Coagulation and Blood Factors and Clinical Disease Indicators in Patients with Chronic Angioedema and Urticaria—A Validation Study

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Please rephrase and combine text in the lines 67-78 as well as 114-124, 79-89 and 125-137, for a better flow of narrative.

Please also indicate the current knowledge gaps and link with the purpose of the study.

In methods please specify the name of guidelines that were used for classification and patient selection (not just a reference) to define diagnostic criteria used.

What was the chronicity of angioedema?  It is important to include all aspects that define patient population.

AECT is control test not a disease severity score - it is important to clarify 

As it is a disease control test and disease specific test it is not usable in the control group.

in the exclusion criteria please specify what medication was on the exclusion list.

Please discuss the gender age mismatch in the groups as it makes significant limitation 

It is better to steer away from analysis of precentage of patients with elevated values, as laboratory cut offs can lead to a loss of important data. It would be better to base analysis on the values of the tests and work out percentage from them.

Please standardise the names and labels of the groups through the manuscript and figures and there is inconsistency in abbreviations and notations.

Discriminatory analysis of correctly classified 54% is a weak result meaning that half of the participants are miscalssified which does not correlate with biomarkers. The interpretation of this parameter should be revised.

This study does not provide evidence for therapeutic efiicacy just association and claims on the therapeutic use of anticoagulants in all forms AE and CU is an overstatement.

The discussion will be strengthen by comparison with the result of mentioned in the beginning pilot study.

Basophil function and IgE receptor antibody  discussion is not substantiated by the data from the study as they were not measured. It is impotent to acknowledge this and provide explanation.

Conclusion needs re-writing with concise summary of finding that D-dimers were significantly different among the groups highliting potential role in pathophysiology but with significant limitations due to groups mismatch and not that further research is needed.

The quality of English needs to be imporoved in spelling and grammar. Correction by a native English speaker of proof reading agency is recommend.

 

 

 

 

 

 

 

Comments on the Quality of English Language

As above requires improvement 

Author Response

Review report in attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

I would like to thank the authors for their valuable work on this clinically relevant topic. This manuscript examines coagulation and inflammatory markers in patients with isolated angioedema, angioedema with urticaria, and healthy controls. The subject is clinically relevant, and the attempt to compare isolated AE with AE/Urt is potentially useful. However, in its current form, the manuscript requires major revision before it can be considered further.

1. Novelty is limited and currently overstated: The main novelty appears to be an expanded sample and additional analyses, not a fundamentally new research question. Coagulation activation in CSU, especially D-dimer, is already well documented in prior literature, as mentioned by the authors. In addition, the authors have already published a 2024 pilot study comparing isolated AE, AE/Urt, and control. The manuscript should therefore be framed as an expanded follow-up/validation study. At present, the Discussion is internally inconsistent: it mentions the prior pilot study, but then states that no study has examined isolated AE, which needs correction/clarification.
2. Abstract suggestions: the abstract should include the age/sex imbalance findings, especially because they affect interpretation.
3. Introduction suggestions:
   
   • A substantial part of the Introduction is repeated (rows 66-85 and 110-129), the repeated paragraph should be removed.
   • CSU abbreviation should be clearly defined. 
   • Introduction should end with a short paragraph summarizing the study's aim.
4. Methods suggestions:
   • The citation to the guideline is formatted differently from the rest of the references (“Zuberbier et al., 2022” rather than bracketed style). This should be standardized.
   • The AECT and cytometry descriptions are too long for a Methods section and read partly like a mini-review. This should be written in a more concise form.
   • Hemoglobin should start a new paragraph for readability.
   • Finally, the statistical analysis should be presented as its own numbered subsection.
5. Results suggestions:
   
