Hepatocellular Function and Injury Correlate Poorly With BDI
Overall, there was a weak correlation between peak ALT concentration in perfusate during NMP, a marker of hepatocellular injury, and the degree of BDI (Spearman r, 0.424; P = 0.044) (Figure 2A). In livers with extremely high ALT levels (>6000 U/L), the BDI score was also high in 7 (88%) of 8 cases. However, when ALT levels were less than 6000 U/L and livers could potentially be considered for transplantation based on an acceptable degree of hepatocellular injury,14 the correlation between ALT and BDI was very poor (Spearman r, 0.192; P = 0.493) with 7 (47%) of 15 livers still having a high BDI score.
Similarly, 4 (44%) of 9 livers with good lactate clearance also had high BDI (Figure 2B). There was no correlation between BDI and lactate concentration (Spearman r, 0.349; P = 0.103). Likewise, 10 (56%) of 18 livers with high bile production also had high BDI. There was no correlation between BDI and cumulative bile production (Spearman r, −0.291; P = 0.178) (Figure 2C). Furthermore, there was no correlation between (cumulative) bile production and biliary pH, bicarbonate, LDH and the glucose and bile/perfusate glucose ratio (Figure S1, SDC, http://links.lww.com/TP/B649).These findings indicate that markers of hepatocellular injury and function poorly predicted the degree of BDI, especially in cases that could potentially be considered for transplantation based on hepatocellular criteria. This supports the need for specific biomarkers of BDI during NMP.
Biliary pH and Bicarbonate Correlate Significantly With BDI
Biliary pH and bicarbonate concentration during NMP were significantly higher in livers with low BDI, compared with livers with high BDI (Figures 3A-B). As expected, biliary pH and bicarbonate were strongly correlated (Figure 3C). The line of best fit (R2 = 0.7377) demonstrated that in the lower range of biliary bicarbonate small increases lead to a relatively large increase in pH, whereas at higher bicarbonate concentrations (>30 mmol/L), pH remained relatively stable.
Because bile production can be low or absent during the first 2 hours of NMP, we only used bile samples collected after 2 hours to determine optimal cutoff values to discriminate livers with low BDI from those with high BDI (Table S1, SDC, http://links.lww.com/TP/B649). Already after 2 hours, a biliary bicarbonate concentration of 18 mmol/L discriminated between low and high BDI with an AUC-ROC of 0.91 (P = 0.002) and a Sens, Spec, PPV, NPV greater than 80% (Figure 3D-E).
The earliest timepoint at which biliary pH discriminated livers with low BDI from those with high BDI was 2.5 hours (Table S1, SDC, http://links.lww.com/TP/B649). At this timepoint, the optimal cutoff value for pH was 7.48, with an AUC-ROC of 0.81 (P = 0.019) (Figure 3D-E).
Biliary Glucose and Bile/Perfusate Glucose Ratio Correlate Significantly With BDI
Glucose concentrations were lower in bile of livers with low BDI, compared with livers with high BDI score (Figure 4A). Although biliary glucose gradually decreased in livers with low BDI, biliary glucose increased in livers with high BDI, generally remaining greater than 20 mmol/L. In livers with high BDI, the median bile/glucose concentration ratio increased to 1 and remained stable throughout perfusion. In contrast, in livers with low BDI the median ratio was always 0.7 or less and gradually declined over time (Figure 4B-C). Another way to study the relation between biliary and perfusate glucose is by calculating the difference between perfusate and biliary glucose concentration (delta).14 In livers with high BDI, the delta was lower compared with livers with low BDI, reaching significance at only 3 and 3.5 hours NMP (Figure 4C). After several hours of NMP, negative delta values were reached.
The earliest timepoint at which biliary glucose and the bile/perfusate glucose ratio could discriminate livers with low BDI from those with high BDI was 2 hours (Table S1, SDC, http://links.lww.com/TP/B649). At this timepoint, the optimal cutoff values were 16 and 0.67 mmol/L, respectively (both AUC 0.82; P = 0.013) (Figure 4F-G). The delta between perfusate and biliary glucose did not result in an AUC greater than 0.80 at any timepoint, though at 3 hours NMP an AUC of 0.79 resulted in a cutoff value of 2.3 mmol/L (P = 0.030, Figure 4F-G, and Table S1, SDC, http://links.lww.com/TP/B649).
Biliary LDH Correlates Significantly With BDI
Biliary LDH concentration, a marker of biliary epithelial cell injury, was more than twofold higher in livers with high BDI, compared with livers with low BDI (Figure 4E). In both groups, biliary LDH concentration declined gradually during NMP. The earliest timepoint with a significant AUC-ROC for biliary LDH was 2 hours NMP (Table S1, SDC, http://links.lww.com/TP/B649), with an optimal cutoff value of 3689 U/L and an AUC of 0.83 (P = 0.014) (Figures 4F-G).
