A detection system, a detection method and a detection kit for detecting platelet function subgroup PAC-1 marker, Annexin-V marker and CD62P marker

Abstract

The present invention provides a detection system for detecting three markers on platelets in whole blood, including: a buffer, a calcium concentrate and an agonist combination; the buffer contains: 130-140mM NaCl, 2.5-4mM KCl, 0.5-2mM MgCl2, 5-12mM HEPES, 5-12mM NaHCO3, 0.1-0.5mM Na2HPO4, 0.1%-0.4% w / v human serum albumin, 2-8mM glucose, pH 7.35-7.45, the agonist contains 1-2mM ADP and 0.1-1mM TRAP6, and the calcium concentrate contains 50-100mM calcium ions. A method for detecting three markers and a kit design are also provided. The buffer provides a physiological condition more similar to plasma, and three markers can be detected simultaneously.

Description

Technical Field

[0001] The present invention belongs to the field of platelet function quality detection in clinical testing and cell therapy production, and particularly relates to a detection system, a detection method and a detection kit for detecting PAC-1 markers, Annexin-V markers and CD62P markers on platelets. Background Art

[0002] Platelets are important cellular components in the blood that mediate hemostasis. Platelets are formed by the lysis of megakaryocyte cytoplasm in the bone marrow. After entering the blood circulation, they continue to mature and lyse to form mature platelets [1]. When bleeding occurs, platelets are activated and interact with the coagulation system to form physiological thrombi to stop bleeding [2]. However, various causes such as hematological malignancies, autoimmune diseases, radiotherapy and chemotherapy affect bone marrow function, peripheral platelet status and platelet clearance, thereby causing thrombocytopenia and bleeding tendency [3, 4]. Therefore, when the indications and transfusion criteria are met, platelet transfusion is an important alternative treatment for supplementing platelets in the body [4-6]. Therefore, platelet function detection methods are very important. They can not only be used to detect the function of transfused platelets, but also provide a basis for the diagnosis and treatment of platelet diseases.

[0003] After activation, platelets undergo three functional changes: 1. aggregation, 2. coagulation, and 3. secretion [2, 7], thereby forming platelet thrombi and promoting hemostasis. Platelet aggregation is determined by the expression level of PAC-1 before and after platelet activation, the coagulation function is determined by the binding of Annexin-V before and after platelet activation, and the secretion function is evaluated by the expression level of CD62P before and after platelet activation [7-11]. The current platelet function test process can be found in Figure 1 In brief, blood is drawn from the human body and stored, and diluted 10 times using a detection buffer (Modified HEPES-Tyrode buffer), and the activators and other additives required for the test are added. After platelet activation is completed, the platelets are fixed, and marker analysis and counting are performed using a flow cytometer [8-10].

[0004] However, the current three-marker detection has the following shortcomings:

[0005] 1. The detection method is defective: As can be seen from Table 4, the three markers have their own detection methods and are not related [8, 9, 11], and cannot reflect platelet function under physiological conditions;

[0006] 2. The detection buffer also has deficiencies: The detection buffer (Modified HEPES-Tyrode Buffer) formula is shown in Table 5, which has the following deficiencies:

[0007] 1. Potassium ion concentration, bicarbonate and calcium ion concentration are different from the plasma electrolyte components under physiological conditions, which may affect the platelet function status;

[0008] 2. HEPES content is high. HEPES is often used in the preparation of artificial tissue buffers because it prevents cell edema and has a strong buffering function. However, it is not a buffering substance naturally present in blood, so its dosage needs to be reduced;

[0009] 3. The colloidal component is bovine serum albumin, which is a heterologous protein component. The heterologous antibodies in it may affect the functional status of platelets.

[0010] The existence of these deficiencies and deviations will affect the accuracy of the test results and cannot reflect the platelet function under normal physiological conditions. Therefore, there is an urgent need in the prior art for a platelet function test system that has correlation and simulates the physiological environment to achieve simple, fast and accurate testing. Summary of the invention

[0011] In view of the shortcomings of the prior art described above, the object of the present invention is to provide a detection system, method and kit for detecting PAC-1 markers, Annexin-V markers and CD62P markers on platelets, as well as a detection buffer having electrolyte components and colloid components similar to those of plasma, so as to solve the problems in the prior art.

[0012] To achieve the above-mentioned purpose and other related purposes, the present invention provides a detection system, method and kit for detecting the PAC-1 marker on platelets, a detection system, method and kit for detecting Annexin-V and CD62P markers on platelets, and a detection system, method and kit for detecting the PAC-1 marker, Annexin-V marker and CD62P marker on platelets.

[0013] The first aspect of the present invention provides a detection system for detecting PAC-1 markers on platelets, the detection system comprising the following components: a buffer, a calcium concentrate and an agonist; the buffer comprises: 130-140mM NaCl, 2.5-4mM KCl, 0.5-2mM MgCl2, 5-12mM HEPES, 5-12mM NaHCO3, 0.1-0.5mM Na2HPO4, 0.1%-0.4% w / v human serum albumin, 2-8mM glucose, pH 7.35-7.45; the calcium concentrate comprises 50-100mM calcium ions; the agonist comprises 1-2mM ADP and 0.1-1mM TRAP6. The NaCl content in the buffer is selected from 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140mM.

[0014] The KCl content is selected from 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0 mM.

