay aux Roses, France
| | - A. Vral
- Ghent University, Radiobiology Research Unit, Gent, Belgium
| | - Y. Abe
- Department of Radiation Biology and Protection, Nagasaki University, Japan
| | - L. Ainsbury
- UK Health Security Agency and Office for Health Improvement and Disparities, Cytogenetics and Pathology Group, Oxfordshire, England
| | - L Alkebsi
- Department of Radiation Measurement and Dose Assessment, National Institute of Radiological Sciences, National Institutes for Quantum Science and Technology, Chiba, Japan
| | - S.A. Amundson
- Columbia University, Irving Medical Center, Center for Radiological Research, New York, New York
| | - C. Badie
- UK Health Security Agency, Radiation, Chemical and Environmental Hazards Division, Oxfordshire, United Kingdom
| | - A. Baeyens
- Ghent University, Radiobiology Research Unit, Gent, Belgium
| | - A.S. Balajee
- Cytogenetic Biodosimetry Laboratory, Oak Ridge Institute for Science and Education, Oak Ridge, Tennessee
| | - K. Balázs
- Radiation Medicine Unit, Department of Radiobiology and Radiohygiene, National Public Health Centre, Budapest, Hungary
| | - S. Barnard
- UK Health Security Agency, Radiation, Chemical and Environmental Hazards Division, Oxfordshire, United Kingdom
| | - C. Bassinet
- Institut de Radioprotection et de Surete Nucleaire, Fontenay aux Roses, France
| | | | - C. Beinke
- Bundeswehr Institute of Radiobiology, Munich, Germany
| | - L. Bobyk
- Institut de Recherche Biomédicale des Armées (IRBA), Bretigny Sur Orge, France
| | | | - K. Brzoska
- Institute of Nuclear Chemistry and Technology, Warsaw, Poland
| | - M. Bucher
- Bundesamt für Strahlenschutz, Oberschleißheim, Germany
| | - B. Ciesielski
- Medical University of Gdansk, Department of Physics and Biophysics, Gdansk, Poland
| | - C. Cuceu
- Genevolution, Porcheville, France
| | - M. Discher
- Paris-Lodron-University of Salzburg, Department of Environment and Biodiversity, 5020 Salzburg, Austria
| | - M.C. D,Oca
- Università Degli Studi di Palermo, Dipartimento di Fisica e Chimica “Emilio Segrè,” Palermo, Italy
| | - I. Domínguez
- Universidad de Sevilla, Departamento de Biología Celular, Sevilla, Spain
| | | | - A. Dumitrescu
- National Institute of Public Health, Radiation Hygiene Laboratory, Bucharest, Romania
| | - P.N. Duy
- Dalat Nuclear Research Institute, Radiation Technlogy & Biotechnology Center, Dalat City, Vietnam
| | - F. Finot
- Genevolution, Porcheville, France
| | - G. Garty
- Columbia University, Irving Medical Center, Center for Radiological Research, New York, New York
| | - S.A. Ghandhi
- Columbia University, Irving Medical Center, Center for Radiological Research, New York, New York
| | - E. Gregoire
- Institut de Radioprotection et de Surete Nucleaire, Fontenay aux Roses, France
| | - V.S.T. Goh
- Department of Radiobiology, Singapore Nuclear Research and Safety Initiative (SNRSI), National University of Singapore, Singapore
| | - I. Güçlü
- TENMAK, Nuclear Energy Research Institute, Technology Development and Nuclear Research Department, Türkey
| | - L. Hadjiiska
- National Centre of Radiobiology and Radiation Protection, Sofia, Bulgaria
| | - R. Hargitai
- Radiation Medicine Unit, Department of Radiobiology and Radiohygiene, National Public Health Centre, Budapest, Hungary
| | - R. Hristova
- National Centre of Radiobiology and Radiation Protection, Sofia, Bulgaria
| | - K. Ishii
- Department of Radiation Measurement and Dose Assessment, National Institute of Radiological Sciences, National Institutes for Quantum Science and Technology, Chiba, Japan
| | - E. Kis
- Radiation Medicine Unit, Department of Radiobiology and Radiohygiene, National Public Health Centre, Budapest, Hungary
| | - M. Juniewicz
- Medical University of Gdansk, Department of Physics and Biophysics, Gdansk, Poland
| | - R. Kriehuber
- Department of Safety and Radiation Protection, Forschungszentrum Jülich, Jülich, Germany
| | - J. Lacombe
- University of Arizona, Center for Applied Nanobioscience & Medicine, Phoenix, Arizona
| | - Y. Lee
- Laboratory of Biological Dosimetry, Korea Institute of Radiological & Medical Sciences, Seoul, Republic of Korea
| | | | - K. Lumniczky
- Radiation Medicine Unit, Department of Radiobiology and Radiohygiene, National Public Health Centre, Budapest, Hungary
| | - T.T. Mai
- Dalat Nuclear Research Institute, Radiation Technlogy & Biotechnology Center, Dalat City, Vietnam
| | - N. Maltar-Strmečki
- Ruðer Boškovic Institute, Division of Physical Chemistry, Zagreb, Croatia
| | - M. Marrale
- Università Degli Studi di Palermo, Dipartimento di Fisica e Chimica “Emilio Segrè,” Palermo, Italy
| | - J.S. Martinez
- Institut de Radioprotection et de Surete Nucleaire, Fontenay aux Roses, France
| | - A. Marciniak
- Medical University of Gdansk, Department of Physics and Biophysics, Gdansk, Poland
| | - N. Maznyk
- Radiation Cytogenetics Laboratory, S.P. Grigoriev Institute for Medical Radiology and Oncology of Ukrainian National Academy of Medical Science, Kharkiv, Ukraine
| | - S.W.S. McKeever
- Radiation Dosimetry Laboratory, Oklahoma State University, Stillwater, Oklahoma
| | | | - M. Milanova
- University of Defense, Faculty of Military Health Sciences, Hradec Králové, Czech Republic
| | - T. Miura
- Institute of Radiation Emergency Medicine, Hirosaki University, Hirosaki, Japan
| | - O. Monteiro Gil
- Instituto Superior Técnico/ Campus Tecnológico e Nuclear, Lisbon, Portugal
| | - A. Montoro
- Servicio de Protección Radiológica. Laboratorio de Dosimetría Biológica, Valencia, Spain
| | - M. Moreno Domene
- Hospital General Universitario Gregorio Marañón, Laboratorio de dosimetría biológica, Madrid, Spain
| | - A. Mrozik
- Institute of Nuclear Physics, Polish Academy of Sciences, Krakow, Poland
| | - R. Nakayama
- Institute of Radiation Emergency Medicine, Hirosaki University, Hirosaki, Japan
| | - G. O’Brien
- UK Health Security Agency, Radiation, Chemical and Environmental Hazards Division, Oxfordshire, United Kingdom
| | - D. Oskamp
- Department of Safety and Radiation Protection, Forschungszentrum Jülich, Jülich, Germany
| | - P. Ostheim
- Bundeswehr Institute of Radiobiology, Munich, Germany
| | - J. Pajic
- Serbian Institute of Occupational Health, Belgrade, Serbia
| | - N. Pastor
- Universidad de Sevilla, Departamento de Biología Celular, Sevilla, Spain
| | - C. Patrono
- Italian National Agency for New Technologies, Energy and Sustainable Economic Development, Rome, Italy
| | | | - M.J. Prieto Rodriguez
- Hospital General Universitario Gregorio Marañón, Laboratorio de dosimetría biológica, Madrid, Spain
| | - M. Repin
- Columbia University, Irving Medical Center, Center for Radiological Research, New York, New York
| | | | - U. Rößler
- Bundesamt für Strahlenschutz, Oberschleißheim, Germany
| | | | - A. Sakai
- Department of Radiation Life Sciences, Fukushima Medical University School of Medicine, Fukushima, Japan
| | - H. Scherthan
- Bundeswehr Institute of Radiobiology, Munich, Germany
| | - S. Schüle
- Bundeswehr Institute of Radiobiology, Munich, Germany
| | - K.M. Seong
- Laboratory of Biological Dosimetry, Korea Institute of Radiological & Medical Sciences, Seoul, Republic of Korea
| | | | - S. Sholom
- Radiation Dosimetry Laboratory, Oklahoma State University, Stillwater, Oklahoma
| | - S. Sommer
- Institute of Nuclear Chemistry and Technology, Warsaw, Poland
| | - Y. Suto
- Department of Radiation Measurement and Dose Assessment, National Institute of Radiological Sciences, National Institutes for Quantum Science and Technology, Chiba, Japan
| | - T. Sypko
- Radiation Cytogenetics Laboratory, S.P. Grigoriev Institute for Medical Radiology and Oncology of Ukrainian National Academy of Medical Science, Kharkiv, Ukraine
| | - T. Szatmári
- Radiation Medicine Unit, Department of Radiobiology and Radiohygiene, National Public Health Centre, Budapest, Hungary
| | - M. Takahashi-Sugai
- Department of Radiation Life Sciences, Fukushima Medical University School of Medicine, Fukushima, Japan
| | - K. Takebayashi
- Institute of Radiation Emergency Medicine, Hirosaki University, Hirosaki, Japan
| | - A. Testa
- Italian National Agency for New Technologies, Energy and Sustainable Economic Development, Rome, Italy
| | - I. Testard
- CEA-Saclay, Gif-sur-Yvette Cedex, France
| | - A. Tichy
- University of Defense, Faculty of Military Health Sciences, Hradec Králové, Czech Republic
| | - S. Triantopoulou
- National Centre for Scientific Research “Demokritos”, Health Physics, Radiobiology & Cytogenetics Laboratory, Agia Paraskevi, Greece
| | - N. Tsuyama
- Department of Radiation Life Sciences, Fukushima Medical University School of Medicine, Fukushima, Japan
| | - M. Unverricht-Yeboah
- Department of Safety and Radiation Protection, Forschungszentrum Jülich, Jülich, Germany
| | - M. Valente
- CEA-Saclay, Gif-sur-Yvette Cedex, France
| | - O. Van Hoey
- Belgian Nuclear Research Center SCK CEN, Mol, Belgium
| | | | - A. Wojcik
- Stockholm University, Stockholm, Sweden
| | - M. Wojewodzka
- Institute of Nuclear Chemistry and Technology, Warsaw, Poland
| | - Lee Younghyun
- Laboratory of Biological Dosimetry, Korea Institute of Radiological & Medical Sciences, Seoul, Republic of Korea
| | - D. Zafiropoulos
- Laboratori Nazionali di Legnaro - Istituto Nazionale di Fisica Nucleare, Legnaro, Italy
| | - M. Abend
- Bundeswehr Institute of Radiobiology, Munich, Germany
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10
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M’Kacher R, Colicchio B, Junker S, El Maalouf E, Heidingsfelder L, Plesch A, Dieterlen A, Jeandidier E, Carde P, Voisin P. High Resolution and Automatable Cytogenetic Biodosimetry Using In Situ Telomere and Centromere Hybridization for the Accurate Detection of DNA Damage: An Overview. Int J Mol Sci 2023; 24:ijms24065699. [PMID: 36982772 PMCID: PMC10054499 DOI: 10.3390/ijms24065699] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2022] [Revised: 03/07/2023] [Accepted: 03/10/2023] [Indexed: 03/19/2023] Open
Abstract
In the event of a radiological or nuclear accident, or when physical dosimetry is not available, the scoring of radiation-induced chromosomal aberrations in lymphocytes constitutes an essential tool for the estimation of the absorbed dose of the exposed individual and for effective triage. Cytogenetic biodosimetry employs different cytogenetic assays including the scoring of dicentrics, micronuclei, and translocations as well as analyses of induced premature chromosome condensation to define the frequency of chromosome aberrations. However, inherent challenges using these techniques include the considerable time span from sampling to result, the sensitivity and specificity of the various techniques, and the requirement of highly skilled personnel. Thus, techniques that obviate these challenges are needed. The introduction of telomere and centromere (TC) staining have successfully met these challenges and, in addition, greatly improved the efficiency of cytogenetic biodosimetry through the development of automated approaches, thus reducing the need for specialized personnel. Here, we review the role of the various cytogenetic dosimeters and their recent improvements in the management of populations exposed to genotoxic agents such as ionizing radiation. Finally, we discuss the emerging potentials to exploit these techniques in a wider spectrum of medical and biological applications, e.g., in cancer biology to identify prognostic biomarkers for the optimal triage and treatment of patients.
