Microvascular Imaging as a Novel Tool for the Assessment of Blood Flow Velocity in Patients with Systemic Sclerosis: A Single-Center Feasibility Study

Round 1

Reviewer 1 Report

The manuscript is clear and relevant for the field and presented in a well-structured manner.

The cited references are current and references are appropriate.

The manuscript’s results are reproducible based on the details given in the methods section.

The figures/tables/images/schemes are appropriate and they properly show the data and result. They are easy to interpret and understand. 

I think the conclusions are consistent with the evidence and arguments presented.

Author Response

Thank you for the positive assessment of our paper.

Reviewer 2 Report

This article describe a tecnique, the  Micro Vascular Imaging (MVI) to assess flow quantification of small fingertip vessels

Althought the article is quite readible and it has a logic construction there are major limitations that cut down the application of MVI in SSC.

• fIrst of all the age of the patients are too different and and therefore this article do not prove a real difference;
• in addition 30% of SSC paties were past or current smorkers 
• too small the sample of the SSc patients
• we do not know how many patients have comorbidities (diabetes, hypertension, renal insufficiency, cholesterol etc)
• The authors should discuss the limitation also regarding the drugs commonly used in SSc that could influence the exam
• Also the authors should discuss why this exam is complementary or better then capillaroscopy.
• The article could have  more scientific sound if there is a correlation with the flow and the SSC ulcers

Author Response

Response to Reviewer #2

This article describe a tecnique, the Micro Vascular Imaging (MVI) to assess flow quantification of small fingertip vessels

Althought the article is quite readible and it has a logic construction there are major limitations that cut down the application of MVI in SSC.

R/ We thank the reviewer for taking the time to review our paper. As a general comment, this is the first study of MVI in this indication. Therefore, we sought to test it in a limited population. Of course, additional patient numbers and other indications need to be investigated.

• fIrst of all the age of the patients are too different and and therefore this article do not prove a real difference;

 R/ thank you for your comment. As mentioned above, this is the first feasibility study describing the method in a healthy and diseased population. We aimed to establish the feasibility and cut-off values between SSc and healthy controls. We feel that it is even an advantage of the HC group that it has a different age and no comorbidities because these could influence the measurement results (and give a false impression on what should be considered “normal”). Further, we are confident that the correlation analysis is the correct way to show a dependency (in this case a non-existent dependency) between age and MVI flow results.

• in addition 30% of SSC paties were past or current smorkers 

R/ We added this point in the discussion as a potential limitation.

• too small the sample of the SSc patients

R/ Thank you for this comment. However, we respectfully disagree with the reviewer that for the purpose of our study aims a larger number of patients is required. This is a hypothesis-generating study without the goal of a confirmatory analysis. Hence, we were not expecting any specific differences between groups and, therefore, did not perform a sample size calculation (technically also impossible without prior knowledge of effect sizes). We added the small sample size as a limitation to the discussion section emphasizing that these first results need to be validated in additional cohorts.

• we do not know how many patients have comorbidities (diabetes, hypertension, renal insufficiency, cholesterol etc)

R/ We agree with the reviewer and added the comorbidities to Table 1.

• The authors should discuss the limitation also regarding the drugs commonly used in SSc that could influence the exam

R/ We added this point in the discussion section as a potentially influencing variable. Nevertheless, since commonly used drugs in SSc and RP have the purpose of vasodilation, the differences between SSc and HC may be even more pronounced in an untreated SSc patient population.

• Also the authors should discuss why this exam is complementary or better then capillaroscopy.

R/ We added this to the discussion. Clearly, NVC is the most frequently used and most widely accepted test to assess the microvasculature in SSc. We expanded the discussion section to clarify that MVI is, at present, not intended to replace NVC as it is a novel modality without longitudinal data. 

• The article could have  more scientific sound if there is a correlation with the flow and the SSC ulcers

R/ We agree with the reviewer. However, in the current cohort, none of the patients had digital ulcers at the time of the investigation. We will expand this a) in a larger cohort b) including patients with DU (and other comorbidities) and c) follow-up over time. 

 

 

Reviewer 3 Report

First, I would like to thank the authors for allowing me to review your manuscript, "Superb Microvascular Imaging as a Novel Tool for the Assessment of Blood Flow Velocity in Patients with Systemic Sclerosis: A Single-Center Feasibility Study."

