Markers for the diagnosis of cerebral major artery occlusion

Abstract

SUMMARY Markers for the diagnosis of cerebral major artery occlusion. The present invention relates to a method for diagnosing cerebral major artery occlusion in a subject suffering from an ischemic stroke episode, comprising determining the level of a specific protein in an isolated sample of the subject. The present invention also relates to a method for distinguishing ischemic stroke from any other condition selected from hemorrhagic stroke, transient ischemic attack and stroke mimicking condition. A simplified kit containing reagents for carrying out the method is also disclosed.

Description

[Technical field]

[0001] This application claims the benefit of European Patent Application No. 21382322.2, filed April 15, 2021.

[0002] Technical Field The present invention relates to the field of diagnosis or companion diagnostics, in particular to a method for diagnosing major cerebral artery occlusion during an ischemic stroke episode, and to the selection of an appropriate treatment depending on said diagnosis. [Background technology]

[0003] Background technology A large vessel occlusion (LVO) is defined as a blockage caused by a blood clot that has migrated from another location in the body and lodged in an artery in the brain. However, sometimes a large vessel occlusion can cause such severe damage to the lining that a clot can form itself in the large blood vessel. A less common example of these is a large vessel occlusion called a thrombotic stroke.

[0004] LVO is associated with adverse outcomes at 3 and 6 months in patients with acute ischemic stroke (AIS) (Gandhi CD, Al Mufti F, Singh iP, et al. Neuroendovascular management of emergent large vessel occlusion: technical aspects and practice update by the Standards and Guidelines Committee of the Society of Neurointerventional Surgery; J Neurointerv Surg 2018;10:315-20). Thus, LVO is one of the causes of worse outcomes in subjects who survive an ischemic stroke episode.

[0005] Lakomkin et al. found that the 16 studies included in their systematic review used nine different definitions of LVO (different combinations of the location of arterial occlusion) and that this may condition the prevalence of LVO presented by Waqas et al. (see 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; and 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). A restrictive definition of LVO discloses occlusion of any of the following arteries or arterial segments: intracranial carotid artery (ICA), basilar artery (BA), and M1 segment of middle cerebral artery occlusion (see Vanacker P, Heldner MR, Amiguet M, et al. Prediction of large vessel occlusions in acute stroke: National institute of Health Stroke Scale is hard to beat. Crit Care Med 2016;44:e336-43). No clear definition of LVO exists, but today it is still classified according to the location of occlusion regardless of etiology.

[0006] Recent clinical trials have shown that endovascular treatment of LVO reduces morbidity and mortality in patients who experience this form of severe acute ischemic stroke. Nevertheless, a minority of patients who experience LVO undergo endovascular treatment, often due to delays in arriving at specialized hospitals that can perform endovascular treatment (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). Patients experiencing acute stroke are often first attended to by emergency medical services (EMS) professionals, and early recognition of LVO stroke in the prehospital setting by EMS professionals can improve timely transport to an endovascular treatment facility and result in better patient outcomes (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.). Different scales used to diagnose LVO were compared by Crowe et al. (op. cit.). They showed that of 2,415 patients experiencing acute ischemic stroke, 26% (n=628) of patients with ischemic stroke were diagnosed with LVO.

[0007] A Cincinnati Prehospital Stroke Scale (CPSS) score of 2 or greater demonstrated a sensitivity of 69% and a specificity of 78% for LVO. A Rapid Arterial Occlusion (RACE) score of 4 or greater demonstrated a sensitivity of 63% and a specificity of 73%. A Los Angeles Motor Scale (LAMS) score of 3 or greater demonstrated a sensitivity of 63% and a specificity of 72%, and a positive VAN score demonstrated a sensitivity of 86% and a specificity of 65%. Comparison of the areas under the receiver operating characteristic curves for each scale did not reveal statistically significant differences in their ability to discriminate between LVO strokes. This highlights the need for reliable LVO markers.

[0008] In an attempt to provide a reliable marker for the rapid diagnosis of LVO, the inventors have already proposed in International Patent Application WO2020229691 to utilize circulating levels of retinol binding protein 4 (RBP4) and N-terminal fragment of B-type natriuretic protein (NT-pro-BNP) in isolated samples of patients affected by stroke. The accuracy of the diagnosis could be improved by adding the levels of glial fibrillary acidic protein (GFAP) and further determining D-dimer in isolated samples of patients.

[0009] Despite all efforts, it is apparent that there is a critical need in the art for improved or alternative tests using biomarkers to overcome the limitations of the methods disclosed in the art for accurate diagnosis of LVO and to determine the best therapeutic approach for patients in the shortest time possible. Moreover, while a clear definition of LVO is established, the need for reliable markers is even more urgent since LVO is a condition that requires specific treatment (i.e., endovascular or thrombectomy procedures that may not be generally available in all local facilities). Summary of the Invention [Problem to be solved by the invention]

[0010] Summary of the Invention The inventors have surprisingly found that determining fatty acid binding proteins (FABPs), such as heart-type fatty acid binding protein (HFABP), alone or in combination with the value of at least one clinical parameter of an individual, can provide an accurate diagnosis of LVO in subjects with an episode of ischemic stroke. Diagnostic methods involving the determination of the level of FABP (e.g., HFABP) give sensitivity values ​​of the order of 40-60%, or higher, with high specificity of approximately 90-97%. Moreover, 100% specificity for LVO can be achieved with a sensitivity of more than 50% in true positive LVO subjects, which is considered to be a high value compared to other markers or decision criteria in this field.

[0011] Thus, the method offers the advantage of being able to detect most LVOs, with either a small number of false positives that are not LVO targets (a concept of sensitivity) or a large number of actual true negatives (a concept of specificity).

[0012] Furthermore, the diagnostic method is easily implementable with a simple kit that can be used at the outpatient level or in an ambulance (i.e., as a point-of-care (POC) device), which allows for a rapid determination of the markers, preferably within the first 2 hours after the onset of stroke symptoms. In addition, a rapid determination of actual LVO allows for a timely decision to transport to an endovascular treatment facility, resulting in a better patient outcome.

[0013] We provide an in vitro method for the diagnosis of LVO, comprising determining the level of one or more FABP members, in particular HFABP, in an isolated sample from a subject. [Means for solving the problem]

[0014] Thus, a first aspect of the present invention provides an in vitro method for the diagnosis of LVO comprising determining the level of a FABP member such as HFABP in an isolated sample from a subject and comparing said level with a reference value or range, - if the reference value or range (i.e. interval) is for a subject suffering from LVO and the level of a FABP, such as HFABP, in the isolated sample is equal to or within said value or range, the subject is diagnosed with LVO; or - the reference value is that of a subject not suffering from LVO (i.e. of a healthy subject), and if the level of a FABP, such as HFABP, in the isolated sample is higher than the reference value (also referred to herein and in this example as cut-off discrimination value), the subject is diagnosed with LVO; This is an in vitro method.

[0015] Fatty acid binding proteins (FABPs) are a family of transport proteins for fatty acids and other lipophilic substances. These proteins are thought to facilitate the transport of fatty acids between the outer and inner cell membranes. The family of FABPs includes 12 (FABP-1 to 12) identified proteins and corresponding genes, known primarily by the tissues in which they are expressed. The best characterized are FABP1 to 7. FABP1 is known as liver-type fatty acid binding protein (LFABP) (human protein with accession number P07148 in the Uniprot KB database). FABP2 is known as intestinal-type fatty acid binding protein (IFABP) (human protein with accession number P12104 in the Uniprot KB database). FABP3 or heart-type fatty acid binding protein (HFABP), also known as mammary-derived growth inhibitor, is a protein that, in humans, is encoded by the FABP3 gene. Its Uniprot KB database accession number is P05413 and has a protein sequence length of 133 amino acids that is processed to the mature form (version 4 of the sequence on January 23, 2007, and version 199 of the database entry on February 10, 2021). FABP4, also known as aP2 (adipocyte protein 2) or adipose-type fatty acid-binding protein (AFABP), is a carrier protein of fatty acids that is expressed primarily in adipocytes and macrophages (human protein with accession number P15090 in the Uniprot KB database). FABP5 is known as epithelial-like fatty acid-binding protein (EFABP) (human protein with accession number Q01469 in the Uniprot KB database). FABP6 or ileal (gastrotropin) (human protein with accession number P51161 in the Uniprot KB database). FABP7 or brain lipid-binding protein (BLBP or B-FABP) is expressed in the brain (human protein with accession number O15540 in the Uniprot KB database).

[0016] It was indeed surprising that members of FABP such as HFABP could provide such an accurate diagnostic method for LVO. In patients with large artery atherosclerosis (LAA) and cardioembolism (CE), for example, HFABP was observed to increase with the level of S100 calcium binding protein B (S100B) in plasma during the acute phase of ischemic stroke, but without statistical significance (So-Ypung et al. (2012). Plasma heart type fatty acid binding protein level in acute ischemic stroke: comparative analysis with plasma S100B level for diagnosis of stroke and prediction of long-term clinical outcome. Clinical Neurology and Neurosurgery 115, pp.: 405-410). However, these diseases (i.e., LAA, CE or SVD) are different events from LVO, and in any case, a non-significant correlation was observed for HFABP. In the international patent application WO2018229144, HFABP (i.e., FABP3) is proposed in combination with other markers to distinguish ischemic stroke (IS) and transient ischemic attack (TIA) from hemorrhagic stroke (HS) (see data in Table 3 of WO2018229144). This document does not address LVO diagnosis or selecting candidates for reperfusion therapy.

[0017] Kits are available for determining proteins even at the ambulance level, so the following example shows the determination of HFABP among the members of the FABP family. Nevertheless, other FABPs, in particular BFABPs, can be determined with appropriate kits or reagents for carrying out the method of the present invention.

[0018] The present invention also provides, in a second aspect, an in vitro method for selecting a subject for reperfusion therapy, said subject suffering from LVO in the course of an ischemic stroke episode, the method comprising determining the level of fatty acid binding protein (FABP), in particular heart-type fatty acid binding protein (HFABP), in an isolated sample from the subject, and then comparing said level with a reference value or range, - if the reference value or range (interval) is for a subject suffering from LVO and the level of FABP, in particular HFABP, in the isolated sample is equal to or within said value or range, the subject is diagnosed with LVO and is a candidate for reperfusion therapy; or - if the reference value or range is for a subject not suffering from LVO and the level of FABP, in particular HFABP, in the isolated sample is higher than the reference value or cut-off discrimination value, the subject is diagnosed with LVO and is a candidate for reperfusion therapy; In vitro methods are also provided.

[0019] This method is therefore encompassed as a comparative diagnostic method.

[0020] Considering that FABP, particularly HFABP, and other biomarkers identified in the present invention allow the diagnosis of LVO, and therefore specific treatments are subsequently applied to these subjects, the present invention also provides the above-mentioned method of selecting subjects for reperfusion therapy, particularly thrombectomy. Thus, both aspects are closely and conceptually related.

[0021] Indeed, the main goal of rapid diagnosis of LVO, ideally at ambulance level, is to enable the determination of the best nearby facility where the subject can receive appropriate treatment. LVO is treated in this specialized facility by thrombectomy, which is a type of reperfusion therapy. In other words, the method of the previous embodiment allows the patient to be transferred to the correct hospital, while gaining time to start the correct treatment.

[0022] Reperfusion therapies are disclosed in more detail below, but they include those interventions that can re-establish blood flow either through or around a blocked artery. Reperfusion therapy can be performed utilizing drugs such as thrombolytics (antithrombotic agents) and fibrinolytics. Reperfusion therapy can also be performed with endovascular procedures such as thrombectomy. Subjects diagnosed with LVO are always treated with thrombectomy. However, by using a reference cut-off that allows 100% specificity for actual LVO, subjects can be selected for thrombolytics while thrombectomy is finally performed. As shown, and contrary to expectations, by determining the levels of the FABP member family, especially HFABP, high sensitivity values ​​are achieved with specificity of even 100% for this condition (i.e., LVO). Thus, by determining the levels of FABP, especially HFABP levels, in the ambulance or outpatient department, the correct interim therapy can be quickly determined.

[0023] Similarly, using a cutoff reference of HFABP as an example of a FABP family member that allows 100% specificity for LVO, subjects can be selected for rapid treatment including neuroprotective drugs while preparing them for their remission thrombectomy.

[0024] Indeed, another aspect of the present invention is an in vitro method for selecting subjects for transfer to a thrombectomy facility and / or for direct transfer to an angiography laboratory (DTAS) within said thrombectomy facility, comprising determining a level of fatty acid binding protein (FABP) in an isolated sample from the subject and comparing said level with a reference value or range, (a) if the level of FABP in the isolated sample is equal to or within the range of a reference value for subjects suffering from LVO, the subject is selected to be transferred to a thrombectomy facility; or (b) if the level of FABP in the isolated sample is higher than the reference value or range for subjects not suffering from LVO, the subject is selected to be transferred to a thrombectomy facility; and / or (c) if the level of FABP in the isolated sample is higher than a reference value that allows for 100% specificity discrimination between subjects suffering from either LVO or hemorrhagic stroke and subjects suffering from either non-LVO ischemic stroke or stroke mimic (i.e., stroke mimicking condition), the subject is selected for direct transfer to an angiography laboratory within the facility for said thrombectomy; This is an in vitro method.

[0025] Another embodiment of the present invention is a kit comprising reagent means for simultaneously detecting a FABP family member and one or more of the levels of BNP, DDi, and GFAP.

[0026] Yet another aspect of the present invention is the use of a kit as defined above for diagnosing LVO in a subject or for selecting patients suffering from ischemic stroke for reperfusion therapy, in particular thrombectomy.

[0027] In yet another aspect, the present invention is also directed to the use of a means for detecting the presence of any of the HFABPs, or any other FABP member of the family, in a test sample, said means being selected from the group consisting of immunoassay, protein migration, chromatography, mass spectrometry, turbidimetry, nephelometry and polymerase chain reaction (PCR) for carrying out a method for diagnosing LVO as defined in the first aspect or for selecting patients who have suffered a stroke for reperfusion therapy, primarily for thrombectomy procedures.