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Affiliation(s)
- Radhia M’Kacher
- Cell Environment DNA Damage R&D, Genopole, 91000 Evry-Courcouronnes, France
- Correspondence: ; Tel.: +33-160878918
| | - Bruno Colicchio
- IRIMAS, Institut de Recherche en Informatique, Mathématiques, Automatique et Signal, Université de Haute-Alsace, 69093 Mulhouse, France
| | - Steffen Junker
- Institute of Biomedicine, University of Aarhus, DK-8000 Aarhus, Denmark
| | - Elie El Maalouf
- Cell Environment DNA Damage R&D, Genopole, 91000 Evry-Courcouronnes, France
| | | | - Andreas Plesch
- MetaSystems GmbH, Robert-Bosch-Str. 6, D-68804 Altlussheim, Germany
| | - Alain Dieterlen
- IRIMAS, Institut de Recherche en Informatique, Mathématiques, Automatique et Signal, Université de Haute-Alsace, 69093 Mulhouse, France
| | - Eric Jeandidier
- Laboratoire de Génétique, Groupe Hospitalier de la Région de Mulhouse Sud-Alsace, 69093 Mulhouse, France
| | - Patrice Carde
- Department of Hematology, Institut Gustave Roussy, 94804 Villejuif, France
| | - Philippe Voisin
- Cell Environment DNA Damage R&D, Genopole, 91000 Evry-Courcouronnes, France
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11
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Shen X, Ma T, Li C, Wen Z, Zheng J, Zhou Z. High-precision automatic identification method for dicentric chromosome images using two-stage convolutional neural network. Sci Rep 2023; 13:2124. [PMID: 36746997 PMCID: PMC9902391 DOI: 10.1038/s41598-023-28456-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2022] [Accepted: 01/18/2023] [Indexed: 02/08/2023] Open
Abstract
Dicentric chromosome analysis is the gold standard for biological dose assessment. To enhance the efficiency of biological dose assessment in large-scale radiation catastrophes, automatic identification of dicentric chromosome images is a promising and objective method. In this paper, an automatic identification method for dicentric chromosome images using two-stage convolutional neural network is proposed based on Giemsa-stained automatic microscopic imaging. To automatically segment the adhesive chromosome masses, a k-means based adaptive image segmentation and watershed segmentation algorithm is applied. The first-stage CNN is used to identify the dicentric chromosome images from all the images and the second-stage CNN works to specifically identify the dicentric chromosome images. This two-stage CNN identification method can effectively detects chromosome images with concealed centromeres, poorly expanded and long-armed entangled chromosomes, and tricentric chromosomes. The novel two-stage CNN method has a chromosome identification accuracy of 99.4%, a sensitivity of 85.8% sensitivity, and a specificity of 99.6%, effectively reducing the false positive rate of dicentric chromosome. The analysis speed of this automatic identification method can be 20 times quicker than manual detection, providing a valuable reference for other image identification situations with small target rates.
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Affiliation(s)
- Xiang Shen
- School of Mechanical Engineering and Automation, Beihang University, Beijing, 100083, China
| | - Tengfei Ma
- School of Mechanical Engineering and Automation, Beihang University, Beijing, 100083, China
| | - Chaowen Li
- Beijing Huironghe Technology Co., Ltd., Beijing, 101102, China
| | - Zhanbo Wen
- Beijing Huironghe Technology Co., Ltd., Beijing, 101102, China
| | - Jinlin Zheng
- Beijing Huironghe Technology Co., Ltd., Beijing, 101102, China
| | - Zhenggan Zhou
- School of Mechanical Engineering and Automation, Beihang University, Beijing, 100083, China. .,Ningbo Institute of Technology, Beihang University, Ningbo, 315800, China.
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12
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Lee Y, Kim SH, Lee YH, Yang SS, Yoon HJ, Wilkins RC, Jang S. Application of a semi-automated dicentric scoring system in triage and monitoring occupational radiation exposure. Front Public Health 2022; 10:1002501. [PMID: 36339161 PMCID: PMC9631783 DOI: 10.3389/fpubh.2022.1002501] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2022] [Accepted: 10/06/2022] [Indexed: 01/27/2023] Open
Abstract
The dicentric chromosome assay (DCA) is considered the gold standard for radiation biodosimetry, but it is limited by its long dicentric scoring time and need for skilled scorers. The automation of scoring dicentrics has been considered a strategy to overcome the constraints of DCA. However, the studies on automated scoring methods are limited compared to those on conventional manual DCA. Our study aims to assess the performance of a semi-automated scoring method for DCA using ex vivo and in vivo irradiated samples. Dose estimations of 39 blind samples irradiated ex vivo and 35 industrial radiographers occupationally exposed in vivo were estimated using the manual and semi-automated scoring methods and subsequently compared. The semi-automated scoring method, which removed the false positives of automated scoring using the dicentric chromosome (DC) scoring algorithm, had an accuracy of 94.9% in the ex vivo irradiated samples. It also had more than 90% accuracy, sensitivity, and specificity to distinguish binary dose categories reflecting clinical, diagnostic, and epidemiological significance. These data were comparable to those of manual DCA. Moreover, Cohen's kappa statistic and McNemar's test showed a substantial agreement between the two methods for categorizing in vivo samples into never and ever radiation exposure. There was also a significant correlation between the two methods. Despite of comparable results with two methods, lower sensitivity of semi-automated scoring method could be limited to assess various radiation exposures. Taken together, our findings show the semi-automated scoring method can provide accurate dose estimation rapidly, and can be useful as an alternative to manual DCA for biodosimetry in large-scale accidents or cases to monitor radiation exposure of radiation workers.
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Affiliation(s)
- Younghyun Lee
- Laboratory of Biological Dosimetry, National Radiation Emergency Medical Center, Korea Institute of Radiological and Medical Sciences, Seoul, South Korea,Department of Biomedical Laboratory Science, College of Medical Sciences, Soonchunhyang University, Asan, South Korea
| | - Seung Hyun Kim
- Laboratory of Biological Dosimetry, National Radiation Emergency Medical Center, Korea Institute of Radiological and Medical Sciences, Seoul, South Korea
| | - Yang Hee Lee
- Laboratory of Biological Dosimetry, National Radiation Emergency Medical Center, Korea Institute of Radiological and Medical Sciences, Seoul, South Korea
| | - Su San Yang
- Laboratory of Biological Dosimetry, National Radiation Emergency Medical Center, Korea Institute of Radiological and Medical Sciences, Seoul, South Korea
| | - Hyo Jin Yoon
- Laboratory of Biological Dosimetry, National Radiation Emergency Medical Center, Korea Institute of Radiological and Medical Sciences, Seoul, South Korea
| | - Ruth C. Wilkins
- Consumer and Clinical Radiation Protection Bureau, Health Canada, Ottawa, ON, Canada
| | - Seongjae Jang
- Laboratory of Biological Dosimetry, National Radiation Emergency Medical Center, Korea Institute of Radiological and Medical Sciences, Seoul, South Korea,*Correspondence: Seongjae Jang
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13
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Bucher M, Weiss T, Endesfelder D, Trompier F, Ristic Y, Kunert P, Schlattl H, Giussani A, Oestreicher U. Dose Variations Using an X-Ray Cabinet to Establish in vitro Dose-Response Curves for Biological Dosimetry Assays. Front Public Health 2022; 10:903509. [PMID: 35655448 PMCID: PMC9152255 DOI: 10.3389/fpubh.2022.903509] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2022] [Accepted: 04/25/2022] [Indexed: 11/13/2022] Open
Abstract
In biological dosimetry, dose-response curves are essential for reliable retrospective dose estimation of individual exposure in case of a radiation accident. Therefore, blood samples are irradiated in vitro and evaluated based on the applied assay. Accurate physical dosimetry of the irradiation performance is a critical part of the experimental procedure and is influenced by the experimental setup, especially when X-ray cabinets are used. The aim of this study was to investigate variations and pitfalls associated with the experimental setups used to establish calibration curves in biological dosimetry with X-ray cabinets. In this study, irradiation was performed with an X-ray source (195 kV, 10 mA, 0.5 mm Cu filter, dose rate 0.52 Gy/min, 1st and 2nd half-value layer = 1.01 and 1.76 mm Cu, respectively, average energy 86.9 keV). Blood collection tubes were irradiated with a dose of 1 Gy in vertical or horizontal orientation in the center of the beam area with or without usage of an additional fan heater. To evaluate the influence of the setups, physical dose measurements using thermoluminescence dosimeters, electron paramagnetic resonance dosimetry and ionization chamber as well as biological effects, quantified by dicentric chromosomes and micronuclei, were compared. This study revealed that the orientation of the sample tubes (vertical vs. horizontal) had a significant effect on the radiation dose with a variation of -41% up to +49% and contributed to a dose gradient of up to 870 mGy inside the vertical tubes due to the size of the sample tubes and the associated differences in the distance to the focal point of the tube. The number of dicentric chromosomes and micronuclei differed by ~30% between both orientations. An additional fan heater had no consistent impact. Therefore, dosimetric monitoring of experimental irradiation setups is mandatory prior to the establishment of calibration curves in biological dosimetry. Careful consideration of the experimental setup in collaboration with physicists is required to ensure traceability and reproducibility of irradiation conditions, to correlate the radiation dose and the number of aberrations correctly and to avoid systematical bias influencing the dose estimation in the frame of biological dosimetry.
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Affiliation(s)
- Martin Bucher
- Department of Effects and Risks of Ionizing and Non-Ionizing Radiation, Federal Office for Radiation Protection (BfS), Oberschleißheim, Germany
| | - Tina Weiss
- Department of Medical and Occupational Radiation Protection, Federal Office for Radiation Protection (BfS), Oberschleißheim, Germany
| | - David Endesfelder
- Department of Effects and Risks of Ionizing and Non-Ionizing Radiation, Federal Office for Radiation Protection (BfS), Oberschleißheim, Germany
| | - Francois Trompier
- Department of External Dosimetry, Institut de Radioprotection et de Sûreté Nucléaire (IRSN), Fontenay-aux-Roses, France
| | - Yoann Ristic
- Department of External Dosimetry, Institut de Radioprotection et de Sûreté Nucléaire (IRSN), Fontenay-aux-Roses, France
| | - Patrizia Kunert
- Department of Medical and Occupational Radiation Protection, Federal Office for Radiation Protection (BfS), Oberschleißheim, Germany
| | - Helmut Schlattl
- Department of Medical and Occupational Radiation Protection, Federal Office for Radiation Protection (BfS), Oberschleißheim, Germany
| | - Augusto Giussani
- Department of Medical and Occupational Radiation Protection, Federal Office for Radiation Protection (BfS), Oberschleißheim, Germany
| | - Ursula Oestreicher
- Department of Effects and Risks of Ionizing and Non-Ionizing Radiation, Federal Office for Radiation Protection (BfS), Oberschleißheim, Germany
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Bucher M, Trinkl S, Endesfelder D, Weiss T, Gomolka M, Pätzold J, Lechel U, Roessler U, de Las Heras Gala H, Moertl S, Giussani A. Radiation field and dose inhomogeneities using an X-ray cabinet in radiation biology research. Med Phys 2021; 48:8140-8151. [PMID: 34655237 DOI: 10.1002/mp.15297] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2021] [Revised: 09/03/2021] [Accepted: 10/10/2021] [Indexed: 01/01/2023] Open
Abstract
PURPOSE X-ray cabinets are replacing 137 Cs/60 Co sources in radiation biology research due to advantages in size, handling, and radiation protection. However, because of their different physical properties, X-ray cabinets are more susceptible to experimental influences than conventional sources. The aim of this study was to examine the variations related to the experimental setups typically used to investigate biological radiation effects with X-ray cabinets. MATERIALS AND METHODS A combined approach of physical dose measurements by thermoluminescence dosimetry and detection of biological effects by quantification of γH2AX and 53BP1 foci was used to analyze field inhomogeneity and evaluate the influence of the components of the experimental setup. RESULTS Irradiation was performed using an X-ray tube (195 kV, 10 mA, 0.5-mm-thick copper filter, dose rate of 0.59 Gy/min). Thermoluminescence dosimetry revealed inhomogeneity and a dose decrease of up to 42.3% within the beam area (diameter 31.1 cm) compared to the dose at the center. This dose decrease was consistent with the observed decline in the number of radiation-induced foci by up to 55.9 %. Uniform dose distribution was measured after reducing the size of the radiation field (diameter 12.5 cm). However, when using 15-ml test tubes placed at different positions within this field, the dose decreased by up to 17% in comparison to the central position. Analysis of foci number revealed significant differences between the tubes for γH2AX (1 h) and 53BP1 (4 h) at different time points after irradiation. Neither removal of some tubes nor of the caps improved the dose decrease significantly. By contrast, when using 1.5-ml tubes, dose differences were less than 4%, and no significant differences in foci number were detected. CONCLUSION X-ray cabinets are user-friendly irradiation units for investigating biological radiation effects. However, field inhomogeneities and experimental setup components considerably affect the delivered irradiation doses. For this reason, strict dosimetric monitoring of experimental irradiation setups is mandatory for reliable studies.
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Affiliation(s)
- Martin Bucher
- Department of Effects and Risks of Ionising and Non-Ionising Radiation, Federal Office for Radiation Protection, Oberschleissheim, Germany
| | - Sebastian Trinkl
- Department of Medical and Occupational Radiation Protection, Federal Office for Radiation Protection, Oberschleissheim, Germany
| | - David Endesfelder
- Department of Effects and Risks of Ionising and Non-Ionising Radiation, Federal Office for Radiation Protection, Oberschleissheim, Germany
| | - Tina Weiss
- Department of Medical and Occupational Radiation Protection, Federal Office for Radiation Protection, Oberschleissheim, Germany
| | - Maria Gomolka
- Department of Effects and Risks of Ionising and Non-Ionising Radiation, Federal Office for Radiation Protection, Oberschleissheim, Germany
| | - Juliane Pätzold
- Department of Effects and Risks of Ionising and Non-Ionising Radiation, Federal Office for Radiation Protection, Oberschleissheim, Germany
| | - Ursula Lechel
- Department of Medical and Occupational Radiation Protection, Federal Office for Radiation Protection, Oberschleissheim, Germany
| | - Ute Roessler
- Department of Effects and Risks of Ionising and Non-Ionising Radiation, Federal Office for Radiation Protection, Oberschleissheim, Germany
| | - Hugo de Las Heras Gala
- Department of Medical and Occupational Radiation Protection, Federal Office for Radiation Protection, Oberschleissheim, Germany
| | - Simone Moertl
- Department of Effects and Risks of Ionising and Non-Ionising Radiation, Federal Office for Radiation Protection, Oberschleissheim, Germany
| | - Augusto Giussani
- Department of Medical and Occupational Radiation Protection, Federal Office for Radiation Protection, Oberschleissheim, Germany
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15
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Inter-laboratory automation of the in vitro micronucleus assay using imaging flow cytometry and deep learning. Arch Toxicol 2021; 95:3101-3115. [PMID: 34245348 PMCID: PMC8380241 DOI: 10.1007/s00204-021-03113-0] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2021] [Accepted: 06/29/2021] [Indexed: 12/31/2022]
Abstract
The in vitro micronucleus assay is a globally significant method for DNA damage quantification used for regulatory compound safety testing in addition to inter-individual monitoring of environmental, lifestyle and occupational factors. However, it relies on time-consuming and user-subjective manual scoring. Here we show that imaging flow cytometry and deep learning image classification represents a capable platform for automated, inter-laboratory operation. Images were captured for the cytokinesis-block micronucleus (CBMN) assay across three laboratories using methyl methanesulphonate (1.25-5.0 μg/mL) and/or carbendazim (0.8-1.6 μg/mL) exposures to TK6 cells. Human-scored image sets were assembled and used to train and test the classification abilities of the "DeepFlow" neural network in both intra- and inter-laboratory contexts. Harnessing image diversity across laboratories yielded a network able to score unseen data from an entirely new laboratory without any user configuration. Image classification accuracies of 98%, 95%, 82% and 85% were achieved for 'mononucleates', 'binucleates', 'mononucleates with MN' and 'binucleates with MN', respectively. Successful classifications of 'trinucleates' (90%) and 'tetranucleates' (88%) in addition to 'other or unscorable' phenotypes (96%) were also achieved. Attempts to classify extremely rare, tri- and tetranucleated cells with micronuclei into their own categories were less successful (≤ 57%). Benchmark dose analyses of human or automatically scored micronucleus frequency data yielded quantitation of the same equipotent concentration regardless of scoring method. We conclude that this automated approach offers significant potential to broaden the practical utility of the CBMN method across industry, research and clinical domains. We share our strategy using openly-accessible frameworks.
