INTRODUCTION
Liver perfusion has become increasingly adopted into clinical practice due to its evident abilities to overcome the many shortcomings of static cold storage (SCS) 1-4. As a consequence of the increasing use of extended criteria donors to meet waitlisting demands, transplant teams are having to assess the optimal ways to implement the various perfusion technologies, hoping to improve graft quality and transplant outcomes 5.
Ex-situ normothermic machine perfusion (NMP) aims to replicate the liver’s normal physiological condition and maintain its metabolic activity during preservation 6. Data collected and the liver’s behaviour can then be used to assess its suitability for transplantation 7-9. Beyond encouraging clinical data, demonstrating a positive impact on liver quality remains challenging. The reassurance provided regarding liver functional activity and the logistical improvements it provides are often considered beneficial enough to establish an NMP programme 2,10-12. The ability to assess the viability of liver grafts has evolved with the perfusion technologies and looks to improve further as our knowledge and experience grows 8.
This review aims to provide information regarding the practical aspects of starting an NMP programme and pragmatic guidance towards assessing liver viability for teams new to this field.
KEY DECISIONS PRIOR COMMENCING NMP PROGRAMME
Machine perfusion evolution and early pathways to clinical adoption
The introduction of liver perfusion into clinical practice was pioneered by several groups around the world, starting by Guarrera and colleagues in New York using hypothermic (4-10°C) machine perfusion (HMP), Dutkovski in Zurich developing hypothermic oxygenated machine perfusion (HOPE), and Friend in Oxford introducing normothermic (36-38°C) machine perfusion 6,13,14. Machine perfusion was adopted by several teams very early on through participation in the initial clinical trials, sharing awareness about the new dynamic liver preservation to the wider transplant community 6,15-21. The teams participating were typically provided with access to the perfusion device, consumables, technical support, and team training that were funded through trials. The developed setup, gained team expertise, and infrastructure facilitated subsequent seamless NMP adoption by those teams 3,22,23. This historical context explains why some perfusion approaches may be predominant in specific regions 3,23,24.
Contrary to the early adopter’s pathway and programmes funding via research resources, the subsequent uptake of the technology has been more challenging 22. It requires evidence-based decision-making, significant resources, and specialised expertise, all of which have not yet been firmly established 25. Whilst the regulatory aspects are beyond the scope of this manuscript, it is worth mentioning that the majority of the commercially available devices are CE marked and there are no barriers to their clinical use in Europe. The funding, however, remains the major hurdle to overcome, in particular on the background of the ongoing debate about the real-world clinical efficacy that is aggravated by the lack of supportive health economic data.
DONOR CHARACTERISTICS, TIMING, AND LOCATION OF THE NMP PROCEDURE
Evidence from completed clinical trials is quickly accumulating and evolving and it has become increasingly apparent that certain donor livers benefit from specific perfusion approaches 24,26-30.
It has been widely accepted that liver cooling and rewarming in the context of concomitant ischaemia triggers the irreversible cascade resulting in organ damage. The team in Guangzhou who pioneered ischaemia-free normothermic perseveration demonstrated that if these events can be avoided then even marginal livers typically deemed untransplantable (e.g. donors with 90% macrosteatosis) can be transplanted successfully 31. Its logistical requirements, however, are currently too challenging for this method to become mainstream 32.
Steatotic organs seem to be most susceptible to damage caused by SCS and benefit most from a normothermic approach with limited cold ischaemia time (CIT). Similarly, in donors after circulatory death (DCD), early intervention seems to be of key importance to minimise risk of developing non-anastomotic biliary strictures (NAS) with normothermic regional perfusion and NMP commenced at the donor centre showing superior stricture outcomes compared to end-ischaemic NMP 26,28,29. When applied to already cold DCDs, and commenced at the transplant centre, these livers benefited from HOPE 3. Whilst in the past hypothermic and normothermic perfusion were seen as mutually exclusive and competitive technologies, it has been recognised that these different approaches can be complimentary 5,28,33. The Groningen team were the first to demonstrate clinical benefits of such combination, treating DCD livers initially with a period of HOPE to minimise the ischaemia-reperfusion injury leading to biliary strictures, followed by functional assessment using NMP 34.
