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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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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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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: 143] [Impact Index Per Article: 28.6] [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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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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Wilde S, Queisser N, Holz C, Raschke M, Sutter A. Differentiation of Aneugens and Clastogens in the In Vitro Micronucleus Test by Kinetochore Scoring Using Automated Image Analysis. ENVIRONMENTAL AND MOLECULAR MUTAGENESIS 2019; 60:227-242. [PMID: 30561837 DOI: 10.1002/em.22259] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/11/2018] [Revised: 09/30/2018] [Accepted: 10/06/2018] [Indexed: 06/09/2023]
Abstract
The in vitro micronucleus test according to OECD Test Guideline 487 (TG 487) is widely used to investigate the genotoxic potential of drugs. Besides the identification of in vitro genotoxicants, the assay can be complemented with kinetochore staining for the differentiation between clastogens and aneugens. This differentiation constitutes a major contribution to risk assessment as especially aneugens show a threshold response. Thus, a novel method for automated MN plus kinetochore (k+) scoring by image analysis was developed based on the OECD TG 487. Compound-induced increases in MN frequency can be detected using the cytokinesis-block (cytochalasin B) method in V79 cells after 24 h in a 96-well format. Nuclei, MN, and kinetochores were labeled with nuclear counterstain and anti-kinetochore antibodies, respectively, to score MN in binuclear or multinuclear cells and to differentiate compound-induced MN by the presence of kinetochores. First, a reference data set was created by manual scoring using two clastogens and aneugens. After developing the automated scoring process, a set of 14 reference genotoxicants were studied. The automated image analysis yielded the expected results: 5/5 clastogens and 6/6 aneugens (sensitivity: 100%) as well as 3/3 non-genotoxicants (specificity: 100%) were correctly identified. Further, a threshold was determined for identifying aneugens. Based on the data for our internally characterized reference compounds, unknown compounds that induce ≥53.8% k+ MN are classified as aneugens. The current data demonstrate excellent specificity and sensitivity and the methodology is superior to manual microscopic analysis in terms of speed and throughput as well as the absence of human bias. Environ. Mol. Mutagen. 60:227-242, 2019. © 2018 Wiley Periodicals, Inc.
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Affiliation(s)
- Sabrina Wilde
- Bayer AG, Investigational Toxicology, Berlin, Germany
- Fraunhofer ITEM, Preclinical Pharmacology and In Vitro Toxicology, Hannover, Germany
| | - Nina Queisser
- Bayer AG, Investigational Toxicology, Berlin, Germany
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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: 25] [Impact Index Per Article: 3.6] [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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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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Manservisi F, Marquillas CB, Buscaroli A, Huff J, Lauriola M, Mandrioli D, Manservigi M, Panzacchi S, Silbergeld EK, Belpoggi F. An Integrated Experimental Design for the Assessment of Multiple Toxicological End Points in Rat Bioassays. ENVIRONMENTAL HEALTH PERSPECTIVES 2017; 125:289-295. [PMID: 27448388 PMCID: PMC5332192 DOI: 10.1289/ehp419] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/12/2015] [Revised: 04/27/2016] [Accepted: 06/20/2016] [Indexed: 05/03/2023]
Abstract
BACKGROUND For nearly five decades long-term studies in rodents have been the accepted benchmark for assessing chronic long-term toxic effects, particularly carcinogenicity, of chemicals. The European Food Safety Authority (EFSA) and the World Health Organization (WHO) have pointed out that the current set of internationally utilized test methods capture only some of the potential adverse effects associated with exposures to these agents over the lifetime. OBJECTIVES In this paper, we propose the adaption of the carcinogenicity bioassay to integrate additional protocols for comprehensive long-term toxicity assessment that includes developmental exposures and long-term outcomes, capable of generating information on a broad spectrum of different end points. DISCUSSION An integrated study design based on a stepwise process is described that includes the priority end points of the Economic Co-operation and Development and the National Toxicology Program guidelines on carcinogenicity and chronic toxicity and developmental and reproductive toxicity. Integrating a comprehensive set of relevant toxicological end points in a single protocol represents an opportunity to optimize animal use in accordance with the 3Rs (replacement, reduction and refinement). This strategy has the potential to provide sufficient data on multiple windows of susceptibility of specific interest for risk assessments and public health decision-making by including prenatal, lactational, neonatal exposures and evaluating outcomes over the lifespan. CONCLUSION This integrated study design is efficient in that the same generational cohort of rats used for evaluating long-term outcomes can be monitored in satellite parallel experiments to measure biomarkers and other parameters related to system-specific responses including metabolic alterations and endocrine disturbances. Citation: Manservisi F, Babot Marquillas C, Buscaroli A, Huff J, Lauriola M, Mandrioli D, Manservigi M, Panzacchi S, Silbergeld EK, Belpoggi F. 2017. An integrated experimental design for the assessment of multiple toxicological end points in rat bioassays. Environ Health Perspect 125:289-295; http://dx.doi.org/10.1289/EHP419.
