Method for measuring platelet activation through in-vitro ADP stimulation and application

Abstract

The invention belongs to the technical field of medical biology, and particularly relates to a method for measuring platelet activation through in-vitro ADP stimulation and application. The method provided by the invention comprises the following steps: S1, respectively adding a buffer solution, an ADP solution and an anticoagulant whole blood sample into a homotype control tube and a tube to be detected; s2, placing the homotype control tube and the to-be-tested tube at 18-28 DEG C for 5-30 min for stimulation; the final concentration of the ADP solution in the step S1 is 5-160 [mu] mol / L; the addition amount of the anticoagulant whole blood sample is 5-10 microliters. According to the method provided by the invention, the operation is more direct, simple and convenient, the stimulation is completed at normal temperature, and the antibody reagent is directly added after the stimulation is completed without additionally taking a flow type tube for secondary split charging of the antibody reagent. In addition, according to the method, the sample dosage is remarkably reduced, and the patient burden is reduced; the dosage of ADP is also greatly reduced, and the dosage of reagents is saved; the collection time is obviously shortened, and the detection efficiency is greatly improved.

Description

Technical Field

[0001] The invention belongs to the field of medical biotechnology, and particularly relates to a method for measuring platelet activation by ADP stimulation in vitro and an application thereof. Background Art

[0002] Adenosine diphosphate (ADP) is a key factor in the platelet activation process. It specifically binds to the P2Y12 receptor on the platelet membrane surface, triggering a series of complex and orderly signaling events, including platelet shape change, aggregation, and release reactions. In vitro, using ADP stimulation to measure the degree of platelet activation is an important experimental method for evaluating platelet function and the efficacy of different antiplatelet treatment regimens. This method simulates the in vivo platelet activation mechanism, using ADP as a key stimulating factor to accurately observe the activation response of platelets under its influence, providing a reliable basis for in-depth understanding of platelet functional status and evaluating the effectiveness of various antiplatelet treatment strategies.

[0003] In the existing technology (Clinical Application of Flow Cytometry, edited by Wu Lijuan, People's Medical Publishing House, 2021, 256-259), flow cytometry is used to carry out the determination of in vitro stimulated activated platelets. However, this method requires a large amount of blood sample during the ADP stimulation determination process. For patients who already have diseases, especially those who need to regularly measure platelets for drug monitoring due to long-term medication, the large amount of blood consumed undoubtedly increases their physical and psychological burden. At the same time, it takes a long time to collect 10,000 platelets, resulting in low measurement efficiency. In addition, the stimulation operation is required to be carried out at a constant temperature of 37°C, and the operating conditions are quite harsh. In the collection gate link, only platelets are circled, and after the stimulation is completed, due to the adhesion of a large number of platelets to white blood cells, the number of platelets is sharply reduced, the collection efficiency is greatly reduced, and the accuracy of the measurement results is poor.

[0004] The study "Effects of Photodynamic Therapy on Thromboxane Release from Rat Platelets" (Zhou Chuan-nong, Chinese Journal of Laser Medicine, Vol. 6, No. 4, 1997) investigated the use of intravascular injection of photosensitizers to activate platelets. However, this approach was insufficient to allow platelets to adequately load the drug, had side effects on other cells in the body, and could not achieve a controlled effect in both time and space. These factors hindered in vitro studies and the selective in vivo use of platelets to treat various diseases.

[0005] Patent CN105806818A discloses a method for detecting platelet NMDA receptor activity and its application, which reflects the platelet NMDA receptor activity by regulating the Ca2+ influx of the platelet NMDA receptor channel and detecting the promoting effect of NMDA receptor on platelet adhesion and aggregation, thereby evaluating platelet function.2+ During the process of influx and testing its promotion of platelet adhesion and aggregation, a large blood sample may be required to isolate sufficient platelets to meet experimental requirements. During sample handling and testing, sample activity can be easily affected by various factors such as temperature, pH, and centrifugal force, leading to changes in platelet activity and interfering with the accurate assessment of NMDA receptor activity. This places high demands on the sample and is highly complex to perform.