[0028] Another aspect of the invention is an in vitro method for selecting a subject for reperfusion therapy and / or treatment with a neuroprotective agent, comprising: (a) determining the level of FABP, particularly HFABP, in an isolated sample from a subject; - one or more clinical parameters selected from the group consisting of blood pressure, including systolic blood pressure (BSP) and / or diastolic blood pressure (DBP) and / or mean blood pressure (mean BP), glycemia, age, stroke-related neurological impairment systematic assessment tool score, time from symptom onset, and sex, and - the level of one or more of the following proteins in an isolated sample of the subject: natriuretic peptides, in particular selected from BNP and ANP; D-dimer (DDi); and GFAP, In combination with: (b) comparing the level of FABP, in particular HFABP, and the level of one or more of the clinical parameters and the levels of other proteins with corresponding reference values ​​or reference intervals of subjects suffering from an ischemic stroke and / or of subjects suffering from a condition selected from hemorrhagic stroke, transient ischemic attack or stroke mimicking conditions, and diagnosing the subject as suffering from an ischemic stroke and as a candidate for reperfusion therapy and / or for treatment with a neuroprotective drug if the level of FABP, in particular HFABP, and likewise the level of one or more of the other determined proteins, and / or the value of one or more of the determined clinical parameters is within the value or interval of values ​​of subjects suffering from an ischemic stroke, It is an in vitro method comprising:

[0029] Similarly, another embodiment of the present invention is an in vitro method for the prognosis of a patient suffering from LVO, comprising determining the level of FABP in an isolated sample of said patient and comparing said level with cut-off values ​​which stratify the patient according to either degree of dependency and / or mortality, wherein if the level of FABP in the sample is higher than the cut-off value, the subject suffering from LVO is: (i) have a prognosis defined by a degree of dependency greater than 2, as determined within 1-5 months after stroke onset according to the modified Rankin Score (mRS); and / or (ii) have a prognosis defined by a 3-month mortality rate of 25% to 35%; This is an in vitro method classified as:

[0030] This prognostic method is of great interest as it will aid future decisions regarding individualized endovascular therapy and the addition of neuroprotective strategies in subjects predicted for poor outcome despite successful reperfusion.

[0031] Yet another aspect of the present invention is a computer implemented method for carrying out the method defined in any of the first and second aspects, wherein after determination of the level of FABP, in particular HFABP, said level is given a value and / or score, optionally calculated with a mathematical formula to obtain a calculated value, and a decision is made between the option of suffering from LVO and / or being a candidate for reperfusion therapy in function of said level, score and / or calculated value. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] Detailed Description of the Invention All terms used herein in this application are to be understood with their ordinary meanings known in the art unless otherwise specified. Other more specific definitions for certain terms used in this application are as set forth below, and are intended to be applied equally throughout the specification and claims, unless another explicitly stated definition provides a broader definition.

[0033] As used herein, the indefinite articles "a" and "an" are synonymous with "at least one" or "one or more." Unless otherwise indicated, definite articles used herein, such as "the," also include a plural of that noun.

[0034] As used herein, the term "patient (or subject)" refers to a person who is presenting with one or more signs or symptoms typically associated with a stroke, such as sudden facial paresis, arm drift, speech abnormalities, and combinations thereof such as FAST (face, arms, speech and time), facial hemiparesis and weakness, numbness, reduced sensory or vibration sensation, spasticity (hypertonia) after initial relaxation (hypotonia), hyperreflexia, obligatory muscle synergies, and especially when they appear on one side of the body (unilateral), changes in smell, taste, hearing, or vision (total or partial), drooping eyelids (ptosis), and weakness of the eye muscles, decreased reflexes (e.g., choking, swallowing, pupillary response to light). "Patient" refers to any subject that exhibits the following symptoms: loss of sensation and facial weakness, loss of balance and nystagmus, changes in respiratory rate and heart rate, sternocleidomastoid muscle weakness with inability to rotate head to one side, tongue weakness (cannot stick out and / or move from side to side), aphasia, dysarthria, apraxia, visual field defects, memory impairment, hemispatial neglect, disorganized thinking, confusion, hypersexual behavior, lack of insight, disability commonly associated with stroke, changes in gait, changes in motor coordination, dizziness, headaches and / or imbalance. The term "patient" as used herein also refers to all animals classified as mammals, including but not limited to domestic and farm animals, primates and humans, such as humans, non-human primates, cows, horses, pigs, sheep, goats, dogs, cats, or rodents. Preferably, the patient is a human, male or female, of any age or race. Preferably, the patient is suffering from stroke.

[0035] The term "selecting a patient for treatment" in this particular description of "thrombectomy" or "endovascular treatment" (used synonymously), optionally in combination with other perfusion techniques such as administration of thrombolytic and fibrinolytic drugs, refers to identifying a patient for treatment designed to cure a disease or alleviate symptoms associated with one or more diseases or conditions. In the particular example of treatment of LVO in a patient suffering from a stroke, any treatment that removes, delays, or reduces symptoms associated with stroke, more particularly ischemic stroke, by removing a thrombus that occludes a large blood vessel, is understood. In effect, the patient is selected for "reperfusion therapy", which refers to a medical procedure that reestablishes blood flow either through or around a blocked artery. Reperfusion therapy includes drugs and endovascular procedures. The drugs are thrombolytic drugs (antithrombotic agents) and fibrinolytic drugs used in a process called thrombolysis. The intervention performed may be a less invasive endovascular procedure (thrombectomy) to remove the clot, possibly using one or more stent retrievers, suction techniques, or alternative devices combining both stent retrievers and suction. Other procedures performed are more invasive bypass surgeries to graft arteries around the blockage. "Mechanical thrombectomy" or simply thrombectomy is an interventional procedure to remove blood clots (thrombi) from blood vessels. It is generally performed in coronary arteries (interventional cardiology), peripheral arteries (interventional radiology), and cerebral arteries (interventional neuroradiology), examples of the latter being referred to in the description herein.

[0036] It is preferred, but not necessary, that the selection or diagnosis of patients is appropriate for 100% of the subjects selected by the first method of the present invention. However, the term requires that a statistically significant proportion of patients is correctly selected. Whether the selection or diagnosis of patients in a subject population is statistically significant can be determined by those skilled in the art using various well-known statistical evaluation tools, such as confidence interval determination, p-value determination, Student's t-test, Mann-Whitney test, etc. Details can be found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 50%, at least more than 60%, at least more than 70%, at least more than 80%, at least more than 90%, or at least 95%. The p-value is preferably 0.01, 0.05, 0.005, 0.001 or lower. In the next section defining the terms "reference value or cutoff" or "reference range / interval", particularly desirable specificity criteria in this field and their impact on the sensitivity of the method are disclosed in more detail.

[0037] The term "ischemic stroke" (abbreviation IS) refers to the physical blocking of blood flow to an area of ​​the brain, causing the death of brain cells in that area. Ischemic stroke can be further divided into thrombotic stroke and embolic stroke. Thrombotic stroke occurs when a cerebral artery is blocked by a blood clot formed in the brain. Embolic stroke is caused by a blood clot formed in a peripheral artery or in the heart that travels to the brain and causes ischemia there. When an embolic or thrombotic stroke blocks a large blood vessel (artery) in the brain, it is also defined as an ischemic stroke with major cerebral artery occlusion (abbreviated as LVO or LVO IS herein). Conversely, non-LVO IS includes any type of ischemic stroke that does not involve major cerebral artery occlusion. Another type of ischemic stroke is lacunar stroke due to occlusion of the cerebellar artery (i.e., these are types that can also be classified as non-LVO IS). Conversely, "hemorrhagic stroke" (abbreviated as ICH for intracerebral hemorrhage) as used herein refers to bleeding into brain tissue due to blood vessel rupture. Stroke mimic, or stroke mimic condition, is defined as a disease or condition that presents a stroke-like clinical picture but does not involve infarction of neural tissue. Multiple clinical syndromes may present symptoms or signs similar to acute ischemic stroke, and thus distinguishing between stroke and stroke mimic is difficult due to the wide variety of overlapping clinical findings. This is a substantial challenge for physicians due to the potential adverse effects of stroke interventional therapy. Today, there are few markers in isolated patient samples that can distinguish between true stroke and mimic.

[0038] The term "expression level of one or more proteins" or "level of one or more in a sample" relates to the amount of protein expressed as a concentration, usually the weight of protein in a volume of sample.

[0039] A "device" or "kit" in the sense of the present invention is an assay or method for determining a biomarker (level of a protein of interest in a sample) or a (combination of) biomarker panel according to the present invention that can be used to perform an assay or method for the diagnosis of LVO or for the selection of patients for thrombectomy or any other reperfusion therapy. Examples are carrier plates, test strips, biochip arrays, or the like, known in the art, that contain reagent means for detecting the presence and level of a protein of interest. A kit as used herein refers to a product that contains the various reagents (or reagent means) required to carry out the method of the present invention, packaged to allow transportation and storage. Materials suitable for packaging the components of the kit include crystal, plastic (e.g., polyethylene, polypropylene, polycarbonate), bottles, vials, paper, or envelopes. Instructions for carrying out the method in different formats are also included in the kit in some embodiments. The particular format of the instructions may be selected from a leaflet, an electronic support capable of storing readable or understandable instructions, such as an electronic storage medium (e.g., magnetic disk, tape), or an optical medium (e.g., CD-ROM, DVD), or audio material.

[0040] A "marker" or "biomarker" or "molecular marker" or "molecular biomarker" or "protein level" or "gene expression level" (all used interchangeably) in the sense of the present invention is any useful biomarker for detecting the presence of LVO in a subject, preferably in a blood, plasma, saliva, tear, CSF or urine sample. The marker (i.e., protein level) is used in a suitable assay setting, in particular whose specificity is set to 90-100% and whose sensitivity is in particular above 40%.

[0041] A marker "panel" in the sense of the present invention is a combination of at least two biomarkers (levels of two proteins in a sample), in particular a combination of two or three or four markers, used in combination in an appropriate setting or device, optionally in combination with a clinical parameter or essential trait of interest.

[0042] As indicated above, in a first aspect, the present invention relates to an in vitro method for the diagnosis of large cerebral artery occlusion (LVO), comprising determining the level of FABP, in particular heart-type fatty acid binding protein (HFABP), in an isolated sample from a subject.

[0043] A LVO, as indicated, is defined as a blockage caused by a blood clot that migrates from elsewhere in the body and lodges in an artery in the brain. Hence, it is truly a LVO.

[0044] The present invention also relates in a second aspect to a direct practical application of the previous aspect, thus to an in vitro method for selecting a subject for reperfusion therapy, said subject suffering from LVO in the course of an ischemic stroke episode, the method comprising determining the level of heart-type fatty acid binding protein (HFABP) in an isolated sample from the subject.

[0045] In certain embodiments of the in vitro method for selecting a subject for reperfusion therapy of the second aspect, the reperfusion therapy is thrombectomy or thrombectomy in combination with prior administration of a thrombolytic and / or fibrinolytic and / or neuroprotective agent.

[0046] In certain embodiments of the in vitro method for selecting a subject for reperfusion therapy of the second aspect, the reperfusion therapy is thrombectomy.

[0047] Therefore, in a more particular manner, a second aspect of the present invention relates to an in vitro method for selecting a subject for thrombectomy, comprising the above-mentioned steps of firstly determining the level of a fatty acid binding protein (FABP), in particular a heart-type fatty acid binding protein (HFABP), in an isolated sample from the subject, and secondly comparing said level with a reference value or range, - if the reference value or range (i.e., interval) is for a subject suffering from LVO, and the level of a FABP, such as HFABP, in the isolated sample is equal to or within said value or range, then the subject is diagnosed with LVO and is a candidate for reperfusion therapy; or - if the reference value or range is for a subject not suffering from LVO and the level of a FABP, such as HFABP, in the isolated sample is higher than the reference value or cut-off discrimination value, the subject is diagnosed with LVO and is a candidate for reperfusion therapy; This is an in vitro method.

[0048] As illustrated in the examples and data obtained by the inventors, the method allows the implementation of a novel strategy to select patients who are directly directed to a thrombectomy facility or to an angiography room where thrombectomy is performed (direct transfer). This novel strategy directly correlates with a better prognosis for patients in terms of the degree of care after a stroke episode. In fact, patients can be appropriately recommended for thrombectomy two hours earlier than would be the case with other protocols (i.e., first directing the patient to the closest facility for stroke treatment, but one that does not allow thrombectomy). If the subject is directly transferred to an angiography room, the patient can be appropriately recommended for thrombectomy two and a half hours earlier than would be the case with other protocols.

[0049] In certain embodiments of either the in vitro method for diagnosis of major cerebral artery occlusion (LVO) or the in vitro method for selecting a subject for reperfusion therapy, LVO manifests as occlusion of one or more of the following arteries or arterial segments: occlusion of the intracranial internal carotid artery (ICA), occlusion of the base of the skull (BA), and occlusion of the M1 segment of the middle cerebral artery.

[0050] Similarly, in another specific embodiment of either the in vitro method for diagnosis of cerebral large artery occlusion (LVO) or the in vitro method for selecting a subject for reperfusion therapy, the FABP is selected from one or more of liver-type fatty acid binding protein (LFABP), intestinal-type fatty acid binding protein (IFABP), heart-type fatty acid binding protein (HFABP), adipocyte-type fatty acid binding protein (AFABP), epithelial-like fatty acid binding protein (EFABP), FABP6 (or ileal or gastrotropin), and brain lipid binding protein (BLBP or B-FABP).

[0051] In a more particular embodiment, the FABP is selected from HFABP and BFABP, and combinations thereof. More particularly, the FABP is HFABP.

[0052] In certain embodiments of either the in vitro method for diagnosing a large vessel occlusion (LVO) or the in vitro method for selecting a subject for reperfusion therapy, the method further included determining one or more clinical parameters.

[0053] The term "clinical parameters" or clinical data as used herein refers to human demographics (age or birth date, race and / or ethnicity), clinical symptoms or signs of a patient related to a disease / condition associated with stroke. The term also includes laboratory parameters such as glycemia determination. These clinical parameters are annotated values ​​of the above features, which are taken into account in the diagnosis determination or treatment selection in combination with the level of FABP, such as HFABP, in the isolated sample of the subject. These clinical parameters are obtained or collected from the subject as normal parameters or information that are taken into account when a stroke is suspected, any parameter that can be measured or determined at the ambulance level (i.e., electrocardiogram, fundus examination, etc.). These annotated values ​​of clinical parameters are calculated in a formula or algorithm, in certain embodiments, together with the detected level of FABP, such as HFABP, and other protein levels, if determined, of the subject suspected of having a stroke. The output of the calculated or applied algorithm provides information for making a decision (i.e., classification for treatment and / or diagnosis of a condition, such as LVO).

[0054] In a more specific embodiment, the subject's clinical parameters are selected from the group consisting of blood pressure, including systolic blood pressure (SBP) and / or diastolic blood pressure (DBP), mean blood pressure (mean BP), glycemia, age, stroke-related neurological disorder assessment tool score, time from symptom onset, sex, presence of atrial fibrillation, and combinations thereof. In an even more specific embodiment, the clinical parameters are selected from stroke-related neurological disorder assessment tool score, blood pressure, including systolic blood pressure (SBP) and / or diastolic blood pressure (DBP), mean blood pressure (mean BP), and combinations thereof.

