An additive for slowing down spontaneous activation of platelets in vitro, and its preparation method and application

Abstract

The present invention provides an additive for slowing down spontaneous activation of platelets in vitro. The additive of the present invention comprises two functional components, a platelet activation inhibitor and a blood anticoagulant, wherein the platelet activation inhibitor is used to slow down spontaneous activation of platelets in vitro, and the blood anticoagulant is used to prevent blood coagulation. The platelet activation inhibitor comprises a certain proportion of theophylline and aspirin. The present invention has found that the combined use of a certain proportion of theophylline and aspirin has a better effect of slowing down spontaneous activation of platelets in vitro than the single use of the two, and has a significant synergistic effect. The blood sample using the above additive can be stored at room temperature for 36 hours to meet clinical detection needs, greatly extend the storage time of the sample, and improve the accuracy of the detection.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro diagnosis, and more specifically, to an additive for slowing down spontaneous activation of platelets in vitro, and a preparation method and application thereof. Background Art

[0002] The physiological hemostatic function of platelets depends on platelet activation. Activated platelets aggregate to form thrombi to exert hemostatic function. Platelets in a resting state have no hemostatic function. However, excessive platelet activation can lead to thrombosis. Assessing the risk of thrombosis by detecting the activation state of platelets is of great significance for the diagnosis of thrombotic diseases, prognosis judgment, and monitoring of drug treatment effects.

[0003] The platelet activation ratio can be used to identify overactive platelet function, predict thrombosis tendency or bleeding risk, guide the prevention or treatment of thrombosis, and improve patients' clinical outcomes. The platelet activation ratio of commercially available blood collection tubes will increase sharply if left at room temperature for 1 to 2 hours after blood collection. The test results cannot reflect the activation status of platelets in vivo or just out of the body. The activation ratio is often higher than the activation status in vivo or just out of the body. The test results fed back to doctors are inaccurate, which affects clinicians' medication and drug efficacy testing, greatly limiting clinical use.

[0004] At present, the existing technology usually uses vacuum blood collection tubes containing blood anticoagulants, such as sodium citrate, for blood collection pretreatment before testing. However, conventional blood anticoagulants are not effective in slowing down the spontaneous activation of platelets in vitro. The level of platelet activation release factors increases rapidly. If the platelet activation level of the patient's blood sample cannot be tested immediately after blood collection, the actual activation state of the platelets in the body cannot be truly reflected, which will affect the clinician's medication, and may cause the patient to develop thrombosis or bleeding symptoms, and may also cause the patient to suffer from serious cardiovascular and cerebrovascular diseases such as stroke. Therefore, there is a need to provide an additive that can slow down the spontaneous activation of platelets in vitro. Summary of the invention

[0005] The purpose of the present invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide an additive for slowing down the spontaneous activation of platelets in vitro.

[0006] The second object of the present invention is to provide a method for preparing the additive for slowing down spontaneous platelet activation in vitro.

[0007] The third object of the present invention is to provide the use of the additive for slowing down spontaneous activation of platelets in vitro.

[0008] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0009] An additive for slowing down spontaneous activation of ex vivo platelets, the additive comprising the following components in parts by mass: 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 protectant, and 3 to 18 parts of a pH regulator, wherein the platelet activation inhibitor is theophylline and aspirin, and the mass ratio thereof is 1:9 to 9:1.

[0010] The additive of the present invention comprises two functional components, a platelet activation inhibitor and a blood anticoagulant, wherein the platelet activation inhibitor is used to slow down the spontaneous activation of platelets in vitro, and the blood anticoagulant is used to prevent blood coagulation; the pH regulator can adjust the pH of the additive to be within the pH range of 7.35 to 7.45 of the blood, and the cell membrane protector can effectively protect the cell membrane of cells in the blood and maintain the integrity of the cells. The platelet activation inhibitor comprises a certain proportion of theophylline and aspirin. The present invention has found that the combined use of equal amounts of theophylline and aspirin has a better effect of slowing down the spontaneous activation of platelets in vitro than the single use of the two, and has a significant synergistic effect.

