WO2022255349A1 - 可溶型ｃｌｅｃ２を用いた出血性脳卒中のリスク評価法 - Google Patents

可溶型ｃｌｅｃ２を用いた出血性脳卒中のリスク評価法 Download PDF

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WO2022255349A1
WO2022255349A1 PCT/JP2022/022077 JP2022022077W WO2022255349A1 WO 2022255349 A1 WO2022255349 A1 WO 2022255349A1 JP 2022022077 W JP2022022077 W JP 2022022077W WO 2022255349 A1 WO2022255349 A1 WO 2022255349A1
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hemorrhagic stroke
concentration
patient
sclec2
soluble
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French (fr)
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英夫 和田
雅英 川村
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LSI Medience Corp
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LSI Medience Corp
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Priority to KR1020237044828A priority Critical patent/KR20240017001A/ko
Priority to US18/565,258 priority patent/US20240264175A1/en
Priority to CN202280038986.4A priority patent/CN117769652A/zh
Priority to EP22816095.8A priority patent/EP4350350A4/en
Priority to JP2023525850A priority patent/JPWO2022255349A1/ja
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/49—Blood
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2871—Cerebrovascular disorders, e.g. stroke, cerebral infarct, cerebral haemorrhage, transient ischemic event
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease

Definitions

  • the present invention relates to methods for diagnosing and monitoring hemorrhagic stroke using soluble CLEC2.
  • Stroke is a group of diseases that includes ischemic stroke, which occurs when a blood vessel in the brain is clogged, that is, cerebral infarction, and hemorrhagic stroke, which occurs when a blood vessel in the brain is cut, that is, cerebral hemorrhage and subarachnoid hemorrhage. It is the second leading cause of death after ischemic heart disease. Stroke is not only a major cause of death, but it often leaves severe aftereffects even after survival, making it a major factor in the need for nursing care. , prevention, early diagnosis, prompt response after diagnosis, and a system that enables care and risk management by a wide range of medical professionals.
  • Hemorrhagic stroke includes subarachnoid hemorrhage caused by rupture of an aneurysm on the surface of the brain, and cerebral hemorrhage caused by rupture of small arteries in the brain.
  • a subarachnoid hemorrhage is bleeding in a blood vessel in the space between the brain and the membrane on the surface of the brain called the arachnoid. Most of the symptoms of subarachnoid hemorrhage are severe headaches, which are described as ⁇ headaches like being hit with a bat'' or ⁇ headaches you have never experienced before.'' Subarachnoid hemorrhage has a mortality rate as high as 30%, is a very serious acute disease that requires immediate treatment, and needs to be diagnosed as soon as possible.
  • CT computed tomography
  • D-dimer measurement was proposed in the past as an attempt to diagnose subarachnoid hemorrhage by blood test.
  • the sensitivity positive rate of patients
  • the sensitivity must be almost 100%, but not only cerebrospinal fluid measurement but also conventional blood test items have achieved such sensitivity. , none of which are currently in actual use.
  • biomarkers for predicting complications of subarachnoid hemorrhage many biomarkers using cerebrospinal fluid samples are known. Many biomarkers in cerebrospinal fluid, such as endothelin, TNF- ⁇ , IL-8, IL-6, thrombin-antithrombin complex, and HMGB1, have been described to be associated with complications in patients with subarachnoid hemorrhage. Since it is necessary to collect cerebrospinal fluid from the lumbar spine, it has not been put to practical use. Even if it were to be carried out, it would be unrealistic to use it to observe changes over time because it would be highly invasive and impose a heavy burden on the patient.
  • cerebral hemorrhage is an acute disease in which blood vessels in the brain rupture for some reason, causing bleeding into the brain. Blood clots from hemorrhage press against the brain, causing swelling in the brain and impairing brain function.
  • the symptoms are said to vary depending on the site and amount of bleeding, but the symptoms vary from sensory symptoms such as headache, nausea, and vomiting to motor symptoms such as motor paralysis, numbness, and gait disturbance in the hands and feet. It is said that it is difficult to distinguish it because it includes symptoms similar to cerebral infarction.
