Method for detecting thrombin generation promotion capability of extracellular vesicles in blood sample

Abstract

The invention relates to the technical field of biology, and discloses a method for detecting thrombin production promotion capability of extracellular vesicles in a blood sample, which comprises the following steps: centrifuging the blood sample collected within 2 hours at 2500g at room temperature for 15 minutes, sucking upper platelet-deficient plasma and placing in a centrifuge tube, centrifuging at 2500g at room temperature again for 15 minutes, and sucking upper platelet-free plasma; centrifuging the obtained platelet-free plasma at 4 DEG C at 20000g for 1 hour; and sucking the upper-layer extracellular vesicle-free plasma for later use, taking the obtained extracellular vesicle precipitate as a to-be-detected sample, and detecting the thrombin generation promotion capability of the extracellular vesicles by using a thrombin generation analyzer. The method provided by the invention can be used for accurately detecting the capability of the extracellular vesicles in the blood sample for promoting thrombin generation, has the advantages of convenience in operation, easiness in obtaining the sample, noninvasiveness, no damage, high detection speed, low cost and the like, has important significance on thrombus risk assessment and prediction, and has better clinical application value.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically to a method for detecting the ability of extracellular vesicles in a blood sample to promote thrombin generation. Background Art

[0002] Extracellular vesicles (EVs) are tiny vesicles released into body fluids by budding from endosomes or cell membranes during cell activation and apoptosis. During the release process of extracellular vesicles, they carry substances of the parental cells, including proteins, lipids, nucleic acids, etc., and can also bind to certain proteins present in body fluids to perform corresponding functions. Therefore, extracellular vesicles can perform functions similar to those of parental cells and functions that parental cells do not possess. Extracellular vesicles can be derived from a variety of cells, and those that have been studied more include platelet-derived extracellular vesicles, red blood cell-derived extracellular vesicles, neutrophil-derived extracellular vesicles, endothelial cell-derived extracellular vesicles, and tumor cell-derived extracellular vesicles. The functions exerted by extracellular vesicles are diverse. In the field of thrombosis and hemostasis, extracellular vesicles play an important role in thrombus formation. Extracellular vesicles carry tissue factor and phosphatidylserine in the coagulation cascade, providing tissue factor for the extrinsic coagulation pathway and a phospholipid surface for both the extrinsic and intrinsic coagulation pathways. In addition to the field of thrombosis and hemostasis, extracellular vesicles are widely involved in the pathophysiological processes of various diseases, such as inflammatory diseases, immune system diseases, and cancers. Extracellular vesicles act as messengers in them, transmitting various signals for material exchange and signal transmission between cells.

[0003] Currently, there are various methods for detecting the procoagulant function of extracellular vesicles, including flow cytometry, immunoassay, confocal microscopy, and other methods.

[0004] Flow cytometry can detect the quantitative differences in extracellular vesicles expressing tissue factor and phosphatidylserine, but it only evaluates the function of extracellular vesicles in terms of quantity and cannot intuitively reflect the function of extracellular vesicles.

[0005] Since extracellular vesicles carry tissue factor and phosphatidylserine on their surfaces, Elisa reagents can be used to detect the expression levels of tissue factor and phosphatidylserine, thereby reflecting the procoagulant activity of extracellular vesicles. However, the Elisa method has poor sensitivity and a high detection limit, and cannot finely evaluate the procoagulant activity of extracellular vesicles.

[0006] Confocal microscopy is another method for evaluating the quantity of extracellular vesicles. The detection effect is similar to that of flow cytometry, and the disadvantage is also that it cannot more intuitively observe the procoagulant function of extracellular vesicles. Summary of the Invention

[0007] (I) Technical Problems to be Solved

[0008] In view of the deficiencies of the prior art, the present invention provides a method for detecting the ability of extracellular vesicles in a blood sample to promote thrombin generation, which has the advantages of simple operation, easy sample acquisition, non-invasive and harmless, fast detection speed, and more comprehensive evaluation.

[0009] (II) Technical solution

[0010] To achieve the above object, the present invention provides the following technical solution: A method for detecting the ability of extracellular vesicles in a blood sample to promote thrombin generation, comprising the following steps:

[0011] Step 1: Centrifuge the blood sample collected within 2 hours at 2500 g for 15 minutes at room temperature, aspirate the upper platelet-poor plasma and place it in a centrifuge tube, then centrifuge it again at 2500 g for 15 minutes at room temperature, aspirate the upper platelet-free plasma, centrifuge the obtained platelet-free plasma at 20000 g for 1 hour at 4 °C, and aspirate the upper extracellular vesicle-free plasma for standby;

[0012] Step 2: Place a mixture containing four types of fluorescence localization microspheres with diameters for positioning the detection gates of 0.1 μm and 1 μm in a flow tube, load the sample, adjust the threshold of the side scatter channel of the flow cytometer, the voltages of the side scatter, forward scatter, and FITC channels, and determine the upper and lower limits of the extracellular vesicle detection gate;