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Rodrigues MA, Probst CE, Zayats A, Davidson B, Riedel M, Li Y, Venkatachalam V. The in vitro micronucleus assay using imaging flow cytometry and deep learning. NPJ Syst Biol Appl 2021; 7:20. [PMID: 34006858 PMCID: PMC8131758 DOI: 10.1038/s41540-021-00179-5] [Citation(s) in RCA: 20] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2020] [Accepted: 02/16/2021] [Indexed: 02/07/2023] Open
Abstract
The in vitro micronucleus (MN) assay is a well-established assay for quantification of DNA damage, and is required by regulatory bodies worldwide to screen chemicals for genetic toxicity. The MN assay is performed in two variations: scoring MN in cytokinesis-blocked binucleated cells or directly in unblocked mononucleated cells. Several methods have been developed to score the MN assay, including manual and automated microscopy, and conventional flow cytometry, each with advantages and limitations. Previously, we applied imaging flow cytometry (IFC) using the ImageStream® to develop a rapid and automated MN assay based on high throughput image capture and feature-based image analysis in the IDEAS® software. However, the analysis strategy required rigorous optimization across chemicals and cell lines. To overcome the complexity and rigidity of feature-based image analysis, in this study we used the Amnis® AI software to develop a deep-learning method based on convolutional neural networks to score IFC data in both the cytokinesis-blocked and unblocked versions of the MN assay. We show that the use of the Amnis AI software to score imagery acquired using the ImageStream® compares well to manual microscopy and outperforms IDEAS® feature-based analysis, facilitating full automation of the MN assay.
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Affiliation(s)
| | | | - Artiom Zayats
- Amnis Flow Cytometry, Luminex Corporation, Seattle, WA, USA
| | - Bryan Davidson
- Amnis Flow Cytometry, Luminex Corporation, Seattle, WA, USA
| | - Michael Riedel
- Amnis Flow Cytometry, Luminex Corporation, Seattle, WA, USA
| | - Yang Li
- Amnis Flow Cytometry, Luminex Corporation, Seattle, WA, USA
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Chaurasia RK, Bhat NN, Gaur N, Shirsath KB, Desai UN, Sapra BK. Establishment and multiparametric-cytogenetic validation of 60Co-gamma-ray induced, phospho-gamma-H2AX calibration curve for rapid biodosimetry and triage management during radiological emergencies. Mutat Res 2021; 866:503354. [PMID: 33985694 DOI: 10.1016/j.mrgentox.2021.503354] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2020] [Revised: 03/12/2021] [Accepted: 03/30/2021] [Indexed: 01/01/2023]
Abstract
Exposure to ionizing radiation is unavoidable to our modern developing society as its applications are widespread and increasing with societal development. The exposures may be planned as in medical applications or may be unplanned as in occupational work and radiological emergencies. Dose quantification of planned and unplanned exposures is essential to make crucial decisions for management of such exposures. This study aims to establish ex-vivo dose-response curve for 60Co-gamma-ray induced gamma-H2AX-foci by immunofluorescence using microscopy and flowcytometry with human lymphocytes. This technique has the potential to serve as a rapid tool for dose estimation and triage application during small to large scale radiological emergencies and clinical exposures. Response curves were generated for the dose range 0-4 Gy (at 1, 2, 4, 8, 16, 24, 48, 72 and 96 h of incubation after irradiation) with microscopy and 0-8 Gy (at 2, 4, 8, 16 and 24 h of incubation after irradiation) with flow cytometry. These curves can be applied for dose reconstruction when post exposure sampling is delayed up to 96 h. In order to evaluate Minimum Detection Limit (MDL) of the assay, variation of background frequency of gamma-H2AX-foci was measured in 12 volunteers. To understand the application window of the assay, gamma-H2AX foci decay kinetics has been studied up to 96 h with microscopy and response curves were generated from 1 to 96 hours post exposure. Gamma-H2AX fluorescence intensity decay kinetics was also studied up to 96 h with flow cytometry and response curves were generated from 2 to 24 hours post irradiation. Established curves were validated with dose blinded samples and also compared with standard cytogenetic assays. An inter-comparison of dose estimates was made among gamma-H2AX assay, dicentric aberrations and reciprocal translocations for application window in various dose ranges and time of blood collection after exposures.
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Affiliation(s)
- Rajesh Kumar Chaurasia
- Radiological Physics and Advisory Division, Bhabha Atomic Research Centre (BARC), Mumbai, India; Homi Bhabha National Institute (HBNI), Mumbai, India.
| | - N N Bhat
- Radiological Physics and Advisory Division, Bhabha Atomic Research Centre (BARC), Mumbai, India; Homi Bhabha National Institute (HBNI), Mumbai, India.
| | - Neeraj Gaur
- Radiological Physics and Advisory Division, Bhabha Atomic Research Centre (BARC), Mumbai, India.
| | - K B Shirsath
- Radiological Physics and Advisory Division, Bhabha Atomic Research Centre (BARC), Mumbai, India.
| | - U N Desai
- Radiological Physics and Advisory Division, Bhabha Atomic Research Centre (BARC), Mumbai, India.
| | - B K Sapra
- Radiological Physics and Advisory Division, Bhabha Atomic Research Centre (BARC), Mumbai, India; Homi Bhabha National Institute (HBNI), Mumbai, India.
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18
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Bucher M, Endesfelder D, Roessler U, Borkhardt A, Dückers G, Kirlum HJ, Lankisch P, Oommen PT, Niehues T, Rübe CE, Baumgartner I, Bunk F, Moertl S, Hornhardt S, Gomolka M. Analysis of chromosomal aberrations and γH2A.X foci to identify radiation-sensitive ataxia-telangiectasia patients. Mutat Res 2020; 861-862:503301. [PMID: 33551102 DOI: 10.1016/j.mrgentox.2020.503301] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2020] [Revised: 11/27/2020] [Accepted: 12/02/2020] [Indexed: 01/08/2023]
Abstract
Ataxia-telangiectasia (AT) is a rare inherited recessive disorder which is caused by a mutated Ataxia-telangiectasia mutated (ATM) gene. Hallmarks include chromosomal instability, cancer predisposition and increased sensitivity to ionizing radiation. The ATM protein plays an important role in signaling of DNA double-strand breaks (DSB), thereby phosphorylating the histone H2A.X. Non-functional ATM protein leads to defects in DNA damage response, unresolved DSBs and genomic instability. The aim of this study was to evaluate chromosomal aberrations and γH2A.X foci as potential radiation sensitivity biomarkers in AT patients. For this purpose, lymphocytes of 8 AT patients and 10 healthy controls were irradiated and induced DNA damage and DNA repair capacity were detected by the accumulation of γH2A.X foci. The results were heterogeneous among AT patients. Evaluation revealed 2 AT patients with similar γH2A.X foci numbers as controls after 1 h while 3 patients showed a lower induction. In regard to DNA repair, 3 of 5 AT patients showed poor damage repair. Therefore, DNA damage induction and DNA repair as detected by H2A.X phosphorylation revealed individual differences, seems to depend on the underlying individual mutation and thus appears not well suited as a biomarker for radiation sensitivity. In addition, chromosomal aberrations were analyzed by mFISH. An increased frequency of spontaneous chromosomal breakage was characteristic for AT cells. After irradiation, significantly increased rates for non-exchange aberrations, translocations, complex aberrations and dicentric chromosomes were observed in AT patients compared to controls. The results of this study suggested, that complex aberrations and dicentric chromosomes might be a reliable biomarker for radiation sensitivity in AT patients, while non-exchange aberrations and translocations identified both, spontaneous and radiation-induced chromosomal instability.
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Affiliation(s)
- Martin Bucher
- Department of Effects and Risks of Ionising and Non-Ionising Radiation, Federal Office for Radiation Protection, Ingolstädter Landstraße 1, 85764, Oberschleißheim, Germany.
| | - David Endesfelder
- Department of Effects and Risks of Ionising and Non-Ionising Radiation, Federal Office for Radiation Protection, Ingolstädter Landstraße 1, 85764, Oberschleißheim, Germany
| | - Ute Roessler
- Department of Effects and Risks of Ionising and Non-Ionising Radiation, Federal Office for Radiation Protection, Ingolstädter Landstraße 1, 85764, Oberschleißheim, Germany
| | - Arndt Borkhardt
- Department of Pediatric Oncology, Hematology and Clinical Immunology, Medical Faculty, Center for Child and Adolescent Health, Heinrich Heine University Düsseldorf, Moorenstraße 5, 40225, Dusseldorf, Germany
| | - Gregor Dückers
- Center for Child and Adolescent Health, HELIOS Hospital Krefeld, Lutherplatz 40, 47805, Krefeld, Germany
| | - Hans-Joachim Kirlum
- Pediatric Surgery and Pediatric Orthopedics in der Au, Kühbachstraße 1, 81543, Munich, Germany
| | - Petra Lankisch
- Department of Pediatric Oncology, Hematology and Clinical Immunology, Medical Faculty, Center for Child and Adolescent Health, Heinrich Heine University Düsseldorf, Moorenstraße 5, 40225, Dusseldorf, Germany
| | - Prasad T Oommen
- Department of Pediatric Oncology, Hematology and Clinical Immunology, Medical Faculty, Center for Child and Adolescent Health, Heinrich Heine University Düsseldorf, Moorenstraße 5, 40225, Dusseldorf, Germany
| | - Tim Niehues
- Center for Child and Adolescent Health, HELIOS Hospital Krefeld, Lutherplatz 40, 47805, Krefeld, Germany
| | - Claudia E Rübe
- Department of Radiotherapy and Radiation Oncology, Saarland University Hospital and Saarland University Faculty of Medicine, Kirrberger Straße, Building 6.5, 66421, Homburg/Saar, Germany
| | - Ingrid Baumgartner
- Department of Effects and Risks of Ionising and Non-Ionising Radiation, Federal Office for Radiation Protection, Ingolstädter Landstraße 1, 85764, Oberschleißheim, Germany
| | - Frank Bunk
- Department of Effects and Risks of Ionising and Non-Ionising Radiation, Federal Office for Radiation Protection, Ingolstädter Landstraße 1, 85764, Oberschleißheim, Germany
| | - Simone Moertl
- Department of Effects and Risks of Ionising and Non-Ionising Radiation, Federal Office for Radiation Protection, Ingolstädter Landstraße 1, 85764, Oberschleißheim, Germany
| | - Sabine Hornhardt
- Department of Effects and Risks of Ionising and Non-Ionising Radiation, Federal Office for Radiation Protection, Ingolstädter Landstraße 1, 85764, Oberschleißheim, Germany
| | - Maria Gomolka
- Department of Effects and Risks of Ionising and Non-Ionising Radiation, Federal Office for Radiation Protection, Ingolstädter Landstraße 1, 85764, Oberschleißheim, Germany
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Shirley BC, Knoll JHM, Moquet J, Ainsbury E, Pham ND, Norton F, Wilkins RC, Rogan PK. Estimating partial-body ionizing radiation exposure by automated cytogenetic biodosimetry. Int J Radiat Biol 2020; 96:1492-1503. [PMID: 32910711 DOI: 10.1080/09553002.2020.1820611] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
PURPOSE Inhomogeneous exposures to ionizing radiation can be detected and quantified with the dicentric chromosome assay (DCA) of metaphase cells. Complete automation of interpretation of the DCA for whole-body irradiation has significantly improved throughput without compromising accuracy, however, low levels of residual false positive dicentric chromosomes (DCs) have confounded its application for partial-body exposure determination. MATERIALS AND METHODS We describe a method of estimating and correcting for false positive DCs in digitally processed images of metaphase cells. Nearly all DCs detected in unirradiated calibration samples are introduced by digital image processing. DC frequencies of irradiated calibration samples and those exposed to unknown radiation levels are corrected subtracting this false positive fraction from each. In partial-body exposures, the fraction of cells exposed, and radiation dose can be quantified after applying this modification of the contaminated Poisson method. RESULTS Dose estimates of three partially irradiated samples diverged 0.2-2.5 Gy from physical doses and irradiated cell fractions deviated by 2.3%-15.8% from the known levels. Synthetic partial-body samples comprised of unirradiated and 3 Gy samples from 4 laboratories were correctly discriminated as inhomogeneous by multiple criteria. Root mean squared errors of these dose estimates ranged from 0.52 to 1.14 Gy2 and from 8.1 to 33.3%2 for the fraction of cells irradiated. CONCLUSIONS Automated DCA can differentiate whole- from partial-body radiation exposures and provides timely quantification of estimated whole-body equivalent dose.