  • the amount of bleeding is large, it may be life-threatening. Like subarachnoid hemorrhage, it is often diagnosed by CT.
  • Cerebral hemorrhage can be stopped by treatment such as lowering blood pressure after diagnosis, but if it does not stop, hematoma in the brain will increase and symptoms will worsen.
  • An increase in hematoma can be confirmed by taking CT images many times, but as described above, it is often difficult to frequently perform CT imaging examinations using a CT examination apparatus having a certain degree of accuracy or higher.
  • biomarkers for monitoring the pathology of cerebral hemorrhage S100 ⁇ , GFAP, etc. are known at the research level, but their clinical usefulness is low and they are not used in actual clinical settings. There is no test that can be easily monitored by tests or the like.
  • C-type lectin-like receptor 2 (CLEC2) was identified on platelets as a receptor for the platelet-activating snake venom rhodocytin.
  • CLEC2 is expressed in a platelet/megakaryocyte-specific manner in humans, and can be said to be a platelet-specific molecule. It has been reported that this sCLEC2 is released into the blood as soluble CLEC2 (hereinafter abbreviated as sCLEC2) when platelets are activated (Patent Document 1, Non-Patent Document 1).
  • Hemorrhagic stroke has a high mortality rate, and since it is a serious acute stage disease that may leave sequelae even if death is avoided, it is extremely important to diagnose and start treatment as soon as possible. If typical symptoms are exhibited, a visit to a medical institution is made shortly after the onset of symptoms, and cerebral hemorrhage is confirmed by imaging tests such as CT and MRI, a diagnosis can be made based on this alone. In some cases, such as when the amount of bleeding is small or the bleeding is mild and arrives at a specialized medical institution after a long time, bleeding cannot be confirmed by CT images. In some countries and regions, medical institutions with highly sensitive CT and MRI are limited.
  • a subarachnoid hemorrhage requires a highly invasive examination of the cerebrospinal fluid by lumbar puncture.
  • Lumbar puncture is not performed for cerebral hemorrhage, but there is a problem that accurate diagnosis is difficult if confirmation cannot be performed with a CT image or the like.
  • the initial treatment is CT, MRI, etc., and then treatment such as lowering blood pressure is started, but there is no biomarker that can monitor whether the treatment is successful and the bleeding is stopped.
  • the present inventors have made intensive studies to solve the above problems. As a result, the inventors found that the blood sCLEC2 concentration in hemorrhagic stroke patients was elevated in almost all patients compared to healthy subjects, leading to the completion of the present invention. That is, the present inventors have found a method of easily diagnosing subarachnoid hemorrhage and cerebral hemorrhage by detecting hemorrhagic stroke patients with a simple blood test, which has excellent sensitivity, which is the positive rate of patients.
  • the blood sCLEC2 concentration threshold for hemorrhagic stroke is preferably 137 pg/mL or less. Based on these findings, the present invention was completed as a method for evaluating the risk of hemorrhagic stroke using sCLEC2.
  • the present invention provides: [1] Assess the risk of hemorrhagic stroke, including the step of measuring the concentration of soluble CLEC2 present in blood collected from patients suspected of having hemorrhagic stroke or diagnosed with hemorrhagic stroke. Method.
  • a method for risk assessment of a patient suspected of having hemorrhagic stroke or a patient diagnosed with hemorrhagic stroke comprising: (1) providing a blood sample from said patient; (2) determining the concentration of soluble CLEC2 in the sample; (3) correlating the soluble CLEC2 concentration with the presence or absence of hemorrhagic stroke or likely outcome in the patient;
  • the method of [1] comprising [3] A method for assessing the risk of a patient suspected of having hemorrhagic stroke or a patient diagnosed with hemorrhagic stroke, wherein the soluble CLEC2 concentration and the presence or absence of hemorrhagic stroke in the patient or wherein the step of correlating likelihood of outcome comprises assessing whether said patient is at risk based on said change in soluble CLEC2 concentration.