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Affiliation(s)
- Fabiana Manservisi
- Cesare Maltoni Cancer Research Center, Ramazzini Institute, Bentivoglio, Bologna, Italy
| | - Clara Babot Marquillas
- Leonardo da Vinci Programme at the Cesare Maltoni Cancer Research Center, Ramazzini Institute, Bentivoglio, Bologna, Italy
| | - Annalisa Buscaroli
- Cesare Maltoni Cancer Research Center, Ramazzini Institute, Bentivoglio, Bologna, Italy
| | - James Huff
- National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina, USA
| | - Michelina Lauriola
- Cesare Maltoni Cancer Research Center, Ramazzini Institute, Bentivoglio, Bologna, Italy
| | - Daniele Mandrioli
- Cesare Maltoni Cancer Research Center, Ramazzini Institute, Bentivoglio, Bologna, Italy
- Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA
| | - Marco Manservigi
- Cesare Maltoni Cancer Research Center, Ramazzini Institute, Bentivoglio, Bologna, Italy
| | - Simona Panzacchi
- Cesare Maltoni Cancer Research Center, Ramazzini Institute, Bentivoglio, Bologna, Italy
| | - Ellen K. Silbergeld
- Leonardo da Vinci Programme at the Cesare Maltoni Cancer Research Center, Ramazzini Institute, Bentivoglio, Bologna, Italy
| | - Fiorella Belpoggi
- Cesare Maltoni Cancer Research Center, Ramazzini Institute, Bentivoglio, Bologna, Italy
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Zhang Y, Sriraman SK, Kenny HA, Luther E, Torchilin V, Lengyel E. Reversal of Chemoresistance in Ovarian Cancer by Co-Delivery of a P-Glycoprotein Inhibitor and Paclitaxel in a Liposomal Platform. Mol Cancer Ther 2016; 15:2282-2293. [PMID: 27466355 DOI: 10.1158/1535-7163.mct-15-0986] [Citation(s) in RCA: 51] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2015] [Accepted: 07/13/2016] [Indexed: 01/23/2023]
Abstract
The overexpression of permeability-glycoprotein (P-gp), an ABC transporter involved in the cellular exclusion of chemotherapeutic drugs, is a major factor in paclitaxel-resistant ovarian cancer. However, in clinical trials, co-administration of P-gp inhibitors and anticancer drugs has not resulted in the efficient reversal of drug resistance. To improve administration, we encapsulated the third-generation P-gp inhibitor tariquidar (XR-9576, XR), alone or in combination with paclitaxel (PCT) in liposomes (LP). After optimization, the liposomes demonstrated favorable physicochemical properties and the ability to reverse chemoresistance in experiments using chemosensitive/chemoresistant ovarian cancer cell line pairs. Analyzing publicly available datasets, we found that overexpression of P-gp in ovarian cancer is associated with a shorter progression-free and overall survival. In vitro, LP(XR) significantly increased the cellular retention of rhodamine 123, a P-gp substrate. LP(XR,PCT) synergistically inhibited cell viability, blocked proliferation, and caused G2-M arrest in paclitaxel-resistant SKOV3-TR and HeyA8-MDR cell lines overexpressing P-gp. Holographic imaging cytometry revealed that LP(XR,PCT) treatment of SKOV3-TR cells induced almost complete mitotic arrest, whereas laser scanning cytometry showed that the treatment induced apoptosis. In proof-of-concept preclinical studies, LP(XR,PCT), when compared with LP(PCT), significantly reduced tumor weight (43.2% vs. 16.9%, P = 0.0007) and number of metastases (44.4% vs. 2.8%, P = 0.012) in mice bearing orthotopic HeyA8-MDR ovarian tumors. In the xenografts, LP(XR,PCT) efficiently induced apoptosis and impaired proliferation. Our findings suggest that co-delivery of a P-gp inhibitor and paclitaxel using a liposomal platform can sensitize paclitaxel-resistant ovarian cancer cells to paclitaxel. LP(XR,PCT) should be considered for clinical testing in patients with P-gp-overexpressing tumors. Mol Cancer Ther; 15(10); 2282-93. ©2016 AACR.
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Affiliation(s)
- Yilin Zhang
- Department of Obstetrics and Gynecology/Section of Gynecologic Oncology, University of Chicago, Chicago, Illinois
| | - Shravan Kumar Sriraman
- Center for Pharmaceutical Biotechnology and Nanomedicine, Northeastern University, Boston, Massachusetts
| | - Hilary A Kenny
- Department of Obstetrics and Gynecology/Section of Gynecologic Oncology, University of Chicago, Chicago, Illinois
| | - Ed Luther
- Center for Pharmaceutical Biotechnology and Nanomedicine, Northeastern University, Boston, Massachusetts
| | - Vladimir Torchilin
- Center for Pharmaceutical Biotechnology and Nanomedicine, Northeastern University, Boston, Massachusetts
| | - Ernst Lengyel
- Department of Obstetrics and Gynecology/Section of Gynecologic Oncology, University of Chicago, Chicago, Illinois.
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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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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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12
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Rawlins CM, Salisbury JP, Feldman DR, Isim S, Agar NYR, Luther E, Agar JN. Imaging and Mapping of Tissue Constituents at the Single-Cell Level Using MALDI MSI and Quantitative Laser Scanning Cytometry. Methods Mol Biol 2015; 1346:133-49. [PMID: 26542720 DOI: 10.1007/978-1-4939-2987-0_10] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/01/2023]
Abstract
For nearly a century, histopathology involved the laborious morphological analyses of tissues stained with broad-spectrum dyes (i.e., eosin to label proteins). With the advent of antibody-labeling, immunostaining (fluorescein and rhodamine for fluorescent labeling) and immunohistochemistry (DAB and hematoxylin), it became possible to identify specific immunological targets in cells and tissue preparations. Technical advances, including the development of monoclonal antibody technology, led to an ever-increasing palate of dyes, both fluorescent and chromatic. This provides an incredibly rich menu of molecular entities that can be visualized and quantified in cells-giving rise to the new discipline of Molecular Pathology. We describe the evolution of two analytical techniques, cytometry and mass spectrometry, which complement histopathological visual analysis by providing automated, cellular-resolution constituent maps. For the first time, laser scanning cytometry (LSC) and matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) are combined for the analysis of tissue sections. The utility of the marriage of these techniques is demonstrated by analyzing mouse brains with neuron-specific, genetically encoded, fluorescent proteins. We present a workflow that: (1) can be used with or without expensive matrix deposition methods, (2) uses LSC images to reveal the diverse landscape of neural tissue as well as the matrix, and (3) uses a tissue fixation method compatible with a DNA stain. The proposed workflow can be adapted for a variety of sample preparation and matrix deposition methods.
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Affiliation(s)
- Catherine M Rawlins
- Barnett Institute of Chemical and Biological Analysis, Northeastern University, Boston, MA, USA.
| | - Joseph P Salisbury
- Barnett Institute of Chemical and Biological Analysis, Northeastern University, Boston, MA, USA.
| | - Daniel R Feldman
- Department of Neurosurgery, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA.
| | - Sinan Isim
- Life Sciences Department, Brandeis University, Waltham, MA, USA.
| | - Nathalie Y R Agar
- Department of Neurosurgery, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA.
| | - Ed Luther
- Department of Pharmaceutical Sciences, Northeastern University, 360 Huntington Avenue, Boston, MA, 02115, USA.
| | - Jeffery N Agar
- Barnett Institute of Chemical and Biological Analysis, Northeastern University, Boston, MA, USA. .,Department of Neurosurgery, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA.
