Predictive Role of Early Blood KLK10 and Other Neuroinflammatory Molecules on Clinical Outcomes of Acute Ischemic Stroke
Conditions: Ischemic Stroke
Sponsor: Ruijin Hospital

Version number: 1.0 Version date: March 11, 2024 
Form No.: KY01003 
v1. 1/ 20200603  
1 
Annex 3. 
 
Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine 
Research projects involving human subjects 
(Applicable to prospective studies) 
 
 
Study Title: 
Predictive Role of Early Blood KLK10 and Other 
Neuroinflammatory Molecules on Clinical 
Outcomes of Acute Ischemic Stroke 
Protocol Number: 
2024092 
Principal Investigator: 
Longxuan Li 
Department: 
Neurology 
Study Period: 
April 2024 - March 2025 
       
 
 
 
 
 
 
Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine 
  March 11, 2024 
Version Number: 1.0 
 
 

Version number: 1.0 Version date: March 11, 2024 
Form No.: KY01003 
v1. 1/ 20200603  
2 
1. Research Abstract 
1.1 Abstract 
Study Name: 
Predictive Role of Early Blood KLK10 and Other Neuroinflammatory 
Molecules on Clinical Outcomes of Acute Ischemic Stroke 
Research Introduction: 
This study aims to investigate the dynamic changes and differential 
expression characteristics of neuroinflammatory molecules such as 
kallikrein-related peptidase 10 (KLK10), soluble triggering receptor 
expressed on myeloid cells 2 (sTREM2), and glial fibrillary acidic protein 
(GFAP) in the plasma of patients with first-ever anterior circulation acute 
ischemic stroke (AIS). It will analyze the correlation between these 
molecules and the severity of neurological deficits, infarct volume, brain 
edema, hemorrhagic transformation, progressive stroke, and clinical 
outcomes at three months. The goal is to assess the predictive value of these 
molecules for AIS prognosis, providing guidance for early treatment and new 
molecular targets for prevention and treatment. 
 
Study Objectives 
 : 
1. To study the correlation between early plasma changes in 
neuroinflammatory molecules like KLK10 and clinical outcomes at three 
months post-AIS. 
2. To investigate the relationship between early plasma changes in these 
molecules and hemorrhagic transformation post-AIS. 
3. To explore the correlation between early plasma changes in these 
molecules and the occurrence of progressive stroke post-AIS. 
 
 
Study subjects: 
182 patients with first-ever anterior circulation AIS, regardless of gender and 
age, along with 91 age- and gender-matched healthy individuals as controls. 
 
Research Site: 
Ruijin Hospital North Campus 
Study Interventions : 
Non-interventional studies 

Version number: 1.0 Version date: March 11, 2024 
Form No.: KY01003 
v1. 1/ 20200603  
3 
Study duration: 
April 2024 - March 2025 
Participant Duration: 
3 months 
1.2 Technology Roadmap 
 
 
 
 
 
 
 
 
 
First survey 
(At the time of admission) 
 
Second survey 
(24 hours after onset) 
 
The third survey 
(48 hours after onset) 
 
 
Fourth survey 
(Within 7 days of onset) 
 
 
Fifth survey 
( 3 months after onset) 
 
control group 
(healthy individuals) 
Informed Consent and Recruitment 
Participants will be recruited based on inclusion and exclusion criteria. 
 
ischemic stroke 
group 
Baseline Assessment 
 Survey on gender, age, medical history, smoking, drinking habits, and current 
medication; NIHSS score at admission; measurement of plasma levels of 
KLK10, sTREM2, and GFAP; collection of blood glucose, creatinine, high-
sensitivity C-reactive protein, blood cell analysis report, and brain MRI or CT 
imaging reports.. 
NIHSS Score and Plasma Levels of KLK10, sTREM2, and GFAP Detection 
NIHSS Score and Plasma Levels of KLK10, sTREM2, and GFAP Detection 
Obtain brain CT or MRI reports to extract information on infarct volume, 
brain edema, and hemorrhagic transformation; perform NIHSS scoring to 
assess the occurrence of progressive stroke; and Final Assessment: 
<Good Functional Outcome or Mortality>e plasma levels of KLK10, 
sTREM2, and GFAP. 
Final Assessment: 
<Good Functional Outcome 
or Mortality> 
Note: The control group will only 
undergo the first survey.  

