Andrew J. Butler

Advantages of normothermic perfusion over cold storage in liver preservation.

Background. To minimize the ischemia-reperfusion injury that occurs to the liver with the current method of preservation and transplantation, we have used an extracorporeal circuit to preserve the liver with normothermic, oxygenated, sanguineous perfusion. In this study, we directly compared preservation by the standard method of simple cold storage in University of Wisconsin (UW) solution with preservation by perfusion. Methods. Porcine livers were harvested from large white sows weighing between 30 and 50 kg by the standard procedure for human retrieval. The livers were preserved for 24 hr by either cold storage in UW solution (n=5) or by perfusion with oxygenated autologous blood at body temperature (n=5). The extracorporeal circuit used included a centrifugal pump, heat exchanger, and oxygenator. Both groups were then tested on the circuit for a 24 hr reperfusion phase, analyzing synthetic function, metabolic capacity, hemodynamics, markers of hepatocyte and reperfusion injury, and histology. Results. Livers preserved with normothermic perfusion were significantly superior (P =0.05) to cold-stored livers in terms of bile production, factor V production, glucose metabolism, and galactose clearance. Cold-stored livers showed significantly higher levels of hepatocellular enzymes in the perfusate and were found to have significantly more damage by a blinded histological scoring system. Conclusions. Normothermic sanguineous oxygenated perfusion is a superior method of preservation compared with simple cold storage in UW solution. In addition, perfusion allows the possibility to assess viability of the graft before transplantation.

Successful extracorporeal porcine liver perfusion for 72 hr.

Background. Improvements in extracorporeal perfusion technology and the production of transgenic pigs resistant to hyperacute rejection have stimulated several groups to re-explore the possibility of supporting patients in hepatic failure with extracorporeal porcine livers. The success of organ transplantation has also stimulated interest in using extracorporeal perfusion as a means of organ preservation and resuscitation of organs from marginal donors. The present study describes a method by which livers can be maintained in a viable condition for a minimum of 72 hr of normothermic, extracorporeal perfusion. Methods. Five extracorporeal porcine liver perfusions were performed, each with a duration of 72 hr. Hepatectomy was performed, followed by cold preservation, cannulation of vessels, and initiation of perfusion with normothermic, oxygenated porcine blood. Organ viability was assessed by metabolic, synthetic, hemodynamic, and histologic parameters. Results. After 72 hr of normothermic, extracorporeal perfusion, the isolated livers demonstrated maintenance of normal physiological levels of pH and electrolytes. Continued hepatic protein synthesis (complement and factor V) was maintained throughout the perfusion. Hemodynamic parameters remained within normal physiological range. Histology demonstrated good preservation of the liver with no overall architectural change. Conclusion. It is possible to maintain a liver in a viable condition for a minimum of 72 hr of extracorporeal perfusion. This technique has been developed primarily as a preclinical model of extracorporeal liver support with the intention of proceeding to a clinical trial in patients with fulminant liver failure. However, it also has potential applications in organ preservation or resuscitation before transplantation and in the experimental study of isolated liver physiology.

In Situ Normothermic Regional Perfusion for Controlled Donation After Circulatory Death—The United Kingdom Experience

Organs recovered from donors after circulatory death (DCD) suffer warm ischemia before cold storage which may prejudice graft survival and result in a greater risk of complications after transplant. A period of normothermic regional perfusion (NRP) in the donor may reverse these effects and improve organ function. Twenty‐one NRP retrievals from Maastricht category III DCD donors were performed at three UK centers. NRP was established postasystole via aortic and caval cannulation and maintained for 2 h. Blood gases and biochemistry were monitored to assess organ function. Sixty‐three organs were recovered. Forty‐nine patients were transplanted. The median time from asystole to NRP was 16 min (range 10–23 min). Thirty‐two patients received a kidney transplant. The median cold ischemia time was 12 h 30 min (range 5 h 25 min–18 h 22 min). The median creatinine at 3 and 12 months was 107 µmol/L (range 72–222) and 121 µmol/L (range 63–157), respectively. Thirteen (40%) recipients had delayed graft function and four lost the grafts. Eleven patients received a liver transplant. The first week median peak ALT was 389 IU/L (range 58–3043). One patient had primary nonfunction. Two combined pancreas–kidney transplants, one islet transplant and three double lung transplants were performed with primary function. NRP in DCD donation facilitates organ recovery and may improve short‐term outcomes.

Preimplant Normothermic Liver Perfusion of a Suboptimal Liver Donated After Circulatory Death.

