Methods for selecting patients for reperfusion therapy

Abstract

The present invention relates to a method for selecting a patient suffering from a stroke for reperfusion therapy, based on determining the levels of retinol binding protein 4 (RBP4) and N-terminal pro-B-type natriuretic peptide (NT-proBNP) in an isolated sample of said patient. The present invention also relates to a method for differentiating between ischemic stroke and hemorrhagic stroke, and to a kit comprising reagents for performing said methods.

Description

[0001] This application claims the benefit of European patent application EP19382384.6, filed on May 16, 2019. Technical Field

[0002] This invention relates to the field of diagnosis or companion diagnosis, particularly to methods for differentiating between ischemic and hemorrhagic stroke, and to selecting appropriate treatment based on the type of stroke event. Background Technology

[0003] Stroke, also known as cerebrovascular disease (CVD), remains one of the most important neurological disorders. It is the second leading cause of preventable death worldwide and a major contributor to impaired productivity. The two main subtypes of stroke are ischemic stroke (IS) and intracerebral hemorrhage (ICH), also known as hemorrhagic stroke. More than 80% to 85% of strokes are caused by occlusion of a cerebral artery, while the remaining 15% to 20% are due to artery rupture, resulting in ICH. Patients with ICH have poorer associated consequences, with a 30-day mortality rate of 37% to 38%, compared to 8% to 12% for IS patients.

[0004] Accurate differentiation between the two subtypes in the acute phase is crucial for developing the most appropriate treatment plan, which is specific and varies greatly between IS and ICH. The primary treatment recommended for acute IS includes reperfusion, i.e., restoring blood flow through medication or endovascular surgery (thrombectomy). The main medications used are thrombolytic agents, such as recombinant tissue plasminogen activator (r-tPA), a serine protease that cleaves clots obstructing cerebral arteries, or tenecteplase (TNK, a recombinant fibrin-specific plasminogen activator derived from natural t-PA through modifications at three sites in its protein structure). The treatment window for thrombolysis is narrow, only 4.5 hours from symptom onset; therefore, rapid identification of IS may allow for early recanalization, enabling tissue recovery from the penumbra and thus improving clinical outcomes. On the other hand, patients with acute ICH typically benefit from lowering blood pressure to slow hematoma growth or prevent edema and rebleeding. Currently, the diagnosis of stroke subtypes is primarily based on brain imaging data from computed tomography (CT) or magnetic resonance imaging (MRI). Therefore, patients suspected of having a stroke must be transferred to a hospital for this neuroimaging technique, wasting valuable time obtaining a CT scan or MRI. Unfortunately, MRI and CT scans are not widely available, especially in underdeveloped areas, where a lack of resources prevents their repeated use in primary care hospitals. Furthermore, some of these techniques may have side effects primarily related to radiation or contrast agent injections. Additionally, errors or uncertainties may occur if the medical personnel performing MRI and CT scans are inexperienced or inadequately trained.

[0005] Among several documents describing the use of biomarkers for rapid differentiation of stroke subtypes, international application WO2016087611 discloses a method for differentiating patients from ischemic stroke and hemorrhagic stroke, as well as a method for selecting stroke patients for treatment with antithrombotic agents or agents that can lower blood pressure, based on the determination of the level of glial fibrillary acidic protein (GFAP) in the patient's sample in combination with one or more biomarkers.

[0006] Another example of a study analyzing biomarkers that may be associated with acute IS can be found in Reynolds et al.'s work, "Early Biomarkers of Stroke". Clinical Chemistry -2003, vol.:49(10), pp.:1733-1739. This paper presents results for S-100B molecules, neurotrophic growth factor B, Von Willebrand factor, matrix metalloproteinase-9 (MMP-9), and chemokine ligand 2 (with CC motif) (CCL-2) (also known as chemokine-1 (MCP-1)) as potential biomarkers in the plasma of stroke patients. The authors conclude that only MCP-1 protein is of significant diagnostic value for acute ischemic stroke, extracted from samples of cerebrospinal fluid from patients, but its serum concentration was not different from that of control patients. Therefore, it is considered a method with low practicality for the accurate identification of this disease.

[0007] Montaner et al., in “Etiologic Diagnosis of Ischemic Stroke Subtypes With Plasma Biomarkers”, Stroke 2008, vol. no. 39, pp. 2280-2287, also disclosed biomarkers for the specific diagnosis of cardioembolic stroke. Brain natriuretic peptide (BNP) and D-dimer (DD) have been proposed for improving the diagnosis of cardioembolic stroke in the acute phase of stroke.

[0008] Due to the severity of the disease, accurate diagnosis between stroke subtypes is crucial, as reperfusion therapy in non-IS patients can be fatal. This subtype diagnosis should ideally be performed as quickly as possible, especially once the patient is identified in the acute phase at home, on the street, or in a GP's office. Therefore, kits or point-of-care facilities that can be easily performed in an ambulance and further validated in a hospital are a significant advantage.

[0009] Other teams are using strategies such as mobile stroke units, which function as ambulances equipped with CT scans, to enable stroke diagnosis and reperfusion therapy as quickly as possible outside of hospitals, thereby improving neurological outcomes for treated patients. However, this strategy is very expensive, with those high-tech ambulances costing a fortune and requiring specialized personnel.

[0010] Recent trials have shown that endovascular treatment of large vessel occlusion (LVO) can reduce morbidity and mortality in patients experiencing this severe acute ischemic stroke. Nevertheless, a small percentage of patients experiencing LVO receive endovascular treatment, often due to delays in reaching specialized hospitals capable of performing endovascular procedures (Rai AT et al., (2017)). Population-based incidence of acute large vessel occlusion and eligibility for thrombectomy suggest significant growth potential for endovascular stroke treatment in the United States (J Neurointerv Surg. 9:722-6). Patients with acute stroke typically first encounter emergency medical services (EMS) professionals, and early identification of LVO stroke in the pre-hospital setting by EMS professionals can improve timely transfer to endovascular centers and lead to 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 EmergCare. 2020 Feb.). 25:1-9.). Crowe et al. (see above) compared different scales used to diagnose LVO. They showed that among 2415 patients with acute ischemic stroke, 26% of the 26% of ischemic stroke patients (n=628) were diagnosed with LVO. A CPSS score of 2 or higher indicated a sensitivity of 69% and a specificity of 78% for LVO. A RACE score of 4 or higher indicated a sensitivity of 63% and a specificity of 73%. A LAMS score of 3 or higher indicated a sensitivity of 63% and a specificity of 72%, while a positive VAN score indicated a sensitivity of 86% and a specificity of 65%. Comparison of the area under the ROC curve for each scale showed no statistically significant difference in their ability to distinguish LVO stroke. This indicates the need for reliable LVO biomarkers.

[0011] Furthermore, LVO was 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: 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 et al. found that 16 studies included in their systematic review used nine different definitions of LVO (different combinations of arterial occlusion locations), which may affect the universality of LVO shown 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).

[0012] Finally, it is worth noting in the field of stroke diagnosis and treatment that it is important to distinguish between so-called stroke mimics and actual strokes. Stroke mimics are defined as diseases or conditions that present with stroke-like clinical manifestations but without neurological infarction. Several clinical syndromes can present with symptoms or signs similar to acute ischemic stroke, making it difficult to differentiate between stroke and stroke mimics due to the wide variety of overlapping clinical manifestations. This poses a real challenge for physicians because interventional stroke treatments have potential side effects. Currently, there are very few biomarkers in isolated patient samples that can distinguish between actual strokes and stroke mimics.

[0013] Therefore, there is a need in the art for alternative tests using biomarkers to overcome the limitations of the methods disclosed in the art, and to reliably distinguish stroke subtypes and exclude simulated scenarios, in order to determine the best treatment for patients in the shortest possible time. Furthermore, while establishing a clear definition of LVO, there is also a need for reliable biomarkers for LVO as a condition requiring specific treatment (i.e., endovascular therapy or thrombectomy). Summary of the Invention

[0014] In a first aspect, the present invention relates to an in vitro method for selecting stroke patients for reperfusion therapy, the method comprising determining the levels of retinol-binding protein 4 (RBP4) and the N-terminal fragment of B-type natriuretic peptide (NT-proBNP) in isolated samples from said patients.

[0015] Therefore, this method includes companion diagnostics.

[0016] The inventors unexpectedly discovered for the first time that a good classification of IS and ICH could be achieved by determining the levels of these two proteins in isolated samples.

[0017] Therefore, another aspect of the present invention is an in vitro method for distinguishing between IS and ICH in patients, comprising determining the levels of RBP4 and NT-proBPN in isolated samples from said patients.

[0018] The levels of NT-proBNP and RBP4 allow for the classification of patients into two groups: those who can receive reperfusion therapy (primarily with antithrombotic agents or thrombectomy), and those who should avoid reperfusion therapy to prevent fatal consequences. In the latter group, ICH patients are more likely to be treated with therapies that lower or optimize blood pressure.

[0019] As demonstrated in the examples below, the combination of NT-proBNP and RBP4 levels allows for patient classification with 100% or near-100% specificity. Therefore, this biomarker combination offers high accuracy, providing a truly safe selection of appropriate treatment modalities (i.e., candidates for reperfusion therapy). To the inventors' knowledge, this is the first time that biomarkers detectable in isolated patient samples (i.e., biofluid samples) have achieved 100% or near-100% specificity. Furthermore, and particularly advantageously, even when measured less than 6 hours or even less than 3 hours after symptom onset, the two biomarkers allow for differentiation between two distinct stroke types. In other words, accurate differentiation can be made at critical moments (hyperacute phase).

[0020] Furthermore, and as illustrated in the examples below, identifying these two proteins in isolated samples also allows for the detection of stroke patients with worse prognoses or outcomes (in terms of their higher mortality rates). Therefore, these patients require prompt treatment to prevent the progression of their adverse outcomes.

[0021] Therefore, the present invention also relates to a method for the prognosis of patients with stroke (particularly ischemic stroke and therefore a candidate for reperfusion therapy), the method comprising determining the expression level of RBP4 in a separated sample of the patient, optionally in conjunction with the expression level of NT-proBNP. To the knowledge of the inventors, this is the first time that such an adverse outcome association has been indicated for RBP4, or RBP4 and NT-proBNP. In a specific embodiment of this prognostic method, the levels of these two proteins in a sample of the patient are determined, said sample in another specific embodiment being a biological fluid sample; more specifically, blood (plasma or serum). In yet another specific embodiment of the prognostic method for patients with stroke, the level of at least RBP4 or both proteins is compared to a reference value, said reference value being selected from values ​​or ranges indicating that the subject has an ischemic stroke.

[0022] In another aspect, the present invention relates to a kit comprising reagent elements for detecting levels of RBP4 and NT-proBNP.

[0023] The present invention also discloses a kit containing reagents for detecting levels of markers selected from GFAP, RBP4, NT-proBNP, or combinations thereof.

[0024] In another aspect, the present invention also relates to the application of means for detecting the presence of either RBP4 or NT-proBNP in a test sample, said means being selected from the group consisting of immunoassay, protein migration, chromatography, mass spectrometry, turbidimetry, scattering turbidimetry, and polymerase chain reaction (PCR), for performing the method for selecting stroke patients for reperfusion therapy as defined in the first aspect; or for distinguishing between ischemic stroke and hemorrhagic stroke in patients. Attached Figure Description

[0025] Figure 1 The figures show: In (A), the cutoff (horizontal black line) levels (Y-axis, pg / mL) of NT-proBNP with 100% specificity for both stroke subtypes; and in (B), the cutoff (horizontal black line) levels of RBP4 (Y-axis, μg / mL). In Figure C, log10 (NT-proBNP) and RBP4 levels are plotted simultaneously against the corresponding previously determined cutoff values ​​for each protein. 100% specificity IS means that all patients with values ​​in this space of the graph (Figure) are ischemic stroke patients.

[0026] Figure 2 Display: In (A), the cutoff level (log(GFAP)) for >325 pg / ml of GFAP in different patient cohorts. In Figure (B), log10(NT-proBNP) and RBP4 levels are plotted simultaneously against the corresponding previously determined cutoff values ​​for each protein in this patient cohort. 100% specific IS has with Figure 1 Same meaning.

[0027] Figure 3 This is a graph obtained by analyzing the retrieved data using a Support Vector Machine (SVM) procedure. Values ​​above the S-curve represent 100% IS, and values ​​below the curve correspond to patients with either ICH or IS subtypes. Y-axis: NT-proBNP level (pg / mL); X-axis: RBP4 level (μg / mL). "Can be introduced in a support vector machine procedure with a Gaussian kernel." 100% specific IS has... Figure 1 Same meaning.

[0028] Figure 4 This is a graph showing the classification of subject groups using logistic model scores as significant predictors of ischemic stroke, which include logarithmic transformations of GFAP (pg / ml), NT-proBNP (pg / ml), and diastolic blood pressure (mmHg).

[0029] Figure 5It is a graph showing the classification of the subject groups, in which marker levels were measured within the first hour after the onset of stroke symptoms, and logistic model scores, including logarithmic transformations of GFAP (pg / ml), NT-proBNP (pg / ml), and diastolic blood pressure (mmHg), were used as significant predictors of ischemic stroke. Detailed Implementation

[0030] Unless otherwise stated, all terms used in this application shall be understood in their ordinary meaning as known in the art. Further more specific definitions of certain terms used in this application are set forth below and are intended to apply uniformly throughout the specification and claims, unless otherwise expressly stated, which provides for a broader definition.

