Extracellular Vesicle Surface Markers In Acute Cerebrovascular Syndromes.
试验速览
- 阶段
- 不适用
- 状态
- 招募中
- 发起方
- 入组人数
- 200
- 试验地点
- 1
- 主要终点
- TIA differentiation from TIA mimics
研究概览
简要总结
Clinical implication of eventual blood biomarkers for stroke diagnosis and prognosis would be limited, mainly because clinical evaluation and scales (providing stroke severity) or neuroimaging (providing accurate size of the lesion) are more reliable predictors for clinical outcome prediction. In clinical practice, it would be more useful to find a biomarker, which can help to orientate the physician in conditions in which the clinical picture and imaging provide a limited support.
Transient Ischemic Attacks (TIAs) represent a classical example for which a biomarker would be of interest to confirm and distinguish a brain ischemic process from a stroke mimic. Diagnostic biomarkers of TIA have been investigated, but none of the potential candidates reached enough accuracy for TIA diagnosis. Our group has found that Extracellular Vesicles (EVs) could be useful as biomarkers for detecting brain ischemia in patients with TIA because the EV-surface antigen profile appears to be different in patients with transient symptoms, adjudicated to be very likely caused by brain ischemia, compared to patients whose symptoms were less likely to due to brain ischemia. Our study has raised interest in the scientific community recognizing the promising role of of blood-derived EVs analysis in expanding the possibilities to correctly diagnose and classify TIA and stroke events, discriminate them from TIA or stroke mimics, with important future implications in management and therapy of the patients with acute ischemic cerebrovascular syndrome. the validity of our approach needs to be tested in a larger, prospective, multicenter study.
详细描述
Characterization of extracellular vesicles Extracellular vesicles (EVs) will be characterized in pre-cleared serum samples (see transfer and managements of blood samples). EVs will be characterized by nanoparticle tracking analysis, western blot, bead-based flow cytometry (direct and inverse approach), and ELISA.
We will evaluate diameter and concentration of serum nanoparticles (NPs) by nanoparticle tracking analysis (NTA) using NanoSight LM10 (Malvern Instruments, United Kingdom) equipped with a 405 nm laser and Nanoparticle Tracking Analysis NTA 2.3 analytic software. A camera will record Brownian movement of EVs and size and number of EVs per mL will be calculated by Stokes-Einstein equation. Three videos of 60 s will be recorded for each analysis.
Western Blot analysis will be performed on protein lysate after EV immuno-capture (using beads covered with antibodies against EV specific tetraspanins, CD9, CD63 and CD81). Blots of representative samples will be incubated with primary antibodies against specific markers expressed on the surface or within EVs (TSG101, Syntenin-1, CD81, Alix) and potential contaminants (GRP94, Apolipoprotein A1, Apolipoprotein B48).
EV profiling will be performed using a standardized multiplex bead-based flow cytometric assay, using MACSPlex Human Exosome Kit (Miltenyi Biotec; Bergisch Gladbach, Germany), as previously described. 22 The detection of 37 different surface antigens commonly expressed on EV membrane (including markers from activated platelets, endothelium, and inflammatory cells) will be simultaneously performed. Background will be subtracted from the MFI value of each marker, and then normalized using by the mean fluorescence intensity levels (MFI) of CD9, CD63, and CD81. Levels of expression will be reported as normalized MFI (nMFI; %) for each EV-surface antigen (median value and interquartile range).
After an initial screening by multiplexed assay, single candidate EV biomarkers will be confirmed by either reverse flow cytometry and ELISA techniques.
研究设计
- 研究类型
- Observational
- 观察模型
- Cohort
- 时间视角
- Prospective
入排标准
- 年龄范围
- 18 Years 至 —（Adult, Older Adult）
- 性别
- All
- 接受健康志愿者
- 否
入选标准
- •Affected by:
- •I. Suspect ischemic minor stroke (acute loss of focal cerebral function of presumed vascular etiology and NIHSS ≤327, 28) within 48 hours of symptoms onset or II. TIA (defined as the acute onset of focal neurological symptoms lasting <24 h and presumed to be caused by brain ischemia at the time of referral)19 III. Symptoms mimicking I. or II. with but with high suspition of a non-ischemic condition being the cause of the event;
- •age ≥18 years; 3) if cerebral magnetic resonance imaging will be performed within 48-72 hours from admission; 4) if baseline CT/MRI is without hemorrhage; 5) if informed consent obtained.
排除标准
- •ocular TIA (amaurosis fugax);
- •contraindications for MRI;
- •concomitant acute/chronic inflammatory disease (e.g., infections, autoimmune disease);
- •concomitant hematological diseases;
- •Central Nervous System infection within 30 days;
- •serious head trauma within 30 days;
- •major surgery within 90 days.
结局指标
主要结局
TIA differentiation from TIA mimics
时间窗: 72 hours from symptoms onset
determine the performance of EV profiling (analysis of antigens expressed on vesicles surface), added to a structured clinical (PREDISC score) and imaging evaluation (brain MRI), to discriminate TIAs from TIA mimics (non ischemic events).
次要结局
- TIA differentiation from Stroke(acute phase, 3 and 12 months)
- Stroke etiology(acute phase, 3 and 12 months)
研究者
Dr. med. Carlo Cereda
Prof. Dr. med.
Ospedale Civico, Lugano