Cytokines Adsorption During Ex Situ Machine Perfusion of Liver Grafts from Elderly Donors: A Pilot, Prospective, Randomized Study

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The article "Cytokines Adsorption During Ex-Situ Machine Perfusion of Very Old Liver Grafts: A Pilot, Prospective, Randomized Study" presents a significant and clinically relevant study investigating the safety and efficacy of cytokine adsorption during ex-situ machine perfusion of elderly liver grafts.

 

It is a well-designed study, employing a prospective randomized approach to explore the potential value of cytokine adsorption during ex-situ machine perfusion. The research question is clear and the methodology is well described. Additionally, the analysis of the data is very comprehensive. Its small sample size is a major limitation, but it is appropriately acknowledged.

 

There are a couple of recommendations, though, in order to improve the overall text and interpretation of the obtained results.

 

In the Abstract, the first sentence should directly state the research background and objective, avoiding lengthy preambles. Also, the results could more concisely summarize key findings, while the conclusion should more explicitly state the potential value of CA and future research directions.

 

The Introduction section of the manuscript is generally well-written but could be more concise, emphasizing the research gap. The authors could consider clearly suggesting the specific objectives and hypotheses of the study at the end of the Introduction section.

 

In the Materials and Methods section, the study design is clearly described, but the authors could consider adding details on randomization. Also, the working principle and scope of application of CA device could be briefly explained.

 

In the Results section of the manuscript, when describing statistical differences, consistently use of terms like "significant" or "trend" to avoid ambiguity would be beneficial. Also, as highlighted in the text, table 4 is missing.

 

The Discussion section of the manuscript is comprehensive but could be further deepened. Specifically, it could be explained why CA was more effective in NMP than in D-HOPE. Also, a more clear highlighting the novelty and clinical significance of this study would also be beneficial. Finally, the authors could also consider providing a deeper interpretation of negative results (e.g., no differences in clinical outcomes), emphasizing the potential impact of the small sample size.

 

The Conclusion section currently overlaps considerably with the Abstract and Discussion. It should be concise and highlight the core messages, such as the fact that CA is safe and feasible during ex-situ liver perfusion, as well as that it effectively reduces inflammatory cytokines, improves vascular flow, and decreases neutrophil infiltration. Finally, it should be mentioned that larger studies are needed to validate its clinical benefits.

 

Other than the mentioned, no further adjustments are necessary.

Author Response

We would like to thank the Reviewer for the thorough evaluation of our manuscript and for the positive assessment of its clinical relevance, methodological rigor, and clarity. We are particularly grateful for the constructive suggestions aimed at improving the presentation and interpretation of our findings. Below, we provide a detailed point-by-point response. We appreciate the Reviewer’s recognition of the strengths of the study design and the comprehensive data analysis, as well as the acknowledgment of the small sample size as a key limitation. In the revised manuscript, we have carefully incorporated the Reviewer’s suggestions to improve clarity, conciseness, and focus, while maintaining a consistently cautious interpretation of the results.

Abstract

We have revised the first sentence of the Abstract to more directly introduce the clinical background and primary objective of the study, avoiding an extended preamble. The Results section of the Abstract has been streamlined to concisely highlight the key findings, and the Conclusion has been revised to more explicitly state the potential value of CA and the need for further studies to confirm its clinical impact.

Introduction

The Introduction has been refocused to better emphasize the existing research gap. In addition, we now clearly state the specific objectives and hypotheses of the study in the final paragraph of the Introduction, thereby improving readability and conceptual clarity.

Materials and Methods

We have added further details regarding the randomization process to enhance transparency. In addition, a brief description of the working principle and scope of application of the cytokine adsorption device has been included to provide context for readers who may be less familiar with this technology.

Results

We have revised the Results section to ensure consistent use of terms such as “significant” and “trend” when describing statistical differences, in order to avoid ambiguity. We also acknowledge the comment and apologize for the confusion; Table 4 does not exist, and the text has been corrected accordingly.

Discussion

The Discussion has been refined and further developed. In particular:

• We have expanded the discussion on the potential reasons why CA appeared more effective during NMP compared to D-HOPE, considering differences in temperature, metabolic activity, and cytokine kinetics.

• The novelty and clinical significance of the study have been more clearly highlighted.

• We have provided a deeper interpretation of negative findings, including the lack of differences in clinical outcomes, emphasizing the likely impact of the limited sample size and the exploratory nature of the study.

Conclusion

The Conclusion section has been substantially revised to reduce overlap with the Abstract and Discussion. It is now more concise and focused on the core messages of the study: the safety and feasibility of CA during ex-situ liver perfusion, its association with reduced inflammatory cytokines, improved vascular flows, and decreased neutrophil infiltration. We also explicitly state that larger, adequately powered studies are required to validate potential clinical benefits.