[0006] Currently, a method for measuring platelet activation by in vitro ADP stimulation has been developed. This method provides accurate results, significantly reduces sample and ADP dosage, and shortens collection time. The method is simple to operate and can be performed at room temperature. It is being used to assess platelet function and the efficacy of different antiplatelet therapies, providing a solution to the aforementioned issues. Summary of the Invention

[0007] In response to the above-mentioned deficiencies, the present invention provides a method and application for determining platelet activation by ADP stimulation in vitro. The method for determining platelet activation by ADP stimulation in vitro provided by the present invention comprises: S1, adding a buffer solution, an ADP solution and an anticoagulated whole blood sample to an isotype control tube and a test tube respectively; S2, placing the isotype control tube and the test tube at 18-28°C for 5-30 minutes for stimulation; the final concentration of the ADP solution described in step S1 is 5-160 μmol / L; the amount of anticoagulated whole blood sample added is 5-10 μL. The method provided by the present invention is more direct and simple to operate. It only needs to complete the stimulation at room temperature. After the stimulation is completed, the antibody reagent is directly added, and there is no need to take out a flow tube for secondary subpackaging and adding the antibody reagent. In addition, the method provided by the present invention significantly reduces the sample usage, reducing the burden on patients; the ADP usage is also greatly reduced, saving the reagent usage; the collection time is significantly shortened, greatly improving the detection efficiency.

[0008] the term:

[0009] As used herein, the term "CD62P+" refers to platelets that express CD62P, an adhesion molecule primarily expressed on the surface of activated platelets and activated endothelial cells. Anti-CD62P antibody labeling can be used to detect CD62P expression on the platelet surface, thereby assessing platelet activation status.

[0010] In the present invention, the term "10,000 platelet collection time" refers to the time required to collect 10,000 platelets.

[0011] As used herein, the term "ADP" stands for adenosine diphosphate (ADP). ADP is a key agonist for platelet activation, binding to P2Y1 and P2Y12 receptors on the platelet surface, triggering platelet shape changes, aggregation, and release reactions. In platelet function tests, ADP is often used as a stimulant to assess platelet reactivity.

[0012] In the present invention, the term "activated platelets" refers to the process in which platelets are transformed from a resting state to a physiologically active state, and plays a key role in physiological and pathological processes such as hemostasis, thrombosis and inflammatory response.

[0013] In the present invention, the term "isotype control" refers to an antibody that has the same species origin, subtype, fluorescent marker, dosage and concentration as the flow cytometry antibody used, but has no specific binding to the target.

[0014] The technical solution of the present invention is:

[0015] In one aspect, the present invention provides a method for measuring platelet activation by ADP stimulation in vitro, the method comprising the following steps:

[0016] S1. Add buffer solution, ADP solution and anticoagulated whole blood sample to the isotype control tube and the test tube respectively;

[0017] Incubate S2, the isotype control tube, and the test tube at 18-28°C for 5-30 minutes.

[0018] S3. Add platelet-specific surface glycoprotein antibody-fluorescent complex and platelet activation marker antibody-fluorescent complex to the test tube and mix well;

[0019] S4. Add platelet-specific surface glycoprotein antibody-fluorescent complex and platelet activation marker isotype control antibody-fluorescent complex to the isotype control tube and mix well;

[0020] S5. After incubating the isotype control tube and the test tube in the dark, add the fixative, mix them separately, and then measure the degree of platelet activation.

[0021] Specifically, the final concentration of the ADP solution in step S1 is 5-160 μmol / L.

[0022] Preferably, the final concentration of the ADP solution in step S1 is 5-10 μmol / L, 10-20 μmol / L, 20-30 μmol / L, 30-40 μmol / L, 40-50 μmol / L, 50-60 μmol / L, 60-70 μmol / L, 70-80 μmol / L, 80-90 μmol / L, 90-100 μmol / L, 100-110 μmol / L, 110-120 μmol / L, 120-130 μmol / L, 130-140 μmol / L, 140-150 μmol / L or 150-160 μmol / L.

[0023] Further preferably, the final concentration of the ADP solution in step S1 is 5 μmol / L, 10 μmol / L, 20 μmol / L, 40 μmol / L, 80 μmol / L or 160 μmol / L.

[0024] Still further preferably, the final concentration of the ADP solution in step S1 is 10 μmol / L.

[0025] Specifically, the amount of anticoagulated whole blood sample added in step S1 is 5-10 μL.

[0026] Preferably, the amount of the anticoagulated whole blood sample added in step S1 is 5-6 μL, 6-7 μL, 7-8 μL, 8-9 μL or 9-10 μL.

[0027] Further preferably, the amount of the anticoagulated whole blood sample added in step S1 is 5 μL or 10 μL.

[0028] Still more preferably, the amount of anticoagulated whole blood sample added in step S1 is 5 μL.

[0029] Specifically, step S2 is to place the isotype control tube and the test tube at 18-28° C. for 5-10 min, 10-15 min, 15-20 min, 20-25 min, or 25-30 min.

[0030] Preferably, step S2 is to place the isotype control tube and the test tube at 18-28° C. for 10-20 minutes.

[0031] Further preferably, in step S2, the isotype control tube and the test tube are placed at 18-28° C. for 10-11 min, 11-12 min, 12-13 min, 13-14 min, 14-15 min, 15-16 min, 16-17 min, 17-18 min, 18-19 min or 19-20 min.

[0032] Furthermore, preferably, in step S2, the isotype control tube and the test tube are placed at 18-28° C. for 10 min, 15 min or 20 min.

[0033] More preferably, in step S2, the isotype control tube and the test tube are placed at 18-28° C. for 10 minutes.

[0034] Specifically, the platelet-specific surface glycoprotein antibody-fluorescence complex in step S3 or step S4 is: a fluorescently labeled platelet-specific surface glycoprotein antibody.

[0035] Preferably, the platelet-specific surface glycoprotein antibody includes any one or more of CD41 antibody, CD42a antibody, CD42b antibody, CD61 antibody, and CD36 antibody.

[0036] Further preferably, the platelet-specific surface glycoprotein antibody is CD41 antibody.

[0037] Preferably, the fluorescent labeling includes labeling with any one or more of FITC, PE, PerCP, Cy5, Cy7, APC, Alexa Fluor 700, Pacific Blue, and Brilliant Violet 421.

[0038] Further preferably, the platelet-specific surface glycoprotein antibody is fluorescently labeled with PerCP.

[0039] Specifically, the platelet activation marker-fluorescence complex in step S3 is: a fluorescently labeled platelet activation marker antibody.

[0040] Preferably, the platelet activation marker antibody is selected from any one or more of CD62P and CD63.

[0041] More preferably, the platelet activation marker antibody is CD62P.

[0042] Preferably, the fluorescent labeling includes labeling with any one or more of FITC, PE, PerCP, Cy5, Cy7, APC, Alexa Fluor 700, Pacific Blue, and Brilliant Violet 421.

[0043] Further preferably, the platelet activation marker antibody is fluorescently labeled with PE.

[0044] Specifically, the platelet activation marker isotype control antibody-fluorescence complex in step S4 is a fluorescently labeled isotype control antibody of the platelet activation marker antibody.

[0045] Preferably, the isotype control antibody of the platelet activation marker antibody is IgG1.

[0046] Preferably, the fluorescent labeling includes labeling with any one or more of FITC, PE, PerCP, Cy5, Cy7, APC, Alexa Fluor 700, Pacific Blue, and Brilliant Violet 421.

[0047] More preferably, the isotype control antibody for the platelet activation marker antibody is fluorescently labeled with PE.

[0048] Specifically, the dark-proof incubation condition in step S5 is incubation at 18-28° C. for 15-30 min.

[0049] Preferably, the dark-proof incubation condition in step S5 is incubation at 18-28° C. for 15 minutes.

[0050] Specifically, the fixative in step S5 is a paraformaldehyde solution.

[0051] Preferably, the concentration of the paraformaldehyde is 1% w / v.

[0052] Specifically, the determination of the platelet activation degree in step S5 includes: determining the percentage of activated platelets or determining the mean fluorescence intensity of activated platelets.

[0053] On the other hand, the present invention provides the use of the above method in evaluating platelet function, evaluating antiplatelet treatment regimens, evaluating platelet-promoting treatment regimens, screening antiplatelet drugs, or screening platelet-promoting drugs.