Regarding transplant logistics and extension of the preservation times for long-distance sharing, this aspect used to be dominated by NMP. There is, however, emerging data that HOPE can maintain perfusion for over 12 hours without detriment to the organ 35. In situations where a liver reaches a transplant centre with excessive CIT causing concern about primary non-function (PNF), the decision about organ usability may be guided by viability assessment by NMP as described later.
Commencing NMP at the donor hospital may be more resource intensive but it follows established organ preservation and allocation pathways and its superiority over SCS has been demonstrated in several randomised trials 23,24.
In the context of skilled personnel shortage, financial restrictions, and achievable short CITs, commencing perfusion at the transplant centre is often easier in the real-world as it provides access to senior surgeons to prepare the liver for perfusion and implantation, including arterial reconstruction if needed, and the expertise for NMP troubleshooting 22,36. The back-to-base approach also does not require complex transportable devices and can be applied to selective livers based only on its perceived quality that helps to keep the cost down. As the organs are already cold it provides opportunity to choose the perfusion temperature, though it is still widely accepted that a liver needs to reach normothermia if viability is to be assessed.
LIVER PERFUSION DEVICES
The number of available perfusion devices is increasing, and the expanding market is likely to continue this trend. The issues about the perfusion and temperature are discussed above. The next important aspect to consider is cost. The manufacturers often provide access to the device within loan or lease schemes that are combined with staff training and pump maintenance. The cost of the perfusion disposable kit, blood, fluids, and drugs are therefore the most obvious expense considered when starting a perfusion programme. These details are outside the scope of this review, but in general, transportable NMP devices are more complex and expensive than non-transportable ones, and hypothermic devices are simpler and cheaper than normothermic variations. Device cost often reflects companies’ investment into the development, registration, clinical testing, customer support, and the biggest players in the field offer pumps used in large randomised controlled trials.
The impact of device simplicity, or a degree of automatisation and user friendliness in more complex machines is often underestimated. This was less of an issue for teams who pioneered the technology and appreciated the opportunity to amend multiple perfusion parameters. For new teams entering the field now, however, this is a key feature in shortening the personnel learning curve and minimising the risk of user error.
A feature less often highlighted is oxygenator specifications. Although many authors challenge using machines solely to improve transplant logistics, having the option of starting procedures semi-electively in the morning often becomes the most adored perfusion features in newly established programmes. To benefit from this possibility for perfused livers, the device needs to support perfusion lasting a minimum of 12 hours.
THE ORGAN RETRIEVAL, PERFUSION, AND TRANSPLANT TEAMS
Dynamic organ preservation is an emerging subspeciality requiring multi-disciplinary skills. The ability to operate and troubleshoot the device is the key requirement to establishing an NMP programme, that needs to be complemented by surgical expertise in multi-organ retrieval, ability to perform hepatic artery reconstructions, and ability to interpret the perfusion and functional parameters 37. The stakes are higher in the NMP approach due to the potential risk of losing the organ, albeit this seems to be significantly lower than initially envisaged and reports of graft loss or device malfunction are extremely rare 38.
Although there is a learning curve with the NMP (around 5-10 procedures) it is often quickly overcome by the team’s enthusiasm. The real-world challenge it to sustain the programmes by appointing personnel running 24/7 rotas as those initially “hidden costs” might be higher than perfusion consumables. Centres with lower transplant volumes might find this more difficult but for them the need might be less urgent. Multi-organ transplant programmes can pool the perfusion specialists into one team as the skill set is similar. For high-volume teams the justification of the additional manpower and other resources may be easier to justify. Further reading on this topic was well covered by Hunt and colleagues 39.