Clinical Validation of Biliary Biomarkers During NMP
Based on the preclinical research data, we included biliary pH as one of the criteria for hepatobiliary viability assessment in a clinical trial of end-ischemic NMP of high-risk livers that were initially declined nationwide for transplantation. So far, 6 DCD livers (median Eurotransplant Donor Risk Index,29 2.9; IQR, 2.7–2.9) have been included in this trial of which 4 (median Eurotransplant Donor Risk Index, 2.9; IQR, 2.8–3.0; median United Kingdom-DCD risk score,30 6.0; IQR, 5.8–7.5) met all selection criteria for transplantation during NMP, including a biliary pH greater than 7.48 within 2.5 hours of NMP. In addition, biliary bicarbonate and glucose bile/perfusate ratio were within the ranges identified in the preclinical study (Figure 5). The recipients of these 4 liver grafts had an uneventful recovery and none of them developed clinical evidence of posttransplant cholangiopathy at a median follow-up of 8.3 months (IQR, 7.6–10.1 months). Of the 2 livers that were declined for transplantation, the first fulfilled all hepatocellular criteria and was declined only on the basis of bile biochemistry (at 2.5 hours NMP: cumulative bile production 45 mL [≥10 mL within 2.5 hours NMP and ≥4 mL in preceding hour]; perfusate lactate of 0.3 mmol/L [<1.7 mmol/L]; perfusate pH 7.38 [7.35–7.45]).The second liver was also declined based on hepatocellular criteria (cumulative bile production, 57 mL; perfusate lactate, 3.5 mmol/L; perfusate pH, 7.25). At 2.5 hours NMP, the perfusate ALT concentration was approximately 4000 and 6000 U/L, respectively. The retrospectively determined median BDI score for the transplanted livers was 3.2 (IQR, 2.8–3.5) and 4.3 (3.0–5.5) for the nontransplanted livers.
In the present study, we have determined the diagnostic accuracy of the previously described biomarkers of biliary injury and viability, which can be easily assessed point of care during ex situ NMP of human donor livers. Biliary bicarbonate concentration greater than 18 mmol/L, biliary pH greater than 7.48, biliary glucose concentration less than 16 mmol/L, bile/perfusate glucose concentration ratio less than 0.67, and biliary LDH concentration less than 3689 U/L within 2.5 hours of NMP were strongly associated with low histological BDI. These findings have important clinical implications as the proposed biomarkers allow transplant teams to stratify livers grafts during ex situ NMP based on the risk of BDI, and potentially also that of posttransplant cholangiopathy. Biliary bicarbonate had the highest predictive value out of all of the studied biomarkers. This is likely because biliary bicarbonate is less influenced by other nonbiliary factors.
Ex situ NMP is increasingly being applied and explored as a tool to assess viability of liver grafts that were initially declined for transplantation based on a perceived high risk of early graft failure.11-14 In most centers, selection criteria are currently based on hepatocellular injury and function. The risk of posttransplant graft failure, however, is not only determined by the degree of hepatocellular injury, but also by the presence of biliary injury. Especially DCD livers have an increased risk of developing biliary complications.31 In a recent clinical study of 12 initially declined liver grafts that were identified as transplantable during end-ischemic NMP, 25% developed posttransplant cholangiopathy despite adequate hepatocellular function during pretransplant ex situ NMP.13 These clinical data are in line with our observation that some livers with a low degree of hepatocellular injury can still have a high degree of BDI, which can be missed when no specific biliary viability criteria are used. This difference in hepatocellular and cholangiocellular injury can be explained by the greater susceptibility of cholangiocytes to ischemia-reperfusion injury and a slower postischemic recovery of intracellular ATP, compared with hepatocytes.23,32
The parameters we have selected as potential biomarkers of biliary viability were all based on the known physiological function of healthy bile duct epithelium. Two important physiological functions of biliary epithelium are active secretion of bicarbonate and reabsorption of glucose, leading to an alkalotic pH and very low biliary glucose concentrations at the level of the extrahepatic bile duct.17,18 We have previously suggested that these parameters could potentially serve as biomarkers of bile duct viability during machine perfusion.26,33,34 In the current study, we demonstrated that biliary pH, bicarbonate and glucose concentration indeed strongly correlate with histological BDI of liver grafts during NMP, confirming observations made earlier this year.14 In 3 independent clinical studies, the histological degree of BDI before liver transplantation has been identified as a significant predictor of the development of NAS after transplantation.8-10
Our findings suggest that biliary epithelial cells of livers with high BDI are unable to secrete sufficient amounts of bicarbonate to raise the biliary pH. Increasing the biliary pH helps biliary epithelial cells to protect themselves against the toxic effects of hydrophobic bile salts, which is also known as the “biliary bicarbonate umbrella”.35 Low biliary pH and bicarbonate, therefore, not only reflect biliary injury/dysfunction, but may also contribute to additional biliary injury due to an absent bicarbonate umbrella.36 Biliary pH and bicarbonate were not linearly correlated, which is explained by the fact pH varies on a logarithmic scale, whereas bicarbonate does not. The pH values we observed in liver grafts with low BDI are within the normal range of biliary pH, which varies between 7.5 and 8.1 in the common bile duct.17 The same is true for biliary bicarbonate concentration, which normally ranges widely between 12 and 55 mmol/L.18 Interestingly, Watson et al14 recently reported 3 patients that developed cholangiopathy after the transplantation of livers that were unable to produce bile with a pH greater than 7.4 during NMP. This preliminary clinical observation is in line with the identified biomarkers of BDI in the current study in preclinical livers as well as the clinical validation cohort.