  • [4] A method for risk assessment of a patient suspected of having hemorrhagic stroke or a patient diagnosed with hemorrhagic stroke, wherein the step of correlating soluble CLEC2 concentration with hemorrhagic stroke, wherein the threshold is 137 pg /mL, the method of [2] or [3].
  • [5] A method for risk assessment of a patient suspected of having hemorrhagic stroke or a patient diagnosed with hemorrhagic stroke, comprising: (1) providing a blood sample from said patient; (2) determining the concentration of soluble CLEC2 in the sample; (3) measuring the platelet count in the sample; (4) dividing the soluble CLEC-2 concentration by the platelet count; (5) correlating the soluble CLEC2 concentration divided by the platelet count with the presence or absence of hemorrhagic stroke in the patient; The method of [1], comprising [6] A method for assessing the risk of hemorrhagic stroke in patients suspected of having hemorrhagic stroke or diagnosed with hemorrhagic stroke, wherein the value obtained by dividing the soluble CLEC2 concentration by the platelet count and the hemorrhagic The method of [5], wherein in the step of correlating stroke, said value threshold is between 0.7 and 1.0.
  • [7] The method of any one of [1] to [6], wherein the hemorrhagic stroke is either subarachnoid hemorrhage or cerebral hemorrhage. [8] of [1] to [7], wherein the step of determining the soluble CLEC2 concentration is a highly sensitive immunoassay, such as a chemiluminescence immunoassay, an electrochemiluminescence immunoassay, or a fluorescence immunoassay; either way.
  • a highly sensitive immunoassay such as a chemiluminescence immunoassay, an electrochemiluminescence immunoassay, or a fluorescence immunoassay; either way.
  • a method for assessing the risk of hemorrhagic stroke by measuring (or determining) the soluble CLEC2 concentration (or the value obtained by dividing the soluble CLEC-2 concentration by the number of platelets) in a sample, A method of measuring (or determining) the soluble CLEC2 concentration (or the value obtained by dividing the soluble CLEC-2 concentration by the platelet count) in a sample to aid in the risk assessment of hemorrhagic stroke; A method of measuring (or determining) the soluble CLEC2 concentration (or the value obtained by dividing the soluble CLEC-2 concentration by the platelet count) in a sample for risk assessment of hemorrhagic stroke,
  • An in vitro risk assessment method for hemorrhagic stroke which comprises measuring (or determining) the soluble CLEC2 concentration (or the value obtained by dividing the soluble CLEC-2 concentration by the platelet count) in a sample, use of an antibody capable of detecting the concentration of soluble CLEC2 in manufacturing a kit for evaluating the
  • FIG. 4 is a diagram comparing the plasma concentration of sCLEC2 between subarachnoid hemorrhage patients and healthy subjects.
  • FIG. 2 is a diagram comparing the plasma concentration of sCLEC2 between cerebral hemorrhage patients and healthy subjects. This is an example of chronological monitoring of a case of subarachnoid hemorrhage. This is an example of continuous monitoring of a case of cerebral hemorrhage.
  • CLEC2 is a platelet-activating receptor belonging to the C-type lectin family, normally present in the platelet membrane, but is released into the blood following platelet activation.
  • soluble CLEC2 sCLEC2
  • CLEC2 or a CLEC2-derived molecule that is released from such platelets and detected in the blood.
  • sCLEC2 is said to include a protein with a molecular weight of about 40 kDa, a protein with a molecular weight of about 32 kDa, a protein with a molecular weight of about 25 kDa, etc. in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions.
  • SDS-PAGE SDS-polyacrylamide gel electrophoresis
  • Non-Patent Document 1 A protein with a molecular weight of about 40 kDa and a protein with a molecular weight of about 32 kDa are present on the platelet membrane surface and are presumed to be released in a state contained in microparticles produced with platelet activation. It is considered that sugar chains are added to these.
  • a protein with a molecular weight of about 25 kDa is considered to be cleaved by protease and released from platelets along with platelet activation.
