Within-individual variation of C-reactive protein (CRP) measurements in primary care: A retrospective cohort study

Alex Gough; Alice Sitch; Tom Marshall

doi:10.1371/journal.pone.0337221

Peer Review History

Original SubmissionAugust 26, 2025
22 Sep 2025 Decision Letter - Marwan Al-Nimer, Editor Dear Dr. Alex Gough, 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. ============================== ACADEMIC EDITOR: Major revision Please submit your revised manuscript by Nov 06 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. 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, Marwan Salih Al-Nimer, MD, PhD 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 https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. We noted in your submission details that a portion of your manuscript may have been presented or published elsewhere. [A version of this research made up a chapter in A. Gough's PhD thesis.] Please clarify whether this [conference proceeding or publication] was peer-reviewed and formally published. If this work was previously peer-reviewed and published, in the cover letter please provide the reason that this work does not constitute dual publication and should be included in the current manuscript. 3. We note that you have indicated that there are restrictions to data sharing for this study. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For more information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Before we proceed with your manuscript, please address the following prompts: a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially identifying or sensitive patient information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., a Research Ethics Committee or Institutional Review Board, etc.). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent. b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of recommended repositories, please see https://journals.plos.org/plosone/s/recommended-repositories. You also have the option of uploading the data as Supporting Information files, but we would recommend depositing data directly to a data repository if possible. We will update your Data Availability statement on your behalf to reflect the information you provide. 4. 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. Academic Editor Lines 58-60: add references about the values of CRP Lines 66-71: Add references about the type of variations Line 117: Check each point of STROBE statement Line 147: Mention the method that used in this study about missing data Recheck the unit of CRP mg/L and mg/l Line 181: there is no zero value for CRP, and a biotransformation way used in this study, i.e., log 0 is....... [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? Reviewer #1: Yes Reviewer #2: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: No Reviewer #2: Yes ****** Reviewer #1: General comments • The study addresses an important aspect in the clinical interpretation of CRP levels for diagnosis and prognosis. The cohort is impressively large and well described. However, the very high missingness in ethnicity (50.7%) and deprivation (14.1%) limits subgroup interpretability (Table 2). Authors should clarify how this was handled and consider analyses restricted to complete cases, as the dataset remains large enough. Methods and cohort description • The flow diagram should be redesigned in a top-down, left-to-right waterfall format, with the number of participants excluded at each step and the justification for exclusion shown clearly on the horizontal branches.Exclusions currently listed in the Statistical Methods (lines 171–173) should be integrated into the flow chart to justify participant losses. • The manuscript reports age at first CRP measurement, implying age stratification is based on baseline age. Given the 7-year median follow-up, participants likely transitioned into older categories and potentially contributed CRP levels in the incorrect age bracket. Authors should state clearly whether age was fixed at baseline or updated, and discuss potential misclassification. • The long follow-up means participants plausibly underwent disease or treatment transitions. Given CRP’s short half-life (~19 hours), much variation likely reflects these transitions. Authors should clarify this in the Discussion and frame the coefficient of variation as an upper-bound real-world estimate, considering analyses that minimise transition effects. • The manuscript mentions STROBE compliance but does not explain how the study size was determined. This should be included. Authors should also justify the inclusion criterion of ≥4 CRP measurements Results • Figures and tables should be fully captioned and units stated (e.g., Age in years, CRP in mg/L). • In Table 2, variable-specific exclusions (age >100, CRP >300) create denominator inconsistencies. Authors should consider defining the analytic cohort a priori (age ≤100, CRP ≤300) so that all analyses share the same denominator, and apply exclusions consistently across all analyses with a clear rationale. • Authors should also define what is meant by the category “Other” under ethnicity. • Age grouping is inconsistent: Table 2 uses 10-year bands, some analyses use ≤10, while Figure 2 collapses to 20–60 vs >60, but do not necessarily align with physiological transitions or clinical relevance for interpreting CRP. CRP is strongly influenced by developmental stage, hormonal status, and comorbidity patterns. More meaningful grouping could strengthen the analysis and clinical interpretability. I suggest that authors harmonise age categories across all analyses. They could consider using commonly applied clinical or epidemiological bands. • Figure 2 functions as a heatmap but lacks a colour key. Authors should add a legend or colour scale bar and state in the caption what the shading corresponds to. • Supplementary Table S2 includes results for participants with only 2–3 CRP tests, although the inclusion criterion was ≥4. Authors should justify that. • Discussion • References to “Chapter 3” (lines 262, 265) should be removed and replaced with appropriate details in the manuscript. • The Discussion section is comparatively brief, while the ‘Strengths and Limitations' section is relatively lengthy. This imbalance underemphasises interpretation, contextualisation, and clinical implications of the findings. Authors should expand the Discussion to integrate findings with prior studies, explore physiological and social demographic factors underlying the high within-individual CRP variability, and outline implications for guideline development and clinical decision-making. The reference list requires revision to align with the PLOS ONE formatting guidelines. For example, reference 9 does Reviewer #2: As I am not specialised in the field of blood tests and analyses, my comment concerns the need to include current references (if available). I hope these suggestions will help you improve your work, which is already excellent. I wish you every success. ****** 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: No Reviewer #2: 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. https://doi.org/10.1371/journal.pone.0337221.r001
Revision 1
31 Oct 2025 Author Response Dear Dr Chenette Thank you for giving us the opportunity to revise our manuscript “Within-individual variation of C-reactive protein (CRP) measurements in primary care: a retrospective cohort study.” We would like to thank the reviewers for their thorough comments, which we think has made the paper much stronger. A point-by-point answer to their queries is appended below. Yours sincerely, Alex Gough MA VetMB PhD Department of Applied Health Research University of Birmingham 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. Author: The file names have been revised. 2. We noted in your submission details that a portion of your manuscript may have been presented or published elsewhere. [A version of this research made up a chapter in A. Gough's PhD thesis.] Please clarify whether this [conference proceeding or publication] was peer-reviewed and formally published. If this work was previously peer-reviewed and published, in the cover letter please provide the reason that this work does not constitute dual publication and should be included in the current manuscript. Author: The work has not been peer-reviewed, published or presented as an abstract elsewhere except as a chapter in A. Gough’s PhD thesis. 3. We note that you have indicated that there are restrictions to data sharing for this study. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For more information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Author: IMRD data used for the study were obtained under licence from IQVIA; pseudonymised participant data are available from IQVIA subject to Scientific Review Committee approval. IMRD-UK data contains electronic health records from UK primary care. In compliance with the UK Data Protection Act and licensing agreements, the data cannot be shared via a public repository. These restrictions aim to protect patient confidentiality. Requests can be submitted to bassam.bafadhal@iqvia.com. 4. 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. Author: N/A Academic Editor 5. Lines 58-60: add references about the values of CRP Author: Added to manuscript 6. Lines 66-71: Add references about the type of variations Author: Added to manuscript 7. Line 117: Check each point of STROBE statement Author: Done 8. Line 147: Mention the method that used in this study about missing data Author: Added to manuscript 9. Recheck the unit of CRP mg/L and mg/l Author: Done 10. Line 181: there is no zero value for CRP, and a biotransformation way used in this study, i.e., log 0 is....... Author: Thanks for pointing out this issue with log 0. On investigation, we discovered that results of 0 were treated as missing values by Stata after log transformation, leading to around 1% of participants being excluded. This led to a very small but noticeable difference in the results. As a consequence, the dataset has been reanalysed using the method described by Rock and Durbin (2003) which involves adding a small constant to the results prior to transformation. Rocke DM, Durbin B. Approximate variance-stabilizing transformations for gene-expression microarray data. Bioinformatics. 2003 May 22;19(8):966-72. doi: 10.1093/bioinformatics/btg107. PMID: 12761059. Reviewer #1: General comments 11. The study addresses an important aspect in the clinical interpretation of CRP levels for diagnosis and prognosis. The cohort is impressively large and well described. However, the very high missingness in ethnicity (50.7%) and deprivation (14.1%) limits subgroup interpretability (Table 2). Authors should clarify how this was handled and consider analyses restricted to complete cases, as the dataset remains large enough. Author: A sensitivity analysis was performed with participants with missing ethnicity or deprivation – see sensitivity analysis in manuscript. Methods and cohort description 12. The flow diagram should be redesigned in a top-down, left-to-right waterfall format, with the number of participants excluded at each step and the justification for exclusion shown clearly on the horizontal branches. Exclusions currently listed in the Statistical Methods (lines 171–173) should be integrated into the flow chart to justify participant losses. Author: Fig 1 corrected. 