Bile proteome reveals biliary regeneration during normothermic preservation of human donor livers.

Normothermic machine perfusion (NMP) after static cold storage is increasingly used for preservation and assessment of human donor livers prior to transplantation. Biliary viability assessment during NMP reduces the risk of post-transplant biliary complications. However, understanding of molecular changes in the biliary system during NMP remains incomplete. We performed an in-depth, unbiased proteomics analysis of bile collected during sequential hypothermic machine perfusion, rewarming and NMP of 55 human donor livers. Longitudinal analysis during NMP reveals proteins reflective of cellular damage at early stages, followed by upregulation of secretory and immune response processes. Livers with bile chemistry acceptable for transplantation reveal protein patterns implicated in regenerative processes, including cellular proliferation, compared to livers with inadequate bile chemistry. These findings are reinforced by detection of regenerative gene transcripts in liver tissue before machine perfusion. Our comprehensive bile proteomics and liver transcriptomics data sets provide the potential to further evaluate molecular mechanisms during NMP and refine viability assessment criteria.

The international normalised ratio to monitor coagulation factor production during normothermic machine perfusion of human donor livers.

BACKGROUND: Normothermic machine perfusion (NMP) of donor livers allows for new diagnostic and therapeutic strategies. As the liver produces most of the haemostatic proteins, coagulation assays such as the International Normalised Ratio (INR) performed in perfusate may be useful to assess hepatocellular function of donor livers undergoing NMP. However, high concentrations of heparin and low levels of fibrinogen may affect coagulation assays. METHODS: Thirty donor livers that underwent NMP were retrospectively included in this study, of which 18 were subsequently transplanted. We measured INRs in perfusate in presence or absence of exogenously added fibrinogen and/or polybrene. Additionally, we prospectively included 14 donor livers that underwent NMP (of which 11 were transplanted) and measured INR using both a laboratory coagulation analyser and a point-of-care device. RESULTS: In untreated perfusate samples, the INR was above the detection limit in all donor livers. Addition of both fibrinogen and polybrene was required for adequate INR assessment. INRs decreased over time and detectable perfusate INR values were found in 17/18 donor livers at the end of NMP. INR results were similar between the coagulation analyser and the point-of-care device, but did not correlate with established hepatocellular viability criteria. CONCLUSIONS: Most of the donor livers that were transplanted showed a detectable perfusate INR at the end of NMP, but samples require processing to allow for INR measurements using laboratory coagulation analysers. Point-of-care devices bypass this need for processing. The INR does not correlate with established viability criteria and might therefore have additional predictive value.

Normothermic Machine-perfused Human Donor Livers Produce Functional Hemostatic Proteins.

BACKGROUND: Normothermic machine perfusion (NMP) is used for the viability assessment of high-risk donor livers before transplantation. The production of hemostatic proteins is one of the major synthetic functions of the liver. The objective of this study was to measure the concentration and functionality of hemostatic proteins concentration in the NMP perfusate of human donor livers. METHODS: Thirty-six livers that underwent NMP for viability assessment were included in this study. Perfusate samples taken during NMP (start, 150 min, and 300 min) were used for the measurement of antigen and activity levels of hemostatic proteins (factors II, VII, and X; fibrinogen; plasminogen; antithrombin; tissue plasminogen activator; von Willebrand factor; and proteins induced by vitamin K absence). The antigen levels were correlated with hepatocellular function according to previously proposed individual hepatocellular viability criteria: lactate clearance and perfusate pH. RESULTS: Antigen levels of hemostatic proteins reached subphysiological levels in the NMP perfusate. Hemostatic proteins that were produced during NMP were at least partially active. All livers produced all hemostatic proteins tested within 150 min of NMP. Hemostatic protein concentrations did not significantly correlate with perfusate lactate and perfusate pH after 150 min of NMP. CONCLUSIONS: All livers produce functional hemostatic proteins during NMP. The generation of a functional hemostatic system in NMP perfusate confirms the need for adequate anticoagulation of the perfusate to avoid generation of (micro)thrombi that may harm the graft.

Restoration of Bile Duct Injury of Donor Livers During Ex Situ Normothermic Machine Perfusion.

BACKGROUND: End-ischemic ex situ normothermic machine perfusion (NMP) enables assessment of donor livers prior to transplantation. The objective of this study was to provide support for bile composition as a marker of biliary viability and to investigate whether bile ducts of high-risk human donor livers already undergo repair during NMP. METHODS: Forty-two livers that were initially declined for transplantation were included in our NMP clinical trial. After NMP, livers were either secondary declined (n = 17) or accepted for transplantation (n = 25) based on the chemical composition of bile and perfusate samples. Bile duct biopsies were taken before and after NMP and assessed using an established histological injury severity scoring system and a comprehensive immunohistochemical assessment focusing on peribiliary glands (PBGs), vascular damage, and regeneration. RESULTS: Bile ducts of livers that were transplanted after viability testing during NMP showed better preservation of PBGs, (micro)vasculature, and increased cholangiocyte proliferation, compared with declined livers. Biliary bicarbonate, glucose, and pH were confirmed as accurate biomarkers of bile duct vitality. In addition, we found evidence of PBG-based progenitor cell differentiation toward mature cholangiocytes during NMP. CONCLUSIONS: Favorable bile chemistry during NMP correlates well with better-preserved biliary microvasculature and PBGs, with a preserved capacity for biliary regeneration. During NMP, biliary tree progenitor cells start to differentiate toward mature cholangiocytes, facilitating restoration of the ischemically damaged surface epithelium.

