Prolonged preservation by hypothermic machine perfusion facilitates logistics in liver transplantation: A European observational cohort study

A short period (1–2 h) of hypothermic oxygenated machine perfusion (HOPE) after static cold storage is safe and reduces ischemia‐reperfusion injury‐related complications after liver transplantation. Machine perfusion time is occasionally prolonged for logistical reasons, but it is unknown if prolonged HOPE is safe and compromises outcomes. We conducted a multicenter, observational cohort study of patients transplanted with a liver preserved by prolonged (≥4 h) HOPE. Postoperative biochemistry, complications, and survival were evaluated. The cohort included 93 recipients from 12 European transplant centers between 2014–2021. The most common reason to prolong HOPE was the lack of an available operating room to start the transplant procedure. Grafts underwent HOPE for a median (range) of 4:42 h (4:00–8:35 h) with a total preservation time of 10:50 h (5:50–20:50 h). Postoperative peak ALT was 675 IU/L (interquartile range 419–1378 IU/L). The incidence of postoperative complications was low, and 1‐year graft and patient survival were 94% and 88%, respectively. To conclude, good outcomes are achieved after transplantation of donor livers preserved with prolonged (median 4:42 h) HOPE, leading to a total preservation time of almost 21 h. These results suggest that simple, end‐ischemic HOPE may be utilized for safe extension of the preservation time to ease transplantation logistics.


| INTRODUC TI ON
The use of machine perfusion to preserve donor livers is one of the most important advances in liver transplantation in the past decade.
Hypothermic oxygenated machine perfusion (HOPE) is performed at 4-12°C with an acellular perfusion solution at low perfusion pressures and flow rates. 1 Hypothermic oxygenation of mitochondria induces metabolic programming within 1 h, thereby decreasing mitochondrial succinate accumulation and uploading adenosine triphosphate levels. 2 Reperfusion of livers treated by end-ischemic HOPE is, therefore, associated with less oxidative injury and mitochondrial damage with subsequently less downstream inflammation. [2][3][4] The results of two recently published randomized controlled trials comparing end-ischemic HOPE versus static cold storage (SCS) confirm the beneficial effects of this technique on clinical outcomes. 5, 6 In the transplantation of donation after circulatory death (DCD) livers, 2 h of HOPE after SCS reduced the incidence of ischemia-reperfusionrelated complications after transplantation, including a 68% reduction in symptomatic nonanastomotic biliary strictures (NAS), when compared to grafts preserved with SCS alone. 5 In the transplantation of livers from high-risk donation after brain death (DBD) donors, approximately 2 h of end-ischemic HOPE reduced the incidence of early allograft dysfunction (EAD) and postoperative complications. 6 Whereas a brief period (usually 1-2 h) of HOPE after SCS improves post-transplant outcomes, machine perfusion time may occasionally be prolonged because of unforeseen transplant logistics. For example, when the donor liver is reallocated to another recipient in the last minute, or in the event of a difficult recipient hepatectomy. [7][8][9] Good outcomes after prolonged normothermic machine perfusion (NMP) up to 20 h have been reported previously, 10 but the use of prolonged HOPE is still unexplored. In a preclinical study of porcine and discarded human livers, HOPE could succesfully be prolonged for up to 24 h, followed by normothermic reperfusion. 11 However, clinical data are currently lacking and it remains unknown whether postoperative outcomes are compromised when HOPE is prolonged beyond 2 h.
The objective of this multicenter study was, therefore, to evaluate outcomes after transplantation of donor livers preserved by prolonged (≥4 h) HOPE. We hypothesize that good outcomes after prolonged HOPE can be achieved, which are comparable to those previously reported for regular HOPE.

| Study design
European liver transplant centers with a clinical HOPE program were approached for participation in this multicenter observational cohort study. All cases of prolonged (≥4 h) HOPE-preserved donor livers

K E Y W O R D S
clinical research/practice, graft survival, ischemia reperfusion injury (IRI), liver allograft function/dysfunction, liver transplantation/hepatology, organ acceptance, organ perfusion and preservation, organ procurement and allocation, solid organ transplantation and recipients were eligible for inclusion in the study. There were no exclusion criteria. The study was designed as a stage 1 study according to the IDEAL-D (Idea, Exploration, Assessment, Long-term study outcomes for Devices) framework. 12-14 IDEAL stage 1 ('Idea') studies describe the first use of a procedure or device, either as a planned or unplanned approach with short-term clinical outcomes as endpoints. The study was approved by the Institutional Review Board of the University Medical Center Groningen (RR 202100366) and adhered to the Declaration of Helsinki and the Declaration of Istanbul. The first and last authors had full access to all data in the study and take responsibility for its integrity and the data analysis.
The study complied with the STROBE (Strengthening the Reporting of Observational Studies in Epidemiology) guidelines. 15

| Liver procurement, preservation, and transplantation
Donor livers were obtained, preserved, and transported to recipient transplant centers according to standard national practice. The transplantation surgery and postoperative care were performed according to standard local practice. According to national legislation, livers from DCD donors in Italy were retrieved with in situ normothermic regional perfusion. The Liver Assist device (XVIVO), VitaSmart

| Survey
An online questionnaire was sent to the program leader from each participating center. The survey included 10 questions about the centers' experience with HOPE and the surgeon's view on prolonged HOPE.

