Peripheral blood sample stabilizing solution for inhibiting platelet activation through multi-target synergistic effect and composite anticoagulant formula and application thereof

Abstract

The invention discloses a peripheral blood sample stabilizing solution for inhibiting platelet activation through a multi-target synergistic effect and also discloses a composite anticoagulant formula containing the stabilizing solution. The composite anticoagulant formula comprises three parts, namely a stabilizing solution, a platelet antibody detection reagent and a fixing agent. The platelet can be protected or platelet activation can be reversibly inhibited, on one hand, the detection time of the platelet resting state can be prolonged, on the other hand, the activation, agglutination and adhesion functions of the platelet are reserved, and convenient conditions are provided for research and detection of in-vitro stimulation of the platelet.

Description

Technical Field

[0001] This invention relates to the field of flow cytometry detection formulations, specifically to a peripheral blood sample stabilizing solution that inhibits platelet activation through multi-target synergistic action, and its compound anticoagulant formulation and application. Background Technology

[0002] Platelets are small cells that play a vital role in many pathophysiological processes, including thrombosis, hemorrhage, inflammation, antimicrobial host defense, wound healing, angiogenesis, tumor growth, and metastasis. Besides primary platelet count and function disorders, platelets also play a crucial role in many other common diseases, including coronary artery disease, stroke, peripheral vascular disease, and diabetes.

[0003] Normal coagulation function depends on the integrity of the blood vessel wall, platelets, coagulation factors, anticoagulant proteins, fibrinolytic system, and the physiological regulation and balance between them. Among them, platelets play an important role in the body's normal hemostasis and coagulation mechanism: after blood vessel injury, platelets rapidly aggregate to form a preliminary "platelet thrombus"; after platelet activation, they release various growth factors and chemical mediators (such as ADP, TXA2, etc.), which promote the activation of local coagulation factors and blood coagulation.

[0004] Platelets are key blood components involved in hemostasis and thrombosis, and their in vitro stability is crucial for clinical testing, transfusion therapy, and biomedical research. In vitro platelet self-activation has long been a technical challenge in the fields of blood sample collection, storage, and testing.

[0005] After platelets leave their physiological microenvironment, they spontaneously initiate an activation cascade reaction due to factors such as contact with non-biocompatible surfaces, limitations of anticoagulant action, and fluctuations in storage conditions. This process manifests as conformational changes in membrane surface receptors (such as GPⅡb / Ⅲa), release of α-granules and dense granule contents (ADP, thromboxane A2, PF4, etc.), and cytoskeleton remodeling. This process leads to:

[0006] 1. The reliability of laboratory test results is greatly reduced: Abnormal expression of activated platelet surface markers (such as CD62P and PAC-1) significantly interferes with the test results of flow cytometry, thromboelastography (TEG), and platelet aggregation rate (LTA), resulting in an increased false positive rate (studies show that the positive rate of CD62P in peripheral blood samples collected by traditional sodium citrate anticoagulation blood collection tubes can reach 25%-40%).

[0007] 2. Decreased efficacy of blood transfusion: Activated platelets undergo accelerated apoptosis during storage (PS eversion, loss of mitochondrial membrane potential), resulting in a shortened survival time in the body after transfusion (<48 hours, while the survival time of normal platelets in the body is about 7-10 days), thus reducing the clinical hemostatic effect.

[0008] 3. Potential clinical risks: Infusion of products containing activated platelets may trigger a cytokine storm (elevation of IL-6 and TNF-α) or microvascular thrombosis in recipients, posing a particular threat to critically ill patients.

[0009] In existing technologies, the mainstream anticoagulant in clinical practice is sodium citrate (3.2% concentration) blood collection tubes or ACD blood collection bags, which work by chelating free calcium ions (Ca). 2+ It blocks thrombin generation and platelet calcium-dependent activation pathways.

[0010] However, platelet autoactivation in vitro involves multiple non-calcium-dependent mechanisms, and sodium citrate can only inhibit calcium... 2 The initial activation phase is dependent on sodium citrate and does not block ADP release, TXA2 synthesis, or ROS accumulation. Studies have shown that in blood collection tubes treated with sodium citrate alone, platelets can still be activated via the aforementioned bypass pathway within 4 hours, with an activation rate exceeding 30%.

[0011] It is evident that existing anticoagulation vacuum blood collection tubes lack a comprehensive solution targeting the inhibition of multiple pathways for platelet activation in vitro.