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Siddiqui MS, François M, Fenech MF, Leifert WR. γH2AX responses in human buccal cells exposed to ionizing radiation. Cytometry A 2014; 87:296-308. [DOI: 10.1002/cyto.a.22607] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2014] [Revised: 10/15/2014] [Accepted: 11/27/2014] [Indexed: 11/06/2022]
Affiliation(s)
- Mohammad Sabbir Siddiqui
- CSIRO Food & Nutrition Flagship; Nutrigenomics & DNA Damage; Adelaide South Australia 5000 Australia
- University of Adelaide, School of Agriculture, Food & Wine; Urrbrae South Australia 5064 Australia
| | - Maxime François
- CSIRO Food & Nutrition Flagship; Nutrigenomics & DNA Damage; Adelaide South Australia 5000 Australia
| | - Michael F. Fenech
- CSIRO Food & Nutrition Flagship; Nutrigenomics & DNA Damage; Adelaide South Australia 5000 Australia
| | - Wayne R. Leifert
- CSIRO Food & Nutrition Flagship; Nutrigenomics & DNA Damage; Adelaide South Australia 5000 Australia
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14
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Rodrigues MA, Beaton-Green LA, Kutzner BC, Wilkins RC. Multi-parameter dose estimations in radiation biodosimetry using the automated cytokinesis-block micronucleus assay with imaging flow cytometry. Cytometry A 2014; 85:883-93. [PMID: 25154929 DOI: 10.1002/cyto.a.22511] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2014] [Revised: 05/08/2014] [Accepted: 07/03/2014] [Indexed: 11/08/2022]
Abstract
The cytokinesis-block micronucleus (CBMN) assay is an established technique in radiation biological dosimetry for estimating the dose to an individual by measuring the frequency of micronuclei (MN) in binucleated lymphocyte cells (BNCs). The assay has been partially automated using slide-scoring algorithms, but an automated multiparameter method without the need of the slide-making procedure would be advantageous to further increase throughput for application in mass casualty events. The development of the ImageStreamX (ISX) imaging flow cytometer has made it possible to adapt the CBMN assay to an automated imaging flow cytometry (FCM) method. The protocol and analysis presented in this work tailor and expand the assay to a multiparameter biodosimetry tool. Ex vivo irradiated whole blood samples were cultured, processed, and analyzed on the ISX and BNCs, MN, and mononuclear cells were imaged, identified, and enumerated automatically and simultaneously. Details on development of the method, gating strategy, and dose response curves generated for the rate of MN per BNC, percentage of mononuclear cells as well as the replication index are presented. Results indicate that adapting the CBMN assay for use in imaging FCM has produced a rapid, robust, multiparameter analysis method with higher throughput than is currently available with standard microscopy. We conclude that the ISX-CBMN method may be an advantageous tool following a radiological event where triage biodosimetry must be performed on a large number of casualties.
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Affiliation(s)
- M A Rodrigues
- Consumer and Clinical Radiation Protection Bureau, Health Canada, Ottawa, Ontario, Canada; Department of Physics, Carleton University, K1S 5B6, Ottawa, Ontario, Canada
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Seager AL, Shah UK, Brusehafer K, Wills J, Manshian B, Chapman KE, Thomas AD, Scott AD, Doherty AT, Doak SH, Johnson GE, Jenkins GJS. Recommendations, evaluation and validation of a semi-automated, fluorescent-based scoring protocol for micronucleus testing in human cells. Mutagenesis 2014; 29:155-64. [DOI: 10.1093/mutage/geu008] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
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16
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Romm H, Barnard S, Boulay-Greene H, De Amicis A, De Sanctis S, Franco M, Herodin F, Jones A, Kulka U, Lista F, Martigne P, Moquet J, Oestreicher U, Rothkamm K, Thierens H, Valente M, Vandersickel V, Vral A, Braselmann H, Meineke V, Abend M, Beinke C. Laboratory Intercomparison of the Cytokinesis-Block Micronucleus Assay. Radiat Res 2013; 180:120-8. [DOI: 10.1667/rr3234.1] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/03/2022]
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Yao J, Gao Q, Mi Q, Li X, Miao M, Cheng P, Luo Y. In vitro micronucleus assay for the analysis of total particulate matter in cigarette smoke: comparison of flow cytometry and laser scanning cytometry with microscopy. Mutat Res 2013; 755:120-5. [PMID: 23770001 DOI: 10.1016/j.mrgentox.2013.05.017] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/19/2012] [Revised: 05/17/2013] [Accepted: 05/26/2013] [Indexed: 10/26/2022]
