An in vitro quantitative detection reagent and detection method for activated platelets in whole blood using flow cytometry

Abstract

The present invention provides a detection reagent and a detection method for in vitro quantitative detection of activated platelets in whole blood by flow cytometry. In the method of the present invention, the blood sample is pre-treated and then detected by flow cytometry. The pre-treatment can effectively slow down the spontaneous activation of platelets. The platelet activation ratio of the blood sample treated by this method is still lower than the upper limit level of 3% of the normal platelet activation ratio of the human body after being stored at room temperature for 36 hours, still meeting the clinical detection requirements of flow cytometry, greatly extending the sample storage time, and greatly prolonging the effective detection time of the blood sample.

Description

Technical Field

[0001] The present invention relates to the field of in vitro diagnostic technologies, and more particularly, to a detection reagent and a detection method for quantitatively detecting activated platelets in whole blood by applying flow cytometry in vitro. Background Art

[0002] Platelets are irregular blood cells produced by mature megakaryocytes, with physiological characteristics such as adhesion, release, and aggregation, and mainly participate in the body's physiological hemostasis activities. When a local blood vessel is damaged or the body is in an inflammatory response, platelets are stimulated to transform into an activated state. Activated platelets will interact with white blood cells, endothelial cells, etc., and then participate in the human hemostasis process and immune regulation activities. However, the excessive aggregation of platelets in blood vessels can also lead to the formation of thrombi. Current studies have shown that the monitoring of activated platelets can effectively evaluate the curative effects and postoperative rehabilitation conditions of patients with cardiovascular and cerebrovascular diseases such as acute cerebral infarction and coronary heart disease, and guide clinical medication. In addition, there are also studies showing that activated platelets will act as a mobile medium for human white blood cells and participate in the body's immune activities. Even activated platelets will bind to tumor cells and participate in the tumor metastasis process. Activated platelets are closely related to human immune injury, thrombus formation, and tumor metastasis. Therefore, the detection of activated platelets has very important clinical significance.

[0003] Clinically, the thromboelastogram (TEG) method is commonly used to evaluate the function of platelets. TEG is a coagulation detection method that simulates the human internal environment in vitro and activates the coagulation system to reflect the dynamic changes of blood coagulation (including the formation rate of fibrin, the activity of coagulation factors, the number and activation function of platelets, etc.), and comprehensively analyzes the whole process of coagulation and fibrinolysis. The TEG method also has certain defects. Since it is a method for simulating the comprehensive coagulation function of patients in vitro, the detection sensitivity of the function of platelets by this method is relatively low, and it cannot be quantified, making it difficult to provide a true and reliable detection result of platelet activation function. Therefore, there is a great necessity for a platelet activation function detection method with high sensitivity and strong specificity clinically.

[0004] As a highly efficient, rapid, and extremely sensitive technology, flow cytometry can clearly judge the activation state of platelets by accurately detecting the changes in specific glycoproteins on the surface of activated platelets. However, at present, the detection of activated platelets by flow cytometry often has high requirements for samples. The traditional method for detecting activated platelets needs to complete blood sampling to detection within 1 to 2 hours, and is often interfered by problems such as spontaneous activation of platelets and difficulty in completing the treatment of blood samples within the specified time. The detection result cannot reflect the activation situation of platelets in vivo or just after leaving the body. The activation ratio is often higher than that in vivo or just after leaving the body, and the detection result feedback to doctors is inaccurate, thus affecting the clinical doctor's medication and the detection of drug efficacy, and greatly limiting the clinical use. Summary of the Invention

[0005] The object of the present invention is to overcome the above-mentioned defects and deficiencies existing in the prior art, and to provide a platelet function detection reagent.

[0006] The second object of the present invention is to provide a method for pre-treating a blood sample. The pre-treatment method can effectively inhibit the spontaneous activation of platelets. The blood sample treated by this method can meet the clinical detection requirements when measured within 36 hours of storage at room temperature, greatly extending the sample storage time, reflecting the activation situation of platelets in vivo or just after leaving the body, and improving the detection accuracy.

[0007] The third object of the present invention is a method for detecting the function of activated platelets for non-disease treatment and diagnosis purposes.

[0008] The above objects of the present invention are achieved by the following technical solutions:

[0009] A platelet function detection reagent, the reagent includes a blood collection tube containing an additive for slowing down the spontaneous activation of ex vivo platelets, an activated platelet labeling reagent, a platelet labeling reagent, and a platelet incubation solution. The activated platelet labeling reagent is an activated platelet marker capture antibody conjugated with a first fluorophore, the platelet labeling reagent is a platelet marker capture antibody conjugated with a second fluorophore, and the platelet incubation solution is a phosphate buffer solution; the additive for slowing down the spontaneous activation of ex vivo platelets includes the following components in parts by mass: 1-10 parts of a platelet activation inhibitor, 10-20 parts of a blood anticoagulant, 3-18 parts of a cell membrane protectant, and 3-18 parts of a pH regulator. The platelet activation inhibitor is theophylline and aspirin, and their mass ratio is 1:9-9:1.

[0010] The blood collection tube in the platelet function detection reagent provided by the present invention contains an additive for slowing down the spontaneous activation of ex vivo platelets. The additive for slowing down the spontaneous activation of ex vivo platelets contains two functional components, namely a platelet activation inhibitor and a blood anticoagulant, as well as a cell membrane protector and a pH regulator. The platelet activation inhibitor is used to inhibit the spontaneous activation of ex vivo platelets, the blood anticoagulant is used to prevent blood coagulation, the pH regulator can adjust the pH of the additive to be similar to the pH of blood, which is 7.35 - 7.45, and the cell membrane protector can effectively protect the cell membranes of cells in the blood and maintain the integrity of the cells. The platelet activation inhibitor contains a certain proportion of theophylline and aspirin. The present invention has found through research that when a certain proportion of theophylline and aspirin are used in combination, compared with their single use, they have a better effect of inhibiting the spontaneous activation of ex vivo platelets and have a significant synergistic effect. The blood treated with the above blood collection tube can be stored at room temperature for 36 hours, and the platelet activation ratio is still lower than the upper limit level of the normal platelet activation ratio in the human body by 3%. It still meets the clinical detection requirements of flow cytometry, greatly extends the sample storage time, and greatly extends the effective detection time of the blood sample. Together with the activated platelet labeling reagent, platelet labeling reagent, and platelet incubation solution in the detection reagent, it can be used for the in vitro quantitative detection of activated platelets in whole blood by flow cytometry.

[0011] The activated platelet labeling reagent is a capture antibody for activated platelet markers conjugated with a first fluorophore; the platelet labeling reagent is a capture antibody for platelet markers conjugated with a second fluorophore; the types and emission wavelengths of the first fluorophore and the second fluorophore are different.

[0012] There are various glycoproteins on the platelet membrane surface, including GPⅠb-Ⅸ-Ⅴ, GPⅡb (CD41), GP-Ⅲa (CD61), CD62p, and CD63, etc. Among them, CD61 is the most abundant adhesion receptor on the platelet membrane and is the integrin β3 chain. It combines with GPⅡb (CD41) to form an integrin. Clinically, abnormalities in the quality or quantity of GPⅡb (CD41) and / or GPIIIa (CD61) can be used for the diagnosis of thrombasthenia. CD62p is a single-chain glycoprotein present on the α-granule membrane of platelets. It is stimulated by strong platelet activation factors (ADP or collagen) and appears on the platelet membrane surface. CD62p is a selectin of platelets. In addition to activated platelets, activated endothelial cells and megakaryocytes are also found. Clinically, it can be used for the identification of activated platelets during thrombosis. For example, the corresponding antibodies of platelet surface antigens CD41 and CD61 can be used in combination for detection to clearly distinguish platelet cell populations. On this basis, the corresponding antibody of CD62p can accurately detect the percentage of activated platelets among them.

[0013] Further, the activated platelet marker is at least one of CD62P, CD63, PAC-1, CD107a, and CD107b; the platelet marker is at least one of CD42a, CD42b, CD41, CD41a, CD41b, and CD61, and contains CD41b or CD61. The method for detecting activated platelets selects a platelet cell population through the combined detection of at least one platelet marker, and selects an activated platelet marker to detect the percentage of activated platelets therein.

[0014] Further, the first fluorophore and the second fluorophore can be excited by at least one of the lasers with wavelengths of 405 nm, 488 nm, and 633 nm.

[0015] Further, the first fluorophore is selected from PE, APC, and FITC, and the second fluorophore is selected from FITC, PE, PE-Cy7, PerCP, APC, and APC-Cy7. The emission wavelengths of the first fluorophore and the second fluorophore are different, which can avoid the mutual interference between the characteristic signals of different fluorophores and improve the accuracy of the detection results.

[0016] Further, the type of the capture antibody for the activated platelet marker is at least one, and the type of the capture antibody for the platelet marker is at least one.