[0011] Furthermore, the mass ratio of theophylline to aspirin is 1:4 to 4:1.

[0012] Preferably, the mass ratio of theophylline to aspirin is 1:1.

[0013] Furthermore, the platelet activation inhibitor is 1 to 5 parts, and the blood anticoagulant is 10 to 16 parts.

[0014] Furthermore, the platelet activation inhibitor is 2 parts, and the blood anticoagulant is 12 parts.

[0015] Furthermore, the blood anticoagulant includes but is not limited to one or more of sodium citrate and EDTA salt.

[0016] Furthermore, the EDTA salt is dipotassium ethylenediaminetetraacetate.

[0017] Furthermore, the cell membrane protective agent is 3 to 10 parts.

[0018] Furthermore, the pH adjuster is 3 to 12 parts.

[0019] Furthermore, the cell membrane protective agent is 10 parts, and the pH regulator is 5 parts.

[0020] Furthermore, the cell membrane protective agent includes but is not limited to one or more of sucrose and glucose.

[0021] 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.

[0022] Furthermore, the platelet activation inhibitor further comprises one or more of clopidogrel, ticlopidine, adenosine and dipyridamole.

[0023] As a preferred implementation method, the additive for slowing down spontaneous activation of platelets in vitro includes the following components in parts by mass: 1 to 5 parts of platelet activation inhibitor, 10 to 16 parts of blood anticoagulant, 3 to 10 parts of cell membrane protectant, and 3 to 12 parts of pH regulator; wherein the platelet activation inhibitor is theophylline and aspirin, and the mass ratio thereof is 1:1.

[0024] Further preferably, the additive for slowing down spontaneous activation of platelets in vitro comprises the following components in parts by mass: 2 parts of platelet activation inhibitor, 12 parts of blood anticoagulant, 10 parts of cell membrane protectant, and 12 parts of pH regulator; wherein the platelet activation inhibitor is theophylline and aspirin, and the mass ratio thereof is 1:1.

[0025] The present invention also provides a method for preparing any of the above-mentioned additives, which comprises mixing the components in the additive to obtain a mixture, adding water and stirring evenly to obtain the additive. That is, the platelet activation inhibitor, the blood anticoagulant, the cell membrane protective agent, and the pH regulator are mixed according to mass fractions to obtain a mixture, and adding water and stirring evenly to obtain the additive.

[0026] The present invention also provides the use of any of the above-mentioned additives in detecting platelet activation state for non-disease treatment and diagnosis purposes or in preparing a vacuum blood collection tube or a kit for detecting platelet activation state.

[0027] Specifically, the use of the additive in the detection of platelet activation status for non-disease treatment and diagnosis purposes is to mix any of the above additives with a blood sample, store it at room temperature within 36 hours after blood collection, and perform platelet activation detection using flow cytometry.

[0028] Preferably, the detection is performed using CD41-PerCP / CD61-APC-Cy7 / CD62P-PE flow cytometry detection reagent.

[0029] The present invention also provides a vacuum blood collection tube, which contains any of the above-mentioned additives.

[0030] A platelet activation detection kit comprises any one of the additives described above.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] (1) The present invention provides an additive for slowing down the spontaneous activation of platelets in vitro, comprising two functional components, a platelet activation inhibitor and a blood anticoagulant, wherein the platelet activation inhibitor is used to slow down the spontaneous activation of platelets in vitro, and the blood anticoagulant is used to prevent blood coagulation. The platelet activation inhibitor contains a certain proportion of theophylline and aspirin. The present invention has found that the combined use of a certain proportion of theophylline and aspirin has a better effect of slowing down the spontaneous activation of platelets in vitro than the use of either alone, and has a significant synergistic effect. Blood samples using the above additives can be stored at room temperature for 36 hours to meet clinical testing needs, greatly extend the sample storage time, reflect the activation of platelets in vivo or just out of the body, and improve the accuracy of the test.

