Review Reports - Indocyanine Green and Technetium-99m: CT-Guided Marking of Lung Nodules Before Robotic-Assisted Lung Resection

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This article is timely because the widespread implementation of lung cancer screening programs has led to a significant increase in the detection of small pulmonary nodules. Robotic thoracic surgery is a state-of-the-art treatment method; however, the lack of tactile feedback makes intraoperative navigation a challenging task. The hybrid approach proposed by the authors, combining radioisotope marking and ICG near-infrared fluorescence, has high clinical potential. However, despite the value of the clinical data presented, the current version of the article has a number of significant shortcomings that prevent its acceptance in its original form.

The Abstract states 82 patients. However, it is further stated that there are 22 cases in the Tech group and 61 cases in the Tech+ICG group (22 + 61 = 83). The Methods section correctly states 83 patients. It is recommended to correct the number in the Abstract to 83.
There is a complete lack of a technical description of the robotic optical system and specifications for the equipment used to detect ICG fluorescence. For a high-quality scientific article, it is not sufficient to simply state “NIR fluorescence imaging”; a detailed technical description and a diagram of the setup are required, as well as the excitation and signal detection parameters (wavelengths), since these directly affect the penetration depth of the optical signal into lung tissue.
The number format is incorrect throughout the text. In English, a period (.) must be used in decimal fractions, not a comma. (For example, it is written as “26,5%,” “21,5mm,” “111,6 ± 45,6.” It should be: “26.5%,” “21.5 mm,” “111.6 ± 45.6”). It is recommended to replace all decimal commas with periods throughout the manuscript. A space should also be added between the number and the units of measurement (not 21,5mm, but 21.5 mm).
The authors report a decrease in the conversion rate from 13.63% to 6.55%, but do not provide the p-value in the Abstract. The text (Line 180) states that the difference is not statistically significant (p=0.375). It is recommended to add the p-value of 0.375 for the conversion rate to the Results section of the Abstract to avoid misleading the reader about the method’s effectiveness. Additionally, the Abstract states a value of 6.55% (Line 28), while the Results section (Line 180) states 6.6%. It is recommended to use a consistent format for decimal fractions.
The quality of the figures (Figures 1 and 2, Lines 259–261) is very poor. The photographs are low-resolution and lack explanatory labels. It is recommended to use higher-quality images. Add arrows to the photographs pointing to the area of nodal fluorescence and the area of ICG leakage, and also add a scale bar if possible. In addition, images related to the study results should be placed in the Results section.
Currently, virtually all study results (patient demographics, lung nodule characteristics, frequency of complications and conversions) are presented only as plain text. This makes it difficult for the reader to quickly grasp the information and compare groups. It is recommended to present key results more clearly using graphs and charts.
In the Discussion section, the authors mention that ICG does not provide information on the depth of the nodule (lines 255–256). It is recommended to briefly supplement the discussion by mentioning the physical limitations of NIR fluorescence in tissues. This will scientifically justify the need to add penetrating gamma radiation from 99mTc-MAA.
The patient groups being compared are extremely uneven in size: the “Tech” control group has only 22 patients (26.5%), while the main “Tech+ICG” group has 61 patients (73.5%). Such a significant numerical imbalance substantially reduces the statistical power of the comparative analysis. A separate structured paragraph dedicated to the limitations of this study (Study Limitations) must be added at the end of the Discussion section.
The submitted manuscript lacks a final “Conclusions” section. According to MDPI journal formatting guidelines, conclusions must be presented in a separate structured section - 5. Conclusions.

Author Response

The Abstract states 82 patients. However, it is further stated that there are 22 cases in the Tech group and 61 cases in the Tech+ICG group (22 + 61 = 83). The Methods section correctly states 83 patients. It is recommended to correct the number in the Abstract to 83.

Answer: the number of patients has been corrected in the Abstract

 There is a complete lack of a technical description of the robotic optical system and specifications for the equipment used to detect ICG fluorescence. For a high-quality scientific article, it is not sufficient to simply state “NIR fluorescence imaging”; a detailed technical description and a diagram of the setup are required, as well as the excitation and signal detection parameters (wavelengths), since these directly affect the penetration depth of the optical signal into lung tissue.

