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.

Graphical abstract

Keywords

DNA double-strand breaks / DNA damage response / H2AX histone / 53BP1 protein / calyculin A / biodosimetry / lymphocytes / phosphatase inhibitors / ionizing radiation / immunofluorescence

1. Introduction

Accidental or deliberate exposure to ionizing radiation (IR) remains a serious public health concern, particularly in the context of nuclear accidents, radiological emergencies, and increasing geopolitical instability. Consequently, rapid, sensitive, and reliable biodosimetric tools are indispensable for population triage following radiological events [1]. Beyond emergency response, biodosimetry is increasingly applied in clinical practice, for example, for individualization of radiotherapy and monitoring of patient responses to IR [2, 3]. In addition, sensitive biodosimetric approaches are required for the detection and evaluation of low-dose radiation exposures originating from diagnostic imaging procedures, including conventional X-rays [4, 5], computed tomography [6, 7], nuclear medicine [8, 9, 10], and other radiological examinations [11, 12, 13, 14, 15, 16].

Among currently available assays, immunofluorescent detection of DNA double-strand break (DSB) repair foci, particularly those marked by phosphorylated histone H2AX (\gammaH2AX) and p53-binding protein 1 (53BP1), represents one of the most sensitive approaches for assessing radiation-induced DNA damage at the single-cell level. These assays exhibit linear dose-response relationships even at low radiation doses, making them attractive candidates for biodosimetric applications [17, 18, 19, 20, 21, 22]. However, despite their analytical sensitivity, several challenges limit their broader implementation, including inter-laboratory variability, rapid dephosphorylation of \gammaH2AX leading to signal loss over time, and interference from apoptotic \gammaH2AX pan-nuclear staining that can confound interpretation by mimicking DNA DSB-induced foci [23, 24, 25, 26, 27]. Consequently, the principal limitation for the broader deployment of \gammaH2AX/53BP1-based biodosimetry is no longer sensitivity per se, but the lack of systematic validation of assay reproducibility under harmonized low-dose protocols.

In an effort to overcome these limitations, pharmacological modulation of DNA damage signaling pathways has been explored [28]. Among several phosphatase inhibitors, including fostriecin and okadaic acid, calyculin A (cal A) has attracted particular attention. Cal A is a potent inhibitor of serine/threonine protein phosphatases 1 and 2A (PP1/PP2A) [29, 30] and has been reported to stabilize \gammaH2AX foci by inhibiting dephosphorylation events [31, 32]. Originally described as a strong inducer of premature chromosome condensation in peripheral blood lymphocytes (PBL) [33], cal A has subsequently been proposed as a modifier of \gammaH2AX signaling, although reported effects vary substantially across experimental workflows. Indeed, several studies reported prolonged persistence of \gammaH2AX foci following irradiation in the presence of cal A, facilitating dose estimation particularly in the low-dose range [31, 34]. Nevertheless, the reported effects of cal A remain highly context-dependent, complicating its routine use in biodosimetric applications [25, 35, 36]. Such discrepancies may reflect differences in experimental conditions, including cell type, cryopreservation protocols, irradiation settings, and subtle methodological variations.

Beyond practical biodosimetric implications, cal A interferes with fundamental DNA damage response mechanisms. Through inhibition of serine/threonine phosphatases, cal A can alter chromatin-associated signaling pathways, complicating the interpretation of radiation-induced foci (IRIF)-based readouts. In particular, apoptosis-associated \gammaH2AX pan-staining, a recognized marker of early apoptotic processes [37, 38], can obscure or mimic genuine IRIF, complicating accurate DSB quantification. Moreover, distinct lymphocyte subsets such as umbilical cord blood lymphocytes (UCBL), hematopoietic stem cells, and PBL, differ in their DNA damage responses and sensitivity to apoptosis, further challenging reproducibility across studies [23, 36]. In this context, cal A is not considered here as an optimization strategy, but rather as a deliberate stress test to probe the biological consistency and robustness of \gammaH2AX/53BP1 foci assays under harmonized experimental conditions.