[0033] (2) The present invention uses fewer types of components, and the additive can be prepared by a one-step method, and the preparation steps are simple to operate. DETAILED DESCRIPTION

[0034] The present invention is further described below with reference to specific examples, but the examples 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 art.

[0035] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0036] Example 1

[0037] Weigh 2g of platelet activation inhibitor, 10g of cell protectant, 5g of pH adjuster, and 12g of anticoagulant, add 400mL of water and stir to dissolve, then add water to make the volume 500mL, stir evenly to obtain a mixture; wherein the platelet activation inhibitor is theophylline and aspirin, and the mass ratio is 1:1, that is, 1g of theophylline and aspirin, the cell membrane protectant is glucose, the pH adjuster is phosphate buffer, and the anticoagulant is EDTA.K2. The mixture is filtered with a filter membrane with a pore size of 0.22μm to obtain a formula additive, and the pH of the formula additive is 7.4, which is similar to the pH of blood.

[0038] Example 2

[0039] Weigh 5g of platelet activation inhibitor, 3g of cell protectant, 3g of pH adjuster, and 10g of anticoagulant, add 400mL of water and stir to dissolve, then add water to make the volume 500mL, stir evenly to obtain a mixture; wherein the platelet activation inhibitor is theophylline and aspirin, and the mass ratio is 1:1, that is, theophylline and aspirin are 2.5g each, the cell membrane protectant is sucrose, the pH adjuster is sodium citrate, and the anticoagulant is sodium citrate. The mixture is filtered with a filter membrane with a pore size of 0.22μm to obtain a formula additive, and the pH of the formula additive is 7.4, which is similar to the pH of blood.

[0040] Example 3

[0041] Weigh 1g of platelet activation inhibitor, 6g of cell protectant, 12g of pH adjuster, and 16g of anticoagulant, add 400mL of water and stir to dissolve, then add water to make the volume 500mL, stir evenly to obtain a mixture; wherein the platelet activation inhibitor is theophylline and aspirin, and the mass ratio is 1:1, that is, theophylline and aspirin are 0.5g each, the cell membrane protectant is sucrose, the pH adjuster is trishydroxymethylaminomethane, and the anticoagulant is EDTA.K2. The mixture is filtered with a filter membrane with a pore size of 0.22μm to obtain a formula additive, and the pH of the formula additive is 7.4, which is similar to the pH of blood.

[0042] Example 4

[0043] Weigh 10g of platelet activation inhibitor, 18g of cell protectant, 18g of pH adjuster, and 20g of anticoagulant, add 400mL of water and stir to dissolve, then add water to make the volume 500mL, stir evenly to obtain a mixture; wherein the platelet activation inhibitor is theophylline and aspirin, and the mass ratio is 1:1, that is, 5g of theophylline and aspirin, the cell membrane protectant is glucose, the pH adjuster is sodium citrate, and the anticoagulant is sodium citrate. The mixture is filtered with a filter membrane with a pore size of 0.22μm to obtain a formula additive, and the pH of the formula additive is 7.4, which is similar to the pH of blood.

[0044] Example 5

[0045] It is basically the same as Example 1, the only difference is that the mass ratio of theophylline and aspirin in the platelet activation inhibitor is 1:9. That is, weigh 2g of platelet activation inhibitor, 10g of cell protectant, 5g of pH adjuster, and 12g of anticoagulant, add 400mL of water and stir to dissolve, then add water to 500mL, stir evenly to obtain a mixture; wherein the platelet activation inhibitor is theophylline and aspirin, and the mass ratio is 1:9, that is, theophylline is 0.2g, aspirin is 1.8g, the cell membrane protectant is glucose, the pH adjuster is phosphate buffer, and the anticoagulant is EDTA.K2. The mixture is filtered with a filter membrane with a pore size of 0.22μm to obtain a formula additive, the pH of the formula additive is 7.4, which is similar to the pH of blood.