We believe that these revisions have significantly improved the clarity, focus, and scientific rigor of the manuscript. We sincerely thank the Reviewer for the valuable feedback, which has helped us strengthen the presentation and interpretation of our work.

Sincerely,
Giulia Cirillo
on behalf of all authors

Reviewer 2 Report

Comments and Suggestions for Authors

This pilot randomized study addresses an important and timely topic, namely the modulation of inflammatory mediators during ex-situ machine perfusion of very old liver grafts. The concept is clinically relevant and the manuscript is generally well written, with a clear description of the perfusion techniques and a thoughtful discussion of the biological rationale. The results regarding cytokine dynamics, vascular flows and neutrophil infiltration are interesting and suggest a potential mechanistic benefit of cytokine adsorption, particularly during NMP.

To further strengthen the manuscript, the authors are encouraged to better frame the exploratory nature of the clinical comparisons, given the extremely small sample size of the MP subgroups (n=3 per arm). The interpretation of clinical outcomes should be consistently cautious throughout the Results and Discussion, avoiding any implication of efficacy beyond feasibility and mechanistic signals. Clarifying the randomization process, the rationale for the chosen cytokine panel, and the handling of multiple comparisons would also improve transparency and reproducibility.

Finally, some sections would benefit from focused streamlining, particularly in the Discussion, where several speculative mechanisms are presented. A more explicit separation between data-driven findings and hypotheses for future work would improve readability and scientific rigor.

 

Α series of to the point proposals follow

• Explicitly state in the Abstract and Discussion that the study is not powered for clinical outcome comparisons and is hypothesis-generating only.

• Add a brief rationale for the selection of IL-1, IL-6, IL-10 and TNF-α, and clarify why other mediators (e.g. DAMPs) were not included.

• Describe in more detail how randomization and allocation concealment were performed, including any stratification.

• Reduce speculative language in the Discussion and clearly distinguish observed results from proposed mechanisms.

• Clarify how multiple statistical comparisons were handled and whether any correction was considered.

• Add a concise limitations paragraph summarizing sample size, single-center design and lack of power for clinical endpoints.

• Consider moving some technical detail from the main text to Supplementary Material to improve flow.

Author Response

We sincerely thank the Reviewer for the careful evaluation of our manuscript and for the constructive and insightful comments. We are pleased that the Reviewer recognizes the clinical relevance of the topic and the potential mechanistic implications of cytokine adsorption during ex-situ machine perfusion of very old liver grafts. Below, we provide a point-by-point response to all comments and suggestions.

General comment

We fully agree with the Reviewer that, given the extremely small sample size of the machine perfusion subgroups (n = 3 per arm), the study must be interpreted as exploratory and hypothesis-generating. In the revised manuscript, we have taken care to consistently frame the clinical comparisons as feasibility and mechanistic observations only, avoiding any implication of clinical efficacy.

Specific comments

1. Exploratory nature and lack of power for clinical outcomes
   We have now explicitly stated in both the Abstract and the Discussion that the study was not powered to detect differences in clinical outcomes and should be considered hypothesis-generating. All language suggesting efficacy has been revised to ensure a consistently cautious interpretation.

2. Rationale for cytokine selection
   A brief rationale has been added to the Methods section explaining the selection of IL-1, IL-6, IL-10, and TNF-α. These cytokines were chosen as representative pro- and anti-inflammatory mediators with established relevance in ischemia–reperfusion injury and liver transplantation. We also clarify that other mediators, including DAMPs, were not included due to the exploratory nature of the study and limitations related to sample volume and assay availability.

3. Randomization and allocation concealment
   We have expanded the description of the randomization process, specifying how allocation was performed and how concealment was ensured. 

4. Speculative mechanisms in the Discussion
   In response to the Reviewer’s comment, we have reduced speculative language in the Discussion . Speculative mechanisms are now explicitly framed as such.

5. Multiple statistical comparisons
   We have clarified how multiple comparisons were handled in the statistical analysis and whether any correction was applied. This information has been added to the Statistical Analysis subsection to improve transparency and reproducibility.

6. Limitations paragraph
   A concise and explicit Limitations paragraph has been added, summarizing the small sample size, single-center design, and lack of power for clinical endpoints.

7. Streamlining and Supplementary Material
   We appreciate the suggestion to move selected technical details to the Supplementary Material to improve readability. However, for reasons of text coherence and flow, we have decided to keep all relevant details in the main manuscript to ensure that the methods and results are fully transparent and accessible to the reader.

We believe that these revisions have substantially strengthened the manuscript and improved its clarity, rigor, and transparency. We are grateful to the Reviewer for the valuable feedback, which has helped us refine both the presentation and interpretation of our work.

Sincerely,

Giulia Cirillo