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Subject and rationale of the research projectLiver Transplantation (LTx) has become the preferred method to treat end-stage liver disease. The persistent donor organ shortage and the mortality on the waiting list encourage an increasing use of Expanded Criteria Donors (ECD), such as donors with hepatic steatosis, and Donors after Cardiac Death (DCD). However; these ECD and DCD livers induce a higher risk of graft dysfunction after LTx compared to “ideal” livers. An accurate and objective evaluation on the viability of these livers prior to LTx is thus necessary to avoid transplantation of livers likely to fail as well as to avoid discarding livers that were transplantable. Currently, the standard evaluation to accept or discard liver grafts for transplantation is the transplant surgeon’s subjective estimation based on donor’s profile and biochemical data, macroscopic appearance of livers, and sometimes microscopic examination. Objective criteria to evaluate liver viability are still lacking. In order to define objective markers reflecting liver viability, we investigated whether - similar to the kidney - such markers could be obtained during Hypothermic Machine Perfusion (HMP) preservation. HMP is regarded as a superior preservation method compared to simple cold storage, the current gold standard preservation for all solid organs (other than kidneys). The aim of this research project was to delineate whether objective markers reflecting liver graft viability could be identified during HMP preservation. For this study, we used DCD livers in a previously validated porcine model and human livers discarded for transplantation. The parameters measured in this study are listed in Table 1. The aims of this study were1. to optimize liver procurement to preserve maximal liver viability (avoiding any bias of procurement on the viability testing),2. to optimize liver HMP preservation (avoiding any bias of the preservation on the viability testing),3. to evaluate DCD liver viability during HMP by using apparent diffusion coefficient, 4. to evaluate DCD liver viability by using proton magnetic resonance spectroscopy on the HMP perfusate, 5. to evaluate DCD liver viability by developing a damage index based on biochemical compounds in perfusate and vascular resistance during HMP,6. to evaluate the viability of human livers discarded for clinical transplantation using the biochemical compounds in perfusate and vascular resistance during HMP. Summary of the results1. Liver procurement was optimized to preserve maximal liver viability by eliminating intra-hepatic air embolism. Leaving hepatic vasculature open to air during procurement and back-table preparation caused a substantial amount of intra-hepatic air emboli resulting in insufficiently perfused liver parenchyma. This phenomenon was not observed when the hepatic vasculature remained closed by vascular clamps after flush-out until the start of the HMP. Consequently, for the experiments on porcine liver viability in this thesis, the hepatic vasculature was left occluded during preservation and back-table preparation to avoid intra-hepatic air embolism compromising liver perfusion and thus viability.2. Liver HMP setting was optimized by adding oxygen to the perfusate and by reducing the flow and pressure in hydrodynamics, applying a mild total hepatic flow of 0.5ml/g liver/min. This resulted in better preservation of intracellular adenosine triphosphate contents and a better preserved liver morphology at the end of HMP. Consequently, this HMP setting was applied for the subsequent experiments on porcine livers.3. Apparent Diffusion Coefficient (ADC) was found to be a sensitive marker for hepatic in vivo Warm Ischemia (WI), in contrast to ex vivo HMP. The decrease of ADC during hepatic WI in vivo was similar (the first 30min of WI period) to the decrease observed during cerebral ischemia. The lack of sensitivity of ADC during HMP ex vivo might be due to the (i) absence of inflammatory cells, (ii) flushed-out congestion and coagulation in the sinusoids, and (iii) artificial maintenance of extracellular space and sinusoid dilatation caused by HMP itself. 4. Alanine and histidine in HMP perfusate were identified to be able to discriminate non-viable DCD livers from viable livers in proton magnetic resonance spectroscopy as a screening tool for the HMP perfusate. These two compounds were significantly higher in the WI-group at the end of HMP, which presumably resulted from the WI-induced hepatocellular damage/leakage. 5. Biochemical compounds - pH, Aspartate Transaminase (AST), Liver-Fatty Acid Binding Protein (L-FABP) - in perfusate and vascular resistance during HMP were clarified as potential markers on DCD liver viability. Curve-fitting of the changing levels of pH, AST, L-FABP and arterial resistance during HMP provided ß-coefficients that can be used as viability indicators. These ß-coefficients also allowed a flexible sampling time during HMP and thus a better clinical applicability. An index, referred to as damage index, combining these ß-coefficients in a mathematical equation [damage index = 37 * ßAST + 257 * ßpH – 2] had higher sensitivity and specificity to discriminate viability of DCD livers compared to single parameter and predicted the risk of subsequent primary non-function as observed previously in our lab. 6. We eventually translated these experimental findings into a clinical setting by testing these parameters for human livers discarded for clinical use (first in man study of its kind). Similar to the porcine model, AST released in the perfusate during HMP was found to discriminate absolutely not from potentially transplantable livers and appeared a promising parameter to evaluate viability during HMP and prior to transp