Method for Selecting Patients for Reperfusion Therapy

Abstract

The present invention relates to a method for selecting patients who have suffered a stroke for reperfusion therapy based on determining the levels of retinol binding protein-4 (RBP4) and the N-terminal fragment of B-type natriuretic peptide (NT-proBNP) in an isolated patient sample. The present invention also relates to a method for distinguishing ischemic from hemorrhagic stroke, and a kit containing reagents for carrying out the method.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of European Patent Application EP19382384.6, filed on May 16, 2019.

[0002] The present invention relates to the field of diagnosis or companion diagnosis, in particular to a method for differentiating ischemic stroke from hemorrhagic stroke, and to the selection of an appropriate therapy according to the type of stroke event.

Background Art

[0003] Stroke, also known as cerebrovascular disease (CVD), is one of the most important neurological diseases. It is the second leading preventable cause of death worldwide and a major cause of reduced productivity. The two main subtypes of stroke are ischemic stroke (IS) and intracerebral hemorrhage (ICH), also called hemorrhagic stroke. More than 80 - 85% of all strokes are IS caused by cerebral artery occlusion, while the remaining 15 - 20% are ICH that appears due to arterial rupture. In contrast to patients suffering from IS, which has a 30 - day mortality rate of 8 - 12%, in ICH patients, the mortality rate 30 days after symptom onset is 37 - 38% and the prognosis is poor.

[0004] In the acute phase, it is important to accurately distinguish between the two subtypes in order to define optimal treatment protocols that are specific and significantly different between IS and ICH. The primary treatment recommended for acute IS includes reperfusion, which is the restoration of blood flow by drug administration or endovascular surgery (thrombectomy). The main drugs used are thrombolytics, such as recombinant tissue plasminogen activator, a serine protease that dissolves blood clots occluding cerebral arteries, or tenecteplase (TNK, a recombinant fibrin-specific plasminogen activator derived from native t-PA by modification at three sites in the protein structure). Thrombolysis has a narrow treatment time frame of only 4.5 hours from symptom onset. By rapidly identifying IS, early blood flow reopening becomes possible, leading to tissue recovery from the periphery and potentially improving the clinical outcome. On the other hand, acute ICH patients are usually managed by reducing blood pressure to slow down hematoma growth or to avoid the appearance of edema and rebleeding. Today, the diagnosis of stroke subtypes is mainly based on brain imaging data by computed tomography (CT) or magnetic resonance imaging (MRI). Therefore, patients suspected of having a stroke have to be transported to a hospital to obtain a CT scan or MRI, losing valuable time. Unfortunately, MRI and CT scans are not widely available, especially in underdeveloped regions, and cannot be reused repeatedly due to resource constraints in primary hospitals. Furthermore, some of these technologies may have side effects mainly related to radiation or contrast agent injection. In addition, MRI and CT are susceptible to errors or uncertainties if the medical staff performing them have no experience or have not received sufficient training.

[0005] Among several documents that describe the use of biomarkers for the rapid discrimination of stroke subtypes, Patent Document 1 discloses a method for differentiating ischemic stroke from hemorrhagic stroke in a patient, and a method for selecting a patient suffering from stroke for therapy with an antithrombotic agent or an agent capable of reducing blood pressure, based on the determination of the level of glial fibrillary acidic protein (GFAP) in a sample of the patient in combination with one or more biomarkers.

[0006] Another example of a study analyzing biomarkers that may be associated with acute IS can be extracted from the literature by Reynolds et al., Non-Patent Document 1. In this literature, results regarding S-100B molecule, nerve growth factor type B, von Willebrand factor, matrix metalloproteinase-9 (MMP-9), and chemokine ligand 2 (having a C-C motif) (CCL-2), also known as chemotactic protein-1 (MCP-1), as potential biomarkers in the plasma of stroke patients are shown. The authors concluded that only the MCP-1 protein had significant value for the diagnosis of acute ischemic stroke, and samples were extracted from the cerebrospinal fluid of the patients, but the serum concentration was not different from that of the control patients. Therefore, it was assumed to be a method with low practicality for the accurate identification of the disease.

[0007] Biomarkers for the specific diagnosis of cardioembolic stroke are also disclosed in Non-Patent Document 2. Brain natriuretic peptide (BNP) and D-dimer (DD) have been proposed to improve the diagnosis of cardioembolic stroke in the acute phase of stroke.

[0008] Strictly speaking, for the severity of the disease, accurate diagnosis between stroke subtypes is important because reperfusion therapy for non-IS patients can be potentially fatal. The diagnosis of this subtype is preferably performed as soon as possible, especially after the patient is found in the acute phase at home, on the road, or in the clinic of a general practitioner. Therefore, it would be even better if there were a kit or point-of-care that could be easily implemented in an ambulance and further verified in the hospital.

[0009] Other teams use strategies such as mobile stroke units, which are ambulances incorporating CT scans, to perform stroke diagnosis outside the hospital and administer reperfusion therapy as soon as possible to improve the neurological outcomes of treated patients. However, this strategy is very expensive, these high-tech ambulances are costly, and specialized personnel are required.

[0010] Recent trials have shown that endovascular treatment for large vessel occlusion (LVO) reduces the morbidity and mortality of patients experiencing this form of severe acute ischemic stroke. Nevertheless, in many cases, a minority of patients experiencing LVO receive endovascular treatment due to delays in arrival at specialized hospitals where endovascular treatment can be performed (Non-Patent Document 3). Emergency medical services (EMS) specialists are often the first to encounter patients experiencing acute stroke, and if LVO stroke is recognized early in the pre-hospital setting by EMS specialists, timely transport to an endovascular center is improved, which may lead to better patient outcomes (Non-Patent Document 4). Crowe et al. compared various scales used for the diagnosis of LVO (see above). Crowe et al. showed that in 2,415 patients experiencing acute ischemic stroke, 26% (n = 628) of ischemic stroke patients were diagnosed with LVO.

[0011] Two or more CPSS scores showed 69% sensitivity and 78% specificity for LVO. Four or more RACE scores showed 63% sensitivity and 73% specificity. Three or more LAMS scores showed 63% sensitivity and 72% specificity, and a positive VAN score showed 86% sensitivity and 65% specificity. When comparing the areas under the ROC curves of each scale, no statistically significant difference in the discriminatory ability for LVO stroke was observed. This reveals the need for a reliable marker for LVO.

[0012] Furthermore, LVO is associated with poor outcomes at 3 and 6 months in patients with acute ischemic stroke (AIS) (Non-Patent Document 5). Lakomkin et al. found that 16 trials included in the systematic review used 9 different definitions of LVO (different combinations of arterial occlusion sites), and as shown by Waqas et al., this may affect the prevalence of LVO (see Non-Patent Documents 6 and 7).

[0013] Finally, in the field of stroke diagnosis and treatment, it is worth noting to distinguish between so-called stroke mimics and actual strokes. A stroke mimic is defined as a disease or condition that presents a clinical picture similar to a stroke but is not accompanied by neurological tissue infarction. Some clinical syndromes may present symptoms or signs similar to acute ischemic stroke, and thus, differentiating between a stroke and a stroke mimic is difficult due to a wide variety of overlapping clinical symptoms. This is a real challenge for physicians due to the potential harmful effects of interventional stroke treatment. Currently, there are few markers that can distinguish between actual strokes and mimics in isolated patient samples.

Prior Art Documents

Patent Documents

[0014]

Patent Document 1

Non-Patent Documents

[0015]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 7

Summary of the Invention

Problems to be Solved by the Invention

[0016] Therefore, in the art, there is a need for alternative tests that overcome the limitations of the methods disclosed in the art and can reliably identify stroke subtypes and mimics that are excluded in order to determine the best treatment approach for patients in the shortest period of time. Furthermore, while a clear definition of LVO has been established, the need for a reliable marker for LVO, a condition that requires specific treatment (i.e., endovascular treatment or thrombus removal), has not been met.

Means for Solving the Problems

[0017] In a first aspect, the present invention relates to an in vitro method for selecting a patient suffering from stroke for reperfusion therapy, the in vitro method comprising determining the level of retinol-binding protein-4 (RBP4) and the level of the N-terminal fragment of B-type natriuretic peptide (NT-proBNP) in an isolated sample of the patient.

[0018] Therefore, this method is included as a companion diagnostic method.

[0019] The inventors have surprisingly discovered for the first time that by determining the levels of these two proteins in an isolated sample, a good classification between IS and ICH is possible.

[0020] Therefore, another aspect of the present invention is an in vitro method for distinguishing IS from ICH in a patient, the in vitro method comprising determining the levels of RBP4 and NT-proBN in an isolated sample of the patient.

[0021] Based on the levels of NT-proBNP and RBP4, patients can be classified into two groups: a group that can mainly receive reperfusion therapy by an anticoagulant or thrombus removal, and a group that should avoid reperfusion therapy to avoid a fatal outcome. Among the latter, ICH patients are likely to be treated by a treatment that reduces or optimizes blood pressure.

[0022] As shown in the following examples, based on the combined levels of NT-proBNP and RBP4, patients can be classified with a specificity of 100% or almost 100%. Therefore, this combination of markers is very accurate, and a truly safe way to select an appropriate therapy (i.e., patients who are candidates for reperfusion therapy) is envisioned. To the best knowledge of the inventors, this is the first time that a marker detectable in an isolated sample of a patient (i.e., a biological fluid sample) has achieved a specificity value of 100% or almost 100%. Furthermore, very advantageously, both markers enable discrimination between different stroke types even when measured within 6 hours or less, or even within 3 hours or less after symptom onset. In other words, accurate discrimination during the critical time (ultra-early stage) is possible.

[0023] Furthermore, as exemplified in the following examples, by determining these two proteins in an isolated sample, it is also possible to detect patients suffering from stroke with a poor prognosis or outcome in the sense of having a relatively high mortality rate. Therefore, these patients need to be treated as quickly as possible to avoid the progression of a poor outcome.

[0024] Accordingly, the present invention also relates to a method for the prognosis of patients suffering from stroke, particularly ischemic stroke, and thus candidates for reperfusion therapy, which method comprises determining the expression level of RBP4, optionally in combination with the expression level of NT-proBNP in an isolated sample of the patient. As far as the inventors know, this is the first time that the association of RBP4, or of RBP4 and NT-proBNP with this poor outcome has been shown. In a particular embodiment of the method for prognosis, the levels of both proteins in the patient's sample are determined, which sample is, in another particular embodiment, a biological fluid sample, more specifically blood (plasma or serum). In yet another particular embodiment of the method for prognosis of patients suffering from stroke, at least the level of RBP4 or of the two proteins is compared to a reference value, which reference value is selected from a value or range of values indicative of the subject suffering from ischemic stroke.

[0025] In yet another aspect, the present invention relates to a kit comprising reagent means for detecting the levels of RBP4 and NT-proBNP.

[0026] The present invention also discloses a kit comprising a reagent for detecting the level of a marker selected from GFAP, RBP4, NT-proBNP or combinations thereof.

[0027] In yet another aspect, the present invention also aims at the use of means for detecting the presence of either RBP4 or NT-proBNP in a test sample, selected from the group consisting of immunoassay, protein migration, chromatography, mass spectrometry, nephelometry, nephrometry and polymerase chain reaction (PCR), for selecting a patient suffering from stroke for reperfusion therapy as defined in the first aspect, or for performing a method for differentiating an ischemic stroke of a patient from a hemorrhagic stroke.

Brief Description of the Drawings

[0028]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Mode for Carrying Out the Invention

[0029] Detailed Description of the Invention All terms used in the specification of this application shall be understood to have their ordinary meanings known in the art, unless otherwise specified. Further specific definitions of particular terms used in this application are set forth below and are intended to be uniformly applied throughout the specification and the claims, unless an explicitly defined definition provides a broader definition.

[0030] As used herein, the term "patient" (or subject) refers to any subject exhibiting one or more signs or symptoms typically associated with stroke, such as sudden onset facial muscle weakness, arm drift, abnormal speech, and combinations thereof, such as FAST (face, arm, speech, and time), facial hemiparesis and muscle weakness, numbness, decreased sensation or vibration sense, initial flaccidity (hypotonia) replaced by spasticity (hypertonia), hyperreflexia, forced synergistic movement, and in particular, when they occur on one side (unilateral) of the body, (total or partial) changes in smell, taste, hearing, or vision, ptosis (drooping) and extraocular muscle weakness, hyporeflexia (e.g., pharyngeal, swallowing, and pupillary responses to light), facial sensory loss and muscle weakness, balance disorders and nystagmus, changes in respiration and heart rate, weakness of the sternocleidomastoid muscle preventing turning the head to one side, tongue weakness (inability to protrude and / or move laterally), aphasia, dysarthria, apraxia, visual field defects, memory impairment, hemineglect, fragmented thinking, confusion, hypersexual gesture, lack of insight, disorders typically associated with stroke, changes in gait pattern, changes in motor coordination, dizziness, headache, and / or balance disorders. As used herein, the term "patient" also refers to any animal classified as a mammal, including but not limited to, domesticated animals and livestock, primates and humans, such as humans, non-human primates, cows, horses, pigs, sheep, goats, dogs, cats, or rodents. Preferably, the patient is a male or female human of any age or ethnicity. Preferably, the patient is suffering from a stroke.

[0031] As used herein, the term "selecting a patient for therapy" relates to the identification of a patient for a therapy designed to treat a disease or alleviate symptoms associated with one or more diseases or conditions. In the specific case of stroke therapy, it is understood as any therapy that eliminates, delays, or reduces symptoms associated with stroke, more specifically, symptoms associated with ischemic stroke or hemorrhagic stroke.