Abstract
The possible genotoxicity of the total particulate matter (TPM) in cigarette smoke has typically been evaluated using the in vitro micronucleus assay. In recent years, automated scoring techniques have been developed to replace the manual counting process in this assay. However, these automated scoring techniques have not been applied in routine genotoxicity assays for the analysis of TPM to improve the assay efficiency. Chinese hamster ovary (CHO) cells were treated with TPM produced from 14 types of cigarettes at five concentrations (25-200μg/ml) without exogenous metabolic activation. The three following methods were used to score the micronucleus (MN) frequency: (a) flow cytometry with SYTOX and EMA dyes, which differentially stain micronuclei and apoptotic/necrotic chromatin to enhance assay reliability; (b) laser scanning cytometry with FITC and PI dyes, which is a system that combines the analytical capabilities of flow and image cytometry; and (c) visual microcopy with Giemsa dye. The test results obtained using the three methods were compared using correlation analysis. The key findings for this set of compounds include the following: (a) both flow cytometry- and laser scanning cytometry-based methods were effective for MN identification, (b) the three scoring methods could detect dose-dependent micronucleus formation for the 14 types of TPM, and (c) the MN frequencies that were measured in the same samples by flow cytometry, laser scanning cytometry, and visual microscopy were highly correlated, and there were no significant differences (p>0.05). In conclusion, both flow cytometry and laser scanning cytometry can be used to evaluate the MN frequency induced by TPM without exogenous metabolic activation. The simpler and faster processing and the high correlation of the results make these two automatic methods appropriate tools for use in in vitro micronucleus assays for the analysis of TPM using CHO cells.
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Affiliation(s)
- Jianhua Yao
- Faculty of Environmental Science and Engineering, Kunming University of Science & Technology, Kunming 650224, China
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Fenech M, Kirsch-Volders M, Rossnerova A, Sram R, Romm H, Bolognesi C, Ramakumar A, Soussaline F, Schunck C, Elhajouji A, Anwar W, Bonassi S. HUMN project initiative and review of validation, quality control and prospects for further development of automated micronucleus assays using image cytometry systems. Int J Hyg Environ Health 2013; 216:541-52. [PMID: 23507632 DOI: 10.1016/j.ijheh.2013.01.008] [Citation(s) in RCA: 56] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2012] [Revised: 01/29/2013] [Accepted: 01/30/2013] [Indexed: 12/31/2022]
Abstract
The use of micronucleus (MN) assays in in vitro genetic toxicology testing, radiation biodosimetry and population biomonitoring to study the genotoxic impacts of environment gene-interactions has steadily increased over the past two decades. As a consequence there has been a strong interest in developing automated systems to score micronuclei, a biomarker of chromosome breakage or loss, in mammalian and human cells. This paper summarises the outcomes of a workshop on this topic, organised by the HUMN project, at the 6th International Conference on Environmental Mutagenesis in Human Populations at Doha, Qatar, 2012. The aim of this paper is to summarise the outcomes of the workshop with respect to the set objectives which were: (i) Review current developments in automation of micronucleus assays by image cytometry; (ii) define the performance characteristics of automated MN scoring using image cytometry and methods of assessment for instrument validation and quality control and (iii) discuss the design of inter-laboratory comparisons and standardisation of micronucleus assays using automated image cytometry systems. It is evident that automated scoring of micronuclei by automated image cytometry using different commercially available platforms [e.g. Metafer (MetaSystems), Pathfinder™ (IMSTAR), iCyte(®) (Compucyte)], particularly for lymphocytes, is at a mature stage of development with good agreement between visual and automated scoring across systems (correlation factors ranging from 0.58 to 0.99). However, a standardised system of validation and calibration is required to enable more reliable comparison of data across laboratories and across platforms. This review identifies recent progress, important limitations and steps that need to be taken into account to enable the successful universal implementation of automated micronucleus assays by image cytometry.
