Work

The caveat of biliary pH as biomarker of bile duct viability during normothermic machine perfusion of donor livers

Year: 2024

Type: article

Abstract: To the editor, Shortage of suitable donor organs remains a major challenge in liver transplantation. This has resulted in an increased utilization of organs from so-called extended criteria donors.1 These organs are of suboptimal quality and have an increased risk of developing post-transplant complications.2,3 Ex situ normothermic machine perfusion (NMP) enables the assessment of livers before transplantation using predetermined hepatobiliary viability criteria. Many groups already include hepatocyte assessment criteria, and there is an increasing interest in biliary pH as a biomarker of cholangiocyte viability.4 However, when measuring biliary pH, we have noted variations in the results depending on the time interval between collection and processing of samples, questioning the stability of biliary pH as a biomarker. The pH of a solution, dependent on the concentration of hydrogen ions (H+), is influenced by the partial pressure of carbon dioxide (pCO2), as reflected by the following equation: CO2+H2O↔H2CO3↔H++HCO3−. When the concentration of dissolved CO2 decreases, the equilibrium shifts, resulting in a reduction of H+ ions and an increase in pH. To minimize CO2 exchange between bile samples and air, we therefore started to collect bile during NMP under a thin layer of mineral oil (MO). MO functions as a physical barrier that prevents CO2 diffusion, avoiding artificial changes in pH. However, bile collection under MO is not standard practice in other centers, which can lead to variations in reported pH values and affect its reliability as a viability criterion, especially when bile is collected in an open system. Therefore, our aim was to determine the impact of CO2 exchange between bile and air on biliary pH and whether the method of bile collection and processing can influence the measured pH value. METHODS We prospectively analyzed the composition of bile samples collected during NMP as a proposed cholangiocyte viability marker and during liver surgery as a reference for in vivo biliary composition. Samples were collected between January and May 2024. Bile from livers during NMP was collected at the University Medical Center Groningen after informed consent for research was provided by the next of kin. Bile samples obtained from the common bile duct (CBD) during open liver surgery were collected at the Erasmus Medical Center after written consent under the approval of the Medical Ethics Review Committee (Erasmus MC Medical Center, MEC-2019-0488). This research was conducted in accordance with the Declarations of Helsinki and Istanbul. In NMP livers, a biliary catheter was placed in the CBD at the start of perfusion and secured with a ligature. Bile samples of 2.0–3.5 mL were collected in plastic tubes with or without MO (Figure 1A). As previous research observed differences in CO2 diffusion between plastic and glass,5 bile was also collected in glass tubes with or without MO (Figure 1A). The bile drain was sequentially placed into the four different tubes. Depending on the bile flow rate, bile collection time was approximately 1–5 minutes for each sample. During liver surgery, 2.0–5.0 mL bile was collected directly from the CBD with a 5.0 mL syringe. Bile samples were immediately distributed into eight plastic tubes, either with or without MO occlusion (Sigma Aldrich, Darmstadt, Germany) (Figure 1B, C).FIGURE 1: Bile collection and preparation for analysis. (A) Bile was collected in four different tubes: glass (G) and plastic (P), with and without MO. The green arrows show the thin layer of MO (A, C, D). (B, C) Bile was collected in four 2 mL tubes without MO (B) and four with MO (C). (D) Bile drain placed underneath MO before the start of bile collection to prevent CO2 exchange from the bile to the ambient air. Only a small amount of MO is needed. (E) For analyses, bile samples were aspirated from the bottom of the tube to prevent the aspiration of MO. Abbreviation: MO, mineral oil.Bile samples were analyzed immediately as well as 5, 10, 15, and 30 minutes after collection. Bile was aspirated from the tubes using a 1.0 mL syringe (Figure 1D, E) and analyzed with a blood gas analyzer (ABL90Flex blood gas meter, Radiometer, Brønhøj, Denmark) for pCO2, pH, bicarbonate (HCO3−) and glucose. Continuous data were presented as the median and interquartile range (IQR). The Mann-Whitney U test was used to compare groups. A p-value <0.05 was considered statistically significant. Analyses were performed using Graphpad Prism v10.0.02 (Graphpad Software, San Diego, CA). A more detailed description of NMP, bile collection, and blood gas analysis is provided