Machine Perfusion of Livers with Moderate Macrosteatosis: Efficacy of HOPE with Controlled Rewarming

Aim. To evaluate the efficacy of dual hypothermic oxygenated machine perfusion (D-HOPE) in reducing ischemia-reperfusion injury (IRI) in human donor livers with moderate macrosteatosis by analysing markers of oxidative stress, antioxidant defence, inflammatory response, morphological changes, and post-transplant clinical outcomes.

Materials and Methods. A prospective pilot study enrolling 7 human donor livers with macrosteatosis of 30–50%, verified by urgent intraoperative histological examination (Oil Red O staining of frozen sections). Organs were sequentially allocated to a control group — static cold storage at 4°C in HTK solution (n=3) — or to the D-HOPE group — dual hypothermic oxygenated perfusion at 10 °C for 4 hours using the Aferetica PerLife system with continuous monitoring of portal resistance (target range  0.15–0.25 mmHg/(ml/min/g)), lactate clearance, and perfusate pH (n=4). Portal flow was maintained at 0.25–0.35 ml/min/g; oxygen partial pressure at 60–80 kPa. Assessed parameters included markers of oxidative stress (malondialdehyde, MDA), antioxidant enzyme activity (superoxide dismutase, catalase, glutathione peroxidase), interleukin-6 (IL-6) concentration in the perfusate, morphological signs of hepatocyte injury (cytoplasmic vacuolisation, karyopyknosis) scored in 10 high-power fields (×400) by two independent blinded pathologists, and clinical outcomes over a 6-month follow-up period. Statistical analysis: one-tailed Mann–Whitney test (a priori directional hypothesis), Spearman correlation analysis; significance threshold p<0.05. Data are presented as M±SEM.

Results. The D-HOPE group demonstrated a statistically significant reduction in MDA at the time of reperfusion (T3): 3.9±0.7 vs. 5.8±1.2 nmol/mg protein in the control group (p=0.029; reduction of 33%). Antioxidant enzyme activity in the D-HOPE group significantly exceeded control values: superoxide dismutase 18.7±2.8 vs. 12.3±2.1 U/mg protein (p=0.029); catalase 203±34 vs. 145±28 U/mg protein (p=0.029); glutathione peroxidase 13.2±2.1 vs. 8.9±1.6 U/mg protein (p=0.029). Perfusate IL-6 concentration at the end of perfusion was 52±14 pg/ml. Hepatocyte cytoplasmic vacuolisation at 2 hours post-reperfusion (T4): 38±9% in D-HOPE vs. 65±11% in controls (p=0.029); karyopyknosis: 11±3% vs. 18±4%. Portal resistance in the D-HOPE group declined by 39% over the 4-hour perfusion period (p=0.063 from baseline). Lactate clearance was 50%. Correlation analysis revealed associations between MDA (T3) and peak ALT (ρ=0.79, p=0.036), between MDA (T3) and serum bilirubin on post-operative day 7 (ρ=0.71, p=0.074), and between vacuolisation (T4) and ICU length of stay (ρ=0.83, p=0.021). Peak ALT values: 820±190 U/L (D-HOPE) vs. 1,180±340 U/L (control), representing a 31% reduction. Total bilirubin on post-operative day 7: 41±11 vs. 62±16 μmol/L. ICU length of stay: 2.8±0.9 vs. 4.3±1.4 days. During the 6-month follow-up, the control group recorded hepatic artery thrombosis (n=1, 33%) and biliary stricture (n=1, 33%); no vascular or biliary complications were observed in the D-HOPE group. All recipients are alive.

Conclusion. Dual hypothermic oxygenated perfusion provides statistically significant protection of moderately macrosteatotic donor livers from IRI, as assessed by biochemical, antioxidant, and morphological criteria. MDA and morphological markers demonstrated predictive value for post-transplant clinical outcomes. These preliminary data support the conduct of multicentre randomised trials to validate the technique and subsequently develop personalised perfusion protocols.

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