Experimental long-term sub-normothermic machine perfusion for non-allocable human liver grafts: first data towards feasibility

After procurement, the organs were used for MP after SCS in Custodiol® (Dr. Franz Köhler Chemie GmbH, Bensheim, Germany) as soon as the study team received the organs from the clinical team. For MP, the hepatic portal vein and hepatic artery were cannulated (25 F portal or 8 F arterial cannula; Organ Assist, Xvivo, Groningen, Netherlands) by fixation with a Vicryl (Ethicon, Johnson & Johnson Medical N.V., Belgium) ligature. To collect the produced bile, the common bile duct was cannulated by use of a polyurethane Nutrifit feeding tube (8 F, 125 cm length; Vygon, Paris, France), which was also fixed by means of a Vicryl ligature. Immediately prior to perfusion start, the organ was weighted and subsequently flushed with 2 L of 4 °C Custodiol®.

For liver perfusion, the Liver Assist® (LiA) machine was used in combination with the LiA disposable set (Organ Assist). The 24-hour perfusion protocol was adapted from [15]. The system was pre-filled with 4 l of Custodiol® or Belzer MPS® (Bridge to Life Europe, London, UK) supplemented with penicillin/streptomycin (Sigma-Aldrich; Merck, Darmstadt, Germany) and amphotericin B (Gibco; Thermo Fisher Scientific, Vienna, Austria) to a final concentration of 40,000 U/l, 0.04 mg/l, and 1 mg/l, respectively. Machine setup was accomplished according to the manufacturer’s protocol, with the temperature set to 21 °C and oxygenation with 100% O₂ at a constant flowrate of 1 l/min. After 6 h of perfusion, 50% of perfusate was replaced by the same type of fresh perfusate to dilute potentially harmful substances from the circulation.

Sampling (perfusate and tissue) was performed hourly for the whole 24 h of MP. Perfusate samples were shock frozen in liquid nitrogen and stored at −80 °C until further use. Bile samples were processed in the same way as perfusate samples if bile was produced in a sufficient quantity.

Aspartate aminotransferase (AST), alanine aminotransferase (ALT), γ‑glutamyltransferase (GGT), alanine phosphatase (AP), lactate (LAC), and lactate dehydrogenase (LDH) were determined from frozen perfusate samples by a cobas® 8000 analyzer (Roche Diagnostics GmbH, Mannheim, Germany) with reagents from the same manufacturer. Since the respective methods were not validated for this extraordinary sample matrix, we evaluated the analytical performance using a standard addition protocol.

A blood plasma pool was constituted from several anonymized leftover samples from laboratory routine. The concentrations of ALP, ALT, AST, GGT, LAC, and LDH were determined as the means of fivefold serial measurement for each marker in both the plasma pool and the pure perfusion solute. Thereafter, pure perfusion solute was mixed with pooled plasma at different proportions (10 + 0, 9 + 1, 8 + 2, 7 + 3, etc.) and every marker was determined for every mixture ratio. We calculated the percentage recovery as the ratio of the measured to the expected result, a value between 85 and 115% was considered acceptable. The best recovery rates were seen for ALT (91–112%), AST (90–106%), and LDH (94–101%). For ALP (67–100%) and GGT (69–103%), recovery was worse at lower concentrations. Linear regression analysis revealed a predominantly negative constant bias for these two markers. Because we only performed longitudinal analyses, we did not exclude these markers from the study, since a constant bias does not tarnish the interpretation of dynamics over time.

Perfusate pH and glucose levels as well as bile pH, glucose, and HCO₃⁻ levels as measures of biliary epithelial integrity [16] were monitored on site by an ePOCT Analyzer (Siemens Healthcare, Vienna, Austria) in combination with BGEM test cards. Perfusate pH was adjusted by addition of 8.4% sodium bicarbonate solution.

Sixteen non-allocable livers from 9 female and 7 male donors with a mean age of 66 years (range 44–81 years) were included into the study. The mean SCS time was 14 h 17 min (range 3–29 h 10 min). Donor characteristics, SCS time prior to SNMP onset, and the perfusate solution used for MP are listed in Table 1.

All liver parameter levels increased at different rates in SNMP, irrespective of the perfusate used, with LDH, AST, and ALT reaching statistical significance when changes in MPS were compared to changes in the Custodiol® group (Fig. 1b). A similar picture is observed when changes in liver parameters are grouped by reasons for rejection (cirrhosis, steatosis, other reasons as described in Table 1). The average slope depicts an increase at different rates in all liver parameters, irrespective of the state of the liver, but only for lactate did the differences reach statistical significance (Fig. 1a). The average amount of sodium bicarbonate added to sustain a stable, physiological pH was similar with respect to reason of rejection (median [Q1, Q3]: 205 ml [153 ml, 320 ml] liver rejected for other reasons, 175 ml [125 ml, 375 ml] steatotic livers, 290 ml [0 ml, 580] cirrhotic livers; n. s.), but was significantly higher in livers perfused with Custodiol® (median [Q1, Q3]: 365 ml [280 ml, 513 ml]) compared to Belzer MPS (median [Q1, Q3]: 140 ml [88 ml, 161 ml]; p = 0.0002). Liver parameters prior to start of SNMP (representing the enzymes produced during SCS), after 1 h of machine perfusion, and after 24 h of SNMP are listed in Table S1 and detailed information on changes over time are shown in Figures S1–S6.

In this study, six (one steatotic in Custodiol®; two steatotic, one cirrhotic, and two livers rejected for other reasons in the Belzer MPS group) out of 16 livers did not produce any bile during 24 h of SNMP. Among those livers producing bile, the total quantity varied between 2.40 and 179 ml (Custodiol®: average 43.69 ml; Belzer MPS: average 17.37 ml). In the hours prior to perfusate renewal at 6 h of SNMP, the average bile production was about 1.69 ml/h (Custodiol®: 2.16 ml/h; Belzer MPS: 0.59 ml/h) compared to about 1.43 ml/h (Custodiol®: 1.71 ml/h; Belzer MPS: 0.77 ml/h) between hours 7 and 24 of SNMP (Table 2). It seems that upon perfusion with Custodiol®, bile production during the first 6 h of SNMP was higher than in the later period, whereas upon perfusion with Belzer MPS, bile production seems stable over the whole period of SNMP. None of the reported differences in bile production reached statistical significance. The ratios of bile/perfusate glucose levels, a surrogate for bile duct injury [17], are close to 1 for all, except for two livers during the whole time of perfusion. The bile/perfusate glucose ratio for L5 is much lower, whereas the cirrhotic liver (L7) revealed a ratio of around 4. For all but one liver, bicarbonate levels increased over time at different rates.