Computational analysis of the number, area and density of γ-H2AX foci in breast cancer cells exposed to<sup>111</sup>In-DTPA-hEGF or γ-rays using Image-J software
Bibliographic record
Abstract
Zhongli Caia, Katherine A. Vallisb & Raymond M. Reilly PhDacd*a Department of Pharmaceutical Sciences, University of Torontob Radiation Oncology and Biology, University of Oxford, Oxford, UKc Toronto General Research Institute, University Health Network, Torontod Department of Medical Imaging, University of Toronto, Toronto, Ontario, Canada† Correspondence: Raymond M. Reilly, Leslie Dan Faculty of Pharmacy, University of Toronto, 144 College Street, Toronto, Ontario, Canada, M5S 3M2 raymond.reilly@utoronto.caPurpose: To develop a simple method for the quantification of γ-H2AX focus number, density and size.Methods: MDA-MB-468 human breast cancer cells were treated overnight with 111In-diethylenetriaminepentaacetic acid human epidermal growth factor (111In-DTPA-hEGF, 0–142 kBq/pmol) or exposed to γ-radiation to induce DNA double strand breaks (DSB). DNA DSB formation was evaluated by detection of phosphorylated histone H2AX on serine 139 (γ-H2AX) using immunofluorescence. Confocal microscopy was used to capture images of γ-H2AX foci and cell nuclei. Image-J software with customized macros was used to quantify γ-H2AX foci.Results: The number of γ-H2AX foci per nucleus scored using Image-J correlated strongly with the number scored using direct visual confirmation (coefficient of determination, R2 = 0.950; 60 nuclei scored). The mean density (grayscale values per pixel), area and integrated density (IntDen) of individual foci increased linearly as the specific radioactivity (SR) increased up to 67 kBq/pmol (R2 values of 0.826, 0.964, 0.978, respectively). The mean number of foci per nucleus, the combined area of γ-H2AX foci per nucleus and the IntDen per nucleus also increased linearly with SR, giving R2 values of 0.926, 0.974 and 0.983, respectively. Similar linear relationships were observed with the γ-ray absorbed dose up to 3.0 Gy.Conclusions: The density, area and IntDen of individual foci, as well as the number of γ-H2AX foci, total focus area and IntDen per nucleus were successfully quantified using Image-J with customized macros.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.001 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.001 |
| Bibliometrics | 0.001 | 0.001 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.001 | 0.000 |
| Open science | 0.001 | 0.000 |
| Research integrity | 0.001 | 0.000 |
| Insufficient payload (model declined to judge) | 0.003 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".