Livers retrieved after circulatory death are associated with an increased incidence of primary nonfunction, early allograft dysfunction, and biliary strictures. The authors report a case of preimplant normothermic perfusion of a suboptimal liver from a 57‐year‐old donor after circulatory death who had been hospitalized for 9 days; predonation alanine transaminase level was 63 IU/L, and the period from withdrawal of life‐supporting treatment to circulatory arrest was 150 minutes. After 5 hours of static cold storage, the liver was subject to normothermic machine perfusion with a plasma‐free red cell–based perfusate. Perfusate lactate level fell from 7.2 to 0.3 mmol/L within 74 minutes of ex situ perfusion, at which point perfusate alanine transaminase level was 1152 IU/L and urea concentration was 9.4 mmol/L. After 132 minutes, normothermic perfusion was stopped and implantation begun. After transplantation, the patient made an uneventful recovery and was discharged on day 8; liver biochemistry was normal by day 19 and has remained normal thereafter. Donor common bile duct excised at implantation showed preservation of peribiliary glands, and cholangiography 6 months posttransplantation showed no evidence of cholangiopathy. Preimplant ex situ normothermic perfusion of the liver appears to be a promising way to evaluate a marginal liver before transplantation and may modify the response to ischemia.

Optimisation of bile production during normothermic preservation of porcine livers.

Machine perfusion of livers may provide a mechanism for extended preservation of marginal donor organs before transplantation, as well as a method for viability assessment. It has proved possible in a series of experimental porcine liver perfusions to maintain liver viability for up to 72 h. However, a reduction in bile production with associated histological evidence of cholestasis was seen after 10 h of perfusion, damaging the biliary canaliculi during the preservation period and leaving these organs in an unacceptable condition for transplantation.

Evidence of macrophage receptors capable of direct recognition of xenogeneic epitopes without opsonization

Abstract:  Background:  We have previously demonstrated that porcine livers perfused with human blood remove most of the erythrocytes from three units of human blood over the course of a 72‐h extracorporeal perfusion. Red blood cell loss did not appear to involve classical complement pathway‐mediated hemolysis, but instead resulted from porcine Kupffer cell phagocytosis.

Prolonged function of extracorporeal hDAF transgenic pig livers perfused with human blood.

Background. The development of genetically modified pigs has renewed interest in the use of porcine liver perfusion in the treatment of acute liver failure. Method. A previously developed model of extracorporeal perfusion has been used to test the function of porcine livers transgenic for human decay accelerating factor when perfused with fresh, whole, human blood. Three experimental groups were studied: alloperfusions (normal pig livers perfused with pig blood) and xenoperfusions of both unmodified and transgenic pig livers with human blood. All livers were perfused for up to 72 hr. Results. Alloperfusion resulted in the maintenance of good function and histological structure. Stable hemodynamic, synthetic, and metabolic parameters were demonstrated in both unmodified and transgenic liver xenoperfusions; hyperacute rejection was not seen. In both groups, however, the measured parameters of liver function deteriorated toward the end of the 72 hr perfusion period; deterioration was more marked in the nontransgenic group. Xenoperfusions were characterized by a progressive and marked decrease in hematocrit of the circulating blood. Histologically, patchy necrosis was noted in both groups and more retained erythrocytes were seen in the sinusoids of nontransgenic livers, but no other consistent differences were apparent. Conclusions. These studies have demonstrated that porcine liver xenoperfusions can be performed for prolonged periods while maintaining good liver function. The use of organs from animals transgenic for a human complement regulator protein confers improvement in some measures of liver function. This preclinical model provides evidence that extracorporeal liver xenoperfusion may be effective in temporary liver support for patients in acute liver failure.

Porcine livers perfused with human blood mount a graft-versus-host reaction

Background. A major focus of xenotransplantation research is the interaction between human immune effector mechanisms and porcine tissues. We present evidence that a transplanted porcine organ might also mount a significant immune response to a human recipient. Methods. Isolated porcine livers were perfused with fresh human blood. Plasma samples were analyzed for complement production by reverse CH50 analysis. ELISA was used to determine the amount and class of porcine immunoglobulin in human blood after xenoperfusion. Flow cytometry was used to determine the specificity and class of porcine immunoglobulin in human blood after xenoperfusion and to determine whether porcine immunoglobulins had bound to the human lymphocytes in the blood perfusing the porcine livers. Electron microscopy was used to evaluate the interaction of porcine Kupffer cells and human erythrocytes. Results. Over the course of 72 hr of extracorporeal perfusion of porcine livers with human blood, the hematocrit fell progressively to as low as 2.5% of starting values, a phenomenon not seen in experiments using porcine blood. We have demonstrated both porcine complement and immunoglobulin in the human blood after xenoperfusion. The porcine antibodies in the human blood have specificity for human lymphocyte antigens. In fact, with increasing duration of perfusion, 40% of the xenoperfusions showed increasing titers of porcine antibodies with specificity for human lymphocyte antigens suggesting a response by primed porcine lymphocytes. However, the majority of erythrocytes are extracted directly by Kupffer cells in the liver. Conclusions. These data demonstrate the ability of porcine livers to generate both a humoral and cellular graft versus host response to human cells.