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Affiliation(s)
| | - Joan H M Knoll
- CytoGnomix Inc., London, Canada.,Department of Pathology and Laboratory Medicine, University of Western Ontario, London, Canada
| | | | | | | | | | | | - Peter K Rogan
- CytoGnomix Inc., London, Canada.,Departments of Biochemistry and Oncology, University of Western Ontario, London, Canada
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20
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Endesfelder D, Kulka U, Einbeck J, Oestreicher U. Improving the accuracy of dose estimates from automatically scored dicentric chromosomes by accounting for chromosome number. Int J Radiat Biol 2020; 96:1571-1584. [PMID: 33001765 DOI: 10.1080/09553002.2020.1829152] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/23/2022]
Abstract
PURPOSE The traditional workflow for biological dosimetry based on manual scoring of dicentric chromosomes is very time consuming. Especially for large-scale scenarios or for low-dose exposures, high cell numbers have to be analyzed, requiring alternative scoring strategies. Semi-automatic scoring of dicentric chromosomes provides an opportunity to speed up the standard workflow of biological dosimetry. Due to automatic metaphase and chromosome detection, the number of counted chromosomes per metaphase is variable. This can potentially introduce overdispersion and statistical methods for conventional, manual scoring might not be applicable to data obtained by automatic scoring of dicentric chromosomes, potentially resulting in biased dose estimates and underestimated uncertainties. The identification of sources for overdispersion enables the development of methods appropriately accounting for increased dispersion levels. MATERIALS AND METHODS Calibration curves based on in vitro irradiated (137-Cs; 0.44 Gy/min) blood from three healthy donors were analyzed for systematic overdispersion, especially at higher doses (>2 Gy) of low LET radiation. For each donor, 12 doses in the range of 0-6 Gy were scored semi-automatically. The effect of chromosome number as a potential cause for the observed overdispersion was assessed. Statistical methods based on interaction models accounting for the number of detected chromosomes were developed for the estimation of calibration curves, dose and corresponding uncertainties. The dose estimation was performed based on a Bayesian Markov-Chain-Monte-Carlo method, providing high flexibility regarding the implementation of priors, likelihood and the functional form of the association between predictors and dicentric counts. The proposed methods were validated by simulations based on cross-validation. RESULTS Increasing dose dependent overdispersion was observed for all three donors as well as considerable differences in dicentric counts between donors. Variations in the number of detected chromosomes between metaphases were identified as a major source for the observed overdispersion and the differences between donors. Persisting overdispersion beyond the contribution of chromosome number was modeled by a Negative Binomial distribution. Results from cross-validation suggested that the proposed statistical methods for dose estimation reduced bias in dose estimates, variability between dose estimates and improved the coverage of the estimated confidence intervals. However, the 95% confidence intervals were still slightly too permissive, suggesting additional unknown sources of apparent overdispersion. CONCLUSIONS A major source for the observed overdispersion could be identified, and statistical methods accounting for overdispersion introduced by variations in the number of detected chromosomes were developed, enabling more robust dose estimation and quantification of uncertainties for semi-automatic counting of dicentric chromosomes.
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Affiliation(s)
- David Endesfelder
- Department of Effects and Risks of Ionising and Non-Ionising Radiation, Federal Office for Radiation Protection, Neuherberg, Germany
| | - Ulrike Kulka
- Department of Effects and Risks of Ionising and Non-Ionising Radiation, Federal Office for Radiation Protection, Neuherberg, Germany.,Department of National and International Cooperation, Federal Office for Radiation Protection, Neuherberg, Germany
| | - Jochen Einbeck
- Department of Mathematical Sciences, Durham University, Durham, UK
| | - Ursula Oestreicher
- Department of Effects and Risks of Ionising and Non-Ionising Radiation, Federal Office for Radiation Protection, Neuherberg, Germany
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21
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Micronucleus Assay: The State of Art, and Future Directions. Int J Mol Sci 2020; 21:ijms21041534. [PMID: 32102335 PMCID: PMC7073234 DOI: 10.3390/ijms21041534] [Citation(s) in RCA: 141] [Impact Index Per Article: 28.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/04/2020] [Revised: 02/13/2020] [Accepted: 02/14/2020] [Indexed: 12/16/2022] Open
Abstract
During almost 40 years of use, the micronucleus assay (MN) has become one of the most popular methods to assess genotoxicity of different chemical and physical factors, including ionizing radiation-induced DNA damage. In this minireview, we focus on the position of MN among the other genotoxicity tests, its usefulness in different applications and visibility by international organizations, such as International Atomic Energy Agency, Organization for Economic Co-operation and Development and International Organization for Standardization. In addition, the mechanism of micronuclei formation is discussed. Finally, foreseen directions of the MN development are pointed, such as automation, buccal cells MN and chromothripsis phenomenon.
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22
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Zaguia N, Laplagne E, Colicchio B, Cariou O, Al Jawhari M, Heidingsfelder L, Hempel WM, Jrad BBH, Jeandidier E, Dieterlen A, Carde P, Voisin P, M’kacher R. A new tool for genotoxic risk assessment: Reevaluation of the cytokinesis-block micronucleus assay using semi-automated scoring following telomere and centromere staining. MUTATION RESEARCH-GENETIC TOXICOLOGY AND ENVIRONMENTAL MUTAGENESIS 2020; 850-851:503143. [DOI: 10.1016/j.mrgentox.2020.503143] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/08/2019] [Revised: 01/15/2020] [Accepted: 01/17/2020] [Indexed: 12/14/2022]
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Wilkins RC, Rodrigues MA, Beaton-Green LA. Automated Identification and Scoring of Micronuclei. THE MICRONUCLEUS ASSAY IN TOXICOLOGY 2019. [DOI: 10.1039/9781788013604-00305] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/30/2023]
Abstract
Micronucleus (MN) assays are used as a measure of cellular damage, and are often preferred over other chromosomal aberration assays since they possess similar statistical robustness for detection of genotoxins and require less technical expertise, making them easier to perform. However, the traditional visual scoring methods are tedious and prone to scorer subjectivity. A number of techniques to automate the MN assays have been developed using a variety of technologies. This chapter will provide an overview of several current methods used to automate MN assays, including automated slide-scoring and laser scanning cytometry, as well as conventional and imaging flow cytometry techniques.
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Affiliation(s)
- R. C. Wilkins
- Consumer and Clinical Radiation Protection Bureau, Health Canada Ottawa Ontario K1A 1C1 Canada
| | - M. A. Rodrigues
- Luminex Corporation 645 Elliott Ave W, Suite 100 Seattle WA 98119 USA
| | - L. A. Beaton-Green
- Consumer and Clinical Radiation Protection Bureau, Health Canada Ottawa Ontario K1A 1C1 Canada
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Ryan TL, Pantelias AG, Terzoudi GI, Pantelias GE, Balajee AS. Use of human lymphocyte G0 PCCs to detect intra- and inter-chromosomal aberrations for early radiation biodosimetry and retrospective assessment of radiation-induced effects. PLoS One 2019; 14:e0216081. [PMID: 31059552 PMCID: PMC6502328 DOI: 10.1371/journal.pone.0216081] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2018] [Accepted: 04/12/2019] [Indexed: 12/13/2022] Open
Abstract
A sensitive biodosimetry tool is required for rapid individualized dose estimation and risk assessment in the case of radiological or nuclear mass casualty scenarios to prioritize exposed humans for immediate medical countermeasures to reduce radiation related injuries or morbidity risks. Unlike the conventional Dicentric Chromosome Assay (DCA), which takes about 3–4 days for radiation dose estimation, cell fusion mediated Premature Chromosome Condensation (PCC) technique in G0 lymphocytes can be rapidly performed for radiation dose assessment within 6–8 hrs of sample receipt by alleviating the need for ex vivo lymphocyte proliferation for 48 hrs. Despite this advantage, the PCC technique has not yet been fully exploited for radiation biodosimetry. Realizing the advantage of G0 PCC technique that can be instantaneously applied to unstimulated lymphocytes, we evaluated the utility of G0 PCC technique in detecting ionizing radiation (IR) induced stable and unstable chromosomal aberrations for biodosimetry purposes. Our study demonstrates that PCC coupled with mFISH and mBAND techniques can efficiently detect both numerical and structural chromosome aberrations at the intra- and inter-chromosomal levels in unstimulated T- and B-lymphocytes. Collectively, we demonstrate that the G0 PCC technique has the potential for development as a biodosimetry tool for detecting unstable chromosome aberrations (chromosome fragments and dicentric chromosomes) for early radiation dose estimation and stable chromosome exchange events (translocations) for retrospective monitoring of individualized health risks in unstimulated lymphocytes.
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Affiliation(s)
- Terri L. Ryan
- Cytogenetic Biodosimetry Laboratory, Radiation Emergency Assistance Center/Training site, Oak Ridge Institute for Science and Education, Oak Ridge Associated Universities, Oak Ridge, Tennessee, United States of America
| | - Antonio G. Pantelias
- Health Physics, Radiobiology & Cytogenetics Laboratory, Institute of Nuclear & Radiological Sciences & Technology, Energy & Safety, National Centre for Scientific Research “Demokritos”, Ag. Paraskevi, Athens, Greece
| | - Georgia I. Terzoudi
- Health Physics, Radiobiology & Cytogenetics Laboratory, Institute of Nuclear & Radiological Sciences & Technology, Energy & Safety, National Centre for Scientific Research “Demokritos”, Ag. Paraskevi, Athens, Greece
| | - Gabriel E. Pantelias
- Health Physics, Radiobiology & Cytogenetics Laboratory, Institute of Nuclear & Radiological Sciences & Technology, Energy & Safety, National Centre for Scientific Research “Demokritos”, Ag. Paraskevi, Athens, Greece
| | - Adayabalam S. Balajee
- Cytogenetic Biodosimetry Laboratory, Radiation Emergency Assistance Center/Training site, Oak Ridge Institute for Science and Education, Oak Ridge Associated Universities, Oak Ridge, Tennessee, United States of America
- * E-mail:
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25
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Wang Q, Rodrigues MA, Repin M, Pampou S, Beaton-Green LA, Perrier J, Garty G, Brenner DJ, Turner HC, Wilkins RC. Automated Triage Radiation Biodosimetry: Integrating Imaging Flow Cytometry with High-Throughput Robotics to Perform the Cytokinesis-Block Micronucleus Assay. Radiat Res 2019; 191:342-351. [PMID: 30779694 DOI: 10.1667/rr15243.1] [Citation(s) in RCA: 36] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
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.
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Affiliation(s)
- Qi Wang
- a Center for Radiological Research, Columbia University Medical Center, New York, New York 10032
| | | | - Mikhail Repin
- a Center for Radiological Research, Columbia University Medical Center, New York, New York 10032
| | - Sergey Pampou
- b Columbia Genome Center High-Throughput Screening Facility, Columbia University Medical Center, New York, New York 10032
| | - Lindsay A Beaton-Green
- d Consumer and Clinical Radiation Protection Bureau, Health Canada, Ottawa K1A 1C1, Canada
| | - Jay Perrier
- a Center for Radiological Research, Columbia University Medical Center, New York, New York 10032
| | - Guy Garty
- a Center for Radiological Research, Columbia University Medical Center, New York, New York 10032
| | - David J Brenner
- a Center for Radiological Research, Columbia University Medical Center, New York, New York 10032
| | - Helen C Turner
- a Center for Radiological Research, Columbia University Medical Center, New York, New York 10032
| | - Ruth C Wilkins
- d Consumer and Clinical Radiation Protection Bureau, Health Canada, Ottawa K1A 1C1, Canada
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26
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Balajee AS, Smith T, Ryan T, Escalona M, Dainiak N. DEVELOPMENT OF A MINIATURIZED VERSION OF DICENTRIC CHROMOSOME ASSAY TOOL FOR RADIOLOGICAL TRIAGE. RADIATION PROTECTION DOSIMETRY 2018; 182:139-145. [PMID: 30247729 DOI: 10.1093/rpd/ncy127] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/30/2018] [Indexed: 06/08/2023]
Abstract
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.
