Ex Situ Normothermic Machine Perfusion of Donor Livers

Summary

Here we present a protocol describing oxygenated ex situ machine perfusion of donor liver grafts. This article contains a step by step protocol to procure and prepare the liver graft for machine perfusion, prepare the perfusion fluid, prime the perfusion machine and perform oxygenated normothermic machine perfusion of the liver graft.

Abstract

In contrast to conventional static cold preservation (0-4 °C), ex situ machine perfusion may provide better preservation of donor livers. Continuous perfusion of organs provides the opportunity to improve organ quality and allows ex situ viability assessment of donor livers prior to transplantation. This video article provides a step by step protocol for ex situ normothermic machine perfusion (37 °C) of human donor livers using a device that provides a pressure and temperature controlled pulsatile perfusion of the hepatic artery and continuous perfusion of the portal vein. The perfusion fluid is oxygenated by two hollow fiber membrane oxygenators and the temperature can be regulated between 10 °C and 37 °C. During perfusion, the metabolic activity of the liver as well as the degree of injury can be assessed by biochemical analysis of samples taken from the perfusion fluid. Machine perfusion is a very promising tool to increase the number of livers that are suitable for transplantation.

Introduction

The current method of organ preservation in liver transplantation is flush out with and subsequent storage of donor livers in cold (0-4 °C) preservation fluid (such as University of Wisconsin solution or Histidine-Tryptophan-Ketoglutarate solution). This method is referred to as static cold storage (SCS). Although the metabolic rate of livers at 0-4 °C is very low, there is still demand for 0.27 µmol oxygen/min/g liver tissue, which cannot be provided during SCS¹. The conventional method of SCS, therefore, results in some degree of (additional) injury of donor livers. While this amount of preservation injury is not a problem in donor livers of good quality,....

Protocol

This protocol has been approved by the Medical Ethical Committee (Medisch Ethische Toetsingscommissie) of the University Medical Center Groningen, the Netherlands.

1. Preparation of the Perfusion Fluid

Note: The total volume of the perfusion fluid prepared for normothermic machine perfusion according to this protocol is 2,233 ml and the targeted osmolarity of the perfusion fluid is 302 mOsmol/L.

1. From the components of the perfusion fluid described in Table 1, keep the human packed red blood cells, fresh frozen plasma and human albumin separated. Mix the rest of the components in a ste....

Results

12 human livers that were declined for transplantation due to various reasons were used after obtaining informed consent for research from donor families. Donor characteristics are described in Table 2. The human donor livers were perfused normothermically for 6 hr by using the protocol described in this paper. The quality of the liver grafts were evaluated by monitoring the macroscopic homogeneity of liver perfusion (Figure 2A-D). The hemodynamics of th.......

Discussion

This video provides a step by step protocol for normothermic machine perfusion of human donor livers using a device that enables pressure controlled dual perfusion through the hepatic artery and portal vein. While following this protocol, technical failures of the perfusion machine did not occur and all grafts were well perfused and well oxygenated. The ex situ perfused livers had stable hemodynamics and were metabolically active, as defined by the production of bile^16,17.

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Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Liver Assist	Organ Assist	OA.Li.Li.140	Perfusion device
Liver Assist disposable package	Organ Assist	OA.Li.DP.540	Disposable set and cannulas
Meredith No.8	Vygon Nederlands B.V.	1362082	Bile duct cannula
Human albumin 200 g/L / ALBUMAN	Sanquin	15522598	100 ml
Modified parenteral nutrition	Baxter Nederland B.V.	N14G30E	7.35 ml
Multivitamins for infusion / CERNEVIT	Baxter International Inc.	9800927	7 μl
Concentrated trace elements for infusion / NUTRITRACE	B. Braun Melsungen AG	14811332	7.35 ml
Metronidazole 5 mg/ml	Baxter Nederland B.V.	98181882	40 ml
Cefazoline / SERVAZOLIN	Sandoz B.V.	15611337	2 ml
Fast acting insulin	various vendors		20 ml
Calcium glubionate, intravenous solution 10%, 137.5 mg/ml	Sandoz	97038695	40 ml
Sterile H2O	Fresenius Kabi Nederland B.V.	98084453	51.3 ml
NaCl 0.9%	Baxter Nederland B.V.	15262510	160 ml
Heparin 5,000 IE/ml for i.v. administration	LEO Pharma B.V.	98026178	4 ml
Sodium bicarbonate 8.4%	B. Braun Melsungen AG	97973874	The amount depends on the pH
Packed red blood cell (in SAGM)	Blood bank (Sanquin)	N0012000	750 ml
Fresh frozen plasma	Blood bank (Sanquin)	N04030A0/N04030B0	900 ml