Key words : Aspartate aminotransferase, Early liver function, Liver function capacity test

Introduction

Liver transplantation remains the only curative therapy for patients with end-stage liver disease. Nevertheless, the successful clinical development of liver transplantation has to contend with severe shortages of donor organs, which is also due to its increasing prevalence.¹ In addition to numerical restrictions, qualitative challenges remain for potential donor organs, which are increasingly affected by mild to moderate hepatic steatosis. In addition, demographic changes have led to a significant increase in the average age of donors and the associated comorbidities. In particular, lifestyle-associated nonal-coholic steatohepatitis has a high prevalence.² However, the condition of donor organs of borderline quality can be significantly improved if the moda-lities of organ preservation are changed or techniques are used that enable revitalization of the ischemic tissue. An increasingly accepted method to better maintain organ viability during the preservation phase is continuous hypothermic machine perfusion of the kidney or liver.

Various studies have shown that transplant recovery can be improved even after prolonged cold ischemic storage if a short period of hypothermic machine perfusion of the liver is performed before transplantation.^3,4 Such a procedure offers the attrac-tive possibility of reconditioning marginal organs in the patient’s own clinic by means of hypothermic machine perfusion before transplantation, even after a sometime unforeseen longer period of static preser-vation. In addition, information on the functional integrity of the organ could be obtained during treatment. For example, the behavior of vascular flow resistance or the determination of perfusate parameters could provide information about the success of the treatment.

Systematic, comparative studies of different modalities of short-term reconditioning before trans-plantation have shown that both constant hypothermic and constant subnormothermic or constant normot-hermic perfusion have advantages over an untreated control group,^5,6 but controlled oxygenated rewarming proved to be significantly superior to all other methods tested.^7,8 Initial testing of the feasibility of imple-menting oxygenated rewar-ming in everyday clinical practice showed that it could be used without complications and that the course of the respective patients was clinically unremarkable.⁹

On the basis of these previous results, a ran-domized controlled pilot study was undertaken with the aim of characterizing the effects of short-term, end-ischemic machine perfusion with slow rewar-ming of the organ on early reperfusion injury after liver transplantation for the first time in a clinical trial approach (ISRCTN 94691167).¹⁰ Machine perfusion can also be used to provide physiological or biochemical data to support the medical decision-making process to assess the transplantability of the organ. In particular, data on vascular conductivity (derived from perfusion pressure and flow values) could be considered, as well as perfusate parameters (eg, lactate), which can be determined promptly in the blood-gas analyzer.

Here, we analyzed various parameters during machine perfusion for their prognostic value in predicting actual liver integrity after transplantation.

Materials and Methods

In this study, we used data obtained in the course of a randomized controlled clinical trial on the role of controlled oxygenated rewarming as novel adjunct in liver transplantation (ISRCTN 94691167).¹⁰ The research ethics board of the University Duisburg-Essen approved this study. Written informed consent was obtained from each patient. All transplants were performed between April 2019 and April 2021 at the University Hospital of Essen, Germany.

The study included organ grafts from deceased, brain dead donors fulfilling the extended donor criteria according to the German medical association. Extended criteria included at least 1 of the following: donor age >65 years, intensive therapy including assisted ventilation of >7 days, obesity of donor (body mass index [calculated as weight in kilograms divided by height in meters squared] >30), serum sodium >165 mol/L, aspartate aminotransferase (AST) or alanine aminotransferase (ALT) >3× of normal, serum bilirubin >3 mg/dL, and histologically proven liver steatosis >40%. All patients listed for a liver transplant at the University Hospital Essen with a minimum age of 18 years and residency in Germany were eligible to participate in the study.

Data obtained during machine perfusion in the treatment arm were used to evaluate their respective correlations with graft integrity after transplantation. For this purpose, graft integrity was judged either by monitoring the peak serum activities of transa-minases (ALT or AST) as conventional markers of ischemic injury or by evaluation of early functional recovery as determined by maximum liver function capacity (LiMAx) test¹¹ on postoperative day 1.

After arrival in the hospital, livers included in the study were put on a machine (Liver Assist, Fa. Organ Assist) and subjected to a controlled oxygenated rewarming on the machine by dual perfusion via the hepatic artery and portal vein in a closed circuit. Temperature was kept hypothermic at 8 °C during the first period of perfusion and then gradually increased to 12, 16, and 20 °C after 30, 45, and 60 minutes, respectively.¹⁰

Perfusate samples were drawn after 90 minutes of machine perfusion, as steady-state conditions had been established for temperature and perfusion characteristics. Lactic acid and pH were determined on an acid base laboratory (Radiometer), and total perfusate flow was recorded by the perfusion machine via electromagnetic flow sensors. Flavin mono-nucleotide (FMN) level was determined with the use of a commercial enzyme-linked immunosorbent assay kit (MyBioSource) from perfusate samples, which were collected during the study and kept frozen at -80 °C until day of analyses.
Perfusion was continued until the recipient operation had advanced to the point that the liver could be implanted. At that time, the liver was taken out of the machine, reflushed via the portal vein, and transplanted with vena cava replacement and end-to-end-anastomosis of portal vein, hepatic artery, and bile duct. All patients were treated in the intensive care unit after transplantation. Postoperatively, tacrolimus (adjusted in accordance to the trough level of the drug) in combination with corticosteroids and mycophe-nolate mofetil were used. Donor risk index of liver grafts was calculated according to Braat and colleagues¹² with “race” being always set to White.