Our previous work directly examined the effects of cal A on \gammaH2AX/53BP1 foci formation in UCBL exposed to low and therapeutic doses in vitro [36]. That study demonstrated no stabilizing effect at either 1 or 10 nM cal A, while the higher concentration induced pronounced cytotoxicity and apoptotic signaling. Building on these findings, the present study was designed as an inter-laboratory evaluation of \gammaH2AX/53BP1-based IRIF assays under harmonized low-dose workflows, using cal A as a test case to assess whether pharmacological modulation improves reproducibility. UCBL samples from a single provider were analyzed independently in two laboratories using identical cell processing, handling, irradiation protocols and cal A from the same supplier, with foci kinetics followed for up to 2 h post-irradiation. To address potential artifacts introduced by cryopreservation, analyses were extended in one laboratory to freshly isolated PBL, in which IRIF kinetics were examined for up to 6 h after irradiation. The study focused primarily on biologically and clinically relevant low-dose ranges, with higher doses included in PBL to explore dose-dependent effects under conditions free of cryopreservation stress. Cal A was restricted to a non-toxic concentration of 1 nM to assess whether foci stabilization could be achieved without compromising cellular viability. By focusing on inter-laboratory reproducibility under fully harmonized low-dose workflows, this work directly addresses a critical barrier to the standardization and translational readiness of \gammaH2AX/53BP1-based biodosimetry.

2. Materials and Methods

All experimental procedures were designed to ensure maximal reproducibility between laboratories. Wherever possible, identical reagents, suppliers, incubation times, irradiation conditions, and image acquisition parameters were used. Methodological details are provided to explicitly document harmonization steps critical for inter-laboratory comparison rather than to serve as a procedural manual.

2.1 Chemicals

Reagent grade chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA), Merck KGaA (Darmstadt, Germany) and Life Technologies (Carlsbad, CA, USA). Cal A was purchased from Abcam Biochemicals (Cambridge, UK) and dissolved in dimethyl sulfoxide (DMSO; SERVA Electrophoresis GmbH, Heidelberg, Germany).

2.2 Cells

UCB mononuclear cells (MNC) were extracted as previously described [27] from 3 healthy newborns after full-term pregnancies and provided by Eurocord-Slovakia, Bratislava, Slovakia. In Laboratory 1, cryopreserved UCB MNC from three donors were thawed using standard protocols and cultured in RPMI 1640-based basal medium supplemented with fetal bovine serum and antibiotics. Monocytes were removed by short-term adherence.

In Laboratory 2, UCB MNC from three independent donors obtained under identical inclusion criteria were analyzed as part of a previously published dataset [36] and used here exclusively for inter-laboratory comparison at matched irradiation doses and time points. Except for fixation timing, identical cell handling and processing steps were applied in both laboratories.

Peripheral blood (PB) MNC were isolated from six healthy adult donors by density-gradient centrifugation as previously described [39]. Monocytes were removed by adherence, yielding predominantly lymphocyte populations. Cell viability exceeded 95% in all experiments as assessed by Trypan blue exclusion.

Cell Authenticity and Quality Control

All experiments were performed using primary human mononuclear cells isolated directly from umbilical cord blood or peripheral blood of healthy donors. These cells were not cultured or passaged and were used immediately after isolation (PBL) or after standardized cryopreservation and thawing (UCBL). Cell identity was ensured by established lymphocyte isolation procedures, donor documentation, and ethical approval. Cell morphology was examined by light microscopy, and cell viability exceeded 95% as assessed by trypan blue exclusion. As the study did not involve immortalized cell lines or long-term cell culture, STR profiling and routine mycoplasma testing were not applicable.

2.3 Cell Treatment

Following equilibration, lymphocytes were divided into three treatment groups: cal A (1 nM), DMSO vehicle control (0.1%), and untreated controls. Cells were incubated on ice for approximately 40 min prior to irradiation to synchronize handling between laboratories. To avoid prolonged exposure-related toxicity, cal A and DMSO were added immediately after irradiation in both laboratories, consistent with our previous observations [36].

2.4 Irradiation

Cells were irradiated on ice using acute \gamma-rays at a dose rate of approximately 45 cGy/min. UCBL were exposed to doses of 10, 50, 100, 500, and 1000 mGy, while PBL were irradiated at 10, 100, and 1000 mGy. Irradiations were performed using ⁶⁰Co sources (Theratron Elite 100, MDS Nordion, Canada; or TERAGAM® K-02, UJP Praha, Czech Republic). Dose deviations did not exceed 5%. After irradiation, cells were rapidly warmed to 37 °C and incubated under standard culture conditions until fixation.

2.5 Immunofluorescence and Image Acquisition

At defined time points post-irradiation (0.5, 1, and 2 h for UCBL; 2, 4, and 6 h for PBL), cells were cytocentrifuged onto poly-L-lysine-coated slides, fixed with paraformaldehyde, permeabilized, and blocked according to standard protocols. \gammaH2AX and 53BP1 were detected using well-characterized primary antibodies provided by Novus Biologicals, Abingdon, UK - monoclonal \gammaH2AX anti-mouse (1:800, NB100-78356) and polyclonal 53BP1 anti-rabbit (1:1000, NB100-304); followed by Alexa Fluor-conjugated secondary antibodies (Invitrogen Molecular Probes, Life Technologies, OR, USA) - 488 IgG (H+L) polyclonal anti-rabbit (1:200, A11029) and 555 IgG (H+L) monoclonal anti-mouse (1:200, 21429). Nuclei were counterstained with DAPI-containing antifade medium.

Image acquisition was performed in both laboratories using Metafer Slide Scanning Systems V 3.9 (MetaSystems GmbH, Altlussheim, Germany) coupled to Zeiss Axio Imager fluorescence microscopes (Carl Zeiss Microscopy GmbH, Gottingen, Germany) under identical acquisition settings. For each condition, at least 600 cells were analyzed. Automated scoring of \gammaH2AX and 53BP1 foci was conducted as previously described [16, 40] and verified by manual inspection. Co-localized foci were defined by spatial overlap within 0.5 µm. \gammaH2AX pan-nuclear staining was quantified in UCBL as an apoptosis-associated endpoint but was not observed in PBL.

2.6 Statistical Analysis

The yields of foci (Y) were expressed per cell. At each experimental dose point in cells of each blood donor the foci-per-cell distribution was checked for compliance with the Poisson model by \chi² test. When individual data were pooled, standard errors (SE), standard deviations (SD) or \pm0.95 confidence intervals (CI) for the mean foci yield were calculated from the dispersion of donors’ individual values within the group. The group data were analyzed using factorial analysis of variance (ANOVA) together with control for the false discovery rate (FDR). For pooled datasets fitting a normal distribution and involving a single categorical independent variable, comparisons between groups were performed by one-way ANOVA, followed by Fisher’s LSD post hoc test. Associations between groups were assessed using the Spearman rank-order correlation test (ROC), while correlations between independent groups were evaluated with the Mann-Whitney U test. These statistical analyses were performed with Statistica 8.0 software (StatSoft Inc., Tulsa, OK, USA). Differences were considered statistically significant at p 0.05.

Dose-response relationships for the foci yield were fitted by the iteratively reweighted least squares method to the linear model:

Y = c + \alpha \times D,

where Y - foci yield per cell; c - the background foci level; \alpha - linear coefficient representing a foci induction rate per unit dose; D - radiation dose in Gy. Goodness of fit was assessed by a linear correlation coefficient (r). The coefficient \alpha was compared between blood donors or laboratories as normally distributed parameter, using the unpaired Student’s t-test. The statistical software package Dose Estimate_v.5.1 [41] was used to perform foci distribution testing and dose-response curve fitting; the latter was carried out assuming Poisson foci-per-cell distribution for calculations of coefficient errors.

3. Results

3.1 Dose-response Characteristics and Assay Sensitivity in UCBL Across Laboratories

For each UCBL donor, the foci-per-cell distributions in unirradiated and irradiated samples were consistent with Poisson statistics, as expected for homogeneous exposure to low-LET radiation (data not shown). Donor-specific regression coefficients are summarized in Supplementary Table 1, and pooled regressions for each laboratory are shown in Table 1 and Fig. 1.

Across both laboratories, \gammaH2AX, 53BP1, and \gammaH2AX/53BP1 co-localized foci displayed strong linear dose-response relationships within the investigated dose range, with excellent goodness-of-fit to the linear model (r \ge 0.94 for all endpoints; Table 1). In Laboratory 1 (UCBL fixed 0.5 h post-irradiation), all three endpoints were significantly elevated already at 50 mGy relative to unirradiated controls, indicating early sensitivity above endogenous foci levels. In Laboratory 2 (UCBL fixed 2 h post-irradiation), co-localized foci remained the most sensitive endpoint (significant induction at 50 mGy), while separate \gammaH2AX and 53BP1 foci reached statistical significance at 100 mGy. This time-dependent shift in the apparent sensitivity pattern is consistent with the known differences in foci kinetics during the first post-exposure hours.

