PONE-D-20-14510

Development of an optimized method of processing Peripheral Blood Mononuclear Cells for 1H-Nuclear Magnetic Resonance based metabolomic profiling

PLOS ONE

Dear Dr. Gómez Archila,

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, there are a number of issues that must be addressed before the manuscript can be considered for publication.

Please submit your revised manuscript by Sep 19 2020 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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We look forward to receiving your revised manuscript.

Kind regards,

Oscar Millet

Academic Editor

PLOS ONE

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2. Please provide further details on participant numbers, demographics and recruitment. Please also provide the IRB approval number in the ethics statement."

3. We note that this submission includes NMR spectroscopy data. We would recommend that you include the following information in your methods section or as Supporting Information files:

a) The make/source of the NMR instrument used in your study, as well as the magnetic field strength. For each individual experiment, please also list: the nucleus being measured; the sample concentration; the solvent in which the sample is dissolved and if solvent signal suppression was used; the reference standard and the temperature.

b. A list of the chemical shifts for all compounds characterised by NMR spectroscopy, specifying, where relevant: the chemical shift (δ), the multiplicity and the coupling constants (in Hz), for the appropriate nuclei used for assignment.

c. The full integrated NMR spectrum, clearly labelled with the compound name and chemical structure.

We also strongly encourage authors to provide primary NMR data files, in particular for new compounds which have not been characterised in the existing literature. Authors should provide the acquisition data, FID files and processing parameters for each experiment, clearly labelled with the compound name and identifier, as well as a structure file for each provided dataset. See our list of recommended repositories here: https://journals.plos.org/plosone/s/recommended-repositories

4. We noticed you have some minor occurrence of overlapping text with previous publications, which needs to be addressed. In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed.

5.

We suggest you thoroughly copyedit your manuscript for language usage, spelling, and grammar. If you do not know anyone who can help you do this, you may wish to consider employing a professional scientific editing service.  

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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: No

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

Reviewer #1: 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: No

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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: No

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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: Gómez Archila et al. have evaluated two new methods for PBMCs metabolomic profiling by using NMR spectroscopy: Ultrasound Method (UM) and Ultrasound and Ultrafiltration Method (UUM). They have compared them against the gold standard Folch method in terms of sensitivity, processing time, spectrum quality, metabolite numbers and reproducibility.

The present study aims to apply a previously validated methodology based on high intensity ultrasounds sample processing to obtain PBMC extracted matrixes for NMR metabolomics profiling. The authors argue a higher degree of robustness and reproducibility by using these new methods, and reduced sample handling time compared to the traditional Folch extraction methods.

The sample size is not described, the conclusions are only qualitative, there’s no statistical treatment of results, as there’s not an analytical approach. The methods are interesting, although they should address considerably the study approach.

My major concern -considering the objective and nature of the study- is the absolutely lack of quantitative comparison.

This study should include the analysis of a sample set representing a wider range of biological conditions, to be able to report the analytical performance of the new approach compared against the gold standard. The authors do not report quantitative measurements of robustness, reproducibility, fold changes, sensibility and variability. Neither replicates… as it appears to be 1 sample analysis.

Minor: There are some aspects deserving a better approach:

In the abstract the authors should include methodological aspects.

Line 68- 70 Please, include references and clarify:

“For example in the case of, immunosuppressive diseases (HIV type), cancer and primary immunodeficiencies the availability of PBMCs will be very low.”

Line 70 -74 To Consider Rewriting: I do not understand the construction.

“Therefore, taking into account cross-sectional or longitudinal studies, death, loss of contact, etc., it would be optimal to work with smaller blood and cell samples by changing the isolation method, the processing time (sample handling) delays and/or the cryopreservation technique that can affect the metabolic profile of the PBMCs obtained”.

Methods: Please, include some details in the Human subjects description. It should include some complementary information. I do understand that healthy volunteers accepted to donate blood for research purposes, but how was created the validation cohort? There was some inclusion / exclusion criteria. % hematocrite, fasting, non-fasting, volume extraction, extraction tubes, etc…

General comments: English review is mandatory

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Reviewer #1: No

[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.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PONE-D-20-14510R1

Development of an optimized method of processing Peripheral Blood Mononuclear Cells for 1H-Nuclear Magnetic Resonance based metabolomic profiling

PLOS ONE

Dear Dr. Gómez Archila,

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.

There are still a number of issues raised by the reviewer that need to be addressed before we can further proceed with the manuscript.

Please submit your revised manuscript by Feb 12 2021 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Oscar Millet

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)

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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

**********

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

Reviewer #1: I Don't Know

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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

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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

**********

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: Gómez Archila et al. have evaluated two new methods for PBMCs metabolomic profiling by using NMR spectroscopy: Ultrasound Method (UM) and Ultrasound and Ultrafiltration Method (UUM). They have compared them against the gold standard Folch method in terms of sensitivity, processing time, spectrum quality, metabolite numbers and reproducibility.

The present study has addressed previous concerns and has really improved validating their methodology for NMR metabolomics profiling. Although some minor questions remained opened:

Line 83: Please, consider clarifying the highlighted term:

For example, with immunosuppressive diseases such as HIV [39], cancer [40] and primary immunodeficiencies, characterized by lower leukocyte production, the availability of PBMCs reduces.

Line 199:

Please indicate the nuclear Overhauser Effect spectroscopy.

Line 218: Please, consider including into the discussion section the limitation associated with integrative methods to obtain metabolite concentration as the one presented in the manuscript; and the potential benefits of new UUM / UM methods on alternative mathematical treatment of the raw spectra based on methods (deconvolution processes). Would this approach differently improve quantification in any of the described experimental methods?

Line 223: The statistical analysis should be carefully reviewed. There are different objectives, as it can be easily seen in the Results, but not in the Data analysis and statistics methods section. I would recommend to emphasize the different objectives (to evaluate the reproducibility, by using the variation coefficient, to evaluate the differences in the number between methods, and also in the area by difference on central tendency, ANOVA, etc.... It looks like it has been evaluated together. So, clarify how do you address the differences between central tendences? And how do you treat the duplicates/triplicates (not as independent / nor paired measurements).

Line 224 Please, consider using the term mean in Table 2 instead of Average.

Line 227: Please review the use of central tendency instead of mean. Mean differences for t-tests, and median differences for Wilcoxon test.

Statistics general comment: There's a problem with this particularly low sample size regarding the power of the test. The authors should state that in the discussion, groups are not large enough to have high power of detecting a meaningful difference say delta between the two means, but artifacts. You should add this limitation for the t test and the normality test.

Line 233: central difference instead of mean (as it depends on the test)

Line 254: From figure it is not easy to identify differences between Ultrasound and Ultrafiltration methods. The two methods are explained or only the UUM? please clarify as there’s a plural form in 256.

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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: Dr. Nuria Amigo Grau

[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.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Development of an optimized method of processing Peripheral Blood Mononuclear Cells for 1H-Nuclear Magnetic Resonance based metabolomic profiling

PONE-D-20-14510R2

Dear Dr. Gómez Archila,

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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Kind regards,

Oscar Millet

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: