Kit for diagnosing immune thrombocytopenia and application thereof
By providing a kit containing specific reagents, it is possible to accurately detect immune thrombocytopenia mediated by heparin or non-heparin-dependent PF4/polyanionic complex antibodies, solving the problem of inaccurate detection in the prior art, achieving the realization of the problem of inaccurate detection Accurate diagnosis and clinical application.
Patent Information
- Application Number
- CN202411460901.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-18
- Publication Date
- 2025-02-11
AI Technical Summary
The detection of immune thrombocytopenia in the prior art is inaccurate, and it is difficult to accurately diagnose immune thrombocytopenia mediated by heparin or non-heparin-dependent PF4/polyanionic complex antibodies.
Provide a kit for diagnosing immune thrombocytopenia, including platelets, PF4 protein, FcγRIIa monoclonal antibody, Annexin V, FITC-Annexin V and loading buffer, through which the kits can be detected Heparin-dependent or non-heparin-based PF4/polyanionic complex antibody-mediated immune thrombocytopenia.
This kit can accurately detect immune thrombocytopenia, overcome the defects of inaccurate detection in the prior art, and correctly identify pathogenic antibodies that rely on heparin or non-heparin. It has clinical significance and wide application prospects.
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Figure CN119414014A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of kits, and in particular relates to a kit for diagnosing immune thrombotic thrombocytopenia and an application thereof. Background Art
[0002] PF4 protein itself is not immunogenic, but because of its positive charge, it can form complexes with negatively charged molecules such as heparin or other glycosaminoglycans (GAG), causing its conformation to change and forming new antigenic epitopes. The PF4 / polyanion complex is a multi-molecular super-complex composed of more than 16 PF4 molecules and polyanions, which can induce the body to produce anti-PF4 / polyanion immune complexes. When the concentration of this antibody immune complex increases significantly, it can bind to the platelet surface receptor FcγIIA in large quantities, causing continuous platelet activation and formation of microthrombi, ultimately leading to heparin-induced thrombocytopenia (HIT) or vaccine-induced immune thrombocytopenia and thrombosis (VITT).
[0003] The clinical diagnosis of HIT is generally based on the 4T'S scoring system and dynamic monitoring of platelet count, combined with HIT antibody detection and (or) platelet function test for exclusion diagnosis and confirmation. The lack of specificity of the 4T'S scoring system can easily lead to overdiagnosis, and it is necessary to use antibody detection and platelet function evaluation for diagnosis. Literature reports that the positive rate of PF4 / heparin complex antibodies in patients treated with heparin is 20% to 60%, but only 5% of patients develop HIT, and most antibodies are not pathogenic. Therefore, platelet function tests are needed to determine whether PF4 / polyanion complex antibodies can activate platelets.
[0004] In recent years, the PF4 / polyanion complex antibodies in cases of autologous HIT and VITT reported here can activate platelets independently of heparin, leading to immune thrombotic thrombocytopenia. In addition, most of the relevant literature published in domestic journals are case reports, and only a few have been tested for PF4 antibodies. All patients have not undergone confirmatory tests. This is because the confirmatory test methods of platelet serotonin release assay (SRA) and heparin-induced platelet aggregation (HIPA) are difficult to promote and apply in various laboratories, which poses challenges to the laboratory diagnosis of this disease. Therefore, the current method for diagnosing immune thrombotic thrombocytopenia has the defect of inaccurate detection of immune thrombotic thrombocytopenia. Summary of the invention
[0005] The technical problem to be solved by the present invention is to overcome the defect of inaccurate detection of immune thrombotic thrombocytopenia in the prior art, and to provide a kit for diagnosing immune thrombotic thrombocytopenia and its application.
[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions.
[0007] The invention provides a kit for diagnosing immune thrombotic thrombocytopenia, comprising: platelets from healthy people, PF4 protein, FcγRIIa monoclonal antibody, annexin V, FITC-annexin V and loading buffer.
[0008] The kit for diagnosing immune thrombotic thrombocytopenia provided by the present invention can detect heparin-dependent or heparin-independent PF4 / polyanion complex antibody-mediated immune thrombotic thrombocytopenia, and can correctly detect immune thrombotic thrombocytopenia, overcoming the defect of inaccurate detection of immune thrombotic thrombocytopenia in the prior art.
[0009] At the same time, based on the above-mentioned kit for diagnosing immune thrombotic thrombocytopenia, the present invention also provides a method for diagnosing PF4 / polyanion complex antibody-mediated immune thrombotic thrombocytopenia based on platelet phosphatidylserine expression. The inventive method can detect both heparin-dependent and heparin-independent mediated immune thrombotic thrombocytopenia, has clinical significance, and has broad application prospects.
