Study Population and Procedure
From August 2018 to July 2019, patients receiving intravenous thrombolysis within 4.5 hours after symptoms onset were consecutively screened to collect blood samples prior to thrombolysis from five pre-set stroke centers in the INTRECIS study. INTRECIS is a nationwide, multicenter, prospective, and registry study of consecutive adult patients who were eligible for treatment with intravenous thrombolysis within 4.5 hours of onset of symptoms. Details of the study design and results of the primary outcomes have been reported recently.14 Briefly, the consecutive patients who received standard dose of alteplase (0.9 mg/kg, maximum 90 mg; manufacturer: Boehringer Ingelheim) within 4.5 hours after symptoms onset were enrolled. The exclusion criteria were as follows: (1) patients received urokinase or non-standard dose of alteplase, (2) patients received endovascular intervention, (3) patients lacked complete clinical data, (4) blood samples were not collected prior to intravenous thrombolysis. All the patients and/or their legally gave written informed consent for data collection. According to the occurrence of ENI, enrolled patients were divided into two groups: (1) ENI group: patients occurred ENI; (2) Non ENI group: patients did not occur ENI. Furthermore, propensity score matching was performed between groups with the ratio 1:1, caliper of 0.1, and a nearest-neighbor matching strategy, and operated with control factors including age, gender, current smoking, alcohol consumption, systolic blood pressure, diastolic blood pressure, blood glucose, symptom onset-to-treatment time, National Institutes of Health Stroke Scale (NIHSS) score at admission, the Trial of Org 10172 in Acute Stroke Treatment (TOAST) classification,15 previous use of antiplatelet, and medical history.
The baseline characteristics and clinical data of recruited patients were obtained from electronic database: age, gender, current smoking, alcohol consumption, systolic blood pressure, diastolic blood pressure, blood glucose, symptom onset-to-thrombolytic time, NIHSS scores, TOAST classification, previous use of antiplatelet, history of stroke, hypertension, diabetes mellitus, atrial fibrillation, and congestive heart failure. ENI was defined as a decrease of ≥4 on the NIHSS scores within 24 hours after thrombolysis from baseline in the present study.2
The study was centrally approved by the Institution Human Research Ethics Committees of General Hospital of Northern Theater Command and performed in accordance with the Declaration of Helsinki.
Blood Sampling and Biomarkers Measurements
About four milliliters of peripheral venous blood samples were collected from each patient just prior to intravenous thrombolysis. The blood samples were centrifuged at 1000 × g for 10 minutes at 4℃, and then transferred into an 1.8 milliliter cryotube and stored at -80℃ until measurement.
According to the manufacturer’s instructions, pre-customized protein microarray analysis (Raybiotech Inc) was used to simultaneously detect and quantify 49 biomarkers in the collected blood samples, which were preset based on published data. Identified biomarkers were defined as those variations with P < 0.05, and fold change > 1.20 or < 0.83. Functional enrichment analysis and protein-protein interactions network were performed to explore the possible mechanism between identified biomarkers
Descriptive statistics was performed to compare variables between two groups. Continuous variables with normal distribution were described as means and standard deviation. Continuous variables included age, systolic blood pressure, diastolic blood pressure, blood glucose, symptom onset to thrombolysis time, NIHSS score, and detected serum concentration of biomarkers. The t tests were used to analyze the normally distributed continuous variables. Categorical variables were described as number and proportions. Categorical variables included gender, current smoking, alcohol consumption, medical history, previous use of antiplatelet, and TOAST classification. The Pearson χ2 tests were used to analyze the categorical variables.
The p value is obtained from the moderated t-statistic with false discovery rate of adjustment for multiple testing. In all analysis, differences were considered statistically significant with a p < 0.05. The free statistical language R (version 3.10.3) was used for the outcomes and graph in the propensity score matching and microarray analysis.