Consecutive patients with AIS were screened and selected from our Acute Ischemic Stroke Registry Program in Shanghai Fifth People’s Hospital (The Fifth People’s Hospital of Shanghai, Fudan university) between August 1, 2017 and September 1, 2019. All included patients were followed up in outpatients to get their mRS scores at 3 months except those who died or reoccurred in this period. Patients were included if they met the following criteria: (1) diagnosed with AIS within 24 hours of onset; (2) aged 18 years or older; and (3) completed a head magnetic resonance imaging (MRI) in 48 hours. AIS was diagnosed if there were new focal neurological deficits explained by relevant lesions detected on head diffusion-weighted imaging (DWI). Patients were excluded if they met the following criteria: (1) patients with previous AIS or current cerebral hemorrhage; (2) pregnant patients; (3) severe heart (with cardiac function in grade Ⅲ or Ⅳ according to New York Heart Association or left ventricular ejection fraction < 40% in echocardiography), lung (with blood oxygen saturation less than 95% and symptoms of shortness of breath, cyanosis, and abnormal blood gas analysis), liver (serum alanine aminotransferase levels > 10-fold upper limit of reference interval), kidney (serum creatinine > 443 µmol/L) and neoplastic diseases; (4) autoimmune diseases; (5) infection symptoms or signs was present at the time of stroke onset. According to the relevant guidelines, if the patients in the cohort meet the criteria of thrombolysis, they will be treated by intravenous alteplase (the dosage = 0. 9 mg/kg × patient’s body weight (kg); 10% of the dosage was injected intravenously within the first minute and 90% dripped intravenously within 1 hour). The inclusion criteria of thrombolysis are as follows: (1) aged within 18 ~ 80; (2) onset of neurological impairment within 4.5 h, head CT or MRI examination excluded bleeding lesions; (3) systolic blood pressure < 180 mmHg or diastolic blood pressure < 110 mmHg before treatment; (4) no hemorrhagic disease and bleeding constitution; (5) patients and their families agree to treatment and sign informed consent. AIS patients with uncertain onset time, suspected subarachnoid hemorrhage, seizures, intracranial aneurysms or arteriovenous malformations or heart, lung, liver, renal insufficiency, or active internal bleeding or arterial puncture treatment within 1 week, surgery within 2 weeks gastrointestinal or systemic hemorrhage within 3 weeks, urological trauma within 3 months or death, or blood glucose < 2.7 mmol/L or > 22.2 mmol/L were excluded for thrombolysis. Figure 1 showed the flow diagram of selecting patients. Written informed consent was obtained from all patients. This study was approved by the Ethical Review Board of Shanghai Fifth People’s Hospital. Baseline data were collected from medical records including age, sex, and history of hypertension, diabetes mellitus (DM), atrial fibrillation (AF), dyslipidemia, NIHSS scores, door-to-needle time (DNT), stroke onset-to-needle time (ONT) (stroke onset-to-second blood sampling time), systolic blood pressure (SBP), history of cigarette smoking, and alcohol drinking.
All blood samples were collected via vacuum tubes, stored at 4 ℃ and tested by clinical laboratory technicians in hospital certificated laboratory within 2 h after collection. White blood count (WBC), neutrophil proportion (N), lymphocyte proportion (L), platelet count (P), serum total bilirubin, serum direct bilirubin, serum glucose, homocysteine (HCY), triglyceride (TG), total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL) concentrations were measured. Neutrophil-lymphocyte ratio (NLR) was calculated as the ratio of neutrophil counts to lymphocyte counts (N/L). Some laboratory indexes, such as NLR, were collected and calculated after thrombolysis in included patients. Only in patients with thrombolysis the blood samples before thrombolysis medication were collected within 1 h at emergency department. The collection time of blood samples, operated by trained nurses, for patients without thrombolysis was the same as that after thrombolysis (6: 00–7:00 on the second day of admission).
Each enrolled patient underwent a head 3T MRI (Siemens, Forchheim, Germany) within 24 hours after AIS onset. AIS patients were divided into 4 subgroups by Trial of Org 10172 in Acute Stroke Treatment (TOAST)(15): large artery atherosclerosis (LAA), cardio-embolism (CE), small artery occlusion (SAO), and others (including others subtypes). Severity was quantified by admission NIHSS scores and 3-month outcome was quantified by 3-month mRS scores into 2 levels: favorable outcome (mRS < 3), and unfavorable outcome (mRS > = 3). Minor stroke was defined as NIHSS score < = 5.
According to inclusion criteria, we selected 450 individuals from the large cohort for preliminary inclusion review, excluding 57 people according to the exclusion criteria, see Fig. 1. Only 2 persons (a male and a female) was lost to follow-up in thrombolysis group, we did not get their mRS.
SPSS 26.0 (IBM Corp., Armonk, NY) and GraphPad Prism 8 (GraphPad Software Inc., La Jolla, CA) were used to perform statistical analysis. Categorical variables were presented as frequency and percentage, and Pearson Chi-square test and Fisher’s exact test were used to compare between these groups. For continuous variables, the Kolmogorov–Smirnov test was used to determine the distribution of data. If the data demonstrated normal distribution, they will be presented with mean ± SD and the Student t test is used for comparison. For data without normal distribution, median and interquartile range are shown and the Mann–Whitney U test is used for comparison. Significance level was set at α = 0.05 (two-tailed). Parameters showing a statistical trend (p < 0.1) or being proved a significant association in previous studies were included in a multivariate model to identify parameters independently associated with functional outcome. The Hosmer–Lemeshow test was used to test the goodness of fit. A receiver operating characteristic (ROC) curve and Youden’s J statistic were used to determine the cutoff value for NLR. Then, patients were dichotomized according to the identified cutoff value of NLR, and 3-month mRS scores of different groups divided by cutoff value of NLR were tested by chi-square test and z test.