-->PONE-D-25-54074-->-->Assessing tear-film parameters in participants with signs and symptoms of dry eye disease using a novel tear-film imaging device-->-->PLOS One

Dear Dr. Antman,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Manuel Garza León

Academic Editor

PLOS One

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6. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

Additional Editor Comments:

Your study addresses an important and clinically relevant topic in tear film imaging. I would encourage you to clarify two methodological aspects that would strengthen the transparency and statistical rigor of the manuscript.

First, please clarify how inter-eye correlation was handled in the statistical analysis. If both eyes from the same subjects were included, appropriate statistical methods that account for within-subject correlation should be applied. Alternatively, selecting a single eye per participant would avoid pseudo-replication. As currently written, the analysis appears to treat eyes as independent observations, which may overestimate statistical significance.

Second, please revise the abstract and methods to clearly state the final number of eyes and subjects included in the analysis, particularly if exclusions occurred due to external interferences or data quality concerns. The current wording suggests that 134 eyes from 79 subjects were analyzed, and any reduction in the effective sample should be transparently reported.

Addressing these two aspects will substantially improve the clarity, statistical validity, and interpretability of the study.

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Reviewers' comments:

Reviewer's Responses to Questions

-->Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. -->

Reviewer #1: Yes

Reviewer #2: Yes

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-->2. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #1: Yes

Reviewer #2: No

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-->3. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: Yes

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-->5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)-->

Reviewer #1: In this different device study the authors researched the novel TFI system to assess differences in the sublayers and characteristics of the human TF in participants with signs and symptoms of DED compared to healthy controls. But I could not understand the device principles I think you should use some pictures of the devices. Also introduction is too long and different from the aim you should reorganize the section. Tables are too busy some results are give in the text.

Reviewer #2: 1. Causal language. The abstract's final sentence could be read as mechanistic inference ("suggests eyes,….. have a thinner LLT/MALT"). Keep strictly descriptive given the cross-sectional design. Rephrase to "were associated with" and move mechanistic speculation to Discussion.

2. Ensure the OSDI validation paper is cited where OSDI is introduced and used (now only referenced generically in Introduction).Add recent work on TFI repeatability and LLT/MALT reproducibility if available, to contextualize measurement precision.

3. EQUATOR-aligned, STROBE-style title options that state the exposure/measure, outcome, population, setting, and design. Pick the one that best matches your manuscript. Eg: Tear Film Imager measurements of lipid and muco-aqueous layers and their association with dry eye signs and symptoms in adults: a prospective cross-sectional study at a tertiary eye clinic

4. Use of both eyes without accounting for within-subject correlation. Both eyes were included whenever “qualified,” yet analyses use Mann–Whitney tests that assume independence. This risks underestimated variance and inflated significance. If reanalysis is infeasible, prespecify and use one eye per subject.

5. Post-hoc exclusions and risk of selection bias. Participants using lotions/makeup were “retrospectively excluded,” and scans shorter than 6 s were removed, leaving 60 eyes of 34 subjects. Criteria introduced after viewing data can bias results. Provide a CONSORT-style flow diagram, justify exclusions a priori, and present sensitivity analyses including the excluded eyes when possible. Define the 6-s threshold’s rationale and show results across alternative thresholds.

6. Confounding by age and environment not controlled. In page 16, Table 2 shows large age difference between groups (62.8 vs 41.9 years) and different room temperatures; page 20, the “DED signs” group is substantially younger, yet age is associated with LLT in prior literature and in your discussion. Temperature also differed. Adjust comparisons for age and temperature using multivariable models, or perform age-matched subgroup analyses with adequate power. Report standardized mean differences to quantify group imbalance.

7. Definition and grading of “DED signs” require clarity. “Signs of DED” are defined as MGD or anterior blepharitis, yet Table 2 labels the group “MGD”. The grading scale, inter-grader reliability, and masking are not reported. Specify diagnostic criteria and grades for MGD and blepharitis, whether a single masked examiner graded all cases, and provide inter-rater or test-retest reliability if applicable.

8. Repeatability and measurement quality metrics are missing. No within-session or between-session repeatability (e.g., ICC, CoV) is provided for MALT, MALTR, or LLT. Without reliability, effect sizes are difficult to interpret. Add a repeatability analysis from duplicate measures or cite device reliability in a comparable setting, and state any quality-control rules used (e.g., blink detection, ROI rejection, minimum frames).

