Immediate outcome prognostic value of plasma factors in patients with acute ischemic stroke after intravenous thrombolytic treatment

Patients with AIS admitted to the Department of Neurology at the People's Hospital of Guangxi Zhuang Autonomous Region for IVT between January 2020 and June 2021 were prospectively entered into the study. The study was performed according to the World Medical Association (WMA) Declaration of Helsinki and approved by the Ethics Committee of Guangxi Zhuang Autonomous Region People's Hospital. Given its observational and anonymous nature, the need for patient consent to participate in this study was waived.

The inclusion criteria were as follows: (1) AIS patients who underwent IVT; (2) age was ≥ 18 years; (3) the initial modified Rankin Score (mRS) on admissions ≤ 2.

The exclusion criteria were as follows: (1) unfavorable outcomes occurred before sampling; (2) the patient exhibited significant pre-stroke disability (pre-stroke mRS ≥ 2); (3) evidence of intracranial hemorrhage, subarachnoid hemorrhage, arteriovenous malformation, aneurysm, or intracranial tumor verified by CT/MRI on admission; (4) the presence of pre-existing neurological or psychiatric disease that would confound the neurological evaluation; (5) active or recent hemorrhage and a history of trauma or surgery within two months before stroke onset; (6) concurrent infection at the time of sample collection; (7) the presence of rheumatoid immune diseases, severe liver or kidney disease, hematopathy, or malignant tumors; (8) suspicion of the presence of an infectious embolus or bacterial endocarditis; (9) a baseline platelet count < 50,000/μL; (10) the patient was pregnant or lactating; (11) missing clinical, imaging, or follow-up data or information; and (12) blood samples were of poor quality.

Ninety-five consecutive patients were included in the study. Of the 95 patients, 15 patients were excluded due to the presence of infection on admission, 2 patients had malignant tumors, 1 patient had a rheumatic immune disease,2 patients had incomplete clinical data and missing follow-up data, and 1 patients’ blood samples were of low quality. The remaining eligible patients (n = 74) were divided into two groups based on their NIHSS scores. One group consisted of patients with a NIHSS score greater than 5 (NIHSS > 5) (n = 36), and the other group of patients had a NIHSS score equal to or less than 5 (NIHSS ≤ 5) (n = 38).

IVT was carried out according to international guidelines. 0.9 mg alteplase/kg body weight was administered with a maximum dose of 90 mg. The drug was administered as an infusion of a 10% bolus dose within 1 min, followed by an infusion of a 90% dose within 60 min. The NIHSS score was assessed at admission and at 24 h and one week after stroke onset to determine neurological function. Baseline demographic information and vascular risk factors were collected from the patients, including baseline stroke severity (NIHSS score at admission), pre-stroke mRS, cerebrovascular risk factors (age, sex, hypertension, diabetes, hyperlipidemia, atrial fibrillation, coronary artery disease, atherosclerosis, as well as smoking and alcohol consumption status). Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured three times within 24 h of admission, and the mean systolic and mean diastolic blood pressures were calculated. Biochemical parameters, including liver function, coagulation, uric acid, creatinine, and other laboratory tests, including blood cell count, were obtained before or 24 h after thrombolytic therapy. Computed tomography, magnetic resonance and digital subtraction angiography, electrocardiography, and carotid ultrasound were used to determine the stroke etiology. The clinical outcome at 90 days after AIS was assessed using the mRS. An mRS ≥ 2 or death was defined as a poor prognosis [8, 9]. The data were obtained using a telephone follow-up interview by a physician who was unaware of the patients’ clinical information and factor levels.

Blood samples were collected before stroke onset (t0), 24 h after thrombolytic therapy (t1), and 72 h after thrombolytic therapy (t2). The samples were immediately centrifuged at 3,000 g for 15 min (Thermo Scientific Haraeus Multifuge 3SR plus centrifuge, US) and stored at -80 °C until analyzed.

The levels of IL-6, MMP-9, TNC, and ADAMTS13 were determined using a Luminex R&D System kit (Labex Bio, Shanghai, China), following the manufacturer’s instructions. This method utilized different antibody factors covalently cross-linked to specifically coded microspheres. Each coded microsphere was dyed with a different fluorochrome in different ratios, resulting in a corresponding fluorescent code and assay. The coefficients of variation (CVs) were 0.56% for IL-6, 0.58% for MMP-9, 0.44% for TNC, and 0.3% for ADAMTS13.

The TRX and GSN antibodies in serum were determined using a quantitative sandwich enzyme immunoassay (ELISA), following the manufacturer's instructions (Service Bio, Wuhan, China). The CVs for the TRX inter-assay and intra-assay ranged from 8 to 10%, respectively. The lower detection limit was 1.172 ng/mL, and the line range was 4.688 ng/ml to 300 ng/ml. The CVs for the GSN inter-assay and intra-assay ranged from 8 to 10%, respectively, and exhibited a range of 3.12 ng/ml to 200 ng/ml with a lower detection limit of 0.78 ng/ml.

Statistical analysis of the data was performed using SPSS version 24.0 and Graphpad prism 8.0.2. Continuous variables that conformed to a normal distribution were expressed as means ± standard deviation. Other continuous variables that did not exhibit a normal distribution were expressed as medians and interquartile ranges (25% to 75%). Categorical variables were expressed as composition ratios. The plasma markers were labeled as continuous variables, log-transformed, and tested using repeated-measures ANOVA. The Pearson's chi-square test was used for categorical variables to test for differences in baseline characteristics. We also used binary logistic regression analysis to detect risk factors affecting the severity of illness and adverse prognosis. After correcting for potential confounders, including age and gender, independent influences associated with the prognosis of patients who underwent intravenous thrombolysis for AIS were screened using univariate and multivariate logistic regression analyses. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated for each endpoint. Subject (receiver) operating characteristic (ROC) curves were used to assess the plasma markers (IL-6, MMP-9, ADAMTS13, TNC, GSN, and TRX) and determine the optimal cut-off point, sensitivity, and calculated specificity.

Ninety-five patients were recruited, including 43 men and 52 women. Only 74 patients (43 males and 31 females) with adequate admission data were included in the study. The patients had a median NIHSS score on admission of 5.5(IQR 4 to 13). Patients were categorized into two subgroups according to their NIHSS scores. One group included patients with a NIHSS score high than 5 (NIHSS > 5; IQR 7.5 to 17.5, median: 13; n = 36). The other group included patients with a NIHSS score equal to or less than 5 (NIHSS ≤ 5; IQR 3 to 5, median: 4; n = 38) (Table 1).

The mean age of the low NIHSS score group was significantly higher than that of the high NIHSS score group (P = 0.022). The median NIHSS score 24 h after admission was 4 (IQR 2 to 8.5). Patients in the high NIHSS score group (IQR 3 to 17, median: 5) had a significantly higher NIHSS score 24 h after admission than those in the low NIHSS score group (IQR 2 to 4.5, median: 3) (P = 0.006). The median mRS at 90 days was 2.5(IQR 2 to 4), and the mRS at 90 days was significantly higher in the high NIHSS score group (IQR 2 to 4, median: 3) than in the low NIHSS score group (IQR 1 to 3, median: 2) (P = 0.008). There were no significant differences in hypertension, diabetes, hyperlipidemia, atrial fibrillation, coronary heart disease, arteriosclerosis, mean systolic and diastolic blood pressure, smoking, and drinking between the two groups.