[0032] The term "reperfusion therapy" relates to medical procedures for restoring blood flow through or around an occluded artery. Reperfusion therapies include medications and endovascular surgeries. The medications are thrombolytic agents (antithrombotic agents) and fibrinolytic agents used in a process called thrombolysis. The interventions performed may be minimally invasive endovascular procedures (thrombectomy) for removing thrombi that use one or more stent retrieval devices, aspiration techniques, or alternative devices that combine both stent retrieval devices and aspiration. Other surgeries performed are more invasive bypass surgeries that graft an artery around the occlusion. "Mechanical thrombectomy" or simply thrombectomy is an interventional procedure for removing blood clots (thrombi) from blood vessels. This is generally performed for coronary arteries (interventional cardiology), peripheral arteries (interventional radiology), and cerebral arteries (interventional neuroradiology). Patient selection, while it is preferred to be so, need not be appropriate for 100% of the subjects selected according to this first method of the present invention. However, this term requires that a statistically significant portion of the subjects be correctly selected. Whether the selection of patients within the subject population is statistically significant can be determined by one skilled in the art using various well-known statistical evaluation tools, such as determination of confidence intervals, p-value determination, Student's t-test, Mann-Whitney test, etc. Details can be found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95%. The p-value is preferably 0.01, 0.05, 0.005, 0.001 or less.

[0033] The term "ischemic stroke" (abbreviated as IS) refers to the physical blockage of blood flow to a certain area of the brain, resulting in the death of brain cells in that area. Ischemic stroke can be further divided into thrombotic stroke and embolic stroke. Thrombotic stroke occurs when a cerebral artery is occluded by a blood clot formed within the brain. Embolic stroke is caused by a blood clot formed in a peripheral artery or the heart that migrates to the brain and causes ischemia. Another type of ischemic stroke is lacunar stroke due to the occlusion of small cerebral arteries.

[0034] As used herein, the term "hemorrhagic stroke" (abbreviated as ICH in the case of intracerebral hemorrhage) refers to bleeding into the brain tissue due to blood vessel rupture.

[0035] The inventors of the present invention have identified RBP4 and BNP as new plasma biomarkers for accurately selecting patients suffering from stroke for reperfusion therapy. Therefore, these markers can be used to differentially diagnose acute IS from ICH. By using some data analysis methods with these two markers, a specificity of 100% was achieved with a sensitivity of 20% - 30%. Furthermore, when the level of a third marker, particularly GFAP, was added, the sensitivity increased by 60% while the specificity was maintained.

[0036] Therefore, in a specific embodiment of the first aspect of selecting a patient suffering from stroke for reperfusion therapy, the method further comprises determining the level of GFAP in an isolated sample of the patient.

[0037] Although an improvement in sensitivity is desirable, the inventors have also developed a simplified kit that only includes means for detecting the levels of RBP4 and NT-proBNP. Using this simplified kit, which can be used in an ambulance when a stroke patient is being cared for, IS and ICH can be accurately discriminated. Thereby, if appropriate, reperfusion therapy can be administered as soon as possible (i.e., an anti-thrombotic agent in the ambulance), and the patient can be treated immediately, thus avoiding adverse outcomes, at least in the case of IS. Furthermore, in the case of ICH, if the blood pressure is optimized, the worsening of symptoms can be avoided.

[0038] In a still more specific embodiment of the first aspect, the method includes comparing the level with a corresponding reference value or reference range for each protein, the reference value or reference range being selected from values or ranges of values obtained from subjects suffering from ischemic stroke, and the subject being classified as a candidate for reperfusion therapy when at least the levels of both RBP4 and NT-proBNP are within the value or range of values obtained from subjects suffering from IS.

[0039] In a further specific embodiment, the reference value or reference range is selected from values or ranges of values obtained from subjects suffering from IS or subjects suffering from ICH, and the subject is classified as a candidate for reperfusion therapy when at least the levels of both RBP4 and NT-proBNP are within the value or range of values obtained from subjects suffering from IS. At both levels within the value or range of values obtained from subjects suffering from IS, a significant clinical sensitivity (about 21%) is achieved for a specificity exceeding 98%, particularly 100% specificity.

[0040] In another specific embodiment of the first aspect, the method includes comparing the levels of RBP4, NT-proBNP, and optionally GFAP when determined, with corresponding reference values or reference ranges for each protein, the reference values or reference ranges being selected from values or ranges of values obtained from subjects suffering from ischemic stroke, and the subject being classified as · a candidate for reperfusion therapy when at least the levels of both RBP4 and NT-proBNP are within the value or range of values obtained from subjects suffering from ischemic stroke, and · having a prognosis defined by a dependency exceeding 2 according to a modified ranking score (mRS) determined within 1 to 5 months after the onset of stroke, and / or having a prognosis defined by a 3-month post-onset mortality rate composed of 20% to 30%.

[0041] In yet another more specific embodiment, the method includes the step of comparing the levels of RBP4, NT-proBNP, and GFAP, and if the subject is classified as a candidate for reperfusion therapy, the subject is also defined by a dependency greater than 2 according to the modified Rankin score (mRS), and within 1 ~ It is classified as having a prognosis determined within 5 months and / or as having a prognosis defined by a 3-month post-onset mortality rate consisting of 20% to 30%.

[0042] The prognosis is defined as a dependency greater than 2 according to the modified Rankin score (mRS) and is determined at least 3 months after the onset of stroke. In a specific embodiment, the 3-month post-onset mortality rate is at least 23%. In another specific embodiment, it is 25%.

[0043] In a specific embodiment where only one of the levels of RBP4 and NT-proBNP is within or within the range of values obtained from a subject suffering from IS, the subject is also classified as a candidate for reperfusion.

[0044] In another specific embodiment of the first aspect, the in vitro method further includes the step of comparing the levels of RBP4, NT-proBNP, and, if determined, GFAP with the corresponding reference cut-off values for each protein, · If only the levels of RBP4 and NT-proBNP are determined, the levels of RBP4 and NT-proBNP simultaneously, the corresponding reference cut-off values Ref1 RBP4 and Ref1 NT-proBNP being above indicates that the patient is a candidate for reperfusion therapy, or · If the levels of RBP4, NT-proBNP, and additional GFAP are determined, in the first step the level of GFAP is below the reference cut-off value Ref GFAP and in the second step the levels of RBP4 and NT-proBNP simultaneously, the corresponding reference cut-off value Ref2 RBP4and Ref2 NT-proBNP If so, the patient is selected as a candidate for reperfusion therapy, and the cut-off value discriminates between ischemic stroke patients and intracerebral hemorrhage patients.

[0045] Indeed, different alternative embodiments of the method of the first aspect when including the option of comparing the test level with each respective cut-off value or reference range are selected taking into account specific values of the desired sensitivity and specificity. Thus, if 100% specificity (accurate classification between two states) is desired, the sensitivity (detection of one state in a target cohort having different states) may decrease. On the other hand, if the specificity is decreased (i.e., about 94% or 98%), the sensitivity of the method can be increased. Thus, the reference value can be varied according to the desired specificity and / or the desired sensitivity.

[0046] In a more specific embodiment of this method including comparison with cut-off values of two or three protein levels, if a subject is classified as a candidate for reperfusion therapy, the subject is also defined by a dependency greater than 2 according to the modified Rankin Scale (mRS) and having a prognosis determined within 1 to 5 months after stroke onset, and / or having a prognosis defined by a 3-month post-onset mortality rate composed of 20% to 30%.

[0047] Also, as another specific embodiment of the method of the first aspect for selecting a patient suffering from stroke for reperfusion therapy, the step of treating the patient with the reperfusion therapy, or, in the case of corresponding reference cut-off values for RBP4 and NT-proBNP, optionally GFAP, further includes the step of classifying the patient as a candidate for reperfusion therapy when at least the levels of RBP4 and NT-proBNP are both within or within the range of values obtained from a subject suffering from IS.

[0048] As shown above, there are several treatment protocols for promoting reperfusion. In a particular embodiment of the first aspect of the present invention, the reperfusion therapy is selected from the group consisting of therapy with antithrombotic agents, thrombus removal, and combinations thereof.

[0049] In a more particular embodiment, the antithrombotic agent is a thrombolytic agent. In an even more particular embodiment, the thrombolytic agent is a plasminogen activator. More specifically, the plasminogen activator is tissue plasminogen activator.

[0050] As used herein, the term "antithrombotic agent" refers to a drug capable of reducing blood clot formation. Suitable antithrombotic agents for use in the present invention include, but are not limited to, thrombolytic agents, antiplatelet agents, and anticoagulant compounds.

[0051] As used herein, the term "thrombolytic agent" refers to a drug capable of dissolving blood clots. All thrombolytic agents are serine proteases that convert plasminogen to plasmin, degrade fibrinogen and fibrin, and dissolve blood clots. Currently available thrombolytic agents include reteplase (r-PA or Retavase), alteplase (t-PA or Activase), urokinase (Abbokinase), prourokinase, anistoylated purified streptokinase activator complex (APSAC), staphylokinase (Sak), tenecteplase (TNK-tPA), atenecteplase (TNKasa), anisoylated streptokinase (Eminase), streptokinase (Kabikinase, Streptase) or uroquinase (Abokinase). Tenecteplase (TNK-tPA) can be administered as a rapid single bolus and can be used at the ambulance level, and is thus used in certain embodiments. TNK has efficacy 1 minute after administration (after injection). The suppliers of TNK are Boehringer Ingelheim (European Union) and Genentech Inc (USA).

[0052] As used herein, the term anticoagulant compound refers to compounds that prevent coagulation and include, but are not limited to, vitamin K antagonists (warfarin, acenocoumarol, fenprocoumon and fenidione), heparin and heparin derivatives, such as low molecular weight heparin, factor Xa inhibitors, such as synthetic pentasaccharides, direct thrombin inhibitors (argatroban, lepirudin, bivalirudin and ximelagatran), and compounds that act by inhibiting platelet aggregation and thus thrombosis, including, but not limited to, cyclooxygenase inhibitors (aspirin), adenosine diphosphate receptor inhibitors (clopidrogrel and ticlopidine), phosphodiesterase inhibitors (cilostazol), glycoprotein IIb / IIIa inhibitors (Abciximab, Eptifibatide, Tirofiban and Defibrotide) and adenosine uptake inhibitors (dipyridamole). In a preferred embodiment, the antithrombotic agent is a thrombolytic agent. In a more preferred embodiment, the thrombolytic agent is a plasminogen activator. In an even more preferred embodiment, the plasminogen activator is tPA (tissue plasminogen activator).

[0053] As used herein, the term "tissue plasminogen activator (t-PA)" refers to a serine protease found on endothelial cells that catalyzes the conversion of plasminogen to plasmin. The complete protein sequence of human t-PA has UniProt KB accession number P00750 (July 11, 2012), sequence number 1. tPA can be produced using recombinant biotechnology techniques, and tPA produced in this way can be referred to as recombinant tissue plasminogen activator (rtPA). Recombinant tissue plasminogen activator (r-tPA) includes the thrombolytic agents alteplase, reteplase, and tenecteplase (also called TNKase, TNK-tPA, sequence number 2). In human t-PA, the amino acids at positions 296-299 are lysine, histidine, and two arginines. In TNK-tPA, these amino acids are replaced by four alanines. This mutation is involved in the increased resistance to plasminogen activator inhibitor 1 (PAI-1).

[0054] Administration of t-PA should be performed within the first 3 hours from the onset of symptoms, or within a maximum of 4.5 hours from the onset of symptoms. Recommended total dose: Infuse 0.9 mg / kg (maximum dose should not exceed 90 mg) over 60 minutes. Administer 0.09 mg / kg (10% of the 0.9 mg / kg dose) as an intravenous bolus over 1 minute, followed by 0.81 mg / kg (90% of the 0.9 mg / kg dose) as a continuous infusion over 60 minutes. In stroke, heparin should not be initiated more than 24 hours after the start of alteplase. The above t-PA is administered intravenously and may, in some cases, be administered directly into an artery and should be administered immediately after the onset of the first symptoms of stroke. The above dose and route of administration apply to any of the embodiments of the first aspect. Also, in particular, in embodiments including the step of treating a patient.

[0055] Single administration of TNK-tPA should be performed as soon as possible after a subject with stroke has been determined to be a candidate for reperfusion therapy and within 3 hours of symptom onset or within a maximum of 4.5 hours from symptom onset, preferably within 1 hour after stroke onset.

[0056] As shown above, the use of TNK-tPA is particularly useful because, for a specific prescription as a rapid single-dose bolus, it has efficacy about 1 minute after administration even at the ambulance level and can be administered at any point of care.

[0057] Patients with stroke who have not been selected for reperfusion therapy are, in certain embodiments, selected for therapies to lower blood pressure. In particular, the above therapies are performed using agents capable of lowering blood pressure.

[0058] "Blood pressure" should be understood herein to refer to blood pressure at sites of central arteries such as the aorta and carotid arteries. Central blood pressure can be suitably measured non-invasively (as described below) at the carotid or radial artery by applanation tonometry. Thus, "blood pressure" as used herein encompasses aortic blood pressure.

