A method for measuring platelet activation in vitro by ADP stimulation and uses thereof

Abstract

The application belongs to the field of medical biotechnology, and particularly relates to a method for in-vitro ADP stimulation determination of platelet activation and application. The method provided by the application comprises: S1, adding buffer solution, ADP solution and anticoagulant whole blood sample into a homologous control tube and a to-be-detected tube respectively; S2, placing the homologous control tube and the to-be-detected 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 micromol / L; and the addition amount of the anticoagulant whole blood sample is 5-10 microliters. The method provided by the application is more direct and simple to operate, and only needs to be completed at normal temperature. After the stimulation is completed, the antibody reagent is directly added without the need of taking a flow tube for secondary dispensing and adding the antibody reagent. In addition, the sample amount of the method provided by the application is significantly reduced, the burden of patients is reduced, the ADP amount is greatly reduced, the reagent amount is saved, the collection time is significantly shortened, and the detection efficiency is greatly improved.

Description

Technical Field

[0001] This invention belongs to the field of medical biotechnology, specifically relating to a method and application for in vitro ADP stimulation to measure platelet activation. Background Technology

[0002] Adenosine diphosphate (ADP) is a key factor in platelet activation. By specifically binding to the P2Y12 receptor on the platelet membrane surface, it triggers a series of complex and ordered signal transduction events, including platelet shape changes, aggregation, and release responses. 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 therapies. 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. This provides a reliable basis for understanding platelet functional status and evaluating the effectiveness of various antiplatelet therapy strategies.

[0003] In existing technologies (Clinical Applications of Flow Cytometry, edited by Wu Lijuan, People's Medical Publishing House, 2021, 256-259), flow cytometry is used to measure in vitro stimulated platelet activation. However, this method requires a large volume of blood sample during ADP stimulation. For patients with pre-existing conditions, especially those who require regular platelet counts for drug monitoring due to long-term medication, this large blood volume undoubtedly increases their physiological and psychological burden. Furthermore, collecting 10,000 platelets is time-consuming, resulting in low measurement efficiency. In addition, the stimulation procedure requires a constant temperature of 37°C, making the operating conditions quite stringent. During the collection gate stage, only platelets are delineated; after stimulation, a large number of platelets adhere to leukocytes, leading to a sharp decrease in platelet count and a significant reduction in collection efficiency, thus resulting in unsatisfactory measurement results.

[0004] The study "The Effect of Photodynamic Therapy on Thromboxane Release from Rat Platelets" (Zhou Chuannong, Chinese Journal of Laser Medicine, Vol. 6, No. 4, 1997) investigated the activation of platelets by injecting photosensitizers into blood vessels. However, this method was insufficient to load platelets with adequate drugs and had side effects on other cells in the body, failing to achieve spatiotemporally controllable effects. These limitations hinder in vitro research and the selective in vivo treatment of various diseases using platelets.

[0005] Patent CN105806818A discloses a method for detecting platelet NMDA receptor activity and its application. This method reflects platelet NMDA receptor activity by regulating Ca2+ influx into platelet NMDA receptor channels and detecting the promoting effect of NMDA receptors on platelet adhesion and aggregation, thereby evaluating platelet function. However, this method has limitations in regulating Ca2+ influx.2+ The process of influx and the detection of its promoting effect on platelet adhesion and aggregation may require a large number of blood samples to separate sufficient platelets to meet experimental needs. During sample processing and detection, the platelets are easily affected by various factors such as temperature, pH, and centrifugation, leading to changes in platelet activity and thus interfering with the accurate assessment of NMDA receptor activity. Therefore, the sample requirements are high, and the operation is complex.

[0006] Currently, a method for in vitro ADP stimulation to measure platelet activation is being developed. This method offers accurate results, significantly reduces sample and ADP usage, shortens collection time, and is simple to operate at room temperature. It can be used to assess platelet function and the efficacy of different antiplatelet therapies, providing a solution to the aforementioned problems. Summary of the Invention

[0007] To address the aforementioned shortcomings, this invention provides a method and application for in vitro ADP stimulation assay of platelet activation. The method for in vitro ADP stimulation assay of platelet activation provided by this invention includes: S1, adding buffer solution, ADP solution, and anticoagulated whole blood sample to a control tube and a test tube, respectively; S2, stimulating the control tube and test tube at 18-28℃ for 5-30 min; the final concentration of the ADP solution in step S1 is 5-160 μmol / L; the amount of anticoagulated whole blood sample added is 5-10 μL. The method provided by this invention is more direct and simpler to operate, requiring only stimulation at room temperature, and antibody reagents can be added directly after stimulation without the need for secondary aliquoting and antibody reagent addition in flow cytometry tubes. Furthermore, the method provided by this invention significantly reduces sample volume, reducing the burden on patients; the amount of ADP used is also greatly reduced, saving reagent consumption; and the collection time is significantly shortened, greatly improving detection efficiency.