   • The Abstract states 102 participants but lists groups of 33 isolated AE, 33 AE/Urt, and 35 controls, which sums to 101. In Results, the groups are 33 AE, 34 AE/Urt, and 35 controls, which sum to 102. This must be corrected throughout the manuscript.
   • Tables numbering starts with Table 3 and figure numbering starts with Figure 5. At the same time, Methods refers to Table 1. The authors need to explain whether Tables 1-2 and Figures 1-4 are missing, misnumbered, or omitted during submission.
   • Figure captions should also be improved. The current captions usually state only that groups connected by a line differ significantly, but they should at least specify the statistical test and short method description.
   • The left non-log-transformed plot of Figure 7 could be moved to supplements.
   • Table terminology should be unified: either “elevated” or “increased”.
   • Also, the female gender label on figures appears to be in Croatian.
   • There are multiple correlations and adjusted analyses, but there is no tables or figures for these results. A correlation table, heatmap, or at least selected scatterplots should be added.
6. Discussion suggestions:
   • The Results show that the control group is younger than both patient groups, and that the AE/Urt group contains more men. The manuscript also shows that coagulation factors and CRP correlate with age, and that some variables differ by sex. This means that the main comparisons are vulnerable to findings and this must be discussed clearly as a study limitation. 
   • The authors do mention their previous pilot study, but this comparison should be expanded and systematized: what is new here beyond larger numbers, what changed analytically, and why do some findings differ from the pilot?
   • In addition, the manuscript needs a short, explicit Limitations paragraph covering: modest sample size, age/gender imbalance, etc.
7. In a few places, bracketed text appears to reflect draft-stage comments, and there seem to be occasional symbols (e.g. "+") and Croatian phrases that may have remained in the text by accident.

Author Response

Review report 2 in attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This cross-sectional study investigates the relationship between coagulation factors, other serum parameters, and clinical disease indicators in patients with chronic angioedema (AE) and urticaria. The authors compared three groups: patients with isolated AE, patients with AE accompanied by urticaria, and healthy controls. The main finding is that D-dimer levels were significantly higher in the AE with urticaria group compared to controls, and elevated D-dimer and CRP were more frequent in both patient groups. 
1.  The study's conclusions are significantly weakened by a major confounding factor: the control group is substantially younger than both patient groups, and age is known to correlate positively with many of the biomarkers studied, including D-dimer, fibrinogen, and CRP. While the authors attempt to address this with some statistical methods, this fundamental mismatch in a key demographic variable casts doubt on whether the observed differences are due to the disease or simply to age. This is a critical design flaw that undermines the validity of the core finding.
2.  The introduction and discussion sections are highly repetitive and poorly structured. Large portions of text are repeated verbatim, particularly the descriptions of the roles of IgE/IgG, the coagulation cascade, and various cytokines. This not only makes the manuscript unnecessarily long but also suggests a lack of careful synthesis of the literature. The authors should consolidate these sections to present a concise, logical, and non-repetitive background and discussion.
3.  A key weakness is the lack of analysis correlating the laboratory findings (D-dimer, CRP, etc.) with the clinical severity scores obtained from the AECT questionnaire. The stated aim includes investigating the relationship between these factors and "clinical disease indicators," yet the results section contains no such correlation analysis. This omission represents a missed opportunity to provide clinically meaningful insights and directly address the study's objective.
4.  The diagnostic criteria for "isolated angioedema" are not sufficiently rigorous. The manuscript does not clearly state how hereditary angioedema (HAE) or angioedema induced by ACE inhibitors was ruled out. Given that the pathophysiology of these conditions differs markedly from histaminergic angioedema, including such patients could introduce significant heterogeneity and confound the results related to coagulation factors. Clearer exclusion criteria and diagnostic workup are essential.
5.  The presentation of statistical results is confusing and sometimes contradictory. For example, the text states that fibrinogen and factor VII values did not differ significantly, yet Table 4 shows that 91.2% of the isolated AE group had elevated Factor VII compared to 71.4% of controls. While not statistically significant, this trend warrants comment, and the lack of significance might be due to insufficient power. Furthermore, the manuscript would benefit from a clearer distinction between group comparisons of mean/median values and comparisons of the frequency of values falling outside the reference range.

Author Response

Review report 3 in attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I'd like to thank the authors for addressing all the main comments. 