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Affiliation(s)
- Michael Fenech
- CSIRO Preventative Health Flagship, PO Box 10041 Adelaide BC, 5000, Australia.
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Abstract
The micronucleus (MN) assay, applied in different surrogate tissues, is one of the best validated cytogenetic techniques for evaluating chromosomal damage in humans. The cytokinesis-block micronucleus cytome assay (CBMNcyt) in peripheral blood lymphocytes is the most frequent method in biomonitoring human populations to evaluate exposure to genotoxic agents, micronutrient deficiency, or excess and genetic instability. Furthermore recent scientific evidence suggests an association between an increased MN frequency in lymphocytes and risk of cancer and other age-related degenerative diseases. The micronucleus cytome assay applied in buccal exfoliated cells (BMNCyt) provides a complementary method for measuring DNA damage and cytotoxic effects in an easily accessible tissue not requiring in vitro culture. The protocol for CBMNcyt described here refers to the use of ex vivo whole blood involving 72 h of culture with the block of cytokinesis at 44 h. BMNCyt protocol reports the established method for sample processing, slide preparation, and scoring.
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Affiliation(s)
- Claudia Bolognesi
- Environmental Carcinogenesis Unit, IRCCS Azienda Ospedaliera, Universitaria San Martino-IST Istituto Nazionale Ricerca sul Cancro, Genova, Italy
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Pozarowski P, Holden E, Darzynkiewicz Z. Laser scanning cytometry: principles and applications-an update. Methods Mol Biol 2012; 931:187-212. [PMID: 23027005 DOI: 10.1007/978-1-62703-056-4_11] [Citation(s) in RCA: 37] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Laser scanning cytometer (LSC) is the microscope-based cytofluorometer that offers a plethora of unique analytical capabilities, not provided by flow cytometry (FCM). This review describes attributes of LSC and covers its numerous applications derived from plentitude of the parameters that can be measured. Among many LSC applications the following are emphasized: (a) assessment of chromatin condensation to identify mitotic, apoptotic cells, or senescent cells; (b) detection of nuclear or mitochondrial translocation of critical factors such as NF-κB, p53, or Bax; (c) semi-automatic scoring of micronuclei in mutagenicity assays; (d) analysis of fluorescence in situ hybridization (FISH) and use of the FISH analysis attribute to measure other punctuate fluorescence patterns such as γH2AX foci or receptor clustering; (e) enumeration and morphometry of nucleoli and other cell organelles; (f) analysis of progeny of individual cells in clonogenicity assay; (g) cell immunophenotyping; (h) imaging, visual examination, or sequential analysis using different probes of the same cells upon their relocation; (i) in situ enzyme kinetics, drug uptake, and other time-resolved processes; (j) analysis of tissue section architecture using fluorescent and chromogenic probes; (k) application for hypocellular samples (needle aspirate, spinal fluid, etc.); and (l) other clinical applications. Advantages and limitations of LSC are discussed and compared with FCM.
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Affiliation(s)
- Piotr Pozarowski
- The Brander Cancer Research Institute, New York Medical College, Valhalla, NY, USA
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Han F, Liang P, Wang F, Zeng L, Zhang B. Automated analysis of time-lapse imaging of nuclear translocation by retrospective strategy and its application to STAT1 in HeLa cells. PLoS One 2011; 6:e27454. [PMID: 22125613 PMCID: PMC3220678 DOI: 10.1371/journal.pone.0027454] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/04/2011] [Accepted: 10/17/2011] [Indexed: 11/19/2022] Open
Abstract
Cell-based image analysis of time-lapse imaging is mainly challenged by faint fluorescence and dim boundaries of cellular structures of interest. To resolve these bottlenecks, a novel method was developed based on “retrospective” analysis for cells undergoing minor morphological changes during time-lapse imaging. We fixed and stained the cells with a nuclear dye at the end of the experiment, and processed the time-lapse images using the binary masks obtained by segmenting the nuclear-stained image. This automated method also identifies cells that move during the time-lapse imaging, which is a factor that could influence the kinetics measured for target proteins that are present mostly in the cytoplasm. We then validated the method by measuring interferon gamma (IFNγ) induced signal transducers and activators of transcription 1 (STAT1) nuclear translocation in living HeLa cells. For the first time, automated large-scale analysis of nuclear translocation in living cells was achieved by our novel method. The responses of the cells to IFNγ exhibited a significant drift across the population, but common features of the responses led us to propose a three-stage model of STAT1 import. The simplicity and automation of this method should enable its application in a broad spectrum of time-lapse studies of nuclear-cytoplasmic translocation.