[0055] The term "systematic assessment tool for stroke-related neurological impairment" refers to a tool designed to measure and evaluate the most common neurological impairments in stroke. Several aspects or parameters are evaluated, such as level of consciousness, visual field, facial weakness, motor ability of the limbs, gaze, sensory loss, coordination (ataxia), language (aphasia), speech (dysarthria), etc. For all of them, a value is given, 0 being normal. Therefore, in most of these tools, the higher the score, the worse the neurological impairment. Those skilled in the art will know the existence of different tools for this purpose.

[0056] Thus, in an even more particular embodiment of either the first or second method of the present invention, the score of the systematic assessment tool of stroke-related neurological deficits is selected from a score of the NIH Stroke Scale (NIHSS), the Rapid Arterial Occlusion Assessment Scale for Stroke (RACE), the Cincinnati Prehospital Stroke Scale compared to Stroke Severity Tool for Prediction of Major Cerebral Artery Occlusion (Cincinnati Score or CPSS), the Los Angeles Motor Scale (LAMS), the Vision-Aphasia-Hemispatial Neglect (VAN), Stroke Triage with Field Assessment for Emergency Delivery (FAST-ED), or the modified Rankin Scale or score (mRS).

[0057] Publications disclosing these tools include, for example, Noorian AR, Sanossian N, Shkirkova K, Liebeskind Ds, Eckstein M, Stratton SJ, Pratt FD, Conwit R, Chatfield F, Sharma LK, Restrepo L, Valdes-Sueiras M, Kim, Tenser M, Starkman S, Saver JL; FAST-MAG Trial Investigators and Coordinators. Los Angeles Motor Scale to Identify Large Vessel Occlusion: Prehospital Validation and Comparison With Other Screens. Stroke. 2018 Mar;49(3):565-572. Another publication is Gropen TI, Boehme A, Martin-Schild S, Albright K, Samai A, Pishanidar S, Janjua N, Brandler ES, Levine SR. Derivation and Validation of the Emergency Medical Stroke Assessment and Comparison of Large Vessel Occlusion Scales. J Stroke Cerebrovasc Dis. 2018 Mar;27(3):806-815.

[0058] The term "NIHSS score" as used in the present invention refers to the score on the NIH Stroke Scale (NIHSS), a systematic assessment tool that provides a quantitative measure of neurological impairment associated with stroke. The NIHSS was initially designed as a research tool to measure baseline data on patients in acute stroke clinical trials. Today, the scale is also widely used as a clinical assessment tool to evaluate the acuity of stroke patients, determine appropriate treatment, and predict patient outcomes. The NIHSS is a 15-item neurological stroke scale used to evaluate the impact of acute cerebral infarction on level of consciousness, language, hemispatial neglect, visual field defects, extraocular movements, motor skills, ataxia, dysarthria, and sensory loss. Trained observers assess the patient's ability to answer questions and perform behaviors. Each item is scored on a grade of 3 to 5, with 0 being normal, and non-testable items are allowed. Levels of stroke severity measured by the NIH Stroke Scale scoring system: 0=no stroke, 1-4=mild stroke, 5-15=moderate stroke, 15-20=moderate / severe stroke, 21-42=severe stroke. In the present invention, the term "higher scores" refers to scores of 5-42 on the NIH Stroke Scale scoring system.

[0059] Similarly, modifications of the NIHSS such as the Rapid Assessment of Arterial Occlusion score for Stroke (RACE) or other scores used to identify ischemic stroke such as the CPSS with major cerebral artery occlusion may be used.

[0060] In another particular embodiment of an in vitro method for the diagnosis of LVO or for selecting a subject for reperfusion therapy, the method comprises determining levels of one or more of the following proteins: natriuretic peptides, including one or more of B-type natriuretic peptide (BNP) and atrial natriuretic peptide (ANP); DDi, and GFAP.

[0061] In certain embodiments, optionally in combination with any of the embodiments of the above or below aspects, the natriuretic peptide is a B-type natriuretic peptide. In even more particular embodiments, the BNP determined in the isolated sample of the subject is a prohormone called N-terminal fragment of B-type natriuretic peptide (NT-proBNP), which is a secreted biologically inactive form of BNP. The N-terminal fragment of B-type natriuretic peptide 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 active B-type natriuretic peptide (BNP). BNP is a hormone secreted by cardiac myocytes in the ventricles in response to the strain caused by an increase in ventricular blood volume. The complete human sequence BNP has Uniprot KB accession number P16860 (August 1, 1990 - sequence version 1, and database publication 187 on May 8, 2019). For simplicity, NT-proBNP and BNP are used herein as synonymous abbreviations, however, in the examples below, NT-proBNP was the form ultimately determined in the isolated samples.

[0062] The term "DDi" refers to D-dimer (or D-dimer), which is a fibrinolysis product (or FDP), a small protein fragment present in the blood after blood clots are broken down by fibrinolysis. It is so named because it contains two D fragments of fibrinoprotein joined by a cross-link. The concentration of D-dimer may be determined by a blood test to aid in the diagnosis of thrombosis.

[0063] As used herein, the term "GFAP" refers to glial fibrillary acidic protein, an intermediate filament protein expressed by numerous cell types in the central nervous system. The complete human sequence for glial fibrillary acidic protein has Uniprot KB accession number P14136 (January 1, 1990 - sequence version 1, and April 7, 2021 database release 221).

[0064] In more particular embodiments of the method, the method comprises determining the level of one or two of the above-listed proteins in combination with the level of FABP, particularly HFABP. In more particular embodiments of these methods that comprise determining the level of FABP, particularly HFABP, and one or two of the other above-listed proteins, the method also comprises determining one or more clinical parameters of the subject.

[0065] The inventors have realized that when the level of HFABP in isolated samples is determined in combination with one or more of the clinical parameters listed above, the value of 100% specificity for correctly classifying LVO subjects from non-LVO subjects can be fixed at a certain cutoff / reference (see examples below). This certain specificity gives a sensitivity of around 50% for detecting positive LVO from a population. In addition, when considering (determining) the level of one or two of the proteins listed above, a specificity of 100% is obtained with a sensitivity of around 75%.

[0066] In another particular embodiment of the method of the first and second aspects of the invention, optionally in combination with any of the above or below specific embodiments, said method comprises determining at least one of the following combinations of markers (i.e. the levels of HFABP and other proteins in an isolated sample of the subject, and / or taking into account one or more clinical parameters of the subject in the determination): (a) HFABP + NIHSS + DBP + age; (b) HFABP + NT-proBNP + NIHSS + DBP + age; (c) HFABP+DDi+NIHSS+DBP+age; (d) HFABP+GFAP+NIHSS+DBP+age; (e) HFABP+NT-proBNP+DDi+NIHSS+DBP+age; (f) HFABP+NT-proBNP+GFAP+NIHSS+DBP+age; (g) HFABP+GFAP+DDi+NIHSS+DBP+age; (h) HFABP + NIHSS + SBP + age; (i) HFABP + NIHSS + mean BP + age; and (j) HFABP + NTproBNP + NIHSS + mean BP + age.

[0067] As illustrated in the Examples, most of these combinations, including determining the levels of HFABP, and specific combinations (a)-(g), provided sensitivities for diagnosing LVO of greater than 40%, greater than 70%, combined with specificities of greater than 90% or 100%.

[0068] Combination of the level of HFABP with the score of another systematic assessment tool for stroke-related neurological disorders (Cincinnati-CPSS) also provides high sensitivity for LVO diagnosis with high specificity. Thus, in another specific embodiment of the method of the first and second aspects of the invention, optionally in combination with any of the specific embodiments described above or below, said method comprises determining at least one of the following combinations of markers (i.e., the level of HFABP and other proteins in an isolated sample of the subject, and / or taking into account one or more clinical parameters of the subject in the determination): (k) HFABP+Cincinnati+DBP+age; (l) HFABP + Cincinnati + SBP + age; (m) HFABP+Cincinnati+mean BP+age; (n) HFABP+NT-proBNP+Cincinnati+DBP+age; (o) HFABP+DDi+Cincinnati+DBP+age; (p)HFABP+GFAP+Cincinnati+DBP+age; (q) HFABP+NT-proBNP+DDi+Cincinnati+DBP+age; (r) HFABP+NT-proBNP+GFAP+Cincinnati+DBP+age; (s) HFABP+GFAP+DDi+Cincinnati+DBP+AGE; and (t)HFABP+NTproBNP+Cincinnati+meanBP+age.

[0069] In yet another particular embodiment, when the in vitro method of the invention comprises determining the level of a natriuretic peptide such as DDi, GFAP, NT-proBNP, etc. in an isolated sample from a subject, and / or determining one or more clinical parameters disclosed above: - if the level of FABP, in particular HFABP, is equal to or within the reference range for subjects affected by LVO and the level of one or more of the other listed proteins is also within the reference range for subjects affected by LVO; and / or - if the level of FABP, in particular HFABP, is equal to or within the reference range for subjects affected by LVO and the level of one or more of the clinical parameters is also within the reference range for subjects affected by LVO, The subject has been diagnosed with LVO; and / or - the method further comprises a step of calculating the determined levels of the proteins in the formula and all values ​​of the determined clinical parameters to obtain a predictor, and diagnosing the subject as suffering from +VO if the predictor is within a reference value (i.e. cut-off) or within an interval for LVO.

[0070] These methods, which involve determining the level of one or more proteins in an isolated sample and / or taking into account one or more clinical parameters of the subject in combination with the level of a FABP, such as HFABP, in said determination, provide more accurate classification of patients, including greater sensitivity and specificity.

[0071] The term "reference value" as used herein refers to a predetermined standard used as a reference for evaluating values ​​or data obtained from samples collected from subjects. Reference values ​​or reference levels can be absolute values ​​(i.e., cut-off values ​​or cut-off discrimination values); relative values; values ​​with upper or lower limits; ranges of values ​​(i.e., ranges of possible cut-off values); representative values; median values; mean values; or values ​​compared to a specific control or baseline value. Reference values ​​or ranges can be based on individual sample values, such as values ​​at earlier time points obtained from samples of subjects to be tested. Reference values ​​or ranges can be based on multiple samples, such as from a population of subjects of a chronological age-matched group, or on a pool of samples that includes or excludes the sample to be tested. Reference values ​​have been determined for the biomarkers of the present invention. Reference values ​​for FABPs, such as HFABP, can be from lower and higher values, as disclosed in light of the following examples. The ranges of values ​​(protein levels) of each biomarker, and the specific combinations of values ​​of different biomarkers provide correct classification of subjects with high sensitivity and specificity.

[0072] In fact, in different alternative embodiments of the method of the disclosed aspect of the present invention, when it includes the option of comparing the tested level with each cut-off value or reference interval, these values ​​or intervals may vary, and they are selected taking into account the specific values ​​of desired sensitivity and specificity. Thus, when 100% specificity (true negative or correct classification between two conditions) is desired, the sensitivity (true positive, or detection of one condition in a cohort of subjects with different conditions) can be reduced. On the other hand, a reduction in specificity (i.e., around 94% or 98%) generally allows the method to have an increased sensitivity. Therefore, the reference value / cut-off can vary according to the desired specificity and / or the desired sensitivity. For example, for a certain pair of specificity and sensitivity, a cut-off will be appropriate, but for a different pair of specificity and sensitivity, a different cut-off will be considered as reference. In the same way, a certain cut-off may indicate or distinguish one condition among a plurality of different conditions, and another cut-off may give additional information, such as prognostic information, about the same condition.

[0073] In the present case, cut-offs for HFABP, as an example of a FABP family member, selected from the interval defined by 1.25 ng / ml to 6.0 ng / ml in blood have been determined to be beneficial for the correct diagnosis of LVO and for different features of this LVO condition, in particular cut-offs for blood HFABP levels of 1.25, 1.50, 1.75, 2.00, 2.25, 2.50, 2.75, 3.0, 3.25, 3.50, 3.75, 4.0, 4.25, 4.50, 4.75, 5.0, 5.25, 5.50, 5.75, 6.0 ng / ml.

[0074] Furthermore, the reference value or reference interval may vary depending on the characteristics of the subject, such as the subject's race or the subject's ethnicity / geographic genetic background. The reference values ​​(i.e., cut-offs) in the following examples are determined for white subjects, particularly from a Spanish cohort.

[0075] In summary, the absolute value of the reference value or the absolute group of values ​​within an interval is adjusted according to a particular parameter, but the validity of determining a FABP such as HFABP alone or in combination with the levels of other parameters or other proteins is such that a determination can be made with confidence by comparing the level of the FABP (i.e. HFABP) with said reference.

[0076] In fact, the accuracy of the statistical methods used according to the present invention can be best described by the receiver operating characteristic (ROC). The ROC curve addresses both the sensitivity of the test, i.e. the number of true positives, and the specificity, i.e. the number of true negatives. Therefore, the sensitivity and specificity values ​​for a given combination of biomarkers are indicators of the accuracy of the assay. For example, if a combination of biomarkers has a sensitivity and specificity value of 80% for LVO, then from the determination of the presence of a particular combination of biomarkers, 80 out of 100 patients with LVO ischemic stroke will be correctly identified as positive for LVO ischemic stroke (LVO IS), and 80 out of 100 patients without LVO ischemic stroke (non-LVO IS) will be correctly tested negative for this disease. In other words, the sensitivity of the method provides information about true positive subjects, or the proportion of subjects with LVO that are correctly detected from a group. The specificity of the method provides information about true negative subjects, or the proportion of subjects that are correctly excluded from a group. Thus, in the context of the present invention, 100% specificity provided by the method means that all non-LVO subjects are excluded, which means that only most (80%) of the actual LVO subjects are correctly selected for specific and specific appropriate treatment, thus not wasting time and effort trying to treat non-LVOs with inappropriate treatments (e.g., non-LVOs do not require thrombectomy).

[0077] A level of a biomarker (in the present invention, the level of HFABP as a FABP family member, or any other protein listed) is considered higher than a reference value or range if the level of the biomarker (in the present invention, the level of HFABP as a FABP family member, or any other protein listed) is at least 1.5%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150% higher or higher than the reference value.

[0078] Similarly, in the context of the present invention, the level of a biomarker in a sample is reduced if said level is lower than a reference value. A level of a biomarker is considered to be lower than a reference value if the level of the biomarker is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150% lower or lower than the reference value.

[0079] The term "predictor" as used herein refers to a factor derived from the determined levels of the above-mentioned proteins, optionally in combination with one or more clinical parameters. The predictor can be calculated by summing the values ​​obtained for each protein and / or the values ​​of the clinical parameters, corrected by a specific coefficient for each of the proteins and / or for the clinical parameters. Statistical methods for calculating such correction coefficients are known to those skilled in the art. This "predictor" can also be obtained by applying different algorithms known to those skilled in the art. Examples of possible algorithms, including statistical / mathematical algorithms, are listed in more detail below.

[0080] The inventors also understand that the level of HFABP (as an example of FABP) in isolated samples indicates that a subject suffers from LVO, and if said level is increased by at least 4 units compared to the cutoff or reference for diagnosing LVO, the subject can be classified as a subject that in fact suffers from LVO and is poorly responsive to treatment and applied or recommended treatment regarding post-stroke dependency.In other words, these subjects with these very high levels of HFABP show a response to applied treatment that corresponds to an increase of 2.006 (p-value=0.040) of the odds ratio of dependency at 3 months, that is, doubling the risk of modified Rankin score (mRS) higher than 2.

[0081] Thus, in a more detailed embodiment of the first aspect, if a subject has been diagnosed with LVO, the subject is also classified as having a degree of dependency greater than 2 according to the modified Rankin Score (mRS) determined within 1 to 5 months after the stroke episode, preferably determined 3 months after the stroke, if the level of HFABP in an isolated sample of the subject diagnosed with LVO is at least 4 units higher than the cutoff or reference for diagnosing LVO.

[0082] This particular embodiment of the diagnostic method is also applied to a method for selecting subjects for thrombectomy (i.e., reperfusion, which is most likely to be successful in these patients). Note that thrombectomy is recommended even in cases of likely poor or no response to treatment.

[0083] In fact, in connection with this observation regarding the type of response to treatment of subjects suffering from ischemic stroke with LVO, the inventors disclose a method for prediction of the response of patients suffering from ischemic stroke with LVO, i.e. candidates for reperfusion therapy, in particular thrombectomy, which method comprises determining the expression level of HFABP (as an example of FABP) in an isolated sample of the patient, then comparing said level with a reference value (i.e. cut-off) to allow classifying the patient as suffering from LVO and a level increased by at least 4 units compared to said cut-off or reference for the diagnosis of LVO indicating a type of response resulting in or progressing to a degree of assistance greater than 2, determined within 1 to 5 months after the onset of the stroke according to the modified Rankin Score (mRS). To the best of the inventors' knowledge, this is the first time that such a poor response to a treatment involving HFABP for LVO has been shown.

[0084] The inventors have also realized that HFABP is a marker that allows for the implementation of rapid decisions for in-hospital intervention in rooms dedicated to thrombectomy, so-called angiography rooms or angiography rooms. Hospital angiography rooms are areas of hospitals where catheter interventions and stent placements are performed. These rooms contain diagnostic imaging equipment that is used to visualize the arteries of the heart or brain and then treat any stenosis or abnormality (i.e., LVO) that is found.

[0085] Thus, as clearly explained above, another aspect of the present invention is an in vitro method for selecting subjects to be transferred to a thrombectomy facility and / or moved directly to an angiography laboratory within said thrombectomy facility, comprising determining the level of fatty acid binding protein (FABP) in an isolated sample from the subject and comparing said level to a reference value or range, (a) if the level of FABP in the isolated sample is equal to or within the range of a reference value for subjects suffering from LVO, the subject is selected to be transferred to a thrombectomy facility; or (b) if the level of FABP in the isolated sample is higher than the reference value or range for subjects not suffering from LVO, the subject is selected to be transferred to a thrombectomy facility; and / or (c) if the level of FABP in the isolated sample is higher than a reference value that allows for 100% specificity in distinguishing between subjects suffering from either LVO or hemorrhagic stroke and subjects suffering from either non-LVO ischemic stroke or stroke mimic, the subject is selected for direct transfer to the angiography laboratory in said facility for thrombectomy. This is an in vitro method.

[0086] In certain embodiments of the method for selecting subjects to be transported to a thrombectomy facility and / or moved directly to an angiography laboratory within said thrombectomy facility, the FABP is selected from HFABP, BFABP, and combinations thereof.

[0087] Thus, in particular, an in vitro method is disclosed for the diagnosis of LVO and for selecting subjects for thrombectomy with prior (i.e. conventional CT) scanning of the brain in an angiography laboratory, comprising the steps of determining the level of HFABP in an isolated sample from a subject and comparing said level with a reference value (i.e. cut-off value) capable of discriminating with 100% specificity between subjects suffering from either LVO or hemorrhagic stroke and subjects suffering from either non-LVO IS or stroke mimic, and if the level is higher than this cut-off, the subject is selected for thrombectomy with prior scanning of the brain in an angiography laboratory.

[0088] The rationale for this method is that subjects with hemorrhagic stroke are not selected for thrombectomy, but the hemorrhage can be detected at least by brain scanning equipment in the angiography room with a regular computed tomography (CT) scan. If no hemorrhage is detected after this scan in the angiography room, the subject has LVO and he / she is already in the appropriate room to perform the thrombectomy. This ultimately results in a quicker treatment, 2 to 2.5 hours faster than if the subject first went to the emergency department of the hospital where he / she first underwent a more complex but accurate brain scanning by cerebral angiography. If the scan in the angiography room reveals the presence of hemorrhage, the subject is then directed to the corresponding neurosurgery or stroke unit area of ​​the hospital.

[0089] Thus, this method for selecting subjects for a thrombectomy facility and / or for direct transfer to an angiography laboratory is included in the methods of either the first and second aspects, which in fact have the property that the selected reference cut-off allows for a classification that facilitates the treatment of actual LVO ischemic stroke.

[0090] In another particular embodiment of any of the methods of the preceding aspects, optionally in combination with any of the above or below embodiments, if the subject is diagnosed with LVO: Subjects were also randomized to receive FABP if their level was higher than the cutoff values ​​that stratified patients according to either degree of dependency and / or mortality. (i) have a prognosis defined by a disability rating of greater than 2 according to the modified Rankin Score (mRS), determined within 1 to 5 months after the onset of the stroke; and / or (ii) have a prognosis defined by a 3-month mortality rate of 25% to 35%; It is classified as follows.

[0091] This prognostic value relative to the diagnosis of LVO is extremely advantageous and is illustrated by the representative cohorts in the Examples.

[0092] The term "sample" as used herein relates to any sample that can be obtained from a patient (subject). The method of the present invention can be applied to any type of biological sample from a patient, such as a biopsy sample, tissue, cell or body fluid (plasma, serum, saliva, semen, sputum, cerebrospinal fluid (CSF), tears, mucus, sweat, breast milk, brain extract and the like).

[0093] In another specific embodiment of any of the methods of the previous aspect, optionally in combination with any of the above or below described embodiments, the isolated sample of the subject (i.e., the patient who has suffered a stroke) is a body fluid. Exemplary and non-limiting body fluids are blood (i.e., whole blood), plasma, serum, saliva, urine or cerebrospinal fluid. In a more preferred embodiment, the body fluid is plasma or serum. In another more preferred embodiment, the body fluid is whole blood obtained either by fingerprinting or by collecting in a test tube.

[0094] Different samples can be used to determine the levels of different markers. Thus, it is not necessary that the levels of all markers according to the method of the present invention are measured in the same type of sample. Thus, in another specific embodiment, the level of HFABP (as an example of FABP) and, if determined, the levels of other proteins listed (i.e., GFAP, DDi, BNP, etc.) are measured in serum or whole blood. In another specific embodiment, the level of any of them is measured in plasma.

[0095] In a preferred embodiment of the method of the present invention, a sample is obtained at baseline.

[0096] "Baseline" as used in the present invention is considered to be any time from the onset of symptoms to the first time the patient is seen by a doctor. This is usually within the first few hours after stroke, usually the first care in an ambulance or in a hospital. In a preferred embodiment, baseline is within the first 4.5 hours after the onset of symptoms, or less than 6 hours after stroke, or in another preferred embodiment, less than 24 hours after the onset of symptoms. In another specific embodiment, the level of markers, particularly one or more of HFABP (as an example of FABP) and NT-proBNP (as an example of natriuretic peptide), DDi, and GFAP, is determined within the first 24 hours after the onset of symptoms, within the first 12 hours, within the first 6 hours, or within the first 3 hours after stroke onset or symptoms. The inventors understand that accurate classification can be obtained by considering the time of measurement of the level of HFABP in isolated samples.

[0097] In another particular embodiment of either the first and second aspect of the invention, or any other aspect set out above, the in vitro method comprises determining the level of FABP, in particular HFABP, and, if determined, the level of one or more of BNP (e.g. NT-proBNP), DDi and GFAP, within the first 2 hours after stroke onset, more in particular within the first hour after stroke onset.

[0098] Thus, after a diagnosis of LVO has been made, in the first and second alternative specific embodiments, the method further comprises the step of recommending reperfusion therapy, in particular thrombectomy, to said diagnosed subject and / or treating said subject with reperfusion therapy, in particular thrombectomy.

[0099] This particular embodiment can be drafted as a method of treating a subject suffering from LVO, comprising carrying out an in vitro method for the diagnosis of LVO in a patient according to the first aspect, and treating the patient diagnosed with LVO with a reperfusion therapy, in particular a thrombectomy. Advantageously, by this method the patient is treated, or recommended to be treated, with the most appropriate therapeutic regimen within the first few hours of symptom onset.

[0100] As another specific embodiment of the method of the first and second aspects of the invention, also optionally in combination with any of the above or below embodiments of these aspects, it is also encompassed herein that if the level of at least a FABP, such as HFABP, is equal to or within a reference range for subjects with LVO or higher than a reference value for subjects not suffering from LVO, the invention further comprises the step of treating the subject with said reperfusion therapy, such as thrombectomy.

[0101] The present disclosure also provides a method for detecting a level of a FABP, such as HFABP, in an isolated sample from a subject who has suffered a stroke during LVO, comprising: (a) obtaining a sample from a subject; and (b) detecting whether a FABP, such as HFABP, is present in the isolated sample by (i) contacting the sample with a means capable of binding to a corresponding expressed FABP (e.g., HFABP) and detecting said binding, or (ii) contacting the sample with a means capable of binding to the corresponding RNA that will be translated into said FABP (e.g., HFABP) and detecting said binding; The present invention also encompasses a method comprising the steps of:

[0102] In certain embodiments, this detection method is also carried out in step (b) by detecting whether one or more of BNPs, such as NT-proBNP, DDi and GFAP, are present in the isolated sample by (i) contacting the sample with a means capable of binding to the corresponding expressed protein and detecting said binding, or (ii) contacting the sample with a means capable of binding to the corresponding RNA, which will be translated into one or more of the corresponding proteins, and detecting said binding.

[0103] The determination of the level of protein (i.e., HFABP and other proteins listed) in the isolated sample in all in vitro methods of the present invention can be performed by qualitative and / or quantitative tests selected from the group consisting of immunological tests, bioluminescence, fluorescence, chemiluminescence, electrochemical tests and mass spectrometry. Certain tests that can be implemented in point-of-care testing format (POCT) are recommended to make the determination of marker levels easy and fast. In certain embodiments, point-of-care testing encompasses lateral flow testing, which is capable of detecting the presence (or absence) of target analytes in liquid samples (matrix) without the need for dedicated expensive equipment, although there are many laboratory-based applications supported by reading equipment.

[0104] Independent of the test format, the particular quantitative test is selected from the group consisting of immunoassays, bioluminescence, fluorescence, chemiluminescence, electrochemical tests and mass spectrometry.

[0105] In one embodiment, the level of expression is determined by immunological techniques such as enzyme-linked immunosorbent assay (ELISA), enzyme immunodot assay, agglutination assay, antibody-antigen-antibody sandwich assay, antigen-antibody-antigen sandwich assay, immunochromatography or other immunoassay formats well known to those skilled in the art such as radioimmunoassay, as well as protein microarray formats such as single molecule assay (SIMOA), Western blot or immunofluorescence.

[0106] Western blot is based on the detection of proteins previously resolved by gel electrophoresis under denaturing conditions and fixed on a membrane, generally nitrocellulose, by incubation with an antibody-specific color-developing system (e.g., chemiluminescent). Analysis by immunofluorescence requires the use of an antibody specific for the target protein for the analysis of expression. ELISA is based on the use of an antigen or antibody labeled with an enzyme, where the conjugate formed between the target antigen and the labeled antibody leads to the formation of an enzymatically active complex. Since one of the components (antigen or labeled antibody) is fixed to a support, the antibody-antigen complex is fixed to the support and thus the complex can be detected by adding a substrate, which is converted by the enzyme into a product that can be detected, for example, by spectrophotometry, fluorimetry, mass spectrometry, or Tandem Mass Spectrometry Tag (TMT). SIMOA is a type of assay that is more sensitive than ELISA, since it uses an array of femtoliter-sized reaction chambers called single molecule arrays (Simoa™), which can isolate and detect single enzyme molecules. The array volume is roughly 2 billion times smaller than conventional ELISA, resulting in rapid build-up of fluorescent product when labeled protein is present. When diffusion collapses, this high local concentration of product can be easily observed. Only a single molecule is required to reach the detection limit. This method has been used to measure proteins in a variety of different matrices (serum, plasma, cerebrospinal fluid, urine, cell extracts, etc.) at femtomolar (fg / mL) concentrations using the same reagents as conventional ELSA, providing roughly a 1000-fold improvement in sensitivity.

[0107] Alternatively, the determination of protein levels can be carried out by constructing tissue microarrays (TMA) containing the assembled subject samples and determining the expression levels of the proteins by techniques well known in the state of the art.

[0108] In a preferred embodiment, the determination of the level of the marker is determined by an immunological technique, hi a more preferred embodiment, the immunological technique is an ELISA.

[0109] When immunological methods are used, any antibody or reagent known to bind with high affinity to the target protein (i.e., HFABP and optionally others listed above) can be used to detect the amount of the target protein. Nevertheless, the use of antibodies, such as polyclonal sera, hybridoma supernatants or monoclonal antibodies, antibody fragments, Fv, Fab, Fab' y F(ab')2, ScFv, diabodies, triabodies, tetrabodies and humanized antibodies is preferred.

[0110] As will be understood by those skilled in the art, the expression levels of FABPs, such as HFABP, and, if determined, the expression levels of other proteins listed, can be detected by measuring the levels of mRNA encoded by the corresponding genes.

