This single-center, observational study was performed in accordance with the ethical standards laid down in the Declaration of Helsinki. Institutional ethics committee (Medical University of Vienna) approval for the study was obtained. Written informed consent was obtained before enrollment into the study.
Twenty-two adult patients scheduled for OLT at the General Hospital of Vienna between August 2014 and August 2015 were enrolled in the study. Exclusion criteria were combined liver-kidney or liver-lung transplantation, and the need of intraoperative veno-venous bypass10. To allow direct comparison between patients with ESLD and healthy individuals, blood samples were additionally obtained form twenty-two healthy age-matched adult volunteers.
Anesthesia, surgery, and immunosuppression
Anesthesia and surgery were performed according to the local standards, which have already been previously described by our research group10,11. The surgical technique was performed pursuant to the local standard technique with cross clamping of the inferior caval vein. All grafts were ABO compatible and matched for the graft-to-recipient-weight ratio. Immunosuppression was initiated already intraoperatively, before graft reperfusion, and continued according to the local standardized protocol for immunosuppression in OLT.
Data and sample collection
Demographic data, primary disease leading to ESLD, the model of end-stage liver disease (MELD) score, surgical reports, the anesthetic report, as well as relevant parameters from the postoperative course on the intensive care unit, including the use of blood and coagulation products were collected for each patient. Diagnosis of clinically significant portal hypertension was established in patients suffering from ESLD fulfilling one or more of the following: hepatic vein pressure gradient >10 mmHg, gastroenteral varices, liver stiffness measurement or imaging showing collateral circulation12,13.
Results from routinely daily measured laboratory parameters were extracted from the patient data management software (PDMS, Phillips, USA). The maximal norepinephrine infusion rate and the total amount of infused norepinephrine during OLT were recorded. In patients scheduled for OLT, blood samples were drawn from an arterial line, which is placed standardly during OLT. Arterial blood samples were collected at the following pre-defined time points (TP): at baseline (BL) prior to surgery, during OLT in the anhepatic phase immediately prior to reperfusion (TP1) and 10 minutes after graft – reperfusion (TP2). Control blood samples were obtained in the morning from the radial artery of fasting adult volunteers. Immediately after collection, plasma samples were centrifuged at 2600 g for 20 minutes and stored at −80 °C until analysis.
Histamine concentrations in plasma were measured using a commercially available enzyme-linked immunosorbent assay (ELISA) (Immunotech, Beckmann Coulter Company, Brea, CA) on a standard curve ranging from 1–90 nM.
Diamine oxidase concentrations were measured using a custom-assembled ELISA as described recently14 using recombinant human diamine oxidase as a standard15. Briefly, a purified monoclonal antibody raised against human DAO isolated from Caco-2 cell supernatant was coated at 5 µg/ml onto white high protein binding microtiter plates (Greiner bio-one 655074) in 50 mM carbonate-bicarbonate coating buffer pH 9.6 overnight at 4 °C. EDTA plasma was diluted 1 to 5 with LowCross-Buffer (CANDOR bioscience 1000500; Germany). Bound human DAO was detected with an anti-DAO polyclonal rabbit IgG serum fraction diluted 1 to 1000. The bound rabbit antibodies were detected with a 1 to 32000 dilution of donkey anti-rabbit IgG HRP-labelled antibodies (Sigma SAB3700928; Austria). Antibodies were incubated for 50 minutes at room temperature. Bound HRP molecules were quantified with the SuperSignal™ ELISA Pico Chemiluminescent Substrate (Thermo Scientific 37070; Vienna) using a standard chemiluminescent reader (Victor2TM 1420 Microtiter Plate Reader (Perkin Elmer). The limit of blank (LOB) and detection (LOD) are 0.27 and 0.48 ng/ml respectively using 42 standard curve determinations. The estimate limit of quantification (eLOQ) is 0.70 ng/ml. A value of 0.48 ng/mL was assigned for values below the detection limit for conservative statistical comparisons.
Mast cell degranulation triggered by anaphylactic reactions is a frequent cause for histamine release into patient’s systemic circulation16, but may also be associated with DAO release17. To exclude possible intraoperative mast cell degranulation as a potential cause for histamine release in our patients, total tryptase concentrations were measured in plasma of patients undergoing OLT (ImmunoCAP Tryptase ELISA Kit, Thermoscientific, Waltham, MA).
Diamine oxidase release is triggered by heparin18. To determine whether endogenous hepariniziation could explain a possible DAO release, we measured aPTT and anti-FXa activities in a subgroup of patients, and performed a thrombelastometric analysis calculating the difference between measurements with and without heparinase (INTEM – HEPTEM).
Statistical analyses were performed using Prism 6.0 (GraphPad Software, La Jolla, CA) and Statistica, (Stat Soft Inc., Tulsa, OK). Results are depicted as median with interquartile ranges (IQR, [25–75%], unless indicated otherwise. A Kolmogorov-Smirnov test was used to verify normal distribution. A Friedman analysis of variance (ANOVA), followed by post-hoc Wilcoxon tests, and U-tests were applied for comparison between groups. Correlations were calculated by the Spearman ranks correlation analysis. Receiver operating characteristic (ROC) curve was used to assess the diagnostic value of plasma histamine and DAO concentrations to discriminate the need for higher dosage of vasopressor support with norepinephrine (≥0.3 mcg/kg/min) during the anhepatic phase and reperfusion period. Statistical significance was set at p < 0.05.
Sample size calculation
Previous studies assessed plasma histamine concentrations in healthy volunteers19, and compared histamine concentrations between cirrhotic patients and healthy individuals20. Based on these data we estimated an intra- and inter-subject coefficient of variation of plasma histamine concentrations of approximately 60% and 90%, respectively. We calculated that 19 subjects in each group would be sufficient to allow detection of a 2-fold greater histamine concentration in patients undergoing OLT with 80% power and an alpha error of <5%. We felt that this effect size is clinically justified because plasma histamine concentrations greater than 2-fold the upper normal limit of 11 nM might be hemodynamically relevant as demonstrated by a 30% increase in the heart rate in healthy volunteers3.
Approval was obtained from the local ethics committee of the Medical University of Vienna. Patients informed consent was obtained before enrollment in the study.