  • the amount of sCLEC2 as described above is measured.
  • sCLEC2 may detect a protein with a molecular weight of about 40 kDa, a protein with a molecular weight of about 32 kDa, and a protein with a molecular weight of about 25 kDa collectively, or may detect only a protein with a molecular weight of about 25 kDa.
  • the sCLEC2 concentration used in the present invention may be the sCLEC2 concentration alone, but preferably the value obtained by dividing the sCLEC2 concentration by the platelet count (hereinafter sometimes referred to as the C2PAC index) is used. You can In the present specification, unless otherwise specified, the sCLEC2 concentration is interpreted to include both the case of using the sCLEC2 concentration and the case of dividing the sCLEC2 concentration by the platelet count.
  • the platelet count is usually measured using an automatic blood cell counter (hematometer), but a blood cell counter and a microscope are used. It is also possible to count using
  • the C2PAC index which expresses the sCLEC2 concentration in plasma, for example, in pg/mL, expresses the blood platelet count, for example, in 1,000/mm 3 , and calculates the sCLEC2 concentration/platelet count.
  • Any unit such as ng/mL or ng/L may be used for the concentration of sCLEC2 used in , and any unit such as 10,000/ mm3 may be used for the platelet count. Units should be used.
  • sCLEC2 concentration/platelet count can take various values, but essentially the concept is the same.
  • ratio calculations will often be performed using measurements from a clinical laboratory instrument that measures sCLEC2 concentrations and from a hemocytometer that measures platelet counts.
  • This calculation is preferably automatically calculated on a system such as a hospital inspection system, a hospital system, or an electronic medical chart connected to both measuring instruments in daily practice.
  • a connecting system may be constructed, or a machine capable of simultaneously measuring sCLEC2 concentration and platelet count may be constructed.
  • you may calculate manually using both data.
  • the plasma sCLEC2 concentration is divided by the blood platelet count to calculate the amount of sCLEC2 released per platelet, and this is used as an index for diagnosing thrombotic diseases
  • the platelet activity is not dependent on the blood platelet count. It is preferable because it makes it possible to evaluate the degree of conversion.
  • the plasma sCLEC2 concentration is expressed as pg/mL (A)
  • the blood platelet count is expressed as 1,000/mm 3 (B)
  • the number obtained by dividing A by B is the number of platelet activation. It can be used as an index.
  • the samples used for measurement are preferably derived from humans, but samples derived from animals other than humans may be used to understand the pathology of experimental animals.
  • Experimental animals include, but are not limited to, guinea pigs, rats, mice, dogs, and the like.
  • the method for detecting the presence of sCLEC2 is not particularly limited, but an immunological method using an antibody that recognizes sCLEC2 is preferred.
  • methods for immunologically detecting proteins include enzyme immunoassay (ELISA method), chemiluminescence immunoassay, electrochemiluminescence immunoassay, fluorescence immunoassay, radioimmunoassay, and immunochromatography. Any method can be used as long as it is an immunoassay method using a labeled antibody such as, Western blotting method, latex agglutination method, immunoturbidimetric method, or the like.
  • An immunoassay method using a labeled antibody is preferably used in terms of simplicity and measurement accuracy.
  • Chemiluminescence immunoassay, immunochromatography, and the like are particularly preferably used for diagnosis in the emergency area because it is desired to obtain results quickly.
  • a sample is collected from the target subject (especially a patient), for example, using a blood collection tube for plasma blood collection.
  • a collection tube containing citric acid, which leaves little residual platelets, is usually suitable, but a tube containing heparin or EDTA is also possible.
  • a blood collection tube containing EDTA is used for blood platelet count measurement, but separate blood collection tubes may be used for simultaneous blood collection.
  • Plasma sCLEC2 concentration is measured, for example, by using plasma that has been centrifuged at 2000 g for about 20 minutes, but the conditions for centrifugation are not limited to this, and a measurement system using whole blood may also be used.
