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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., (© 2018 The Authors Liver Transplantation published by Wiley Periodicals, Inc. on behalf of American Association for the Study of Liver Diseases.)

Background: Lack of oxygen and biomechanical stimulation during static cold storage (SCS) of donor livers compromises endothelial cell function. We investigated the effect of end-ischemic oxygenated hypothermic machine perfusion (HMP) on endothelial cell function of extended criteria donor (ECD) livers., Methods: Eighteen livers, declined for transplantation, were transported to our center using static cold storage (SCS). After SCS, 6 livers underwent two hours of HMP, and subsequent normothermic machine perfusion (NMP) to assess viability. Twelve control livers underwent NMP immediately after SCS. mRNA expression of transcription factor Krüppel-like-factor 2 (KLF2), endothelial nitric oxide synthase (eNOS), and thrombomodulin (TM) was quantified by RT-PCR. Endothelial cell function and injury were assessed by nitric oxide (NO) production and release of TM into the perfusate., Results: In HMP livers, mRNA expression of KLF2 (p = 0.043), eNOS (p = 0.028), and TM (p = 0.028) increased significantly during NMP. In parallel, NO levels increased during NMP in HMP livers but not in controls. At the end of NMP cumulative TM release was significantly lower HMP livers, compared to controls (p = 0.028)., Conclusion: A short period of two hours oxygenated HMP restores endothelial cell viability after SCS and subsequent normothermic reoxygenation of ECD livers., (Copyright © 2017 International Hepato-Pancreato-Biliary Association Inc. Published by Elsevier Ltd. All rights reserved.)

Background: Ex situ normothermic machine perfusion (NMP) can be performed after traditional static cold preservation to assess graft function and viability before transplantation. It is unknown whether this results in activation of coagulation and fibrinolysis, as may occur upon graft reperfusion in vivo., Methods: Twelve donor livers declined for transplantation underwent 6 hours of end-ischemic NMP using a heparinized plasma-based perfusion fluid. Concentration of prothrombin fragment F1 + 2 (marker of coagulation activation), D-dimer, plasmin-antiplasmin complex, tissue plasminogen activator and plasminogen activator inhibitor-1 (markers for fibrinolysis) and alanine aminotransferase (ALT) (marker of ischemia-reperfusion [I/R] injury) were measured in perfusion fluid at regular intervals. Liver biopsies were examined for the presence of fibrin, using light microscopy after Maurits, Scarlet and Blue staining., Results: No significant increase in prothrombin F1 + 2 was noted during NMP. D-dimer and plasmin-antiplasmin complex levels increased soon after start of NMP and D-dimer concentrations correlated significantly with levels of tissue plasminogen activator. In livers displaying good function during NMP, perfusate levels of ALT and D-dimers were low (≤3500 ng/mL), whereas significantly higher D-dimer levels (>3500 ng/mL) were in found in livers with poor graft function. Activation of fibrinolysis correlated significantly with the degree of I/R injury, as reflected by ALT levels., Conclusions: End-ischemic ex situ NMP results in activation of fibrinolysis, but not of coagulation. Markers of fibrinolysis activation correlate significantly with markers of I/R injury. High concentrations of D-dimer early after start of NMP can be considered a marker of severe I/R injury and a predictor of poor liver graft function.

Bile duct injury may occur during liver procurement and transplantation, especially in livers from donation after circulatory death (DCD) donors. Normothermic machine perfusion (NMP) has been shown to reduce hepatic injury compared to static cold storage (SCS). However, it is unknown whether NMP provides better preservation of bile ducts. The aim of this study was to determine the impact of NMP on bile duct preservation in both DCD and non-DCD livers. DCD and non-DCD livers obtained from Lewis rats were preserved for 3 hours using either SCS or NMP, followed by 2 hours ex vivo reperfusion. Biomarkers of bile duct injury (gamma-glutamyltransferase and lactate dehydrogenase in bile) were lower in NMP-preserved livers compared to SCS-preserved livers. Biliary bicarbonate concentration, reflecting biliary epithelial function, was 2-fold higher in NMP-preserved livers (P < 0.01). In parallel with this, the pH of the bile was significantly higher in NMP-preserved livers (7.63 ± 0.02 and 7.74 ± 0.05 for non-DCD and DCD livers, respectively) compared with SCS-preserved livers (7.46 ± 0.02 and 7.49 ± 0.04 for non-DCD and DCD livers, respectively). Scanning and transmission electron microscopy of donor extrahepatic bile ducts demonstrated significantly decreased injury of the biliary epithelium of NMP-preserved donor livers (including the loss of lateral interdigitations and mitochondrial injury). Differences between NMP and SCS were most prominent in DCD livers. Compared to conventional SCS, NMP provides superior preservation of bile duct epithelial cell function and morphology, especially in DCD donor livers. By reducing biliary injury, NMP could have an important impact on the utilization of DCD livers and outcome after transplantation. Liver Transplantation 22 994-1005 2016 AASLD., (© 2016 American Association for the Study of Liver Diseases.)

Background: The mechanism through which oxygenated hypothermic machine perfusion (HMP) improves viability of human extended criteria donor (ECD) livers is not well known. Aim of this study was to examine the benefits of oxygenated HMP after static cold storage (SCS)., Methods: Eighteen ECD livers that were declined for transplantation underwent ex situ viability testing using normothermic (37 °C) machine perfusion (NMP) after traditional SCS (0 °C-4 °C) for 7 to 9 hours. In the intervention group (n = 6), livers underwent 2 hours of oxygenated HMP (at 12 °C) after SCS and before NMP. Twelve control livers underwent NMP without oxygenated HMP after SCS., Results: During HMP, hepatic ATP content increased greater than 15-fold, and levels remained significantly higher during the first 4 hours of NMP in the HMP group, compared with controls. Cumulative bile production and biliary secretion of bilirubin and bicarbonate were significantly higher after HMP, compared with controls. In addition, the levels of lactate and glucose were less elevated after HMP compared with SCS preservation alone. In contrast, there were no differences in levels of hepatobiliary injury markers AST, ALT, LDH, and gamma-GT after 6 hours of NMP. Hepatic histology at baseline and after 6 hours of NMP revealed no differences in the amount of ischemic necrosis between both groups., Conclusions: Two hours of oxygenated HMP after traditional SCS restores hepatic ATP levels and improves hepatobiliary function but does not reduce (preexisting) hepatobiliary injury in ECD livers.

A short period of oxygenated machine perfusion (MP) after static cold storage (SCS) may reduce biliary injury in donation after cardiac death (DCD) donor livers. However, the ideal perfusion temperature for protection of the bile ducts is unknown. In this study, the optimal perfusion temperature for protection of the bile ducts was assessed. DCD rat livers were preserved by SCS for 6 hours. Thereafter, 1 hour of oxygenated MP was performed using either hypothermic machine perfusion, subnormothermic machine perfusion, or with controlled oxygenated rewarming (COR) conditions. Subsequently, graft and bile duct viability were assessed during 2 hours of normothermic ex situ reperfusion. In the MP study groups, lower levels of transaminases, lactate dehydrogenase (LDH), and thiobarbituric acid reactive substances were measured compared to SCS. In parallel, mitochondrial oxygen consumption and adenosine triphosphate (ATP) production were significantly higher in the MP groups. Biomarkers of biliary function, including bile production, biliary bicarbonate concentration, and pH, were significantly higher in the MP groups, whereas biomarkers of biliary epithelial injury (biliary gamma-glutamyltransferase [GGT] and LDH), were significantly lower in MP preserved livers. Histological analysis revealed less injury of large bile duct epithelium in the MP groups compared to SCS. In conclusion, compared to SCS, end-ischemic oxygenated MP of DCD livers provides better preservation of biliary epithelial function and morphology, independent of the temperature at which MP is performed. End-ischemic oxygenated MP could reduce biliary injury after DCD liver transplantation., (© 2015 American Association for the Study of Liver Diseases.)

Background: Livers derived from donation after circulatory death (DCD) are increasingly accepted for transplantation. However, DCD livers suffer additional donor warm ischemia, leading to biliary injury and more biliary complications after transplantation. It is unknown whether oxygenated machine perfusion results in better preservation of biliary epithelium and the peribiliary vasculature. We compared oxygenated hypothermic machine perfusion (HMP) with static cold storage (SCS) in a porcine DCD model., Methods: After 30 min of cardiac arrest, livers were perfused in situ with HTK solution (4°C) and preserved for 4 h by either SCS (n = 9) or oxygenated HMP (10°C; n = 9), using pressure-controlled arterial and portal venous perfusion. To simulate transplantation, livers were reperfused ex vivo at 37°C with oxygenated autologous blood. Bile duct injury and function were determined by biochemical and molecular markers, and a systematic histological scoring system., Results: After reperfusion, arterial flow was higher in the HMP group, compared to SCS (251±28 vs 166±28 mL/min, respectively, after 1 hour of reperfusion; p = 0.003). Release of hepatocellular enzymes was significantly higher in the SCS group. Markers of biliary epithelial injury (biliary LDH, gamma-GT) and function (biliary pH and bicarbonate, and biliary transporter expression) were similar in the two groups. However, histology of bile ducts revealed significantly less arteriolonecrosis of the peribiliary vascular plexus in HMP preserved livers (>50% arteriolonecrosis was observed in 7 bile ducts of the SCS preserved livers versus only 1 bile duct of the HMP preserved livers; p = 0.024)., Conclusions: Oxygenated HMP prevents arteriolonecrosis of the peribiliary vascular plexus of the bile ducts of DCD pig livers and results in higher arterial flow after reperfusion. Together this may contribute to better perfusion of the bile ducts, providing a potential advantage in the post-ischemic recovery of bile ducts.