[0012] To overcome the above-mentioned shortcomings, such anticoagulation vacuum blood collection tubes need to complete the sample processing and experimental analysis for platelet function and activation detection within 2 hours. Moreover, most existing technologies achieve platelet stability by permanently fixing platelets, but this approach is irreversible. Once platelets are fixed, they cannot be subjected to further in-depth studies such as in vitro platelet stimulation. Summary of the Invention

[0013] In order to overcome the above-mentioned defects of the prior art, the purpose of this invention is to provide a peripheral blood sample stabilizing solution that inhibits platelet activation through multi-target synergistic action, and its compound anticoagulant formulation and application.

[0014] To achieve the objectives of this invention, the following technical solution is adopted:

[0015] A peripheral blood sample stabilizing solution that inhibits platelet activation through multi-target synergistic action, the stabilizing solution being pre-packaged in vacuum blood collection tubes, and further comprising, by weight percentage, the following components.

[0016] 1%-4% trisodium citrate;

[0017] 0.3%-2% citric acid;

[0018] 1%-4% glucose;

[0019] 0.0002%-0.009% of prostaglandin E1;

[0020] 0.0002%-0.008% ibuprofen;

[0021] 0.0002%-0.006% ticagrelor;

[0022] 0.0002%-0.006% theophylline;

[0023] 0.0002%-0.005% ADP decomposing agent;

[0024] The remainder is water, and the sum of the proportions of the above components is 100%.

[0025] The volume ratio of the stabilizing solution to the blood sample is 1:9 to 1:19.

[0026] A compound anticoagulant formulation for peripheral blood sample stabilizing solution that inhibits platelet activation through multi-target synergistic action, comprising:

[0027] The stabilizer is pre-packaged in vacuum blood collection tubes;

[0028] At least one platelet antibody detection reagent, wherein the platelet antibody detection reagent is any one or more of CD41, CD42a, CD61, CD42b, and CD62P antibody reagents that are fluorescently labeled by staining with fluorescent dyes;

[0029] At least one fixative, said fixative being paraformaldehyde or glutaraldehyde at a concentration of 0.5-5%.

[0030] In a preferred embodiment of the present invention, the fluorescent dye is a fluorescent dye excited by laser light of at least one or more wavelengths selected from 405nm, 488nm, and 638nm.

[0031] In a preferred embodiment of the present invention, the concentration of the fixative, when it is paraformaldehyde, is 0.5-4%. Preferably, it is a 1% concentration of paraformaldehyde phosphate buffer.

[0032] In a preferred embodiment of the present invention, the concentration of the fixative, when it is glutaraldehyde, is 1-5%.

[0033] In a preferred embodiment of the present invention, the ADP decomposing agent is C 221 H 342 N 54 O 61 .

[0034] In a preferred embodiment of the present invention, the ratio of the total volume of the stabilizing fluid and the blood sample in a single vacuum blood collection tube to the total volume of the platelet antibody detection reagent used for the blood sample is 1:5 to 1:1.

[0035] In a preferred embodiment of the present invention, the ratio of the total volume of the stabilizing fluid in a single vacuum blood collection tube to the total volume of the blood sample to the total volume of the fixative used for the blood sample is 1:500-1:10.

[0036] An application of a compound anticoagulant formulation for stabilizing peripheral blood samples by inhibiting platelet activation through multi-target synergistic action, wherein the application is to protect platelets or reversibly inhibit platelet activation.

[0037] The protection of platelets or reversible inhibition of platelet activation specifically involves covering key pathways of platelet activation through multiple targets, including calcium signaling, ADP / TXA2, cAMP regulation, and energy metabolism pathways, forming a multi-level synergistic inhibitory network, significantly reducing in vitro platelet self-activation, and keeping platelets in the collected samples in or close to a resting state.

[0038] The beneficial effects of this invention are as follows:

[0039] This invention can protect platelets or reversibly inhibit platelet activation. On the one hand, it can prolong the detection time of platelet resting state, and on the other hand, it retains the functions of platelet activation, aggregation and adhesion, providing convenient conditions for the research and detection of in vitro platelet stimulation. When in vitro platelet stimulation experiments are required, it is only necessary to wash the whole blood sample once with phosphate buffer by centrifugation before the in vitro platelet stimulation experiment can be performed. Attached Figure Description

[0040] Figure 1 This is a schematic diagram of the structure of the ADP decomposition agent of the present invention.

[0041] Figure 2 This is a data analysis illustration of Embodiment 1 of the present invention. Figure 1 .