Importantly, because the two laboratories quantified foci at different post-irradiation times (0.5 h vs 2 h), absolute foci yields between laboratories are not interpreted as direct head-to-head “inter-laboratory disagreement”. Instead, the inter-laboratory comparison is focused on (i) the reproducibility of the dose-response form (linearity), (ii) the similarity of regression slopes (\alpha), and (iii) the consistency of conclusions regarding cal A effects under harmonized processing and scoring workflows. In this context, pooled regression analysis indicated highly comparable \gammaH2AX induction rates per unit dose between laboratories, whereas \alpha values for 53BP1 and co-localized foci were numerically lower in Laboratory 2; these differences did not reach statistical significance (Table 1). Overall, the narrow range of \alpha values supports reproducible dose-response relationships despite differences in fixation time and donor sets.

Together, these results establish that the \gammaH2AX/53BP1 IRIF assay provides robust, quantitative, and highly sensitive detection of radiation-induced DNA damage in UCBL within the low-dose range, and that dose-response relationships remain reproducible across independent laboratories when protocols are workflow-aligned and key steps are harmonized.

3.2 UCBL: Effects of Dose, Treatment, and Post-irradiation Time in Laboratory 1

Laboratory 1 assessed the effects of irradiation dose (0, 10, 50, 100 mGy), treatment condition (cal A, DMSO vehicle, untreated), and post-irradiation time (0.5, 1, 2 h) on \gammaH2AX, 53BP1, and co-localized foci, as well as on the frequency of \gammaH2AX pan-nuclear staining in UCBL (Table 2). Multivariate ANOVA identified irradiation dose as the only significant factor across foci endpoints. Treatment condition and time within the first 2 h did not reach statistical significance, indicating that foci levels were primarily determined by dose under the applied experimental conditions.

Univariate testing confirmed a highly significant dose dependence for \gammaH2AX, 53BP1, and co-localized foci (all FDR-adjusted p 0.00001; Table 2). In contrast, neither treatment (cal A or DMSO) nor post-irradiation time significantly altered foci yields. \gammaH2AX pan-staining showed no significant dependence on dose, treatment, or time within this experimental window (Table 2), supporting its interpretation as a distinct endpoint not tracking with repair foci formation under low-dose conditions.

Vehicle Control and Cal A Effect in Laboratory 1

Untreated UCBL were compared with matched DMSO-treated samples at identical doses and time points. No significant differences were detected, validating DMSO as an appropriate vehicle control. For clarity, untreated data were omitted from Fig. 2, which focuses on direct cal A vs DMSO comparisons. As shown in Fig. 2, foci yields are presented across multiple doses, time points, and treatments to illustrate overall assay behavior under the experimental design; no direct quantitative comparison between post-irradiation time points is intended.

As shown in Fig. 2, \gammaH2AX, 53BP1, and co-localized foci increased with irradiation dose at all time points (0.5, 1, 2 h). Critically, cal A at 1 nM did not produce a systematic increase in foci yield or altered kinetics relative to DMSO controls at any dose or time point examined. \gammaH2AX pan-staining remained low and comparable between treatments. Strong positive correlations were observed among \gammaH2AX, 53BP1, and co-localized foci, whereas pan-staining did not correlate with foci-based endpoints, consistent with an apoptosis-associated signal distinct from DSB repair foci formation. Representative immunofluorescence images are provided in Fig. 3.

3.3 Inter-laboratory Comparison of UCBL at the Matched 2 h Time Point

To enable a time-matched inter-laboratory comparison, we evaluated UCBL data acquired at 2 h post-irradiation (Laboratory 1:2 h time point from the present study; Laboratory 2:2 h dataset reported previously and included here specifically for comparative evaluation). A three-way factorial ANOVA (Laboratory, Treatment, Dose) was performed (Table 3).