9. Primary outcome and multiplicity not prespecified, there were multiple endpoints tested with Mann–Whitney. The study tests many TF parameters and subgroupings without a declared primary endpoint or multiple-testing control. Prespecify a single primary endpoint (for example, LLT difference between MGD vs no MGD), define secondary endpoints, and apply Holm or Benjamini–Hochberg adjustments. Report adjusted p values and 95% CIs.

10. Tables 2–4: Results rely on means, SDs, and p values only. Clinical interpretation would benefit from effect sizes. Add mean differences with 95% CIs, and where non-normal, provide Hodges–Lehmann median differences. Include standardized effect sizes.

11. The data availability statement mentioned on the opening pages and the manuscript contradict, please check.

12. Study design description in p.11, lines 49–52: The design is described as “prospective cross-sectional,” which can confuse readers. Suggested fix: State “prospective, cross-sectional observational study.”

13. Sample size rationale (Methods overall). No precision or feasibility justification is provided. The authors need to add a brief rationale for the planned sample size or acknowledge as a limitation.

14. Use “muco-aqueous” consistently and define abbreviations once. Standardize the use of MALT and MALTR. MALTR has not been described in the paper. Figures have a heatmap. Does the heatmap signify MALT, MALTR, or TLL? Please mention in the figure notes. Figure 1A shows that there is no MALTR, and how missing data was accounted for

15. Page 11: “eyes presenting with signs of DED have a thinner LLT, whereas eyes with symptoms of DED have a thinner MALT.” Suggested change “Eyes with clinical signs of DED showed thinner LLT, while symptomatic eyes showed thinner MALT.”

16. pg.12, “Due to the subjective nature… patients frequently grouped into only DED positive or negative categories.” Suggested change “Because symptoms are subjective and signs vary, subtyping is difficult, and patients are often grouped simply as DED positive or negative.”

17. Page19: “The significance of this finding is unclear and further investigation is warranted.” Revision: “The clinical significance is unclear; please explore this in adjusted models or acknowledge as hypothesis-generating.”

18. Page 21: “We also found statistically significant differences in the room temperature…unlikely to significantly affect the results.” Change to: “Room temperature differed slightly between groups. Although the absolute difference was small, please adjust for temperature to exclude residual confounding.”

19. References that would improve the discussion: A recent overview of environmental influences and chamber conditions on tear metrics to contextualize temperature/humidity effects.

20. A statistical reference for analyzing paired-eye data in ophthalmic studies. If available, more recent TFI reproducibility or validation studies in symptomatic DED cohorts.

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Reviewer #1: No

Reviewer #2: No

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NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications.

-->-->PONE-D-25-54074R1-->

Assessing tear-film parameters in participants with signs and symptoms of dry eye disease using a novel tear-film imaging device

PLOS One

Dear Dr. Antman,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Jun 04 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'

-->

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

As the corresponding author, your ORCID iD is verified in the submission system and will appear in the published article. PLOS supports the use of ORCID, and we encourage all coauthors to register for an ORCID iD and use it as well. Please encourage your coauthors to verify their ORCID iD within the submission system before final acceptance, as unverified ORCID iDs will not appear in the published article. Only  the individual author can complete the verification step; PLOS staff cannot  verify ORCID iDs on behalf of authors.

We look forward to receiving your revised manuscript.

Kind regards,

Manuel Garza León

Academic Editor

PLOS One

Journal Requirements:

1. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

2. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments:

The authors have responded thoughtfully to the reviewers’ suggestions and provided useful clarifications regarding the statistical limitations. The acknowledgment of the exploratory nature of the study is appreciated. Nevertheless, the choice to treat each eye as an independent observation without accounting for inter-eye correlation remains a methodological limitation that could affect the interpretation of the results. To ensure transparency and to help readers properly contextualize the findings, I recommend explicitly identifying the work as a pilot study in the title. For example: Tear-film imaging measurements of the lipid and muco-aqueous layers and their association with dry eye disease signs and symptoms in adults: A pilot, prospective, cross-sectional study at a tertiary eye care center. This adjustment would align the title with the authors’ own description of the study as preliminary and exploratory, and would guide readers to interpret the results appropriately.

The abstract reports results based on 134 eyes from 79 participants, but after exclusions the final analysis included 60 eyes from 34 subjects. This should be stated clearly in the abstract to avoid confusion and ensure consistency with the methods and results sections.

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-->-->

Tear-film imaging measurements of the lipid and muco-aqueous layers and their association with dry eye disease signs and symptoms in adults: A pilot, prospective, cross-sectional study at a tertiary eye care center

PONE-D-25-54074R2

Dear Dr. Antman,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Manuel Garza León

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments: