Retrospective Analysis of Hematological Parameter Changes in DMARD-Naive Rheumatoid Arthritis Patients Treated with Methotrexate: Correlation with Disease Activity and Treatment Outcomes

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript “Retrospective analysis of hematological parameter changes in DMARD-naive rheumatoid arthritis patients treated with methotrexate: correlation with disease activity and treatment outcomes” by ESRA DILSAT IMRAK and colleague aims to evaluate changes in hematological indices after 12 weeks of starting methotrexate (MTX) and analyze their correlation and predictive value regarding treatment response in patients with rheumatoid arthritis (RA).

Although the study is interesting, I am attaching a series of observations that should be taken into account in order to improve the scientific quality of the manuscript:

• The section “Materials and methods” should mention whether the study was approved by an ethics committee, as well as the reference for the work.
• The section “Study design and participants” should mention whether the participants signed an informed consent form.
• The methodology used in “Data collection” should be specified in more detail, as in the case of ELISAs, where the protocol used should be briefly discussed.
• The captions and axes of the graphs in Figures 1, 2, and 3 are very small and difficult to read.
• The bibliography is not up to date; at least 40% of the references should have been published in the last 5 years.
• The bibliography does not follow the journal's guidelines. The authors are advised to follow the guidelines in the “Instructions for Authors.”

Author Response

Reviewer 1

Comment: The section “Materials and methods” should mention whether the study was approved by an ethics committee, as well as the reference for the work.

Response: ethics approval number is added to methods section. Approval number and date is also available in data statement.

Comment: The section “Study design and participants” should mention whether the participants signed an informed consent form.

Response: participants provided verbal informed consent (I mentioned it in the method section). We did nor performed Written informed concent because of the retropective design of the study

Comment: The methodology used in “Data collection” should be specified in more detail, as in the case of ELISAs, where the protocol used should be briefly discussed.

Response: I have edded the detailed indormation for RF, CCP, CRP and CBC in the data collection section

Comment: The captions and axes of the graphs in Figures 1, 2, and 3 are very small and difficult to read.

Response: Figure sizes have been increased for easier reading. I can enlarge them further if needed and if it's suitable for the journal.

Comment: The bibliography is not up to date; at least 40% of the references should have been published in the last 5 years.

Response: References have been updated, especially in the Introduction section

Comment: The bibliography does not follow the journal's guidelines. The authors are advised to follow the guidelines in the “Instructions for Authors.”

Response: references are edited

Reviewer 2 Report

Comments and Suggestions for Authors

The present retrospective article addresses the implications of MTX on hematological parameters changes in DMARD-naïve RA patients. The topic is relevant, but highly studied and major deficiencies identified in both content and form need to be addressed, especially the real contribution and the novelty of the study:

1. Why were only these specific hematological parameters selected?
2. Consecutive multiple references such as [5-8], [12-14], [15–18], [22-26] etc., should be avoided, as they make it difficult to correlate the information with the specific sources. Instead, insert citations directly after each relevant piece of information. Revise this practice throughout the entire manuscript.
3. The 2010 ACR/EULAR criteria used for the diagnosis should be presented in greater detail.
4. The analyses should be stratified by seropositive vs. seronegative status, given that the study focuses exclusively on patients undergoing MTX monotherapy.
5. Briefly discuss the pre-RA phase and the potential implications, particularly for seronegative patients who are often diagnosed later due to existing classification systems.
6. The discussion section should be improved by comparing the obtained data with other similar studies.
7. The fact that MTX can induce hematological alterations is well-known. What is the novelty of this study, and what specific scientific gap does it fill? It is currently unclear what new insights it provides compared to existing literature.

 

Author Response

Reviewer 2

Comment: Why were only these specific hematological parameters selected?

Response: I have expanded the introduction (Lines 102-116) to explicitly justify the selection. The chosen parameters (WBC, NEU, LYM, MONO, Hb, PLT, MPV, RDW, NLR, PLR) represent a comprehensive panel of routine, low-cost CBC indices. They were selected because each has a known or theoretical link to the pathophysiology of RA (e.g., IL-6 driving thrombocytosis, anemia of chronic disease) and/or is a known target of MTX's pharmacological effects (e.g., folate antagonism impacting RBC indices, potential myelosuppression). This makes them ideal candidates for exploring their utility as accessible biomarkers of treatment response.

Comment: Consecutive multiple references such as [5-8], [12-14], [15–18], [22-26] etc., should be avoided, as they make it difficult to correlate the information with the specific sources. Instead, insert citations directly after each relevant piece of information. Revise this practice throughout the entire manuscript.

Response: I agree with you. I reduced the number of references in the introduction section and replaced them with more up-to-date ones.

Comment: The 2010 ACR/EULAR criteria used for the diagnosis should be presented in greater detail.

Response: Frankly, the 2010 ACR diagnostic criteria are a very well-known set of criteria that don't require further explanation in the articles. However, if you still think an explanation is necessary, I will add a detailed explanation to the methods section.

Comment: The analyses should be stratified by seropositive vs. seronegative status, given that the study focuses exclusively on patients undergoing MTX monotherapy.

Response: I have fully addressed this. The new Supplementary Tables 1 and 2, have been incorporated. Their content is now summarized in the Results section and discussed in the Discussion. This analysis confirms that baseline characteristics and 12-week hematological changes were largely similar between seropositive and seronegative patients, strengthening the validity of our main, unstratified findings.

Comment: Briefly discuss the pre-RA phase and the potential implications, particularly for seronegative patients who are often diagnosed later due to existing classification systems.

Response: A new paragraph has been added to the Discussion to address this important point. It acknowledges that our study starts at the point of DMARD initiation, which may be a late stage in the disease continuum, especially for seronegative patients who can face diagnostic delays. It discusses the potential implications of this for the generalizability of our findings and suggests that future research should investigate hematological changes in the pre-RA phase.

Comment: The discussion section should be improved by comparing the obtained data with other similar studies.

Response: The Discussion has been significantly strengthened by adding direct comparisons with other studies. We now contrast our findings regarding reductions in NLR, PLR and, crucially, we directly address the discrepancy between our negative findings for NLR/PLR as predictors and positive results from some cross-sectional studies. We explicitly state that our longitudinal, multivariate design likely accounts for this difference

Comment: The fact that MTX can induce hematological alterations is well-known. What is the novelty of this study, and what specific scientific gap does it fill? It is currently unclear what new insights it provides compared to existing literature.

Response: The novelty is now clearly articulated. We emphasize that this is the first study to perform a comprehensive, longitudinal evaluation of a broad panel of hematological parameters in a well-defined cohort of DMARD-naïve RA patients initiating MTX monotherapy. Previous studies were largely cross-sectional, included patients on various treatments, or lacked true baseline data. By using a rigorous longitudinal design and multivariate analysis, we are able to distinguish changes due to disease modification from those that are direct drug effects, and we robustly demonstrate that these parameters are not independent predictors of early response. This fills the gap by definitively testing, and refuting, the hypothesis that these simple indices can serve as standalone prognostic tools in this specific, clinically relevant setting. This is stated in the abstract and reinforced in the introduction and discussion

 

Reviewer 3 Report

Comments and Suggestions for Authors

In this study, the authors conducted a retrospective analysis aimed at identifying relatively simple and cost-effective hematological predictors of therapeutic response in DMARD-naive patients with rheumatoid arthritis (RA) receiving initial methotrexate therapy. Conventional clinical and laboratory rheumatological parameters were assessed concurrently, and the DAS28-CRP index was calculated.

Recommendations:

1. The table should include a "p-value" label, either within the table legend or directly in the table itself. Although this information is provided in the main text, tables should be self-contained and interpretable without reference to the manuscript body.
2. In the Results section, I recommend presenting the regression equations along with their corresponding plots. Alternatively, these may be provided in the Supplementary Materials.
3. It would be advisable to visualize the predictive performance of the binary classifier (not only via ROC curves) and to present a collinearity matrix to assess interrelationships among the investigated variables. These additions may also be included in the Supplementary Materials.

Overall, the manuscript is logically structured, employs a sound analytical approach, and appropriately addresses study limitations in the Discussion section. However, I have concerns regarding the novelty of the present work. The hematological parameters and variables examined herein have been extensively investigated over an extended period. Consequently, more robust composite indices such as DAS28 or DAS28-CRP have been established and are widely accepted as superior reflectors of disease activity compared to individual hematological variables. While the identification of simple, inexpensive predictors of RA course and outcomes remains an important and clinically relevant objective for rheumatologists, in my view, this study does not substantially advance existing knowledge nor propose a novel contribution to the field.