[0042] Figure 3 This is a data analysis illustration of Embodiment 1 of the present invention. Figure 2 .

[0043] Figure 4 This is a data analysis illustration of Embodiment 1 of the present invention. Figure 3 . Detailed Implementation

[0044] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. However, it should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of the invention. Furthermore, in the following descriptions, well-known structures and technologies are omitted to avoid unnecessarily obscuring the concept of the invention.

[0045] The main difficulties faced by existing technologies are:

[0046] Because platelets are highly activated after venipuncture and removal from the body, which is not conducive to platelet-related function testing, the laboratory industry standard stipulates that the first 2-3 ml of whole blood collected after venipuncture cannot be used for platelet testing, which has caused great inconvenience to the patients being tested and the blood collection personnel.

[0047] However, in the first 2-3 mL of whole blood, platelets are in a reversible semi-activated state. The formula of this invention can restore platelets in the semi-activated state to the resting state. Therefore, using the formula of this invention in conjunction with the sampling method can prevent the in vitro self-activation of peripheral blood samples, thereby allowing direct extraction of the patient's whole blood for the detection of platelet function or activation.

[0048] This invention can protect platelets or reversibly inhibit platelet activation. On the one hand, it can prolong the detection time of platelet resting state, and on the other hand, it retains the functions of platelet activation, aggregation and adhesion, providing convenient conditions for the research and detection of in vitro platelet stimulation. When in vitro platelet stimulation experiments are required, it is only necessary to wash the whole blood sample once with phosphate buffer by centrifugation before the in vitro platelet stimulation experiment can be performed.

[0049] This invention covers key pathways of platelet activation (calcium signaling, ADP / TXA2, cAMP regulation, and energy metabolism) through multiple targets, forming a multi-level synergistic inhibitory network. This allows platelets in collected whole blood samples to be in a near-resting state, achieving relative stability of clinical peripheral blood samples for platelet function and activation assessment within 10 hours (i.e., clinical samples processed within 10 hours yield reliable experimental results; currently used ACD blood collection tubes or sodium citrate vacuum blood collection tubes require platelet function-related biological indicators to be detected within 2 hours).

[0050] This invention effectively prevents platelet activation during the experimental reaction by adding a fixative at the beginning of the detection reaction.

[0051] The multi-target synergistic inhibition of platelet activation referred to in this invention means:

[0052] By covering key pathways of platelet activation (calcium signaling, ADP / TXA2, cAMP regulation, and energy metabolism) through multiple targets, a multi-level synergistic inhibitory network is formed, which significantly reduces platelet self-activation in vitro, so that platelets in the collected samples are in or close to a resting state.

[0053] The citrate buffer solution, composed of trisodium citrate and citric acid, is a blood anticoagulant. This citrate buffer solution possesses a unique anticoagulant effect, chelating calcium ions in the blood and rendering them inactive, thereby preventing the initiation of the coagulation process. This anticoagulant mechanism is reversible; by adjusting the calcium ion concentration, coagulation function can be restored, which is crucial in coagulation testing.

[0054] In this invention, glucose is used as a protectant for cell membranes in whole blood.

[0055] In this invention, prostaglandin E1 activates adenylate cyclase in platelets, increasing the concentration of cyclic adenosine monophosphate (cAMP), thereby inhibiting the release and aggregation of calcium ions in platelets while preserving the platelet aggregation function.

[0056] The ibuprofen used in this invention is a nonsteroidal anti-inflammatory drug (NSAID) with anti-inflammatory, analgesic, and antipyretic effects. Ibuprofen inhibits platelet activation and aggregation by inhibiting the activity of cyclooxygenase (COX) and reducing the synthesis of prostaglandins and thromboxane A2 (TXA2).

[0057] The ticagrelor in this invention rapidly and reversibly inhibits platelet activation and aggregation by directly inhibiting the platelet P2Y12 receptor.

[0058] The theophylline and ADP-degrading agent (scavadp) in this invention are non-selective phosphodiesterase (PDE) inhibitors that increase the level of intracellular cyclic adenosine monophosphate (cAMP) by inhibiting PDE activity.

[0059] Increased cAMP levels can inhibit platelet aggregation and activation. The molecular formula of the ADP-degrading agent (scavadp) is: C 221 H 342 N 54 O 61 Its structural formula is Figure 1 As shown.

[0060] The steps for using this invention are as follows:

[0061] Blood collection steps:

[0062] 1. Patient Confirmation: Verify the patient's identity information to ensure that the blood collection is accurate.

