Combined Subcutaneous Fat Aspirate and Skin Tru-Cut Biopsy for Amyloid Screening in Patients with Suspected Systemic Amyloidosis

Round 1

Reviewer 1 Report

molecules-1153347

The authors present a communication regarding the use of methodologies to accurately diagnose amyloidosis. This document is what is seen as a compete story as there is the authors present a unique question and definitive answer to said question. From the results, it is clear that a single screening of skin biopsies is NOT a method that will provide a concrete answer; on top of that, the authors present the case that at the moment this method is the most frequently used to diagnose patients. This manuscript should become the go to reference for all practitioners who assist in diagnosing patients with amyloidosis. This reviewer sees no objection publishing the manuscript as is; however, providing a more detailed table or graph regarding the results could help strengthen their findings.

Author Response

Reviewer 1

The authors present a communication regarding the use of methodologies to accurately diagnose amyloidosis. This document is what is seen as a compete story as there is the authors present a unique question and definitive answer to said question. From the results, it is clear that a single screening of skin biopsies is NOT a method that will provide a concrete answer; on top of that, the authors present the case that at the moment this method is the most frequently used to diagnose patients. This manuscript should become the go to reference for all practitioners who assist in diagnosing patients with amyloidosis. This reviewer sees no objection publishing the manuscript as is; however, providing a more detailed table or graph regarding the results could help strengthen their findings.

Answer: We thank the reviewer for the comment, and we agree. We have included a new Table 1 with patient specific data presentation and Table 2 summarizing findings and statistics.

Reviewer 2 Report

The paper titled “Combined Subcutaneous Fat Aspirate and Skin Tru-Cut Biopsy 2 for Amyloid Screening in Patients with Suspected Systemic 3 Amyloidosis” of N. Abildgaard and co-workers deals with the comparison between two different test and combination thereof to screen amyloidosis in patients. In particular, the sensitivity both fat aspirates and skin tru-cut biopsies in the amyloidosis detection were evaluated in a group of patients, in order to test a valid approach to overcome the problems related to the biopsy of the involved organs. The amyloidosis detection was matched to the microscopic and spectrometric data, obtained by using immunoelectron microscopy (IEM) and mass spectrometry (MS).

The investigation was carried out by using several methodological approaches. However, the general congruence of the experimental results notwithstanding, some issues must be better addressed. A major revision is therefore needed to make the manuscript suitable of publication in Molecules, on the basis of the following observations:

• The number of patients the author are referred to, seems quite small. Can the author better argue how the statistic is important enough to support the conclusion?
• The molecular criteria and the parameters used to assign the amyloidosis by IEM and MS are not sufficiently described in the introduction
• Even though the manuscript was submitted in a communication form, reporting only figure to show all the data is not enough. All the data regarding the screening tests, as well as the raw data of IMS and MS must be included in the main test (some of them) and in the Supporting information section (most of them), the latter being incredibly absent

Author Response

Reviewer 2

The paper titled “Combined Subcutaneous Fat Aspirate and Skin Tru-Cut Biopsy for Amyloid Screening in Patients with Suspected Systemic Amyloidosis” of N. Abildgaard and co-workers deals with the comparison between two different test and combination thereof to screen amyloidosis in patients. In particular, the sensitivity both fat aspirates and skin tru-cut biopsies in the amyloidosis detection were evaluated in a group of patients, in order to test a valid approach to overcome the problems related to the biopsy of the involved organs. The amyloidosis detection was matched to the microscopic and spectrometric data, obtained by using immunoelectron microscopy (IEM) and mass spectrometry (MS).

The investigation was carried out by using several methodological approaches. However, the general congruence of the experimental results notwithstanding, some issues must be better addressed. A major revision is therefore needed to make the manuscript suitable of publication in Molecules, on the basis of the following observations:

• The number of patients the author are referred to, seems quite small. Can the author better argue how the statistic is important enough to support the conclusion?

Answer: We agree that the number of the patients with diagnosed systemic amyloidosis is rather small (n=24 out of 76 screened patients). However, we observed a huge difference in the sensitivity of the two procedures, fat aspirate vs. skin biopsy, overall 58.3% vs 20.8%, which was statistically significant using Fiscer Exact test. Statistical analysis data have been included in the text and in new Table 2. Because of the huge difference and observed no extra value of performing both procedures, we stopped routine use of skin tru-cut biopsies in our diagnostic workup. We believe this was the medical and ethical correct thing to do.

• The molecular criteria and the parameters used to assign the amyloidosis by IEM and MS are not sufficiently described in the introduction.

Answer: We have included a more thorough description of the criteria for amyloid subclassification by IEM and MS in the introduction as well as in an extended methods section.  We agree that this is an important issue for assessment of the screening sensitivity according to amyloid subtype.

• Even though the manuscript was submitted in a communication form, reporting only figure to show all the data is not enough. All the data regarding the screening tests, as well as the raw data of IMS and MS must be included in the main test (some of them) and in the Supporting information section (most of them), the latter being incredibly absent.

Answer: We agree that raw data presentation would strengthen our results and conclusion. They were merely not included because of the manuscript format. We have included two tables, Table 1 summarizingIEM and MS analysisdata, and Table 2 with descriptive analyses and statistics. Instead, we then believe that Figure 1 could be omitted.

Reviewer 3 Report

The work by Hansen et al. focus on a possible increase in diagnostic sensitivity of systemic amyloidosis by a combined use of fat aspirate and tru-cut skin biopsy in human patients. The study is carefully performed, however, it does not bring any new methodologic or scientific finding for the field despite its importance for diagnosis of this disease. Introduction and Discussion should be improved and bring more relevant information.

Author Response

Reviewer 3

The work by Hansen et al. focus on a possible increase in diagnostic sensitivity of systemic amyloidosis by a combined use of fat aspirate and tru-cut skin biopsy in human patients. The study is carefully performed, however, it does not bring any new methodologic or scientific finding for the field despite its importance for diagnosis of this disease. Introduction and Discussion should be improved and bring more relevant information.

Answer: The reviewer acknowledge that our findings are important in the planning of optimal screening procedures in suspected systemic amyloidosis, and this is exactly our message. We agree that we do not present new technologies or explorative data in the field of amyloidosis. However, to perform the best screening procedures are important in optimizing patient care.

Round 2

Reviewer 2 Report

I thank the authors about the improvements they have made on the manuscript. Results are now supproted by the tables that make more clear them for the reader. However, I don't think that the communication form the paper has been submitted as, excludes the inclusion of the raw data and detailed experimental features of the IEM and MS analysis in the supporting information. For this reason, I reccommand the publication in Molecules after providing these data

Author Response

As proposed we have now included the raw data for IEM and MS as supplementary files: supplementary Figure 1 for IEM data, and supplementary Table 1 for MS data.

Moreover we have included "supplementary methods" describing the IEM and MS methods used in detail. In stead, we have reduced the methods section in the main manuscript, not to double some of the information.   

Reviewer 3 Report

The work has been significantly improved; however, the introduction still does not contain enough information for a reader to understand results. Please, add information on types of amyloidosis and explain the abbreviations used in the text.

Author Response

Thanks to the reviewer for pointing out the missings in the Introduction. We agree that we have missed to include a more detailed introduction to the different amyloid subtypes, and to the used abbreviations. We have included that.