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Thanks for a quick turnover of this one.
I concur with the reviewers that this method has considerable merit, but that slightly more detail on the method and in particular the analysis and reporting of accuracy could be improved.
Good, no comment
Good with minor comments:
For blood sampling, please clarify how many of the panel of healthy donors was used for this study. Also there is repetition regarding control and reference samples.
Please identify the antibody ID number for the gH2AX antibody as well as for the lyse/fix buffer from BD Biosciences. Better describe the 96 deep well plates used for shape/volume or provide the ID/company where they were purchased from.
The opening sentence(s) of the first paragraph should be edited to highlight the fact that Figure 1 also provides a colour- coded key for the identification of the protocol steps.
Good. No comment.
In the context of this study, this work compares blood dose estimates for the two different protocols as opposed to whole body dose estimates as written in the text (line 78) and Figure 5 title.
Since this is useful protocol for quick screening of the damage, it has relevance for the accidental exposure situations. Certainly it overcomes the time consuming protocol and worth pursing.
The protocol is excellent, and achievable in any adverse conditions.
In my opinion, this manuscript is acceptable and it should be published in your journal.
See general and specific comments below.
General Comments. The authors address modifications to their routine laboratory blood leukocytes gamma-H2AX assay to permit enhancing throughput for use in large scale radiation accidents. They compared a modified lyse/fixation technique using 96-well deep-well plates with the routine leukocytes isolation using Histopaque 1077 solution in centrifuge tubes for rapid processing of blood samples. Sample processing and scoring times were compared. Accuracy of scoring was determined in samples exposed to 250-kVp x rays with manual (30 min repair after 0.5 Gy) and automated (1 h repair after 0 and 1 Gy) scoring. Accuracy for manual scoring was also determined at doses of 0, 0.2, 0.6, 1.1, 1.2, 2.1, and 4.3 Gy with 2 h repair. The authors discuss the utility of their “lyse/fix protocol” to facilitate high throughput processing for gamma-H2AX biodosimetry in large-scale radiation incidents.
Overall the study is well designed and results presented will be of high interest in the biodosimetry community. Major issues of concern are:
Small blood volume. Where is the evidence that sampling 0.1 ml of peripheral blood from an individual will provide comparable yields of gammaH2AX foci in leukocytes following radiation exposure when compared with larger volume obtained by venipuncture? Qualify text in “Abstract” to clarify.
Methodology. Additional details regarding methodology are needed to address: a) radiation exposure setup and dosimetry, b) statistical analysis.
Lyse/Fix method. Recommend the authors consider incorporating the concept that 96-well plates are used in the processing of samples using their proposed “lyse/fix method” to enhance throughput by considering renaming the method: “96-well plate lyse/fix” or “96 lyse/fix” as an abbreviation.
Page 1, Abstract, line 9. …where lymphocytes separated by Ficoll density density gradient .. ==[suggested change]=> …where lymphocytes in larger blood volumes (> 1 ml) are typically separated by Ficoll density gradient ..
Page 1, Abstract. “Accuracy” findings are presented in the Abstract as “mean absolute differences”, however, the radiation quality, dose rate, repair time, doses tested are not provided. The use of “mean absolute difference” as a measure for accuracy is quite restricted here since it’s value increases as a function of the magnitude of the doses tested, which are not shown in the abstract. Recommend use of an alternative measure to assess accuracy to permit inter-comparison of your findings with others, who may not have selected the specific doses you used in this study.
Page 1, Abstract. Briefly provide information about the doses (and radiation quality, dose rate, repair time) used to test accuracy.
Page 1, Abstract. Provide SE for “mean absolute differences”.
Page 3, Material and Methods. Briefly described the setup for radiation exposures (i.e., blood tubes exposed free-in-air or with 1-cm tissue equivalent shielding) and the method of dosimetry.
Page 4, Materials and Methods. Clarify that diluted blood was layered onto Histopaque solution in “15-ml conical centrifuge tubes”.
Page 4, Materials and Methods. Briefly describe methodology for performance of statistical analysis of results.
Page 5, Results and Discussion, “Scoring Time”. Provide a table that shows “scoring 20 cells in 96 samples times” for each method and dose point.
Page 6, Results and Discussion, line 157. Is there any differences in scoring foci per cells for the data shown in Figure 4? Statistically validate the statement that “automated foci counts are comparable between methods (Figure 4).
Page 6, Results and Discussion, “Mean absolute differences”. See comment above regarding use of “mean absolute differences to assess accuracy. Use a conventional measure of “accuracy” for dose assessment spanning the dose range tested (0 to 4.3 Gy) for the two methods. Provide the slope ± SE and correlation coefficient (r2) for a comparison of measured and actual doses?
Page 6, Results and Discussion. If you use “mean absolute difference” then provide standard errors.
Page 8. Conclusion. Conclusion paragraph should include comment about “false negatives” rate when using automated scoring.
Figure 1 legend. Add statement in figure legend to indicate that experiment was performed one time (16 samples using lyse/fix; 8 samples by histopaque) and scaling estimates are used for the timing for the handling of 96 samples.
Page 3, line 9. Horn & Rothkamm, 20111 => Horn & Rothkamm, 2011
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