[0054] The beneficial effects of the present invention are:

[0055] The in vitro ADP stimulation method for determining platelet activation provided by the present invention significantly reduces sample volume, reducing the burden on patients; the amount of ADP used is also greatly reduced, saving reagent usage; and the collection time is significantly shortened, greatly improving detection efficiency. In addition, the in vitro ADP stimulation method for determining platelet activation provided by the present invention is more direct and simple to operate. During stimulation, stimulation only needs to be completed at room temperature, not at 37°C. After stimulation is completed, the antibody reagent is directly added, eliminating the need for a separate flow tube for secondary subpackaging and adding the antibody reagent. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 The results of platelet activation assay with different final concentrations of ADP are shown.

[0057] Figure 2The results of platelet activation assays with different sample sizes are shown in Figure 1. A represents the time required to collect 10,000 platelets with different sample sizes; and B represents the CD62P+ expression level with different sample sizes.

[0058] Figure 3 The results of platelet activation determination at different stimulation times. DETAILED DESCRIPTION

[0059] The present invention will be further clarified and fully described below by way of examples. The following examples are only a portion of the present invention and are not intended to limit the present invention, but are merely for illustration. The experimental methods used in the following examples are all routine experiments unless otherwise specified, and the materials and reagents used in the following examples are all commercially available unless otherwise specified.

[0060] Example 1 A method for measuring platelet activation by ADP stimulation in vitro

[0061] S1. Take two flow cytometry tubes and label them "isotype control tube" and "test tube." Add 195 μl of 1× PBS, 5 μl of 410 μmol / L ADP solution, and 5 μl of sodium citrate anticoagulated whole blood to the isotype control tube and the test tube, respectively, to achieve a final ADP concentration of 10 μmol / L.

[0062] S2. Place at room temperature (18-28°C) for 10 minutes.

[0063] S3. Add 5 μl each of CD41-PerCP and IgG1-PE antibody reagents to the isotype control tube; add 5 μl each of CD41-PerCP and CD62P-PE antibody reagents to the test tube.

[0064] S4. Vortex mix for 2-3 seconds and incubate at room temperature (18-28°C) in the dark for 15 minutes.

[0065] S5. Add 1 ml of 1% paraformaldehyde to the isotype control tube and the test tube in turn for fixation, and vortex mix to obtain the test solution.

[0066] The CD62P+ expression level of the test fluid was measured using a flow cytometer, and the time required to collect 10,000 platelets was recorded. The results are shown in Table 1.

[0067] Table 1

[0068] Example 1 Operation method Sample volume (μl) 10 Platelet collection time (s) 23 CD62P+(%) 85.3

[0069] Example 2 Effects of different factors on platelet activation assay results

[0070] 2.1 Effects of different final concentrations of ADP on platelet activation

[0071] The 410 μmol / L ADP solution in step S1 of Example 1 was replaced with 0 μmol / L, 25.625 μmol / L, 51.25 μmol / L, 102.5 μmol / L, 205 μmol / L, 820 μmol / L, 1640 μmol / L, or 3280 μmol / L ADP solution, respectively, to achieve a final ADP concentration of 0 μmol / L, 0.625 μmol / L, 1.25 μmol / L, 2.5 μmol / L, 5 μmol / L, 20 μmol / L, 40 μmol / L, 80 μmol / L, or 160 μmol / L.

[0072] The CD62P+ expression level of the test solution was determined by flow cytometry. The results of platelet activation by different final concentrations of ADP are shown in Table 2 and Figure 1 The results show that at a final ADP concentration of 10 μmol / L, CD62P+ expression reached 87.2 μmol / L. After reaching 10 μmol / L, CD62P+ expression did not increase significantly with ADP concentration. Therefore, a final ADP concentration of 10 μmol / L achieved optimal platelet activation, at the lowest possible ADP concentration.

[0073] Table 2

[0074] Final ADP concentration (μmol / L) CD62P+(%) 0 23.9 0.625 71.9 1.25 79 2.5 83.9 5 85.9 10 87.2 20 87.3 40 87.6 80 87.3 160 87.4

[0075] 2.2 Effect of different sample sizes on platelet activation assay results

[0076] Groups A and D were set up to investigate the effect of different sample volumes (the amount of sodium citrate anticoagulated whole blood added) on the platelet activation assay results. Groups A, C, and D differed from Example 1 only in step S1. Group B was the same as Example 1.