Version number: 1.0 Version date: March 11, 2024 
Form No.: KY01003 
v1. 1/ 20200603  
4 
2. Study background 
2.1 Research Significance 
Stroke is the leading cause of death and disability in China. Intravenous thrombolysis or thrombectomy 
are currently the only approved early treatments for AIS (acute ischemic stroke). However, due to the 
limited time window, the number of patients benefiting from these treatments is restricted, leaving many 
patients with cerebral infarction. Studies have shown that neuroinflammatory molecules rapidly activate 
post-infarction and are involved in the pathogenesis of cerebral infarction. Moderate inflammation helps 
clear necrotic tissue and plays a crucial role in tissue repair and remodeling, while excessive 
inflammation can exacerbate ischemic neuronal damage and impair recovery (Lancet Neurol, 2019, 
18(11): 1058-1066). Inflammatory markers in the blood rise quickly after 2 hours of stroke onset and 
continue to increase within 24 hours (Stroke.2022;53(7):2249-2259), making early detection of blood 
neuroinflammatory markers significant for predicting clinical outcomes in AIS. This study aims to 
dynamically measure changes in a set of neuroinflammatory molecules in the plasma, including 
KLK10, and analyze their correlation with the severity of acute neurological deficits, infarct volume, 
brain edema, hemorrhagic transformation, progressive stroke, and clinical outcomes at three months. 
Given the time-sensitive and challenging nature of AIS management, confirming that a set of 
neuroinflammatory molecules like KLK10 can accurately predict post-stroke clinical outcomes would 
have significant scientific value for guiding early treatment of AIS and could provide new molecular 
targets for stroke prevention and treatment. 
2.2 Research Background 
Microglia, the primary immune cells in the brain, are among the first cells to activate after AIS, playing 
a critical role in accelerating inflammation, increasing or reducing neuronal apoptosis, and regulating 
the post-stroke inflammatory microenvironment, thus affecting stroke prognosis (J Neuroinflammation. 
2022;19(1):88). KLK10, also known as normal epithelial cell-specific molecule-1, is one of the 15 
secreted serine proteases in the kallikrein family. Previous studies have shown that KLK10 is expressed 
in endothelial cells, where it participates in regulating endothelial inflammation and atherosclerosis 
(Elife. 2022;11:e72579). Our recent systematic observations of KLK10's spatial-temporal expression 
and cellular distribution in the brain after ischemia revealed that this molecule is expressed not only in 
cerebral endothelial cells but also in microglia and astrocytes. While it did not significantly increase in 

Version number: 1.0 Version date: March 11, 2024 
Form No.: KY01003 
v1. 1/ 20200603  
5 
the ischemic penumbra in the early stages (2-4 days post-ischemia), it was highly expressed in the 
infarct core area at 2 days post-ischemia and significantly co-labeled with microglia. The specific high 
expression of KLK10 in microglia in specific brain regions post-AIS suggests that it may be involved 
in early pathological damage after cerebral ischemia. Given that the permeability of the blood-brain 
barrier (BBB) changes within 6 hours of stroke onset (Front Neurol. 2020;11:594672), and since 
KLK10 is a secreted molecule, it is likely that its expression can be detected in the blood of AIS patients. 
Whether plasma KLK10 levels can reflect changes in neuroinflammatory damage in AIS patients and 
serve as a specific biomarker for predicting AIS prognosis remains unclear and warrants further 
investigation. 
TREM2 is an innate immune receptor in the brain, consisting of an extracellular immunoglobulin-like 
domain, a transmembrane domain, and a cytoplasmic portion. Post-stroke, TREM2 is activated by 
nucleotides, lipid mediators, and other key factors released by apoptotic neurons, cells/myelin debris, 
playing an important role in regulating neuronal damage (Front Immunol, 2019, 10: 1668). Literature 
shows that TREM2 molecules are mainly expressed in microglia and can reflect their activation status. 
Elevated plasma sTREM2 levels are closely associated with increased risk of death and cardiovascular 
events one year after AIS onset (J Neuroinflammation. 2022;19(1):88). 
Astrocytes, which also play a role in regulating brain inflammation, are the main glial cells in the central 
nervous system and are widely distributed throughout the brain. In their resting state, astrocytes provide 
structural and functional support to neurons, regulate synaptic activity by releasing gliotransmitters, and 
participate in synapse formation and remodeling (Neurochem Int. 2023;162:105456. Neuroscience, 
2019, 396:73-78). After cerebral ischemia, astrocytes become activated and proliferate, limiting 
immune cell infiltration, repairing damaged BBB, and restoring stability in the brain's internal 
environment (Front Immunol, 2020, 11:1024. Neurosci Lett, 2019; 689: 45-55). Our studies and others 
have shown that astrocytes are extensively lost in the infarct core after cerebral ischemia, peaking at 
48-98 hours post-stroke. The GFAP expressed by astrocytes can be released into peripheral blood, and 
its levels in the blood can reflect cell necrosis and BBB disruption (Transl Stroke Res. 2010;1(4):246-
51) and can serve as molecular markers for distinguishing ischemic and hemorrhagic strokes 
(Diagnostics (Basel). 2023 Aug 25;13(17):2757). 