Answer: The robotic platform used is the Da Vinci Xi. The optical system used is the Firefly mode integrated into the system. The operator cannot in any way alter the wavelength detection, nor, of course, is it possible to alter the spectrum of radiation emitted by indocyanine green. The characterization of wavelengths is beyond the scope of this scientific article, and for the reasons mentioned above, it is exclusively a matter of Physics. In any case, the specification of the platform and technology used has been added to the text.

 The number format is incorrect throughout the text. In English, a period (.) must be used in decimal fractions, not a comma. (For example, it is written as “26,5%,” “21,5mm,” “111,6 ± 45,6.” It should be: “26.5%,” “21.5 mm,” “111.6 ± 45.6”). It is recommended to replace all decimal commas with periods throughout the manuscript. A space should also be added between the number and the units of measurement (not 21,5mm, but 21.5 mm).

Answer: format correction has been made.

 The authors report a decrease in the conversion rate from 13.63% to 6.55%, but do not provide the p-value in the Abstract. The text (Line 180) states that the difference is not statistically significant (p=0.375). It is recommended to add the p-value of 0.375 for the conversion rate to the Results section of the Abstract to avoid misleading the reader about the method’s effectiveness. Additionally, the Abstract states a value of 6.55% (Line 28), while the Results section (Line 180) states 6.6%. It is recommended to use a consistent format for decimal fractions.

Answer: the percentages have been checked and reconciled throughout the text

 The quality of the figures (Figures 1 and 2, Lines 259–261) is very poor. The photographs are low-resolution and lack explanatory labels. It is recommended to use higher-quality images. Add arrows to the photographs pointing to the area of nodal fluorescence and the area of ICG leakage, and also add a scale bar if possible. In addition, images related to the study results should be placed in the Results section.

Answer: Unfortunately, the digital image capture system is currently out of service, which explains the poor image quality. Captions have been added below each image. We believe that including these images in the “Results” section is helpful to the discussion, as they serve to illustrate the result of a correctly performed marking procedure compared to a failed marking due to dye diffusion within the pleural cavity. We remain open to further discussions

 Currently, virtually all study results (patient demographics, lung nodule characteristics, frequency of complications and conversions) are presented only as plain text. This makes it difficult for the reader to quickly grasp the information and compare groups. It is recommended to present key results more clearly using graphs and charts.

Answer: a summary table has been added

 In the Discussion section, the authors mention that ICG does not provide information on the depth of the nodule (lines 255–256). It is recommended to briefly supplement the discussion by mentioning the physical limitations of NIR fluorescence in tissues. This will scientifically justify the need to add penetrating gamma radiation from 99mTc-MAA.

Answer: ICG fluorescence is visible on the pleural surface because, physically speaking, the lung is an opaque structure and cannot be penetrated by electromagnetic radiation in the near-visible spectrum. What is visible on the visceral pleura is therefore the dye that has been drained by the lymphatic system and injected near the nodule, as stated in the paper. Measuremente of radioactivity of 99mTc-MAA can be differently detected by applying pressure on the lung surface, depending on the deep of the nodule (lines 81-89, 268-274 in last version)

 The patient groups being compared are extremely uneven in size: the “Tech” control group has only 22 patients (26.5%), while the main “Tech+ICG” group has 61 patients (73.5%). Such a significant numerical imbalance substantially reduces the statistical power of the comparative analysis. A separate structured paragraph dedicated to the limitations of this study (Study Limitations) must be added at the end of the Discussion section.

Answer: a “Study limitation” paragraph has been added to the paper.

 The submitted manuscript lacks a final “Conclusions” section. According to MDPI journal formatting guidelines, conclusions must be presented in a separate structured section - 5. Conclusions.

Answer: as stated in MDPI website in “Instructions for Authors” (https://www.mdpi.com/journal/diagnostics/instructions#preparation), the conclusion section is not mandatory. Anyway a Conclusion section has been added.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript addresses a clinically relevant topic. The study provides valuable real-world experience comparing two CT-guided marking techniques (99mTc-MAA alone versus 99mTc-MAA combined with ICG). However, several issues should be addressed to strengthen scientific rigor and improve interpretation of the findings. Here are my comments:

The reasoning behind comparing 99mTc-MAA alone with the combined 99mTc-MAA and ICG approach should be explained more fully in the Introduction. The expected benefits of the dual-tracer technique remain unclear.

Please describe the criteria used to divide patients into the Tech and Tech+ICG groups. It would be helpful to clarify whether the choice of localization technique was based on surgeon preference, radiologist preference, institutional protocol, or time period, as this may introduce selection bias.