Transplant professionals are highly motivated individuals and cover commitments often exceeding those in other specialities. In the context of the COVID-19 pandemic, the critical shortage of healthcare workforce and burnout, team resilience and well-being, staff recruitment, and retention are becoming issues needing increasing attention. By extension of the liver preservation times the machine perfusion can significantly improve transplant procedure logistics, making it a semi-elective daytime procedure 2,22. The novelty of the machine perfusion often provides opportunities for research that can further attract new young colleagues into our speciality. These benefits might be most noticeable in large volume units and whilst difficult to quantify there is evidence demonstrating increased safety for the liver recipients 22,40,41.
VIABILITY ASSESSMENT PROTOCOLS
The ability to monitor liver metabolic activity during NMP provides opportunities to evaluate suitability for transplantation. For this review, we define the viability assessment as an approach directed only to selected livers – in contrast to systematic use in all grafts and applied post-hoc when the ideal time to intervene was lost and the organ reached the transplant team preserved by SCS 27,42,43.
There have been several proposed viability protocols, that were all derived from small cohorts of perfusions, different type donors, with different key objectives 16,44,45. To ensure recipients safety, all protocols included multiple measures with favourable readings along those observed physiologically in-vivo conditions.
With increasing confidence in the technology and learning from suboptimal outcomes it becomes apparent that strengths and limitations of each protocol should be evaluated in the context of the diagnostic dilemma they are meant to answer. Also, most authors acknowledge that with increasing experience the comfort zone to use suboptimal livers increases and the viability assessment cut off values are being relaxed as described below 44,46.
The functional assessment of donor organs covers two compartments, hepatocytes and cholangiocytes, and these are predominantly responsible for initial liver function and late biliary strictures, respectively.
PRIMARY NON-FUNCTION AND SEVERE GRAFT DYSFUNCTION
The key question – is this liver going to work in the next 7 days? – can be predicted very early on from the consumption of several perfusate substrates, e.g. lactate, glucose, and oxygen or by measurement of synthetic activity e.g. urea, clotting factors, and bile volume. The incidence of PNF in the published NMP series is extremely low. In the first multicentre randomised study comparing NMP commenced in the donor hospital with SCS the authors reported one such event in a liver that did not clear lactate levels below 5 mmol/L 23. The trial, however, was conducted before any viability criteria were defined and functional assessment taken into the decision making about usability for transplantation.
Lactate clearance
Perfusate lactate clearance is a widely accepted marker of good hepatocyte function. Livers exposed to prolonged SCS, especially steatotic organs, may be slower to recover their function. This difference is demonstrated in Figure 1, showing fast lactate clearance (levels below 2.0 mmol/L within 60-90 minutes in livers put on the device in the donor hospital) in contrast to a slower decline in high-risk livers exposed to CIT around 10 hours (meeting similar lactate levels in 2-4 hours).
The timeframe to assess liver viability is a less defined area which demonstrates the learning curve and increasing confidence with the technology, though this change was facilitated by transition to a device with improved oxygenator specifications allowing longer perfusions. Our initial protocol in pilot discarded liver series assessed viability parameters at 2 hours, it was subsequently validated in the VITTAL trial with assessment at 4 hours and most recently we assess based on readings at 6 hours (Fig. 2). The rate of lactate clearance (or any other viability marker) is perhaps a good gauge of liver quality and livers meeting the parameters quickly are less likely to develop any degree of graft dysfunction.
Bile production
Presence of good quality bile is convincing and perhaps the most sensitive marker for liver viability, offering the possibility to assess the functioning of both hepatocytes and cholangiocytes. An hourly bile production > 10 mL is the frequently quoted volume anticipated in transplantable livers 45,47. Bile production is a complex metabolic process and if used beyond simple volume production measurement it provides more accurate information about the organ’s overall condition. Importantly, bile composition may help to identify livers at risk of developing late biliary NAS that is of key importance in DCDs. Its role in assessment of DBD livers is of lesser importance, with the main drawbacks being the delayed production and possible failure due to technical complications 10,27,48. A lag in recovery of the production can be often observed in steatotic livers or organs following extensive CIT with the times being beyond the widely accepted 2-4 hours viability assessment timeframe. To prevent technical problems, bile is best collected by thin tube (8-12 Fr, e.g. paediatric feeding tube, ideally placed under mineral oil to prevent oxidation and changes in bile pH), the cystic duct should be ligated, with the tube tip positioned well below hepatic ducts confluence, and fixed without obstructing its lumen 1.
