D-dimer, glial fibrillary acidic protein (GFAP), osteoprotegerin (OPG) and osteopontin (OPN) as biomarkers for stroke caused by large vessel occlusion

Abstract

The present invention relates to the diagnosis of stroke resulting from the occlusion of one or more large blood vessels in the brain, and in particular to the diagnosis of stroke resulting from the occlusion of one or more large blood vessels in the brain using one or more biomarkers.

Description

Technical Field

[0001] The present invention relates to the diagnosis of stroke resulting from occlusion of one or more large blood vessels in the brain, and particularly to the diagnosis of stroke resulting from occlusion of one or more large blood vessels in the brain using one or more biomarkers.

Background Art

[0002] Every year, 16 million people worldwide suffer from stroke. More than 30% of these patients die, and 90% of the survivors become permanently disabled. Stroke is a clinical syndrome consisting of the rapid onset of clinical signs of focal (or, in the case of coma, global) impairment of cerebral function, defined as symptoms lasting 24 hours or more, or leading to death without an obvious cause other than a vascular origin. Thus, the definition of stroke excludes transient ischemic attacks (TIAs), which present with the same or similar symptoms as stroke but are defined as a temporary interruption in blood supply to a part of the brain that lasts less than 24 hours; and non-vascular conditions presenting with neurological deficits mimicking stroke, including seizure and post-ictal paralysis (stroke mimics); toxic metabolic disorders; brain tumors; infections; and migraine.

[0003] There are two major types of stroke: ischemic stroke and hemorrhagic stroke. The treatment management for each is very different due to their different etiologies.

[0004] Hemorrhagic stroke occurs due to rupture of blood vessels within the brain and the resulting bleeding. There are two major types of hemorrhagic stroke: intracerebral hemorrhage (bleeding within the brain itself, either parenchymal hemorrhage (bleeding within brain tissue) or intraventricular hemorrhage (bleeding within the ventricular system of the brain)); and subarachnoid hemorrhage (bleeding within the skull but outside the brain tissue; more precisely, bleeding between the arachnoid and pia mater). Hemorrhagic stroke accounts for approximately 15% of strokes seen in patients.

[0005] Ischemic stroke is defined as a physical obstruction of blood flow to a region of the brain and accounts for approximately 85% of strokes seen in patients.

[0006] One type of ischemic stroke is stroke resulting from small vessel occlusion (SVO) (also referred to as lacunar stroke). SVO stroke or lacunar stroke is caused by the occlusion of one or more small blood vessels, typically those supplying the deep brain structures.

[0007] However, the most lethal type of ischemic stroke is stroke resulting from large vessel occlusion (LVO), or simply LVO stroke. LVO stroke results from the occlusion of one or more large blood vessels within the brain, including the common carotid artery, basilar artery, vertebral artery, middle cerebral artery, anterior cerebral artery, posterior cerebral artery, external carotid artery, internal carotid artery, and / or anterior choroidal artery 1 。 Strokes resulting from LVO contribute disproportionately to stroke-related disability and death 1 。

[0008] A typical treatment for patients identified as having or having had an LVO stroke is mechanical thrombectomy (MT). MT is a surgical procedure that mechanically removes blood clots via a probe inserted at the groin level. MT is typically only useful for treating LVO stroke, as the vessels involved in other types of ischemic stroke (e.g., SVO stroke) are too deep within the brain and / or too small to reach with a mechanical probe. Treatment of LVO patients with MT significantly increases the probability of survival and reduces the degree of disability 2~4 。 Mechanical thrombectomy has been proven to be a safe and effective treatment for LVO stroke up to 24 hours from stroke onset 3 , and detection of LVO stroke within hours of onset has been shown to significantly assist the stroke treatment pathway. Improved speed and / or accuracy in identifying individuals presenting with stroke symptoms who have or have had an LVO stroke is very important to ensure that the patient is treated with MT and achieves reperfusion.

[0009] However, ischemic and hemorrhagic strokes typically present with similar symptoms. In addition, various types of ischemic strokes can present with similar symptoms. To further compound these issues, patients presenting with stroke-like symptoms may be suffering from a TIA or stroke mimics, and / or may be suffering from two or more pathologies. These factors mean that an accurate diagnosis of the type of stroke can be difficult.

[0010] Typically, the diagnosis of a stroke is achieved or reported using a computed tomography (CT) scan of the head. CT is very accurate for the detection of brain hemorrhage but can be very inaccurate for the detection of ischemic strokes, and particularly LVO strokes. Also, an MRI scan can be performed to assist in the diagnosis of ischemic strokes and the types of ischemic strokes. If an LVO stroke is suspected, the patient typically undergoes a further procedure called CT angiography. Subsequently, examination of the CTA and / or MRI images by a neuroradiologist is required to diagnose an LVO stroke.

[0011] Therefore, the diagnosis of an LVO stroke in an individual presenting with stroke symptoms can be a time-consuming procedure and requires the involvement of specialists (neuroradiologists). Delays in diagnosis and treatment negatively impact the patient's outcome. A diagnosis that can identify or assist in the identification of individuals suffering from an LVO stroke can expedite the treatment of these patients, thereby reducing mortality and disability rates.

[0012] Several studies have investigated the ability of clinical scores based on a patient's symptoms to assist in the diagnosis of an LVO stroke. 5~10 Such clinical scores are generally based on a simplified version of the widely applied National Institutes of Health Stroke Scale (NIHSS) system, but none of them have shown an improvement in diagnostic performance for the identification of LVO strokes compared to the NIHSS system. 11 The NIHSS system has shown promising results in the identification of LVO strokes, but 12The cut-off values applied to real-world clinical scenarios show insufficient diagnostic performance. 13 This indicates that clinical scores alone are not sufficient to diagnose LVO stroke.

[0013] There are several documents reporting the use of biomarker measurements for differential diagnosis of stroke subtypes. WO2012009567 describes the measurement of an expression profile to determine whether a patient has ischemic stroke. WO2016087611 describes a method for differentiating ischemic and hemorrhagic stroke subtypes by measuring a panel of blood biomarkers. Other studies have evaluated the ability of blood biomarkers to distinguish stroke subtypes and assist in acute stroke diagnosis. Sharma et al. measured 262 proteins and found that a panel of 5 proteins could identify any type of stroke and similar symptoms, 14 but there were no biomarkers suggested for differentiating between stroke subtypes. Bustamante et al. tested a panel of 21 biomarkers for differentiating stroke from similar symptoms and between ischemic and hemorrhagic stroke. 15 From their study, it was concluded that the tested biomarkers were not sufficient for accurate diagnosis of stroke or stroke subtypes in the acute clinical setting. Other studies have attempted to identify biomarkers for diagnosing LVO stroke. Wang et al. found a significant relationship between plasma soluble CD40L / P-selectin and LVO stroke patients. 16 Arenillas and co-workers observed an increase in the amount of blood C-reactive protein (CRP) in patients with intracranial large artery occlusion stroke. 17 Chang et al. reported an association between cardiac biomarkers and LVO stroke. 18 However, there is currently no means, especially for diagnosing stroke arising from LVO.

Prior Art Documents

Patent Documents

[0014] [Patent Document 1] WO2012009567 [Patent Document 2] WO2016087611 [Patent Document 3] U.S. Patent No. 5,475,096 [Patent Document 4] U.S. Patent No. 5,670,637 [Patent Document 5] U.S. Patent No. 5,696,249 [Patent Document 6] U.S. Patent No. 5,270,163 [Patent Document 7] U.S. Patent No. 5,707,796 [Patent Document 8] U.S. Patent No. 5,595,877 [Patent Document 9] U.S. Patent No. 5,660,985 [Patent Document 10] U.S. Patent No. 5,567,588 [Patent Document 11] U.S. Patent No. 5,683,867 [Patent Document 12] U.S. Patent No. 5,637,459 [Patent Document 13] U.S. Patent No. 6,011,020 [Non-Patent Document]

[0015] [Non-Patent Document 1] Konig [Journal fur Praktische Chemie, 1904, 70, 19 - 56] [Non-Patent Document 2] Konig [Journal fur Praktische Chemie, 1904, 69, 105 - 137] [Non-Patent Document 3] Johnson et al. "Magnetic resonance angiography: A review" Academic radiology 5, Vol. 4 (1998): 289 - 305

Non-Patent Document 4

Non-Patent Document 5

Summary of the Invention

Means for Solving the Problems

[0016] According to a first aspect, a method for diagnosing a stroke as a result of large vessel occlusion (LVO), comprising the step of determining the amounts of at least two biomarkers in a sample taken from an individual who has or has been identified as having or suspected of having a stroke, wherein the biomarkers are selected from succinate, succinate - glutathione, N - acetyl - aspartate, propionyl - carnitine, glutamate, heart - type fatty acid - binding protein (H - FABP), brain - type fatty acid - binding protein (B - FABP), beta - amyloid (Abeta) 1 - 40, osteoprotegerin (OPG), soluble tumor necrosis factor - like weak inducer of apoptosis (sTWEAK), pro - vWF, retinol - binding protein 4 (RBP4), ADAMTS13, NMDA receptor 2 peptide (NR2 peptide), 20 - HETE, bilirubin, brain natriuretic peptide (BNP), glial fibrillary acidic protein (GFAP), D - dimer, C - reactive protein (CRP), matrix metalloproteinase 9 (MMP9), interleukin 6 (IL - 6), osteopontin (OPN), troponin I, s100b, von Willebrand factor (vWF) or P - selectin.

[0017] A method of identifying individuals who have suffered a stroke as a result of LVO from a group of individuals who have or are identified as having had or suspected of having had a stroke, the method comprising determining the amounts of at least two biomarkers in a sample taken from each individual, wherein the biomarkers are selected from succinate, succinate-glutathione, N-acetyl-aspartate, propionyl-carnitine, glutamate, heart fatty acid binding protein (H-FABP), brain fatty acid binding protein (B-FABP), Abeta 1-40, osteoprotegerin (OPG), soluble tumor necrosis factor-like weak apoptosis inducer (sTWEAK), pro-vWF, retinol binding protein 4 (RBP4), ADAMTS13, NMDA receptor 2 peptide (NR2 peptide), 20-HETE, bilirubin, brain natriuretic peptide (BNP), glial fibrillary acidic protein (GFAP), D-dimer, C-reactive protein (CRP), matrix metalloproteinase 9 (MMP9), interleukin 6 (IL-6), osteopontin (OPN), troponin I, s100b, von Willebrand factor (vWF) or P-selectin.

[0018] In some embodiments, the method comprises determining the amounts of at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 or 12 or more biomarkers.

[0019] In some embodiments, the method comprises comparing the amount of each biomarker in an individual who has or is identified as having had or suspected of having had a stroke or in a sample taken from each individual to the amount of the same biomarker in one or more of a control, a positive standard, one or more positive criteria or a negative criterion.

[0020] In some embodiments, an individual is diagnosed as having or having had a stroke as a result of LVO if the amount of one or more of the measured biomarkers is different from the amount of the same biomarker in a control or equivalent to the amount of the same biomarker in a positive standard.

[0021] In some embodiments, the method includes converting the amount of one or more biomarkers measured in a sample taken from an individual or individuals in which a stroke is identified or suspected to have occurred into an LVO stroke score.

[0022] In some embodiments, the method includes comparing an LVO stroke score generated using the measured biomarker amount in a sample taken from an individual in which a stroke is identified or suspected to have occurred to an LVO stroke score generated using the biomarker amount from a control or a positive standard.

[0023] In some embodiments, an individual is diagnosed as having or having had a stroke as a result of LVO if the LVO stroke score generated using the measured biomarker amount in a sample taken from an individual in which a stroke is identified or suspected to have occurred is equivalent to the LVO stroke score generated using the biomarker amount from a positive standard, or if the LVO stroke score generated using the measured biomarker amount in a sample taken from an individual in which a stroke is identified or suspected to have occurred is different from the LVO stroke score generated using the biomarker amount from a control.

[0024] In some embodiments, the sample from the individual is one or more of blood, plasma, cerebrospinal fluid, or saliva.

[0025] In some embodiments, the amount of biomarker is determined using an agent that binds to the biomarker. In some such embodiments, detection and / or quantification of the amount of biomarker(s) includes the use of ELISA. In some such embodiments, detection and / or quantification of the amount of biomarker(s) includes the use of a lateral flow assay.

[0026] In some embodiments, the method further includes consideration of data and / or information from one or more clinical evaluations received by the individual. In some such embodiments, the clinical evaluation is a CT or CTA scan, an MRA or MRI scan, or one or more of the individual's NIHSS, FAST, ABCD, ABCD2 Rosier, TOAST, EMSA, PASS, VAN, RACE, FAST_ED or CPSS scores.

[0027] In some embodiments, the amount of biomarker in a sample taken from an individual having or suspected of having had a stroke is determined using an agent that binds to the biomarker, preferably the amount of biomarker is determined using ELISA or lateral flow.

[0028] In some embodiments, the method is performed within about 24 hours of the onset of stroke symptoms in the individual. In some embodiments, the method is performed within about 17 hours of the onset of stroke symptoms in the individual. In some embodiments, the method is performed within about 6 hours of the onset of stroke symptoms in the individual.

[0029] Use of two or more biomarkers for detecting stroke resulting from large vessel occlusion, wherein the biomarkers are selected from succinate, succinate-glutathione, N-acetyl-aspartate, propionyl-carnitine, glutamate, heart-type fatty acid binding protein (H-FABP), brain-type fatty acid binding protein (B-FABP), Abeta 1-40, OPG, soluble tumor necrosis factor-like weak inducer of apoptosis (sTWEAK), pro-vWF, retinol binding protein 4 (RBP4), ADAMTS13, NMDA receptor 2 peptide (NR2 peptide), 20-HETE, bilirubin, brain natriuretic peptide (BNP), glial fibrillary acidic protein (GFAP), D-dimer, C-reactive protein (CRP), matrix metalloproteinase 9 (MMP9), interleukin 6 (IL-6), osteopontin (OPN), troponin I, s100b, von Willebrand factor (vWF), osteoprotegerin (OPG) or P-selectin is provided.

[0030] In some embodiments, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 or 12 or more biomarkers are used to identify stroke resulting from large vessel occlusion.

[0031] In a sixth aspect, a kit for performing the method according to the first or second aspect of the present invention, the kit comprising: a container for holding a sample from an individual having or suspected of having had a stroke; means for determining the amount of a target biomarker; and one or more of instructions for use on how to perform the reaction of the sample and the means provided for determining the amount of the target biomarker.

[0032] For a better understanding of the present invention and to show how embodiments of the present invention may be carried out, reference is now made, by way of example, to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0033]

Figure 1

Figure 2A

Figure 2B

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Mode for Carrying Out the Invention

[0034] Detection of a specific amount of a specific biomarker in a sample from an individual has been found to be able to determine or report a determination as to whether the individual has suffered a stroke resulting from LVO.

[0035] Accordingly, in a first aspect, a method of diagnosing stroke as a result of large vessel occlusion (LVO), comprising determining the amounts of at least two biomarkers in a sample taken from an individual who has or is identified as having or suspected of having had a stroke, wherein the biomarkers are selected from succinate, succinate-glutathione, N-acetyl-aspartate, propionyl-carnitine, glutamate, heart fatty acid binding protein (H-FABP), brain fatty acid binding protein (B-FABP), Abeta 1-40, OPG, soluble tumor necrosis factor-like weak inducer of apoptosis (sTWEAK), pro-vWF, retinol binding protein 4 (RBP4), ADAMTS13, NMDA receptor 2 peptide (NR2 peptide), 20-HETE, bilirubin, brain natriuretic peptide (BNP), glial fibrillary acidic protein (GFAP), D-dimer, C-reactive protein (CRP), matrix metalloproteinase 9 (MMP9), interleukin 6 (IL-6), osteopontin (OPN), troponin I, s100b, von Willebrand factor (vWF), osteoprotegerin (OPG) or P-selectin.

[0036] The phrase "stroke as a result of large vessel occlusion (LVO)" is also referred to herein as "LVO stroke" and means a stroke resulting from occlusion of one or more of the common carotid artery, basilar artery, vertebral artery, middle cerebral artery, anterior cerebral artery, posterior cerebral artery, external carotid artery, internal carotid artery and / or anterior cerebral artery.

[0037] As used herein, the phrase "an individual who has or is suspected of having had a stroke" refers to an individual who exhibits one or more signs or symptoms associated with a stroke. This can include an individual suffering from a TIA or stroke-like symptoms. The phrase "an individual who has or is identified as having had a stroke" as used herein refers to an individual who exhibits one or more signs or symptoms associated with a stroke and, in addition, has been diagnosed as having or having had a stroke.

[0038] Symptoms or signs associated with stroke include arm flapping, sudden onset facial weakness, facial hemiparesis and muscle weakness, speech abnormalities and combinations thereof, such as the FAST test, reduced perception, sensory or vibratory sensation, numbness, initial flaccidity (hypotonia), replacement by spasticity (hypertonia), obligatory synergy, and especially when they appear on one side of the body (unilateral), changes in smell, taste, hearing or vision (whole or in part), hyperreflexia, reduced reflexes (e.g., choking, swallowing, pupillary response to light), reduced facial sensation and muscle weakness, eyelid drooping (ptosis) and extraocular muscle weakness, balance disorder and nystagmus, aphasia, dysarthria, apraxia, changes in respiration and heart rate, atrial fibrillation, weakness of the sternocleidomastoid muscle that cannot turn the head to one side, weakness of the tongue (inability to protrude and / or move from side to side), visual field defect, hemispatial neglect, confused thinking, memory impairment, disorientation, lack of insight into the disorders associated with the subject's usual stroke, changes in motor coordination, changes in gait, dizziness, hypersexual behavior, headache and / or balance disorder.

[0039] The method can be performed in vivo, in vitro or ex vivo. Preferably, the method is performed in vitro or ex vivo. Most preferably, the method is performed in vitro.

[0040] In some embodiments, the method includes determining the amount of at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 or 12 or more biomarkers.

[0041] As described in the examples and shown in FIGS. 2A and 3, the inventors have shown for the first time that the biomarkers OPN, OPG and D-dimer are surprisingly effective in determining whether a subject has suffered a stroke as a result of large vessel occlusion (LVO).

[0042] Therefore, preferably, the method includes a step of determining the amount of at least one biomarker selected from D-dimer, OPN, and OPG. More preferably, the method includes a step of determining the amount of at least two biomarkers selected from D-dimer, OPN, and OPG. Most preferably, the method includes a step of determining the amounts of D-dimer, OPN, and OPG.

[0043] Preferably, the method includes a step of determining the amount of D-dimer. Preferably, the method includes a step of determining the amount of OPN. Preferably, the method includes a step of determining the amount of OPG.

[0044] According to further research by the present inventors, as summarized in FIGS. 5 and 14, the inclusion of GFAP, an additional biomarker, has been shown to provide even higher accuracy in diagnosing stroke as a result of large vessel occlusion (LVO). This finding suggests that the inclusion of GFAP in the biomarker panel may assist in excluding hemorrhagic stroke from the population of patients suspected of having a stroke.

[0045] Therefore, preferably, the method includes a step of determining the amount of at least one biomarker selected from D-dimer, OPN, OPG, and GFAP. More preferably, the method includes a step of determining the amount of at least two biomarkers selected from D-dimer, OPN, OPG, and GFAP. Even more preferably, the method includes a step of determining the amount of at least three biomarkers selected from D-dimer, OPN, OPG, and GFAP. Most preferably, the method includes a step of determining the amounts of the biomarkers D-dimer, OPN, OPG, and GFAP.

[0046] As shown in FIG. 14, surprisingly, a specific combination of D-dimer and GFAP results in a diagnostic specificity of over 90%.

[0047] Therefore, preferably, the method includes a step of determining the amounts of D-dimer and GFAP.

[0048] In one embodiment, the D-dimer protein sequence can be represented by Entrez Gene ID 2243, provided herein as SEQ ID NO: 1.

[0049]

Chemical formula

[0050] Thus, preferably, the D-dimer comprises or consists of substantially the amino acid sequence shown in SEQ ID NO: 1 or a fragment or variant thereof.

[0051] In one embodiment, the D-dimer can be encoded by the nucleotide sequence provided herein as SEQ ID NO: 2.

[0052]

Chemical formula

[0053]

Chemical formula

[0054] Thus, preferably, the D-dimer comprises or consists of substantially the nucleotide sequence shown in SEQ ID NO: 2 or a fragment or variant thereof.

[0055] In one embodiment, the OPN protein sequence can be represented by Entrez Gene ID 6696, provided herein as SEQ ID NO: 3.

[0056]

Chemical formula

[0057] Thus, preferably, OPN comprises or consists of substantially the amino acid sequence shown in SEQ ID NO: 3 or a fragment or variant thereof.

[0058] In one embodiment, OPN can be encoded by the nucleotide sequence provided herein as SEQ ID NO: 4, as follows.

[0059]

Chemical formula

[0060]

Chemical formula

[0061]

Chemical formula

[0062] Thus, preferably, OPN comprises or consists of substantially the nucleotide sequence shown in SEQ ID NO: 4 or a fragment or variant thereof.

[0063] In one embodiment, the OPG protein sequence can be represented by Entrez Gene ID 4982 provided herein as SEQ ID NO: 5, as follows.

[0064]

Chemical formula

[0065] Thus, preferably, OPG comprises or consists of substantially the amino acid sequence shown in SEQ ID NO: 5 or a fragment or variant thereof.

[0066] In one embodiment, OPG can be encoded by the nucleotide sequence provided herein as SEQ ID NO: 6, as follows.

[0067] [Chemistry]

[0068] Thus, preferably, OPG comprises or consists of a nucleotide sequence substantially represented by SEQ ID NO: 6 or a fragment or variant thereof.

[0069] In one embodiment, the GFAP protein sequence can be represented by Entrez Gene No. 2670, provided herein as SEQ ID NO: 7, as follows.

[0070] [Chemistry]

[0071] Thus, preferably, GFAP comprises or consists of an amino acid sequence substantially represented by SEQ ID NO: 7 or a fragment or variant thereof.

[0072] In one embodiment, GFAP can be encoded by the nucleotide sequence provided herein as SEQ ID NO: 8, as follows.

[0073] [Chemistry]

[0074] [Chemistry]

[0075] [Chemistry]

[0076] Thus, preferably, GFAP comprises or consists of a nucleotide sequence substantially represented by SEQ ID NO: 8 or a fragment or variant thereof.

[0077] One of ordinary skill in the art can understand that there are some natural variants of the biomarkers mentioned, and that the method may be extended to the detection of any of these naturally occurring variants of D-dimer, OPN, OPG, and GFAP.

[0078] The sample can be any fluid and / or tissue collected from or derived from an individual who has had a stroke or is identified or suspected of having had a stroke. In some embodiments, the sample is one or more of cells, tissue, cerebrospinal fluid (CSF), whole blood, serum, plasma, cytoplasmic fluid, urine, feces, gastric juice, tears, digestive fluid, saliva, nasal or other airway fluid, vaginal fluid, or semen. In some embodiments, two or more samples can be collected from an individual. In some embodiments, two or more samples can be collected from an individual. In some such embodiments, the samples can be different, for example, the samples can be whole blood and CSF; saliva and whole blood; whole blood, CSF, and tears. In some embodiments, the sample(s) from an individual is one or more of blood, plasma, cerebrospinal fluid, or saliva. Preferably, the sample is a whole blood or plasma sample. More preferably, the sample is a plasma sample. Most preferably, the sample is a whole blood sample.

[0079] In some embodiments, the sample(s) collected from an individual is processed prior to analysis to detect and / or quantify any biomarker(s) in the sample. The need for processing and / or the type of sample processing required depends on the biomarker(s) to be detected and / or the assay used to detect the biomarker(s). In some embodiments, the sample may need to be diluted, for example, with saline, to obtain a concentration appropriate for the biomarker detection assay.

[0080] A whole blood sample can be processed using standard procedures in the art to obtain plasma and / or serum. For example, a whole blood sample can be centrifuged at 2000 g for 15 minutes at 4°C to obtain plasma, which can then be used directly or diluted with saline as needed for the biomarker detection assay.

[0081] Thereafter, the sample is analyzed to detect the presence of a target biomarker in the sample and / or to determine the amount of the target biomarker in the sample. If two or more samples are taken from an individual, the analysis of the samples can be performed simultaneously or sequentially. Samples from two or more individuals may be analyzed simultaneously and / or in the same assay.

[0082] The analysis can be performed by any suitable means. For example, the analysis can be performed by one or more of ELISA, lateral flow immunoassay (lateral flow), radioimmunoassay, radioassay, enzyme activity assay, cell assay, Western blot, Southern blot, Northern blot, immunoprecipitation, immunofluorescence, liquid chromatography, high performance liquid chromatography, positron emission tomography, mass spectrometry, RT-PCR, PCR, mass spectrometry analysis, gel electrophoresis, massensing BioCD protein array, electrochemical immunoassay, surface enhanced Raman spectroscopy, fluorescence-based detection, flow cytometry, nanoparticle-based detection, quantum dot technology, and protein microarray.

[0083] In some embodiments, the analysis technique involves the use of one or more agents that recognize and bind to a biomarker in the sample. The agent(s) may have the ability to bind to two or more biomarkers.

[0084] In some embodiments, the agent is a monoclonal antibody or a polyclonal antibody. The agent may be an intact antibody, a fragment thereof (e.g., Fab or F(ab')2), or an engineered variant (e.g., sFv). Such antibodies may be antibodies of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. Suitable antibodies are commercially available, for example, from Abcam (Cambridge, UK).

[0085] The agent may be labeled to enable detection. Suitable detectable labels are known in the art. As an example, conjugation of an agent with horseradish peroxidase (HRP) enables detection of the agent by addition of a suitable substrate that can be converted by the enzymatic action of HRP to produce a color change. Other examples of detectable labels include alkaline phosphatase, peroxidase, colloidal gold, fluorescent compounds, biotin, radioisotopes, luminescent compounds, magnetic particles, and other enzymes. Detectable labels and labeling kits are commercially available, for example, from Expedeon Ltd, Cambridge, UK.

[0086] Also, the primary / secondary antibody system can be used to detect and / or determine the amount of a biomarker in a sample. In such a system, the sample is contacted with a primary antibody that specifically recognizes and binds one or more biomarkers. Subsequently, a secondary antibody with a suitable label that recognizes the species or isotype of the primary antibody is contacted with the sample. Detection of one or more biomarkers in the sample is achieved by measuring the signal generated by the labeled molecule conjugated to the secondary antibody.

[0087] Antibodies produced in various species can be used to detect biomarkers to which the first antibody binds. For example, a first antibody produced in a rabbit; and a secondary anti-species antibody that can recognize and bind the primary antibody produced in a rabbit.

[0088] The agent may have the ability to bind to two or more biomarkers, one or more of which binds to a substrate. In such embodiments, the agents can be bound with spatial overlap. In some such embodiments, the agents can be differentially labeled to enable detection. For example, the use of color intensity-based labels enables detection and quantification of each spatially overlapping bound biomarker.

[0089] In some embodiments, the sample can be immobilized on a suitable substrate, such as a physical substrate, such as a nitrocellulose or PVDF membrane, a robust substrate made from polystyrene or other plastic polymers, such as a microtiter plate. The substrate can then be contacted with an antibody that specifically recognizes and binds to the first biomarker. Washing of the substrate aids in ensuring specific binding of the antibody to the target biomarker. The antibody can be labeled, for example, through conjugation with a detectable label, such as an enzyme, a fluorophore, or a radioisotope. Detection of the label and quantification of the amount of binding can be achieved by suitable means. Alternatively, a secondary antibody conjugated to a detectable label that binds to the complex of the biomarker and the first antibody can be added, followed by washing to remove excess antibody, ensuring that the presence of the detectable label on the substrate indicates a sample containing the biomarker being measured.

[0090] Alternatively, the biomarker-specific antibody can be immobilized on the substrate, and then the substrate can be contacted with the biomarker, which itself is conjugated to a detectable label. Contact of the labeled biomarker with the antibody results in the antibody binding to the labeled biomarker. The sample is then contacted with the substrate under conditions that allow binding of the biomarker to the antibody. Since the biomarker in the sample is not labeled, replacement of the labeled biomarker from the antibody results in a reduction in the amount of detectable label on the substrate after washing, indicating that the sample contains the biomarker.

[0091] Alternative agents include peptides, aptamers, enzymes or small molecules that specifically bind to a target biomarker. For example, an aptamer that specifically binds to H-FABP may be used. An aptamer is a nucleic acid-based molecule that binds to a specific ligand. Methods for generating aptamers with specific binding specificities are known as detailed in U.S. Patent Nos. 5,475,096, 5,670,637, 5,696,249, 5,270,163, 5,707,796, 5,595,877, 5,660,985, 5,567,588, 5,683,867, 5,637,459 and 6,011,020.

[0092] Quantification of the amount of biomarker in a sample can be performed using any suitable assay. For example, an antibody-based assay or immunosorbent assay such as ELISA, lateral flow, radioimmunoassay or immunoprecipitation. In some embodiments, the analysis is performed using ELISA. In some embodiments, the analysis is performed using a lateral flow assay.

[0093] Alternative and / or complementary methods can be used to detect non-immunogenic molecules or compounds such as metabolites, catabolites, peptides, hormones, signaling molecules, small molecules, drugs, or any other compound or molecule that may not be recognized or bound by antibodies in their native form. Such methods include colorimetric, enzymatic, gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS), high performance liquid chromatography (HPLC) and high performance liquid chromatography-mass spectrometry (HPLC-MS), nuclear magnetic resonance (NMR) mass spectrometry, Raman mass spectrometry. For example, a suitable colorimetric method can be based on a chemical reaction similar to the chemical reactions first described by Konig [Journal fur Praktische Chemie, 1904, 70, 19 - 56 and Journal fur Praktische Chemie, 1904, 69, 105 - 137].

[0094] Furthermore, techniques for measuring biomarkers in vivo involve introducing into a patient a binding agent that specifically recognizes and binds to the biomarker.

[0095] In some embodiments, the amount of biomarker in a sample taken from an individual having or suspected of having had a stroke is compared to a negative standard and / or one or more positive standards.

[0096] The negative standard is a research fluid, such as saline or buffer, in which the absence of the target biomarker or an undetectable amount of the target biomarker is known. The positive standard is a research fluid, such as saline or buffer, that contains the target biomarker in a known amount. The biomarker used to create the positive and / or negative standards may be an isolated target biomarker or a variant thereof, such as a recombinant or synthetic variant of the biomarker.

[0097] In some embodiments, the amount of biomarker in a sample taken from an individual having or suspected of having had a stroke can be compared to a negative standard and / or a range of positive standards that contain various known amounts of the target biomarker.

[0098] In some embodiments, the difference between the amount of biomarker in a sample taken from an individual having or suspected of having had a stroke and the negative standard indicates that the individual has suffered a stroke as a result of LVO. In some embodiments, a statistically significant difference between the amount of biomarker in a sample taken from an individual having or suspected of having had a stroke and the negative standard indicates that the individual has suffered a stroke as a result of LVO. In some embodiments, the correlation between the amount of biomarker in a sample taken from an individual having or suspected of having had a stroke and the positive standard determines or provides an indication of the amount of that biomarker in the sample from the individual.

[0099] Alternatively or in addition, the amount of a biomarker in a sample taken from an individual having or suspected of having had a stroke can be compared to the amount of the same biomarker in a control.

[0100] The control is a sample or data generated from a sample taken from one or more healthy individuals. As used herein, the term "healthy individual" means an individual who does not have signs or symptoms of a stroke and does not have a history of stroke. Alternatively or in addition, the control can be a sample or data generated from a sample taken from one or more individuals having or having had a TIA, stroke-like symptoms, hemorrhagic stroke, or stroke resulting from a small vessel occlusion. As used herein, the term "individual having or having had a TIA, stroke-like symptoms, hemorrhagic stroke, or stroke resulting from a small vessel occlusion" means an individual determined or determined by clinical evaluation to be suffering from a TIA, stroke-like symptoms, hemorrhagic stroke, or stroke resulting from a small vessel occlusion. Such a patient can be included in the control group (i.e., contribute to the sample for inclusion in the control) until the neurological symptoms associated with the TIA, stroke-like symptoms, hemorrhagic stroke, or stroke resulting from a small vessel occlusion have ended. Preferably, the control is a sample or data taken from one or more patients not suffering from LVO stroke. Preferably, the control is a sample or data taken from patients suffering from non-LVO stroke. Preferably, the control is a sample or data taken from patients with ischemic non-LVO stroke, hemorrhagic stroke, and / or non-stroke patients.

[0101] In some embodiments, a difference between the amount of a biomarker in a sample taken from an individual having or suspected of having had a stroke and a control indicates that the individual has suffered a stroke as a result of LVO. In some embodiments, a statistically significant difference between the amount of a biomarker in a sample taken from an individual having or suspected of having had a stroke and a control indicates that the individual has suffered a stroke as a result of LVO.

[0102] Alternatively or in addition, the amount of biomarker in a sample from an individual can be compared to the amount of the same biomarker in a positive standard. The positive standard exhibits one or more signs or symptoms associated with stroke and, in addition, is a sample or data generated from a sample taken from an individual who has been diagnosed as having or having had a stroke as a result of LVO.

[0103] In some embodiments, equality of the amount of biomarker in a sample taken from an individual identified or suspected as having had a stroke, to the positive standard, indicates that the individual has suffered a stroke as a result of LVO. In some embodiments, a statistically significant difference between the amount of biomarker in a sample taken from an individual identified or suspected as having had a stroke, and the positive standard, indicates that the individual has suffered a stroke as a result of LVO.

[0104] In some embodiments, a ratio between two or more biomarkers may be calculated, where the ratio is the amount of one biomarker (the "target biomarker") relative to the amount of another biomarker (the "reference biomarker"). In some embodiments, the reference biomarker is the reference biomarker in a control, positive reference, or positive standard, and for example, the amount of a biomarker in a sample taken from an individual suspected of having an LVO stroke may be compared to a sample or data generated from a sample taken from one or more healthy individuals. In some embodiments, the target and reference biomarkers are the target and reference biomarkers from the same sample, and for example, the amount of succinate may be compared to the amount of GFAP in a sample taken from an individual suspected of having an LVO stroke. Alternatively or in addition, the levels of the biomarkers may be analyzed in samples taken from the same individual at different times, and for example, the amount of GFAP may be compared in two samples taken from an individual before and after the individual has a stroke. In some embodiments, a positive ratio indicates that the individual has had a stroke as a result of an LVO, while a negative ratio indicates that the individual has not had a stroke as a result of an LVO, where the phrase "positive ratio" indicates an increase in the amount of the test biomarker compared to the reference biomarker, and "negative ratio" refers to a decrease in the amount of the test biomarker compared to the amount of the reference biomarker. An intermediate ratio accounts for no or virtually no difference in the amount of the test biomarker and the amount of the reference biomarker.

[0105] Preferably, an increase in the amount of one or more of the biomarkers selected from D-dimer, OPN, and OPG, when compared to a healthy or non-LVO control or a negative criterion, indicates that the individual has suffered a stroke as a result of LVO. Preferably, an increase in the amount of two or more of the biomarkers selected from D-dimer, OPN, and OPG, when compared to a healthy or non-LVO control or a negative criterion, indicates that the individual has suffered a stroke as a result of LVO. Preferably, an increase in the amount of the biomarker selected from D-dimer, OPN, and OPG, when compared to a healthy or non-LVO control or a negative criterion, indicates that the individual has suffered a stroke as a result of LVO. Preferably, an increase in the amount of D-dimer, OPN, and / or OPG, when compared to a healthy or non-LVO control or a negative criterion, indicates that the individual has suffered a stroke as a result of LVO. Preferably, an increase in the amount of D-dimer, OPN, and OPG, when compared to a healthy or non-LVO control or a negative criterion, indicates that the individual has suffered a stroke as a result of LVO.

[0106] Preferably, a decrease in the amount of GFAP, when compared to a healthy or non-LVO control or a negative criterion, indicates that the individual has suffered a stroke as a result of LVO. Preferably, a decrease in the amount of GFAP, when compared to a healthy or hemorrhagic stroke control or a negative criterion, indicates that the individual has suffered a non-hemorrhagic stroke, preferably a stroke as a result of LVO. Preferably, an increase in the amount of GFAP, when compared to a healthy or non-hemorrhagic control or a negative criterion, indicates that the individual has suffered a hemorrhagic stroke.

[0107] Preferably, an increase in the amount of D-dimer, OPN, and / or OPG, and a decrease in the amount of GFAP, when compared to a healthy or non-LVO control or negative criterion, indicate that the individual has suffered a stroke as a result of LVO. Preferably, an increase in the amount of D-dimer, OPN, and OPG, and a decrease in the amount of GFAP, when compared to a healthy or non-LVO control or negative criterion, indicate that the individual has suffered a stroke as a result of LVO.

[0108] The amount of biomarker may be related to the concentration of the biomarker polypeptide sequence. Preferably, the concentration of the biomarker polypeptide sequence is related to the concentration of the biomarker in whole blood. Preferably, the concentration of the biomarker polypeptide sequence is related to the concentration of the biomarker in plasma.

[0109] Preferably, a concentration of D-dimer protein of at least 0.5 μg / ml indicates that the individual has suffered a stroke as a result of LVO. Preferably, a concentration of D-dimer protein of at least 1 μg / ml indicates that the individual has suffered a stroke as a result of LVO. Preferably, a concentration of D-dimer protein of at least 1.2 μg / ml indicates that the individual has suffered a stroke as a result of LVO. Preferably, a concentration of D-dimer protein of at least 1.3 μg / ml indicates that the individual has suffered a stroke as a result of LVO. Preferably, a concentration of D-dimer protein of at least 1.4 μg / ml indicates that the individual has suffered a stroke as a result of LVO. Preferably, a concentration of D-dimer protein of at least 1.5 μg / ml indicates that the individual has suffered a stroke as a result of LVO.

[0110] Preferably, a concentration of OPG protein of at least 100 pg / ml indicates that the individual has suffered a stroke as a result of LVO. Preferably, a concentration of OPG protein of at least 105 pg / ml indicates that the individual has suffered a stroke as a result of LVO. Preferably, a concentration of OPG protein of at least 110 pg / ml indicates that the individual has suffered a stroke as a result of LVO. Preferably, a concentration of OPG protein of at least 120 pg / ml indicates that the individual has suffered a stroke as a result of LVO. Preferably, a concentration of OPG protein of at least 125 pg / ml indicates that the individual has suffered a stroke as a result of LVO. Preferably, a concentration of OPG protein of at least 150 pg / ml indicates that the individual has suffered a stroke as a result of LVO. Preferably, a concentration of OPG protein of at least 200 pg / ml indicates that the individual has suffered a stroke as a result of LVO.

[0111] Preferably, a concentration of OPN protein of at least 1 ng / ml indicates that the individual has suffered a stroke as a result of LVO. Preferably, a concentration of OPN protein of at least 1.2 ng / ml indicates that the individual has suffered a stroke as a result of LVO. Preferably, a concentration of OPN protein of at least 1.4 ng / ml indicates that the individual has suffered a stroke as a result of LVO. Preferably, a concentration of OPN protein of at least 1.6 ng / ml indicates that the individual has suffered a stroke as a result of LVO. Preferably, a concentration of OPN protein of at least 1.8 ng / ml indicates that the individual has suffered a stroke as a result of LVO. Preferably, a concentration of OPN protein of at least 2 ng / ml indicates that the individual has suffered a stroke as a result of LVO. Preferably, a concentration of OPN protein of at least 2.5 ng / ml indicates that the individual has suffered a stroke as a result of LVO. Preferably, a concentration of OPN protein of at least 5 ng / ml indicates that the individual has suffered a stroke as a result of LVO.

[0112] Preferably, a concentration of GFAP protein of at least 100 pg / ml indicates that the individual has suffered a hemorrhagic stroke. Preferably, a concentration of GFAP protein of at least 150 pg / ml indicates that the individual has suffered a hemorrhagic stroke. Preferably, a concentration of GFAP protein of at least 200 pg / ml indicates that the individual has suffered a hemorrhagic stroke. Preferably, a concentration of GFAP protein of at least 250 pg / ml indicates that the individual has suffered a hemorrhagic stroke. Preferably, a concentration of GFAP protein of at least 260 pg / ml indicates that the individual has suffered a hemorrhagic stroke. Preferably, a concentration of GFAP protein of at least 265 pg / ml indicates that the individual has suffered a hemorrhagic stroke.

[0113] Preferably, a concentration of GFAP protein of less than 100 pg / ml indicates that the individual has suffered a non-hemorrhagic stroke. Preferably, a concentration of GFAP protein of less than 150 pg / ml indicates that the individual has suffered a non-hemorrhagic stroke. Preferably, a concentration of GFAP protein of less than 200 pg / ml indicates that the individual has suffered a non-hemorrhagic stroke. Preferably, a concentration of GFAP protein of less than 250 pg / ml indicates that the individual has suffered a non-hemorrhagic stroke. Preferably, a concentration of GFAP protein of less than 260 pg / ml indicates that the individual has suffered a non-hemorrhagic stroke. Preferably, a concentration of GFAP protein of less than 265 pg / ml indicates that the individual has suffered a non-hemorrhagic stroke.

[0114] Preferably, a concentration of GFAP protein of less than 100 pg / ml indicates that the individual has suffered a stroke as a result of LVO. Preferably, a concentration of GFAP protein of less than 150 pg / ml indicates that the individual has suffered a stroke as a result of LVO. Preferably, a concentration of GFAP protein of less than 200 pg / ml indicates that the individual has suffered a stroke as a result of LVO. Preferably, a concentration of GFAP protein of less than 250 pg / ml indicates that the individual has suffered a stroke as a result of LVO. Preferably, a concentration of GFAP protein of less than 260 pg / ml indicates that the individual has suffered a stroke as a result of LVO. Preferably, a concentration of GFAP protein of less than 265 pg / ml indicates that the individual has suffered a stroke as a result of LVO.

[0115] In some embodiments, one or more of the measured biomarker amounts in the sample are converted to an LVO stroke score. In some embodiments, the LVO stroke score includes correlating the amount of biomarker in a sample from an individual with one or more of a negative reference, a positive reference, a control, or a positive standard. For example, an LVO stroke score of 0-100 may be established where 0 corresponds to the biomarker value in the control and 100 corresponds to the biomarker value in the positive standard. A weighting factor may be assigned to one or more of the biomarkers. For example, the biomarkers may be weighted such that biomarkers with higher diagnostic relevance contribute more to the LVO stroke score. For example, a biomarker may be a biomarker with higher diagnostic relevance if the amount of that biomarker is significantly different between individuals diagnosed as having or having had LVO and the control. In contrast, a biomarker may be a biomarker with less diagnostic relevance if the difference in the amount of that biomarker in individuals diagnosed as having or having had LVO and the control is small. Such weighting factors modulate the contribution of the biomarker to the diagnosis of LVO stroke. The weighting factor may be introduced, for example, in a calculation step using an algorithm, or when the binding of a drug to the biomarker is detected and measured in a detection tool such as an optical reader.

[0116] In some embodiments, an LVO stroke score generated using the measured amount of a biomarker in a sample taken from an individual who has had or is identified as having had or suspected of having a stroke can be compared to an LVO stroke score generated using the amount of the biomarker from a control or a positive standard.

[0117] In some embodiments, an LVO score generated using the measured amount of a biomarker in a sample taken from an individual who has had or is identified as having had or suspected of having a stroke, which is equivalent to the LVO score generated using the amount of the biomarker from a positive standard, indicates that the individual has suffered a stroke as a result of LVO. In some embodiments, an LVO score generated using the measured amount of a biomarker in a sample taken from an individual who has had or is identified as having had or suspected of having a stroke, which is statistically equivalent to the LVO score generated using the amount of the biomarker from a positive standard, indicates that the individual has suffered a stroke as a result of LVO.

[0118] In some embodiments, the difference between an LVO score generated using the measured amount of a biomarker in a sample taken from an individual who has had or is identified as having had or suspected of having a stroke and a control indicates that the individual has suffered an LVO stroke. In some embodiments, a statistically significant difference between an LVO score generated using the measured amount of a biomarker in a sample taken from an individual who has had or is identified as having had or suspected of having a stroke and a control indicates that the individual has suffered an LVO stroke.

[0119] In some embodiments, a defined value or range (“cut-off value”) is provided for one or more of the biomarkers and / or for the LVO stroke score. In some such embodiments, an LVO stroke score or amount of biomarker that exceeds the defined value indicates that the individual has suffered a stroke resulting from LVO. In other such embodiments, an LVO stroke score or amount of biomarker that is less than the defined value indicates that the individual has not suffered a stroke resulting from LVO.

[0120] In some embodiments, the method can be performed within about 72 hours, about 48 hours, or about 24 hours of the onset of stroke symptoms in the individual. In some embodiments, the method is performed within about 24 hours or within 24 hours of the onset of stroke symptoms in the individual. In some embodiments, the method is performed within about 17 hours or within 17 hours of the onset of stroke symptoms in the individual. In some embodiments, the method is performed within about 10 hours or within 10 hours of the onset of stroke symptoms in the individual. In some embodiments, the method is performed within about 6 hours or within 6 hours of the onset of stroke symptoms in the individual.

[0121] Preferably, the method is performed on a sample obtained from the individual within about 24 hours or within 24 hours of the onset of stroke symptoms in the individual. More preferably, the method is performed on a sample obtained from the individual within about 17 hours or within 17 hours of the onset of stroke symptoms in the individual. Even more preferably, the method is performed on a sample obtained from the individual within about 10 hours or within 10 hours of the onset of stroke symptoms in the individual. Most preferably, the method is performed on a sample obtained from the individual within about 6 hours or within 6 hours of the onset of stroke symptoms in the individual.

[0122] As shown in FIGS. 9 and 14, combining biomarker expression with the stroke severity score significantly improves the sensitivity and specificity of the method.

[0123] Thus, in some embodiments, the method further includes consideration of data and / or information from one or more clinical evaluations received by the individual. Clinical evaluations can include one or more of CT or CTA, MRA, MRI, diffusion weighted imaging, cerebral angiography, electrocardiogram, atrial fibrillation evaluation, FAST (Face, Arm, Speech Test), ABCD, ABCD2 and California Prediction Rule Rosier (Recognition of Stroke in the Emergency Room) stroke scale, stroke scale and TOAST (trial of ORG. 10172 in Acute Stroke Treatment) classification, OCSP (the Oxford Community Stroke Project classification), National Institutes of Health Stroke Scale (NIHSS), Emergency Medical Stroke Assessment (EMSA), Prehospital Acute Stroke Severity (PASS) scale, Vision, Aphasia, Neglect (VAN) evaluation, Rapid Arterial Occlusion Evaluation (RACE), Field Assessment Stroke Triage for Emergency Destination (FAST-ED) scale, Cincinnati pre-hospital Stroke Severity Scale (CPSSS), Doppler ultrasound examination, carotid echo ultrasound examination, angiography or arteriography. In some embodiments, the method further includes consideration of data and / or information from angiographic evaluation by CTA and / or MRA.

[0124] Preferably, the method further includes determining a stroke severity score for the individual. Preferably, the stroke severity score is selected from NIHSS, FAST, FAST-ED, RACE, C-STAT and EMSA. Such scores are well known in the art and the methods by which the scores are calculated may be known to those skilled in the art.

[0125] Preferably, one or more NIHSS scores indicate that the individual has suffered a stroke as a result of LVO.

[0126] Preferably, one or more FAST scores indicate that the individual has suffered a stroke as a result of LVO.

[0127] Preferably, one, two, three, four, or five or more FAST-ED scores indicate that the individual has suffered a stroke as a result of LVO. Preferably, two or more FAST-ED scores indicate that the individual has suffered a stroke as a result of LVO. More preferably, three or more FAST-ED scores indicate that the individual has suffered a stroke as a result of LVO. More preferably, four or more FAST-ED scores indicate that the individual has suffered a stroke as a result of LVO. More preferably, five or more FAST-ED scores indicate that the individual has suffered a stroke as a result of LVO.

[0128] Preferably, one, two, three, four, five, six, or seven or more RACE scores indicate that the individual has suffered a stroke as a result of LVO. Preferably, one or more RACE scores indicate that the individual has suffered a stroke as a result of LVO. Preferably, two or more RACE scores indicate that the individual has suffered a stroke as a result of LVO. Preferably, three or more RACE scores indicate that the individual has suffered a stroke as a result of LVO. Preferably, three or more RACE scores indicate that the individual has suffered a stroke as a result of LVO. Preferably, four or more RACE scores indicate that the individual has suffered a stroke as a result of LVO. Preferably, five or more RACE scores indicate that the individual has suffered a stroke as a result of LVO. Preferably, six or more RACE scores indicate that the individual has suffered a stroke as a result of LVO. Preferably, seven or more RACE scores indicate that the individual has suffered a stroke as a result of LVO.

[0129] Preferably, one or more C-STAT scores indicate that the individual has suffered a stroke as a result of LVO. Preferably, two or more C-STAT scores indicate that the individual has suffered a stroke as a result of LVO.

[0130] Preferably, three, four, or five or more EMSA scores indicate that the individual has suffered a stroke as a result of LVO. Preferably, three or more EMSA scores indicate that the individual has suffered a stroke as a result of LVO. Preferably, four or more EMSA scores indicate that the individual has suffered a stroke as a result of LVO. Preferably, five or more EMSA scores indicate that the individual has suffered a stroke as a result of LVO.

[0131] Preferably, the stroke severity score is the NIHSS. Preferably, the stroke severity score is the FAST. Preferably, the stroke severity score is the FAST-ED. Preferably, the stroke severity score is the RACE. Preferably, the stroke severity score is the C-STAT. Preferably, the stroke severity score is the EMSA.

[0132] Most preferably, the stroke severity score is the FAST, the FAST-ED, or the EMSA.

[0133] In the most preferred embodiment, the biomarkers are D-dimer and GFAP, and the stroke severity score is the FAST, the FAST-ED, or the EMSA.

[0134] Preferably, the method includes combining the amount of one or more biomarkers measured in a sample taken from an individual who has, has been identified as having had, or is suspected of having had a stroke, with the stroke severity score obtained from the individual.

[0135] Preferably, the method further comprises producing an LVO stroke score by combining the amount of one or more biomarkers measured in a sample taken from an individual having, identified as having had, or suspected of having a stroke, with a stroke severity score obtained from the individual.

[0136] Preferably, the method includes comparing an LVO stroke score generated using the measured biomarker amount in a sample taken from an individual having, identified as having had, or suspected of having a stroke, and the stroke severity score taken from the individual having, identified as having had, or suspected of having a stroke, with an LVO stroke score generated using the biomarker amount and the stroke severity score from a control or a positive standard.

[0137] Preferably, the individual has an LVO stroke severity score generated using the measured biomarker amount in a sample taken from an individual having, identified as having had, or suspected of having a stroke, and the LVO stroke score taken from the individual having, identified as having had, or suspected of having a stroke, that is equivalent to a positive standard; or the individual has an LVO stroke severity score generated using the measured biomarker amount in a sample taken from an individual having, identified as having had, or suspected of having a stroke, and the LVO stroke severity score taken from the individual having, identified as having had, or suspected of having a stroke, that is different from an LVO stroke score generated using the biomarker amount and the stroke severity score from a control, and is diagnosed as having or having had a stroke as a result of LVO.

[0138] The LVO stroke score can be calculated by processing the amount of one or more biomarkers obtained from an individual and the stroke severity score using linear regression, random forest, neural network, support vector machine, Bayesian linear regression, Bayesian logistic regression, or partial least squares regression.

[0139] Preferably, the LVO stroke score is calculated by processing the amounts of one or more biomarkers and the stroke severity score obtained from an individual using a regression model, preferably a multivariate logistic regression model.

[0140] Preferably, the LVO stroke score is given by the following formula: Y = a*X1, b*X2, ···, n*X n and is determined by solving the equation, where a, b, ··· n represent the multiplication factors for each biomarker or stroke severity score, and X1, X2 ··· X n represents the value of the biomarker measured or obtained from the patient or the stroke severity score.

[0141] Preferably, the multiplication factor for each measured biomarker or stroke severity score is as listed in FIG. 13.

[0142] The LVO stroke score can be obtained for each patient by transforming the value of Y obtained by solving the above equation by a logistic transformation known to those skilled in the art.

[0143] Thus, preferably, the LVO stroke score is calculated by solving a linear equation based on the coefficients listed in FIG. 17 and the measured values of each corresponding biomarker and the stroke severity score collected for any given patient suspected of having a stroke.

[0144] In one embodiment, the LVO stroke score can have a value between 0 and 1 for any subject. The threshold of the LVO stroke score that can be used to diagnose LVO patients can vary depending on which combination of biomarkers and stroke severity scores is selected. Preferably, the threshold of the LVO stroke score used to diagnose LVO patients is selected from among the values listed in FIG. 14.

[0145] Preferably, an LVO stroke score exceeding 0.20 indicates an individual suffering from LVO stroke. Preferably, an LVO stroke score exceeding 0.24 indicates an individual suffering from LVO stroke. Preferably, an LVO stroke score exceeding 0.30 indicates an individual suffering from LVO stroke. Preferably, an LVO stroke score exceeding 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39 or 0.40 indicates an individual suffering from LVO stroke.

[0146] More preferably, an LVO stroke score exceeding 0.33 indicates an individual suffering from LVO stroke. More preferably, an LVO stroke score exceeding 0.34 indicates an individual suffering from LVO stroke. More preferably, an LVO stroke score exceeding 0.35 indicates an individual suffering from LVO stroke. Most preferably, an LVO stroke score exceeding 0.39 indicates an individual suffering from LVO stroke.

[0147] CTA is a computed tomography reconstruction imaging technique used to visualize arteries and veins at the level of the head, neck and / or brain. By applying a contrast agent, CTA enables visualization of vascular occlusions, aneurysms, dissections and stenoses. A contrast agent is any radiopaque compound that blocks the X-ray part of the electromagnetic spectrum and can therefore result in opaque regions in the calculated image.

[0148] MRA refers to any magnetic resonance imaging-based diagnostic technique used to generate an image of blood vessels that visualizes vascular occlusions, aneurysms, dissections and stenoses. A contrast agent, such as a gadolinium-based contrast agent, is applied to generate the image and various different techniques can be used (Johnson et al., "Magnetic resonance angiography: A review", Academic radiology 5, volume 4 (1998): 289-305).

[0149] Identification of an individual suffering from or having suffered a stroke as a result of LVO enables selection of an appropriate further clinical investigation and / or treatment pathway for that individual.

[0150] Thus, in a second aspect, a method for identifying individuals who have suffered a stroke as a result of LVO from a group of individuals who have or are identified as having had or are suspected of having had a stroke, the method comprising determining the amount of at least two biomarkers in a sample taken from each individual, wherein the biomarkers are selected from succinate, succinate-glutathione, N-acetyl-aspartate, propionyl-carnitine, glutamate, heart fatty acid binding protein (H-FABP), brain fatty acid binding protein (B-FABP), Abeta 1-40, osteoprotegerin (OPG), soluble tumor necrosis factor-like weak apoptosis inducer (sTWEAK), pro-vWF, retinol binding protein 4 (RBP4), ADAMTS13, NMDA receptor 2 peptide (NR2 peptide), 20-HETE, bilirubin, brain natriuretic peptide (BNP), glial fibrillary acidic protein (GFAP), D-dimer, C-reactive protein (CRP), matrix metalloproteinase 9 (MMP9), interleukin 6 (IL-6), osteopontin (OPN), troponin I, s100b, von Willebrand factor (vWF) or P-selectin is provided.

[0151] However, preferably, the method of the second aspect comprises determining the amount of at least one biomarker selected from D-dimer, OPN and OPG. More preferably, the method comprises determining the amount of at least two biomarkers selected from D-dimer, OPN and OPG. Most preferably, the method comprises determining the amounts of D-dimer, OPN and OPG. Preferably, the method comprises determining the amount of at least one biomarker selected from D-dimer, OPN, GFAP and OPG. More preferably, the method comprises determining the amount of at least two biomarkers selected from D-dimer, OPN, GFAP and OPG. Most preferably, the method comprises determining the amounts of D-dimer, OPN, GFAP and OPG.

[0152] Therefore, preferably, the step of determining the biomarker, the amount and concentration of the biomarker can be as defined in the first aspect.

[0153] The method can be performed in vivo, in vitro or ex vivo. Preferably, the method is performed in vitro or ex vivo. Most preferably, the method is performed in vitro.

[0154] The biomarker can be detected using any method disclosed herein. In addition, the amount of the biomarker can be determined using any method disclosed herein.

[0155] In some embodiments, the method according to the second aspect includes the step of determining the amount of at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 or 12 or more biomarkers.

[0156] The method according to the second aspect includes the step of determining the presence and / or amount of one or more biomarkers according to any embodiment of the first aspect.

[0157] In some embodiments according to the second aspect, the method comprises comparing the amount of each biomarker in a sample taken from each individual having or suspected of having had a stroke, as disclosed above according to the first aspect, with the amount of the same biomarker in one or more of a control, a positive standard, one or more positive criteria or a negative criterion.

[0158] In some embodiments according to the second aspect, an individual having a difference in the amount of one or more of the measured biomarkers compared to the amount of the same biomarker in the control is identified as having or having had a stroke as a result of LVO. In some embodiments according to the second aspect, an individual having an equivalent amount of one or more of the measured biomarkers compared to a positive standard is identified as having or having had a stroke as a result of LVO.

[0159] In some embodiments, the method includes converting the amount of one or more biomarkers measured in a sample taken from an individual identified as having or suspected of having had a stroke, as disclosed above according to the first aspect, into an LVO stroke score.

[0160] In some embodiments according to the second aspect, an individual having an LVO score equivalent to the LVO score created using the biomarker amount from a positive standard is identified as having or having had a stroke as a result of LVO. In some embodiments, an individual having an LVO score statistically equivalent to the LVO score created using the biomarker amount from a positive standard is identified as having or having had a stroke as a result of LVO. In some embodiments, an individual having an LVO score different from the LVO score created using the biomarker amount from a control is identified as having or having had a stroke as a result of LVO. In some embodiments, an individual having an LVO score statistically different from the LVO score created using the biomarker amount from a control is identified as having or having had a stroke as a result of LVO.

[0161] In some embodiments, the method according to the second aspect further comprises determining an individual's stroke severity score as defined in the first aspect. In some embodiments, the method according to the second aspect comprises combining the amount of one or more biomarkers measured in a sample taken from an individual having or suspected of having had a stroke, as defined in the first aspect, with the stroke severity score obtained from the individual.

[0162] In some embodiments, the method according to the second aspect further comprises, as described above, taking into account data and / or information from one or more clinical evaluations received by each individual, as disclosed according to the first aspect. In some such embodiments, the clinical evaluation is one or more of a CT or CTA, MRA or MRI scan, or the individual's NIHSS, FAST, ABCD, ABCD2 Rosier, TOAST, EMSA, PASS, VAN, RACE, FAST_ED or CPSS score.

[0163] Appropriate further clinical investigations for individuals identified as having or having had a stroke as a result of LVO include computed tomography angiography and / or magnetic resonance angiography. Appropriate treatment pathways for individuals identified as having or having had a stroke as a result of LVO may include treatment with antithrombotic agents and / or mechanical thrombectomy.

[0164] Use of two or more biomarkers for diagnosing stroke resulting from large vessel occlusion, wherein the biomarkers are selected from succinate, succinate-glutathione, N-acetyl-aspartate, propionyl-carnitine, glutamate, heart fatty acid binding protein (H-FABP), brain fatty acid binding protein (B-FABP), Abeta 1-40, osteoprotegerin (OPG), soluble tumor necrosis factor-like weak apoptosis inducer (sTWEAK), pro-vWF, retinol binding protein 4 (RBP4), ADAMTS13, NMDA receptor 2 peptide (NR2 peptide), 20-HETE, bilirubin, brain natriuretic peptide (BNP), glial fibrillary acidic protein (GFAP), D-dimer, C-reactive protein (CRP), matrix metalloproteinase 9 (MMP9), interleukin 6 (IL-6), osteopontin (OPN), troponin I, s100b, von Willebrand factor (vWF) or P-selectin is provided.

[0165] The biomarker and the concentration of the biomarker can be as defined in the first aspect.

[0166] In some embodiments, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 or 12 or more biomarkers are used to identify stroke resulting from large vessel occlusion.

[0167] In some embodiments, the biomarker is present in a sample taken from an individual having or identified as having or suspected of having a stroke. The biomarker can be detected using any method disclosed herein. Additionally, the amount of the biomarker can be determined using any method disclosed herein.

[0168] As described in the Examples and above, the inventors have identified biomarkers that can be used to diagnose stroke as a result of LVO with a high degree of sensitivity and specificity.

[0169] Accordingly, in a fourth aspect of the invention, D-dimer, OPN, OPG and / or GFAP are provided for use in diagnosing stroke as a result of large vessel occlusion (LVO).

[0170] In one embodiment, D-dimer is provided for use in diagnosing stroke as a result of large vessel occlusion (LVO). In one embodiment, OPN is provided for use in diagnosing stroke as a result of large vessel occlusion (LVO). In one embodiment, OPN is provided for use in diagnosing stroke as a result of large vessel occlusion (LVO). In one embodiment, GFAP is provided for use in diagnosing stroke as a result of large vessel occlusion (LVO). In one embodiment, D-dimer and GFAP are provided for use in diagnosing stroke as a result of large vessel occlusion (LVO).

[0171] In one embodiment, D-dimer, OPN and OPG are provided for use in diagnosing stroke as a result of large vessel occlusion (LVO).

[0172] The biomarker can be detected using any method disclosed herein. In addition, the amount of the biomarker can be determined using any method disclosed herein.

[0173] Preferably, the biomarker, the amount of the biomarker associated with stroke as a result of LVO, the step of determining the concentration, and the sample are as defined in the first aspect.

[0174] In a fifth aspect of the invention, GFAP is provided for use in diagnosing hemorrhagic stroke.

[0175] Preferably, a step of determining the amount and concentration associated with stroke as a result of LVO, and the sample is as defined in the first aspect.

[0176] Also, in a sixth aspect, a kit for performing the methods described in the first, second, and third aspects of the present invention is provided. The kit includes a container for holding a sample from an individual having or identified as having or suspected of having a stroke; means for determining the amount of a target biomarker; and instructions for how to perform the reaction of the provided sample and means for determining the amount of the target biomarker, for example, one or more of printed instructions.

[0177] In some embodiments, the means for determining the amount of the target biomarker may include one or more agents disclosed herein. The agent may be an antibody. The agent may be immobilized on a substrate and / or labeled with at least one detectable label. In some embodiments, each agent is labeled with a different detectable label.

[0178] The method described in the present invention enables patients suffering from stroke resulting from LVO to be identified quickly and easily, and appropriate treatment options to be selected.

[0179] Thus, in a seventh aspect, a method of treating an individual suffering from a stroke as a result of large vessel occlusion (LVO), a) a step of determining the amount of at least one biomarker in a sample taken from each individual, wherein the biomarker is selected from osteoprotegerin (OPG), glial fibrillary acidic protein (GFAP), D-dimer, and osteopontin (OPN), and the amount of the biomarker indicates that the individual is suffering from a stroke as a result of large vessel occlusion (LVO); b) administering or having administered to the individual a therapeutic agent for treating a stroke as a result of large vessel occlusion (LVO) A method comprising is provided.

[0180] Biomarkers can be detected using any method disclosed herein. In addition, the amount of a biomarker can be determined using any method disclosed herein. Preferably, the biomarker, the amount of the biomarker associated with stroke as a result of LVO, the step of determining the concentration of the biomarker, and the sample are as defined in the first aspect.

[0181] Preferably, the treatment includes mechanical thrombectomy (MT) and / or intravenous or intra-arterial thrombolysis treatment.

[0182] Without being bound by any theory, the accuracy of detecting stroke resulting from LVO in the methods described in the present invention is suggested to result from the detection of a specific amount of a specific biomarker or a combination of specific biomarkers in a sample from an individual having or suspected of having had a stroke, and the level of the specific biomarker or the combination of specific biomarkers is altered as a result of biological changes occurring as a result of LVO stroke. Such markers are suggested to be associated with cerebral ischemia and / or cerebral cell death, both of which increase in LVO compared to other types of stroke. Ischemia can lead to damage to tissues and cells, which results in the release and / or upregulation of any biomarker from the damaged cells or tissue itself or from tissues or cells in communication with the damaged cells.

[0183] Accordingly, some embodiments of the present invention provide methods, kits, and uses for indicating a stroke and for identifying or assisting in identifying an individual suffering from or having suffered from a stroke resulting from LVO. Such embodiments enable the determination of such individuals with improved accuracy and / or speed, thereby providing an opportunity to improve the treatment pathway for the individual.

[0184] It is understood that the present invention extends to any nucleic acid or peptide or variant, derivative or analog thereof that substantially comprises an amino acid or nucleic acid sequence of any of the sequences referred to herein and variants or fragments thereof. The terms "substantially amino acid / nucleotide / peptide sequence", "variant" and "fragment" can be sequences having at least 40% sequence identity with an amino acid / nucleotide / peptide sequence of any of the sequences referred to herein, for example, a sequence identified as SEQ ID NOs: 1-8 etc. and having 40% identity.

[0185] Also contemplated are amino acid / polynucleotide / polypeptide sequences having a sequence identity of more than 65%, more preferably more than 70%, even more preferably more than 75%, and still more preferably more than 80% sequence identity with any of the sequences referred to. Preferably, the amino acid / polynucleotide / polypeptide sequence has at least 85% identity with any of the sequences referred to, more preferably at least 90% identity with any of the sequences referred to herein, even more preferably at least 92% identity, even more preferably at least 95% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, and most preferably at least 99% identity.

[0186] One of ordinary skill in the art understands how to calculate the percentage identity between two amino acid / polynucleotide / polypeptide sequences. To calculate the percentage identity between two amino acid / polynucleotide / polypeptide sequences, an alignment of the two sequences must first be prepared, followed by calculating the value of sequence identity. The percentage identity for two sequences depends on (i) the method used to align the sequences, such as ClustalW, BLAST, FASTA, Smith-Waterman (implemented in various programs) or structural alignment from 3D comparison; and (ii) the parameters used by the alignment method, such as local alignment versus global alignment, the substitution score matrix used (e.g., BLOSUM62, PAM250, Gonnet, etc.), and the gap penalty, which can take different values depending on, for example, the functional form and constants.

[0187] After creating the alignment, there are a variety of different ways to calculate the percentage identity between two sequences. For example, the number of identities can be divided by (i) the length of the shortest sequence, (ii) the length of the alignment, (iii) the average value of the lengths of the sequences, (iv) the number of non-gap positions, or (v) the number of equivalent positions excluding overhangs. Additionally, it is understood that the percentage identity is strongly dependent on length. Therefore, it can be predicted that higher sequence identity will occur by chance for shorter sequence pairs.

[0188] Therefore, it is understood that the accurate alignment of protein or DNA sequences is a complex process. The general multiple alignment program ClustalW (Thompson et al., 1994, Nucleic Acids Research, 22, 4673-4680; Thompson et al., 1997, Nucleic Acids Research, 24, 4876-4882) is a preferred method for generating multiple alignments of proteins or DNA according to the present invention. Suitable parameters for ClustalW can be a gap open penalty = 15.0, a gap extension penalty = 6.66, and a matrix = Identity for DNA alignment. For protein alignment, the gap open penalty = 10.0, the gap extension penalty = 0.2, and the matrix = Gonnet can be used. For DNA and protein alignment, ENDGAP = -1, and GAPDIST = 4 can be used. Those skilled in the art will recognize that it may be necessary to vary these and other parameters for optimal sequence alignment.

[0189] Preferably, subsequently, the calculation of the percentage identity between two amino acid / polynucleotide / polypeptide sequences may be calculated as (N / T)*100 from such an alignment, where N is the number of positions at which the sequences share identical residues, and T is the total number of positions compared including gaps and including or excluding overhangs. Preferably, overhangs are included in the calculation. Therefore, the most preferred method for calculating the percentage identity between two sequences comprises (i) preparing a sequence alignment using a ClustalW program using a suitable set of parameters, such as those shown above, and (ii) inserting the values of N and T into the formula [sequence identity = (N / T)*100].

[0190] Alternative methods for identifying similar sequences are known to those skilled in the art. For example, substantially similar nucleotide sequences are encoded by sequences that hybridize to a DNA sequence or its complement under stringent conditions. By stringent conditions, we mean that the nucleotides hybridize to the filter-bound DNA or RNA, in approximately 45°C in 3x sodium chloride / sodium citrate (SSC), followed by at least one wash in approximately 20 - 65°C in 0.2x SSC / 0.1% SDS. Alternatively, substantially similar polypeptides may differ by at least 1, but less than 5, 10, 20, 50 or 100 amino acids from the sequences shown in SEQ ID NOs: 1 - 8, for example.

[0191] Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence described herein can be varied or changed without substantially affecting the sequence of the protein encoded thereby, and can provide functional variants thereof. Preferred nucleotide variants are variants having a sequence that has been altered by substitution of different codons within the sequence that encode the same amino acid, and thus produce silent (synonymous) changes. Other preferred variants are all sequences or portions of sequences that have the same nucleotide sequence but are altered by substitution of different codons that encode amino acids having side chains with biophysical properties similar to the substituted amino acid, producing conservative changes. For example, small molecule nonpolar hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine. Large molecule nonpolar hydrophobic amino acids include phenylalanine, tryptophan, and tyrosine. Polar neutral amino acids include serine, threonine, cysteine, asparagine, and glutamine. Positively charged (basic) amino acids include lysine, arginine, and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Therefore, it is understood that amino acids may be substituted with amino acids having similar biophysical properties, and those skilled in the art know the nucleotide sequences that encode these amino acids.

[0192] All of the features described herein (including any appended claims, abstract, and drawings) and / or all of the steps of any method or process so disclosed may be combined in any combination with any of the above aspects, except combinations where at least some of such features and / or steps are mutually exclusive.

[0193] The following examples are provided to illustrate the invention and should not be construed as limiting the invention.

Examples

[0194] (Example 1) (Example 1A) Sample Preparation Blood samples may be collected from human subjects via venipuncture, collected in tubes containing sodium EDTA, and immediately placed on ice. Alternatively, CSF samples may be obtained by lumbar puncture.

[0195] The need for and type of sample processing prior to biomarker detection depends on the biomarker to be detected and / or the assay used to detect the biomarker. If whole blood is required for analysis, the sample can be used as obtained. Alternatively, the sample may be diluted with saline to obtain a biomarker concentration appropriate for compatibility with the required type of biomarker detection assay.

[0196] If plasma is required for analysis, whole blood samples may be centrifuged, for example at 2000 g at 4 °C for 15 minutes to obtain plasma. The plasma may then be withdrawn and used directly or diluted with saline as required for the biomarker detection assay.

[0197] (Example 1b) Assay Optimization Rabbit monoclonal antibodies are purchased from Abcam, Cambridge, UK. Isolated or recombinant antibodies of known concentration are applied to a preliminary assay to determine the specificity of the antibody that recognizes and binds to the target biomarker. To determine the optimal concentration of the antibody, an indirect ELISA is performed in which the isolated or recombinant biomarker protein is bound to an ELISA microtiter plate. Rabbit monoclonal anti-human biomarker antibodies are coated onto the microtiter plate to determine the antibody concentration required to obtain a maximum signal and to enable determination of the lower limit of detection of the indirect ELISA for each antibody. After incubating the diluted sample with the rabbit monoclonal anti-human biomarker antibody for 2 hours, appropriate washing is performed, followed by addition of a monoclonal anti-human biomarker antibody labeled with biotin and incubation with the captured biomarker. A number of subsequent washing steps are then performed, followed by addition of horseradish peroxidase conjugated to streptavidin. After 1 hour incubation of the reagents, a further washing step is performed, followed by contacting the resulting conjugate with hydrogen peroxide tetramethylbenzidine to obtain a yellow product. The reaction is stopped by addition of an acidic solution, and the reaction signal is erroneously measured by absorbance at 450 nanometers, which is proportional to the concentration of the biomarker. A standard curve is obtained by plotting the absorbance values as a function of the measured amount of biomarker in samples of known biomarker concentration. The standard curve is then used to determine the amount of the target biomarker in unknown samples.

[0198] (Example 1C) Materials for Biomarker Analysis Exemplary reagents used in the practice of the present invention include sodium bicarbonate (Sigma catalog number C-3041), blocking buffer (Startingblock T20-TBS) (Pierce catalog number 37543), Tris-buffered saline with Tween 20 (TBST; Sigma catalog number T-9039). Phosphate-buffered saline (PBS; Sigma catalog number P-3813); Tween 20 (Sigma catalog number P5927); Ultra TMB ELISA (Pierce catalog number 34028); and Nunc maxisorp ELISA plates (Fisher). Monoclonal and polyclonal antibodies against heart fatty acid-binding protein (H-FABP), brain fatty acid-binding protein (B-FABP), Abeta 1-40, OPG, soluble tumor necrosis factor-like weak apoptosis inducer (sTWEAK), pro-vWF, retinol-binding protein 4 (RBP4), ADAMTS13, NMDA receptor 2 peptide (NR2 peptide), 20-HETE, brain natriuretic peptide (BNP), glial fibrillary acidic protein (GFAP), D-dimer, C-reactive protein (CRP), matrix metalloproteinase 9 (MMP9), interleukin 6 (IL-6), osteopontin (OPN), troponin I, s100b, von Willebrand factor (vWF), P-selectin are purchased from Abcam, Cambridge, UK. Labels for many subtypes of antibodies are available from Expedeon Ltd, Cambridge, UK. Protein concentration in the sample is determined by albumin standard using the bicinchoninic acid microprotein assay (Pierce Inc., Rockford, IL, USA). All other necessary reagents and materials are known to those of skill in the art and can be readily identified. Colorimetric assay kits for bilirubin, succinate, N-acetylaspartate, propionyl-L-carnitine, glutamate are available from Abcam, Cambridge, UK and Biovision Inc, Milpitas, CA.

[0199] (Example 2) Method Patient recruitment and sample processing Patients suspected of having a stroke within 18 hours "since last known well" were retrospectively recruited after arrival at the emergency departments (ED) of Freeman Hospital and Royal Victoria Infirmary in Newcastle upon Tyne (UK). A 4 mL venous blood sample was drawn within 30 minutes of arrival and immediately stored at +4°C in the dark until processing. Plasma was obtained by centrifugation of whole blood at 2000 x g for 15 minutes at +4°C. After processing, the plasma samples were immediately frozen at -80°C until biomarker measurement.

[0200] Diagnostic adjudication 170 patients suspected of having a stroke were identified by emergency medical technicians and ED clinicians. Adjudication of stroke subtype, stroke mimics, or TIA diagnosis was based on brain imaging results and neurologist reports. Diagnosis of hemorrhagic stroke was based on results from computed tomography (CT) brain scans, while adjudication of stroke mimics or transient ischemic attack (TIA) was based on neurologist reports. Ischemic stroke patients were subdivided into three categories based on neurologist reports, CT angiography (CTA) imaging, and NIHSS scores. Ischemic stroke patients labeled "unknown" were excluded from the following analysis due to diagnostic uncertainty. Exclusion of 23 cases of "unknown" ischemic stroke and utilization of 19 samples for immunoassay testing yielded a final patient cohort of 128 patients suspected of having a stroke, which was subjected to biomarker measurement and statistical analysis.

[0201] Derivation of pre-hospital stroke scale from NIHSS score The FAST score was calculated by assigning 1 point for any presence of facial palsy (NIHSS item 4), 1 point for any arm weakness (NIHSS items 5a / b), and 1 point for any speech impairment (NIHSS item 9). The FAST-ED was calculated as described by Lima et al. 5 and Perez de la Ossa 7Calculate the RACE score as described by, and Katz et al. 19 Calculate the CPSS as described by, and Gropen et al. 10 Calculate the EMSA as described by.

[0202] Measurement of blood biomarkers Plasma biomarkers were measured by commercially available enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions. The ELISA kits or matching antibody pairs, D-dimer (product number: ab196269), OPN (product number: ab100618), OPG (product number: ab100617), GFAP (product number: ab222279), vWF (product number: ab223864), and ADAMTS13 (product number: ab234559) were purchased from Abcam (Cambridge, UK). Plasma sample dilutions for the detection of each biomarker were D-dimer (1:80), OPN (1:2), OPG (1:6), GFAP (1:2), vWF (1:4000), ADAMTS13 (1:800). Samples were diluted in dilution buffer as described by the manufacturer's instructions. All samples were analyzed in duplicate, and the mean value was used for quantification. For all biomarkers, the mean coefficient of variation was less than 10%. Biomarker quantification was performed by linear or quadratic polynomial interpolation against a calibration curve obtained with each analyte of known concentration. GraphPad Prism version 8.4.3 was used for biomarker quantification.

[0203] Statistical analysis For univariate analysis of individual blood biomarkers or clinical variables, the normality of the distribution was evaluated by the Shapiro–Wilk test. For continuous variables, normally distributed (Shapiro–Wilk p-value > 0.1) variables were analyzed by the Student's t-test to obtain mean and standard deviation (SD) values, while for variables with non-normal distributions, the Wilcoxon Mann–Whitney U test was used and the median and interquartile range (IQR) were reported. Pearson's chi-squared test for categorical variables was used to evaluate differences between groups. When testing more than 10 variables simultaneously, multiple hypothesis correction was performed by the Benjamini–Hochberg method. Analysis of variance was used to evaluate the interaction between all blood biomarkers and subtypes of suspected stroke. When overall significance was confirmed, pairwise comparisons were made by Tukey's test.

[0204] To identify the optimal panel of blood biomarkers for LVO prediction, we used multivariate logistic regression with diagnosis (LVO vs. non-LVO) as the outcome variable and plasma levels of D-dimer, GFAP, OPN, OPG, vWF, and ADAMTS13 as exploratory variables. Bidirectional stepwise deletion based on the Akaike information criterion (AIC) level was used for model selection. Linearity between predictor variables and outcome measurements was evaluated through logarithmic and quadratic transformations. The transformations were selected based on the AIC.

[0205] To investigate whether the addition of blood biomarkers improved the accuracy of stroke severity scales for LVO identification, we used a second multivariate logistic with diagnosis as the outcome variable and the optimal panel of stroke severity scales (FAST, FAST-ED, RACE, C-STAT, or EMSA) and one of the stroke severity scales as exploratory variables. Since the scales were highly correlated, we used this approach, which reduced the level of collinearity in the model since comparison of different severity scales was outside the scope of this work.

[0206] To evaluate the fitness of blood biomarker panels and stroke scales, likelihood ratio tests (LR) and AIC were used. The area under the receiver operating characteristic curve (AUC) with 95% CI was used as a measure of discrimination. Also, sensitivity, specificity, positive likelihood ratio (LR+), and negative likelihood ratio (LR-) were assessed at the selected cut-off points. For each model, the cut-off point was selected by maximizing the specificity for LVO prediction while maintaining a minimum specificity level of 90% according to our power test. Correction for optimistic prediction was performed through the bootstrap method with 2000 resamples and shown with confidence intervals (CI).

[0207] All analyses were performed using R version 3.6.2 with the assistance of RStudio version 1.2.5033, using the packages nnet, ROCR, caret, tidyverse, oddsratio, lmtest, and OptimalCutpoints.

[0208] (Example 2A) Cohort description and clinical variable analysis In this study, the inventors retrospectively recruited 170 patients with suspected stroke. After adjudication (method) of the diagnosis, the inventors obtained a final cohort of 128 patients with suspected stroke, which consisted of stroke subtypes of hemorrhagic stroke (n = 16, 12.5%), LVO ischemic stroke (n = 23, 18%), non-LVO ischemic stroke (n = 42, 33%), stroke-like symptoms (n = 31, 24%), and transient ischemic attack (n = 16, 12.5%). The median time from stroke onset to blood collection (OBT) for the cohort was 158 minutes (IQR = 161.5 minutes).

[0209] The inventors evaluated the differences between LVO patients and non-LVO patients (including ischemic non-LVO, hemorrhagic, and non-stroke patients) for 42 clinical variables associated with each patient (Figure 1). After adjustment for multiple hypothesis testing, the inventors found that the NIHSS score (p-value = 7.04 e-08; median LVO vs. non-LVO: 18 vs. 3; IQR LVO vs. non-LVO: 8.5 vs. 5), presence of atrial fibrillation (p-value = 0.0002; absence / presence LVO vs. non-LVO: 11 / 12 vs. 94 / 11), and systolic blood pressure (p-value = 0.033; mean LVO vs. non-LVO: 140 vs. 157; SD LVO vs. non-LVO: 22 vs. 29). In our cohort, age and sex were not significantly associated with the diagnosis of LVO. Notably, we found no difference in the time from stroke onset to blood draw between LVO and non-LVO patients.

[0210] (Example 2B) Blood Biomarkers for LVO Identification Subsequently, the inventors initiated an investigation of the levels of a panel of blood proteins in a patient cohort. The inventors measured the levels of D-dimer, osteopontin (OPN), osteoprotegerin (OPG), von Willebrand factor (vWF), ADAMTS13 (a disintegrin and a metalloproteinase with a thrombospondin type I motif, member 13), and glial fibrillary acidic protein (GFAP) in the plasma of LVO and non-LVO patients (FIGS. 2A, 7, and 11). The inventors found statistically significant differences between LVO and non-LVO patients for the blood biomarkers D-dimer (p-value < 0.001; mean LVO vs. non-LVO: 1.31 vs. 0.42 μg / mL; SD LVO vs. non-LVO: 2.00 vs. 0.45 μg / mL), OPN (p-value < 0.01; mean LVO vs. non-LVO: 1.71 vs. 1.16 ng / mL; SD LVO vs. non-LVO: 1.05 vs. 1.09 ng / mL), and OPG (p-value < 0.01; mean LVO vs. non-LVO: 125.24 vs. 97.96 pg / mL; SD LVO vs. non-LVO: 60.96 vs. 54.29 pg / mL). Without being bound by any particular theory, the inventors believe this data suggests that among the biomarkers tested in the panel, D-dimer, OPN, and OPG may assist in the identification of LVO stroke.

[0211] Subsequently, the inventors constructed a logistic regression model based on the levels of each significant biomarker. The inventors estimated the area under the ROC curve for each model and found that D-dimer yielded an AUC of 0.80, while the AUCs associated with OPN and OPG were 0.66 and 0.67, respectively (Figure 2). By ROC curve analysis, we estimated the cut-off levels for D-dimer, OPN, and OPG by maximizing the diagnostic specificity for LVO identification while maintaining a minimum level of 50% sensitivity (Table 2). All three biomarkers achieved a specificity of over 70% for LVO identification, and D-dimer achieved a specificity of nearly 90% (Figure 4). Notably, D-dimer, OPN, and OPG were independent predictors of the LVO state, with ORs of 10.34 (CI95%: 4.65 - 20), 3.05 (CI95%: 1.48 - 5.63), and 3.36 (CI95%: 1.68 - 6.32), respectively.

[0212] GFAP was included in the inventors' panel because it is known to be associated with hemorrhagic stroke. 20~22 Consistent with this hypothesis, GFAP levels were increased in hemorrhagic stroke compared to non-hemorrhagic stroke and non-stroke patients (Figure 1B; p-value = 0.007; mean hemorrhagic vs. non-hemorrhagic / non-stroke: 1043.46 vs. 66.10 pg / mL; SD hemorrhagic vs. non-hemorrhagic: 2581.28 vs. 127.24 pg / mL). The inventors constructed a logistic regression model based on the GFAP plasma model for the identification of hemorrhagic stroke in our cohort. Applying ROC curve analysis, the inventors found that a cut-off value of 265 pg / mL for GFAP levels could identify hemorrhagic patients with 88% accuracy, 30% sensitivity, and 96% specificity. Notably, GFAP levels higher than 265 pg / mL were an independent predictor of hemorrhagic stroke in our cohort (OR: 12.27; CI95%: 2.87 - 52.52; p-value = 0.0015).

[0213] The inventors' discovery suggests that a biomarker panel (OOD) consisting of OPN, OPG, and D-dimer can assist in the identification of LVO. The inventors constructed a logistic model based on the levels of the OOD biomarkers and applied ROC curve analysis to estimate the logistic model threshold with the highest accuracy for the identification of LVO (Figure 5). The inventors found that a model threshold of 0.69 can identify LVO patients with 83% accuracy (CI95%: 79 - 88%), 57% sensitivity (CI95%: 42 - 71%), 90% specificity (CI95%: 86 - 93%), a positive likelihood ratio of 5.7 (CI95%: 3.47 - 9.39), and a negative likelihood ratio of 0.48 (CI95%: 0.32 - 0.64). Notably, patients scored with a model value lower than 0.69 had a 12-fold higher likelihood of suffering from LVO stroke compared to patients with higher model values (OR 12.56; CI95%: 5.48 - 27.71). This discovery indicates that a biomarker panel (OOD) consisting of OPN, OPG, and D-dimer can assist in the identification of LVO stroke.

[0214] In addition, the inventors' findings suggest that the inclusion of GFAP in the biomarker panel aids in excluding hemorrhagic stroke from a population of patients suspected of having a stroke and may potentially improve LVO identification. To test this hypothesis, the inventors estimated the LVO diagnostic performance of a biomarker panel (GOOD) consisting of GFAP, OPN, OPG, and D-dimer and compared its performance to an OOD model (Figure 5). Maximizing accuracy by ROC curve analysis estimated a logistic model threshold of 0.59, which enabled the identification of LVO patients with 86% accuracy (CI95%: 82 - 90%), 52% sensitivity (CI95%: 38 - 67%), 94% specificity (CI95%: 91 - 97%), a positive likelihood ratio of 10.14 (CI95%: 5.09 - 21.67), and a negative likelihood ratio of 0.51 (CI95%: 0.35 - 0.66). Notably, the inclusion of GFAP in the OOD panel nearly doubled the likelihood of LVO patients testing positive (OR 21.06; CI95%: 8.17 - 50). Application of the logistic model threshold at the highest predicted accuracy (cut-off = 0.59) identified patients with biomarker levels in the range of GFAP: 17.43 - 184.27 pg / mL; OPN: 0.71 - 1.86 ng / mL; OPG: 73.34 - 205.47 pg / mL; D-dimer: 2.42 - 4.65 μg / mL.

[0215] The inventors estimated the various levels of diagnostic performance obtained by varying the logistic model threshold of the GOOD biomarker model (Figure 6). The inventors divided the model value distribution into deciles and found that the first two deciles (logistic model values: 0.18 - 0.71) were the most significant for LVO identification. Indeed, when the logistic model threshold was set at the first and second deciles, the odds ratios of LVO patients having a positive test were 16.67 (CI95%: 5.44 - 51.4; p-value = 0.00011) and 5.55 (CI95%: 2.11 - 12.50; p-value = 0.0129), respectively (Figure 6).

[0216] These data indicate that a biomarker panel (GOOD) consisting of GFAP, OPN, OPG, and D-dimer can provide a highly specific tool for the identification of LVO in a population of patients suspected of having pre-hospital stroke.

[0217] The inventors performed additional multivariate analyses and surprisingly found that the optimal combination of blood biomarkers for LVO prediction was D-dimer (OR 15.44, 95% CI 4.91 - 57.6; p-value < 0.0011) and GFAP (OR 0.83, 95% CI 0.5 - 0.99; p-value = 0.03) (Figure 10). The AUC of the model using D-dimer and GFAP for LVO prediction was 81% (95% CI 74 - 88%; Figure 9), and the accuracy was 85% (95% CI 81 - 89%; Table 5). The inventors estimated the model threshold to maximize specificity and obtained a sensitivity level of 56% (95% CI 34 - 76%), a specificity of 91% (95% CI 84 - 96%), an LR+ of 6.99 (95% CI 3.29 - 14.88), and an LR- of 0.47 (95% CI 0.3 - 0.76) (Figure 14).

[0218] (Example 2D) Integration of Biomarkers with Field Stroke Scales Several pre-hospital scales based on patient symptoms have been developed for the identification of LVO stroke in the field and show a high level of diagnostic sensitivity for LVO but lack diagnostic specificity. 5,7,10,19,23. The present inventors' findings suggest that a GOOD biomarker panel can provide a highly specific tool for LVO identification. Therefore, the present inventors hypothesized whether the inclusion of a blood biomarker panel could improve the diagnostic specificity of the pre-hospital stroke scales for LVO. The present inventors used the NIHSS score items to derive the stroke scales FAST, FAST-ED, RACE, CPSS, and EMSA and estimated their diagnostic performance at established scale thresholds (Figure 7). In the cohort, FAST-ED and RACE achieved the highest diagnostic performance for LVO identification, with accuracies of 84% (CI95%: 80-89) and 86% (CI95%: 82-91), respectively, and ORs of 32.11 (12.86-81.4) and 24.54 (10.73-52.78).

[0219] The present inventors then combined the blood biomarkers with the pre-hospital stroke scales and found that the GOOD panel improved the diagnostic performance of all the tested stroke scales (Figures 8 and 14). The highest diagnostic performance for LVO prediction was obtained by the combination of GOOD and FAST-ED, which achieved an accuracy of 96% (CI95%: 94-98), a sensitivity of 91% (CI95%: 83-100), and a specificity of 97% (CI95%: 95-99). According to this model, LVO patients had a 39 (CI95%: 18.2-96.09) times higher likelihood of testing positive compared to non-LVO patients. The present inventors found a significant association between our biomarkers and the FAST-ED scale (GFAP: p-value = 0.00139; OPN: p-value = 0.01321; OPG: p-value = 0.04144; D-dimer: p-value = 0.00002), but after adjustment for such associations, the logistic model constructed for the GOOD biomarkers was still highly significant (Fisher's exact p-value = 6.53 e-12 ).

