Combination of Low-Temperature Electrosurgical Unit and Extractive Electrospray Ionization Mass Spectrometry for Molecular Profiling and Classification of Tissues

Round 1

Reviewer 1 Report

Comments in the Report

Comments for author File: Comments.pdf

Author Response

Please see attached file.

Author Response File: Author Response.pdf

Reviewer 2 Report

This manuscript describes the incorporation of a low temperature electrosurgery unit, commercially available, to a home built extractive electrospray ionization source for direct analysis of tissue samples. The motivation behind this study is to reduce tissue damage as well as molecular degradation caused by high temperature electrocautery, employed by other mass spectrometry-based methods for tissue analysis, such as the iKnife. The authors tested and optimized this method on chicken skin, and then used it to analyze several areas within five breast cancer specimens to evaluate differences in the molecular profiles obtained and develop classification models.

Even though the goal of the study is of interest, data demonstrating the reduction in tissue damage and molecular fragmentation is needed to support the claims discussed. Moreover, a big concern is the lack of a proper introduction to the field, discussing previous work done with intraoperative mass spectrometry-based methods. Overall, the authors need to be more careful with the claims and conclusions, since this manuscript describes data more suitable for a proof of concept study rather than demonstrating the capabilities of this system for tissue diagnosis in surgery. Thus, we recommend the paper to be toned down and that the title of the manuscript be changed to reflect that, since no surgeries were performed using the device. Moreover, it is unclear what system was used for the analyses, since the use of a PTFE tubing for remote analyses is described, but the setup shown in the manuscript does not use a transfer line, and instead requires positioning of the sample in front of the mass spectrometer, not suitable for surgical use. Overall, the study could benefit from improved descriptions, more clear and detailed interpretations of the data provided, and improved quality of the figures.

More specific comments below:

Comments:

Abstract:

·        “Thus, determination of the molecular tissue composition at a particular location point by surgical smoke analysis is becoming routine nowadays.” – This sentence can be misleading to the reader, since molecular analysis of surgical smoke is not yet used in surgeries routinely. Please reword.

Introduction:

·        The first paragraph of the introduction does a very poor job acknowledging other techniques in the field of intraoperative analysis using mass spectrometry, as well as explaining how they work. Contributions provided by other approaches, such as PIRL, SPME, etc, should be mentioned, as there are more than “two approaches” used for “MS-based molecular visualization”. Moreover, it is important to provide the reader with a better understanding of the principal of operation of these techniques. For example: the authors describe that “The first approach is the direct MS analysis of molecular smoke released upon electrosurgery [1]. The second approach is to use a special MS-based instrument which allows one to scan the tissue in real time [2,3]”. Citations 2 and 3 refer to two different methods, one using a laser probe for molecular desorption, while the other method relies on a solvent based extraction upon contact with the tissue sample. These should be better described and properly referenced. The description of “a special MS-based instrument” is not very informative.  

·        Moreover, the integration of electrosurgery with electrospray has also been previously described by the Takats group, as shown in reference 12 in the manuscript. This work should also be acknowledged in the introduction as well.

·        Line 39-40 “The chemical composition of smoke produced by high-frequency electrosurgery has emerged since 1995”. Please reword.

·        Line 46-47 “analyzed directly without auxiliary ionization, such as in REIMS”. Yet, most REIMS approaches do also rely on ionization happening at the inlet by heated collision. This sentence should be rephrased.

·        Line 62-63: “…analysis of biological tissues both in vivo and ex vivo and its potential for intraoperative use”. Data is not provided in the manuscript for in vivo analysis, so this sentence should be edited to reflect that.

Materials and methods:

·        The authors mention that a “ventilation line” using a PTFE tubing was used to transfer the sample particles from the surgical site to the mass spectrometer. This statement implies that the experiments were performed in a surgery room, which does not appear to be the case. Please clarify.

·        What were the dimensions of the PTFE line? Moreover, that setup is not the same as the one showed in Figure 1. The system shown in Figure 1 appears to require the positioning of the tissue specimen in front of the mass spectrometer for analysis, and no transfer line is used. Was that the system used for the results shown for the breast tissue analysis? If that is the case, how is that translatable to surgical analysis?

·        Please label all setup components, as well as where the sample is located in Figure 1.

·        The authors mention that the breast samples were “obtained during surgical operation”.  Were the samples collected after surgery and analyzed in the lab? Were the samples analyzed fresh or were they thawed prior to analysis? Please specify. 

·        How was the pathology validated for these samples? Were serial sections used? This needs to be explained.

·        The authors describe that for data analysis, a table including the m/z values and sample IDs was created. Was the intensity of the peaks not taken into consideration for building the models? If that is the case, why was the Pareto scaling method used? This needs to be clarified.

·        It is also unclear which samples, and how many spectra, were used for building the classification models, and which ones were used for testing. The possibility of spectra from same sample being used for both training and validation is concerning. Please clarify.

Results and discussion:

·        Figure 2 shows differences in the temperatures provided by the two electrosurgery units. These differences would be easier to visualize if the two images were set to the same scale.

·        Since the goal of using the cold electrosurgical tool is to reduce tissue damage, as well as minimize molecular degradation, data should be provided to demonstrate improvements in this matter comparing the two systems.

·        Line 95-96. What to the authors mean by the “formation of ions in the process of cutting or coagulation is almost impossible”? Not favorable? Why do the authors believe that is the case?

·        It is concerning that the statement in line 101-102 “when the ion current produced by the applied electrosurgical method is not sufficient for MS analysis” is directly copied from the abstract of the manuscript cited in reference 12. Please rephrase. Plagiarism is a serious scientific misconduct.

·        Figure 3A shows the spectrum obtained without the extractive electrospray applied. The authors claim that “ions were not generated in the LTEU desorption process”. Yet, there are still some ions detected in the spectrum. What are those species attributed to?

·        Figure 3B – Further descriptions of the species detected in the spectrum of chicken skin with the electrospray applied would be helpful. What are the species between m/z 850-1000 attributed to?

·        The authors describe that the “proper geometry of the setup” was determined using the chicken meat samples. Is that referring to the conformation between the electrosurgical probe and the extractive electrospray source? Again, if a certain conformation is needed, how do they authors envision translating this to the surgical space where increased mobility of the probe is required?

·        Line 127 “These peaks mostly correspond to glycero- and phospholipids according to mass defect”. Please clarify what is meant by mass defect.

·        Figure 5 should be further edited for improved visualization of the results. The labels for colors red and blue are not shown in the figure. Are “pathological” tissues cancer samples?

·        Figure 6 should also be further edited. The mass spectra are blurry, and the m/z values in are hard to see.

·        Please label both axis in Figure 7. What is the number of samples/spectra used to report the 97% sensitivity and 94% specificity values?

Author Response

Please see attched file.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Thank you for your responses to comments. I wish you the best for continuing the development of this valuable system for intraoperative tissue characterization