Are Sputum High Mobility Group Box 1 and D-Dimer Changes Relevant Markers of Tissue Damage and Fibrinolysis in Cystic Fibrosis?

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this manuscript, the authors aim to determine the levels of HMGB1 and D-dimer in sputum from CF patients through two protocols. In the first protocol, the sample was treated with DTT and centrifuged to collect the supernatant. In the second, the sample was centrifuged to collect the supernatant, then the pellet was treated with DTT and centrifuged again. ELISA assays were performed on all samples.
The scope is interesting, and the main idea is important to the sample management.
I have some comments for the authors to address before making a final decision on its suitability and potential acceptance.

In the Materials and Methods section, in addition to the trademark, please include the catalog numbers, detection ranges, and sensitivity values for the HMGB1 and D-dimer biomarkers.

In Section 3.2, HMGB1 and D-dimer levels, it is stated: "In our experimental procedure, some sputum samples..." Please clarify how many samples were included in the SE, SPE, and SE procedures.

Please add a table or describe the quantitative data for both biomarkers in the text; Figure 1b does not clearly identify each value. Also, reformulate the last paragraph of the Results section, "D-dimers SED correlated posi-160 tively with FEV1 and monocytes (both rs = 0.9; p = 0.083)." since this p-value is not statistically significant.

Author Response

In this manuscript, the authors aim to determine the levels of HMGB1 and D-dimer in sputum from CF patients through two protocols. In the first protocol, the sample was treated with DTT and centrifuged to collect the supernatant. In the second, the sample was centrifuged to collect the supernatant, then the pellet was treated with DTT and centrifuged again. ELISA assays were performed on all samples.
The scope is interesting, and the main idea is important to the sample management.

I have some comments for the authors to address before making a final decision on its suitability and potential acceptance.

In the Materials and Methods section, in addition to the trademark, please include the catalog numbers, detection ranges, and sensitivity values for the HMGB1 and D-dimer biomarkers.

As requested, we have revised the methods section by including the detection range, assay sensitivity and catalog number for each ELISA kit used in this study. The corresponding information has been added to the revised manuscript.

The observation that several samples approached or exceeded the upper quantification limit of D-dimer levels suggests that D-dimer concentrations in cystic fibrosis sputum may be higher than anticipated and warrants further investigation using assays specifically validated for this biological matrix.

In Section 3.2, HMGB1 and D-dimer levels, it is stated: "In our experimental procedure, some sputum samples..." Please clarify how many samples were included in the SE, SPE, and SE procedures.

We have clarified the number of samples in Section 2.

Please add a table or describe the quantitative data for both biomarkers in the text; Figure 1b does not clearly identify each value. Also, reformulate the last paragraph of the Results section, "D-dimers SED correlated positively with FEV1 and monocytes (both rs = 0.9; p = 0.083)." since this p-value is not statistically significant.

We have described the quantitative data for both biomarkers in the new Table 2. We have reformulated this paragraph in the following manner: “In both cases, a strong trend toward a positive correlation was observed between the two parameters (rs = 0.90), which, however, did not reach statistical significance (p = 0.08), likely due to the limited sample size. Figure 2 represents these correlations.” Thus, we have also introduced the new Figure 2.

Reviewer 2 Report

Comments and Suggestions for Authors

This is an original research communication article investigating the HMGB1 and d-dimers as potential biomarkers in sputum in cystic fibrosis as well as comparing two distinct protocols for their measurement. Despite the overall originality and clinically meaningful idea, this work has serious flaws, which require attention:

1. The research project is actually focusing on two different aspects of the topic: i) the potential roles of HMGB1 and d-dimers as biomarkers; and ii) the different assays for their measurement. Those two aspects are not well distinguished in the article.
2. The rationale behind the investigation of the different protocols of measurement is not well presented.
3. The data processing and statistical analysis are not well explained, and results are only partially reported. Normality distribution tests are not described, and data are presented as means with standard errors instead of medians with interquartile ranges, which are the appropriate statistics for non-normally distributed variables. The results from Kruskal–Wallis tests should be reported in more detail beyond Figure 1 (i.e., the exact levels of each biomarker and the exact p-values). Also, Spearman correlations are not illustrated at all. It is surprising that “D-dimers SED correlated positively with FEV1 and monocytes (both rs = 0.9; p = 0.083)” with exactly the same rho and p-value.
4. The sample size is very small, making any statistically significant results not generalisable, even if the study was methodologically appropriate.
5. Other issues also exist (e.g., ELISA assays are not described in detail, clinical features are only partially reported, presence of bronchiectasis in participants is not discussed at all, minor linguistic errors, etc.)

Author Response

This is an original research communication article investigating the HMGB1 and d-dimers as potential biomarkers in sputum in cystic fibrosis as well as comparing two distinct protocols for their measurement. Despite the overall originality and clinically meaningful idea, this work has serious flaws, which require attention:

1. The research project is actually focusing on two different aspects of the topic: i) the potential roles of HMGB1 and d-dimers as biomarkers; and ii) the different assays for their measurement. Those two aspects are not well distinguished in the article.

 

We thank the reviewer for this comment. We agree that the biological and methodological objectives of the study were not sufficiently distinguished in the original version of the manuscript. To address this point, we have revised the introduction and explicitly stated the two aims of the study: (i) the evaluation of HMGB1 and D-dimers as biomarkers of tissue damage and fibrinolysis in CF sputum, and (ii) the assessment of the potential impact of DTT treatment on their measurement. We believe that this revision has improved the clarity of the study design and objectives.

