Stroke subtype
Typical clinical management
Large artery atherosclerosis
Antiplatelet therapy, major risk factor modification (hypertension, diabetes, hyperlipidemia, smoking cessation)
Cardioembolic
Anticoagulation
Small vessel occlusion (lacunar)
Antiplatelet therapy, major risk factor modification (hypertension, diabetes, hyperlipidemia, smoking cessation)
Stroke of other determined etiology
Interventions specific to cause
Stroke of undetermined etiology (two or more causes identified; negative evaluation; incomplete evaluation)
Antiplatelet therapy (aspirin), further diagnostic testing
Suggested diagnostic evaluation of ischemic stroke to determine etiologya
Diagnostic evaluation | Suggested approach |
|---|---|
Brain imaging | • Brain MRI in patients with cryptogenic stroke • Brain CT when stroke mechanisms known |
Cardiac imaging | • TTE on all patients with AIS • TEE with bubble study on patients <50 years old if TTE nondiagnostic |
Cardiac monitoring | • Thirty day noninvasive cardiac monitoring for patients with cryptogenic stroke and ≥40 years old • Implantable cardiac monitor if 30 day monitor does not reveal atrial fibrillation or flutter |
Hypercoagulable testing | • Serum hypercoagulable workup in patients with no or minimal risk factors |
Malignancy screening | • Age appropriate screening • CT of chest/abdomen/pelvis when systemic symptoms suggestive of cancer |
Vascular imaging | • Intracranial and extracranial vascular imaging in all patients with AIS • MRA with fat-suppressed images if cervical artery dissection suspected |
The pursuit of blood-borne stroke diagnostic tests for diagnosing acute ischemic stroke and determining stroke etiology has spanned decades. Many have investigated individual or combinations of serum proteins and yet no study has demonstrated sufficient diagnostic robustness to be useful in routine clinical practice [3]. More recent studies have suggested whole blood RNA expression may help differentiate ischemic stroke mechanisms [4–8]. The majority of RNA in whole blood is from circulating leukocytes, monocytes, and neutrophils. In very early samples obtained close to stroke ictus, these circulating immune cells likely reflect prestroke biologic activity, including inflammatory states, immune modulation, states of coagulation balance, and overall signaling. Samples obtained later in the course of acute ischemic stroke likely reflect more common pathways associated with response to the infarction. Thus, understanding the time course of gene expression is critical to be able to use the signatures for a specific clinical question.
Jickling and colleagues were amongst the first to perform detailed whole blood gene expression studies in patients with acute ischemic stroke [5]. In an early study, a 40-gene profile differentiated large-vessel stroke from cardioembolic stroke with reported 95% sensitivity and specificity. In the same study, a different 37-gene expression signature further classified patients with cardioembolic stroke due to atrial fibrillation vs. other cardioembolic sources with >90% sensitivity and specificity. More recently, Jickling and colleagues looked at the differential gene expression with the additional information of physical infarct location to identify the likely etiology of stroke previously classified as cryptogenic [9]. Using Affymetrix® U133 Plus 2.0 microarrays (Affymetrix, Thermo Fisher Scientific, Waltham, MA) and the sample profiles from the prior study, combining stroke location with the gene expression signatures identified 58% of patients previously classified with cryptogenic stroke as being cardioembolic, 18% to be large vessel stroke, 12% lacunar, and 12% unclassified. These findings encouraged further study.
Based on these promising studies, the Biomarkers of Acute Stroke Etiology (BASE) study was initiated by Ischemia Care (ISCDX, Oxford, OH, USA) and partnered with leading academic medical centers for study sites. BASE (NCT02014896) is a multicenter observational study utilizing RNA gene expression from the Ischemia Care diagnostic platform to identify the etiology of acute ischemic stroke. As noted before when stroke or TIA occur, the immune system changes gene expression in multiple cell types, thus activating innate and adaptive immune responses. Previous studies suggest that differential gene expression profiles are a function of stroke subtype, with each subtype producing a unique gene expression “signature” [4–8]. The Ischemia Care diagnostic platform consists of whole blood biomarker tests to determine the etiology of ischemic stroke (ISCDX, Oxford, OH, USA) by measuring acute ischemic stroke gene expression changes. For example, the ISCDX test based on previous gene signatures distinguishes between cardioembolic and large artery, as well as lacunar, atherosclerotic stroke using a signature of 40 unique genes. A patient’s pattern of gene regulation can determine if the stroke etiology is that of a cardioembolic or large artery atherosclerotic source. Further, a separate 37 gene signature can differentiate cardioembolic strokes caused by atrial fibrillation (AF) or other cardioembolic sources. Ultimately, for most patients, the diagnostic expression pattern clearly identifies stroke etiology.
The primary objective of the BASE study is to confirm the diagnostic accuracy of the ISCDX test to identify stroke subtypes in patients with acute ischemic stroke. This manuscript describes the methodology employed in the BASE study to identify stroke etiology in patients presenting with acute stroke.
2 Methods
Inclusion and exclusion criteria for BASE
Inclusion criteria |
• Suspected acute ischemic stroke within 24 (± 6) h of last known normal or symptom onset • Baseline CT normal, without hemorrhage or alternate explanation for symptoms • >18 years old • Informed consent obtained |
Exclusion criteria |
• Central nervous system infection within 30 days • Serious head trauma within 30 days • Any ischemic or hemorrhagic stroke within 30 days • Active cancer (not in remission) • Autoimmune disease (e.g., lupus) • Acute systemic infection • Major surgery within 90 days |
BASE initially enrolled acute stroke patients within 8 h of symptom onset or the time of last known normal. However, after patient enrollment reached 650, evaluation time was lengthened to 24 (± 6) h. This was from a planned interim data analysis determining the 24 (± 6) h window from symptom onset was most predictive for identifying stroke cause using blood biomarkers, was most consistent with the time a stroke patient would present, and represented the window for which a blood test for stroke would be used clinically.
Typically, prior to enrollment, patients are evaluated by the local stroke team or ED physicians, have undergone baseline laboratory testing and cerebrovascular imaging, and may receive intravenous thrombolysis and/or endovascular therapies. Approximately 2.5 mL of blood is drawn into two PreAnalytiX® PAXgene® blood RNA tubes (Qiagen, Venlo, Netherlands) within 24 (± 6) h of stroke onset. Additional draws occur at 24 (± 6), and 48 (± 6) h, or at ED/hospital discharge, whichever comes first. Longer collection periods were considered but were challenged by the amount of RNA response making it difficult to identify diagnostic patterns consistent with the primary objective of this study. Eligible control subjects are patients presenting without a potentially neurologic complaint and have blood drawn within 6 h of ED presentation.
PAXgene® tubes can be kept at room temperature for up to 24 h, and then are frozen at −20 °C, until shipped on dry ice to the Ischemia Care CLIA laboratory (Middletown, OH) where the ISCDX testing is performed. The entire sample from one tube will be used to perform the ISCDX test. The second tube is stored at −80 °C for future testing.
Analysis for RNA expression is performed by Affymetrix® human gene ST array plates. These provide whole-genome coverage, including protein coding and long intergenic non-coding RNA (lincRNA) transcripts. Whole genome arrays thus have the ability to provide a complete profile of mRNA expression.
- 1.
RNA extraction: Total RNA is extracted from blood collected in PAXgene® RNA tubes which are used to specifically preserve the integrity of the RNA.
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