Assessment of liver graft quality during hypothermic oxygenated perfusion: The first international validation study

Abstract

Background & aims: While it is currently assumed that liver assessment is only possible during normothermic machine perfusion, there is uncertainty regarding a reliable and quick prediction of graft injury during ex situ hypothermic oxygenated perfusion (HOPE). We therefore intended to test, in an international liver transplant cohort, recently described mitochondrial injury biomarkers measured during HOPE before liver transplantation.

Methods: Perfusate samples of human livers from ten centers in seven countries with HOPE experience were analyzed for released mitochondrial compounds, i.e. flavin mononucleotide (FMN), NADH, purine derivatives and inflammatory markers. Livers deemed unsuitable for transplantation served as negative controls.

Results: We collected 473 perfusate samples of human donation after cardiac death (n = 315) and donation after brain death (n = 158) livers. Fluorometric assessment of FMN in perfusate was validated by mass spectrometry (R = 0.7011, p <0.0001). Graft loss due to primary non-function or cholangiopathy was predicted by perfusate FMN values (c-statistic mass spectrometry 0.8418, 95% CI 0.7466-0.9370, p <0.0001; c-statistic fluorometry 0.7733, 95% CI 0.7006-0.8461, p <0.0001). Perfusate FMN values were also significantly correlated with symptomatic non-anastomotic strictures and kidney failure, and superior for the prediction of graft loss than conventional scores derived from donor and recipient parameters, such as the donor risk index and the balance of risk score. Mitochondrial FMN values in liver tissues of non-utilized livers were low, and inversely correlated to high perfusate FMN values and purine metabolite release.

Conclusions: This first international study validates the predictive value of the mitochondrial cofactor FMN, released from complex I during HOPE, and may therefore contribute to a better risk stratification of injured livers before implantation.

Impact and implications: Analysis of 473 perfusates, collected from ten international centers during HOPE (hypothermic oxygenated perfusion), revealed that mitochondria-derived flavin mononucleotide values in perfusate are predictive of graft loss, cholangiopathy, and kidney failure after liver transplantation. This result is of high clinical relevance, as recognition of graft quality is urgently needed to improve the safe utilization of marginal livers. Ex situ machine perfusion approaches, such as HOPE, are therefore likely to increase the number of useable liver grafts.

Keywords: hypothermic machine perfusion; liver transplantation; liver utilization; mitochondria; outcome; viability assessment.

Figure 1.. Study design, correlation of fluorescence and mass spectrometry.

The study refers to 473 machine liver perfusates, 435 livers were implanted and 38 livers were discarded. All perfusates were analyzed fluorometrically for FMN, 163 perfusates were additionally analyzed by mass spectrometry. (A). Ten centers from 7 countries participated in this study (B). Calibration of fluorometric measurements allowed calculation of μg FMN/ml perfusate (C). Next, MRM transitions for FMN are shown with overlay of 3 chromatograms for FMN specific ion transitions in positive ionization mode: m/z 457→ (439, 359, 243)(D). The corresponding fragment structures are shown on the right. The areas above the red lines were used for peak integrations (Gaussian smoothing width: 2 points. MRM, multiple reaction monitoring; m/z, mass over charge). Mass spectrometry for FMN correlated with fluorometric values (E)(Pearson’s parametric correlation). The single values of all centers are visualized with medians and IQR in scatter plots for fluorescence and mass spectrometry (F, G, respectively). P values are two taild and refer to discarded livers (Mann-Whiney U test, levels of significance p < 0.01.

Figure 2.. Predictive capacity of perfusate FMN by fluorescence and mass spectrometry.

Receiver operating characteristic (ROC) curves based thresholds for clinical decisions on perfused liver grafts are illustrated for fluorometric perfusate FMN measurements or mass spectrometry for graft loss (A and B), non-anastomotic strictures (C and D) and renal replacement therapy (E and F). The area under the curve (AUC) represents the concordance statistic (C statistic), quantifying the overall ability of the test to discriminate between positive and negative values. The reported p values test the null hypothesis that the AUC equally 0.5, according to Berrar D et al (supplementary reference 1). Confidence intervals are calculated by Prism using the hybrid Wilson/Brown method (supplementary reference 2–3).

Figure 3.. Determination of thresholds for accepting or discarding liver grafts during HOPE.

Proposed acceptance, risk and discard thresholds determined by the maximal Youden index of the receiver operating characteristics (ROC) curves by fluorescence (A) and mass spectrometry (B). ).

Figure 4.. Correlation of mitochondrial FMN and perfusate FMN, correlation of perfusate FMN and complex I function.

Perfusate FMN inversely correlated with mitochondrial FMN (A), and mitochondrial FMN correlated with mitochondrial content of ATP and IMP (B). Consistently, complex I bound mitochondrial FMN correlated with complex I activity (C), and perfusate FMN was associated with perfusate NADH (D). Perfusates collected within the first hour of HOPE showed an increasing level of purine metabolites, underlining mitochondrial activity under hypothermic conditions (E, F). P values are two taild (Mann-Whiney U test, levels of significance p < 0.01 (A,F). Pearson’s parametric correlation was used in B, C, D, levels of significance p < 0.01, two-tailed p values.

Figure 5.. Perfusate FMN and purine metabolites.

Perfusate FMN measure by mass spectrometry shows positive correlations with perfusate (A) NAD, (B) succinate, (C) ADP and (D) IMP. Pearson’s parametric correlation was used in A-D, levels of significance p < 0.01, two-tailed p values.

Figure 6.. Mitochondrial injury and inflammation in implanted and discarded livers (liver tissue samples).

Liver tissue of discarded and implanted livers were compared for changes at the NDUFS1 region of complex I, for NFκB, CD 68, and for vWF (A) with respective quantifications (B). P values are two taild (Mann-Whiney U test, levels of significance p < 0.01 (B).

Figure 7.. Mitochondrial injury and inflammation in implanted and discarded livers (perfusate analysis).

Schematic of mitochondrial-related inflammatory cascades (A). Inflammatory markers differed significantly between perfusates of discarded and implanted livers (B). P values are two taild (Mann-Whiney U test, levels of significance p < 0.01 (B).