Although biliary glucose also correlated significantly with the degree of biliary injury, the interpretation of glucose values is slightly more complex. Postischemic reperfusion of a liver graft almost universally results in a pronounced increase of glucose levels in the perfusion fluid due to glycogenolysis.37 This contributes to a higher glucose concentration in the primary bile produced by hepatocytes, which affects the reabsorption of glucose from bile when it passes from the canaliculi to the common bile duct. Even when the biliary epithelium is intact, glucose reabsorption via SGLT1 and GLUT1 becomes insufficient when biliary glucose concentrations are too high, a phenomenon analogous to the renal threshold for glucose in kidney tubuli.22,38 In other words, high glucose concentration in the perfusion fluid (which is frequently seen during NMP) may affect the biliary reabsorption of glucose. Biliary glucose levels should therefore be viewed in relation to glucose levels in the perfusion fluid. To illustrate this, we have correlated glucose concentrations in bile with those in the perfusion fluid. The ratio between glucose in bile and perfusion fluid was almost 1 in livers with high BDI, whereas it was 0.7 or less in livers with low BDI. Others have reported the delta between perfusate and biliary glucose levels,14 though in our hands this did not result in a usable criterion. Our findings regarding biliary glucose are in line with an in vitro study with rat cholangiocytes, in which ATP-depleted cholangiocytes showed prolonged dysfunction of biliary glucose transporter SGLT1 and diminished glucose reabsorption.23 Similarly, in an experimental study in rabbits, higher glucose concentrations were found in bile of livers that had suffered from warm and cold ischemia, compared with livers that had not been ischemic.39
The observations made for biliary bicarbonate, pH, and glucose were supported by the data on biliary LDH concentrations. Based on animal experiments, biliary LDH has previously been proposed as a marker of biliary epithelial injury.24 In the current study, biliary LDH was indeed significantly higher in livers with high BDI, compared with livers with low BDI. In all livers, however, biliary LDH concentration declined gradually during NMP, which can be explained by an early washout of LDH from dead cholangiocytes when bile flow recurs. This is in line with previous research, where biliary LDH correlated with the length of cold ischemia in a rat liver model and previous studies that reported lower LDH concentrations in machine perfused livers compared with cold stored livers.25,27,40
This study has some limitations. We took biopsies from the extrahepatic bile duct to assess the degree of histological injury, whereas the intrahepatic bile ducts can also be involved in posttransplant cholangiopathy. However, in a previous study, we have demonstrated that histological injury (in particular injury to the peribiliary vascular plexus, extramural peribiliary glands and stroma necrosis) assessed at the level of the extrahepatic bile duct also reflects the degree of injury in the intrahepatic bile ducts, as deep as the segmental bile ducts.41 Furthermore, there may have been some degree of sampling error in bile duct biopsies. To minimize this, the average BDI of 2 biopsies was taken. Furthermore, our preclinical data included 5 DBD livers, whereas the clinical livers were all DCD. However, when we excluded the DBD livers in the preclinical study, similar results were obtained. Lastly, some livers produce very little or no recorded bile during NMP. This raises the question as to whether or not such livers can be transplanted safely. We would advocate an approach tailored to the specific donor liver and recipient. For example, when assessing a liver with a high risk of developing NAS, production of good quality bile during NMP would be essential to allow cholangiocyte viability assessment before transplantation. On the other hand, for livers with a low risk of NAS, cholangiocyte viability assessment may not always be necessary, as has been reported in the literature.14
Implementation of the identified biomarkers in larger clinical trials of NMP with longer follow-up, in which both DCD and DBD livers are included, is necessary to confirm their utility in decreasing the incidence of posttransplant cholangiopathy. Machine perfusion has the potential to provide opportunities to ameliorate BDI, for instance through stem cell therapy. These cholangiocellular criteria could be applied to identify livers that require additional pharmacological interventions, and be used to assess the efficacy of such treatments. In conclusion, biliary bicarbonate, pH, glucose, and LDH during ex situ NMP are accurate biomarkers of clinically relevant, histological biliary injury of livers grafts. These biomarkers can be easily determined point-of-care, making them suitable for the assessment of bile duct viability during NMP and the potential identification of donor livers with a low risk of developing posttransplant cholangiopathy.
The authors would like to thank all donors and donor relatives, as well as the Dutch Transplantation Society and its staff, for enabling the use of donor livers for research.
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