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Affiliation(s)
- Adayabalam S Balajee
- Cytogenetics Biodosimetry Laboratory, Radiation Emergency Assistance Center and Training Site, Oak Ridge Institute for Science and Education, Oak Ridge Associated Universities, 1299, Bethel Valley Road, Oak Ridge, TN, USA
| | - Tammy Smith
- Cytogenetics Biodosimetry Laboratory, Radiation Emergency Assistance Center and Training Site, Oak Ridge Institute for Science and Education, Oak Ridge Associated Universities, 1299, Bethel Valley Road, Oak Ridge, TN, USA
| | - Terri Ryan
- Cytogenetics Biodosimetry Laboratory, Radiation Emergency Assistance Center and Training Site, Oak Ridge Institute for Science and Education, Oak Ridge Associated Universities, 1299, Bethel Valley Road, Oak Ridge, TN, USA
| | - Maria Escalona
- Cytogenetics Biodosimetry Laboratory, Radiation Emergency Assistance Center and Training Site, Oak Ridge Institute for Science and Education, Oak Ridge Associated Universities, 1299, Bethel Valley Road, Oak Ridge, TN, USA
| | - Nicholas Dainiak
- Cytogenetics Biodosimetry Laboratory, Radiation Emergency Assistance Center and Training Site, Oak Ridge Institute for Science and Education, Oak Ridge Associated Universities, 1299, Bethel Valley Road, Oak Ridge, TN, USA
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27
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Oestreicher U, Endesfelder D, Gomolka M, Kesminiene A, Lang P, Lindholm C, Rößler U, Samaga D, Kulka U. Automated scoring of dicentric chromosomes differentiates increased radiation sensitivity of young children after low dose CT exposure in vitro. Int J Radiat Biol 2018; 94:1017-1026. [PMID: 30028637 DOI: 10.1080/09553002.2018.1503429] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2018] [Revised: 05/28/2018] [Accepted: 06/29/2018] [Indexed: 12/12/2022]
Abstract
PURPOSE Automated detection of dicentric chromosomes from a large number of cells was applied to study age-dependent radiosensitivity after in vitro CT exposure of blood from healthy donors. MATERIALS AND METHODS Blood samples from newborns, children (2-5 years) and adults (20-50 years) were exposed in vitro to 0 mGy, 41 mGy and 978 mGy using a CT equipment. In this study, automated scoring based on 13,000-31,000 cells/dose point/age group was performed. Results for control and low dose points were validated by manually counting about 26,000 cells/dose point/age group. RESULTS For all age groups, the high number of analyzed cells enabled the detection of a significant increase in the frequency of radiation induced dicentric chromosomes in cells exposed to 41 mGy as compared to control cells. Moreover, differences between the age groups could be resolved for the low dose: young donors showed significantly increased risk for induced dicentrics at 41 mGy compared to adults. CONCLUSIONS The results very clearly demonstrate that the automated dicentric scoring method is capable of discerning radiation induced biomarkers in the low dose range (<100 mGy) and thus may open possibilities for large-scale molecular epidemiology studies in radiation protection.
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Affiliation(s)
- Ursula Oestreicher
- a Federal Office for Radiation Protection (BfS) , Oberschleissheim , Germany
| | - David Endesfelder
- a Federal Office for Radiation Protection (BfS) , Oberschleissheim , Germany
| | - Maria Gomolka
- a Federal Office for Radiation Protection (BfS) , Oberschleissheim , Germany
| | | | - Peter Lang
- c Department of Radiation Oncology , University Hospital, LMU , Munich , Germany
| | - Carita Lindholm
- d Radiation and Nuclear Safety Authority, STUK , Helsinki , Finland
| | - Ute Rößler
- a Federal Office for Radiation Protection (BfS) , Oberschleissheim , Germany
| | - Daniel Samaga
- a Federal Office for Radiation Protection (BfS) , Oberschleissheim , Germany
- e Research Unit Radiation Cytogenetics , Helmholtz Zentrum Muenchen , Oberschleissheim , Germany
| | - Ulrike Kulka
- a Federal Office for Radiation Protection (BfS) , Oberschleissheim , Germany
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28
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Rodrigues MA. Automation of the in vitro micronucleus assay using the Imagestream ® imaging flow cytometer. Cytometry A 2018; 93:706-726. [PMID: 30118149 PMCID: PMC6174940 DOI: 10.1002/cyto.a.23493] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2017] [Revised: 04/16/2018] [Accepted: 04/25/2018] [Indexed: 12/02/2022]
Abstract
The in vitro micronucleus (MN) assay is a well‐established test for evaluating genotoxicity and cytotoxicity. The use of manual microscopy to perform the assay can be laborious and often suffers from user subjectivity and interscorer variability. Automated methods including slide‐scanning microscopy and conventional flow cytometry have been developed to eliminate scorer bias and improve throughput. However, these methods possess several limitations such as lack of cytoplasmic visualization using slide‐scanning microscopy and the inability to visually confirm the legitimacy of MN or storage of image data for re‐evaluation using flow cytometry. The ImageStreamX® MK II (ISX) imaging flow cytometer has been demonstrated to overcome all of these limitations. The ISX combines the speed, statistical robustness, and rare event capture capability of conventional flow cytometry with high resolution fluorescent imagery of microscopy and possesses the ability to store all collected image data. This paper details the methodology developed to perform the in vitro MN assay in human lymphoblastoid TK6 cells on the ISX. High resolution images of micronucleated mono‐ and bi‐nucleated cells as well as polynucleated cells can be acquired at a high rate of capture. All images can then be automatically identified, categorized and enumerated in the data analysis software that accompanies the ImageStream, allowing for the scoring of both genotoxicity and cytotoxicity. The results demonstrate that statistically significant increases in MN frequency when compared with solvent controls can be detected at varying levels of cytotoxicity following exposure to well‐known aneugens and clastogens. This work demonstrates a fully automated method for performing the in vitro micronucleus assay on the ISX imaging flow cytometry platform. © 2018 The Author. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of ISAC.
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29
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Ridinger J, Koeneke E, Kolbinger FR, Koerholz K, Mahboobi S, Hellweg L, Gunkel N, Miller AK, Peterziel H, Schmezer P, Hamacher-Brady A, Witt O, Oehme I. Dual role of HDAC10 in lysosomal exocytosis and DNA repair promotes neuroblastoma chemoresistance. Sci Rep 2018; 8:10039. [PMID: 29968769 PMCID: PMC6030077 DOI: 10.1038/s41598-018-28265-5] [Citation(s) in RCA: 38] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2017] [Accepted: 06/15/2018] [Indexed: 12/19/2022] Open
Abstract
Drug resistance is a leading cause for treatment failure in many cancers, including neuroblastoma, the most common solid extracranial childhood malignancy. Previous studies from our lab indicate that histone deacetylase 10 (HDAC10) is important for the homeostasis of lysosomes, i.e. acidic vesicular organelles involved in the degradation of various biomolecules. Here, we show that depleting or inhibiting HDAC10 results in accumulation of lysosomes in chemotherapy-resistant neuroblastoma cell lines, as well as in the intracellular accumulation of the weakly basic chemotherapeutic doxorubicin within lysosomes. Interference with HDAC10 does not block doxorubicin efflux from cells via P-glycoprotein inhibition, but rather via inhibition of lysosomal exocytosis. In particular, intracellular doxorubicin does not remain trapped in lysosomes but also accumulates in the nucleus, where it promotes neuroblastoma cell death. Our data suggest that lysosomal exocytosis under doxorubicin treatment is important for cell survival and that inhibition of HDAC10 further induces DNA double-strand breaks (DSBs), providing additional mechanisms that sensitize neuroblastoma cells to doxorubicin. Taken together, we demonstrate that HDAC10 inhibition in combination with doxorubicin kills neuroblastoma, but not non-malignant cells, both by impeding drug efflux and enhancing DNA damage, providing a novel opportunity to target chemotherapy resistance.
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Affiliation(s)
- Johannes Ridinger
- Preclinical Program, Hopp Children's Cancer Center at NCT Heidelberg (KiTZ), Heidelberg, Germany.,Clinical Cooperation Unit Pediatric Oncology, German Cancer Research Center (DKFZ), and German Cancer Consortium (DKTK), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany.,Faculty of Biosciences, University of Heidelberg, Heidelberg, Germany
| | - Emily Koeneke
- Preclinical Program, Hopp Children's Cancer Center at NCT Heidelberg (KiTZ), Heidelberg, Germany.,Clinical Cooperation Unit Pediatric Oncology, German Cancer Research Center (DKFZ), and German Cancer Consortium (DKTK), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany.,University of Heidelberg, Heidelberg, Germany
| | - Fiona R Kolbinger
- Preclinical Program, Hopp Children's Cancer Center at NCT Heidelberg (KiTZ), Heidelberg, Germany.,Clinical Cooperation Unit Pediatric Oncology, German Cancer Research Center (DKFZ), and German Cancer Consortium (DKTK), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany
| | - Katharina Koerholz
- Preclinical Program, Hopp Children's Cancer Center at NCT Heidelberg (KiTZ), Heidelberg, Germany.,Clinical Cooperation Unit Pediatric Oncology, German Cancer Research Center (DKFZ), and German Cancer Consortium (DKTK), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany
| | - Siavosh Mahboobi
- Institute of Pharmacy, University of Regensburg, Regensburg, Germany
| | - Lars Hellweg
- Research Group Cancer Drug Development, German Cancer Research Center, Heidelberg, Germany
| | - Nikolas Gunkel
- Research Group Cancer Drug Development, German Cancer Research Center, Heidelberg, Germany
| | - Aubry K Miller
- Research Group Cancer Drug Development, German Cancer Research Center, Heidelberg, Germany
| | - Heike Peterziel
- Preclinical Program, Hopp Children's Cancer Center at NCT Heidelberg (KiTZ), Heidelberg, Germany.,Clinical Cooperation Unit Pediatric Oncology, German Cancer Research Center (DKFZ), and German Cancer Consortium (DKTK), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany
| | - Peter Schmezer
- Division of Epigenomics and Cancer Risk Factors, German Cancer Research Center, Heidelberg, Germany
| | - Anne Hamacher-Brady
- Johns Hopkins University, Bloomberg School of Public Health, Baltimore, United States
| | - Olaf Witt
- Preclinical Program, Hopp Children's Cancer Center at NCT Heidelberg (KiTZ), Heidelberg, Germany.,Clinical Cooperation Unit Pediatric Oncology, German Cancer Research Center (DKFZ), and German Cancer Consortium (DKTK), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany.,Department of Pediatric Oncology, Hematology and Immunology, University Hospital Heidelberg, Heidelberg, Germany
| | - Ina Oehme
- Preclinical Program, Hopp Children's Cancer Center at NCT Heidelberg (KiTZ), Heidelberg, Germany. .,Clinical Cooperation Unit Pediatric Oncology, German Cancer Research Center (DKFZ), and German Cancer Consortium (DKTK), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany.
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30
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Rodrigues MA, Beaton-Green LA, Wilkins RC, Fenech MF. The potential for complete automated scoring of the cytokinesis block micronucleus cytome assay using imaging flow cytometry. MUTATION RESEARCH-GENETIC TOXICOLOGY AND ENVIRONMENTAL MUTAGENESIS 2018; 836:53-64. [PMID: 30389163 DOI: 10.1016/j.mrgentox.2018.05.003] [Citation(s) in RCA: 43] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/14/2018] [Revised: 03/27/2018] [Accepted: 05/03/2018] [Indexed: 12/21/2022]
Abstract
The lymphocyte Cytokinesis-Block Micronucleus (CBMN) assay was originally developed for the measurement of micronuclei (MN) exclusively in binucleated (BN) cells, which represent the population of cells that can express MN because they completed nuclear division. Recently the assay has evolved into a comprehensive cytome method to include biomarkers that measure chromosomal instability and cytotoxicity by quantification of nuclear buds (NBUDs), nucleoplasmic bridges (NPBs) and apoptotic/necrotic cells. Furthermore, enumeration of mono- and polynucleated cells allows for computation of the nuclear division index (NDI) to assess mitotic activity. Typically performed by manual microscopy, the CBMN cytome assay is laborious and subject to scorer bias and fatigue, leading to inter- and intra-scorer variability. Automated microscopy and conventional flow cytometry methods have been developed to automate scoring of the traditional and cytome versions of the assay. However, these methods have several limitations including the requirement to create high-quality microscope slides, lack of staining consistency and sub-optimal nuclear/cytoplasmic visualization. In the case of flow cytometry, stripping of the cytoplasmic membrane makes it impossible to measure MN in BN cells, calculate the NDI or to quantify apoptotic or necrotic cells. Moreover, the absence of cellular visualization using conventional flow cytometry, makes it impossible to quantify NBUDs and NPBs. In this review, we propose that imaging flow cytometry (IFC), which combines high resolution microscopy with flow cytometry, may overcome these limitations. We demonstrate that by using IFC, images from cells in suspension can be captured, removing the need for microscope slides and allowing visualization of intact cytoplasmic membranes and DNA content. Thus, mono-, bi- and polynucleated cells with and without MN can be rapidly and automatically identified and quantified. Finally, we present high-resolution cell images containing NBUDs and NPBs, illustrating that IFC possesses the potential for completely automated scoring of all components of the CBMN cytome assay.
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Affiliation(s)
| | - Lindsay A Beaton-Green
- Consumer and Clinical Radiation Protection Bureau, Health Canada, Ottawa, Ontario, K1A 1C1, Canada
| | - Ruth C Wilkins
- Consumer and Clinical Radiation Protection Bureau, Health Canada, Ottawa, Ontario, K1A 1C1, Canada
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Shirley B, Li Y, Knoll JHM, Rogan PK. Expedited Radiation Biodosimetry by Automated Dicentric Chromosome Identification (ADCI) and Dose Estimation. J Vis Exp 2017. [PMID: 28892030 PMCID: PMC5619684 DOI: 10.3791/56245] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Biological radiation dose can be estimated from dicentric chromosome frequencies in metaphase cells. Performing these cytogenetic dicentric chromosome assays is traditionally a manual, labor-intensive process not well suited to handle the volume of samples which may require examination in the wake of a mass casualty event. Automated Dicentric Chromosome Identifier and Dose Estimator (ADCI) software automates this process by examining sets of metaphase images using machine learning-based image processing techniques. The software selects appropriate images for analysis by removing unsuitable images, classifies each object as either a centromere-containing chromosome or non-chromosome, further distinguishes chromosomes as monocentric chromosomes (MCs) or dicentric chromosomes (DCs), determines DC frequency within a sample, and estimates biological radiation dose by comparing sample DC frequency with calibration curves computed using calibration samples. This protocol describes the usage of ADCI software. Typically, both calibration (known dose) and test (unknown dose) sets of metaphase images are imported to perform accurate dose estimation. Optimal images for analysis can be found automatically using preset image filters or can also be filtered through manual inspection. The software processes images within each sample and DC frequencies are computed at different levels of stringency for calling DCs, using a machine learning approach. Linear-quadratic calibration curves are generated based on DC frequencies in calibration samples exposed to known physical doses. Doses of test samples exposed to uncertain radiation levels are estimated from their DC frequencies using these calibration curves. Reports can be generated upon request and provide summary of results of one or more samples, of one or more calibration curves, or of dose estimation.
