Investigation of Protein Heterogeneity in Extracellular Vesicles Derived From Different Human Blood Circulatory Regions
Conditions: Subarachnoid Aneurysm Hemorrhage, Unruptured Intracranial Aneurysm
Sponsor: The First Hospital of Jilin University

Study Protocol 
Version: 2.0 
Version Date: 05-07-2025 
 
1. Title: Investigation of Protein Heterogeneity in Extracellular Vesicles Derived from 
Different Human Blood Circulatory Regions (DBCR-EV-p) 
 
2. Research Background and Rationale 
Extracellular vesicles (EVs) are small, lipid bilayer-enclosed vesicles that are 
broadly secreted by nearly all cell types into the extracellular microenvironment. They 
carry biomolecules such as proteins, lipids, mRNA, and miRNA, and play key roles in 
intercellular communication and disease regulation. Due to their stability and specificity, 
EVs have become a major focus in the study of disease diagnosis and prognosis¹. 
Blood samples are widely used for clinical EV detection due to their accessibility. 
Over the past five years, one-third of the EV studies included in the EV-TRACK 
knowledgebase have used blood-derived EVs². EV detection from blood is increasingly 
applied in the diagnosis and treatment of neurological diseases. Neurons can release 
EVs that cross the blood-brain barrier (BBB) into the bloodstream, reflecting the 
pathological changes of the central nervous system (CNS). Therefore, blood-derived 
EVs are considered potential biomarkers for the development and progression of 
neurological diseases. 
For instance, abnormal aggregation of α-synuclein (α-Syn) is a hallmark of 
Parkinson’s disease. Elevated levels of α-Syn in blood-derived exosomes of Parkinson’s 
disease patients suggest its potential as a diagnostic biomarker³. In patients with 
ischemic stroke, studies have shown that surface antigens on blood-derived EVs from 
patients with transient ischemic attack (TIA) differ from those in healthy controls. 
Markers such as CD8, CD2, and CD62P can help distinguish TIA patients from healthy 
individuals⁴. 
In addition, blood-derived EVs can cross the BBB to exert effects within the 
nervous system and are closely associated with neurological prognosis. For example, 

in ischemic stroke patients treated intravenously with mesenchymal stem cells (MSCs), 
circulating EV levels increased fivefold and correlated with improvements in motor 
function and brain plasticity as observed by MRI⁵. The concentration of EVs in the 
blood also reflects the pathological state of the brain. Studies have found that EV levels 
increase during inflammation and are associated with neural injury and disease⁶. 
Furthermore, since EVs carry surface markers and cargo that reflect their parent cells, 
identifying the temporal release patterns of neuron-specific EVs may provide new 
insights into the pathophysiological changes following stroke. 
Currently, most blood-derived EV samples in existing studies are collected from 
peripheral veins, such as the brachial vein. However, there has been no investigation 
into the heterogeneity of EV contents across different vascular regions such as arteries 
and deep veins of the head and neck. EVs are rapidly cleared from circulation—studies 
in mice have shown that 70% of intravenously administered EVs are eliminated from 
the bloodstream within 2 minutes.⁷ Therefore, EVs extracted from blood in the internal 
jugular vein (which directly drains metabolic products from brain tissue) and from the 
internal carotid artery (which directly supplies blood to the brain) may more accurately 
reflect the concentration and content changes of CNS-derived EVs following 
neurological pathology, making them better sources for diagnostic and prognostic 
biomarker analysis. 
Neurointerventional surgery is a minimally invasive, image-guided medical 
technique. Using microcatheters and microwires, the procedure enables vascular access 
for imaging or interventional treatment of cerebrovascular diseases. During surgery, 
blood from head and neck arteries or veins can be collected. The standard procedure 
involves: after general anesthesia in the neurointerventional suite, the patient undergoes 
groin disinfection. A femoral artery puncture is performed using the Seldinger 
technique, followed by insertion of a 6F or 8F femoral arterial sheath. A 6F or 8F 
guiding catheter is then advanced over a guidewire through the femoral artery, iliac 
artery, abdominal aorta, thoracic aorta, aortic arch, and common carotid artery into the 
internal carotid artery or from the subclavian artery into the vertebral artery. A 
microcatheter is then guided into the aneurysm for treatment with coiling, stent-assisted 