Hypothermic machine perfusion (HMP) provides better protection against ischemic damage of the kidney compared to cold-storage. The required perfusion pressures needed for optimal HMP of the liver are, however, unknown. Rat livers were preserved in University of Wisconsin organ preservation solution enriched with acridine orange (AO) to stain viable cells and propidium iodide (PI) to detect dead cells. Perfusion pressures of 12.5%, 25% or 50% of physiologic perfusion pressures were compared. Intravital fluorescence microscopy was used to assess liver perfusion by measuring the percentage of AO staining. After 1-h, the perfusion pressure of 12.5% revealed 72% +/- 3% perfusion of mainly the acinary zones one and two. The perfusion pressure of 25% and 50% showed complete perfusion. Furthermore, 12.5% showed 14.7 +/- 3.6, 25% showed 3.7 +/- 0.9, and 50% showed 11.2 +/- 1.4 PI positive cells. One hour was followed by another series of experiments comprising 24-h preservation. In comparison with 24-h cold-storage, HMP at 25% showed less PI positive cells and HMP at 50% showed more PI positive cells. In summary, perfusion at 25% showed complete perfusion, demonstrated by AO staining, with minimal cellular injury, shown with PI. This study indicates that fine-tuning of the perfusion pressure is crucial to balance (in)complete perfusion and endothelial injury.

In kidney transplantation, microthrombi and fibrin deposition may lead to local perfusion disorders and subsequently poor initial graft function. Microthrombi are often regarded as donor-derived. However, the incidence, time of development, and potential difference between living donor kidneys (LDK) and deceased donor kidneys(DDK), remains unclear. Two open-needle biopsies, taken at preimplantation and after reperfusion, were obtained from 17 LDK and 28 DDK transplanted between 2005 and 2008. Paraffin-embedded sections were immunohistochemically stained with anti-fibrinogen antibody. Fibrin deposition intensity in peritubular capillaries(PTC) and glomeruli was categorized as negative, weak, moderate or strong and the number of microthrombi/mm2 was quantified. Reperfusion biopsies showed more fibrin deposition (20% to 100% moderate/strong, p < 0.001) and more microthrombi/mm2 (0.97 ± 1.12 vs. 0.28 ± 0.53, p < 0.01) than preimplantation biopsies. In addition, more microthrombi/mm2 (0.38 ± 0.61 vs. 0.09 ± 0.22, p = 0.02) and stronger fibrin intensity in glomeruli (28% vs. 0%, p < 0.01) and PTC (14% vs. 0%, p = 0.02) were observed in preimplantation DDK than LDK biopsies. After reperfusion, microthrombi/mm2 were comparable (p = 0.23) for LDK (0.09 ± 0.22 to 0.76 ± 0.49, p = 0.03) and DDK (0.38 ± 0.61 to 0.90 ± 1.11, p = 0.07). Upon reperfusion, there is an aggravation of microthrombus formation and fibrin deposition within the graft. The prominent increase of microthrombi in LDK indicates that they are not merely donor-derived. [ABSTRACT FROM AUTHOR]

Although normothermic machine perfusion of donor livers may allow assessment of graft viability prior to transplantation, there are currently no data on what would be a good parameter of graft viability. To determine whether bile production is a suitable biomarker that can be used to discriminate viable from non-viable livers we have studied functional performance as well as biochemical and histological evidence of hepatobiliary injury during ex vivo normothermic machine perfusion of human donor livers. After a median duration of cold storage of 6.5 h, twelve extended criteria human donor livers that were declined for transplantation were ex vivo perfused for 6 h at 37°C with an oxygenated solution based on red blood cells and plasma, using pressure controlled pulsatile perfusion of the hepatic artery and continuous portal perfusion. During perfusion, two patterns of bile flow were identified: (1) steadily increasing bile production, resulting in a cumulative output of ≥30 g after 6 h (high bile output group), and (2) a cumulative bile production <20 g in 6 h (low bile output group). Concentrations of transaminases and potassium in the perfusion fluid were significantly higher in the low bile output group, compared to the high bile output group. Biliary concentrations of bilirubin and bicarbonate were respectively 4 times and 2 times higher in the high bile output group. Livers in the low bile output group displayed more signs of hepatic necrosis and venous congestion, compared to the high bile output group. In conclusion, bile production could be an easily assessable biomarker of hepatic viability during ex vivo machine perfusion of human donor livers. It could potentially be used to identify extended criteria livers that are suitable for transplantation. These ex vivo findings need to be confirmed in a transplant experiment or a clinical trial. [ABSTRACT FROM AUTHOR]