Ignite Creation Date:
2024-05-06 @ 8:15 PM
Last Modification Date:
2024-10-26 @ 3:24 PM
Study NCT ID:
NCT06319742
Status:
RECRUITING
Last Update Posted:
2024-04-18
First Post:
2024-03-13
Brief Title:
Extracellular Vesicle Surface Markers In Acute Cerebrovascular Syndromes
Sponsor:
Ospedale Civico Lugano
Organization:
Ospedale Civico Lugano
Study Overview
Official Title:
Extracellular Vesicle Surface Markers In Acute Cerebrovascular Syndromes
Status:
RECRUITING
Status Verified Date:
2024-04
Last Known Status:
None
Delayed Posting:
No
If Stopped, Why?:
Not Stopped
Has Expanded Access:
False
If Expanded Access, NCT#:
N/A
Has Expanded Access, NCT# Status:
N/A
Acronym:
ElViS-ACS
Brief Summary:
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
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
Detailed Description:
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 23 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
The reverse flow cytometric assay will be performed by isolating EVs by capture beads coated with antibodies against CD9-CD63-CD81 EpCam JSR Micro and then incubated with fluorochrome-conjugated antibodies against single EV surface antigens associated to patient diagnosis during the initial screening by MACSPlex assay Beads incubated with isotype-matched antibodies and beads incubated in absence of vesicles will serve as controls Candidate EV biomarkers will be confirmed also by ELISA technique using specific antibodies as per manufacturers instructions
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 23 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
The reverse flow cytometric assay will be performed by isolating EVs by capture beads coated with antibodies against CD9-CD63-CD81 EpCam JSR Micro and then incubated with fluorochrome-conjugated antibodies against single EV surface antigens associated to patient diagnosis during the initial screening by MACSPlex assay Beads incubated with isotype-matched antibodies and beads incubated in absence of vesicles will serve as controls Candidate EV biomarkers will be confirmed also by ELISA technique using specific antibodies as per manufacturers instructions
Study Oversight
Has Oversight DMC:
None
Is a FDA Regulated Drug?:
False
Is a FDA Regulated Device?:
False
Is an Unapproved Device?:
None
Is a PPSD?:
None
Is a US Export?:
None
Is an FDA AA801 Violation?:
None