[0046] Example 6

[0047] It is basically the same as Example 1, the only difference is that the mass ratio of theophylline and aspirin in the platelet activation inhibitor is 1:4. That is, weigh 2g of platelet activation inhibitor, 10g of cell protectant, 5g of pH adjuster, and 12g of anticoagulant, add 400mL of water and stir to dissolve, then add water to make the volume to 500mL, and stir evenly to obtain a mixture; wherein the platelet activation inhibitor is theophylline and aspirin, and the mass ratio thereof is 1:4, that is, theophylline is 0.4g, aspirin is 1.6g, the cell membrane protectant is glucose, the pH adjuster is phosphate buffer, and the anticoagulant is EDTA.K2. The mixture is filtered with a filter membrane with a pore size of 0.22μm to obtain a formula additive, the pH of the formula additive is 7.4, which is similar to the pH of blood.

[0048] Example 7

[0049] It is basically the same as Example 1, the only difference is that the mass ratio of theophylline and aspirin in the platelet activation inhibitor is 4:1. That is, weigh 2g of platelet activation inhibitor, 10g of cell protectant, 5g of pH adjuster, and 12g of anticoagulant, add 400mL of water and stir to dissolve, then add water to make the volume to 500mL, and stir evenly to obtain a mixture; wherein the platelet activation inhibitor is theophylline and aspirin, and the mass ratio thereof is 4:1, that is, theophylline is 1.6g, aspirin is 0.4g, the cell membrane protectant is glucose, the pH adjuster is phosphate buffer, and the anticoagulant is EDTA.K2. The mixture is filtered with a filter membrane with a pore size of 0.22μm to obtain a formula additive, the pH of the formula additive is 7.4, which is similar to the pH of blood.

[0050] Example 8

[0051] It is basically the same as Example 1, the only difference is that the mass ratio of theophylline and aspirin in the platelet activation inhibitor is 9:1. That is, weigh 2g of platelet activation inhibitor, 10g of cell protectant, 5g of pH adjuster, and 12g of anticoagulant, add 400mL of water and stir to dissolve, then add water to 500mL, stir evenly to obtain a mixture; wherein the platelet activation inhibitor is theophylline and aspirin, and the mass ratio is 9:1, that is, theophylline is 1.8g, aspirin is 0.2g, the cell membrane protectant is glucose, the pH adjuster is phosphate buffer, and the anticoagulant is EDTA.K2. The mixture is filtered with a filter membrane with a pore size of 0.22μm to obtain a formula additive, the pH of the formula additive is 7.4, which is similar to the pH of blood.

[0052] Comparative Example 1

[0053] It is basically the same as Example 1, except that it does not contain a platelet activation inhibitor. That is, 10 g of a cell protectant, 5 g of a pH adjuster, and 12 g of an anticoagulant are weighed, added to 400 mL of water and stirred to dissolve, then water is added to make the volume 500 mL, and stirred evenly to obtain a mixture; wherein the cell membrane protectant is glucose, the pH adjuster is phosphate, and the anticoagulant is EDTA.K2. The mixture is filtered with a filter membrane with a pore size of 0.22 μm to obtain a formula additive, the pH of which is 7.4, which is similar to the pH of blood.

[0054] Comparative Example 2

[0055] It is basically the same as Example 1, the only difference is that the platelet activation inhibitor is theophylline. That is, weigh 2g of platelet activation inhibitor, 10g of cell protectant, 5g of pH adjuster, and 12g of anticoagulant, add 400mL of water and stir to dissolve, then add water to make the volume to 500mL, stir evenly to obtain a mixture; wherein the platelet activation inhibitor is theophylline, the cell membrane protectant is glucose, the pH adjuster is phosphate buffer, and the anticoagulant is EDTA.K2. The mixture is filtered with a filter membrane with a pore size of 0.22μm to obtain a formula additive, the pH of the formula additive is 7.4, which is similar to the pH of blood.