Major issues:

• The authors write that SSc patients (median 60 years) were significantly older than HCs patients (median 26 years). By means of a linear regression it should be shown that there is no correlation between age and flow (Figure 4). I am not convinced by the graph at all. In Figure 4A you can see 4 subjects around 55 years (HCs patients) and on 4B you can clearly see that the majority is over 55 years (SSc patients). Therefore, I don't trust the statistics either. Physiological changes due to age could also be a plausible explanation for the measurement differences. The authors must include more healthy patients who are over and arround 60 years old so that a pure age difference can be excluded. The authors themselves write that the measurement takes just under 15 minutes. This is quite manageable, and should therefore be carried out.  
• Why was a patient included in the SSc without skin involvement? Wouldn't that mean that there are no changes in the skin vessels there? The authors, should explain that in the discussion, what it could mean for the measurements. If it has implications, why not exclude the individual patient and include someone else with SSc?
• Why was Youlden used as the optimal point? The authors should think about what they want to show with their test. This is not only about sensitivity and specificity, but also about positive and negative predictive value. For such a test to be clinically "useful", clear criteria must be set as a basis. Currently it only seems like "we did a ROC, look what we found". There is already a nailfold video capillaroscopy test. What is the sensitivity and specificity there? This should be compared to that and clear criteria should be set BEFORE what you want to achieve with the test. AND also be discussed!
• What is the explanation for the lack of correlation of flow between fingers? There are anatomical differences (A. ulnaris vs A. radialis), especially in the supply of DI and DII compared to DIII to DV. Can this be an explanation? For me it is not understandable why there are such differences. Were the measurements possibly wrong? Was the measurement always performed by one person? I recommend the authors to test 3 healthy patients by 3 investigators, to make one measurement each at DII to DV and see if the results correlate strongly. If not, it could be an error in the measurement. Too much pressure?
• It is not clear in the manuscript where the PD US measurements are? Figure 5 could be compared with the PD US measurements. In any case, this should be presented transparently, since the authors write that MVI is not better than PD US.

Minor issues:

• Please split Figure 4 into A-D. Simultaneous presentation of peak sysolic and end diastolic confuses.
• Please shorten the statistics section a bit. Much is standard knowledge and does not need to be explicitly mentioned. Only distracts from the topic.

Overall, a good manuscript with an interesting methodology. However, the discussion is much too short, but the statistics section is almost half a page long. Therefore, shorten statistics description a bit and make the discussion critical and much longer. Statistics can never compensate for a clever and good study design.

I think the manuscript, after adopting the proposed changes would be much better and above all more credible. Therefore, until then MAJOR REVISION. 

Author Response

Response to Reviewer #3

First, I would like to thank the authors for allowing me to review your manuscript, "Superb Microvascular Imaging as a Novel Tool for the Assessment of Blood Flow Velocity in Patients with Systemic Sclerosis: A Single-Center Feasibility Study."

R/ Thank you for the time you have taken to review our paper. As a general comment before replying to the specific points, we would like to point out that this is the first study of MVI in this indication. Therefore, we sought to test it in a limited population as a feasibility study and not with the purpose to test diagnostic properties. Of course, additional patient numbers and other indications need to be investigated, which we are currently performing in an extended, longitudinal study.

Major issues:

• The authors write that SSc patients (median 60 years) were significantly older than HCs patients (median 26 years). By means of a linear regression it should be shown that there is no correlation between age and flow (Figure 4). I am not convinced by the graph at all. In Figure 4A you can see 4 subjects around 55 years (HCs patients) and on 4B you can clearly see that the majority is over 55 years (SSc patients). Therefore, I don't trust the statistics either. Physiological changes due to age could also be a plausible explanation for the measurement differences. The authors must include more healthy patients who are over and arround 60 years old so that a pure age difference can be excluded. The authors themselves write that the measurement takes just under 15 minutes. This is quite manageable, and should therefore be carried out.  

R/ We understand the point raised and this was well discussed among us. Nevertheless, findings in NVC do not correlate with age, either, but rather are influenced by disease-specific factors (activity, disease duration, etc.). This was added to the discussion section.

