Objective: The aim of this study was to evaluate sequential hypothermic and normothermic machine perfusion (NMP) as a tool to resuscitate and assess viability of initially declined donor livers to enable safe transplantation.Summary Background Data: Machine perfusion is increasingly used to resuscitate and test the function of donor livers. Although (dual) hypothermic oxygenated machine perfusion ([D]HOPE) resuscitates livers after cold storage, NMP enables assessment of hepatobiliary function.Methods: In a prospective clinical trial, nationwide declined livers were subjected to ex situ NMP (viability assessment phase), preceded by 1-hour DHOPE (resuscitation phase) and 1 hour of controlled oxygenated rewarming (COR), using a perfusion fluid containing an hemoglobin-based oxygen carrier. During the first 2.5 hours of NMP, hepatobiliary viability was assessed, using predefined criteria: perfusate lactate 10 mL, and bile pH >7.45. Livers meeting all criteria were accepted for transplantation. Primary endpoint was 3-month graft survival.Results: Sixteen livers underwent DHOPE–COR–NMP. All livers were from donors after circulatory death, with median age of 63 (range 42–82) years and median Eurotransplant donor risk index of 2.82. During NMP, all livers cleared lactate and produced sufficient bile volume, but in 5 livers bile pH remained <7.45. The 11 (69%) livers that met all viability criteria were successfully transplanted, with 100% patient and graft survival at 3 and 6 months. Introduction of DHOPE–COR–NMP increased the number of deceased donor liver transplants by 20%.Conclusions: Sequential DHOPE–COR–NMP enabled resuscitation and safe selection of initially declined high-risk donor livers, thereby increasing the number of transplantable livers by 20%.Trial registration: www.trialregister.nl; NTR5972.

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.

Ex situ dual hypothermic oxygenated machine perfusion (DHOPE) and normothermic machine perfusion (NMP) of donor livers may have a complementary effect when applied sequentially. While DHOPE resuscitates the mitochondria and increases hepatic ATP content, NMP enables hepatobiliary viability assessment prior to transplantation. In contrast to DHOPE, NMP requires a perfusion solution with an oxygen carrier, for which red blood cells (RBC) have been used in most series. RBC, however, have limitations and cannot be used cold. We, therefore, established a protocol of sequential DHOPE, controlled oxygenated rewarming (COR), and NMP using a new hemoglobin‐based oxygen carrier (HBOC)‐based perfusion fluid (DHOPE‐COR‐NMP trial, NTR5972). Seven livers from donation after circulatory death (DCD) donors, which were initially declined for transplantation nationwide, underwent DHOPE‐COR‐NMP. Livers were considered transplantable if perfusate pH and lactate normalized, bile production was ≥ 10 ml and biliary pH >7.45 within 150 min of NMP. Based on these criteria five livers were transplanted. The primary endpoint, 3‐month graft survival, was a 100%. In conclusion, sequential DHOPE‐COR‐NMP using an HBOC‐based perfusion fluid offers a novel method of liver machine perfusion for combined resuscitation and viability testing of suboptimal livers prior to transplantation.

Dual hypothermic oxygenated machine perfusion (DHOPE) of the liver has been advocated as a method to reduce ischemia/reperfusion injury (IRI). This study aimed to determine whether DHOPE reduces IRI of the bile ducts in donation after circulatory death (DCD) liver transplantation. In a recently performed phase 1 trial, 10 DCD livers were preserved with DHOPE after static cold storage (SCS; www.trialregister.nl NTR4493). Bile duct biopsies were obtained at the end of SCS (before DHOPE; baseline) and after graft reperfusion in the recipient. Histological severity of biliary injury was graded according to an established semiquantitative grading system. Twenty liver transplantations using DCD livers not preserved with DHOPE served as controls. Baseline characteristics and the degree of bile duct injury at baseline (end of SCS) were similar between both groups. In controls, the degree of stroma necrosis (P = 0.002) and injury of the deep peribiliary glands (PBG; P = 0.02) increased after reperfusion compared with baseline. In contrast, in DHOPE‐preserved livers, the degree of bile duct injury did not increase after reperfusion. Moreover, there was less injury of deep PBG (P = 0.04) after reperfusion in the DHOPE group compared with controls. In conclusion, this study suggests that DHOPE reduces IRI of bile ducts after DCD liver transplantation. Liver Transplantation 24 655–664 2018 AASLD.

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.