coiling, or flow diverters. After successful embolization, all catheters and sheaths are 
withdrawn, and hemostasis is achieved with compression. 
This project aims to collect blood from the femoral vein, internal jugular vein, and 
internal carotid artery during neurointerventional surgery under general anesthesia for 
EV extraction and proteomic differential analysis. The study will characterize the 
heterogeneity of EVs across different vascular regions in the human body and explore 
diagnostic and prognostic biomarkers for aneurysmal subarachnoid hemorrhage. 
 
3. Objectives of the Study 
 
1. To reveal the proteomic heterogeneity of extracellular vesicles (EVs) across 
different vascular regions in the human body 
 
2. To characterize the differences in EV proteomics within cerebral feeding 
arteries and draining veins between patients with aneurysmal subarachnoid hemorrhage 
and those without hemorrhage 
 
3. To explore EV-derived protein biomarkers for the diagnosis and prognosis of 
subarachnoid hemorrhage 
 
4. Study Design 
4.1 Study Site and Study Population 
This is a prospective, single-center clinical study. Eligible participants will be 
selected from patients admitted to the Department of Neurovascular Surgery at the First 
Hospital of Jilin University who are undergoing neurointerventional surgery under 
general anesthesia for either ruptured or unruptured intracranial aneurysms. During the 
procedure, blood samples will be collected from the femoral vein, internal jugular vein, 
and internal carotid artery for subsequent proteomic analysis. 
Inclusion Criteria: 
 
1. Aged between 18 and 60 years, regardless of gender; 
 
2. Diagnosed with intracranial aneurysm confirmed by MRA, CTA, or DSA; 
 
3. For patients with aneurysmal subarachnoid hemorrhage, diagnosis must be 
confirmed by head CT imaging, and blood sample collection must occur within 3 days 

of symptom onset; 
 
4. Scheduled to undergo neurointerventional surgery for intracranial aneurysm 
under general anesthesia, with the procedure starting between 8:00 AM and 12:00 PM; 
 
5. Willing to comply with the study protocol and data collection procedures; 
 
6. Able to understand and sign the informed consent form. 
 
Exclusion Criteria: 
 
1. Modified Rankin Scale (MRS) score ≥ 2 prior to onset in hemorrhagic cases 
or preoperatively in non-hemorrhagic cases; 
 
2. Presence of other neurological diseases such as Parkinson’s disease, 
Alzheimer’s disease, cerebral infarction, brain tumors, or epilepsy; 
 
3. Presence of other systemic diseases such as diabetes, coronary artery disease, 
cancer, infections, hematological disorders, or severe metabolic diseases that may 
significantly affect the evaluation of EVs in blood; 
 
4. History of severe hepatic or renal dysfunction (ALT > 3× upper limit of normal; 
creatinine > 225 μmol/L); 
 
5. Inability to tolerate anesthesia or anticoagulant therapy, or presence of 
coagulation disorders; 
 
6. History of severe allergic reactions to contrast agents; 
 
7. Pregnant women; 
 
8. Participation in other clinical trials with incomplete follow-up 
 
4.2 Sample Size Determination Method 
This is an exploratory study, and there are currently no similar reports in domestic 
or international literature. As the primary outcome is proteomic differences, potentially 
involving hundreds of differential results, the sample size has been preliminarily set 
based on the planned research budget and clinical experience. A total of 60 cases of 
aneurysmal subarachnoid hemorrhage and 60 cases of unruptured intracranial 
aneurysms will be enrolled. Proteomic analysis of extracellular vesicles from the first 
30 patients will be used to identify intergroup differential markers of interest, which 

will inform potential adjustments to the sample size in the subsequent phase of the study. 
 