Regarding the statistics, we did not detect any obvious outliers or changes within the SSc groups nor within the HC group. We are confident that the linear regression convincingly shows no influence of age. We are not sure that adding older “healthy” patients serves this purpose as these may have potentially confounding comorbidities. Further, as we pointed out earlier, this is a hypothesis-generating study for the establishment of the method. We agree, however, that additional patient(s) cohorts, as was done with nailfold capillaroscopy, will be required to investigate. We added age, among other factors, as a potential confounding variable to the discussion.

• Why was a patient included in the SSc without skin involvement? Wouldn't that mean that there are no changes in the skin vessels there? The authors, should explain that in the discussion, what it could mean for the measurements. If it has implications, why not exclude the individual patient and include someone else with SSc?

R/ The absence of skin fibrosis does not necessarily indicate the absence of microangiopathic changes (i. e. Raynaud’s phenomenon). Rather, we believe that the findings of MVI in SSc are detectable with this method because microangiopathy is so universal in SSc patients. We added this to the discussion section.

• Why was Youlden used as the optimal point? The authors should think about what they want to show with their test. This is not only about sensitivity and specificity, but also about positive and negative predictive value. For such a test to be clinically "useful", clear criteria must be set as a basis. Currently it only seems like "we did a ROC, look what we found". There is already a nailfold video capillaroscopy test. What is the sensitivity and specificity there? This should be compared to that and clear criteria should be set BEFORE what you want to achieve with the test. AND also be discussed!

R/ we agree with the reviewer that one has to think about the right test before performing it. However, this is a very early clinical study. Therefore, we performed ROC analyses with the specific purpose to establish cut-off values between diseased and healthy controls. Sensitivity and specificity, in this case, are not to be misunderstood as diagnostic properties of MVI as all patients already had a known diagnosis of SSc. If we had performed a test without giving any real measurement data (and cut-off values) we would have been able only to state that there is a difference between SSc and healthy controls, which is of no practical value while performing these measurements. If MVI is performed, examiners are interested in knowing which value is considered abnormal. Furthermore, calculating PPV and NPV would have been biased because the prevalence is influenced by choosing the groups sizes and does not reflect any real prevalence.

• What is the explanation for the lack of correlation of flow between fingers? There are anatomical differences (A. ulnaris vs A. radialis), especially in the supply of DI and DII compared to DIII to DV. Can this be an explanation? For me it is not understandable why there are such differences. Were the measurements possibly wrong? Was the measurement always performed by one person? I recommend the authors to test 3 healthy patients by 3 investigators, to make one measurement each at DII to DV and see if the results correlate strongly. If not, it could be an error in the measurement. Too much pressure?

R/ As suggested by the reviewer, we added an interrater agreement (intraclass correlation coefficient) for 3 controls. ICC was excellent (above 0.9) for individual and average class correlations. This has been added to the methods and results sections.  

• It is not clear in the manuscript where the PD US measurements are? Figure 5 could be compared with the PD US measurements. In any case, this should be presented transparently, since the authors write that MVI is not better than PD US.

R/ Again, displaying PD measurements here was not the purpose of this feasibility study of MVI. This would require a thorough and more extensive analysis which would make the results section unnecessarily longer. We decided, therefore, to not compare these results for this paper (another reason being that there is no accepted standard ultrasound technique for the assessment of RP). We agree with the reviewer that it may be confusing to the reader. Therefore, we deleted this part from the methods section.

Minor issues:

• Please split Figure 4 into A-D. Simultaneous presentation of peak sysolic and end diastolic confuses.

R/ done as suggested.

• Please shorten the statistics section a bit. Much is standard knowledge and does not need to be explicitly mentioned. Only distracts from the topic.

R/ The statistics section was shortened.  

Overall, a good manuscript with an interesting methodology. However, the discussion is much too short, but the statistics section is almost half a page long. Therefore, shorten statistics description a bit and make the discussion critical and much longer. Statistics can never compensate for a clever and good study design.

I think the manuscript, after adopting the proposed changes would be much better and above all more credible. Therefore, until then MAJOR REVISION. 

 

 

Round 2

Reviewer 2 Report

The authors discussed the major limitation and they tried to improve the manuscript. The major limitation is stil the age and the limit number of subject involved but this is a pilot study and therefore a think study is acceptable 

Reviewer 3 Report

I am satisfied with the revision and recommend ACCEPT.