[0059] The "agent capable of reducing blood pressure" used in the present invention relates to any drug that can lower blood pressure by various means. The most widely used agents include thiazide diuretics [furosemide, nitroprusside, hydralazine, etc.]; ACE inhibitors, calcium channel blockers (such as nifedipine or nimodipine); adrenergic receptor antagonists (α-adrenergic antagonists, urapidil, etc.), or a combination of α-blockers and β-blockers (labetalol and nitroglycerin); and angiotensin II receptor antagonists (ARBs). Exemplary and non-limiting examples of agents that can lower or reduce blood pressure include α-methyldopa (Aldomet), 11,17α-dimethoxy-18β-[(3,4,5-trimethoxy-benzoyl)oxyl)]-3p,2a-yohimbane-16β-carboxylic acid methyl ester (Reserpine) or 2-(2,6-dichlorophenylamino)2-imidazoline hydrochloride (clonidine hydrochloride), relgotrile, or, that is, 2-chloro-6-methylergoline-8β-acetonitrile disclosed in European Patent No. 0005074. The reference values used to lower blood pressure in ischemic stroke, ischemic stroke treated with thrombolytic agents, or hemorrhagic stroke are the reference values recommended by clinical practice guidelines because these values may be updated. Today, in ischemic patients, when the systolic blood pressure reaches 220 - 120 mmHg and in hemorrhagic patients when the systolic blood pressure reaches 180 - 100 mmHg, it is targeted to reduce the treatment method for lowering blood pressure. In a preferred embodiment, blood pressure can be reduced by intravenous administration of an agent capable of reducing blood pressure and simultaneous administration of an oral antihypertensive agent. The reference values used to lower blood pressure in ischemic stroke, ischemic stroke treated with thrombolytic agents, or hemorrhagic stroke are the reference values recommended by clinical practice guidelines because these values may be updated.

[0060] Using any method suitable for measuring arterial pressure, it can be determined whether the agent can lower blood pressure, and a decrease in arterial pressure is detected after administration of the agent. Exemplary and non-limiting examples of methods for measuring arterial pressure include non-invasive techniques such as, by way of non-limiting example, palpitation, auscultation, oscillometry, and non-invasive continuous blood pressure (CNAP).

[0061] As used herein, the term "reference value" relates to a predetermined criterion used as a reference for evaluating a value or data obtained from a sample collected from a subject. The reference value or reference level can be an absolute value; a relative value; a value having an upper or lower limit; a range of values; an average value; a median value, a midpoint value, or a value compared to a specific control value or baseline value. The reference value can be based on an individual sample value, such as a value obtained from a sample obtained from the subject to be tested at a relatively early time point. The reference value can be based on a large number of samples obtained from a population of subjects with matched chronological age, etc., or on a pool of samples that includes or excludes the sample to be tested. A reference value for the biomarker of the present invention has been determined. The reference value for each of RBP4, NT-proBNP, and GFAP can be from a lower limit value and an upper limit value, as disclosed considering the following examples. The range of values (protein levels) for each biomarker, and specific combinations of values of different biomarkers, accurately classify the subject with high sensitivity and specificity.

[0062] When the level of a biomarker (in the present invention, any one of NT-proBNP, RBP4, and GFAP) is at least 1.5%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150% or higher than the reference value, it is considered to be higher than the reference value.

[0063] Similarly, in the context of the present invention, the level of the biomarker decreases when the level of the biomarker in the sample is lower than the reference value. When the level of the biomarker is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150% or lower than the reference value, it is considered to be lower than the reference value.

[0064] In a specific embodiment of the first aspect, when only the levels of RBP4 and NT-proBNP in the biological fluid sample are determined and compared with the corresponding reference cut-off values (referred to as Ref1 RBP4 and Ref1 NT-proBNP in this specification), these reference cut-off values are for RBP4 52 μg / ml and NT-proBNP 4062 pg / ml. These specific reference cut-off values are, in a specific embodiment, for an isolated plasma sample and for the case when assayed by enzyme-linked immunosorbent assay (ELISA).

[0065] In another specific embodiment of the first aspect, the levels of RBP4, NT-proBNP, and GFAP are determined and compared with the corresponding reference cut-off values (referred to herein as Ref2 RBP4 , Ref2 NT-proBNP and Ref GFAP ). These reference cut-off values are for RBP4 38 μg / ml, BNP1 305 pg / ml, and GFAP 0.325 ng / ml. These specific reference cut-off values are, in a more specific embodiment, for isolated plasma samples. When GFAP is assayed, the level of this marker is determined by a relatively sensitive (picomolar level) single molecule assay (SIMOA: single molecular assay), and the others are determined by ELISA assay. At these cut-off levels, a specific method is performed in two separate steps or conditions. In the first step, the level of GFAP is determined, and if it is below the reference cut-off value Ref GFAP , the patient is considered to be suffering from ischemic stroke. Further, in the second step, the levels of RBP4 and NT-proBNP are simultaneously determined, and if they are above the corresponding reference cut-off values Ref2 RBP4 and Ref2 NT-proBNP , the patient is considered a candidate for reperfusion.

[0066] As shown by way of example, in ICH patients, GFAP levels were higher and RBP4 and NT-proBNP were lower than in IS. A combination of RBP4 > 52 μg / mL and GFAP > 0.18 ng / mL yielded an accurate diagnosis of 6.5% of IS and 34.3% of ICH. Adding NT-proBNP maintained 100% specificity for IS and improved sensitivity by up to 20% (31 / 155) using RBP4 > 52 μg / mL and a BNP cut-off > 4060 pg / mL.

[0067] As shown above, it is widely known that the reference values can vary depending on the determination of the exclusion criteria of the clinical protocol. Additionally, depending on the variables considered to be diagnostic, these values can also always be adjusted to increase sensitivity while maintaining the specificity, which is very important in stroke.

[0068] Furthermore, appropriate classification of patients according to the levels of the detected proteins can be performed using computational methods, whereby the determined values of the proteins are calculated by an equation that gives a predictor, said predictor being calculated based on the expression level of the protein, and said expression level being corrected by a specific coefficient. In other computational methods (such as one of those exemplified herein and called the support vector machine method), it becomes possible to disclose a function that appropriately classifies patients considering the levels of any determined protein.

[0069] All of these reference values shown in certain embodiments were determined for isolated plasma samples from subjects suffering from stroke. A person skilled in the art knows how to find the corresponding ones in serum and other biological fluids.

[0070] As used herein, the term "sample" relates to any sample that can be obtained from a patient. The methods of the present invention can be applied to any type of biological sample obtained from a patient, such as a biopsy sample, tissue, cell or biological fluid (e.g., plasma, serum, saliva, semen, sputum, cerebrospinal fluid (CSF), tears, mucus, sweat, milk, brain extract, etc.).

[0071] Thus, in another specific embodiment, optionally in combination with any of the above or below embodiments, the isolated sample of the subject (i.e., a patient suffering from stroke) is a biological fluid. Exemplary and non-limiting biological fluids are blood, plasma, serum, saliva, urine or cerebrospinal fluid. In a more preferred embodiment, the biological fluid is plasma or serum.

[0072] In a preferred embodiment of the method of the present invention, the sample is obtained at baseline.

[0073] For determining the levels of various markers, various samples can be used. Thus, it is not necessary for the levels of any marker according to the method of the present invention to be measured for the same type of sample. Thus, in another preferred embodiment, the levels of RBP4, NT-proBNP and GFAP are measured for serum. In another preferred example, their levels are measured for plasma.

[0074] The "baseline" used in the present invention is considered at any point in time from the onset of symptoms until the patient is first examined. This is usually within the first few hours after a stroke and is usually first noticed in an ambulance or at the hospital. In a preferred embodiment, the baseline is within the first 4.5 hours from the onset of symptoms, or less than 6 hours after a stroke, or in another preferred embodiment, less than 24 hours from the onset of symptoms.

[0075] In another specific embodiment of this aspect, the step of determining the levels of RBP4 and NT-proBNP, and GFAP if determined, is performed within the first 2 hours after the onset of a stroke. As shown in the following examples, the earlier the determination of the marker in the isolated sample is made, the more the accuracy and sensitivity of the method are improved. In another specific embodiment of this aspect, the step of determining the levels of RBP4 and NT-proBNP, and GFAP if determined, is performed within the first 1 hour after the onset of a stroke.

[0076] In another embodiment of the first aspect of the method for selecting a patient suffering from a stroke for reperfusion therapy, it further comprises determining one or more clinical parameters. Thus, the method of the present invention comprises determining the levels of RBP4 and BNP and one or more clinical parameters.

[0077] As used herein, the term "clinical parameter" or clinical data refers to an individual's background factors (age or date of birth, race and / or ethnicity), the patient's clinical symptoms, or signs associated with stroke-related diseases / conditions. This term also includes test parameters such as the determination of d-dimer or blood glucose.

[0078] In certain embodiments, the clinical parameter is hypertension, and if a patient has hypertension, it indicates that the patient has suffered an ischemic stroke or is a candidate for reperfusion therapy.

[0079] In another specific embodiment of the first aspect, the in vitro method further includes determining a clinical parameter selected from the group consisting of blood pressure including systolic blood pressure and / or diastolic blood pressure, blood glucose, age, a score from a systematic assessment tool stroke-related neurological disorder, such as the NIHSS score, gender, and combinations thereof. The values of any of these parameters are used in combination with the levels of RBP4, NT-proBNP, and optionally GFAP in an appropriate algorithm for accurately classifying a patient as a candidate for reperfusion therapy in certain embodiments. For example, blood pressure within a specific range in combination with specific levels of two or three proteins is used in a determination protocol for accurate classification. In yet another more specific embodiment, the values are introduced into the equation of a regression model to give a score or final value that enables such classification.

[0080] "Hypertension," which may also be called arterial hypertension, should be understood as a chronic condition in which blood pressure in the arteries rises. Normal blood pressure at rest is in the range of 100 - 140 mmHg (upper measured value) for systolic blood pressure and 60 - 90 mmHg (lower measured value) for diastolic blood pressure. If it persists at 140 / 90 mmHg or higher, it is said to have hypertension. As shown above, in ischemic stroke, ischemic stroke treated with a thrombolytic agent, or hemorrhagic stroke, the reference values used to lower blood pressure are the reference values recommended by clinical guidelines because these values may be updated, and today, in ischemic patients, when the systolic blood pressure reaches 220 - 120 mmHg and in hemorrhagic patients when it reaches 180 - 100 mmHg, they are the reference values accepted as suitable treatment methods for lowering blood pressure.

[0081] The term "systematic assessment tool for stroke-related neurological disorders" relates to tools designed to measure and evaluate the neurological disorders most frequently seen in stroke. Several aspects or parameters are evaluated, such as level of consciousness, visual fields, facial muscle weakness, limb motor ability, gaze, sensory impairment, coordination (ataxia), speech (aphasia), and speech production (dysarthria). A value is given for each of these, which is 0 if normal. Thus, in most of these tools, the higher the score, the worse the neurological disorder. Those familiar with it know the existence of various tools for this purpose, such as the National Institutes of Health Stroke Scale (NIHSS), the Rapid Arteria occlusion evaluation scale for stroke (RACE), the Cincinnati Prehospital Stroke Scale Compared to Stroke Severity Tools for Large Vessel Occlusion Stroke Prediction (Cincinnati score), the Los Angeles Motor Scale (LAMS), or the modified Rankin Scale or Score (mRS). All of these scales are designed to provide a rapid and standardized assessment of initial neurological function after stroke. The modified Rankin Scale or Score (mRS) is also a scale for evaluating the degree of disability after the onset of stroke. It is mainly applied at the time of discharge and 3 months after the onset.

[0082] In another preferred embodiment, the clinical parameters are selected from age, NIHSS score, gender, systolic blood pressure, and combinations thereof. The term "NIHSS score" as used in the present invention refers to the National Institutes of Health Stroke Scale (NIHSS) score, which is a systematic assessment tool that provides a quantitative measurement of stroke-related neurological deficits (Adams HP Jr Neurology. 1999 Jul 13;53(1):126-31). The NIHSS was originally designed as a research tool to measure baseline data on patients in clinical trials of acute stroke. Currently, this scale is also widely used as a clinical assessment tool to evaluate the severity of stroke patients, determine appropriate treatment, and predict patient outcomes. The NIHSS is a 15-item neurological examination stroke scale used to evaluate the impact of acute cerebral infarction on levels of consciousness, language, hemispatial neglect, visual field defects, extraocular movements, motor strength, ataxia, dysarthria, and sensory loss. Trained observers evaluate the patient's ability to answer questions and perform activities. Each item is scored on a scale of 3 to 5, with normal being 0, and there is an acceptable range for items that are impossible to examine. Levels of stroke severity measured by the NIH stroke scale scoring system: 0 = no stroke, 1-4 = mild stroke, 5-15 = moderate stroke, 15-20 = moderate / severe stroke, 21-42 = severe stroke. In the present invention, the term "relatively high score" refers to a score of 5 to 42 in the NIH stroke scale scoring system.

[0083] Also, modifications of the NIHSS, such as the Rapid Arteria occlusion evaluation scale (RACE) for stroke, or other scores used to identify ischemic stroke with large vessel occlusion, may also be used.

[0084] Furthermore, in another specific embodiment of the first aspect, optionally, in combination with any of the above or below embodiments, when the subject is classified as a candidate for reperfusion therapy, the subject is also diagnosed with having a large vessel occlusion.

[0085] As a second aspect, the present invention relates to an in vitro method for distinguishing a patient's IS from ICH, the in vitro method comprising determining the levels of RBP4 and NT-proBPN in an isolated sample of the patient.

[0086] In a particular embodiment of the second aspect, the method comprises determining the level of GFAP.

[0087] In yet another more particular embodiment of the second aspect, the method further comprises comparing the levels of RBP4, NT-proBNP, and, if determined, GFAP, with corresponding reference values, · If only the levels of RBP4 and BNP are determined, the levels of RBP4 and BNP being simultaneously higher than the corresponding reference values Ref1 RBP4 and Ref1 NT-proBNP indicates that the patient is an IS patient, or · If the levels of RBP4, BNP, and GFAP are determined, the levels of RBP4 and BNP being simultaneously higher than the corresponding reference values Ref2 RBP4 and Ref2 NT-proBNP and the level of GFAP being lower than the reference value Ref GFAP indicates that the patient is an IS patient.

[0088] In another particular embodiment of the second aspect, when the levels of RBP4 and BNP, and optionally GFAP, are determined and the subject is classified as having an ischemic stroke, the subject is also defined by a dependency greater than 2 according to the modified Rankin Scale (mRS) and having a prognosis determined within 1 to 5 months after stroke onset, and / or having a prognosis defined by a 3-month post-onset mortality rate composed of 20% to 30%.

[0089] In another particular embodiment of the second aspect, it further comprises the step of selecting a therapy, particularly a reperfusion therapy, as shown in the first aspect and its particular embodiments.

[0090] Accordingly, after a differential diagnosis has been achieved, in another specific embodiment of the second aspect, it further includes the steps of recommending reperfusion therapy to a patient diagnosed with IS and / or treating the patient diagnosed with IS by reperfusion therapy mainly by antithrombotic agents or thrombus removal. Alternatively, a patient diagnosed with ICH who should avoid reperfusion therapy to avoid a fatal outcome is recommended or treated with a therapy that reduces or optimizes blood pressure in another specific embodiment.

[0091] This specific embodiment can be formulated as a method for treating a patient suffering from a stroke, the method comprising performing an in vitro method for differentiating a patient's IS from ICH according to the second aspect, and treating a patient diagnosed with IS by reperfusion therapy mainly by antithrombotic agents or thrombus removal, or treating a patient diagnosed with ICH by a therapy that reduces or optimizes blood pressure. Advantageously, in this method, the patient is treated or recommended to be treated with the most appropriate treatment regimen within the first few hours from the onset of symptoms.

[0092] Any particular embodiment previously disclosed for the first aspect is also applicable to this second aspect. In particular, those preferred reference values, types of isolated samples, and options for further determining one or more clinical parameters. Thus, in another particular embodiment of the second aspect, the in vitro method further comprises determining clinical parameters selected from the group consisting of blood pressure, including systolic blood pressure and / or diastolic blood pressure, blood glucose, age, score from a systematic assessment tool stroke-related neurological disorder, such as the NIHSS score, gender, and combinations thereof. As before, the values of any of these parameters are, in certain embodiments, used in combination with the levels of RBP4, NT-proBNP, and optionally GFAP in an appropriate algorithm for accurately classifying a patient as a candidate for reperfusion therapy. For example, blood pressure within a particular range in combination with specific levels of two or three proteins is used in a determination protocol for accurate classification. In another even more particular embodiment, the values are introduced into the equation of a regression model to give a score or final value that enables such classification.

[0093] Also, in another particular embodiment of the second aspect, determining the levels of RBP4 and NT-proBNP, and optionally GFAP if determined, is performed within the first 2 hours after the onset of stroke. More specifically, within the first 1 hour. This means, as shown for the first aspect, the advantage of increasing the sensitivity of the method.

[0094] Furthermore, in another particular embodiment of this second aspect, optionally in combination with any of the above or below embodiments, if the subject is classified as having an ischemic stroke, the subject is also diagnosed as suffering from a large vessel occlusion.

[0095] The present invention is also a method for detecting the levels of RBP4 and NT-proBPN in an isolated sample of a subject suffering from a stroke, optionally in combination with the level of GFAP, comprising (a) obtaining a sample from the subject, and (b) (i) contacting the sample with means capable of binding to the corresponding expressed protein and detecting the binding, or (ii) contacting the sample with means capable of binding to the corresponding RNA translated into one or more of the corresponding proteins and detecting the binding, generally encompasses detecting whether one or more of the proteins are present in the isolated sample.

[0096] As used herein with respect to the second aspect, the term "distinguish" relates to the determination of different states. As will be understood by those skilled in the art, the distinction, although preferably so, need not be accurate for 100% of the subject being diagnosed or evaluated. However, this term requires that a statistically significant portion of the subject can be identified as being likely to have one of two types of stroke. Whether the subject is statistically significant can be determined by those skilled in the art without further difficulty using various well-known statistical evaluation tools, such as determination of confidence intervals, p-value determination, Student's t-test, Mann-Whitney test, etc. For details, see Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95%. The p-value is preferably 0.05, 0.01, 0.005 or less.

[0097] Since the biomarkers identified in the present invention enable distinguishing a patient's IS from ICH, considering that different therapies (antithrombotic agents for patients suffering from ischemic stroke and agents capable of reducing blood pressure for patients suffering from hemorrhagic stroke) are applied to these two types of patients (see Tsivgoulis G. et al, Neurology. 2014 Sep 19), the present invention also provides the above first method for the selection of therapy for patients suffering from stroke. Thus, both aspects are closely and conceptually related.

[0098] When RBP4 is mentioned in the present invention, it refers to retinol-binding protein 4, which belongs to the lipocalin family and is a specific carrier of retinol in the blood, i.e., plasma. The complete sequence of human retinol-binding protein 4 has the UniProtKB accession number P02753 (August 8, 2013) and SEQ ID NO: 3.

[0099] As used herein, the term "GFAP" refers to glial fibrillary acidic protein, an intermediate filament protein expressed by a number of cell types in the central nervous system. The complete human sequence of glial fibrillary acidic protein has the UniProtKB accession number P14136 (August 8, 2013) and SEQ ID NO: 4.

[0100] The N-terminal fragment of B-type natriuretic peptide (NT-proBNP) (SEQ ID NO: 5) is a 76-amino acid N-terminal fragment of the B-type natriuretic peptide prohormone. Cleavage of pro-BNP results in the NT-proBNP fragment and the active form B-type natriuretic peptide (BNP). BNP is a hormone secreted by ventricular cardiomyocytes in response to stretch caused by an increase in ventricular blood volume. The complete human sequence of BNP has the UniProt KB accession number P16860 (August 1, 1990 - version 1 of the sequence, and database release 187 on May 8, 2019).

[0101] All of these proteins have homology in other mammalian species (such as cats, dogs, mice, rats, etc.). Those skilled in the art can search for the corresponding complete sequences in public databases.

[0102] As will be understood by those skilled in the art, the expression levels of NT-proBNP, RBP4 and / or GFAP can be determined by measuring the level of mRNA encoded by the corresponding gene, or by measuring the level of the protein encoded by the above gene or a variant thereof.

[0103] As a non-limiting example, the expression level is determined by quantification of the level of mRNA encoded by the gene. The latter can be determined by conventional methods, such as methods involving amplification of mRNA and quantification of the amplification products of the mRNA, such as by using electrophoresis and staining, or by using Northern blot and a suitable probe, Northern blot and a specific probe for the mRNA of the gene of interest, or a specific probe for their corresponding cDNA / cRNA, mapping by SI nuclease, RT-PCR, hybridization, microarray, etc. Similarly, the level of cDNA / cRNA corresponding to the mRNA encoded by the marker gene can also be quantified by using conventional techniques. In this case, the method of the present invention includes the steps of synthesizing the corresponding cDNA by reverse transcription (RT) of the corresponding mRNA, followed by synthesis (RNA polymerase) and amplification of cRNA complementary to the cDNA. Conventional methods for quantifying expression levels can be found in laboratory manuals.

[0104] To normalize the mRNA expression values between different samples, it is possible to compare the expression level of the mRNA of interest in the test sample with the expression of a control RNA. As used herein, "control RNA" relates to RNA whose expression level does not change or changes only in limited amounts. Preferably, the control RNA is derived from a housekeeping gene, is constitutively expressed, and is an mRNA encoding a protein that performs an essential cellular function. Preferred housekeeping genes for use in the present invention include 18-S ribosomal protein, β-2-microglobulin, ubiquitin, cyclophilin, GAPDH, PSMB4, tubulin, and β-actin.

[0105] Alternatively, since the amount of the corresponding protein increases when the gene expression increases and the amount of the corresponding protein decreases when the gene expression decreases, the expression level of the marker gene can also be determined by determining the expression level of the protein encoded by the gene.

[0106] The determination of the protein expression level can be performed by qualitative and / or quantitative tests selected from the group consisting of immunological tests, bioluminescence, fluorescence, chemiluminescence, electrochemistry, and mass spectrometry. To perform the determination of the marker level easily and rapidly, specific tests that can be implemented in a point of care test format (POCT) are recommended. In certain embodiments, the point of care test includes a lateral flow test that enables the detection of the presence (or absence) of a target analyte in a liquid sample (matrix) without the need for special and expensive equipment, although there are many laboratory-based applications assisted by reading devices.

[0107] As shown in the example, specific POCTs have been developed and tested in ambulances and helicopters. This POCT has achieved a high sensitivity rate with 100% specificity for ischemic stroke, which has enabled pre-hospital reperfusion therapy to be initiated much more rapidly in selected cases than using standard techniques.

[0108] Regardless of the test format, specific quantitative tests are selected from the group consisting of immunological tests, bioluminescence, fluorescence, chemiluminescence, electrochemistry, and mass spectrometry.

[0109] In one embodiment, the expression level is determined by immunological techniques such as enzyme-linked immunosorbent assay (ELISA), enzyme immunodot assay, agglutination assay, antibody-antigen-antibody sandwich assay, antigen-antibody-antigen sandwich assay, immunochromatography, or other immunoassay formats well known to those of ordinary skill in the art, such as radioimmunoassay, and protein microarray formats such as single molecule assay (SIMOA), Western blot, or immunofluorescence.

[0110] Western blotting is based on the detection of nitrocellulose by incubation with antibodies specific to proteins that have been previously separated by gel electrophoresis under denaturing conditions and immobilized on a membrane, and a detection system (e.g., chemiluminescence). For analysis by immunofluorescence, it is necessary to use an antibody specific to the target protein for analysis of expression. ELISA is based on the use of an antigen or an antibody labeled with an enzyme, so that the conjugate formed between the target antigen and the labeled antibody forms an enzymatically active complex. Since one of the components (antigen or labeled antibody) is immobilized on a support, the antibody-antigen complex is immobilized on the support, and thus, for example, by adding a substrate that can be converted by the enzyme into a product detectable by spectrophotometry, fluorometry, mass spectrometry or tandem mass tag (TMT), the antibody-antigen complex can be detected. SIMOA is a type of assay with higher sensitivity than ELISA because it uses an array of femtoliter-sized reaction chambers called single molecule arrays (Simoa™) that can isolate and detect single enzyme molecules. Since the volume of the array is about one two-billionth of that of a conventional ELISA, rapid accumulation of fluorescent products occurs when labeled proteins are present. When diffusion is blocked, this high local concentration of products can be easily observed. Only one molecule is required to reach the detection limit. Using the same reagents as in conventional ELISA, when this method was used to measure proteins in various different matrices (serum, plasma, cerebrospinal fluid, urine, cell extracts, etc.) at femtomolar (fg / mL) concentrations, the sensitivity was improved by about 1000-fold.

[0111] On the other hand, determination of protein expression levels can be performed by constructing a tissue microarray (TMA) containing pooled subject samples and determining the protein expression levels by techniques well known in the art.

[0112] In a preferred embodiment, determination of the level of the marker is determined by an immunological technique. In a more preferred embodiment, the immunological technique is ELISA.

[0113] When using immunological methods, any antibody or reagent known to bind to the target protein with high affinity can be used to detect the amount of the target protein. Nevertheless, the use of antibodies, such as polyclonal sera, hybridoma supernatants or monoclonal antibodies, antibody fragments, Fv, Fab, Fab´ and F(ab’)2, ScFv, diabodies, triabodies, tetra-bodies as well as humanized antibodies is preferred.

[0114] As cited above, the expression levels of NT-proBNP and / or RBP4 and / or GFAP can be determined by measuring both the protein level and the levels of its variants, such as fragments, isoforms, analogs and / or derivatives.

[0115] The term "functionally equivalent variant" is always understood to mean any protein derived from the sequence of NT-proBNP and / or RBP4 and / or GFAP by modification, insertion and / or deletion, or one or more amino acids, provided that the function of the variant is substantially maintained. Preferably, variants of NT-proBNP and / or RBP4 and / or GFAP are (i) polypeptides in which one or more amino acid residues are substituted by conserved or non-conserved amino acid residues (preferably conserved amino acid residues), such substituted amino acids may or may not be encoded by the genetic code, (ii) polypeptides in which there are one or more modified amino acid residues, e.g., residues modified by substituent attachment, (iii) polypeptides resulting from alternative processing of similar mRNAs, (iv) polypeptide fragments and / or (v) polypeptides resulting from the fusion of NT-proBNP and / or RBP4 and / or GFAP, or other polypeptides, e.g., secretion leader sequences, or sequences used for purification (e.g., His tag) or detection (e.g., Sv5 epitope tag), which are polypeptides defined in (i)-(iii). Fragments include polypeptides produced by proteolytic cleavage (including multi-site proteolysis) of the original sequence. Variants can be post-translationally modified or chemically modified. Such variants should be apparent to those skilled in the art.

[0116] As is known in the art, the "similarity" between two proteins is determined by comparing the amino acid sequence of one protein and its conserved amino acid substitutions with the sequence of a second protein. Variants are defined to include polypeptide sequences that differ from the original sequence, preferably polypeptide sequences in which less than 40% of the residues per related segment differ from the original sequence, more preferably polypeptide sequences in which less than 25% of the residues per related segment differ from the original sequence, more preferably polypeptide sequences in which less than 10% of the residues per related segment differ from the original sequence, more preferably polypeptide sequences in which only a few residues per related segment differ from the original sequence, and at the same time, polypeptide sequences that are sufficiently homologous to the original sequence to retain the functionality of the original sequence. Variants according to the invention include amino acid sequences that are at least 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 90% or 95% similar or identical to the original amino acid sequence. The degree of identity between two proteins is determined using computer algorithms and methods that are well known to those skilled in the art. The identity between two amino acid sequences is preferably determined using the BLASTP algorithm [BLAST Manual, Altschul, S., et al, NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al, J. Mol. Biol. 215:403-410 (1990)].