[0015] The MgCl2 content is selected from 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, and 2mM.

[0016] The HEPES content is selected from 5, 6, 7, 8, 9, 10, 11, 12 mM.

[0017] The NaHCO3 content is selected from 5, 6, 7, 8, 9, 10, 11, 12 mM.

[0018] The Na2HPO4 content is selected from 0.1, 0.2, 0.3, 0.4, and 0.5 mM.

[0019] The human serum albumin content is selected from 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4% w / v.

[0020] The glucose content is selected from 2, 3, 4, 5, 6, 7, 8 mM.

[0021] The pH is selected from 7.35, 7.36, 7.37, 7.38, 7.39, 7.40, 7.41, 7.42, 7.43, 7.44, 7.45.

[0022] The ADP content is selected from 1.0, 1.01, 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, 1.1, 1.11, 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, 1.18, 1.19, 1.2, 1.21, 1.22, 1.23, 1 .24, 1.25, 1.26, 1.27, 1.28, 1.29, 1.3, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, 1.4, 1.41, 1.42, 1.43, 1.44, 1.45, 1.46, 1.47, 1.48, 1.49 , 1.5, 1.51, 1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.6, 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.7, 1.71, 1.72, 1.73, 1.74, 1.7 5. 1.76, 1.77, 1.78, 1.79, 1.8, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1. 88, 1.89, 1.9, 1.91, 1.92, 1.93, 1.94, 1.95, 1.96, 1.97, 1.98, 1.99, 2.0mM.

[0023] TRAP6 content is selected from 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.51, 0.52, 0.53, 0.54 , 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, 1.0mM.

[0024] The calcium concentrate content is selected from 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 mM.

[0025] The PAC-1 marker is a platelet membrane glycoprotein IIb / IIIa complex, which contains specific sites for binding to adhesion proteins such as fibrinogen (Fg), fibronectin (Fn), and vWF factor. Under normal circumstances, it exists in a monomeric form and has no ability to bind to ligands. When platelets are activated, the conformation changes, and the receptors on the PAC-1 molecules are exposed. It specifically binds to a variety of adhesion proteins, accelerates platelet fibrinogen and platelet aggregation, and is the final common pathway for causing platelet thrombosis.

[0026] Annexin V is widely used in biomedical research due to its ability to bind to phosphatidylserine. Phosphatidylserine is a phospholipid that is usually located in the inner layer of the cell membrane. In some activated platelets, phosphatidylserine moves to the outer layer of the cell membrane. Annexin V can recognize and bind to the phosphatidylserine exposed on the surface, becoming one of the markers for detecting procoagulant platelets.

[0027] The CD62P marker is a membrane glycoprotein located in the special granules of platelets and the weibe1-palade bodies of vascular endothelial cells. Under normal circumstances, CD62P is only expressed in small amounts on the surface of platelets. When platelets are stimulated, they are rapidly and massively expressed on the surface of platelet membranes. Through the action of the N-terminal agglutinin domain, it mediates the adhesion function of activated endothelial cells with monocytes, neutrophils, etc. These cells not only promote fibrin deposition, but also participate in inflammatory reactions and thrombus formation.

[0028] ADP, also known as adenosine diphosphate or adenosine diphosphate, is a very important platelet activating substance in the body and plays an important role in the function of platelets. This is mainly because ADP is usually present in high-density granules in platelet cells. At the same time, there is ATPase on platelets, and ADP can inhibit ATPase activity. After being released by platelets, ADP can participate in inducing platelet aggregation, thereby promoting hemostasis.

[0029] TRAP6 is a peptide fragment of protease-activated receptor 1 (42-47 residues) and a PAR1 hexapeptide agonist. TRAP6 can stimulate the transient mobilization of free intracellular calcium, platelet aggregation (EC50 = 0.8 μM) and rapidly phosphorylate PDE3A.

[0030] As certain embodiments of the present invention, the buffer comprises: the buffer comprises: 137 mM NaCl, 4 mM KCl, 1 mM MgCl2, 5 mM HEPES, 10 mM NaHCO3, 0.3 mM Na2HPO4, 0.35% w / v human serum albumin, 5.5 mM glucose, pH 7.4;

[0031] The agonist contained 1.34 mM ADP and 0.54 mM TRAP6;

[0032] The calcium concentrate contains 100 mM calcium ions.

[0033] As certain embodiments of the present invention, the detection system further comprises Pacific Blue anti-human CD41 PerCP anti-human CD42b As certain embodiments of the present invention, the anti-PAC-1 marker antibody is selected from FITC anti-human PAC-1 or FITC anti-human PAC-1

[0034] As certain embodiments of the present invention, the detection system further comprises a detection device or a detection reagent for detecting a combination of the expressed PAC-1 marker and an antibody against the PAC-1 marker. As certain embodiments of the present invention, the detection device comprises a flow cytometer, and the detection reagent is a fluorescent detection reagent. As certain embodiments of the present invention, the calcium ion is derived from calcium chloride or calcium gluconate.

[0035] The present invention also provides a method for detecting the PAC-1 marker on platelets using the detection system, the detection method comprising the following steps: (1) diluting the sample to be detected with a buffer; (2) mixing the sample diluted in step (1) with an agonist and an antibody against the PAC-1 marker, incubating, and quantitatively detecting; (3) mixing the sample diluted in step (1) with a calcium concentrate, an agonist, and an antibody against the PAC-1 marker, incubating, and quantitatively detecting; and (4) calculating the ratio of the detection value in step (3) to the detection value in step (2).

[0036] As certain embodiments of the present invention, the sample to be detected in step (1) is diluted 10 to 40 times with a buffer. As certain embodiments of the present invention, the sample to be detected in step (1) is diluted 20 times with a buffer.

[0037] As certain embodiments of the present invention, the incubation conditions in steps (2) and (3) are incubation at room temperature for 15-30 minutes. As certain embodiments of the present invention, the incubation conditions in steps (2) and (3) are incubation at room temperature for 20 minutes.

[0038] As certain embodiments of the present invention, the volume ratio of the diluted sample, agonist, and antibody in step (2) is 50:10:6.

[0039] As certain embodiments of the present invention, the volume ratio of the diluted sample, agonist, antibody and calcium concentrate in step (3) is 50:10:6:0.3-1. The present invention also provides a detection kit for detecting PAC-1 marker on platelets, and the detection kit includes the components.

[0040] The present invention also provides the use of the detection system or the detection kit in preparing a detection reagent for detecting the PAC-1 marker on platelets.

[0041] Another aspect of the present invention is to provide a detection system for detecting Annexin-V and CD62P markers on platelets, the detection system comprising the following components: a buffer, a calcium concentrate and an agonist; the buffer comprises: 130-140mM NaCl, 2.5-4mM KCl, 0.5-2mM MgCl2, 5-12mM HEPES, 5-12mM NaHCO3, 0.1-0.5mMNa2HPO4, 0.1%-0.4% w / v human serum albumin, 2-8mM glucose, pH 7.35-7.45; the calcium concentrate comprises 50-100mM calcium ions; the agonist comprises 1-2mM ADP and 0.1-1mM TRAP6.

[0042] As certain embodiments of the present invention, the buffer comprises: 137mM NaCl, 4mM KCl, 1mM MgCl2, 5mM HEPES, 10mM NaHCO3, 0.3mM Na2HPO4, 0.35% w / v human serum albumin, 5.5mM glucose, pH 7.4; the agonist comprises 1.34mM ADP and 0.54mM TRAP6; and the calcium concentrate comprises 100mM calcium ions.

[0043] As certain embodiments of the present invention, the detection system further comprises Pacific Blue anti-human CD41 PerCP anti-human CD42b As well as antibodies against Annexin-V marker and antibodies against CD62P marker.

[0044] As certain embodiments of the present invention, the antibody against Annexin-V marker is selected from PE / Cy7-Annexin V

[0045] As certain embodiments of the present invention, the anti-CD62P marker antibody is selected from APC anti-humanCD42b

[0046] As certain embodiments of the present invention, the detection system also includes a detection device or a detection reagent for detecting a combination of the expressed Annexin-V marker and an antibody against the Annexin-V marker, and a detection device or a detection reagent for detecting a combination of the expressed CD62P marker and an antibody against the CD62P marker.

[0047] As some embodiments of the present invention, the detection device comprises a flow cytometer, and the detection reagent comprises a fluorescent detection reagent. As some embodiments of the present invention, the calcium ions are derived from calcium chloride or calcium gluconate.

[0048] The present invention also provides a method for detecting Annexin-V markers and CD62P markers on platelets using the detection system, the detection method comprising the following steps: (1) diluting the sample to be detected with the buffer; (2) mixing the sample diluted in step (1) with a calcium concentrate, an agonist, and an antibody against the Annexin-V marker, incubating, and quantitatively detecting; (3) mixing the sample diluted in step (1) with a calcium concentrate, an agonist, and an antibody against the CD62P marker, incubating, and quantitatively detecting.

[0049] As certain embodiments of the present invention, the sample to be detected in step (1) is diluted 10 to 40 times with a buffer. As certain embodiments of the present invention, the sample to be detected in step (1) is diluted 20 times with a buffer. As certain embodiments of the present invention, the incubation conditions in steps (2) and (3) are incubation at room temperature for 15-30 minutes. As certain embodiments of the present invention, the incubation conditions in steps (2) and (3) are incubation at room temperature for 20 minutes.

[0050] As certain embodiments of the present invention, the volume ratio of the diluted sample, agonist, antibody, and calcium concentrate in steps (2) and (3) is 50:10:5-6:1. The present invention also provides a method for simultaneously detecting Annexin-V markers and CD62P markers on platelets, wherein the method is used to simultaneously detect Annexin-V markers and CD62P markers, and the detection device is a flow cytometer.

[0051] The present invention also provides a detection kit for detecting Annexin-V markers and CD62P markers on platelets, and the detection kit includes the components.

[0052] The present invention also provides the use of the detection system or the detection kit in preparing a detection reagent for detecting Annexin-V markers and CD62P markers on platelets.

[0053] Another aspect of the present invention is to provide a detection system for detecting PAC-1 markers, Annexin-V markers and CD62P markers on platelets, the detection system comprising the following components: a buffer, a calcium concentrate and an agonist; the buffer comprises: 130-140mM NaCl, 2.5-4mM KCl, 0.5-2mM MgCl2, 5-12mM HEPES, 5-12mMNaHCO3, 0.1-0.5mM Na2HPO4, 0.1%-0.4% w / v human serum albumin, 2-8mM glucose, pH 7.35-7.45; the calcium concentrate comprises 50-100mM calcium ions; the agonist comprises 1-2mM ADP and 0.1-1mM TRAP6.