[0031] As used herein, the term “patient” (or subject) means any subject exhibiting one or more signs or symptoms commonly associated with stroke, such as sudden onset of facial weakness, arm drift, speech abnormalities, and combinations thereof, such as FAST (facial, arm, speech, and time), facial hemiplegia and muscle weakness, numbness, decreased sensory or vibratory sensation, initial relaxation (hypoosmolarity), replaced by spasticity (hyperosmolarity), hyperreflexia, compulsive synergy, and specifically, when these occur on one side of the body (unilateral), alterations in smell, taste, hearing, or vision (whole or part). Symptoms include: ptosis (drooping eyelid) and oculomotor muscle weakness, diminished reflexes (e.g., vomiting, swallowing, pupillary light reflex), facial sensory and muscle weakness, balance problems and nystagmus, changes in respiration and heart rate, sternocleidomastoid muscle weakness, inability to turn the head to one side, tongue weakness (inability to protrude and / or move from side to side), aphasia, dysarthria, apraxia, visual field defects, memory deficits, unilateral neglect, confusion, altered libido, lack of self-awareness (often associated with stroke), disability, gait changes, altered motor coordination, dizziness, headache, and / or imbalance. As used herein, the term "patient" 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, cattle, horses, pigs, sheep, goats, dogs, cats, or rodents. Preferably, the patient is a male or female of any age or race. Preferably, the patient has suffered a stroke.

[0032] As used herein, the term "patient for therapy selection" refers to the identification of a patient in relation to a therapy designed to cure a disease or alleviate symptoms associated with one or more diseases or conditions. In the specific context of stroke treatment, it should be understood as any therapy that eliminates, delays, or reduces symptoms associated with stroke (more specifically, with ischemic stroke or with hemorrhagic stroke).

[0033] The term "reperfusion therapy" refers to medical treatments that restore blood flow to or around an artery that is blocked. Reperfusion therapy includes medication and endovascular procedures. The medications are thrombolytic agents (antithrombotic agents) and fibrinolytic agents used in a procedure called thrombolysis. The intervention performed may be a minimally invasive endovascular procedure (thrombectomy) to remove the thrombus (possibly using one or more stent retrievers), aspiration techniques, or an alternative device that combines stent retrievers and aspiration. Other procedures performed are more invasive bypass surgeries that graft arteries around the blockage. "Mechanical thrombectomy," or simply thrombectomy, is an interventional procedure that removes a blood clot (thrombus) from a blood vessel. It is typically performed in the coronary arteries (interventional cardiology), peripheral arteries (interventional radiology), and cerebral arteries (interventional neuroradiology).

[0034] Patient selection is not necessary to be sufficient for 100% of the subjects selected according to the first method of the invention, but is preferred. However, this terminology requires the proper selection of a statistically significant subset of subjects. Those skilled in the art can use various well-known statistical assessment tools (e.g., determining confidence intervals, p-value determination, Student t-test, Mann-Whitney test, etc.) to determine whether the selection of patients in the subject population is statistically significant. Details can be found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. P-values ​​are preferably less than 0.01, 0.05, 0.005, or 0.001.

[0035] The term "ischemic stroke" (IS) refers to a physical blockage of blood flow to a region of the brain, leading to the death of brain cells in that region. Ischemic stroke can be further divided into thrombotic stroke and embolic stroke. A thrombotic stroke occurs when a blood clot forms in the brain and blocks a cerebral artery. An embolic stroke is caused by a blood clot that forms in a peripheral artery or heart, travels to the brain, and causes ischemia there. Another type of ischemic stroke is lacunar stroke, caused by occlusion of a cerebellar artery.

[0036] As used in this article, the term "hemorrhagic stroke" (abbreviated as ICH if it is intracerebral hemorrhage) refers to bleeding into brain tissue due to the rupture of a blood vessel.

[0037] The inventors of this invention have identified RBP4 and BNP as novel plasma biomarkers for the accurate selection of stroke patients for reperfusion therapy. Therefore, these biomarkers can differentiate between acute ischemic stroke (IS) and acute hemorrhage (ICH). Using several data analysis methods with these two biomarkers, 100% specificity and 20%-30% sensitivity were achieved. Furthermore, the addition of a third biomarker, specifically GFAP levels, increases sensitivity to 60% while maintaining specificity.

[0038] Therefore, in a specific embodiment of the first aspect of selecting a patient with stroke for reperfusion therapy, the method further includes determining the GFAP level in a separated sample of the patient.

[0039] While aiming for improved sensitivity, the inventors also developed a simplified kit containing only tools for detecting RBP4 and NT-proBNP levels. This simplified kit can be used in ambulances when treating stroke patients, accurately distinguishing between ischemic stroke (IS) and intracerebral hemorrhage (ICH). If appropriate, this allows for rapid administration of reperfusion therapy (i.e., antithrombotic agents in the ambulance); and avoids poor outcomes, at least in IS, as the patient receives treatment quickly. Furthermore, in ICH, symptom worsening can be prevented if blood pressure can be optimized.

[0040] In yet another, more specific embodiment of the first aspect, the method includes the step of comparing the level with a corresponding reference value or reference range for each protein, the reference value or range being selected from values ​​or ranges of values ​​of a subject with ischemic stroke, and wherein the subject is classified as a candidate for reperfusion therapy when the levels of at least RBP4 and NT-proBNP fall within the values ​​or ranges of values ​​of a subject with IS.

[0041] In a more specific implementation, the reference value or range is selected from the values ​​or ranges of values ​​of subjects with IS or ICH, and the subject is classified as a candidate for reperfusion therapy when at least the levels of RBP4 and NT-proBNP fall within the values ​​or ranges of values ​​of subjects with IS. Meaningful clinical sensitivity (approximately 21%) is achieved with a specificity exceeding 98%, particularly 100%, when both levels fall within the values ​​or ranges of values ​​of subjects with IS.

[0042] In another specific embodiment of the first aspect, the method includes the step of comparing the levels of RBP4, NT-proBNP, and optionally GFAP (if determined) with corresponding reference values ​​or reference intervals for each protein, said reference values ​​or intervals being selected from values ​​or value intervals from subjects with ischemic stroke, and wherein the subject is classified as follows when at least the levels of RBP4 and NT-proBNP are within the values ​​or value intervals from subjects with ischemic stroke:

[0043] - Candidates for reperfusion therapy, and

[0044] - A prognosis defined by a dependence level greater than 2 according to the Modified Ranking Scale (mRS), which is determined within 1 to 5 months after stroke onset; and / or a prognosis defined by a 3-month mortality rate of 25% to 30% after onset.

[0045] In another, more specific embodiment, the method includes the step of comparing the levels of RBP4, NT-proBNP, and GFAP, and wherein if a subject is classified as a candidate for reperfusion therapy, it is also classified as having a prognosis defined by a dependence level greater than 2 according to a modified ranking scale (mRS), which is determined within 1 to 5 months after stroke onset; and / or having a prognosis defined by a 20% to 30% mortality rate within 3 months after onset.

[0046] Prognosis can also be defined as a prognosis determined by a dependence of the Modified Ranking Scale (mRS) greater than 2, and which is determined at least 3 months after stroke onset. In one specific embodiment, the mortality rate at three months post-stroke is at least 23%. In another specific embodiment, it is 25%.

[0047] In some implementations, where only one of the RBP4 and NT-proBNP levels is within the value or range of the subject with IS, the subject is also classified as a candidate for reperfusion.

[0048] In another specific embodiment of the first aspect, the in vitro method further includes the step of comparing the levels of RBP4, NT-proBNP, and, if determined, GFAP with a corresponding reference cutoff value for each protein, wherein:

[0049] - If only the levels of RBP4 and NT-proBNP are determined, the levels of both RBP4 and NT-proBNP must be equal to or higher than the corresponding reference cutoff value Ref1 for each protein. RBP4 and Ref1 NT-proBNP This indicates that the patient is a candidate for reperfusion therapy, where the cutoff value distinguishes between patients with ischemic stroke and patients with intracerebral hemorrhage; or

[0050] - If the levels of RBP4, NT-proBNP, and additional GFAP are determined, the patient is selected as a candidate for reperfusion therapy if: in the first step, the GFAP level is equal to or below the reference cutoff value Ref. GFAP Furthermore, in the second step, the levels of RBP4 and NT-proBNP are simultaneously equal to or higher than the corresponding reference cutoff value Ref2. RBP4 and Ref2 NT-proBNP The cutoff value distinguishes between patients with ischemic stroke and patients with intracerebral hemorrhage.

[0051] In practice, considering the specific values ​​of desired sensitivity and specificity, different alternative implementations of the first aspect of the method are chosen when the option of comparing the tested levels with their respective cutoff values ​​or reference intervals is included. Therefore, if 100% specificity (correct classification between two conditions) is required, sensitivity (detecting one condition in a group of subjects with different conditions) can be reduced. On the other hand, reducing specificity (i.e., approximately 94% or 98%) can increase the sensitivity of the method. Therefore, the reference values ​​can vary depending on the desired specificity and / or the desired sensitivity.

[0052] In a more specific embodiment of the method, which includes comparing cutoff values ​​for two or three protein levels, if a subject is classified as a candidate for reperfusion therapy, they are also classified as having a prognosis defined by a dependence level greater than 2 according to a modified ranking scale (mRS), determined within 1 to 5 months after stroke onset; and / or having a prognosis defined by a 20% to 30% mortality rate within 3 months after onset.

[0053] This also includes, as a further specific embodiment of the method for selecting patients with stroke for reperfusion therapy as a first aspect, the method further comprising the step of treating the patient with said reperfusion therapy if at least the levels of RBP4 and NT-proBNP are within the values ​​or ranges of values ​​for a subject with IS, or if reference cutoff values ​​for the corresponding RBP4 and NT-proBNP (and optionally GFAP) classify the patient as a candidate for reperfusion therapy.

[0054] As described above, there are several treatment options that promote reperfusion. In a specific embodiment of the first aspect of the invention, the reperfusion therapy is selected from treatments employing antithrombotic agents, thrombectomy, and combinations thereof.

[0055] In a more specific embodiment, the antithrombotic agent is a thrombolytic agent. In yet another more specific embodiment, the thrombolytic agent is a plasminogen activator. More specifically, the plasminogen activator is a tissue plasminogen activator.

[0056] As used herein, the term "antithrombotic agent" refers to a drug that can reduce clot formation. Suitable antithrombotic agents for use in this invention include, but are not limited to, thrombolytic agents, antiplatelet agents, and anticoagulant compounds.

[0057] As used herein, the term "thrombolytic" refers to a drug that can dissolve clots. All thrombolytics are serine proteases that convert plasminogen into plasmin, which breaks down fibrinogen and fibrin and dissolves clots. Currently available thrombolytics include reteplase (r-PA or Retavase), alteplase (t-PA or Activase), urokinase, prourokinase, benzoyl purified streptokinase activator complex (APSAC), staphylococcal kinase (Sak), tenecteplase (TNK-tPA), alteplase (TNKasa), eminase, streptase, or abokinase. Tenecteplase (TNK-tPA) is used in certain embodiments because it can be administered as a rapid single bolus and can be used at the ambulance level. TNK takes effect 1 minute after administration (injection). The suppliers of TNK are Boehringer Ingelheim (EU) and Genentech (USA).

[0058] As used herein, the term anticoagulant compound refers to compounds that prevent blood clotting, including but not limited to vitamin K antagonists (warfarin, acetocoumarin, phenylpropargyl coumarin, and fenidone), heparin and heparin derivatives (e.g., low molecular weight heparin), factor Xa inhibitors (e.g., synthetic pentasaccharides), direct thrombin inhibitors (argatroban, lepirudine, bivalirudin, and cimetidine), and antiplatelet compounds that inhibit thrombus formation by inhibiting platelet aggregation, including but not limited to cyclooxygenase inhibitors (aspirin), adenosine diphosphate receptor inhibitors (clopidogrel and ticlopidine), phosphodiesterase inhibitors (cilostazol), glycoprotein IIB / IIIA inhibitors (abcitumab, eptifibatide, tirofiban, and defibrinolytic peptide), and adenosine uptake inhibitors (depidamo). In a preferred embodiment, the antithrombotic agent is a thrombolytic agent. In a more preferred embodiment, the thrombolytic agent is a plasminogen activator. In an even more preferred embodiment, the plasminogen activator is tPA (tissue plasminogen activator).

[0059] As used herein, the term "tissue plasminogen activator (t-PA)" refers to a serine protease found on endothelial cells that catalyzes the conversion of plasminogen to plasmin. The complete protein sequence of human t-PA has UniProtKB accession number P00750 (July 11, 2012), SEQ ID NO: 1. tPA can be manufactured using recombinant biotechnology, and tPA created in this manner can be referred to as recombinant tissue plasminogen activator (rtPA). Recombinant tissue plasminogen activator (r-tPA) includes the thrombolytic agents alteplase, reteplase, and tenecteplase (TNKase, also known as TNK-tPA, SEQ ID NO: 2). In human t-PA, amino acids 296-299 are lysine, histidine, and two arginines. In TNK-tPA, these amino acids have been replaced by four alanines. This mutation results in increased resistance to plasminogen activator inhibitor 1 (PAI-1).

[0060] The t-PA dose should be administered within 3 hours before symptom onset or within 4.5 hours after symptom onset. Recommended total dose: 0.9 mg / kg (maximum dose should not exceed 90 mg), infused over 60 minutes. Alternatively, administer a 1-minute intravenous bolus of 0.09 mg / kg (10% of the 0.9 mg / kg dose), followed by a continuous infusion of 0.81 mg / kg (90% of the 0.9 mg / kg dose) over 60 minutes. Heparin should not be started 24 hours or longer after initiating alteplase treatment for stroke. The t-PA is administered intravenously, and in some cases may be administered directly into the artery, and should be administered immediately after the onset of initial stroke symptoms. The dosage and route of administration are applicable to any implementation of the first aspect. Furthermore, particularly in implementations involving the treatment of a patient.

[0061] Once a subject with stroke is identified as a candidate for reperfusion therapy, a single dose of TNK-tPA should be administered as soon as possible, within 3 hours before symptom onset or up to 4.5 hours after symptom onset, preferably within the first hour after stroke onset.

[0062] As mentioned above, the use of TNK-tPA is particularly useful because, as a specific formulation for rapid, single-dose bolus application, it can be administered at any point of care, even at the ambulance level, and takes effect in about one minute after administration.

[0063] In this specific implementation, stroke patients who were not selected for reperfusion therapy were chosen for a blood pressure-lowering therapy. Specifically, this therapy was administered using a medication capable of lowering blood pressure.