A significant laboratory factor was detected for \gammaH2AX, 53BP1, and co-localized foci (all p 0.00001), indicating systematic differences in absolute foci yields between laboratories. In contrast, no laboratory effect was observed for pan-staining (p = 0.234). The treatment factor (1 nM cal A vs 0.1% DMSO) was not significant for any foci endpoint (all p > 0.3). For pan-staining, the treatment effect showed a near-threshold trend (p = 0.078) that did not remain significant after correction, suggesting at most a weak influence under these conditions. Irradiation dose remained the dominant determinant of foci induction (all foci endpoints p 0.00001 after FDR adjustment), while pan-staining again showed no dose dependence (p = 0.546).

Fig. 4 visualizes the 2 h comparison and confirms three core outcomes: (i) robust dose-dependent induction of \gammaH2AX, 53BP1, and co-localized foci in both laboratories, (ii) systematic inter-laboratory offsets in absolute foci yields, and (iii) the absence of a detectable stabilizing effect of 1 nM cal A relative to DMSO. Correlation analyses supported high internal consistency among the three foci endpoints in each laboratory, while pan-staining remained uncorrelated. Non-parametric Mann-Whitney testing confirmed significant inter-laboratory differences in absolute foci counts for all three foci endpoints, but not for pan-staining.

Taken together, the time-matched comparison shows that while absolute yields can differ between laboratories, dose-response relationships and the qualitative treatment conclusion (no cal A stabilization at 1 nM) are reproducible under harmonized workflows.

3.4 PBL Responses to Dose, Treatment, and Time (Laboratory 2)

To assess whether findings in cryopreserved UCBL generalize to fresh adult lymphocytes and to reduce potential freeze-thaw confounding, peripheral blood lymphocytes (PBL) were analyzed in Laboratory 2 across doses and time points (Table 4; Fig. 5). Factorial ANOVA demonstrated highly significant dose effects for all foci endpoints at 2 h (FDR-adjusted p 0.001) and similarly when including extended time points (2, 4, 6 h) at 0 and 100 mGy (FDR-adjusted p 0.005). Treatment and post-irradiation time did not significantly affect foci levels (FDR-adjusted p > 0.3 and p > 0.4, respectively).

Notably, \gammaH2AX pan-nuclear staining was not detected in PBL under any tested condition, in contrast to UCBL where pan-staining was consistently measurable. This difference indicates that pan-staining is not an inherent feature of lymphocytes under low-dose irradiation but depends on cell system and/or pre-analytical handling such as cryopreservation.

Vehicle Control Validation and cal A Effect in PBL

Inclusion of untreated irradiated PBL alongside DMSO- and cal A-treated samples confirmed that DMSO did not alter foci induction compared with untreated controls, supporting its use as vehicle control (Fig. 5). At low doses (0, 10, 100 mGy) and across time points (2, 4, 6 h), cal A did not significantly modify foci induction relative to DMSO or untreated controls.

A significant treatment-dependent effect emerged only at the highest dose tested: at 1000 mGy, co-localized \gammaH2AX/53BP1 foci were significantly reduced in cal A-treated samples compared with DMSO controls (p 0.0014; Fig. 5). This observation indicates that phosphatase inhibition does not universally preserve repair foci and, under high-dose conditions, may be associated with reduced co-localization persistence.

3.5 Cell-type-specific Response Patterns: PBL Versus UCBL Across Laboratories

Under comparable experimental conditions, both UCBL (in both laboratories) and PBL exhibited dose-dependent induction of \gammaH2AX, 53BP1, and co-localized foci. However, two consistent differences were observed. First, \gammaH2AX pan-staining was present in UCBL but absent in PBL, suggesting that apoptotic \gammaH2AX signaling is influenced by cell type and/or cryopreservation-related pre-analytical factors. Second, cal A at 1 nM did not produce stabilizing effects in either system across the low-dose range; the only statistically significant treatment-related effect was a reduction of co-localized foci in PBL at 1000 mGy.

Overall, these results support high robustness of IRIF dose-response relationships under harmonized workflows while indicating that the impact of cal A is strongly context-dependent and not reliably stabilizing under the tested conditions.