[0077] Step S1 of Group A is as follows: take two flow cytometry tubes and label them "isotype control tube" and "test tube", and add 197 μl of 1× PBS, 5 μl of 410 μmol / L ADP solution, and 3 μl of sodium citrate anticoagulated whole blood to the isotype control tube and the test tube in sequence; so that the final ADP concentration is 10 μmol / L.

[0078] Step S1 of Group B is as follows: take two flow cytometry tubes and label them "isotype control tube" and "test tube", and add 195 μl of 1× PBS, 5 μl of 410 μmol / L ADP solution, and 5 μl of sodium citrate anticoagulated whole blood to the isotype control tube and the test tube in sequence; so that the final ADP concentration is 10 μmol / L.

[0079] Step S1 of Group C is as follows: take two flow cytometry tubes and label them "isotype control tube" and "test tube", and add 190 μl of 1× PBS, 5 μl of 410 μmol / L ADP solution, and 10 μl of sodium citrate anticoagulated whole blood to the isotype control tube and the test tube in sequence; so that the final ADP concentration is 10 μmol / L.

[0080] Step S1 of Group D is as follows: take two flow cytometry tubes and label them "isotype control tube" and "test tube", and add 180 μl of 1× PBS, 5 μl of 410 μmol / L ADP solution, and 20 μl of sodium citrate anticoagulated whole blood to the isotype control tube and the test tube in sequence; so that the final ADP concentration is 10 μmol / L.

[0081] The CD62P+ expression level of the test fluid was measured using flow cytometry, and the time required to collect 10,000 platelets was recorded. The effects of different sample volumes on the platelet activation assay results are shown in Table 3 and Figure 2 shown.

[0082] Table 3

[0083] Group Group A Group B Group C Group D Sample volume (μL) 3 5 10 20 1×PBS sample volume (μL) 197 195 190 180 Platelet collection time (s) 48 23 20 30 CD62P+(%) 82.2 85.9 86.2 78.1

[0084] When the sample volume added is 3μL, the collection time is seriously prolonged, which reduces the collection efficiency; when the sample volume added is 20μL, the collection time is slightly extended, but the reagent antibody fails to completely bind to the activated platelets, resulting in a low activation rate and inaccurate results; when the sample volume added is 5-10μL, the results are basically consistent, but considering that the amount of blood used should be as small as possible, the optimal sample volume is 5μL.

[0085] 2.3 Effects of different stimulation times on platelet activation assay results

[0086] The 10 min in step S2 of Example 1 was replaced by 0 min, 15 min, and 20 min, respectively. The CD62P+ expression level was measured by flow cytometry to investigate the effect of different stimulation times on the platelet activation assay results. The assay results are shown in Table 4 and Figure 3 As shown in Figure 2, a final ADP concentration of 10 μmol / L and stimulation for 5-20 minutes achieved high CD62P+ expression. After 10 minutes of stimulation, CD62P+ expression remained largely unchanged over time. A 10-minute stimulation time achieved high CD62P+ expression with the shortest stimulation time.

[0087] Table 4

[0088] Stimulation time CD62P+(%) 0min (no stimulation) 3.28 5min 82.9 10min 85.9 15min 86.1 20min 85.8

[0089] Comparative Example 1 Existing Technology Determination Method

[0090] 1. Take 450 μl of sodium citrate anticoagulated blood sample and add it to a test tube containing 50 μl of 400 mmol / L ADP (final ADP concentration: 40 μmol / L). Cover the tube, mix gently, and place at 37°C for 5 minutes. This is the ADP-treated blood sample.

[0091] 2. Take two flow cytometry tubes and label them as the control tube and the tube to be tested;

[0092] (1) Add 40 μl of 1× PBS, 50 μl of ADP-treated blood sample, 10 μl of CD41-PerCP, and 10 μl of IgG1-PE to an isotype control tube (isotype control);

[0093] (2) Add 40 μl of 1× PBS, 50 μl of ADP-treated blood sample, 10 μl of CD41-PerCP, and 10 μl of CD62P-PE to the test tube;

[0094] 3. Shake the test tube once and place it at room temperature (18-22°C) in the dark for 15-18 minutes.

