The current prospective study was approved by the Ethics Committee of the Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, Zhejiang, China, and the protocol was registered in the ClinicalTrials.gov Registry (NCT03916120). Besides, the study adheres to STROBE guidelines, checklist of items that should be included in reports of observational studies. All subjects signed informed consent documents prior to enrollment.
The study subjects were recruited from consecutive patients undergoing selective lung section with single-port VATS performed by one attending surgeon at the Second Affiliated Hospital of Zhejiang University School of Medicine between July 2018 and January 2019. The detailed surgical procedure was previously described. The criteria for inclusion in the study were age from 18 to 70, ASA classification Ⅰ to Ⅲ, and voluntarily received patient-controlled intravenous (PCIA) treatment. The exclusion criteria included the following: (1) history of mental illness, chronic pain, and alcohol or drug abuse; (2) remarkably abnormal liver and/or kidney function (more than two times of the normal); (3) allergy to related opioid drugs; (4) women during pregnancy or lactation.
All patients received general anesthesia under standard protocol. Specifically, general anesthesia was induced with midazolam (0.2 mg*kg-1), sufentanil (10 μg*kg-1), and etomidate (0.3 mg*kg-1). Cisatracurium besilate (0.15 mg*kg-1) was administered to induce a neuromuscular blockade for tracheal intubation. Anesthesia was continuously maintained with sevoflurane, propofol, and remifentanil. Cisatracurium was bloused as needed. During the surgery, standardized monitoring and bispectral index were applied. Central venous catheterization (CVC) and A-line were implemented for each patient. Before closure of the thoracic incision, surgeons performed a three-site intercostal nerve block with 0.75% 10 mL ropivacaine under thoracoscope. At the end of surgery, pentazocine 5 mg and tropisetron 5 mg were administered by the anesthetist. Immediately after surgery, PCIA was connected to the CVC. Then, patients were transferred to postanesthesia care unit (PACU) for recovery where their vital signs were continuously monitored.
Postoperative pain management
Each subject was extubated at PACU when vital signs stabilized. Patients were asked every 10-15 min after they were awake enough whether they needed pain medication until they became conscious enough to use the PCIA. If the patients felt moderate or severe pain (visual analog scale [VAS] 40–100, 0=no pain to 100=intense pain), they were given 40 mg dynastat until their VAS was ≤ 30. Patients were excluded if they received dynastat as rescue analgesia at PACU. PCIA was administered with a bolus doses of 0.002mg/kg hydromorphone permitted every 8 minutes. In case of PCIA analgesic inadequate (VAS ≥ 40), dynastat 40 mg would be administered as an alternative rescue modality. Tropisetron 5 mg or palonosetron 0.25 mg could be administered to combat postoperative nausea and vomiting.
Data collection and follow-up
During the preoperative interview, demographic characteristics, educational background, work type, and history of cigarette smoking and alcohol consumption were recorded. Besides, the general sleep quality within one month was recorded by a scale with three levels (poor, fair, and good). At the same time, patients were instructed on how to use the VAS to describe the pain they were experiencing, and how to use the PCIA device to control the pain when necessary. After surgery, the intraoperative parameters including surgery type and duration, anesthesia duration, lymphadenectomy, adhesion loosening, and pathologic diagnosis were also recorded.
During the follow-up period, VAS at rest and during coughing was recorded on the first morning (8:00 a.m.) after surgery. In the meantime, the use of rescue analgesia, postoperative sleep quality, and the degree of satisfaction (bad, fair, good, and excellent) to the pain management were recorded.
The primary outcome was the occurrence of postoperative inadequate analgesia. Once patient experience at least one of the following situations during the first night and morning after surgery: require extra analgesic drug; report moderate-to-severe pain (VAS ≥ 4) at rest; report poor sleep quality; report bad satisfaction with pain control, they were defined as postoperative inadequate analgesia.
Blood samples were collected in tubes containing ethylenediaminetetraacetic acid 1 hour after CVC was implemented and were then stored at −80°C. Genomic DNA was extracted from whole blood for genetic analysis by using Blood Genomic DNA Mini Kit (Biomed Corporation, China) according to the manufacturer’s recommendations. DNA samples were then stored at −20°C. SNPs were genotyped using a KASP™ genotyping assay (Rui Biotechnology, Beijing, China) as previously described.[29, 30]
Quality control was performed to ensure the robust genetic association: SNPs with call rates of < 95%, Minor Allele Frequency (MAF) < 0.05, or Hardy-Weinberg equilibrium (HWE) of p < 0.05 were excluded. Linkage disequilibrium (LD) was calculated from the patients’ genotypes. When strong LD (r2 > 0.9) was present in one gene, we only included one SNP from each pairs of SNP in the association study. Finally, there were 28 SNPs among the 17 candidate genes passed all quality control filters. (See Table 1)
Statistical analysis was completed with the SPSS 24.0 (SPSS Inc., Chicago, IL). Continuous variables were expressed as means and standard deviations (SDs) or as medians and interquartile range, and categorical variables as counts and percentages. Differences between two groups were evaluated by Student’s t-test or the Mann-Whitney test for continuous variables, and Chi-squared test or Fisher’s exact test for categorical variables. For analyzing the association between SNPs and inadequate analgesia, odds ratios (ORs) and 95% confidence intervals (CI) were calculated by logistic regression analysis adjusted for potential risk factors. Four genetic models (co-dominant, dominant, recessive and overdominant) were evaluated for association of polymorphisms with risk of inadequate analgesia. HWE was assessed by SNPStats software. The linkage disequilibrium and pairwise LD coefficients were implemented with Haploview 4.2 (Daly Lab: Cambridge, MA, USA, 2008). P value < 0.05 was considered significant.
Power analysis was done using QUANTO (University of Southern California, Los Angeles, CA). For the analyses of associations with postoperative inadequate analgesia, with the sample size of 198 and a modest Type I error rate of 5%, the analysis had more than 90% power to detect an OR of 2.15 for SNPs with an MAF ≥ 0.11 under dominant model and more than 99% power to detect an OR of 0.41 for SNPs with an MAF ≥ 0.26 under recessive model.