journal article
Open Access
Feb 24, 2026
Inter-laboratory Evaluation of γH2AX/53BP1 DNA Double-strand Break Foci Assays in Human Lymphocytes Under Low-dose Irradiation: Implications for Calyculin A
Abstract
Background: Accurate biodosimetry is essential for effective radiological triage, precise clinical monitoring, and assessment of risks from diagnostic exposures. Immunofluorescent detection of phosphorylated histone H2AX (γH2AX) and p53-binding protein 1 (53BP1) DNA double-strand break repair foci provides high sensitivity for radiation dose assessment within the first hours after exposure. However, inter-laboratory reproducibility of γH2AX/53BP1-based biodosimetry remains limited, and the contribution of pharmacological modifiers is unresolved. Methods: Here, we conducted an inter-laboratory comparison of in vitro radiation dose–response relationships of foci yields measured in cryopreserved umbilical cord blood lymphocytes (UCBLs) and freshly isolated peripheral blood lymphocytes (PBLs) from healthy donors using fluorescent microscopy with emphasis on workflow harmonization and reproducibility across laboratories. Results: Under low-dose γ irradiation, both UCBLs and PBLs exhibited a strong linear, dose-dependent induction of γH2AX, 53BP1, and co-localized foci, with co-localization emerging as the most sensitive endpoint. γH2AX pan-nuclear staining was observed exclusively in UCBLs and functioned as a distinct endpoint under the examined conditions. Under harmonized low-dose conditions, calyculin A at a non-toxic concentration of 1 nM did not provide measurable stabilization or enhancement of ionizing radiation-induced foci (IRIF) yields. Although IRIF yields differed between the two laboratories, dose–response slopes were highly concordant, demonstrating reproducibility under harmonized experimental conditions. Conclusions: These findings demonstrate that inter-laboratory reproducibility of γH2AX/53BP1-based biodosimetry is achieved primarily through disciplined workflow harmonization rather than through pharmacological enhancement. By reinforcing assay reproducibility and biological consistency, this work supports the translational applicability of IRIF-based biodosimetry for broader application in radiation exposure assessment.
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- Published
- Feb 24, 2026
- Vol/Issue
- 31(2)
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Funding
Recovery and Resilience Plan of the Slovakia
Award: CU-ESG-2024-05-PD
Grant Agency of the Slovakia
Award: 2/0140/23
Cite This Article
Lucián Zastko, Lukáš Jakl, Jana Kružliaková, et al. (2026). Inter-laboratory Evaluation of γH2AX/53BP1 DNA Double-strand Break Foci Assays in Human Lymphocytes Under Low-dose Irradiation: Implications for Calyculin A. Frontiers in Bioscience-Landmark, 31(2). https://doi.org/10.31083/fbl47070
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