[0056] Comparative Example 3

[0057] It is basically the same as Example 1, except that the platelet activation inhibitor is aspirin. That is, 2g of platelet activation inhibitor, 10g of cell protectant, 5g of pH adjuster, and 12g of anticoagulant are weighed, 400mL of water is added and stirred to dissolve, and then water is added to make the volume to 500mL, and stirred evenly to obtain a mixture; wherein the platelet activation inhibitor is aspirin, the cell membrane protectant is glucose, the pH adjuster is phosphate buffer, and the anticoagulant is EDTA.K2. The mixture is filtered with a filter membrane with a pore size of 0.22μm to obtain a formula additive, and the pH of the formula additive is 7.4, which is similar to the pH of blood.

[0058] Comparative Example 4

[0059] It is basically the same as Example 1, the only difference is that the mass ratio of theophylline and aspirin in the platelet activation inhibitor is 1:10. That is, weigh 2g of platelet activation inhibitor, 10g of cell protectant, 5g of pH adjuster, and 12g of anticoagulant, add 400mL of water and stir to dissolve, then add water to 500mL, stir evenly to obtain a mixture; wherein the platelet activation inhibitor is theophylline and aspirin, and the mass ratio is 1:10, that is, theophylline is 0.182g, aspirin is 1.818g, the cell membrane protectant is glucose, the pH adjuster is phosphate buffer, and the anticoagulant is EDTA.K2. The mixture is filtered with a filter membrane with a pore size of 0.22μm to obtain a formula additive, the pH of the formula additive is 7.4, which is similar to the pH of blood.

[0060] Comparative Example 5

[0061] It is basically the same as Example 1, the only difference is that the mass ratio of theophylline and aspirin in the platelet activation inhibitor is 10:1. That is, weigh 2g of platelet activation inhibitor, 10g of cell protectant, 5g of pH adjuster, and 12g of anticoagulant, add 400mL of water and stir to dissolve, then add water to 500mL, stir evenly to obtain a mixture; wherein the platelet activation inhibitor is theophylline and aspirin, and the mass ratio is 10:1, that is, theophylline is 1.818g, aspirin is 0.182g, the cell membrane protectant is glucose, the pH adjuster is phosphate buffer, and the anticoagulant is EDTA.K2. The mixture is filtered with a filter membrane with a pore size of 0.22μm to obtain a formula additive, the pH of the formula additive is 7.4, which is similar to the pH of blood.

[0062] Test Case

[0063] The additives provided in Examples 1 to 8 and Comparative Examples 1 to 5 were added to vacuum blood collection tubes respectively, peripheral venous blood from the same healthy volunteer was collected, and the blood samples to be tested were prepared after being fully and gently inverted and mixed. The blood samples of Group 1 were preserved with the additive of Example 1, the blood samples of Group 2 were preserved with the additive of Example 2, the blood samples of Group 3 were preserved with the additive of Example 3, the blood samples of Group 4 were preserved with the additive of Example 4, the blood samples of Group 5 were preserved with the additive of Example 5, the blood samples of Group 6 were preserved with the additive of Example 6, the blood samples of Group 7 were preserved with the additive of Example 7, the blood samples of Group 8 were preserved with the additive of Example 8, the blood samples of Group 9 were preserved with the additive of Comparative Example 1, the additive of Group 10 was preserved with the additive of Comparative Example 2, the additive of Group 11 was preserved with the additive of Comparative Example 3, the additive of Group 12 was preserved with the additive of Comparative Example 4, and the additive of Group 13 was preserved with the additive of Comparative Example 5. In each group, the volume ratio of blood sample to additive was 9:1.