[0117] Proteins can be post-translationally modified. For example, post-translational modifications within the scope of the present invention include signal peptide cleavage, glycosylation, acetylation, isoprenylation, proteolytic myristoylation, protein folding, and proteolytic processing. Further, proteins can contain non-natural amino acids formed by post-translational modification or by introduction of non-natural amino acids during translation.

[0118] In another specific embodiment of the in vitro method of the present invention for providing differential diagnosis and information for selecting a therapy, they further comprise (i) a step of collecting diagnostic information, and (ii) a step of storing the information on a data carrier.

[0119] In the context of the present invention, a "data carrier" should be understood as any means containing significant information data for the differential diagnosis of IS and ICH and / or for the selection of candidates for reperfusion therapy, for example, paper. The carrier can also be any physical object or device capable of carrying differential diagnosis data or information for selecting a therapy. For example, the carrier can include a storage medium such as a ROM, for example, a CD ROM or a semiconductor ROM, or a magnetic recording medium, for example, a floppy disk or a hard disk. Furthermore, the carrier can be a transmissible carrier such as an electrical signal or an optical signal, which can be transmitted via an electrical cable or an optical cable, or wirelessly or by other means. If the diagnostic / therapy selection data is embodied in a signal that can be directly transmitted by a cable or other device or means, the carrier can be constituted by such a cable or other device or means. Other carriers are related to USB devices and computer archives. Examples of suitable data carriers include paper, CDs, USBs, computer archives within a PC, or voice recordings using the same information.

[0120] The present invention also encompasses an in vitro method for the prognosis of a patient suffering from ischemic stroke, the method comprising determining the level of retinol-binding protein-4 (RBP4) and the level of the N-terminal fragment of B-type natriuretic peptide (NT-proBNP) in an isolated sample of the patient.

[0121] In certain embodiments of the in vitro method for prognosis, at least the levels of RBP4 or two proteins are compared with a reference value, which is selected from a value or range of values that indicates or confirms that the subject is suffering from ischemic stroke. In another more specific embodiment, the prognosis is defined by a dependency greater than 2 on the modified Rankin score (mRS), determined within 1 to 5 months after the onset of stroke, and / or by the 3-month post-onset mortality rate consisting of 20% to 30%.

[0122] As shown above, the invention also relates to a kit comprising reagent means for detecting the levels of RBP4 and NT-proBNP.

[0123] In certain embodiments of the kit of the invention, they further comprise reagent means for detecting the level of GFAP.

[0124] As used herein, the term "kit" refers to a product containing various reagents (or reagent means) necessary to perform the method of the invention, packaged to enable their transportation and storage. Materials suitable for packaging the components of the kit include crystals, plastics (e.g., polyethylene, polypropylene, polycarbonate), bottles, vials, paper, or envelopes.

[0125] Furthermore, the kit of the invention can include instructions for using the various components within the kit simultaneously, sequentially, or separately. The instructions can be in the form of a printed matter or an electronic support that can store an easy-to-read or easy-to-understand instruction, such as an electronic storage medium (e.g., magnetic disk, tape), or an optical medium (e.g., CD-ROM, DVD), or an audio material. Additionally, or alternatively, the medium can include an Internet address that provides the above instructions.

[0126] The reagent means (or simply reagent) of the kit comprises a compound that specifically binds to a marker protein. Preferably, the compound is an antibody, an aptamer or a fragment thereof.

[0127] In a preferred embodiment, the reagent is an antibody or a fragment thereof. Thus, the reagent means is one or more antibodies that specifically recognize the protein of interest (i.e., NT-proBNP, RBP4, and GFAP if determined). The antibodies of the kit of the present invention can be used according to techniques known in the art for determining protein expression levels, such as, for example, techniques based on the use of flow cytometry, Western blot, ELISA, RIA, competitive EIA, DAS-ELISA, protein microarray, or assay of colloidal precipitation within a reactive strip.

[0128] The antibody can be immobilized on a solid support such as a membrane, plastic or glass, and optionally can be treated to facilitate immobilization of the antibody to the support. The solid support includes at least a set of antibodies that can specifically recognize a marker (i.e., the protein of interest) and be used to detect the expression level of the marker.

[0129] Furthermore, the kit of the present invention includes a reagent for detecting a protein encoded by a constitutive gene. The availability of the additional reagent makes it possible to normalize measurements made on different samples (e.g., the sample to be analyzed and a control sample) to exclude the possibility that differences in biomarker expression are due to different amounts of total protein in the sample rather than actual differences in relative levels of expression. In the present invention, a constitutive gene is a gene that is always active or constantly transcribed, constitutively expressed, and encodes a protein that performs an essential cellular function. Proteins that are constitutively expressed and can be used in the present invention include, but are not limited to, β-2-microglobulin (B2M), ubiquitin, 18-S ribosomal protein, cyclophilin, GAPDH, PSMB4, tubulin and actin.

[0130] In a preferred embodiment, the reagent means for assaying the levels of various biomarkers comprises at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% of the total amount of reagents for assaying the biomarkers that form the kit. Thus, in certain cases of kits containing reagents for assaying the levels of RBP4, NT-proBNP and optionally GFPA, the reagents specific for the above biomarkers (i.e., antibodies that specifically bind to RBP4, NT-proBNP and optionally GFPA) comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% of the antibodies present in the kit. Thus, these kits are primarily simplified kits containing reagent means for detecting the levels of two (or three) proteins.

[0131] In another specific embodiment, the kits of the present invention are contemplated for point-of-care testing. More specifically, they are in the form of lateral flow assays.

[0132] In another specific embodiment, the kit according to the present invention comprises a support for depositing a biological fluid sample, particularly whole blood, and one or more sample inlet ports; and a reaction region containing means / reagents that specifically bind to marker proteins, particularly antibodies, wherein the sample inlet ports are connected to the reaction region. In yet another more specific embodiment, the kit comprises the same number of sample inlet ports as the marker(s) (one, two, or three) to be detected and corresponding reaction regions connected thereto. In another embodiment, the kit comprises a single inlet port as a capillary track connecting to the same number of reaction regions, and the capillary track guides a portion of the sample to each corresponding connected reaction region. A kit containing multiple reaction regions is a composite kit.

[0133] In another aspect, the invention relates to the use of the kit of the invention for distinguishing IS from ICH or for selecting a patient suffering from stroke for reperfusion therapy, which in certain embodiments is selected from the group consisting of therapy with an anti-thrombotic agent, thrombus removal, and combinations thereof.

[0134] Accordingly, in certain embodiments, the invention relates to the use of the kit of the invention in any of the methods of the invention.

[0135] As shown in the following examples, the inventors have surprisingly found that by determining the levels of RBP4 and NT-proBNP, optionally in combination with the levels of GFAP and / or certain additional clinical parameters, it is possible to diagnose patients suffering from stroke with large vessel occlusion (LVO). LVO may be involved in part in the poor outcome or prognosis noted above for patients with ischemic stroke.

[0136] Accordingly, in relation to this selection and accurate classification of patients suffering from stroke, the invention also relates to an in vitro method for the diagnosis of LVO, the in vitro method comprising determining the levels of RBP4 and NT-proBNP in an isolated sample of a subject. In certain embodiments, the subject is a patient with ischemic stroke.

[0137] In certain embodiments of the in vitro method for the diagnosis of LVO, it further comprises determining the level of GFAP. In another more specific embodiment, the method further comprises comparing the levels of RBP4, NT-proBNP, and, if determined, GFAP with corresponding reference values, wherein the reference values or reference ranges are selected from values or ranges of values obtained from subjects suffering from LVO, and the subject is diagnosed as having LVO when at least one of the levels of RBP4, NT-proBNP, and GFAP is within the value or range of values obtained from subjects suffering from LVO. Further, in a more specific embodiment of the in vitro method for the diagnosis of LVO, it comprises determining one or more clinical parameters. These clinical parameters are selected from the group consisting of, in particular, blood pressure including systolic blood pressure and / or diastolic blood pressure, blood glucose, the level of blood d-dimer, age, the score from a systematic evaluation tool for stroke-related neurological disorders, gender, and combinations thereof. In a more specific embodiment, the in vitro method for the diagnosis of LVO comprises determining the levels of RBP4, NT-proBNP, and GFAP in an isolated sample, blood glucose, d-dimer, and further diastolic blood pressure, and a baseline score from a systematic evaluation tool for stroke-related neurological disorders, such as the NIHSS score, RACE, Cincinnati, LAMS, etc. As with other aspects and embodiments, the isolated sample is preferably a biological fluid, and more specifically, is selected from plasma and serum. Also, as disclosed for other aspects of the present invention, the values of the clinical parameters can be used in combination with the levels of RBP4, NT-proBNP, and optionally GFAP in an appropriate algorithm for accurately classifying a patient as suffering from LVO. In another specific embodiment of the in vitro method for the diagnosis of LVO, the determination of RBP4 and NT-proBNP, and, if determined, the level of GFAP is performed within the first 2 hours after the onset of stroke, and more specifically, within the first 1 hour after the onset of stroke.

[0138] According to the findings of the present inventors, it is for the first time that a combination of markers in serum and / or plasma provides reliable information for the accurate diagnosis of LVO (high sensitivity among IS patients). This is another true contribution to the art as these are currently far from the value of 100% sensitivity and clinical scores or clinical protocols with inadequate accuracy are used, which is why they are not fully implemented in clinical practice.

[0139] The high sensitivity in the diagnosis of LVO in ischemic stroke patients, which is related to the determination of the above levels of RBP4, NT-proBNP and optionally GFAP, enables the referral of these patients to the nearest referral hospital where mechanical thrombectomy can be applied.

[0140] Therefore, using the levels of RBP4, NT-proBNP and optionally GFAP to rapidly and well classify patients presenting with stroke symptoms, for example, at the ambulance level, if possible within the first 2 hours after onset and even within the first 1 hour, patients can be classified as candidates for reperfusion, initially administered antithrombotic agents by ambulance, and referred to a hospital with a facility for treating LVO.

[0141] Also disclosed herein is an in vitro method for differentiating a patient's ischemic stroke from a hemorrhagic stroke or for selecting a patient suffering from a stroke for reperfusion therapy, the method comprising determining the levels of NT-proBNP and GFAP in an isolated sample of the patient, optionally in combination with clinical parameters selected from the group consisting of blood pressure, including systolic blood pressure and / or diastolic blood pressure, blood glucose, age, NIHSS score from a systematic assessment tool for stroke-related neurological deficits, gender, and combinations thereof. More specifically, the method comprises determining the levels of NT-proBNP and GFAP in an isolated sample in combination with the patient's blood pressure. This particular combination enables discrimination with high sensitivity and specificity, as shown in the examples below. In another more specific embodiment, the method further comprises determining the level of RBP4 in the isolated sample. More specifically, the isolated sample is selected from a biopsy sample, tissue, cells, or a biological fluid (plasma, serum, saliva, semen, sputum, cerebrospinal fluid (CSF), tears, mucus, sweat, milk, and brain extract). Particularly serum or plasma.

[0142] Any particular embodiments disclosed above for the first and second aspects are also applicable to this method for differentiating a patient's ischemic stroke from a hemorrhagic stroke and / or for selecting a patient suffering from a stroke for reperfusion therapy, the method comprising the step of comparing the levels of NT-proBNP and GFAP.

[0143] In a more specific embodiment, an in vitro method for differentiating a patient's ischemic stroke from a hemorrhagic stroke and / or for selecting a patient suffering from a stroke for reperfusion therapy includes the step of comparing the levels of NT-proBNP and GFAP with corresponding reference values or reference ranges for each protein, wherein the reference values or reference ranges are selected from values or ranges of values obtained from subjects suffering from ischemic stroke, the subjects having at least both the levels of GFAP and NT-proBNP within the values or ranges obtained from subjects suffering from ischemic stroke, and optionally, the blood pressure also being within the values of subjects suffering from ischemic stroke, are classified as candidates for reperfusion therapy. In another specific embodiment, as previously shown for other aspects of the present invention, the values of the levels of NT-proBNP and GFAP in an isolated sample are used in combination with a determined blood pressure in a suitable algorithm for accurately classifying a patient as a candidate for reperfusion therapy. A blood pressure within a specific range in combination with specific levels of the two proteins is used in a decision protocol for accurate classification and selection of therapy. In another specific embodiment, the values are introduced into an equation of a regression model to give a score or final value that enables such classification. To increase the sensitivity of the method, the determination of the levels of NT-proBNP and GFAP is performed within the first 2 hours after the onset of the stroke. More specifically, within the first 1 hour. In another specific embodiment, when the levels of NT-proBNP and GFAP are determined and the values are used in combination with clinical variables such as baseline NIHSS score and / or levels of d-dimer in the blood and / or blood pressure values, a high sensitivity for the detection of ischemic stroke with large vessel occlusion as a candidate for a specific thrombectomy therapy technique is achieved.

[0144] An in vitro method for distinguishing a patient's ischemic stroke from a hemorrhagic stroke or for selecting a patient suffering from a stroke for reperfusion therapy, the method comprising determining the levels of NT-proBNP and GFAP in an isolated sample of the patient. In another particular embodiment of the in vitro method, the levels are measured using a POCT that includes (comprises) reagent means for the analysis of these two proteins in the isolated sample. More specifically, the kit includes only means for detecting the level of one or both of NT-proBNP and GFAP (either the protein level or the mRNA level) among the reagent means.

[0145] Throughout the description and claims, the word "comprising" and variations of the word are not intended to exclude other technical features, additives, components or steps. Further, the word "comprising" encompasses the case of "consisting of". Additional objects, advantages and features of the present invention will become apparent to those skilled in the art by examining the description or can be learned by practicing the present invention. The following examples are provided by way of illustration and are not intended to limit the present invention. Further, the present invention encompasses all possible combinations of the specific preferred embodiments described herein.

