Dual Hypothermic Oxygenated Machine Perfusion in Liver Transplantation Using Allografts From Donors After Brain Death
Dual Hypothermic Oxygenated Machine Perfusion in Liver Transplantation Using Allografts From Donors After Brain Death
This will be a randomized study on the effects of hypothermic oxygenated machine perfusion in
 patients undergoing liver transplantations from donors after brain death with allocation of
 patients to either end-ischemic hypothermic oxygenated machine perfusion group (at least 2
 hours of allograft perfusion at 12 degrees Celsius though hepatic artery and portal vein
 prior to implantation) or simple cold storage group in a 1:3 ratio. The primary outcome
 measure of the study will be model for early graft dysfunction (MEAF) score. A total number
 of 104 patients, including 26 in the hypothermic perfusion group and 78 in simple cold
 storage group will be included. Data on potential risk factors for worse allograft function
 and increased ischemia-reperfusion injury will be collected perioperatively. Circulating
 levels of proinflammatory cytokines (IL-2, IL-10, TNFα), nuclear damage (HMGB-1, 8-OHdG),
 serum activity of transaminases, gamma-glutamyl-transpeptidase, bilirubin concentration, and
 INR will be assessed in the perioperative period. Wedge allograft biopsies will be performed
 90 minutes post-reperfusion to evaluate activation of innate immunity (TLR4), activation of
 endothelium (vWF, P-selectin), hepatocyte necrosis, hepatocyte apoptosis (TUNEL assay), ATP
 content, and oxidative damage (malondialdehyde content). Further, wedge biopsies will be
 performed at the end of simple cold storage and at the beginning and after two hours of
 perfusion to determine steatosis and ATP content. During the perfusion, perfusate samples
 will be periodically tested for lactate, sodium, and potassium concentration, CO2 partiall
 pressure, and flavin mononucleotide concentration. Patients will be closely monitored in the
 postoperative period for allograft function and secondary end-points: 2-year recipient and
 graft survival, 2-year incidence of biliary complications, and 90-day complication rate. Both
 groups will be compared with respect to the primary and secondary end-points, circulating
 levels of IL-2, IL-10, TNFα, HMGB-1, 8-OHdG, activity of transaminases and
 gamma-glutamyl-transpeptidase, and findings in post-reperfusion allograft biopsies. Further,
 changes of hepatic steatosis and hepatic ATP content during perfusion will be evaluated, and
 the results of perfusate analyses will be tested as predictors of allograft function in the
 post-transplant period.
This will be a parallel, randomized controlled study on the effects of end-ischemic
 hypothermic oxygenated machine perfusion in liver transplantations from donors after brain
 death. Patients will be randomized to either hypothermic perfusion or simple cold storage
 (1:3 ratio). The primary end-point will be model for early graft dysfunction (MEAF) score, a
 recently validated continuous measure of allograft function in the early period after liver
 transplantation, calculated using serum alanine transaminase (ALT) activity, international
 normalized ratio for prothrombin time (INR), and serum bilirubin concentration within 3 first
 postoperative days. In the original study, the mean MEAF score was 5.01 with a standard
 deviation of 1.99. The primary hypothesis of this study is reduction of the mean MEAF score
 from 5 to 3.5 using hypothermic oxygenated machine perfusion. With the thresholds for type I
 error of 0.05 and power of 0.90, respectively, 1:3 allocation ratio, and a standard deviation
 of 1.99, the number of patients required to detect such difference is 26 in the hypothermic
 perfusion group and 78 in the simple cold storage group (104 in total). The inclusion
 criteria will comprise age >18 years, deceased-donor liver transplantation, and provision of
 informed consent. Exclusion criteria will comprise donation after cardiac death and either
 reduced or split graft. Secondary outcome measures will include 2-year recipient and graft
 survival, 2-year incidence of biliary complications, and complications in the 90-day
 postoperative period classified by type and their severity according to the Clavien-Dindo
 grading system.

