Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Thank you for the opportunity to review this manuscript that introduces a novel secondary perfusion protocol for donor livers declined after normothermic machine perfusion (NMP), with the aim of better modeling post-transplant performance and refining viability criteria. The work is highly relevant to the field, addresses a clear clinical gap, and provides detailed, reproducible methodology.

The authors propose and describe a secondary perfusion protocol subjecting livers already declined after NMP to an additional cycle of cold ischemia and reperfusion. They report a pilot case study that failed primary NMP viability but underwent secondary perfusion, demonstrating persistent dysfunction but also some unexpected metabolic changes. The authors argue that this model could help validate NMP criteria and reduce unnecessary graft discard.

Major Comments

1. The manuscript is based on one case. While the authors acknowledge this, the abstract and discussion sometimes imply broader validation of secondary perfusion. Please emphasize more clearly that this is a protocol description and proof-of-concept only, not a validated outcome study.
2. The discussion at times overinterprets single findings (e.g., lactate clearance dynamics, “zone 1” liver function). Please distinguish carefully between observations and hypotheses. Statements suggesting confirmation of viability criteria or prediction of primary non-function should be framed as preliminary.
3. The primary perfusion was performed on the OrganOx metra, whereas the secondary perfusion was performed on the XVIVO Liver Assist. This is acknowledged, but a more detailed discussion is needed on how device-specific flow/pressure thresholds may have influenced outcomes and comparability.
4. Figures 2–4 are useful but could be improved with: a) A side-by-side table or figure directly comparing primary and secondary perfusion outcomes (flows, lactate, pH, glucose, bile output); b) If available, histology images or post-secondary perfusion tissue analysis would further strengthen the data presentation.
5. While the concept of secondary perfusion as a research tool is compelling, the manuscript should more strongly clarify that this is not proposed as a clinical rescue pathway at this stage. Explicitly note that secondary perfusion is intended for research/validation of viability criteria, not for re-offering grafts for transplantation.

Minor Comments

1. Abstract: Please reframe the conclusion to highlight that this is a protocol and proof-of-concept, not yet a validated strategy.
2. Introduction: The introduction is comprehensive; however, it could briefly summarize why prior studies of discarded livers (e.g., VITTAL trial, RESTORE) differ fundamentally from the current approach of testing grafts already declined after NMP.
3. Methods: Some details (e.g., choice of perfusate composition, anticoagulation, device-specific settings) could be consolidated into a supplementary table for easier reproducibility across centers.
4. Discussion: Consider expanding on how this protocol could be standardized for multicenter validation and whether a registry of secondary perfusion outcomes might be feasible.
5. References: Ensure that all references are formatted according to journal style. For example, references [22, 23] appear duplicated in both methods and discussion.

This manuscript presents a valuable and innovative contribution. With clarifications and more cautious interpretation, it will serve as a useful reference protocol for the transplantation community.

 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The authors present a protocol for secondary NMP of a donor liver allograft. This protocol is novel in the setting livers on NMP are declined after failing viability testing.

1. However, it would be still be helpful if the authors could provide more biologic rationale and data to support this approach to "stress testing." i.e why is this method better than other possible approaches (In this paper they note the procuring surgeon had concerns about the allograft-would in situ NRP followed by machine perfusion-another "two hit" model been any better than what the authors propose?).
2. In addition the authors note the limitations of current methods of viability testing.  It would be helpful if the authors could provide what percentage of livers at their center failed viability testing and roughly how many of those would have been retested along the protocol they propose and if methods such as FMN would have altered those data. 
3. Finally, the OrganOx metra device differs from other NMP devices in the readouts and technical approach to perfusing the allograft as well as back to base vs perfusion on site. As other NMP devices are currently in use, is this approach applicable to those devices?

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I appreciate the author's responses, however, It is still unclear as to how this protocol would fit into current biological understanding of IR injury and organ preservation. I am presuming the authors are using the notion of repeated ishemic conditioning/preconditioning with the idea that repeated arrest of cellular respiration and secondary reperfusion on an NMP circuit would be the "stress test" the authors are alluding to (lines 82-83). Although there is limited data on retesting of livers, the following data from van Leeuwen and colleagues is important to reference and acknowledge.  Annals of Surgery 270(5):p 906-914, November 2019. | DOI: 10.1097/SLA.0000000000003540 as it represents one of the few trials examining multiple rounds of perfusion to rescue allografts. Albeit this is DHOPE combined with NMP and may not be exactly similar, it is critical for the authors to provide a biological justification for this approach.  It is understandable that the data from van Leeuwen et al is difficult to replicate given the complexity of reperfusion methods but there is biological justification for their approach. There are other references from this group as well that should be included as part of the discussion/references

Sequential hypothermic and normothermic machine perfusion enables safe transplantation of high-risk donor livers.

van Leeuwen OB, Bodewes SB, Lantinga VA, Haring MPD, Thorne AM, Brüggenwirth IMA, van den Berg AP, de Boer MT, de Jong IEM, de Kleine RHJ, Lascaris B, Nijsten MWN, Reyntjens KMEM, de Meijer VE, Porte RJ.Am J Transplant. 2022 Jun;22(6):1658-1670. doi: 10.1111/ajt.17022. Epub 2022 Apr 18.   Transplantation of high-risk donor livers after resuscitation and viability assessment using a combined protocol of oxygenated hypothermic, rewarming and normothermic machine perfusion: study protocol for a prospective, single-arm study (DHOPE-COR-NMP trial). de Vries Y, Berendsen TA, Fujiyoshi M, van den Berg AP, Blokzijl H, de Boer MT, van der Heide F, de Kleine RHJ, van Leeuwen OB, Matton APM, Werner MJM, Lisman T, de Meijer VE, Porte R.BMJ Open. 2019 Aug 15;9(8):e028596. doi: 10.1136/bmjopen-2018-028596.   I am not asking the authors to conduct extensive studies on their approach as it relates to basic cellular or molecular effects of repeat cycles of NMP on liver allografts, however, I would like their to be some insight into why this approach was taken/justified. I would suggest using these references as a guide to providing that biological justification

• 10.Robertson F.P., Magill L.J., Wright G.P., Fuller B., Davidson B.R. A systematic review and meta-analysis of donor ischaemic preconditioning in liver transplantation. Transpl. Int. 2016 doi: 10.1111/tri.12849. [DOI] [PubMed] [Google Scholar]
• An Evaluation of Ischaemic Preconditioning as a Method of Reducing Ischaemia Reperfusion Injury in Liver Surgery and Transplantation. Robertson FP, Fuller BJ, Davidson BR.J Clin Med. 2017 Jul 14;6(7):69. doi: 10.3390/jcm6070069.

The authors could simply say that this model may represent repeated cycles of ischemia and reperfusion akin to (but not exactly) ischemic preconditioning which may reveal recovery of previously non viable liver allografts on nmp. This models value may be supported by reports of how ishemic preconditioning can improve liver viability and the value of multimodal/multistep perfusion cycles may rescue unviable livers.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf