Ionizing radiation induced foci (IRIF) are considered the most sensitive indicator for DNA double-strand break (DSB) detection. Monitoring DSB induction by low doses of ionizing radiation is important due to the increasing exposure in the general population. cH2AX and 53BP1 are commonly used molecular markers for in situ IRIF assessment. Imaging flow cytometry (IFC) via ImageStream system provides a new opportunity in this field. We analyzed the formation of 53BP1, cH2AX foci and their co-localization induced by c-rays (2, 5, 10, 50, 200 cGy) in human lymphocytes using ImageStream and the automated microscopic system Metafer. We observed very similar sensitivity of both systems for the detection of endogenous and low-dose-induced IRIF. Statistically significant induction of cH2AX foci was found at doses of 2 and 10 cGy using ImageStream and Metafer, respectively. Statistically significant induction of 53BP1 foci was evident at doses !5 cGy when analyzed by IFC. Analysis of the colocalizing foci by ImageStream and Metafer showed statistical significance at doses !2 cGy, suggesting that foci co-localization is a sensitive parameter for DSB quantification. Assessment of cH2AX, 53BP1 foci and their co-localization by Metafer and ImageStream showed similar linear dose responses in the low-dose range up to 10 cGy, although IFC showed slightly better resolution for IRIF in this dose range. At higher doses, IFC underestimated IRIF numbers. Using the imaging ability of ImageStream, we introduced an optimized assay by gating cH2AX foci positive (with 1 or more cH2AX foci) and negative (cells without foci) cells. This assay resulted in statistically significant IRIF induction at doses ! 5cGy and a linear dose response up to 50 cGy. In conclusion, we provide evidence for the use of IFC as an accurate high throughput assay for the prompt detection and enumeration of endogenous and low-dose induced IRIF. V C 2015 International Society for Advancement of Cytometry Key terms imaging flow cytometry; ImageStream; Metafer; human lymphocytes; cH2AX; 53BP1; ionizing radiation DNA double strand breaks (DSB) are known as the most deleterious type of DNA damage induced by ionizing radiation and other genotoxic agents. Misrepaired or unrepaired DSB lead to genome rearrangements and contribute to carcinogenesis. Currently, the most sensitive and specific method for DSB analysis is enumeration of small repair centers formed at the locations of DSB. These centers are known as ionizing radiation induced foci (IRIF) or DNA repair foci. IRIF are dynamic structures containing thousands of copies of proteins involved in various aspects of DSB repair and DNA damage response. Phosphorylated histone H2AX variant (cH2AX) and p53 binding protein 1 (53BP1) are commonly accepted molecular markers of IRIF (1,2). Upon DSB induction, histone H2AX molecules become phosphorylated within

Despite widely accepted notion that many childhood leukemias are likely developed from hematopoietic stem/progenitor cells (HSPC) with pre-leukemic fusion genes (PFG) formed in embryonic/fetal development, the data on PFG incidence in newborns are contradictive. To provide a better understanding of a prenatal origin of leukemia, umbilical cord blood from 500 newborns was screened for the presence of the most frequent PFG associated with pediatric B-cell acute lymphoblastic leukemia. This screening revealed relatively high incidence of ETV6-RUNX1, BCR-ABL1 (p190) and MLL-AF4 at very low frequencies, averaging ~14 copies per 100,000 cells. We assume that most of these PFG might originate relatively late in embryonic/fetal development and will be eliminated later during postnatal development. The obtained results suggested that higher PFG copy numbers originating in specific time windows of the hematopoietic stem cell hierarchy may define a better prognostic tool for the assessment of leukemogenic potential. We have observed no significant effect of low-copy PFG on radiation-induced DNA damage response, accumulation of endogenous DNA double-stranded breaks, and apoptosis in either lymphocytes or HSPC. Imaging flow cytometry showed lower level of γH2AX foci in HSPC in comparison to lymphocytes suggesting better protection of HSPC from DNA damage.

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.