Transaminases levels
Contrary to the lactate clearance and bile production, the perfusate release of transaminases provides a snapshot of the damage the liver sustained prior to retrieval, during preservation, and following ischaemia-reperfusion injury. The cut-off values are less well-defined, and this parameter cannot show an improvement over the course of perfusion, however, an exponential rise of the levels is an ominous sign. The Cambridge team reported favourable outcomes if ALT levels were < 6,000 IU/L and this is aligned with our retrospective observations from the VITTAL perfusion cohort 44.
Other parameters
Many other parameters can be measured from the perfusate including urea, clotting factors, C-reactive protein, and albumin 49. The discriminative power for viable or non-viable livers is, however, lower compared to lactate and therefore most livers deemed viable perform favourably in these markers, and any cut-off values are yet to be defined.
Failure to metabolise glucose is a very rare but concerning feature. Perfusate pH is another important parameter to consider, particularly the liver ability to maintain pH within a range of 7.2-7.4 over the course of a perfusion. Its close correlation with the lactate levels, or correction with sodium bicarbonate are other factors to consider when interpreting the results.
The above markers are based on traditional blood gas, bile, and biochemical perfusate analyses. Surprisingly, the new generation of biomarkers based on advanced omics methods are still scarcely reported 49. Other approaches, for example indocyanine green or methicillin clearance were proposed but not yet reported from clinical series 50-52.
Liver biopsy used to be the gold standard in liver transplantability assessment. With the advancement in NMP viability assessment this situation has changed, however, the perfusion may provide time for more detailed histology evaluation, for example, to assess the pre-perfusion bile duct damage that might contribute to decision to discard DCD livers with worrying features, or to evaluate suspicious extra-hepatic lesions discovered unexpectedly during procurement 53,54.
NON-ANASTOMOTIC BILIARY STRICTURES
Hilar or intra-hepatic biliary strictures are among the most troublesome post-transplant complications, occurring almost exclusively in DCD livers (the incidence in DBD is 1-2%) 55. There are several markers related with favourable post-transplant outcomes (Tab. I). Whilst the cut-off values may vary between protocols, to minimise risk of NAS DCD livers should produce over 20 mL of alkalotic bile with low glucose and high bicarbonate concentrations within the first 2-4 hours of perfusion.
The Groningen team suggests bile volume ≥ 10 ml/hr with pH > 7.45, and similarly the Cambridge group reported bile pH > 7.50, glucose level ≤ 3 mmol/L (respective ≥ 10 mmol/L lower than the perfusate) as favourable features for the liver longevity 34,44. The VITTAL trial did not include bile into the transplantability assessment, and some DCD livers developed aggressive features of NAS complications requiring early re-transplantation 27. Retrospective analyses of collected samples confirmed those were present in livers without bile production, low bile pH or bile bicarbonate. Overall, bile production and bile analysis seem to be of paramount importance to assess biliary integrity to predict DCD livers longevity and avoid NAS 8,48.
REAL-WORLD APPROACH TO THE LIVER VIABILITY TESTING
Even when applied to the most marginal livers widely deemed not suitable for transplantation, a high proportion of those organs will meet some of the viability criteria. For example, in the VITTAL trial, requiring lactate clearance below 2.5 mmol/L within 4 hours, 71% livers met the benchmark and were successfully transplanted with 100% 3-month graft and patient survival 27. Livers most likely to fail were those with severe macrosteatosis or those exposed to a prolonged CIT. Through our learning with the viability assessment, we extended the assessment period and dropped some markers included earlier in the programme 7,16,27,46. Indeed, in our series the livers meeting the lactate criteria also uniformly met vascular flows and macroscopic appearance criteria so their diagnostic value is limited. Regarding the bile assessment, our group has successfully transplanted DBD livers meeting the lactate criteria in the absence of bile production 2,27. We acknowledge, however, that a minority of those organs went on to develop early allograft dysfunction or post-reperfusion syndrome and may not be suitable for all recipients.
