PONE-D-21-38473A prophylactic dose of Tinzaparin does not influence qPCR-based assessment of circulating levels of miRNA in humansPLOS ONE

Dear Dr. von Walden,

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.

Specifically:  After reviewing the manuscript, I have several concerns regarding the presentation of the manuscript that I deemed not suitable for review at its present form (see specific comments below). One of the main concerns is the authors does not clearly explain the results in a comprehensive way which gave the impression that the results presented were very preliminary and does not meet the publication criteria.

Specific comments:

1. Introduction reads like a run-on paragraph, suggest breaking into three paragraphs with line 67 – 72 as first paragraph, 72 – 82 as second paragraph, 82 – 90 as third paragraph. Paragraph breaks will provide readers better thought process regarding the rationale of the study.
2. Methods:  Line 165 – 166 belongs to the results section. Suggest moving line 165 – 166 as the opening sentences for Effect of prophylactic Tinzaparin treatment on RNA extraction from plasma.  Line 166 – 171 should be at the beginning of Effect of prophylactic Tinzaparin treatment on cDNA synthesis and qPCR.  While line 171 – 174 should lead the results of Effect of prophylactic Tinzaparin treatment on quantification of different endogenous circulating miRNAs.
3. Results:  This section was very sparse, the authors need to interpret the results in a more comprehensible way and guide the readers through and expand explanation of every table, every figure, and every scheme. For example, explain the concentration difference of three uniSp2, 4, 5 used and the outlier on Fig. 2

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We look forward to receiving your revised manuscript.

Kind regards,

Baochuan Lin, Ph.D.

Academic Editor

PLOS ONE

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"This work was supported by Linköping University, Region Östergötland, the Medical Research Council of Southeast Sweden (JS, grant no. FORSS-852501), Futurum – the Academy for Health and Care, Region Jönköping County, Sweden (BA, grant no. FUTURUM-937508 and 870471) and the Swedish Kidney Foundation (FvW, F2019-0048). Furthermore, we thank the Knut and Alice Wallenberg Foundation for generous support to JS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

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PONE-D-21-38473R1A prophylactic dose of Tinzaparin does not influence qPCR-based assessment of circulating levels of miRNA in humansPLOS ONE

Dear Dr. von Walden,

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. Both reviewers agreed that your paper addresses an interesting question, however, they both stated several concerns about your study and did not recommend publication in its present form.  Please see reviewers' insightful comments below.

Please submit your revised manuscript by Aug 12 2022 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 rebuttal 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.

We look forward to receiving your revised manuscript.

Kind regards,

Baochuan Lin, Ph.D.

Academic Editor

PLOS ONE

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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: (No Response)

Reviewer #2: (No Response)

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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: Partly

Reviewer #2: Partly

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

Reviewer #1: No

Reviewer #2: N/A

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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: No

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 of the manuscript entitled: “A prophylactic dose of Tinzaparin does not influence qPCR-based assessment of circulating levels of miRNA in humans” report the results of the study that aimed to investigate whether a prophylactic dose of the LMWH Tinzaparin influences qPCRbased quantification of circulating miRNAs in blood samples of patients enrolled previously into an ongoing study on acute compartment syndrome (BioFACTS, Clinicaltrials.gov, Identifier: NCT04674592).

In the group of 16 patients with traumatic tibial fractures on tinzaparin (vs. 16 of healthy controls no significant difference was observed for cDNA synthesis or qPCR when comparing tinzaparin treated patients with untreated controls. Among the tinzaparin treated patients, plasma levels of six endogenous miRNAs (hsa-let-7i-5p, hsa-miR-30e-5p, hsa-miR-222-3p, hsa-miR-1-3p, hsa-miR-133a-3p, hsa-miR-133b) were measured before and 1–6 hours after a subcutaneous injection of Tinzaparin 4500IU revealing no significant effect for any of the investigated miRNAs.

The topic is of interest, however, there are some relevant issues related to the manuscript.

In this small sample size study (groups 16 vs 16 patients) the large difference in the age of patients in the study and control group (tinazaparin 48 (15-67) years, control 32 (18-50) appears problematic, the authors should have at least attempted to adjust their analyzes for some baseline confounders.

This is further exacerbated by the fact that the patients in the study group were sampled at different timepoints after the fracture.

How can the differences be attributed directly to the fact of tinzaparin therapy ? What could be the impact of comorbidities?

The authors should further explain the rationale for selecting the described group of microRNA for analysis in their study.

The methodology of sample and microRNA analysis appears appropriate and sufficiently described.

The authors state at the end of the discussion “Our results are important for clinical scientist conducting biomarker-focused research in clinical settings. As of today, many hospitalized patients receive venous thromboembolism prophylaxis, often with a similar regimen as in the current study. Moreover, the ongoing Sars-Cov2 pandemic has contributed to an increase in LMWH-treated patients, given the strong treatment recommendations for thromboembolism prophylaxis [19]. Our results provide guidance when miRNA analysis is performed in patients on Tinzaparin treatment.”

The authors should more clearly and specifically explain to the reader why their results are reportedly “important for clinical scientists”.

The authors should explicitly list the limitations of their study in the discussion section.

A serious linguistic editing is required before the manuscript could be considered for further review and consideration for publication.

Reviewer #2: The topic of the study is interesting. It focuses on an important aspect that should be considered when miRNAs are studied from plasma.

However, I cannot understand some points.

First, UniSp2-4-5 have been added before RNA extraction to verify that Tinzaparin does not affect this step (as demonstrated in Fig.2). Then, UniSp6 and UniSp5 have been used to test cDNA synthesis step. Don’t UniSp2-4-5 already answer to this question? Indeed, since with the first step the authors conclude that Tinzaparin treatment does not affect RNA extraction also the following steps are included. Vice versa, cDNA synthesis step would be analyzed separately in the case of Tinzaparin treatment would affect the first result. To rescue the data about the cDNA synthesis, maybe, the data could be presented in a different order: first cDNA synthesis then RNA extraction.

Finally, in fig. 4 miR-1-3p, miR-133a-3p and miR133b are characterized by a high standard deviation. A different graph for all the six miRNAs should be also presented. Particularly, the segment that joins the first point to the second point should be shown for each patient.

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Reviewer #1: No

Reviewer #2: Yes: Vitiello Marianna

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[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

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A prophylactic subcutaneous dose of the anticoagulant tinzaparin does not influence qPCR-based assessment of circulating levels of miRNA in humans

PONE-D-21-38473R2

Dear Dr. von Walden,

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,

Baochuan Lin, 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

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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 sufficiently addressed all the issues raised in the review. I suggest to accept the manuscript.

Reviewer #2: the authors have adequately addressed my comments raised in a previous round of review. the manuscript is now acceptable for publication.

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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: Dominika Klimczak-Tomaniak

Reviewer #2: Yes: Marianna Vitiello

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