title: Fully automated analysis of chemically induced γH2AX foci in human peripheral blood mononuclear cells by indirect immunofluorescence.
abstract: Analysis of phosphorylated histone protein H2AX (γH2AX) foci is currently the most sensitive method to detect DNA double-strand breaks (DSB). This protein modification has the potential to become an individual biomarker of cellular stress, especially in the diagnosis and monitoring of neoplastic diseases. To make γH2AX foci analysis available as a routine screening method, different software approaches for automated immunofluorescence pattern evaluation have recently been developed. In this study, we used novel pattern recognition algorithms on the AKLIDES® platform to automatically analyze immunofluorescence images of γH2AX foci and compared the results with visual assessments. Dose- and time-dependent γH2AX foci formation was investigated in human peripheral blood mononuclear cells (PBMCs) treated with the chemotherapeutic drug etoposide (ETP). Moreover, the AKLIDES system was used to analyze the impact of different immunomodulatory reagents on γH2AX foci formation in PBMCs. Apart from γH2AX foci counting the use of novel pattern recognition algorithms allowed the measurement of their fluorescence intensity and size, as well as the analysis of overlapping γH2AX foci. The comparison of automated and manual foci quantification showed overall a good correlation. After ETP exposure, a clear dose-dependent increase of γH2AX foci formation was evident using the AKLIDES as well as Western blot analysis. Kinetic experiments on PBMCs incubated with 5 μM ETP demonstrated a peak in γH2AX foci formation after 4 to 8 h, while a removal of ETP resulted in a strong reduction of γH2AX foci after 1 to 4 h. In summary, this study demonstrated that the AKLIDES system can be used as an efficient automatic screening tool for γH2AX foci analysis by providing new evaluation features and facilitating the identification of drugs which induce or modulate DNA damage.
authors.affiliation: Institute of Molecular and Clinical Immunology, Otto-von-Guericke-University, Magdeburg, Germany.
authors.forename: Annika
authors.initials: A
authors.lastname: Willitzki
authors.forename: Sebastian
authors.initials: S
authors.lastname: Lorenz
authors.forename: Rico
authors.initials: R
authors.lastname: Hiemann
authors.forename: Karina
authors.initials: K
authors.lastname: Guttek
authors.forename: Alexander
authors.initials: A
authors.lastname: Goihl
authors.forename: Roland
authors.initials: R
authors.lastname: Hartig
authors.forename: Karsten
authors.initials: K
authors.lastname: Conrad
authors.forename: Eugen
authors.initials: E
authors.lastname: Feist
authors.forename: Ulrich
authors.initials: U
authors.lastname: Sack
authors.forename: Peter
authors.initials: P
authors.lastname: Schierack
authors.forename: Lisa
authors.initials: L
authors.lastname: Heiserich
authors.forename: Caroline
authors.initials: C
authors.lastname: Eberle
authors.forename: Vanessa
authors.initials: V
authors.lastname: Peters
authors.forename: Dirk
authors.initials: D
authors.lastname: Roggenbuck
authors.forename: Dirk
authors.initials: D
authors.lastname: Reinhold
mesh_terms: D053903:DNA Breaks, Double-Stranded / Q000187:drug effects; D004249:DNA Damage / Q000187:drug effects / Q000235:genetics; D005047:Etoposide / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D006657:Histones / Q000097:blood / Q000302:isolation & purification*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000187:drug effects* / Q000276:immunology
chemical_list: C468783:H2AX protein, human; D006657:Histones; D005047:Etoposide
keywords: DNA double-strand breaks; automated microscopy; etoposide; human PBMCs; image analysis; γH2AX foci