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Affiliation(s)
| | | | - Joan H M Knoll
- CytoGnomix Inc.; Department of Pathology and Laboratory Medicine, Western University
| | - Peter K Rogan
- CytoGnomix Inc.; Department of Biochemistry, Western University;
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32
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Bahreyni Toossi MT, Azimian H, Sarrafzadeh O, Mohebbi S, Soleymanifard S. Automatic detection of micronuclei by cell microscopic image processing. Mutat Res 2017; 806:9-18. [PMID: 28934716 DOI: 10.1016/j.mrfmmm.2017.07.012] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2017] [Revised: 06/15/2017] [Accepted: 07/28/2017] [Indexed: 01/19/2023]
Abstract
With the development and applications of ionizing radiation in medicine, the radiation effects on human health get more and more attention. Ionizing radiation can lead to various forms of cytogenetic damage, including increased frequencies of micronuclei (MNi) and chromosome abnormalities. The cytokinesis block micronucleus (CBMN) assay is widely used method for measuring MNi to determine chromosome mutations or genome instability in cultured human lymphocytes. The visual scoring of MNi is time-consuming and scorer fatigue can lead to inconsistency. In this work, we designed software for the scoring of in vitro CBMN assay for biomonitoring on Giemsa-stained slides that overcome many previous limitations. Automatic scoring proceeds in four stages as follows. First, overall segmentation of nuclei is done. Then, binucleated (BN) cells are detected. Next, the entire cell is estimated for each BN as it is assumed that there is no detectable cytoplasm. Finally, MNi are detected within each BN cell. The designed Software is even able to detect BN cells with vague cytoplasm and MNi in peripheral blood smear. Our system is tested on a self-provided dataset and is achieved high sensitivities of about 98% and 82% in recognizing BN cells and MNi, respectively. Moreover, in our study less than 1% false positives were observed that makes our system reliable for practical MNi scoring.
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Affiliation(s)
| | - Hosein Azimian
- Medical Physics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Omid Sarrafzadeh
- Department of Biomedical Engineering, Mashhad Branch, Islamic Azad University, Mashhad, Iran.
| | - Shokoufeh Mohebbi
- Medical Physics Department, Reza Radiation Oncology Center, Mashhad, Iran
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33
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Finot F, Kaddour A, Morat L, Mouche I, Zaguia N, Cuceu C, Souverville D, Négrault S, Cariou O, Essahli A, Prigent N, Saul J, Paillard F, Heidingsfelder L, Lafouge P, Al Jawhari M, Hempel WM, El May M, Colicchio B, Dieterlen A, Jeandidier E, Sabatier L, Clements J, M'Kacher R. Genotoxic risk of ethyl-paraben could be related to telomere shortening. J Appl Toxicol 2016; 37:758-771. [DOI: 10.1002/jat.3425] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2016] [Revised: 11/01/2016] [Accepted: 11/01/2016] [Indexed: 01/22/2023]
Affiliation(s)
- F. Finot
- Covance Laboratory; 78440 Porcheville France
- Cell Environment; Paris France
| | - A. Kaddour
- Cell Environment; Paris France
- Tunis El Manar University; School of Medicine; Tunis Tunisia
| | - L. Morat
- Radiology and Oncology Laboratory, IRCM, DSV; Commissariat à l'energie atomique (CEA); Fontenay-aux Roses France
| | - I. Mouche
- Covance Laboratory; 78440 Porcheville France
- Cell Environment; Paris France
| | - N. Zaguia
- Radiology and Oncology Laboratory, IRCM, DSV; Commissariat à l'energie atomique (CEA); Fontenay-aux Roses France
| | - C. Cuceu
- Radiology and Oncology Laboratory, IRCM, DSV; Commissariat à l'energie atomique (CEA); Fontenay-aux Roses France
| | | | - S. Négrault
- Covance Laboratory; 78440 Porcheville France
| | - O. Cariou
- Covance Laboratory; 78440 Porcheville France
| | - A. Essahli
- Covance Laboratory; 78440 Porcheville France
| | - N. Prigent
- Covance Laboratory; 78440 Porcheville France
| | - J. Saul
- Covance Laboratories; Yorkshire HG3 1PY UK
| | - F. Paillard
- Covance Laboratory; 78440 Porcheville France
| | | | - P. Lafouge
- Covance Laboratory; 78440 Porcheville France
| | | | - W. M. Hempel
- Radiology and Oncology Laboratory, IRCM, DSV; Commissariat à l'energie atomique (CEA); Fontenay-aux Roses France
| | - M. El May
- Tunis El Manar University; School of Medicine; Tunis Tunisia
| | - B. Colicchio
- Laboratoire MIPS - Groupe IMTI Université de Haute-Alsace; F-68093 Mulhouse France
| | - A. Dieterlen
- Laboratoire MIPS - Groupe IMTI Université de Haute-Alsace; F-68093 Mulhouse France
| | - E. Jeandidier
- Service de génétique Groupe Hospitalier de la Région de Mulhouse et Sud Alsace; 68070 Mulhouse France
| | - L. Sabatier
- Radiology and Oncology Laboratory, IRCM, DSV; Commissariat à l'energie atomique (CEA); Fontenay-aux Roses France
| | | | - R. M'Kacher
- Cell Environment; Paris France
- Radiology and Oncology Laboratory, IRCM, DSV; Commissariat à l'energie atomique (CEA); Fontenay-aux Roses France
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Romm H, Beinke C, Garcia O, Di Giorgio M, Gregoire E, Livingston G, Lloyd DC, Martìnez-Lopez W, Moquet JE, Sugarman SL, Wilkins RC, Ainsbury EA. A New Cytogenetic Biodosimetry Image Repository for the Dicentric Assay. RADIATION PROTECTION DOSIMETRY 2016; 172:192-200. [PMID: 27412509 DOI: 10.1093/rpd/ncw158] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/06/2023]
Abstract
The BioDoseNet was founded by the World Health Organization as a global network of biodosimetry laboratories for building biodosimetry laboratory capacities in countries. The newly established BioDoseNet image repository is a databank of ~25 000 electronically captured images of metaphases from the dicentric assay, which have been previously analysed by international experts. The detailed scoring results and dose estimations have, in most cases, already been published. The compilation of these images into one image repository provides a valuable tool for training and research purposes in biological dosimetry. No special software is needed to view and score the image galleries. For those new to the dicentric assay, the BioDoseNet Image Repository provides an introduction to and training for the dicentric assay. It is an excellent instrument for intra-laboratory training purposes or inter-comparisons between laboratories, as recommended by the International Organization for Standardisation standards. In the event of a radiation accident, the repository can also increase the surge capacity and reduce the turnaround time for dose estimations. Finally, it provides a mechanism for the discussion of scoring discrepancies in difficult cases.
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Affiliation(s)
- Horst Romm
- Bundesamt fuer Strahlenschutz, Neuherberg, Salzgitter, Germany
| | | | - Omar Garcia
- Centro de Protección e Higiene de las Radiaciones, Havana, Cuba
| | | | - Eric Gregoire
- Institut de Radioprotection et de Sureté Nucléaire, Fontenay-aux-Roses, France
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Rogan PK, Li Y, Wilkins RC, Flegal FN, Knoll JHM. Radiation Dose Estimation by Automated Cytogenetic Biodosimetry. RADIATION PROTECTION DOSIMETRY 2016; 172:207-217. [PMID: 27412514 DOI: 10.1093/rpd/ncw161] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/06/2023]
Abstract
The dose from ionizing radiation exposure can be interpolated from a calibration curve fit to the frequency of dicentric chromosomes (DCs) at multiple doses. As DC counts are manually determined, there is an acute need for accurate, fully automated biodosimetry calibration curve generation and analysis of exposed samples. Software, the Automated Dicentric Chromosome Identifier (ADCI), is presented which detects and discriminates DCs from monocentric chromosomes, computes biodosimetry calibration curves and estimates radiation dose. Images of metaphase cells from samples, exposed at 1.4-3.4 Gy, that had been manually scored by two reference laboratories were reanalyzed with ADCI. This resulted in estimated exposures within 0.4-1.1 Gy of the physical dose. Therefore, ADCI can determine radiation dose with accuracies comparable to standard triage biodosimetry. Calibration curves were generated from metaphase images in ~10 h, and dose estimations required ~0.8 h per 500 image sample. Running multiple instances of ADCI may be an effective response to a mass casualty radiation event.
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Affiliation(s)
- Peter K Rogan
- Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario N6A 5C1, Canada
- Cytognomix Inc., 60 North Centre Rd., Box 27052, London, Ontario N5X 3X5, Canada
| | - Yanxin Li
- Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario N6A 5C1, Canada
| | - Ruth C Wilkins
- Consumer and Clinical Radiation Protection Bureau, Health Canada, 775 Brookfield Rd., K1A 1C1, Ottawa, Ontario, Canada
| | - Farrah N Flegal
- Canadian Nuclear Laboratories, Radiobiology & Health, Plant Road, Chalk River, Ontario, K0J 1J0, Canada
| | - Joan H M Knoll
- Cytognomix Inc., 60 North Centre Rd., Box 27052, London, Ontario N5X 3X5, Canada
- Department of Pathology and Laboratory Medicine, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario N6A 5C1, Canada
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Rodrigues MA, Probst CE, Beaton-Green LA, Wilkins RC. THE EFFECT OF AN OPTIMIZED IMAGING FLOW CYTOMETRY ANALYSIS TEMPLATE ON SAMPLE THROUGHPUT IN THE REDUCED CULTURE CYTOKINESIS-BLOCK MICRONUCLEUS ASSAY. RADIATION PROTECTION DOSIMETRY 2016; 172:223-229. [PMID: 27421474 DOI: 10.1093/rpd/ncw160] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/06/2023]
Abstract
In cases of overexposure to ionizing radiation, the cytokinesis-block micronucleus (CBMN) assay can be performed in order to estimate the dose of radiation to an exposed individual. However, in the event of a large-scale radiation accident with many potentially exposed casualties, the assay must be able to generate accurate dose estimates to within ±0.5 Gy as quickly as possible. The assay has been adapted to, validated and optimized on the ImageStreamX imaging flow cytometer. The ease of running this automated version of the CBMN assay allowed investigation into the accuracy of dose estimates after reducing the volume of whole blood cultured to 200 µl and reducing the culture time to 48 h. The data analysis template used to identify binucleated lymphocyte cells (BNCs) and micronuclei (MN) has since been optimized to improve the sensitivity and specificity of BNC and MN detection. This paper presents a re-analysis of existing data using this optimized analysis template to demonstrate that dose estimations from blinded samples can be obtained to the same level of accuracy in a shorter data collection time. Here, we show that dose estimates from blinded samples were obtained to within ±0.5 Gy of the delivered dose when data collection time was reduced by 30 min at standard culture conditions and by 15 min at reduced culture conditions. Reducing data collection time while retaining the same level of accuracy in our imaging flow cytometry-based version of the CBMN assay results in higher throughput and further increases the relevancy of the CBMN assay as a radiation biodosimeter.
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Affiliation(s)
- M A Rodrigues
- Environmental and Radiation Health Sciences Directorate, Health Canada, Ottawa, Ontario K1A 1C1, Canada
| | - C E Probst
- Amnis, a part of MilliporeSigma, 645 Elliott Avenue West, Suite 100, Seattle, WA 98119, USA
| | - L A Beaton-Green
- Environmental and Radiation Health Sciences Directorate, Health Canada, Ottawa, Ontario K1A 1C1, Canada
| | - R C Wilkins
- Environmental and Radiation Health Sciences Directorate, Health Canada, Ottawa, Ontario K1A 1C1, Canada
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Garty G, Turner HC, Salerno A, Bertucci A, Zhang J, Chen Y, Dutta A, Sharma P, Bian D, Taveras M, Wang H, Bhatla A, Balajee A, Bigelow AW, Repin M, Lyulko OV, Simaan N, Yao YL, Brenner DJ. THE DECADE OF THE RABiT (2005-15). RADIATION PROTECTION DOSIMETRY 2016; 172:201-206. [PMID: 27412510 PMCID: PMC5225976 DOI: 10.1093/rpd/ncw172] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/06/2023]
Abstract
The RABiT (Rapid Automated Biodosimetry Tool) is a dedicated Robotic platform for the automation of cytogenetics-based biodosimetry assays. The RABiT was developed to fulfill the critical requirement for triage following a mass radiological or nuclear event. Starting from well-characterized and accepted assays we developed a custom robotic platform to automate them. We present here a brief historical overview of the RABiT program at Columbia University from its inception in 2005 until the RABiT was dismantled at the end of 2015. The main focus of this paper is to demonstrate how the biological assays drove development of the custom robotic systems and in turn new advances in commercial robotic platforms inspired small modifications in the assays to allow replacing customized robotics with 'off the shelf' systems. Currently, a second-generation, RABiT II, system at Columbia University, consisting of a PerkinElmer cell::explorer, was programmed to perform the RABiT assays and is undergoing testing and optimization studies.