[0010] At the same time, it should be noted that the reagents in the above-mentioned kit for diagnosing immune thrombotic thrombocytopenia provided by the present invention together constitute a kit, and each reagent is indispensable when used, and can be used to detect non-heparin-dependent mediated immune thrombotic thrombocytopenia. The dosage of each reagent is determined according to the requirements of the actual detection process.
[0011] In some embodiments of the present invention, the platelets from healthy people are mixed with PF4 protein, and the final concentration of the PF4 protein is 10 μg / mL to 37.5 μg / mL.
[0012] In some embodiments of the present invention, the platelet concentration of healthy people ranges from 300×10 9 pcs / L~500×10 9 Pieces / L.
[0013] In some embodiments of the present invention, the method for preparing platelets from healthy people comprises: adding an anticoagulant to the blood of healthy people in vitro and then centrifuging to obtain platelet-rich plasma, wherein the volume ratio of the blood of healthy people in vitro to sodium citrate is 1:8-10.
[0014] A prostaglandin E1 stock solution with a final concentration of 8 μg / mL to 52 μg / mL is added to the platelet-rich plasma, and then a PBS / ACD / BSA buffer is added and centrifuged. After the precipitate is collected, it is washed with a PBS / ACD / BSA buffer and resuspended to obtain platelets from healthy people.
[0015] The volume of the added PBS / ACD / BSA buffer is 1 to 3 times the volume of the platelet-rich plasma.
[0016] The PBS / ACD / BSA buffer solution is prepared by mixing a 0.01M phosphate solution and a sodium citrate solution at a volume ratio of 8.5 to 9.5:1 to obtain a mixed solution; adjusting the pH of the mixed solution to 7.3 to 7.5, and then adding bovine serum albumin to the mixed solution to make the final concentration of the bovine serum albumin 1.5% w to 2.5% wt, thereby obtaining the PBS / ACD / BSA buffer solution. The English abbreviation of phosphate solution is PBS; the English abbreviation of sodium citrate solution is ACD; and the English abbreviation of bovine serum albumin is BSA.
[0017] In some embodiments of the present invention, the prostaglandin E1 stock solution is prepared using dimethyl sulfoxide as a solvent to obtain a solution with a final concentration of 10 μg / mL to 50 μg / mL.
[0018] Among them, the English name of dimethyl sulfoxide is DMSO.
[0019] In some embodiments of the present invention, the anticoagulant is sodium citrate.
[0020] In some embodiments of the present invention, the loading buffer is Binding Buffer.
[0021] In some embodiments of the present invention, negative serum tested negative for PF4 / polyanion complex antibodies and positive serum tested positive for PF4 / polyanion complex antibodies are also included.
[0022] The present invention also provides the use of the kit in preparing a diagnostic or prognostic product for immune thrombotic thrombocytopenia.
[0023] Compared with the prior art, the beneficial effects of the present invention include: in the present invention, by using a monoclonal antibody against FcγRIIa to bind to the FcγRIIa receptor on the platelet to observe whether it can inhibit the activation of platelets by the PF4 / polyanion complex antibody, the pathogenicity of the antibody is evaluated, and PF4 / polyanion complex antibody-mediated immune thrombotic thrombocytopenia is diagnosed. The kit for diagnosing immune thrombotic thrombocytopenia provided by the present invention can detect heparin-dependent or non-heparin-dependent PF4 / polyanion complex antibody-mediated immune thrombotic thrombocytopenia, and can correctly detect immune thrombotic thrombocytopenia, overcoming the defect of inaccurate detection of immune thrombotic thrombocytopenia in the prior art.
[0024] The kit for diagnosing immune thrombotic thrombocytopenia provided by the present invention can be used for the detection of immune thrombotic thrombocytopenia. The method provided by the present invention can detect both heparin-dependent and non-heparin-dependent mediated immune thrombotic thrombocytopenia, has clinical significance and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 This is an example of the NC1-1 test results; Figure 1 A in the figure indicates that after the negative control serum of the kit of the present invention reacts with the platelets and PF4 protein of the blood donor, a platelet scatter plot is obtained by forward angle (FSC) and side angle (SSC) under the LOG state of the flow cytometer, and a gate is set to determine the platelets; Figure 1 B in the figure represents a scatter plot of the fluorescence intensity of FITC-Annexin V on platelets detected by flow cytometry; Figure 1 C shows a histogram of the fluorescence intensity of FITC-Annexin V on platelets detected by flow cytometry.
[0026] Figure 2 This is an example of the NC1-2 test results; Figure 2 A in the figure represents the platelet scatter plot obtained by forward angle (FSC) and side angle (SSC) in the LOG state of the flow cytometer after the negative control serum of the kit of the present invention reacts with the platelets of the blood donor, PF4 protein and FcγRIIa inhibitor, and the platelets are determined by setting a gate; Figure 2 B in the figure represents a scatter plot of the fluorescence intensity of FITC-Annexin V on platelets detected by flow cytometry; Figure 2 C shows a histogram of the fluorescence intensity of FITC-Annexin V on platelets detected by flow cytometry.
[0027] Figure 3 This is an example of the PC1-1 test result; Figure 3 A in the figure represents the platelet scatter plot obtained by forward angle (FSC) and side angle (SSC) under the LOG state of the flow cytometer after the positive control serum of the kit of the present invention reacts with the platelets and PF4 protein of the blood donor, and the gate is set to determine the platelets; Figure 3 B in the figure represents a scatter plot of the fluorescence intensity of FITC-Annexin V on platelets detected by flow cytometry; Figure 3 C shows a histogram of the fluorescence intensity of FITC-Annexin V on platelets detected by flow cytometry.
[0028] Figure 4 This is an example of the PC1-2 test results; Figure 4A in the figure represents the platelet scatter plot obtained by forward angle (FSC) and side angle (SSC) in the LOG state of the flow cytometer after the positive control serum of the kit of the present invention reacts with the platelets of the blood donor, PF4 protein and FcγRIIa inhibitor, and the gate is set to determine the platelets; Figure 4 B in the figure represents a scatter plot of the fluorescence intensity of FITC-Annexin V on platelets detected by flow cytometry; Figure 4 C shows a histogram of the fluorescence intensity of FITC-Annexin V on platelets detected by flow cytometry.
[0029] Figure 5 This is an example diagram of the S1-1 test results; Figure 5 A in the figure indicates the platelet scatter plot obtained by forward angle (FSC) and side angle (SSC) in the LOG state of the flow cytometer after the patient's serum reacted with the donor's platelets and PF4 protein, and the gate was set to determine the platelets; Figure 5 B in the figure represents a scatter plot of the fluorescence intensity of FITC-Annexin V on platelets detected by flow cytometry; Figure 5 C shows a histogram of the fluorescence intensity of FITC-Annexin V on platelets detected by flow cytometry.
[0030] Figure 6 This is an example diagram of the S1-2 test results; Figure 6 A in the figure indicates the platelet scatter plot obtained by forward angle (FSC) and side angle (SSC) in the LOG state of the flow cytometer after the patient's serum reacted with the donor's platelets, PF4 protein and FcγRIIa inhibitor, and the platelets were determined by setting a gate; Figure 6 B in the figure represents a scatter plot of the fluorescence intensity of FITC-Annexin V on platelets detected by flow cytometry; Figure 6 C shows a histogram of the fluorescence intensity of FITC-Annexin V on platelets detected by flow cytometry. DETAILED DESCRIPTION
[0031] The present invention is described in detail below in conjunction with specific examples, but it should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.
[0032] The present invention proposes a method for diagnosing PF4 / polyanion complex antibody-mediated immune thrombotic thrombocytopenia based on platelet phosphatidylserine expression. PF4 / polyanion complex antibody-mediated immune thrombotic thrombocytopenia is a unique syndrome that causes both thrombocytopenia and thrombosis, mainly including heparin-induced thrombocytopenia (HIT) and SARS-CoV-2 vaccine-induced immune thrombocytopenia and thrombosis (VITT). The PF4 / polyanion complex is a multi-molecular super-large complex assembled by more than 16 PF4 molecules and polyanions, which can induce the body to produce PF4 / polyanion IgG immune complexes. When the concentration of PF4-heparin-IgG immune complexes in the blood increases significantly, they can bind to the platelet surface receptor FcγIIa in large quantities, causing continuous activation of platelets to form microthrombi and release platelet microparticles. Platelet emboli can bind to the heparan sulfate-PF4 complex on the surface of vascular endothelial cells through PF4 / polyanion antibodies, fix to the blood vessel wall to form wall-attached emboli, and activate endothelial cells. On the other hand, PF4-heparin-IgG immune complex can also bind to and stimulate monocytes to release tissue factor through Fc RI receptors, activate the coagulation pathway, generate a large amount of thrombin, and finally form fibrin thrombi. Thus, normal platelets (PRP) of blood donors are incubated with patient serum in the presence of PF4 protein, and when pathogenic antibodies are present in the patient serum, normal platelet phosphatidylserine (PS) of blood donors can be induced to flip from the inside of the cell membrane lipid bilayer to the outside. In the present invention, by using a monoclonal antibody against FcγRIIa to bind to the FcγRIIa receptor on the platelets, it is observed whether the PF4 / polyanion complex antibody can inhibit the activation of platelets, thereby evaluating the pathogenicity of the antibody and diagnosing PF4 / polyanion complex antibody-mediated immune thrombotic thrombocytopenia. The method of the invention can detect both heparin-dependent and non-heparin-dependent mediated immune thrombotic thrombocytopenia, has clinical significance, and has broad application prospects.
[0033] Embodiment 1:
[0034] 1. The selection criteria for platelet donors are in accordance with the "Health Examination Requirements for Blood Donors" (GB 18467-2011). All participants in the present invention provide written consent. All blood samples in the present invention are from Nanning Central Blood Station. The preparation method of platelets is as follows:
[0035] Preparation of platelets from healthy blood donors:
[0036] (1) Preparation of platelet-rich plasma: Collect 5 mL of venous blood from 5 type O blood donors and anticoagulate with sodium citrate anticoagulant. Gently invert the tube to mix the blood with sodium citrate. Centrifuge at 150g for 15 minutes to obtain platelet-rich plasma. The platelet-rich plasma (PRP) of the 5 blood donors was mixed for testing.
[0037] Among them, the volume ratio of blood to sodium citrate is 1:9.
[0038] (2) Platelet-rich plasma (PRP) was added with a prostaglandin E1 stock solution with a final concentration of 50 ug / mL to inhibit platelet activation, and the platelets were washed once by centrifugation with PBS / ACD / BSA buffer. The centrifugation conditions were: 150 g for 15 min.
[0039] The prostaglandin E1 stock solution uses dimethyl sulfoxide as a solvent, and the final concentration of the prepared solution is 10 μg / mL to 50 μg / mL.
[0040] Among them, the English name of dimethyl sulfoxide is DMSO.
[0041] The volume of PBS / ACD / BSA buffer added was twice the volume of platelet-rich plasma.
[0042] The preparation method of PBS / ACD / BSA buffer is as follows: 0.01M phosphate solution and sodium citrate solution are mixed at a volume ratio of 9:1 to obtain a mixed solution; the pH of the mixed solution is adjusted to 7.4, and then bovine serum albumin is added to the mixed solution to make the final concentration of the bovine serum albumin 2%wt, thereby obtaining the PBS / ACD / BSA buffer.
[0043] Among them, the English abbreviation of phosphate solution is PBS; the English abbreviation of sodium citrate solution is ACD; and the English abbreviation of bovine serum albumin is BSA.
[0044] (3) The platelets were resuspended in PBS / ACD / BSA buffer at a platelet concentration of 300 × 10 9 Pieces / L.
[0045] 2. Selection of NC and PC
[0046] Negative serum (NC): serum from AB-type male blood donors who tested negative for PF4 / polyanion complex antibodies by ELISA. The subjects were blood donors from Nanning Central Blood Station, including 5 males.
[0047] Positive serum (PC): serum from patients with HIT that was confirmed positive by heparin-induced platelet aggregation (HIPA) after testing PF4 / polyanion complex antibodies by ELISA. The subjects were from the Nanning Central Blood Station, and the serum from patients with HIT was confirmed by the above methods, including 2 males and 2 females.
[0048] Experimental group: patients with PF4 / polyanion complex antibodies detected by ELISA but not further diagnosed by HIPA and other confirmatory tests. The subjects were patients suspected of HIT due to thrombocytopenia caused by heparin treatment who were sent to Nanning Central Blood Station for testing. The sera of patients with PF4 / polyanion complex antibodies detected by ELISA but not further diagnosed by HIPA confirmatory tests were 2 males and 2 females.
[0049] 3. 1×10 platelets from healthy blood donors prepared in step 1 6 / L was mixed with PF4 protein to make the final concentration of PF4 protein 37.5μg / mL, the total volume was 40μL, and 10μL of patient serum in the experimental group was added thereto, mixed well, incubated at room temperature for 60min, and marked as S1-1.
[0050] Among them, PF4 protein mixture: product of abcam, product number: ab81754.