[0095] 4. Add 500 μl of 1% paraformaldehyde to each test tube in turn for fixation and mix well to obtain the test solution.

[0096] The percentage of activated platelets and the expression level of CD62P+ in the test solution were determined using flow cytometry, and the time required to collect 10,000 platelets was recorded. The results are shown in Table 5.

[0097] Table 5

[0098] Comparative Example 1 Operation Method Sample volume (μl) 450 Platelet collection time (s) 90 CD62P+(%) 80

[0099] The results show that compared with Comparative Example 1, the in vitro ADP stimulation method for determining platelet activation provided by the present invention is more direct and simple to operate. During stimulation, stimulation only needs to be completed at room temperature, not at 37°C. After the stimulation is completed, the antibody reagent is directly added, and there is no need to take a flow tube for secondary subpackaging and adding the antibody reagent. In addition, the in vitro ADP stimulation method for determining platelet activation provided by the present invention significantly reduces the sample amount, reducing the burden on patients; the ADP amount is also greatly reduced, saving the reagent amount; the collection time is significantly shortened, greatly improving the detection efficiency.

[0100] Comparative Example 2 A method for determining platelet activation by ADP stimulation in vitro

[0101] The only difference between Comparative Example 2 and Example 1 is that step S1 is different. Step S1 of Comparative Example 2 is:

[0102] S1. Take two flow cytometry tubes and label them "isotype control tube" and "test tube." Add 195 μl of 1× PBS, 5 μl of 8200 μmol / L ADP solution, and 5 μl of sodium citrate anticoagulated whole blood to the isotype control tube (isotype control) and the test tube, respectively, to achieve a final ADP concentration of 200 μmol / L.

[0103] Comparative Example 3: A method for determining platelet activation by ADP stimulation in vitro

[0104] The only difference between Comparative Example 3 and Example 1 is that step S2 is different. Step S2 of Comparative Example 3 is: standing at 37° C. for 10 minutes.

[0105] Comparative Example 4: A method for determining platelet activation by ADP stimulation in vitro

[0106] The only difference between Comparative Example 4 and Example 1 is that step S1 is different. Step S1 of Comparative Example 4 is:

[0107] S1. Take two flow cytometry tubes and label them "isotype control tube" and "test tube." Add 180 μl of 1× PBS, 5 μl of 8200 μmol / L ADP solution, and 20 μl of sodium citrate anticoagulated whole blood to the isotype control tube (isotype control) and the test tube, respectively, to achieve a final ADP concentration of 200 μmol / L.

[0108] Experimental Example 1 Comparative Example 1-Comparative Example 4 Effect Verification

[0109] The CD62P+ expression levels of the test solutions of Comparative Examples 2 to 4 were measured using a flow cytometer, and the time required to collect 10,000 platelets was recorded. The effects of Comparative Examples 2 to 4 on the platelet activation measurement results are shown in Table 6.

[0110] Table 6

[0111] Comparative Example 2 Comparative Example 3 Comparative Example 4 Platelet collection time (s) 37 29 42 CD62P+(%) 82.4 82.1 76.4

[0112] The measurement results showed that the time for collecting 10,000 platelets in Comparative Examples 2 to 4 exceeded 25 minutes, and the collection time was significantly prolonged; and the CD62P+ expression levels did not reach 85%.

[0113] Comparative Example 4 differs from Example 1 in that the final ADP concentration is different (final concentration is 200 μmol / L) and the amount of sodium citrate anticoagulated whole blood added is different (20 μl of sodium citrate anticoagulated whole blood). Using Comparative Example 4 as a comparison basis, the synergistic effect of the in vitro ADP stimulation platelet activation assay of the present invention was demonstrated by comparing Comparative Example 2 (final ADP concentration is 200 μmol / L) and Group D in "2.2 Effect of Different Sample Sizes on Platelet Activation Assay Results" (20 μl of sodium citrate anticoagulated whole blood).

[0114] In comparative example 4, the collection time for 10,000 platelets was 42 seconds, and the CD62P+ expression level was 76.4%;

[0115] In comparative example 2, the collection time for 10,000 platelets was 37 seconds, and the CD62P+ expression level was 82.4%. Compared with comparative example 4, the collection time for 10,000 platelets was shortened by 5 seconds, and the CD62P+ expression level was increased by 6%.