  • the measurement of plasma sCLEC2 concentration will be described as an example, but the present invention is not limited to this.
  • hemorrhagic stroke which is the target of risk assessment, includes cerebral hemorrhage occurring inside the brain (sometimes referred to as intracerebral hemorrhage) and pia mater, which is the inner layer of the tissue covering the brain, and arachnoid membrane, which is the outer layer. It means subarachnoid hemorrhage, hemorrhage that occurs in between, but does not include cerebral hemorrhage caused by head injury.
  • Known causes of cerebral hemorrhage are those derived from hypertension, cerebral aneurysm rupture, cerebral arteriovenous malformation rupture, venous sinus obstruction, dural arteriovenous fistula, amyloid deposition, moyamoya disease, and the like.
  • the target cerebral hemorrhage is not limited to that caused by a specific cause.
  • the blood sCLEC2 concentration value of patients suspected of having hemorrhagic stroke is compared with healthy subjects or neurological disease groups without intracerebral hemorrhage or infarction. Based on such a comparison, sCLEC2 levels can be used to diagnose hemorrhagic stroke (subarachnoid hemorrhage or intracerebral hemorrhage).
  • the threshold value is set by comparing the sCLEC2 concentration in the patient-derived sample and the sCLEC2 concentration in the non-hemorrhagic stroke patient-derived sample. It may be set as appropriate and used, or may be evaluated when a significant change in sCLEC2 concentration is detected from the chronological record of sCLEC2 concentration before onset in the same patient.
  • a threshold value was appropriately set by comparing the sCLEC2 concentration in the patient-derived sample and the sCLEC2 concentration in the healthy subject-derived sample.
  • a method for calculating the threshold an analysis is performed to create a ROC curve (Receiver Operating Characteristic Curve) from the measured sCLEC2 value in plasma, and the sensitivity and specificity of diagnosis are both 80% or more. Concentration can be used as a threshold.
  • ROC curve Receiveiver Operating Characteristic Curve
  • the threshold for sCLEC2-induced hemorrhagic stroke it is preferable to select an arbitrary value of 137 pg/mL or less as the threshold for sCLEC2-induced hemorrhagic stroke.
  • a similar analysis can be performed to calculate a threshold even when using a C2PAC index, which is a value obtained by dividing the sCLEC2 concentration by the platelet count.
  • the threshold when using the C2PAC index can be arbitrarily set between 0.7 and 1.0.
  • the blood sCLEC2 concentration increases with platelet activation, it is suggested that the blood sCLEC2 concentration is related to the formation of thrombus, and it is reported that it can be used for the diagnosis of cerebral infarction and myocardial infarction.
  • hemorrhagic stroke there is no known association with hemorrhagic stroke, and it was surprising that measurement of sCLEC2 concentration would be useful for risk assessment.
  • lumbar puncture and cerebrospinal fluid examination in which a needle is inserted into the lumbar vertebrae to collect cerebrospinal fluid, are performed, but these are highly invasive.
  • Treatments for the diagnosis of subarachnoid hemorrhage mainly include the following treatments.
  • diuretics such as mannitol and glyceol are used to reduce cerebrospinal fluid.
  • careful judgment is required, such as considering the possibility of increased cerebral ischemia due to a decrease in cerebral perfusion pressure due to hypotension.
  • the patient's condition can be monitored by collecting blood samples over time from a patient diagnosed with subarachnoid hemorrhage and measuring sCLEC2. Since sCLEC2 is said to represent the state of platelet activation, if sCLEC2 maintains a high value, it can be expected that bleeding will continue and the formation of a hemostatic thrombus will continue, and if the sCLEC2 value decreases, hemostatic thrombosis will occur. A decrease in formation can be expected. Cerebral vasospasm, which is a serious complication, is said to be induced by blood leaking from blood vessels, so it is important and useful to understand the state of bleeding using blood sCLEC2.