The roughly defined cut-off values for some parameters might be re-defined by more sophisticated definitions. For example, the slope of lactate clearance per kg of liver mass per hour can provide more granularity to liver function assessment 44. In the real-world, however, these are not user friendly and their benefit beyond the initial 1-2 hours would be limited. On this note it is important to mention that all the protocols were developed to ensure patient safety, and hence some safety margins were knowingly included. Teams commencing viability assessment programmes may be overwhelmed by the complexity of decision making and lack of universally accepted transplantability criteria 8. The starting point is always to improve patient’s safety. Cautiously planning to overcome the learning curve by applying more conservative criteria, considering preferably livers of favourable macroscopic appearance exposed to CIT, and selecting lower risk recipient seem to be reasonable starting points prior to proceeding to programme expansion. The initial avoidance of livers considered far beyond a teams’ comfort zone may allow faster programmes growth as it prevents hiccups in outcomes and resources wasted from failed perfusions.
ONGOING AUDIT OF NMP PROGRAMMES
Every introduction of new technology needs to be monitored and its outcomes closely evaluated. The NMP programmes are likely to bring multiple benefits for the wider transplant teams, including daytime operating with improved support to deal with any complications, smoothen the re-perfusion phase, and overall improve early post-transplant patients’ recovery. Liver transplantation is, however, a highly complex technical procedure whose outcome is clearly influenced by various factors including recipient condition. Occurrence of specific complications, e.g. hepatic artery thrombosis or anastomotic biliary strictures are unlikely to be caused by NMP, as those would have already been exposed in closely scrutinised data from the clinical trials. Technical complication related to the machine use may still appear, for example from a misplaced arterial cannula or biliary drain damaging the structures’ lumen above the level of anastomosis. Regular review of the outcomes and critical appraisal of any complications, in particular those unexpected and rarely observed by the programme have an important role for long-term success. In view to excellent outcomes from up-to-date published NMP series the boundaries of the technology use are most likely to come from reflective learning from failures and complications along with the aspects contributing to improved outcomes.
CONCLUSIONS
Machine perfusion is a technology that overcomes the many shortcomings of SCS and is becoming a widely adopted method of liver preservation by transplant teams worldwide. One of the advantages of using NMP is the facilitation of transplanting extended criteria organs with increased confidence in positive outcomes. It is a resource-intensive intervention and the most optimal ways for adoption have yet to be established. Despite its relatively steep learning curve, the setting up of a new NMP brings benefits to both patients and transplant teams alike, and benefits from a multi-disciplinary approach. The NMP approach allows detailed liver functional assessment and objective parameters to determine organ transplantability. The risk of PNF can be predicted from hepatocellular function that is well reflected by perfusate lactate clearance, with other markers including bile volume, perfusate transaminases, or glucose utilisation. Assessment of DCD livers should include analysis of bile composition to exclude organs at increased risk of developing NAS, characterised by a failure to produce alkalotic bile, high glucose, and low bicarbonate concentrations. NMP programmes should monitor outcomes and learn from any complications that might be related to the procedure learning curve.
Acknowledgements
We gratefully acknowledge the generous NMP research support provided by all team members of the Liver Unit at Queen Elizabeth Hospital Birmingham. We also gratefully acknowledge the Welcome Trust for funding that supported the VITTAL trial.
Conflict of interest
The Authors declare no conflict of interest.