[0111] As a non-limiting example, the expression level is determined by quantifying the level of the mRNA encoded by said gene. The latter can be quantified by using conventional methods, such as methods involving the amplification of mRNA and the quantification of the amplified product of said mRNA, such as electrophoresis and staining, or using Northern blot and a suitable probe, using Northern blot and a specific probe of the mRNA or the corresponding cDNA / cRNA of the gene of interest, mapping with SI nuclease, RT-PCR, hybridization, microarray, etc. Similarly, the level of cDNA / cRNA corresponding to said mRNA encoded by a marker gene can also be quantified by utilizing conventional techniques, but in this case, the method of the present invention includes a step of synthesizing the corresponding cDNA by reverse transcription (RT) of the corresponding mRNA, and then a step of synthesizing (RNA polymerase) and amplifying cRNA complementary to said cDNA. Conventional methods for quantifying expression levels can be found in laboratory manuals.

[0112] To normalize the value of mRNA expression in different samples, it is possible to compare the expression level of the mRNA of interest in the test sample with that of a control RNA. As used herein, "control RNA" refers to an RNA whose expression level does not change or changes only in a limited amount. Preferably, the control RNA is an mRNA derived from a housekeeping gene and encodes a protein that is constitutively expressed and performs essential cellular functions. Preferred housekeeping genes for use in the present invention include 18-S ribosomal protein, beta-2-microglobulin, ubiquitin, cyclophilin, GAPDH, PSMB4, tubulin, and beta-actin.

[0113] In another particular embodiment of the in vitro method of the present invention that provides diagnosis of LVO and information for selecting a treatment, the method further comprises the steps of (i) collecting diagnostic information and (ii) storing the information on a data carrier.

[0114] A "data carrier" in the sense of the present invention should be understood as any means, such as paper, that contains meaningful information data for the diagnosis of LVO and / or for the selection of candidates for reperfusion therapy (e.g. thrombectomy). The carrier can be any object or device capable of carrying diagnostic data or information for selecting a treatment. For example, the carrier may include a storage medium such as a ROM, e.g. a CD ROM or a semiconductor ROM, or a magnetic recording medium, e.g. a floppy disk or a hard disk. Furthermore, the carrier may be a transmissible carrier, such as an electric or optical signal that can be conveyed via an electric or optical cable or by wireless or other means. In case the diagnostic / treatment selection data is embodied in a signal that can be directly transmitted by a cable or other device or means, the carrier may be constituted by such a cable or other device or means. Other carriers concern USB devices and computer archives. Examples of suitable data carriers are paper, CD, USB, computer archive in a PC, or acoustic registration of the information.

[0115] As indicated above, the present invention also relates to kits comprising reagent means for detecting the levels of one or more members of the FABP family.

[0116] More particularly, the kit comprises reagent means for simultaneously detecting the levels of one or more members of the FABP family and one or more levels of natriuretic peptides such as ANP or BNP (particularly NT-proBNP), DDi, and GFAP.

[0117] In certain embodiments of the kit, the kit comprises reagent means for simultaneously detecting the level of HFABP and one or more of the levels of NT-proBNP, DDi, and GFAP.

[0118] In addition, the kits of the invention may contain instructions for simultaneous, sequential or separate use of the different components in the kit. The instructions may be in the form of printed material or in the form of an electronic support capable of storing easily readable or understandable instructions, such as, for example, an electronic storage medium (e.g., magnetic disk, tape), or an optical medium (e.g., CD-ROM, DVD) or an audio material. Additionally or alternatively, the medium may include an internet address providing the instructions.

[0119] The reagent means (or simply, the reagents) of the kit comprises a compound which specifically binds to a protein of interest whose level in an isolated sample is to be determined. In another more particular embodiment of the kit of the present invention, said compound which specifically binds to a protein is selected from an antibody, an aptamer, a fragment of either an antibody or an aptamer, and combinations thereof.

[0120] These antibodies or aptamers, or their fragments, specifically recognize FABP, such as HFABP.Other antibodies of the kit specifically recognize other proteins, as determined in the corresponding specific embodiments of the method of the first and second aspects.Antibodies of the kit of the present invention can be used according to the techniques known in the art for determining protein expression levels, such as flow cytometry, Western blot, ELISA, RIA, competitive EIA, DAS-ELISA, techniques based on the use of biochips, protein arrays, or colloidal precipitation assays in reaction strips, etc.

[0121] The antibodies may be immobilized on a solid support, such as a membrane, plastic or glass, optionally treated to facilitate immobilization of the antibodies to the support, which contains at least one set of antibodies that specifically recognize a marker (i.e., a protein of interest) and can be used to detect the expression level of the marker.

[0122] In addition, the kit of the present invention includes a reagent for detecting the protein encoded by a constitutive gene. The availability of said additional reagent allows the normalization of measurements carried out in different samples (e.g., the sample to be analyzed and the control sample) to exclude cases where the difference in expression of biomarkers is due to different amounts of total protein in the samples that exceeds the actual difference in relative expression level. The constitutive genes of the present invention are always active or constantly transcribed genes, which are constitutively expressed and encode proteins that perform essential cellular functions. Proteins that are constitutively expressed and can be used in the present invention include, but are not limited to, beta-2-microglobulin (B2M), ubiquitin, 18-S ribosomal protein, cyclophilin, GAPDH, PSMB4, tubulin and actin.

[0123] In a preferred embodiment, the reagent means for assaying the levels of different biomarkers (proteins) constitutes at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% of the total amount of reagents for assaying the biomarkers forming the kit. Thus, in a particular example of a kit comprising reagents for assaying the levels of HFABP (or any other FABP member of the family) and one or more of the natriuretic peptides (e.g. BNP such as NT-proBNP and / or ANP), DDi and GFAP, the reagents specific for said biomarkers (i.e. antibodies that specifically bind to the proteins) constitute at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% of the antibodies present in the kit. Therefore, these kits are simplified kits that mainly comprise the reagent means for detecting the level of HFABP (or any other FABP member of the family) and the level of one or more of the following natriuretic peptides: ANP and BNP, such as NT-proBNP; DDi and GFAP.In particular, the reagent means for assaying the level of HFABP and the level of one or two of the following natriuretic peptides (e.g., NT-proBNP), DDi and GFAP.

[0124] In a particular embodiment of the kit, the kit comprises, as a single reagent means (i.e., antibody, aptamer, fragment) for detecting proteins, a compound that specifically binds to HFABP (or any other FABP member of its family) and a compound that specifically binds to DDi (or any detectable isoform of DDi). In another embodiment, the kit comprises, as a single reagent means for detecting proteins, a compound that specifically binds to HFABP (or any other FABP member of its family) and a compound that specifically binds to GFAP (or any detectable isoform of GFAP). In another embodiment, the kit comprises, as a single reagent means for detecting proteins, a compound that specifically binds to HFABP (or any other FABP member or isoform of its family) and a compound that specifically binds to a natriuretic peptide, in particular selected from BNP and ANP. In another embodiment, the kit comprises, as a single reagent means for detecting proteins, a compound that specifically binds to HFABP (or any other FABP member of its family), a compound that specifically binds to DDi (or any detectable isoform), and a compound that specifically binds to a natriuretic peptide, in particular selected from BNP and ANP. In another embodiment, the kit comprises, as a single reagent means for detecting proteins, a compound that specifically binds to HFABP (or any other FABP member or isoform thereof), a compound that specifically binds to DDi (or any detectable isoform thereof), and a compound that specifically binds to GFAP (or any detectable isoform thereof). In another embodiment, the kit comprises, as a single reagent means for detecting proteins, a compound that specifically binds to HFABP (or any other detectable FABP member or isoform thereof), a compound that specifically binds to GFAP, and a compound that specifically binds to a natriuretic peptide or any isoform thereof, in particular selected from BNP and ANP.

[0125] In the previous section, the isoforms of the proteins listed relate to any form of the expressed protein of interest for which an assay is available, while this isoform is applicable to methods for diagnosis or therapeutic selection in the sense that it provides relevant information.

[0126] In another particular embodiment, the kits of the invention are considered as point-of-care tests, more particularly they are in the form of lateral flow tests.

[0127] In another particular embodiment, the kit according to the invention comprises a carrier, one or more sample inlets for receiving a body fluid sample, in particular whole blood, and a reaction area comprising means / reagents for specifically binding to a marker protein, in particular an antibody, the sample inlets being linked to the reaction areas. In another more particular embodiment, the kit comprises as many sample inlets as there are markers to be detected (one, two or three) and corresponding reaction areas linked thereto. In another embodiment, the kit comprises one single inlet and a capillary track linked to the same number of reaction areas, said capillary track directing a portion of the sample to each corresponding linked reaction area. A kit comprising more than one type of reaction area is a multiplex kit.

[0128] In another embodiment, the present invention relates to the use of the kit of the present invention for diagnosing LVO or for selecting stroke-affected patients for thrombectomy or any other appropriate reperfusion therapy, such as treatment with antithrombotic agents.

[0129] Thus, in a particular embodiment, the present invention relates to the use of the kit of the present invention in any of the methods of the present invention.

[0130] The present inventors have also determined another combination of markers that allows accurate diagnosis of LVO in subjects suffering from ischemic stroke. This combination includes determining the levels of natriuretic peptides, particularly BNP (e.g. NT-proBNP), GFAP and DDi in isolated samples from subjects. High sensitivity with high specificity has been determined.

[0131] In a more specific embodiment of this practical combination of markers, the levels of NT-proBNP, GFAP and DDi are determined in isolated samples, and then calculated in combination with one or more clinical parameters.In particular, they are calculated in combination with the values ​​of NIHSS, diastolic blood pressure (DBP) and subject's age.A specific cutoff of any of the proteins, higher than the reference value of non-LVO subjects (i.e., healthy subjects) for each of the proteins, achieves a sensitivity of around 80%, with a specificity of around 95%.More particularly, the cutoffs and the values ​​of NIHSS and age and DBP are as follows: Thresholds (cutoffs): NT-proBNP>1385.5, DDI>359.289, GFAP>52.1995, NIHSS>9.5, age>84.5, DBP<90.5, SE=78.9%, SP=94.3%.

[0132] This combination therefore also allows the selection of subjects to be selected for reperfusion therapy, in particular thrombectomy. Accordingly, kits and means for carrying out the method for the diagnosis of LVO by this combination are also disclosed.

[0133] The inventors have also determined that the level of HFABP in combination with certain clinical parameters can accurately distinguish between subjects suffering from ischemic stroke and subjects suffering from any other condition selected from hemorrhagic stroke, transient ischemic attack or stroke mimicking condition.This accurate classification also allows for a method for selecting candidates for reperfusion therapy when a subject is classified as suffering from ischemic stroke.

[0134] Therefore, also disclosed is an in vitro method for discriminating ischemic stroke from any other condition selected from hemorrhagic stroke, transient ischemic attack or stroke mimicking conditions in a subject, comprising determining the level of FABP, in particular HFABP, in an isolated sample of the subject in combination with one or more clinical parameters selected from the group consisting of blood pressure including systolic blood pressure (BSP) and / or diastolic blood pressure (DBP), mean blood pressure (mean BP), glycemia, age, score from a systematic assessment tool for stroke-related neurological disorders, time from symptom onset and gender, the method further comprising comparing said level of FABP, in particular HFABP, and the level of one or more of the clinical parameters with those of a subject affected by ischemic stroke. The method comprises the step of comparing the level of FABP, particularly HFABP, and one or more values ​​of the determined clinical parameters with corresponding reference values ​​or intervals of subjects suffering from either an ischemic stroke and / or a hemorrhagic stroke, a TIA or a stroke mimic (i.e. a stroke mimicking condition), and if the level of FABP, particularly HFABP, and one or more values ​​of the determined clinical parameters are within the value or interval of values ​​of subjects suffering from an ischemic stroke, the subject is diagnosed as suffering from an ischemic stroke, or if the level of FABP, particularly HFABP, and one or more values ​​of the determined clinical parameters are within the value or interval of values ​​of subjects suffering from any of the other conditions, the subject is diagnosed with any other condition selected from hemorrhagic stroke, a transient ischemic attack or a stroke mimicking condition.

[0135] Another aspect of the invention is an in vitro method for distinguishing ischemic stroke from any other condition selected from hemorrhagic stroke, transient ischemic attack or stroke mimicking conditions, comprising: (a) determining the level of FABP, particularly HFABP, in an isolated sample from a subject; - one or more clinical parameters selected from the group consisting of blood pressure, including systolic blood pressure (BSP) and / or diastolic blood pressure (DBP) and / or mean blood pressure (mean BP), glycemia, age, a score from the Systematic Assessment Tool for Stroke-Related Neurological Disorders, time from symptom onset, and sex, and - the level of one or more of the following proteins in an isolated sample of the subject: natriuretic peptides, in particular selected from BNP and ANP; D-dimer (DDi); and GFAP, In combination with the above, (b) comparing the level of FABP, in particular HFABP, and the level of one or more of the clinical parameters and the level of the other proteins with corresponding reference values ​​or intervals of subjects suffering from ischemic stroke and / or of subjects suffering from either hemorrhagic stroke, TIA or stroke mimic, and if the level of FABP, in particular HFABP, and the level of one or more of the other determined proteins and / or the value of one or more of the determined clinical parameters are within the value or interval of values ​​of subjects suffering from ischemic stroke, the subject is diagnosed as suffering from ischemic stroke, or if the level of FABP, in particular HFABP, and the level of one or more of the other determined proteins and / or the value of one or more of the determined clinical parameters are within the value or interval of values ​​of subjects suffering from any of the other conditions, the subject is diagnosed as suffering from any other condition selected from hemorrhagic stroke, transient ischemic attack or stroke mimic condition, It is an in vitro method comprising:

[0136] In certain embodiments of the in vitro method for distinguishing ischemic stroke from these other previously listed conditions, the method further comprises the steps of calculating all of the determined levels of the protein and the determined clinical parameter values ​​in a form to obtain a predictor, and diagnosing the subject as suffering from ischemic stroke and therefore as a candidate for reperfusion if this predictor is within a reference value or interval for ischemic stroke.

[0137] The step of calculating the values ​​of the determined levels of proteins and all of the determined values ​​of clinical parameters is also an alternative step (b) of the in vitro method for distinguishing ischemic stroke from any of the other conditions listed above (i.e. hemorrhagic stroke, TIA or mimic) according to the present invention.

[0138] In another more particular embodiment of the in vitro method for distinguishing ischemic stroke from other conditions listed above (i.e. hemorrhagic stroke, TIA or mimics) according to the present invention, optionally in combination with any of the above or below specific embodiments, the method comprises determining at least one of the following combinations of markers (i.e. levels of HFABP and other proteins) in an isolated sample from the subject and / or taking into account one or more clinical parameters of the subject in said determination: (a) HFABP + NIHSS + DBP + age (b) HFABP + NIHSS + mean BP + age (c) HFABP + NT-proBNP + NIHSS + DBP + age (d) HFABP + DDi + NIHSS + DBP + age (e) HFABP + GFAP + NIHSS + DBP + age (f) HFABP+NT-proBNP+DDi (g) HFABP + NT-proBNP + DDi + NIHSS + mean BP + age (h)HFABP+NT-proBNP+GFAP+NIHSS+average BP+age (i) HFABP + GFAP + DDi + NIHSS + mean BP + age (j)HFABP+NT-proBNP+DDi+NIHSS+DBP+age; (k) HFABP+NT-proBNP+GFAP+NIHSS+DBP+age; (l) HFABP + GFAP + DDi + NIHSS + DBP + age (m) HFABP+NIHSS+SBP+age; (n) HFABP+Cincinnati+DBP+age; (o) HFABP+Cincinnati+SBP+age; (p)HFABP+Cincinnati+mean BP+age; (q) HFABP+NT-proBNP+Cincinnati+DBP+age; (r)HFABP+DDi+Cincinnati+DBP+age; (s) HFABP+GFAP+Cincinnati+DBP+age; (t)HFABP+NT-proBNP+DDi+Cincinnati+DBP+age; (u) HFABP+NT-proBNP+GFAP+Cincinnati+DBP+age; (v) HFABP+GFAP+DDi+Cincinnati+DBP+age; (w) HFABP + NTproBNP + NIHSS + mean BP + age; and (x) HFABP+NTproBNP+Cincinnati+mean BP+age.

[0139] As illustrated in the examples, all of these combinations, including determining the level of HFABP, made it possible to provide a sensitivity of 50-90% for differential diagnosis when a specificity of 100% was fixed. Considering the seriousness of false positives of ischemic stroke (i.e., true patients with hemorrhagic stroke) being finally treated with reperfusion therapy, these are advantageous features of the proposed method for differential diagnosis.

[0140] All embodiments disclosed for the previous aspects relating to the in vitro method for diagnosis of LVO or the in vitro method for selecting subjects for thrombectomy also apply to this aspect of the differential diagnosis between ischemic and hemorrhagic stroke.

[0141] Following from this embodiment, there is also provided an in vitro method for selecting a subject for reperfusion therapy and / or for treatment with a neuroprotective drug, the method comprising: (a) determining the level of FABP, particularly HFABP, in an isolated sample from a subject; - one or more clinical parameters selected from the group consisting of blood pressure, including systolic blood pressure (BSP) and / or diastolic blood pressure (DBP) and / or mean blood pressure (mean BP), glycemia, age, score from the Systematic Assessment Tool for Stroke-Related Neuropathy, time from symptom onset, and sex, and - the level of one or more of the following proteins in an isolated sample of the subject: natriuretic peptides, in particular selected from BNP and ANP; D-dimer (DDi); and GFAP, In combination with: (b) comparing the level of FABP, in particular HFABP, and the level of one or more of the clinical parameters and the levels of other proteins with corresponding reference values ​​or intervals of subjects suffering from ischemic stroke and / or subjects suffering from hemorrhagic stroke, transient ischemic attack or stroke mimicking conditions, and diagnosing the subject as suffering from ischemic stroke and as a candidate for reperfusion therapy and / or treatment with a neuroprotective drug if the level of FABP, in particular HFABP, and the level of one or more of the other determined proteins and / or the value of one or more of the determined clinical parameters is within the value or interval of values ​​of subjects suffering from ischemic stroke, Also disclosed is an in vitro method comprising:

[0142] Neuroprotective therapy, which is mainly performed by neuroprotective drugs, is used in subjects diagnosed with real ischemic stroke (including LVO-IS and non-LVO-IS), even at the ambulance level, with the following drugs: NA-1 (nerinetide), uric acid, activated protein C or 3K3A-APC-activated protein C, alpha-1-antitrypsin, fingolimod, metmorfin, glyburide, glibenclamide, TLR-4 inhibitors, IL1 inhibitors (IL1-RA, anakinra), nitric oxide donors, glyceryl trinitrate (GTN), cilostazol, ginkgo biloba extracts and derivatives, antioxidants, edaravone and edaravone derivatives, resveratrol, melatonin, NAD, anti-intercellular adhesion molecule-1 (ICAM-1) antibodies, enlimomab, calcium stabilizers, and anti-excitotoxic agents, MaxiPost (B MS-204352), nalmefene (Cerven), fosphenytoin, enoxaparin, trafermin, ancrod, magnesium, UK-279,276, ONO-2506, dipyridamole, repinotan, simvastatin, lubelozole, buspirone, nimodipine, heparin, YM872, aptiganel (CNS-1102, Cerestat), diazepam, clomethiazole, natalizumab, ebselen, flunarizine, pentoxifylline, abciximab, pethidine, dextromethorphan, granulocyte-colony stimulating factor (G-CSF) and other growth factors, pituitary adenylate cyclase-activating polypeptide, MMP-9 inhibitors, minocycline, or combinations thereof, or combinations of these and other related neuroprotective agents. Neuroprotective therapy also includes recommendations to administer one or more of the compounds listed above in combination with other neuroprotective strategies such as hypothermia, remote limb ischemic postconditioning (RIPC), increasing collateral cerebral blood flow, blood pressure manipulation, etc., which may be performed in the ambulance when diagnostic testing is performed.

[0143] There are several treatment protocols for promoting reperfusion. In certain embodiments of the first aspect of the invention, the reperfusion therapy is selected from the group consisting of antithrombotic treatment, thrombectomy, and combinations thereof.

[0144] In a more particular embodiment, the antithrombotic agent is a thrombolytic agent. In an even more particular embodiment, the thrombolytic agent is a plasminogen activator. More particularly, the plasminogen activator is tissue plasminogen activator.

[0145] The term "antithrombotic agent" as used herein refers to a drug capable of reducing blood clot formation. Suitable antithrombotic agents for use in the present invention include, but are not limited to, thrombolytic agents, antiplatelet agents, and anticoagulant compounds.

[0146] The term "thrombolytic drug" as used herein refers to a drug capable of dissolving blood clots. All thrombolytic drugs are serine proteases that convert plasminogen to plasmin, which breaks down fibrinogen and fibrin, and dissolves blood clots. Thrombolytic drugs available today include reteplase (r-PA or Retavase), alteplase (t-PA or Activase), urokinase (Abbplomase), pro-urokinase, anisoylated purified streptokinase activator complex (APSAC), staphylokinase (Sak), tenecteplase (TNKase® or TNK-tPA by Genenthec), atenecteplase (TNKasa), anistreplase (Eminase), streptokinase (Kabikinase, Streptase) or urokinase (Abokinase). Tenecteplase (TNK-tPA) is used in certain embodiments because it can be administered as a fast-acting single bolus and can be used at ambulance level. TNK is effective one minute after administration (after injection). Suppliers of TNK are Boehringer Ingelheim (European Union) and Genentech Inc (USA).

[0147] The term anticoagulant compound as used herein refers to compounds that prevent clotting, including, but not limited to, vitamin K antagonists (warfarin, acenocoumarol, phenprocoumon, and phenidione), heparin and heparin derivatives such as low molecular weight heparins, synthetic pentasaccharides, factor Xa inhibitors such as direct thrombin inhibitors (argatroban, lepirudin, bivalirudin, and ximelagatran), and antiplatelet compounds that act by inhibiting platelet aggregation, i.e., thrombus formation, including, but not limited to, cyclooxygenase inhibitors (aspirin), adenosine diphosphate receptor inhibitors (cyclopidrogel and ticlopidine), phosphodiesterase inhibitors (cilostazol), glycoprotein IIB / IIIA inhibitors (abuximab, eptifibatide, tirofiban, and defilotide), and adenosine uptake inhibitors (dipyridamole). In a preferred embodiment, the antithrombotic agent is a thrombolytic drug. In a more preferred embodiment, the thrombolytic agent is a plasminogen activator. In an even more preferred embodiment, the plasminogen activator is tPA (tissue plasminogen activator).

[0148] The term "tissue plasminogen activator (t-PA)" as used herein refers to a serine protease found on endothelial cells that catalyzes the conversion of plasminogen to plasmin. The complete protein sequence of human t-PA has UniProt KB Accession No. P00750 (July 11, 2012). tPA may be produced using recombinant biotechnology techniques, and tPA made in this manner may be referred to as recombinant tissue plasminogen activator (rtPA). Recombinant tissue plasminogen activators (r-tPA) include the thrombolytic agents alteplase, reteplase, and tenecteplase (also referred to as TNKase®, TNK-tPA). In human t-PA, the amino acids at positions 296-299 are a lysine, a histidine, and two arginines. In TNK-tPA, these amino acids are replaced with four alanines. This mutation may be responsible for high resistance to plasminogen activator inhibitor 1 (PAI-1).

[0149] The dose of t-PA should be given within the first 3 hours of symptom onset or up to 4.5 hours from symptom onset. Recommended total dose: 0.9 mg / kg infused over 60 minutes (maximum dose should not exceed 90 mg). Loading of 0.09 mg / kg (10% of the 0.9 mg / kg dose) as an intravenous bolus over 1 minute, followed by 0.81 mg / kg (10% of the 0.9 mg / kg dose) as a continuous infusion over 60 minutes. Heparin should not be started for more than 24 hours after starting alteplase for stroke. The t-PA is given intravenously, and in some cases may be given directly into an artery, and should be given immediately after the first symptoms of stroke occur. The doses and routes of administration apply to any of the embodiments of the first aspect. Also includes the step of treating a patient in certain embodiments.

[0150] After determining that a subject who has suffered a stroke is a candidate for reperfusion therapy, a single dose of TNK-tPA should be given as soon as possible and within the first 3 hours of symptom onset or up to 4.5 hours from symptom onset, preferably within the first hour after stroke onset.

[0151] As indicated above, the use of TNK-tPA is particularly useful because, in its particular embodiment as a fast-acting, single-application bolus, it can be administered at any point of care, even at ambulance level, and is effective approximately one minute after administration.

[0152] Those patients who have suffered a stroke who are not selected for perfusion therapy are, in certain embodiments, selected for a treatment to reduce blood pressure, in particular said treatment being carried out with a drug capable of reducing blood pressure.

[0153] "Blood pressure" should be understood herein to refer to blood pressure at central arterial sites such as the aorta and carotid arteries. Central blood pressure may be measured non-invasively at the carotid or radial arteries, suitably by applanation tonometry (described below). Thus, the term "blood pressure" as used herein includes aortic blood pressure.

[0154] "Drug capable of reducing blood pressure" as used in the present invention relates to any drug that reduces blood pressure by different means, the most widespread of which are thiazide diuretics (furosemide, nitroprusside, hydralazine, etc.); ACE inhibitors that are calcium channel blockers (such as nicardipine or nimodipine); adrenergic receptor antagonists (such as the alpha adrenergic antagonist urapidil) or combinations of alpha and beta blockers (labetalol and nitroglycerin); and angiotensin II receptor antagonists (ARBs). Illustrative, non-limiting examples of drugs capable of lowering or reducing blood pressure are alpha-methyldopa (Aldomet), 11,17-alpha-dimethoxy-18β[(3,4,5-trimethoxy-benzoyl)oxyl)]-3p,2a-yohimban-16β-carboxylic acid methyl ester (reserpine) or 2-(2,6-dichlorophenylamino)2-imidazole hydrochloride (clodinine hydrochloride), lergotrile, i.e., 2-chloro-6-methylergoline-8β-acetonitrile, as disclosed in European Patent 0005074. Reference values ​​used to lower blood pressure in ischemic stroke, ischemic stroke treated with thrombolytic drugs or hemorrhagic stroke may be updated and thus may be recommended by clinical practice guidelines. Today, treatment modalities for lowering blood pressure aim to achieve a systolic blood pressure between 220 and 120 mmHg in ischemic patients and between 180 and 100 mmHg in hemorrhagic patients. In a preferred embodiment, blood pressure may be reduced by intravenous administration of a drug capable of reducing blood pressure and co-administration of oral antihypertensive drug(s). The reference values ​​used to reduce blood pressure in ischemic stroke, ischemic stroke treated with thrombolytic drugs or hemorrhagic stroke may be updated and therefore will be those recommended by clinical practice guidelines.

[0155] A reduction in arterial pressure shall be detected after administration of the drug, and any suitable method for measuring arterial pressure may be used to determine whether the drug is capable of reducing blood pressure. Illustrative and non-limiting examples of methods for measuring arterial pressure are non-invasive techniques such as palpitation, auscultation, oscillometry, and continuous non-invasive arterial pressure (CNAP), as illustrative and non-limiting examples.

[0156] Thus, after making a discriminatory diagnosis, in another particular embodiment of this discriminatory method for ischemic stroke and / or reperfusion and / or neuroprotective therapy, the method further comprises the step of recommending reperfusion therapy and / or treatment with neuroprotective drugs to the patient diagnosed with ischemic stroke, and / or treating said patient diagnosed with ischemic stroke with reperfusion therapy, mainly antithrombotic drugs or thrombectomy. Alternatively, those patients diagnosed with any other condition, such as ICH, where reperfusion therapy should be avoided to avoid fatal outcomes, are in another particular embodiment recommended or treated with a therapy that reduces or optimizes blood pressure.

[0157] This particular embodiment may be drafted as a method of treating a patient who has suffered a stroke, said method comprising carrying out an in vitro method for distinguishing IS from other conditions such as ICH in a patient as disclosed above, and treating patients diagnosed with IS with reperfusion therapy, primarily antithrombotic agents, or utilizing thrombectomy, or treating patients diagnosed with other conditions, particularly patients diagnosed with ICH, with therapies that reduce or optimize blood pressure.

[0158] Advantageously, this method allows the patient to be treated, or recommended to be treated, with the most appropriate therapeutic regimen within the first few hours of symptom onset.

[0159] As previously reviewed for the methods of the first and second aspects, in certain embodiments of the in vitro method for distinguishing ischemic stroke from hemorrhagic stroke, transient ischemic attack or any other condition selected from stroke mimicking conditions according to the present invention, the method further comprises the steps of (i) collecting diagnostic information and (ii) storing the information on a data carrier, which is as defined above.

[0160] Similarly, another embodiment of the present invention is an in vitro method for prognosis of a patient suffering from LVO, comprising determining the level of FABP in an isolated sample of said patient and comparing said level with cut-off values ​​which stratify the patient according to either degree of dependency and / or mortality, wherein if the level of FABP in the sample is higher than the cut-off value, the subject also suffers from LVO. (i) have a prognosis defined by a disability rating of greater than 2 according to the modified Rankin Score (mRS), determined within 1 to 5 months after the onset of the stroke; and / or (ii) have a prognosis defined by a 3-month mortality rate of 25% to 35%; This is an in vitro method classified as:

[0161] In certain embodiments of the in vitro method for determining the prognosis of a patient suffering from LVO according to the preceding aspects, the FABP is selected from HFABP, BFABP, and combinations thereof.

[0162] Similarly, in another particular embodiment of the in vitro method for prognosis, the method further comprises determining one or more clinical parameters and / or characteristics of the subject, said parameters being in particular selected from the group consisting of blood pressure including systolic blood pressure (SBP) and / or diastolic blood pressure (DBP), mean blood pressure (mean BP), glycemia, age, score from the Systematic Assessment Tool for Stroke-Related Neuropathy, time from symptom onset, gender, and combinations thereof.

[0163] As indicated for other aspects of the invention in which a diagnosis of LVO is performed, in certain embodiments of the prognostic method, the score from a systematic assessment tool of stroke-related neurological deficits is selected from the NIH Stroke Scale (NIHSS) score, Rapid Arterial Occlusion for Stroke (RACE), Cincinnati Prehospital Stroke Scale compared to Stroke Severity Tool for Prediction of Major Cerebral Artery Occlusion (Cincinnati Score or CPSS), Los Angeles Motor Scale (LAMS), Vision-Aphasia-Hemispatial Neglect (VAN), Stroke Triage by Field Assessment for Emergency Transport (FAST-ED), or modified Rankin Scale or score (mRS).

[0164] Similarly, in another particular embodiment of the in vitro method for prognosis of a subject previously diagnosed with LVO by determination of FABP, particularly HFABP, the method further comprises determining the level of one or more of the following natriuretic peptides in an isolated sample from the subject: NT-proBNP; D-dimer (DDi); and glial fibrillary acidic protein (GFAP).

[0165] All of the embodiments that apply to the first and second aspects of the invention regarding measurement times and sample types apply equally to this other aspect of the in vitro method for the prognosis of patients affected by LVO.

[0166] Finally, an algorithm is provided herein for carrying out either the method of diagnosis and / or selection of patients for treatment and / or prognosis as defined in the previous embodiment. The term "algorithm" in the sense of the present invention is also synonymous with a panel or decision diagram, predictor, combinatory of data for the correct selection of individual samples.

[0167] According to aspects and embodiments of the present invention, diagnosis of LVO and / or selection for reperfusion therapy and / or discrimination of stroke type and subtype is performed using a mathematical algorithm that evaluates detectable levels of FABP, such as HFABP, and other proteins, if determined, detected by the means disclosed above (i.e., antibodies or fragments thereof), in conjunction with or independently of other clinical parameters, and correctly classifies individual samples as being obtained from healthy patients (i.e., mimics), patients with ischemic stroke with LVO, patients with ischemic stroke without LVO (non-LVO IS), patients with hemorrhagic stroke.

[0168] A classification algorithm can be as simple as determining whether the amount of a specific biomarker or a subset of measured biomarkers is higher than or lower than a certain cutoff number (or absolute value). When multiple biomarkers are used, the classification algorithm can be a linear regression formula. Alternatively, the classification algorithm can be a product of any of a number of learning algorithms. For complex classification algorithms, a computer, for example a programmable digital computer, may be used to perform the algorithm on the data and thereby determine the classification. For example, and as previously revealed, the algorithm is for performing the steps of calculating all of the determined levels of protein and the values ​​of clinical parameters determined by the formula to obtain a predictor, and if this predictor is within a reference value (i.e., cutoff) or interval for LVO, diagnosing the subject as suffering from LVO, and then selecting the subject for perfusion therapy, particularly thrombectomy; or if the predictor is within a reference value (i.e., cutoff) or interval for ischemic stroke, diagnosing the subject as suffering from ischemic stroke and not suffering from hemorrhagic stroke, TIA or mimic. In either case, the condition can then be recorded in a tangible medium, for example in a computer-readable form, such as a memory drive or disk, or simply printed on paper.Results can also be reported on a computer screen.This algorithm is used as a diagnostic and / or prognostic method, and it is particularly part of a kit for carrying out the method disclosed in the former embodiment.

[0169] Those skilled in the art know many suitable methods for developing and applying statistical algorithms, all of which are within the scope of the present invention. Examples of suitable classification algorithms include, as indicated, linear regression such as logistic regression after stepwise variable selection, classification and regression trees, threshold-based algorithms such as the Panelomix (Panelomix) algorithm, Naive Bayes and Random Forest classifiers, among others. The inventors have found that logistic regression after stepwise variable selection, classification and regression trees, and threshold-based algorithms can achieve similar performance in the context of the present invention, suggesting that the analyte (i.e., biomarker) used in the method of the present invention is more important than the method used to create the algorithmic model.

[0170] As indicated before, another aspect of the present invention is also a computer implemented method for performing the method defined in any of the first and second aspects, wherein after determining the level of HFABP, said level is given a value and / or score, optionally which is calculated in a mathematical formula to obtain a calculated value, and a decision is made between the option of suffering from LVO and / or the option of being a candidate for reperfusion therapy in function of said level, score and / or calculated value.

[0171] In a particular embodiment, the formula is implemented by a particular algorithm.

[0172] Similarly, there is provided a computer-implemented method for performing a method for diagnosis of LVO or for selecting a subject for perfusion therapy, comprising: after determining in an isolated sample of the subject a level of a FABP, such as HFABP, and, if determined, a level of one or more of a natriuretic peptide selected from B-type natriuretic peptide (BNP) and / or atrial natriuretic peptide (ANP); DDi; and GFAP, and / or, if determined, a level of one or more clinical parameters of the subject; Disclosed herein is a method in which said level(s) are given a value and / or score, optionally calculated in a mathematical formula to obtain a calculated value, and depending on said level(s), score(s) and / or calculated value(s), a decision is made between an option of suffering from LVO and / or an option of being a candidate for reperfusion therapy, and / or an option of suffering from an ischemic stroke or an option of suffering from any other condition selected from hemorrhagic stroke, transient ischemic attack (TIA) and stroke mimic.

[0173] Throughout the specification and claims, the language "comprises" and variations of that language do not exclude other technical features, additives, ingredients, or steps. Furthermore, the language "comprises" encompasses the example of "consisting of." Additional objects, advantages, and features of the present invention will become apparent to those skilled in the art upon examination of the specification or may be learned by practice of the present invention. The following examples are provided by way of illustration and are not intended to be limiting of the present invention. Furthermore, the present invention includes all possible combinations of the specific and preferred embodiments described herein.

[0174] Working Example Example 1 1. A cohort of patients assayed for the diagnosis of LVO-ischemic stroke (abbreviation LVO-IS or LVO) [Table 1]

[0175] 2. Methods for the two cohorts and statistics Emergency hospital (N=197) Patients with suspected stroke (<6 hours) were enrolled upon arrival to the emergency room (ED). Blood samples were collected on admission and HFABP, NT-proBNP, D-dimer, GFAP and RBP-4 were measured by immunoassay and POCT.

[0176] Ambulance - outside hospital (N=182) Patients with suspected stroke (<6 hours) were enrolled in the ambulance network. Blood samples were collected at the patient's home or in the ambulance, and HFABP, NT-proBNP, D-dimer, and GFAP were measured by immunoassay and POCT.

[0177] To investigate the classification performance of the markers and the panel feasibility, multiple combinations of interesting markers were tested using three different approaches: logistic regression after stepwise variable selection, the CART classification trees of Breiman et al. (1984), and Panelomix, a threshold-based algorithm for creating panels of biomarkers by Robin et al. (2013) (2.). Output models were validated from the three methods with measurements from separate cohorts. All analyses were performed with R (1.) statistical software, version 4.0.2, using the implementations available in the Panelomix and rpart libraries of Robin et al. (2013) and Thermoteau et al. (2019) (3.). 1. Core Team (2020). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL https: / / www.R-project.org / . 2. Xavier Robin, Natacha Turck, Alexandre Hainard, Natalia Tiberti, Frederique Lisacek, Jean-Charles Sanchez and Markus Muller (2011). PanelomiX: A threshold-based algorithm to create panels of biomarkers. Translational Proteomics, 1(1), p. 57-64. DOI:10.1016 / j.trprot.2013.04.003 <http: / / dx.doi.org / 10.1016 / j.trprot.2013.04.003> 3. Terry Therneau and Beth Atkinson (2019). rpart: Recursive Partitioning and Regression Trees. R package version 4.1-15. https: / / CRAN.R-project.org / package=rpart

[0178] 3.Results 3.1. HFABP in combination with clinical parameters and possibly other proteins provides high specificity with high sensitivity for screening patients for LVO and other stroke-related conditions The following Tables 1 and 2 show the marker classification performance and panel feasibility of different combinations including the determination of HFABP levels in isolated samples together with data derived from clinical parameters (i.e., blood pressure, age, scores from the Systematic Assessment Tool for Stroke-Related Neuropathy, and levels of other proteins in the samples). Table 1 includes the combination with the Cincinnati score tool, and Table 2 includes the combination with the NIHSS score tool. Sensitivity (SE) and specificity (SP) are: - LVO ischemic stroke diagnosis (column 2); - Classification or diagnosis of ischemic stroke associated with other conditions, including stroke (hemorrhagic), mimic, and TIA (column 3) - Classification of patients with either hemorrhagic stroke associated with LVO or mimic or non-LVO ischemic stroke We will show the combinations that make this possible. [Table 2] [Table 3]

[0179] In these tables 1 and 2, when BNP is mentioned, it refers to the determination of NT-proBNP fragments in isolated samples. As previously disclosed, cut-off values ​​may vary according to the type of population, race or region depending on the genetic background, so tables 1 and 2 do not indicate the cut-offs for each combination. However, the main goal demonstrated by the data in the tables is that HFABP in combination with one or more clinical parameters and / or scores obtained from multiple systematic assessment tools of stroke-related neurological disorders allows the correct diagnosis and classification of patients most relevant to the pathological scenario under evaluation with almost 100% specificity, advantageously achieved with high sensitivity.

[0180] In any case, the following Table 3 shows, for some of the combinations of the present invention (some of which are also listed in Tables 1 and 2), particular cut-offs in the levels of proteins (including HFABP), score values ​​from the Cincinnati or NIHSS tools, age, blood pressure (mean, systolic or diastolic) and optionally cut-offs in the levels of other proteins in the isolated samples, which gave such high specificity and sensitivity.

[0181] In principle, the inventors understand that a cut-off level of HFABP chosen in the range of 1.25 ng / ml to 6 ng / ml allows a very good classification of these for the diagnosis of LVO. [Table 4]

[0182] 3.2. Prediction of type of response to treatment (thrombectomy) As indicated, the inventors also understand that the level of HFABP in the isolated sample indicates that the subject is suffering from LVO, and that if said level is significantly elevated compared to a cutoff or reference for diagnosing LVO, it may indicate that the subject will have a poor response to treatment related to the degree of assistance following a stroke episode.

[0183] The next section discloses the use of the level of HFABP in an isolated patient sample as a marker for predicting response to commonly applied treatments. (A) HFABP and age are non-independent predictors of response to treatment measured as functional outcome 3 months after stroke. Logistic regression was performed to measure the impact of HFABP on patient dependency 3 months after stroke. To avoid confounding effects, the model was adjusted for patient dependency status and age at admission. Results showed that a statistically significant model X 2(3)=13.75, 0.003, with a moderate fit as indicated by the Nagelkerke R2 of 0.129, which correctly classifies 66.4% of cases. According to the fit, a 4-unit increase in HFABP (ng / ml) measured by POCT increases the odds ratio of dependency at 3 months by 2.006 (p-value=0.040), thereby doubling the risk of an mRS score higher than 2.

[0184] (B) HFABP, GFAP, and age are non-independent predictors of response to treatment measured as functional outcome 3 months after stroke. Logistic regression was performed to measure the effect of HFABP and GFAP on patient dependency 3 months after stroke. To avoid confounding effects, the model was adjusted for patient dependency status and age at admission. Results showed that statistically significant models X 2(4)=20.76, <0.001, with a moderate fit as indicated by the Nagelkerke R2 of 0.190, which correctly classifies 72.3% of cases. According to the fit, a 4-unit increase in HFABP ng / ml measured by POCT raises the odds ratio of dependency at 3 months by 2.006 (p-value=0.041), thereby doubling the risk of mRS score higher than 2. Meanwhile, a 700-unit increase in GFAP to pg / ml while holding other variables produces a similar risk shift, giving an odds ratio of 2.014 (p-value=0.034).

[0185] 3.3. Determination of HFABP in combination with other markers can improve the percentage of patient independence after LVO ischemic stroke. Make important decisions and take prompt action 2 hours earlier The blood biomarker results in this section are derived from previous findings of testing performed on samples from patients with suspected stroke arriving at the emergency room. Results have been replicated in real life using samples obtained in ambulances (BIO-FAST study, Biomarkers for Initiating Onsite and Faster Ambulance Stroke Therapies, ClinicalTrials.gov identifier: NCT04612218).

[0186] By analyzing the time these patients underwent thrombectomy and when biomarker information was already available in the ambulance, we realize that the decision to move directly to a thrombectomy facility is typically made 2 hours earlier.

[0187] However, the inventors went one step further than the decision, which was the clinical benefit of the treatment occurring 2 hours earlier in the patient group. This was done using several calculator-type prediction models, such as the model developed in one of the first and largest trials of thrombectomy in stroke (MR CLEAN), called MR PREDICT (https: / / mrpredicts.shinyapps.io / RRRR_1 / ), which includes the patient's clinical data and images at the start of the treatment with thrombectomy. Using these calculators, the inventors were able to perform a simulation to see what would have happened if the patient had been previously treated by directing the patient with LVO from the ambulance to the thrombectomy facility. According to the obtained data, the absolute improvement in % of unassisted patients (mRS 0-2) would have been 15% using the selected biomarkers (i.e., in combination with HFABP).

[0188] The absolute benefit of thrombectomy has been shown to increase by almost 15% in independent patients 3 months after the procedure. Thus, for example, in some of the large thrombectomy trials, such as the one carried out in Catalonia (REVASCAT), independence went from 28.2% at 3 months in the untreated group to 43.7% in the mechanical thrombectomy group (15.5% absolute benefit); in ESCAPE, it went from 29.3% to 53% (23.7% absolute benefit); in MR CLEAN, it went from 19.1% to 36.6% (17.5% absolute benefit), and in the recent Brazilian trial (RESILIENT), it went from 20% to 35% (15% absolute benefit).