[0064] In this article, “blood pressure” is understood to refer to blood pressure at central arterial sites, such as the aorta and carotid arteries. Central blood pressure can be appropriately measured non-invasively at the carotid or radial artery using applanation tonometry (described below). Therefore, “blood pressure” as used in this article includes aortic blood pressure.

[0065] The term "medicinal agent capable of lowering blood pressure" as used in this invention refers to any drug that lowers blood pressure through different mechanisms. The most widely used include thiazide diuretics [such as furosemide, sodium nitroprusside, hydralazine]; ACE inhibitors, calcium channel blockers (such as nicardipine or nimodipine); adrenergic receptor antagonists (such as alpha-adrenergic antagonists, urapidil), or combinations of alpha- and beta-receptor blockers (labeprolol and nitroglycerin); and angiotensin II receptor antagonists (ARBs). Illustrative (non-limiting) examples of agents that can lower or reduce blood pressure are α-methyldopa (Aldomet), methyl 11,17α-dimethoxy-18β-[(3,4,5-trimethoxy-benzoyl)oxy)]-3p,2a-yohimbine-16β-carboxylate (Reserpine), or 2-(2,6-dichlorophenylamino)2-imidazoline hydrochloride (Clonidine hydrochloride), ergonitrile, or 2-chloro-6-methylergolin-8β-acetonitrile, as disclosed in EP0005074. Reference values ​​for lowering blood pressure in ischemic stroke, ischemic stroke treated with thrombolytic agents, or hemorrhagic stroke are those recommended by clinical practice guidelines (these values ​​are subject to update). Currently, if the systolic blood pressure in ischemic patients reaches 220–120 mmHg, and in hemorrhagic patients it reaches 180–100 mmHg, the goal is to reduce the use of antihypertensive treatments. In a preferred embodiment, blood pressure can be lowered by intravenous administration of a blood pressure-lowering agent and co-administration of an oral antihypertensive agent. Reference values ​​for lowering blood pressure in ischemic stroke, ischemic stroke treated with thrombolytic agents, or hemorrhagic stroke will be those recommended by clinical practice guidelines (these values ​​may be updated).

[0066] Any suitable method for measuring arterial pressure can be used to determine whether a drug can lower blood pressure, wherein the decrease in arterial pressure is detected after administration of the drug. Illustrative (non-limiting) examples of methods for measuring arterial pressure are non-invasive techniques, such as palpitations, auscultation, oscillography, and continuous non-invasive arterial pressure (CNAP) as illustrative non-limiting examples.

[0067] As used herein, the term "reference value" refers to a predetermined standard used as a reference for evaluating values ​​or data obtained from samples collected from subjects. A reference value or reference level can be an absolute value; a relative value; a value with an upper or lower limit; a range of values; a mean; a median, average, or a value compared to a specific control or baseline value. Reference values ​​can be based on individual sample values, for example, values ​​obtained from samples from test subjects (but obtained at an earlier time point). Reference values ​​can be based on a large sample, such as a population of subjects from age-matched groups, or on a sample library that includes or excludes samples to be tested. Reference values ​​for the biomarkers of the present invention have been determined. Reference values ​​for each of RBP4, NT-proBNP, and GFAP can be derived from lower and upper limits, as disclosed in the following examples. The range of values ​​(protein levels) for each biomarker and specific combinations of values ​​for different biomarkers provide accurate classification of subjects with high sensitivity and specificity.

[0068] A biomarker (in this invention, any one of NT-proBNP, RBP4, or GFAP) is considered to be above its reference value when its level 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%, or at least 150% above its reference value.

[0069] Similarly, in the context of this invention, the level of a biomarker is reduced when the level of the biomarker in a sample is below a reference value. A biomarker is considered to be below its reference value when its level 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%, or at least 150% below its reference value.

[0070] In a specific embodiment of the first aspect, when only the levels of RBP4 and NT-proBNP in biological fluid samples are measured and compared with the corresponding reference cutoff values ​​(hereinafter referred to as Ref1) RBP4 and Ref1 NT-proBNPWhen comparing these values, the reference cutoff values ​​are 52 μg / ml for RBP4 and 4062 pg / ml for NT-proBNP. In specific embodiments, these particular reference cutoff values ​​are for isolated plasma samples and for measurements performed using enzyme immunosorbent assay (ELISA).

[0071] In another specific embodiment of the first aspect, when the levels of RBP4, NT-proBNP, and GFAP are determined and compared with the corresponding reference cutoff values ​​(hereinafter referred to as Ref2) RBP4 Ref2 NT-proBNP and Ref GFAP When comparing, these reference cutoff values ​​are: 38 μg / ml for RBP4, 1305 pg / ml for BNP, and 0.325 ng / ml for GFAP. In a more specific implementation, these specific reference cutoff values ​​are for isolated plasma samples, and when GFAP is measured, the level of this marker is determined using a more sensitive (picomolar level) single-molecule assay (SIMOA), while others are determined using ELISA. With these cutoff values, a specific method can be performed in two separate steps or under specific conditions. In the first step, the level of GFAP is determined, and if it is equal to or below the reference cutoff value Ref GFAP Furthermore, if in the second step it is further determined that RBP4 and NT-proBNP are simultaneously equal to or higher than the corresponding reference cutoff value Ref2 RBP4 and Ref2 NT-proBNP If the result is positive, the patient is considered to have ischemic stroke and is a candidate for reperfusion.

[0072] As will be illustrated by examples, ICH patients have higher GFAP levels and lower RBP4 and NT-proBNP levels compared to IS. The combination of RBP4 > 52 μg / mL and GFAP > 0.18 ng / mL resulted in accurate diagnoses of 6.5% of IS and 34.3% of ICH. By using RBP4 > 52 μg / mL and a BNP cutoff value > 4060 pg / mL, the addition of NT-proBNP maintained 100% specificity and improved sensitivity against IS by up to 20% (31 / 155).

[0073] As mentioned above, reference values ​​can vary depending on the exclusion criteria of the clinical protocol. Furthermore, these values ​​can always be adjusted based on the variables considered in the diagnosis to improve sensitivity while maintaining specificity, which is crucial for stroke.

[0074] Furthermore, computational methods can be used to appropriately classify patients based on detected protein levels. These methods calculate determined protein values ​​in a formula that provides predictive factors based on protein expression levels corrected for by specific coefficients. Other computational methods (such as the support vector machine method illustrated herein) allow for the disclosure of a function that takes into account the levels of all determined proteins for appropriate patient classification.

[0075] All these reference values ​​indicated in this particular implementation are values ​​determined in isolated plasma samples from subjects suffering from stroke. Those skilled in the art know how to find the corresponding values ​​in serum and other biological fluids.

[0076] As used herein, the term “sample” refers to any sample that can be obtained from a patient. This method is applicable to any type of biological sample from a patient, such as biopsy samples, tissues, cells, or biological fluids (plasma, serum, saliva, semen, sputum, cerebrospinal fluid (CSF), tears, mucus, sweat, breast milk, brain extracts, etc.).

[0077] Therefore, in another specific embodiment, optionally in combination with any of the embodiments above or below, the isolated sample of the subject (i.e., a patient with a stroke) is a biological fluid. Illustrative, non-limiting biological fluids include blood, plasma, serum, saliva, urine, or cerebrospinal fluid. In a more preferred embodiment, the biological fluid is plasma or serum.

[0078] In a preferred embodiment of the method of the present invention, the sample is obtained at baseline.

[0079] Different samples can be used to determine the levels of different biomarkers. Therefore, it is not necessary to measure the levels of all biomarkers according to the method of the present invention in the same type of sample. Therefore, in another preferred embodiment, the levels of RBP4, NT-proBNP, and GFAP are measured in serum. In another preferred embodiment, their levels are measured in plasma.

[0080] The “baseline” used in this invention is considered to be any time from the onset of symptoms to the first visit to the patient. This typically occurs within the first few hours after a stroke, usually at the first point of concern in an ambulance or hospital. In a preferred embodiment, the baseline is within the first 4.5 hours after the onset of symptoms, or less than 6 hours after the stroke, or in another preferred embodiment, less than 24 hours after the onset of symptoms.

[0081] In another specific embodiment of this aspect, the steps of determining the levels of RBP4 and NT-proBNP are performed within the first two hours after the stroke. As shown in the examples below, the earlier the determination of the markers in the isolated sample is completed, the better the accuracy and sensitivity of the method. In another specific embodiment of this aspect, the steps of determining the levels of RBP4 and NT-proBNP (and, if determined, GFAP) are performed within the first hour after the stroke.

[0082] In another embodiment of the first aspect of the method for selecting patients with stroke for reperfusion therapy, it further includes determining one or more clinical parameters. Therefore, the method of the present invention includes determining the levels of RBP4 and BNP, as well as one or more clinical parameters.

[0083] As used herein, the term "clinical parameter" or clinical data refers to personal statistics (age or date of birth, race and / or ethnicity), or clinical symptoms or signs in patients associated with stroke-related disease / condition. The term also includes laboratory parameters such as D-dimer or blood glucose measurements.

[0084] In a specific implementation, the clinical parameter is hypertension, and if the patient has hypertension, it indicates that the patient has an ischemic stroke or is a candidate for reperfusion therapy.

[0085] In another specific embodiment of the first aspect, the in vitro method further includes determining a selection from blood pressure, including systolic and / or diastolic blood pressure, blood glucose, age, scores from a systematic assessment tool for stroke-related neurological deficits (e.g., NIHSS score), sex, and combinations thereof. In a specific embodiment, the values ​​of all these parameters are combined with levels of RBP4, NT-proBNP, and optionally GFAP in an appropriate algorithm to correctly classify a patient as a candidate for reperfusion therapy. For example, blood pressure at specific intervals combined with specific levels of two or three proteins is used in the decision-making process for correct classification. In another, more specific embodiment, these values ​​are incorporated into the formula of a regression model to give a score or final value that allows for such classification.

[0086] "Hypertension," sometimes also called arterial hypertension, should be understood as a chronic medical condition characterized by elevated blood pressure in the arteries. Normal resting blood pressure ranges from 100-140 mmHg systolic (top reading) and 60-90 mmHg diastolic (bottom reading). Hypertension is defined as blood pressure consistently at or above 140 / 90 mmHg. As noted above, the reference values ​​for lowering blood pressure in ischemic stroke, ischemic stroke treated with thrombolytics, or hemorrhagic stroke will be those recommended by clinical practice guidelines (these values ​​are subject to update). Currently, the accepted treatment modality for lowering blood pressure is 220-120 mmHg systolic in ischemic patients and 180-100 mmHg in hemorrhagic patients.

[0087] The term "systematic assessment tool for stroke-related neurological deficits" refers to tools designed to measure and quantify the most common neurological deficits following stroke. Several aspects or parameters are assessed, such as level of consciousness, visual field, facial weakness, motor function of the limbs, gaze deficits, sensory deficits, coordination (ataxia), language (aphasia), speech (dysarthria), etc. These are all assigned values, with 0 indicating normality. Therefore, in most of these tools, higher scores indicate more severe neurological deficits. Technicians are aware of various tools for this purpose, such as the National Institutes of Health Stroke Scale (NIHSS) score, the Rapid Arterial Occlusion Assessment Scale (RACE) for Stroke, the Cincinnati Pre-Admission Stroke Scale (Cincinnati Score) compared to tools predicting stroke severity in strokes with large vessel occlusion, the Los Angeles Motor Scale (LAMS), or the Modified Ranking Scale or Rating Scale (mRS). All of these scales are designed for rapid and standardized assessment of early neurological function following stroke. The Modified Ranking Scale (mRS) is also a scale for assessing the degree of disability following a stroke. It is typically used at discharge and 3 months after the stroke.

[0088] In another preferred embodiment, the clinical parameters are selected from age, NIHSS score, sex, systolic blood pressure, and combinations thereof. The term “NIHSS score” as used in this invention refers to the National Institutes of Health Stroke Scale (NIHSS) score, a systematic assessment tool that provides a quantitative measure of stroke-related neurological deficits (Adams HP Jr Neurology. 1999 Jul 13; 53(1):126-31). The NIHSS was originally designed as a research tool to measure baseline data of patients in clinical trials of acute stroke. Now, it is also widely used as a clinical assessment tool to evaluate the alertness of stroke patients, determine appropriate treatment, and predict patient prognosis. The NIHSS is a 15-item neurological examination stroke scale used to assess the impact of acute stroke on levels of consciousness, language, neglect, visual field loss, extraocular movements, motor intensity, ataxia, dysarthria, and sensory loss. Trained observers score patients’ ability to answer questions and perform activities. Each item is scored on a scale of 3 to 5, with 0 being normal, and a margin is left for items that cannot be tested. Stroke severity levels are measured using 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 this invention, the term "higher score" refers to a score of 5 to 42 on the NIH Stroke Scale scoring system.

[0089] Variations of the NIHSS, such as the Rapid Arterial Occlusion Assessment Scale for Stroke (RACE) or other scores used to identify ischemic stroke with large vessel occlusion, can also be used.

[0090] In another specific embodiment of the first aspect, optionally in combination with any of the embodiments above or below, if the subject is classified as a candidate for reperfusion therapy, he / she is also diagnosed with large vessel occlusion.

[0091] As a second aspect, the present invention relates to an in vitro method for distinguishing between IS and ICH in patients, comprising determining the levels of RBP4 and NT-proBPN in isolated samples from said patients.

[0092] In a specific implementation of the second aspect, the method includes determining the level of GFAP.

[0093] In another, more specific embodiment of the second aspect, the step of comparing the levels of RBP4, NT-proBNP, and, if determined, GFAP with corresponding reference values ​​is also included, wherein:

[0094] - If only the levels of RBP4 and BNP are determined, then the levels of both RBP4 and BNP are simultaneously higher than the corresponding reference values ​​Ref1.RBP4 and Ref1 NT-proBNP This indicates that the patient is an IS patient; or

[0095] - If the levels of RBP4, BNP, and GFAP are determined, and the levels of RBP4 and BNP are simultaneously higher than the corresponding reference values ​​Ref2 RBP4 and Ref2 NT-proBNP Furthermore, the level of GFAP is lower than the reference value Ref. GFAP This indicates that the patient is an IS patient.

[0096] In another specific embodiment of the second aspect, if a subject is classified as having ischemic stroke when determining the levels of RBP4 and BNP, and optionally GFAP, they are also classified as having a prognosis defined by a dependence of greater than 2 according to the modified ranking scale (mRS), which is determined within 1 to 5 months after the stroke onset; and / or having a prognosis defined by a 20% to 30% mortality rate within 3 months after the onset.

[0097] In another specific embodiment of the second aspect, it further includes the step of selecting a therapy, particularly a reperfusion therapy, as described in the first aspect and its specific embodiments.

[0098] Therefore, after the differential diagnosis is completed, in another specific embodiment of the second aspect, it further includes the step of recommending reperfusion therapy to patients diagnosed with IS and / or treating said patients diagnosed with IS with reperfusion therapy (primarily using antithrombotic agents or with the aid of thrombectomy). Alternatively, in another specific embodiment, those patients diagnosed with ICH who should avoid reperfusion therapy to avoid fatal consequences are recommended for or treated with treatments to lower or optimize blood pressure.

[0099] This particular embodiment can be conceived as a method for treating patients with stroke, the method comprising performing an in vitro method to differentiate between IS and ICH according to the second aspect, and treating patients diagnosed with IS with reperfusion therapy (primarily using antithrombotic agents or with the aid of thrombectomy); or treating patients diagnosed with ICH with treatments to lower or optimize blood pressure. Advantageously, using this method allows patients to be treated or recommended to receive the most appropriate treatment regimen within the first few hours of symptom onset.

[0100] All the specific embodiments previously disclosed for the first aspect also apply to this second aspect. In particular, there are preferred reference values, types of isolated samples, and options for further determining one or more clinical parameters. Therefore, in another specific embodiment of the second aspect, the in vitro method further includes determining clinical parameters selected from blood pressure, including systolic and / or diastolic blood pressure, blood glucose, age, scores from stroke-related neurological deficit assessment tools (e.g., NIHSS scores), sex, and combinations thereof. As previously stated, in specific embodiments, the values ​​of all these parameters are combined with RBP4, NT-proBNP, and optionally GFAP levels in an appropriate algorithm to correctly classify the patient as a candidate for reperfusion therapy. For example, blood pressure at specific intervals combined with specific levels of two or three proteins is used in the decision-making process for correct classification. In another, more specific embodiment, these values ​​are incorporated into the formula of a regression model to give a score or final value that allows for such classification.

[0101] Furthermore, in another specific embodiment of the second aspect, the levels of RBP4 and NT-proBNP (and, if determined, GFAP) are determined within the first two hours after the stroke onset. More specifically, within the first hour. This implies the advantage of increased sensitivity of the method, as explained in the first aspect.

[0102] However, in another specific embodiment of this second aspect, optionally in combination with any of the embodiments above or below, if the subject is classified as having ischemic stroke, he / she is also diagnosed with large vessel occlusion.

[0103] The present invention also generally includes a method for detecting RBP4 and NT-proBPN levels, and optionally in combination with GFAP levels, in isolated samples from subjects suffering from stroke, the method comprising:

[0104] (a) Obtaining samples from subjects; and

[0105] (b) The presence of one or more proteins in an isolated sample is detected as follows: (i) the sample is contacted with a tool capable of binding the corresponding expressed protein and the binding is detected; or (ii) the sample is contacted with a tool capable of binding the corresponding RNA that will be translated into one or more corresponding proteins and the binding is detected.

[0106] As used in the second aspect of this document, the term "differentiation" refers to the identification of different conditions. As those skilled in the art will understand, differentiation need not be accurate for 100% of the subjects to be diagnosed or evaluated, but is preferred. However, the term requires that subjects be able to be identified with an increased probability of having one of the two types of stroke. Those skilled in the art can readily determine whether a subject is statistically significant using a variety of well-known statistical assessment tools, such as the determination of confidence intervals, p-value determination, Student's t-test, Mann-Whitney test, etc. Detailed information can be found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. P-values ​​are preferably less than 0.05, 0.01, or 0.005.

[0107] Because the biomarkers identified in this invention allow for the differentiation between ischemic stroke (IS) and hemorrhagic stroke (ICH), and considering the application of different therapies for these two types of patients (antithrombotic drugs in ischemic stroke patients and blood pressure-lowering drugs in hemorrhagic stroke patients) (see Tsivgoulis G. et al., Neurology. 2014 Sep 19), this invention also provides the first method described above for selecting treatment for patients with stroke. Therefore, the two aspects are conceptually closely related.

[0108] In this invention, when RBP4 is mentioned, it refers to plasma retinol-binding protein 4, which belongs to the lipid transporter family and is a specific carrier of retinol in the blood. The complete sequence of human retinol-binding protein 4 has the UniProtKB accession number P02753 (August 8, 2013) and SEQ ID NO:3.

[0109] As used herein, the term “GFAP” refers to glial fibrillary acidic protein, an intermediate filament protein expressed by multiple cell types in the central nervous system. The complete human sequence of glial fibrillary acidic protein is available in UniProtKB accession number P14136 (August 8, 2013), SEQ ID NO:4.

[0110] The N-terminal fragment of B-type natriuretic peptide (NT-proBNP) (SEQ ID NO:5) is the 76-amino acid N-terminal fragment of pro-B-type natriuretic peptide hormone. Cleavage of pro-BNP yields the NT-proBNP fragment and the active B-type natriuretic peptide (BNP). BNP is a hormone secreted by cardiomyocytes in the ventricles in response to stretching caused by increased ventricular blood volume. The complete human sequence of BNP is available in UniProt KB accession number P16860 (August 1, 1990: Sequence version 1, and database version 187, May 8, 2019).

[0111] All of these proteins have homologs in other mammalian species (cats, dogs, mice, rats, etc.). Technicians can retrieve the corresponding complete sequences from public databases.

[0112] As those skilled in the art will understand, the expression levels of NT-proBNP, RBP4, and / or GFAP can be determined by measuring the mRNA levels encoded by the respective genes or by measuring the protein levels encoded by the genes and their variants.

[0113] By way of non-limiting illustration, the expression level is determined by quantifying the level of mRNA encoded by the gene. The latter can be quantified using conventional methods, such as methods involving mRNA amplification and quantification of the amplified products, e.g., electrophoresis and staining, or alternatively, by Northern blotting with suitable probes, Northern blotting with specific probes of the mRNA of the gene of interest or its corresponding cDNA / cRNA, SI nuclease mapping, RT-PCR, hybridization, microarrays, etc. Similarly, the cDNA / cRNA corresponding to the mRNA encoded by the marker gene can also be quantified using conventional techniques; in this case, the method of the present invention includes the steps of synthesizing the corresponding cDNA by reverse transcription (RT) of the corresponding mRNA and subsequently synthesizing (RNA polymerase) and amplifying the cRNA complementary to the cDNA. Conventional methods for quantifying expression levels can be found in laboratory manuals.

[0114] To standardize mRNA expression values ​​across different samples, the expression levels of the mRNA of interest in the test sample can be compared to those of a control RNA. As used herein, "control RNA" refers to RNA whose expression level is unchanged or changed only in a limited amount. Preferably, the control RNA is an mRNA derived from a housekeeping gene that encodes a constitutively expressed protein that performs a basic cellular function. Preferred housekeeping genes used in this invention include 18-S ribosomal protein, β-2-microglobulin, ubiquitin, cyclophilin, GAPDH, PSMB4, tubulin, and β-actin.

[0115] Alternatively, the expression level of a marker gene can be determined by determining the expression level of the protein encoded by the gene, since an increase in gene expression should result in an increase in the amount of the corresponding protein, and a decrease in gene expression should result in a decrease in the amount of the corresponding protein.

[0116] Protein expression levels can be determined by qualitative and / or quantitative assays selected from immunological assays, bioluminescence, fluorescence, chemiluminescence, electrochemistry, and mass spectrometry. It is suggested that specific assays be implemented in point-of-care testing (POCT) formats to easily and rapidly determine biomarker levels. In specific implementations, point-of-care testing includes lateral flow assays, which allow the detection of the presence (or absence) of a target analyte in a liquid sample (matrix) without the need for specialized and expensive equipment, although many laboratory-based applications are supported by reading devices.

[0117] As illustrated in the examples, a specific point-of-care testing (POCT) was developed and tested in ambulances and helicopters. This POCT achieved useful high sensitivity for ischemic stroke with 100% specificity, enabling the initiation of pre-admission reperfusion therapy in selected cases much faster than using standard techniques.

[0118] Independent of the testing method, the specific quantitative test is selected from immunological tests, bioluminescence, fluorescence, chemiluminescence, electrochemistry, and mass spectrometry.

[0119] In one implementation, the expression level is determined by immunological techniques, such as enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay, agglutination assay, antibody-antigen-antibody sandwich assay, antigen-antibody-antigen sandwich assay, immunochromatography, or other forms of immunoassay known to those skilled in the art, such as radioimmunoassay, and protein microarray forms, such as single-molecule assay (SIMOA), Western blotting, or immunofluorescence.

[0120] Western blot is based on the detection of proteins previously resolved by gel electrophoresis under denaturing conditions and immobilized on a membrane (typically nitrocellulose) by incubation with specific antibodies and a imaging system (e.g., chemiluminescence). Immunofluorescence assays require the use of antibodies specific to the target protein for expression analysis. ELISA is based on the use of antigens or enzyme-labeled antibodies, so that the conjugate formed between the target antigen and the labeled antibody results in the formation of an enzyme-active complex. Since one component (antigen or labeled antibody) is immobilized on a support, the antibody-antigen complex is immobilized on the support and can therefore be detected by adding a substrate, which is converted by the enzyme into a product that can be detected by, for example, spectrophotometry, fluorescence, mass spectrometry, or tandem mass tagging (TMT). SIMOA is a more sensitive assay than ELISA because it uses a femtoliter-sized array of reaction chambers called a single-molecule array (Simoa™), which can separate and detect individual enzyme molecules. Because the array volume is approximately 2 billion times smaller than that of a conventional ELISA, a rapid accumulation of fluorescent products occurs if a labeled protein is present. This high local concentration of the product can be easily observed in the absence of diffusion. Only one molecule is needed to reach the detection limit. Using the same reagents as conventional ELISA, this method has been used to measure proteins in a variety of matrices (serum, plasma, cerebrospinal fluid, urine, cell extracts, etc.) at femtomolar (fg / mL) concentrations, providing approximately 1000-fold improvement in sensitivity.

[0121] On the other hand, the determination of protein expression levels can be performed by constructing a tissue microarray (TMA) containing an assembled subject sample and measuring the protein expression level using techniques known in the art.

[0122] In a preferred embodiment, the biomarker level is determined using an immunological technique. In a more preferred embodiment, the immunological technique is ELISA.

[0123] When using immunological methods, any antibody or reagent known to bind to the target protein with high affinity can be used to detect the amount of the target protein. However, antibodies are preferred, such as polyclonal serum, hybridoma supernatant or monoclonal antibodies, antibody fragments, Fv, Fab, Fab', F(ab')2, ScFv, biantibodies, triantibodies, tetraantibodies and humanized antibodies.

[0124] As previously mentioned, the expression levels of NT-proBNP and / or RBP4 and / or GFAP can be determined by measuring the levels of the protein and its variants (e.g., fragments, isotypes, analogs, and / or derivatives).

[0125] The term "functionally equivalent variant" should be understood to refer to all those proteins derived from the NT-proBNP and / or RBP4 and / or GFAP sequences by modification, insertion, and / or deletion of one or more amino acids, provided that the function of the variant is substantially maintained. Preferably, variants of NT-proBNP and / or RBP4 and / or GFAP are (i) polypeptides in which one or more amino acid residues are substituted with conserved or non-conserved amino acid residues (preferably conserved amino acid residues), and the substituted amino acid may or may not be encoded by the genetic code, (ii) polypeptides in which one or more modified amino acid residues (e.g., residues modified by substitution bonds), (iii) polypeptides produced by substitution processing similar to mRNA, (iv) polypeptide fragments, and / or (v) polypeptides produced by fusion of NT-proBNP and / or RBP4 and / or GFAP or polypeptides defined in (i) to (iii) with another polypeptide (e.g., a secretion leader sequence or a sequence for purification (e.g., a His tag) or for detection (e.g., an Sv5 epitope tag)). Fragments include polypeptides derived from proteolytic cleavage (including multisite proteolysis) of the original sequence. Variants may be post-translational or chemically modified. These variants should be obvious to those skilled in the art.

[0126] As is known in the art, the “similarity” between two proteins is determined by comparing the amino acid sequence of one protein and its conserved amino acid substitutes with the sequence of a second protein. A variant is defined as comprising a polypeptide sequence different from the original sequence, preferably with each relevant fragment differing from the original sequence by less than 40% residues, more preferably by less than 25% residues, even more preferably by less than 10% residues, and even more preferably by only a few residues, while being sufficiently homologous to preserve the functionality of the original sequence. Variants according to the invention comprise amino acid sequences having at least 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 90%, or 95% similarity or identity to the original amino acid sequence. The degree of identity between the two proteins is determined using computer algorithms and methods known to those skilled in the art. The identity between two amino acid sequences is preferably determined by using the BLASTP algorithm [BLASTP, Altschul, S. et al., NCBI NLM NIHBethesda, Md. 20894, Altschul, S. et al., J.Mol.Biol. 215: 403-410 (1990)].

[0127] Proteins can be post-translational modified. For example, post-translational modifications falling within the scope of this invention include signal peptide cleavage, glycosylation, acetylation, isoprenelation, myristylation, protein folding, and protein hydrolysis. Additionally, proteins may include non-natural amino acids formed through post-translational modifications or by introducing non-natural amino acids during translation.

[0128] In another specific embodiment of the in vitro methods of the present invention that provide differential diagnostic and information for selecting treatment, they further include the steps of: (i) collecting diagnostic information, and (ii) storing the information in a data carrier.

[0129] In the context of this invention, "data carrier" should be understood as any means, such as paper, that contains meaningful informational data for differentiating between IS and ICH diagnoses and / or for selecting candidates for reperfusion therapy. The carrier can also be any entity or device capable of carrying differential diagnostic data or information for treatment selection. For example, the carrier may include storage media (e.g., ROM, such as CD ROM or semiconductor ROM) or magnetic recording media (e.g., floppy disk or hard disk). Furthermore, the carrier can be a transmissible carrier, such as an electrical or optical signal, which can be transmitted via cable or optical fiber or via radio or other means. When diagnostic / treatment selection data is embodied in a signal that can be directly transmitted by cable or other means, the carrier may be constituted by such cable or other means. Other carriers include USB devices and computer files. Examples of suitable data carriers are paper, CDs, USB drives, computer files in a PC, or audio recordings containing the same information.

[0130] The present invention also includes an in vitro method for the prognosis of patients with ischemic stroke, comprising determining the levels of retinol-binding protein-4 (RBP4) and the N-terminal fragment of B-type natriuretic peptide (NT-proBNP) in isolated samples from said patients.

[0131] In a specific embodiment of the in vitro method for prognosis, the levels of at least RBP4 or two proteins are compared to reference values, wherein the reference values ​​are selected from values ​​or ranges that indicate or confirm ischemic stroke in the subject. In another, more specific embodiment, the prognosis is defined as follows: a dependence of greater than 2 on a modified ranking scale (mRS) determined within 1 to 5 months after stroke onset; and / or a prognosis defined by a 20% to 30% 3-month mortality rate after onset.

[0132] As previously stated, the present invention also relates to a kit comprising reagent elements for detecting RBP4 and NT-proBNP levels.

[0133] In one specific embodiment of the kit of the present invention, it further includes reagent elements for detecting GFAP levels.

[0134] As used herein, the term "reagent kit" refers to a product containing the various reagents (or reagent elements) necessary for carrying out the methods of the present invention, packaged to allow for transport and storage. Suitable materials for packaging reagent kit components include crystals, plastics (e.g., polyethylene, polypropylene, polycarbonate), bottles, vials, paper, or envelopes.

[0135] Furthermore, the kit of the present invention may include instructions for using the different components of the kit simultaneously, sequentially, or individually. These instructions may be in the form of printed material or an electronic medium capable of storing easily readable or understandable instructions, such as electronic storage media (e.g., magnetic disks, magnetic tapes), optical media (e.g., CD-ROMs, DVDs), or audio material. Additionally, or alternatively, the medium may contain an internet address providing the instructions.

[0136] The reagent elements (or simply reagents) of the kit include compounds that specifically bind to the labeled protein. Preferably, the compound is an antibody, an aptamer, or a fragment thereof.

[0137] In a preferred embodiment, the reagent is an antibody or a fragment thereof. Therefore, the reagent element is one or more antibodies that specifically recognize the protein of interest (i.e., NT-proBNP, RBP4, and, if identified, GFAP). The antibodies in the kit of the present invention can be used according to techniques known in the art for determining protein expression levels, such as flow cytometry, Western blotting, ELISA, RIA, competitive EIA, DAS-ELISA, techniques based on the use of biochips, protein microarrays, or colloidal precipitation assays in reaction strips.

[0138] Antibodies can be immobilized onto a solid support, such as a membrane, plastic, or glass, optionally treated to facilitate antibody immobilization. The solid support contains at least a set of antibodies that specifically recognize a biomarker (i.e., the protein of interest) and can be used to detect the expression level of that biomarker.

[0139] Furthermore, the kit of the present invention includes reagents for detecting proteins encoded by constitutive genes. The availability of these additional reagents allows for the standardization of measurements performed in different samples (e.g., the sample to be analyzed and control samples) to exclude differences in biomarker expression caused by variations in the total protein content in the samples that outweigh the actual differences in relative expression levels. Constitutive genes in this invention are genes that are always active or continuously transcribed, encoding constitutively expressed proteins that perform essential cellular functions. Constitutively expressed proteins that can be used in this invention include, but are not limited to, β-2-microglobulin (B2M), ubiquitin, 18-S ribosomal protein, cyclic proteins, GAPDH, PSMB4, tubulin, and actin.

[0140] In a preferred embodiment, the reagent elements for determining different biomarker levels comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of the total amount of reagents used to determine the biomarkers forming the kit. Therefore, in specific cases where kits contain reagents for determining RBP4, NT-proBNP, and optionally GFPA levels, the reagents specific to said biomarkers (i.e., antibodies that specifically bind to RBP4, NT-proBNP, and optionally GFPA) comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of the antibodies present in the kit. Thus, these kits are simplified kits that primarily comprise reagent elements for detecting two (or three) protein levels.

[0141] In another specific embodiment, the kits of the present invention are considered to be point-of-care testing. More specifically, they are in the form of lateral flow testing.

[0142] In another specific embodiment, the kit according to the invention includes a support and one or more inlets for depositing biological fluid samples, particularly whole blood; a reaction zone including a device / reagent for specific binding to a biomarker protein, particularly an antibody; wherein the inlet is connected to the reaction zone. In another more specific embodiment, the kit includes as many inlets as there are biomarkers (one, two, or three) to be detected and corresponding reaction zones connected thereto. In another embodiment, the kit includes a single inlet and the same number of capillaries connected to multiple reaction zones, the capillaries guiding a portion of the sample to each correspondingly connected reaction zone. A kit containing more than one reaction zone is a multiplex kit.

[0143] On the other hand, the present invention relates to the use of the kit of the present invention for distinguishing between IS and ICH or for selecting patients with stroke for reperfusion therapy, wherein, in a specific embodiment, the reperfusion therapy is selected from the group consisting of antithrombotic therapy, thrombectomy, and combinations thereof.

[0144] Therefore, in specific embodiments, the present invention relates to the application of the reagent kit of the present invention in any method of the present invention.

[0145] As illustrated in the examples below, the inventors have surprisingly discovered that determining the combination of levels of RBP4 and NT-proBNP, optional GFAP, and / or certain additional clinical parameters allows for the diagnosis of stroke patients with large vessel occlusion (LVO). LVO may contribute in part to the adverse outcomes or poor prognosis in patients with ischemic stroke as described above.

[0146] Therefore, in relation to this selection and proper classification of stroke patients, the present invention also relates to an in vitro method for diagnosing LVO, which includes determining the levels of RBP4 and NT-proBNP in a separated sample of a subject. In a specific embodiment, the subject is an ischemic stroke patient.

[0147] In one specific embodiment of the in vitro method for diagnosing LVO, it further includes determining the level of GFAP. In another, more specific embodiment, the method further includes the step of comparing the levels of RBP4, NT-proBNP, and, if determined, GFAP with corresponding reference values, wherein the reference values ​​or ranges are selected from values ​​or ranges of values ​​from subjects with LVO, and wherein a subject is diagnosed with LVO when the level of at least one of RBP4, NT-proBNP, and GFAP is within the value or range of values ​​from subjects with LVO. In a more specific embodiment of the in vitro method for diagnosing LVO, it includes determining one or more clinical parameters. These clinical parameters are specifically selected from groups consisting of blood pressure, including systolic and / or diastolic blood pressure, blood glucose, blood d-dimer levels, age, scores from a systematic assessment tool for stroke-related neurological deficits, sex, and combinations thereof. In a more specific embodiment, the in vitro method for diagnosing LVO includes determining the levels of RBP4, NT-proBNP, and GFAP, blood glucose, d-dimer, and diastolic blood pressure in an isolated sample, as well as baseline scores from a systematic assessment tool for stroke-related neurological deficits, such as the NIHSS score, RACE, Cincinnati, LAMS, etc. As in other aspects and embodiments, the isolated sample is preferably a biological fluid, more specifically selected from plasma and serum. Furthermore, as disclosed in other aspects of the invention, values ​​of clinical parameters can be combined with the levels of RBP4, NT-proBNP, and optionally GFAP to correctly classify the patient as having LVO in a suitable algorithm. In another specific embodiment of the in vitro method for diagnosing LVO, the determination of RBP4 and NT-proBNP levels, and if determined, GFAP levels, is performed within the first two hours after stroke onset, more specifically within the first hour after stroke onset.

[0148] To the inventors' knowledge, this is the first time that a combination of markers in serum and / or plasma has been provided with reliable information for the accurate diagnosis of LVO (highly sensitive in patients with IS). This is another real contribution to the field, as currently used clinical scores or procedures fall far short of 100% sensitivity and lack adequate accuracy, which is why they are not well implemented in clinical practice.

[0149] The high sensitivity of LVO diagnosis in patients with ischemic stroke, combined with the above-mentioned determination of RBP4, NT-proBNP and optional GFAP levels, allows these patients to be transferred to the nearest reference hospital where mechanical thrombectomy can be performed.

[0150] Therefore, by rapidly classifying patients exhibiting stroke symptoms using RBP4, NT-proBNP, and optional GFAP levels (e.g., at the ambulance level, and if possible, within the first two hours after onset, or even within the first hour), patients can be classified as reperfusion candidates so that they can first receive antithrombotic agents in the ambulance and then be transported to a hospital with facilities for treating LVO.

[0151] This article also discloses an in vitro method for differentiating between ischemic and hemorrhagic stroke in patients or for selecting stroke patients for reperfusion therapy. This method includes determining the levels of NT-proBNP and GFAP in a patient's isolated sample, optionally in combination with clinical parameters selected from blood pressure, including systolic and / or diastolic blood pressure, blood glucose, age, NIHSS score from a stroke-related neurological deficit assessment tool, sex, and combinations thereof. More specifically, the method includes determining the levels of NT-proBNP and GFAP in the isolated sample in combination with the patient's blood pressure. This specific combination allows for differentiation with high sensitivity and specificity, as illustrated in the examples below. In another, more specific embodiment, the method further includes determining the level of RBP4 in the isolated sample. More specifically, the isolated sample is selected from biopsy samples, tissues, cells, or biological fluids (plasma, serum, saliva, semen, sputum, cerebrospinal fluid (CSF), tears, mucus, sweat, breast milk, and brain extracts). Serum or plasma is particularly preferred.

[0152] All the specific embodiments previously disclosed with respect to the first and second aspects are also applicable to this method of distinguishing between ischemic and hemorrhagic stroke patients and / or selecting patients with stroke for reperfusion therapy, the method comprising comparing the levels of NT-proBNP and GFAP.

[0153] In a more specific embodiment of an in vitro method for differentiating between ischemic and hemorrhagic stroke and / or selecting stroke patients for reperfusion therapy, the method includes the step of comparing the levels of NT-proBNP and GFAP with corresponding reference values ​​or intervals for each protein, said reference values ​​or intervals being selected from values ​​or intervals of values ​​of subjects with ischemic stroke, and wherein a subject is classified as a candidate for reperfusion therapy when at least the levels of GFAP and NT-proBNP are within the values ​​or intervals of values ​​of subjects with ischemic stroke and optionally blood pressure is also within the values ​​of subjects with ischemic stroke. In another specific embodiment, and as previously noted for other aspects of the invention, the values ​​of the levels of NT-proBNP and GFAP in isolated samples are used in combination with determined blood pressure to correctly classify patients as candidates for reperfusion therapy in an appropriate algorithm. The decision scheme uses a combination of blood pressure within a specific interval and specific levels of the two proteins to correctly classify and select treatment. In another specific embodiment, these values ​​are incorporated into the formula of a regression model to give a score or final value that allows such classification. To improve the sensitivity of this method, NT-proBNP and GFAP levels are measured within the first two hours after stroke onset. More specifically, within the first hour. In another embodiment, high sensitivity for detecting ischemic stroke with large vessel occlusion is achieved when NT-proBNP and GFAP levels are determined and the values ​​are combined with clinical variables such as baseline NIHSS score and / or d-dimer levels and / or blood pressure values, thus making it a candidate for specific thrombectomy treatments.

[0154] In another specific embodiment of an in vitro method for differentiating between ischemic and hemorrhagic stroke in patients, or for selecting stroke patients for reperfusion therapy, it includes determining the levels of NT-proBNP and GFAP in a patient's isolated sample, said levels being measured using a point-of-care testing (POCT) comprising reagent elements for analyzing these two proteins in the isolated sample. More specifically, the kit includes only those devices in the reagent elements for detecting the levels (protein level or mRNA level) of one or both of NT-proBNP and GFAP.

[0155] Throughout the specification and claims, the word "comprising" and its variations are not intended to exclude other technical features, additives, ingredients, or steps. Furthermore, the word "comprising" covers situations where it means "composed of". Other objects, advantages, and features of the invention will become apparent to those skilled in the art upon review of the specification, or may be learned through practice of the invention. The following examples are provided as illustrations and are not intended to limit the invention. Moreover, the invention covers all possible combinations of the specific and preferred embodiments described herein.

[0156] Example

[0157] To provide a method for pre-admission differentiation between ischemic stroke (IS) and intracerebral hemorrhage (ICH) using blood biomarkers, the inventors conducted an in-depth analysis of their existing stroke patient samples. The primary objective was to identify reliable biomarkers that would inform the initiation of reperfusion therapy (primarily intravenous thrombolysis) without the need for neuroimaging techniques.

[0158] Example 1. Patient triage and treatment selection in two different groups (Group 1 had 190 patients; Group 2 had 67 patients).

[0159] Materials and methods

[0160] From December 2013 to July 2014, suspected stroke patients admitted within 4.5 hours of stroke onset were recruited. Blood samples were collected at admission (baseline). Biomarkers were primarily determined by ELISA and SIMOA. Stroke subtypes were confirmed by neuroimaging techniques. Biomarkers were dichotomized by cutoff values, with the highest sensitivity (100% specificity) selected to minimize any errors from administering tPA to ICH patients.

[0161] The patient groups are as follows:

[0162] Group 1 (ELISA group): 190 patients with stroke (155 ischemic and 35 hemorrhagic).

[0163] Group 2 (SIMOA group): 67 patients with stroke (33 ischemic and 34 hemorrhagic).

[0164] Kits for biomarker analysis:

[0165] -For RBP4-->catalog number DRB400, Quantikine R&D Systems;

[0166] -For GFAP (measured using the Simoa suite and the name of the SIMOA group assigned to group 2)-->Catalog number 102336 and consumables:

[0167] -Simoa Accelerator-1 Plate Lab Service Fee, Catalog No. 100835

[0168] -Accelerator Consumables and Reagents, Catalog No. ACC1001

[0169] -For NT-proBNP-->Used in automation The following reference catalog reactants in the system (4842464130-proBNP GEN.2ELECSYS; 4917049922-Precicontrol cardiac G4; 4842472190-CALSET proBNO GEN.2ELECSYS)

[0170] These groups 1 and 2 were selected after excluding simulated cases (those patients who did not have a stroke but had clinical indications for stroke).

[0171] In all patients, the expression levels of GFAP, NT-proBNP, and RBP4 at baseline were measured using ELISA (for NT-proBNP and RBP4; see above) or SIMOA (for GFAP; see above).

[0172] Different methods for analyzing and retrieving data were determined: basic cutoff (method 1), principal component analysis (PCA) (method 2), improved (more rounds) PCA; and support vector machine program (SVM) (method 4).

[0173] Of all the approaches, the focus here is on finding the optimal cutoff values ​​for these blood biomarker values, which would indicate the exact value of each biomarker, thus enabling patients to be classified with 100% accuracy to avoid any errors, as administering reperfusion therapy (i.e., tPA or TNK) to ICH patients can produce fatal side effects.

[0174] result

[0175] Method 1 (Basic cutoff)

[0176] In this method, the simplified cutoff values ​​of unique biomarkers in the training group are used, and then all cutoff values ​​are combined to obtain the final classification.

[0177] In group 1 (ELISA group), ischemic patients can be detected by observing those with high levels of NT-proBNP or RBP4. Therefore, with 100% specificity as the goal, patients are classified as ischemic if they have the following characteristics:

[0178] -NT-proBNP >4062 pg / mL; achieved 100% specificity and 14.3% sensitivity (detected in 22 / 155 ischemic patients at no risk) (see [link to relevant documentation]). Figure 1 (A) or RBP4 > 52 μg / mL; achieving 100% specificity and 6.5% sensitivity (detecting 10 / 155 ischemic patients in a risk-free setting) (see [link]). Figure 1 (B))

[0179] Only one of these two conditions needs to be met. Finally, if both conditions are met, 31 / 155 ischemic patients (20.1%) are classified with 100% specificity (see 1(C)).

[0180] Data plotted Figure 1 In the figure, the cutoff values ​​of 4062 pg / mL for NT-proBNP (the black lines in Figures A and B) or 52 μg / mL for RBP4 enable reliable differentiation between IS and ICH. In Figure C, the log10 (NT-proBNP) and RBP4 levels are plotted against the corresponding previously determined cutoff values ​​for each protein.

[0181] For cohort 2 (67 patients) (also referred to herein as the SIMOA cohort) which included the use of the SIMOA test for GFAP to identify GFAP, the first approach of combining GFAP with other biomarkers was explored to enable safer and better detection in ischemic patients.

[0182] The classification process had two phases. First, patients with GFAP values ​​<325 ng / mL (Case 1) were selected as potential ischemic candidates. Twenty-seven patients with hemorrhagic conditions, along with six ischemic patients from the potential ischemic group, were excluded. A visualization of this first differentiation step is shown in [image / plot]. Figure 2 In (A), the three-dimensional plot categorizes patients based on detected GFAP levels (as log(GFAP), NT-proBNP levels (as log(NT-proBNP), and RBP4 levels). Values ​​below the squares indicating GFAP < 325 ng / mL (arrows in the three-dimensional space) correspond to patients selected as ischemic candidates in the first phase.

[0183] In the second category, ischemic patients are those who meet one of the following criteria:

[0184] -NT-proBNP >1305 pg / mL; achieved 100% specificity and 30.3% sensitivity (detected in 10 / 33 ischemic patients), or

[0185] -RBP4>38μg / mL; 100% specificity and 30.3% sensitivity were obtained (detected in 10 / 33 ischemic patients).

[0186] If GFAP was no longer considered among the potential ischemic candidates selected in the first phase, and patients with NT-proBNP > 1305 pg / mL and RBP4 > 38 μg / mL were considered candidates, then 17 / 33 ischemic patients (51.5%) were detected without any risk (100% specificity). Data plotted on Figure 2 (B)

[0187] Several analyses were performed at different specificity values ​​(other than 100%).

[0188] With 97% specificity, ischemic patients are those with GFAP <325 ng / mL and who also meet the following criteria:

[0189] -NT-proBNP >600 pg / mL; achieved 100% specificity and 51.5% sensitivity (detected in 17 / 33 ischemic patients).

[0190] -RBP4>36.6μg / mL; 97% specificity and 39.4% sensitivity were achieved (detected in 10 / 33 ischemic patients).

[0191] This enabled the detection of 23 / 33 ischemic patients (69.7%) with 97% specificity.

[0192] With 94% specificity, ischemic patients are those with GFAP <325 ng / mL and who also meet the following criteria:

[0193] -NT-proBNP >147 pg / mL; achieved 94% specificity and 69.7% sensitivity (detected in 23 / 33 ischemic patients).

[0194] -RBP4>31μg / mL; 94% specificity and 51.5% sensitivity were achieved (detected in 17 / 33 ischemic patients).

[0195] This enabled the detection of 26 / 33 ischemic patients (78.7%) with 94% specificity.

[0196] Cutoff values ​​always indicate a bias or some variability caused by different factors. All of the cutoff values ​​mentioned here relate to a specific adjusted prediction accuracy (typically IC 95%), thus falling within the range of values ​​that are also considered positive or discriminative.

[0197] The optimal cutoff values ​​for reliable classification of stroke patients using Method 1 are the single cutoff values ​​for the ELISA cohort (RBP4 > 52 μg / ml and NT-proBNP > 4062.0 pg / ml).

[0198] Method 2 (Principal Component Analysis)

[0199] Using principal component analysis (PCA), we determined which of the three biomarkers explained the greatest variability with fewer principal components (Jolliffe, IT (2002). Principal Component Analysis, 2nd Edition (Springer), ISBN 0-387-95442-2). The procedure includes performing principal component analysis. After calculation, we examined which variables contributed most to the principal components. The results showed that GFAP was most correlated with PC1, and RBP4 was most correlated with PC2. Therefore, the optimal order for classifying patients is GFAP > RBP4 > NT-proBNP.

[0200] In short, Principal Component Analysis (PCA) is a statistical procedure that uses orthogonal transformations to convert observations of a set of potentially correlated variables (entities that take different values) into a set of linearly uncorrelated variables called principal components. If there are n observations of p variables, the number of distinct principal components is min(n-1, p). This transformation is defined as follows: the first principal component has the largest possible variance (i.e., explains as much of the variability in the data as possible), and each subsequent component has the largest possible variance in turn, provided it is orthogonal to the preceding components. The resulting vectors (each a linear combination of variables containing n observations) are an uncorrelated orthogonal basis set. PCA is sensitive to the relative scaling of the original variables. PCA is primarily used as a tool for exploratory data analysis and building predictive models. Mathematically, PCA is defined as an orthogonal linear transformation that converts data to a new coordinate system such that the largest variance of a projection of the data lies on the first coordinate (called the first principal component), the second largest variance on the second coordinate, and so on.

[0201] This order of cutoff biomarkers (GFAP > RBP4 > NT-proBNP) is used for patient classification. The first GFAP cutoff value is used to classify the maximum number of patients with 100% sensitivity or 100% specificity. Then, the RBP4 cutoff value is used to classify patients who could not be classified in the previous cutoffs with 100% sensitivity or 100% specificity. Finally, the NT-proBNP cutoff value is used to classify unclassified patients with 100% sensitivity or 100% specificity. Once these three cutoff values ​​have been applied, the first round of cutoffs is considered complete.

[0202] The same analysis was performed again using only two biomarkers (RBP4 and NT-proBNP).

[0203] In this case, when using GFAP, the cutoff values ​​are shown in Table 1:

[0204] Table 1. Patient classification using PCA analysis (Round 1)

[0205]

[0206] The cutoff values ​​in cohort 1 (190 patients) were more robust than those seen in cohort 2, which used the SIMOA test to determine GFAP in addition to the EILSA test for NT-proBNP and RBP4 (i.e., the SIMOA cohort (67 patients)).

[0207] Using the cutoff value of the ELISA group (group 1), 23.16% of the entire group can be classified with 100% sensitivity or 100% specificity (37.14% for hemorrhagic patients and 20% for ischemic patients).

[0208] The cutoff value using the SIMOA group (group 2) can classify the entire group with 100% sensitivity or 100% specificity, representing 31% (38% for hemorrhagic patients and 24% for ischemic patients).

[0209] Using a small number of individuals in group 2 allowed for better differentiation (more than 1% of hemorrhagic patients and 4% of ischemic patients were classified).

[0210] The cutoff values ​​for using only RBP4+NT-proBNP are those in Table 2:

[0211] Table 2: Patient classification using PCA analysis (Round 1)

[0212]

[0213] Group A (190 patients) was again more robust than Group 2 (67 patients) (same cutoff value as above).

[0214] The cutoff value for using the ELISA group (group 1) can be classified with 100% sensitivity or 100% specificity for 16.8% of the entire group (3% for hemorrhagic patients and 20% for ischemic patients).

[0215] The cutoff value using the SIMOA group (group 2) can classify the entire group with 100% sensitivity or 100% specificity at 13.4% (3% for hemorrhagic patients and 24% for ischemic patients).

[0216] Using a small number of individuals in the SIMOA group allows for better differentiation (extraclassifying 4% of ischemic patients).

[0217] Method 3 (Principal Component Analysis)

[0218] This method is an extension of method 2, in which more rounds are computed with unclassified individuals.

[0219] After the first round, logistic regression is performed to check if any trends in certain biomarkers associated with the outcome still exist. Then, cutoff values ​​are calculated again in the same order for patients who have not yet been classified. This method is performed until no trend is observed or more individuals cannot be classified with 100% sensitivity or 100% specificity. It is important to note that once an individual has been classified, the order from the first to the last cutoff value must be followed.

[0220] Method 3 also uses only two biomarkers (RBP4 and NT-proBNP).

[0221] Using the ELISA cohort (cohort 1) and the following cutoff values ​​were obtained using GFAP, RBP4, and NT-proBNP as biomarkers:

[0222] Table 3: Patient classification using PCA analysis (more than one round)

[0223]

[0224] Using this method, 51% of individuals were classified with 100% sensitivity or 100% specificity (60% hemorrhagic and 49% ischemic).

[0225] For the ELISA cohort (cohort 1), RBP4 and NT-proBNP were used as biomarkers, and the following cutoff values ​​were obtained:

[0226] Table 4: Patient classification using PCA analysis (more than one round)

[0227]

[0228] 36.5% of individuals were classified with 100% sensitivity or 100% specificity (hemorrhagic 6%, ischemic 44%).

[0229] Method 4 (SVM)

[0230] RBP4 and NT-proBNP levels were computed using a Support Vector Machine (SVM) procedure to maximize the number of well-classified IS patients while achieving 100% specificity for all ICH patients (see “A User’s Guide to Support Vector Machines”, Article in Methods in Molecular Biology, (Clifton N.J), 2010, Asa Ben-Hur and Jason Weston). Radial kernel analysis was employed with the following parameters: c = 100, sigma = 0.05. To obtain a classifier with 100% specificity for ICH data, the decision value should be increased by 0.71 at each point (the intuition behind the decision value is that the larger the positive value, the less chance there is of classifying an ICH patient as IS while losing the chance of classifying an IS patient as IS).

[0231] Using this method, a sensitivity of 29.7% was achieved for IS.

[0232] Data plotted Figure 3 In the diagram, the value above the "sigmoidal curve" represents 100% IS (indicated by an arrow), while the value below the curve corresponds to patients with either ICH or IS subtypes.

[0233] As from Figure 3 It can be deduced that the combination of RBP4 and NT-proBNP levels allows for good patient classification and appropriate treatment selection. These levels can be introduced into a support vector machine process with a Gaussian kernel to obtain results such as... Figure 3 The diagram shown illustrates how the value of one protein, along with the value of another protein, will determine the correct classification of the patient as either IS or ICH.

[0234] According to SVM, once deterministic values ​​are obtained for the test samples, they are fed into a trained SVM, which returns a decision value that allows the test sample to be classified into one category (IS) or another category (ICH). Specifically, the decision value is typically 0.5, and it can be adapted if needed to improve the correct classification of the subject. Depending on the trained machine, patients exceeding this value are classified into one category, while those below are classified into another. The decision value can be adapted.

[0235] in conclusion:

[0236] Using Method 1 (baseline cutoff) in conjunction with the ELISA cohort (cohort 1) and employing RBP4 and NT-proBNP separately allowed for correct differentiation of 6.5% and 14.2% of IS patients, respectively, with 100% specificity for ICH. When used together, the differentiation increased to 20% of IS patients with 100% specificity for ICH, which is less than the sum of the two biomarkers individually (because one individual was correctly classified by both biomarkers). In the SIMOA cohort (cohort 2): when the maximum number of ICH and IS patients were separated using the GFAP cutoff and then an attempt was made to classify IS patients at the bottom of the GFAP cutoff, the sensitivity for IS patients increased (51.5%) while maintaining 100% specificity for ICH patients.

[0237] Using Method 2 (also known as Class-Delete) and Method 3 (also known as Class-Delete-Duplicate), it was observed that adding the GFAP biomarker helped correctly classify ICH patients (60% with GFAP in the ELISA cohort versus 6% without GFAP, compared to 37% with GFAP and 3% without GFAP in the SIMOA cohort). It is also important to note that there was no significant loss of ability to classify ICH patients without GFAP (49% vs. 44% with and without the GFAP biomarker, and 24% vs. 24% in the SIMOA cohort).

[0238] Including GFAP does not affect the ability to differentiate IS patients.

[0239] Finally, the method 4 (SVM) classifier, a more sophisticated method, was used, which correctly distinguished 29.7% of IS patients and had 100% specificity for ICH.

[0240] All this data also clearly demonstrates that the reference value for correctly classifying patients is a dynamic value, which can be a function of several parameters, such as the number of other markers identified simultaneously, or the techniques used for analyzing patient cohort data. On the other hand, a fixed amount or level of one protein can differ from the amount of the first other markers identified simultaneously and allowing for good classification, and can be represented by several mathematical functions or models used (i.e., basic cutoff, ROC curve, PCA, SVM, etc.).

[0241] Analysis of different data from two different patient groups according to Example 1 of this specification clearly demonstrates that the levels of RBP4 and NT-proBNP, as well as the optional GFAP, allow for good differentiation between IS and ICH and safe selection of reperfusion therapy candidates. Reference intervals or values ​​for proper classification will be adapted based on several variables calculated in the methods. In any case, these values ​​should be provided to the personnel who must perform these methods.

[0242] Using these two or three markers, a global picture of well-classified patients was obtained, making them available for testing the correct classification of patients. Furthermore, it is of particular interest that ischemic patients correctly classified using the RBP4 and NT-proBNP biomarkers are those who would have worse outcomes.

[0243] As shown in Table 5 below, ischemic stroke (IS) identified using these two biomarkers has a higher mortality rate, and their independence decreases three months after stroke.

[0244] This is another reason to treat these patients as quickly as possible using reperfusion techniques to avoid fatal consequences if they were treated later using today's standard methods.

[0245] Table 5. Data from IS patients analyzed

[0246]

[0247]

[0248]

[0249] In layman's terms, this technique, which measures two biomarkers, increases the chances of receiving treatment within the "golden hour" (the first 60 minutes after an ischemic stroke), which nearly doubles the chance of becoming asymptomatic, triples the chance of being isolated, and quadruples the chance of survival (see Kunz et al., "Effects of Ultraearly Intravenous Thrombolysis on Outcomes in Ischemic Stroke: The STEMO (Stroke Emergency Mobile) Group", Circulation-2017 May 2; 135(18):1765-1767, for more information on the "golden hour" and treatment action plans).

[0250] Example 2.32 Patient classification and treatment selection in different groups

[0251] The dataset includes 32 patients (18 with hemorrhagic disease and 14 with ischemic disease). The goal is to separate patients in both categories as much as possible without any risk of misclassification.

[0252] To achieve this, four different GFAP biomarker technologies were used. Therefore, a program was performed for each different technology.

[0253] The procedure involves finding the optimal cutoff value for GFAP, removing patients classified as 100% sensitivity or 100% specificity, and calculating the next cutoff value for RBP4 biomarker data using the remaining data (unclassified / removed with GFAP, therefore incompletely classified), again classifying patients as 100% sensitivity or 100% specificity and removing them. Finally, the process is repeated using NT-proBNP biomarker data.

[0254] The cutoff values ​​for each technique used in GFAP determination are listed below.

[0255] GFAP DxSYS_CLIA (DxSYS Inc. Chemiluminescent Immunoassay): Using a cutoff value of >80.6 pg / ml, 15 patients with bleeding disorders (83% of all bleeding patients) were correctly classified.

[0256] GFAP DxSYS_TMB (DxSYS Inc. using 3,3',5,5'-tetramethylbenzidine (TMB): Using a cutoff value of >88.685 pg / ml, 14 patients with bleeding disorders (78% of all bleeding patients) were correctly classified. The same results were obtained using a cutoff value of 100 pg / ml.

[0257] GFAP Using cutoff values ​​of >2066.078 pg / ml and <166.67 pg / ml, 14 patients with hemorrhagic disease and 3 patients with ischemic disease (78% of all patients with hemorrhagic disease and 21.4% of all patients with ischemic disease) were correctly classified.

[0258] GFAP Elisa (Elisa kit catalog number RD192072200, BioVendor): Using a cutoff value of >50.5 pg / ml, 11 patients with bleeding disorders (61.1% of all patients with bleeding disorders) were correctly classified.

[0259] After applying all these cutoff values ​​and processing RBP4 and NT-proBNP as described above, 17 / 18 hemorrhagic patients and 12 / 14 ischemic patients were ultimately correctly classified.

[0260] Therefore, this procedure demonstrates that identifying three biomarkers in consecutive steps can accurately classify patients to determine the appropriate treatment plan (reperfusion therapy for ischemic stroke). Table 6 describes a summary of the procedure for each analytical technique.

[0261] Table 6. Cutoff values ​​for correctly classified markers

[0262]

[0263] Example 3. Combining biomarkers with clinical data improves the accuracy of identifying ischemic stroke requiring reperfusion therapy.

[0264] Data for the following patients and cutoff values ​​were analyzed:

[0265] Hemorrhagic (n=35) and ischemic (n=155)

[0266] The combination of three biomarkers with cutoff values ​​of GFAP (pg / ml) <97.03, NT-proBNP (pg / ml) >4076.50, and RBP-4 (μg / ml) >52.52 showed a sensitivity of 0.32, a specificity (100%) of 1.00, a positive predictive value (PPV) of 1.00, and a negative predictive value (NPV) of 0.25.

[0267] Adding clinical data (particularly blood pressure and blood glucose levels) to this base improved sensitivity while maintaining 100% specificity. In fact, the sensitivity was 0.45 and the specificity was 1.00 for GFAP (pg / ml) <97.03 and NT-proBNP (pg / ml) >4076.50, systolic blood pressure (SBP) mmHg <119.00, diastolic blood pressure (DBP) (mmHg) <60.50, RBP-4 (μg / ml) >52.52, and blood glucose (mg / dl) <83.50, with PPV = 1.00 and NPV = 0.29.

[0268] Logistic regression-based models:

[0269] To find feasible data transformations that could improve classification accuracy and robustness while limiting overfitting, multiple multilogistic regression models were tested on pooled data from two cohorts (original cohort 1 n = 189 and duplicate cohort n = 300, with hemorrhagic n = 51 and ischemic n = 249 in Example 1). The tested models included a set of biomarkers and relevant clinical variables. A model was selected using the Akaike Information Criterion to classify individuals between ischemic and hemorrhagic stroke.

[0270] The selected model incorporates logarithmic transformations of GFAP (pg / ml), NT-proBNP (pg / ml), and diastolic blood pressure (mmHg) as significant predictors of ischemic stroke, and their combination is as follows:

[0271] -1.56log(GFAP(pg / ml))+0.0008NT-proBNP(pg / ml)-0.041DBP(mmhg)

[0272] This linear combination produces an estimated log-dominance score, which can be considered a composite biomarker. A threshold can be set in this score to maximize the required sensitivity and specificity for classifying individuals between the two groups. When the specificity is above 95%, the weighting and accumulation of the biomarkers improves the classification sensitivity relative to the original biomarkers for both groups.

[0273] In the original group, the sensitivity, specificity, and accuracy of the model were 0.60, 1.00, and 0.68, respectively.

[0274] Figure 4 The classification of subjects using the logical model is illustrated graphically.

[0275] The following lists the characteristics of repeating groups (n=300):

[0276] In cases of suspected stroke (ischemic or hemorrhagic stroke), blood samples are collected within 3 hours of the stroke onset. Diagnostic and treatment examinations are similar to those used in the initial group 1 (n=189) of the patients studied in Example 1 of these documents, which are among the several embodiments.

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

[0283] Example 4. Improved accuracy in detecting ischemic stroke patients undergoing reperfusion therapy at the very early time point.

[0284] In the original cohort 1 (n=189) of Example 1, the performance of the biomarker was evaluated in relation to the time from the onset of symptom onset. Surprisingly, the earlier the test was performed, the better the accuracy of the test:

[0285] (i) 0 to 2 hours (hemorrhagic n=11 and ischemic n=82)

[0286] GFAP (pg / ml) <175.85, NT-proBNP (pg / ml) >3916.50, and RBP-4 (μg / ml) >38.15. Sensitivity = 0.70, Specificity = 1.00, PPV = 1.00, and NPV = 0.31.

[0287] (ii) 2 to 3 hours (hemorrhagic n=13 and ischemic n=35)

[0288] GFAP (pg / ml) < 94.37, NT-proBNP (pg / ml) > 1289.50, and RBP-4 (μg / ml) > 46.55. Sensitivity = 0.60, Specificity = 1.00, PPV = 1.00, and NPV = 0.48.

[0289] (iii) 3 to 4.5 hours (hemorrhagic n=11 and ischemic n=38)

[0290] GFAP (pg / ml) < 98.96, NT-proBNP (pg / ml) > 4254.50, and RBP-4 (μg / ml) > 53.34.

[0291] Sensitivity = 0.34, Specificity = 1.00, PPV = 1.00 and NPV = 0.31.

[0292] In the replicate cohorts (n=300, hemorrhagic, n=51 and ischemic, n=249), patients who arrived at the hospital very early were selected, and blood samples were collected within the first hour of stroke onset. Hemorrhagic n=8 and ischemic n=33.

[0293] In this subgroup, GFAP (pg / ml) <300.03, NT-proBNP (pg / ml) >1033.01, and RBP-4 (μg / ml) >32.93 exhibited excellent accuracy, with sensitivity = 0.91, specificity = 1.00, PPV = 1.00, and NPV = 0.73.

[0294] This was further improved and the test performed perfectly when some clinical variables (clinical parameters) were added: GFAP (pg / ml) <300.03, DBP (mmhg) <82.00, SBP (mmhg) <143.00, NT-proBNP (pg / ml) >2741.31, and blood glucose (mgdl) <108.00. This combination yielded sensitivity = 1.00, specificity = 1.00, PPV = 1.00, and NPV = 1.00.

[0295] Logistic regression was also performed on this group of patients, with samples obtained within the first hour of symptom onset.

[0296] The selected model incorporates logarithmic transformations of GFAP (pg / ml), NT-proBNP (pg / ml), and diastolic blood pressure (mmHg) as significant predictors of ischemic stroke, combined as follows:

[0297] -1.56log(GFAP(pg / ml))+0.0008NT-proBNP(pg / ml)-0.041DBP(mmhg)

[0298] This linear combination yielded an estimated log-odds ratio score, which can be considered a composite marker. When applied to a repeat cohort of patients who presented within the first hour of stroke onset, the model demonstrated sensitivity = 0.79, specificity = 1.00, and accuracy = 0.83.

[0299] Figure 5 The illustration shows the classification of subjects using the logic model for scoring, as well as the isolated sample data in the first hour after a stroke.

[0300] All of these data in Example 4 demonstrate that the selected biomarkers RBP4, NT-proBNP, and GFAP allow for high sensitivity if they can be measured shortly after stroke onset, especially if they can be identified within the first and second hours after onset.

[0301] The earlier the testing is performed, the better the sensitivity to fixed specificities can be achieved, which is a surprising benefit in stroke, where biomarkers typically only provide appropriate signals much longer after the onset of the illness. This is far from a drawback, but rather a goal of this pathology, as it allows for rapid and reliable triage of patients at critical stages of the disease (e.g., at the ambulance level). This allows for the making of the best and most appropriate decisions at those critical moments, which can then be validated with other biomarkers (e.g., once the patient arrives at the hospital).

[0302] Example 5. Rapid, immediate blood test performed in an ambulance to select ischemic stroke patients who should undergo reperfusion therapy from any other condition similar to acute ischemic stroke (stroke mimicry and intracerebral hemorrhage).

[0303] The set of biomarkers (RBP4, NT-proBNP, and GFAP) included in this invention are validated using point-of-care testing (POCT) for ambulances, which can identify ischemic stroke patients using blood samples so that reperfusion therapy (thrombolysis) can be started in the ambulance or the patient can be transferred to a suitable hospital for optimal reperfusion therapy (thrombolysis or mechanical thrombectomy).

[0304] method:

[0305] Patients suspected of having a stroke (<6 hours) were enrolled in the BIO-FAST study (Biomarkers for Initiating On-Site and Rapid Ambulance Stroke Treatment) through a network of more than 20 ambulances and helicopters in the Seville region of southern Spain. Blood samples were collected by ambulances that used rapid point-of-care testing (POCT) (results in 10 to 15 minutes) to measure RBP-4 / NT-pro-BNP, while GFAP was measured using the SIMOA-Quanterix technology.

[0306] Inclusion criteria: Patients >18 years old; stroke code activated by the coordination center; symptom onset <6 hours. In cases of stroke with an indeterminate timeline or awakening stroke, the last moment when the patient appeared to be well was considered the initial time.

[0307] Exclusion criteria: Different from the pre-admission diagnosis of stroke; unable to obtain pre-admission blood samples and refuse to provide informed consent from the patient / family.

[0308] Sample type

[0309] One EDTA tube of blood sample (10 mL) was extracted for the biobank, and one EDTA tube of blood sample (2 mL) was extracted for the point of consumption (POC). In accordance with the established requirements in RD1716 / 2011, the samples were included in the registry set at Valle d'Hebron Hospital with code C.0003176 until they were fully used for biomarker discovery studies.

[0310] -RBP4 Quick Test. RBP4 POC Lateral Flow Handling.

[0311] -NT-proBNP rapid testing. Nt-proBNP POC lateral flow treatment.

[0312] -GFAP SIMOA-Quanterix technology

[0313] result:

[0314] This included 20 patients (10 with ischemic stroke, 3 with intracranial hemorrhage, and 7 with stroke simulations). Running POCT in an ambulance is feasible, and there was even one case where it was performed in a helicopter without any incidents.

[0315] Rapid point-of-care testing (POCT) with selected cutoff values ​​for these biomarkers accurately identified 50% of intracranial strokes (IS) without misclassifying any intracranial hemorrhage (ICH) or simulated conditions (100% specificity, 50% sensitivity). When excluding awake stroke, POCT accurately identified 62.5% of IS without misclassifying any ICH or simulated conditions (100% specificity, 62.5% sensitivity).

[0316] Using blood samples stored from these patients, GFAP was measured using the SIMOA-Quanterix technique to explore how point-of-care testing (POCT) that may contain GFAP, RBP-4, and NT-proBNP could improve outcomes. Four patients were identified as having very high GFAP levels: two with ICH, one with IS, and one in a simulated scenario. Excluding these four cases improved sensitivity to over 70% while maintaining specificity at 100%.

[0317] 20% of ischemic attacks (IS) may be treated with point-of-care testing (POCT) within the first 30 minutes of symptom onset. Furthermore, because POCT may be negative for IS, a suspected patient may be spared from receiving tPA. By using the test in the ambulance, patients receiving tPA treatment may receive the medication 1 hour and 30 minutes earlier—3 out of 30 ischemic patients may have already entered the 4.5-hour testing window and exceeded that time by the time they undergo a CT scan at the hospital.

[0318] in conclusion:

[0319] A group of biomarkers, including RBP-4, NT-proBNP, and GFAP, provides useful sensitivity with 100% specificity for ischemic stroke. This could potentially transform standard clinical practice through point-of-care testing (POCT), which allows for faster initiation of pre-admission reperfusion therapy in selected cases compared to standard techniques.

[0320] Data obtained through POCT allows for the determination of RBP4 and NT-proBNP levels in the blood, which can be further refined using GFAP. GFAP should be the first-line test in real-world scenarios, where patients with IS and ICH should be screened in a simulated condition (non-stroke patients with stroke-like symptoms). In this real-world scenario, the sensitivity is even higher than some of the aforementioned examples (over 50%), while maintaining 100% specificity. This is an unexpected advantage of adding to the biomarker set, allowing for good and reliable classification, which translates into appropriate selection of treatments that can be administered before arrival at the hospital.

[0321] Example 6. RBP-4, NT-proBNP, and GFAP were used to identify ischemic stroke patients with large vessel occlusion (LVO) who required mechanical thrombectomy and transfer to a reference center where this therapy could be administered.

[0322] The identification of LVO in the two stroke patient cohorts (n=189 and n=300) disclosed above was based on the presence and location of cerebral artery occlusion on CT angiography (CTA) performed upon arrival at the hospital. Following the restrictive definition of LVO, it is described as an occlusion of any of the following arteries or arterial segments: intracranial carotid artery (ICA), basilar artery (BA), and M1 segment occlusion of the middle cerebral artery.

[0323] (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).

[0324] The same biomarkers shown below can also be used to identify LVO with less stringent criteria, including occlusion of more distal portions of the middle cerebral artery (MCA), such as M2 (LVO is defined as occlusion of the ICA, M1, M2, or BA), only by using different cutoff values.

[0325] A. Initial group 1 of Example 1 (N = 189):

[0326] Prediction for LVO patients (134 non-LVO vs. 56 LVO):

[0327] Sensitivity = 0.98, specificity = 0.18, PPV = 0.33, and NPV = 0.96 for GFAP (pg / ml) < 694.48, NT-proBNP (pg / ml) > 1764.50, and RBP-4 (μg / ml) > 35.22. Improvements were observed when clinical and laboratory variables (e.g., baseline NIHSS score > 11 points and d-dimer (ng / ml) > 1432.43) were added, with sensitivity = 1.00, specificity = 0.46, PPV = 0.43, and NPV = 1.00.

[0328] When blood samples were collected within 2 hours of symptom onset, those with a baseline NIHSS score >11 and RBP-4 (μg / ml) >51.53 performed better, with sensitivity = 1.00, specificity = 0.40, PPV = 0.46 and NPV = 1.00.

[0329] Furthermore, in patients undergoing thrombectomy (165 without thrombectomy and 22 with thrombectomy), the biomarkers GFAP (pg / ml) >209.43 and NT-proBNP (pg / ml) <848.15 had a sensitivity of 1.00, a specificity of 0.23, a PPV of 0.15, and an NPV of 1.00. Improvements were observed when clinical variables (e.g., baseline NIHSS score >11 and GFAP (pg / ml) <54.84) ​​were added, with a sensitivity of 1.00, a specificity of 0.43, a PPV of 0.19, and an NPV of 1.00.

[0330] When blood samples were collected within 2 hours of symptom onset, NT-proBNP and d-dimer performed better, with sensitivity = 1.00, specificity = 0.70, PPV = 0.39 and NPV = 1.00.

[0331] B. Repeating groups (n=300)

[0332] LVO (restrictive definition) non-LVO = 215 relative to LVO = 85

[0333] Biomarkers GFAP (pg / ml) <153.18, NT-proBNP (pg / ml) >692.60, and RBP-4 (μg / ml) <39.72 showed a sensitivity of 1.00, a specificity of 0.09, a PPV of 0.30, and an NPV of 1.00. These results were improved by adding clinical data to baseline NIHSS score >11 and RBP-4 (μg / ml) <29.03 and blood glucose (mg / dl) <71.50, showing a sensitivity of 1.00, a specificity of 0.20, a PPV of 0.33, and an NPV of 1.00.

[0334] When blood samples were collected within 2 hours of the onset of the illness, the baseline NIHSS score and RBP-4 results were even better, with sensitivity = 1.00, specificity = 0.45, PPV = 0.43 and NPV = 1.00.

[0335] In patients who underwent thrombectomy (n=264 without thrombectomy and n=35 with thrombectomy), the biomarkers GFAP (pg / ml) <153.18 and NT-proBNP (pg / ml) <2049.01 had a sensitivity of 1.00, a specificity of 0.14, a PPV of 0.13, and an NPV of 1.00 to correctly identify these patients. This was improved by adding clinical and laboratory variables, as GFAP (pg / ml) <153.18 and d-dimer (ng / ml) >7.29 and diastolic blood pressure (mmHg) >89.50 had a sensitivity of 0.97, a specificity of 0.33, a PPV of 0.16, and an NPV of 0.99.

[0336] Reference List

[0337] Patent documents

[0338] -WO2016087611

[0339] Non-patent literature

[0340] -Reynolds et al., "Early Biomarkers of Stroke", Clinical Chemistry-2003, vol.:49(10), pp.:1733-1739

[0341] -Montaner et al., "Etiologic Diagnosis of Ischemic Stroke Subtypes WithPlasma Biomarkers", Stroke 2008, vol.no.39, pp.:2280-2287

[0342] -Adams HP Jr Neurology.1999Jul 13;53(1):126-31.

[0343] -Tsivgoulis G. et al. Neurology.2014Sep 19

[0344] -BLASTManual, Altschul, S., et al, NCBI NLM NIH Bethesda, Md. 20894, Altschul, S. et al, J. Mol. Biol. 215: 403-410 (1990)

[0345] -Jolliffe, IT (2002). Principal Component Analysis, 2nd Edition (Springer), ISBN 0-387-95442-2.

[0346] - Kunz et al., "Effects of Ultraearly Intravenous Thrombolysis on Outcomes in Ischemic Stroke: The STEMO (Stroke Emergency Mobile) Group", Circulation - May 2, 2017; 135(18): 1765 - 1767.

[0347] - 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.

[0348] - 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.

[0349] - Crowe RP, Myers JB, Fernandez AR, Bourn S, McMullan JT.. Prehosp Emerg Care. 2020 Feb 25: 1 - 9.

[0350] -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).

[0351] -Lakomkin N, Dhamoon M, Carroll K, et al., Prevalence of large vessel occlusion in patients presenting with acute ischemic stroke: a 10-yearsystematic review of the literature. J Neurointerv Surg 2019;11:241–5.

[0352] -Waqas M et al., Effect of definition and methods on estimates of prevention of large vessel occlusion in acute ischemic stroke: a systematic review and meta-analysis. 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Ser Ala Tyr Arg Gly Thr His 180 185 190 Ser Leu Thr Glu Ser Gly Ala Ser Cys Leu Pro Trp Asn Ser Met Ile 195 200 205 Leu Ile Gly Lys Val Tyr Thr Ala Gln Asn Pro Ser Ala Gln Ala Leu 210 215 220 Gly Leu Gly Lys His Asn Tyr Cys Arg Asn Pro Asp Gly Asp Ala Lys 225 230 235 240 Pro Trp Cys His Val Leu Lys Asn Arg Arg Leu Thr Trp Glu Tyr Cys 245 250 255 Asp Val Pro Ser Cys Ser Thr Cys Gly Leu Arg Gln Tyr Ser Gln Pro 260 265 270 Gln Phe Arg Ile Lys Gly Gly Leu Phe Ala Asp Ile Ala Ser His Pro 275 280 285 Trp Gln Ala Ala Ile Phe Ala Ala Ala Ala Ala Ser Pro Gly Glu Arg 290 295 300 Phe Leu Cys Gly Gly Ile Leu Ile Ser Ser Cys Trp Ile Leu Ser Ala 305 310 315 320 Ala His Cys Phe Gln Glu Arg Phe Pro Pro His His Leu Thr Val Ile 325 330 335 Leu Gly Arg Thr Tyr Arg Val Val Pro Gly Glu Glu Glu Gln Lys Phe 340 345 350 Glu Val Glu Lys Tyr Ile Val His Lys Glu Phe Asp Asp Asp Thr Tyr 355 360 365 Asp Asn Asp Ile Ala Leu Leu Gln Leu Lys Ser Asp Ser Ser Arg Cys 370 375 380 Ala Gln Glu Ser Ser Val Val Arg Thr Val 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Arg Ala Ser Glu Arg Ala Glu Met Met Glu Leu Asn Asp Arg Phe 65 70 75 80 Ala Ser Tyr Ile Glu Lys Val Arg Phe Leu Glu Gln Gln Asn Lys Ala 85 90 95 Leu Ala Ala Glu Leu Asn Gln Leu Arg Ala Lys Glu Pro Thr Lys Leu 100 105 110 Ala Asp Val Tyr Gln Ala Glu Leu Arg Glu Leu Arg Leu Arg Leu Asp 115 120 125 Gln Leu Thr Ala Asn Ser Ala Arg Leu Glu Val Glu Arg Asp Asn Leu 130 135 140 Ala Gln Asp Leu Ala Thr Val Arg Gln Lys Leu Gln Asp Glu Thr Asn 145 150 155 160 Leu Arg Leu Glu Ala Glu Asn Asn Leu Ala Ala Tyr Arg Gln Glu Ala 165 170 175 Asp Glu Ala Thr Leu Ala Arg Leu Asp Leu Glu Arg Lys Ile Glu Ser 180 185 190 Leu Glu Glu Glu Ile Arg Phe Leu Arg Lys Ile His Glu Glu Glu Val 195 200 205 Arg Glu Leu Gln Glu Gln Leu Ala Arg Gln Gln Val His Val Glu Leu 210 215 220 Asp Val Ala Lys Pro Asp Leu Thr Ala Ala Leu Lys Glu Ile Arg Thr 225 230 235 240 Gln Tyr Glu Ala Met Ala Ser Ser Asn Met His Glu Ala Glu Glu Trp 245 250 255 Tyr Arg Ser Lys Phe Ala Asp Leu Thr Asp Ala Ala Ala Arg Asn Ala 260 265 270 Glu Leu Leu Arg Gln Ala Lys His Glu Ala Asn Asp Tyr Arg Arg Gln 275 280 285 Leu Gln Ser Leu Thr Cys Asp Leu Glu Ser Leu Arg Gly Thr Asn Glu 290 295 300 Ser Leu Glu Arg Gln Met Arg Glu Gln Glu Glu Arg His Val Arg Glu 305 310 315 320 Ala Ala Ser Tyr Gln Glu Ala Leu Ala Arg Leu Glu Glu Glu Gly Gln 325 330 335 Ser Leu Lys Asp Glu Met Ala Arg His Leu Gln Glu Tyr Gln Asp Leu 340 345 350 Leu Asn Val Lys Leu Ala Leu Asp Ile Glu Ile Ala Thr Tyr Arg Lys 355 360 365 Leu Leu Glu Gly Glu Glu Asn Arg Ile Thr Ile Pro Val Gln Thr Phe 370 375 380 Ser Asn Leu Gln Ile Arg Glu Thr Ser Leu Asp Thr Lys Ser Val Ser 385 390 395 400 Glu Gly His Leu Lys Arg Asn Ile Val Val Lys Thr Val Glu Met Arg 405 410 415 Asp Gly Glu Val Ile Lys Glu Ser Lys Gln Glu His Lys Asp Val Met 420 425 430 <210> 5 <211> 76 <212> PRT <213> Homo sapiens <400> 5 His Pro Leu Gly Ser Pro Gly Ser Ala Ser Asp Leu Glu Thr Ser Gly 1 5 10 15 Leu Gln Glu Gln Arg Asn His Leu Gln Gly Lys Leu Ser Glu Leu Gln 20 25 30 Val Glu Gln Thr Ser Leu Glu Pro Leu Gln Glu Ser Pro Arg Pro Thr 35 40 45 Gly Val Trp Lys Ser Arg Glu Val Ala Thr Glu Gly Ile Arg Gly His 50 55 60 Arg Lys Met Val Leu Tyr Thr Leu Arg Ala Pro Arg 65 70 75

Claims

1. The use of reagents for detecting GFAP and NT-proBNP levels in the preparation of a diagnostic kit for diagnosing large vessel occlusion (LVO) or for patients with stroke who are selected for thrombectomy.

2. The application as described in claim 1, wherein, GFAP and NT-proBNP levels were obtained from patient samples within the first 2 hours after stroke onset.

3. The application as described in claim 2, wherein, The sample is a biological fluid selected from blood, plasma, or serum.

4. The application as described in claim 1, wherein, The reagents include compounds that specifically bind to NT-proBNP and GFAP.

5. The application as described in claim 4, wherein, The compound that specifically binds to NT-proBNP and GFAP is an antibody or antibody fragment.

6. The application of claim 1, further comprising the use of reagents for detecting levels of retinol-binding protein 4 (RBP4) and / or d-dimer.

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