Plasma protein panels to estimate stroke onset time and differentiate stroke mimics (STROKE-CLOCK study)

One-third of patients with acute ischemic stroke (AIS) present to the emergency department (ED) with an unknown symptom onset time, making them ineligible for intravenous thrombolysis. Blood-based biomarkers predictive of time from symptom-onset, can make patients with unknown stroke onset times eligible for thrombolysis. Majority (>50%) of patients evaluated in the ED for acute neurological symptoms concerning for stroke, are also eventually diagnosed with not having stroke (stroke mimics) after comprehensive evaluation. Many stroke mimic patients also receive thrombolysis in the ED, posing a significant burden via healthcare resource utilization and exposing these individuals to unnecessary bleeding risk. Blood-based biomarkers that can differentiate stroke mimics from AIS, can potentially minimize unnecessary interventions and optimize resource allocation. Currently, no validated blood-based biomarker exists to address these urgent clinical gaps in acute stroke care. The goal of the STROKE-CLOCK study is to identify a blood-based biomarker panel capable of estimating stroke onset time and distinguishing early-onset AIS from stroke mimics. We will perform a single- site proteomics study using plasma samples collected in the ED, from patients who present with acute neurological symptoms concerning for stroke and have a clearly defined time of symptom onset. We will leverage plasma samples from the Emergency Medicine Specimen Biobank (EMSB) at Yale University and will include adults (age ≥18 years) presenting with a suspected stroke to the ED with a clearly defined onset time with blood (plasma) samples collected in the ED prior to any stroke intervention. We will perform mass spectrometry (MS)- based proteomics to identify and validate protein biomarkers that distinguish patients with early-onset AIS (≤4.5 hours) from late-onset AIS (>4.5 hours) (Aim 1) and distinguish early-onset AIS from stroke mimics within 4.5 hours (Aim 2). In both aims, we will first perform data-independent acquisition (DIA)-MS proteomics in a derivation cohort (2 groups, N=30/group) to nominate differentially enriched proteins (DEPs). We will then verify these in an independent set of samples (2 groups, N=30/group). Proteins nominated from untargeted studies will be validated on the same samples using targeted parallel reaction monitoring (PRM)-MS and then validated in an independent cohort of samples (2 groups, N=50/group). We will estimate performance metrics, including sensitivity, specificity, and negative and positive predictive values. Multivariable prediction models will be constructed, and performance of biomarker panels will be evaluated using area under the receiver operating characteristic curves. If successful, this R21 proposal will lay the foundation for future definitive studies on large- scale validation, temporal profiling, and clinical implementation of this biomarker panel to guide acute stroke triage and treatment decisions. Project Number: 1R21NS149205-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of Neurological Disorders and Stroke (NINDS) | Principal Investigator: Srikant Rangaraju (+1 co-PI) | Institution: YALE UNIVERSITY, NEW HAVEN, CT | Award Amount: $251,813 | Activity Code: R21 | Study Section: Brain Injury and Neurovascular Disorders Study Section[BIND] View on NIH RePORTER: https://reporter.nih.gov/project-details/11368419