Serum bilirubin levels are associated with poor functional outcomes in patients with acute ischemic stroke or transient ischemic attack

Data were derived from the Third China National Stroke Registry (CNSR-III), which is a nationwide prospective registry for patients presented to hospitals with acute ischemic cerebrovascular events between August 2015 and March 2018 in China. Participants were consecutively enrolled if meeting the following criteria: (1) age 18 years or older; (2) diagnosis within 7 days of the index event of ischemic stroke or TIA; (3) informed consent from participant or legally authorized representative. The detailed, rationale, and basic description of the CNSR-III have been published previously [11]. A total of 15,166 patients were included from 201 hospitals of 22 provinces. The study protocol was approved by the ethics committee of all participating study centers according to the principles expressed in the Declaration of Helsinki. Written informed consent was provided by all participants or their legal proxies.

Trained research coordinators at each site collected baseline data through a direct interview or medical records, including age, sex, body mass index (BMI, calculated as the weight in kilograms divided by the square of the height in meters), smoking status, medical history, primary diagnosis, the aetiological classification conducted by the Trial of Org 10,172 in Acute Stroke Treatment (TOAST) criteria [12], medication in hospital, and National Institutes of Health Stroke Scale (NIHSS) score.

Blood samples were collected within 24 h of admission. Plasma specimens were extracted, aliquoted and transported through cold chain to the central laboratory in Beijing Tiantan Hospital and stored at -80℃refrigerator until tests were performed centrally and blindly.

TBIL, DBIL, IBIL, total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), triglyceride (TG), fasting blood glucose (FBG), serum creatinine, high-sensitivity C-reactive protein (hs-CRP), alanine aminotransferase (ALT), and aspartate aminotransferase (ALT) were tested in the central laboratory of Beijing Tiantan Hospital using Hitachi 7600 automatic biochemistry analyzer (Hitachi, Tokyo, Japan). The estimated glomerular filtration rate (eGFR) was calculated by Chronic Kidney Disease Epidemiology Collaboration equations [13].

Patients were followed up for clinical outcomes at 3 months and 1year after symptom onset. They were interviewed face to face at 3 months and contacted over the telephone by trained research coordinators at 1 year. The poor functional outcomes included modified Rankin Scale (mRS) score 2–6/3–6 at 3 months and 1 year [14‐16]. All events were collected by trained research coordinators who were blinded to subjects’ baseline characteristics.

Participants were classified into four groups by TBIL quartiles, DBIL quartiles, and IBIL quartiles, respectively. Continuous variables are presented as median (interquartile range [IQR]) because of the skewed distribution. Categorical variables are presented as frequency and percentage. The non-parametric Wilcoxon or Kruskal-Wallis tests is used to compare group differences for continuous variables and chi-square test is used for categorical variables.

Ordinal logistics regression was applied to estimate the common odds ratio (OR) for a shift in the direct of worse outcome on the mRS score according to serum bilirubin. The association between serum bilirubin and poor functional outcomes were assessed with multivariable binary logistic regression. Variables with P < 0.05 and the well-established predictors were selected as confounding variables in the multivariable analyses. The lowest quartile was defined as the reference group. Unadjusted and adjusted ORs and their 95 % confidence intervals (CIs) were calculated. Because 201 hospitals participated in the study, the hospital was treated as clusters in the model and the sandwich estimated were used to account for the correlations. Trends tests were performed using quartiles as ordinal variables. In addition, we used restricted cubic splines to examine the shape of the association between bilirubin and outcomes with five knots (at the 5th, 25th, 50th, 75th, and 95th percentiles) [17]. Reference points for TBIL, DBIL, and IBIL were the median (8.10, 2.05, and 5.10 µmol/L, respectively) of the reference group (the lowest quartile) and OR was adjusted for variables in the multivariable analysis. Additionally, we used C statistics, integrated discrimination improvement (IDI), and net reclassification index (NRI) to evaluate the incremental predictive value of TBIL, DBIL, and IBIL beyond conventional risk factors. To test the robustness of the findings, sensitivity analysis was also performed by excluding patients with cardioembolism or TIA. Considering stroke severity at admission was a strong confounding factor the prognosis of stroke patients, subgroup analysis stratified by stroke severity measured by NIHSS at baseline (NIHSS ≤ 3 for minor ischemic stroke and NIHSS > 3 for moderated to several ischemic stroke) was performed [18], interactions between subgroups were tested using likelihood ratio tests.