[0063] 2. Patient notification: Explain the blood collection process to the patient in detail and obtain their consent.

[0064] 3. Selecting a blood collection site: Choose a suitable blood collection site, usually the vein on the inside of the patient's upper arm.

[0065] 4. Handwashing: Medical staff must strictly follow hand hygiene requirements to wash their hands, or use hand sanitizer and alcohol for disinfection.

[0066] 5. Wear gloves: Wear disposable gloves to avoid cross-infection.

[0067] 6. Preparation site: Clean the blood collection site with an alcohol swab or sterile swab, wiping gently from the inside to the outside.

[0068] 7. Positioning: Use the thumb and forefinger of one hand to fix the blood collection site and keep the vein full.

[0069] 8. Puncture: Hold the lancet with your other hand and insert it into the vein at an angle of 30 degrees or less, being careful not to penetrate the vein. (Note: Do not squeeze the tubing of the lancet during the collection process to avoid artificially activating platelets.)

[0070] 9. Blood Sample Collection: While the blood is flowing back, connect the blood collection tube to the lancet and gently pull the lancet backward to allow blood to enter the blood collection tube. (After blood collection, gently shake to mix (invert 180 degrees, 3-4 times). Do not shake vigorously to avoid artificially activating platelets.)

[0071] 10. Seal the blood collection point: After blood collection, gently seal the blood collection point with a bandage or adhesive tape to prevent bleeding.

[0072] 11. Dispose of waste: Place used blood collection needles and tubes into a waste container.

[0073] 12. Handwashing and disinfection: After the procedure, medical staff must thoroughly disinfect their hands.

[0074] 13. Gently shake to mix within 1 minute after blood collection (invert 180 degrees, 3-4 times). Do not shake vigorously to avoid artificially activating platelets.

[0075] 14. Avoid vigorous shaking during the transport of blood collection tubes to the laboratory. Store them in a refrigerator at 2-8℃ for the shortest possible time.

[0076] Experimental steps:

[0077] Add 5 μL of platelet antibody detection reagent to each test tube and mix well;

[0078] Add 2-5 μL of whole blood sample mixed with stabilizer;

[0079] Add 50 μL of fixative and mix well;

[0080] Let the reaction stand at room temperature in the dark for 30 minutes;

[0081] Add another 1 mL of fixative and mix well;

[0082] On-machine testing and analysis.

[0083] Compared with traditional blood collection methods, the present invention is characterized by:

[0084] 1. Because platelets are highly activated after venous puncture, making detection difficult, reagent manufacturers stipulate that the first 2-3 ml of whole blood cannot be used for platelet testing, causing great inconvenience to both patients and blood collection personnel. However, in the first 2-3 ml of whole blood, platelets are in a reversible semi-activated state. The stabilizing solution in the blood collection tube of this invention can restore the semi-activated platelets to a resting state. Therefore, the blood collection tube of this invention can be used to directly draw whole blood from patients for platelet testing.

[0085] 2. This invention can protect platelets or reversibly inhibit platelet activation. On the one hand, it can prolong the detection time of platelets in the resting state, and on the other hand, it retains the functions of platelet activation, aggregation and adhesion, providing convenient conditions for the research and detection of in vitro platelet stimulation. When it is necessary to conduct in vitro platelet stimulation experiments, it is only necessary to wash the whole blood sample once with phosphate buffer by centrifugation before conducting in vitro platelet stimulation experiments.

[0086] 3. Adding a fixative at the beginning of the test reaction can effectively prevent platelet activation during the experimental reaction.

[0087] Example 1

[0088] The reagents used in this embodiment are as follows:

[0089] The selection of platelet antibody detection reagents is as follows:

[0090] The platelet activation marker CD62P antibody was stained with fluorescent dye, which was phycoerythrin PE.

[0091] The platelet marker CD42b antibody was obtained by fluorescent staining, with isothiocyanate (FITC) as the fluorescent dye.

[0092] The fixative is 1% paraformaldehyde phosphate buffer, which refers to a fixative solution prepared by dissolving 1g of paraformaldehyde in 100mL of phosphate buffer (PBS).

