title: THE EFFECT OF AN OPTIMIZED IMAGING FLOW CYTOMETRY ANALYSIS TEMPLATE ON SAMPLE THROUGHPUT IN THE REDUCED CULTURE CYTOKINESIS-BLOCK MICRONUCLEUS ASSAY.
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 ImageStreamXimaging 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.
authors.affiliation: Environmental and Radiation Health Sciences Directorate, Health Canada, Ottawa, Ontario K1A 1C1, Canada.
authors.forename: M A
authors.initials: MA
authors.lastname: Rodrigues
authors.affiliation: Amnis, a part of MilliporeSigma, 645 Elliott Avenue West, Suite 100, Seattle, WA 98119, USA.
authors.forename: C E
authors.initials: CE
authors.lastname: Probst
authors.affiliation: Environmental and Radiation Health Sciences Directorate, Health Canada, Ottawa, Ontario K1A 1C1, Canada.
authors.forename: L A
authors.initials: LA
authors.lastname: Beaton-Green
authors.affiliation: Environmental and Radiation Health Sciences Directorate, Health Canada, Ottawa, Ontario K1A 1C1, Canada ruth.wilkins@hc-sc.gc.ca.
authors.forename: R C
authors.initials: RC
authors.lastname: Wilkins
mesh_terms: D001681:Biological Assay / Q000379:methods; D002469:Cell Separation / Q000379:methods; D058948:Cell Tracking / Q000379:methods; D002478:Cells, Cultured; D002869:Chromosome Aberrations / Q000528:radiation effects*; D048749:Cytokinesis / Q000235:genetics* / Q000528:radiation effects; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007089:Image Enhancement / Q000379:methods*; D015162:Micronucleus Tests / Q000379:methods*; D008853:Microscopy / Q000379:methods*; D011829:Radiation Dosage; D011874:Radiometry / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity