Method for detecting platelet stress status using kit

Abstract

Provided is a method for detecting platelet stress status using a kit. The kit comprises a platelet diluent, an EAAT2 polyclonal primary antibody, a fluorescent secondary antibody for flow cytometry, an EAAT2 isotype control, a monoclonal antibody directly conjugated to a PE fluorescent dye against CD61, and 1% paraformaldehyde. The EAAT2 polyclonal primary antibody recognizes at least one of amino acid sequences as set forth in SEQ ID No. 1 to SEQ ID No. 5. The method comprises: preparing a platelet suspension using the platelet diluent; sequentially incubating the platelet suspension with the EAAT2 polyclonal primary antibody, the monoclonal antibody directly conjugated to the PE fluorescent dye against CD61, and the fluorescent secondary antibody for flow cytometry; adding the paraformaldehyde to fix the sample to form a sample to be tested; detecting a specific signal of the sample by means of a flow cytometer; and performing statistical analysis onthe specific signal to confirm the stress status of the sample. The reagents to be used in the entire flow cytometry process are rationally proportioned to form the kit, which is conducive to standardizing the detection method.

Description

Method for detecting stress condition of platelet by using kit TECHNICAL FIELD

[0001] The present application relates to the technical field of medical examination, in particular to a method for detecting stress condition of platelet by using kit. BACKGROUND

[0002] Platelets are anucleate membrane structure cells, which belong to non-traditional cells and are easy to be stimulated by extracellular microenvironment to reorganize skeleton in different degrees. Platelet cell phenotype change can reflect the stress state of human body to a certain extent. EAAT2 (Excitatory Amino Acid Transporter 2) of platelets dominates the uptake of glutamate, which is mainly distributed on the membrane of platelets and the membrane of alpha granules of platelets. When the real-time state of platelets changes, the distribution of EAAT2 membrane protein will change significantly with the reorganization of platelet skeleton. The existing technology usually uses immunoelectron microscopy (IEM) and immunofluorescence (IF) to detect EAAT2 membrane protein, but the processing process of blood samples by these two technologies is very tedious. Not only the detection period is long, but also the experimental scheme is not flexible. Moreover, too complicated or too much intervention operation can greatly affect the detection result of EAAT2 membrane protein, so the detection result is not accurate enough and is not suitable for promotion in clinical laboratory. SUMMARY

[0003] The purpose of the present application is to at least partially overcome the deficiencies of the prior art, and to provide a method for detecting stress condition of platelet by using kit, which has high timeliness and simple operation.

[0004] In order to achieve the above technical purpose, the technical scheme adopted by the present application is as follows:

[0005] The present application provides a method for detecting stress condition of platelet by using kit, wherein the kit comprises platelet diluent, EAAT2 polyclonal primary antibody, flow fluorescence secondary antibody, EAAT2 homologous control, monoclonal antibody directly coupled with CD61 and 1% paraformaldehyde; the EAAT2 polyclonal primary antibody recognizes at least one of the amino acid sequences shown as SEQ ID No. 1-SEQ ID No. 5;

[0006] The detection method comprises the following steps:

[0007] Preparation of platelet suspension by using the platelet diluent;

[0008] incubating the platelet suspension with the EAAT2 polyclonal primary antibody, the monoclonal antibody directly coupled with PE fluorescent dye of CD61 and the flow fluorescent secondary antibody in sequence;

[0009] adding the paraformaldehyde to the sample to form a sample to be detected;

[0010] detecting the specific signal of the sample by flow cytometry;

[0011] statistically analyzing the specific signal to confirm the stress condition of the sample.

[0012] Optionally, the platelet diluent comprises 10mM HEPES, 135-145mM NaCl, 2.6-2.8mM KCl, 11-12mM NaHCO3, 1-2mM MgCl2, 0.4-0.5mM Na2HPO4, 5-6mM glucose, 0.2-0.4% bovine serum albumin, and the pH is 7.2-7.4.

[0013] Further optionally, the flow fluorescent secondary antibody comprises a color developing marker, and is a specific antibody against the EAAT2 polyclonal primary antibody, which is derived from goat anti-rabbit; and the isotype control of the EAAT2 is rabbit-derived IgG ISOtype.

[0014] Further, the kit further comprises other platelet-related marker antibodies, including specific antibodies of CD41, CD42b, CD62P, CD63 and / or PAC-1.

[0015] Specifically, the incubation of the platelet suspension with the EAAT2 polyclonal primary antibody, the monoclonal antibody directly coupled with PE fluorescent dye of CD61 and the flow fluorescent secondary antibody in sequence comprises:

[0016] first incubating the platelet suspension with the EAAT2 polyclonal primary antibody, the monoclonal antibody directly coupled with PE fluorescent dye of CD61 at room temperature in the dark for 15-30min;

[0017] then incubating the platelet suspension with the flow fluorescent secondary antibody at room temperature in the dark for 15-20min.

[0018] Further optionally, the kit further comprises a stress regulator of platelets, which comprises an activator, an inhibitor, an interfering agent or an anti-interfering agent for the platelets; and the stress regulator is added according to the need of statistical analysis before the incubation of the platelet suspension with the EAAT2 polyclonal primary antibody, the monoclonal antibody directly coupled with PE fluorescent dye of CD61 and the flow fluorescent secondary antibody in sequence.

[0019] Optionally, the stress modulator includes glutamic acid, arachidonic acid, adenosine diphosphate, collagen, adrenaline, thrombin, thrombin receptor activating enzyme or lipopolysaccharide as an activator, an excitatory amino acid transporter inhibitor as an inhibitor, and an inhibitor for preventing fibrin polymerization to prevent blood clot formation.

[0020] Optionally, the method for preparing the platelet suspension is:

[0021] Freshly collected blood is subjected to anticoagulation treatment;

[0022] Centrifugation at 200g for 10-15min at room temperature;

[0023] After harvesting the plasma, let it stand for 30min to obtain platelet-rich plasma;

[0024] Take 5μL of the platelet-rich plasma and add 95μL of the platelet diluent to prepare a platelet suspension.

[0025] Further, the specific signal correlates the relationship between excitatory amino acid transporter 2 and platelets, and the cell analysis results of the flow cytometer are counted to confirm the cell phenotype of platelets under different stress conditions.

[0026] Compared with the prior art, the present application has the following advantages:

[0027] (1) The method of the present application utilizes the advantages of flow cytometry, which can analyze individual platelets, thereby enabling statistical analysis of the overall stress of the platelets of the individual corresponding to the blood sample, providing reference data for subsequent determination and prediction of the condition of the patient; and the reagents used in the entire flow cytometry are reasonably proportioned to form a kit, which is conducive to the standardization of the detection method;

[0028] (2) The method of the present application utilizes the correlation between platelets and EAAT2 membrane proteins, providing a blood detection method that can detect stroke, neurodegenerative diseases and the like in real time, thereby improving the possibility of clinical application;

[0029] (3) The sample preparation process of the method of the present application is simple, the interference with platelets is reduced to the lowest possible level, and the state of the cells under stress is maintained as much as possible, which is conducive to reflecting more realistic detection results, improving the detection efficiency, and facilitating the application in clinical detection;

[0030] (4) The sample prepared by the method of the present application can simulate the stress or drug intervention scenario by adding a stress modulator, thereby providing more abundant reference data for subsequent determination and prediction of the condition of the patient, which is conducive to improving the accuracy of judgment and prediction. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 shows the distribution of four different platelet subpopulations in a sample, as determined by the method for detecting platelet stress conditions using a kit according to the present application.

[0032] Figure 2 shows the expression pattern of platelet EAAT2 membrane protein, as determined by the method for detecting platelet stress conditions using a kit according to the present application, and mainly shows the detection results of negative homologous controls and positive results.

[0033] Figure 3 shows the expression characteristics of platelet EAAT2 membrane protein in healthy people, as determined by the method for detecting platelet stress conditions using a kit according to the present application, wherein A shows the basal state, B shows the activated state, and C shows the state after being activated after being treated with an inhibitor.

[0034] Figure 4 shows the results of detecting specific samples by the method for detecting platelet stress conditions using a kit according to the present application, and mainly shows the expression characteristics of platelet EAAT2 membrane protein in AIS patients with bleeding transformation.

[0035] Figure 5 shows the detection results of distinguishing CD61 low-fluorescence platelet subpopulations and CD61 high-fluorescence platelet subpopulations by the method for detecting platelet stress conditions using a kit according to the present application.

[0036] Figure 6 shows the Annexin V expression results of four platelet phenotypes labeled by EAAT2-CD61 combination by the method for detecting platelet stress conditions using a kit according to the present application.

[0037] Figure 7 shows the results of confirming that the expression of EAAT2 membrane protein is closely related to the increase in platelet PAC-1 expression by the method for detecting platelet stress conditions using a kit according to the present application. DETAILED DESCRIPTION

[0038] The present application will be further described in detail below in combination with the accompanying drawings and specific embodiments.

[0039] Previous studies have found that platelet activation requires up-regulation of the expression of excitatory amino acid transporters (EAATs) membrane protein to enhance the uptake of glutamate. Under high-concentration glutamate incubation conditions and during platelet activation, the expression of EAAT2 membrane protein (the main subtype of platelet EAATs) can be up-regulated to varying degrees, suggesting that EAAT2 membrane protein is closely related to the response and activation mechanism of platelets, and that EAAT2 membrane protein is a biomarker with significant clinical transformation value.

[0040] EAATs are high-affinity glutamate transporters, which transport three Na + and one H + per cycle, and one K + per cycle. Due to its special molecular transport mechanism, EAATs can form a transmembrane concentration gradient of glutamate as high as one million times between the two sides of the cell membrane at the resting membrane potential level of the cell, and can continuously perform transport in a wide concentration range. Since EAAT2 is a key subtype of EAATs protein on platelets, they must be correctly located on the cell membrane to exert the biological activity of transporting glutamate, therefore, if the expression changes of EAAT2 membrane protein on platelets can be dynamically monitored, it will not only help to analyze the individual platelet uptake capacity, but also help to understand the real-time activation status of platelets, and even evaluate the response strength of platelets to target activators. The present application intends to provide a flow cytometry detection kit capable of specifically recognizing platelet EAAT2 (Excitatory Amino Acid Transporter 2) membrane protein by selecting a flow cytometry antibody specifically recognizing the extracellular segment of EAAT2 membrane protein.

[0041] The prior art uses other techniques to detect EAAT2 membrane proteins of peripheral blood cells (including platelets), such as Immunoelectron microscopy (IEM), immunofluorescence (IF) techniques, and Western blot techniques combined with surface biotin labeling. Immunoelectron microscopy (IEM) is a technique that combines electron microscopy techniques and immunological principles, researchers can observe the location and distribution of EAAT2 protein in cells by using antibodies labeled with gold particles or other electron-dense substances in the sample. Immunofluorescence (IF) techniques can be used to detect EAAT2 protein in cells or tissues. In its experimental process, the sample needs to be fixed, sectioned and stained first, then the EAAT2 protein is combined with antibodies labeled with fluorescent dyes, and the location and distribution of EAAT2 protein are determined by observing the fluorescent signal through a fluorescence microscope. Western blot (WB) techniques are often used to study the expression level of proteins and the presence of specific proteins in cells or tissues. Western blot techniques combined with surface biotin labeling can improve the sensitivity and accuracy of detecting EAAT2 proteins located on the cell membrane surface by combining surface biotin-labeled antibodies with EAAT2 membrane proteins, and then using surface biotin ligase and substances such as fluorescein for detection. However, the above techniques have not been able to quantify or visualize the expression changes of platelet EAAT2 protein at the membrane protein level. In contrast, flow cytometry-based EAAT2 membrane protein detection methods not only clearly present the expression of platelet EAAT2 membrane protein, but also can be used to distinguish various phenotypes related to platelet activation and evaluate the degree of platelet heterogeneity.

[0042] The advantages of IEM are high sensitivity and resolution, which can detect low concentrations of proteins and determine their location at the cellular or subcellular level. However, it requires specialized equipment and techniques, is complex and time-consuming to operate, and has strict requirements for sample processing, which can not only cause protein inactivation or cell membrane structure damage, but also cannot ensure that the measured signal is only the result of membrane surface proteins. The advantages of IF are higher sensitivity and specificity, which can be used to detect the distribution and localization of proteins in cells, and the operation is relatively simple, and the results can be obtained quickly. However, it is not accurate for quantitative analysis, and may be interfered by background fluorescence, which requires specialized equipment and more complex technical optimization. Western blot alone cannot specifically detect EAAT2 protein located on the cell membrane surface, so it is only suitable for analyzing the total EAAT2 protein level in platelet homogenate. In theory, Western blot combined with surface biotin labeling combines the quantitative advantages of Western blot and the high sensitivity of surface biotin labeling, which can be used to accurately detect the expression level of membrane proteins and their differences in different samples. However, its operation is complex, requires additional reagents and equipment support, and the specificity of the results is relatively poor, especially not suitable for platelets, which are highly sensitive cells easily stimulated.

[0043] In general, the above existing technologies not only require expensive professional equipment such as electron microscopes and laser confocal microscopes, but also need to go through long and tedious antibody incubation and / or light fixation steps. These processes are highly stimulating to platelets, and the exogenous stimulation caused by tedious operations can significantly change the expression level of platelet EAAT2 membrane proteins, resulting in a serious deviation of the results from the true level. In addition, due to the long detection period and lack of flexibility in experimental design, these technologies lack supporting commercial detection kits, and therefore are not suitable for clinical laboratory application, but only for research laboratories.

[0044] Flow cytometry has significant advantages in analyzing platelet quantity, size, morphology, surface molecule expression and activity. Its operation is simple, including centrifugal separation of platelet-rich plasma, staining, fixation, machine detection, small stimulation to platelets, experimental results can be obtained within 2h, which can better reflect the real-time characteristics of platelets of the subjects. Therefore, in order to detect the dynamic expression of platelet EAAT2 membrane protein, the present application selects a polyclonal flow antibody which can specifically recognize the extracellular segment of EAAT2 membrane protein, and develops a flow detection kit suitable for quantitative detection of platelet EAAT2 membrane protein in vitro. The experimental results prove that the kit and detection method of the present application are sensitive and efficient, and the results are stable and reliable. In addition, on the basis of the simple EAAT2 membrane protein detection scheme, other marker antibodies of platelets can be combined for multiplex staining, and then different control groups or intervention groups can be added according to different experimental purposes, so as to realize the personalized and accurate diagnosis and analysis of platelets of the subjects, and finally achieve the purpose of providing multi-dimensional information of platelets of the subjects for the clinic.

[0045] The kit for detecting the stress condition of platelets according to the method of the present application, wherein the kit comprises a platelet diluent, an EAAT2 polyclonal primary antibody, a flow fluorescence secondary antibody, an EAAT2 homologous control, a monoclonal antibody directly coupled with PE fluorescent dye of CD61 and 1% paraformaldehyde; the EAAT2 polyclonal primary antibody recognizes at least one of the amino acid sequences shown as SEQ ID No. 1-SEQ ID No. 5;

[0046] The detection method comprises the following steps:

[0047] The platelet suspension is prepared by using the platelet diluent;

[0048] The platelet suspension is incubated by using the EAAT2 polyclonal primary antibody, the monoclonal antibody directly coupled with PE fluorescent dye of CD61 and the flow fluorescence secondary antibody in sequence;

[0049] The paraformaldehyde is added to fix the sample to form a sample to be detected;

[0050] The specific signal of the sample is detected by flow cytometry;

[0051] The stress condition of the sample is confirmed by statistical analysis of the specific signal.

[0052] More specifically, the basic components of the kit of the present application include:

[0053] ① Platelet dilution solution, modified HT buffer: 10 mM HEPES, 135-145 mM NaCl, 2.6-2.8 mM KCl, 11-12 mM NaHCO3, 1-2 mM MgCl2, 0.4-0.5 mM Na2HPO4, 5-6 mM glucose, 0.2-0.4% bovine serum albumin, pH 7.2-7.4;

[0054] ② EAAT2 polyclonal primary antibody and its flow fluorescence secondary antibody; wherein the EAAT2 polyclonal primary antibody can specifically recognize the extracellular epitope fragments of platelets, and the extracellular epitope fragments of the platelets are the amino acid sequences shown in SEQ ID No. 1-SEQ ID No. 5, and the EAAT2 polyclonal primary antibody can be prepared according to at least one of the extracellular epitope fragments; correspondingly, the flow fluorescence secondary antibody includes a fluorescent developing marker that can be excited in a flow cytometer, and also includes a segment that can be specifically recognized by the EAAT2 polyclonal primary antibody, and the flow fluorescence secondary antibody in the embodiment is derived from a goat anti-rabbit polyclonal antibody;

[0055] ③ EAAT2 isotype control, used as a negative control, using rabbit-derived IgG ISOtype;

[0056] ④ CD61-PE, i.e., a monoclonal antibody of CD61 directly coupled with PE fluorescent dye, which can also include other platelet-related marker antibodies,

[0057] ⑤ Fixing solution, 1% paraformaldehyde is used in the embodiment.

[0058] Among them, the EAAT2 / GLT-1 antibody is a mouse-derived polyclonal antibody, which can recognize the EAAT2 membrane protein of human, monkey, rat and mouse cells, and the recognition site is located in the extracellular segment of the EAAT2 membrane protein, such as at least one of the amino acid sequences shown in SEQ ID No. 1-SEQ ID No. 5; the secondary antibody thereof is Anti-rabbit IgG (H+L); and the isotype control of the primary antibody is rabbit-derived IgG ISOtype. CD61 in CD61-PE is also called GPIIIa, which can form GPIIb / IIIa complex with CD41, participates in platelet aggregation, and is the most commonly used pan-platelet marker, which is used to identify cells as platelets and to perform phenotyping of platelets in combination with EAAT2 membrane protein.

[0059] Referring to FIG. 1, according to the changes generated after platelet stress, the scatter plot clusters more than two groups of cells, and further classification can classify these cells into four regions, namely Q1 region in the upper left, Q2 region in the upper right, Q3 region in the lower right, and Q4 region in the lower left: the number of cells in the Q1 region sharply increases after the transition from the inactivated state to the activated state, and there is a certain increase in the state of first inhibition and then activation; the number of cells in the Q2 region also increases after the transition from the inactivated state to the activated state, and the increase is quite significant in the state of first inhibition and then activation. According to the detection results, it is considered that the Q1 region clusters over-stressed platelets with enhanced expression of EAAT2 membrane protein, the Q2 region clusters stressed platelets with enhanced expression of EAAT2 membrane protein, the Q3 region clusters resting platelets (inactivated state), and the Q4 region clusters disintegrated platelets with sharp reduction of EAAT2 membrane protein expression. Thus, the present application quantifies four different platelet subpopulations labeled by CD61 (pan-platelet marker) and EAAT2 membrane protein by flow cytometry, which can directly present four different stages of cell phenotypes related to the basic state and activated state of platelets: resting state (stable), stressed state (hyperactive), over-stressed state (excessive reaction or necrosis), and disintegrated state (degradation or fragmentation). According to the proportion of cells in the four regions, the condition of the individual or patient corresponding to the sample can be determined and predicted.

[0060] Referring to FIG. 2, the use of negative controls makes the positive results more significant, which is beneficial to improve the detection sensitivity of the kit of the present application. Specifically, the Q1 region and the Q2 region differ significantly between the negative control and the positive control.

[0061] Using the detection method of the kit of the present application, the basal level of platelet EAAT2 membrane protein of the subject can be effectively detected and analyzed by using EAAT2 and CD61 as double labels. EAAT2 and CD61 double labels can be further used to analyze the distribution and proportion of different platelet subtypes related to platelet activation of the subject.

[0062] The kit of the present application can also add flow antibodies for labeling other relevant markers of platelets; other platelet-related markers include CD41, CD42b, CD62P, CD63, and PAC-1, etc. Among them, CD41 is also known as GPIIb: an integrin on the surface of platelets, which is involved in platelet aggregation and thrombus formation; CD42b is also known as GPIb: a von Willebrand factor receptor on the surface of platelets, which is involved in the adhesion of platelets to the damaged blood vessel wall; CD62P is also known as P-selectin: which is transferred from the alpha granules inside the platelets to the surface of the platelets after activation, and is involved in the adhesion of platelets to leukocytes and inflammatory response; CD63: a marker of granules (alpha granules and dense granules) inside the platelets, and the increase of its surface expression is related to platelet activation; PAC-1: an antibody that binds to active GPIIb / IIIa integrin, which is used to determine the state of platelet aggregation.

[0063] The kit of the present application also includes a stress modulator of platelets, which includes an activator, an inhibitor, an interfering agent or an anti-interfering agent for the platelets; the activator includes various activators that can be used to activate platelets, such as arachidonic acid (AA), adenosine diphosphate (ADP), collagen (Coll), epinephrine (EPI), thrombin, thrombin receptor activating enzyme (TRAP), lipopolysaccharide (LPS), etc.; the inhibitor includes GPRP tetrapeptide used to inhibit fibrin polymerization and prevent platelet aggregation in the activation experiment. The above stress modulators should be intervened or pretreated on the sample before the EAAT2 polyclonal primary antibody and CD61-PE are added.

[0064] By selecting the polyclonal flow primary antibody Rabbit Polyclonal EAAT2 / GLT1 Antibody and the secondary antibody Anti-rabbit IgG (H+L), F(ab’)2 Fragment (Alexa Fluor Conjugate) that can specifically recognize the extracellular segment of EAAT2, and the pan-platelet marker CD61 direct antibody (CD61-PE), a flow detection kit for specifically recognizing the platelet EAAT2 membrane protein is provided. By using this kit to process the blood sample of the subject, and by means of flow cytometry detection, the intuitive results of the real-time expression of platelet EAAT2 membrane protein of the subject can be obtained by flow analysis software. In addition, by observing the expression of platelet EAAT2 membrane protein and analyzing the correlation changes of other platelet markers under different intervention conditions, it is not only helpful to analyze the basic state of platelets, but also helpful to evaluate the strength of the reaction of platelets to different stimuli and to analyze different subtypes and proportions of platelets.

[0065] In one possible implementation, the responsiveness and regulation potential of platelets to plasma glutamate signals and to activators can be further evaluated by using high concentration of glutamate to incubate platelets, using platelet activators (such as Thrombin in FIG. 3) to induce platelets, or superimposing EAATs inhibitors (such as DL-TBOA in FIG. 3) to pre-treat platelets (aiming to block all transport channels of EAAT proteins on the platelet membrane).

[0066] Under normal conditions, the EAATs uptake activity of resting platelets is low; when exposed to a high concentration of glutamate microenvironment, the EAATs uptake activity of platelets can be enhanced by about 2 times; when stimulated by thrombin, the immunogenicity of platelet EAATs is enhanced, and the glutamate uptake activity can be enhanced by 5-9 times. These results show that the activity of platelet EAATs in taking up glutamate is significantly different under different conditions, and the associated biological effects are also obviously different. Previous studies have found that platelet activation depends on the enhanced uptake of glutamate by EAATs, and since EAAT2 dominates the uptake of glutamate by platelets, the membrane protein level of EAAT2 can reflect the glutamate uptake activity of platelets to some extent. Monitoring the expression level of platelet EAAT2 membrane protein not only helps to evaluate the glutamate uptake activity of platelets under different physiological and pathological conditions, but also helps to understand the real-time activation status of platelets.

[0067] The specific detection steps of the kit of the present application are as follows:

[0068] Blood sample collection: 2 mL of venous blood was collected in a sodium citrate anticoagulant tube and gently mixed.

[0069] Centrifugal separation of platelet-rich plasma (PRP): centrifuged at 200g for 10-15 min at room temperature, and PRP was separated after standing for 30 min.

[0070] Blood sample dilution: 5 μL of PRP was taken and added to 95 μL of platelet diluent to form a reaction system.

[0071] Pre-treatment: three identical samples were prepared, the first sample was only added with thrombin (containing calcium) with a final concentration of 0.25-0.5 IU to stimulate platelets for 2 min, aiming to evaluate the response degree of platelets to thrombin stimulation through EAAT2 membrane protein expression; the second sample was added with DL-TBOA with a final concentration of 4 mM to pre-treat platelets for 10 min, and then added with thrombin (containing calcium) with a final concentration of 0.25-0.5 IU to stimulate platelets for 2 min, aiming to evaluate the response potential of platelets to thrombin stimulation through EAAT2 membrane protein expression; the third sample was not treated.

[0072] Staining: Add 1 μL of EAAT2 / GLT-1 antibody (primary antibody) and 1 μL of CD61-PE, incubate at room temperature in the dark for 15–30 min, then add 1 μL of EAAT2 secondary antibody—Anti-rabbit IgG (H+L), Fragment (Alexa) Continue incubation at room temperature in the dark for 15–20 minutes (using Conjugate).

[0073] Fixation: Add 1 mL of fixative and fix for 30 min.

[0074] Analyze the data using a CytoFLEX flow cytometer. Analytical conditions: Select the PE channel for CD61 fluorescence and the APC channel for EAAT2 fluorescence. APC gain should be between 20 and 350, and PE around 300. Adjust the compensation as needed based on the actual situation. If the prepared blood sample cannot be immediately analyzed by flow cytometry, protect it from light, store it at 4°C, and complete the analysis within 24 hours. Finally, analyze the data using CytExport software.

[0075] In other possible embodiments, the amounts of primary antibody, secondary antibody, and CD61-PE added can be adjusted within the range of 0.2–5 μL as needed; when flow cytometry antibodies for other platelet-related markers need to be added, the amounts of various antibodies added can also be adjusted within the range of 0.2–5 μL as needed; it should be understood that the amount of each antibody added should be adapted to the concentration of the antibody stock solution so that the final concentration of the antibody in the reaction system reaches the preset value. Further, through experiments, the reference values ​​for the final concentrations of various stress modulators in the reaction system are as follows: thrombin: 0.25–2 U / mL; collagen: 1–20 μg / mL; adenosine diphosphate (ADP): 10–40 μM; receptor activator of thrombin (TRAP): 50–200 μM; GPRP: 0.01–5 mM.

[0076] Referring to Figure 4, the summary results of platelet EAAT2 membrane protein expression in a patient with AIS (Acute Ischemic Stroke) and hemorrhagic transformation were analyzed. By analyzing the expression of EAAT2 membrane protein and its phenotypic changes in platelets on the first, second, and seventh days after the stroke, it was found that the inhibitory effect of DL-TBOA on platelet aggregation function increased from weak to strong. Figure 4A-I, flow cytometry contour plots with CD61 and EAAT2 membrane protein as the x and y axes, show that under basal conditions, during thrombin activation, and after thrombin reactivation following DL-TBOA treatment, the patient's platelets exhibited various activation-related phenotypes that changed over time.

[0077] In one possible embodiment, the CD61-PE in the kit can also help to distinguish or exclude non- or weakly-reactive platelet populations.

[0078] Referring to FIG. 5, FIG. 5A-5B are flow cytometry scatter plots showing the expression of CD61 and EAAT2 membrane proteins on healthy human platelets under basal conditions (Basal) and under thrombin activation (0.5 U Thrombin). FIG. 5C-5F show that the platelets can be broadly classified into three states based on the cell size, activation characteristics of the CD61 low-fluorescence platelet subpopulation (CD61+) and the CD61 high-fluorescence platelet subpopulation (CD61++): low-activated state (CD61++, PAC-1 low / CD62P low), high-activated state (CD61++, PAC-1 high / CD62P high) and weakly-reactive state (CD61+, PAC-1 low / CD62P low). FIG. 5G-5I show the expression of EAAT2 membrane proteins on the CD61+ platelet subpopulation and the CD61++ platelet subpopulation under basal conditions and under thrombin stimulation. (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).

[0079] The results of FIG. 5 show that CD61 can separate the platelets into two populations: CD61 low-fluorescence (CD61+) and CD61 high-fluorescence (CD61++). Using the activation markers PAC-1 and CD62P, the CD61++ platelets can be further classified into low-activated platelets (CD61++, PAC-1 low / CD62P low) and high-activated platelets (CD61++, PAC-1 high / CD62P high). The CD61+ platelets show no significant difference or only a weak increase in PAC-1 and CD62P signals before and after thrombin activation, and thus can be determined as non- or weakly-reactive platelets.

[0080] Accordingly, it is speculated that the CD61+ platelets can be composed of damaged platelets and platelet fragments. Therefore, the four platelet phenotypes were further verified using Annexin V, which is a Ca 2+ dependent phospholipid-binding protein commonly used to identify early apoptotic or necrotic cells. The Annexin V expression characteristics of the four platelet phenotypes using the EAAT2-CD61 combined markers.

[0081] The results are shown in Figure 6, Figure 6A is a platelet flow density chart of EAAT2 membrane protein negative homotypic control, and Figures 6B-6C are platelet flow scatter charts before and after thrombin activation. The vertical coordinates of Figures 6A-6K represent the expression level of EAAT2 membrane protein, the horizontal coordinates of Figures 6A-6C represent the expression level of CD61, and the horizontal coordinates of Figures 6D-6K represent the expression level of Annexin V.

[0082] The P4 region platelets show low expression of Annexin V, while the P1, P2 and P3 regions show high expression. Accordingly, it is believed that the P1-P3 platelets are mainly platelets in early apoptosis or necrosis and platelet debris, and the P4 region platelets are mainly resting platelets. The CD61 low fluorescence platelets (i.e. P1 and P2 region platelets) significantly increase after thrombin activation, and can be further divided into two phenotypes of high EAAT2 expression and low EAAT2 expression. Considering that platelets contain a large amount of EAAT2 protein, they can only be recognized by EAAT2 membrane protein antibodies after being localized on the cell membrane surface or cell membrane damage. It is inferred that the P1 region platelets are necrotic platelets with intracellular and extracellular EAAT2 protein labeled after platelet membrane damage; the CD61, EAAT2 and Annexin V signals of the P2 region platelets are significantly reduced, so they are likely to be mainly platelet debris or fragments that have lost a large amount of membrane protein after activation.

[0083] Further, the correlation between the expression of EAAT2 membrane protein and the increase of platelet PAC-1 expression was investigated. Figure 7A is a flow scatter chart of unactivated platelets stained with EAAT2-APC\CD61-PE\PAC-1-FITC\TLR4-PC7 four-way staining. The P1 region corresponds to the aforementioned resting platelets, and the P2 region corresponds to the aforementioned stressed platelets. Figure 7B is a comparison of the average fluorescence intensity of PAC-1 in P1 and P2 platelets.

[0084] The CD61 high fluorescence portion of the platelets can be further divided into EAAT2 low expression platelets (corresponding to resting platelets) and EAAT2 high expression platelets (stress platelets). The difference in activation level of the two can be seen in Figure 7, and the P2 region platelets have a significantly higher PAC-1 fluorescence signal than the P1 region platelets. PAC-1 is used to describe the aggregation state of platelets, and the higher the PAC-1 signal, the higher the degree of activation of the platelets. The present application can accurately obtain various parameters of the cells (such as target protein fluorescence signal, cell size and granularity, etc.) from different dimensions with the detection advantages of flow cytometry, and the obtained data is rich and reliable. In addition, according to the actual needs, the visualization chart of multiple parameters can be easily realized. In addition to this, the operation of the kit is simple and the experimental period is short, which can not only clearly show the EAAT2 membrane protein expression of resting platelets and activated platelets, but also can flexibly add an intervention group to obtain the results after different interventions under the same experimental conditions, greatly widening the research space and application range of this index. The experimental period can be completed within two hours, which can meet the needs of clinicians to grasp the platelet stress condition of patients, and is suitable for popularization and application in clinical medical treatment, and can be further applied to diagnosis and treatment related to stroke, neurodegenerative diseases, autoimmune diseases and cancer, etc.