Please provide additional methodological details, including the rationale and references for the localization timing, the selected ICG dose, and the experience of the two radiologists performing the procedures.

Please recheck that the statistics section may appear incorrect regarding mean/SD and median/IQR. Lines 143-144

The results section would benefit from the inclusion of summary tables presenting patient demographics, nodule characteristics, histopathological findings, surgical outcomes, and procedure-related complications. The current format makes comparisons between groups difficult and limits readability.

The discussion provides an extensive overview of the literature but would benefit from being more concise, focusing more on interpreting the current findings and their potential clinical implications.

The interpretation of the findings should be more considered. Although the Tech+ICG group showed lower failure rates, no statistically significant differences were observed between groups. Therefore, statements suggesting superiority or enhanced effectiveness of the dual-tracer approach should be interpreted with caution. Lines 291-299

Minor comments

The scientific value of Figures 1 and 2 is unclear. The authors should clarify how these images support the study objectives and findings.
Consider moving the limitations paragraph before the conclusion so that the manuscript ends with a clear concluding statement.
Please ensure consistency in numerical formatting throughout the manuscript. Decimal values should be reported using a period (.) rather than a comma (,).

Author Response

1.The reasoning behind comparing 99mTc-MAA alone with the combined 99mTc-MAA and ICG approach should be explained more fully in the Introduction. The expected benefits of the dual-tracer technique remain unclear.

Answer: a sentence has been added to the introduction. The benefits of the dual-tracer technique are specified in lines 231-233 and 250-256.

2. Please describe the criteria used to divide patients into the Tech and Tech+ICG groups. It would be helpful to clarify whether the choice of localization technique was based on surgeon preference, radiologist preference, institutional protocol, or time period, as this may introduce selection bias.

Answer: a specification has been added to the introduction.

3. Please provide additional methodological details, including the rationale and references for the localization timing, the selected ICG dose, and the experience of the two radiologists performing the procedures.

Answer: the timing of marking was chosen on the basis of our previuos experiences (lines 116-119). The experience of the radiologists are specified in the paper

4. Please recheck that the statistics section may appear incorrect regarding mean/SD and median/IQR. Lines 143-144

Answer: the typing mistake has been corrected

5. The results section would benefit from the inclusion of summary tables presenting patient demographics, nodule characteristics, histopathological findings, surgical outcomes, and procedure-related complications. The current format makes comparisons between groups difficult and limits readability.

Answer: A summary table has been added at the end of results section.

6.The discussion provides an extensive overview of the literature but would benefit from being more concise, focusing more on interpreting the current findings and their potential clinical implications.

Answer: we added a brief sentence (lines 252-253). We believe that the literature review serves to provide background on the marking methods already in use and the results of past experiences. To our knowledge the combination of technetium and ICG has never been published before, so the first clinical implication is provided by our paper.

7. The interpretation of the findings should be more considered. Although the Tech+ICG group showed lower failure rates, no statistically significant differences were observed between groups. Therefore, statements suggesting superiority or enhanced effectiveness of the dual-tracer approach should be interpreted with caution. Lines 291-299

Answer: thank you for the comment. A clarification regarding the interpretation of the data has been added to the paper

Minor comments

1. The scientific value of Figures 1 and 2 is unclear. The authors should clarify how these images support the study objectives and findings.

Answer: the captions have been expanded. The figures provided are intended to illustrate a successful and a failed marking procedure, respictively.

2. Consider moving the limitations paragraph before the conclusion so that the manuscript ends with a clear concluding statement.

Answer: the order of the paragraph has been inverted.

3. Please ensure consistency in numerical formatting throughout the manuscript. Decimal values should be reported using a period (.) rather than a comma (,).

Answer: the corrections have been made.

Reviewer 3 Report

Comments and Suggestions for Authors

Reviewer Comments to Authors

Title: Indocyanine Green and Technetium-99m CT-Guided Marking of Lung Nodules before Robotic-Assisted Lung Resection

Dear Authors,

I would like to congratulate you on conducting a very practical and clinically relevant study. The intraoperative localization of small, peripheral, or subsolid pulmonary nodules has become a critical bottleneck in the era of parenchymal-sparing surgery and Robotic-Assisted Thoracic Surgery (RATS). Using Technetium (Tc-99m) injection for lung nodule localization is a relatively novel, highly promising, and continuously evolving field of study. Your effort to evaluate a hybrid technique (99mTc-MAA + ICG) to exploit both deep gamma penetration and real-time near-infrared visualization is commendable.

