2.1 Ethical statement
The study was performed according to the declaration of Helsinki and approved by the Ethics Committee of the Nanjing Brain Hospital Institutional Review Board, Nanjing Medical University (Nanjing, China). All participants received information on the study and provided written informed consent to participate.
2.2 High risk group of stroke individuals
Screening records between 2012 and 2016 were provided by CNSSPP, a nationally ongoing community-based study, which was conducted by the National Project Office of Stroke Prevention and Control. Screenings were performed throughout the city of Nanjing at 21 communities.
Individuals were selected cluster sampling. They should meet these criterions: Age≥40 years old, living in the community above 9 month per year, and above 85% population were included.
According to the standard which is defined by The CNSSPP Committee, high risk group of stroke were interpreted at least 40 years of age and had at least three of the following risk factors: hypertension, atrial fibrillation, smoking, dyslipidemia, diabetes mellitus, physical inactivity, overweight (BMI ≥26 kg/m2), and family history of stroke. People who has the history of stroke or transient ischemic attack also equivalent to high-risk population management.
Hypertension was defined as systolic blood pressure ≥140 mm Hg, diastolic blood pressure ≥90 mmHg, self-reported hypertension diagnosed by a physician, or use of antihypertensive medications. Atrial fibrillation was defined as ECG examination indicates atrial fibrillation, self-reported diagnosis of atrial fibrillation, or use of anticoagulant medications. Smoking status was classified as smoking(current smoking or had a history of smoke for more than one year) and never smoking(never smoking or had a history of smoke for less than one year). Dyslipidemia was defined as having one or more of the following results: triglyceride ≥2.26 mmol/L, total cholesterol ≥6.22 mmol/L, high-density lipoprotein cholesterol <1.04 mmol/L, lowdensity lipoprotein cholesterol ≥4.14 mmol/L, self-reported diagnosis of dyslipidemia, or taking cholesterol-lowering medications. Diabetes mellitus was defined as fasting plasma glucose ≥7.0 mmol/L, self-reported diagnosis of diabetes mellitus, or use of oral hypoglycemic agents or insulin injection. Physical activity was defined as regular physical exercise >3 times/week for at least 30 minutes per session. Body mass index was calculated as body weight (in kg) divided by the square of height (in m; kg/m2). Overweight was defined as body mass index ≥26kg/m2, according to the guidelines of the Working Group on Obesity in China. A family history of stroke was defined as the occurrence of stroke in a participant’s parents, brothers, or sisters.
Before carotid duplex scans, candidates were asked to complete a standardized CNSSPP questionnaires, providing demographic information, lifestyle risk factors, medical history, and a family history of stroke, which were collected through face-to-face interviews by trained staff. Individuals fasting blood were obtained to test for fasting plasma glucose (FPG), homocysteine (Hcy), Total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), Hemoglobin A1C (HbA1C), triglyceride (TG).
2.3 Blood test
Venous blood (5mL) was collected from all patients before breakfast. Serum samples were obtained by centrifugation for 10 min at 3000g, placed in freezing tubes, and preserved at –80°C until subsequent analysis. Serum FPG, HCY and blood lipids levels were determined using OLYMPUS AU5400 (OLYMPUS, Japan). EDTA anticoagulated whole blood samples (2ml) from the same patients were used to measure HbA1c expression by TOSOH G8 (TOSOH, Japan).
2.4 Carotid artery ultrasound screening protocol
According to Chinese stroke vascular ultrasound examination guidelines, the duplex scan consisted of ultrasound imaging of the distal common carotid artery, bulb, and proximal internal and external carotid arteries, with evaluation of a Doppler signal for 3 to 5 beats in each location on both sides. Plaque was interpreted as greater than 1.5mm of IMT based on Doppler-derived. Color Doppler ultrasonography of the carotid artery indicates plaque with hypoechoic, mixed echoes, or plaque indicating ulceration, defined as instability plaque. Carotid duplex examinations were performed by four experienced registered vascular technicians in Nanjing Brain Hospital,which is a Stroke Screening and Training Base Hospital. All of the vascular technicians were independent of other clinical information. Screening work has to comply with the criteria established by CNSSPP Committee.
2.5 Predictive model evaluation
Continuous variables were presented as mean (Standard deviation, SD), and categorical variables were presented as percentages. The whole sample was randomly divided into a model derivation set and a model validation set, which consisted of approximately two-thirds and one-third of the sample, respectively. A comparison was performed between the two groups with t-test of derivation set for continuous variables and with χ2 tests for categorical variables. Univariate logistic regression was taken for each risk factors. When the value of P is less than 0.1, the variable was entered into the multivariable logistic regression model (Stepwise forward). Those variables which P less than 0.05 were kept in. According to the previous study, we also created a scoring system based on the regression coefficients. The lowest coefficient in absolute value was used as denominator. The coefficient of each independence risk factor was divided by the absolute value of the lowest coefficient and then rounded to the nearest integer. The sum of the scores from each individuals were taken into receiver operator characteristic (ROC) curve, generated to determine the prediction power of carotid instability plaque, and the best cutoff scores by Youden index.
We used validation set to evaluate the ability of the classification scheme to discriminate between persons with and without carotid instability plaque, which was also assessed using a ROC curve with SYSSTAT (SPSS Inc, Chicago, IL). A ROC curve plots the true-positive rate (test sensitivity) for a given threshold on the y-axis and the corresponding false-positive rate (one-test specificity) on the x-axis. The area under the resulting fitted curve represents the discriminating ability of that particular screening method and is assumed to be normally distributed. An area of 50% represents a non-discriminant screening test in which the true-positive rate equals the false-positive rate. The area under the ROC curve for excellent test and poor test approximates 100% and 50%, respectively.
All statistical analyses were performed using the SPSS version20.0 software for Windows (SPSS, Inc, Chicago, IL, USA). In all Statistical analyses, a P value <0.05 was considered statistically significant.