[0008] the term:

[0009] In this invention, the term "CD62P+" refers to platelets expressing the CD62P molecule. CD62P is an adhesion molecule mainly expressed on the surface of activated platelets and activated endothelial cells. By labeling with anti-CD62P antibodies, the expression level of CD62P on the platelet surface can be detected, thereby assessing the activation status of platelets.

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

[0011] In this invention, the term "ADP" stands for Adenosine diphosphate. ADP is a key agonist for platelet activation. It binds to P2Y1 and P2Y12 receptors on the platelet surface, triggering changes in platelet shape, aggregation, and release. In platelet function assays, ADP is often used as a stimulant to assess platelet reactivity.

[0012] In this invention, the term "activated platelets" refers to the process by which platelets transform from a resting state to a physiologically active state, playing a crucial role in physiological and pathological processes such as hemostasis, thrombosis, and inflammatory responses.

[0013] In this invention, the term "isotype control" refers to an antibody that has the same species origin, subtype, fluorescent label, dosage, and concentration as the flow cytometry antibody used, but does not specifically bind to the target site.

[0014] The technical solution of this invention is as follows:

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

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

[0017] S2, the same type control tube and the test tube are placed at 18-28℃ 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 incubation in the dark with the isotype control tube and the test tube, fixative was added to each tube, and after mixing, the degree of platelet activation was measured.

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

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

[0026] Preferably, the amount of 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] More preferably, the amount of anticoagulated whole blood sample added in step S1 is 5 μL or 10 μL.

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

[0029] Specifically, step S2 involves placing the control tube and the test tube at 18-28℃ for 5-10 min, 10-15 min, 15-20 min, 20-25 min, or 25-30 min.

[0030] Preferably, step S2 involves placing the control tube and the test tube at 18-28°C for 10-20 minutes.

[0031] More preferably, step S2 involves placing the control tube and the test tube 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] In another preferred step, step S2 involves placing the control tube and the test tube at 18-28°C for 10 min, 15 min, or 20 min.

[0033] More preferably, step S2 involves placing the control tube and the test tube at 18-28°C for 10 minutes.

[0034] Specifically, the platelet-specific surface glycoprotein antibody-fluorescent complex mentioned 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 the following: CD41 antibody, CD42a antibody, CD42b antibody, CD61 antibody, and CD36 antibody.

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

[0037] Preferably, the fluorescent labeling includes labeling using any one or more of FITC, PE, PerCP, Cy5, Cy7, APC, Alexa Fluor700, Pacific Blue, and Brilliant Violet 421.

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

[0039] Specifically, the platelet activation marker-fluorescent complex mentioned 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 using any one or more of FITC, PE, PerCP, Cy5, Cy7, APC, Alexa Fluor700, Pacific Blue, and Brilliant Violet 421.

[0043] More preferably, the platelet activation marker antibody is fluorescently labeled using PE.

[0044] Specifically, the platelet activation marker isotype control antibody-fluorescent complex mentioned 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 using any one or more of FITC, PE, PerCP, Cy5, Cy7, APC, Alexa Fluor700, Pacific Blue, and Brilliant Violet 421.

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

[0048] Specifically, the light-protected incubation conditions described in step S5 are 18-28℃ for 15-30 minutes.

[0049] Preferably, the light-protected incubation conditions described in step S5 are 18-28℃ for 15 minutes.