[0093] The stabilizer was obtained by the following preparation method:

[0094] Weigh out 22g of trisodium citrate, 8g of citric acid, 24.5g of glucose, 3.0mg of prostaglandin E1, 4.0mg of ibuprofen, 4.0mg of ticagrelor, 15mg of theophylline, and 40mg of ADP decomposition agent. Add 800mL of purified water and stir to dissolve. Then add water to make up to 1L and stir evenly to obtain crude sample stabilized solution. Adjust the pH to 7.4, which is similar to the pH of blood. Filter the crude sample stabilized solution through a filter membrane with a pore size of 0.44um and store at 2-8℃ to obtain the stabilized solution.

[0095] The application method of Embodiment 1 of the present invention is as follows:

[0096] (1) Pretreatment: Whole blood was obtained by venipuncture. Blood samples were collected using blood collection tubes containing the above-mentioned sample stabilizing solution. The volume ratio of whole blood to stabilizing solution was 9:1-19:1 to obtain a whole blood sample that could be stably preserved.

[0097] (2) After blood collection, gently invert and mix thoroughly within 1 minute (3-4 times). Avoid violent shaking during the transportation of the blood collection tube to the laboratory.

[0098] (3)(2) Detection: Add 5uL of platelet antibody detection reagent to each test tube and mix well;

[0099] (4) Add 2-5 μL of whole blood sample mixed with stabilizer;

[0100] Add 50 μL of fixative and mix well;

[0101] Let the reaction stand at room temperature in the dark for 30 minutes;

[0102] Add another 1 mL of fixative and mix well.

[0103] The test results will output a report showing the percentage of target platelets in the total platelet count.

[0104] The data analysis steps are as follows:

[0105] Using a Focytos TL08-0001 flow cytometer from Hunan Fuliusite Technology Co., Ltd., we first used an FSC / SSC scatter plot to circle the gating cells and platelets (see...). Figure 2 Then use a CD42b / SSC scatter plot (FITC channel) to circle CD42b+ platelets. Figure 3 Select CD42b+ platelets, set the CD62P / SSC scatter plot (PE channel), and thus obtain the platelet activation ratio. Figure 4 ).

[0106] Comparative Example 1

[0107] The results are basically the same as in Example 1, except that conventional commercially available ACD blood collection tubes are used. The blood collection tubes in Comparative Example 1 do not contain the sample stabilizing solution of Example 1 of this invention, and the detection method and other detection reagents are exactly the same as in Example 1.

[0108] The comparison data between Example 1 and Comparative Example 1 are shown in Table 1 below:

[0109]

[0110] According to the data in Table 1, blood samples from blood collection tubes using the composite anticoagulant formulation of this invention can be stably preserved for at least 10 hours at 2-8°C.

[0111] Example 2:

[0112] This is basically the same as Example 1, except for the difference in the formulation of the stabilizer:

[0113] The stabilized liquid of Example 2 was obtained by the following preparation method:

[0114] Weigh out 22g of trisodium citrate, 8g of citric acid, 24.5g of glucose, 20.0mg of prostaglandin E1, 10.0mg of ibuprofen, 4.0mg of ticagrelor, 20mg of theophylline, and 10mg of ADP decomposition agent. Add 800mL of purified water and stir to dissolve. Then add water to make up to 1L and stir evenly to obtain crude sample stabilized solution. Adjust the pH to 7.4, which is similar to the pH of blood. Filter the crude sample stabilized solution through a filter membrane with a pore size of 0.44um and store at 2-8℃ to obtain the stabilized solution of Example 2.

[0115] The data for Example 2 are shown in Table 2 below:

[0116] Table 2

[0117]

[0118] According to the data in Table 2, the blood sample in the blood collection tube of Example 2 can be stably preserved for at least 10 hours under the temperature conditions of 2-8℃.

[0119] Example 3:

[0120] The operation steps are basically the same as in Example 1, except that:

[0121] The platelet activation marker CD62P antibody was detected by fluorescent staining, with phycoerythrin-anthocyanin 7 (PE-Cy7) as the fluorescent dye.

[0122] The platelet marker CD42b antibody was obtained by fluorescent staining, with isothiocyanate (FITC) as the fluorescent dye.

[0123] The stabilized liquid of Example 3 was obtained by the following preparation method:

[0124] Weigh out 22g of trisodium citrate, 8g of citric acid, 24.5g of glucose, 150.0mg of prostaglandin E, 20.0mg of ibuprofen, 10.0mg of ticagrelor, 10mg of theophylline, and 5mg of ADP decomposition agent. Add 800mL of purified water and stir to dissolve. Then add water to make up to 1L and stir evenly to obtain crude sample stabilized solution. Adjust the pH to 7.4, which is similar to the pH of blood. Filter the crude sample stabilized solution through a filter membrane with a pore size of 0.44um and store at 2-8℃ to obtain the stabilized solution of Example 3.