[0017] Further, both the capture antibody for the activated platelet marker and the capture antibody for the platelet marker are monoclonal antibodies, which can target specific antigen epitopes and improve the accuracy of the detection results.

[0018] Further, the additive for slowing down the spontaneous activation of ex vivo platelets includes the following components in parts by mass: 1-5 parts of a platelet activation inhibitor, 10-16 parts of a blood anticoagulant, 3-10 parts of a cell membrane protectant, and 3-12 parts of a pH regulator.

[0019] Further, the mass ratio of the theophylline to aspirin is 1:4 to 4:1.

[0020] Preferably, the mass ratio of the theophylline to aspirin is 1:1.

[0021] Further, the additive for slowing down the spontaneous activation of ex vivo platelets includes the following components in parts by mass: 2 parts of a platelet activation inhibitor, 12 parts of a blood anticoagulant, 10 parts of a cell membrane protectant, and 12 parts of a pH regulator.

[0022] Further, the blood anticoagulant includes, but is not limited to, one or more of sodium citrate and EDTA salts.

[0023] Further, the EDTA salt is dipotassium ethylenediaminetetraacetate.

[0024] Furthermore, the cell membrane protective agent includes but is not limited to one or more of sucrose and glucose.

[0025] Furthermore, the pH adjuster includes but is not limited to one or more of tris(hydroxymethyl)aminomethane and hydrochloric acid, citric acid and citrate, phosphate, carbonate, acetic acid and acetate.

[0026] Furthermore, the platelet incubation solution is a phosphate buffer. The platelet incubation solution may contain a fixative, and the fixative is selected from one or more of formaldehyde, paraformaldehyde, ethanol solution, methanol solution, and acetone.

[0027] The present invention also provides a blood sample pretreatment method, which is to collect blood samples using a blood collection tube containing an additive that slows down spontaneous platelet activation in vitro in any of the above-mentioned detection reagents, and to fully and gently invert and mix the additive and the blood sample for pretreatment.

[0028] The present invention also provides the use of any of the above detection reagents or the blood sample pretreatment methods in platelet activation function detection.

[0029] The present invention also provides a method for detecting activated platelet function for non-disease treatment and diagnosis purposes using any of the above detection reagents, comprising the following steps:

[0030] S1. Collect blood samples using a blood collection tube containing an additive that slows down spontaneous platelet activation in vitro, and mix thoroughly and gently by inverting;

[0031] S2. Mix the activated platelet labeling reagent, the platelet labeling reagent and the blood sample collected in step S1 in a flow test tube;

[0032] S3. Add platelet incubation solution, mix well, and incubate at room temperature in the dark;

[0033] S4. Add platelet incubation solution and mix well to obtain a sample to be tested;

[0034] S5. Detect the sample to be tested by flow cytometry and analyze the platelet function in the blood sample;

[0035] Preferably, the amount of the blood sample in step S1 is no more than 10 μL and no less than 3 μL.

[0036] Preferably, the light-proof incubation time in step S2 is 15 to 30 minutes.

[0037] Preferably, the amount of the platelet incubation solution in step S2 is 5 to 10 times the amount of the platelet incubation solution in step S3.

[0038] Preferably, during the blood sample collection process, do not squeeze the pipeline of the blood collection needle. After collecting the blood sample, gently invert and mix it thoroughly to avoid artificial activation of platelets.

[0039] Preferably, the collected blood sample is venous blood.

[0040] Furthermore, in the detection step, an isotype control antibody reagent can also be set.

[0041] Preferably, the isotype control antibody reagent is fluorescein-labeled IgG1. The labeled IgG1 fluorescein is selected from PE, APC, FITC, and is of the same type as the first fluorescein.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] The present invention uses an additive that slows down the spontaneous activation of ex vivo platelets to pre-treat the collected blood sample. The additive contains a certain proportion of theophylline and aspirin. Compared with the single use of the two, it has a better effect of slowing down the spontaneous activation of ex vivo platelets and has a significant synergistic effect. After the blood sample of healthy people pre-treated by the method of the present invention is stored at room temperature for 36 hours, the platelet activation ratio is still lower than the upper limit level of the normal platelet activation ratio by 3%, still meeting the clinical detection requirements of flow cytometry, greatly extending the sample storage time, and improving the detection accuracy. Description of the Drawings

[0044] Figure 1 A scatter plot with an irregular gate set for the FSC / SSC scatter plot.

[0045] Figure 2 A CD41 / CD61 scatter plot.

[0046] Figure 3 An isotype control IgG1 scatter plot.

[0047] Figure 4 A CD62p / SSC scatter plot. Detailed Embodiments

[0048] The following further illustrates the present invention in conjunction with the drawings of the specification and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0049] Unless otherwise specified, the reagents and materials used in the following embodiments are all commercially available.

[0050] Example 1 A method for detecting platelet activation function

[0051] 1. Reagents

[0052] The activated platelet marker selected was CD62P, the platelet markers were CD61 and CD41 respectively, the first fluorophore was PE, and the second fluorophores were APC-Cy7 and PerCP respectively, that is, CD41-PerCP / CD61-APC-Cy7 / CD62P-PE was used for detection.

[0053] The platelet incubation solution was phosphate buffer.

[0054] The preparation method of the additive for slowing down the spontaneous activation of ex vivo platelets was to weigh 2 g of platelet activation inhibitor, 10 g of cell protector, 5 g of pH regulator, and 12 g of anticoagulant, add 400 mL of water and stir to dissolve, then add water to make up the volume to 500 mL and stir evenly to obtain a mixture; among them, the platelet activation inhibitor was theophylline and aspirin, and their mass ratio was 1:1, that is, 1 g of each of theophylline and aspirin, the cell membrane protector was glucose, the pH regulator was phosphate buffer, and the anticoagulant was EDTA.K 2 The mixture was filtered through a filter membrane with a pore size of 0.22 μm to obtain the additive for slowing down the spontaneous activation of ex vivo platelets. The pH of the additive was 7.4, which was similar to the pH of blood.

[0055] 2. Method

[0056] (1) Pretreatment: Select a venous puncture whole blood sample, collect the blood sample with a blood collection tube containing the above-mentioned additive for slowing down the spontaneous activation of ex vivo platelets. The volume ratio of the blood sample to the additive was 9:1 to obtain the pretreated blood sample. During the collection process, do not squeeze the pipeline of the blood collection needle to avoid artificial activation of platelets; within 1 minute after blood collection, gently invert and mix well (invert 180°, 3 - 4 times), and avoid violent shaking during the transportation of the blood collection tube to the laboratory.

[0057] (2) Detection: Mix 5 μL each of the activated platelet labeling reagent, platelet labeling reagent, and the pretreated blood sample in a flow tube, then add 200 μL of platelet incubation solution, mix well and incubate in the dark for 15 minutes, then add 1 - 2 mL of platelet incubation solution to obtain the sample to be tested. The sample to be tested was detected and data analyzed by a flow cytometer, and the test results were reported as the percentage of target platelets in platelets. The data analysis included first setting an irregular gate with an FSC / SSC scatter plot to circle out platelets (see Figure 1 ); then using a CD41 / CD61 scatter plot to circle out CD41 + CD61 + platelets (see Figure 2 ); finally, select to display CD61 + CD41 + platelets, set a CD62p / SSC scatter plot, and set CD62p according to the isotype control IgG1+ ranges (see Figure 3 , Figure 4 ), and thus the platelet activation ratio is calculated.

[0058] Example 2 A method for detecting platelet activation function

[0059] It is basically the same as Example 1, and the difference lies in that the preparation method of the additive for slowing down the spontaneous activation of ex vivo platelets is as follows: Weigh 5 g of platelet activation inhibitor, 3 g of cell protector, 3 g of pH regulator, and 10 g of anticoagulant, add 400 mL of water and stir to dissolve, then add water to make up the volume to 500 mL, and stir evenly to obtain a mixture; among them, the platelet activation inhibitor is theophylline and aspirin, and their mass ratio is 1:1, that is, 2.5 g each of theophylline and aspirin, the cell membrane protector is sucrose, the pH regulator is sodium citrate, and the anticoagulant is sodium citrate. The mixture is filtered through a filter membrane with a pore size of 0.22 μm to obtain the additive for slowing down the spontaneous activation of ex vivo platelets. The pH of the additive is 7.4, which is similar to the pH of blood.

[0060] Example 3 A method for detecting platelet activation function

[0061] It is basically the same as Example 1, and the difference lies in that the preparation method of the additive for slowing down the spontaneous activation of ex vivo platelets is as follows: Weigh 1 g of platelet activation inhibitor, 6 g of cell protector, 12 g of pH regulator, and 16 g of anticoagulant, add 400 mL of water and stir to dissolve, then add water to make up the volume to 500 mL, and stir evenly to obtain a mixture; among them, the platelet activation inhibitor is theophylline and aspirin, and their mass ratio is 1:1, that is, 0.5 g each of theophylline and aspirin, the cell membrane protector is sucrose, the pH regulator is tris(hydroxymethyl)aminomethane, and the anticoagulant is EDTA.K 2 . The mixture is filtered through a filter membrane with a pore size of 0.22 μm to obtain the additive for slowing down the spontaneous activation of ex vivo platelets. The pH of the additive is 7.4, which is similar to the pH of blood.

[0062] Example 4 A method for detecting platelet activation function

[0063] It is basically the same as Example 1, except that: the preparation method of the additive for slowing down the spontaneous activation of ex vivo platelets is as follows: Weigh 10 g of platelet activation inhibitor, 18 g of cell protector, 18 g of pH regulator, and 20 g of anticoagulant, add 400 mL of water and stir to dissolve, then add water to make up the volume to 500 mL, and stir evenly to obtain a mixture; the platelet activation inhibitor is theophylline and aspirin, and their mass ratio is 1:1, that is, 5 g of theophylline and 5 g of aspirin each, the cell membrane protector is glucose, the pH regulator is sodium citrate, and the anticoagulant is sodium citrate. The mixture is filtered through a filter membrane with a pore size of 0.22 μm to obtain the additive for slowing down the spontaneous activation of ex vivo platelets, and the pH of the additive is 7.4, which is similar to the pH of blood.

[0064] Example 5

[0065] It is basically the same as Example 1, the only difference being that the mass ratio of theophylline to aspirin in the platelet activation inhibitor is 1:9. That is, weigh 2 g of platelet activation inhibitor, 10 g of cell protector, 5 g of pH regulator, and 12 g of anticoagulant, add 400 mL of water and stir to dissolve, then add water to make up the volume to 500 mL, and stir evenly to obtain a mixture; the platelet activation inhibitor is theophylline and aspirin, and their mass ratio is 1:9, that is, 0.2 g of theophylline and 1.8 g of aspirin, the cell membrane protector is glucose, the pH regulator is phosphate buffer, and the anticoagulant is EDTA.K 2 The mixture is filtered through a filter membrane with a pore size of 0.22 μm to obtain the additive for slowing down the spontaneous activation of ex vivo platelets, and the pH of the additive is 7.4, which is similar to the pH of blood.

[0066] Example 6

[0067] It is basically the same as Example 1, the only difference being that the mass ratio of theophylline to aspirin in the platelet activation inhibitor is 1:4. That is, weigh 2 g of platelet activation inhibitor, 10 g of cell protector, 5 g of pH regulator, and 12 g of anticoagulant, add 400 mL of water and stir to dissolve, then add water to make up the volume to 500 mL, and stir evenly to obtain a mixture; the platelet activation inhibitor is theophylline and aspirin, and their mass ratio is 1:4, that is, 0.4 g of theophylline and 1.6 g of aspirin, the cell membrane protector is glucose, the pH regulator is phosphate buffer, and the anticoagulant is EDTA.K 2 The mixture is filtered through a filter membrane with a pore size of 0.22 μm to obtain the additive for slowing down the spontaneous activation of ex vivo platelets, and the pH of the additive is 7.4, which is similar to the pH of blood.

[0068] Example 7

[0069] It is basically the same as Example 1, with the only difference being that the mass ratio of theophylline to aspirin in the platelet activation inhibitor is 4:1. That is, 2 g of the platelet activation inhibitor, 10 g of the cell protector, 5 g of the pH regulator, and 12 g of the anticoagulant are weighed, added to 400 mL of water and stirred until dissolved, and then the volume is made up to 500 mL with water and stirred evenly to obtain a mixture; the platelet activation inhibitor is theophylline and aspirin, and their mass ratio is 4:1, that is, 1.6 g of theophylline and 0.4 g of aspirin, the cell membrane protector is glucose, the pH regulator is phosphate buffer, and the anticoagulant is EDTA.K 2 The mixture is filtered through a filter membrane with a pore size of 0.22 μm to obtain the additive for slowing down the spontaneous activation of ex vivo platelets. The pH of the additive is 7.4, which is similar to the pH of blood.

[0070] Example 8

[0071] It is basically the same as Example 1, with the only difference being that the mass ratio of theophylline to aspirin in the platelet activation inhibitor is 9:1. That is, 2 g of the platelet activation inhibitor, 10 g of the cell protector, 5 g of the pH regulator, and 12 g of the anticoagulant are weighed, added to 400 mL of water and stirred until dissolved, and then the volume is made up to 500 mL with water and stirred evenly to obtain a mixture; the platelet activation inhibitor is theophylline and aspirin, and their mass ratio is 9:1, that is, 1.8 g of theophylline and 0.2 g of aspirin, the cell membrane protector is glucose, the pH regulator is phosphate buffer, and the anticoagulant is EDTA.K 2 The mixture is filtered through a filter membrane with a pore size of 0.22 μm to obtain the additive for slowing down the spontaneous activation of ex vivo platelets. The pH of the additive is 7.4, which is similar to the pH of blood.