[0051] 1×10 platelets from healthy blood donors prepared in step 1 6 / L was mixed with PF4 protein to make the final concentration of PF4 protein 37.5μg / mL, the total volume was 40μL, and 10μL of patient serum in the experimental group was added thereto, mixed well, and incubated at room temperature for 60min, and then FcγRIIa monoclonal antibody was added thereto to make the final concentration 5μg / mL, mixed well, and incubated at room temperature for 20min, to obtain a sample tube using FcγRIIa receptor inhibition, marked as S1-2;
[0052] 1×10 platelets from healthy blood donors prepared in step 1 6 10 μL of negative serum (NC) was added to the mixture, mixed well, and incubated at room temperature for 60 min as a negative control tube, marked as NC1-1;
[0053] The platelets (1×10 6) was mixed with PF4 protein, the final concentration of PF4 protein was 37.5 μg / mL, the total volume was 40 μL, and 10 μL of negative serum (NC) was added thereto, mixed, and incubated at room temperature for 60 min, and FcγRIIa monoclonal antibody (final concentration 5 μg / mL) was added thereto, mixed, and incubated at room temperature for 20 min to obtain a negative control tube using FcγRIIa receptor inhibition, marked as NC1-2;
[0054] 1×10 platelets from healthy blood donors prepared in step 1 6 / L was mixed with PF4 protein, the final concentration of PF4 protein was 37.5μg / mL, the total volume was 40μL, and 10μL of positive serum (PC) was added to the test tube, marked as PC1-1;
[0055] 1×10 platelets from healthy blood donors prepared in step 1 6 / L was mixed with PF4 protein, the final concentration of PF4 protein was 37.5μg / mL, and the total volume was 40μL), and 10μL of positive serum (PC) was added to the test tube, and FcγRIIa monoclonal antibody (final concentration 5μg / mL) was added thereto, mixed, and incubated at room temperature for 20min to obtain a positive control tube using FcγRIIa receptor inhibition, which was marked as PC1-2.
[0056] 4. Add 100 μL of Binding Buffer (1x) to each tube, add 5 μL of FITC-Annexin V reagent, mix gently, and incubate at room temperature in the dark for 15 minutes. Among them, BD product, catalog number: 556420.
[0057] 5. Add 400 μl 1X Binding Buffer to all the above test tubes and negative and positive control tubes. After mixing, adjust the voltage, threshold and other parameters in the flow cytometer under the logarithmic (LOG) state, obtain platelet particles by forward angle (FSC) and side angle (SSC), and set the gate. Obtain the mean fluorescence intensity (MFI) of FITC-Annexin V in the test tubes and control tubes.
[0058] Obtain the mean fluorescence intensity value of the negative control tube (NC1-1), recorded as MFI NC1-1 value;
[0059] Obtain the mean fluorescence intensity value of the negative control tube (NC1-2) using FcγRIIa receptor inhibition and record it as MFI NC1-2 value;
[0060] Obtain the mean fluorescence intensity value of the positive control tube (PC1-1), recorded as MFI PC1-1 value;
[0061] Obtain the mean fluorescence intensity value of the positive control tube (PC1-2) using FcγRIIa receptor inhibition, recorded as MFI PC1-2 value;
[0062] Get the mean fluorescence intensity value of the sample tube (S1-1), recorded as MFI S1-1 value;
[0063] Obtain the mean fluorescence intensity value of the sample tube (S1-2) using FcγRIIa receptor inhibition, recorded as MFI S1-2 value;
[0064] Inhibition rate calculation formula: Inhibition % = [1-(MFI S1-2 -MFI NC1-2 ) / (MFI S1-1 -MFI NC1-1 )]×100
[0065] When the inhibition rate is greater than 50%, the sample to be tested is positive. When the PC inhibition rate is less than 50%, the test result is invalid.
[0066] Experimental results:
[0067] 1. Negative control test results
[0068] like Figure 1 As shown in the figure, after the negative control serum reacted with the platelets and PF4 protein of the blood donor, the average fluorescence intensity of FITC-Annexin V on the platelets was 45. Figure 2 As shown, after the negative control serum was reacted with donor platelets, PF4 protein and FcγRIIa inhibitor, the average fluorescence intensity of FITC-Annexin V on the platelets was 38.
[0069] 2. Positive control test results
[0070] The results are as follows Figure 3 As shown in the figure, after the positive control serum reacted with the platelets and PF4 protein of the blood donor, the average fluorescence intensity of FITC-Annexin V on the platelets was 1147. Figure 4 As shown, after the positive control serum was reacted with donor platelets, PF4 protein and FcγRIIa inhibitor, the average fluorescence intensity of FITC-Annexin V on the platelets was 495.
[0071] 3. Sample test results
[0072] The results are as follows Figure 5 As shown in the figure, after the patient's serum reacted with the donor's platelets and PF4 protein, the average fluorescence intensity of FITC-annexin V on the platelets was 848. Figure 4As shown, after the patient's serum reacted with the donor's platelets, PF4 protein and FcγRIIa inhibitor, the average fluorescence intensity of FITC-annexin V on the platelets was 206. The FcγRIIa receptor inhibition rate was calculated according to the inhibition rate calculation formula, and the calculated FcγRIIa receptor inhibition rate was 79.08%. When the FcγRIIa receptor inhibition rate of the sample is >50%, it means that the sample to be tested is positive.
[0073] FcγRIIa receptor inhibition rate = [1-(MFI S1-2 -MFI NC1-2 ) / (MFI S1-1 -MFI NC1-1 )]×100=79.08%
[0074] Detection accuracy:
[0075] The heparin-induced platelet aggregation assay (HIPA [Barouqa M, Matta M, Reyes-Gil M. Performance evaluation of heparin-induced platelet aggregation vs serotonin release assay. Am J Clin Pathol. 2024 Feb 1; 161 (2): 122-129. doi: 10.1093 / ajcp / aqad117. PMID: 37936260.] was used to verify the detection results of the present invention, and the detection results of the two methods were consistent. Among them, in the verification experiment, the sample size used in the present invention was 4 people.
[0076] From the above, it can be seen that the kit for diagnosing immune thrombotic thrombocytopenia provided by the present invention can detect heparin-dependent or heparin-independent PF4 / polyanion complex antibody-mediated immune thrombotic thrombocytopenia, and can correctly detect immune thrombotic thrombocytopenia, overcoming the defect of inaccurate detection of immune thrombotic thrombocytopenia in the prior art.
[0077] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.
[0078] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.
Claims
1. A kit for diagnosing immune thrombotic thrombocytopenia, characterized in that: include: Platelets from healthy people, PF4 protein, FcγRIIa monoclonal antibody, Annexin V, FITC-Annexin V and loading buffer.
2. The kit according to claim 1, characterized in that The platelets from healthy people are mixed with PF4 protein, and the final concentration of the PF4 protein is 10 μg / mL to 37.5 μg / mL.
3. The kit according to claim 2, characterized in that The platelet concentration range of healthy people is 300×10 9 pcs / L~500×10 9 Pieces / L.
4. The kit according to claim 3, characterized in that The method for preparing platelets from healthy people comprises: adding an anticoagulant to the blood of healthy people in vitro and then centrifuging to obtain platelet-rich plasma; wherein the volume ratio of the blood of healthy people in vitro to sodium citrate is 1:8-10; Adding a prostaglandin E1 stock solution with a final concentration of 8 μg / mL to 52 μg / mL to the platelet-rich plasma, then adding PBS / ACD / BSA buffer and centrifuging, collecting the precipitate, washing it with PBS / ACD / BSA buffer, and resuspending it to obtain the platelets of the healthy population; The volume of the added PBS / ACD / BSA buffer is 1 to 3 times the volume of the platelet-rich plasma; The PBS / ACD / BSA buffer solution is prepared by mixing a 0.01M phosphate solution and a sodium citrate solution at a volume ratio of 8.5 to 9.5:1 to obtain a mixed solution; adjusting the pH of the mixed solution to 7.3 to 7.5, and then adding bovine serum albumin to the mixed solution to make the final concentration of the bovine serum albumin 1.5% w to 2.5% wt, thereby obtaining the PBS / ACD / BSA buffer solution.
5. The kit according to claim 4, characterized in that The prostaglandin E1 stock solution uses dimethyl sulfoxide as a solvent, and the prepared solution has a final concentration of 10 μg / mL to 50 μg / mL.
6. The kit according to claim 4, characterized in that The anticoagulant is sodium citrate.
7. The kit according to claim 1, characterized in that The loading buffer is Binding Buffer.
8. The kit according to claim 1, wherein Also included are negative sera that test negative for PF4 / polyanion complex antibodies and positive sera that test positive for PF4 / polyanion complex antibodies.
9. Use of the kit according to claim 1 in preparing a product for diagnosis or prognosis of immunothrombotic thrombocytopenia.