Method of selecting patient for reperfusion therapy

Abstract

To provide a method of selecting a patient suffering stroke for a reperfusion therapy, a method of differentiating ischemic stroke from hemorrhagic stroke, and to kits comprising reagents for implementing the methods.SOLUTION: A method of selecting a patient suffering stroke for a reperfusion therapy is provided, the method comprising determining the level of retinol-binding protein-4 (RBP4) and the level of N-terminal fragment of B-type natriuretic peptide (NT-proBNP) in an isolated sample of a patient. The method further comprises determining the level of glial fibrillary acid protein (GFAP) in the isolated sample.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of European patent application EP19382384.6 filed on May 16, 2019. Claim the benefits of

[0002] The present invention relates to the field of diagnostics or companion diagnostics, in particular to the differentiation of ischemic stroke from hemorrhagic stroke. methods for differentiation and selection of appropriate therapy depending on the type of stroke event. [Background technology]

[0003] Stroke, also known as cerebrovascular disease (CVD), is one of the most important neurological disorders. It is the second leading cause of preventable death in the world and a leading cause of lost productivity. The two main subtypes are ischemic stroke (IS), also called hemorrhagic stroke, and cerebral Over 80-85% of all strokes are caused by cerebral artery blockage. The remaining 15-20% are ICH caused by arterial rupture. In contrast to patients with IS, who have a 30-day mortality rate of 8-12%, patients with ICH Patients with this condition have a poor outcome, with a 37-38% mortality rate 30 days after symptom onset.

[0004] In the acute phase, optimal treatment protocols are defined that are specific and significantly different between IS and ICH. It is important to accurately distinguish between these two subtypes in order to treat acute IS. Subsequent treatments include reperfusion, which is the restoration of blood flow through medication or endovascular surgery (thrombus removal). The main drugs used are thrombolytics, e.g., drugs that dissolve blood clots blocking cerebral arteries. Recombinant tissue plasminogen activator, or tenectepla, a serine protease TNK, derived from natural t-PA by modification at three sites in the protein structure. Thrombolysis is a treatment that reduces the risk of symptoms. Rapid identification of IS due to a narrow treatment window of only 4.5 hours from presentation This allows for early revascularization, leading to tissue recovery from the surrounding area and improving clinical outcomes. On the other hand, patients with acute ICH usually require a bolus of 100 mg / kg / day to slow the growth of the hematoma. or the appearance of edema, and is managed by lowering blood pressure to avoid rebleeding. Today, the diagnosis of stroke subtypes is based on computed tomography (CT) or magnetic resonance imaging. The data is mainly based on brain imaging data from magnetic resonance imaging (MRI). Patients with such neurological conditions lose valuable time to obtain a CT scan or MRI. The patient must be transported to a hospital for imaging procedures. Unfortunately, MRI and and CT scans are not widely available, especially in less developed areas, and primary hospitals are resource-constrained. They cannot be used repeatedly for the feet. Furthermore, some of these techniques There may be side effects primarily related to the radiation or contrast injection. If the medical personnel performing the CT are inexperienced or inadequately trained, RI and CT may be subject to error or uncertainty.

[0005] Several studies have described the use of biomarkers to rapidly differentiate stroke subtypes. Among the documents, Patent Document 1 discloses a method for distinguishing ischemic stroke from hemorrhagic stroke in a patient, and and detecting glial fibrillary acidic protein in the patient sample in combination with one or more biomarkers. Based on the determination of the level of GFAP, antithrombotic drugs or blood pressure lowering drugs can be administered. The present invention discloses a method for selecting patients suffering from stroke for therapy with a drug that can do.

[0006] Another example of a test analyzing biomarkers potentially relevant to acute IS is the Reyno This can be extracted from the literature by IDS et al., Non-Patent Document 1. In this literature, S-100B molecule, type B neurotrophic growth factor, was identified as a potential biomarker in the plasma of patients with glaucoma. Child, von Willebrand factor, matrix metalloproteinase-9 (MMP-9), and chemokine ligand 2 (C), also known as chemotactic protein-1 (MCP-1). The results for CCL-2 (which has a -C motif) are presented. Only the α-1 protein has significant value in diagnosing acute ischemic stroke and can be detected from the cerebrospinal fluid of patients. Samples were extracted and it was concluded that serum concentrations were not different from those in control patients. , and was assumed to be an impractical method for accurate disease identification.

[0007] Biomarkers for the specific diagnosis of cardiogenic cerebral embolism are also disclosed in Non-Patent Document 2. To improve the diagnosis of cardiogenic cerebral embolism in the acute phase of stroke, Urinary peptide (BNP) and D-dimer (DD) have been proposed.

[0008] Strictly speaking, due to the severity of the disease, reperfusion therapy would be fatal for non-IS patients. Accurate diagnosis of stroke subtypes is important because they may differ in severity. The diagnosis of HIV is often made after the patient is found in the acute stage at home, on the street, or in a general practitioner's office. It is preferable that this be done as quickly as possible, so it can be easily done in an ambulance. It would be even better if there was a kit or point-of-care that could be further validated in the hospital. stomach.

[0009] Other teams are working outside the hospital to diagnose strokes and administer reperfusion therapy as quickly as possible to improve recovery. To be able to improve the neurological outcome of treated patients, CT scans They use strategies such as mobile stroke units, which are built-in ambulances. The strategy is very expensive, these high-tech ambulances cost a lot of money and require specialized personnel. requires.

[0010] Recent studies have shown that endovascular treatment of large vessel occlusion (LVO) can improve this form of severe It has been shown to reduce morbidity and mortality in patients experiencing acute ischemic stroke. Nevertheless, in many cases, patients are unable to travel to specialized hospitals that can perform endovascular treatment. Due to delayed arrival, a minority of patients experiencing LVO receive endovascular treatment (non- Patients experiencing an acute stroke should be first treated by emergency medical services (EMS) professionals. LVO stroke is frequently encountered and recognized early in the prehospital setting by EMS professionals. This could improve timely transport to endovascular centers and lead to better patient outcomes. It is possible that the endoscopic technique can be used to diagnose LVO (Non-Patent Document 4). Various scales have been compared (see above). Crowe et al. In 2,415 patients, 26% (n=628) of patients with ischemic stroke were diagnosed with LVO This shows that.

[0011] A CPSS score of 2 or greater had a sensitivity of 69% and a specificity of 78% for LVO. A RACE score of 4 or greater had a sensitivity of 63% and a specificity of 73%. The LAMS score showed a sensitivity of 63% and a specificity of 72%, and a positive VAN score ( The tive VAN score showed a sensitivity of 86% and a specificity of 65%. When comparing the areas under the ROC curves of the two methods, statistically significant discriminatory ability was observed for LVO stroke. No significant differences were observed, highlighting the need for reliable markers of LVO.

[0012] Furthermore, LVO was associated with a 3-month and 6-month mortality in patients with acute ischemic stroke (AIS). It is associated with better outcomes (Non-Patent Document 5). Lakomkin et al. Sixteen studies used nine different definitions of LVO (different combinations of arterial occlusion sites). This may be a factor in the prevalence of LVO, as shown by Waqas et al. It has been found that this has the potential to be effective (see Non-Patent Documents 6 and 7).

[0013] Finally, in the field of stroke diagnosis and treatment, it is worth noting the so-called stroke mimics The goal is to distinguish between stroke mimics and actual strokes. is defined as a disease or condition that presents with stroke-like clinical features but is not accompanied by neurological tissue infarction. Several clinical syndromes present with symptoms or signs similar to acute ischemic stroke. Therefore, the distinction between stroke and stroke mimics is difficult to distinguish between a wide variety of overlapping clinical conditions. This is difficult due to the potential adverse effects of interventional stroke treatments. Currently, it is difficult to distinguish between actual strokes and mimics in isolated patient samples. There are very few markers that can do this. [Prior art documents] [Patent documents]

[0014] [Patent Document 1] International Publication No. WO2016 / 087611 [Non-patent literature]

[0015] [Non-Patent Document 1] “Early Biomarkers of Stroke”,Clinical Chemistry-2003,vol.:49(10),pp.:1733-1739 [Non-patent document 2] Montaner et al. “Etiologic Diagnosis of Ischemic Stroke Subtypes With Plasma Biomarkers”, Stroke2008, vol.no.39, pp.:2280-2287 [Non-patent document 3] Rai AT et al.(2017).A population-based incidence of acute large vessel occlusions and thrombectomy eligible patients indicates significant potential for growth of endovascular stroke therapy in the USA.J Neurointerv Surg.9:722-6 [Non-patent document 4] Crowe RP, Myers JB, Fernandez AR, Bourn S, McMullan JT. The Cincinnati Prehospital Stroke Scale Compared to Stroke Severity Tools for Large Vessel Occlusion Stroke Prediction. Prehosp Emerg Care. 2020 Feb 25:1-9 [Non-Patent Document 5] 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 [Non-Patent Document 6] 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 [Non-Patent Document 7] 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 Summary of the Invention [Problem to be solved by the invention]

[0016] Therefore, there are many methods in the art to determine the best treatment approach for a patient in the shortest time period. To overcome the limitations of methods disclosed in the art and to identify stroke subtypes and excluded There is a need for alternative tests using biomarkers that can reliably distinguish between the original and potential mimics. Furthermore, while a clear definition of LVO has been established, specific treatment options are not available. A reliable guide to LVO, a condition requiring endovascular treatment or thrombectomy The need for workers is also not being met. [Means for solving the problem]

[0017] In a first aspect, the present invention provides a method for selecting a patient suffering from a stroke for reperfusion therapy. an in vitro method for detecting retinol binding protein-4 in an isolated sample from said patient, The levels of RBP4 and the N-terminal fragment of B-type natriuretic peptide (NT-pr and determining the level of oBNP.

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

[0019] The inventors were surprised to find that the levels of these two proteins in isolated samples were significantly higher than those in wild-type mice. We found for the first time that accurate classification between IS and ICH is possible by using this method.

[0020] Therefore, another aspect of the present invention provides an in vitro method for distinguishing IS from ICH in patients. determining the levels of RBP4 and NT-proBPN in an isolated sample from said patient, The in vitro method includes determining

[0021] NT-proBNP and RBP4 levels are primarily associated with antithrombotic or thrombectomy therapy. and those who could receive reperfusion therapy to avoid fatal outcomes. Patients can be divided into two groups: those with ICH and those without. These are easily treatable by treatments that mitigate or optimize disease progression.

[0022] As shown in the example below, the combined levels of NT-proBNP and RBP4 This allows for classification of patients with 100% or nearly 100% specificity. This combination of markers is highly accurate and can identify patients who are candidates for appropriate therapy (i.e., reperfusion therapy). To the best of the inventor's knowledge, a truly safe method of selecting patients is envisaged. (i.e., biological fluid samples) with 100% or near 100% specificity for the markers detectable in the sample. Moreover, very advantageously, both markers are If measured within 6 hours or less, or even 3 hours or less, after onset In other words, it allows for differentiation between different stroke types even in the presence of a critical time (ultra-rapid Accurate identification during the sexual phase is possible.

[0023] Furthermore, as illustrated in the following examples, the two proteins in the isolated samples were determined. By determining whether or not a brain tumor has a poor prognosis or outcome in the sense of having a relatively high mortality rate, It can also detect patients who have suffered a stroke, and therefore, these patients are at a higher risk of developing a poor outcome. It needs to be treated as quickly as possible to avoid progression of the disease.

[0024] Therefore, the present invention also relates to a method for treating patients suffering from stroke, particularly ischemic stroke, and therefore 2. A method for prognosis of a patient candidate for reperfusion therapy, comprising: - including determining the expression level of RBP4 in combination with the expression level of proBNP To the best of the inventor's knowledge, no method has been developed that involves the synthesis of RBP4, or RBP4 and NT-pr This is the first time that oBNP has been associated with this poor outcome. In certain embodiments, the levels of both proteins are determined in a patient sample, which sample comprises: In another particular embodiment, the sample is a biological fluid, more particularly blood (plasma or serum). In yet another particular embodiment of the method for prognosis of a patient suffering from stroke, The levels of at least RBP4 or two proteins are compared to a reference value, which reference value is The value or range of values ​​is selected to indicate that the subject has suffered an ischemic stroke.

[0025] In yet another embodiment, the present invention provides a method for detecting levels of RBP4 and NT-proBNP. The present invention relates to a kit containing reagent means for:

[0026] The present invention also provides a method for treating a cancer cell line comprising administering to a patient a cancer-related disease selected from GFAP, RBP4, NT-proBNP, or a combination thereof. Kits containing reagents for detecting the levels of selected markers are disclosed.

[0027] In a further aspect, the present invention also provides a method for treating stroke for reperfusion therapy as defined in the first aspect. to select patients with ischemic stroke or to distinguish patients with ischemic from hemorrhagic stroke Immunoassays, protein transfer, chromatography, and other methods for identifying , consisting of mass spectrometry, turbidimetry, nephelometry, and polymerase chain reaction (PCR) Detect the presence of either RBP4 or NT-proBNP in a test sample selected from the group The purpose is to use the means to [Brief explanation of the drawings]

[0028] [Figure 1] In (A), the cutoff (horizontal black line) levels of NT-proBNP (y-axis in pg / mL) for 100% specificity for the two stroke subtypes are shown, and in (B), the cutoff (horizontal black line) levels of RBP4 (y-axis in μg / mL) are shown. In panel C, log10(NT-proBNP) and RBP4 levels are plotted simultaneously with the corresponding cutoffs previously determined for each protein. 100% specificity IS means that all patients with values ​​in that space on the graph (figure) had ischemic stroke. [Figure 2] In (A), cutoff levels of GFAP (log(GFAP)) >325 pg / ml are shown in different patient cohorts. In panel (B), log10(NT-proBNP) and RBP4 levels are plotted simultaneously with the corresponding cutoffs previously determined for each protein in this patient cohort. 100% specificity IS has the same meaning as in Figure 1. [Figure 3] This is a graph obtained from the analysis of data retrieved using a support vector machine procedure (SVM). Values ​​above the sigmoidal curve are 100% IS, and values ​​below the curve correspond to patients with either ICH or IS subtypes. Y-axis levels of NT-proBNP (pg / mL); X-axis levels of RBP4 (μg / mL). "Can be introduced into the support vector machine procedure with a Gaussian kernel." 100% specificity IS has the same meaning as in Figure 1. [Figure 4] Graph showing classification of subject cohort using logistic model scores including logarithmic transformation of GFAP (pg / ml), NT-proBNP (pg / ml) and diastolic blood pressure (mmHg) as significant predictors of ischemic stroke status. [Figure 5]This graph shows the classification of a subject cohort using a logistic model score including logarithmic transformation of GFAP (pg / ml), NT-proBNP (pg / ml) and diastolic blood pressure (mmHg) as significant predictors of ischemic stroke status, with marker levels determined within the first hour after stroke symptom onset. DETAILED DESCRIPTION OF THE INVENTION

[0029] Detailed Description of the Invention All terms used herein in this application are those known in the art unless otherwise specified. Certain terms used in this application are to be understood in their ordinary and prevailing sense. More specific definitions are provided below, and other explicitly stated definitions are broader. Unless definitions are provided, they are intended to be applied uniformly throughout the specification and claims. is doing.

[0030] As used herein, the term "patient" (or subject) refers to one of the following conditions typically associated with stroke: Any of the above signs or symptoms, such as sudden onset of facial weakness or arm unsteadiness, drift), abnormal speech and their combinations, e.g., FAST (face, arm, speech and facial hemiplegia and weakness, numbness, decreased sensation or vibration sensation, spasticity (tension) early relaxation (hypotonia) replaced by hypertonia, hyperreflexia, forced synkinesis, and especially If they appear on one side of the body, they may affect the sense of smell, taste, hearing or vision (total). or partial) changes in the eyelids (ptosis) and weakness of the eye muscles, hyporeflexia (e.g., pharyngeal , swallowing, and pupillary response to light), facial loss of sensation and weakness, balance problems, and Nystagmus, changes in breathing and heart rate, and sternocleidomastoid weakness, which can cause the head to turn to one side Below, tongue weakness (inability to protrude and / or move side to side), aphasia, dysarthria, apraxia visual field defects, memory loss, hemi-neglect, disorganized thinking, confusion, hypersexual behavior (hypers visual gesture), lack of insight, impairments commonly associated with stroke, gait pattern This refers to any subject who exhibits changes in motor coordination, dizziness, headaches and / or balance problems. As used herein, the term "patient" refers to any animal classified as a mammal, Non-limiting examples include domestic animals and livestock, primates and humans, e.g., humans, This includes primates other than the primate, cow, horse, pig, sheep, goat, dog, cat or rodent. Preferably, the patient is a human being, male or female, of any age or race. The patient has suffered a stroke.

[0031] As used herein, the term "selecting a patient for therapy" refers to treating a disease or , a patient for a therapy designed to alleviate symptoms associated with one or more diseases or conditions. In the particular case of stroke therapy, it concerns the identification of stroke-related symptoms, as well as Specifically, it may eliminate or delay symptoms associated with ischemic or hemorrhagic stroke. It is understood as any therapy that prevents or reduces the

[0032] The term "reperfusion therapy" refers to a procedure to restore blood flow through or around a blocked artery. Reperfusion therapy involves drugs and endovascular procedures. Drugs can help reduce blood clots. It is a thrombolytic (antithrombotic) and fibrinolytic agent used in a process called lysis The intervention performed may involve one or more of a stent retrieval device, aspiration techniques, or a stent retrieval device. To remove blood clots, an alternative device combining both suction and suction may be used. Other procedures that may be performed include minimally invasive endovascular procedures (thrombectomy) around the blockage. A more invasive bypass procedure involves grafting an artery. Aortic ablation is an interventional procedure to remove a blood clot (thrombus) from a blood vessel. This is typically done in the coronary arteries ( Interventional Cardiology), Peripheral Arteries (Interventional Radiology) (interventional neuroradiology) and cerebral arteries (interventional neuroradiology). The selection of patients is preferably based on the first method of the present invention. The term does not necessarily apply to 100% of the subjects studied. However, the term The selection of patients within the target population must be statistically significant. Significance can be determined using various well-known statistical evaluation tools, such as determining confidence intervals and p-values. The results can be determined by those skilled in the art using a standard deviation test, Student's t-test, Mann-Whitney test, etc. For more information, see Dowdy and Wearden, Statistical cs 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 It is less than that.

[0033] The term "ischemic stroke" (abbreviated IS) refers to a condition in which blood flow to an area of ​​the brain is physically blocked, This refers to the death of brain cells in that area. Ischemic stroke is divided into thrombotic stroke and embolic stroke. It can be further divided into: Thrombotic stroke occurs when a blood clot forms in the brain and blocks a cerebral artery. An embolic stroke occurs when an embolic bolus forms in a peripheral artery or in the heart and travels to the brain. It is caused by a blood clot that causes ischemia. Another type of ischemic stroke is a small stroke in the brain. It is a pit stroke caused by arterial blockage.

[0034] As used herein, the term "hemorrhagic stroke" (abbreviated as ICH for intracerebral hemorrhage) refers to a stroke caused by blood This refers to bleeding into brain tissue caused by a ruptured vessel.

[0035] The inventors of the present invention have developed a method for accurately selecting patients suffering from stroke for reperfusion therapy. We identified RBP4 and BNP as new plasma biomarkers for this purpose. These two markers can help differentiate acute IS from ICH. Several data analysis methods using Furthermore, when a third marker, specifically GFAP levels, was added, the sensitivity increased by 6. 0% increase while specificity was maintained.

[0036] Therefore, the first aspect of selecting a patient suffering from a stroke for reperfusion therapy is In certain embodiments, the method comprises determining the level of GFAP in an isolated sample from said patient. Further includes:

[0037] Although improved sensitivity is desirable, we have not yet determined whether RBP4 and NT-proBNP levels are sufficient to assess the sensitivity of the assay. A simplified kit containing only the means to detect the stroke was also developed. This simplified kit can be used in ambulances when IS and This allows for accurate identification of ICH and, if appropriate, re-injection as soon as possible. Patients are treated immediately with perfusion therapy (i.e., antithrombotic agents in the ambulance), resulting in fewer In ICH, even if blood pressure is optimal, it is possible to avoid poor outcomes. If this is done, the worsening of symptoms can be avoided.

[0038] In a still more particular embodiment of the first aspect, the method comprises determining the levels and for each protein: and comparing the results of the analysis with a corresponding reference value or reference range for the respective The range is selected from a value or range of values ​​obtained from subjects suffering from ischemic stroke, Elephants with IS had at least two elevated levels of RBP4 and NT-proBNP. Subjects were classified as candidates for reperfusion therapy if their values ​​were within or within a range of values ​​obtained from subjects with will be done.

[0039] In a more particular embodiment, the reference value or reference range is determined from a subject suffering from IS or A value or range of values ​​is selected from a value or range of values ​​obtained from subjects suffering from ICH, and the subjects Both RBP4 and NT-proBNP levels were significantly elevated in subjects with IS. If the patient's score is within the range of values ​​obtained, they are classified as a candidate for reperfusion therapy. More than 98% of both levels were within or within the range of values ​​obtained from subjects with Meaningful clinical sensitivity (approximately 21%) has been achieved for specificity, especially 100% specificity. can be.

[0040] In another particular embodiment of the first aspect, the method comprises: and optionally, the levels of GFAP and the corresponding reference values ​​for each protein. or a reference range, wherein the reference value or reference range is a value that indicates a risk of ischemic stroke. A value or range of values ​​is selected from values ​​or ranges of values ​​obtained from diseased subjects, the subjects At least RBP4 and NT-proBNP levels were associated with ischemic stroke If the value is within the range of values ​​obtained from the subject, Candidates for reperfusion therapy, and Modified Ranking Score (mRS) ) defined by a dependency rating of greater than 2 and determined within 1 to 5 months after stroke onset. The prognosis is poor and / or mortality is 20% to 30% within 3 months after onset. The patients are classified as having a prognosis defined by the rate.

[0041] In another more specific embodiment, the method comprises administering to a subject a therapeutically effective amount of RBP4, NT-proBNP, and GFA. and if the subject is classified as a candidate for reperfusion therapy, comparing the levels of P in the subject. is also defined by a dependency score greater than 2 according to the modified ranking score (mRS), 1 day after stroke ~ As having a prognosis determined within 5 months and / or 20% Classification as having a prognosis defined by a 3-month mortality rate of 30% will be done.

[0042] Prognosis was defined as a dependency of more than 2 according to the modified ranking score (mRS), The mortality rate is determined at least 3 months after stroke onset. In certain embodiments, the mortality rate is determined at least 3 months after stroke onset. is at least 23%. In another particular embodiment, it is 25%.

[0043] Only one of the RBP4 and NT-proBNP levels was elevated in patients with IS. In certain embodiments, within a value or range of values ​​obtained from a subject, the subject also has a reperfusion are classified as candidates for

[0044] In another particular embodiment of the first aspect, the in vitro method comprises: NP, and GFAP levels, if determined, along with the corresponding baseline values ​​for each protein. a cutoff value; If only RBP4 and NT-proBNP levels are determined, RBP4 and NT-proBNP levels simultaneously distinguish between patients with ischemic stroke and those with intracerebral hemorrhage. The corresponding reference cutoff value Ref1 for each of the proteins RBP4 and Ref1 N T-proBNP or greater indicates that the patient is a candidate for reperfusion therapy; or · If the levels of RBP4, NT-proBNP and additional GFAP are determined, In step 1, the GFAP level is the reference cutoff value Ref GFAP The second step is as follows: The levels of RBP4 and NT-proBNP were simultaneously compared with the corresponding reference cut-off values ​​Re f2 RBP4 and Ref2 NT-proBNP If so, the patient is a candidate for reperfusion therapy. The cutoff value was chosen as a complement to distinguish between patients with ischemic stroke and those with intracerebral hemorrhage. .

[0045] In fact, it includes the option to compare test levels with their respective cutoff values ​​or reference ranges. Different alternative embodiments of the method of the first aspect may involve determining specific values ​​of desired sensitivity and specificity. Therefore, 100% specificity (correct classification between two states) is If desired, the sensitivity (detection of one condition in a cohort of subjects with a different condition) may be reduced. On the other hand, decreasing the specificity (i.e., about 94% or 98%) reduces the sensitivity of the method. Therefore, the reference value can be increased to achieve the desired specificity and / or the desired sensitivity. You can change it accordingly.

[0046] Further specificity of this method involves comparison with two or three protein level cutoff values. In certain embodiments, if a subject is classified as a candidate for reperfusion therapy, the subject also receives a modified label. Dependence was defined as a level of dependence greater than 2 according to the modified rating scale (mRS) and was assessed by a mean of 1 year after stroke onset. As having a prognosis determined within ~5 months and / or consisting of 20%-30% Patients are classified as having a poor prognosis defined by the 3-month mortality rate after onset.

[0047] Also, the method of the first aspect for selecting a patient suffering from a stroke for reperfusion therapy. In another particular embodiment of the method, the levels of at least RBP4 and NT-proBNP are are both within or within a range of values ​​obtained from subjects with IS, Treating the patient with reperfusion therapy, or RBP4 and NT-proBNP and optionally, corresponding baseline cutoff values ​​of GFAP, the patient is considered a candidate for reperfusion therapy. The method further includes the step of classifying the sample by the number of samples.

[0048] As indicated above, several treatment protocols exist to promote reperfusion. In certain embodiments of the first aspect of the invention, reperfusion therapy includes antithrombotic therapy, thrombectomy, and combinations thereof.

[0049] In more particular embodiments, the antithrombotic agent is a thrombolytic agent. In this case, thrombolytic agents are plasminogen activators. More specifically, plasminogen The plasminogen activator is tissue plasminogen activator.

[0050] As used herein, the term "antithrombotic agent" refers to a drug that can reduce blood clot formation. Antithrombotic agents suitable for use in the present invention include, but are not limited to, thrombolytic agents, These include 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. It converts fibrinogen into fibrin and dissolves the clot. Fibrinolytic agents include reteplase (r-PA or Retavase), alteplase (t- PA or Activase), urokinase (Abbokinase), prourokinase Purified streptokinase activator complex (anisoylated purified streptokinase activator complete x) (APSAC), staphylokinase (Sak), tenecteplase (TNK-tP A), atenecteplase (TNKasa), Anist Eminase, streptokinase (Kabikinase, Streptases) or urokinase (uroquina Tenecteplase (TNK-tPA) is a rapid It can be administered as a rapid single bolus and can be used at ambulance level, In certain embodiments, TNK is effective one minute after administration (post-injection). The supplier of this product is Boehringer Ingelheim (European Union) n) and Genentech Inc (USA).

[0052] The term anticoagulant compound as used herein refers to compounds that prevent clotting, but is not limited to However, vitamin K antagonists (warfarin, acenocoumarol, phenprocoumon) (fenprocoumon and fenidione), heparin and heparin derivatives, e.g., low molecular weight heparin, factor Xa inhibitors, e.g., synthetic pentaerythritol Sugars, direct thrombin inhibitors (argatroban, lepirudin, bivalirudin, and ximera) It acts by inhibiting platelet aggregation and therefore thrombus formation, and Although not limited to, cyclooxygenase inhibitors (aspirin), adenosine diphosphate Acid receptor inhibitors (clopidrogrel and ticlopidine) , phosphodiesterase inhibitor (cilostazol), glycoprotein IIB / IIIA inhibitor drugs (Abciximab, Eptifibatide, Tirofiban and Def Antiplatelet drugs containing ribrotide and adenosine uptake inhibitors (dipyridamole) In a preferred embodiment, the antithrombotic agent is a thrombolytic agent. In an embodiment, the thrombolytic agent is a plasminogen activator. In its form, plasminogen activator is tPA (tissue plasminogen activator). is.

[0053] As used herein, the term "tissue plasminogen activator (t-PA)" refers to a plasminogen activator (tPA) that activates plasminogen A serine protease found on endothelial cells that catalyzes the conversion of plasminogen to plasmin The complete protein sequence of human t-PA is available in UniProt KB accession no. The tPA is a recombinant protein. It can be produced using biotechnology techniques, and the tP produced in this way A can be referred to as recombinant tissue plasminogen activator (rtPA). r-tPA includes the thrombolytic agents alteplase and reteplase. ase and tenecteplase (TNKase, also known as TNK-tPA, SEQ ID NO: 2) In human t-PA, the amino acids at positions 296 to 299 are lysine, histidine, and In TNK-tPA, these amino acids are replaced by four alanines. This mutation is responsible for the plasminogen activator inhibitor 1 (PAI-1) It is involved in increasing resistance to

[0054] t-PA should be administered within the first 3 hours of symptom onset or up to 4.5 hours after symptom onset. This should be done within 2 hours. Recommended total dose: 0.9 mg / kg (maximum dose is 90 mg) Infuse 0.09 mg / kg (0.9 mg / kg) over 60 minutes. 10% of the dose given as an intravenous bolus over 1 minute, followed by 0.81 mg / kg (90% of the 0.9 mg / kg dose) administered as a continuous infusion over 60 minutes. Heparin should not be initiated more than 24 hours after starting alteplase. t-PA is administered intravenously, or sometimes directly into an artery, to treat the initial stage of a stroke. The dose and route of administration should be administered as soon as possible after the onset of symptoms. It applies to any of the embodiments, and particularly to embodiments involving treating a patient. .

[0055] A single dose of TNK-tPA was administered to subjects who had suffered a stroke and were candidates for reperfusion therapy. As soon as possible after the decision is made and within 3 hours of the onset of symptoms or up to 4.5 hours after the onset of symptoms This should be done within 24 hours, preferably within 1 hour of the onset of the stroke.

[0056] As indicated above, the use of TNK-tPA is a specific treatment option as a rapid single-dose bolus. For those who require ambulance care, it takes effect within about one minute of administration and can be administered at any point of time. This is particularly useful because it can be administered in the clinic.

[0057] Patients who have suffered a stroke and are not selected for reperfusion therapy are considered in certain embodiments. In particular, the therapy is selected for the treatment of lowering blood pressure. This is done using drugs that can

[0058] "Blood pressure" as used herein refers to blood pressure at the site of central arteries such as the aorta and carotid arteries Central blood pressure is measured in the carotid or radial artery by applanation tonometry. It can be suitably measured non-invasively (as described below). "Blood pressure" referred to includes aortic blood pressure.

[0059] The "drug capable of lowering blood pressure" used in the present invention is a drug capable of lowering blood pressure by various means. It refers to any drug that reduces blood pressure. The most widely used drugs include thiazide diuretics [ furosemide, nitroprusside, hydralazine, etc.]; ACE inhibitors, calcium channel blockers (such as nicardipine or nimodipine); adrenergic receptor antagonists (alpha-adrenergic receptor antagonists) phosphate antagonists, urapidil, etc.), or a combination of alpha-blockers and beta-blockers (labetalol and and nitroglycerin); and angiotensin II receptor blockers (ARBs). Illustrative, non-limiting examples of agents that can lower or reduce blood pressure include alpha-methyldodecanoate, Aldomet, 11,17α-dimethoxy-18β-[(3,4,5-trimetho[ 2a-Yohimban-16β-carboxylate methyl ester Reserpine ester or 2-(2,6-dichlorophenylamino) 2- Imidazoline hydrochloride (clonidine hydrochloride), lergotrile, or i.e., European Patent No. 2-chloro-6-methylergoline-8β-aminobenzoates disclosed in US Pat. No. 5,074,444 Ischemic stroke, ischemic stroke treated with thrombolytic agents, and The reference values ​​used to lower blood pressure in hemorrhagic stroke are as follows: This is the standard value recommended by clinical practice guidelines. In patients with hemorrhage, the systolic blood pressure reaches 220-120 mmHg, and in patients with bleeding, the systolic blood pressure reaches 180-180 mmHg. If blood pressure reaches 100mmHg, the goal is to reduce blood pressure treatment. In a preferred embodiment, blood pressure is measured by intravenous administration of a drug capable of lowering blood pressure. It can be reduced by administration of acetaminophen and concomitant administration of oral antihypertensive drugs. To reduce blood pressure in ischemic stroke treated with thrombolytic agents or in hemorrhagic stroke The reference values ​​used for clinical practice guidelines are subject to change as these values ​​may be updated. This is the more recommended standard value.

[0060] Any suitable method for measuring arterial blood pressure may be used to determine whether the drug is capable of lowering blood pressure. It can be determined whether the drug can be administered and a decrease in arterial pressure is detected after administration of the drug. Illustrative, non-limiting examples of methods for measuring heart rate include, but are not limited to, palpitations, auscultation, and the like. There are non-invasive techniques such as oscillometry and continuous non-invasive blood pressure (CNAP).

[0061] As used herein, the term "reference value" refers to a value obtained from a sample collected from a subject or refers to a predetermined criterion used as a basis for evaluating the data. Reference levels can be absolute values; relative values; values ​​with upper or lower limits; a range of values; an average value; or a median value. The reference value may be a value relative to a specific control or baseline value, a mean value, or a reference value relative to a specific control or baseline value. The values ​​may be, for example, values ​​obtained from a sample obtained at a relatively early time point from the subject being tested. Reference values ​​may be derived from a population of subjects, such as a chronological age-matched group. The sample size can be based on a large number of samples or on a pool of samples that includes or excludes the sample being tested. Reference values ​​for the biomarkers of the present invention have been determined. The reference values ​​for each of NP and GFAP are the lower and upper limits, as disclosed in the following examples. The range of values ​​(protein levels) for each biomarker can be from the upper and lower limits. and specific combinations of different biomarker values ​​allow accurate targeting with high sensitivity and specificity. It is classified as follows.

[0062] Biomarkers (in the present invention, any of NT-proBNP, RBP4, and GFAP) ) levels are at least 1.5%, at least 2%, at least 5%, or 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 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 more higher than the reference value. It is considered to be high.

[0063] Similarly, in the context of the present invention, the level of a biomarker refers to the level of said biomarker in a sample. The level of a biomarker is lower than the reference level. 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%, less 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 at least 95%, at least 100%, at least 110%, at least 120%, at least If the It is considered to be lower than the standard value.

[0064] In certain embodiments of the first aspect, RBP4 and NT-proBNP in a biological fluid sample are Only the level of RBP4 oh and Ref1 NT-proBNP When compared with these reference cutoffs, The values ​​were for RBP4 52 μg / ml and NT-proBNP 4062 pg / ml. These particular reference cutoff values ​​are, in certain embodiments, based on the isolated plasma samples. For samples and when assayed by enzyme-linked immunosorbent assay (ELISA) This is what we do.

[0065] In another particular embodiment of the first aspect, RBP4, NT-proBNP and GFAP The level of α-glucan is determined and the corresponding reference cut-off value (referred to herein as Ref2 RBP4 ,Re f2 NT-proBNP and Ref GFAP When compared with The reference cutoff values ​​were RBP4 38 μg / ml, BNP1 305 pg / ml, and These specific reference cutoff values ​​are for GFAP 0.325ng / ml. In a more specific embodiment, the method is for an isolated plasma sample, and GFAP is detected. When assayed, the levels of this marker are relatively sensitive (picomolar level) single-molecule assays. determined by single molecular assay (SIMOA) others are determined by ELISA assay. In this example, a particular method is carried out in two distinct steps or conditions. In the first step, the GFA The level of P is determined and it is the reference cut-off value Ref GFAP If the patient: It is believed that the brain is affected by ischemic stroke, and in a second step, RBP4 and NT-p The roBNP level was simultaneously compared with the corresponding reference cutoff value Ref2 RBP4 and Ref 2 NT-proBNP If so, they are considered candidates for reperfusion.

[0066] As shown by the example, ICH patients had higher GFAP levels and RB than IS patients. P4 and NT-proBNP were low. RBP4>52μg / mL and GFAP>0. In combination with 18 ng / mL, accurate diagnosis of IS was achieved in 6.5% of cases and ICH in 34.3% of cases. The addition of NT-proBNP resulted in a significant improvement in RBP4 > 52 μg / mL and BNP clusters. By using a cutoff >4060 pg / mL, 100% specificity for IS was ensured. The sensitivity improved by up to 20% (31 / 155).

[0067] As noted above, baseline values ​​may change depending on the exclusion criteria determined in the clinical protocol. In addition, depending on the variables that are considered to be diagnostic, The value of β-glucan is also important in stroke, as it increases sensitivity while maintaining specificity. can be adjusted to

[0068] Furthermore, the appropriate classification of patients according to the levels of detected proteins can be determined using computational methods. This can be done by calculating the determined value of the protein with a formula that gives a predictor. and the predictor is calculated based on the expression level of a protein, the expression level being The correction is made by a specific coefficient. Other calculation methods (such as those exemplified in this specification, In some methods, such as the so-called machine method, the levels of all determined proteins are considered. This allows functions to be developed that appropriately classify patients taking into account the above.

[0069] In certain embodiments, these reference values ​​are all based on the individual stroke subjects. The results were determined for isolated plasma samples. We know how to find their counterparts in liquids.

[0070] The term "sample" as used herein refers to any sample that can be obtained from a patient. The methods of the present invention can be used with any type of biological sample obtained from a patient, for example, a biopsy sample, Tissues, cells, or biological fluids (plasma, serum, saliva, semen, sputum, cerebrospinal fluid (CSF), tears, mucus) , sweat, milk, brain extract, etc.

[0071] Thus, optionally in combination with any of the above or below embodiments, another particular In this embodiment, the isolated sample from the subject (i.e., the patient suffering from stroke) is a biological fluid. Exemplary, 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, a sample is obtained at baseline.

[0073] Different samples can be used to determine the levels of different markers. It is not necessary that the levels of all markers measured by the methods of the invention be measured on the same type of sample. Therefore, in another preferred embodiment, RBP4, NT-proBNP and G In another preferred embodiment, the levels of FAP are measured in serum. is measured against

[0074] As used herein, "baseline" refers to the time from the onset of symptoms until the patient is first seen. This is usually within the first few hours after a stroke and is usually considered at any time after rescue In a preferred embodiment, the baseline is the initial Within the first 4.5 hours of symptom onset or less than 6 hours after stroke, or another preferred method In embodiments, less than 24 hours from symptom onset.

[0075] In another particular embodiment of this aspect, RBP4 and NT-proBNP, and If so, the step of determining the level of GFAP is performed within the first two hours after stroke onset. As shown in the example below, the earlier the determination of the marker in the isolated sample is performed, the more efficient the method. In another specific embodiment of this aspect, RBP4 and NT- Determining the levels of proBNP, as well as GFAP if determined, may also be used to assess stroke onset. This will be done within the first hour after the event.

[0076] Another aspect of the first embodiment of the method for selecting a patient suffering from a stroke for reperfusion therapy. In this embodiment, it further comprises determining one or more clinical parameters. Thus, the methods of the present invention involve combining the levels of RBP4 and BNP with one or more clinical parameters. This includes determining:

[0077] As used herein, the term "clinical parameters" or clinical data refers to an individual's background factors (e.g., age or date of birth, race and / or ethnicity), the patient's clinical symptoms, or stroke-related This term refers to symptoms related to a disease / condition, such as d-dimer or blood glucose determinations. The test parameters are also included.

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

[0079] In another particular embodiment of the first aspect, the in vitro method comprises measuring systolic blood pressure and / or Blood pressure (including diastolic blood pressure), blood glucose, age, and scores from the Systematic Assessment Tool for Stroke-Related Neurological Disability clinical characteristics selected from the group consisting of, for example, NIHSS score, gender, and combinations thereof The values ​​of any of these parameters may be determined for a particular implementation. In an embodiment, a suitable algorithm is used to accurately classify patients as candidates for reperfusion therapy. In the rhythm, in combination with the levels of RBP4, NT-proBNP, and possibly GFAP, For example, in combination with specific levels of two or three proteins. The blood pressure within a specific range is used in the determination protocol for accurate classification. In certain embodiments, the values ​​are introduced into the equation of the regression model to allow such classification. The score or final value is given.

[0080] Hypertension, sometimes called arterial hypertension, is a chronic condition in which blood pressure in the arteries is elevated. It should be understood as a medical condition. Normal resting blood pressure is a systolic blood pressure of 100-140 mmHg. Hg (upper measurement), and diastolic blood pressure ranges from 60 to 90 mmHg (lower measurement). If your blood pressure is consistently above 140 / 90mmHg, you are said to have high blood pressure. ischemic stroke, ischemic stroke treated with thrombolytic agents, or hemorrhagic stroke In the future, the reference values ​​used to lower blood pressure may be subject to change. Therefore, it is the standard value recommended by clinical practice guidelines, and today, Systolic blood pressure is 220-120 mmHg in patients with hemorrhage and 180-100 mmHg in patients with bleeding. g, which is the accepted benchmark for suitable treatment to lower blood pressure. .

[0081] The term "Stroke-related Neurological Deficits" refers to the most frequent neurological deficits seen after stroke. It concerns tools designed to measure and assess harm: level of consciousness, visual field, facial muscle strength Decreased motor skills, gaze, sensory impairment, coordination (ataxia), language (aphasia), speech (construction) Several aspects or parameters are assessed, including: A value is given, which is 0 if normal. Therefore, most of these tools The higher the core, the worse the neuropathy. National Institutes of Health Stroke Scale (NIHSS) score, brain Rapid Arterial Occlusion Evaluation for Stroke Scale (RACE), Cincinnati Prehospital Stroke Scale Compared to Stroke Severity Tools for Large Vessel Occlusion Stroke Predi ction (Cincinnati Score), Los Angeles Motor S scale (LAMS), or modified Rankin Scale or Score (mR As a S), I know that there are various tools for this purpose. Both are designed to provide a rapid, standardized assessment of early neurological function after stroke. The modified Rankin Scale or Score (mRS) is a measure of the patient's condition after a stroke. It is also a scale for assessing the degree of disability. It is mainly used at the time of discharge and three months after the onset of the condition. Applies at the time.

[0082] In another preferred embodiment, the clinical parameters include age, NIHSS score, sex, contractility, The term "NIHSS score" as used herein is selected from the group consisting of NIHSS score, NIHSS score (NIHSS score 0.01), ... and combinations thereof. NatioA is a systematic assessment tool that provides quantitative measurements of stroke-related neurological impairment. nal Institutes of Health Stroke Scale(NI Refers to the HSS score (Adams HP Jr Neurology. 1999 Ju l 13;53(1):126-31). The NIHSS was originally a clinical trial for acute stroke. It was designed as a research tool to measure baseline data for patients in This scale is used to assess the acuity of stroke patients, determine appropriate treatment, and predict patient outcomes. The NIHSS is also widely used as a clinical assessment tool to measure consciousness, language, and hemispatial function. Levels of neglect, visual field defects, extraocular movements, motor strength, ataxia, dysarthria and sensory loss Stroke Schedule: A 15-item neurological examination used to assess the impact of acute cerebral infarction on Trained observers answer questions and assess the patient's ability to perform activities. Items are scored on a 3-5 scale, with 0 representing normal and 0 representing untestable items. Stroke severity as measured by the NIH Stroke Scale scoring system Levels: 0 = no stroke, 1-4 = mild stroke, 5-15 = moderate stroke, 15-2 0 = moderate / severe stroke, 21-42 = severe stroke. "Score" refers to a score of 5 to 42 on the NIH stroke scale scoring system. vinegar.

[0083] Also, Rapid Arterial Occlusion Evaluator for Stroke ation scale (RACE), or to identify ischemic stroke with large vessel occlusion Variations of the NIHSS, such as other scores used in the NIHSS, may also be used.

[0084] Furthermore, in another particular embodiment of the first aspect, optionally, the above or below embodiments In combination with any of the above conditions, if the subject is classified as a candidate for reperfusion therapy, the subject will He was also diagnosed with large vessel occlusion.

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

[0086] In certain embodiments of the second aspect, the method comprises determining the level of GFAP.

[0087] In another more particular embodiment of the second aspect, the method comprises: and comparing the level of GFAP, if determined, with a corresponding reference value. Including, If only the levels of RBP4 and BNP are determined, At the same time, the corresponding reference value Ref1 RBP4 and Ref1 NT-proBNP than A high level indicates that the patient has IS, or If the levels of RBP4, BNP and GFAP are determined, At the same time, the corresponding reference value Ref2 RBP4 and Ref2 NT-proBNP GFAP levels are higher than the reference value RefGFAP The lower the mean, the more likely the patient is to have IS. Indicates that the person is a

[0088] In another particular embodiment of the second aspect, RBP4 and BNP, and optionally G If a subject is classified as having an ischemic stroke when determining the level of FAP, the subject also Stroke incidence was defined by a dependency score of more than 2 according to the modified ranking score (mRS). with a prognosis determined within 1-5 months after onset and / or 20%-30% Patients are classified as having a prognosis defined by the 3-month mortality rate after onset, which is composed of:

[0089] In another particular embodiment of the second aspect, it is As shown in , further comprising the step of selecting a therapy, particularly a reperfusion therapy.

[0090] Thus, after a differential diagnosis has been achieved, in another particular embodiment of the second aspect, , recommend reperfusion therapy for patients diagnosed with IS and / or primarily use antithrombotic or hemostatic agents. The method further includes treating the patient diagnosed with IS by thrombus removal reperfusion therapy. Alternatively, patients diagnosed with ICH who should avoid reperfusion therapy to avoid fatal outcomes should be included. In another particular embodiment, the subject is recommended or receives a therapy to reduce or optimize blood pressure. This is used for treatment.

[0091] This particular embodiment is envisioned as a method of treating a patient who has suffered a stroke. and the method may, according to a second aspect, include an index for distinguishing IS from ICH in a patient. In vitro methods and reperfusion therapy, primarily with antithrombotic agents or thrombectomy, are used. , treating patients diagnosed with IS or with therapies that lower or optimize blood pressure. Advantageously, this method involves treating a patient diagnosed with ICH with Patients should be treated with the most appropriate treatment regimen within the first few hours of symptom onset. or treatment is recommended.

[0092] Any specific embodiments disclosed above for the first aspect also apply to this second aspect. In particular, their preferred reference values, the type of isolation sample, and one or more clinical parameters Therefore, in another particular embodiment of the second aspect, In vitro methods include blood pressure, including systolic and / or diastolic blood pressure, blood glucose, age, and systematic Scores from assessment tools for stroke-related neurological disorders, e.g., NIHSS score, gender and other The method further comprises determining a clinical parameter selected from the group consisting of any combination thereof. Similarly, the values ​​of any of these parameters may be used in certain embodiments to determine whether the patient is receiving reperfusion therapy. In an appropriate algorithm, RBP4, NT - used in combination with proBNP and, optionally, GFAP levels. Blood pressure within a certain range, combined with specific levels of two or three proteins, is positive. In another more particular embodiment, the value is used in a decision protocol for accurate classification. introduced into the regression model equation to give a score or final value that allows such classification .

[0093] In another particular embodiment of the second aspect, RBP4 and NT-proBNP, etc. Furthermore, determining the level of GFAP is important for the first 2 hours after stroke onset. More specifically, within the first hour. This is shown for the first aspect. As mentioned above, this represents an advantage in increasing the sensitivity of the method.

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

[0095] The present invention also provides a method for determining the level of GFAP in subjects who have suffered a stroke. 1. A method for detecting levels of RBP4 and NT-proBPN in an isolated elephant sample, comprising: (a) obtaining a sample from a subject; and (b)(i) contacting the sample with a means capable of binding to the corresponding expressed protein; (ii) translating the polypeptide into one or more of the corresponding proteins and detecting said binding; contacting the sample with a means capable of binding to the corresponding RNA to be translated, and By detecting one or more of the proteins, it is possible to determine whether they are present in the isolated sample. Generally, this term encompasses the act of releasing information.

[0096] The term "distinguish" as used herein with respect to the second aspect refers to the determination of different states. As will be appreciated by those skilled in the art, differentiation may be, but is preferably, not diagnostic. It does not have to be accurate for 100% of the subjects being evaluated. A statistically significant portion of elephants are likely to have one of two types of stroke. Whether a subject is statistically significant or not can be determined by various well-known statistical methods. Statistical evaluation tools, e.g., confidence interval determination, p-value determination, Student's t-test, Mann-Heinz test This can be determined without further ado by those skilled in the art using a Totney test or the like. 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%, and at least 70%. , at least 80%, at least 90% or at least 95%. The range is usually 0.05, 0.01, 0.005 or less.

[0097] The biomarkers identified in this study make it possible to distinguish IS from ICH in patients. Therefore, different therapies are used for these two types of patients (for patients with ischemic stroke, Antithrombotic drugs, and drugs that can lower blood pressure in patients who have had a hemorrhagic stroke Considering that the application of ology.2014 Sep 19), the present invention also relates to a method for treating stroke patients. The present invention provides the first method for selecting a therapy for a cancer. are conceptually related.

[0098] In the present invention, when RBP4 is mentioned, it belongs to the lipocalin family and is present in blood. It refers to retinol-binding protein 4, a specific carrier of retinol, in plasma. The complete sequence of IL-binding protein 4 is available under UniProtKB accession number P0275 3 (August 8, 2013), having SEQ ID NO:3.

[0099] As used herein, the term "GFAP" refers to a protein expressed by multiple cell types in the central nervous system. It refers to glial fibrillary acidic protein, an intermediate filament protein that is found in glial fibrillary fibers. The complete human sequence of fibrous acidic protein is available at UniProtKB accession number P14 136 (August 8, 2013), having SEQ ID NO:4.

[0100] 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 produces the NT-proBNP fragment and the active B-type NADH. BNP is stimulated by an increase in ventricular blood volume. BN is a hormone secreted by ventricular myocytes in response to induced stretch. The complete human sequence of P is available at UniProt KB accession number P16860 (199 August 1, 2000 - sequence version 1, and database release May 8, 2019 187).

[0101] All of these proteins have been shown to be homologous to other mammalian species (cats, dogs, mice, rats, etc.). The skilled person can search for the corresponding complete sequence in public databases. It is possible.

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

[0103] As a non-limiting example, the expression level may be determined by measuring the level of mRNA encoded by the gene. The latter can be determined by conventional methods, e.g., amplification of mRNA, and using methods including quantification of the mRNA amplification products, such as electrophoresis and staining; Alternatively, by Northern blot and the use of suitable probes, Northern blot and specific probes for the mRNA of the gene of interest, or their corresponding cDNA Use of cRNA-specific probes, mapping with SI nuclease, and RT-PCR , hybridization, microarrays, etc. At the level of cDNA / cRNA corresponding to the above mRNA encoded by the marker gene, The amount of iodine can also be determined by using conventional techniques. The method involves reverse transcription (RT) of the corresponding mRNA to synthesize the corresponding cDNA, followed by the above-mentioned It involves the synthesis (RNA polymerase) and amplification of cRNA complementary to the cDNA. Conventional methods for quantifying current levels can be found in laboratory manuals.

[0104] To normalize mRNA expression values ​​between different samples, the expression level of the mRNA of interest in the test sample was calculated. The expression level can be compared to the expression of a control RNA. A "control RNA" is an RNA whose expression level does not change or changes only in limited amounts. Preferably, the control RNA is derived from a housekeeping gene and is constitutively expressed. It is mRNA that encodes proteins that perform essential cellular functions. Preferred housekeeping genes for ribosomal protein synthesis include 18-S ribosomal protein, β-2 -Microglobulin, ubiquitin, cyclophilin, GAPDH, PSMB4, tubules These include phospholipid and β-actin.

[0105] Alternatively, increased gene expression may increase the amount of the corresponding protein, and gene expression When the amount of the corresponding protein decreases, the amount of the protein encoded by the gene decreases. Determining the expression level of the marker gene by determining the expression level of the protein It is also possible.

[0106] Protein expression levels can be determined using immunoassays, bioluminescence, fluorescence, chemiluminescence, electrophoresis, and other methods. and quantitative analysis by qualitative and / or quantitative tests selected from the group consisting of chemistry and mass spectrometry. To make the determination of the marker level easier and faster, Conducted in a point of care test format (POCT) In certain embodiments, point-of-care testing is recommended. The presence (or absence) of target analytes in a liquid sample (matrix) can be detected without the need for expensive equipment. lateral flow tests that allow the detection of the presence or absence of There are many laboratory-based applications that can be assisted.

[0107] As an example, specific POCTs have been developed and tested in ambulances and helicopters. This POCT demonstrated a useful high sensitivity rate with 100% specificity for ischemic stroke. This can be achieved in selected cases much more quickly than using standard techniques. It became possible to start prehospital reperfusion therapy.

[0108] Regardless of the test format, certain quantitative tests are available using immunoassays, bioluminescence, fluorescence, and chemiluminescence. The method is selected from the group consisting of optical, electrochemical and mass spectrometry.

[0109] In one embodiment, the expression level is measured using immunological techniques, such as enzyme-linked immunosorbent assay (ELISA). LISA), enzyme immunoassay, 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 radioimmunoassays, and Protein microarray formats, e.g., single molecule assay (SIMOA), Western Determined by blot or immunofluorescence.

[0110] Western blots are prepared by first separating the proteins by gel electrophoresis under denaturing conditions and then running them on a membrane. incubation with an antibody specific for the protein immobilized on the It is based on the detection of nitrocellulose by a development system (e.g., chemiluminescence). Optical analysis requires the use of antibodies specific to the target protein for expression analysis. ELISA is based on the use of antigens or antibodies labeled with enzymes. Therefore, the conjugate formed between the target antigen and the labeled antibody is enzymatically active. A complex is formed when one of the components (antigen or labeled antibody) is immobilized on a support. Therefore, the antibody-antigen complex is immobilized on the support and can therefore be detected by, for example, spectrophotometry. , to products detectable by fluorometry, mass spectrometry or tandem mass tagging (TMT). By adding a substrate that is converted by the enzyme, the antibody-antigen complex can be detected. SIMOA is a single molecule array (SIMOA) that can isolate and detect single enzyme molecules. It uses an array of femtoliter-sized reaction chambers called moa™ Therefore, it is a type of assay that is more sensitive than ELISA. The volume of the array is Approximately 2 billion times less than ISA, so in the presence of labeled proteins, Rapid accumulation occurs. When diffusion is blocked, this high local concentration of product can be easily observed. Only one molecule is needed to reach the limit of detection. The same reagents as in conventional ELISA are used. This method was used to measure a variety of different matrix metalloproteinases at femtomolar (fg / mL) concentrations. When proteins in fluids (serum, plasma, cerebrospinal fluid, urine, cell extracts, etc.) were measured, , the sensitivity was improved by about 1000 times.

[0111] On the other hand, determination of protein expression levels is performed using tissue microarrays containing aggregated subject samples. A TMA was constructed and protein expression levels determined by techniques well known in the art. This can be done by specifying

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

[0113] When immunological methods are used, a protein known to bind with high affinity to the target protein is Any antibody or reagent available for detecting the amount of target protein can be used. Nevertheless, antibodies, e.g., polyclonal sera, hybridoma supernatants, or monoclonal antibodies, may be used. Clonal antibodies, antibody fragments, Fv, Fab, Fab' and F(ab')2, ScFv The use of antibodies, diabodies, triabodies, tetrabodies as well as humanized antibodies is preferred.

[0114] As previously cited, NT-proBNP and / or RBP4 and / or GFA The expression level of P can be measured by the level of the protein and its variants, e.g., the fragment Determined by measuring both the nucleotides, isoforms, analogs and / or derivatives It is possible.

[0115] The term "functionally equivalent variant" refers to a variant of a gene that is a member of a family of genes, wherever the function of said variant is substantially maintained. , modifications, insertions and / or deletions that alter NT-proBNP and / or RBP4 and and / or any protein derived from the sequence of GFAP, or one or more amino acids thereof. Preferably, NT-proBNP and / or RBP4 and and / or variants of GFAP, such as (i) conserved or non-conserved amino acid residues; one or more amino acid residues are replaced by a group (preferably a conserved amino acid residue) Such substituted amino acids may or may not be encoded by the genetic code. (ii) a polypeptide that may contain one or more modified amino acid residues, e.g., a substituent group bond (iii) polypeptides in which modified residues are present due to alternative processing of similar mRNAs; (iv) a polypeptide resulting from the synthesis; (v) a polypeptide fragment; and / or ) Polypeptides resulting from the fusion of NT-proBNP and / or RBP4 and / or GFAP or other polypeptides, such as secretory leader sequences, or purification (e.g. , His tag) or detection (e.g., Sv5 epitope tag). A fragment is a polypeptide as defined in (i) to (iii) of the original sequence. Polypeptides generated by proteolytic cleavage (including multisite proteolysis) The variants may be post-translationally modified or chemically modified. Such variants include , should be apparent to one skilled in the art.

[0116] As is known in the art, "similarity" between two proteins is a measure of the similarity between two proteins. The amino acid sequence of a protein and its conserved amino acid substitutes, and the sequence of a second protein A variant is a polypeptide sequence that differs from the original sequence, preferably Preferably, the polypeptides differ from the original sequence by fewer than 40% of the residues per relevant segment. and more preferably, less than 25% of the residues per related segment are identical to the original sequence. Different polypeptide sequences, more preferably 10% of the residues per related segment A polypeptide sequence that differs from the original sequence by less than one segment per A polypeptide sequence that differs from the original sequence by only a few residues, and at the same time, is sufficiently similar to the original sequence. is defined to include polypeptide sequences that are homologous to and retain the functionality of the original sequence. The variants according to the present invention have at least 60%, 65%, 70%, 72% or more amino acid sequence identical to the original. , 74%, 76%, 78%, 80%, 90% or 95% similar or identical The degree of identity between two proteins can be determined using control sequences well known to those skilled in the art. The identity between two amino acid sequences is determined using computer algorithms and methods. is preferably determined using the BLASTP algorithm [BLASTManu al,Altschul,S.,et al,NCBI NLM NIH Bethes da,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 schizophenone-3, schizophenone-4, schizophenone-5, schizophenone-6, schizophenone-7, schizophenone-8, schizophenone-9, schizophenone-10, schizophenone-11, schizophenone-12, schizophenone- Signal peptide cleavage, glycosylation, acetylation, isoprenylation, proteolysis These include stoylation, protein folding and proteolytic processing. Additionally, proteins can incorporate unnatural amino acids either post-translationally or during translation. The amino acid sequence may include unnatural amino acids formed by the introduction of amino acids.

[0118] Another aspect of the in vitro method of the present invention provides a differential diagnosis and information for selecting a therapy. In certain embodiments, they comprise steps of (i) collecting diagnostic information, and (ii) analyzing the information. The step of storing the data on a data carrier is further included.

[0119] In the sense of the present invention, a "data carrier" is a device for the differential diagnosis of IS and ICH and and / or any means containing meaningful information data for the selection of candidates for reperfusion therapy, e.g. For example, it should be understood as a paper. The carrier also provides a differential diagnosis for selecting a therapy. It can be any entity or device capable of carrying data or information. The rear is a storage medium such as a ROM, for example, a CD ROM or a semiconductor ROM, or a magnetic The recording medium may include a magnetic recording medium, such as a floppy disk or a hard disk. The carrier may be a transmissible carrier such as an electrical or optical signal; Optical signals may be transmitted via electrical or optical cables, or by radio or other means. Diagnostic / therapy selection data may be transmitted by cable or other device or means. When embodied in a signal capable of being transmitted directly by such cable or It may be constituted by other devices or means. Other carriers include USB devices and computers. Examples of suitable data carriers include paper, CD, USB, PC There is a computer archive in the UK, or an audio registration using the same information.

[0120] The present invention also provides an in vitro method for the prognosis of a patient suffering from ischemic stroke. The levels of retinol-binding protein-4 (RBP4) in isolated samples from the above patients and type B Determine the levels of N-terminal fragment of natriuretic peptide (NT-proBNP) The present invention also encompasses in vitro methods comprising:

[0121] In certain embodiments of the in vitro method for prognosis, at least RBP4 or two The protein level is compared to a reference value, which indicates whether the subject has suffered an ischemic stroke. In another more particular embodiment, the value or range of values ​​is selected from a range of values ​​that indicates or confirms that the In this study, prognosis was defined by a dependency score of more than 2 according to the modified ranking score (mRS). determined within 1-5 months after stroke onset and / or comprised 20%-30% It is defined by the 3-month mortality rate after onset.

[0122] As previously indicated, the present invention also detects levels of RBP4 and NT-proBNP. The present invention relates to a kit containing reagent means for:

[0123] In certain embodiments of the kits of the present invention, they comprise a marker for detecting the level of GFAP. Further comprising a reagent means.

[0124] As used herein, the term "kit" refers to a device or kits that are suitable for transport and storage thereof. A packaged product containing the various reagents (or reagent means) necessary to carry out the method of the present invention. Suitable materials for packaging the components of the kit include crystal, plastic (e.g. , polyethylene, polypropylene, polycarbonate), bottles, vials, paper or seals Includes barrel.

[0125] Furthermore, the kits of the present invention allow the various components contained in the kit to be simultaneously, sequentially, or The instructions may be stored, for example, on an electronic storage medium. on a hard disk (e.g., magnetic disk, tape), or optical medium (e.g., CD-ROM, DVD) ) or audio material, or other instructions that are easy to read or understand, Additionally or alternatively, the information may be in the form of a printed matter or an electronic support. The media may include an internet address providing the instructions.

[0126] The reagent means (or simply, reagent) of the kit comprises a compound that specifically binds to the 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. Drug means are directed against proteins of interest (i.e., NT-proBNP, RBP4, and The antibodies in the kit of the present invention are one or more antibodies that specifically recognize GFAP (GFAP in the case of GFAP). , e.g., flow cytometry, Western blot, ELISA, RIA, competitive ELISA A, DAS-ELISA, a technology based on the use of biochips, protein microarrays or assays of colloidal precipitation within reactive strips to determine protein expression levels. The method can be used in accordance with techniques known in the art for determining the activity of the antibody.

[0128] The antibody may be immobilized on a solid support such as a membrane, plastic, or glass, and in some cases The solid support may be treated to facilitate immobilization of the antibody to the support. At least, it specifically recognizes a marker (i.e., a protein of interest) and It includes a set of antibodies that can be used to detect expression levels.

[0129] Furthermore, the kit of the present invention can be used to detect proteins encoded by the constituent genes. The availability of these additional reagents may allow for the differential expression of biomarkers to be compared with the relative expression of This is due to different amounts of total protein in the samples rather than actual differences in relative levels. For different samples (e.g., the sample to be analyzed and the control sample) to exclude In the present invention, a constitutive gene is a gene that is constantly active. proteins that are constantly transcribed, constitutively expressed, and perform essential cellular functions It is a gene that encodes a protein that is constitutively expressed and can be used in the present invention. Examples include, but are not limited to, beta-2-microglobulin (B2M), ubiquitin, 18 -S ribosomal proteins, cyclophilin, GAPDH, PSMB4, tubulin and and actin.

[0130] In a preferred embodiment, the reagent means for assaying the levels of the various biomarkers comprises , at least 10% of the total amount of reagents for assaying the biomarkers forming the kit; At least 20%, at least 30%, at least 40%, at least 50%, at least At least 60%, at least 70%, at least 80%, at least 90% or at least 1 00%. Therefore, RBP4, NT-proBNP and sometimes GFPA In the particular case of a kit comprising reagents for assaying the levels of the biomarkers described above, Specific reagents (i.e., specific for RBP4, NT-proBNP, and possibly GFPA) The antibody (antibody that binds heterologously) should be at least 10%, at least 20% of the antibody present in the kit. , at least 30%, at least 40%, at least 50%, at least 60%, less Both account for 70%, at least 80%, at least 90% or at least 100%. Therefore, these kits mainly detect the levels of two (or three) proteins. It is a simplified kit containing reagent means for:

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

[0132] In another particular embodiment, the kit according to the invention is adapted to deposit a biological fluid sample, in particular whole blood. and one or more sample inlet ports; specific for a marker protein, in particular an antibody a reaction area containing a binding means / reagent, and a sample inlet port connected to the reaction area. In another more specific embodiment, the kit comprises a marker to be detected (one, two or 3) and corresponding reaction areas connected thereto. In an embodiment, the kit comprises one single capillary track connecting to the same number of reaction areas. The capillary tracks each include one inlet port, and each is connected to a corresponding one of the sample ports. A kit containing multiple reaction regions is a multiplex kit.

[0133] In another aspect, the present invention provides a method for distinguishing IS from ICH or for treating stroke. and the use of the kit of the present invention for selecting a patient undergoing reperfusion therapy, said patient undergoing reperfusion therapy. Therapy, in certain embodiments, comprises antithrombotic therapy, thrombectomy, and combinations thereof. is selected from the group consisting of:

[0134] Thus, in certain embodiments, the present invention provides a method for treating a pulmonary arthritis with a pulmonary arthritis virus (PAS) virus in a patient receiving the treatment of a pulmonary arthritis ... Regarding the use of the kit.

[0135] As shown in the examples below, the inventors have surprisingly found that RBP4 and NT-pro Levels of BNP, and optionally GFAP and / or certain additional clinical parameters By determining the combination of the drugs, patients with large vessel occlusion (LVO) and those who have suffered a stroke can be LVO is a previously noted condition in patients with ischemic stroke. These findings may be partly responsible for the poor outcome or poor prognosis associated with cerebrovascular disease.

[0136] Therefore, in relation to this selection and accurate classification of patients suffering from stroke, the present invention The disclosure also provides an in vitro method for diagnosing LVO, comprising: detecting RBP4 in an isolated sample from a subject; and an in vitro method comprising determining the level of NT-proBNP. In one embodiment, the subject is an ischemic stroke patient.

[0137] In certain embodiments of the in vitro method for the diagnosis of LVO, it involves measuring the level of GFAP. In another more specific embodiment, the method further comprises determining the RBP4, N Compare levels of T-proBNP, and GFAP, if determined, with corresponding baseline values The method further comprises a step of measuring the reference value or the reference range obtained from a subject suffering from LVO. and the subject has at least one of RBP4, NT-proBN, The levels of both P and one of GFAP were obtained from subjects suffering from LVO. If the value is within the range of values ​​specified, LVO is diagnosed. In a more particular embodiment of the in vitro method for, it comprises measuring one or more clinical parameters. These clinical parameters include, inter alia, determining systolic blood pressure and / or blood pressure including diastolic blood pressure, blood glucose, blood d-dimer level, age, stroke-related neurological deficits The score is selected from the group consisting of a score from a systematic assessment tool, gender, and combinations thereof. In a more particular embodiment, the in vitro method for diagnosing LVO comprises detecting RB in an isolated sample. P4, NT-proBNP and GFAP levels, blood glucose, d-dimer, and further diastolic blood pressure, and baseline scores from the Systematic Assessment Tool for Stroke-Related Neurological Disability For example, to determine NIHSS score, RACE, Cincinnati, LAMS, etc. As with other aspects and embodiments, the isolated sample is preferably a biological fluid. , more specifically selected from plasma and serum. As disclosed, the values ​​of the clinical parameters accurately classify patients as suffering from LVO. A suitable algorithm for this would include RBP4, NT-proBNP, and possibly G Other in vitro methods for diagnosing LVO may be used in combination with FAP levels. In certain embodiments, RBP4 and NT-proBNP, and if determined, G Determining the level of FAP is important within the first 2 hours after stroke onset, and more specifically It is performed within the first hour after the stroke occurs.

[0138] According to the inventors' findings, a combination of markers in serum and / or plasma may be a positive indicator of LVO. This is the first time that reliable information for accurate diagnosis has been provided (high incidence in IS patients). This is the reason why they are not widely implemented in clinical practice today. , which is far from a 100% sensitivity value and does not have adequate accuracy. Since floor protocols are used, we envision another real contribution to the art.

[0139] Related to determining the above levels of RBP4, NT-proBNP and possibly GFAP The high sensitivity in diagnosing LVO in patients with ischemic stroke suggests that these patients should be treated with mechanical thrombectomy. This allows the patient to be directed to the nearest reference hospital where the procedure may be applied.

[0140] Therefore, the levels of RBP4, NT-proBNP, and possibly GFAP were used For example, at the ambulance level, within the first two hours after the onset of symptoms, if possible, and within the first Rapid and successful classification of patients presenting with stroke symptoms within hours identifies patients as candidates for reperfusion. Patients were classified according to their condition, and antithrombotic drugs were administered first in the ambulance, and they were then referred to a hospital with facilities for treating LVO. It can be guided.

[0141] Also provided herein are methods for distinguishing ischemic from hemorrhagic stroke in a patient, or 1. An in vitro method for selecting a patient suffering from a stroke for reperfusion therapy. blood pressure, including systolic and / or diastolic blood pressure, blood glucose, age, systematic evaluation NIHSS score, gender, and their combinations from the estimator tool stroke-related neurological impairment and determining whether or not NT-proB is present in the isolated patient sample in combination with a clinical parameter selected from the group consisting of: Also disclosed are in vitro methods that include determining levels of NP and levels of GFAP. More specifically, the method involves measuring the level of NT-proB in an isolated sample in combination with the patient's blood pressure. This involves determining the level of NP and the level of GFAP. As shown in the following examples, discrimination with high sensitivity and specificity is possible. In certain embodiments, the method also includes determining the level of RBP4 in the isolated sample. Specifically, the isolated sample may be a biopsy sample, tissue, cell, or biological fluid (plasma, serum, saliva, sperm, etc.). fluid, sputum, cerebrospinal fluid (CSF), tears, mucus, sweat, milk and brain extract. Serum or plasma.

[0142] Any of the specific embodiments disclosed above for the first and second aspects may be used in conjunction with a method for treating ischemia in a patient. to distinguish hemorrhagic from hemorrhagic stroke and / or in patients who have had a stroke This method also applies to the selection of NT-proBN for reperfusion therapy. The method includes comparing the levels of P and GFAP.

[0143] In a more specific embodiment, a method for distinguishing ischemic from hemorrhagic stroke in a patient is provided. and / or in vitro diagnostics for selecting patients suffering from stroke for reperfusion therapy. The Toro method measures the levels of NT-proBNP and GFAP and the corresponding the step of comparing the reference value or reference range with a reference value or reference range corresponding to the ischemic The subject is selected from a value or range of values ​​obtained from subjects suffering from a stroke, At least both GFAP and NT-proBNP levels were significantly elevated in patients with ischemic stroke. and optionally, blood pressure is also within or within a range of values ​​obtained from subjects with ischemic stroke. If the value is within the range of the values ​​for affected subjects, the subject is classified as a candidate for reperfusion therapy. In embodiments, NT-proBNP and The value of GFAP levels in isolated samples is important for accurately classifying patients as candidates for reperfusion therapy. The two timescales are used in combination with the determined blood pressure in an appropriate algorithm for Blood pressure within a specific range combined with specific levels of protein allows for accurate classification and treatment of therapies. In another particular embodiment, the values ​​are used in a decision protocol for selection. is introduced into the formula to give a score or final value that allows such classification. To improve efficacy, the determination of NT-proBNP and GFAP levels was performed after stroke onset. Within the first two hours, and more specifically within the first hour. In this condition, levels of NT-proBNP and GFAP were determined and values ​​were compared to the baseline NI Clinical information such as HSS score and / or blood d-dimer level and / or blood pressure value When used in combination with floor variables, it may be possible to identify patients with large vessel obstruction as candidates for specific thrombectomy approaches. High sensitivity for the detection of ischemic stroke with occlusion is achieved.

[0144] To distinguish ischemic stroke from hemorrhagic stroke in patients, or who have suffered a stroke 1. An in vitro method for selecting a patient for reperfusion therapy, comprising: In vitro methods including determining levels of NT-proBNP and levels of GFAP In another particular embodiment of the method, the levels are determined by the analysis of these two proteins in the isolated sample. The measurement is carried out using a POCT that includes (is equipped with) a reagent means for the measurement. The kit contains, in a reagent means, levels of NT-proBNP and / or GFAP. It only contains a means to detect the protein or mRNA level. nothing.

[0145] Throughout the description and claims, the word "comprises" and variations of that word include other technical It is not intended to exclude any feature, additive, component or step. The word "comprises" encompasses the case of "consisting of." Additional objects, advantages, and features of the present invention are: These and other features will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples are provided by way of illustration and are not intended to limit the invention. Furthermore, the present invention does not encompass all of the specific preferred embodiments described herein. All possible combinations are included. [Example]

[0146] Prehospital differentiation of ischemic stroke (IS) and intracerebral hemorrhage (ICH) using blood biomarkers To provide a method for distinguishing between the two, the inventors conducted extensive analysis of available stroke patient samples. The main objective was to investigate the effectiveness of reperfusion therapy (mainly intravenous thrombus) without the need for neuroimaging techniques. The goal was to discover a reliable marker that would provide information to initiate lysis.

[0147] Example 1: Two different cohorts (Cohort 1 containing 190 patients; Cohort 2 containing 67 patients) Patient classification and treatment selection for hort 2

[0148] material and method From December 2013 to July 2014, patients were admitted within 4.5 hours of stroke onset. Patients with suspected stroke were enrolled. Blood samples were collected at the time of admission (baseline). Biomarkers were primarily measured by ISA and SIMOA. Stroke subtypes were confirmed by the tPA administration in ICH patients. To minimize this, it was selected as having the highest sensitivity with 100% specificity for IS. Selected biomarkers were dichotomized by cutoff.