 

1. The rationale behind the investigation of the different protocols of measurement is not well presented.

 

We thank the reviewer for this observation. To better clarify the rationale for investigating different measurement protocols, we have expanded the introduction by emphasizing that sputum samples require homogenization before analysis and that reducing agents such as DTT have been repotrted to affect the detection of several biomarkers. We also highlight that the effect of DTT on D-dimer measurement in CF sputum has not been previously investigated. Therefore, evaluating the impact of DTT represents a necessary methodological step to ensure the reliability of biomarker quantification in this biological matrix.

 

1. The data processing and statistical analysis are not well explained, and results are only partially reported. Normality distribution tests are not described, and data are presented as means with standard errors instead of medians with interquartile ranges, which are the appropriate statistics for non-normally distributed variables. The results from Kruskal–Wallis tests should be reported in more detail beyond Figure 1 (i.e., the exact levels of each biomarker and the exact p-values). Also, Spearman correlations are not illustrated at all. It is surprising that “D-dimers SED correlated positively with FEV1 and monocytes (both rs = 0.9; p = 0.083)” with exactly the same rho and p-value.

 

We have now better defined the normality distribution test, presented the data as medians with interquartile ranges, and explained the statistical test for non-normally distributed variables. Also, we have reported the quantitative data in the new Table 2. Finally, we now show the correlations in the new Figure 2. The identity of the rho and p-value between the two correlations is purely coincidental.

 

1. The sample size is very small, making any statistically significant results not generalisable, even if the study was methodologically appropriate.

 

We thank the Reviewer for her/his appreciation of the methodological appropriateness of our work. We have now highlighted the issue of low sample size in the Discussion section. However, it is worth to say that even with a very low sample size (n=5) some strong correlations emerged, suggesting an interesting biological and clinical mindful relationship between D-dimer levels and FEV1/monocytes.

 

1. Other issues also exist (e.g., ELISA assays are not described in detail, clinical features are only partially reported, presence of bronchiectasis in participants is not discussed at all, minor linguistic errors, etc.)

 

ELISA assays specifics have been described.

We report other clinical parameters in Table 1, i.e. the age at the diagnosis and the age of beginning of the triple therapy, i.e. Trikafta.

We now report in Table 1 that all the considered patients presented bronchiectasis.

We have done our best to correct all linguistic errors.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Thank you for addressing my previous comments.

According to your response, it is important to include a sentence noting that several samples approached or exceeded the upper quantification limit for D-dimer levels, suggesting that D-dimer concentrations in cystic fibrosis sputum may be higher than anticipated and warrant further investigation using assays specifically validated for this biological matrix.

Also, describe it in the limitations section.

The conclusion should be fitted, I mean, consider that the conclusions are direct answers to the stated objectives. The text regarding the potential utility of sputum-based biomarker analysis and the warranted future studies is a perspective and should be moved to the last paragraph of the discussion. Also, avoid being repetitive; the "Conclusions" section doesn't need to start with "In conclusion..."

Author Response

Thank you for addressing my previous comments.

According to your response, it is important to include a sentence noting that several samples approached or exceeded the upper quantification limit for D-dimer levels, suggesting that D-dimer concentrations in cystic fibrosis sputum may be higher than anticipated and warrant further investigation using assays specifically validated for this biological matrix.

Also, describe it in the limitations section.

We have now included a sentence in the Results section and discussed these results in the limitation section at the end of the Discussion.

The conclusion should be fitted, I mean, consider that the conclusions are direct answers to the stated objectives. The text regarding the potential utility of sputum-based biomarker analysis and the warranted future studies is a perspective and should be moved to the last paragraph of the discussion. Also, avoid being repetitive; the "Conclusions" section doesn't need to start with "In conclusion..."

We have modified accordingly the conclusion to be fitted better with the stated objectives. The text indicated by the Reviewer has been moved to the last paragraph of the discussion. We have also deleted “in conclusion” from the beginning of the “Conclusions” section.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors are congratulated for their efforts to improve the manuscript. Most of my comments have been addressed. However, my opinion is that the very small sample size is a serious issue of the study, making any statistically significant results not generalisable and potentially leading to wrong conclusions. Considering that the overall idea is original and clinically meaningful and that the preliminary results are promising, the authors are advised to include additional participants in the study (either from their centre or in collaboration with other centres, considering that cystic fibrosis is a rare disease) to conclude to more robust findings.

Author Response

The authors are congratulated for their efforts to improve the manuscript. Most of my comments have been addressed. However, my opinion is that the very small sample size is a serious issue of the study, making any statistically significant results not generalisable and potentially leading to wrong conclusions. Considering that the overall idea is original and clinically meaningful and that the preliminary results are promising, the authors are advised to include additional participants in the study (either from their centre or in collaboration with other centres, considering that cystic fibrosis is a rare disease) to conclude to more robust findings.

We fully agree with the reviewer’s assessment. However, as the reviewer correctly notes, cystic fibrosis is a rare disease; consequently, gathering additional sputum samples —either from our initiating center or through a multicenter collaboration— would require several months. Given that D-dimer levels in cystic fibrosis sputum have never been evaluated before, we kindly ask the reviewer to consider the inherent novelty of our study, that, although preliminar, allows us to establish priority in this poorly understood aspect of cystic fibrosis lung pathology. To convey this message, we have modified the title of our manuscript to “Are Sputum High Mobility Group Box 1 and D-dimer changes relevant markers of tissue damage and fibrinolysis in cystic fibrosis?”.