Funding
This manuscript did not require any external funding. This paper presents the authors’ views and experience gained from research supported by the University Hospitals Birmingham Liver Charities and NIHR Birmingham Biomedical Research Centre at the UHB NHS Foundation Trust and the University of Birmingham. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR, or the Department of Health. The VITTAL trial was an independent, academic research project funded by the Welcome Trust [200121/Z/15/Z].
Author contributions
HM: conceived the manuscript concept; GC, HM: prepared the manuscript draft; GC, JM: contributed equally to drafting tables, figures, and paper editing; all Authors were involved in the research underpinning the presented data; NMP: expertise, and reviewed the final manuscript version.
Ethical consideration
Not applicable.
Figures and tables
Figure 1. Lactate clearance characteristics. The figure outlines different lactate clearance characteristics according to the NMP approach. The blue curve shows rapid lactate decline typically seen during normothermic preservation, when the NMP procedure is commenced in the donor hospital. For viability assessment following a period of static cold storage, the slope of lactate clearance is usually slower, with most livers meeting the 2.5 mmol/L threshold between 2-4 hours as illustrated by the green curve. The clearance curve of non-viable livers may vary widely, but most livers demonstrate some degree of lactate metabolism and reach levels between 5-10 mmol/L as shown by the red curve (The figure was created in the BioRender.com application).
Figure 2. Refinement of the lactate clearance criteria. The initial viability criteria were derived from a small experimental NMP series. The lactate clearance threshold 2.5 mmol/l was reached by all livers that were able to maintain 6-hour perfusion. For our pilot clinical series, the viability was assessed at 2 hours. Along the learning curve and increased confidence with the technology (60 liver transplants using NMP liver) the VITTAL trial assessed the viability within 4 hours. Following analysis of the trial results, the assessment timeframe was extended to 6 hours though applied exclusively to DBD livers. Such extension does have only limited impact on yield of transplantable livers regarding the lactate clearance assessment, however, the time extension allows for more livers to produce bile (The figure was created in the BioRender.com application).
| Established viability testing protocols | |||
|---|---|---|---|
| Assessment period | Birmingham 1 | Cambridge 2 | Groningen3 |
| 240 minutes | 120-240 minutes* | 150 minutes | |
| Favourable parameters | |||
| Hepatocyte function | Lactate < 2. 5mmol/L Evidence of bile production Perfusate pH > 7.30 | Peak lactate fall ≥ 4.4 mmol/L/kg/hr Falling glucose beyond 2 hrs < 10 mmol/L ALT < 6,000 UI/L at 2 hrs Perfusate pH > 7.20 | Lactate ≤ 1.7 mmol/L Cumulative bile production > 10 mL Perfusate pH 7.35-7.45 |
| Biliary parameters | No biliary parameters | Biliary pH > 7.50 Bile glucose ≤ 3 mmol/L (or ≥ 10 mmol less than perfusate glucose) | Biliary pH > 7.45 |
| Other parameters | Stable arterial flow > 15 0 mL/min Stable portal flow > 500 mL/min Homogeneous perfusion with soft liver consistency | ||
| Pragmatic approach to viability assessment | |||
| Essential markers | Desirable markers | Other markers | |
| DBD livers - assessment at 4 hrs | Lactate < 2.5 mmol/L within 4 hrs | Cumulative bile production > 2 5mL ALT < 6,000 UI/L Perfusate pH > 7.20 Falling glucose | Stable vascular flows Homogeneous perfusion with soft liver consistency Biliary pH > 7.50 Bile glucose ≤ 3 mmol/L (or ≥ 10 mmol less than perfusate glucose) Bile bicarbonate > 25 mmol/L |
| DCD livers – assessment at 4-6 hrs | Lactate < 2.5 mmol/L within 4 hours Bile production > 25 mL Bile pH > 7.50 Bile glucose ≤ 3 mmol/L (or ≥ 10 mmol less than perfusate glucose) Bile bicarbonate > 25 mmol/L | ALT < 6,000 UI/L Perfusate pH > 7.20 Falling glucose | Stable vascular flows Homogeneous perfusion with soft liver consistency |
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