[0149] The patient cohorts were as follows: Cohort 1 (ELISA cohort): 190 patients with stroke (15 ischemic strokes) 5 cases, hemorrhagic 35 cases). Cohort 2 (SIMOA cohort): 67 patients with stroke (33 ischemic) , 34 hemorrhagic cases).

[0150] The kits for marker analysis were as follows: For RBP4 -> Catalog number DRB400, Quantikine R&D S ystems; GFAP (measured using the Simoa kit and included the SIMOA cohort in cohort 2) (Name the port) -> Catalog Number 102336 and Consumables: ·Simoa Accelerator-1 Plate Lab Service Fee, Catalog Number 100835 Accelerator Consumables Kit, catalog number ACC1 001 For NT-proBNP -> When used with the automated Roche® system , the following reference catalogue reactant (4842464130-proBNP GEN.2 EL ECSYS;4917049922-Precicontrol cardiac G4 ;4842472190-CALSET proBNO GEN.2 ELECSYS)

[0151] We excluded mimics, which are patients who did not suffer from stroke but had clinical signs of stroke. These cohorts 1 and 2 were subsequently selected.

[0152] All patients were randomly assigned to ELISA techniques (for NT-proBNP and RBP4, see above). GFAP, NT- Baseline expression levels of proBNP and RBP4 were measured.

[0153] Various methods for analyzing the retrieved data were assessed: basic cutoffs ( Method 1), Principal Component Analysis (PCA) (Method 2), and refinement of PCA (more rounds); and support vector machine procedure (SVM) (Method 4).

[0154] Either way, the focus here is on reperfusion therapy (i.e., tPA or To avoid any errors, the provision of HIV / AIDS or TNK may have fatal side effects. To achieve this, we can guarantee that patients are classified with 100% accuracy for each biomarker. To find the optimal cutoff values ​​of these blood biomarkers that can accurately indicate the It was.

[0155] result Method 1 (basic cutoff) This method uses the simplest cutoff for the unique biomarkers in the training cohort. All cutoffs were then combined to obtain the final classification.

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

[0157] Therefore, aiming for 100% specificity, patients were classified as ischemic if: NT-proBNP > 4062 pg / mL, with 100% specificity and 14.3% sensitivity. The results showed that 22 / 155 ischemic patients were detected without risk (Figure 1(A)). ), or RBP4 > 52 μg / mL. 100% specificity and 6.5% sensitivity were obtained (1 0 / 155 ischemic patients were detected without risk (see Figure 1(B)).

[0158] Only one of these two conditions had to be met. When these criteria were met, 31 / 155 ischemic patients were classified with 100% specificity (20 .1%) (see Figure 1(C)).

[0159] The data are shown in Figure 1, with a cutoff of 4062 pg / ml for NT-proBNP. (dark lines in both panels A and B), or a cutoff of 52 μg / mL for RBP4 allowed reliable discrimination between IS and ICH. In panel C, log10(N T-proBNP and RBP4 levels were compared using the corresponding The values ​​were plotted simultaneously along with the corresponding cutoffs.

[0160] Cohort 2 ( The SIMOA cohort (67 patients) demonstrated safe and effective treatment for ischemic patients. We performed the first approach to combine GFAP with other biomarkers for better detection. Ta.

[0161] The classification had two stages. First, GFAP < 325 ng / mL was defined as the first criterion. Individuals with a value were selected as possible ischemic candidates. 27 patients with bleeding and occult Only six patients with localized ischemic disease were excluded. The results are plotted in Figure 2(A), where the 3D graph shows the detected levels of GFAP. The levels of NT-proBNP (log(NT-pro Patients were classified according to their levels of GFAP<32 and RBP4. Values ​​below the square that defines the 5 ng / mL value (3D space indicated by the arrow) are the first This corresponds to the value of patients selected as ischemic candidates at the stage 1.

[0162] As a second condition, patients were considered ischemic if they had any of the following: NT-proBNP > 1305 pg / mL. 100% specificity and 30.3% sensitivity. A diagnosis was obtained (10 / 33 ischemic patients were detected), or RBP4 > 38 μg / mL. 100% specificity and 30.3% sensitivity were achieved ( 10 / 33 ischemic patients were detected).

[0163] GFAP is no longer considered for potential ischemic candidates selected in the first stage. If not, NT-proBNP > 1305 pg / mL and RBP4 > 38 μg / mL were present. When considering patients with this condition as candidates, 17 / 33 cases were considered to be at no risk (100% specificity). Ischemic patients (51.5%) were detected. The data are shown in Figure 2(B).

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

[0165] With a specificity of 97%, ischemic patients were those with GFAP levels <325 ng / mL. and NT-proBNP >600pg / mL, with 100% specificity and 51.5% sensitivity (17 / 33 ischemic patients were detected) RBP4 > 36.6 μg / mL, with a specificity of 97% and a sensitivity of 39.4%. (13 / 33 ischemic patients were detected)

[0166] This allowed us to detect 23 / 33 ischemic patients (69.7%) with a specificity of 97%. This was possible.

[0167] With a specificity of 94%, ischemic patients were those with GFAP levels <325 ng / mL. and NT-proBNP > 147 pg / mL, with a specificity of 94% and a sensitivity of 69.7% Obtained (23 / 33 ischemic patients were detected) RBP4 > 31 μg / mL. 94% specificity and 51.5% sensitivity were obtained (1 7 / 33 ischemic patients were detected)

[0168] This allowed us to detect 26 / 33 ischemic patients (78.7%) with a specificity of 94%. This was possible.

[0169] The cut-off value always implies deviation or a certain variability due to various factors. All of the published data relate to a specific fixed prediction accuracy (usually IC95%), so These are also within the range that is considered a positive or discriminant value.

[0170] Using method 1, the best cutoff for reliably classifying stroke patients was ELI The individual cutoffs for the SA cohort were RBP4 > 52 μg / ml and NT-pr oBNP>4062.0pg / ml)

[0171] Method 2 (principal component analysis) Using PCA calculations, we identified which of these three biomarkers was a relatively minor major component. It has been shown that the most variability in the 002).Principal Component Analysis,second edition (Springer), ISBN0-387-95442-2). Procedure This consisted of conducting a principal component analysis. After calculation, it was determined which variables contributed most to the principal components. The results showed that GFAP was the variable most correlated with PC1, and RBP4 was the variable most correlated with PC2. were also correlated variables. Therefore, the best order to follow for classifying patients is G FAP>RBP4>NT-proBNP.

[0172] In summary, principal component analysis (PCA) uses orthogonal transformations to separate potentially correlated variables. A set of observations of numbers (entities that each take on a range of values) is called a principal component. It is a statistical procedure that transforms a set of values ​​of variables that are not linearly correlated into a set of n values ​​of p variables. If there are observations, the number of distinct principal components is min(n-1,p). This transformation is The first principal component has the greatest possible variance (i.e., minimizes the variability of the data as much as possible). (explained in detail), each subsequent component is the best possible component under the constraint that it is orthogonal to the preceding component. The resulting vectors (each of which is a linear representation of the variables) are defined to have a consistent variance in order. The eigenvalues ​​(which are eigenvalues ​​and contain n observations) are uncorrelated orthogonal basis sets. It is sensitive to the relative scaling of variables. PCA is primarily used as a tool for exploratory data analysis. PCA is used to create some projection of the data. The maximum variance due to is on the first coordinate (called the first principal component), and the second maximum variance is on the Orthogonal linear transformations are used to transform the data into the new coordinate system so that it is on coordinate system 2, and so on. It is mathematically defined as a

[0173] Using this cutoff biomarker sequence (GFAP>RBP4>NT-proBNP), First, a GFAP cutoff was used to classify patients with 100% sensitivity or classified the largest number of patients with 100% specificity. Then, using the RBP4 cutoff, , 100% sensitivity or 100% specificity for patients who could not be classified using previous cutoffs Finally, NT-proBNP cutoffs were used to classify patients who were not yet classified. Patients were classified with either 100% sensitivity or 100% specificity. After use, the first round of cutoffs was considered complete.

[0174] The same analysis was performed using only two biomarkers (RBP4 and NT-proBNP). I went again.

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

[0176] The cutoff for Cohort 1 (190 patients) was NT-pro using ELISA testing. In addition to BNP and RBP4 determination, GFAP was also determined using the SIMOA test. The results were more robust than those found using cohort 2 (i.e., the SIMOA cohort (67 patients)). It was a prison.

[0177] Using the cutoff for the ELISA cohort (cohort 1), the overall cohort was 23.1 6% (37.14% in hemorrhagic patients and 20% in ischemic patients) as a sensitivity of 100% or 1 Classification was possible with a specificity of 0.00%.

[0178] Using the cutoff for the SIMOA cohort (cohort 2), 31% of the entire cohort ( 38% hemorrhagic patients, 24% ischemic patients) with 100% sensitivity or 100% specificity I was able to do it.

[0179] Cohort 2 uses fewer individuals to better differentiate them. This makes it possible to classify approximately 1% more bleeding patients and 4% more ischemic patients.

[0180] Using only RBP4 + NT-proBNP, the cutoffs were as follows in Table 2: [Table 2]

[0181] Again, cohort a (190 patients) was more robust than cohort 2 (67 patients). (same cutoff as above)

[0182] Using the cutoff from the ELISA cohort (cohort 1), 16% of the total cohorts were .8% (3% in hemorrhagic patients and 20% in ischemic patients) as 100% sensitivity or 100% The results were classified based on the specificity of the

[0183] Using the cutoff from the SIMOA cohort (cohort 2), 13% of the total cohort 0.4% (3% hemorrhagic patients, 24% ischemic patients) with 100% sensitivity or 100% specificity could be classified as follows.

[0184] The SIMOA cohort uses fewer individuals, allowing for better differentiation between them This makes it possible to classify 4% more ischemic patients.

[0185] Method 3 (principal component analysis) This method is an extension of Method 2, where unclassified individuals are used for many more rounds. was calculated.

[0186] After the first round was completed, logistic regression was performed to identify any biases associated with the outcome. We then compared the results with patients who had not yet been classified. The cutoffs were recalculated in the same order.

[0187] This method is not suitable for cases where no trend is observed or where there is no sensitivity or specificity of 100%. This was continued until it was no longer possible to classify more individuals. Once the individuals were classified, the first Please note that the cutoff order from round to last round must be followed. It is important to do so.

[0188] Method 3 uses only two biomarkers (RBP4 and NT-proBNP). went.

[0189] In the ELISA cohort (cohort 1), biomarkers were GFAP, RBP4, and Using NT-proBNP and NT-proBNP, the following cutoffs were obtained: [Table 3]

[0190] This method classified 51% of individuals with 100% sensitivity or specificity (for bleeding). 60% and 49% for ischemic.

[0191] In the ELISA cohort (cohort 1), RBP4 and NT-p were used as biomarkers. Using roBNP, the following cutoffs were obtained: [Table 4]

[0192] 100% sensitivity or specificity in 36.5% of individuals (6% for hemorrhagic and 44% for ischemic) ).

[0193] Method 4 (SVM) Maximize the number of well-classified IS patients in classifying all ICH patients with 100% specificity To identify the RBP4 and NT genes, a support vector machine procedure (SVM) was used. -proBNP levels were calculated (see "A User's Guide to Supp ort Vector Machines”,Article in Methods in Molecular Biology,(Clifton NJ),2010, (See Asa Ben-Hur and Jason Weston). Radial kernel The ICH analysis was used with the following parameters: c=100 and σ=0.05. For the data, each point of 0.71 is needed to obtain a classifier with 100% specificity. (The intuition behind the decision is that positive values ​​are The more patients with ICH are classified as IS, the less likely they are to be classified as IS. It's a missed opportunity.)

[0194] This method achieved a sensitivity of 29.7% for IS.

[0195] The data are shown in Figure 3, where values ​​on the "S-curve" are 100% IS (indicated by the arrow). The value under the curve is used for patients with either ICH or IS subtypes. handle.

[0196] As can be seen from Figure 3, the combination of RBP4 and NT-proBNP levels This allows for a better classification of patients and an appropriate selection of therapy. This is introduced in the support vector machine procedure using kernels, giving a graph like that in Figure 3. Depending on the value of one protein, the value of the other protein can determine whether the patient is IS or not. is correctly classified as ICH.

[0197] According to SVM, once the determined values ​​of the test samples are obtained, they can be used to classify the test samples into one A decision that allows classification into one category (IS) or another category (ICH) In particular, the above decision value is usually 0.5, and if necessary, It can be adjusted accordingly to improve the accuracy of classification of objects. According to the synthase, above this value the patient is placed in one category, below this value , falls into another category. The judgment value can be adjusted.

[0198] Conclusion: The ELISA cohort (cohort 1) used method 1 (basic cutoff), and RB When used separately, P4 and NT-proBNP were shown to be 100% specific for ICH. The combination of the two methods successfully identified 6.5% and 14.2% of IS, respectively. The specificity of ICH rose to 20% in IS patients with 100% specificity for ICH, which was a distinct The number of cases was less than the sum of the biomarkers (because one case was adequately evaluated by both biomarkers). (This is because they are classified as

[0199] In the SIMOA cohort (Cohort 2), GFAP cutoffs were used to identify ICH patients and The maximum number of patients with GFAP and IS was then isolated, and the IS patients were then separated within the lower limit of the GFAP cutoff. When we attempted to classify patients, the sensitivity increased for IS patients (51.5%) but not for ICH patients. The specificity for HIV-1 remained at 100%.

[0200] Method 2 (also known as sort-exclude) and Method 3 (also known as sort-exclude-repeat) Using the GFAP biomarker, the addition of the GFAP biomarker may be beneficial in appropriately classifying ICH patients. (In the ELISA cohort, we used GFAP.) 60% with GFAP vs. 6% without, SIMOA cohort values 37% with GFAP vs. 3% without GFAP Furthermore, even when GFAP was not used, the high ability to classify IS patients was lost. It is also important to note that the results were not based on the use of the GFAP biomarker. Regardless, the values ​​for the ELISA cohort were 49% vs. 44%, and the values ​​for the SIMOA cohort were 49% vs. 44%. is 24% vs. 24%).

[0201] The inclusion of GFAP does not affect the ability to identify IS patients.

[0202] Finally, a more sophisticated method, Method 4 (SVM) classifier, was used to compare the ICH Using this method, we were able to properly identify 29.7% of IS patients with a specificity of 100%.

[0203] Both of these data will be used to simultaneously determine reference values ​​for accurate classification of patients. Several factors, such as the number of markers in a given sample or the techniques used to analyze data from a cohort of patients, are important. It has also been shown that the dynamic value of the protein is a function of the parameters For one fixed amount or level of The amount of other simultaneous markers that allow classification varies and some mathematical methods are used. It can be represented by a function or model (i.e., basic cutoff, ROC curve, P CA, SVM, etc.).