Author Response

Thank you very much, I hope the work has been improved.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript attempts to validate associations between coagulation factors, inflammatory markers, and clinical disease control in patients with angioedema and urticaria. While the authors have made some revisions in response to previous reviewer comments, the core methodological and analytical flaws have not been adequately resolved. 
1. The most critical flaw, as noted by the previous reviewer, is the significant age mismatch between the control group and the patient groups. Age is a well-established confounding factor for the key biomarkers studied, such as D-dimer, fibrinogen, and CRP. The authors' response states they have "modified and explained" this, but the revised manuscript fails to provide a convincing solution. Simply using age as a covariate in an ANCOVA does not rectify a fundamental design flaw in a cross-sectional study, especially given the modest sample size. The core finding of elevated D-dimer in the AE/Urt group remains confounded by age, and the authors have not provided a sensitivity analysis or age-matched subgroup analysis to demonstrate that the findings are independent of this significant demographic imbalance.
2. The authors claim to have added an analysis correlating laboratory findings with clinical disease control (AECT) in response to the reviewer's comment. However, the presented analysis is inadequate. The manuscript states no linear correlation was found, but then presents a multiple linear regression model identifying gender and erythrocyte count as predictors of disease control. This analysis does not directly address the study's stated aim of investigating the relationship between coagulation factors, inflammatory markers, and clinical disease indicators. The model is poorly specified, includes variables with no clear a priori hypothesis, has a very low adjusted R² of 0.154, and lacks a biological rationale for why erythrocyte count would be a primary predictor of angioedema control.
3. The diagnostic criteria for isolated angioedema remain insufficiently rigorous, despite the authors' claim of revision. The manuscript does not provide clear evidence on how hereditary angioedema and angiotensin-converting enzyme inhibitor-induced angioedema were ruled out. The pathophysiology of these conditions is distinct from histaminergic angioedema, and including them would introduce significant heterogeneity. Without a detailed description of the diagnostic workup, including specific laboratory tests such as C4 levels or C1-inhibitor function, the homogeneity of the patient groups cannot be assured, which severely compromises the validity of the study's conclusions.
4. There are inconsistencies in the reported data that undermine the manuscript's reliability. The sample size is stated as 101 participants in the methods section but is reported as 102 in the results section. Furthermore, the group numbers are inconsistent between the text and the figures. Such discrepancies suggest a lack of meticulous data handling and must be reconciled throughout the entire manuscript. Additionally, the results section contains vague statements, such as a trend for worse disease control in the AE/Urt group, without providing the supporting statistical data or p-values.

Author Response

Review Report attached.

Author Response File: Author Response.pdf

Round 3

Reviewer 3 Report

Comments and Suggestions for Authors

The study addresses a clinically relevant topic, and the authors have made efforts to respond to previous reviewer comments. However, three fundamental issues remain that must be corrected before acceptance. These relate to unresolved age confounding, lack of valid clinical correlation, and inconsistent data reporting.
1. The age mismatch between control and patient groups remains the most serious methodological flaw. Age is a known major confounder for D-dimer, fibrinogen, and CRP. The authors’ solution of using a 50-year cutoff and analyzing participants aged ≤49 years showed no between-group differences, which actually demonstrates that the original positive findings are likely driven entirely by age, not disease. The authors must either provide an age-matched subgroup analysis (e.g., selecting older controls or younger patients to balance groups) or explicitly state in the abstract and conclusion that the observed differences disappear after age stratification, and revise their conclusions accordingly to avoid overstatement.
2. The authors deleted regression analyses after reviewer criticism but did not provide any valid替代 analysis to assess the relationship between biomarkers and clinical disease control (AECT scores). The stated aim of the study includes investigating this relationship, yet the current version contains no such analysis beyond a simple statement of “no linear correlation.” The authors must perform at least a basic Spearman correlation between AECT scores and key biomarkers (D-dimer, CRP, fibrinogen) and report the coefficients and p-values, or explicitly state in the results and abstract that this analysis was not performed due to lack of association, and remove the corresponding aim from the introduction.
3. There are clear inconsistencies in reported sample sizes and group numbers. The abstract states 101 participants while the results section says 102. Group sizes are reported as 33, 34, and 35 in some places but differ elsewhere. The authors must carefully reconcile all numbers throughout the manuscript, including the abstract, methods, results, tables, and figure legends. Every mention of participant counts must be identical. This is essential for manuscript credibility.

Author Response

Review report attached. 

Author Response File: Author Response.pdf