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Affiliation(s)
- G Garty
- Center for Radiological Research, Columbia University, VC11-230, 630 West 168th Street, New York, NY 10032, USA
| | - H C Turner
- Center for Radiological Research, Columbia University, VC11-230, 630 West 168th Street, New York, NY 10032, USA
| | - A Salerno
- Department of Mechanical Engineering, Columbia University, 500 West 120th Street, New York, NY 10027, USA
- Present address: Pratt & Whitney Canada Corp., 1000 Marie-Victorin, Longueil, QC, Canada J4G 1A1
| | - A Bertucci
- Center for Radiological Research, Columbia University, VC11-230, 630 West 168th Street, New York, NY 10032, USA
| | - J Zhang
- Department of Mechanical Engineering, Columbia University, 500 West 120th Street, New York, NY 10027, USA
- Present address: Auris Surgical Robotics Inc., 125 Shoreway Rd, San Carlos, CA 94070, USA
| | - Y Chen
- Department of Mechanical Engineering, Columbia University, 500 West 120th Street, New York, NY 10027, USA
| | - A Dutta
- Center for Radiological Research, Columbia University, VC11-230, 630 West 168th Street, New York, NY 10032, USA
- Present address: BioReliance Corp., 9630 Medical Center Dr, Rockville, MD 20850, USA
| | - P Sharma
- Center for Radiological Research, Columbia University, VC11-230, 630 West 168th Street, New York, NY 10032, USA
| | - D Bian
- Department of Mechanical Engineering, Columbia University, 500 West 120th Street, New York, NY 10027, USA
| | - M Taveras
- Center for Radiological Research, Columbia University, VC11-230, 630 West 168th Street, New York, NY 10032, USA
| | - H Wang
- Department of Mechanical Engineering, Columbia University, 500 West 120th Street, New York, NY 10027, USA
- Present address: General Motors Co., 30500 Mound Road, Warren, MI 48090, USA
| | - A Bhatla
- Department of Mechanical Engineering, Columbia University, 500 West 120th Street, New York, NY 10027, USA
- Present address: Curiosity Lab Inc., 54 Mallard Pl. Secaucus, NJ, 07094, USA
| | - A Balajee
- Center for Radiological Research, Columbia University, VC11-230, 630 West 168th Street, New York, NY 10032, USA
- Present address: Cytogenetic Biodosimetry Laboratory, Radiation Emergency Assistance Center and Training Site, Oak Ridge Institute for Science and Education, Oak Ridge Associated Universities, Building SC-10, 1299, Bethel Valley Road, Oak Ridge, TN, 37830, USA
| | - A W Bigelow
- Center for Radiological Research, Columbia University, VC11-230, 630 West 168th Street, New York, NY 10032, USA
| | - M Repin
- Center for Radiological Research, Columbia University, VC11-230, 630 West 168th Street, New York, NY 10032, USA
| | - O V Lyulko
- Center for Radiological Research, Columbia University, VC11-230, 630 West 168th Street, New York, NY 10032, USA
| | - N Simaan
- Department of Mechanical Engineering, Columbia University, 500 West 120th Street, New York, NY 10027, USA
- Present address: Department of Mechanical Engineering, Vanderbuilt University, PMB 351592, Nashville, TN, 37235, USA
| | - Y L Yao
- Department of Mechanical Engineering, Columbia University, 500 West 120th Street, New York, NY 10027, USA
| | - D J Brenner
- Center for Radiological Research, Columbia University, VC11-230, 630 West 168th Street, New York, NY 10032, USA
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Depuydt J, Baeyens A, Barnard S, Beinke C, Benedek A, Beukes P, Buraczewska I, Darroudi F, De Sanctis S, Dominguez I, Monteiro Gil O, Hadjidekova V, Kis E, Kulka U, Lista F, Lumniczky K, M’kacher R, Moquet J, Obreja D, Oestreicher U, Pajic J, Pastor N, Popova L, Regalbuto E, Ricoul M, Sabatier L, Slabbert J, Sommer S, Testa A, Thierens H, Wojcik A, Vral A. RENEB intercomparison exercises analyzing micronuclei (Cytokinesis-block Micronucleus Assay). Int J Radiat Biol 2016; 93:36-47. [DOI: 10.1080/09553002.2016.1206231] [Citation(s) in RCA: 29] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/21/2022]
Affiliation(s)
- Julie Depuydt
- Faculty of Medicine and Health Sciences, Ghent University, Gent, Belgium
| | - Ans Baeyens
- National Research Foundation (NRF) iThemba LABS, Somerset West, South Africa
| | - Stephen Barnard
- Public Health England, Centre for Radiation Chemical and Environmental Hazards, Chilton, UK
| | - Christina Beinke
- Bundeswehr Institut für Radiobiology, Universität Ulm, Munich, Germany
| | - Anett Benedek
- National Public Health Centre – National Research Directorate for Radiobiology and Radiohygiene, Budapest, Hungary
| | - Philip Beukes
- National Research Foundation (NRF) iThemba LABS, Somerset West, South Africa
| | | | | | | | | | - Octávia Monteiro Gil
- Centro de Ciências e Tecnologias Nucleares, Instituto Superior Técnico, Universidade de Lisboa, Bobadela-LRS, Portugal
| | | | - Enikő Kis
- National Public Health Centre – National Research Directorate for Radiobiology and Radiohygiene, Budapest, Hungary
| | - Ulrike Kulka
- Bundesamt für Strahlenschutz, Department Radiation Protection and Health, Oberschleissheim, Germany
| | - Florigio Lista
- Army Medical and Veterinary Research Center, Rome, Italy
| | - Katalin Lumniczky
- National Public Health Centre – National Research Directorate for Radiobiology and Radiohygiene, Budapest, Hungary
| | - Radhia M’kacher
- Laboratoire de Radiobiologie et Oncologie, Commissariat à l’Energy Atomique, France
| | - Jayne Moquet
- Public Health England, Centre for Radiation Chemical and Environmental Hazards, Chilton, UK
| | - Doina Obreja
- Institutul National de Sanatate Publica, Bucuresti, Romania
| | - Ursula Oestreicher
- Faculty of Medicine and Health Sciences, Ghent University, Gent, Belgium
| | - Jelena Pajic
- Serbian Institute of Occupational Health “Dr Dragomir Karajovic”, Radiation Protection Center, Belgrado, Serbia
| | | | - Ljubomira Popova
- National Center for Radiobiology and Radiation Protection, Sofia, Bulgaria
| | | | - Michelle Ricoul
- Laboratoire de Radiobiologie et Oncologie, Commissariat à l’Energy Atomique, France
| | - Laure Sabatier
- Laboratoire de Radiobiologie et Oncologie, Commissariat à l’Energy Atomique, France
| | - Jacobus Slabbert
- National Research Foundation (NRF) iThemba LABS, Somerset West, South Africa
| | | | - Antonella Testa
- Agenzia Nazionale per le Nuove Tecnologie, l'Energia e lo Sviluppo Economico Sostenibile, Rome, Italy
| | - Hubert Thierens
- Faculty of Medicine and Health Sciences, Ghent University, Gent, Belgium
| | - Andrzej Wojcik
- Institute Molecular Biosciences, Stockholm University, Stockholm, Sweden
| | - Anne Vral
- Faculty of Medicine and Health Sciences, Ghent University, Gent, Belgium
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39
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VRAL ANNE, DECORTE VEERLE, DEPUYDT JULIE, WAMBERSIE ANDRÉ, THIERENS HUBERT. A semi‑automated FISH‑based micronucleus‑centromere assay for biomonitoring of hospital workers exposed to low doses of ionizing radiation. Mol Med Rep 2016; 14:103-10. [PMID: 27176451 PMCID: PMC4918626 DOI: 10.3892/mmr.2016.5265] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2015] [Accepted: 03/10/2016] [Indexed: 12/02/2022] Open
Abstract
The aim of the present study was to perform cytogenetic analysis by means of a semi‑automated micronucleus‑centromere assay in lymphocytes from medical radiation workers. Two groups of workers receiving the highest occupational doses were selected: 10 nuclear medicine technicians and 10 interventional radiologists/cardiologists. Centromere‑negative micronucleus (MNCM‑) data, obtained from these two groups of medical radiation workers were compared with those obtained in matched controls. The blood samples of the matched controls were additionally used to construct a 'low‑dose' (0‑100 mGy) MNCM‑ dose‑response curve to evaluate the sensitivity and suitability of the micronucleus‑centromere assay as an 'effect' biomarker in medical surveillance programs. The physical dosimetry data of the 3 years preceding the blood sampling, based on single or double dosimetry practices, were collected for the interpretation of the micronucleus data. The in vitro radiation results showed that for small sized groups, semi‑automated scoring of MNCM‑ enables the detection of a dose of 50 mGy. The comparison of MNCM‑ yields in medical radiation workers and control individuals showed enhanced MNCM‑ scores in the medical radiation workers group (P=0.15). The highest MNCM‑ scores were obtained in the interventional radiologists/cardiologists group, and these scores were significantly higher compared with those obtained from the matched control group (P=0.05). The higher MNCM‑ scores observed in interventional radiologists/cardiologists compared with nuclear medicine technicians were not in agreement with the personal dosimetry records in both groups, which may point to the limitation of 'double dosimetry' procedures used in interventional radiology/cardiology. In conclusion, the data obtained in the present study supports the importance of cytogenetic analysis, in addition to physical dosimetry, as a routine biomonitoring method in medical radiation workers receiving the highest occupational radiation burdens.
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Affiliation(s)
- ANNE VRAL
- Department of Basic Medical Sciences, Ghent University, B-9000 Ghent, Belgium
| | - VEERLE DECORTE
- Scientific Committee Centre de Services Interentreprises, External Service For Prevention and Protection at Work, Catholic University of Louvain, St-Luc Clinical University, B-1200 Brussels, Belgium
| | - JULIE DEPUYDT
- Department of Basic Medical Sciences, Ghent University, B-9000 Ghent, Belgium
| | - ANDRÉ WAMBERSIE
- Institute of Experimental and Clinical Research, Molecular Imaging, Radiotherapy and Oncology Unit, Catholic University of Louvain, St-Luc Clinical University, B-1200 Brussels, Belgium
| | - HUBERT THIERENS
- Department of Basic Medical Sciences, Ghent University, B-9000 Ghent, Belgium
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40
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Rodrigues MA, Probst CE, Beaton-Green LA, Wilkins RC. Optimized automated data analysis for the cytokinesis-block micronucleus assay using imaging flow cytometry for high throughput radiation biodosimetry. Cytometry A 2016; 89:653-62. [PMID: 27272602 PMCID: PMC5089661 DOI: 10.1002/cyto.a.22887] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2015] [Revised: 05/05/2016] [Accepted: 05/10/2016] [Indexed: 01/23/2023]
Abstract
The cytokinesis-block micronucleus (CBMN) assay is a well-established technique that can be employed in triage radiation biodosimetry to estimate whole body doses of radiation to potentially exposed individuals through quantitation of the frequency of micronuclei (MN) in binucleated lymphocyte cells (BNCs). The assay has been partially automated using traditional microscope-based methods and most recently has been modified for application on the ImageStream(X) (IS(X) ) imaging flow cytometer. This modification has allowed for a similar number of BNCs to be automatically scored as compared to traditional microscopy in a much shorter time period. However, the MN frequency measured was much lower than both manual and automated slide-based methods of performing the assay. This work describes the optimized analysis template which implements newly developed functions in the IDEAS(®) data analysis software for the IS(X) that enhances specificity for BNCs and increases the frequency of scored MN. A new dose response calibration curve is presented in which the average rate of MN per BNC is of similar magnitude to those presented in the literature using automated CBMN slide scoring methods. In addition, dose estimates were generated for nine irradiated, blinded samples and were found to be within ±0.5 Gy of the delivered dose. Results demonstrate that the improved identification accuracy for MN and BNCs in the IS(X) -based version of the CBMN assay will translate to increased accuracy when estimating unknown radiation doses received by exposed individuals following large-scale radiological or nuclear emergencies. © 2016 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of ISAC.
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Affiliation(s)
- M A Rodrigues
- Amnis - MilliporeSigma, Seattle, WA, 98119.,Environmental and Radiation Health Sciences Directorate, Health Canada, Ottawa, Ontario, Canada
| | - C E Probst
- Amnis - MilliporeSigma, Seattle, WA, 98119
| | - L A Beaton-Green
- Environmental and Radiation Health Sciences Directorate, Health Canada, Ottawa, Ontario, Canada
| | - R C Wilkins
- Environmental and Radiation Health Sciences Directorate, Health Canada, Ottawa, Ontario, Canada
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41
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HERD OLIVIA, FRANCIES FLAVIA, KOTZEN JEFFREY, SMITH TRUDY, NXUMALO ZWIDE, MULLER XANTHENE, SLABBERT JACOBUS, VRAL ANNE, BAEYENS ANS. Chromosomal radiosensitivity of human immunodeficiency virus positive/negative cervical cancer patients in South Africa. Mol Med Rep 2016; 13:130-6. [PMID: 26549042 PMCID: PMC4686097 DOI: 10.3892/mmr.2015.4504] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2014] [Accepted: 07/28/2015] [Indexed: 12/01/2022] Open
Abstract
Cervical cancer is the second most common cancer amongst South African women and is the leading cause of cancer-associated mortality in this region. Several international studies on radiation‑induced DNA damage in lymphocytes of cervical cancer patients have remained inconclusive. Despite the high incidence of cervical cancer in South Africa, and the extensive use of radiotherapy to treat it, the chromosomal radiosensitivity of South African cervical cancer patients has not been studied to date. Since a high number of these patients are human immunodeficiency virus (HIV)‑positive, the effect of HIV infection on chromosomal radiosensitivity was also investigated. Blood samples from 35 cervical cancer patients (20 HIV‑negative and 15 HIV‑positive) and 20 healthy controls were exposed to X‑rays at doses of 6 MV of 2 and 4 Gy in vitro. Chromosomal radiosensitivity was assessed using the micronucleus (MN) assay. MN scores were obtained using the Metafer 4 platform, an automated microscopic system. Three scoring methods of the MNScore module of Metafer were applied and compared. Cervical cancer patients had higher MN values than healthy controls, with HIV‑positive patients having the highest MN values. Differences between groups were significant when using a scoring method that corrects for false positive and false negative MN. The present study suggested increased chromosomal radiosensitivity in HIV-positive South African cervical cancer patients.