Version number: 1.0 Version date: March 11, 2024 
Form No.: KY01003 
v1. 1/ 20200603  
6 
The pathological damage mechanism of stroke is highly complex. Single neuroinflammatory molecules 
in plasma, whether sTREM2 or GFAP, are insufficient for predicting AIS prognosis. However, 
measuring a set of neuroinflammatory molecule levels may more accurately reflect the degree of 
neuropathological damage in AIS. Therefore, we propose that "early changes in plasma levels of 
KLK10, sTREM2, and GFAP, among other neuroinflammatory molecules, can effectively predict the 
clinical prognosis of AIS." 
2.3 Expected Outcomes 
To reveal whether early plasma levels of KLK10 and other neuroinflammatory molecules can serve as 
significant molecular markers for predicting the prognosis of AIS patients. 
2.4 Risk/Benefit Assessment 
2.4.1 Known Potential Risks 
This study requires collecting 10ml of peripheral blood from AIS patients, with a total of three 
collections, and from healthy individuals, with a total of one collection. There is no significant potential 
risk to patients or healthy individuals, with the rare occurrence of fainting after blood draw. 
2.4.2 Known Potential Benefits 
The study does not provide significant benefits to patients, but the new findings from this study may 
help guide future treatment of stroke. 
2.4.2 Potential Risk/Benefit Assessment 
Given the rare occurrence of fainting in some patients after peripheral blood collection, symptomatic 
treatment will be administered. The success of this project has significant scientific value for guiding 
early treatment of AIS and providing new molecular targets for stroke prevention and treatment, 
potentially bringing important economic value. 
 
 
 
 
 
 
 
 

Version number: 1.0 Version date: March 11, 2024 
Form No.: KY01003 
v1. 1/ 20200603  
7 
3. Principal Investigator Information 
3.1 Principal Investigator's Name, Qualifications, and Contact Information 
Longxuan Li, Professor, Chief Physician 
Phone: 13611649930 
Email: llx12472@rjh.com.cn 
3.2 Main Participants 
Serial 
number 
Name 
gender 
age 
job title 
Specialt
y 
Whether GCP 
Training 
Roles in 
the 
research 
( eg. PI , 
sub-I, 
CRC) 
1 
Longxuan 
Li 
male 
51 
Chief 
Physician 
Neurolo
gy 
yes 
PI 
2 
Bean YIn 
male 
38 
Deputy 
Chief 
Physician 
Neurolo
gy 
yes 
sub-I 
3 
Wei Jin 
male 
38 
Indications 
Physician 
Neurolo
gy 
yes 
sub-I 
4 
Yi Zhang 
male 
33 
Indications 
Physician 
Neurolo
gy 
yes 
CRC 
5 
Pingchen 
Zhang 
female 
29 
Hospitalizat
ion 
Physician 
Neurolo
gy 
yes 
CRC 
 
4. Research Objectives 
1) 
To determine whether early KLK10 and a group of neuroinflammatory molecules in plasma can 
serve as molecular markers for predicting clinical prognosis in AIS (Acute Ischemic Stroke). 
2) 
To determine whether early KLK10 and a group of neuroinflammatory molecules in blood can 
serve as molecular markers for predicting hemorrhagic transformation in AIS. 
 
