Blood proteomic markers of stroke and methods thereof

Abstract

Provided are compositions and methods for evaluating patients with suspected stroke in order to diagnostically distinguish acute ischemic stroke and its subgroups from intracerebral hemorrhage, transient ischemic attack, and stroke mimics; to predict responsiveness to therapy and adverse reactions to therapy; and to predict the likelihood of subsequent stroke, quality of life, and survival.

Description

Technical Field

[0001] This disclosure relates to methods for diagnosing and treating subjects with suspected stroke. This disclosure also relates to a set of biomedical biomarkers associated with a high probability of stroke in the subject. Background Technology

[0002] Stroke affects 17 million people globally each year, and these individuals could benefit from improved diagnosis. Blood biomarkers have the potential to improve the early detection, diagnosis, and treatment of acute stroke, including acute ischemic stroke (AIS) and intracerebral hemorrhage (ICH), in patients with suspected stroke. Similar to their role in patients with chest pain, biomarkers in stroke care can improve healthcare efficiency and save millions of lives and unnecessary costs. These biomarkers will be patented and used to develop simple, inexpensive, and accurate point-of-care tests (POCTs) for use in emergency departments (EDs), pre-hospital settings, outpatient clinics, and hospital wards. The global acute ischemic stroke diagnosis and treatment industry is estimated to reach a market size of $2.4 billion by 2027.

[0003] In 2020, the global incidence of stroke was 11.71 million, of which AIS accounted for 7.59 million (65%), ICH for 3.41 million (29%), and subarachnoid hemorrhage (SAH) for 710,000 [1, 2]. The number of deaths attributable to stroke was 7.08 million, of which AIS accounted for 3.48 million and ICH for 3.25 million [2]. Of the 89.13 million global prevalences across all stroke subtypes, AIS accounted for 68.16 million and ICH for 18.88 million [2]. It is the third leading cause of residue-adjusted life years [3] and results in more than 143 million lost healthy life years each year [1]. In Hong Kong, it is the fourth leading cause of death [4].

[0004] Clinicians diagnose stroke based on clinical history, neurological examination, and cerebrovascular imaging (computed tomography (CT) and / or magnetic resonance imaging (MRI)). The reference criteria for including / excluding stroke are imaging (CT / MRI). Figure 1 [5]. However, many challenges can affect early stroke management.

[0005] When imaging (CT) is not available in an acute context, it can be difficult to distinguish between stroke and ICH, stroke mimicry (SM), and transient ischemic attack (TIA).

[0006] When imaging (CT) is available in the acute phase and ICH is ruled out, the clinical ability to differentiate AIS from SM / TIA is limited, leading to many SMs being inappropriately diagnosed as AIS and treated as AIS in the early stages. Non-contrast CT read by an experienced radiologist or neurologist has 96% specificity for inclusion of ICH in the first few hours, but poor specificity for inclusion of AIS[5]. Non-contrast CT has limited ability to differentiate AIS from SM[6]. Diffusion-weighted MRI is more sensitive than CT because it shows fresh ischemic lesions as early as 2 hours after symptom onset[7]. However, it is usually unavailable in the first few hours. 25% of patients receiving intravenous thrombolysis (IVT) have SM and are inappropriately treated[8]. In addition, CT and MRI provide limited information on cellular and molecular pathophysiology[9]. Diagnosing stroke subtypes (e.g., diagnosing large vessel occlusion (LVO) versus non-LVO) can also be challenging when diagnosing AIS.

[0007] In the early diagnosis and treatment of stroke, timing is critical: rtPA is most effective within 3 hours of symptom onset, although it remains effective within 4.5 hours, and is expected to be effective up to 9 hours in some subgroups. Urokinase thrombolysis, arterial thrombolysis, and anterior circulation thrombectomy all have a 6-hour time window. For subgroups with LVO, endovascular therapy is effective within 24 hours, although there may be some potential use within the extended 24–48 hour time window.

[0008] Therefore, additional, complementary, and alternative diagnostic strategies are needed.

[0009] Biomarkers for acute myocardial infarction (AMI) and mild traumatic brain injury (mTBI)

[0010] In patients with chest pain and suspected AMI, the introduction of blood-based biomarkers has altered diagnostic accuracy, disease assessment, and treatment, and has given patients extended and better quality of life

[10] . In patients with mTBI, a combination of brain-enriched proteins measured in serum and plasma during the acute phase—glial fibrillary acidic protein (GFAP) and ubiquitin C-terminal hydrolase L1 (UCH-L1)—can safely rule out abnormalities on CT with a sensitivity of 96.7% and a negative predictive value of 99.5%, thus improving healthcare efficiency[11,12]. ALINITY iTBI (Abbott) is the first CE-marked, laboratory-based, FDA-approved GFAP / UCH-L1 blood test that enables physicians to objectively rule out the presence of intracranial lesions and makes CT unnecessary

[13] .

[0011] Pathophysiology

[0012] Stroke is a complex and heterogeneous disease with a variety of tissue and biologically specific pathologies and adaptive response pathways to brain injury. However, identifiable patterns exist to guide diagnostic biomarker strategies

[14] . The early stages of acute stroke involve vascular occlusion or rupture, thrombosis, and endothelial injury, which leads to ischemia and / or hemorrhage, neuronal death, apoptosis and necrosis, and leakage of the blood-brain barrier (BBB). This is followed by adaptive repair mechanisms, including immune inflammation and repair responses [15,16].

[0013] TIA and most SMs differ from stroke in that they exhibit multiple pathophysiological patterns [6,17]. ICH differs from AIS in that the former involves vascular rupture, hemorrhage, ischemia, and significantly more neuronal death. This is evidenced by neuronal-rich biomarkers (e.g., GFAP), which rise to much higher plasma levels in ICH than in AIS within the first 6 hours. Figure 2 [14,18]. Therefore, different levels of biomarkers reflect different pathophysiological stages and mechanisms of suspected stroke.

[0014] The diagnosis, risk stratification, prediction, and prognosis of stroke rely on clinical assessment and imaging.

[0015] In a significant proportion of cases, clinical assessment alone is not sensitive enough for accurate diagnosis of stroke. While clinical stroke scales with predictive potential exist (such as the National Institutes of Health Stroke Scale, NIHSS), these scales are subjective, and scores can vary between assessors.

[0016] Radiological techniques such as CT are very accurate in ruling out intracranial hemorrhage, but they cannot accurately classify AIS or differentiate it from SM or TIA in the early stages. MRI has limited availability (depending on whether the patient is good enough for the scan) and may miss small infarcts in critical areas.

[0017] Therefore, a relatively simple, rapid, accurate, non-invasive, and inexpensive blood test may have great clinical practicality and commercial value. Summary of the Invention

[0018] This invention relates to methods for diagnosing and treating subjects with suspected stroke. The invention also relates to a set of biomedical biomarkers associated with a high probability of stroke in a subject. Furthermore, methods, kits, and medical decision support systems are provided for detecting, diagnosing, monitoring, or predicting medical conditions, or for detecting, diagnosing, monitoring, or predicting a subject's responsiveness to treatments for said medical conditions, as well as for classifying subjects.

[0019] This disclosure provides a group of blood protein biomarkers, individually or in combination, that enable rapid diagnosis, risk classification, and prognosis in patients suspected of having stroke. Patients from the community, emergency department, or hospital with symptoms and signs indicative of stroke require accurate early assessment. Patients suspected of having stroke may have AIS (60%), ICH (20%), TIA (10%), or SM (10%).

[0020] In one implementation, a group of multiple biomarkers is provided, wherein the multiple biomarkers are selected to selectively determine the occurrence or non-occurrence of stroke in subjects exhibiting one or more symptoms associated with a stroke diagnosis, wherein the biomarkers are selected to distinguish the occurrence of stroke in the subject from one or more stroke mimicry conditions.

[0021] In some implementations, one or more stroke simulation conditions are selected from the group consisting of: brain tumor, aneurysm, electric shock, burn, infection, cerebral hypoxia, head injury, stress, dehydration, nerve palsy, hypoglycemia, migraine, multiple sclerosis, peripheral vascular disease, peripheral neuropathy, epileptic seizure, subdural hematoma, syncope, and transient unilateral weakness.

[0022] In one implementation, a group of multiple biomarkers is provided, which are selected to selectively determine the occurrence or non-occurrence of acute stroke in subjects exhibiting one or more symptoms associated with a stroke diagnosis.

[0023] In one implementation, a group of multiple biomarkers is provided, which are selected to selectively determine the occurrence or non-occurrence of non-acute stroke in subjects exhibiting one or more symptoms associated with a stroke diagnosis.

[0024] In one implementation, one or more markers from the group are selected to distinguish whether the stroke occurred within 3 hours.

[0025] In one implementation, one or more markers from the group are selected to distinguish whether the stroke occurred within 4.5 hours.

[0026] In one implementation, one or more markers from the group are selected to distinguish whether the stroke occurred within 6 hours.

[0027] In one implementation, one or more markers from the group are selected to distinguish whether the stroke occurred within 9 hours.

[0028] In one implementation, one or more markers from the group are selected to distinguish whether the stroke occurred within a 24-hour window.

[0029] In one implementation, one or more markers from the group are selected to distinguish whether the stroke occurred within a window of 24 to 48 hours.

