Automation of the Cytokinesis-Block Micronucleus Assay Using Imaging Flow Cytometry for High Throughput Radiation Biodosimetry
Bibliographic record
Abstract
This thesis contains three manuscripts that, when taken together as a whole, outline the development, optimization and validation of automating the CBMN assay using imaging flow cytometry (FCM) for applications in radiation biodosimetry.Each manuscript, presented as a separate chapter in this thesis, has been expanded upon to include more details regarding the rationale, experimental methods and pertinent results.Chapter 1 contains a general introduction to mass casualty events involving radiation, the biological and cellular effects of ionizing radiation, radiation biodosimetry and both traditional and imaging FCM.It also discusses the rationale for high-throughput automated methods for radiation biodosimetry.Chapter 2 presents background information on the CBMN assay and standard protocols and procedures as applied to radiation biodosimetry.Also presented is an introduction to the principles and concepts of both traditional and imaging flow cytometry.Chapter 3 is based on the manuscript entitled "Automated analysis of the cytokinesisblock micronucleus assay for radiation biodosimetry using imaging flow cytometry" published in Radiation and Environmental Biophysics (53(2), 273-282, 2014).Additional sections describing the procedure used to stain cells for DNA content, the use of cell surface markers and cytoplasm stains as well a more detailed description of the masking strategy used in IDEAS® are included.Chapter 4 is based on the manuscript entitled "Multi-Parameter Dose Estimations in Radiation Biodosimetry using the Automated Cytokinesis-Block Micronucleus Assay with Imaging Flow Cytometry" published in Cytometry Part A (85(10), 883-893, 2014).Additional sections describing results on receiver operating characteristics (ROC) analysis of the automated scoring method in IDEAS® as well as individual donor variability are included.v Chapter 5 is based on the methods developed and results obtained for the manuscript entitled "Validation of the Cytokinesis-Block Micronucleus Assay using Imaging Flow Cytometry for High Throughput Triage Radiation Biodosimetry" submitted to Health Physics (March, 2015).Additional sections detailing the methods used to determine the minimum blood volume required to score sufficient BNCs, 0-10 Gy dose response calibration curves for the rate of MN per BNC for the 72 h, 200 µL; 48 h, 2 mL and 48h, 200 µL culture conditions as well as dose estimations on all blinded samples examined between 0-10 Gy are presented.Chapter 6 contains a summary of the thesis findings and general conclusions on the advancements made in automating the CBMN assay using imaging FCM presented in this thesis as well as a short discussion on possible future work to be performed in this area of automated radiation biodosimetry For consistency, the references have been unified throughout the thesis and are listed at the end of this thesis.
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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 | 0.003 | 0.004 |
| Meta-epidemiology (narrow) | 0.001 | 0.001 |
| Meta-epidemiology (broad) | 0.001 | 0.001 |
| Bibliometrics | 0.001 | 0.001 |
| Science and technology studies | 0.000 | 0.001 |
| Scholarly communication | 0.001 | 0.001 |
| Open science | 0.001 | 0.001 |
| Research integrity | 0.001 | 0.002 |
| Insufficient payload (model declined to judge) | 0.004 | 0.004 |
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".