[0072] Comparative Example 1

[0073] It is basically the same as Example 1, except that a conventional blood anticoagulant is used and it does not contain a platelet activation inhibitor. That is, 10 g of the cell protector, 5 g of the pH regulator, and 12 g of the anticoagulant are weighed, added to 400 mL of water and stirred until dissolved, and then the volume is made up to 500 mL with water and stirred evenly to obtain a mixture; the cell membrane protector is glucose, the pH regulator is phosphate buffer, and the anticoagulant is EDTA.K 2 The mixture is filtered through a filter membrane with a pore size of 0.22 μm to obtain an additive. The pH of the additive is 7.4, which is similar to the pH of blood.

[0074] Comparative Example 2

[0075] It is basically the same as Example 1, except that the platelet activation inhibitor is only theophylline. That is, 2 g of platelet activation inhibitor, 10 g of cytoprotectant, 5 g of pH regulator, and 12 g of anticoagulant are weighed, added to 400 mL of water and stirred until dissolved, and then the volume is made up to 500 mL with water and stirred evenly to obtain a mixture; the platelet activation inhibitor is theophylline, the cell membrane protectant is glucose, the pH regulator is phosphate buffer solution, and the anticoagulant is EDTA.K 2 . The mixture is filtered through a filter membrane with a pore size of 0.22 μm to obtain an additive, and the pH of the additive is 7.4, which is similar to the pH of blood.

[0076] Comparative Example 3

[0077] It is basically the same as Example 1, except that the platelet activation inhibitor is only aspirin. That is, 2 g of platelet activation inhibitor, 10 g of cytoprotectant, 5 g of pH regulator, and 12 g of anticoagulant are weighed, added to 400 mL of water and stirred until dissolved, and then the volume is made up to 500 mL with water and stirred evenly to obtain a mixture; the platelet activation inhibitor is aspirin, the cell membrane protectant is glucose, the pH regulator is phosphate buffer solution, and the anticoagulant is EDTA.K 2 . The mixture is filtered through a filter membrane with a pore size of 0.22 μm to obtain an additive, and the pH of the additive is 7.4, which is similar to the pH of blood.

[0078] Comparative Example 4

[0079] It is basically the same as Example 1, the only difference being that the mass ratio of theophylline to aspirin in the platelet activation inhibitor is 1:10. That is, 2 g of platelet activation inhibitor, 10 g of cytoprotectant, 5 g of pH regulator, and 12 g of anticoagulant are weighed, added to 400 mL of water and stirred until dissolved, and then the volume is made up to 500 mL with water and stirred evenly to obtain a mixture; the platelet activation inhibitor is theophylline and aspirin, and their mass ratio is 1:10, that is, 0.182 g of theophylline and 1.818 g of aspirin, the cell membrane protectant is glucose, the pH regulator is phosphate buffer solution, and the anticoagulant is EDTA.K 2 . The mixture is filtered through a filter membrane with a pore size of 0.22 μm to obtain an additive, and the pH of the additive is 7.4, which is similar to the pH of blood.

[0080] Comparative Example 5

[0081] Basically the same as Example 1, the only difference is that the mass ratio of theophylline to aspirin in the platelet activation inhibitor is 10:1. That is, 2 g of the platelet activation inhibitor, 10 g of the cytoprotectant, 5 g of the pH regulator, and 12 g of the anticoagulant are weighed, added to 400 mL of water and stirred to dissolve, and then the volume is fixed to 500 mL with water and stirred evenly to obtain a mixture; the platelet activation inhibitor is theophylline and aspirin, and their mass ratio is 10:1, that is, 1.818 g of theophylline and 0.182 g of aspirin, the cell membrane protectant is glucose, the pH regulator is phosphate buffer, and the anticoagulant is EDTA.K 2 The mixture is filtered through a filter membrane with a pore size of 0.22 μm to obtain an additive, and the pH of the additive is 7.4, which is similar to the pH of blood.