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

Ex situ normothermic machine perfusion (NMP) is increasingly used for viability assessment of high-risk donor livers, whereas dual hypothermic oxygenated machine perfusion (DHOPE) reduces ischemia-reperfusion injury. We aimed to resuscitate and test the viability of initially-discarded, high-risk donor livers using sequential DHOPE and NMP with two different oxygen carriers: an artificial hemoglobin-based oxygen carrier (HBOC) or red blood cells (RBC). In a prospective observational cohort study of 54 livers that underwent DHOPE-NMP, the first 18 procedures were performed with a HBOC-based perfusion solution and the subsequent 36 procedures were performed with an RBC-based perfusion solution for the NMP phase. All but one livers were derived from extended criteria donation after circulatory death donors, with a median donor risk index of 2.84 (IQR 2.52-3.11). After functional assessment during NMP, 34 livers (63% utilization), met the viability criteria and were transplanted. One-year graft and patient survival were 94% and 100%, respectively. Post-transplant cholangiopathy occurred in 1 patient (3%). There were no significant differences in utilization rate and post-transplant outcomes between the HBOC and RBC group. Ex situ machine perfusion using sequential DHOPE-NMP for resuscitation and viability assessment of high-risk donor livers results in excellent transplant outcomes, irrespective of the oxygen carrier used.

Oxygen Transport during Ex Situ Machine Perfusion of Donor Livers Using Red Blood Cells or Artificial Oxygen Carriers.

Oxygenated ex situ machine perfusion of donor livers is an alternative for static cold preservation that can be performed at temperatures from 0 °C to 37 °C. Organ metabolism depends on oxygen to produce adenosine triphosphate and temperatures below 37 °C reduce the metabolic rate and oxygen requirements. The transport and delivery of oxygen in machine perfusion are key determinants in preserving organ viability and cellular function. Oxygen delivery is more challenging than carbon dioxide removal, and oxygenation of the perfusion fluid is temperature dependent. The maximal oxygen content of water-based solutions is inversely related to the temperature, while cellular oxygen demand correlates positively with temperature. Machine perfusion above 20 °C will therefore require an oxygen carrier to enable sufficient oxygen delivery to the liver. Human red blood cells are the most physiological oxygen carriers. Alternative artificial oxygen transporters are hemoglobin-based oxygen carriers, perfluorocarbons, and an extracellular oxygen carrier derived from a marine invertebrate. We describe the principles of oxygen transport, delivery, and consumption in machine perfusion for donor livers using different oxygen carrier-based perfusion solutions and we discuss the properties, advantages, and disadvantages of these carriers and their use.

Extended hypothermic oxygenated machine perfusion enables ex situ preservation of porcine livers for up to 24 hours.

BACKGROUND & AIMS: End-ischemic hypothermic oxygenated machine perfusion (HOPE) of the donor liver for 1-2 h mitigates ischemia-reperfusion injury during subsequent liver transplantation. Extended preservation time may be preferred to facilitate difficult recipient hepatectomy or to optimize logistics. We therefore investigated whether end-ischemic dual HOPE (DHOPE) could extend preservation time for up to 24 h using a porcine liver reperfusion model. METHODS: Following 30 min warm ischemia, porcine livers were subjected to 2 h static cold storage (SCS), followed by 2 h, 6 h, or 24 h DHOPE (n = 6 per group). Subsequent normothermic reperfusion was performed for 4 h using autologous blood. Two livers preserved by 24 h SCS served as additional controls. A proof of principle confirmation was carried out in 2 discarded human livers subjected to extended DHOPE. Hepatocellular and cholangiocyte injury and function were assessed. Oxidative stress levels and histology were compared between groups. RESULTS: Perfusion flows remained stable during DHOPE, regardless of duration. After normothermic reperfusion, livers perfused for 24 h by DHOPE had similar lactate clearance, blood pH, glucose, and alanine aminotransferase levels, and biliary pH, bicarbonate, and LDH levels, as livers perfused for 2 h and 6 h. Levels of malondialdehyde and high-mobility group box 1 in serum and liver parenchyma were similar for all groups. Histological analysis of bile ducts and liver parenchyma revealed no differences between the groups. Extended DHOPE in discarded human livers preserved hepatocellular and cholangiocyte function and histology after reperfusion. In contrast, livers preserved by 24 h SCS were non-functioning. CONCLUSION: Extended end-ischemic DHOPE enabled successful preservation of porcine and discarded human donor livers for up to 24 h. Extended DHOPE enables safe extension of preservation time, which may facilitate allocation and transplantation from a logistical perspective, and further expand the donor pool. LAY SUMMARY: It has been suggested that preserving liver grafts with a technique called (dual) hypothermic oxygenated machine perfusion ([D]HOPE) leads to better outcomes after transplantation than if livers are stored on ice, especially if an organ is of lesser quality. In this study, we showed that DHOPE could be used to preserve liver grafts for up to 24 h. This extended procedure could be used globally to facilitate transplantation and expand the donor pool.