Additionally, the reported lack of prognostic significance for NLR and PLR appears unexpected, given that these markers are currently regarded in the literature as negative predictors of RA disease course. This discrepancy warrants further discussion or methodological clarification.

It appears that substantial revision of the manuscript, particularly through the incorporation of additional clinical and laboratory data or the inclusion of complementary patient cohorts, could enhance the novelty and scientific merit of the study.

Author Response

Reviewer 3

Comment: The table should include a "p-value" label, either within the table legend or directly in the table itself. Although this information is provided in the main text, tables should be self-contained and interpretable without reference to the manuscript body.

Response: p value labels are added

Comment: In the Results section, I recommend presenting the regression equations along with their corresponding plots. Alternatively, these may be provided in the Supplementary Materials.

Response: I have added a note in the Results section  stating that the detailed regression parameters, including equations, are provided in the supplementary materials. I have created a new Supplementary File that contains the regression equations for the significant correlations shown in Figures 1 and 2.

Comment: It would be advisable to visualize the predictive performance of the binary classifier (not only via ROC curves) and to present a collinearity matrix to assess interrelationships among the investigated variables. These additions may also be included in the Supplementary Materials.

Response: I have created a new Supplementary Table 3, which is a collinearity matrix (Variance Inflation Factor - VIF) for the variables included in the multivariate logistic regression models. This confirms that multicollinearity was not a significant issue in our analysis. This is now referenced in the Statistical Analysis section and the Results section. The ROC curves already visualize the poor predictive performance, but we now explicitly link this to the VIF analysis to show the independence of the predictors.

Comment: Overall, the manuscript is logically structured, employs a sound analytical approach, and appropriately addresses study limitations in the Discussion section. However, I have concerns regarding the novelty of the present work. The hematological parameters and variables examined herein have been extensively investigated over an extended period. Consequently, more robust composite indices such as DAS28 or DAS28-CRP have been established and are widely accepted as superior reflectors of disease activity compared to individual hematological variables. While the identification of simple, inexpensive predictors of RA course and outcomes remains an important and clinically relevant objective for rheumatologists, in my view, this study does not substantially advance existing knowledge nor propose a novel contribution to the field.

Additionally, the reported lack of prognostic significance for NLR and PLR appears unexpected, given that these markers are currently regarded in the literature as negative predictors of RA disease course. This discrepancy warrants further discussion or methodological clarification.

It appears that substantial revision of the manuscript, particularly through the incorporation of additional clinical and laboratory data or the inclusion of complementary patient cohorts, could enhance the novelty and scientific merit of the study.

Response: This point is now thoroughly addressed in the Discussion. We explain that the discrepancy is likely due to differences in study design: previous studies were often cross-sectional (comparing active vs. inactive disease at one time point) and did not control for confounders like concurrent therapies. Our longitudinal design, with multivariate adjustment, provides a more rigorous test of their predictive value for future response, rather than just their association with current disease activity.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have significantly improved the manuscript based on the suggestions received.

Author Response

Dear Reviewer 2

Thank you four you comment 'The authors have significantly improved the manuscript based on the suggestions received.'

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have made substantial revisions, and all of my comments and concerns have been thoroughly addressed. The statistical metrics that I had previously identified as missing have been incorporated into both the Supplementary Materials and the main text of the manuscript. Furthermore, the authors have provided a more detailed discussion of the study's strengths and have clarified the discrepancies with prior research.

Overall, in my assessment, the manuscript in its current form demonstrates significant improvement.

Author Response

Dear Reviewer 3,

Thank you for your comment; 'The authors have made substantial revisions, and all of my comments and concerns have been thoroughly addressed. The statistical metrics that I had previously identified as missing have been incorporated into both the Supplementary Materials and the main text of the manuscript. Furthermore, the authors have provided a more detailed discussion of the study's strengths and have clarified the discrepancies with prior research.

Overall, in my assessment, the manuscript in its current form demonstrates significant improvement.'