[0082] Test results

[0083] Peripheral venous blood of the same healthy volunteer is collected, pretreated according to the methods of Examples 1 to 8 and Comparative Examples 1 to 5, and stored at room temperature. Platelet activation tests are carried out at 0 h (that is, within 30 minutes after blood collection), 2 h, 4 h, 8 h, and 12 h after blood collection and pretreatment respectively. Group 1 is detected by the method of Example 1, Group 2 is detected by the method of Example 2, Group 3 is detected by the method of Example 3, Group 4 is detected by the method of Example 4, Group 5 is detected by the method of Example 5, Group 6 is detected by the method of Example 6, Group 7 is detected by the method of Example 7, Group 8 is detected by the method of Example 8, Group 9 is detected by the method of Comparative Example 1, Group 10 is detected by the method of Comparative Example 2, Group 11 is detected by the method of Comparative Example 3, Group 12 is detected by the method of Comparative Example 4, and Group 13 is detected by the method of Comparative Example 5. The test results of the platelet activation ratio of each pretreatment method are shown in Table 1.

[0084] Table 1 Statistical table of platelet activation ratio

[0085] Duration 0h 4h 8h 24h 36h Group 1 0.20％ 0.41％ 0.73％ 1.21％ 2.28％ Group 2 0.24％ 0.48％ 0.86％ 1.32％ 2.43％ Group 3 0.25％ 0.53％ 1.18％ 1.68％ 2.56％ Group 4 0.28％ 0.52％ 1.25％ 1.81％ 2.72％ Group 5 0.26％ 0.54％ 1.03％ 1.63％ 2.60％ Group 6 0.23％ 0.50％ 0.99％ 1.54％ 2.53％ Group 7 0.22％ 0.49％ 0.97％ 1.52％ 2.51％ Group 8 0.27％ 0.55％ 1.05％ 1.67％ 2.64％ Group 9 0.40％ 3.22％ 5.43％ 10.57％ 18.67％ Group 10 0.35％ 1.21％ 2.52％ 4.65％ 7.25％ Group 11 0.30％ 0.98％ 2.03％ 3.67％ 6.36％ Group 12 0.32％ 1.13％ 2.32％ 4.06％ 6.78％ Group 13 0.34％ 1.19％ 2.44％ 4.49％ 7.01％

[0086] It can be seen from Table 1 that by using the pretreatment methods described in Examples 1-8 (Groups 1-8) of the present invention, the platelet activation ratio is relatively low, less than 2%, within 24 hours. At 36 hours after blood collection, the platelet activation ratio is still lower than the upper limit level of 3% of the normal platelet activation ratio in the human body. That is, the platelet activation function detection methods described in Groups 1-8 can still meet the clinical requirements of flow cytometry within 36 hours of normal temperature storage of blood samples. Moreover, the inhibitory effect of the pretreatment methods described in Examples 1-8 of the present invention on platelet activation is significantly better than that of Comparative Example 1 (Group 9). At the same time, the effects of Groups 1-3 are better than those of Group 4, and the effects of Groups 6-7 are better than those of Groups 5 and 8. The detection method of Group 9 has a platelet activation ratio exceeding the upper limit level of 3% of the normal platelet activation ratio in the human body at 4 hours at normal temperature. Therefore, it is necessary to complete the detection within 2 hours by using the pretreatment method of Comparative Example 1. Although the activation ratios of the pretreatment methods of Comparative Examples 10-13 (Groups 10-13) are lower than the upper limit level of 3% of the normal platelet activation ratio in the human body at 8 hours at normal temperature, the platelet activation ratios at 24 hours after blood collection all exceed the upper limit level of 3% of the normal platelet activation ratio in the human body. Therefore, it is necessary to complete the detection within 8 hours by using the pretreatment methods described in Comparative Examples 10-13. In addition, from the comparison results of Groups 1, 5-8 and Groups 10-13, it can be seen that when the total amount of platelet activation inhibitors is the same, when the platelet activation inhibitors are a compound of theophylline and aspirin and the mass ratio of theophylline to aspirin is 1:9-9:1 (Groups 1, 5-8), the method has a better technical effect of inhibiting platelet ex vivo activation compared with the method without platelet activation inhibitors (Group 9), the single use of theophylline and aspirin (Groups 10-11), or the compound of theophylline and aspirin but with an inappropriate dosage ratio (Groups 12-13). Among them, the technical effect of the method of Group 1 is the best, indicating that the combined use of theophylline and aspirin in a specific ratio has a synergistic effect in slowing down platelet ex vivo activation.

