Blood-Based Molecular Clock Biomarkers Predict Acute Ischemic Stroke Onset: A Prospective Observational Study (wakeupstroke)

March 5, 2026 updated by: Ejder Saylav BORA, Izmir Katip Celebi University

The Role of Blood-Based Molecular Clock Biomarkers in Predicting the Onset Time of Acute Ischemic Stroke: A Prospective Observational Study

Acute ischemic stroke (AIS) is a major cause of mortality and long-term neurological disability worldwide. The effectiveness of reperfusion therapies such as intravenous thrombolysis and mechanical thrombectomy is highly dependent on the time elapsed since symptom onset. However, in approximately 15-25% of patients, the exact onset time cannot be determined because symptoms begin during sleep (wake-up stroke) or the onset is otherwise unclear. This uncertainty often prevents patients from receiving time-dependent reperfusion treatments.

Currently, imaging-based approaches such as diffusion-weighted imaging-fluid attenuated inversion recovery (DWI-FLAIR) mismatch are used to estimate the biological stage of ischemia in patients with unknown onset time. However, advanced imaging techniques may not be available in all centers and interpretation may vary.

This study aims to evaluate the diagnostic performance of a multi-biomarker panel representing different biological components of ischemic brain injury, including glial, neuronal, axonal, cellular stress, and vascular responses. Blood samples obtained at admission will be analyzed for glial fibrillary acidic protein (GFAP), ubiquitin C-terminal hydrolase L1 (UCH-L1), serum neurofilament light chain (sNfL), phosphatidylethanolamine-binding protein 1 (PEBP1), and matrix metalloproteinase-9 (MMP-9).

The primary objective is to determine whether this biomarker panel can distinguish patients presenting within ≤4.5 hours from those presenting after >4.5 hours of symptom onset. Biomarker findings will be compared with imaging-based reference methods to explore the feasibility of a blood-based "molecular clock" approach for estimating stroke timing in patients with uncertain onset.

Study Overview

Status

Active, not recruiting

Conditions

Detailed Description

Acute ischemic stroke (AIS) is one of the leading causes of mortality and long-term neurological disability worldwide. The effectiveness of reperfusion therapies, including intravenous thrombolysis and mechanical thrombectomy, is highly dependent on the time elapsed since symptom onset. Determination of the symptom onset time is therefore a critical component of treatment eligibility. However, a substantial proportion of patients present with stroke symptoms of unknown onset, most commonly because the symptoms develop during sleep (wake-up stroke) or because the exact onset time cannot be reliably established. This uncertainty may prevent eligible patients from receiving time-dependent reperfusion therapies.

Current clinical decision-making in patients with uncertain onset time relies largely on imaging-based approaches that attempt to estimate the biological stage of ischemia. The diffusion-weighted imaging-fluid attenuated inversion recovery (DWI-FLAIR) mismatch concept is the most widely used method and is based on the hypothesis that tissue signal evolution reflects the duration of ischemia. Advanced perfusion imaging techniques have also been incorporated into clinical practice. However, these imaging methods require specialized equipment and expertise, may not be available in all centers, and their interpretation may vary between institutions. Consequently, there is increasing interest in identifying circulating biomarkers that can reflect the biological timing of ischemic injury.

Ischemic brain injury initiates a complex cascade of pathophysiological processes involving astroglial activation, neuronal membrane disruption, axonal injury, inflammatory signaling, cellular stress responses, and vascular dysfunction. These processes evolve dynamically over time and lead to the release of measurable molecular components into the systemic circulation. Biomarkers reflecting different cellular compartments of the neurovascular unit may therefore provide complementary information regarding the temporal evolution of ischemic injury.

Several candidate biomarkers have been identified that represent different biological aspects of this process. Glial fibrillary acidic protein (GFAP) is an astrocyte-specific structural protein that reflects astroglial injury and early glial activation following acute brain damage. Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a neuron-specific cytoplasmic enzyme released after neuronal membrane disruption and has been proposed as an indicator of acute neuronal injury. Serum neurofilament light chain (sNfL) is a structural component of the axonal cytoskeleton and is considered a marker of axonal degeneration and structural neuronal damage. Phosphatidylethanolamine-binding protein 1 (PEBP1), also known as Raf kinase inhibitory protein, is involved in the regulation of cellular signaling pathways and stress responses and may reflect cellular adaptation to ischemic stress. Matrix metalloproteinase-9 (MMP-9) is associated with inflammatory activation, extracellular matrix degradation, and blood-brain barrier dysfunction during the evolution of ischemic brain injury.

The combined evaluation of biomarkers representing multiple biological layers of the neurovascular unit may provide a more comprehensive representation of the temporal dynamics of ischemic injury than single-marker approaches. A multi-biomarker strategy has the potential to capture different phases of the ischemic cascade, including glial activation, neuronal damage, axonal injury, cellular stress response, and vascular dysfunction.

The present study aims to investigate whether a multi-compartment biomarker panel can contribute to the estimation of the biological timing of acute ischemic stroke. By evaluating circulating biomarkers representing different components of the neurovascular unit, the study seeks to explore the feasibility of a blood-based molecular signature that reflects the temporal evolution of ischemic injury.

The findings of this study may contribute to the development of a biologically informed approach for estimating stroke timing and may support clinical decision-making in patients presenting with uncertain symptom onset, particularly in settings where advanced imaging techniques are not readily available.

Study Type

Observational

Enrollment (Estimated)

80

Contacts and Locations

This section provides the contact details for those conducting the study, and information on where this study is being conducted.

Study Locations

  • Turkey (Türkiye)
      • Izmir, Turkey (Türkiye), 35220
        • Izmir Katip Çelebi University Ataturk Research and Training Hospital

Participation Criteria

Researchers look for people who fit a certain description, called eligibility criteria. Some examples of these criteria are a person's general health condition or prior treatments.

Eligibility Criteria

Ages Eligible for Study

  • Adult
  • Older Adult

Accepts Healthy Volunteers

No

Sampling Method

Probability Sample

Study Population

Patients with acute ischemic stroke whose onset time is unknown or who present with a wake-up stroke.

Serum/plasma samples obtained from venous blood samples taken during routine clinical practice in all patients will be analyzed for GFAP, UCH-L1, sNfL, PEBP1, and MMP-9 levels. Patients' demographic data, clinical characteristics, NIHSS scores, and magnetic resonance imaging (MRI) findings (particularly DWI-FLAIR mismatch status) will be recorded from the electronic patient record system. The performance of the multi-compartment molecular panel formed by the obtained biomarker levels in distinguishing the biological time window of acute ischemic stroke (≤4.5 hours vs. >4.5 hours) will be evaluated by comparing it with the imaging-based reference method.

Description

Inclusion Criteria:

  1. Be 18 years of age or older
  2. Presence of acute focal neurological deficit
  3. Presentation within ≤24 hours of the last time the patient was well

Exclusion Criteria:

  1. Intracranial hemorrhage
  2. Active infection or sepsis
  3. Major surgery/trauma within the last 14 days
  4. Active autoimmune disease
  5. Stroke within the last 3 months
  6. Immunosuppressive therapy

Study Plan

This section provides details of the study plan, including how the study is designed and what the study is measuring.

How is the study designed?

Design Details

Cohorts and Interventions

Group / Cohort
ACUTE STROKE PRESENTING TO THE EMERGENCY DEPARTMENT-1
ACUTE STROKE PRESENTING TO THE EMERGENCY DEPARTMENT (WITNESSED STROKE, STROKE WITH KNOWN OCCURRENCE)
ACUTE WAKE UP STROKE PRESENTING TO THE EMERGENCY DEPARTMENT-2
WAKE UP STROKE (Without Witnesses and Unknown Start Time)

What is the study measuring?

Primary Outcome Measures

Outcome Measure
Measure Description
Time Frame
Diagnostic performance of the multi-biomarker panel in identifying the biological time window of acute ischemic stroke (≤4.5 hours vs >4.5 hours from symptom onset)
Time Frame: At emergency department admission.
The primary outcome will be the ability of the combined biomarker panel (GFAP, UCH-L1, sNfL, PEBP1, and MMP-9) measured from admission blood samples to discriminate patients presenting within ≤4.5 hours versus >4.5 hours after symptom onset. Diagnostic performance will be evaluated using receiver operating characteristic (ROC) curve analysis and reported as area under the curve (AUC), sensitivity, specificity, and optimal cut-off values.
At emergency department admission.

Secondary Outcome Measures

Outcome Measure
Measure Description
Time Frame
Diagnostic performance of individual biomarkers
Time Frame: At emergency department admission.
The diagnostic accuracy of each biomarker (GFAP, UCH-L1, sNfL, PEBP1, and MMP-9) will be evaluated individually for distinguishing ≤4.5 hours versus >4.5 hours from symptom onset using ROC curve analysis.
At emergency department admission.
Association between biomarker levels and imaging-based reference methods
Time Frame: Baseline imaging obtained at hospital admission.
Circulating biomarker levels will be compared with imaging findings used to estimate stroke timing (e.g., DWI-FLAIR mismatch or other clinically available imaging parameters) to assess concordance between molecular and imaging-based indicators of ischemic timing.
Baseline imaging obtained at hospital admission.
Incremental diagnostic value of the combined biomarker model
Time Frame: At emergency department admission.
We will assess multivariable models that include the biomarker panel to see if analyzing the biomarkers together offers better diagnostic accuracy than looking at each biomarker separately.
At emergency department admission.
Correlation between circulating biomarker levels and infarct volume on diffusion-weighted imaging (DWI)
Time Frame: Baseline imaging obtained within the initial diagnostic evaluation after hospital admission.
The relationship between admission biomarker concentrations (GFAP, UCH-L1, sNfL, PEBP1, and MMP-9) and infarct volume measured on diffusion-weighted magnetic resonance imaging (DWI) will be evaluated to explore the association between molecular markers of ischemic injury and the extent of cerebral infarction. Correlation analyses will be performed to determine whether biomarker levels reflect infarct burden.
Baseline imaging obtained within the initial diagnostic evaluation after hospital admission.

Collaborators and Investigators

This is where you will find people and organizations involved with this study.

Sponsor

Izmir Katip Celebi University

Investigators

  • Study Director: BORA, MD PhD, Izmir Katip Çelebi University Faculty of Medicine

Publications and helpful links

The person responsible for entering information about the study voluntarily provides these publications. These may be about anything related to the study.

General Publications

Helpful Links

Study record dates

These dates track the progress of study record and summary results submissions to ClinicalTrials.gov. Study records and reported results are reviewed by the National Library of Medicine (NLM) to make sure they meet specific quality control standards before being posted on the public website.

Study Major Dates

Study Start (Actual)

March 1, 2026

Primary Completion (Estimated)

March 1, 2027

Study Completion (Estimated)

April 1, 2027

Study Registration Dates

First Submitted

March 1, 2026

First Submitted That Met QC Criteria

March 2, 2026

First Posted (Actual)

March 5, 2026

Study Record Updates

Last Update Posted (Actual)

March 6, 2026

Last Update Submitted That Met QC Criteria

March 5, 2026

Last Verified

March 1, 2026

More Information

Terms related to this study

Keywords

Additional Relevant MeSH Terms

Other Study ID Numbers

  • IZMIR-KCU-02

Plan for Individual participant data (IPD)

Plan to Share Individual Participant Data (IPD)?

NO

Drug and device information, study documents

Studies a U.S. FDA-regulated drug product

No

Studies a U.S. FDA-regulated device product

No

This information was retrieved directly from the website clinicaltrials.gov without any changes. If you have any requests to change, remove or update your study details, please contact register@clinicaltrials.gov. As soon as a change is implemented on clinicaltrials.gov, this will be updated automatically on our website as well.

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