Normothermic Perfusion in the Assessment and Preservation of Declined Livers Before Transplantation: Hyperoxia and Vasoplegia-Important Lessons From the First 12 Cases.

Background A program of normothermic ex situ liver perfusion (NESLiP) was developed to facilitate better assessment and use of marginal livers, while minimizing cold ischemia. Methods Declined marginal livers and those offered for research were evaluated. Normothermic ex situ liver perfusion was performed using an erythrocyte-based perfusate. Viability was assessed with reference to biochemical changes in the perfusate. Results Twelve livers (9 donation after circulatory death [DCD] and 3 from brain-dead donors), median Donor Risk Index 2.15, were subjected to NESLiP for a median 284 minutes (range, 122-530 minutes) after an initial cold storage period of 427 minutes (range, 222-877 minutes). The first 6 livers were perfused at high perfusate oxygen tensions, and the subsequent 6 at near-physiologic oxygen tensions. After transplantation, 5 of the first 6 recipients developed postreperfusion syndrome and 4 had sustained vasoplegia; 1 recipient experienced primary nonfunction in conjunction with a difficult explant. The subsequent 6 liver transplants, with livers perfused at lower oxygen tensions, reperfused uneventfully. Three DCD liver recipients developed cholangiopathy, and this was associated with an inability to produce an alkali bile during NESLiP. Conclusions Normothermic ex situ liver perfusion enabled assessment and transplantation of 12 livers that may otherwise not have been used. Avoidance of hyperoxia during perfusion may prevent postreperfusion syndrome and vasoplegia, and monitoring biliary pH, rather than absolute bile production, may be important in determining the likelihood of posttransplant cholangiopathy. Normothermic ex situ liver perfusion has the potential to increase liver utilization, but more work is required to define factors predicting good outcomes.BACKGROUND A programme of normothermic ex situ liver perfusion (NESLiP) was developed to facilitate better assessment and use of marginal livers, while minimising cold ischaemia. METHODS Declined marginal livers and those offered for research were evaluated. NESLiP was performed using an erythrocyte-based perfusate. Viability was assessed with reference to biochemical changes in the perfusate. RESULTS 12 livers (9 from circulatory death (DCD) and 3 from brain-dead donors), median Donor Risk Index 2.15, were subjected to NESLiP for a median 284 minutes (range 122-530) after an initial cold storage period of 427 minutes (range 222-877). The first 6 livers were perfused at high perfusate oxygen tensions, and the subsequent 6 at near-physiologic oxygen tensions. After transplantation, 5 of the first 6 recipients developed postreperfusion syndrome and 4 had sustained vasoplegia; 1 recipient experienced primary nonfunction in conjunction with a difficult explant. The subsequent 6 liver transplants, with livers perfused at lower oxygen tensions, reperfused uneventfully. Three DCD liver recipients developed cholangiopathy, and this was associated with an inability to produce an alkali bile during NESLiP. CONCLUSIONS NESLiP enabled assessment and transplantation of 12 livers that may otherwise not have been used. Avoidance of hyperoxia during perfusion may prevent postreperfusion syndrome and vasoplegia, and monitoring biliary pH, rather than absolute bile production, may be important in determining the likelihood of posttransplant cholangiopathy. NESLiP has the potential to increase liver utilization, but more work is required to define factors predicting good outcomes.This is an open access article distributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Normothermic regional perfusion for donation after circulatory death without prior heparinization.

Background Over 40% of deceased donors in the UK donate after circulatory death (DCD). Normothermic regional perfusion has been reported to improve outcomes in such donors in Europe and the United States. Unlike the United States, legal and professional requirements in the UK prevent cannulation and heparinization before verification of death, which must be a minimum of 5 min after circulatory arrest. We developed a novel protocol for in situ normothermic regional perfusion (NRP) which complied with these requirements. Methods NRP was achieved by cannulating the aorta and vena cava after death. Donor blood was then warmed and oxygenated using a bespoke extracorporeal membrane oxygenator circuit before return to the donor. A shunt was incorporated into the extracorporeal circuit to permit heparin mixing before oxygenation and warming was commenced to prevent thrombosis of the oxygenator. Normothermic perfusion was continued for 2 hr before in situ cold perfusion with preservation fluid. All organs were subject to static cold storage after recovery. Results Eight controlled DCD donors underwent NRP from which 3 livers, 2 pancreases, and 14 kidneys were transplanted. Four livers were not used because of biochemical evidence of hepatocellular damage and one because of cirrhosis. Two kidneys were lost from venous thrombosis before function returned and two developed delayed graft function; all transplanted livers and pancreases had primary function. Conclusions Cannulation and heparinization after circulatory arrest does not prevent successful normothermic regional perfusion. The technique permits evaluation of donor organs before implantation and may improve short-term outcomes.