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

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

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

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

[0054] The beneficial effects of this invention are as follows:

[0055] The method for in vitro ADP stimulation assay of platelet activation provided by this invention significantly reduces sample volume, thus reducing the burden on patients; the amount of ADP used is also greatly reduced, saving reagent consumption; the collection time is significantly shortened, greatly improving detection efficiency. Furthermore, the method for in vitro ADP stimulation assay of platelet activation provided by this invention is more direct and simple to operate. Stimulation can be completed at room temperature, eliminating the need for stimulation at 37°C. After stimulation, antibody reagent is added directly, eliminating the need for secondary dispensing and addition of antibody reagent in a separate flow cytometer. Attached Figure Description

[0056] Figure 1 The results show the platelet activation assay at different final concentrations of ADP.

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

[0058] Figure 3 The results show the platelet activation assay at different stimulation times. Detailed Implementation

[0059] The present invention will be further clearly and completely illustrated below through embodiments. These embodiments are only some examples of the present invention and are not intended to limit the present invention, but are only for illustrating the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are all conventional experiments, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0060] Example 1: A method for in vitro ADP stimulation to determine platelet activation

[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, so that the final concentration of ADP is 10 μmol / L.

[0062] S2. Place at room temperature (18-28℃) 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, then incubate at room temperature (18-28℃) in the dark for 15 minutes.

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

[0066] The expression level of CD62P+ in the test solution was determined by flow cytometry, 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 Specimen volume (μl) 10 Collection time (s) for 10,000 platelets 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 ADP solutions of 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, respectively, to achieve final ADP concentrations 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 expression level of CD62P+ in the test solution was determined by flow cytometry. The results of platelet activation assays with different final concentrations of ADP are shown in Table 2 and 3. Figure 1 As shown in the figure, the results indicated that when the final ADP concentration was 10 μmol / L, the expression level of CD62P+ reached 87.2 μmol / L. After the final ADP concentration reached 10 μmol / L, the increase in CD62P+ concentration with further increases in ADP concentration was not significant. Therefore, a final ADP concentration of 10 μmol / L achieved the best platelet activation effect at the lowest possible 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 through D were set up to investigate the effect of different sample sizes (the amount of sodium citrate anticoagulated whole blood added) on platelet activation assay results. The only difference between groups A, C, and D and Example 1 was step S1. Group B was the same as in Example 1.

[0077] Step S1 for Group A is as follows: Take two flow cytometry tubes and label them "Isotype Control Tube" and "Test Tube". 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 respectively, so that the final concentration of ADP is 10 μmol / L.

[0078] Step S1 for Group B is as follows: 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, so that the final concentration of ADP is 10 μmol / L.

[0079] Step S1 for Group C is as follows: Take two flow cytometry tubes and label them "Isotype Control Tube" and "Test Tube". 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 respectively, so that the final concentration of ADP is 10 μmol / L.

[0080] Step S1 for Group D is as follows: Take two flow cytometry tubes and label them "Isotype Control Tube" and "Test Tube". 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 respectively, so that the final concentration of ADP is 10 μmol / L.

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

[0082] Table 3

[0083] Group Group A Group B Group C Group D Sample size (μL) 3 5 10 20 1×PBS loading volume (μL) 197 195 190 180 Collection time (s) for 10,000 platelets 48 23 20 30 CD62P+(%) 82.2 85.9 86.2 78.1

[0084] When the sample volume is 3 μL, the collection time becomes significantly longer, reducing the collection efficiency. When the sample volume is 20 μL, the collection time is slightly longer, 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 is 5-10 μL, the results are basically consistent, but considering that the amount of blood used should be as small as possible, the optimal sample volume is 5 μL.

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

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

[0087] Table 4

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

[0089] Comparative Example 1: Existing Technical Measurement 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). Cap the tube, mix gently, and place at 37°C for 5 min to obtain the ADP-treated blood sample.

[0091] 2. Take two flow cytometer tubes and label them with the same type of control tube and the tube to be tested;

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

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

[0094] 3. Shake the test tube once by hand, and place it at room temperature (18-22℃) in the dark for 15-18 minutes;

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

[0096] The percentage of activated platelets and the expression level of CD62P+ in the test solution were determined by 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 Specimen volume (μl) 450 Collection time (s) for 10,000 platelets 90 CD62P+(%) 80

[0099] The results showed that, compared with Comparative Example 1, the in vitro ADP stimulation method for measuring platelet activation provided by this invention is more direct and simpler to operate. Stimulation can be performed at room temperature, eliminating the need for stimulation at 37°C. After stimulation, antibody reagents are added directly without the need for secondary aliquoting in flow cytometry tubes. Furthermore, the in vitro ADP stimulation method for measuring platelet activation provided by this invention significantly reduces sample volume, reducing the burden on patients; the amount of ADP used is also greatly reduced, saving reagent consumption; and the collection time is significantly shortened, greatly improving detection efficiency.

