title: Validation of JCountPro software for efficient assessment of ionizing radiation-induced foci in human lymphocytes.
abstract: Ionizing radiation-induced foci (IRIF) known also as DNA repair foci represent the most sensitive and specific assay for assessing DNA double-strand break (DSB). IRIF are usually visualized and enumerated with the aid of fluorescence microscopy using antibodies to phosphorylated γH2AX and 53BP1. Although several approaches and software packages were developed for quantification of IRIF, not one of them was commonly accepted and inter-laboratory variability in the outputs was reported. In this study, JCountPro software was validated for IRIF enumeration in two independent laboratories.
  
  
  
  Human lymphocytes were γ-irradiated at doses of 0, 2, 5, 10 and 50 cGy. The cells were fixed, permeabilized and IRIF were immunostained using appropriate antibodies. Cell images were acquired with automatic Metafer system. Endogenous and radiation-induced γH2AX and 53BP1 foci were enumerated using JCountPro. This analysis was performed from the same cell galleries by the researchers from two laboratories. Yield of foci was analyzed by either arithmetic mean (AM) value (foci/cell) or principal average (PA) derived from the approximation of foci distribution with Poisson statistics. Statistical analysis was performed using factorial ANOVA.
  
  
  
  Enumeration of 53BP1, γH2AX and co-localized 53BP1/γH2AX foci by JCountPro was essentially the same between laboratories. IRIF were detected at all doses and linear dose response was obtained in the studied dose range. PA values from Poisson distribution fitted the data better as compared to AM values and were more powerful and sensitive for IRIF analysis than the AM values. All JCountPro data were confirmed by visual focus enumeration.
  
  
  
  We concluded that the JCountPro software was efficient in objectively enumerating IRIF regardless of an individual researcher's bias and has a potential for usage in clinics and molecular epidemiology.
authors.affiliation: a Laboratory of Radiobiology , Cancer Research Institute, Biomedical Research Centre SAS, Slovak Academy of Sciences , Bratislava , Slovakia.
authors.forename: Lukáš
authors.initials: L
authors.lastname: Jakl
authors.affiliation: b Molecular Radiation Biology Laboratory , Peter MacCallum Cancer Centre , Melbourne , Australia.
authors.forename: Pavel
authors.initials: P
authors.lastname: Lobachevsky
authors.affiliation: a Laboratory of Radiobiology , Cancer Research Institute, Biomedical Research Centre SAS, Slovak Academy of Sciences , Bratislava , Slovakia.|c Institute of Physiology, Faculty of Medicine Comenius University , Bratislava , Slovakia.
authors.forename: Lenka
authors.initials: L
authors.lastname: Vokálová
authors.affiliation: a Laboratory of Radiobiology , Cancer Research Institute, Biomedical Research Centre SAS, Slovak Academy of Sciences , Bratislava , Slovakia.
authors.forename: Matúš
authors.initials: M
authors.lastname: Durdík
authors.affiliation: a Laboratory of Radiobiology , Cancer Research Institute, Biomedical Research Centre SAS, Slovak Academy of Sciences , Bratislava , Slovakia.
authors.forename: Eva
authors.initials: E
authors.lastname: Marková
authors.affiliation: a Laboratory of Radiobiology , Cancer Research Institute, Biomedical Research Centre SAS, Slovak Academy of Sciences , Bratislava , Slovakia.|d Laboratory of Radiobiology , General Physics Institute, Russian Academy of Science , Moscow , Russia.
authors.forename: Igor
authors.initials: I
authors.lastname: Belyaev
mesh_terms: D002478:Cells, Cultured; D004249:DNA Damage; D004307:Dose-Response Relationship, Radiation; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007090:Image Interpretation, Computer-Assisted / Q000379:methods; D008214:Lymphocytes / Q000473:pathology* / Q000528:radiation effects*; D008856:Microscopy, Fluorescence / Q000379:methods*; D011829:Radiation Dosage; D011839:Radiation, Ionizing; D012984:Software; D012986:Software Validation
keywords: DNA double-strand break; DNA repair foci; ionizing radiation