Examples

[0146] To provide a method for differentiating ischemic stroke (IS) from intracerebral hemorrhage (ICH) before admission using blood biomarkers, the inventors conducted an extensive analysis of samples from stroke patients at hand. The main objective was to find a reliable biomarker that provides information for initiating reperfusion therapy (mainly intravenous thrombolysis) without the need for neuroimaging techniques.

[0147] Example 1; Classification and therapy selection for patients in two different cohorts (cohort 1 including 190 patients; cohort 2 including 67 patients)

[0148] Materials and methods From December 2013 to July 2014, patients suspected of having a stroke who were admitted within 4.5 hours after the onset of stroke were enrolled. Blood samples were collected at the time of admission (baseline). Biomarkers were mainly measured by ELISA and SIMOA. Stroke subtypes were confirmed by neuroimaging techniques. To minimize the error of administering tPA to ICH patients, the biomarker selected as having the highest sensitivity with 100% specificity for IS was dichotomized by a cut-off.

[0149] The patient cohorts were as follows: Cohort 1 (ELISA cohort): 190 patients with stroke (155 ischemic and 35 hemorrhagic). Cohort 2 (SIMOA cohort): 67 patients with stroke (33 ischemic and 34 hemorrhagic).

[0150] The kits for marker analysis were as follows: · For RBP4 -> Catalog number DRB400, Quantikine R&D Systems; · For GFAP (measured using the Simoa kit and naming the SIMOA cohort as Cohort 2) -> Catalog number 102336 and consumables: · Simoa Accelerator-1 Plate Lab Service Fee, Catalog number 100835 · Accelerator Consumables Kit, Catalog number ACC1001 · For NT-proBNP -> When used in the automated Roche® system, the following reference catalog reactants (4842464130-proBNP GEN.2 ELECSYS; 4917049922-Precicontrol cardiac G4; 4842472190-CALSET proBNO GEN.2 ELECSYS)

[0151] After excluding mimics who were patients without stroke but with clinical signs of stroke, these Cohorts 1 and 2 were selected.

[0152] For all patients, the baseline expression levels of GFAP, NT-proBNP, and RBP4 were measured using ELISA technology (see above for NT-proBNP and RBP4) or SIMOA (see above for GFAP).

[0153] We assayed various methods for analyzing the retrieved data: basic cut-off (Method 1), principal component analysis (PCA) (Method 2), modified PCA (more rounds); and support vector machine procedure (SVM) (Method 4).

[0154] With any method, the concern here was that since the provision of reperfusion therapy (i.e., tPA or TNK) to ICH patients can have lethal side effects, the optimal cut-off values of these blood biomarker values that can indicate the exact values of each biomarker, ensuring that patients can be classified with 100% accuracy to avoid any errors, were sought.

[0155] Results Method 1 (basic cut-off) In this method, the simplest cut-off of the individual biomarker in the training cohort was used, and then all cut-offs were combined to obtain the final classification.

[0156] In Cohort 1 (ELISA cohort), ischemic patients could be detected by examining patients with high levels of NT-proBNP or RBP4.

[0157] Thus, seeking 100% specificity, patients were classified as ischemic in the following cases: · NT-proBNP > 4062 pg / mL. 100% specificity and 14.3% sensitivity were obtained (22 / 155 ischemic patients were detected without risk) (see Figure 1(A)), or · RBP4 > 52 μg / mL. A 100% specificity and 6.5% sensitivity were obtained (10 / 155 ischemic patients were detected without risk) (see Figure 1(B)).

[0158] Only one of these two conditions needed to be met. Finally, when both conditions were met simultaneously, 31 / 155 ischemic patients were classified with 100% specificity (20.1%) (see Figure 1(C)).

[0159] Data are shown in Figure 1, where a cut-off of 4062 pg / ml for NT-proBNP (dark lines in both Panels A and B), or a cut-off of 52 μg / mL for RBP4, enabled reliable discrimination between IS and ICH. In Panel C, log10(NT-proBNP) and RBP4 levels were plotted simultaneously with the corresponding cut-offs determined previously for each protein.

[0160] In Cohort 2 (67 patients) (also called the SIMOA cohort here) that included determination of GFAP using the SIMOA assay for this protein, the first approach of combining GFAP with other biomarkers was performed to achieve safe and good detection of ischemic patients.

[0161] The classification had two steps. First, as a first condition, individuals with a GFAP < 325 ng / mL value were selected as potential ischemic candidates. Only 27 hemorrhagic patients and 6 ischemic patients out of the potential ischemic patients were excluded. The visual representation of this first discrimination step is plotted in Figure 2(A), where the 3D graph classifies patients according to the detected levels of GFAP (as log(GFAP)), NT-proBNP (as log(NT-proBNP)), and RBP4 levels. The values below the square defining the GFAP < 325 ng / mL value (3D space indicated by the arrow) correspond to the values of the patients selected as ischemic candidates at the first step.

[0162] As a second condition, the patients with ischemia were patients having any of the following: · NT-proBNP > 1305 pg / mL. A specificity of 100% and a sensitivity of 30.3% were obtained (10 / 33 ischemic patients were detected), or · RBP4 > 38 μg / mL. A specificity of 100% and a sensitivity of 30.3% were obtained (10 / 33 ischemic patients were detected).

[0163] When GFAP is no longer considered for the potential ischemic candidates selected in the first stage, considering patients having NT-proBNP > 1305 pg / mL and RBP4 > 38 μg / mL as candidates, 17 / 33 ischemic patients (51.5%) were detected without risk (100% specificity). The data are shown in Figure 2(B).

[0164] Several analyses were performed with different specificity values (different from 100%).

[0165] At a specificity of 97%, ischemic patients were patients having a value of GFAP < 325 ng / mL and · NT-proBNP > 600 pg / mL. A specificity of 100% and a sensitivity of 51.5% were obtained (17 / 33 ischemic patients were detected) · RBP4 > 36.6 μg / mL. A specificity of 97% and a sensitivity of 39.4% were obtained (13 / 33 ischemic patients were detected)

[0166] Thereby, 23 / 33 ischemic patients (69.7%) could be detected at a specificity of 97%.

[0167] At a specificity of 94%, ischemic patients were patients having a value of GFAP < 325 ng / mL and · NT-proBNP > 147 pg / mL. A specificity of 94% and a sensitivity of 69.7% were obtained (23 / 33 ischemic patients were detected) · RBP4 > 31 μg / mL. A specificity of 94% and a sensitivity of 51.5% were obtained (17 / 33 ischemic patients were detected)

[0168] As a result, 26 / 33 ischemic patients (78.7%) could be detected with a specificity of 94%.

[0169] Cut-off values always imply deviations or certain variabilities due to various factors. All those shown here are also within the range considered to be positive values or discriminant values because they are related to a specific fixed prediction accuracy (usually IC95%).

[0170] Using Method 1, the best cut-off for reliably classifying stroke patients was the individual cut-off of the ELISA cohort (RBP4 > 52 μg / ml and NT-proBNP > 4062.0 pg / ml)

[0171] Method 2 (principal component analysis) Using the calculation of PCA, it was shown which of these three biomarkers explained the greatest variability in relatively few principal components (Jolliffe, I.T. (2002). Principal Component Analysis, second edition (Springer), ISBN0-387-95442-2). The procedure consisted of performing principal component analysis. After the calculation, it was found which variable contributed most to the principal components, and the results were that GFAP was the variable most correlated with PC1 and RBP4 was the variable most correlated with PC2. Therefore, the best order to follow for classifying patients was GFAP > RBP4 > NT-proBNP.

[0172] In summary, principal component analysis (PCA) is a statistical procedure that uses an orthogonal transformation to convert a set of observations of possibly correlated variables (entities each taking on various numerical values) into a set of values of linearly uncorrelated variables called principal components. If there are n observations with p variables, the number of distinct principal components is min(n - 1, p). This transformation is defined such that the first principal component has the largest possible variance (i.e., explains as much of the variability of the data as possible), and each subsequent component has the highest possible variance in turn, subject to the constraint that it is orthogonal to the preceding components. The resulting vectors (each being a linear combination of the variables and containing n observations) form an uncorrelated orthogonal basis set. PCA is sensitive to the relative scaling of the original variables. PCA is mainly used as a tool for exploratory data analysis and for creating predictive models. Mathematically, PCA is defined as an orthogonal linear transformation that transforms the data into a new coordinate system such that the maximum variance due to any projection of the data lies on the first coordinate (called the first principal component), the second largest variance lies on the second coordinate, and so on.

[0173] Using this order of cutoff biomarkers (GFAP > RBP4 > NT-proBNP), the patients were classified. First, the GFAP cutoff was used to classify the maximum number of patients with either 100% sensitivity or 100% specificity. Next, the RBP4 cutoff was used to classify the patients who could not be classified with the previous cutoff with either 100% sensitivity or 100% specificity. Finally, the NT-proBNP cutoff was used to classify the patients who had not yet been classified with either 100% sensitivity or 100% specificity. After the application of these three cutoffs, the first round of cutoffs was considered complete.

[0174] The same analysis was repeated using only two biomarkers (RBP4 and NT-proBNP).

[0175] In this case of using GFAP, the cutoffs are shown in Table 1:

Table 1

[0176] The cut-off for cohort 1 (190 patients) was more robust than that found using cohort 2 (i.e., the SIMOA cohort (67 patients)) in which GFAP was also determined using the SIMOA assay in addition to the determination of NT-proBNP and RBP4 using the ELISA assay.

[0177] Using the cut-off of the ELISA cohort (cohort 1), 23.16% of the entire cohort (37.14% in hemorrhagic patients and 20% in ischemic patients) could be classified with 100% sensitivity or 100% specificity.

[0178] Using the cut-off of the SIMOA cohort (cohort 2), 31% of the entire cohort (38% in hemorrhagic patients and 24% in ischemic patients) could be classified with 100% sensitivity or 100% specificity.

[0179] In cohort 2, using a smaller number of individuals made it possible to distinguish them even better (classifying almost 1% more hemorrhagic patients and 4% more ischemic patients).

[0180] When using only RBP4 + NT-proBNP, the cut-off was as shown in Table 2:

Table 2

[0181] Again, cohort a (190 patients) was more robust than cohort 2 (67 patients). (Same cut-off as above)

[0182] Using the cut-off from the ELISA cohort (cohort 1), 16.8% of the entire cohort (3% in hemorrhagic patients and 20% in ischemic patients) could be classified with 100% sensitivity or 100% specificity.

[0183] Using the cut-off from the SIMOA cohort (cohort 2), 13.4% of the entire cohort (3% hemorrhagic patients, 24% ischemic patients) could be classified with 100% sensitivity or 100% specificity.

[0184] Using a small number of individuals in the SIMOA cohort made it possible to distinguish them even better (classifying 4% more ischemic patients).

[0185] Method 3 (principal component analysis) This method is an extension of method 2 and calculated more rounds using the unclassified individuals.

[0186] After the completion of the first round, logistic regression was performed to check if there was still any tendency of some biomarker related to the outcome. Then, the cut-off was recalculated in the same order as the patients who were still unclassified.

[0187] This method was performed until no tendency was observed or until it was no longer possible to classify more individuals with 100% sensitivity or 100% specificity. It is important to note that once an individual is classified, the order of the cut-offs from the first round to the last round must be followed.

[0188] Method 3 was performed using only two biomarkers (RBP4 and NT-proBNP).

[0189] In the ELISA cohort (cohort 1), using GFAP, RBP4, and NT-proBNP as biomarkers, the following cut-offs were obtained:

Table 3

[0190] With this method, 51% of the individuals were classified with 100% sensitivity or specificity (60% hemorrhagic, 49% ischemic).

[0191] In the ELISA cohort (cohort 1), using RBP4 and NT-proBNP as biomarkers, the following cut-offs were obtained:

Table 4

[0192] 36.5% of individuals (6% hemorrhagic, 44% ischemic) with 100% sensitivity or specificity.

[0193] Method 4 (SVM) To maximize the number of IS patients that were well classified when classifying all ICH patients with 100% specificity, the levels of RBP4 and NT-proBNP were calculated using the support vector machine procedure (SVM) (“A User’s Guide to Support Vector Machines”, Article in Methods in Molecular Biology, (Clifton N.J), 2010, see Asa Ben-Hur and Jason Weston). Radial kernel analysis was used and the parameters were as follows: c = 100 and σ = 0.05. For the data on ICH, to obtain a classifier with 100% specificity, the decision value had to be increased by 0.71 at each point (the intuition behind the decision value is that the larger the positive value, the less chance there is of classifying an ICH patient as an IS, and the more chance there is of losing the opportunity to classify an IS patient as an IS).

[0194] With this method, a sensitivity of 29.7% was achieved for IS.

[0195] The data are shown in Figure 3, where the values on the “S-shaped curve” are 100% IS (indicated by the arrow), and the values below the curve correspond to patients with either ICH or an IS subtype.

[0196] As can be derived from this Figure 3, the combination of the levels of RBP4 and NT-proBNP enables good classification of patients and appropriate selection of therapies. The above levels are introduced into a support vector machine procedure using a Gaussian kernel and can give a graph like that in Figure 3, and depending on the value of one protein, patients can be accurately classified as IS or ICH from the value of the other protein.

[0197] According to the SVM, when the determined values of the test samples are obtained, they are introduced into a trained SVM that returns a decision value that enables the test samples to be classified into one category (IS) or another category (ICH). In particular, the above decision value is usually 0.5 and can be adjusted as necessary to improve the accurate classification of the subject. Depending on the trained machine, if this value is exceeded, the patient is classified into one category, and if this value is below, the patient is classified into another category. The decision value can be adjusted.