[0064] The 13 groups of samples were placed vertically, and one blood collection tube sample was taken from each group at 0h after blood collection (i.e., within 30 minutes after blood collection), 4h after blood collection, 8h after blood collection, 24h after blood collection, and 36h after blood collection. Platelet activation test was performed on a flow cytometer using CD41-PerCP / CD61-APC-Cy7 / CD62P-PE flow detection reagent. The platelet activation ratio test results of each sample are shown in Table 1.

[0065] Table 1 Statistical table of platelet activation ratio

[0066] 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％

[0067] It can be concluded from Table 1 that the platelet activation ratio of Group 1 to 8 additives provided in Examples 1 to 8 is lower than 2% within 24 hours, and the platelet activation ratio is still lower than the upper limit of the normal platelet activation ratio of human body at 36 hours after blood collection, that is, the blood samples using Group 1 to 8 additives can still meet clinical needs within 36 hours of storage at room temperature, and the effect of slowing down platelet activation is significantly better than that of Group 9, while Group 1 to 3 is better than Group 4, and Group 6 to 7 is better than Group 5 and Group 8. The platelet activation ratio of Group 9 additives at room temperature for 4 hours has exceeded the upper limit of the normal platelet activation ratio of healthy people by 3%, and the platelet activation ratios of Groups 10 to 13 at 24 hours after blood collection have also exceeded the upper limit of the normal platelet activation ratio of healthy people by 3%, resulting in inaccurate test results. From the comparison results of Group 1, Groups 5-8 and Groups 10-13, it can be seen that when the total amount of platelet activation inhibitor is the same, when the platelet activation inhibitor is a compound of theophylline and aspirin and the mass ratio of theophylline and aspirin is 1:9-9:1 (Group 1, Group 5-8), the additive has a better technical effect of slowing down platelet activation in vitro than the additives without platelet activation inhibitor (Group 9), theophylline and aspirin are used alone (Groups 10-11) or theophylline and aspirin are compounded but the dosage ratio is not appropriate (Groups 12-13), especially Group 1 has the best technical effect of slowing down platelet activation in vitro. The above results show that the combination of theophylline and aspirin in a certain proportion has a synergistic effect in slowing down platelet activation in vitro, and the determination within 36 hours of storage at room temperature can meet the clinical detection needs, greatly extend the storage time of the sample, and improve the accuracy of the detection.

[0068] Example 9

[0069] It is basically the same as Example 1, except that 1g of adenosine and 1g of dipyridamole are also added; that is, 4g of platelet activation inhibitor, 10g of cell protectant, 5g of pH adjuster, and 12g of anticoagulant are weighed, 400mL of water is added and stirred to dissolve, and then water is added to make the volume to 500mL, and the mixture is stirred evenly to obtain a mixture; wherein the platelet activation inhibitor is theophylline, aspirin, adenosine, and dipyridamole, and the mass ratio is 1:1:1:1, that is, theophylline, aspirin, adenosine, and dipyridamole are 1g each, the cell membrane protectant is glucose, the pH adjuster is phosphate buffer, and the anticoagulant is EDTA.K2. The mixture is filtered with a filter membrane with a pore size of 0.22μm to obtain a formula additive, and the pH of the formula additive is 7.4, which is similar to the pH of blood.

[0070] Referring to the above test example method, peripheral venous blood from healthy volunteers was collected by blood collection tubes, and the blood samples to be tested were prepared after thorough inversion and mixing. The blood samples were stored for 36 hours using the additive of this example, and the platelet activation ratio was measured to be 2.04%.