[0220] Further analysis by the inventors has found that the addition of D-dimer and GFAP results in an improvement in goodness-of-fit (i.e., lower AIC), an improvement in LVO prediction (i.e., higher AUC), and a significant LR test for each stroke scale tested, compared to using the stroke scale alone (Figures 9, 10, and 14). The combination of D-dimer and GFAP with FAST-ED or EMSA resulted in the highest LR+ for LVO prediction (22.6 and 17.22, respectively, 95% CI 8.58 to 59.51 and 7.22 to 41.04), LR- of 0.09 (95% CI 0.02 to 0.34) or 0.14 (95% CI 0.05 to 0.39), sensitivity of 91% (95% CI 71 to 98) or 86% (95% CI 66 to 97), and specificity of 95% (95% CI 89 to 98) or 94% (95% CI 88 to 98), respectively.

[0221] Without being bound by any particular theory, the inventors' findings indicate that the combination of D-dimer, OPN, OPG, and GFAP, particularly D-dimer and GFAP, with a pre-hospital stroke scale can provide a high-precision tool and significantly improve the identification of LVO stroke.

[0222] Discussion The inventors have identified, for the first time, OPN, OPG, and D-dimer as biomarkers for LVO stroke. The inventors have also demonstrated that a biomarker panel (GOOD) consisting of GFAP, OPN, OPG, and D-dimer can provide a useful tool for the highly specific (94%) identification of stroke patients with large vessel occlusion (LVO) from a population of patients suspected of having a stroke. Furthermore, the inventors have shown that combining the GOOD biomarker panel with a pre-hospital stroke scale based on the patient's symptoms can result in unprecedented diagnostic accuracy (greater than 95%) for LVO identification.

[0223] The inventors analyzed the plasma of 128 patients suspected of having a stroke. The observed proportion of LVO stroke compared to all confirmed ischemic strokes was as previously reported1 was 32%, which was consistent. In addition, the percentage of stroke-like symptoms we observed (29%) was comparable to other studies 67 .

[0224] The inventors found that a cut-off value of 265 pg / mL for GFAP levels can identify hemorrhagic patients with 30% sensitivity and 96% specificity. To the inventors' knowledge, no study has directly addressed the ability of GFAP measurements as a diagnostic tool for identifying LVO patients. In addition, previous studies have not addressed the ability of plasma GFAP measurements to identify LVO patients.

[0225] The inventors' study demonstrates that GFAP can significantly improve LVO identification by excluding hemorrhagic patients from the population suspected of having a stroke when measured together with other blood biomarkers. The inventors provide the first evidence that plasma GFAP measurements can be used as a tool for diagnosing and triaging LVO patients.

[0226] Also, the inventors' results show that not only do OPN and OPG individually increase in LVO vs. non-LVO patients, but both markers significantly contribute to LVO identification, either individually or in combination with the inventors' biomarker panel. Previous published studies have focused on comparing D-dimer levels between subtypes of ischemic stroke or between ischemic stroke and control subjects, while the inventors' study evaluates D-dimer levels in LVO patients compared to a heterogeneous group of patients suspected of having a stroke, including hemorrhagic stroke, non-LVO ischemic stroke, stroke-like symptoms, and TIA, and thus simulates a realistic clinical scenario.

[0227] To the inventors' knowledge, this is the first evidence of an increase in D-dimer levels in LVO patients compared to a population of patients suspected of having a stroke that closely resembles the population found in the pre-hospital setting.

[0228] The inventors have shown that combining a large number of biomarkers into a panel can achieve higher diagnostic performance for LVO identification compared to the use of individual biomarkers. In the inventors' study, the combination of GFAP, OPN, OPG, and D-dimer enabled the identification of LVO stroke with 86% accuracy, which is higher compared to the individual biomarker with the highest score (i.e., D-dimer). Nevertheless, the measurement of each biomarker alone also provides an accurate means of diagnosing LVO stroke. In particular, D-dimer alone results in a diagnostic accuracy of 83%, with clinically acceptable levels of specificity and sensitivity (89% and 52%, respectively), and the combination of D-dimer and GFAP results in even higher accuracy, with specificity and sensitivity of 91% and 56%, respectively.

[0229] These findings suggest that more complex biomarker panels can enable more accurate identification of LVO stroke, but the measurement of biomarkers alone, and particularly D-dimer, can be selected in specific clinical scenarios, such as scenarios where blood collection techniques do not allow for the collection of large volumes of blood (e.g., collection by pricking a fingertip at the scene), or where simplicity and rapidity of testing are prioritized (e.g., in ambulances).

[0230] Several stroke scales based on patient symptoms have been developed to identify LVO stroke patients in the pre-hospital setting. In this study, the inventors derived the LVO pre-hospital stroke scales FAST-ED, RACE, CPSS, and EMSA. In addition, since the FAST score is the currently most widely applied pre-hospital scale, the inventors derived the FAST score. The inventors estimated the diagnostic measurements of these scales for LVO identification and observed that their predictive performance was higher compared to previous studies 5~10 This may be due to the fact that the inventors derived these stroke scales from the NIHSS scores of each patient taken by neurologists rather than by trained paramedics.

[0231] The overall goal of identifying LVO patients in the field is to guide the decision of ambulance staff regarding the transfer of patients suspected of having a stroke to a specialized stroke center with dedicated capabilities. Since LVO stroke patients require treatment by endovascular thrombectomy (EVT), the identification of these patients in the field can direct their transfer to an EVT-capable center, even if it is not the closest stroke center. Direct transfer of LVO patients to an EVT-capable center has been shown to 24 reduce the time to treatment and patient outcome compared to interfacility transfer. Nevertheless, the use of prehospital stroke scales in the field has spread across the country, and their clinical value remains to be confirmed and validated. As previously suggested 25 , this may be due to the limited diagnostic specificity of LVO demonstrated so far. In fact, to modify the emergency transport itinerary from transfer to the closest stroke center to transfer to the closest EVT-capable center 26 , a high level of specificity (or positive predictive value) may be required.

[0232] In this study, the inventors propose a combination of blood biomarkers and prehospital stroke scales for the identification of LVO. The inventors have identified novel biomarkers for LVO stroke and demonstrated that a model constructed for the GOOD biomarker panel together with prehospital stroke scales can provide even higher predictive ability for LVO compared to the use of blood biomarkers or stroke scales alone. The inventors observed that the combination of the GOOD biomarker panel and FAST-ED provides the highest diagnostic accuracy (96%) for LVO, while the combination with RACE provides the highest sensitivity (81%). Notably, the highest diagnostic specificity (98%) was obtained when the GOOD panel was combined with either FAST or EMSA. The inventors also showed that these findings indicate that combining biomarker panels with prehospital stroke scales can provide the diagnostic performance highly necessary for confidently triaging LVO patients in the field.

[0233] To address various problems and advance the art, the present disclosure as a whole shows, by way of example, various embodiments in which the claimed invention may be practiced to provide an excellent diagnostic test for stroke resulting from large vessel occlusion. The advantages and features of the present disclosure are those of only representative samples of the embodiments and are not comprehensive and / or exclusive. They are shown only to assist in understanding and teaching the features of the claims. It should be understood that the advantages, embodiments, examples, functions, features, structures, and / or other aspects of the present disclosure are not to be considered as limitations on the present disclosure as defined by the claims or on equivalents of the claims, and that other embodiments may be utilized and modifications may be made without departing from the scope and / or spirit of the present disclosure. The various embodiments may preferably comprise, consist of, or consist essentially of various combinations of the disclosed elements, components, features, parts, steps, means, etc. Additionally, the present disclosure includes other inventions that may not be currently claimed but may be claimed in the future. (References) TIFF0007711092000014.tif224170TIFF0007711092000015.tif238170TIFF0007711092000016.tif59170

Claims

**Claim 1** A method for identifying stroke as a result of large vessel occlusion (LVO), comprising the step of determining the amounts of at least two biomarkers in a sample taken from an individual who has, has been identified as having, or is suspected of having had a stroke, and comparing the amount of each biomarker in said sample taken from said individual who has, has been identified as having, or is suspected of having had a stroke, with the amount of the same biomarker in one or more of a control and a positive standard, wherein said biomarkers are D-dimer and glial fibrillary acidic protein (GFAP), wherein said control means data on the amounts of said at least two biomarkers obtained from samples taken from one or more healthy individuals, and wherein said individual is identified as having had or having stroke as a result of LVO if the amount of one or more of said measured biomarkers is different from the amount of the same biomarker in the control or equivalent to the amount of the same biomarker in the positive standard. **Claim 2** A method for identifying individuals suffering from stroke as a result of LVO from a group of individuals who have, have been identified as having, or are suspected of having had a stroke, comprising the step of determining the amounts of at least two biomarkers in a sample taken from each individual, and comparing the amount of each biomarker in said sample taken from said individual who has, has been identified as having, or is suspected of having had a stroke, with the amount of the same biomarker in one or more of a control and a positive standard, wherein said biomarkers are D-dimer and glial fibrillary acidic protein (GFAP), wherein said control means data on the amounts of said at least two biomarkers obtained from samples taken from one or more healthy individuals, and wherein said individual is identified as having had or having stroke as a result of LVO if the amount of one or more of said measured biomarkers is different from the amount of the same biomarker in the control or equivalent to the amount of the same biomarker in the positive standard. **Claim 3** The method according to claim 1 or 2, comprising the step of determining the amounts of up to two additional biomarkers, wherein said up to two additional biomarkers are osteopontin (OPN) and / or osteoprotegerin (OPG). **Claim 4**: The method according to claim 1, wherein an increase in the amount of D-dimer when compared to a control indicates that the individual has suffered a stroke as a result of LVO. **Claim 5**: The method according to claim 3, wherein an increase in the amount of OPN and / or OPG when compared to a control indicates that the individual has suffered a stroke as a result of LVO. **Claim 6**: The method according to claim 1, wherein a decrease in the amount of GFAP when compared to a control indicates that the individual has suffered a stroke as a result of LVO. **Claim 7** The method according to any one of claims 1 to 6, wherein the sample from the individual is one or more of blood, plasma, cerebrospinal fluid or saliva. **Claim 8** The method according to any one of claims 1 to 7, further comprising the step of determining a stroke severity score from the individual. **Claim 9**: The method according to claim 8, wherein the stroke severity score is selected from NIHSS, FAST, FAST-ED, RACE, C-STAT and EMSA. **Claim 10** The method according to any one of claims 1 to 9, comprising the step of combining the amount of one or more biomarkers measured in the sample taken from an individual identified as having or suspected of having had a stroke with the stroke severity score obtained from the individual to form an LVO stroke score. **Claim 11** The method according to claim 10, comprising the step of comparing the LVO stroke score generated from an individual identified as having or suspected of having had a stroke with the LVO stroke score generated from a control or a positive standard. **Claim 12**: The method according to claim 11, wherein the LVO stroke score generated using the amount of the biomarker measured in the sample taken from an individual identified as having or suspected of having had a stroke is equal to the LVO stroke score generated using the amount of the biomarker from a positive standard; or if the LVO stroke score generated using the amount of the biomarker measured in the sample taken from an individual identified as having or suspected of having had a stroke is different from the LVO stroke score generated using the amount of the biomarker from a control, indicating that the individual has or has had a stroke as a result of LVO. **Claim 13** The method according to any one of claims 1 to 12, wherein the amount of the biomarker in the sample taken from the individual having or suspected of having had a stroke is determined using an agent that binds to the biomarker.

14. The method according to claim 13, wherein the amount of the biomarker is determined using ELISA or a lateral flow assay.

15. The method according to any one of claims 1 to 14, which is performed within 24 hours of the onset of stroke symptoms in the individual.

16. The method according to claim 15, which is performed within 10 hours of the onset of stroke symptoms in the individual.

17. The method according to claim 16, which is performed within 6 hours of the onset of stroke symptoms in the individual.

18. Use of at least two biomarkers for predicting stroke resulting from large vessel occlusion (LVO) in an individual having or suspected of having had a stroke, wherein the biomarkers are D-dimer and glial fibrillary acidic protein (GFAP).

19. Up to two additional biomarkers are used to predict stroke resulting from LVO in an individual having or suspected of having had a stroke, and the up to two additional biomarkers are osteopontin (OPN) and / or osteoprotegerin (OPG). The use according to claim 18.

20. A kit for performing the method according to any one of claims 1 to 12, comprising: a container for holding a sample from the individual having or suspected of having had a stroke; means for determining the amount of the biomarker; and one or more of instructions for use on how to perform the reaction between the sample and the means provided for determining the amount of the biomarker.

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