[0125] The data for Example 3 are shown in Table 3 below:

[0126] Table 3

[0127]

[0128] According to the data in Table 3, the blood sample in the blood collection tube of Example 3 can be stably preserved for at least 10 hours under the temperature conditions of 2-8℃.

[0129] The foregoing has shown and described the basic principles and main features of the invention and the advantages of the invention.

[0130] Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the present invention. Various changes and modifications can be made to the present invention without departing from its spirit and scope. All such changes and modifications fall within the scope of the present invention as claimed, which is defined by the appended claims and their equivalents.

Claims

1. A peripheral blood sample stabilizing solution that inhibits platelet activation through multi-target synergistic action, characterized in that, The stabilizing solution is pre-packaged using vacuum blood collection tubes, and further comprises, by weight percentage, the following components. 1%-4% trisodium citrate; 0.3%-2% citric acid; 1%-4% glucose; 0.0002%-0.009% of prostaglandin E1; 0.0002%-0.008% ibuprofen; 0.0002%-0.006% ticagrelor; 0.0002%-0.006% theophylline; 0.0002%-0.005% ADP decomposing agent; The remainder is water, and the sum of the proportions of the above components is 100%. The volume ratio of the stabilizing solution to the blood sample is 1:9 to 1:

19.

2. A formulation of a peripheral blood sample stabilizing solution with a compound anticoagulant that inhibits platelet activation through multi-target synergistic action, comprising: The stabilizer pre-packaged via vacuum blood collection tube as described in claim 1; At least one platelet antibody detection reagent, wherein the platelet antibody detection reagent is any one or more of CD41, CD42a, CD61, CD42b, and CD62P antibody reagents that are fluorescently labeled by staining with fluorescent dyes; At least one fixative, said fixative being paraformaldehyde or glutaraldehyde at a concentration of 0.5-5%.

3. The formulation of a peripheral blood sample stabilizing solution with a compound anticoagulant that inhibits platelet activation through multi-target synergistic action as described in claim 2, characterized in that... The fluorescent dye is a fluorescent dye excited by laser light of at least one or more wavelengths selected from 405nm, 488nm, and 638nm.

4. The formulation of a peripheral blood sample stabilizing solution with a compound anticoagulant that inhibits platelet activation through multi-target synergistic action as described in claim 2, characterized in that... When the fixative is paraformaldehyde, the addition concentration is 0.5-4%.

5. The formulation of a peripheral blood sample stabilizing solution with a compound anticoagulant that inhibits platelet activation through multi-target synergistic action as described in claim 2, characterized in that, When the fixative is glutaraldehyde, the concentration is 1-5%.

6. The formulation of a peripheral blood sample stabilizing solution with a compound anticoagulant that inhibits platelet activation through multi-target synergistic action as described in claim 2, characterized in that, The ADP decomposing agent is C. 221 H 342 N 54 O 61 .

7. The formulation of a peripheral blood sample stabilizing solution with a compound anticoagulant that inhibits platelet activation through multi-target synergistic action as described in claim 2, characterized in that, The ratio of the total volume of the stabilizing fluid and the blood sample in a single vacuum blood collection tube to the total volume of the platelet antibody detection reagent used for that blood sample is 1:5 to 1:

1.

8. The formulation of a peripheral blood sample stabilizing solution with a compound anticoagulant that inhibits platelet activation through multi-target synergistic action as described in claim 2, characterized in that, The ratio of the total volume of the stabilizing fluid in a single vacuum blood collection tube to the total volume of the blood sample to the total volume of the fixative used for that blood sample is 1:500-1:

10.

9. The application of a peripheral blood sample stabilizing solution compound anticoagulant formulation that inhibits platelet activation through multi-target synergistic action as described in any one of claims 1-8, characterized in that, The application is to protect platelets or reversibly inhibit platelet activation.

10. The application of the peripheral blood sample stabilizing solution compound anticoagulant formulation that inhibits platelet activation through multi-target synergistic action as described in claim 9, characterized in that... The protection of platelets or reversible inhibition of platelet activation specifically involves covering key pathways of platelet activation through multiple targets, including calcium signaling, ADP / TXA2, cAMP regulation, and energy metabolism pathways, forming a multi-level synergistic inhibitory network, significantly reducing in vitro platelet self-activation, and keeping platelets in the collected samples in or close to a resting state.

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