[0085] In summary, the present application provides a method for detecting platelet stress condition using a kit, wherein the kit comprises platelet diluent, EAAT2 polyclonal antibody, flow cytometry fluorescence secondary antibody, EAAT2 homologous control, CD61 directly coupled PE fluorescent dye monoclonal antibody and 1% paraformaldehyde; the EAAT2 polyclonal antibody recognizes at least one of the amino acid sequences shown as SEQ ID No. 1-SEQ ID No. 5; the detection method comprises the following steps: preparing platelet suspension using the platelet diluent; sequentially incubating the platelet suspension with the EAAT2 polyclonal antibody, the CD61 directly coupled PE fluorescent dye monoclonal antibody and the flow cytometry fluorescence secondary antibody; adding the paraformaldehyde to fix the sample to form a sample to be detected; detecting the specific signal of the sample by flow cytometry; and statistically analyzing the specific signal to confirm the stress condition of the sample. The method of the present application utilizes the advantages of flow cytometry, which can analyze individual platelets, thereby analyzing the overall stress condition of the platelets of the individual corresponding to the blood sample from a statistical point of view, providing reference data for subsequent judgment and prediction of the patient's condition; and the reagents used in the entire flow cytometry are reasonably matched to form a kit, which is conducive to the standardization of the detection method.

[0086] The above embodiments are the preferred embodiments of the present application, but are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application shall be equivalent replacement manners and shall be included in the protection scope of the present application.

Claims

1. A method for detecting a stress condition of platelets using a kit, characterized by, The kit comprises a platelet diluent, an EAAT2 polyclonal primary antibody, a flow fluorescence secondary antibody, an EAAT2 isotype control, a monoclonal antibody directly coupled with PE fluorescence dye of CD61 and 1% paraformaldehyde; the EAAT2 polyclonal primary antibody recognizes at least one of the amino acid sequences shown in SEQ ID No. 1-5; The detection method comprises the following steps: Preparation of a platelet suspension by using the platelet diluent; Incubation of the platelet suspension by using the EAAT2 polyclonal primary antibody, the monoclonal antibody directly coupled with PE fluorescence dye of CD61 and the flow fluorescence secondary antibody in sequence; Addition of the paraformaldehyde to fix the sample to form a sample to be detected; Detection of specific signals of the sample by using a flow cytometer; Statistical analysis of the specific signals to confirm the stress condition of the sample.

2. The method of claim 1, wherein, The platelet diluent comprises 10 mM HEPES, 135-145 mM NaCl, 2.6-2.8 mM KCl, 11-12 mM NaHCO3, 1-2 mM MgCl2, 0.4-0.5 mM Na2HPO4, 5-6 mM glucose, 0.2-0.4% bovine serum albumin, and the pH is 7.2-7.

4.

3. The method of claim 1, wherein, The flow fluorescence secondary antibody comprises a chromogenic marker and is an antibody specifically recognizing the EAAT2 polyclonal primary antibody, which is derived from goat anti-rabbit; the EAAT2 isotype control is rabbit-derived IgG ISOtype.

4. The method of claim 1, wherein, The kit further comprises other platelet-related marker antibodies, including specific antibodies of CD41, CD42b, CD62P, CD63 and / or PAC-1.

5. The method of claim 1, wherein, The incubation of the platelet suspension by using the EAAT2 polyclonal primary antibody, the monoclonal antibody directly coupled with PE fluorescence dye of CD61 and the flow fluorescence secondary antibody in sequence comprises: First, the platelet suspension is incubated at room temperature and away from light by using the EAAT2 polyclonal primary antibody and the monoclonal antibody directly coupled with PE fluorescence dye of CD61 for 15-30 min; Then, the platelet suspension is incubated at room temperature and away from light by using the flow fluorescence secondary antibody for 15-20 min.

6. The method of claim 1, wherein, The kit further comprises a stress regulator of platelets, and the stress regulator comprises an activator, an inhibitor, an interfering agent or an anti-interfering agent for the platelets; the stress regulator is added according to the need of statistical analysis before the incubation of the platelet suspension by using the EAAT2 polyclonal primary antibody, the monoclonal antibody directly coupled with PE fluorescence dye of CD61 and the flow fluorescence secondary antibody in sequence.

7. The method of claim 6, wherein, The stress regulator comprises glutamic acid, arachidonic acid, adenosine diphosphate, collagen, adrenaline, thrombin, thrombin receptor activating enzyme or lipopolysaccharide as an activator, an excitatory amino acid transporter inhibitor as an inhibitor, and an inhibitor for preventing fibrin polymerization to prevent blood clot formation.

8. The method of claim 1, wherein, The preparation method of the platelet suspension comprises: Anticoagulation treatment of freshly collected blood; Centrifugation at 200g at room temperature for 10-15 min; After harvesting the blood plasma, let it stand for 30 minutes to obtain the platelet-rich plasma; 5 μL of the platelet-rich plasma is taken and added to 95 μL of the platelet diluent to prepare a platelet suspension.

9. The method of claim 1, wherein, The specific signal is associated with the relationship between excitatory amino acid transporter 2 and platelets, and the cell analysis results of the flow cytometer are counted to confirm the cell phenotype of the platelets under different stress conditions.

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