[0204] Different data provided by two different cohorts of patients according to Example 1 of this description. Data analysis revealed levels of RBP4 and NT-proBNP, and possibly GFA. The level of P allows for better discrimination between IS and ICH and also for the safety of candidates for reperfusion therapy. It has been shown that the reference interval or criteria for accurate classification can be used to The reference value is calculated in this way and adjusted according to several variables. These values ​​are shown to personnel who must perform the method.

[0205] These two or three markers provided a good overall picture of the patients classified , which were useful for accurate classification of study patients.

[0206] Furthermore, of particular interest, the use of RBP4 and NT-proBNP biomarkers The bottom line is that properly classified ischemic patients are those with poor outcomes.

[0207] As shown in Table 5 below, ischemic strokes identified using these two biomarkers Intermediate-stage (IS) patients have higher mortality rates and reduced independence at 3 months after stroke.

[0208] This is to avoid fatal outcomes if treated later according to today's standard pathways. , another reason to treat these patients with reperfusion techniques as soon as possible.

[0209] [Table 5] JPEG2025143285000007.jpg223170

[0210] In short, this technique, based on the determination of both biomarkers, is a "golden hour" (virtual Increases the chances of treatment within 60 minutes of the onset of a stroke, which is a significant factor in asymptomatic strokes. nearly doubles the probability of becoming a resident, triples the probability of independence, and quadruples the probability of survival ( For more information about the "Golden Hour" and therapeutic action protocols, please see Kunz e t al. “Effects of Ultraearly Intravenous Thrombolysis on Outcomes in Ischemic Str oke:The STEMO(Stroke Emergency Mobile)Gr oup”,Circulation-2017May2;135(18):1765-1 (See 767).

[0211] Example 2: Patient classification and therapy selection for a different cohort of 32 patients.

[0212] The dataset consisted of 32 patients (18 hemorrhagic and 14 ischemic). The goal was to separate the largest number of patients from both classes without risking classification.

[0213] To do so, we used four different methods for the GFAP biomarker. Therefore, we calculated the steps for each different method.

[0214] The procedure was to find the best cutoff for GFAP and achieve 100% sensitivity or 100% The purpose of this study was to exclude patients classified with a specificity of 0.05, and to exclude the remaining data (classified using GFAP). RBP4 biomarker data were used to identify the phenotypes of phenotypes that were not excluded / excluded and therefore not fully classified. The following cutoffs based on the data were calculated to re-identify patients with 100% sensitivity or 100% specificity: Finally, we used the NT-proBNP biomarker data to identify patients with pulmonary embolism and to classify them as pulmonary embolism. Repeat the procedure.

[0215] Below we include the cutoff values ​​used for each method for GFAP determination.

[0216] GFAP DxSYS_CLIA (DxSYS Inc. Chemiluminescent Immunoassay): Using a cutoff of >80.6 pg / ml, 15 bleeding patients (83% of all bleeding patients) ) were correctly classified. GFAP DxSYS_TMB (3,3',5,5'-tetramethylbenzidine (TM B) Using DxSYS Inc): Using a cutoff of >88.685 pg / ml 14 bleeding patients (78% of all bleeding patients) were correctly classified. The same results were obtained using cutoffs. GFAP Quanterix®: >2066.078 pg / ml and < Using a cutoff of 166.67 pg / ml, 14 hemorrhagic patients and 3 ischemic patients were included. Cases (78% of all hemorrhagic patients and 21.4% of ischemic patients) were correctly classified. GFAP Elisa (Elisa kit catalog number RD192072200, Bi oVendor): Using a cutoff of >50.5 pg / ml, 11 bleeding patients (total 61.1% of bleeding patients were correctly classified.

[0217] Applying all these cutoffs, we assessed RBP4 and NT-proBNP as previously described. After further investigation, 17 / 18 hemorrhagic patients and 12 / 14 ischemic patients were ultimately successful. was accurately classified.

[0218] Therefore, this procedure is suitable for determining three biomarkers in a sequential manner. Accurate patient assessment to determine appropriate medical regimen (reperfusion therapy for ischemic stroke) Table 6 shows an overview of the procedures for each analytical method.

[0219] [Table 6]

[0220] Example 3: Combining biomarkers and clinical data predicts ischemic stroke requiring reperfusion therapy Improve accuracy of identifying the inside

[0221] Data were analyzed using the following patients and cutoffs: Hemorrhagic n=35 and ischemic n=155

[0222] The cutoff values ​​for GFAP (pg / ml) < 97.03 and NT-proBNP (pg / m l) In combination with >4076.50 and RBP-4 (μg / ml) >52.52 The three biomarkers had a sensitivity of 0.32, a specificity of 1.00 (100%), and a positive predictive value ( The PPV was 1.00 and the negative predictive value (NPV) was 0.25.

[0223] Adding clinical data (especially blood pressure and blood glucose levels) on top of that maintains 100% specificity. In fact, GFAP (pg / ml) < 97.03 and NT-pr oBNP (pg / ml)>4076.50 and systolic blood pressure (SBP)mmhg<119 .00, and diastolic blood pressure (DBP) (mmHg) < 60.50 and RBP-4 (u g / ml)>52.52 and blood glucose (mg / dl)<83.50 had a sensitivity of 0.45 and It had a specificity of 1.00, with a PPV=1.00 and NPV=0.29.

[0224] Logistic regression-based models: Feasible data sets can increase classification accuracy and robustness while limiting overfitting. To find the conversion, both cohorts (original cohort 1 in Example 1 n=189, as well as bleeding Pooled data from a replication cohort (n=300) of anemic (n=51) and ischemic (n=249) patients Several multiple logistic regression models were tested on the data. The model included a panel of markers and relevant clinical variables. The patients were selected according to the formation criteria, and were classified into ischemic stroke, hemorrhagic stroke, and was used to classify individuals between

[0225] The selected model included GFAP (pg / ml) as a significant predictor of ischemic stroke status. ), NT-proBNP (pg / ml) and diastolic blood pressure (mmHg) were logarithmically transformed as follows: Included in combination:

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

[0227] This linear combination produces an estimated log-odds ratio sequence that can be treated as a compound marker. The core was obtained by maximizing the desired sensitivity and specificity requirements for classifying individuals between the two groups. A threshold could be placed on this score. Marker weighting and aggregation allowed for a particular When the heterogeneity was above 95%, the classification sensitivity was improved over the raw markers in both cohorts.

[0228] In the original cohort, application of this model yielded sensitivity = 0.60, specificity = 1.00, and accuracy = 0.60. The degree was 0.68.

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

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

[0231] In cases of suspected stroke (ischemic or hemorrhagic stroke), the 3-hour period from the onset of the stroke Blood samples were obtained within 24 hours. Diagnostic and therapeutic workup was performed in several cases (n=189). The data are similar to those in the initial cohort 1 of Example 1 of the study patients used in these files. It was.

[0232]

number

[0233] Example 4: Improving the accuracy of detecting ischemic stroke patients candidates for reperfusion therapy at very early time points above

[0234] The original cohort 1 (n=189) in Example 1 was used to examine the relationship between the time from symptom onset to blood sampling. Surprisingly, the earlier the test was performed, the better the performance of the biomarkers. Improved accuracy of:

[0235] (i) 0–2 hours (hemorrhagic n = 11 and ischemic n = 82) GFAP (pg / ml) < 175.85 and NT-proBNP (pg / ml) > 3 916.50 and RBP-4 (ug / ml)>38.15. Sensitivity=0.70, specificity=1.00, PPV=1.00 and NPV0.31. (ii) 2–3 hours (hemorrhagic n = 13 and ischemic n = 35) GFAP (pg / ml) < 94.37 and NT-proBNP (pg / ml) > 12 89.50 and RBP-4(ug / ml)>46.55. Sensitivity=0.60, specificity=1.00, PPV=1.00 and NPV0.48. (iii) 3–4.5 hours (hemorrhagic n = 11 and ischemic n = 38) GFAP (pg / ml) < 98.96 and NT-proBNP (pg / ml) > 42 54.50 and RBP-4 (ug / ml) > 53.34. Sensitivity=0.34, specificity=1.00, PPV=1.00 and NPV0.31.

[0236] In the replication cohort (n=300, hemorrhagic n=51 and ischemic n=249), Early arriving patients were always selected and blood samples were taken within the first hour of stroke onset. Hemorrhagic n=8 and ischemic n=33.

[0237] In that subcohort, GFAP (pg / ml) < 300.03 and NT-proB NP (pg / ml) > 1033.01 and RBP-4 (ug / ml) > 32.93 are excellent. It had a high accuracy with sensitivity = 0.91, specificity = 1.00, PPV = 1.00 and NPV = 0.00. The figure was .73.

[0238] Several clinical variables (clinical parameters), i.e., 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 (mgdl)<1 Adding 08.00 improved it even more and the test worked perfectly.

[0239] This combination yielded a sensitivity of 1.00, specificity of 1.00, PPV of 1.00, and NP V1.00 was obtained.

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

[0241] The selected model included GFAP (pg / m l), NT-proBNP (pg / ml) and diastolic blood pressure (mmHg) were logarithmically transformed as follows: Included in the following combinations:

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

[0243] This linear combination produces an estimated log-odds ratio sequence that can be treated as a compound marker. A core was obtained for the replication cohort of patients who were cared for within the first hour of stroke onset. Application of this model had sensitivity = 0.79, specificity = 1.00 and accuracy = 0.83. Ta.

[0244] Figure 5 shows this logistics data with data from isolated samples within the first hour after stroke onset. Figure 11 graphically illustrates the classification of subjects using the metric model scores.

[0245] These data in Example 4 all show that the selected biomarkers RBP4, NT-pr If oBNP and GFAP could be measured soon after the onset of stroke, they would have high sensitivity. In particular, they are determined within 1 and 2 hours after the onset of symptoms. If you get.

[0246] In stroke, the earlier the test is performed, the better for a fixed specificity. The sensitivity that can be achieved is surprising, and in general, biomarkers are useful for post-onset comparisons. Give appropriate cues after a reasonably long period of time. ) allows for a good and reliable classification of patients quickly, which is not possible in this condition. This is not just a profit, it is a goal. This allows us to choose the best and most appropriate strategy for these critical moments. A decision can be made that can later be verified using additional biomarkers ( For example, after the patient arrives at the hospital).

[0247] Example 5: Other diseases that mimic acute ischemic stroke (stroke mimicking conditions and intracerebral hemorrhage) performed in ambulances to select ischemic stroke patients eligible for reperfusion therapy. Rapid point-of-care blood testing

[0248] The ambulance will be used to initiate reperfusion therapy (thrombolysis) or to transport this patient to an appropriate hospital. blood samples to determine the best reperfusion therapy (thrombolysis or mechanical thrombectomy) Point-of-care testing for ambulances that can identify patients with ischemic stroke T) was used to measure the panel of biomarkers included in the present invention (RBP4, NT-proBN P and GFAP) were examined.

[0249] method: A network of over 20 ambulances and helicopters in the Seville region of southern Spain The BIO-FAST study (Biomarkers for Initiation g Onsite and Faster Ambulance Stroke The Patients with suspected stroke (<6 hours) were enrolled in the rapid point-of-care (POCT) study. (10-15 minutes until discharge) to measure RBP-4 / NT-pro-BNP Blood samples were collected by car and GFAP was analyzed using SIMOA-Quanterix technology. Measured.

[0250] Inclusion criteria: patients over 18 years of age; activated by a coordinating center Stroke code, and symptom onset less than 6 hours. Stroke with uncertain timeline or morning stroke In the case of a stroke, the first hour is considered the last moment the patient appeared well.

[0251] Exclusion criteria: pre-hospital diagnosis other than stroke; impossibility to obtain pre-hospital blood sample; and refusal by the patient / next of kin to provide informed consent.

[0252] Sample type One EDTA tube of blood sample (10 mL) for biobanking + one blood sample for POC Extract one EDTA tube of sample (2 mL). Samples are used for biomarker discovery studies. Until full use, Vall, in accordance with the requirements established in RD 1716 / 2011, Placed in the collection registered under code C.0003176 within d'Hebron Hospital Included in the collection.

[0253] RBP4 rapid test. RBP4 POC lateral flow cytometry positive (dispo sitive). Rapid NT-proBNP test. Nt-proBNP POC lateral flow disposable Gitive. ·GFAP SIMOA-Quanterix technology

[0254] result: Twenty patients were included (10 with ischemic stroke, 3 with intracerebral hemorrhage, and 7 with stroke mimics). It is possible to perform POCT in an ambulance, and in one case it was performed in a helicopter. The event was held without incident.

[0255] Rapid POCT (RBP-4) using selected cutoffs for these biomarkers -NT-proBNP) showed a significant difference of 50% of IS without misclassifying ICH or mimic. % were correctly identified (100% specificity, 50% sensitivity). In this case, POCT was performed with 62.5% correct IS without misclassifying it as ICH or mimic. was accurately identified (100% specificity, 62.5% sensitivity).

[0256] Using stored blood samples from these patients, we investigated the expression of GFAP, RBP-4, and NT- Explore how outcomes can be improved using POCT that can incorporate proBNP To assess the effect of GFAP on the GFAP signaling pathway, we used SIMOA-Quanterix technology. Four patients with very high levels were identified: two with ICH, one with IS, and one with mimic By excluding these four cases, the sensitivity increased to over 70% and the specificity increased to 100%. We were able to maintain it at %.

[0257] 20% of IS could be treated within the first 30 minutes of symptom onset using POCT Furthermore, one patient was administered tPA because the POCT was negative for IS. Patients treated with tPA were more likely to be diagnosed with pulmonary embolism in the ambulance than those treated with tPA. By using a diagnostic test, it is possible that the drug was administered 1 hour and 30 minutes before the initial dose. Ischemic patients (30%) could have been in the 4.5-hour window for testing. , but it was outside of those hours when the CT scan was performed at the hospital.

[0258] Conclusion: A panel of biomarkers including RBP-4, NT-proBNP, and GFAP was used to assess ischemia provides a useful sensitivity rate with 100% specificity for acute stroke, which is comparable to standard techniques. Initiating prehospital reperfusion therapy in selected cases much more quickly than is currently used The use of point-of-care testing (POCT) has the potential to change standard clinical practice. .

[0259] By POCT allowing determination of blood levels of RBP4 and NT-proBNP The data obtained and further complemented by GFAP determination were used in IS and ICH patients. However, screening in the presence of pre-existing mimics (non-stroke patients with stroke-like symptoms) This was intended as the first test in a real-world situation where While maintaining a specificity of 100%, the sensitivity was also higher than some of the previous examples (50% This adds to the panel of biomarkers that allows for good and reliable classification. An unexpected benefit of biomarkers is the ability to better select therapies that can be administered before arrival at the hospital. is converted to a choice.