in the Supplemental Methods, https://links.lww.com/LVT/A868.5–7 RESULTS Bile was collected during seven NMP procedures and from five patients undergoing liver surgery. There were no significant differences in biliary pCO2, pH, HCO3− and glucose in bile samples collected during NMP and liver surgery, and the same trends for the different parameters were observed (Supplemental Figure S1, https://links.lww.com/LVT/A869). Therefore, measurements were combined in further analyses. Likewise, measurements of bile collected in glass and plastic tubes were combined, as no differences were observed (Supplemental Figure S2, https://links.lww.com/LVT/A869). In bile collected without MO, biliary pCO2 decreased with increasing time between collection and analysis (Figure 2A). In parallel, biliary pH increased (Figure 2B). In bile collected under MO, the decrease in biliary pCO2 was less pronounced, and biliary pH remained stable (Figure 2A, B). The difference in biliary pCO2 and pH was already statistically significant after 5 minutes and increased further when the time between collection and analysis increased up to 30 minutes. These significant differences in biliary composition were seen in the combined analysis, as well as in the analysis of NMP cases only. The difference in biliary pH was clinically meaningful, as biliary pH was 7.47 (7.43–7.58) after 30 minutes in samples collected under MO (borderline acceptance according to viability criteria), compared to 7.70 (7.58–7.90) when samples were collected without MO (definitive acceptance according to viability criteria). In contrast to pCO2 and pH, biliary HCO3− and glucose values remained stable over time in both groups, and no significant differences were observed between the two sampling methods (Figure 2C, D).FIGURE 2: Biliary pCO2, pH, HCO3 −, and glucose. Biliary pCO2, pH, HCO3 −, and glucose values after collection with and without MO. Samples were analyzed immediately (T0) and at 5, 10, 15, and 30 minutes after collection. Presented are the median with interquartile ranges. Significant differences between bile samples collected with or without MO per timepoint are indicated. *, **, ***, and **** indicate a statistically significant difference of p<0.05, p<0.01, p<0.001, and p<0.0001, respectively. The horizontal interrupted black line in the pH figure indicates the pH value (7.45), which is considered as the lower limit of this parameter in the assessment of biliary viability. Abbreviations: HCO3 −, bicarbonate; MO, mineral oil; pCO2, partial pressure of carbon dioxide.DISCUSSION This study indicates that biliary pH is critically influenced by the diffusion of CO2 from bile samples after collection. With increasing time between sample collection and analytic processing, the impact on biliary pH became more pronounced. The collection of bile under a thin layer of MO mitigates the diffusion of CO2 and prevents the artificial increase in pH. We found similar changes in bile composition between samples collected during NMP and during liver surgery, indicating that the changes in pCO2 and pH during NMP do not only occur in bile collected ex situ but also in bile collected in vivo. Although CO2 diffusion through the plastic biliary drain was not assessed, the lack of significant differences in baseline pH between bile collected directly from the CBD and from the biliary catheter during NMP suggests minimal CO2 diffusion. Additionally, no significant differences in change of bile composition were found between glass or plastic tubes, indicating equal suitability for bile collection. The finding that biliary pH, an increasingly used biomarker of cholangiocyte viability during liver NMP, is highly dependent on the method of sample collection and timing of the analysis, points to a potential caveat in the measurement of this biomarker. A minimal biliary pH of 7.45 is commonly proposed as a cutoff value of bile duct viability during NMP.4 Our data now show that this value can be “artificially” reached within 5 minutes when a sample is not collected and stored under MO. Additionally, our data indicate that biliary HCO3− and glucose, other proposed viability markers, are not susceptible to gas exchange and might serve as more stable biomarkers of cholangiocyte viability. In conclusion, if biliary pH is used as a viability marker during liver NMP, it is crucial to prevent interface contact between bile and ambient air. We advocate to collect bile under MO and to analyze samples as quickly as possible, preferably within 5 minutes. For accurate comparisons between centers and studies, a detailed description of the bile collection method is essential to identify and understand discrepancies in reported values.

Source: Liver Transplantation

DOI: 10.1097/lvt.0000000000000538