[0087] Example 9 A method for detecting platelet activation function

[0088] It is basically the same as Example 1, except that the activated platelet marker is CD62P, the platelet markers are CD42a and CD42b respectively, the first fluorophore is PE, and the second fluorophores are FITC and APC respectively. That is, the peripheral venous blood of healthy volunteers (36 hours after blood collection) is detected by using CD42a-FITC / CD42b-APC / CD62P-PE detection reagent, and the calculated platelet activation ratio is 2.35%.

[0089] Example 10 A method for detecting platelet activation function

[0090] Basically the same as Example 1, except that the activated platelet marker is PAC-1, the platelet markers are CD61 and CD41 respectively, the first fluorophore is FITC, and the second fluorophores are APC-Cy7 and PerCP respectively. That is, the peripheral venous blood of healthy volunteers (36 hours after blood collection) was detected using the CD41-PerCP / CD61-APC-Cy7 / PAC-1-FITC detection reagent, and the platelet activation ratio was calculated to be 2.49%.

Claims

1. A platelet function detection reagent, characterized in that: The reagent comprises a blood collection tube filled with an additive for slowing down spontaneous activation of ex vivo platelets, an activated platelet labeling reagent, a platelet labeling reagent, and a platelet incubation solution, wherein the activated platelet labeling reagent is an activated platelet marker capture antibody coupled with a first fluorescein, the platelet labeling reagent is a platelet marker capture antibody coupled with a second fluorescein, and the platelet incubation solution is a phosphate buffer solution; the additive for slowing down spontaneous activation of ex vivo platelets comprises the following components in mass fractions: 1 to 10 parts of a platelet activation inhibitor, 10 to 20 parts of a blood anticoagulant, 3 to 18 parts of a cell membrane protective agent, and 3 to 18 parts of a pH regulator, and the platelet activation inhibitor is theophylline and aspirin, and the mass ratio thereof is 1:9 to 9:

1.

2. The detection reagent according to claim 1, characterized in that The additive for slowing down the spontaneous activation of isolated platelets comprises the following components in parts by weight: 1 to 5 parts of a platelet activation inhibitor, 10 to 16 parts of a blood anticoagulant, 3 to 10 parts of a cell membrane protective agent, and 3 to 12 parts of a pH regulator.

3. The detection reagent according to claim 1 or 2, characterized in that The blood anticoagulant is one or more of sodium citrate and EDTA salt; the cell membrane protectant is one or more of sucrose and glucose; the pH adjuster is one or more of tris(hydroxymethyl)aminomethane and hydrochloric acid, citric acid and citrate, phosphate, carbonate, acetic acid and acetate.

4. A blood sample pretreatment method, characterized in that: A blood sample is collected by using a blood collection tube containing an additive for slowing down spontaneous activation of platelets in vitro in the detection reagent of any one of claims 1 to 3, and the additive and the blood sample are thoroughly and gently inverted and mixed for pretreatment.

5. Use of the detection reagent according to any one of claims 1 to 3 or the blood sample pretreatment method according to claim 4 in the detection of platelet activation function.

6. A method for detecting activated platelet function for non-disease treatment and diagnosis purposes using the detection reagent according to any one of claims 1 to 3, characterized in that: The steps include: S1. Collect blood samples using a blood collection tube containing an additive that slows down spontaneous platelet activation in vitro, and mix thoroughly and gently by inverting; S2. Mix the activated platelet labeling reagent, the platelet labeling reagent and the blood sample collected in step S1 in a flow test tube; S3. Add platelet incubation solution, mix well, and incubate at room temperature in the dark; S4. Add platelet incubation solution and mix well to obtain a sample to be tested; S5. Detect the sample to be tested by flow cytometry and analyze the platelet function in the blood sample; The first fluorescein and the second fluorescein in the activated platelet labeling reagent and the platelet labeling reagent are different in type and emission wavelength.

7. The method according to claim 6, characterized in that: The activated platelet marker is at least one of CD62P, CD63, PAC-1, CD107a and CD107b; the platelet marker is at least one of CD42a, CD42b, CD41, CD41a, CD41b and CD61, and contains CD41b or CD61.

8. The method according to claim 6, characterized in that: The first fluorescent substance and the second fluorescent substance can be excited by at least one laser with a wavelength of 405nm, 488nm and 633nm.

9. The method according to claim 6, characterized in that: The first fluorescent substance is selected from PE, APC, and FITC, and the second fluorescent substance is selected from FITC, PE, PE-Cy7, PerCP, APC, and APC-Cy7.

10. The method according to claim 6, characterized in that: It also includes setting up isotype control antibody reagents for detection.

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