  • sCLEC2 concentration can be compared before and after treatment for hemorrhagic stroke patients and used as risk prediction. For example, if the sCLEC2 concentration decreases due to treatment, platelet activation tends to be suppressed, but if the sCLEC2 concentration remains high or increases, bleeding may continue. If the treatment with antihypertensive is not effective, it is possible to provide materials for considering the selection of therapeutic methods such as further lowering blood pressure. During the process of monitoring, fluctuations in the blood platelet count may be observed, so monitoring using the C2PAC index is also more suitable for observing the patient's condition.
  • sCLEC2 is said to represent the state of platelet activation, if sCLEC2 maintains a high value, it can be expected that bleeding will continue and the formation of a hemostatic thrombus will continue, and if the sCLEC2 value decreases, hemostatic thrombosis will occur. A decrease in formation can be expected. If the bleeding continues, intracerebral hematoma increases, compressing the brain and exacerbating the symptoms. Therefore, it is important and useful to understand the bleeding status using blood sCLEC2.
  • sCLEC2 concentration before and after treatment for hemorrhagic stroke patients can be compared and used as risk prediction. For example, if the sCLEC2 concentration decreases due to treatment, platelet activation tends to be suppressed, but if the sCLEC2 concentration continues to be high or increases, reassessment of antihypertensive therapy can be considered. During the process of monitoring, fluctuations in the blood platelet count may be observed, so monitoring using the C2PAC index is also more suitable for observing the patient's condition.
  • blood sCLEC2 concentration should be measured in patients who describe symptoms that may be suspected of hemorrhagic stroke (subarachnoid hemorrhage or cerebral hemorrhage), and if the value is in healthy subjects, etc. If the value is higher than , it can be judged that the possibility of hemorrhagic stroke is high.
  • the patient's condition can be monitored by sampling blood over time from a patient diagnosed with hemorrhagic stroke, measuring the sCLEC2 concentration, and comparing the results over time.
  • the correlation between the measured sCLEC2 concentration in the patient-derived sample and the possibility of hemorrhagic stroke can be determined by examining the sCLEC2 concentration in the patient-derived sample and non-hemorrhagic stroke.
  • a threshold value may be appropriately set and used by comparing the sCLEC2 concentration in a human-derived sample, or from the time-lapse recording of the sCLEC2 concentration before onset in the same patient, when a significant change in the sCLEC2 concentration is detected. An evaluation may be performed.
  • sCLEC2 is released into the blood with platelet activation.
  • Existing platelet activation markers such as platelet factor 4 (PF4) and ⁇ -thromboglobulin ( ⁇ TG)
  • PF4 platelet factor 4
  • ⁇ TG ⁇ -thromboglobulin
  • sCLEC2 is a signaling-dependent release mechanism that triggers platelet activation, and can be a marker that more accurately reflects platelet activation in vivo.
  • CLEC2 since CLEC2 is almost exclusively expressed in the platelet/megakaryocyte system in humans, it can be a platelet-specific marker with less false positives. Therefore, by measuring sCLEC2, it becomes possible to estimate the activation state of platelets at an early stage, and it can be used for diagnosing hemorrhagic stroke and monitoring pathology.
  • sCLEC2 concentration in plasma was measured as follows. (Preparation of measurement reagent and preparation of test sample) ⁇ Specimen diluent: 2% sodium octanoate and 0.5% n-octyl- ⁇ -D-glucoside (OG) using 0.1 mol/L HEPES buffer (pH 7.5) containing preservatives They were combined to obtain a specimen diluent.
  • the antibody contained in the reagent was prepared as follows using the antibody described in Examples of Japanese Patent No. 6078845.
  • ⁇ First antibody solution A mouse monoclonal antibody (11D5) that recognizes sCLEC2 was bound to magnetic latex particles (JSR) and dispersed in a 0.01 mol/L MES buffer solution (pH 6.0) containing a preservative. . -Second antibody solution: Another mouse monoclonal antibody (11E6) that recognizes sCLEC2 is labeled with alkaline phosphatase (ALP) by the maleimide method and dispersed in 0.01 mol/L MES buffer (pH 6.5) containing a preservative.