4.3 Data Collection 
Eligible patients will be screened based on inclusion and exclusion criteria. After 
obtaining informed consent, the following data will be collected: enrollment date, group 
classification, age, sex, Hunt-Hess grade, Fisher grade, Glasgow Coma Scale (GCS) 
score, time interval from hemorrhage onset to blood collection, complete blood count, 
blood biochemistry, perioperative medications, history of hypertension, smoking 
history, and modified Rankin Scale (mRS) score at discharge (as a prognostic indicator). 
During neurointerventional surgery under general anesthesia, 5 mL of blood will be 
collected from each of the femoral vein, internal jugular vein, and internal carotid artery 
for subsequent proteomic detection and analysis. 
 
Blood Collection Procedure: 
 
1. After general anesthesia takes effect and vital signs are stable, the primary 
surgeon performs femoral vein puncture and inserts a vascular sheath. 
 
2. Under guidance with a guidewire, a catheter is advanced to the internal jugular 
vein on the aneurysm side. Through the catheter, 5 mL of blood is collected from the 
internal jugular vein, and the guiding catheter is then withdrawn. 
 
3. Another 5 mL of blood is collected from the femoral vein through the vascular 
sheath. After collection, the sheath is removed and the puncture site is compressed for 
3–5 minutes. If active bleeding is observed, compression is continued for an additional 
3–5 minutes until hemostasis is achieved. 
 
4. The vascular sheath and catheter are thoroughly flushed with heparinized 
saline. 
 
5. Femoral artery puncture is then performed to insert an arterial sheath. Under 
guidewire guidance, a catheter is advanced to the internal carotid artery on the 
aneurysm side. Five milliliters of blood are collected from the internal carotid artery 
via the guiding catheter, followed by thorough flushing with heparinized saline. 
 
6. A microwire and microcatheter are then delivered through the catheter into the 

intracranial vasculature for interventional treatment of the aneurysm. 
Blood is collected using EDTA tubes. After collection, each tube should be gently 
inverted 10 times to prevent coagulation. 
 
4.4 Data Management and Statistical Analysis Plan 
Data Management: 
A database will be established using electronic spreadsheets. 
Statistical Analysis: 
Continuous variables will be expressed as mean ± standard deviation (SD), and 
categorical variables will be presented as absolute numbers and percentages. Statistical 
analysis will be performed using SPSS version 26.0 (IBM Corp, Armonk, NY). 
Comparisons between two groups for continuous variables will be conducted using the 
independent two-tailed Student’s t-test. For comparisons involving more than two 
groups, one-way analysis of variance (ANOVA) will be applied, followed by Newman–
Keuls post hoc multiple comparisons test. Differences in categorical variables between 
groups will be assessed using the chi-square test. A p-value of < 0.05 will be considered 
statistically significant. 
Handling of Missing or Erroneous Data: 
For subjects with incomplete observations, the Last Observation Carried Forward 
(LOCF) method will be applied. If necessary, alternative imputation methods may be 
used for sensitivity analysis. For erroneous or implausible data, a root cause analysis 
will be conducted, and data may be excluded if necessary. 
 
4.5 Bias Control 
To reduce confounding caused by age and comorbid systemic conditions, patients 
will be strictly screened according to the inclusion and exclusion criteria, enrolling only 
those aged 18–60 years without serious systemic diseases other than intracranial 
aneurysm or subarachnoid hemorrhage. To minimize the impact of time-dependent 
changes in EV protein composition, only patients whose blood samples are collected 
within 3 days of hemorrhage onset and whose surgeries are performed between 8:00 

AM and 12:00 PM will be included. 
To reduce variability in blood lipoprotein levels caused by diet, only patients who 
have fasted for at least 8 hours before surgery under general anesthesia will be included. 
Plasma will be prepared within 1 hour of blood collection to minimize the confounding 
effect of platelet-derived EV release. EVs will be extracted within 12 hours of plasma 
preparation, and EV suspensions will be snap-frozen in liquid nitrogen and stored at –
80°C for subsequent proteomic analysis. This approach aims to reduce changes in EV 
content due to processing delays. 
 