[0071] Example 10

[0072] It is basically the same as Example 1, except that 1g of clopidogrel is also added; that is, 3g of platelet activation inhibitor, 10g of cell protectant, 5g of pH adjuster, and 12g of anticoagulant are weighed, 400mL of water is added and stirred to dissolve, and then water is added to make the volume to 500mL, and stirred evenly to obtain a mixture; wherein the platelet activation inhibitor is theophylline, aspirin, and clopidogrel, and the mass ratio thereof is 1:1:1, that is, theophylline, aspirin, and clopidogrel are 1g respectively, the cell membrane protectant is glucose, the pH adjuster is phosphate buffer, and the anticoagulant is EDTA.K2. The mixture is filtered with a filter membrane with a pore size of 0.22μm to obtain a formula additive, and the pH of the formula additive is 7.4, which is similar to the pH of blood.

[0073] Referring to the above test example method, peripheral venous blood from healthy volunteers was collected by blood collection tubes, and the blood samples to be tested were prepared after thorough inversion and mixing. The blood samples were stored for 36 hours using the additive of this example, and the platelet activation ratio was measured to be 2.15%.

[0074] Embodiment 11

[0075] It is basically the same as Example 1, except that 1g of ticlopidine is also added; that is, 3g of platelet activation inhibitor, 10g of cell protectant, 5g of pH adjuster, and 12g of anticoagulant are weighed, 400mL of water is added and stirred to dissolve, and then water is added to make the volume to 500mL, and stirred evenly to obtain a mixture; wherein the platelet activation inhibitor is theophylline, aspirin, and ticlopidine, and the mass ratio thereof is 1:1:1, that is, theophylline, aspirin, and ticlopidine are 1g respectively, the cell membrane protectant is glucose, the pH adjuster is phosphate buffer, and the anticoagulant is EDTA.K2. The mixture is filtered with a filter membrane with a pore size of 0.22μm to obtain a formula additive, and the pH of the formula additive is 7.4, which is similar to the pH of blood.

[0076] Referring to the above test example method, peripheral venous blood from healthy volunteers was collected by blood collection tubes, and the blood samples to be tested were prepared after thorough inversion and mixing. The blood samples were stored for 36 hours using the additive of this example, and the platelet activation ratio was measured to be 2.20%.

[0077] Examples 9 to 11 show that the addition of other platelet activation inhibitors to the additives in the formula of Example 1 can further slow down the platelet activation rate.

[0078] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An additive for slowing down spontaneous activation of platelets in vitro, characterized in that: The invention comprises the following components in parts by weight: 1 to 10 parts of platelet activation inhibitor, 10 to 20 parts of blood anticoagulant, 3 to 18 parts of cell membrane protective agent and 3 to 18 parts of pH regulator. The platelet activation inhibitor is theophylline and aspirin, and the mass ratio thereof is 1:9 to 9:

1.

2. The additive according to claim 1, characterized in that: The mass ratio of theophylline to aspirin is 1:4 to 4:

1.

3. The additive according to claim 1, characterized in that: The blood anticoagulant is one or more of sodium citrate and EDTA salt, and the cell membrane protective agent is one or more of sucrose and glucose.

4. The additive according to claim 1, characterized in that: The pH regulator is one or more of tris(hydroxymethyl)aminomethane and hydrochloric acid, citric acid and citrate, phosphate, carbonate, acetic acid and acetate.

5. The additive according to any one of claims 1 to 4, characterized in that The platelet activation inhibitor is 1 to 5 parts, and the blood anticoagulant is 10 to 16 parts.

6. The additive according to any one of claims 1 to 4, characterized in that The cell membrane protective agent is 3 to 10 parts.

7. The additive according to any one of claims 1 to 4, characterized in that The pH adjuster is 3 to 12 parts.

8. The additive according to any one of claims 1 to 4, characterized in that The platelet activation inhibitor further comprises one or more of clopidogrel, ticlopidine, adenosine and dipyridamole.

9. Use of the additive according to any one of claims 1 to 8 in the detection of platelet activation state for purposes other than disease treatment and diagnosis or in the preparation of a vacuum blood collection tube for the detection of platelet activation state.

10. A vacuum blood collection tube, characterized in that: The vacuum blood collection tube contains the additive according to any one of claims 1 to 8.