[0054] Preferably, the buffer comprises: 137 mM NaCl, 4 mM KCl, 1 mM MgCl2, 5 mM HEPES, 10 mM NaHCO3, 0.3 mM Na2HPO4, 0.35% w / v human serum albumin, 5.5 mM glucose, pH 7.4. The agonist comprises 1.34 mM ADP and 0.54 mM TRAP6; and the calcium concentrate comprises 100 mM calcium ions.

[0055] As certain embodiments of the present invention, the detection system further comprises Pacific Blue anti-human CD41 PerCP anti-human CD42b As well as antibodies against PAC-1 marker, antibodies against Annexin-V marker and antibodies against CD62P marker.

[0056] As certain embodiments of the present invention, the anti-PAC-1 marker antibody is selected from FITC anti-humanPAC-1 or FITC anti-human PAC-1

[0057] As certain embodiments of the present invention, the antibody against Annexin-V marker is selected from PE / Cy7-Annexin V

[0058] As certain embodiments of the present invention, the anti-CD62P marker antibody is selected from APC anti-humanCD62P

[0059] As certain embodiments of the present invention, the detection system also includes a detection device or a detection reagent for detecting a combination of the expressed PAC-1 marker and an antibody against the PAC-1 marker, a detection device or a detection reagent for detecting a combination of the expressed Annexin-V marker and an antibody against the Annexin-V marker, and a detection device or a detection reagent for detecting a combination of the expressed CD62P marker and an antibody against the CD62P marker.

[0060] As certain embodiments of the present invention, the detection device comprises a flow cytometer, and the detection reagent comprises a fluorescent detection reagent.

[0061] As certain embodiments of the present invention, the calcium ions are derived from calcium chloride or calcium gluconate.

[0062] The present invention also provides a method for detecting PAC-1 marker, Annexin-V marker and CD62P marker on platelets using the detection system, the detection method comprising the following steps: (1) diluting the sample to be detected with a buffer solution; (2) mixing the sample diluted in step (1) with an agonist and an antibody against the PAC-1 marker, incubating, and quantitatively detecting; (3) mixing the sample diluted in step (1) with a calcium concentrate, an agonist, and an antibody against the PAC-1 marker, incubating, and quantitatively detecting; (4) calculating the ratio of the detection value in step (3) to the detection value in step (2) to detect the PAC-1 marker; (5) mixing the sample diluted in step (1) with a calcium concentrate, an agonist, and an antibody against the Annexin-V marker, incubating, and quantitatively detecting to detect the Annexin-V marker; and (6) mixing the sample diluted in step (1) with a calcium concentrate, an agonist, and an antibody against the CD62P marker, incubating, and quantitatively detecting to detect the CD62P marker.

[0063] As certain embodiments of the present invention, the sample to be detected in step (1) is diluted 10 to 40 times with a buffer. As certain embodiments of the present invention, the sample to be detected in step (1) is diluted 20 times with a buffer.

[0064] As certain embodiments of the present invention, the incubation conditions in step (2), step (3), step (5) and step (6) are incubation at room temperature for 15-30 minutes. As certain embodiments of the present invention, the incubation conditions in step (2), step (3), step (5) and step (6) are incubation at room temperature for 20 minutes.

[0065] As certain embodiments of the present invention, the volume ratio of the diluted sample, agonist, and antibody in step (2) is 50:10:6.

[0066] As certain embodiments of the present invention, the volume ratio of the diluted sample, agonist, antibody, and calcium concentrate in steps (3), (5), and (6) is 50:10:5-6:1.

[0067] The present invention also provides a method for simultaneously detecting PAC-1 marker, Annexin-V marker and CD62P marker on platelets. The method is used to simultaneously detect PAC-1 marker, Annexin-V marker and CD62P marker, and the detection equipment is selected from flow cytometer.

[0068] The present invention also provides a detection kit for detecting PAC-1 marker, Annexin-V marker and CD62P marker on platelets, and the detection kit includes the above components. The present invention also provides the use of the above detection system or the above detection kit in preparing a detection reagent for detecting PAC-1 marker, Annexin-V marker and CD62P marker on platelets.

[0069] Another invention of the present invention is to provide a buffer for detecting PAC-1 markers, Annexin-V markers and CD62P markers on platelets, the buffer comprising: 130-140mM NaCl, 2.5-4mM KCl, 0.5-2mM MgCl2, 5-12mM HEPES, 5-12mM NaHCO3, 0.1-0.5mM Na2HPO4, 0.1%-0.4% w / v human serum albumin, 2-8mM glucose, pH 7.35-7.45. The agonist comprises 1-2mM ADP and 0.1-1mM TRAP6; the buffer does not contain calcium ions or also contains 1.5mM calcium ions.

[0070] As certain embodiments of the present invention, the buffer comprises: 137 mM NaCl, 4 mM KCl, 1 mM MgCl2, 5 mM HEPES, 10 mM NaHCO3, 0.3 mM Na2HPO4, 0.35% w / v human serum albumin, 5.5 mM glucose, pH 7.4. The agonist comprises 1.34 mM ADP and 0.54 mM TRAP6; the buffer does not contain calcium ions or further contains 1.5 mM calcium ions.

[0071] As described above, the detection system, method and kit for detecting the PAC-1 marker on platelets, the detection system, method and kit for detecting Annexin-V and CD62P markers on platelets, and the detection system, method and kit for detecting the PAC-1 marker, Annexin-V marker and CD62P marker on platelets of the present invention have the following characteristics:

[0072] Beneficial effects:

[0073] Provides a platelet function test method that is relevant and simulates physiological environments to evaluate platelet function in vivo;

[0074] Provides a buffer with electrolyte and colloid components similar to plasma;

[0075] The buffer provided can detect three markers: PAC-1, Annexin-V, and CD62P;

[0076] A method for simultaneously detecting PAC-1 markers, Annexin-V markers and CD62P markers in samples is provided, which can quickly and efficiently detect the three markers;

[0077] A basic buffer and calcium concentrate are provided to enable detection of three markers. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 The current platelet activation state detection workflow is shown in the prior art;

[0079] Figure 2 The optimized platelet activation state detection process established for the present invention is shown. The innovative points have been highlighted in red;

[0080] Figure 3 The display shows the detection design of platelet activation state of the present invention. In the figure, the calcium ion concentration changes with the change of the detection purpose; WB is whole blood, PLT is platelet;

[0081] Figure 4The present invention shows the average levels of PAC-1, Annexin-V and CD62P after activation in HT buffer 1 and HT buffer 2 in the range of calcium ion concentration from 0 mM to 1.5 mM;

[0082] Figure 5 Shown are the average levels of PAC-1 after activation in HT buffer 1 (HT1) and HT buffer 2 (HT2) of the present invention;

[0083] Figure 6 Shown are the levels of Annexin-V after activation in HT buffer 1 (HT1) and HT buffer 2 (HT2) of the present invention;

[0084] Figure 7 Shown are the CD62P levels of the present invention after activation in HT buffer 1 (HT1) and HT buffer 2 (HT2);

[0085] Figure 8 Shown are the normalized levels of PAC-1 and Annexin-V after activation of the present invention in HT buffer 1 (HT1) and HT buffer 2 (HT2). DETAILED DESCRIPTION

[0086] The present invention is further described below in conjunction with specific examples, and it should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0087] It should be understood that the terms used in the examples of the present invention are intended to describe specific embodiments rather than to limit the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated, the singular forms "a", "an" and "the" include plural forms. When the examples give numerical ranges, it should be understood that unless otherwise specified in the present invention, both endpoints of each numerical range and any value between the two endpoints can be selected.

[0088] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, any methods, equipment, and materials of the prior art that are similar or equivalent to the methods, equipment, and materials in the embodiments of the present invention may be used to implement the present invention, based on the prior art mastery of those skilled in the art and the description of the present invention.

[0089] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in the technical field and related fields. DETAILED DESCRIPTION OF THE INVENTION

[0091] The platelet activation function detection process developed by the present invention is as follows Figure 2 As shown. With the ethical approval of the cooperating hospital, blood was collected using blood collection tubes. The blood collection tube was pre-filled with ACD-A anticoagulant, 15 ml of fresh venous blood was collected, and the HEPES-Tyrode buffer developed by the present invention was used as a buffer (the formula is shown in Table 1) to dilute the whole blood sample 20 times. After that, the detection buffer developed by the present invention was used to prepare the activation reaction system, and the HEPES-Tyrode buffer (HT2, each component is shown in Table 1) was used as the detection buffer or as the main component of the detection buffer:

[0092] Activation reaction system = diluted blood sample + activator + antibody + calcium concentrate

[0093] The volumes of diluted blood sample, activator, antibody, and calcium concentrate are 50 μL, 10 μL, and the volume of antibody is added according to Table 2, 0-1 μL. The activator is used in combination with ADP and TRAP6, and calcium chloride is added to the activation reaction system to adjust the calcium ion concentration to between 0 mM and 1.5 mM for testing. The activation and antibody (primary antibody) incubation are carried out simultaneously at room temperature for 20 minutes, and then HBS buffer is added for direct quantitative analysis without fixation, and flow cytometry is used for counting and analysis, such as Figure 3 shown.

[0094] Table 1 Composition of the optimized HEPES-Tyrode buffer (HT2) of the present invention

[0095] Components Concentration (mM) NaCl 137 KCl 4 <![CDATA[MgCl2]]> 1 HEPES 5 <![CDATA[NaHCO3]]> 10 <![CDATA[Na2HPO4]]> 0.3 Human serum albumin 0.35%(w / v) glucose 5.5 pH 7.4

[0096] The antibodies tested and their concentrations are shown in Table 2.

[0097] Table 2 Antibodies used in the assay

[0098] Antibody Name Manufacturer Volume (μL) Working concentration (μg / mL) Pacific Blue anti-human CD41 Biolegend 2 2.5 PerCP anti-human CD42b Biolegend 2 12 FITC anti-human PAC-1 Invitrogen 0.5 NA FITC anti-human PAC-1 Biolegend 2 3 PE / Cy7-Annexin V Biolegend 1 0.2 APC anti-human CD62P Biolegend 2 6

[0099] The technology of the present invention aims to solve the above-mentioned deficiencies of the prior art to improve the quality of platelet function testing, and mainly overcomes two problems:

[0100] Provides a platelet function test method that is relevant and simulates physiological environment to evaluate platelet function in vivo;

[0101] Provides assay buffers with electrolyte and colloid compositions similar to plasma.

[0102] Based on the above experimental design and experimental results, this patent is expected to develop a platelet activation function detection kit.

[0103] Table 3 Components of platelet activation state detection kit

[0104]

[0105] The kit includes a buffer, a platelet activator, and a calcium ion concentrate, as shown in Table 3. After the blood is collected through a blood collection tube, the blood sample is diluted 20 times with a buffer. The diluted blood sample is then added to different marker buffers and a platelet activator and a corresponding marker antibody (not included in the kit) and incubated at room temperature for 20 minutes before flow cytometry detection and analysis.

[0106] Example 1

[0107] For the convenience of comparison, the conventional Modified HEPES-Tyrode buffer (referred to as HT1 in the specification, and its composition and concentration are shown in Table 5) is used as a control, and the optimized buffer developed by the present invention, HEPES-Tyrode buffer, is referred to as HT2 in the specification (its composition and concentration are shown in Table 1). Table 4 shows the detection system of the three existing markers PAC-1, Annexin-V and CD62P. The following detection test results are detailed for the three markers:

[0108] Table 4 Current methods for detecting platelet activation markers[8,9,11]

[0109]

[0110] Table 5 Composition of existing Modified HEPES-Tyrode buffer (HT1)

[0111]

[0112]

[0113] 1. Determination of the detection method of the present invention

[0114] PAC-1

[0115] PAC-1 is used to detect the conformational changes of αIIbβIII after platelet activation and to evaluate the aggregation function of platelets. Figure 4 and Figure 5As shown, the expression level of PAC-1 is affected by the calcium ion level in the buffer, and the level of PAC-1 activated in platelets is the highest in the environment of 0mM calcium (calcium ion concentration in the traditional ModifiedHT buffer). When using HT1, the expression ratio (%) of PAC-1 at calcium ion concentrations of 0mM, 0.5mM, 1mM, and 1.5mM was 54.6±20.8%, 38.5±17.1%, 35.5±17.4% and 33.9±17%, respectively, and in HT2 at calcium ion concentrations of 0mM, 0.5mM, 1mM, and 1.5mM was 56.2±22.5%, 39.1±17.1%, 37.2±18.3% and 36.2±17.1%, respectively.

[0116] In HT1 and HT2, there was a significant difference in PAC-1 expression between the 0 mM calcium group and the 1.5 mM calcium group (p<0.05, **). Figure 8 However, considering that the environment of platelets is blood and 0 mM calcium ion is a non-physiological state, the ratio of 1.5 mM calcium ion PAC-1% to 0 mM calcium ion PAC-1% is proposed, namely PAC-1@[1.5Ca 2+ ] / PAC-1@[0Ca 2+ ] to comprehensively evaluate the expression of PAC-1 in the reserve state and the near-physiological state, rather than just reflecting the functional state under a single test. This ratio was 0.62 in HT1 and 0.64 in HT2.

[0117] Annexin-V

[0118] Annexin-V is used to detect phosphatidylserine after platelet activation. The expression of Annexin-V is related to the calcium ion level. Figure 4 , Figure 6 As shown, the expression level of Annexin-V is affected by the calcium ion level in the buffer, and the level of Annexin-V activated platelets is the highest in a high calcium environment. Using HT1, the expression ratio (%) of Annexin-V at calcium ion concentrations of 0mM, 0.5mM, 1mM, and 1.5mM was 14.8±1.3%, 19.6±4%, 18.1±2.1%, and 21.3±3.4%, respectively, and in HT2 at calcium ion concentrations of 0mM, 0.5mM, 1mM, and 1.5mM was 13.8±1%, 18±2%, 18.7±3.3%, and 20.6±1.5%, respectively.

[0119] In HT1, there was a significant difference in the expression of Annexin-V between the 0 mM calcium group and the 1.5 mM calcium group (p<0.05, *), and in HT2, there was a significant difference in the expression of Annexin-V between the 0 mM calcium group and the 1.5 mM calcium group (p<0.05, ***), see Figure 8 However, considering that platelets are located in blood and 0 mM calcium ions are a non-physiological state, the detection of Annexin-V needs to be performed at a calcium ion concentration of 1.5 mM, which is close to the physiological state.

[0120] CD62P

[0121] CD62P is expressed on platelet granule vesicles. When platelets are activated, the granule vesicles fuse with the cell membrane to release active substances. Therefore, the expression level of CD62P before and after platelet activation is used to evaluate platelet secretory function. Figure 4 , Figure 7 As shown, the expression level of CD62P is affected by the calcium ion level in the buffer, and the level of activated platelet CD62P is lowest under the 0mM calcium environment. Using HT1, the CD62P expression ratio (%) at calcium ion concentrations of 0mM, 0.5mM, 1mM, and 1.5mM was 87.1±4%, 89.6±2.2%, 90.2±0.2% and 89.9±0.1%, respectively, and in HT2 at calcium ion concentrations of 0mM, 0.5mM, 1mM, and 1.5mM was 88.7±2%, 90.5±1.1%, 90.9±0.3% and 90.9±0.3%, respectively.

[0122] However, considering that the environment of platelets is blood and 0 mM calcium ions is a non-physiological state, the detection of CD62P needs to be carried out at a calcium ion concentration of 1.5 mM, which is close to the physiological state.

[0123] 2. Comparison of the detection effects of the prior art HT1 activation system and the present invention HT2 activation system

[0124] HT1 and HT2 were evaluated based on the expression levels of PAC-1, Annexin-V and CD62P. Figure 4 , Figure 5 The relationship between the expression trend of CD62P and PAC-1 and calcium ions is less related to the HT type. However, the expression trend of Annexin-V in HT1 is more variable and oscillating, while in HT2, the trend is linear and gentle. In order to detect the three in the same system, the present invention selects HT2 buffer as the test buffer according to the test results. The detection system of the three activation markers is shown in Table 6.

[0125] Table 6 Detection systems for PAC-1, Annexin-V and CD62P

[0126] Markers Buffer Agonists Calcium ion concentration (mM) PAC-1 HT2 ADP+TRAP 6 0，1.5 Annexin-V HT2 ADP+TRAP 6 1.5 CD62P HT2 ADP+TRAP 6 1.5

[0127] Based on the above results, the method of the present invention is compared with the existing traditional Modified HT buffer (HT1) and activation system. By improving the buffer composition to make it closer to the physiological state and setting different calcium ion concentrations in the activation reaction system, the expression of PAC-1, Annexin-V and CD62P can be detected respectively, so as to facilitate the evaluation of platelet aggregation, coagulation and secretion function.

[0128] The above is only a preferred embodiment of the present invention, and is not any formal or substantial limitation of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention. Any technician familiar with this profession, without departing from the spirit and scope of the present invention, can make some changes, modifications and evolutions of the technical content disclosed above, which are equivalent embodiments of the present invention; at the same time, any changes, modifications and evolutions of any equivalent changes made to the above embodiments based on the essential technology of the present invention are still within the scope of the technical solution of the present invention.

[0129] References

[0130] 1. Machlus, KR and JEI Taliano, Jr., The incredible journey: From megakaryocyte development to platelet formation. J Cell Biol, 2013.201(6):p.785-96.

[0131] 2.Gremmel, T., ALFrelinger, 3rd, and ADMichelson, Platelet Physiology. Semin Thromb Hemost, 2016.42(3):p.191-204.

[0132] 3.Jinna S,KPThrombocytopenia.2022 2022-07-05;Available from:https: / / www.ncbi.nlm.nih.gov / books / NBK542208 / .

[0133] 4.Santoshi,R.K.,et al.,A·Comprehensive Review of ThrombocytopeniaWith aSpotlight on Intensive Care Patients.Cureus,2022.14(8):p.e27718.

[0134] 5.Anwer,A.I.K.F.Platelet Transfusion.2022 2022-05-01;Available from:https: / / www.ncbi.nlm.nih.gov / books / NBK560632 / .

[0135] 6.Marino,P.L.,Platelets and plasma,in The ICU Book,K.Donnellan,Editor.2014,Wolters Kluwer.p.369-387.

[0136] 7.van der Meijden,P.E.J.and J.W.M.Heemskerk,Platelet biology andfunctions:new concepts and clinical perspectives.Nat Rev Cardiol,2019.16(3):p.166-179.

[0137] 8.Krueger,L.A.,et al.,Immunophenotypic analysis of platelets.CurrProtoc Cytom,2002.Chapter 6:p.Unit 6.10.

[0138] 9.Spurgeon,B.E.J.,et al.,Immunophenotypic Analysis of Platelets byFlow Cytometry.Curr Protoc,2021.1(6):p.e178.

[0139] 10.Paul,D.S.and W.Bergmeier,Novel Mouse Model for Studying HemostaticFunction of Human Platelets.Arterioscler Thromb Vasc Biol,2020.40(8):p.1891-1904.

[0140] 11.Sugimoto,N.,et al.,Production and nonclinical evaluation of anautologous iPSC-derived platelet product for the iPLAT1 clinical trial.BloodAdv,2022.6(23):p.6056-6069.

Claims

1. A method for detecting PAC-1 marker on platelets using a detection system, characterized in that: The detection system includes the following components: a buffer, a calcium concentrate and an agonist; The buffer contains: 130-140 mM NaCl, 2.5-4 mM KCl, 0.5-2 mM MgCl2, 5-9 mM HEPES, 5-10 mM NaHCO3, 0.1-0.5 mM Na2HPO4, 0.1% -0.4% w / v human serum albumin, 2-8 mM glucose, pH 7.35-7.45; Agonists contained 1–2 mM ADP and 0.1–1 mM TRAP6; Calcium concentrate contains 50-100 mM calcium ions; The method comprises the following steps: (1) Dilute the sample to be tested with buffer; (2) mixing the diluted sample in step (1), the agonist, and the anti-PAC-1 marker antibody, incubating, and quantitatively detecting; (3) mixing the sample diluted in step (1) with an agonist, a calcium concentrate, and an antibody against a PAC-1 marker, incubating, and quantitatively detecting; (4) calculating the ratio of the detection value in step (3) to the detection value in step (2); The calcium concentrate is tested by adjusting the calcium ion concentration in the reaction system to 1.0 mM-1.5 mM.

2. The method according to claim 1, characterized in that The buffer contains: 137 mM NaCl, 4 mM KCl, 1 mM MgCl2, 5 mM HEPES, 10 mM NaHCO3, 0.3 mM Na2HPO4, 0.35% w / v human serum albumin, 5.5 mM glucose, pH 7.4; The agonist contained 1.34 mM ADP and 0.54 mM TRAP6; Calcium concentrate contains 100 mM calcium ion.

3. The method according to claim 1, characterized in that The detection system also includes Pacific Blue anti-human CD41, PerCP anti-human CD42b and anti-PAC-1 marker antibody FITC anti-human PAC-1 or FITC anti-human PAC-1.

4. The method according to claim 3, characterized in that The anti-PAC-1 marker antibody is selected from FITCanti-human PAC-1.

5. The method according to claim 1, characterized in that The detection system further comprises a detection device or a detection reagent for detecting the combination of the expressed PAC-1 marker and the anti-PAC-1 marker antibody.

6. The method according to claim 5, characterized in that The detection equipment includes a flow cytometer, and the detection reagent includes a fluorescent detection reagent.

7. The method according to claim 1, characterized in that The calcium ions are derived from calcium chloride and calcium gluconate.

8. The method according to claim 1, characterized in that In step (1), the sample to be tested is diluted 10 to 40 times with a buffer solution; and in steps (2) and (3), the incubation condition is to incubate at room temperature for 15 to 30 minutes.

9. The method according to claim 8, characterized in that In step (1), the sample to be tested is diluted 20 times with a buffer solution; and in steps (2) and (3), the incubation condition is to incubate at room temperature for 20 minutes.

10. A method for detecting PAC-1 marker, Annexin-V marker and CD62P marker on platelets using a detection system, characterized in that: The detection system includes the following components: a buffer, a calcium concentrate and an agonist; The buffer comprises: 130-140 mM NaCl, 2.5-4 mM KCl, 0.5-2 mM MgCl2, 5-9 mM HEPES, 5-10 mM NaHCO3, 0.1-0.5 mM Na2HPO4, 0.1% -0.4% w / v human serum albumin, 2-8 mM glucose, pH 7.35-7.45; Agonists contained 1-2 mM ADP and 0.1-1 mM TRAP6; Calcium concentrate contains 50-100 mM calcium ions; The method comprises the following steps: (1) Dilute the sample to be tested with buffer; (2) mixing the sample agonist diluted in step (1) and the anti-PAC-1 marker antibody, incubating, and quantitatively detecting; (3) mixing the diluted sample in step (1) with a calcium concentrate, an agonist, and an antibody against a PAC-1 marker, incubating, and quantitatively detecting; (4) calculating the ratio of the detection value in step (3) to the detection value in step (2) to detect the PAC-1 marker; (5) mixing the sample diluted in step (1) with a calcium concentrate, an agonist, and an antibody against the Annexin-V marker, incubating, and quantitatively detecting to achieve detection of the Annexin-V marker; (6) mixing the sample diluted in step (1) with a calcium concentrate, an agonist, and an antibody against a CD62P marker, incubating, and quantitatively detecting to achieve detection of the CD62P marker; The calcium concentrate is tested by adjusting the calcium ion concentration in the reaction system to 1.0 mM or 1.5 mM.

11. The method according to claim 10, characterized in that The buffer contains: 137 mM NaCl, 4 mM KCl, 1 mM MgCl2, 5 mM HEPES, 10 mM NaHCO3, 0.3 mM Na2HPO4, 0.35% w / v human serum albumin, 5.5 mM glucose, pH 7.4; the agonist contains 1.34 mM ADP and 0.54 mM TRAP6; and the calcium concentrate contains 100 mM calcium ions.

12. The method according to claim 10, characterized in that The detection system also includes Pacific Blue anti-human CD41, PerCP anti-human CD42b, and antibodies against PAC-1 markers, antibodies against Annexin-V markers, and antibodies against CD62P markers.

13. The method according to claim 12, characterized in that The anti-PAC-1 marker antibody is selected from FITCanti-human PAC-1.

14. The method according to claim 12, characterized in that The antibody against Annexin-V marker is selected from PE / Cy7-Annexin V.

15. The method according to claim 12, characterized in that The anti-CD62P marker antibody is selected from APC anti-human CD62P.

16. The method according to claim 10, characterized in that The detection system further comprises a detection device or a detection reagent for detecting a combination of the expressed PAC-1 marker and an antibody against the PAC-1 marker, a detection device or a detection reagent for detecting a combination of the expressed Annexin-V marker and an antibody against the Annexin-V marker, and A detection device or a detection reagent for detecting the combination of the expressed CD62P marker and the anti-CD62P marker antibody.

17. The method according to claim 16, characterized in that The detection equipment includes a flow cytometer, and the detection reagent includes a fluorescent detection reagent.

18. The method according to claim 10, characterized in that The calcium ions are derived from calcium chloride and calcium gluconate.

19. The method according to claim 10, characterized in that The sample to be tested in step (1) is diluted 10 to 40 times with a buffer solution; The incubation conditions in step (2), step (3), step (5) and step (6) are incubation at room temperature for 15-30 minutes.

20. The method according to claim 19, characterized in that In step (1), the sample to be tested is diluted 20 times with a buffer solution; and in step (2), step (3), step (5) and step (6), the incubation condition is incubation at room temperature for 20 minutes.

21. A method for simultaneously detecting PAC-1 marker, Annexin-V marker and CD62P marker on platelets, characterized in that: The method according to any one of claims 10 to 20 is used to simultaneously detect PAC-1 marker, Annexin-V marker and CD62P marker, wherein the detection equipment of the method is selected from flow cytometer.

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