-->PONE-D-25-46295-->-->High-quality histochemistry, immunohistochemistry, and immunofluorescence on xylene- and formalin-free paraffin-embedded tissues-->-->PLOS ONE

Dear Dr. Hoogland,

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.

In your revised manuscript, please ensure that you address the following concerns raised by Reviewers 1 and 2. While both reviewers agree that your study presents an innovative, well-written, and highly relevant advance in formalin- and xylene-free tissue processing, several revisions are necessary to strengthen methodological rigor, reproducibility, and quantitative support for your conclusions. While you should address all reviewer comments completely, I will point out the following critical comments:

• Specify the number of biological and technical replicates analyzed for each staining approach.
• Identify who performed morphological assessments (e.g., pathologists, trained reviewers) and state whether evaluations were blinded.
• Include counts or percentages of marker-positive cells (e.g., P63⁺), signal intensity measures, or reproducibility scoring.
• Include appropriate statistical analyses to support statements of equivalence or similarity between FFPE, NFPE, and XFPE groups.
• Discuss the diagnostic implications of markers that failed in NFPE (CD68, PAX-8, TDT) and note possible solutions such as alternative antibody clones.
• Provide commentary on the altered collagen morphology observed in NFPE skin samples, and if possible, evaluate whether picrosirius red with polarized light can distinguish collagen subtypes in NFPE-processed tissues.
• Improve your methods section to provide sufficient methodological detail to allow replication in other laboratories.

Please submit your revised manuscript by Nov 30 2025 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.

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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.

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We look forward to receiving your revised manuscript.

Kind regards,

Jordan Robin Yaron, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

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2. Thank you for stating the following in the Competing Interests section:

I have read the journal's policy and the authors of this manuscript have the following competing interests:

Agnes Marije Hoogland is included as one of the inventors in the Tispa patent WO 2024/117901 A1 of Tispa Medical B.V. and Stichting Isala Klinieken on the Non-Fixed Paraffin-Embedded (NFPE) technique, with additional patents pending. The other authors do not report a conflict of interest.

Please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials, by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests). If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.

Please include your updated Competing Interests statement in your cover letter; we will change the online submission form on your behalf.

3. Please amend the manuscript submission data (via Edit Submission) to include author Marije Hoogland

4. Please amend your authorship list in your manuscript file to include author Agnes Marije Hoogland

5. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

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.

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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: Partly

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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?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.-->

Reviewer #1: Yes

Reviewer #2: Yes

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-->4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.-->

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: This manuscript presents a novel Non-Fixed Paraffin Embedding (NFPE) method for pathological tissues that eliminates the use of both formalin and xylene, instead utilizing supercritical CO₂. The authors conduct a comprehensive evaluation of this technique across histochemistry, immunohistochemistry, and immunofluorescence, using a wide array of markers. The study is timely, relevant, and has significant implications for laboratory safety, environmental sustainability, and molecular diagnostics.

**Strengths:**

- The work clearly represents original research. While prior studies have explored either xylene-free or formalin-free approaches, this paper uniquely integrates both methods and validates the approach across a broad range of stains.

- The breadth of testing is impressive, encompassing 11 histochemical stains, 31 immunohistochemical markers, and 7 immunofluorescence markers.

- The results are generally strong: morphology is well-preserved, DNA quality is likely enhanced, and most markers functioned well with minimal protocol adjustments.

- The clinical and public health relevance is high, as the study provides a pathway to reduce exposure for pathology staff to toxic and carcinogenic substances.

**Concerns / Limitations:**

- **Marker Gaps:** Three immunohistochemical markers (CD68, PAX-8, TDT) did not produce reliable staining with NFPE. This limitation should be explicitly discussed in terms of diagnostic consequences and potential solutions, such as alternative antibody clones.

- **Residual Formalin Use:** The reticulin staining required a brief dip in formalin. While this amount is minimal compared to traditional FFPE workflows, it undermines the claim of a completely formalin-free process. This nuance should be highlighted in the conclusions.

- **Statistical Analysis:** The evaluation is primarily descriptive and based on visual assessment. Incorporating quantitative scoring (e.g., inter-observer agreement, reproducibility measures, signal-to-noise quantification) would strengthen the robustness of the findings.

- **Protocol Reproducibility:** While the authors note that protocols must be optimized for each laboratory, providing more detailed methodological information would assist other labs in reproducing and validating NFPE more effectively.

- **Generalizability:** The study was conducted at a single institution. Broader validation across multiple pathology laboratories would be important before clinical adoption.

**Ethics, Integrity, and Reporting:**

- Ethical standards were met; residual anonymized tissue was utilized with confirmation from the ethics committee.

- The manuscript is well-written in clear English and is easy to follow.

- Data availability complies with journal standards, with supplementary data provided.

**Overall Recommendation:**

This is an important and highly original contribution that advances the field of pathology towards safer and more sustainable practices. With minor revisions—particularly clarifying limitations, expanding the discussion on marker gaps, and enhancing details on reproducibility—the manuscript will be a strong candidate for publication.

Reviewer #2: Niemantsverdriet et al. developed a novel method of tissue processing that eliminates the need for toxic chemicals xylene and formalin. They compared the efficacy of staining in non-formalin fixed tissues to the tissues processed with standard formalin-fixed methods. They tested variety of stains in different organs and they claim that they found comparable results in most of them. Overall, the manuscript is structured well. The methods need more detail, quantifiable parameters need to be used, and statistical analyses need to be performed to compare different groups. Because of lack of quantifiable measures, the conclusions drawn cannot be supported with data, despite histological images showing similar morphology. I have following questions and recommendations:

1. It is recommended that authors describe the NFPE processing method in this manuscript as well.

2. Visual appearance of histological images stained with two different methods need not draw the same conclusions. I suggest adding a quantifiable metric to compare different methods. For example, do you see similar number of P63 positive cells in a tissue processed with FFPE and NFPE? Alternatively, you can develop a visual analog scale and have a blind reviewer assess the histological images of tissues with pathological conditions and confirm if similar inferences are made.

3. The authors should mention who drew the conclusions ‘Good morphology’. Were the conclusions drawn in a double-blinded manner by independent reviewers? Were they experts in identifying the morphology of the tissues? Were they pathologists?

4. In NFPE H&E Skin images, collagen fibrils seem to have lost morphology. Is it consistent in all replicates? Will this technique (NFPE) work with picrosirius red staining with polarized light imaging? Will it be able to separate thin (green) and thicker (red) bundles?

5. I could not see information about the replicates. How many replicates were included per group?

6. There needs to be more discussion about XFPE. Clearly mention the significance and relevance of this group.

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-->6. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .-->

Reviewer #1: Yes: SADIK BAY

Reviewer #2: No

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High-quality histochemistry, immunohistochemistry, and immunofluorescence on xylene- and formalin-free paraffin-embedded tissues

PONE-D-25-46295R1

Dear Dr. Hoogland,

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.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Jordan Robin Yaron, Ph.D.

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

-->Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.-->

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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-->2. 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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-->3. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #1: Yes

Reviewer #2: Yes

**********

-->4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.-->

Reviewer #1: Yes

Reviewer #2: Yes

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-->5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.-->

Reviewer #1: Yes

Reviewer #2: Yes

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-->6. 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: The authors have been improved the manuscript as my suggestions. I am happy to advice to accept the manuscript

Reviewer #2: The authors have addressed all the comments satisfactorily. I think authors need to discuss the quantification results (Figure S4) in the "Results" section.

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-->7. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .-->

Reviewer #1: Yes: SADIK BAY

Reviewer #2: No

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