<abstract xmlns="http://www.w3.org/1999/xhtml">

<bold>Purpose</bold>: Ionizing radiation induced foci (IRIF) known also as DNA repair foci represent the most sensitive endpoint for assessing DNA double strand breaks (DSB). IRIF are usually visualized and enumerated with the aid of fluorescence microscopy using antibodies to γH2AX and 53BP1. Although several approaches and software packages were developed for the quantification of IRIF, not one of them was commonly accepted and inter-laboratory variability in the outputs was reported. In this study, the sensitization of Metafer software to counting also small appearing IRIF was validated.
<bold>Materials and Methods</bold>: Human lymphocytes were γ-irradiated at a dose of 2 Gy. The cells were fixed at 0.5, 1, 2, and 18 hours post-irradiation, permeabilized and IRIF were immunostained using appropriate antibodies. Cell images were acquired with the automatic Metafer system. Radiation-induced γH2AX and 53BP1 foci were enumerated using either manual counting (JCountPro program) or the Metafer software (after its classifier optimization has been done) and compared. The statistical analysis was performed using One-way ANOVA.
<bold>Results</bold>: The enumeration of 53BP1, γH2AX foci manually by JCountPro did not statistically significantly differ from the automatic one performed with the optimized Metafer classifier. A detailed step-by-step protocol of this successful optimization is described in this study.
<bold>Conclusions</bold>: We concluded that the Metafer software after the optimization was efficient in objectively enumerating IRIF, having a potential for usage in clinics and molecular epidemiology.
</abstract>

Purpose: Ionizing radiation induced foci (IRIF) known also as DNA repair foci represent the most sensitive endpoint for assessing DNA double strand breaks (DSB). IRIF are usually visualized and enumerated with the aid of fluorescence microscopy using antibodies to γH2AX and 53BP1. Although several approaches and software packages were developed for the quantification of IRIF, not one of them was commonly accepted and inter-laboratory variability in the outputs was reported. In this study, the sensitization of Metafer software to counting also small appearing IRIF was validated.
Materials and Methods: Human lymphocytes were γ-irradiated at a dose of 2 Gy. The cells were fixed at 0.5, 1, 2, and 18 hours post-irradiation, permeabilized and IRIF were immunostained using appropriate antibodies. Cell images were acquired with the automatic Metafer system. Radiation-induced γH2AX and 53BP1 foci were enumerated using either manual counting (JCountPro program) or the Metafer software (after its classifier optimization has been done) and compared. The statistical analysis was performed using One-way ANOVA.
Results: The enumeration of 53BP1, γH2AX foci manually by JCountPro did not statistically significantly differ from the automatic one performed with the optimized Metafer classifier. A detailed step-by-step protocol of this successful optimization is described in this study.
Conclusions: We concluded that the Metafer software after the optimization was efficient in objectively enumerating IRIF, having a potential for usage in clinics and molecular epidemiology.

Classifier Spot Count Optimization of Automated Fluorescent Slide Scanning System

Department of Laboratory Medicine, Faculty of Health Care

Department of Radiological Technology, Faculty of Health Care

Acta Medica Martiniana

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Purpose: Ionizing radiation induced foci (IRIF) known also as DNA repair foci represent the most sensitive endpoint for assessing DNA double strand breaks...

Classifier Spot Count Optimization of Automated Fluorescent Slide Scanning System

Pubblicato online: 09 mag 2022

Pagine: 24 - 33

Ricevuto: 01 feb 2022

Accettato: 24 feb 2022

DOI: https://doi.org/10.2478/acm-2022-0004

Parole chiave
DNA double-strand breaks, DNA repair foci, ionizing radiation

© 2022 Zastko Lucian et al., published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Classifier Spot Count Optimization of Automated Fluorescent Slide Scanning System

Pubblicato online: 09 mag 2022

Pagine: 24 - 33

Ricevuto: 01 feb 2022

Accettato: 24 feb 2022

DOI: https://doi.org/10.2478/acm-2022-0004

Parole chiaveDNA double-strand breaks, DNA repair foci, ionizing radiation

© 2022 Zastko Lucian et al., published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Parole chiave
DNA double-strand breaks, DNA repair foci, ionizing radiation