Biliary Bicarbonate, pH, and Glucose Are Suitable Biomarkers of Biliary Viability During Ex Situ Normothermic Machine Perfusion of Human Donor Livers

Abstract

Background: Ex situ normothermic machine perfusion (NMP) can be used to assess viability of suboptimal donor livers before implantation. Our aim was to assess the diagnostic accuracy of bile biochemistry for the assessment of bile duct injury (BDI).

Methods: In a preclinical study, 23 human donor livers underwent 6 hours of end-ischemic NMP to determine biomarkers of BDI. Livers were divided into groups with low or high BDI, based on a clinically relevant histological grading system. During NMP, bile was analyzed biochemically and potential biomarkers were correlated with the degree of BDI. Receiver operating characteristics curves were generated to determine optimal cutoff values. For clinical validation, identified biomarkers were subsequently included as viability criteria in a clinical trial (n = 6) to identify transplantable liver grafts with low BDI.

Results: Biliary bicarbonate and pH were significantly higher and biliary glucose was significantly lower in livers with low BDI, compared with high BDI. The following cutoff values were associated with low BDI: biliary bicarbonate greater than 18 mmol/L (P = 0.002), biliary pH greater than 7.48 (P = 0.019), biliary glucose less than 16 mmol/L (P = 0.013), and bile/perfusate glucose ratio less than 0.67 (P = 0.013). In the clinical trial, 4 of 6 livers met these criteria and were transplanted, and none developed clinical evidence of posttransplant cholangiopathy.

Conclusions: Biliary bicarbonate, pH, and glucose during ex situ NMP of liver grafts are accurate biomarkers of BDI and can be easily determined point of care, making them suitable for the pretransplant assessment of bile duct viability. This may improve graft selection and decrease the risk of posttransplant cholangiopathy.

FIGURE 1.

Representative histological H&E staining of an extrahepatic bile duct biopsy with a low histological BDI score (A) and a high BDI score (B). A, Intact extramural peribiliary glands (encircled), intact vasculature (eg, arrowhead pointing to vital arteriole in stroma) and no signs of stroma necrosis. Note that also the periluminal peribiliary glands are largely intact. B, Severe injury to the extramural peribiliary glands with loss of cells (encircled), necrotic (arrowhead) or absent vessels and diffuse stroma necrosis. Note the denuded epithelial lining of the bile duct lumen (asterisk), as is the case in >90% of all donor bile ducts before transplantation.^{- BDI, bile duct injury.}

FIGURE 2.

Poor correlation between hepatocellular injury and function and BDI. A, Peak ALT concentration in perfusate during NMP correlated poorly with BDI. This was especially true for livers with ALT <6000 U/L, which are potentially transplantable based on this hepatocellular criterion, of which nearly half had simultaneous high BDI. Livers with very high ALT (>6000 U/L) frequently had very high BDI scores. B, Nearly half of the livers with good lactate clearance, also had a high BDI score. C, There was no correlation between bile production and BDI, with over half of livers with high bile production also having high BDI. ALT, alanine aminotransferase; BDI, bile duct injury; NMP, normothermic machine perfusion.

FIGURE 3.

Biliary bicarbonate and pH produced during NMP correlated significantly with BDI. Biliary bicarbonate concentration (A) and biliary pH (B) were significantly higher in livers with a low BDI score, compared with livers with a high BDI score at each timepoint during NMP. C, Biliary pH and bicarbonate concentration were strongly correlated with each other. The line of best fit shows that in the lower range of biliary bicarbonate, small increases in biliary bicarbonate led to relatively large increases in biliary pH. At biliary bicarbonate greater than 30 mmol/L, biliary pH remained relatively stable despite further increases in bicarbonate concentration. (D) ROC curves and (E) statistical analyses of biliary bicarbonate and pH used to discriminate high and low BDI at the earliest timepoints. * P < 0.05. More detailed results, including calculations for all timepoints of bile collection during 6 hours of normothermic machine perfusion (NMP), are provided in Table S1, SDC (http://links.lww.com/TP/B649). AUC-ROC, area under the ROC curve; BDI, bile duct injury; CI, confidence interval; LR, likelihood ratio; NPV, negative predictive value; PPV, positive predictive value; ROC, receiver operating characteristics.

FIGURE 4.

Biliary glucose, bile/perfusate glucose ratio and biliary LDH correlated significantly with BDI during NMP. A, Biliary glucose concentration, inversely reflecting biliary epithelial cell function, was significantly lower in livers with low BDI, compared with livers with high BDI at nearly each timepoint during NMP. B, In livers with high BDI, the bile/perfusate glucose concentration ratio was higher compared with livers with low BDI. C, The delta between perfusate and biliary glucose concentration was lower in livers with high BDI, even reaching negative values at the end of NMP. D, Livers with high BDI have relatively higher biliary glucose in relation to perfusate glucose concentrations, compared with livers with a low BDI. This resulted in a steeper slope in high BDI livers (slope 0.88), compared with low BDI livers (slope, 0.62). E, Biliary LDH concentration, a marker of biliary epithelial cell injury, was significantly higher at each timepoint in livers with a high BDI score compared with livers with a low BDI score. (F) ROC curves and (G) Statistical analyses of biliary glucose, bile/perfusate glucose ratio, perfusate-biliary glucose delta and biliary LDH used to discriminate high and low BDI at the earliest timepoints. * P < 0.05. More detailed results, including calculations for all timepoints of bile collection during 6 hours of NMP, are provided in Table S1, SDC (http://links.lww.com/TP/B649). AUC-ROC, area under the ROC curve; BDI, bile duct injury; CI, confidence interval; LDH, lactate dehydrogenase; LR, likelihood ratio; NMP, normothermic machine perfusion; NPV, negative predictive value; PPV, positive predictive value; ROC, receiver operating characteristics.

FIGURE 5.

Clinical validation of biliary biomarkers during ex situ NMP of transplanted and nontransplanted livers. Six livers that were declined for transplantation nationwide underwent end-ischemic NMP to assess their viability for transplantation in a clinical trial. According to protocol, viability assessment occurred within 2.5 hours NMP. Four livers fulfilled the hepatobiliary criteria and were transplanted. There was no clinical evidence of posttransplant cholangiopathy at a median of 8.3 months (IQR, 7.6-10.1) follow-up. Dotted lines indicate biliary biomarker cutoff values established in the preclinical study. IQR, interquartile range; NMP, normothermic machine perfusion.