[0198] Conclusion: In the ELISA cohort (cohort 1), when using method 1 (basic cut-off) and using RBP4 and NT-proBNP separately, it was possible to appropriately identify 6.5% and 14.2% of IS for ICH with 100% specificity respectively. When used in combination, the identification increased to 20% of IS patients with 100% specificity for ICH, which was less than the sum of both individual biomarkers (the reason being that 1 case was appropriately classified by both biomarkers).

[0199] In the SIMOA cohort (cohort 2), when using the GFAP cut-off to separate the maximum number of ICH patients and IS patients and then trying to classify IS patients within the lower limit of the GFAP cut-off, the sensitivity for IS patients increased (51.5%), while the specificity for ICH patients was maintained at 100%.

[0200] Using Method 2 (also known as Classification-Exclusion) and Method 3 (also known as Classification-Exclusion-Repeat), it was observed that the addition of the GFAP biomarker was exactly useful for appropriately classifying ICH patients (in the ELISA cohort, 6% when GFAP was not used compared to 60% when GFAP was used; in the SIMOA cohort, 3% when GFAP was not used compared to 37% when GFAP was used). It is also important to note that the high ability to classify IS patients was not lost even when GFAP was not used (49% vs. 44% in the ELISA cohort values and 24% vs. 24% in the SIMOA cohort values, regardless of the use or non-use of the GFAP biomarker).

[0201] Including GFAP does not affect the ability to identify IS patients.

[0202] Finally, using Method 4 (SVM) classifier, a more refined method, 29.7% of IS patients could be appropriately identified for ICH with 100% specificity.

[0203] All these data also clarify that the reference values for accurate patient classification can be dynamic values that are a function of several parameters, such as the number of other markers determined simultaneously or the technology used for data analysis from the patient cohort. On the other hand, for one fixed amount or level of a protein, the amount of other co-existing markers that are determined by the first and enable good classification can vary and can be represented by several mathematical functions or models used (i.e., basic cut-off, ROC curve, PCA, SVM, etc.).

[0204] From different data analyses provided by two different cohorts of patients according to Example 1 of this description, it has been revealed that the levels of RBP4 and NT-proBNP, and optionally the level of GFAP, enable good discrimination between IS and ICH and also enable a safe selection of candidates for reperfusion therapy. The reference ranges or reference values for accurate classification are adjusted according to several variables calculated in this way. In any case, these values are shown to the person in charge who must perform this method.

[0205] These two or three markers were useful for the accurate classification of the test patients because an overall picture of the well-classified patients was obtained.

[0206] Furthermore, of particular interest is the fact that ischemic patients appropriately classified using the RBP4 and NT-proBNP biomarkers are patients with poor outcomes.

[0207] As shown in Table 5 below, ischemic stroke (IS) identified using these two biomarkers has a higher mortality rate and a lower independence at the three-month time point after stroke.

[0208] This is another reason to treat these patients as early as possible using reperfusion techniques to avoid lethal outcomes when treated later according to today's standard pathways.

[0209]

Table 5

[0210] Put simply, this technology based on the determination of both biomarkers increases the probability of treatment within the "golden hour" (less than 60 minutes from the onset of ischemic stroke), and the fact almost doubles the probability of becoming asymptomatic, triples the independent probability, and quadruples the probability of survival (for details regarding the "golden hour" and the treatment action protocol, see Kunz et al. “Effects of Ultraearly Intravenous Thrombolysis on Outcomes in Ischemic Stroke: The STEMO (Stroke Emergency Mobile) Group”, Circulation - 2017 May 2; 135(18):1765 - 1767).

[0211] Example 2: Classification of patients and selection of therapies for 32 different cohorts of patients.

[0212] The dataset existed in 32 patients (18 hemorrhagic and 14 ischemic). The aim was to separate the maximum number of patients in both classes without the risk of misclassification.

[0213] To do so, four different methods for the GFAP biomarker were used. Thus, the procedure was calculated for each different method.

[0214] The procedure consisted of finding the best cut-off for GFAP and excluding patients classified with 100% sensitivity or 100% specificity, and using the remaining data (not classified / excluded using GFAP and thus not fully classified) to calculate the next cut-off with the RBP4 biomarker data, re-classifying the patients with 100% sensitivity or 100% specificity and excluding them. Finally, the procedure was repeated using the NT-proBNP biomarker data.

[0215] Below, the cut-off values used for each method for GFAP determination are included.

[0216] GFAP DxSYS_CLIA (DxSYS Inc. Chemiluminescent Immunoassay): Using a cutoff > 80.6 pg / ml, 15 hemorrhagic patients (83% of all hemorrhagic patients) were accurately classified. GFAP DxSYS_TMB (DxSYS Inc. using 3,3’,5,5’-tetramethylbenzidine (TMB)): Using a cutoff > 88.685 pg / ml, 14 hemorrhagic patients (78% of all hemorrhagic patients) were accurately classified. The same result was obtained using a cutoff of 100 pg / ml. GFAP Quanterix®: Using cutoffs of > 2066.078 pg / ml and < 166.67 pg / ml, 14 hemorrhagic patients and 3 ischemic patients (78% of all hemorrhagic patients and 21.4% of ischemic patients) were accurately classified. GFAP Elisa (Elisa kit catalog number RD192072200, BioVendor): Using a cutoff > 50.5 pg / ml, 11 hemorrhagic patients (61.1% of all hemorrhagic patients) were accurately classified.

[0217] After applying all these cutoffs and proceeding with RBP4 and NT-proBNP as described above, finally, 17 / 18 hemorrhagic patients and 12 / 14 ischemic patients were accurately classified.

[0218] Therefore, this procedure demonstrated that by determining three biomarkers in a series of steps, it is possible to accurately classify patients for determining an appropriate medical regimen (reperfusion therapy for ischemic stroke). The outline of the procedure for each analytical method is shown in Table 6.

[0219]

Table 6

[0220] Example 3: The combination of biomarkers and clinical data improves the accuracy of identifying ischemic stroke patients in need of reperfusion therapy

[0221] Data analysis was performed using the following patients and cutoffs: Hemorrhagic n = 35 and ischemic n = 155

[0222] In the combination using cutoffs GFAP (pg / ml) < 97.03 and NT-proBNP (pg / ml) > 4076.50 and RBP-4 (μg / ml) > 52.52, the three biomarkers showed a sensitivity of 0.32, specificity of 1.00 (100%), positive predictive value (PPV) of 1.00 and negative predictive value (NPV) of 0.25.

[0223] Adding clinical data (especially blood pressure and blood glucose levels) on top of that improved the sensitivity while maintaining 100% specificity. In fact, GFAP (pg / ml) < 97.03 and NT-proBNP (pg / ml) > 4076.50 and systolic blood pressure (SBP) mmHg < 119.00, and diastolic blood pressure (DBP) (mmHg) < 60.50 and RBP-4 (ug / ml) > 52.52 and blood glucose (mg / dl) < 83.50 had a sensitivity of 0.45 and specificity of 1.00, with PPV = 1.00 and NPV = 0.29.

[0224] Logistic regression-based model: To find realizable transformations of the data that can improve classification accuracy and robustness while limiting overfitting, several multiple logistic regression models were tested on the pooled data obtained from both cohorts (the original cohort 1 of Example 1 n = 189, as well as the replicated cohort n = 300 with hemorrhagic n = 51 and ischemic n = 249). The models tested included a panel of markers and related clinical variables. The models were selected according to the Akaike Information Criteria and used to classify individuals between ischemic stroke and hemorrhagic stroke.

[0225] The selected model included the log-transformations of GFAP (pg / ml), NT-proBNP (pg / ml) and diastolic blood pressure (mmHg) as significant predictors of the ischemic stroke state in the following combination:

[0226] -1.56 log(GFAP (pg / ml)) + 0.0008 NT-proBNP (pg / ml) - 0.041 DBP (mmHg)

[0227] By this linear combination, an estimated log odds ratio score that can be treated as a compound marker was obtained. A threshold that maximizes the desired sensitivity and specificity requirements for classifying individuals between two groups could be placed on this score. By weighting and aggregating the markers, when the specificity exceeded 95%, the classification sensitivity was improved compared to the raw markers of both cohorts.

[0228] In the original cohort, the application of this model had a sensitivity = 0.60, specificity = 1.00, and accuracy = 0.68.

[0229] Figure 4 graphically shows the classification of subjects using this logistic model score.

[0230] The characteristics of the replication cohort (n = 300) are listed below:

[0231] In cases suspected of stroke (ischemic stroke or hemorrhagic stroke), blood samples were obtained within 3 hours of stroke onset. Diagnostic and therapeutic workups were, in some cases (n = 189), similar to those of the initial cohort 1 of Example 1 of the test subjects used in these files.

[0232]

Number

[0233] Example 4: Improvement in accuracy for detecting candidates for reperfusion therapy in ischemic stroke patients at a very early stage

[0234] For the original cohort 1 (n = 189) of Example 1, the performance of the biomarker was evaluated in relation to the time of blood sampling from symptom onset. Surprisingly, the earlier the test was performed, the higher the accuracy of the test:

[0235] (i) 0 - 2 hours (hemorrhagic n = 11 and ischemic n = 82) GFAP (pg / ml) < 175.85 and NT - proBNP (pg / ml) > 3916.50 and RBP - 4 (μg / ml) > 38.15. Sensitivity = 0.70, Specificity = 1.00, PPV = 1.00 and NPV 0.31. (ii) 2 - 3 hours (hemorrhagic n = 13 and ischemic n = 35) GFAP (pg / ml) < 94.37 and NT - proBNP (pg / ml) > 1289.50 and RBP - 4 (μg / ml) > 46.55. Sensitivity = 0.60, Specificity = 1.00, PPV = 1.00 and NPV 0.48. (iii) 3 - 4.5 hours (hemorrhagic n = 11 and ischemic n = 38) GFAP (pg / ml) < 98.96 and NT - proBNP (pg / ml) > 4254.50 and RBP - 4 (μg / ml) > 53.34. Sensitivity = 0.34, Specificity = 1.00, PPV = 1.00 and NPV 0.31.

[0236] In the replication cohort (n = 300, hemorrhagic n = 51 and ischemic n = 249), patients who arrived at the hospital very early were selected and blood samples were taken within the first hour of stroke onset. There were hemorrhagic n = 8 and ischemic n = 33.

[0237] In that sub-cohort, GFAP (pg / ml) < 300.03 and NT-proBNP (pg / ml) > 1033.01 and RBP-4 (ug / ml) > 32.93 had excellent accuracy, with sensitivity = 0.91, specificity = 1.00, PPV = 1.00 and NPV 0.73.

[0238] Adding several clinical variables (clinical parameters), namely GFAP (pg / ml) < 300.03 and DBP (mmhg) < 82.00 and SBP (mmhg) < 143.00 and NT-proBNP (pg / ml) > 2741.31 and blood glucose (mg / dl) < 108.00, it was further improved and the test worked perfectly.

[0239] This combination yielded sensitivity = 1.00, specificity = 1.00, PPV = 1.00 and NPV 1.00.

[0240] In this cohort of patients, logistic regression was also performed using samples obtained within the first hour of symptom onset.

[0241] The selected model included the log-transformations of GFAP (pg / ml), NT-proBNP (pg / ml) and diastolic blood pressure (mmhg) as significant predictors of ischemic stroke status in the following combination:

[0242] -1.56 log(GFAP (pg / ml)) + 0.0008 NT-proBNP (pg / ml) - 0.041 DBP (mmhg)

[0243] This linear combination yielded an estimated log odds ratio score that can be treated as a compound marker. The application of this model to patients in a replicated cohort nursed within the first hour of stroke onset had sensitivity = 0.79, specificity = 1.00 and accuracy = 0.83.

[0244] Figure 5 graphically shows the classification of subjects using this logistic model score together with data from isolated samples within the first hour of stroke onset.

[0245] All of these data from Example 4 indicate that the selected biomarkers RBP4, NT-proBNP, and GFAP would allow for high sensitivity if measured soon after the onset of stroke, particularly if they can be determined within 1 and 2 hours after onset.

[0246] In stroke, it is a surprising effect that the earlier the test is performed, the better the sensitivity can be achieved for a fixed specificity. Generally, biomarkers give an appropriate signal after a relatively long period after onset. In this medical condition, this is not a disadvantage but rather a goal, as it enables rapid and reliable classification of patients during the critical phase of the disease (e.g., at the ambulance level). This allows for the best and most appropriate decisions to be made at these important moments and later verified using additional biomarkers (e.g., after the patient arrives at the hospital).

[0247] Example 5: A rapid point-of-care blood test performed in the ambulance to select ischemic stroke patients worthy of receiving reperfusion therapy from other diseases similar to acute ischemic stroke (stroke mimicking conditions and intracerebral hemorrhage)

[0248] A point-of-care test (POCT) for ambulances was used to verify a panel of biomarkers (RBP4, NT-proBNP, and GFAP) included in the present invention, which can identify patients with ischemic stroke using a blood sample to initiate reperfusion therapy (thrombolysis) in the ambulance or to send this patient to an appropriate hospital for the best reperfusion therapy (thrombolysis or mechanical thrombectomy).

[0249] Method: More than 20 ambulances and a helicopter network in the Seville region of southern Spain registered patients suspected of having a stroke (less than 6 hours) in the BIO-FAST trial (Biomarkers for Initiating Onsite and Faster Ambulance Stroke Therapies). Blood samples were collected by ambulances using rapid POCT (results in 10 - 15 minutes) to measure RBP-4 / NT-pro-BNP, and GFAP was measured using SIMOA-Quanterix technology.

[0250] Inclusion criteria: Patients over 18 years old; a stroke code activated by the coordinating center, and less than 6 hours from symptom onset. In the case of a stroke with an uncertain time series or a stroke upon waking up, the first hour is considered the last moment the patient appeared well.

