Trial design and patient enrollment
This prospective, double-blind, randomized intervention trial was approved by the Ethical Clearance Committee on Human Rights Related to Research Involving Human Subjects, Faculty of Medicine Ramathibodi Hospital, Mahidol University (protocol ID 045823) and the protocol was registered to ClinicalTrials.gov (identifier NCT02559297) on September 22, 2015. Patients aged ³18 years old who received their first living donor kidney transplantation at Ramathibodi Hospital were included in the study. Patients were excluded for hyperacute graft rejection, currently on immunosuppressive drugs due to underlying diseases, receiving blood products during 24-h perioperative period, or patient refusal to participate in the study at any time point. Informed consent was obtained from all subjects. No interim analysis was performed during the trial. This study followed the CONSORT reporting guideline.27
Randomization was generated in a 1:1 allocation with a block size of 8 and the random number was put in a sealed envelope. Patients were randomly assigned to either desflurane or sevoflurane intervention by drawing a sealed envelope. Randomization took place on the day of surgery just prior to initiation of anesthesia.
Subjects and outcome assessors (including laboratory technicians and all investigators except the designated research coordinator) were blinded to group allocation throughout surgery, laboratory investigation and data collection. Blinding was uncovered at the time of data analysis.
Patients were randomly assigned to receive desflurane or sevoflurane for the maintenance phase of anesthesia. In addition to the randomized inhalation agents, patients received the same regimen of 1-2 mg of midazolam for premedication and intravenous anesthetic agents including 1-2 mcg kg-1 of fentanyl, 1-2 mg kg -1 of propofol and 0.5-0.6 mg kg -1 of atracurium for induction of anesthesia and intubation. A balance anesthesia technique was used for maintenance phase. The inhalation agent (sevoflurane or desflurane) was used in conjunction with 50% nitrous oxide in oxygen. Ventilation was adjusted to maintain normocarbia. End-tidal anesthetic gas monitoring was used to ensure 1.0 - 1.5 minimum alveolar concentration (MAC) of the inhalation agent during maintenance phase in both groups.
During anesthesia, blood pressure, heart rate, oxygen saturation, ETCO2, and temperature were monitored and recorded. Blood pressure was maintained within 20% of baseline values. Hypotension was managed by intravenous fluid and ephedrine IV bolus as needed. Total doses of intravenous medications were recorded. All patients in both interventions were transferred to the kidney transplant unit for postoperative care.
Blood sample collection
Venipuncture was performed at three time-points; pre-exposure (0-h) and post-exposure (2-h, and 24-h) to inhalation agents. Two tubes of 0.5-ml EDTA blood were collected at each time point, one for Treg enumeration and the other for cytokine measurement.
The primary outcome was the absolute change in number of peripheral blood CD4+CD25+FoxP3+Tregs, which was measured by flow cytometry and expressed as the percentage of the total population of CD4+ T lymphocytes at pre-exposure (0-h) and post-exposure (2-h and 24-h) to anesthetic gas. A secondary outcome was the plasma level of anti-inflammatory cytokine IL-10 (the major cytokine produced by Tregs), TGF-β1 (anti-inflammatory cytokines produced by many types of cells and required for Tregs differentiation), and pro-inflammatory cytokines produced by T helper (Th) 1/Th2, i.e., GM-CSF, IFN-ɣ, IL-2, IL-4, IL-5, IL-12, IL-13 and TNF-a, which measured by multiplex immunoassay. All measurements were performed in triplicate.
Treg enumeration by flow cytometry
Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation. Approximately 5x105 cells were suspended in 20 µL phosphate buffer saline (PBS) in the presence of cell surface marker antibodies (APC-CD4, PE-Cy7-CD25) (#MHCD0404, #25-0259-41; ThermoFisher, Florence, KY), mixed well and incubated at room temperature for 15 minutes. Thereafter, cells were permeabilized and intracellularly stained using FoxP3-FITC antibody (#11-4776-42; ThermoFisher). Flow cytometry (BD FACSVerse with BD FACSuite software; BD Bioscience, San Jose, CA) was used to measure the number of Tregs, expressed as a percentage of CD4+CD25+FoxP3+ T cells among the CD4+ cell population. The estimated number of CD4+cells and Treg were calculated by determining the ratio of CD4+cell count and CD4+CD25+FoxP3+cell count, respectively, to the total count in the flow cytometry, and then multiplied by the number of white blood cells measured from the complete blood count (CBC) which ordered at the pre-operative and post-operative evaluations.
Cytokine measurement by multiplex immunoassay
Multiplex cytokine immunoassay was performed by BioPlex-200 system (Bio-Rad, Hercules, CA). GM-CSF, IFN-ɣ, IL-2, IL-4, IL-5, IL-10, IL-12, IL13, and TNF-a were detected by BioPlex Pro human cytokine Th1/Th2 assay (Bio-Rad), and TGF-β1 was measured by single-plex custom assay (Bio-Rad) as the manufacturer’s instruction.
Sample size estimation and statistical analysis
There was no data related to sevoflurane and desflurane anesthesia on Treg immunomodulation available at the initiation of the study. Nevertheless, Pirbudak Cocelli L et al.,21 showed that sevoflurane and desflurane anesthesia caused a significant difference in total lymphocyte count at 2-h post-induction in patients undergoing abdominal surgery. Since Treg is a subset of lymphocytes, our study then adopted mean difference and standard deviation to calculate the effect size. The nQuery Advisor program was applied for sample size calculation. Accordingly, at least 40 patients (20 patients per group) were required to determine statistically significant mean difference between groups (the effect size of 0.915, alpha = 0.05 and power = 80%).
Statistical analysis was performed by Excel and R packages. Data were reported in number, percentage, mean±SD (or SEM) or median [IQR] as appropriate. Parametric and non-parametric tests were used, as appropriate, to determine difference between groups. ANOVA with Tukey post-hoc test was performed for multiple comparison. P-value <0.05 was considered to be statistically significant.