[0189] Therefore, having a marker that allows a rapid decision to move to the appropriate facility and to the appropriate intervention room (e.g., angiography room) would be of enormous clinical benefit. HFABP, as an example of a FABP family member, makes this possible, enhancing and encouraging the application of this novel clinical strategy (i.e., detecting LVO patients and sending them to angiography room as soon as possible).

[0190] Example 2 A rapid point-of-care blood test to diagnose LVO patients and directly refer them to a thrombectomy center would enhance prognostic information regarding the success of endovascular reperfusion therapy A rapid blood test measuring FABP (i.e., HFABP) / NT-proBNP used in this field among patients with suspected stroke would allow identifying patients with LVO with fine specificity / sensitivity and referring them to a thrombectomy center or attempting direct transfer to an angiography laboratory (BIOFAST, NCT04612218). In addition to diagnostic accuracy, the prognostic value of the LVO check was evaluated among confirmed LVO patients (n=320) undergoing angioplasty.

[0191] method: Consecutive patients with confirmed LVO were tested by rapid testing for both HFABP and NT-proBNP in blood samples obtained before thrombectomy. Functional outcome at 3 months was the primary endpoint (dependent: mRS 0-2 vs. dependent: mRS 3-6); mortality at discharge was also assessed.

[0192] result: A total of 320 patients were enrolled. Regarding functional outcome after thrombectomy (n=45 missing data), 142 (51.6%) were still independent at 3 months and 133 (48.4%) were dependent. Clinical factors significantly associated with poor outcome were age, baseline NIHSS, diabetes mellitus, and chronic kidney disease. Both HFABP and NT-proBNP were elevated among dependent patients (p<0.001). In logistic regression [OR (2.5%-97.5%)], baseline NIHSS 1.11 / point (1.07-1.16; p<0.001), FABP 1.17 / unit (1.05-1.32; p=0.008), and diabetes 1.65 (0.94-2.89; p=0.079) were the major non-independent predictors of outcome. Using Panelomix software, optimal cut-offs were identified for age, NIHSS score, and FABP to predict poor outcome with good specificity (specificity=99.30% and sensitivity=21.10%). Regarding mortality (n=2 missing data), 289 patients (90.9%) were alive at the time of discharge, and 29 patients (9.1%) died in the hospital. Both HFABP and NT-proBNP were significantly elevated among deceased patients (p<0.001). Using Panelomix software, optimal cut-offs were identified for age, NIHSS score, and FABP to predict mortality with good specificity (specificity=99.70% and sensitivity=25.90%). Optimal cut-offs were also identified for age, NIHSS score, systolic blood pressure, and FABP to predict mortality with good sensitivity (specificity=50.90% and sensitivity=100%).

[0193] Conclusion: Apart from their diagnostic ability for LVO, FABP and NT-proBNP may predict outcome among those patients transferred to endovascular thrombectomy centers. This may aid future decisions regarding the individualization of endovascular therapy and the addition of neuroprotective strategies in patients predicted to have poor outcomes despite successful perfusion.

[0194] List of References Patent Literature - WO2020229691 issue - WO2018229144 Non-patent literature - Gandhi CD, Al Mufti F, Singh iP, et al. Neuroendovascular management of emergent large vessel occlusion: update on the technical aspects and standards of practice by the Standards and Guidelines Committee of the Society of Neurointerventional Surgery. J Neurointerv Surg 2018;10:315-20. - Lakomkin N, Dhamoon M, Carroll K, et al. Prevalence of large vessel occlusion in patients presenting with acute ischemic stroke: a 10-year systematic review of the literature. J Neurointerv Surg 2019;11:241-5. - Waqas M, et al. Effect of definition and methods on estimates of prevalense of large vessel occlusion in acute ischemic stroke: a systematic review and meta-analysis. J Neurointerv Surg. 2020 Mar;12(3):260-265). - Vanacker P, Heldner MR, Amiguet M, et al. Prediction of large vessel occlusions in acute stroke: National institute of Health Stroke Scale is hard to beat. Crit Care Med 2016;44:e336-43. - 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. - 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. - So-Young et al. (2012). Plasma heart type fatty acid binding protein level in acute ischemic stroke: comparative analysis with plasma S100B level for diagnosis of stroke and prediction of long-term clinical outcome. Clinical Neurology and Neurosurgery 115, pp.: 405-410. - Noorian AR, Sanossian N, Shkirkova K, Liebeskind DS, Eckstein M, Stratton SJ, Pratt FD, Conwit R, Chatfield F, Sharma LK, Restrepo L, Valdes-Sueiras M, Kim, Tenser M, Starkman S, Saver JL; FAST-MAG Trial Investigators and Coordinators. Los Angeles Motor Scale to Identify Large Vessel Occlusion: Prehospital Validation and Comparison With Other Screens. Stroke. 2018 Mar;49(3):565-572 - Gropen TI, Boehme A, Martin-Schild S, Albright K, Samai A, Pishanidar S, Janjua N, Brandler ES, Levine SR. Derivation and Validation of the Emergency Medical Stroke Assessment and Comparison of Large Vessel Occlusion Scales. J Stroke Cerebrovasc Dis. 2018 Mar;27(3):806-815. - Core Team (2020). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL https: / / www.R-project.org / . - Xavier Robin, Natacha Turck, Alexandre Hainard, Natalia Tiberti, Frederique Lisacek, Jean-Charles Sanchez and Markus Muller (2011). PanelomiX: A threshold-based algorithm to create panels of biomarkers. Translational Proteomics, 1(1), p. 57-64. DOI: 10.1016 / j.trprot.2013.04.003 (http: / / dx.doi.org / 10.1016 / j.trprot.2013.04.003> - Terry Therneau and Beth Atkinson (2019). rpart: Recursive Partitioning and Regression Trees. R package version 4.1-15. https: / / CRAN.R-project.org / package=rpart.

Claims

1. 1. An in vitro method for diagnosing large cerebral artery occlusion (LVO), comprising determining the levels of one or more of heart-type fatty acid binding protein (HFABP), N-terminal fragment of B-type natriuretic peptide (NT-proBNP), D-dimer (DDi), and glial fibrillary acidic protein (GFAP) in an isolated sample from a subject, and comparing said levels with corresponding reference values ​​or ranges, - if said reference value or range is for a subject suffering from LVO, and said level of HFABP, and one or more of NT-proBNP, DDi, and GFAP in said isolated sample is equal to or within said value or range, said subject is diagnosed with LVO; or - said reference value is a cut-off value that distinguishes LVO from any other condition selected from one or more of non-LVO ischemic stroke, hemorrhagic stroke, mimic, and healthy subject, and said subject is diagnosed with LVO if said levels of HFABP, and one or more of NT-proBNP, DDi, and GFAP in said isolated sample are higher than said cut-off; or - said reference value is for a subject not suffering from LVO, and if said level of HFABP, and one or more of NT-proBNP, DDi and GFAP in said isolated sample is higher than said reference value, said subject is diagnosed with LVO; In vitro methods.

2. 1. An in vitro method for selecting a subject for reperfusion therapy, wherein the subject suffers from LVO during an ischemic stroke episode, the method comprising determining levels of HFABP, and one or more of NT-proBNP, DDi, and GFAP in an isolated sample from the subject, and comparing the levels to corresponding reference values ​​or ranges; - if said reference value or range is for a subject suffering from LVO, and said level of HFABP, and one or more of NT-proBNP, DDi, and GFAP in said isolated sample is equal to or within said value or range, said subject is diagnosed with LVO and is a candidate for reperfusion therapy; or - if said reference value or range is for a subject not suffering from LVO, and said level of HFABP, and one or more of NT-proBNP, DDi, and GFAP in said isolated sample is higher than said reference value, said subject is diagnosed with LVO and is a candidate for reperfusion therapy; An in vitro method, wherein said reperfusion therapy is thrombectomy or thrombectomy in combination with prior administration of a thrombolytic and / or fibrinolytic and / or neuroprotective agent.

3. An in vitro method for selecting a subject to be transferred to a thrombectomy facility and / or for direct transfer to an angiography laboratory within said facility for thrombectomy, comprising determining levels of HFABP, and one or more of NT-proBNP, DDi, and GFAP in an isolated sample from the subject, and comparing said levels to corresponding reference values ​​or ranges; (a) if the levels of HFABP, and one or more of NT-proBNP, DDi, and GFAP in the isolated sample are equal to the reference values ​​or within the ranges of subjects suffering from LVO, the subject is selected to be transferred to a thrombectomy facility; or (b) if the levels of HFABP, and one or more of NT-proBNP, DDi, and GFAP in the isolated sample are higher than the reference values ​​or ranges of subjects not suffering from LVO, the subject is selected to be transferred to a thrombectomy facility; and / or (c) if the levels of HFABP, and one or more of NT-proBNP, DDi, and GFAP in the isolated sample are higher than reference values ​​that allow for 100% specificity discrimination between subjects suffering from either LVO or hemorrhagic stroke and subjects suffering from either non-LVO ischemic stroke or stroke mimic, the subject is selected for direct transfer to an angiography laboratory within the facility for said thrombectomy; In vitro methods.

4. If the subject is diagnosed with LVO, and the levels of HFABP, and one or more of NT-proBNP, DDi, and GFAP are higher than cutoff values ​​that stratify patients according to either degree of dependency and / or mortality, the subject also (i) having a prognosis defined by a disability rating of greater than 2, determined within 1-5 months after stroke onset according to the modified Rankin Score (mRS); and / or (ii) having a prognosis defined by a 3-month mortality rate of 25% to 35%; are classified as: An in vitro method according to any one of claims 1 to 3.

5. An in vitro method for determining the prognosis of a patient suffering from LVO, comprising determining the levels of HFABP, and one or more of NT-proBNP, DDi, and GFAP in an isolated sample from said patient, and comparing said levels to cut-off values ​​which stratify patients according to either degree of dependency and / or mortality, wherein if the level of HFABP in the sample is higher than the cut-off value, then the subject suffering from LVO is also (i) having a prognosis defined by a disability rating of greater than 2 according to the modified Rankin Score (mRS) and determined within 1 to 5 months after the onset of the stroke; and / or (ii) having a prognosis defined by a 3-month mortality rate of 25% to 35%; are classified as: In vitro methods.

6. Further comprising determining one or more clinical parameters of the subject, said clinical parameters and / or characteristics of said subject are selected from the group consisting of blood pressure including systolic blood pressure (SBP) and / or diastolic blood pressure (DBP), mean blood pressure (mean BP), glycemia, age, score on the NIH Stroke Scale (NIHSS), score from a systematic assessment tool of stroke-related neurological deficits selected from the Rapid Arterial Occlusion Assessment Scale (RACE) for stroke, the Cincinnati Prehospital Stroke Scale compared to the Stroke Severity Tool for prediction of major cerebral artery occlusion (Cincinnati score or CPSS), the Los Angeles Motor Scale (LAMS), acuity-aphasia-hemispatial neglect (VAN), Stroke Triage with Field Assessment for Emergency Transport (FAST-ED), or the modified Rankin Scale or score (mRS), time from symptom onset, sex, and combinations thereof; An in vitro method according to any one of claims 1 to 5.

7. A combination of at least the following markers: (j) HFABP+NT-proBNP+NIHSS+average BP+age 7. The in vitro method according to any one of claims 1 to 6, comprising determining NT-proBNP, wherein NT-proBNP is the N-terminal fragment of B-type natriuretic peptide.

8. The combination of the following markers in said isolated sample of said subject: (a) HFABP + NIHSS + DBP + age; (b) HFABP + NT-proBNP + NIHSS + DBP + age; (c) HFABP + DDi + NIHSS + DBP + age; (d) HFABP + GFAP + NIHSS + DBP + age; (e) HFABP + NT-proBNP + DDi + NIHSS + DBP + age; (f) HFABP + NT-proBNP + GFAP + NIHSS + DBP + age; (g) HFABP+GFAP+DDi+NIHSS+DBP+age; (h) HFABP + NIHSS + SBP + age; (i) HFABP + NIHSS + mean BP + age; (k) HFABP+Cincinnati+DBP+age; (l) HFABP + Cincinnati + SBP + age; (m) HFABP + Cincinnati + mean BP + age; (n) HFABP + NT-proBNP + Cincinnati + DBP + age; (o) HFABP+DDi+Cincinnati+DBP+age; (p) HFABP+GFAP+Cincinnati+DBP+age; (q) HFABP + NT-proBNP + DDi + Cincinnati + DBP + age; (r) HFABP + NT-proBNP + GFAP + Cincinnati + DBP + age; (s) HFABP+GFAP+DDi+Cincinnati+DBP+age; and (t) HFABP + NTproBNP + Cincinnati + mean BP + age wherein the combining comprises determining a level of HFABP. An in vitro method according to any one of claims 1 to 7.

9. The in vitro method according to any one of claims 1 to 8, wherein determining the level of HFABP is performed within the first 6 hours after the occurrence of a stroke.

10. The in vitro method according to any one of claims 1 to 9, wherein the isolated sample is a body fluid.

11. A kit comprising reagent means for simultaneously detecting HFABP and one or more levels of a natriuretic peptide, DDi and GFAP. (a) diagnosing LVO, (b) selecting a subject to undergo thrombectomy or thrombectomy in combination with prior administration of a thrombolytic and / or fibrinolytic and / or neuroprotective agent, wherein the subject suffers from LVO during the course of an ischemic stroke episode; (c) selecting a subject for transfer to a thrombectomy facility and / or direct transfer to an angiography laboratory within said facility for thrombectomy; or (d) Determining the prognosis of patients with LVO The present invention relates to use of HFABP or a combination of HFABP with one or more of NT-proBNP, DDi, and GFAP for the treatment of rheumatoid arthritis.

13. 12. A computer-implemented method for performing the method defined in any of claims 1 to 11, wherein after determining the level of HFABP, and one or more of NT-proBNP, DDi, and GFAP in said isolated sample of said subject and / or when the levels of said one or more clinical parameters of said subject are determined, said level(s) are given a value and / or score, optionally calculated with a mathematical formula to obtain a calculated value, and depending on said level(s), score(s) and / or calculated value(s) a decision is made between an option of suffering from LVO and / or an option of being a candidate for reperfusion therapy and / or a discrimination is made between an option of suffering from an ischemic stroke or an option of suffering from a condition selected from hemorrhagic stroke, transient ischemic attack or a stroke mimicking condition.