4.6 Quality Management 
The principal investigator is responsible for overall quality control and for clearly 
defining and executing the responsibilities of all study personnel. Prior to study 
initiation, all investigators will be trained on the study protocol by the study team. All 
research staff involved in this study must have valid GCP (Good Clinical Practice) 
certification and adhere strictly to the clinical trial protocol, using standardized 
operating procedures to ensure quality control and assurance. 
All source data must comply with GCP requirements, and laboratory test results 
must be accurate and reliable. The principal investigator will conduct regular and 
systematic reviews of trial-related activities and documentation to assess whether the 
study is being conducted in accordance with the protocol, standard operating 
procedures, and applicable regulations, and to ensure that study data are recorded in a 
timely, truthful, accurate, and complete manner. 
 
4.7 Safety Evaluation 
This study involves patients undergoing neurointerventional procedures under 
general anesthesia. Blood from the internal carotid artery will be collected directly 
during surgery, posing no additional safety risks. Prior to arterial intervention, femoral 
vein puncture and sheath placement will be performed to obtain blood samples from 
the femoral and internal jugular veins. 
Femoral vein catheterization is a standard neurointerventional procedure, 

commonly used in venous access for treating cerebrovascular malformations or dural 
sinus diseases. It is also the gold standard for venous blood sampling in the diagnosis 
of Cushing’s syndrome. The complication rate for femoral vein catheterization for 
cerebral venous sampling is extremely low. Although in theory there may be risks such 
as infection, excessive bleeding, thromboembolism, or vascular injury, these are rarely 
encountered in actual practice. 
In a clinical study involving 246 patients with Cushing’s syndrome undergoing 
femoral vein catheterization for cerebral venous blood sampling, the only observed 
complication was mild femoral vein hematoma in a few cases, with no major procedure-
related adverse events reported8. 
 
4.8 Ethical Review and Informed Consent 
Ethical Approval: 
This study will adhere to the ethical principles outlined in the Declaration of 
Helsinki and comply with the “Good Clinical Practice for Medical Device Trials” and 
relevant national regulations. The study protocol will be submitted for approval by the 
hospital’s ethics committee prior to implementation. If amendments to the protocol are 
required during the course of the study, they must be reviewed and approved by the 
ethics committee before implementation. 
Informed Consent: 
Before the clinical trial begins, investigators must provide detailed information 
about the study to the prospective participants or their legal guardians (authorized 
representatives). The informed consent form must include the following: the name and 
contact information of the investigator, the name of the trial site, the title, purpose, 
methods, and procedures of the study, trial duration, funding sources, potential conflicts 
of interest, expected benefits and known or foreseeable risks, and potential adverse 
events. 
Participation in the study must be voluntary, and participants have the right to 
withdraw at any time without discrimination or retaliation, and without affecting their 
medical care or rights. Participants must be informed that their personal data will be 

kept confidential, but may be accessed by the ethics committee, health authorities, or 
researchers when required according to standard procedures. 
In the event of any study-related harm, participants will be eligible for treatment 
and financial compensation. During the study, participants have the right to access 
relevant information about themselves and may receive other related compensation or 
support. The clinical trial may only proceed after the participant or their legal 
representative has fully understood the study, agreed to participate, signed and dated 
the informed consent form, and the investigator or their delegate has also signed and 
dated the form. For each participant, accurate contact details must be recorded. The 
investigator must also provide their contact information to the participant or their legal 
representative to ensure that they can be reached promptly in the event of any medical 
changes. This also facilitates the investigator’s ability to monitor clinical progress in 
real time. 
 
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