[0116] In “2.2 Effect of different sample sizes on platelet activation assay results”, the collection time of 10,000 platelets in Group D was 30 seconds, and the CD62P+ expression level was 78.1%. Compared with the control base example 4, the collection time of 10,000 platelets was shortened by 12 seconds, and the CD62P+ expression level increased by 1.7%.

[0117] In Example 1 of the present invention, the collection time of 10,000 platelets is 23 seconds, and the CD62P+ expression level is 85.3%; compared with the comparative example 4, the collection time of 10,000 platelets is shortened by 19 seconds, and the CD62P+ expression level is increased by 8.9%.

[0118] It can be seen that the in vitro ADP stimulation method for measuring platelet activation provided in Example 1 of the present invention has achieved synergistic effects in shortening the collection time of 10,000 platelets (19s>5s+12s) and increasing the expression of CD62P+ (8.9%>6%+1.7%).

[0119] The above detailed description is a specific description of one feasible embodiment of the present invention and is not intended to limit the scope of the present invention. It should be noted that any equivalent implementation or modification that does not depart from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the scope of protection of the patent of this invention should be based on the attached requirements.

Claims

1. A method for measuring platelet activation by ADP stimulation in vitro, characterized in that: The method comprises the following steps: S1. Add buffer solution, ADP solution and anticoagulated whole blood sample to the isotype control tube and the test tube respectively; Incubate S2, the isotype control tube, and the test tube at 18-28°C for 5-30 minutes. S3. Add platelet-specific surface glycoprotein antibody-fluorescent complex and platelet activation marker antibody-fluorescent complex to the test tube and mix well; S4. Add platelet-specific surface glycoprotein antibody-fluorescent complex and platelet activation marker isotype control antibody-fluorescent complex to the isotype control tube and mix well; S5. After incubating the isotype control tube and the test tube in the dark, add the fixative, mix them, and then measure the platelet activation degree; The final concentration of the ADP solution in step S1 is 5-160 μmol / L; The amount of the anticoagulated whole blood sample added in step S1 is 5-10 μL.

2. The method according to claim 1, characterized in that The final concentration of the ADP solution in step S1 is 10 μmol / L; and step S2 is to place the isotype control tube and the test tube at 18-28° C. for 10 minutes.

3. The method according to claim 1, characterized in that The platelet-specific surface glycoprotein antibody-fluorescent complex described in step S3 or step S4 is: a fluorescently labeled platelet-specific surface glycoprotein antibody; The platelet activation marker-fluorescence complex described in step S3 is: a fluorescently labeled platelet activation marker antibody; The platelet activation marker isotype control antibody-fluorescence complex described in step S4 is a fluorescently labeled isotype control antibody of the platelet activation marker antibody.

4. The method according to claim 3, characterized in that The fluorescent labeling includes labeling with any one or more of FITC, PE, PerCP, Cy5, Cy7, APC, Alexa Fluor 700, Pacific Blue, and Brilliant Violet 421.

5. The method according to claim 4, characterized in that The fluorescent labeling includes labeling with PerCP or PE.

6. The method according to claim 3, characterized in that The platelet-specific surface glycoprotein antibodies include any one or more of CD41 antibody, CD42a antibody, CD42b antibody, CD61 antibody, and CD36 antibody.

7. The method according to claim 3, characterized in that The platelet activation marker antibody is selected from any one or more of CD62P and CD63.

8. The method according to claim 3, characterized in that The isotype control antibody of the platelet activation marker antibody is IgG1.

9. The method according to claim 1, characterized in that The dark-protected incubation condition in step S5 is 18-28° C. for 15-30 min; The fixative in step S5 is a paraformaldehyde solution, and the concentration of the fixative is 1% w / v; Determining the degree of platelet activation in step S5 includes determining the percentage of activated platelets or determining the mean fluorescence intensity of activated platelets.

10. Use of the method according to any one of claims 1 to 9 in evaluating platelet function, evaluating antiplatelet therapy regimens, evaluating platelet-stimulating therapy regimens, screening antiplatelet drugs, or screening platelet-stimulating drugs.

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