Study design and occupational exposure
This study was approved by the Human Medical Research Ethics Review Board, Puren Hospital (prll2018001) prior to its initiation. All subjects who participated in this study provided written informed consent, and we adhered to the stringent ethical requirements for research on human subjects. All participants answered a standardized and detailed questionnaire that included demographic data, medical history, lifestyle and anesthetic exposure before each blood sample was collected. Pregnant women, individuals with any chronic infectious or inflammatory disease, and participants using illicit substances, medications, vitamins, and/or antioxidant supplements, and those who had recently been exposed to radiation (within six month), and those who had medical or family history of blood diseases were excluded from the study to avoid the effects of possible confounding factors. The exposed group consisted of 28 anesthetists mainly exposed to sevoflurane at least 24 months of exposure. The control group comprised 28 residents from internal medicine who were not exposed to waste anesthetic gases or other pollutants. The exposed group was age- and sex-matched with the unexposed group. The biological sampling was performed from 2019 to 2020.
Measurement of waste anesthetic gases in the breathing zone of medical residents during surgeries
There were 13 operation rooms which have an average area of 25 square meters without a scavenging system but with vertical laminar flow in Puren Hospital. All the medical residents from the exposed group worked in the 13 operating rooms in the hospital. All the WAGs were collected in the breathing zone of medical residents during surgeries under inhalation anesthesia in all the operating rooms. The collection time lasted for one hour. The measurements were performed by using a GilAir-5 sampler (Sensidyne, USA) and Agilent 7890BGas Chromatograph (Agilent Technologies, USA) according to the instruction of the manufacturer. The averages gas concentration of sevoflurane was calculated and shown in ppm. Measurement requirement was in accordance with National Institute Occupational Safety and Health (NIOSH) approved procedure[13].
Peripheral blood lymphocyte preparation
EDTA anticoagulated venous blood samples were collected from all participants. Peripheral blood lymphocytes were separated within subsequent 10-20 min with a standard method by centrifugation over lymphocyte separation medium at 400×g for 20 min (lymphoprepTM, STEMCELL technologies, Canada). The cells were washed with PBS (250×g, 10 min). Subsequently, 5×105 /ml. PBMCs suspended in the PBS were used in all the experiments.
Assessments of apoptosis
The percentages of viable (annexin-/propidium iodide-PI-) and early apoptotic (annexin+/PI−) cells were quantified by using annexin V-fluorescein isothiocyanate (FITC) staining, which was used to detect phosphatidylserine that is externalized in the early phases of apoptosis. Annexin V is an important marker of early apoptosis in which changes in externalized phosphatidylserine levels occur prior to DNA fragmentation[14] .We used the Annexin V-FITC Apoptosis Analysis Kit according to the manufacturer’s instructions (Tianjin Sungene Biotech Co., Ltd., China); 1×105 mononuclear cells were labeled, incubated in the dark for 15 min and immediately sorted by flowcytometry (BD FACSAriaTMш, USA). Marked annexin V-FITC staining (green) was analyzed using the FL-1channel, while PI staining (red) was analyzed using the FL-2 channel. The data were analyzed by using the flowjo software on a BD FACS Aria™ cytometer (BD BioSciences, USA).
Cell cycle analysis
Cell cycle was analyzed by flow cytometry according to a previous report[15]. Briefly, cells (1× 106) were harvested and washed with 10 ml PBS by centrifugation for 5 min at 300×g, and then re-suspended in 0.5 ml PBS. They were fixed by adding 4.5 ml pre-chilled 70% ethanol while vortexing. After incubation for 2 hours at 4°C, residual ethanol was eliminated by centrifugation for 5 min at 300×g. Supernatants were removed and discarded. Cells were washed with 5 ml FACS buffer twice by centrifugation for 5 min at 200×g. Cells were stained using 0.5 ml propidium iodide staining solution and kept in dark. Afterwards, they were incubated for 20 min at room temperature and the fluorescence analyzed using the ModFit LT software on BD FACSCalibur cytometry (BD, BioSciences USA).
Analysis of subpopulations of lymphocyte by Flow cytometry
Freshly collected EDTA-anticoagulated whole blood were processed within 2 h of collection by ten-color flow cytometry (BD FACSCantoTM, USA) to analyze the lymphocyte subsets as previously described[16, 17]. The lymphocyte subsets were identified using the following monoclonal antibodies: anti-CD3-APC-H7, anti-CD8-PerCP-cy5.5, anti-CD4-BV605, anti-CD25-PE, anti-CD56-BV510, anti-CD19-APC, and anti-ᵧᵟ-BV421 from BD Biosciences; anti-CD28-PE-CY7 and anti-CD127-FITC from Biolegend. The cell suspension was incubated at room temperature in the dark for 30 min. Red blood cells were removed using 500 μl of lysis buffer at room temperature in the dark for 10 min. Finally, the cells were analyzed by using FACS Canto flow cytometry and flowjo software (BD BioSciences, USA).
Immunoglobulin quantification by immunoturbidimetry
The blood of the elbow vein was collected into coagulation-promoting blood vessel, and the content of immunoglobulins (IgA, IgG, IgM) in the serum was detected by Immunoturbidimetric Assay according to the instructions of immunoglobulin assay kit (Shanghai Fosun Long March Medical Science co., LTD) on an automatic biochemical analyzer (beckmancoulterAU5800, USA)
Statistical analysis
Data were analyzed using an SPSS software version 25. The sample size for this study was calculated based on a pilot study, with a test power of 80% and level of 5% of significance (mean expected differences of 7.02 and 7.85 standard deviations between two groups), and was determined to be 52 subjects. By adding a 10% sample shedding rate, finally, the sample size was 56. Independent student’s t-test was used for comparing the mean of quantitative variables and chi-square for comparing the mean of qualitative variables. P-Values less than 0.05 were considered significant.