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Affiliation(s)
- Fujun Han
- Laboratory for RNA Chemical Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, People's Republic of China
- Department of Otorhinolaryngology, People's Liberation Army No. 458 Hospital, Guangzhou, China
- School of Biochemistry, Medical Sciences, University Walk, Bristol, United Kingdom
- * E-mail: (FH); (BZ)
| | - Peizhou Liang
- Laboratory for RNA Chemical Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, People's Republic of China
| | - Feifei Wang
- Laboratory for RNA Chemical Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, People's Republic of China
| | - Lingyun Zeng
- Department of Neuroendocrine, People's Liberation Army No. 458 Hospital, Guangzhou, China
| | - Biliang Zhang
- Laboratory for RNA Chemical Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, People's Republic of China
- State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Respiratory Diseases, Guangzhou, China
- * E-mail: (FH); (BZ)
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Fenech M. Current status, new frontiers and challenges in radiation biodosimetry using cytogenetic, transcriptomic and proteomic technologies. RADIAT MEAS 2011. [DOI: 10.1016/j.radmeas.2011.01.016] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/18/2022]
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Darzynkiewicz Z, Traganos F, Zhao H, Halicka HD, Li J. Cytometry of DNA replication and RNA synthesis: Historical perspective and recent advances based on "click chemistry". Cytometry A 2011; 79:328-37. [PMID: 21425239 DOI: 10.1002/cyto.a.21048] [Citation(s) in RCA: 48] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2011] [Revised: 02/10/2011] [Accepted: 02/17/2011] [Indexed: 01/04/2023]
Abstract
This review covers progress in the development of cytometric methodologies designed to assess DNA replication and RNA synthesis. The early approaches utilizing autoradiography to detect incorporation of (3) H- or (14) C-labeled thymidine were able to identify the four fundamental phases of the cell cycle G(1) , S, G(2) , and M, and by analysis of the fraction of labeled mitosis (FLM), to precisely define the kinetics of cell progression through these phases. Analysis of (3) H-uridine incorporation and RNA content provided the means to distinguish quiescent G(0) from cycling G(1) cells. Subsequent progress in analysis of DNA replication was based on the use of BrdU as a DNA precursor and its detection by the quenching of the fluorescence intensity of DNA-bound fluorochromes such as Hoechst 33358 or acridine orange as measured by flow cytometry. Several variants of this methodology have been designed and used in studies to detect anticancer drug-induced perturbations of cell cycle kinetics. The next phase of method development, which was particularly useful in studies of the cell cycle in vivo, including clinical applications, relied on immunocytochemical detection of incorporated halogenated DNA or RNA precursors. This approach however was hampered by the need for DNA denaturation, which made it difficult to concurrently detect other cell constituents for multiparametric analysis. The recently introduced "click chemistry" approach has no such limitation and is the method of choice for analysis of DNA replication and RNA synthesis. This method is based on the use of 5-ethynyl-2'deoxyuridine (EdU) as a DNA precursor or 5-ethynyluridine (EU) as an RNA precursor and their detection with fluorochrome-tagged azides utilizing a copper (I) catalyzed [3+2] cycloaddition. Several examples are presented that illustrate incorporation of EdU or EU in cells subjected to DNA damage detected as histone H2AX phosphorylation that have been analyzed by flow or laser scanning cytometry.
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Affiliation(s)
- Zbigniew Darzynkiewicz
- Brander Cancer Research Institute and Department of Pathology, New York Medical College, Valhalla, New York 10595, USA.
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Laser Scanning Cytometry and Its Applications: A Pioneering Technology in the Field of Quantitative Imaging Cytometry. Methods Cell Biol 2011; 102:161-205. [DOI: 10.1016/b978-0-12-374912-3.00007-9] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
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