[0100] Comparative Example 2: A method for in vitro ADP stimulation to determine platelet activation

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

[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 and the test tube respectively, so that the final concentration of ADP is 200 μmol / L.

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

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

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

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

[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 and the test tube respectively, so that the final concentration of ADP is 200 μmol / L.

[0108] Experiment Example 1: Verification of the effects of Comparative Examples 1-4

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

[0110] Table 6

[0111] Comparative Example 2 Comparative Example 3 Comparative Example 4 Collection time (s) for 10,000 platelets 37 29 42 CD62P+(%) 82.4 82.1 76.4

[0112] The results showed that the collection time for 10,000 platelets in Comparative Examples 2-4 exceeded 25 minutes, indicating a significant prolongation of collection time; the CD62P+ expression level did not reach 85%.

[0113] The difference between Comparative Example 4 and Example 1 lies in the final ADP concentration (200 μmol / L) and the amount of sodium citrate anticoagulated whole blood added (20 μl). Using Comparative Example 4 as a basis for comparison, the synergistic effect of the in vitro ADP stimulation method for platelet activation assay of this invention is demonstrated through Comparative Example 2 (final ADP concentration of 200 μmol / L) and Group D in "2.2 Effect of Different Sample Amounts on Platelet Activation Assay Results" (20 μl of sodium citrate anticoagulated whole blood).

[0114] In Comparative Example 4, 10,000 platelets were collected in 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 the baseline Comparative Example 4, the collection time for 10,000 platelets was shortened by 5 seconds, and the CD62P+ expression level increased by 6%.

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

[0117] In Example 1 of this invention, the collection time for 10,000 platelets was 23 seconds, and the CD62P+ expression level was 85.3%. Compared with the baseline control example 4, the collection time for 10,000 platelets was shortened by 19 seconds, and the CD62P+ expression level increased by 8.9%.

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

[0119] The above detailed description is a specific illustration of one feasible embodiment of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or modifications made without departing from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of the present invention should be determined by the appended claims.

Claims

1. A method for measuring platelet activation by in vitro ADP stimulation, characterized in that, The method includes the following steps: S1. Add buffer solution, ADP solution and anticoagulated whole blood sample to the control tube and the test tube, respectively. S2, the same type control tube and the test tube are placed at 18-28℃ for 10-20 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 incubation in the dark with the isotype control tube and the test tube, fixative was added to each tube, and after mixing, the degree of platelet activation was measured. The final concentration of the ADP solution mentioned in step S1 is 5-160 μmol / L; The amount of anticoagulated whole blood sample added in step S1 is 5-10 μL; The anticoagulated whole blood sample mentioned in step S1 is sodium citrate anticoagulated whole blood.

2. The method according to claim 1, characterized in that, The final concentration of the ADP solution mentioned in step S1 is 10 μmol / L; step S2 involves placing the control tube and the test tube at 18-28℃ for 10 min.

3. The method according to claim 1, characterized in that, The platelet-specific surface glycoprotein antibody-fluorescent complex mentioned in step S3 or step S4 is: a fluorescently labeled platelet-specific surface glycoprotein antibody; The platelet activation marker-fluorescent complex mentioned in step S3 is: a fluorescently labeled platelet activation marker antibody; The platelet activation marker isotype control antibody-fluorescent complex mentioned 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 using 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 using 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 the following: 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 for the platelet activation marker antibody is IgG1.

9. The method according to claim 1, characterized in that, The conditions for incubation in the dark as described in step S5 are 18-28℃ for 15-30 minutes; The fixative mentioned in step S5 is a paraformaldehyde solution, and the concentration of the fixative is 1% w / v; The determination of platelet activation level in step S5 includes: determining the percentage of activated platelets or determining the average fluorescence intensity of activated platelets.

10. The use of the method according to any one of claims 1-9 in the preparation of products for evaluating platelet function, evaluating antiplatelet therapy regimens, evaluating platelet-promoting therapy regimens, screening antiplatelet drugs, or screening platelet-promoting drugs.

Images