5. Study Design 
5.1 Overall Design 
A total of 182 patients with first-ever anterior circulation AIS (Acute Ischemic Stroke) hospitalized in 
the Neurology Department at the Northern Campus of our hospital will be selected as the observation 
group, and 91 healthy individuals undergoing physical examination will be selected as the control 
group. For AIS patients, 10 ml of elbow venous blood will be collected at admission and then at 24 
hours and 7 days after stroke onset. For healthy individuals, 10 ml of fasting elbow venous blood will 

Version number: 1.0 Version date: March 11, 2024 
Form No.: KY01003 
v1. 1/ 20200603  
8 
be collected once. The dynamic changes and differential expression characteristics of a group of 
neuroinflammatory molecules, including KLK10, sTREM2, and GFAP in plasma will be observed. 
The early changes (at admission and 24 hours after onset) of these neuroinflammatory molecules in 
plasma will be compared and analyzed in relation to the severity of acute neurological deficits 
(NIHSS score), infarct volume, brain edema, hemorrhagic transformation, occurrence of progressive 
stroke, and clinical prognosis at 3 months (mRS score), to evaluate the predictive value of these 
molecules for clinical outcomes after AIS. 
  
5.2 Defining study endpoints 
The study is completed when patients have completed blood sample collection according to the study 
protocol and follow-up at 3 months after stroke onset. For healthy controls, the study is completed 
after the initial blood sampling and baseline investigation.   
 
5.3 Determining sample size 
Assuming a 40% probability of poor prognosis at 3 months after AIS, with an OR of 2.0 for high 
KLK10 expression and a correlation coefficient of 0.5 between KLK10 and other influencing factors, 
a significance level of 0.05, and a test power of 0.8, the required sample size is calculated using 
PASS11 software's Logistic Regression module. A total of 182 AIS cases are needed. Additionally, 
91 age- and gender-matched individuals will be selected as healthy controls in a 2:1 ratio. 
 
 
6. Research subjects 
6.1 Inclusion Criteria 
Inclusion criteria for AIS patients: 
• 
Selection of 182 patients with first-ever acute ischemic stroke (AIS) who are hospitalized at our 
hospital, aged ≥18 years, with no restriction on gender; 
• 
Patients must be admitted within 24 hours of the onset of AIS; 
• 
Diagnosis must be confirmed by cranial magnetic resonance imaging (MRI) or computed 
tomography (CT); 
• 
All enrolled patients must provide written informed consent. 
Inclusion criteria for healthy controls: 

Version number: 1.0 Version date: March 11, 2024 
Form No.: KY01003 
v1. 1/ 20200603  
9 
• 
Age and gender matched; 
• 
No organic diseases; 
• 
Written informed consent must be signed 
 
6.2 Exclusion criteria 
• 
Intracranial hemorrhage; 
• 
Pregnancy; 
• 
Stroke with unknown onset time; 
• 
Malignant tumors; 
• 
Hematologic disorders; 
• 
Severe liver or kidney dysfunction; 
• 
Recent myocardial infarction (less than 3 months); 
• 
Ongoing anti-inflammatory drug treatment 
 
6.3 Recruitment of research subjects 
• Source of subjects: AIS patients will be recruited from inpatients in the Department of 
Neurology at the Northern Campus; healthy controls will be selected from outpatient 
health check-up populations who have undergone routine blood tests, biochemical tests, 
cranial CT, or MRI 
• Recruitment site: Ruijin Hospital Northern Campus 
 
6.4 Method of allocating research subjects 
not applicable 
 
7. Study Intervention 
7.1 Administration of Study Intervention 
not applicable 
7.1.1 Description of study intervention 
not applicable 
 
7.1.2 Dosage and administration method 
not applicable 
 
7.1.3 Establishment, Storage, Unblinding Method, and Emergency Unblinding 
Procedure for the Trial Drug Code 
not applicable 
 

Version number: 1.0 Version date: March 11, 2024 
Form No.: KY01003 
v1. 1/ 20200603  
10 
7.1.4 Items and frequency of clinical and laboratory examinations to be conducted 
1) Collect reports of complete blood count and biochemical tests upon admission: including 
neutrophil and lymphocyte counts, blood glucose, creatinine, and high-sensitivity C-
reactive protein levels. 
2) Collect blood pressure information upon admission. 
3) Collect reports of cranial MRI or CT scans upon admission, and record the Alberta 
Stroke Program Early CT Score (ASPECTS) (0-10 points). 
4) Measure levels of neuroinflammatory markers KLK10, sTREM2, and GFAP in plasma 
at admission, and at 24 hours and 7 days after stroke 
5) Assess the degree of neurological impairment using the National Institutes of Health 
Stroke Scale (NIHSS) at admission, and at 24 hours, 4 days, 7 days, and discharge after 
stroke. 
6) Collect information on infarct volume within 7 days after stroke, measured using 
diffusion-weighted imaging (DWI) (J Neuroinflammation. 2020 Apr 7;17(1):107). 
7) Collect information on brain edema grading within 7 days after stroke, measured using 
non-contrast cranial CT scans. The severity of brain edema in AIS patients is assessed 
with a score of 0-3 based on our established method (Clin Neurol Neurosurg. 2022 Nov 
2;223:107507). 
8) Collect information on hemorrhagic transformation within 7 days after stroke, measured 
using non-contrast cranial CT scans. 
9) Record the Modified Rankin Scale (mRS) score at 3 months after stroke. 
7.2 Preparation/Handling/Storage/Responsibility 
not applicable 
 
7.3 Measures to reduce bias: 
not applicable 
 
7.4 Follow-up and Compliance 
3 months mRS Score: 0-2 indicates a good outcome, 4-6 indicates a poor outcome 
Grading Description: 
• 
0: No symptoms at all 

Version number: 1.0 Version date: March 11, 2024 
Form No.: KY01003 
v1. 1/ 20200603  
11 
• 
1: No significant disability despite symptoms; able to carry out all usual duties and 
activities 
• 
2: Slight disability; unable to carry out all previous activities but able to look after 
own affairs without assistance 
• 
3: Moderate disability; requiring some help but able to walk without assistance 
• 
4: Severe disability; unable to walk without assistance and unable to attend to own 
bodily needs without assistance 
• 
5: Very severe disability; bedridden, incontinent, and requiring constant nursing care 
and attention 
• 
6: Death 
 
7.5 Research Intervention Commitment 
not applicable 
 
7.6 Research Plan 
Visit 
Screeni
ng 
period 
Observation period 
Follow-up 
period 
1 day 
after 
onset 
On admission 
24 hours 
after 
onset 
24 
hours 
after 
onset 
7 days 
after  
onset 
3 months 
after onset 
Informed consent 
× 
 
 
 
 
 
Inclusion/Exclusion Criteria 
× 
 
 
 
 
 
Basic information collection 
× 
 
 
 
 
 
Peripheral venous blood collection 
 
× 
× 
× 
× 
 
NIHSS score 
 
× 
× 
× 
× 
 
blood sugar 
 
× 
 
 
 
 
KLK10, sTREM2 and GFAP 
detection 
 
× 
× 
× 
× 
 
MRI 
 
 
 
 
× 
 
CT 
× 
 
 
 
× 
 
mRS score 
 
 
 
 
 
× 
 

Version number: 1.0 Version date: March 11, 2024 
Form No.: KY01003 
v1. 1/ 20200603  
12 
8. Discontinuation of study intervention and discontinuation/withdrawal of study 
subjects    
8.1 Discontinuation of Study Intervention 
not applicable 
8.2 Discontinuation/Withdrawal of Study Subjects 
not applicable 
8.3 Loss to follow-up 
To reduce loss of follow-up and missing data, patients who were not followed up on time 
were contacted by telephone.  
 
9. Study endpoint evaluation 
9.1 Primary Endpoint Evaluation 
The study examines the association between plasma levels of KLK10, sTREM2, and GFAP 
with favorable functional outcomes at 3 months after stroke onset. 
9.2 Secondary Endpoint Evaluation 
1) Investigate the correlation between plasma KLK10, sTREM2, and GFAP levels and 
mortality at 3 months after stroke onset. 
2) Examine the correlation between plasma KLK10, sTREM2, and GFAP levels and 
hemorrhagic transformation after infarction, based on brain CT scans to evaluate 
hemorrhagic transformation of cerebral infarction within 7 days after stroke onset. 
3) Explore the association between plasma KLK10, sTREM2, and GFAP levels and the 
occurrence of progressive stroke, with neurological deficits assessed using the NIHSS 
score. Progressive stroke is diagnosed if the NIHSS score increases by 2 points within 7 
days after stroke onset.  
 
9.2 Safety and other evaluations 
not applicable 
 
9.3 Adverse Events and Serious Adverse Events 
not applicable 
 
10. Statistical Analysis 
10.1 General Methods 

Version number: 1.0 Version date: March 11, 2024 
Form No.: KY01003 
v1. 1/ 20200603  
13 
For descriptive statistical analysis, measurement data conforming to a normal distribution are 
expressed as mean ± standard deviation; measurement data not conforming to a normal 
distribution are expressed as median (interquartile range); count data are expressed as rates or 
proportions. For comparing two groups of measurement data with normal distribution and 
equal variance, an independent two-sample t-test is used; for comparing two groups of 
measurement data not conforming to normal distribution and equal variance, a non-parametric 
Mann-Whitney U test is used. For count data in contingency tables, Pearson's chi-square test is 
used if conditions are met; otherwise, Pearson's chi-square test with continuity correction or 
Fisher’s exact test is applied. All tests are two-tailed, with P<0.05 considered statistically 
significant. 
 
10.2 Primary Endpoint Analysis 
Investigate the association between plasma KLK10, sTREM2, and GFAP levels and functional 
prognosis at 3 months: Univariate and multivariate logistic regression models are used to 
evaluate independent predictors, and odds ratios (OR) with 95% confidence intervals (CI) are 
calculated. ROC curves are plotted and the area under the curve (AUC) is calculated. 
 
10.3 Analysis of Secondary Study Endpoints 
1) The correlation between plasma KLK10, sTREM2, and GFAP levels and hemorrhagic 
transformation and mortality at 3 months was investigated: univariate and multivariate 
logistic regression models were used to evaluate independent predictors, and ORs and 
95% CIs were calculated. ROC curves were drawn and AUCs were calculated. 
2) The association between plasma KLK10, sTREM2 and GFAP levels and the occurrence 
of progressive stroke was studied: univariate and multivariate logistic regression models 
were used to evaluate independent predictive factors , calculate OR and 95%CI. ROC 
curves were drawn and AUC was calculated. 
 
10.4 Security Analysis 
not applicable 
 
10.5 Baseline Descriptive Analysis 

Version number: 1.0 Version date: March 11, 2024 
Form No.: KY01003 
v1. 1/ 20200603  
14 
Descriptive statistics are used to compare demographic characteristics and laboratory 
indicators at baseline between groups. Continuous variables such as age, blood pressure, blood 
glucose levels, infarct volume, ASPECTS score, neurological function (NIHSS) score, and 
levels of neuroinflammatory molecules (KLK10, sTREM2, and GFAP) are expressed as mean 
(standard deviation) or median (interquartile range). Categorical variables such as the presence 
of hypertension, diabetes, hyperlipidemia, history of stroke, and occurrence of malignant brain 
edema are expressed as n (percentage). Fisher’s exact test is used to assess differences in 
categorical variables, and the Kruskal-Wallis Rank Sum test is used to detect differences in 
continuous variables.. 
 
10.6 Subgroup Analysis 
not applicable 
 
11. Supporting documents and notes 
11.1 Informed Consent Process 
Before the study begins, the researcher must obtain approval from the Ruijin Hospital Ethics 
Committee. An informed consent form should be provided to the research subjects. The procedure for 
obtaining informed consent should be described, including how the consent is obtained, the 
environment during the signing, and issues such as signing on behalf of the subject by a guardian. 
Informed consent should be completed before the research subject or their legal guardian agrees to 
participate in the study and should continue throughout the study. The informed consent form, 
approved by the ethics committee, should be read by the research subject. The researcher will explain 
the study process, answer any questions from the subjects, and inform them of potential risks and their 
rights. Subjects may discuss participation with their family or guardian before agreeing. The researcher 
must inform the subjects that participation is voluntary and that they may withdraw from the study at 
any time. A copy of the informed consent form may be provided to the subjects for their records. The 
rights and welfare of the subjects will be protected, and it will be emphasized that their quality of 
medical care will not be affected if they refuse to participate. 
 
11.2 Privacy Protection 
Electronic files should be password protected and stored on a computer, while paper files should be 
kept in a locked cabinet. All data is accessible only to the researchers, and no research information can 
be disclosed to unauthorized third parties without approval. 
 
