Introduction: Quantitative assessment in pre-transplants of biochemical indicators has already been indicated during Normothermic Machine Perfusion (NMP). Also, during Hypothermic Oxygenated Machine Perfusion (HOPE), flavin mononucleotide (FMN) is proposed as an assessment method. In addition, however, there is a global demand for integrated assessment methods during HOPE. In this study, we proposed a fluorescence-based measurement method focusing on metabolic indicators of oxygen consumption and FMN using perfusate fluorescence measurement and image-analysis indicators using Indocyanine green (ICG) images during HOPE.
Method: Porcine livers were harvested after warm ischemia time (WIT) 45min (n=4) or 0 min (n=5). We performed 4h HOPE (6-8 ℃) using HTK+. As fluorescence-based measurement methods, combined fluorescence measurements of oxygen, FMN and ICG were proposed and validated during HOPE. This method can measure non-invasive and rapid assessment of liver function during perfusion. In the image-analysis of ICG, we focused on fluorescence distribution and time variation. Finally, we evaluated ischemic reperfusion injury using 2h ex-vivo isolated reperfusion model (IRM) at normothermic temperature (37 ℃) with diluted autologous blood. Resistances of portal vein and hepatic artery, oxygen consumption, pH, lactate, and enzymes (AST･LDH) were measured during the IRM.
Results: Oxygen consumption at 30min of HOPE was 1.57±0.38 vs 2.29±0.33 [mg/L/100g liver] (WIT45 vs WIT0, p<0.05). FMN concentrations were 0.038±0.019 vs 0.040±0.024 [mg/L/100g liver] at 30 min of HOPE (WIT45 vs WIT0, ns). At 240 min of HOPE, it showed 0.058±0.020 vs 0.035±0.040 [mg/L/100g liver] (WIT45 vs WIT0, ns). ICG fluorescence images-analysis focusing on time variation were 83.1±16.5 vs 54.2±6.9 [-] (WIT45 vs WIT0, p<0.01). During IRM, WIT45 vs WIT0, AST time variations were 1500±1138 vs 199±133 [IU/L] and hepatic artery resistances were 0.87±0.38 vs 0.77±0.37 [mmHg/mL/min]. These results measured during HOPE correlated AST time variation and hepatic artery resistance during IRM (FMN-AST: r=0.505, Oxygen consumption-AST: r=-0.839, ICG time variation-hepatic artery resistance: r=0.503). By focusing on metabolic indicators, it is possible to assess hepatocellular function, while vascular dynamics can be assessed through image-analysis indicators during HOPE.
Conclusion: Integrated assessment with fluorescence-based quantification of these indicators enables early prediction of liver damage of IRM. This integrated assessment provides DCD liver during HOPE.