Consecutive patients admitted to the Department of Neurology of Vittoria Hospital (Italy) with objectively diagnosed stroke between January 1st, 2006 and April 15th, 2009 were included in this prospective cohort study. Patients with transient ischemic attack, cerebral venous thrombosis and subarachnoid hemorrhage on admission were excluded. All patients were assessed by a neurologist to determine the diagnosis of stroke (neurological deficit lasted >24 h) and its pathological and etiological subtypes [10]. On admission, stroke severity was assessed using the NIH (National Institute of Health) Stroke Scale. The NIHSS is a clinical assessment tool that is widely used in clinical trials and practice to evaluate stroke-related neurological deficits. It assesses speech, language cognition, inattention, visual field abnormalities, motor and sensory impairments, and ataxia. The maximum possible score, in case of severe stroke, is 42.
The study centre provided stroke unit standard care for each patient. All patients were monitored for blood pressure, temperature, glucose level, heart rate and blood gases in the first days after stroke.
In all patients cerebral Computed Tomography (CT) examination without contrast was performed on admission and after 48–72 h from stroke onset to define both topography and extension of the ischemic or hemorrhagic lesion. Patients were admitted to the hospital within 12 h from the stroke onset.
Based on standard templates the size of the lesion was quantified as: a) lacunar (lesion in the anterior or posterior circulation <1.5 cm), b) non lacunar (lesion in the anterior or posterior circulation >1.5 cm).
Stroke risk factors
Data was collected on stroke risk factors: age, gender, history of hypertension (blood pressure of >140/90 mmHg at least twice before stroke or already under treatment with antihypertensive drugs), history of diabetes mellitus (glucose level >126 mg/dL preprandial on 2nd examination, glucose level >200 mg/dL postprandial, or HbA1c > 8.5 %, or under hypoglycemic treatment), current cigarette smoking, history of hyperlipidemia (total cholesterol concentration >200 mg/dL and/or triglyceride concentration >140 mg/dL the day after admission or already under lipid lowering therapy), history of symptomatic ischemic heart disease (proven myocardial infarction, history of angina or existence of multiple lesions on thallium heart isotope screen or evidence of coronary disease on coronary angiography), atrial fibrillation, or previous stroke/transient ischemic attacks (TIAs). Other baseline variables that were obtained at admission for each patient included homocysteinemia (normal values ≤15 μmol/l) and PCR (normal values ≤10 mg/dL).
All cases had ultrasonography examination of the carotid and vertebral arteries. The degree of stenosis on ultrasonography was evaluated on a duplex sonography with power Doppler imaging; linear stenosis, area stenosis and peak systolic velocity (PSV) were measured in the most stenotic part of the ICA or vertebral artery [11, 12]. Patients were considered to have carotid atherosclerosis if at least one vessel had stenosis ≥50 % or atherosclerotic occlusion. Occlusion of an artery was defined as an absence of flow and when there was the presence of a visible plaque, it was diagnosed to be caused by atherosclerosis.
Evaluation of outcome
Patients were followed up prospectively by face-to face or telephone interview. Study outcome was 120-day mortality. The time of occurence and the cause of death were recorded. The causes of death were divided into: neurological (recurrence of stroke, status epilepticus, edema, herniation), cardiovascular (myocardial infarction, heart failure, sudden death, other cardiovascular disease) and other causes (pneumonia, cancer, pulmonary embolus and other causes).
Measurement of biomarkers with Triage Stroke Panel
The analysis of the biochemical markers (BNP, D-Dimers, MMP-9, S 100 B, MMX) was done by Sandwich-Fluorescence-Immunoassay-Technology of the so-called Triage® Stroke Panel using blood from an EDTA sample taken within 15 min after admission.
Principles of the test procedure
The test procedure involves the addition of several drops of an EDTA whole blood or plasma specimen to the sample port on the Test Device. After addition of the sample, the cells are automatically separated from the plasma via a filter contained in the Test Device. The sample reacts with fluorescent antibody conjugates within the reaction chamber and flows down the Test Device detection lane by capillary action. Complexes of each fluorescent antibody conjugate are captured on discrete zones resulting in binding assays that are specific for each biomarker. The Test Device is inserted into the Triage MeterPlus (hereafter referred to as Meter) and results are measured and displayed on the screen. These results are then presented as a single composite MMX result that is calculated automatically. This composite MMX result is the test result that is used as an aid in the diagnosis and assessment of ischemic stroke [9]. All analyte concentrations and the MMX result are stored in the Meter memory and are available on demand. The MMX result reportable range is 0 to 10. The manufacturer’s recommended cutoffs are 1.3 and 5.9. Using these cutoffs the interpretation is as follows: MMX results less than or equal to 1.3 represent a low probability of stroke; MMX results greater than 1.3 are considered abnormal and suggest the need for further evaluation; MMX results greater than 5.9 represent a high probability of stroke. However, it is recommended that each laboratory should determine if these cutoffs are appropriate for the patient population that is to be evaluated. We have used a score of > 4 because our statistical analysis detects that values higher than 4 correspond with a greater degree of disability and increased mortality.
Statistical analysis
A receiver operating characteristic (ROC) curve was applied to determine a cut-off point of the panel that could best predict the occurrence of death within 120 days. After the determination of cut-off point, outcomes in patients with Stroke Panel >4 and ≤4 were compared by Χ2 test. The first step of analysis was aimed at identifying predictors of adverse outcome (death) at 120 days including biomarkers tested with Triage Stroke Panel. Univariate tests were used to compare clinical characteristics on admission, preexisting risk factors for stroke and CT findings in comparison to patients who died. All variables (age, sex, vascular risk factors, stroke severity on admission, PCR and Stroke Panel > 4) were subjected to multiple logistic regression analysis to identify independent predictors for death.
The second step of analysis was aimed at identifying predictors of the increase of biomarkers (Stroke Panel > 4) among baseline findings. Univariate tests were applied to compare clinical characteristics on admission, preexisting risk factors for stroke and CT findings to patients with or without an increase of biomarkers. All variables were subjected to multiple logistic regression analysis to identify independent predictors for the increase of biomarkers.
Data was analyzed with the SPSS/PC Win package 19.0 [13].