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Affiliation(s)
- OLIVIA HERD
- Department of Radiation Biophysics, NRF-iThemba LABS, Somerset West 7129, South Africa
- Department of Radiation Sciences, University of Witwatersrand, Johannesburg 2193, South Africa
| | - FLAVIA FRANCIES
- Department of Radiation Biophysics, NRF-iThemba LABS, Somerset West 7129, South Africa
- Department of Radiation Sciences, University of Witwatersrand, Johannesburg 2193, South Africa
| | - JEFFREY KOTZEN
- Department of Radiation Oncology, Charlotte Maxeke Johannesburg Academic Hospital, Johannesburg 2193, South Africa
| | - TRUDY SMITH
- Department of Obstetrics and Gynaecology, Charlotte Maxeke Johannesburg Academic Hospital, Johannesburg 2193, South Africa
| | - ZWIDE NXUMALO
- Department of Obstetrics and Gynaecology, Charlotte Maxeke Johannesburg Academic Hospital, Johannesburg 2193, South Africa
| | - XANTHENE MULLER
- Department of Radiation Biophysics, NRF-iThemba LABS, Somerset West 7129, South Africa
| | - JACOBUS SLABBERT
- Department of Radiation Biophysics, NRF-iThemba LABS, Somerset West 7129, South Africa
| | - ANNE VRAL
- Department of Basic Medical Sciences, Ghent University, Ghent B-9000, Belgium
| | - ANS BAEYENS
- Department of Radiation Biophysics, NRF-iThemba LABS, Somerset West 7129, South Africa
- Department of Radiation Sciences, University of Witwatersrand, Johannesburg 2193, South Africa
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42
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Rodrigues MA, Beaton-Green LA, Wilkins RC. Validation of the Cytokinesis-block Micronucleus Assay Using Imaging Flow Cytometry for High Throughput Radiation Biodosimetry. HEALTH PHYSICS 2016; 110:29-36. [PMID: 26606062 DOI: 10.1097/hp.0000000000000371] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/05/2023]
Abstract
The cytokinesis-block micronucleus assay can be employed in triage radiation biodosimetry to determine the dose of radiation to an exposed individual by quantifying the frequency of micronuclei in binucleated lymphocyte cells. Partially automated analysis of the assay has been applied to traditional microscope-based methods, and most recently, the assay has been adapted to an automated imaging flow cytometry method. This method is able to automatically score a larger number of binucleated cells than are typically scored by microscopy. Whole blood samples were irradiated, divided into 2 mL and 200 μL aliquots, cultured for 48 h and 72 h, and processed to generate calibration curves from 0-4 Gy. To validate the method for use in radiation biodosimetry, nine separate whole blood samples were then irradiated to known doses, blinded, and processed. Results indicate that dose estimations can be determined to within ±0.5 Gy of the delivered dose after only 48 h of culture time with an initial blood volume of 200 μL. By performing the cytokinesis-block micronucleus assay using imaging flow cytometry, a significant reduction in the culture time and volume requirements is possible, which greatly increases the applicability of the assay in high throughput triage radiation biodosimetry.
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Affiliation(s)
- Matthew A Rodrigues
- *Consumer and Clinical Radiation Protection Bureau, Health Canada, 775 Brookfield Rd., K1A 1C1, Ottawa, Ontario, Canada; †Department of Physics, Carleton University, 1125 Colonel By Drive, K1S 5B6, Ottawa, Ontario, Canada
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Baeyens A, Herd O, Francies FZ, Cairns A, Katzman G, Murdoch M, Padiachy D, Morford M, Vral A, Slabbert JP. The influence of blood storage time and general anaesthesia on chromosomal radiosensitivity assessment. Mutagenesis 2015; 31:181-6. [DOI: 10.1093/mutage/gev072] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/14/2022] Open
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Perumal V, Sekaran TSG, Raavi V, Basheerudeen SAS, Kanagaraj K, Chowdhury AR, Paul SFD. Radiation signature on exposed cells: Relevance in dose estimation. World J Radiol 2015; 7:266-278. [PMID: 26435777 PMCID: PMC4585950 DOI: 10.4329/wjr.v7.i9.266] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/25/2015] [Revised: 07/03/2015] [Accepted: 08/03/2015] [Indexed: 02/06/2023] Open
Abstract
The radiation is considered as a double edged sword, as its beneficial and detrimental effects have been demonstrated. The potential benefits are being exploited to its maximum by adopting safe handling of radionuclide stipulated by the regulatory agencies. While the occupational workers are monitored by personnel monitoring devices, for general publics, it is not a regular practice. However, it can be achieved by using biomarkers with a potential for the radiation triage and medical management. An ideal biomarker to adopt in those situations should be rapid, specific, sensitive, reproducible, and able to categorize the nature of exposure and could provide a reliable dose estimation irrespective of the time of the exposures. Since cytogenetic markers shown to have many advantages relatively than other markers, the origins of various chromosomal abnormalities induced by ionizing radiations along with dose-response curves generated in the laboratory are presented. Current status of the gold standard dicentric chromosome assay, micronucleus assay, translocation measurement by fluorescence in-situ hybridization and an emerging protein marker the γ-H2AX assay are discussed with our laboratory data. With the wide choice of methods, an appropriate assay can be employed based on the net.
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45
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Lue SW, Repin M, Mahnke R, Brenner DJ. Development of a High-Throughput and Miniaturized Cytokinesis-Block Micronucleus Assay for Use as a Biological Dosimetry Population Triage Tool. Radiat Res 2015; 184:134-42. [PMID: 26230078 DOI: 10.1667/rr13991.1] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/03/2022]
Abstract
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.
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Affiliation(s)
- Stanley W Lue
- a Center for Radiological Research, Department of Radiation Oncology, Columbia University Medical Center, New York, New York 10032; and
| | - Mikhail Repin
- a Center for Radiological Research, Department of Radiation Oncology, Columbia University Medical Center, New York, New York 10032; and
| | - Ryan Mahnke
- b Northrop Grumman, Elkridge, Maryland 21075
| | - David J Brenner
- a Center for Radiological Research, Department of Radiation Oncology, Columbia University Medical Center, New York, New York 10032; and
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Ramakumar A, Subramanian U, Prasanna PGS. High-throughput sample processing and sample management; the functional evolution of classical cytogenetic assay towards automation. MUTATION RESEARCH-GENETIC TOXICOLOGY AND ENVIRONMENTAL MUTAGENESIS 2015; 793:132-41. [PMID: 26520383 DOI: 10.1016/j.mrgentox.2015.07.011] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/20/2015] [Accepted: 07/23/2015] [Indexed: 10/23/2022]
Abstract
High-throughput individual diagnostic dose assessment is essential for medical management of radiation-exposed subjects after a mass casualty. Cytogenetic assays such as the Dicentric Chromosome Assay (DCA) are recognized as the gold standard by international regulatory authorities. DCA is a multi-step and multi-day bioassay. DCA, as described in the IAEA manual, can be used to assess dose up to 4-6 weeks post-exposure quite accurately but throughput is still a major issue and automation is very essential. The throughput is limited, both in terms of sample preparation as well as analysis of chromosome aberrations. Thus, there is a need to design and develop novel solutions that could utilize extensive laboratory automation for sample preparation, and bioinformatics approaches for chromosome-aberration analysis to overcome throughput issues. We have transitioned the bench-based cytogenetic DCA to a coherent process performing high-throughput automated biodosimetry for individual dose assessment ensuring quality control (QC) and quality assurance (QA) aspects in accordance with international harmonized protocols. A Laboratory Information Management System (LIMS) is designed, implemented and adapted to manage increased sample processing capacity, develop and maintain standard operating procedures (SOP) for robotic instruments, avoid data transcription errors during processing, and automate analysis of chromosome-aberrations using an image analysis platform. Our efforts described in this paper intend to bridge the current technological gaps and enhance the potential application of DCA for a dose-based stratification of subjects following a mass casualty. This paper describes one such potential integrated automated laboratory system and functional evolution of the classical DCA towards increasing critically needed throughput.
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Affiliation(s)
- Adarsh Ramakumar
- Scientific Research Department, Armed Forces Radiobiology Research Institute, 8901 Wisconsin Avenue, Bldg. 42, Bethesda, MD 20889, USA.
| | - Uma Subramanian
- Scientific Research Department, Armed Forces Radiobiology Research Institute, 8901 Wisconsin Avenue, Bldg. 42, Bethesda, MD 20889, USA
| | - Pataje G S Prasanna
- Scientific Research Department, Armed Forces Radiobiology Research Institute, 8901 Wisconsin Avenue, Bldg. 42, Bethesda, MD 20889, USA
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Roemer E, Zenzen V, Conroy LL, Luedemann K, Dempsey R, Schunck C, Sticken ET. Automation of the in vitro micronucleus and chromosome aberration assay for the assessment of the genotoxicity of the particulate and gas-vapor phase of cigarette smoke. Toxicol Mech Methods 2015; 25:320-33. [PMID: 25986082 DOI: 10.3109/15376516.2015.1037413] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
Total particulate matter (TPM) and the gas-vapor phase (GVP) of mainstream smoke from the Reference Cigarette 3R4F were assayed in the cytokinesis-block in vitro micronucleus (MN) assay and the in vitro chromosome aberration (CA) assay, both using V79-4 Chinese hamster lung fibroblasts exposed for up to 24 h. The Metafer image analysis platform was adapted resulting in a fully automated evaluation system of the MN assay for the detection, identification and reporting of cells with micronuclei together with the determination of the cytokinesis-block proliferation index (CBPI) to quantify the treatment-related cytotoxicity. In the CA assay, the same platform was used to identify, map and retrieve metaphases for a subsequent CA evaluation by a trained evaluator. In both the assays, TPM and GVP provoked a significant genotoxic effect: up to 6-fold more micronucleated target cells than in the negative control and up to 10-fold increases in aberrant metaphases. Data variability was lower in the automated version of the MN assay than in the non-automated. It can be estimated that two test substances that differ in their genotoxicity by approximately 30% can statistically be distinguished in the automated MN and CA assays. Time savings, based on man hours, due to the automation were approximately 70% in the MN and 25% in the CA assays. The turn-around time of the evaluation phase could be shortened by 35 and 50%, respectively. Although only cigarette smoke-derived test material has been applied, the technical improvements should be of value for other test substances.
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Affiliation(s)
- Ewald Roemer
- a Philip Morris Products S.A., Philip Morris International R&D , Neuchâtel , Switzerland
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Tamizh Selvan G, Chaudhury N, Venkatachalam P. Comparison of results of the manual and automated scoring of micronucleus frequencies in 60 Co-irradiated peripheral blood lymphocytes for triage dosimetry. Appl Radiat Isot 2015; 97:70-77. [DOI: 10.1016/j.apradiso.2014.12.018] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2014] [Revised: 12/11/2014] [Accepted: 12/18/2014] [Indexed: 11/22/2022]
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Exit from dormancy provokes DNA-damage-induced attrition in haematopoietic stem cells. Nature 2015; 520:549-52. [PMID: 25707806 DOI: 10.1038/nature14131] [Citation(s) in RCA: 480] [Impact Index Per Article: 48.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2014] [Accepted: 12/02/2014] [Indexed: 01/03/2023]
Abstract
Haematopoietic stem cells (HSCs) are responsible for the lifelong production of blood cells. The accumulation of DNA damage in HSCs is a hallmark of ageing and is probably a major contributing factor in age-related tissue degeneration and malignant transformation. A number of accelerated ageing syndromes are associated with defective DNA repair and genomic instability, including the most common inherited bone marrow failure syndrome, Fanconi anaemia. However, the physiological source of DNA damage in HSCs from both normal and diseased individuals remains unclear. Here we show in mice that DNA damage is a direct consequence of inducing HSCs to exit their homeostatic quiescent state in response to conditions that model physiological stress, such as infection or chronic blood loss. Repeated activation of HSCs out of their dormant state provoked the attrition of normal HSCs and, in the case of mice with a non-functional Fanconi anaemia DNA repair pathway, led to a complete collapse of the haematopoietic system, which phenocopied the highly penetrant bone marrow failure seen in Fanconi anaemia patients. Our findings establish a novel link between physiological stress and DNA damage in normal HSCs and provide a mechanistic explanation for the universal accumulation of DNA damage in HSCs during ageing and the accelerated failure of the haematopoietic system in Fanconi anaemia patients.
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Automation of the cytokinesis-block micronucleus cytome assay by laser scanning cytometry and its potential application in radiation biodosimetry. Biotechniques 2014; 57:309-12. [PMID: 25495731 DOI: 10.2144/000114239] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2014] [Accepted: 09/26/2014] [Indexed: 11/23/2022] Open
Abstract
Here we describe the adaptation of laser scanning cytometry (LSC) to measure micronuclei (MN) automatically in lymphocytes. MN frequencies were determined in irradiated human lymphocytes using the cytokinesis-block technique, and the results from LSC were compared with visual scoring results obtained from slides of cells stained using Fast Green and 4',6-diamidino-2-phenylindole (DAPI). This fluorescent approach allowed clear identification of binucleated cells and detection of MN. The dose responses measured visually and by LSC showed similar trends and correlated positively (r = 0.9689; P < 0.0001). High-content analysis was developed to further automatically score MN within mono-, tri- and tetra-nucleated cells and to determine the nuclear division index and nuclear circularity values. The high-throughput nature of LSC can provide unique advantages in future DNA damage diagnostics in experimental and epidemiological studies. Importantly, it allows for co-detection of other biomarkers of interest within a single lymphocyte, and further development of this capability is anticipated.
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