Objectives and endpoints
All patients were observed for 7 days posttransplant on a daily basis. Serum peak value of AST or ALT during the first 3 days after transplant were assessed enzymatically using absorption spectrometry for the kinetic reduction of NADH as indicator reaction at the laboratory center of the University Hospital. Functional recovery of livers was determined with the LiMAx test,¹³ which measured the metabolism of 13C-methacetin as dynamic functional parameter on postoperative day 1 while the patient was still intubated.

Statistical analyses
Sample size calculation was performed with the goal to detect significant correlations between machine data and posttransplant results with a power of 0.8 and a correlation coefficient of 0.6 or better considered as clinically relevant. A sample size of 19 was needed; thus, the inclusion of 20 patients was deemed adequate. GraphPad Prism version 8.0.0 for Windows (GraphPad Software; www.graphpad.com) was used for statistical analyses and calculation of Pearson correlation coefficient (r). P < .05 was considered statistically significant.

Results

Significant correlations with peak transaminase levels after transplant were found for perfusate levels of AST, pH, and lactate, with best correlations evident for lactate (r2 = 0.67) (Figure 1).

Combining 2 of the predictive biomarkers ob-tained during machine perfusion could further increase the degree of correlation with posttransplant peak levels of transaminases. Thus, an optimal coefficient of correlation could be disclosed for the product of serum concentrations of lactate and AST activity (r² = 0.86; P < .001).

Other parameters often used for liver evaluation before or during machine perfusion, such as cold ischemia time, donor risk index, perfusate flow, and hepatic release of FMN, did not disclose significant correlations with peak transaminase levels after transplantation (Figure 2). Of note, early liver function (LiMAx) after transplantation did not significantly correlate with any of the above parameters. The best prognostic factor was the perfusate activity of AST, which showed a correlation factor of r² = 0.11 (P = .10) ((Figure 3) and (Figure 4)).

Readout of morphological reperfusion injury, as represented by peak transaminase levels, did not at all correlate with readout of early hepatic functional recovery (LiMAx test values) (Figure 5). The cor-relation coefficient between the 2 parameters showed r² = 0.13 (not significant).

Discussion

Pushing the limits of acceptance criteria for donor organs can only be successful if evaluative tools are developed to support the surgeon to appropriately estimate the quality of organ grafts before transplantation.

In the present study, we analyzed data from the recently reported CORNET trial (ISRCTN 94691167) to approximate the value of several biomarkers measured during short-term machine perfusion before transplantation for prediction of hepatic outcome after liver engraftment. As previously shown, levels of transaminases, measured during hypothermic machine perfusion of liver grafts, are significantly correlated with peak activity of AST after transplantation.¹⁴

In our study, which used only 90 minutes of rewarming machine perfusion up to mid-thermia (eg, 20 °C), we observed significant correlations to peak transaminase levels after engraftment for perfusate activities of AST, lactate concentrations, and pH during pretransplant machine perfusion. The accuracy to predict peak transaminase levels after engraftment could even be increased to r² = 0.86 when the product of the 2 best corresponding parameters (ie, AST activity and lactic acid concentration) was plotted against the postoperative outcome.

Of note, a comparably predictive potential for conventional descriptive parameters like overall cold ischemic time or donor risk index according to Braat and colleagues was shown.¹² However, the focus of our investigation was whether peak transaminase levels after transplant would actually represent the most adequate endpoint to be predicted by prior machine perfusion parameters. The appropriate definition of liver graft function by clinical or biochemical parameters may indeed be seen as controversial.¹⁵ A more dynamic testing approach was shown by the metabolic demethylation of 13C-methacetin by the microsomal cytochrome P450 1A2 system of the hepatocytes.¹⁵

This LiMAx test has been reported to validly determine liver function capacity in liver surgery¹⁶ and to closely reflect different stages of chronic liver disease.¹⁷ Therefore, we sought to use this test as an alternative endpoint of graft recovery after liver transplant in conjunction with the predictive parameters obtained during pretransplant machine perfusion. We found that peak AST levels, cold ischemia time, and donor risk index did not exhibit any valuable correlation with the postoperative LiMAx value. Most surprisingly, however, the machine perfusion parameters also did not allow for prediction of LiMAx results after transplantation.

Because of the relatively limited number of patients in our study, we could not formally rule out the possibility of missing significant correlations (type 2 error); however, the observed correlations were so weak (below 0.2) that they fell short of being clinically relevant. This points to a possibly relevant difference between functional and biochemical parameters in the judgment of liver recovery, which is neatly reflected by the missing correlation between the 2 endpoints observed in our study. The release of transaminases may primarily reflect hepatic injury, whereas LiMAx evaluated residual metabolic function, disturbation of which was often unparalleled by morphological manifestations of cellular injury.^18,19

Conclusions

Posttransplant release of transaminases might not qualify as a unique criterion to reflect functional reco­very after transplantation, and biomarkers validated against this parameter should not be considered as sole argument for discard or acceptance of a graft.

Because functional integrity might not easily be prognosticated by predominantly nonfunctional injury parameters, further development and refine-ment of functional investigations of the organ during machine perfusion are needed. A first approach to do so has been reported by Schurink and associates. In a proof-of-concept study on human discard livers, the investigators showed that liver function can be quantified by using the LiMAx test already during normothermic machine perfusion.²⁰ Experimental data have shown that such kind of functional testing on the machine corresponded quite well to different degrees of liver alteration by warm ischemia.^21,22 However, to date, this approach has not yet been validated compared with posttransplant recovery data; such a study would seem highly warranted in light of our results.

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