| Data collection
Data were collected at each center and anonymously stored in a single electronic database. Donor and recipient characteristics included age, the body mass index (BMI), the model for end-stage liver disease (MELD) score, the donor risk index (DRI), the Eurotransplant DRI (ET-DRI), and the balance of risks (BAR) score. The DRI is calculated based on the following donor characteristics: age, race, height, cause of death, DCD, and whether it was a partial or split graft. 16 By adding the latest laboratory gamma-glutamyl transferase (yGT) of the donor and rescue allocation, the ET-DRI was developed. 17 The BAR score is calculated based on the following variables: MELD score, retransplantation, whether the recipient was on life support preoperatively, recipient age, cold ischemia time, and donor age. 18

| Definitions
Post-reperfusion syndrome was defined as (1) a decrease in mean arterial blood pressure ≥30 mmHg below baseline, lasting for ≥1 min, Biliary leakage was defined as fluid with an elevated bilirubin level in the abdominal drain or intra-abdominal fluid on or after postoperative day three or the need for radiological intervention owing to biliary collections or relaparotomy due to biliary peritonitis. 24 Acute kidney injury was defined as (1) increase serum creatinine by ≥0.3 mg/dl within 48 h after transplantation or, (2) increase in serum creatinine ≥1.5 times baseline, or (3) urine volume <0.5 ml/ kg/h for 6 h. 25 Graft survival was defined as the time between liver transplantation and retransplantation or death. Graft survival was censored for patients dying with a functional graft. Patient survival was defined as the time between liver transplantation and all-cause death.

| Survey outcomes and center characteristics
Twelve transplant centers in 6 European countries (the Netherlands, Germany, Belgium, Italy, Switzerland, and France) contributed to the study. The median number of liver transplantations performed per year in the participating centers was 80 (60-130) ( Table S1). In 5 centers, both single and dual vessel HOPE were performed, and in the other centers either single HOPE (n = 4) or dual HOPE (n = 3) was performed. In the participating centers, the median proportion of livers preserved by HOPE was 30% (8%-100%) for DCD livers and 23% (16%-29%) for DBD livers. A dedicated on-call organ perfusionist team was available for machine perfusion in 8 out of 12 centers.
Among the surveyed surgeons, the reported duration up to which they would currently feel comfortable perfusing a liver with HOPE averaged 6 h. For prolonged machine perfusion in particular, dual HOPE was preferred over single HOPE by 8 out of 12 centers.
Levels of yGT peaked around postoperative day 7 and were low at 1 and 3 months after liver transplantation. Bilirubin levels were low at 1 and 3 months after liver transplantation. The incidence of PRS was 12% ( Table 2). Twenty-four hours after reperfusion, median In a subanalysis, outcomes after prolonged HOPE of 43 DCD versus 50 DBD grafts were compared ( Table 3). Inherent to DCD liver transplantation, the incidence of PRS was higher, when compared to transplantation of DBD grafts, albeit not reaching significance (19% vs. 6%, p = .053). There were no major differences in other postoperative outcomes between recipients of DCD or DBD livers, including PNF (0% vs. 2%, p = .35), EAD (40% vs. 32%, p = .45), and NAS (0% vs. 2%, p = .35). One DCD liver recipient underwent retransplantation versus four DBD liver recipients (p = .23). When comparing outcomes after transplantation of livers preserved by prolonged HOPE or DHOPE, no major differences were found (Table S2).  (Tables S3 and S4). Outcomes after prolonged HOPE are also comparable to the benchmark outcome values in DBD (Table S3) and DCD ( Table S4) liver transplantation of non-machine perfused grafts. 29,30 While prolonged cold ischemia is a well-known risk factor for postoperative complications in DCD liver transplantation, the results of this study suggest HOPE can be used to prolong preservation time without impairing postoperative outcome.

| DISCUSS ION
The results of the present study show that the combination of SCS and prolonged HOPE is safe in liver grafts with high risks and achieves comparable outcomes as low risk benchmark cohorts, which may have several clinical implications. In clinical practice, there is a need for an extension of the preservation time due to the lack of intensive care beds or to bridge theater capacity (frequently from early morning hours to mornings or noon), where the here described approach with a median overall preservation time of 11 h appears very beneficial. Additionally, the indication for machine perfusion could be recipient-orientated rather than based on donor characteristics only. This way, grafts can be preserved by HOPE during a difficult recipient hepatectomy with less time pressure. Answers to the survey in this study include the potential of prolonged HOPE to accept two livers at the same time (so that one graft is preserved longer with HOPE), or to enable transplantation at daytime.
Prolonged preservation has previously been achieved clinically using NMP, or by supercooling in an experimental setting. 10,31,32 Previous studies showed that NMP enables a prolongation of liver preservation and overnight organ care. 10,33 The results of the present study suggest that similar results can be achieved with HOPE.
Prolonged preservation by HOPE compared with NMP, can be advantageous since the organ is maintained in a hypometabolic state with minimal production of coagulation factors and waste products, of human livers with their custom-made normothermic perfusion device. 32 Both techniques, however, are still in a preclinical phase.
A limitation of this study is its retrospective design. Based on this, there are inherent differences in organ procurement and implantation techniques between the centers contributing to this study. Machine perfusion-early allograft dysfunction d 7 (14%) 6 (14%) .995