4. Discussion

4.1 Summary of Principal Findings

Across two independent laboratories, \gammaH2AX, 53BP1, and \gammaH2AX/53BP1 co-localized foci in UCBL showed reproducible linear dose-response relationships under harmonized low-dose workflows. \gammaH2AX pan-nuclear staining represented a biologically distinct endpoint, uncoupled from dose-response behavior and repair foci formation under the examined low-dose conditions. Importantly, under harmonized low-dose conditions, cal A (1 nM) did not provide any measurable stabilization or enhancement of IRIF yields. Where data are shown across multiple post-irradiation times, comparisons are interpreted qualitatively and within each time point, rather than as direct kinetic analyses.

Although absolute IRIF yields differed between laboratories, the form and linearity of the dose-response relationships were preserved across endpoints. Crucially, the qualitative conclusion regarding cal A was consistent across laboratories. Although these findings confirm the expected low-dose sensitivity of \gammaH2AX/53BP1 foci induction, the principal contribution of this study lies in demonstrating that harmonized experimental workflows yield reproducible dose-response relationships across independent laboratories. These findings show that inter-laboratory reproducibility of IRIF-based biodosimetry depends primarily on workflow harmonization rather than on pharmacological modulation of DNA damage signaling.

Analysis of freshly isolated PBL served as a within-laboratory control to assess whether conclusions derived from cryopreserved UCBL were workflow-dependent. PBL showed consistent dose-dependent induction of all IRIF endpoints, with foci yields at 100 mGy exceeding those observed in UCBL from Laboratory 1 and closely matching UCBL data from Laboratory 2. Importantly, \gammaH2AX pan-staining was absent in PBL under all experimental conditions, underscoring system-dependent differences between UCBL and PBL under the examined conditions and minimizing apoptosis-related confounding in fresh cells. At the highest dose tested (1000 mGy), cal A treatment in PBL was associated with a reduction in \gammaH2AX/53BP1 co-localized foci relative to DMSO controls, a pattern consistent with altered repair kinetics or apoptosis-related chromatin changes rather than foci stabilization. Under the tested low-dose, early post-irradiation conditions, this study rules out cal A as a generally applicable strategy for improving IRIF-based biodosimetric robustness.

4.2 Positioning Within the Literature

Phosphatase inhibition by cal A has been reported to affect \gammaH2AX signaling in several experimental systems, although with substantial context dependence [32, 34]. In adult PBL, Kuefner et al. (2013) [31] reported prolonged \gammaH2AX foci persistence after low-dose irradiation in the presence of cal A, while effects on 53BP1 were limited. Importantly, these experiments differed from the present study in post-irradiation timing, workflow structure, and analytical endpoints. By contrast, under harmonized low-dose workflows optimized for early biodosimetric readout, the present study did not detect a consistent stabilizing effect of calyculin A on \gammaH2AX or 53BP1 foci. These discrepancies are more plausibly explained by differences in workflow design, post-irradiation timing, dose range, and analytical thresholds than by cal A efficacy per se. Recent reviews emphasize that such context dependency is a central challenge in biodosimetry assay standardization [17, 42].

The PBL dataset was included to test the internal consistency of conclusions under conditions free of cryopreservation-related stress. While UCBL showed no cal A effect across the examined dose range, PBL displayed a reduction in \gammaH2AX/53BP1 co-localization at high dose under cal A treatment. This observation supports the notion that cal A’s impact depends not only on timing and workflow but also on intrinsic biological properties of the cell system. The results do not contradict earlier reports of cal A effects, but delimit their applicability: under rapid, harmonized low-dose biodosimetry workflows, pharmacological phosphatase inhibition does not improve assay robustness.

Comparison With Our Previous Study

The present findings directly extend and reinforce our earlier observations in UCBL [36]. In that study, \gammaH2AX, 53BP1, and co-localized foci exhibited highly linear dose-response relationships, with co-localization representing the most sensitive endpoint. The current inter-laboratory design reproduces these patterns and further demonstrates that sensitivity and linearity are preserved across laboratories and fixation times.

Inclusion of freshly isolated PBL adds an important dimension, indicating that donor variability, fixation timing, and cryopreservation stress contribute to absolute yield differences [43, 44, 45].

Across both studies, DMSO was confirmed as a neutral vehicle control, and cal A at 1 nM consistently failed to stabilize IRIF. Higher concentrations previously shown to induce cytotoxicity and apoptosis [36] were deliberately excluded from the present comparison. Together, these datasets converge on a coherent interpretation in which low, non-toxic concentrations of cal A do not measurably enhance IRIF stability, whereas higher concentrations trigger apoptosis-related chromatin alterations that confound biodosimetric readouts.

