Multi‐parameter dose estimations in radiation biodosimetry using the automated cytokinesis‐block micronucleus assay with imaging flow cytometry

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. © 2014 International Society for Advancement of Cytometry

[1]  W. Vogel,et al.  An automated scoring procedure for the micronucleus test by image analysis. , 2004, Mutagenesis.

[2]  Andrzej Wojcik,et al.  Validation of the micronucleus-centromere assay for biological dosimetry , 2000 .

[3]  W. Müller,et al.  The micronucleus assay in human lymphocytes after high radiation doses (5-15 Gy). , 2002, Mutation research.

[4]  D. Lloyd,et al.  The role of cytogenetics in early triage of radiation casualties. , 2000, Applied radiation and isotopes : including data, instrumentation and methods for use in agriculture, industry and medicine.

[5]  Individual variations in the micronucleus assay for biological dosimetry after high dose exposure. , 2013, Mutation research.

[6]  The yield of radiation-induced micronuclei in early and late-arising binucleated cells depends on radiation quality. , 2010, Mutation research.

[7]  M. Fenech,et al.  Cytokinesis-block micronucleus method in human lymphocytes: effect of in vivo ageing and low dose X-irradiation. , 1986, Mutation research.

[8]  W Suter,et al.  Evaluation of a new procedure for the flow cytometric analysis of in vitro, chemically induced micronuclei in V79 cells , 1998, Environmental and molecular mutagenesis.

[9]  J. Lillard,et al.  The ImageStream System: a key step to a new era in imaging. , 2007, Folia histochemica et cytobiologica.

[10]  Robert M. Haralick,et al.  Textural Features for Image Classification , 1973, IEEE Trans. Syst. Man Cybern..

[11]  M. Fenech,et al.  Measurement of micronuclei in lymphocytes. , 1985, Mutation research.

[12]  John F Koerner,et al.  Assessment of Biodosimetry Methods for a Mass-Casualty Radiological Incident: Medical Response and Management Considerations , 2013, Health physics.

[13]  R. Wilkins,et al.  Automated analysis of the cytokinesis-block micronucleus assay for radiation biodosimetry using imaging flow cytometry , 2014, Radiation and environmental biophysics.

[14]  Leonora Marro,et al.  Validation of the cytokinesis-block micronucleus (CBMN) assay for use as a triage biological dosimetry tool. , 2009, Radiation protection dosimetry.

[15]  M. Nüsse,et al.  Flow cytometric analysis of micronuclei found in cells after irradiation. , 1984, Cytometry.

[16]  Stephen D Dertinger,et al.  In vitro micronucleus scoring by flow cytometry: Differential staining of micronuclei versus apoptotic and necrotic chromatin enhances assay reliability , 2006, Environmental and molecular mutagenesis.

[17]  Claudia Bolognesi,et al.  HUMN project initiative and review of validation, quality control and prospects for further development of automated micronucleus assays using image cytometry systems. , 2013, International journal of hygiene and environmental health.

[18]  Stephen D Dertinger,et al.  In vitro micronucleus assay scored by flow cytometry provides a comprehensive evaluation of cytogenetic damage and cytotoxicity. , 2007, Mutation research.

[19]  Hongliang Wang,et al.  The RABiT: A Rapid Automated Biodosimetry Tool for radiological triage. II. Technological developments , 2011, International journal of radiation biology.

[20]  Hubert Thierens,et al.  Automated micronucleus (MN) scoring for population triage in case of large scale radiation events , 2010, International journal of radiation biology.

[21]  J. Hendry,et al.  Radiobiology for the Radiologist , 1979, British Journal of Cancer.

[22]  M. Fenech.  Cytokinesis-block micronucleus cytome assay , 2007, Nature Protocols.

[23]  Jian Zhang,et al.  THE RABIT: A RAPID AUTOMATED BIODOSIMETRY TOOL FOR RADIOLOGICAL TRIAGE , 2010, Health physics.

[24]  D. Brenner,et al.  Fast Image Analysis for the Micronucleus Assay in a Fully Automated High-Throughput Biodosimetry System , 2014, Radiation research.

[25]  M. Nüsse,et al.  Flow cytometric analysis of micronuclei in cell cultures and human lymphocytes: advantages and disadvantages. , 1997, Mutation research.

[26]  Michael Fenech,et al.  THE LYMPHOCYTE CYTOKINESIS-BLOCK MICRONUCLEUS CYTOME ASSAY AND ITS APPLICATION IN RADIATION BIODOSIMETRY , 2010, Health physics.

[27]  Michael Fenech,et al.  Laser scanning cytometry for automation of the micronucleus assay. , 2012, Mutagenesis.

[28]  Michael Fenech,et al.  The micronucleus assay as a biological dosimeter of in vivo ionising radiation exposure. , 2011, Mutagenesis.

[29]  T. Lörch,et al.  New developments in automated cytogenetic imaging: unattended scoring of dicentric chromosomes, micronuclei, single cell gel electrophoresis, and fluorescence signals , 2004, Cytogenetic and Genome Research.

[30]  M. Fenech,et al.  Automation of the buccal micronucleus cytome assay using laser scanning cytometry. , 2011, Methods in cell biology.

[31]  M. Nüsse,et al.  Antikinetochore antibodies and flow karyotyping: new techniques to detect aneuploidy in mammalian cells induced by ionizing radiation and chemicals. , 1987, Molecular toxicology.