Study procedures

After the liver of a donor in the Netherlands is declined by all three centres for regular transplantation following SCS alone, it will again be offered to our centre for inclusion in this protocol. In compliance with ET rules, livers will be allocated only to patients who are identified in the ET match list, based on the ET allocation rules. If a liver is not accepted for a patient who has given informed consent for the DHOPE-COR-NMP trial, it will be allocated to another centre in one of the ET member states, according to ET allocation rules. ET and the national competent authority, the Dutch Transplantation Foundation (Nederlandse Transplantatie Stichting) have agreed that this protocol does not interfere with the regular allocation rules within the Netherlands or ET.

The donor liver will be procured by one of the national multiorgan procurement teams. A standard surgical technique of in situ cold flush via the aorta with 4–7 L of University of Wisconsin (UW) cold storage solution (0°C–4°C), supplemented with 50 000 IU of heparin, will be used. If possible, the liver will be procured with a segment of 3–5 cm supratruncal aorta left attached to the coeliac trunk. The portal vein and common bile duct will be kept as long as possible. After procurement, the liver will be flushed via the portal vein with at least 1 L of UW cold storage solution. The cystic duct will be ligated, and the bile duct will be gently flushed with Belzer UW cold storage solution. The liver will be kept on ice (SCS) during transport to the UMCG where cannulation of the supratruncal aorta, portal vein, and bile duct will be performed. Furthermore, a small catheter will be inserted into the caval vein to collect venous perfusate samples.

The perfusion solution containing HBOC-201 (table 2) and taurocholic acid for continuous infusion will be prepared under sterile conditions. The perfusion fluid was developed by our research group and has been used successfully in a preclinical study and the first clinical patients.^{24 25} For the purpose of this study, the perfusion fluid was evaluated and tested for stability and compatibility by the pharmacy of the UMCG. Sterile preparation of the perfusion fluid will be conducted by the pharmacy of the UMCG. Taurocholic acid will be bought from Sigma Aldrich (Saint Louis, USA) and subsequently prepared for clinical use by pharmacy A 15 (the Netherlands), according to good manufacturing practice (GMP). Taurocholic acid will be continuously infused into the perfusion solution at a rate of 7.7 mg/hour from the start of the NMP phase. Furthermore, sodium bicarbonate can be added to the perfusion solution to correct a low pH.

Composition of the HBOC-201-based perfusion solution

HBOC, haemoglobin-based oxygen carrier.

The Liver Assist (Organ Assist, Groningen, the Netherlands) is a CE marked (European Union Certification of Safety, Health and Environmental Requirements) machine perfusion device for ex situ perfusion of donor livers. The Liver Assist enables perfusion of the liver via the portal vein and hepatic artery, using two centrifugal pumps to provide continuous and pulsatile flow, respectively. The system is pressure controlled, which provides auto regulation of the flow through the liver. The temperature can be set from 8°C to 37°C, and the preservation solution can be oxygenated by two hollow fibre membrane oxygenators.

The Liver Assist will be primed with the HBOC-201-based perfusion solution. The machine perfusion protocol consists of 1 hour of DHOPE, followed by 1 hour of COR, and a minimum of 2.5 hours of NMP. During DHOPE, the fraction of inspired oxygen (FiO₂) will be set at a 100% and the O₂ flow at 1 L, as described previously.¹⁰ During COR and NMP, the FiO₂ and O₂ flow will be adjusted according to arterial partial pressure of oxygen (pO₂) and venous saturation at the level of the suprahepatic inferior vena cava, where arterial pO₂ should be 10.0–13.3 kPa and venous saturation 55%–75%. During COR, the temperature and arterial and portal pressure will be gradually increased, as depicted in figure 2.

Liver Assist pressure and temperature settings during DHOPE, COR and NMP. COR, controlled oxygenated rewarming; DHOPE, dual hypothermic oxygenated perfusion; HA, hepatic artery; NMP, normothermic machine perfusion; PV, portal vein.

During the first 2.5 hours of NMP, viability assessment will be carried out. The liver should match all of the criteria as described in box 1. These criteria were derived from literature and our own experience and reflect both hepatocellular and biliary viability.^{12 13 15 17 29} If the liver meets the predefined viability criteria, the recipient operation will be started. NMP of the liver will continue until recipient hepatectomy is nearly complete. The liver will then be disconnected from the machine and immediately flushed with 2 L cold Belzer UW cold storage solution to remove the HBOC-201-based perfusion fluid. If the liver does not meet the predefined viability criteria, the liver will be offered back to ET for re allocation or secondary discard.

After transplantation, patients will be monitored and treated according to standard post-transplant care. After discharge from the hospital, patients will be evaluated in the outpatient clinic up to 3 months. A summary of outcome parameters is shown in box 2.

Aspartate aminotransferase.

Alanine aminotransferase.

Alkaline phosphatase.

Gamma-glutamyl transferase total bilirubin.

Lactate dehydrogenase.

Creatinine.

Platelets.

International normalised ratio.