A proof of concept study on real-time LiMAx CYP1A2 liver function assessment of donor grafts during normothermic machine perfusion

Abstract

No single reliable parameter exists to assess liver graft function of extended criteria donors during ex-vivo normothermic machine perfusion (NMP). The liver maximum capacity (LiMAx) test is a clinically validated cytochromal breath test, measuring liver function based on 13CO2 production. As an innovative concept, we aimed to integrate the LiMAx breath test with NMP to assess organ function. Eleven human livers were perfused using NMP. After one hour of stabilization, LiMAx testing was performed. Injury markers (ALT, AST, miR-122, FMN, and Suzuki-score) and lactate clearance were measured and related to LiMAx values. LiMAx values ranged between 111 and 1838 µg/kg/h, and performing consecutive LiMAx tests during longer NMP was feasible. No correlation was found between LiMAx value and miR-122 and FMN levels in the perfusate. However, a significant inverse correlation was found between LiMAx value and histological injury (Suzuki-score, R = - 0.874, P < 0.001), AST (R = - 0.812, P = 0.004) and ALT (R = - 0.687, P = 0.028). Furthermore, a significant correlation was found with lactate clearance (R = 0.683, P = 0.043). We demonstrate, as proof of principle, that liver function during NMP can be quantified using the LiMAx test, illustrating a positive correlation with traditional injury markers. This new breath-test application separates livers with adequate cytochromal liver function from inadequate ones and may support decision-making in the safe utilization of extended criteria donor grafts.

Figure 1

Overview of performing the LiMAx test in normothermic machine perfusion. (1) The LiMAx analyzer is connected to the air outlet of the membrane oxygenator. All extra air openings should be closed. (2) Then a baseline ¹³CO₂:¹²CO₂ ratio is measured, followed by administering the ¹³C-methacetin. (3) The ¹³C-methacetin is metabolized in the hepatocytes into ¹³CO₂ and paracetamol. (4) The ¹³CO₂ is excreted by the oxygenator from the perfusate into the “exhaled” air. (5) The LiMAx analyzer analyses the “exhaled” air, produces a curve illustrating delta over baseline (DoB) against the time in minutes. The LiMAx curve first presents the baseline that is being established (A), and then the ¹³C-methacetin is administered (B), followed by a delayed signal time (the time until the first increase in ¹³CO₂ is measured by the analyzer; (C). Then, there is an acceleration phase (D), followed by the DoB_max (E), and finally resulting in an elimination phase (F) in which the signal slowly returns to the baseline.

Figure 2

Histologic evaluation of biopsies from machine perfused livers stained with CYP1A2 immunohistochemistry. (A,B) Present liver H8 and panels C and D present liver H4. (A) and (C) consist of a low magnification picture (× 100). (B) and (D) display a high magnification (× 400). The portal vein (in the portal tract area) is represented by P, Cv represents the central vein, and L represents lymphocytic infiltrate. Zone 1, 2, and 3 represent the hepatic metabolic zones (as defined by the acinus of Rappaport). Zone 1 is the peri-portal zone, zone 2 is the intermediate zone, and zone 3 is the peri-central zone. H8 has normal liver microscopy; in this liver, it is visible that CYP1A2 is the cytoplasmatic hepatocellular stain highly present in zone 2 and 3, and it is less prominent in zone 1. H4 is microscopically a relatively injured graft. In this graft, lymphocytic infiltrates are present, causing hepatocyte loss and, therefore, loss of CYP1A2 positive cells.

Figure 3

Feasibility of LiMAx testing during normothermic machine perfusion. Panel (A) displays the curve of the ¹³CO₂:¹²CO₂ ratio LiMAx curve as measured as a delta over baseline (DOB) during NMP. In the beginning, there is a flat line, which indicates the baseline, followed by the acceleration phase where the DoB increases until it reaches its maximum point (DoB_max). Then, there is a decline of the DoB in the elimination phase. Panel (B) presents the fit of formula 4 on the elimination phase of the curve, which is fitted with an R² of 0.998. The K of the fitted formula was 3.633 × 10^–2, which translates to a half-life time of 19 min.

Figure 4

Consecutive LiMAx testing during normothermic machine perfusion. (A) Four sequentially repeated LiMAx tests over a period of 30 h of liver H11 were performed. Every 6 h, a new bolus methacetin was injected, resulting in a new DoB peak. (B) Before administering a new bolus, the DoB has not returned to the baseline, which should be corrected. The corrected DoB_max is made by calculating the remnant DoB of the previous bolus of ¹³C-methacetin and subtracting this from the new DoB_max.. (C) This illustrates the different LiMAx values in time during long-term machine perfusion. (D) This presents the different ¹³C-Methacetin half-life time during long-term machine perfusion.

Figure 5

LiMAx value correlates to liver damage biomarkers. (A) Displays a significant correlation with lactate clearance. (B) Displays a significant correlation between LiMAx and ALT. (C) Expresses a significant correlation with AST.

Figure 6

Histologic evaluation of biopsies from machine perfused livers with the Suzuki score. In (A–C), liver biopsies are stained with H and E staining from grafts with low, moderate, and high Suzuki scores. The pictures are magnified 400 times and the high magnification image is magnified 1,200 times. Panel A illustrates liver H8, which has a Suzuki score of 0, indicating normal liver microscopy with no congestion, vacuolization, or necrosis. (B) Illustrated H9, which has a Suzuki score of 2; some necrotic cells are indicated by the black arrows in the high magnification image. Furthermore, minimal vacuolization was identified. (C) Presents H4, which has a Suzuki score of 5. This liver was deliberately stored on ice for over 60 h. A moderate amount of congestion was found, as seen in the high magnification window. Furthermore, minimal vacuolization and some single-cell necrosis was noticed. (D) Presents a significant correlation of the Suzuki score with LiMAx score.