Mitochondrial respiratory chain and Krebs cycle enzyme function in human donor livers subjected to end-ischaemic hypothermic machine perfusion.

INTRODUCTION: Marginal human donor livers are highly susceptible to ischaemia reperfusion injury and mitochondrial dysfunction. Oxygenation during hypothermic machine perfusion (HMP) was proposed to protect the mitochondria but the mechanism is unclear. Additionally, the distribution and uptake of perfusate oxygen during HMP are unknown. This study aimed to examine the feasibility of mitochondrial function analysis during end-ischaemic HMP, assess potential mitochondrial viability biomarkers, and record oxygenation kinetics. METHODS: This was a randomised pilot study using human livers retrieved for transplant but not utilised. Livers (n = 38) were randomised at stage 1 into static cold storage (n = 6), hepatic artery HMP (n = 7), and non-oxygen supplemented portal vein HMP (n = 7) and at stage 2 into oxygen supplemented and non-oxygen supplemented portal vein HMP (n = 11 and 7, respectively). Mitochondrial parameters were compared between the groups and between low- and high-risk marginal livers based on donor history, organ steatosis and preservation period. The oxygen delivery efficiency was assessed in additional 6 livers using real-time measurements of perfusate and parenchymal oxygen. RESULTS: The change in mitochondrial respiratory chain (complex I, II, III, IV) and Krebs cycle enzyme activity (aconitase, citrate synthase) before and after 4-hour preservation was not different between groups in both study stages (p > 0.05). Low-risk livers that could have been used clinically (n = 8) had lower complex II-III activities after 4-hour perfusion, compared with high-risk livers (73 nmol/mg/min vs. 113 nmol/mg/min, p = 0.01). Parenchymal pO2 was consistently lower than perfusate pO2 (p ≤ 0.001), stabilised in 28 minutes compared to 3 minutes in perfusate (p = 0.003), and decreased faster upon oxygen cessation (75 vs. 36 minutes, p = 0.003). CONCLUSIONS: Actively oxygenated and air-equilibrated end-ischaemic HMP did not induce oxidative damage of aconitase, and respiratory chain complexes remained intact. Mitochondria likely respond to variable perfusate oxygen levels by adapting their respiratory function during end-ischaemic HMP. Complex II-III activities should be further investigated as viability biomarkers.

Reliability and Accuracy of Clinical Assessment and Digital Image Analysis for Steatosis Evaluation in Discarded Human Livers.

BACKGROUND: Accurate assessment of steatosis in procured livers is crucial to reduce the poor outcome associated with high-grade steatosis and to optimize the utilization of donor grafts. Clinical examination and digital image analysis (DIA) have been used for steatosis evaluation, but the validity of these methods is debated. This study aimed to compare these methods with standard histology for assessment of steatosis severity in human livers and to evaluate a revised classification system for automated fat measurement. METHODS: Clinical assessment of liver steatosis at time of retrieval and automated measurement were compared with standard histology in paraffinized and hematoxylin and eosin-stained slides, using a 4-grade scale for ordinal data and percentages for numerical values. RESULTS: Analysis of 42 human livers that were retrieved but not transplanted showed that clinical examination was not reliable for assigning steatosis grades (κw, 0.12; 95% CI, -0.06 to 0.30), overestimated steatosis severity, and had an accuracy of 67% for discriminating low- and high-grade steatosis. Digital image analysis had a substantial agreement on absolute fat percentage (intraclass correlation coefficient, 0.76; 95% CI, 0.63-0.84) and steatosis grades (κw, 0.70; 95% CI, 0.57-0.82), with 88% accuracy using the revised classification system. CONCLUSIONS: Clinical assessment of steatosis is inaccurate, and relying on this method alone could result in unnecessary discard of livers. Digital image analysis is feasible with higher accuracy and reliability, but further clinical studies are required to evaluate its clinical validity.