11.3 Collection and use of specimens and data 
Sample collection :  

Version number: 1.0 Version date: March 11, 2024 
Form No.: KY01003 
v1. 1/ 20200603  
15 
1) Blood collection and separation method: After the subjects sign the informed consent form, 10ml 
of peripheral elbow vein blood is collected using EDTA tubes at the time of admission and at 
follow-up points. The sample should be gently inverted 5-10 times immediately after collection to 
ensure thorough mixing and reduce the risk of hemolysis. Let it stand at room temperature for 30 
minutes. Centrifuge at 2500 rpm for 15 minutes in a high-speed centrifuge. The supernatant is 
collected, and the serum is stored at -80°C for subsequent analysis.  
2) Basic information and labeling rules for sample submission: For samples submitted for testing 
KLK10, sTREM2, and GFAP levels in blood from AIS patients and healthy controls, basic 
information such as the patient's name, age, and date of submission must be provided. The sample 
numbering principle is: the subject's initials (e.g., Zhang San Si, ZSS), research center number, 
subject number (e.g., 0001), and follow-up number (e.g., a for admission, b for 24h after onset, and 
so on). 
3) Sample testing: ELISA is used to detect plasma levels of KLK10, sTREM2, and GFAP, following 
the specific methods in the kit instructions. 
After the study ends, the remaining biological samples will be directly destroyed. Imaging data and 
other data will be stored in electronic files, which should be password protected and stored on a 
computer. Paper files should be kept in a locked cabinet, and all data is accessible only to the 
researchers. No research information can be disclosed to unauthorized third parties without approval. 
All study data and original documents will generally be retained permanently, with permission required 
before destruction. 
 
11.4 Quality Control and Quality Assurance 
All personnel involved in the study should establish a quality assurance system, perform their 
respective duties, and strictly follow the clinical trial protocol and corresponding standard operating 
procedures to ensure the implementation of the quality control and quality assurance system for the 
study. The study will adhere to the relevant SOPs. 
Quality control during the study: The personnel assessing the study site should evaluate the basic 
conditions for participating in the study to ensure they meet protocol requirements. Before initiation, 
the main investigators at the study site should thoroughly train the study staff on the protocol, ensuring 
a clear understanding of inclusion and exclusion criteria and the specific content of various indicators. 
During the study, staff should carefully execute the enrollment and follow-up procedures as required by 
the protocol, and record data accurately, timely, completely, and in a standardized manner. Quality 
control personnel will conduct quality checks on the trial process and corresponding original records. 
After the trial, the study site will organize the relevant project files, which will be checked by quality 
control personnel before archiving. 
 

Version number: 1.0 Version date: March 11, 2024 
Form No.: KY01003 
v1. 1/ 20200603  
16 
Quality control during sample testing: The testing laboratory should have a strict quality assurance 
system and strictly follow domestic and international technical guidelines, laboratory standard 
operating procedures, and quality control procedures. This includes, but is not limited to, personnel 
training and authorization, sample management, standard management, equipment verification, 
calibration and maintenance, method validation, sample testing, and data verification. 
 
Quality control of data entry: Level 1 quality control: The information automation judgment system 
on the user side of EDC will initially check the logic and accuracy of the input information, and 
perform routine logic checks when entering the software (for example, age cannot be filled in as 200, 
etc.), reminding the data entry personnel to implement corrections before entering the data. Level 2 
quality control: The sponsor will send verification personnel to conduct consistency verification of on-
site original data and EDC data from time to time. Level 3 quality control: Quality control is carried out 
by professionals, mainly including the investigation and correction of routine errors in risk assessments 
such as medicine and epidemiology, to avoid errors in assessment reports. Questionnaires with 1% 
positive assessment results and 0.5% negative assessment results will be reviewed and re-evaluated to 
monitor the quality of the assessment. 
 
11.5 Data Processing and Record Keeping 
11.5.1 Data Collection and Management 
1) 
Data entry and management: managed by a dedicated person, research staff should ensure the 
completeness, authenticity and accuracy of the trial data. This study uses the electronic case 
reporting system (EDC) to collect data from included subjects, and all data should be derived from 
original records. The original records of this study, including complete examination reports for 
each follow-up, and clinical diagnosis and treatment information related to stroke, are stored in the 
research center information system. 
2) 
Clinical data will be stored in a database, which should be password protected and a logical 
proofreading procedure should be established when the database is created. 
 
11.5.2 Research Data Retention 
All research data and original documents will generally be retained permanently and permission 
should be obtained before they are destroyed. 
 
11.6 Publication and Data Sharing Agreement 
not applicable 

Version number: 1.0 Version date: March 11, 2024 
Form No.: KY01003 
v1. 1/ 20200603  
17 
 
11.7 Conflict of Interest Statement 
not applicable