4.3 Inter-laboratory Variability and Methodological Robustness

Systematic offsets in absolute IRIF yields between laboratories are a well-recognized feature of multicenter biodosimetry studies and do not, by themselves, indicate methodological failure [15, 17, 46]. In the present study, such offsets did not alter dose-response linearity or qualitative treatment conclusions. These findings are directly relevant to ongoing biodosimetry standardization efforts within international networks such as RENEB and EURADOS, which increasingly prioritize workflow harmonization over absolute inter-laboratory yield matching. Accordingly, the objective of inter-laboratory comparison in this study was not to eliminate absolute yield differences, but to determine whether harmonized workflows preserve interpretable and reproducible dose-response behavior. Recent methodological analyses converge on the same conclusion: reproducibility in IRIF-based biodosimetry is achieved through disciplined workflow alignment rather than through incremental assay sensitivity gains [47, 48].

4.4 Biological Interpretation and Implications

Consistent correlations among \gammaH2AX, 53BP1, and co-localized foci support co-localization as the most robust readout for low-dose DSB detection under harmonized assay conditions. By contrast, \gammaH2AX pan-nuclear staining did not track with IRIF-based endpoints and should be interpreted separately from repair-associated foci under low-dose conditions [16, 26, 27, 37, 38]. This distinction is particularly relevant when evaluating cal A, as phosphatase inhibition can alter chromatin-associated signaling without improving repair foci-based readouts.

Standardized IRIF assays without pharmacological modifiers provide reliable and interpretable dose-response information in the low-dose range relevant for biodosimetry [2, 6, 49, 50]. While cal A remains useful in specific radiobiological applications, such as premature chromosome condensation assays [51, 52, 53, 54], its routine use as an enhancer of IRIF-based biodosimetry is not supported under harmonized low-dose workflows.

Future work should focus on validating IRIF-based biodosimetry under realistic operational constraints, including delayed processing and inter-operator variability, rather than on pharmacological signal amplification [55]. From a translational perspective, these findings argue for abandoning cal A as a default ‘fix’ for inter-laboratory variability and instead prioritizing disciplined workflow harmonization.

5. Conclusions

This study provides an inter-laboratory evaluation of \gammaH2AX/53BP1-based IRIF responses of human normal G₀ lymphocytes to low-dose IR under harmonized low-dose biodosimetry workflows. By integrating analyses of cryopreserved UCBL and freshly isolated PBL, the study separates workflow-dependent effects from biologically interpretable IRIF responses.

Our results demonstrate that \gammaH2AX, 53BP1, and co-localized foci exhibit robust, linear dose-response relationships across both systems, while \gammaH2AX pan-staining represents a distinct apoptosis-associated endpoint. At a non-toxic concentration of 1 nM, cal A does not provide a consistent benefit for IRIF-based biodosimetry under the tested low-dose conditions. Under higher-dose conditions, phosphatase inhibition altered IRIF patterns without improving interpretability for biodosimetric purposes.

Collectively, these findings indicate that cal A should not be regarded as a default enhancer of \gammaH2AX/53BP1-based biodosimetry. Instead, its effects are context-dependent, influenced by cell type, irradiation dose, and pre-analytical workflow. Under harmonized and timely laboratory conditions, cal A offers no measurable advantage at non-toxic concentrations.

By demonstrating reproducibility across laboratories and biological systems, this work strengthens confidence in IRIF-based biodosimetry as a standardized and translatable tool. These results further support integration of IRIF-based biodosimetry into automated and predictive scoring platforms for radiation preparedness and clinical triage. Recent integrative analyses further underscore that such progress toward automation and predictive biodosimetry critically depends on rigorously validated, reproducible experimental foundations rather than on incremental assay sensitivity gains alone. Instead, reproducibility of IRIF-based biodosimetry is best achieved through disciplined workflow harmonization rather than through pharmacological intervention.

Availability of Data and Materials

The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.

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Funding

Recovery and Resilience Plan of the Slovakia(CU-ESG-2024-05-PD)

Grant Agency of the Slovakia (VEGA)(2/0140/23)