 Following provision of informed consent, recipients will be randomly assigned in a 1:3 ratio
 to either hypothermic perfusion group or simple static cold storage group. In the hypothermic
 perfusion group, allografts will be subject to end-ischemic hypothermic oxygenated perfusion
 at 12 degrees Celsius through both hepatic artery and portal vein after a period of simple
 cold storage at 4 degrees Celsius and immediately prior to implantation. The perfusion will
 last at least 2 hours and the period will be prolonged in case of ongoing hepatectomy, in
 order to perform graft implantation immediately after perfusion. Two wedge allograft biopsies
 will be performed at the beginning and after 2 hours of perfusion for the assessment of
 micro- and macrovesicular steatosis and ATP content. Samples of the perfusate will be taken
 at the beginning and every 30 minutes for the assessment of sodium, potassium, and lactate
 concentration, CO2 partial pressure, and FMN. Perfusate temperature, portal and arterial
 flow, and portal and arterial resistance will be closely monitored during the procedure. In
 the simple cold storage group, the allografts will be stored in perfusate at 4 degrees
 Celsius from the procurement until implantation. At the end of simple cold storage, a wedge
 biopsy will be taken for the assessment of micro- and macrovesicular steatosis and ATP
 content. In both groups during the back-table procedure, the allograft will be flushed with 1
 litre of perfusate through the portal vein. At the end of flushing, a sample of perfusate
 will be taken from the right hepatic vein for assessment of FMN and lactate concentration.

 In both groups, clinical, anthropometric, and laboratory pre-transplant data will be
 collected. This includes: indication for transplantation, Child-Turcotte-Pugh classification,
 presence of hepatitis B and C virus infection, model for end-stage liver disease (MELD) score
 (without exception points), patient weight, height, body mass index, and waist circumference,
 serum bilirubin, creatinine, glucose and albumin concentration, INR, and serum activity of
 transaminases. Relevant donor and procurement data will be collected, including donor age,
 height, body mass index, cause of death, ethnicity, serum sodium and bilirubin concentration,
 serum activity of transaminases, INR, and extraction time. Donor risk index will be
 calculated in every case. Duration of cold ischemia and several intraoperative parameters
 will be collected, including: duration of warm ischemia, intraoperative transfusions, serum
 sodium and potassium changes, occurrence of post-reperfusion syndrome, and duration of
 operation. A wedge biopsy will be performed in all patients 90 minutes after portal
 reperfusion for histological assessment of ischemia-reperfusion injury (Suzuki score),
 apoptosis, endothelial activation (vWF and P-selectin), activation of innate immunity (TLR4)
 , oxidative injury (MDA), and ATP content. Blood samples will be collected immediately prior
 to reperfusion, 90 minutes after reperfusion, and at first postoperative day for the
 assessment of proinflammatory cytokines (TNFα, IL-2, IL-10), nuclear damage (HMGB1, 8-OHdG)
 and hepatocyte and cholangiocyte injury (transaminases and GGT activity). Following
 transplantation, all patients will be strictly followed according to the centre protocol with
 the assessment of serum activity of transaminases and GGTP, bilirubin and creatinine
 concentration, and INR twice daily within the first 3 postoperative days and daily or once
 every 2-3 days (according to the clinical assessment) thereafter. All complications occurring
 during the 90-postoperative period will be recorded, including primary non-function, early
 allograft dysfunction, biliary leaks, rejection episodes, and vascular complications. After
 hospital discharge, patients will be followed-up according to the center protocol with
 regular appointments in the outpatient clinic, every 2 weeks within first 2 months
 post-transplantation, every month up to 6 months, every 2-3 months up to 1 year, and every
 3-6 months thereafter. Occurrence of all complications, particularly biliary complications,
 will be recorded. Both groups will be compared with respect to primary and secondary outcome
 measures, other pre-defined clinical outcomes, allograft injury and ATP content before
 implantation, and signs of ischemia-reperfusion injury, oxidative damage, apoptosis, ATP
 content, activation of innate immunity, and endothelial activation in allograft biopsies
 after reperfusion. Comparisons will also include pro-inflammatory cytokine release,
 circulating markers of nuclear injury, and other laboratory markers of allograft function and
 injury. Analyses of perfusate samples and allograft biopsies obtained before and after
 reperfusion is planned in order to determine both the impact of hypothermic perfusion on
 hepatic ATP content and mitochondrial injury (FMN). Further, results of perfusate analyses
 (particularly FMN) will be evaluated for potential association with clinical outcome
 measures, results of post-reperfusion allograft biopsies assessment, and serum markers of
 allograft function, injury, and inflammatory response in order to determine its role in
 pre-transplant assessment of allograft viability.
Inclusion Criteria:

 -  age >18 years

 -  deceased-donor liver transplantation

 -  provision of informed consent

 Exclusion Criteria:

 -  donation after cardiac death

 -  either reduced or split graft