[0251] Exclusion criteria: A pre-hospital diagnosis different from a stroke; inability to obtain a pre-hospital blood sample, and refusal by the patient / next of kin to provide informed consent.

[0252] Sample type Extraction of one EDTA tube of blood sample (10 mL) for the biobank + one EDTA tube of blood sample (2 mL) for POC. Samples were included in the collection registered under code C.0003176 at the Vall d’Hebron Hospital in accordance with the requirements established in RD 1716 / 2011 until they were fully used for biomarker discovery research.

[0253] · RBP4 rapid test. RBP4 POC lateral flow dispositive. · NT-proBNP rapid test. Nt-proBNP POC lateral flow dispositive. · GFAP SIMOA-Quanterix technology

[0254] Results: Including 20 patients (10 had ischemic stroke, 3 had intracerebral hemorrhage, and 7 had stroke mimics). POCT could be performed by ambulance, and 1 was done in a helicopter without incident.

[0255] Using the cut-offs selected for these biomarkers, rapid POCT (RBP-4-NT-proBNP) accurately identified 50% of IS without misclassifying ICH or mimics (100% specificity, 50% sensitivity). Excluding wake-up strokes, POCT accurately identified 62.5% of IS without misclassifying ICH or mimics (100% specificity, 62.5% sensitivity).

[0256] Using the SIMOA-Quanterix technology to measure GFAP, stored blood samples from these patients were used to investigate how results could be improved using a POCT that could incorporate GFAP, RBP-4, and NT-proBNP. Four patients with very high GFAP levels were identified, 2 with ICH, 1 with IS, and 1 with a mimic. Excluding these 4 patients increased the sensitivity to over 70% while maintaining 100% specificity.

[0257] Twenty percent of IS could potentially be treated within the first 30 minutes from symptom onset using POCT. Additionally, 1 mimic who was given tPA was potentially avoided because POCT was negative for IS. Patients treated with tPA could potentially have received this drug 1 hour 30 minutes earlier by using in-ambulance testing. Three ischemic patients (30%) could potentially have fallen within the 4.5-hour time frame for testing but were outside that time when a CT scan was done at the hospital.

[0258] Conclusion: A panel of biomarkers including RBP-4, NT-proBNP, and GFAP provides a sensitivity rate useful with 100% specificity for ischemic stroke. This could change standard clinical practice by using a POCT that allows starting pre-hospital reperfusion therapy for cases selected much more rapidly than using standard techniques.

[0259] Data obtained by a POCT enabling determination of blood levels of RBP4 and NT-proBNP and further finished by determination with GFAP were envisioned for the first test in the realistic situation where IS and ICH patients are screened in the presence of existing mimics (non-stroke patients with stroke-like symptoms). In this realistic situation, while fixing 100% specificity, the sensitivity was also higher (above 50%) than some of the previous examples. This is an unexpected advantage of the biomarker to be added to the panel of biomarkers enabling a good and reliable classification and is translated into an appropriate selection of therapies that can be administered before arrival at the hospital.

[0260] Example 6: RBP-4, NT-proBNP, and GFAP identify ischemic stroke patients with large vessel occlusion (LVO) who require mechanical thrombectomy and need to be transferred to a reference facility if this therapy is available.

[0261] In two previously disclosed cohorts of stroke patients (n = 189 and n = 300), identification of LVO was based on the presence and location of occluded cerebral arteries in CT angiography (CTA) performed upon arrival at the hospital. It followed a limited definition of LVO described as occlusion of any of the following arteries or arterial segments, namely, the intracranial internal carotid artery (ICA), the basilar artery (BA), and the M1 segment of the middle cerebral artery occlusion.

[0262] (See Vanacker P, Heldner MR, Amiguet M, et al. Prediction of large vessel occlusions in acute stroke: National institute of Health Stroke Scale is hard to beat. Crit Care Med 2016;44: e336-43).

[0263] The same biomarkers shown below are also useful for identifying LVO with less stringent criteria, including occlusions in more distal portions of the middle cerebral artery (MCA), such as M2 (LVO defined as occlusion of the ICA, M1, M2, or BA), simply by using different cut-offs.

[0264] A. Initial cohort 1 of Example 1 (N = 189):

[0265] Prediction of LVO patients (134 without LVO vs. 56 with LVO):

[0266] GFAP (pg / ml) < 694.48 and NT-proBNP (pg / ml) > 1764.50 and RBP-4 (μg / ml) > 35.22 had a sensitivity = 0.98, specificity = 0.18, PPV = 0.33 and NPV 0.96; this was improved by adding clinical variables and test variables such as baseline NIHSS score > 11 points and d-dimer (ng / ml) > 1432.43, having a sensitivity = 1.00, specificity = 0.46, PPV = 0.43 and NPV 1.00.

[0267] When blood samples were obtained within 2 hours of symptom onset, at baseline NIHSS score > 11 points and RBP-4 (ug / ml) > 51.53, the marker ability was improved with sensitivity = 1.00, specificity = 0.40, PPV = 0.46 and NPV 1.00.

[0268] Furthermore, in patients who underwent thrombectomy (165 cases without thrombectomy and 22 cases with thrombectomy), the biomarkers GFAP (pg / ml) > 209.43 and NT-proBNP (pg / ml) < 848.15 had a sensitivity = 1.00, specificity = 0.23, PPV = 0.15, and NPV = 1.00.

[0269] This was improved by adding clinical variables such as baseline NIHSS score > 11 points and GFAP (pg / ml) < 54.84, and had a sensitivity = 1.00, specificity = 0.43, PPV = 0.19, and NPV = 1.00.

[0270] When blood samples were obtained within 2 hours of symptom onset, NT-proBNP with D-dimer had improved ability, with a sensitivity = 1.00, specificity = 0.70, PPV = 0.39, and NPV = 1.00.

[0271] B. Replication cohort (n = 300)

[0272] LVO (limited definition), no LVO = 215 cases vs. LVO = 85 cases

[0273] The biomarkers GFAP (pg / ml) < 153.18, NT-proBNP (pg / ml) > 692.60, and RBP-4 (μg / ml) < 39.72 showed a sensitivity = 1.00, specificity = 0.09, PPV = 0.30, and NPV = 1.00.

[0274] These results were improved by adding baseline NIHSS score > 11 points, RBP-4 (ug / ml) < 29.03, and blood glucose (mg / dl) < 71.50 to the clinical data, and a sensitivity = 1.00, specificity = 0.20, PPV = 0.33, and NPV = 1.00 was shown.

[0275] These results were even better for baseline NIHSS score and RBP-4 when blood samples were obtained within 2 hours after onset, with a sensitivity = 1.00, specificity = 0.45, PPV = 0.43, and NPV = 1.00.

[0276] In patients who underwent thrombectomy (no thrombectomy n = 264 and thrombectomy n = 35), biomarker GFAP (pg / ml) < 153.18 and NT-proBNP (pg / ml) < 2049.01 had a sensitivity = 1.00, specificity = 0.14, PPV = 0.13, and NPV 1.00 for accurately identifying these patients. This was improved by adding clinical and test variables, and GFAP (pg / ml) < 153.18 and d-dimer (ng / ml) > 7.29 and diastolic blood pressure (mmhg) > 89.50 had a sensitivity = 0.97, specificity = 0.33, PPV = 0.16, and NPV 0.99.

[0277] Cited References Patent Documents - International Publication WO2016 / 087611 Non-Patent Documents - Reynolds et al., “Early Biomarkers of Stroke”, Clinical Chemistry - 2003, vol.: 49 (10), pp.: 1733 - 1739 - Montaner et al. “Etiologic Diagnosis of Ischemic Stroke Subtypes With Plasma Biomarkers”, Stroke 2008, vol. no. 39, pp.: 2280 - 2287 - Adams HP Jr Neurology. 1999 Jul 13;53(1): 126 - 31. - Tsivgoulis G. et al, Neurology. 2014 Sep 19 - BLAST Manual, Altschul, S., et al, NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al, J. Mol. Biol. 215: 403 - 410 (1990) -Jolliffe, I.T. (2002). Principal Component Analysis, second edition(Springer), ISBN 0-387-95442-2. -Kunz et al. “Effects of Ultraearly Intravenous Thrombolysis onOutcomes in Ischemic Stroke: The STEMO (Stroke Emergency Mobile) Group”,Circulation- 2017 May 2;135(18):1765-1767. -Vanacker P, Heldner MR, Amiguet M, et al. Prediction of largevessel occlusions in acute stroke: National institute of Health Stroke Scale ishard to beat. Crit Care Med 2016;44:e336-43. -Rai AT et al. (2017). A population-based incidence of acutelarge vessel occlusions and thrombectomy eligible patients indicatessignificant potential for growth of endovascular stroke therapy in the USA. JNeurointerv Surg. 9:722-6. -Crowe RP, Myers JB, Fernandez AR, Bourn S, McMullan JT.. Prehosp Emerg Care. 2020 Feb 25:1-9. -Gandhi CD, Al Mufti F, Singh iP, et al. Neuroendovascular management of emergent large vessel occlusion: update on the technical aspects and standards of practice by the Standards and Guidelines Committee of the Society of Neurointerventional Surgery. J Neurointerv Surg 2018;10:315-20). -Lakomkin N, Dhamoon M, Carroll K, et al. Prevalence of large vessel occlusion in patients presenting with acute ischemic stroke: a 10-year systematic review of the literature. J Neurointerv Surg 2019;11:241-5. -Waqas M, et al. Effect of definition and methods on estimates of prevalence of large vessel occlusion in acute ischemic stroke: a systematic review and meta-analysis. J Neurointerv Surg. 2020 Mar;12(3):260-265

Claims

1. An in vitro method for differentiating ischemic stroke from hemorrhagic stroke in a patient or for selecting a patient suffering from stroke for reperfusion therapy, comprising: (a) determining the levels of NT-proBNP and GFAP in an isolated sample from said patient; and (b) comparing the levels of said NT-proBNP and GFAP with corresponding reference ranges for each protein; wherein said reference ranges are selected from the ranges of values obtained from subjects suffering from ischemic stroke, and said patient is classified as a candidate for reperfusion therapy or suffering from ischemic stroke when at least both the levels of said NT-proBNP and GFAP are within said ranges of values obtained from subjects suffering from ischemic stroke, said sample is a biological fluid selected from blood, plasma or serum, and determining the levels of NT-proBNP and GFAP in step (a) is performed within the first 2 hours after the onset of stroke, an in vitro method.

2. The in vitro method according to claim 1, further comprising determining clinical parameters selected from the group consisting of blood pressure including systolic blood pressure and / or diastolic blood pressure, blood glucose, age, score obtained from a systematic evaluation tool for stroke-related neuropathy, gender, and combinations thereof.

3. The in vitro method according to claim 2, wherein said clinical parameter is blood pressure.

4. The in vitro method according to any one of claims 1 to 3, further comprising determining the level of retinol-binding protein-4 (RBP4).

5. The in vitro method according to any one of claims 1 to 4, wherein said reperfusion therapy is selected from the group consisting of therapy with an antithrombotic agent, thrombus removal, and combinations thereof.

6. The in vitro method according to claim 5, wherein said antithrombotic agent is a thrombolytic agent.

7. The method according to claim 6, wherein said thrombolytic agent is a plasminogen activator.

8. The method according to claim 7, wherein said plasminogen activator is tissue plasminogen activator.

9. The method according to claim 8, wherein said thrombolytic agent is a recombinant tissue plasminogen activator selected from the group consisting of alteplase, reteplase and tenecteplase and combinations thereof.

10. The method according to any one of claims 1 to 9, which is a method for detecting ischemic stroke with large vessel occlusion, in which the levels of NT-proBNP and GFAP are determined and used in combination with clinical variables.

11. The method according to claim 10, wherein the clinical variables are the baseline NIHSS score and / or the level of d-dimer in the blood and / or blood pressure values.

12. A kit for differentiating ischemic stroke from hemorrhagic stroke in a patient within the first 2 hours after the onset of stroke or for selecting a patient within the first 2 hours after the onset of stroke suffering from stroke for reperfusion therapy, comprising means for detecting both the level of NT-proBNP and the level of GFAP.

13. Use of a kit for differentiating ischemic stroke from hemorrhagic stroke in a patient or for selecting a patient suffering from stroke for reperfusion therapy, wherein the kit comprises reagent means and a solid support for detecting both the level of NT-proBNP and the level of GFAP, the reagent means comprising a compound that specifically binds to the NT-proBNP and GFAP, and the detection of the level of NT-proBNP and the level of GFAP is performed within the first 2 hours after the onset of stroke.

14. The use according to claim 13, wherein the compound that specifically binds to the NT-proBNP and GFAP is an antibody or a fragment thereof.

15. Use of a combination of NT-proBNP and GFAP as biomarkers for differentiating ischemic stroke from hemorrhagic stroke in a patient within the first 2 hours after the onset of stroke or for selecting a patient within the first 2 hours after the onset of stroke suffering from stroke for reperfusion therapy.

16. An in vitro method for differentiating ischemic stroke from hemorrhagic stroke in a patient or for selecting a patient suffering from stroke for a therapy to lower or optimize blood pressure, (a) determining the levels of NT-proBNP and GFAP in an isolated sample of the patient; and (b) comparing the levels of NT-proBNP and GFAP with corresponding reference ranges for each protein; comprising, wherein the reference range is selected from the range of values obtained from subjects suffering from ischemic stroke. When at least both the level of said NT-proBNP and the level of GFAP are within the range of said values obtained from a subject suffering from hemorrhagic stroke, said patient is classified as a candidate for a therapy to lower or optimize blood pressure or as suffering from hemorrhagic stroke, An in vitro method, wherein said sample is a biological fluid selected from blood, plasma or serum, and wherein determination of the level of NT-proBNP and the level of GFAP in said step (a) is performed within the first 2 hours after the onset of stroke.

Images