[0030] Currently, there are no nationally approved blood-based biomarkers that can be used in the first few hours to confirm a stroke diagnosis, classify stroke types, guide treatment, and predict outcomes. Imaging (CT scan or MRI) is the next best option, but its applicability is limited. Early CT scans can diagnose some SMs (e.g., brain cancer) and rule out hemorrhage. However, in the first 12 hours, it only determines the presence and location of AIS in about 20% of subjects. It provides little information about the pathology or timing of the stroke onset. Early CT scans are often not readily available, especially outside of hospitals, and when negative, they offer no guidance on the disease or its progression. MRI is more accurate but still provides limited information about the pathology, is often unavailable in most hospitals at the early stages, and is contraindicated in some patients (e.g., with metallic implants or instability).

[0031] In one implementation, a patient suspected of having a stroke can provide a blood sample for immediate testing. This test determines the presence of biomarkers, quantifies their quantity, and guides the probability of diagnosis, the need for specific treatment, the likely response to treatment, and long-term survival and quality of life.

[0032] In one respect, this disclosure addresses an unmet clinical need for early, objective, and accurate diagnosis of stroke, its subtype, or its analogue in patients with suspected stroke in the community or hospital. It also improves prognosis, risk classification, and responsiveness to treatment.

[0033] In one implementation, blood biomarkers will serve as bedside, point-of-care testing. Abnormal concentrations of biomarkers will indicate the presence of stroke and its subtype. Normal or slightly elevated or decreased biomarker levels will rule out stroke with a high degree of accuracy, thus eliminating the need for further investigation and treatment. Quantitative changes in blood biomarkers within the first few hours confirm whether a stroke has occurred, the type of stroke, whether the stroke will benefit from treatment (e.g., thrombolysis or thrombectomy), and, in patients with unclear onset time, the likely timing of the stroke.

[0034] In one implementation, protein biomarkers can be used to screen patients suspected of having a stroke.

[0035] In one implementation, protein biomarkers are highly diagnostic for stroke.

[0036] In one implementation, protein biomarkers differentiate IS from ICH at an early stage.

[0037] In one implementation, protein biomarkers are useful for disease monitoring and progression.

[0038] Groups of proteins are provided that are biologically and temporally relevant and have sufficiently high sensitivity and specificity to be considered individually and in combination to meet unmet clinical needs.

[0039] Identified blood proteins can be used alone or in combination for the diagnosis, risk stratification, prediction, and prognosis of patients exhibiting stroke-like symptoms.

[0040] Broadly speaking, there are unmet clinical needs for biomarkers and biomarker sets in acute stroke assessment within pre-hospital, outpatient, emergency (or equivalent) and hospital settings. These needs include:

[0041] Diagnostic biomarkers

[0042] a) In situations where imaging is unavailable (e.g., pre-hospital, outpatient, or developing world environments),

[0043] a. Differentiate between stroke (AIS or ICH) and non-stroke (SM or TIA);

[0044] b. Differentiate between AIS and HS; and

[0045] c. Differentiate AIS from low-risk SM (e.g., migraine, depression, dehydration, somnolence);

[0046] b) In cases where imaging (CT) is available in the early stages of stroke (in a hospital or mobile stroke unit),

[0047] a. Differentiate AIS from low-risk SM (e.g., migraine, depression, dehydration, somnolence) in CT-negative suspected stroke cases;

[0048] b. Differentiate between AIS and TIA

[0049] c. In AIS, distinguish between LVO and non-LVO.

[0050] This disclosure relates to the field of diagnosis and treatment of subjects suspected of having stroke, and particularly to a set of biomarker genes for prognostic assessment of stroke. Primary culture and sequencing analysis of tissue samples revealed that a series of genes were significantly upregulated / downregulated in samples from subjects with stroke compared to samples from control subjects; results using these genes as prognostic biomarkers for stroke were interpreted objectively and with high precision. The genomes can be used to develop RT-qPCR kits for in vitro prognostic assessment of stroke.

[0051] This method offers the advantage of providing predictive information in the early stages of disease development, such as stroke. It has been successfully used to identify stratified genes in stroke patients.

[0052] This disclosure provides biomedical biomarkers or groups of biomedical biomarkers that are highly associated with a subject suffering from stroke, wherein the biomedical biomarkers or groups of biomedical biomarkers comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14 biomarkers or all biomarkers.

[0053] This article provides a measure for detecting, diagnosing, classifying, monitoring, or predicting medical conditions, or for detecting, diagnosing, monitoring, or predicting a subject's responsiveness to treatments for said medical conditions (particularly stroke).

[0054] This document provides corresponding methods and medical decision support systems for classifying subjects. In a further aspect, this disclosure relates to the determination of a subject's responsiveness to a therapy for the medical condition (preferably stroke), for detecting, diagnosing, monitoring, or predicting a medical condition, comprising at least the following steps: (a) testing the expression of a group of biomarkers or biomedical biomarkers in samples obtained from the subject; (b) testing the expression of the same biomarker or group of biomarkers in (a) in a control sample; (c) determining the difference in biomarker expression between steps (a) and (b); and (d) based on the results obtained in step (c), determining the presence or stage of the medical condition in the subject receiving the therapy for the medical condition (preferably stroke).

[0055] In one embodiment, this disclosure relates to a method for classifying subjects, comprising: (a) providing a dataset of subjects containing data on gene expression of a hierarchical biomedical marker or group of said markers obtained by the method defined above or as defined in a list or group of biomedical markers as described above or below; (b) accessing a database containing database values ​​for hierarchical biomedical markers or groups of said markers as defined above or as described above or below; and (c) calculating a classification score for the subjects based on the differences between the databases resulting from steps (a) and (b).

[0056] In one embodiment, this disclosure relates to a medical decision support system comprising: input for providing a subject dataset containing data on gene expression of a hierarchical biomedical marker or group of said markers obtained by the methods defined above or as defined in a list or group of biomedical markers as described above; a computer program product for enabling a processor to perform a method for classifying subjects, the method including the methods defined above; and output for outputting subject classification scores.

[0057] The term "predictive value of a medical condition" refers to a value that allows for the assessment of a medical condition or the future development of said medical condition, such as within 1-2 hours, 2-4 hours, 4-6 hours, 6-8 hours, 8-12 hours, 12-24 hours, 1-2 days, 2-4 days, 4-6 days, 1-2 weeks, 2-3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1, 2, 3, 4, 5, 6, 7, 10 years or more, or any other time period. This term also includes all factors associated with said medical condition, such as treatment outcomes, responsiveness to treatment, etc.

[0058] In one embodiment, this disclosure relates to compositions for diagnosing, detecting, monitoring, or predicting a disease (e.g., stroke) in vivo or in vitro, or for diagnosing, detecting, monitoring, or predicting the likelihood of a subject's responsiveness to a therapy. In one embodiment, the therapy is used for stroke. Such compositions may alternatively or additionally comprise thrombolytic therapy or antibodies against any of the aforementioned biomarkers. In one embodiment, a nucleic acid affinity ligand or peptide affinity ligand is modified to function as an imaging contrast agent.

[0059] In one implementation, a method is provided for determining a subject’s eligibility for a therapy to prevent stroke, the method comprising: (a) testing in a sample obtained from the subject a parameter associated with a marker or group of markers as described above; (b) classifying the levels of the tested parameter; and (c) determining whether an individual is eligible to receive the therapy, wherein the subject’s sample is classified as having abnormal expression of one or more of the markers described above.

[0060] In one implementation, expression can be tested by any suitable means known to those skilled in the art, such as room temperature polymerase chain reaction (RT-PCR), RNA sequencing, or gene expression detection on a microarray.

[0061] In one embodiment, this disclosure relates to a medical decision support system, which is a molecular stroke assay decision-making workstation. The decision-making workstation can be used to decide on the initiation and / or continuation of therapy for a subject. In one embodiment, the decision-making workstation is used to determine the probability and likelihood of responsiveness to the therapy. Attached Figure Description

[0062] The patent or application documents contain at least one color drawing. A color copy of the drawings from this patent or patent application publication will be provided by the Patent Office upon request and at the cost of the necessary payment.

[0063] Figure 1 CT images of two patients with suspected stroke. (A) Patient 1 with ICH (arrow). Patient 2 with AIS: The first CT was negative within 6 hours (B), and the second CT showed the lesion 24 hours after the onset of symptoms (arrow) (C).

[0064] Figure 2 Temporal variation of GFAP circulating levels. Plotting GFAP concentrations (combined median with 95% CI). The plot shows that in patients with ICH, GFAP increases rapidly from 3 hours after symptom onset, peaks at 4.5 hours after symptom onset, and then declines steadily until 24 hours. In patients with AIS, GFAP remains at a lower level than in patients with ICH.

[0065] Figure 3. Research workflow for selecting novel proteins with high potential for efficacy in stroke.

[0066] (A) shows a heatmap comparing differentially expressed circulating proteins between stroke and non-stroke patients. (B) shows a volcano plot comparing differentially expressed circulating proteins between stroke and non-stroke patients, and (C) AIS versus control. Example box plots of brain-enriched proteins EHD3(D) and THY1(E) comparing early AIS, ICH, and control within 6 hours of stroke onset. Example box plot of PTPN6(F) comparing stroke and non-stroke patients within 6 hours of stroke onset. Example box plots of differentially expressed CTTN(G), ADGRL4(H), and NIF3L1(I) comparing AIS versus ICH within 6 hours of stroke onset. Example box plots of THY1(J), CTTN(K), and NIF3L1(L) comparing small and large infarct volumes.

[0067] Figure 4A -F. Exemplary timeline of discovery of novel proteins within 24 hours of stroke.

[0068] Based on our preliminary data, we identified distinct patterns of acute changes in circulating proteins over 24 hours compared to age-, sex-, and comorbidity-matched non-stroke controls (dashed horizontal line). Shaded areas represent 95% confidence intervals (CI). From the onset of stroke, some brain-enriched proteins exhibited an (A) upward trend (THY1), (B) a downward trend (EHD3), and (C) a biphasic trend (LEPR). Similar patterns were observed in some adaptive response proteins: PTPN6 (D), BCAM (E), and TAGLN (F).

[0069] Figure 5 The future clinical pathway using novel diagnostic biomarkers.

[0070] 4.1 Definition

[0071] As used herein, the terms “patient” or “subject” are used interchangeably and refer to mammals, including but not limited to human or non-human mammals such as cattle, horses, dogs, sheep, or cats. Preferably, the subject is a human.

[0072] As used in this article, the term "increase or decrease" refers to the ability to cause an overall increase or decrease of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or greater. A decrease can refer to symptoms of a condition being treated, or the presence of a stroke.

[0073] The term "treatment" or "curing" for a condition, symptom, or illness includes:

[0074] (1) To prevent or delay the development of clinical symptoms of a condition, illness, or disease in persons who may have or are susceptible to such condition but have not yet experienced or displayed clinical symptoms of such condition; or

[0075] (2) A state, symptom, or condition that prevents, reduces, or delays the development of a disease or its recurrence (in the presence of maintenance therapy) or at least one of its clinical symptoms, signs, or tests; or

[0076] (3) Relief of disease, that is, the elimination of at least one of the states, symptoms or conditions or clinical or subclinical symptoms or signs.

[0077] The benefits to the subjects treated were statistically significant, or at least perceptible to the patients or physicians.

[0078] "Effective dose for prevention" refers to the amount that effectively achieves the desired preventive outcome within the necessary dosage and time period. Typically, because the preventive dose is administered to the subject before or in an early stage of the disease, the effective dose for prevention will be less than the effective dose for treatment.

[0079] Acceptable excipients, diluents, and carriers for therapeutic use are well known in the pharmaceutical field and described, for example, in Remington: The Science and Practice of Pharmacy. Lippincott Williams & Wilkins (AR Gennaro edit. 2005). The selection of drug excipients, diluents, and carriers can be made with regard to the intended route of administration and standard pharmaceutical practice.

[0080] "Therapeutic effective dose" refers to the amount of a compound that, when administered to an animal for the treatment of a condition, symptom, or illness, is sufficient to affect that treatment. "Therapeutic effective dose" can vary depending on the compound, the disease and its severity, and the age, weight, physical condition, and responsiveness of the animal being treated.

[0081] The compositions disclosed herein may include the compounds described herein in a “therapeutic effective amount” or a “preventive effective amount.” A “therapeutic effective amount” refers to the amount that effectively achieves the desired therapeutic outcome within the necessary dose and time period. Therapeutic effective amounts of antibodies or antibody portions can vary depending on factors such as an individual’s disease state, age, sex, and weight, and the ability of the antibody or antibody portion to elicit the desired response in the individual. A therapeutic effective amount is also the amount in which any toxic or adverse effects of the compounds are outweighed by the beneficial therapeutic effects. A “preventive effective amount” refers to the amount that effectively achieves the desired preventive outcome within the necessary dose and time period. Typically, because the preventive dose is administered to the subject before or at an early stage of the disease, the preventive effective amount will be less than the therapeutic effective amount.

[0082] As used herein, the terms “screen” and “screening” refer to testing subjects or patients to determine whether they have or may have a particular illness or disease, or a particular manifestation of an illness or disease. The term also means testing a drug to determine whether it has a particular effect or efficacy.

[0083] As used herein, the terms "identification," "identify," and "identifying" refer to the identification of a disease state or the clinical manifestations or severity of a disease state in a subject or patient. The term is also used to describe a test drug and its ability to have a specific effect or efficacy.

[0084] The terms “prediction,” “predict,” and “predicting” used in this article refer to prior information based on specific knowledge.

[0085] The terms “prevent” and “prevention” refer to taking action before a disease becomes apparent in order to prevent its development, minimize its severity, or slow its progression.

[0086] As used herein, the term "pharmaceutical" means a substance that produces or is capable of producing an effect, and may include, but is not limited to, chemicals, drugs, biological agents, small organic molecules, antibodies, nucleic acids, peptides, and proteins.

[0087] The terms “about” or “approximately” mean within an acceptable margin of error for a particular value as determined by one of ordinary skill in the art (this will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system), i.e., the precision required for a particular purpose (e.g., pharmaceutical formulation). For example, according to practice in the art, “about” may mean within one or more standard deviations. Alternatively, “about” may mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. Or, particularly with respect to biological systems or processes, the term may mean within orders of magnitude of the value, preferably within 5 times, more preferably within 2 times. Where a particular value is described in this application and claims, unless otherwise stated, the term “about” should be assumed to mean within an acceptable margin of error for the particular value. Invention Details

[0088] Currently, there are no blood tests available for diagnosing stroke in the first few hours after onset. Two blood biomarkers have been used to exclude the need for CT scans in patients with mild traumatic brain injury (mTBI) – GFAP and UCH. These biomarkers are FDA-approved for mTBI but not for stroke. Neither has been rigorously studied in patients with suspected stroke and is not approved for inclusion or exclusion of stroke. We have demonstrated that GFAP can differentiate between AIS and HS within the first six hours.

[0089] Our diagnostic meta-analysis of circulating GFAP levels showed that, within the first 24 hours, especially within 4.5 hours, patients with ICH had significantly higher GFAP levels than those with AIS. Figure 2 Therefore, GFAP can be used as a standalone or supplemental test to exclude ICH from IVT, especially when CT is not routinely available

[20] . More evidence is needed to evaluate the efficacy of GFAP in distinguishing IS from SM, and its efficacy when combined with other biomarkers, such as the NR2A / 2B subunits of NMDAR

[21] . Therefore, we selected GFAP as a potential protein biomarker for further validation.

[0090] Compositions and methods are provided for evaluating patients with suspected stroke in order to first differentiate acute ischemic stroke and its subgroups (e.g., LVO, cardioembolic stroke, small vessel occlusion, atherosclerotic thrombotic stroke, stroke of unknown etiology) from ICH, TIA, and SM in terms of diagnosis.

[0091] High-throughput proteomics study: Recently, we conducted a preliminary high-throughput proteomics study using liquid chromatography-mass spectrometry (LC-MS / MS) on 30 AIS subjects, 6 ICH subjects, and 14 age-, sex-, and comorbidity-matched controls (see Figure 3). We identified 13 novel brain-specific / enriched or biologically relevant proteins that demonstrated the highest early diagnostic efficacy in patients with suspected stroke (see Table 1). We compared these to GFAP, currently the most studied and promising biomarker, selected from our systematic review on early stroke diagnosis. Table 1 summarizes the biological plausibility and early diagnostic efficacy of each of these 14 proteins.

[0092] Brain-specific / enriched proteins: We have identified three brain-specific / enriched proteins with diagnostic potential.

[0093] EH domain-containing protein 3 (EDH3) is an ATP and membrane-bound protein that controls membrane remodeling / tubulation during ATP hydrolysis and is highly expressed in the brain and heart

[25] . It participates in the retrograde dendritic transport of endocytosed basal cell adhesion molecule (BACE1) in a unidirectional manner and in the efficient sorting of BACE1 to axons. This suggests that it is involved in neuronal ATP processing and apoptosis, the latter playing a major role in stroke

[25] . Our previous studies have shown that circulating levels of this protein are increased in patients with AIS in the early stages compared with ICH and controls, with 100% sensitivity and specificity in distinguishing AIS from ICH in the hyperacute phase, and 85% sensitivity and 100% specificity in identifying AIS compared with controls (Figure 4 and Table 1).

[0094] Receptor protein tyrosine phosphatase ζ (PTPRZ1) is a cell surface receptor and chondroitin sulfate (CS) proteoglycan that is highly enriched in the brain and is expressed primarily on neural progenitor cells and glial cells

[26] . It is essential for the normal differentiation of progenitor cells into mature oligodendrocytes and plays a role in protecting oligodendrocytes from apoptosis

[26] . Our preliminary data showed that plasma PTPRZ1 was elevated in patients with acute stroke compared to controls (Figure 4 and Table 1).

[0095] THY1 (CD90) is a glycosylphosphatidylinositol-anchored (GPI-anchored) glycoprotein and is highly expressed in neurons. THY1 has multiple physiological functions, including intercellular signaling, cell differentiation, cell adhesion, and direct involvement in Fas-mediated apoptosis

[27] . Our data showed that THY1 was elevated in patients with ICH compared to AIS and controls (Figure 3). These brain-specific / enriched proteins are released into circulation after stroke and are potential diagnostic biomarkers (Table 1).

[0096] Adaptive response proteins: Other biologically relevant proteins also have high potential for use in the diagnosis of stroke. Gamma-glutamyl cyclase (GGCT) is an essential enzyme of the gamma-glutamyl cycle and plays a crucial role in maintaining glutathione (GSH) homeostasis during normal metabolism by catalyzing the exchange between GSH and gamma-glutamyl amino acid dipeptides

[28] . GGCT levels are positively correlated with GSH levels and are associated with their antioxidant function after cerebral ischemia-reperfusion

[29] . Our preliminary data showed that it was significantly increased in the plasma of patients with AIS, especially in the hyperacute phase (sensitivity 100%, specificity 95%, used to differentiate AIS from controls (Table 1)). Elevated GGCT levels may be a self-protective mechanism that helps scavenge oxygen free radicals, thereby mitigating damage to brain cells.

[0097] BCAM is a cell surface glycoprotein belonging to the immunoglobulin superfamily that functions as a receptor for laminin and adhesion molecules [30, 31] and can protect cells from apoptosis

[32] . It is significantly elevated in circulation in patients with AIS (Figure 4 and Table 1). One possible explanation is that after cerebral infarction, apoptosis increases, and leukocytes migrate into the lesion. Upregulation of BCAM may help reduce apoptosis and promote leukocyte migration.

[0098] NIF3-like protein 1 (NIF3L1) is a cytoplasmic protein that is highly conserved from bacteria to mammals. Its transcript is expressed throughout mouse embryonic development

[33] . It is involved in neuronal differentiation

[34] . Our data showed that it was downregulated in patients with stroke compared to controls (Figure 3, Table 1).

[0099] All 13 proteins identified in our previous studies in human plasma are genetically relevant in mice and can be experimentally studied in a mouse stroke model

[35] .

[0100] Key challenges and unmet clinical needs

[0101] The key challenges in early (<24 hours) stroke diagnosis can be divided into two broad scenarios:

[0102] a) In situations where imaging is unavailable (e.g., pre-hospital, outpatient, some ED / hospital),

[0103] b) Where imaging (CT) is available.

[0104] In situations where imaging is unavailable, there is no accurate method for diagnosing stroke.

[0105] Therefore, there is an unmet clinical need for biomarkers for early detection and diagnosis to differentiate stroke (AIS or ICH) from low-risk SM (e.g., migraine, depression, dehydration, somnolence) and TIA. For example, some patients may not require referral to a hospital.

[0106] In cases where CT imaging is available (e.g., in a hospital or mobile stroke unit), biomarkers are needed to differentiate AIS from low-risk SM (e.g., migraine, depression, dehydration, somnolence) and TIA in patients with CT-negative suspected stroke. Figure 1 ).

[0107] Potential future solutions

[0108] Figure 5 This demonstrates a novel stroke pathway that can integrate new biomarker strategies.

[0109] Bradford Hill Causality Standard

[0110] In this proposal, we will address the analytical aspects that are crucial to strengthening causal evidence in molecular epidemiology and research, namely the Bradford Hill criteria, including association strength, consistency, specificity, timeliness, biological gradient, plausibility, coherence, experimental and analogical evidence

[36] .

[0111] This disclosure provides a highly efficient early blood biomarker for diagnosing stroke and non-stroke conditions, which can be used in clinical settings, including emergency departments (EDs), pre-hospital settings, and general medical settings.

[0112] The selected diagnostic blood biomarkers will be validated in suspected stroke patients and mouse models.

[0113] In some implementations, certain circulating biomarkers, alone or in combination, are used in adult patients suspected of having a stroke who develop symptoms within 24 hours of symptom onset to diagnose acute stroke (AIS, ICH) versus non-stroke (TIA and SM) before imaging, and to diagnose AIS versus non-stroke (SM and TIA) when CT is negative.

[0114] In some implementations, single and consecutive blood samples are collected from a patient population suspected of having a stroke. In some implementations, biomarkers are correlated with clinical findings, including but not limited to following up appropriately for stroke survival and assessing quality of life.

[0115] The earlier stroke is diagnosed and patients with ICH and AIS are differentiated and treated, the more brain cells are preserved and the better the outcome. As a result of pathophysiological responses, protein biomarkers are differentially released into the bloodstream early in the acute phase of stroke (AIS and ICH). The pathology of stroke has been briefly described above and involves encephalopathy, ischemia, stress, necrosis, apoptosis, and BBB damage, which are caused by differential arterial occlusion (as in AIS) or vascular rupture (as in ICH). Although adaptive responses may not be specific to stroke, they are highly correlated when interpreted in the appropriate clinical context (i.e., suspected stroke with chest pain and abdominal inflammation).

[0116] In some embodiments, this disclosure also provides methods for stratifying subjects and / or monitoring their response to treatment prior to treatment, such as the administration of oral and topical medications, lifestyle modifications (such as diet and exercise), and non-traditional treatments (such as acupuncture). This is useful in both patient care and clinical trials.

[0117] In some embodiments, the method includes obtaining the expression level of at least one gene (or corresponding protein) in at least one genetic signature in a subject prior to any treatment. In alternative embodiments, the method includes obtaining the expression level of at least one gene (or corresponding protein) in at least one genetic signature in a normal subject, which serves as a reference expression value. Following a treatment process over a specific time period as can be determined by a person skilled in the art, a biomarker profile or an expression measurement of one or more desired genes (or corresponding proteins) is measured, and differential expression or protein levels, when compared to a reference (e.g., prior to treatment or control levels), may indicate that the subject has a suspected stroke. In some embodiments, the expression level of at least one gene (or corresponding protein) in at least one genetic signature is measured before or after treatment. In alternative embodiments, more than one gene (or corresponding protein) from each signature is measured.

[0118] This disclosure also provides methods for identifying target genes or proteins for drug development.

[0119] This disclosure also considers that, for example, the protein product of any of the genes in the gene signature found in the dataset and / or described in any of the tables or figures herein may have diagnostic value, as well as serve as a potential therapeutic target for patient monitoring, stratification, or drug development.

[0120] Determination and methods for protein detection

[0121] In some implementations, samples of biological tissues or bodily fluids from a subject with the condition to be tested are obtained.

[0122] In some embodiments, the protein level of the sample is tested (a protein inference for any of the biomarkers described herein). The protein sample can be obtained from any biological tissue. In some embodiments, the biological tissue includes, but is not limited to, biopsy tissue, epidermis, whole blood, and plasma. The protein sample can be obtained from any biological fluid. In some embodiments, the fluid includes, but is not limited to, plasma, saliva, and urine. Proteins can be isolated and / or purified from the sample using any method known in the art, including but not limited to immunoaffinity chromatography.

[0123] While any method known in the art may be used, preferred methods for detecting and measuring increased levels of protein in a protein sample include quantitative Western blotting, immunoblotting, quantitative mass spectrometry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradioassay (IRMA), and immunoenzyme assay (IEMA), as well as other sandwich assays, using monoclonal and polyclonal antibodies.

[0124] Antibodies are methods for detecting and measuring target or desired proteins in samples. Such antibodies are commercially available or can be prepared using conventional methods known in the art. These antibodies can be monoclonal or polyclonal, fragments thereof, and their immunobinding equivalents. The term "antibody" refers to homologous molecular entities and mixtures, such as serum products composed of several homologous molecular entities.

[0125] In one implementation, such antibodies will immunoprecipitate the desired protein from solution and react with the desired / target protein in Western blotting, immunoblotting, ELISA, and other assays listed above.

[0126] Antibodies used for these assays can be covalently or non-covalently labeled with a pharmaceutical agent that provides a detectable signal. Any labeling and conjugation method known in the art can be used. Labels include, but are not limited to, enzymes, fluorescent agents, radiolabels, substrates, inhibitors, cofactors, magnetic particles, and chemiluminescent agents. Many fluorescent materials are known and can be used as detectable labels. These include, for example, fluorescein, rhodamine, auramine, Texas red, AMCA blue, and fluorescein yellow. A specific detection material is an anti-rabbit antibody prepared in goats and conjugated to fluorescein via isothiocyanate. Any desired target or binding coupler can also be labeled with a radioactive element or with an enzyme. Radiolabels can be detected by any of the currently available counting procedures. Preferred isotopes can be selected from 3H, 14C, 32P, 35S, 36Cl, 51Cr, 57Co, 58Co, 59Fe, 90Y, 125I, 131I, and 186Re. Enzyme labels are also useful and can be detected by any of currently used colorimetric, spectrophotometric, fluorescence spectrophotometric, amperometric, or gaseous methods. The enzyme is coupled to selected particles by reacting with bridging molecules such as carbodiimide, diisocyanate, glutaraldehyde, etc. Many enzymes that can be used in these methods are known and can be utilized. In embodiments, the enzyme is a peroxidase, β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, glucose oxidase plus peroxidase, and alkaline phosphatase. U.S. Patent Nos. 3,654,090; 3,850,752; and 4,016,043 are cited by way of example because they disclose alternative labeling materials and methods.

[0127] As used herein, the term "sample" or "biological sample" refers to a sample of biological fluid, tissue, or cells obtained from a subject in a healthy and / or pathological state. Such samples include, but are not limited to, blood, bronchoalveolar lavage fluid, sputum, saliva, urine, amniotic fluid, lymph, tissue or fine-needle biopsy samples, peritoneal fluid, cerebrospinal fluid, and supernatants from cell lysates, lysed cells, cell extracts, and nuclear extracts. In some embodiments, whole blood samples are further processed into serum or plasma samples. In some embodiments, the sample includes a blood spot test.

[0128] Reagent test kit

[0129] All assays disclosed herein (e.g., components used to determine the biomarker profile of a sample) may be available in kit form for use by healthcare providers and / or diagnostic laboratories.

[0130] In some embodiments, this disclosure provides a kit comprising one or more probes and / or antibodies for detecting the expression levels of one or more biomarkers as described herein.

[0131] Assays for detecting and quantifying the signature profile of one or more biomarkers can be incorporated into a kit. Such kits may include probes for one or more proteins from one or more biomarkers, as described herein, reagents for isolating and purifying the proteins, instructions for use, and reference values ​​or means for obtaining reference values ​​for the included genes in a control sample.

[0132] Preferred kits for patient classification based on disease activity and clinical presentation will include probes for at least one protein from each of the signatures described herein.

[0133] In another embodiment, the kit will include, for example, reagents for testing biomarkers. Such kits may include antibodies that identify proteins of interest, reagents for isolating and / or purifying proteins from biological tissues or body fluids, reagents for measuring the isolated and purified proteins, instructions for use, and reference values ​​or means for obtaining reference values ​​for the quantity or level of peptides in control samples.

[0134] Preferred kits for detecting and using disease activity will include probes from at least one gene signed by each of the biomarkers described herein. Such kits may include antibodies that identify proteins of interest, reagents for isolating and / or purifying proteins from biological tissues or body fluids, reagents for measuring the isolated and purified proteins, instructions for use, and reference values ​​or means for obtaining reference values ​​for the number or level of peptides in control samples.

[0135] In one implementation, a kit for diagnosing or predicting stroke will include probes targeting at least one gene from each determinant signature, such as any combination of biomarkers as described herein, or the corresponding protein.

[0136] In a further embodiment, a commercially available test kit suitable for use by a specialist physician can be prepared to determine the presence or amount of a desired protein or protein activity, expression, or signature amplification in samples from suspected stroke subjects. One class of such kits may contain at least a labeled target or its binding partner, such as an antibody specific to it, and an indicator, depending on the chosen method, such as a “competitive”, “sandwich”, or similar assay. The kit may also contain accompanying reagents, such as buffers, stabilizers, etc. In one embodiment, the kit comprises one or more antibodies described herein.

[0137] Therefore, a test kit for identifying and quantifying a desired target or protein in cells or samples can be prepared, comprising:

[0138] (a) A predetermined amount of at least one labeled immunochemically reactive component, which is obtained by directly or indirectly attaching a target or its specific binding partner to a detectable label;

[0139] (b) Other reagents; and

[0140] (c) Instructions for use of the kit.

[0141] More specifically, diagnostic test kits may contain:

[0142] (a) A known amount of the target (or binding coupler) as described above, which is typically bound to a solid to form an immunoadsorbent, or, in an alternative, bound to a suitable tag or multiple such end products (or their binding couplers), one copy of each.

[0143] (b) Other reagents, if necessary; and

[0144] (c) Instructions for use of the test kit.

[0145] In a further variation, a test kit can be prepared and used for the above-mentioned purposes, and includes:

[0146] (a) A labeled component obtained by coupling a target to a detectable marker;

[0147] (b) One or more other immunochemical reagents, wherein at least one reagent is a ligand or an immobilized ligand selected from the group consisting of:

[0148] (i) a ligand capable of binding to the labeled component (a);

[0149] (ii) A ligand capable of binding to the binding partner of the labeled component (a);

[0150] (iii) a ligand capable of binding to at least one component to be identified; and

[0151] (iv) a ligand capable of binding to at least one binding pair of at least one component to be identified; and

[0152] (c) Instructions for performing a protocol for detecting and / or determining an immunochemical reaction between a target and its specific binding partner.

[0153] As cited herein, a “target” may include any of the following: any of the genes (including any single or combination) of biomarkers described herein, any corresponding proteins of those genes; alone or in combination with one or more biomarkers.

[0154] Molecular biology

[0155] According to this disclosure, many tools and techniques may exist within the scope of the art, such as those commonly used in molecular immunology, cellular immunology, pharmacology, and microbiology. See, e.g., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY; Ausubel et al. eds. (2005) Current Protocols in Molecular Biology. John Wiley and Sons, Inc.: Hoboken, NJ; Bonifacino et al. eds. (2005) Current Protocols in Cell Biology. (2005) Current Protocols in Microbiology, John Wiley and Sons, Inc.: Hoboken, NJ; Coligan et al. eds. (2005) Current Protocols in Immunology, John Wiley and Sons, Inc.: Hoboken, NJ; Coico etal. eds. ProteinScience, John Wiley and Sons, Inc.: Hoboken, NJ; and Enna et al. eds. (2005) Current Protocols in Pharmacology, John Wiley and Sons, Inc.: Hoboken, NJ.

[0156] In the context of this invention and the specific context in which each term is used, the terms used herein generally have their common meaning in the art. Certain terms are discussed below or elsewhere in the specification to provide additional guidance to practitioners in describing the methods of the invention and how to use them. Furthermore, it will be understood that if the same thing can be expressed in more than one manner. Therefore, alternative language and synonyms may be used for any one or more terms discussed herein, and no particular meaning should be assigned to terms whether or not they are elaborated or discussed herein. Synonyms for certain terms are provided. The recitation of one or more synonyms does not preclude the use of other synonyms. The use of examples anywhere in the specification, including examples of any terms discussed herein, is merely illustrative and in no way limits the scope and meaning of the invention or any exemplary terminology. Likewise, the invention is not limited to its preferred embodiments.

[0157] The invention will be better understood from the experimental details below. However, those skilled in the art will readily understand that the specific methods and results discussed are merely illustrative examples of the invention as more fully described in the appended claims.

[0158] 6. Examples

[0159] Bradford Hill Causal Evidence 1: In order to establish biological rationale, we have and will further investigate the role of each protein (refer to the Human Protein Atlas

[37] ) in brain specificity, enrichment and stroke adaptive responses (e.g., apoptosis, inflammation) through literature and bioinformatics analysis.

[0160] 6.1 Clinical studies of the selected target protein

[0161] Study Design: In a prospective extrapolation and validation cohort study, we will recruit 250 consecutive suspected stroke patients within 24 hours of symptom onset and 100 controls from the ED of Queen Mary Hospital, Hong Kong, over a two-year period from January 1, 2025 to December 31, 2027. The cohort may be divided into two phases.

[0162] Recruitment: We will recruit 125 suspected stroke patients as the extrapolated cohort in the first year of this study. We will recruit two types of controls – a) 25 healthy, age- and sex-matched controls, and b) 25 age-, sex-, and comorbidity-matched controls without acute disease (e.g., diabetes, hypertension, smoking). A neurologist will confirm the stroke (AIS and ICH).

[0163] Inclusion criteria: Adults ≥18 years of age; suspected acute stroke defined as FAST positive, LAPSS positive, or ROSIER >0; symptoms within 24 hours of onset; and informed consent provided. Control subjects were non-neurological patients (matched for age, race, sex, and smoking plus one or more of the following vascular risk factors: diabetes, hypertension, atrial fibrillation, hyperlipidemia) or a) relatives or other adults.

[0164] Data: On day one, we will assess the National Institutes of Health Stroke Scale (NIHSS)

[38] . Study nurses will collect demographic data, medical history, risk factors, medications, time elapsed since symptom onset, laboratory and imaging data.

[0165] Outcomes: The primary outcome was the efficacy of biomarkers for diagnosing acute stroke (AIS or ICH) versus non-stroke (SM, TIA, and control) within 6 hours of symptom onset, expressed as proportion, AUC, specificity, and sensitivity. Secondary outcomes included the temporal and dynamic changes of biomarkers within 24 hours in patients with AIS, ICH, SM, and TIA.

[0166] Indicator testing: The indicator standard is any one or a group of 14 biomarkers. The initial pre-specified cutoff levels are detailed in Table 1. The derivation cohort is semi-exploratory, as it will determine which of the 14 biomarkers has the highest efficacy accuracy.

[0167] Reference criteria: The reference criteria will be the diagnosis of stroke (AIS and ICH) and non-stroke (TIA, SM) made by a neurologist consultant and based on clinical history, neurological examination and neuroimaging (CT; and / or MRI).

[0168] Biological specimen (blood) collection, processing, storage and analysis: We will collect 20 ml of blood from each patient into an EDTA bottle within 24 h of symptom onset. The sample will be centrifuged at 1600 xg at 4°C for 15 minutes within 15 minutes after collection. The plasma fractions will be aliquoted and stored at -80°C. We will use a commercial ELISA kit for the measurement of the target protein

[39] , which is based on the sandwich principle.

[0169] Location: Queen Mary Hospital receives 800 suspected stroke patients annually. In the year prior to this proposed study, we recruited 160 suspected stroke patients (AIS 60%; ICH 10%; TIA 14%; SM 16%) and 25 controls. Prior to starting this study, our goal was to establish a biobank of 250 suspected stroke patients and 50 controls.

[0170] Sample size: During the discovery phase, when the target AUC was 0.9, the specificity was > / = 0.99, and the sensitivity was > / = 0.8, the sample size used to compare the two groups (i.e. stroke and non-stroke) was 53 people in each group (106 people in total) [40,41].

[0171] Bradford Hill Causal Evidence 2: To determine the strength of associations between AIS lesion volume, NIHSS, and each biomarker, we will use samples from 500 patients with suspected stroke (250 from a clinical cohort and 250 from an archived biobank), and present the results as correlation coefficients. The same procedure will be repeated in our mouse study.

[0172] Bradford Hill Causal Evidence 3: To investigate the consistency between 14 biomarkers and the diagnosis of stroke type (AIS and ICH) versus non-stroke (TIA and SM) in patients with suspected stroke, we will run derivation, internal validation, and external validation studies (Figure 6) that compare the sensitivity, specificity, AUC, and relative expression of each biomarker individually and in combination.

[0173] Phase 1A Derivation and Internal Validation Queue 1

[0174] Using data from our preliminary study, we will evaluate our 14 biomarkers, individually and in combination (Table 1), to identify potential models for stroke diagnosis. While our preliminary data provide initial insights into novel protein biomarkers ( Figure 2 -Figure 4 and Table 1), but we need to establish the efficacy of each biomarker in a larger, more robust dataset. We will run a clinical extrapolation study to optimize the results. This initial dataset (Year 1) will undergo internal validation via bootstrapping prior to subsequent validation in the second cohort (Year 2 below), consistent with the Bradford Hill causal evidence mentioned above.3

[0175] Data Analysis: In this phase, we will compare the diagnostic efficacy of each target protein and select individual proteins or groups of proteins (see the statistical plan below).

[0176] Phase 1B Validation Queue 2

[0177] In this phase, we will validate single proteins or groups of proteins selected from phase 1A in an independent cohort (N=125). During this phase, three consecutive blood samples will be collected within 72 hours of symptom onset. At the end of this phase, we will confirm the diagnostic efficacy of the biomarker signature.

[0178] Bradford Hill Causal Evidence 4: To determine the specificity of each biomarker, alone or in combination, in diagnosing AIS and ICH, we will use subdivided SM (low risk (e.g., migraine, psychosis, dehydration) and high risk (e.g., cancer, myelodegenerative disease)), TIA, age-sex-healthy controls, and non-acute comorbidity matched controls (Figure 3).

[0179] Bradford Hill Causal Evidence 5: To establish temporal relationships and dynamic changes, we will use single (within 24 hours) and consecutive (within 72 hours) blood samples from patients with suspected stroke and controls, at which point quantitative changes will be correlated with the timing of symptom onset. We will establish different temporal profiles (Figure 4). Compared to controls, an upward trend in brain enrichment markers and an upward / downward trend in adaptive response markers would be most typical of stroke, while a flat trend may indicate pre-stroke pathology.

[0180] Bradford Hill Causal Evidence 6: We will investigate the biological gradient in human studies by comparing the relationships between normal, mild, moderate and severe stroke types (determined by NIHSS score, infarct or hemorrhage volume and mRS 3 months after stroke).

[0181] 6.2 Mouse experimental model

[0182] Bradford Hill Causal Evidence 7: We will use experiments to establish causal links between circulating proteins and time-varying patterns in our AIS (middle cerebral artery occlusion, MCAO) and ICH mouse models (see below). Such studies are not possible in humans. We have already demonstrated that 14 brain-specific and adaptive response proteins identified in our previous studies have mouse homologs

[35] .

[0183] Procedure: We will use MCAO and ICH models in mice.

[0184] i) Phase 2A AIS (MCAO) model; based on the procedure of our previous study

[42] .

[0185] ii) Stage 2B ICH (hemorrhagic) model; based on previously reported procedures

[43] .

[0186] Experimental Overview: In phase 2A, BALB / c mice were randomly assigned to six groups at 1 h, 3 h, 4.5 h, 6 h, 12 h, and 24 h post-MCAO surgery (N=6 per group, total 36 mice). Phase 2B will include similar grouping (6 groups, N=6 per group, total 36 mice). Sham-operated mice will undergo simplified surgery without vascular occlusion or hemorrhage and will be assigned to six groups (N=6 per group, total 36 mice) based on the aforementioned sacrifice time points. A blank control group (n=6) without any damage will also be included. The total number of mice is 112.

[0187] Biological sample collection, processing, and storage: Blood samples and brains will be collected from mice in the MCAO, hemorrhage, and sham-operated groups at 1 h, 3 h, 4.5 h, 6 h, 12 h, and 24 h post-operation. The same sample collection will also be performed from blank mice to obtain baseline information. 1 mL of blood will be taken from each mouse, centrifuged as in the clinical phase, and plasma will be stored for later analysis. Brain tissue will be frozen for infarct volume, BBB leakage analysis, and immunofluorescence staining (see Methods below). We will evaluate the relationship between circulating levels of biomarkers and infarct volume and BBB leakage. Immunohistochemical analysis of brain tissue will be performed to compare protein levels of biomarkers on the ipsilateral and contralateral sides of the surgical lesion, with changes in ipsilateral stained cells expected to be lower than contralateral. We will also assess the relationship between circulating levels of biomarkers and changes in the number of stained cells from the brain.

[0188] Bradford Hill Causal Evidence 8: In our experimental mouse study, we will investigate consistency through immunohistochemical analysis and immunofluorescence of mouse brain tissue.

[0189] Analysis of mouse brain tissue: Infarct volume will be calculated based on previous reports

[42] . For assessment of BBB integrity, immunoglobulin G (IgG) will be visualized by immunofluorescence staining

[44] . Sections will be examined by fluorescence microscopy. Images will be analyzed using NIH ImageJ software. For immunohistochemical staining of biomarkers in brain tissue, brain sections were first fixed with 4% paraformaldehyde for 10 minutes, blocked with BSA (10%) for 1 h, and then processed as described above

[44] .

[0190] 6.3 Statistical Analysis

[0191] All statistical analyses will be performed using Prism 9 (GraphPad Software Inc. La Jolla, CA92037 USA). Non-normally distributed data will be log-transformed before analysis and expressed as mean ± SD. Statistical significance will be assessed by Student's t-test or one-way ANOVA, with Bonferroni correction for multiple comparisons. We will use the Pearson correlation test to determine the correlation between human plasma biomarker levels and infarct volume and NIHSS score. We will also use the Pearson correlation test to evaluate the correlation between mouse plasma biomarker levels and infarct volume, BBB leakage, and ipsilateral fluorescence density. A p-value < 0.05 will be considered statistically significant. Diagnostic efficacy will be presented as AUC, sensitivity, and specificity. In the preliminary analysis, we will derive a potential model adjusted for age, sex, and comorbidities. We will use univariate analysis followed by multivariate logistic regression. This will be supplemented by machine learning

[45] . Our derived set will determine the optimal cutoff values ​​with sensitivity, specificity, and 95% CI. Our first internal validation will use a bootstrap method. Our second external validation will confirm the efficacy characteristics of the protein model. Indeterminate indicators and reference standards will be tested for sensitivity based on a 95% CI. We will report the time to symptom onset and its relationship with reference standards, as well as adverse events. Results will be reported in accordance with the Transparent Reporting of Individual Prognostic or Diagnostic Multivariate Predictive Models (TRIPOD) statement

[46] and the Standards for Reporting Diagnostic Studies (STARD) 2015 Guidelines for Reporting Diagnostic Accuracy Studies

[47] .

[0192] Point-of-care testing (POCT) is provided for patients with suspected stroke. Figure 5 In cases where imaging is unavailable, it serves as a diagnostic test to guide decisions on whether to refer the patient to a hospital and to support clinical conclusions in the presence of uncertain or negative imaging results.

[0193] Exemplary products, systems, and methods are listed below:

[0194] The foregoing description of the specific embodiments so fully reveals the general nature of this disclosure that others, by applying knowledge within the art of the relevant field (including the content of documents cited and incorporated herein), can readily modify and / or adapt such specific embodiments to various applications without departing from the overall conception of this disclosure, without excessive experimentation. Therefore, based on the teachings and guidance presented herein, such modifications and alterations are intended to be within the meaning and scope of equivalents of the disclosed embodiments. It should be understood that the wording or terminology herein is for descriptive rather than limiting purposes, and that the terminology or terminology in this specification will be interpreted by those skilled in the art based on the teachings and guidance presented herein and the knowledge of those skilled in the art.

[0195] While various embodiments of this disclosure have been described above, it should be understood that they have been presented by way of example, not limitation. It will be apparent to those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of this disclosure. Therefore, this disclosure should not be limited to any of the exemplary embodiments described above, but should be defined solely by the following claims and their equivalents.

[0196] All references cited in this article are incorporated herein by reference in their entirety and for all purposes to the same extent that each individual publication or patent or patent application is specifically and individually indicated to be incorporated herein by reference in its entirety for all purposes.

[0197] Table 1. Newly discovered biomarkers and GFAP with high potential for diagnosis of stroke

[0198]

[0199]

[0200] References

[0201] 1. Feigin VL, Brainin M, Norrving B, et al. World Stroke Organization(WSO): Global Stroke Fact Sheet 2022. Int J Stroke 2022;17:18-29.

[0202] 2. Tsao CW, Aday AW, Almarzooq ZI, et al. Heart Disease and StrokeStatistics-2022 Update: A Report From the American Heart Association.Circulation 2022;145:e153-e639.

[0203] 3. Diseases GBD, Injuries C. Global burden of 369 diseases andinjuries in 204 countries and territories, 1990-2019: a systematic analysisfor the Global Burden of Disease Study 2019. Lancet 2020;396:1204-22.

[0204] 4. Centre for Health Protection. Death Rates by Leading Causes ofDeath, 2001 – 2019. https: / / www.chp.gov.hk / en / statistics / data / 10 / 27 / 117.html[Accessed on 22nd September 2023]

[0205] 5. Kidwell CS, Chalela JA, Saver JL, et al. Comparison of MRI and CTfor detection of acute intracerebral hemorrhage. JAMA 2004;292:1823-30.

[0206] 6. B HB, Akhtar N, Alrohimi A, et al. Stroke mimics: incidence,aetiology, clinical features and treatment. Ann Med 2021;53:420-36.

[0207] 7. Brazzelli M, Sandercock PA, Chappell FM, et al. Magnetic resonanceimaging versus computed tomography for detection of acute vascular lesions inpatients presenting with stroke symptoms. Cochrane Database Syst Rev 2009:CD007424.

[0208] 8. Liberman AL, Choi HJ, French DD, et al. Is the Cost-Effectivenessof Stroke Thrombolysis Affected by Proportion of Stroke Mimics? Stroke 2019;50:463-8.

[0209] 9. Serhal M MR. Evaluation of Cryptogenic Stroke. https: / / www.acc.org / latest-in-cardiology / articles / 2019 / 10 / 10 / 23 / 20 / evaluation-of-cryptogenic-stroke [Accessed on 22nd September 2023].

[0210] 10. Shah ASV, Anand A, Strachan FE, et al. High-sensitivity troponinin the evaluation of patients with suspected acute coronary syndrome: astepped-wedge, cluster-randomised controlled trial. Lancet 2018;392:919-28.

[0211] 11. Diaz-Arrastia R, Wang KKW, Papa L, et al. Acute Biomarkers ofTraumatic Brain Injury: Relationship between Plasma Levels of Ubiquitin C-Terminal Hydrolase-L1 and Glial Fibrillary Acidic Protein. J Neurotraum 2014;31:19-25.

[0212] 12. Papa L, Brophy GM, Welch RD, et al. Time Course and DiagnosticAccuracy of Glial and Neuronal Blood Biomarkers GFAP and UCH-L1 in a LargeCohort of Trauma Patients With and Without Mild Traumatic Brain Injury. JamaNeurol 2016;73:551-60.

[0213] 13. Alinity I TBI. Abbott Ireland.https: / / www.globalpointofcare.abbott / en / lp / apoc / overcoming-challenges-evaluation-mild-traumatic-brain-injury.html [Accessed on 22nd September 2023].

[0214] 14. di Biase L, Bonura A, Pecoraro PM, et al. Unlocking the Potentialof Stroke Blood Biomarkers: Early Diagnosis, Ischemic vs. HaemorrhagicDifferentiation and Haemorrhagic Transformation Risk: A Comprehensive Review.Int J Mol Sci 2023;24.

[0215] 15. Woodruff TM, Thundyil J, Tang SC, et al. Pathophysiology,treatment, and animal and cellular models of human ischemic stroke. MolNeurodegener 2011;6:11.

[0216] 16. Magid-Bernstein J, Girard R, Polster S, et al. CerebralHemorrhage: Pathophysiology, Treatment, and Future Directions. Circ Res 2022;130:1204-29.

[0217] 17. Simmatis LER, Scott SH, Jin AY. The Impact of Transient IschemicAttack (TIA) on Brain and Behavior. Front Behav Neurosci 2019;13:44.

[0218] 18. Abdelhak A, Foschi M, Abu-Rumeileh S, et al. Blood GFAP as anemerging biomarker in brain and spinal cord disorders. Nature ReviewsNeurology 2022;18:158-72.

[0219] 19. Li Q, Zhao L, Chan CL, et al. Multi-Level Biomarkers for EarlyDiagnosis of Ischaemic Stroke: A Systematic Review and Meta-Analysis.International Journal of Molecular Sciences 2023;24.

[0220] 20. Jaeger HS, Tranberg D, Larsen K, et al. Diagnostic performance ofGlial Fibrillary Acidic Protein and Prehospital Stroke Scale foridentification of stroke and stroke subtypes in an unselected patient cohortwith symptom onset < 4.5 h. Scand J Trauma Resusc Emerg Med 2023;31:1.

[0221] 21. Stanca DM, Marginean IC, Soritau O, et al. GFAP and antibodiesagainst NMDA receptor subunit NR2 as biomarkers for acute cerebrovasculardiseases. J Cell Mol Med 2015;19:2253-61.

[0222] 22. Leung LY, Chan CP, Leung YK, et al. Comparison of miR-124-3p andmiR-16 for early diagnosis of hemorrhagic and ischemic stroke. Clin Chim Acta2014;433:139-44.

[0223] 23. Rainer TH, Leung LY, Chan CPY, et al. Plasma miR-124-3p and miR-16 concentrations as prognostic markers in acute stroke. Clin Biochem 2016;49:663-8.

[0224] 24. Rainer TH, Wong LK, Lam W, et al. Prognostic use of circulatingplasma nucleic acid concentrations in patients with acute stroke. Clin Chem2003;49:562-9.

[0225] 25. Lu Q, Insinna C, Ott C, et al. Early steps in primary ciliumassembly require EHD1 / EHD3-dependent ciliary vesicle formation. Nat Cell Biol2015;17:228-40.

[0226] 26. Dwyer CA, Katoh T, Tiemeyer M, et al. Neurons and glia modifyreceptor protein-tyrosine phosphatase zeta (RPTPzeta) / phosphacan with cell-specific O-mannosyl glycans in the developing brain. J Biol Chem 2015;290:10256-73.

[0227] 27. Morris RJ. Thy-1, a Pathfinder Protein for the Post-genomic Era.Front Cell Dev Biol 2018;6:173.

[0228] 28. Oakley AJ, Yamada T, Liu D, et al. The identification andstructural characterization of C7orf24 as gamma-glutamyl cyclotransferase. Anessential enzyme in the gamma-glutamyl cycle. J Biol Chem 2008;283:22031-42.

[0229] 29. Zhang X, Wang X, Khurm M, et al. Alterations of BrainQuantitative Proteomics Profiling Revealed the Molecular Mechanisms ofDiosgenin against Cerebral Ischemia Reperfusion Effects. J Proteome Res 2020;19:1154-68.

[0230] 30. Zen Q, Cottman M, Truskey G, et al. Critical factors in basalcell adhesion molecule / lutheran-mediated adhesion to laminin. J Biol Chem1999;274:728-34.

[0231] 31. Rettig WJ, Garin-Chesa P, Beresford HR, et al. Cell-surfaceglycoproteins of human sarcomas: differential expression in normal andmalignant tissues and cultured cells. Proc Natl Acad Sci U S A 1988;85:3110-4.

[0232] 32. Drewniok C, Schon M, Schon MP. Basal cell adhesion molecule isinversely associated with apoptosis, but plays a limited role for protectionagainst apoptotic stimuli. Skin Pharmacol Phys 2004;17:304-9.

[0233] 33. Tascou S, Uedelhoven J, Dixkens C, et al. Isolation andcharacterization of a novel human gene, NIF3L1, and its mouse ortholog,Nif3l1, highly conserved from bacteria to mammals. Cytogenet Cell Genet 2000;90:330-6.

[0234] 34. Akiyama H, Fujisawa N, Tashiro Y, et al. The role oftranscriptional corepressor Nif3l1 in early stage of neural differentiationvia cooperation with Trip15 / CSN2. J Biol Chem 2003;278:10752-62.

[0235] 35. Human Protein Atlas. https: / / www.proteinatlas.org / humanproteome / brain / mouse+brain [Accessed on 22nd September 2023].

[0236] 36. Fedak KM, Bernal A, Capshaw ZA, et al. Applying the Bradford Hillcriteria in the 21st century: how data integration has changed causalinference in molecular epidemiology. Emerg Themes Epidemi 2015;12.

[0237] 37. Human Protein Atlas.https: / / www.proteinatlas.org / [Accessed on22nd September 2023].

[0238] 38. Guterud M, Fagerheim Bugge H, Roislien J, et al. Prehospitalscreening of acute stroke with the National Institutes of Health Stroke Scale(ParaNASPP): a stepped-wedge, cluster-randomised controlled trial. LancetNeurol 2023;22:800-11.

[0239] 39. Bustamante A, Penalba A, Orset C, et al. Blood Biomarkers toDifferentiate Ischemic and Hemorrhagic Strokes. Neurology 2021;96:e1928-e39.

[0240] 40. Flahault A, Cadilhac M, Thomas G. Sample size calculation shouldbe performed for design accuracy in diagnostic test studies. J Clin Epidemiol2005;58:859-62.

[0241] 41. Obuchowski NA, McClish DK. Sample size determination fordiagnostic accuracy studies involving binormal ROC curve indices. Stat Med1997;16:1529-42.

[0242] 42. Li QY, Tang GH, Xue SH, et al. Silica-coated superparamagneticiron oxide nanoparticles targeting of EPCs in ischemic brain injury.Biomaterials 2013;34:4982-92.

[0243] 43. Xie B, Miao P, Sun Y, et al. Micro-computed tomography forhemorrhage disruption of mouse brain vasculature. Transl Stroke Res 2012;3:174-9.

[0244] 44. Liao B, Geng L, Zhang F, et al. Adipocyte fatty acid-bindingprotein exacerbates cerebral ischaemia injury by disrupting the blood-brainbarrier. Eur Heart J 2020;41:3169-80.

[0245] 45. Tiedt S, Brandmaier S, Kollmeier H, et al. CirculatingMetabolites Differentiate Acute Ischemic Stroke from Stroke Mimics. AnnNeurol 2020; 88:736-46.

[0246] 46. Collins GS, Reitsma JB, Altman DG, et al. Transparent Reportingof a multivariable prediction model for Individual Prognosis or Diagnosis(TRIPOD): the TRIPOD statement. Ann Intern Med 2015; 162:55-63.

[0247] 47. Bossuyt PM, Reitsma JB, Bruns DE, et al. STARD 2015: An UpdatedList of Essential Items for Reporting Diagnostic Accuracy Studies. Radiology2015; 277:826-32.

[0248] 48. Li Q, Zhao L, Chan CL, Zhang Y, Tong SW, Zhang X, Ho JWK, Jiao Y,Rainer TH. Multi-Level Biomarkers for Early Diagnosis of Ischaemic Stroke: ASystematic Review and Meta-Analysis. Int J Mol Sci. 2023 Sep 7;24(18):13821.

[0249] 49. US Patent publication 20230332236A1.

Claims

1. The group comprising one or more biomarkers for selectively determining the occurrence or non-occurrence of stroke in subjects exhibiting one or more symptoms associated with a diagnosis of stroke, wherein the one or more biomarkers are selected from the group consisting of: receptor tyrosine protein phosphatase ζ ("PTPRZ1"), protein 3 containing an EH domain ("EHD3"), Thy-1 membrane glycoprotein ("THY1"), gamma-glutamyl cyclase ("GGCT"), basal cell adhesion molecule ("BCAM"), interleukin-1 receptor type 2 ("IL1R2"), transcollin ("TAGLN"), annexin A7 ("ANXA7"), tyrosine protein phosphatase non-receptor type 6 ("PTPN6"), Src substrate cortactin ("CTTN"), NIF3-like protein 1 ("NIF3L1"), and leptin receptor ("LEPR") and adhesion-type G protein-coupled receptor L4 ("ADGRL4").

2. The group according to claim 1, wherein the one or more symptoms associated with the diagnosis of stroke are selected from the group consisting of: a) sudden numbness or weakness in the face, arm, or leg, especially on one side of the body; b) sudden confusion, difficulty speaking, or incomprehensible speech; c) sudden visual impairment in one or both eyes; d) sudden, severe headache of unknown cause; e) sudden dizziness, loss of balance, or incoordination; f) sudden difficulty walking or lack of coordination; g) other symptoms from the group consisting of: brain tumor, aneurysm, electric shock, burn, infection, cerebral hypoxia, head injury, stress, dehydration, nerve palsy, hypoglycemia, migraine, multiple sclerosis, peripheral vascular disease, peripheral neuropathy, seizure, subdural hematoma, syncope, and transient unilateral weakness.

3. The group according to any one of the preceding claims, wherein the one or more biomarkers selectively determine the occurrence or non-occurrence of acute stroke in subjects exhibiting one or more symptoms associated with a stroke diagnosis, and wherein the biomarkers further comprise glial fibrillary acidic protein ("GFAP").

4. The group according to any one of the preceding claims, wherein the one or more biomarkers selectively determine the occurrence or non-occurrence of non-acute stroke in subjects exhibiting one or more symptoms associated with the stroke diagnosis, and wherein the biomarkers further comprise glial fibrillary acidic protein ("GFAP").

5. The group according to any one of the preceding claims, wherein one or more markers are selected to distinguish whether the onset of the stroke occurs within 3-4.5 hours, 4.5 to 6 hours, 6-9 hours, 9-12 hours, 12-24 hours, or 24-48 hours.

6. The group according to any one of the preceding claims, wherein one or more markers from the group are selected to distinguish whether the stroke occurred within 3 hours.

7. The group according to any one of the preceding claims, wherein one or more markers from the group are selected to distinguish whether the stroke occurred within 4.5 hours.

8. The group according to any one of the preceding claims, wherein one or more markers from the group are selected to distinguish whether the stroke occurred within a 24 to 48-hour window.

9. A method for diagnosing stroke in a first subject, the method comprising: One or more biomarkers were measured from samples from the subject, the biomarkers being selected from the group consisting of: receptor tyrosine protein phosphatase ζ ("PTPRZ1"), protein 3 containing the EH domain ("EHD3"), Thy-1 membrane glycoprotein ("THY1"), γ-glutamyl cyclase ("GGCT"), basal cell adhesion molecule ("BCAM"), interleukin-1 receptor type 2 ("IL1R2"), transcollin ("TAGLN"), annexin A7 ("ANXA7"), tyrosine protein phosphatase non-receptor type 6 ("PTPN6"), Src substrate cortactin ("CTTN"), and NIF3-like protein 1 ("NIF3L1"), wherein an increase in the expression of one or more biomarkers compared to the expression of the same biomarker in a control sample from a second subject who did not have a stroke indicated that the first subject had a stroke.

10. The method of claim 9, further comprising measuring one or more biomarkers from a sample of the first subject, said biomarkers being selected from the group consisting of leptin receptor ("LEPR") and adhesion-type G protein-coupled receptor L4 ("ADGRL4"), wherein a decrease in the expression of said one or more biomarkers, compared to the expression of the same biomarker in a control sample of a second subject who has not suffered a stroke, indicates that the first subject has suffered a stroke.

11. A method for diagnosing stroke in a first subject, the method comprising: One or more biomarkers were measured from samples from the first subject, the biomarkers being selected from the group consisting of: protein 3 containing the EH domain ("EHD3"), γ-glutamyl cyclase ("GGCT"), basal cell adhesion molecule ("BCAM"), NIF3-like protein 1 ("NIF3L1"), interleukin-1 receptor type 2 ("IL1R2"), and Src substrate cortactin ("CTTN"), wherein an increase in the expression of the one or more biomarkers compared to the expression of the same biomarkers in control samples from a second subject who did not have a stroke indicated that the first subject had AIS.

12. The method of claim 11, further comprising measuring the adhesion-type G protein-coupled receptor L4 ("ADGRL4") in a sample from the first subject, wherein a decrease in ADGRL4 expression compared to the expression of ADGRL4 in a control sample from a second subject who has not suffered a stroke indicates that the subject has AIS.

13. The method of claim 9, wherein an increase in THY1 expression compared to the expression of ADDRL4 in a control sample from a second subject who has not suffered a stroke indicates that the subject has an intracerebral hemorrhage ("ICH").

14. The method of claim 13, further comprising measuring glial fibrillary acidic protein ("GFAP") in the sample from the first subject, wherein higher GFAP expression in the first 24 hours compared to a subject with AIS indicates that the subject has ICH.

15. A method for determining stroke in a subject, the method comprising: (a) Samples were obtained from subjects suspected of having a stroke. (b) Determine the level of one or more biomarkers in the sample, said biomarkers being selected from the group consisting of: receptor tyrosine-protein phosphatase ζ ("PTPRZ1"), protein 3 containing an EH domain ("EHD3"), Thy-1 membrane glycoprotein ("THY1"), gamma-glutamyl cyclase ("GGCT"), basal cell adhesion molecule ("BCAM"), interleukin-1 receptor type 2 ("IL1R2"), transcollin ("TAGLN"), annexin A7 ("ANXA7"), tyrosine protein phosphatase non-receptor type 6 ("PTPN6"), Src substrate cortactin ("CTTN"), and NIF3-like protein 1 ("NIF3L1"), leptin receptor ("LEPR"), and adhesion-type G protein-coupled receptor L4 ("ADGRL4"). (c) Compare the levels of one or more biomarkers in the sample with those in a control sample, wherein the control sample is from a subject who has not suffered a stroke.

16. The method of claim 15, wherein the sample from the subject is selected from the group consisting of blood, urine, plasma, serum, cerebrospinal fluid, saliva, sweat, or brain tissue or derivatives thereof, and wherein the marker further comprises glial fibrillary acidic protein ("GFAP").

17. The method of claim 16, wherein multiple blood samples are obtained over a period of time.

18. The method of any one of the preceding claims, further comprising the step of treating the patient with thrombolytic therapy or endovascular therapy.

19. The method of claim 18, wherein the thrombolytic therapy is the administration of tissue plasminogen activator ("TPA") or tenepase.

20. The method of claim 9, wherein the assay is performed using quantitative Western blotting, immunoblotting, quantitative mass spectrometry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradioassay (IRMA), and immunoenzyme assay (IEMA) or sandwich assay with monoclonal and polyclonal antibodies.

21. A method for determining the presence or risk of bleeding in a subject, the method comprising: Test samples were obtained from the subjects; The presence or amount of the following substances in the obtained test sample shall be analyzed: (1) one or more biomarkers selected from the group consisting of PTPRZ1, EHD3, THY1, GGCT, BCAM, IL1R2, TAGLN, ANXA7, PTPN6, CTTN, NIF3L1, LEPR, ADGRL4 and GFAP; and (2) one or more additional proteomics biomarkers and non-proteomics biomarkers, or the mass spectrometry peak levels of the following: specific and non-specific biomarkers of apoptosis, cell adhesion, cell damage, coagulation, glial cell activation, inflammation-mediated, myelination, thrombosis, vascular injury and brain injury; and clinical patient information associated with brain injury in order to infer the probability of the subject's current or future bleeding risk.

22. A kit comprising one or more probes and / or antibodies for detecting the expression levels of biomarkers selected from the group consisting of: receptor-type tyrosine protein phosphatase ζ ("PTPRZ1"), protein 3 containing an EH domain ("EHD3"), Thy-1 membrane glycoprotein ("THY1"), gamma-glutamyl cyclase ("GGCT"), basal cell adhesion molecule ("BCAM"), interleukin-1 receptor type 2 ("IL1R2"), transcollin ("TAGLN"), annexin A7 ("ANXA7"), tyrosine protein phosphatase non-receptor type 6 ("PTPN6"), Src substrate cortactin ("CTTN"), and NIF3-like protein 1 ("NIF3L1"), leptin receptor ("LEPR"), and adhesion-type G protein-coupled receptor L4 ("ADGRL4").

23. A method for treating stroke in a subject, the method comprising: (a) Obtaining samples from subjects suspected of having a stroke; (b) Determine the RNA level of one or more biomarkers in the sample, said biomarkers being selected from the group consisting of: receptor tyrosine protein phosphatase ζ ("PTPRZ1"), protein 3 containing the EH domain ("EHD3"), Thy-1 membrane glycoprotein ("THY1"), γ-glutamyl cyclase ("GGCT"), basal cell adhesion molecule ("BCAM"), interleukin-1 receptor type 2 ("IL1R2"), transcollin ("TAGLN"), annexin A7 ("ANXA7"), tyrosine protein phosphatase non-receptor type 6 ("PTPN6"), Src substrate cortactin ("CTTN"), and NIF3-like protein 1 ("NIF3L1"); (c) Comparing the RNA levels of one or more biomarkers in the sample with those of one or more biomarkers in a control sample, wherein the control sample is from a control subject without stroke; and (d) Treat the subject with one or more thrombolytic or endovascular therapies.

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