13. The manuscript reports age at first CRP measurement, implying age stratification is based on baseline age. Given the 7-year median follow-up, participants likely transitioned into older categories and potentially contributed CRP levels in the incorrect age bracket. Authors should state clearly whether age was fixed at baseline or updated, and discuss potential misclassification. Author: Corrected in manuscript. 14. The long follow-up means participants plausibly underwent disease or treatment transitions. Given CRP’s short half-life (~19 hours), much variation likely reflects these transitions. Authors should clarify this in the Discussion and frame the coefficient of variation as an upper-bound real-world estimate, considering analyses that minimise transition effects. Author: Corrected in manuscript. 15. The manuscript mentions STROBE compliance but does not explain how the study size was determined. This should be included. Authors should also justify the inclusion criterion of ≥4 CRP measurements. Author: Corrected in manuscript Results 16. Figures and tables should be fully captioned and units stated (e.g., Age in years, CRP in mg/L). Author: Corrected in manuscript 17. In Table 2, variable-specific exclusions (age >100, CRP >300) create denominator inconsistencies. Authors should consider defining the analytic cohort a priori (age ≤100, CRP ≤300) so that all analyses share the same denominator, and apply exclusions consistently across all analyses with a clear rationale. Author: Corrected in manuscript 18. Authors should also define what is meant by the category “Other” under ethnicity. Author: Addressed in manuscript 19. Age grouping is inconsistent: Table 2 uses 10-year bands, some analyses use ≤10, while Figure 2 collapses to 20–60 vs >60, but do not necessarily align with physiological transitions or clinical relevance for interpreting CRP. CRP is strongly influenced by developmental stage, hormonal status, and comorbidity patterns. More meaningful grouping could strengthen the analysis and clinical interpretability. I suggest that authors harmonise age categories across all analyses. They could consider using commonly applied clinical or epidemiological bands. Author: Age grouping altered for table 2 and fig 2. 20. Figure 2 functions as a heatmap but lacks a colour key. Authors should add a legend or colour scale bar and state in the caption what the shading corresponds to. Author: Colour key added to legend. 21 Supplementary Table S2 includes results for participants with only 2–3 CRP tests, although the inclusion criterion was ≥4. Authors should justify that. Author: Justification added to legend for Fig S12 Discussion 22. References to “Chapter 3” (lines 262, 265) should be removed and replaced with appropriate details in the manuscript. Author: Corrected in manuscript 23. The Discussion section is comparatively brief, while the ‘Strengths and Limitations' section is relatively lengthy. This imbalance underemphasises interpretation, contextualisation, and clinical implications of the findings. Authors should expand the Discussion to integrate findings with prior studies, explore physiological and social demographic factors underlying the high within-individual CRP variability, and outline implications for guideline development and clinical decision-making. Author: Discussion revised in manuscript 24. The reference list requires revision to align with the PLOS ONE formatting guidelines. For example, reference 9 does Author Reference list corrected. Reviewer #2: As I am not specialised in the field of blood tests and analyses, my comment concerns the need to include current references (if available). I hope these suggestions will help you improve your work, which is already excellent. I wish you every success. Author: A new reference [4] was added. Attachments Attachment Submitted filename: Rebuttal letter CRP TM.docx https://doi.org/10.1371/journal.pone.0337221.r002
5 Nov 2025 Decision Letter - Marwan Al-Nimer, Editor Within-individual variation of C-reactive protein (CRP) measurements in primary care: a retrospective cohort study PONE-D-25-45864R1 Dear Dr. % Alex Gough, 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. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact billing support . 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, Marwan Salih Al-Nimer, MD, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0337221.r003
Formally Accepted
Acceptance Letter - Marwan Al-Nimer, Editor PONE-D-25-45864R1 PLOS ONE Dear Dr. Gough, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. You will receive an invoice from PLOS for your publication fee after your manuscript has reached the completed accept phase. If you receive an email requesting payment before acceptance or for any other service, this may be a phishing scheme. Learn how to identify phishing emails and protect your accounts at https://explore.plos.org/phishing. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Professor Marwan Salih Al-Nimer Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0337221.r004

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .