Identification of Ischemic Stroke Patients Based on Plasma Concentrations of Extracellular Vesicles

Translational Stroke Research (2025) 16:2082–2092 https://doi.org/10.1007/s12975-025-01371-z RESEARCH Identification of Ischemic Stroke Patients Based on Plasma Concentrations of Extracellular Vesicles Naomi C. Buntsma1,2,3,4,5 · Chi M. Hau1 · Mandy Los1 · Vivien M. Chen10 · Ton G. van Leeuwen3,5,7 Yvo B. W. E. M. Roos2,4,5 · Rienk Nieuwland1,6,7 · Aleksandra Gasecka1,8,9 · Edwin van der Pol1,3,5 · Received: 28 March 2025 / Revised: 4 July 2025 / Accepted: 14 July 2025 / Published online: 15 August 2025 © The Author(s) 2025 Abstract Patients presenting with stroke symptoms suffer from either ischemic stroke, hemorrhagic stroke, transient ischemic attacks (TIA), or “stroke mimics,” which include benign headaches, epilepsy, and vestibular disorders. As ischemic and hemorrhagic stroke patients require different medical treatments, early identification of the underlying cause of symptoms is essential for tailored and urgent medical intervention. This study investigates whether extracellular vesicles (EVs), present in peripheral blood of patients presenting with stroke symptoms, can be used to identify patients with ischemic stroke. Blood was collected from 155 patients presenting with stroke symptoms in the emergency room and analyzed for EVs by flow cytometry (ethics approval number NL72929.018.20). The primary endpoint was to compare platelet EV concentrations between patients with (n = 66) and without (n = 89) ischemic stroke. Concentrations of EVs from both activated platelets and leukocytes were lower in patients presenting with ischemic stroke compared to other patients (p = 0.038 and p = 0.015, respectively). No significant differences in other EV types were observed. In addition, ischemic stroke patients were older and had a higher diastolic blood pressure compared to patients with other diagnoses. In a multivariable analysis, leukocyte EVs and diastolic blood pressure were independent indicators of ischemic stroke. To conclude, this study demonstrates that the plasma concentration of leukocyte EVs can be useful to identify ischemic stroke patients in an emergency setting. Keywords Acute cerebrovascular accident · Biomarkers · Extracellular vesicles · Flow cytometry · Stroke * Naomi C. Buntsma 5 Amsterdam Cardiovascular Sciences, Atherosclerosis and Ischemic Syndromes, Amsterdam, The Netherlands * Edwin van der Pol 6 Amsterdam Cardiovascular Sciences, Microcirculation, Amsterdam, The Netherlands 7 Cancer Center Amsterdam, Imaging and Biomarkers, Amsterdam, The Netherlands 8 Chair and Department of Cardiology, Medical University of Warsaw, Warsaw, Poland 9 Department of Cardiology, Onze Lieve Vrouwe Gasthuis, Amsterdam, The Netherlands 10 Concord Clinical School, Faculty of Medicine and Health, University of Sydney, Sydney Australia Haematology, Concord Hospital and ANZAC Research Institute, Sydney Local Health District, Sydney, Australia 1 Laboratory of Experimental Clinical Chemistry and Amsterdam Vesicle Center, Laboratory Specialized Diagnostics & Research, Department of Laboratory Medicine, Amsterdam UMC, University of Amsterdam, Meibergdreef 9, Amsterdam, The Netherlands 2 Department of Neurology, Amsterdam UMC, University of Amsterdam, Meibergdreef 9, Amsterdam, The Netherlands 3 Biomedical Engineering & Physics, Amsterdam UMC, University of Amsterdam, Meibergdreef 9, Amsterdam, The Netherlands 4 Amsterdam Neuroscience, Neurovascular Disorders, Amsterdam, The Netherlands Vol:.(1234567890) Translational Stroke Research (2025) 16:2082–2092 2083 Introduction Methods Stroke is one of the leading causes of disability worldwide, with about 15 million people suffering from stroke annually [1]. There are two major subtypes of stroke: ischemic and hemorrhagic stroke. Ischemic stroke is caused by a cerebrovascular occlusion, whereas hemorrhagic stroke is caused by a ruptured cerebral blood vessel. Although the underlying causes differ, the symptoms of patients presenting with ischemic or hemorrhagic stroke are often indistinguishable. This similarity of symptoms complicates the tailored and urgent medical treatment. At present, patients presenting with stroke symptoms are immediately transported to the nearest hospital for a computed tomography (CT) scan. In case of an ischemic stroke caused by a cerebrovascular occlusion, either tissue plasminogen activator (tPA) is administered or intra-arterial thrombectomy is performed, which is a method available only in specialized stroke centers [2–4]. In case of a cerebral hemorrhage, next to blood pressure control and regulation of blood coagulation, patients may qualify for surgical clipping or endovascular coiling if a cerebral aneurysm is identified as the cause of the hemorrhage. In addition to ischemic or hemorrhagic stroke, patients can present with stroke symptoms but in reality suffer from a transient ischemic attack (TIA) or “stroke mimics,” such as benign headaches, epilepsy, or vestibular disorders. Since ischemic and hemorrhagic stroke as well as stroke mimics all require different treatment, the ability to diagnose the underlying cause of stroke-like symptoms at an early stage is of utmost importance. Currently, no clinically applicable diagnostic test is available for differential diagnosis in home or ambulance settings. Hence, there is a quest for biomarkers, present in liquid biopsies such as blood, that can be used to speed up the appropriate medical intervention in patients with stroke-like symptoms. Especially biomarkers that can discriminate ischemic stroke from all other diagnoses in home or ambulance settings are of interest, as ischemic stroke patients benefit most from urgent medical interventions. EVs are membrane-delimited particles that are released by probably all cell types. Their biochemical composition, cellular origin, and function reflect real-time changes in an individual’s health status. Several studies measured the concentration of EVs in plasma of stroke patients. However, these concentrations were compared to those in plasma from healthy individuals [5–10], which does not address the clinical reality. Moreover, previously published data are irreproducible as data are in arbitrary units and detection limits unreported [11, 12]. Here, we addressed these questions by investigating whether ischemic stroke patients can be identified in a clinical setting by measuring (reproducible) concentrations of EVs in their blood plasma. Patient Inclusion This observational study is a preliminary analysis of the study Circulating Nanotraces to Identify the Cause of Stroke (CINTICS), which aims to identify nanotraces, including small RNAs and extracellular vesicles (EVs), that can identify stroke and discriminate between ischemic and hemorrhagic stroke. The study was approved by the Ethical Review Board of Amsterdam UMC, location AMC, in combination with a deferred consent process (approval number NL72929.018.20). Patients were included from October 2020 to November 202 (...truncated)