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

[0261] In two previously published cohorts of stroke patients (n=189 and n=300), L Identification of VO was based on the detection of occluded cerebral arteries on CT angiography (CTA) performed on arrival at the hospital. The criteria were based on the presence and location of: occlusion of any of the following arteries or arterial segments: occlusion, i.e., intracranial carotid artery (ICA), basilar (BA), and M1 segment of middle cerebral artery occlusion. We followed a restrictive definition of LVO described as occlusion of a segment of the esophagus.

[0262] (Vanacker P, Heldner MR, Amiguet M, et al. Prediction of large vessel occlusions in acute stroke:National institute of Heal th Stroke Scale is hard to beat.Crit Car See e Med2016;44:e336-43).

[0263] The same biomarkers shown below were defined as occlusion of M2 (ICA, M1, M2 or BA). This includes occlusions in the more distal part of the middle cerebral artery (MCA), such as LVO (defined as Less strict criteria may be used to identify LVO simply by using different cutoffs. is also useful.

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

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

[0266] GFAP (pg / ml) < 694.48 and NT-proBNP (pg / ml) > 1 764.50 and RBP-4 (μg / ml) > 35.22 had a sensitivity of 0.98 and a specificity of = 0.18, PPV = 0.33 and NPV 0.96; IHSS score >11 points and d-dimer (ng / ml) >1432.43, etc. The addition of clinical and laboratory variables improved the sensitivity to 1.00 and specificity to 0.46, P It had a PV=0.43 and NPV1.00.

[0267] If blood sample obtained within 2 hours of symptom onset, baseline NIHSS score > For 11 pts and RBP-4 (ug / ml) > 51.53, sensitivity = 1.00, specificity The marker's performance improved with a coefficient of 0.40, PPV=0.46, and NPV=1.00.

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

[0269] This included baseline NIHSS score >11 points and GFAP (pg / ml) Adding clinical variables such as <54.84 improved the sensitivity to 1.00 and specificity to 0.43. It had a PPV=0.19 and NPV1.00.

[0270] NT-pro with d-dimer if blood sample obtained within 2 hours of symptom onset For BNP, sensitivity = 1.00, specificity = 0.70, PPV = 0.39 and NPV 1.0 0 and improved ability.

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

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

[0273] Biomarkers GFAP (pg / ml) < 153.18 and NT-proBNP (pg / ml)>692.60 and RBP-4(μg / ml)<39.72, sensitivity=1.0 0, specificity=0.09, PPV=0.30 and NPV1.00.

[0274] These results were consistent with clinical data including baseline NIHSS scores >11 points and R Add BP-4 (ug / ml) < 29.03 and blood glucose (mg / dl) < 71.50 The sensitivity was improved by 1.00, specificity by 0.20, PPV by 0.33, and NP by 0.33. V1.00 was indicated.

[0275] These results suggest that when blood samples were obtained within 2 hours of symptom onset, baseline NIHS It was even better for S-score and RBP-4, with sensitivity=1.00 and specificity=0. 45, had a PPV=0.43 and NPV1.00.

[0276] In patients who underwent thrombectomy (n=264 without thrombectomy and n=35 with thrombectomy), Omnimarkers GFAP (pg / ml) <153.18 and NT-proBNP (pg / ml )<2049.01 has a sensitivity of 1.00 and specificity of 0.01 for accurately identifying these patients. The mean PPV was 0.14, PPV=0.13, and NPV=1.00. The results improved with the addition of the examination variables, with GFAP (pg / ml) < 153.18 and d -Dimer (ng / ml) > 7.29 and diastolic blood pressure (mmHg) > 89.50 are sensitive = 0.97, specificity = 0.33, PPV = 0.16 and NPV 0.99.

[0277] References Patent documents -International Publication No. WO2016 / 087611 Non-patent literature -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 -BLASTManual, Altschul, S., et al, NCBI NLMNIH 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 Isc hemic 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 occlu sions and thrombectomy eligible patients indicatessignificant potential for grow th 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. 2 020 Feb 25:1-9. -Gandhi CD, Al Mufti F, Singh iP, et al. Neuroendovascular managementof emergen t large vessel occlusion: update on the technical aspects andstandards of practi ce by the Standards and Guidelines Committee of the Societyof Neurointerventiona l Surgery. J Neurointerv Surg 2018;10:315-20). -Lakomkin N, Dhamoon M, Carroll K, et al. Prevalence of large vesselocclusion i n patients presenting with acute ischemic stroke: a 10-yearsystematic review of the literature. J Neurointerv Surg 2019;11:241-5. -Waqas M, et al. Effect of definition and methods on estimates ofprevalence of large vessel occlusion in acute ischemic stroke: a systematicreview and meta-ana lysis. J Neurointerv Surg. 2020 Mar;12(3):260-265

Claims

1. An in vitro method for selecting patients suffering from stroke for reperfusion therapy. and the level of retinol binding protein-4 (RBP4) in the isolated sample from the patient and type B and the level of N-terminal fragment of natriuretic peptide (NT-proBNP). An in vitro method comprising:

2. The method further comprises determining the level of glial fibrillary acidic protein (GFAP). Item 1. The in vitro method according to Item 1.

3. The levels of RBP4, NT-proBNP, and, if determined, GFAP, and the levels of each protein The method further comprises comparing the quality of the sample with a corresponding standard value or standard range for the quality of the sample, or the reference interval is from a value or range of values ​​obtained from subjects who have suffered an ischemic stroke and the subject has at least elevated levels of both RBP4 and NT-proBNP. If the value is within or within the range of values ​​obtained from subjects suffering from ischemic stroke, 3. The method of claim 1, wherein the patient is classified as a candidate for reperfusion therapy. In vitro method.

4. The levels of RBP4, NT-proBNP, and, if determined, GFAP, and the levels of each protein and comparing the measured values ​​with corresponding reference cutoff values ​​for the respective proteins; If only RBP4 and NT-proBNP levels are determined, NT-proBNP levels simultaneously distinguish between patients with ischemic stroke and those with intracerebral hemorrhage. The corresponding reference cutoff value Ref1 for each of said proteins ＲＢＰ４ and Ref 1 ＮＴ－ｐｒｏＢＮＰ or more indicates that the patient is a candidate for reperfusion therapy. ,or If the levels of RBP4, NT-proBNP and additional GFAP are determined, In step 1, the GFAP level is the reference cutoff value Ref ＧＦＡＰ The second step is as follows: The levels of RBP4 and NT-proBNP were simultaneously compared with the corresponding reference cut-off values ​​Re f2 ＲＢＰ４ and Ref2 ＮＴ－ｐｒｏＢＮＰ If so, the patient is eligible for reperfusion therapy. and the cutoff value is selected as a candidate for distinguishing between ischemic stroke patients and intracerebral hemorrhage patients.

3. The in vitro method according to claim 1 , wherein

5. If the subject is classified as a candidate for reperfusion therapy, the subject is also evaluated for the modified ranking score. Dependence defined as a score of ≥2 according to the mRS Core and occurring ≥1-5 months after stroke As having a determined prognosis within and / or incidence comprised 25% to 30% 5. The method of claim 1, wherein the patient is classified as having a prognosis defined by 3-month mortality after administration of the method of claim 1.

10. The in vitro method according to any one of claims 1 to 9.

6. If the subject is classified as a candidate for reperfusion therapy, the subject also has a large vessel occlusion. The in vitro method of claim 1 , wherein the patient is diagnosed with the disease.

7. The reperfusion therapy is selected from the group consisting of antithrombotic therapy, thrombectomy, and combinations thereof.

7. The in vitro method according to any one of claims 1 to 6, wherein the method is selected from:

8. 8. The in vitro method of claim 7, wherein the antithrombotic agent is a thrombolytic agent.

9. 9. The method of claim 8, wherein the thrombolytic agent is a plasminogen activator.

10. 9. The method of claim 8, wherein the plasminogen activator is tissue plasminogen activator. The method described below.

11. The thrombolytic agent is alteplase, reteplase, and tenecteplase, and a recombinant tissue plasminogen activator selected from the group consisting of: The method of claim 10.

12. The method of claim 1 , wherein the sample is a biological fluid.

13. 13. The method of claim 12, wherein the biological fluid is plasma or serum.

14. Determine levels of RBP4 and NT-proBNP, and GFAP, if determined.

14. Any one of claims 1 to 13, wherein said step (a) is performed within the first two hours after the onset of stroke.

10. The method according to claim 1.

15. 1. An in vitro method for distinguishing ischemic from hemorrhagic stroke in a patient, comprising: and determining the levels of RBP4 and NT-proBPN in an isolated patient sample. In vitro method.

16. 16. The in vitro method of claim 15, further comprising determining the level of GFAP.

17. The levels of RBP4, NT-proBNP, and GFAP, if determined, and the corresponding A step of comparing with a reference value, The reference value or reference range is for subjects suffering from ischemic stroke and / or those who have not. a value or range of values ​​obtained from a subject suffering from a hemorrhagic stroke, However, both RBP4 and NT-proBNP levels were significantly elevated in patients with ischemic stroke. ischemic stroke if the value is within the range of values ​​obtained from the affected subject. Classified as moderate or low in RBP4 and NT-proBNP levels At least one of the values ​​is within or obtained from a subject suffering from a hemorrhagic stroke. or alternatively classifying the stroke as hemorrhagic if the match is within the range of Levels of RBP4, NT-proBNP, and GFAP, if determined, and each comparing the RBP4 and RBP5 protein levels to corresponding reference cutoff values ​​for the protein, If only the levels of RBP4 and NT-proBNP are determined, The level of NP simultaneously distinguishes between patients with ischemic stroke and patients with intracerebral hemorrhage. The corresponding reference cutoff value Ref1 for each ＲＢＰ４ and Ref1 ＮＴ－ｐｒｏ ＢＮＰ or more, indicating that the patient has suffered an ischemic stroke; or (b) when the levels of RBP4, NT-proBNP and additional GFAP are determined; In the first step, the level of GFAP is determined to be equal to or less than the reference cutoff value Ref ＧＦＡＰ The second step is as follows: At this stage, the levels of RBP4 and NT-proBNP simultaneously reach the corresponding reference cut-off values ​​R ef2 ＲＢＰ４ and Ref2 ＮＴ－ｐｒｏＢＮＰ If the above condition is met, the patient is diagnosed with ischemic cerebral and the cut-off value is used to classify patients as having ischemic stroke and those having intracerebral hemorrhage. The method of any one of claims 15 to 16, further comprising the step of identifying the patient. In vitro method.

18. If the subject is classified as having an ischemic stroke, the subject is given a modified ranking score (m Dependence is defined as a score of greater than 2 according to the RS (Reporting by Patient Recognition and Assessment) and is determined within 1 to 5 months after stroke onset. and / or 3 months after onset consisting of 25% to 30% 18. The method of claim 15, wherein the patient is classified as having a prognosis defined by mortality. The in vitro method according to any one of claims 1 to 4.

19. If the subject is classified as having an ischemic stroke, the subject may also have suffered from a large vessel occlusion. The in vitro method of any one of claims 15 to 18, wherein the patient is diagnosed with the disease.

20. Determine levels of RBP4 and NT-proBNP, and GFAP, if determined.

20. The method of any one of claims 15 to 19, wherein said step of administering to said subject is performed within the first two hours after the onset of stroke.

1. The in vitro method according to claim 1.

21. 15. Any of claims 1 to 14, further comprising determining one or more clinical parameters. The in vitro method for selecting patients suffering from stroke for reperfusion therapy according to claim 1 or a method according to any one of claims 15 to 20, wherein the ischemic stroke in the patient is treated with a hemorrhagic stroke. In vitro methods for distinguishing between

22. The clinical parameters include blood pressure, including systolic blood pressure and / or diastolic blood pressure, blood glucose, age, , scores from the Systematic Assessment Tool for Stroke-Related Neurological Disability, gender, and combinations thereof 22. The in vitro method of claim 21 , wherein the antibody is selected from the group consisting of:

23. An in vitro method for the diagnosis of large vessel occlusion (LVO) comprising: In vitro methods comprising determining the levels of BP4 and NT-proBNP.

24. The method further comprises determining the level of glial fibrillary acidic protein (GFAP). Item 24. An in vitro method for diagnosing large vessel occlusion according to Item 23.

25. The levels of RBP4, NT-proBNP, and GFAP, if determined, and the corresponding The method further comprises a step of comparing the reference value or the reference range with a reference value, and wherein the subject has at least one RBP4 The levels of NT-proBNP and one of GFAP were both significantly elevated in patients with LVO. If the value is within the above range or within the above range obtained from subjects who have been diagnosed with LVO, 25. The in vitro method according to any one of claims 23 to 24, wherein

26. 26. Any of claims 23 to 25, further comprising determining one or more clinical parameters.

1. The in vitro method according to claim 1.

27. The clinical parameters include blood pressure, including systolic blood pressure and / or diastolic blood pressure, blood glucose, blood d -dimer level, age, score from the Systematic Assessment Tool for Stroke-Related Neurological Disability, and sex 27. The in vitro method of claim 26, wherein the compound is selected from the group consisting of: 。

28. The levels of RBP4, NT-proBNP and GFAP in the isolated samples, blood glucose, d- Dimers, and further diastolic blood pressure, and a systematic assessment tool for stroke-related neurological disorders? 28. The method of any one of claims 23 to 27, comprising determining a baseline score from In vitro methods described.

29. Determine levels of RBP4 and NT-proBNP, and GFAP, if determined.

29. The method of claim 23, wherein the step of administering the cerebral infarction inhibitor is performed within the first two hours after the onset of the stroke.

1. The in vitro method according to claim 1.

30. 1. An in vitro method for the prognosis of a patient suffering from ischemic stroke, comprising: Retinol-binding protein-4 (RBP4) levels and type B natriuresis in isolated samples and determining the level of the N-terminal fragment of the peptide (NT-proBNP). In vitro methods.

31. The level of at least RBP4 or two proteins is compared to a reference value, and the reference value is from a value or range of values ​​that indicates or confirms that the subject has suffered an ischemic stroke The in vitro method for prognosis according to claim 30, wherein the selected

32. The prognosis is defined by a dependency of more than 2 according to the modified ranking score (mRS). determined within 1-5 months after stroke onset and / or comprised 25%-30% 32. The method according to claim 30, wherein the method is defined by a 3-month mortality rate after onset. In vitro methods for prognosis of the above.

33. To distinguish ischemic stroke from hemorrhagic stroke in patients, or who have suffered a stroke 1. An in vitro method for selecting a patient for reperfusion therapy, optionally comprising: Systematic assessment of blood pressure, including blood pressure and / or diastolic blood pressure, blood glucose, age, and stroke-related neurological impairment a clinical score selected from the group consisting of a score obtained from a clinical assessment tool, gender, and a combination thereof In combination with the determination of the parameter, the level of NT-proBNP in the isolated sample of said patient. and determining the level of GFAP.

34. Reagent means for detecting levels of RBP4 and NT-proBNP, and optionally and a reagent means for detecting the level of GFAP.

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