The manuscript is well-structured, and the clinical rationale is clearly stated. However, several points need to be clarified or amended to enhance the scientific rigor and readability of the paper.

Major and Minor Comments:

Introduction & Rationale:

* The introduction smoothly transitions from the clinical need to the technical solutions. To strengthen it further, could you briefly expand on why the lack of tactile feedback in RATS makes depth estimation uniquely difficult compared to VATS, beyond just missing the visual marker?

Materials and Methods:

* Lines 95-96: You mention enrolling 83 patients retrospectively from January 2019 to February 2026. Could you clarify the exact sample size breakdown in the text? (From the results, it seems Tech Group n=22 and Tech+ICG Group n=61. Please state this explicitly in the Methods section).

* Line 113-117: You state that marking was performed ~20 hours before surgery based on previous experience. This is a very interesting protocol. Did you notice any significant ICG diffusion or "blurring" of the pleural surface after 20 hours compared to immediate surgery, or did the dual-tracer injection completely mitigate this risk? A brief comment on this would be highly valuable for readers attempting to replicate your protocol.

* Lines 123-126: You note that direct intra-nodular injection caused neutrophilic inflammatory infiltration, leading you to prefer peritumoral injection. This is an excellent, practical safety tip. Did this change in technique alter the background radioactivity or fluorescence signals during the surgery?

Results & Statistical Analysis:

* In the comparison of the two groups (lines 176-180), you report that the groups are homogeneous for diameter, distance, SUV max, and operative time. It would be helpful to present these baseline comparative data in a unified table (e.g., Table 1) to make the comparison visually clear and scannable for the readers.

* The reduction in conversion rate in the hybrid group (6.55% vs 13.63%) is clinically meaningful, despite the lack of statistical significance (p = 0.375). Please explicitly emphasize that the study might be underpowered to detect this specific difference due to the sample size layout.

Discussion & Limitations:

* Lines 255-257: You mention that the most common scenario for ICG failure is leakage into the pleural cavity, which causes indiscriminate marking. Could you provide a brief recommendation on how the surgical team should troubleshoot or handle this specific complication intraoperatively when using the gamma probe as a backup?

* The limitations section is honest and well-formulated. However, please also add that the uneven distribution of patients between the two groups (22 vs. 61) is a limitation of the retrospective study design over time.

Language and Typography:

* There are minor grammatical slip-ups and formatting errors (e.g., missing spaces in "March 13rd" on line 101, which should be "March 13th"; and "0,3 mL" on line 121, which should use a decimal point "0.3 mL" for English nomenclature). A thorough English proofreading is advised.

Overall, this is a very high-quality and thought-provoking paper addressing a rapidly developing frontier in thoracic surgery. I look forward to seeing the revised version.

Kind regards,

Author Response

Introduction & Rationale:

Answer: A sentence has been added to clarify technical aspects.

2.Materials and Methods:

Answer: The breakdown is explained in the Methods section.

Answer: A clarification regarding our preference has been added. In introduction section you can find a further explaination of dye concentration mechanism.

Answer: thank you for the comment. A comment has been added to the paper.

3.Results & Statistical Analysis:

Answer: a summary table has been added to the paper.

Answer: the limitations of the study have been explicited

4. Discussion & Limitations:

Answer: a recommendation has been added to the text

Answer: a precisation has been added to the text to clarify this point

Language and Typography:

Answer: a revision has been made.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Despite the authors' efforts to revise the manuscript, the fundamental methodological flaws remain unaddressed. The main conclusions are not supported by the data, as the observed differences in conversion rates lack statistical significance (p=0.375). The study is underpowered and relies on a non-randomized, historical control design with imbalanced groups (n=22 vs n=61). The authors did not provide a subgroup analysis based on nodule depth or histology (e.g., benign vs malignant), which significantly affects the interpretation of the marking accuracy.

Author Response

We provided a subgroup analys based on the distance from visceral pleura. The conclusion and discussion have been revised to present the results in a more cautious manner, given that statistical significance was not achieved. Thank you for your suggestions

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have addressed all of my comments. I have no further comments.

Author Response

Thank you for your suggestions

Reviewer 3 Report

Comments and Suggestions for Authors

Thank you for your answers. The revisions have significantly improved the clarity, methodological transparency, and overall quality of the study.

Author Response

Thank you for your suggestions