  • ALP alkaline phosphatase
  • Luminescent substrate solution 2-chloro-5-(4-methoxyspiro ⁇ 1,2-dioxetane-3,2′-(5′-chloro)-tricyclo[3.3.1.13,7]decane ⁇ - 4-yl)-1-phenylphosphate disodium (CDP-Star®: Applied Biosystems) was used.
  • - B/F wash solution A buffer solution containing 0.1 mol/L citric acid (pH 6.5), 0.15 mol/L NaCl, and 0.1% Triton X-100 was used.
  • ⁇ Test sample Recombinant hCLEC2 protein diluted with buffer (0.025 mol/L HEPES, 0.14 mol/L NaCl, 0.1% sodium octanoate, 0.3% BSA) Sample 1, diluted with citrated plasma, was used as test sample 2.
  • a fully automatic clinical examination system STACIA registered trademark, manufactured by LSI stipulatece
  • the sample diluent, the first antibody solution (magnetic latex reagent), and the second antibody solution (enzyme-labeled antibody reagent) were filled in STACIA-dedicated bottles and set in the apparatus. Measurements were carried out according to the operating method of the apparatus described below. Specifically, 40 ⁇ L of the specimen diluent was added to 10 ⁇ L of the sample and heated at 37° C. for several minutes, then 25 ⁇ L of the first antibody solution (magnetic latex reagent) was added and heated at 37° C. for several minutes.
  • FIG. 1 shows a standard curve prepared using the hsCLEC2 protein as a standard.
  • Example 2 Measurement of sCLEC2 in plasma specimens of subarachnoid hemorrhage patients, cerebral hemorrhage patients and healthy subjects>>> Plasma sCLEC2 concentration, platelet level, and sCLEC2/platelet ratio (C2PAC index) were measured by the method of Example 1 using citrated plasma obtained from patients diagnosed with subarachnoid hemorrhage or cerebral hemorrhage by outpatient examination. (Tables 1 and 2). In addition, sCLEC concentrations in healthy subjects were also measured (Table 3).
  • the measured sCLEC2 values (pg/mL) of hemorrhagic stroke patients with both subarachnoid hemorrhage and cerebral hemorrhage were significantly higher than those of healthy subjects (P ⁇ 0.0001) (Table 4). Each measured value is shown in FIG. 2 (subarachnoid hemorrhage) and FIG. 3 (cerebral hemorrhage). In addition, the upper limit of the C2PAC index measured separately for healthy subjects was 0.55.
  • ⁇ Example 3 Sensitivity and specificity of diagnosis of hemorrhagic stroke (subarachnoid hemorrhage and cerebral hemorrhage) by sCLEC2>>
  • the threshold is set at 120 pg/mL
  • the measurement of sCLEC2 and the sCLEC2/platelet ratio (C2PAC index) are means for providing simple and accurate data for diagnosing hemorrhagic stroke.
  • Example 4 Monitoring of patients with subarachnoid hemorrhage by sCLEC2>> Various test values on the 1st, 8th, and 10th days of illness in one case diagnosed with subarachnoid hemorrhage are shown (Fig. 4). sCLEC2 slightly increased from day 1 to day 8, but the platelet count also increased greatly, so the C2PAC index showed a slight downward trend.
  • Example 5 Monitoring of cerebral hemorrhage patients with sCLEC2>> Various test values on the 1st, 2nd, 12th, and 14th days of illness in one case diagnosed with cerebral hemorrhage are shown (FIG. 5). sCLEC2, and C2PAC index increased on day 12 and decreased on day 14, whereas D-dimer increased from day 12 to day 14.
  • the measured value of blood sCLEC2 and the sCLEC2/platelet ratio in the present invention are clinical tests that can be used to diagnose hemorrhagic stroke, subarachnoid hemorrhage, and cerebral hemorrhage.
  • sCLEC2 measurement is useful as a risk assessment method for hemorrhagic stroke patients because it can also be used to monitor the condition of patients with cerebral hemorrhage.

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