[0013] Step 3: Add normal human pooled plasma to the extracellular vesicle precipitate obtained in Step 1 to prepare an extracellular vesicle resuspension, shake and mix well, place it in a TruCount absolute counting tube, load the sample for flow cytometry detection, and calculate the content of the obtained extracellular vesicle resuspension according to the known number of counting fluorescence microspheres in the TruCount absolute counting tube and the flow cytometry results;

[0014] Step 4: According to the content of the extracellular vesicles obtained in Step 3, adjust the test samples with different extracellular vesicle contents to the same concentration with normal human pooled extracellular vesicle-free plasma;

[0015] Step 5: Add the test samples with the same concentration to a 96-well plate and detect the ability of the extracellular vesicles to promote thrombin production on a machine. This well is the sample well;

[0016] Step 6: Add the extracellular vesicle-free plasma obtained in Step 1 to a 96-well plate as a standard well;

[0017] Step 7: After loading the sample well and the standard well, detect the ability of the extracellular vesicles to promote thrombin production on a machine;

[0018] Preferably, in Step 1, the blood sample is freshly collected peripheral venous blood, and the anticoagulant used is sodium citrate.

[0019] Preferably, in step two, the four diameters are 0.22 μm, 0.45 μm, 0.88 μm, and 1.34 μm respectively.

[0020] Preferably, in step three, the calculation formula for the extracellular vesicle resuspension is: EVs / μL = total counting microspheres in the absolute counting tube × number of collected extracellular vesicles / (number of collected counting microspheres × sample volume in μL).

[0021] Preferably, step two and step three are continuously adjusted to each other to jointly obtain the voltage and threshold of this process, specifically as follows: the threshold of the FSC and SSC channels is 200 volts, the voltage of the SSC channel is 566 volts, the voltage of the FSC channel is 780 volts, and the voltage of the FITC channel is 500 volts.

[0022] Preferably, in step four, serial dilution is preferably used to adjust the concentration of extracellular vesicles, and a maximum dilution of 10 - fold is allowed. Dilutions exceeding 10 - fold will be regarded as having large errors and not meeting the experimental requirements.

[0023] Preferably, to avoid experimental errors, 3 replicate wells are set for the sample wells and the standard wells in steps five and six. 80 μl of the extracellular vesicle resuspension with the same concentration obtained in step four is added to each sample well, and then 20 μl of PBS solution is added respectively. At this time, the total volume of the solution in each well is 100 μl; 3 replicate wells are set for the standard wells to avoid experimental errors; 80 μl of cell - free plasma obtained in step one is added to each standard well, and then 20 μl of thrombin calibrator is added respectively. At this time, the total volume of the solution in each well is 100 μl; the 96 - well plate is required to be a transparent round - bottom plate and cannot be covered when loaded onto the machine. After sample addition is completed, it can be loaded onto the machine for detection.

[0024] Preferably, in step 7, after the sample wells and standard wells are loaded onto the machine, they are first incubated at 37 °C for 10 min. The fluorescent buffer is placed in a 37 °C water bath. After incubation, the fluorescent buffer is taken out from the 37 °C water bath, 40 μl of fluorescent substrate is added, and it is pipetted and mixed well to form fluca. Then 20 μl of fluca is added to each of the standard wells and sample wells by the machine. At this time, the total volume of the solution in the sample wells and standard wells is both 120 μl.

[0025] Preferably, in step 7, the ability of extracellular vesicles to promote thrombin production is evaluated according to the following four indicators:

[0026] Indicator 1: Lagtime, the time from the start of the reaction to the start of thrombin generation. The shorter the time, the stronger the ability to promote thrombin generation;

[0027] Indicator 2: ETP, thrombin generation potential, the amount of thrombin generated per minute. The higher the content, the stronger the ability to promote thrombin generation;

[0028] Index 3: Peak, the maximum peak value of thrombin generation, the maximum amount of thrombin generated. The higher the peak value, the stronger the ability to promote thrombin generation;

[0029] Index 4: ttPeak, the time required from the start of thrombin generation to reaching the highest point of thrombin generation. The shorter the time, the stronger the ability to promote thrombin generation;

[0030] Record the results of extracellular vesicle Lagtime, ETP, Peak, and ttPeak in different wells for data analysis.

[0031] Compared with the prior art, the present invention provides a method for detecting the ability of extracellular vesicles in a blood sample to promote thrombin generation, having the following beneficial effects: establishing a relatively standardized method with good sensitivity, which can detect the ability of any extracellular vesicles to promote thrombin production, and having the advantages of simple operation, easy availability of samples, non-invasive and non-destructive, fast detection speed, and more comprehensive evaluation. By detecting the ability of extracellular vesicles in blood to promote thrombin production, the procoagulant intensity in patients with different diseases can be evaluated, which is of great significance for thrombus risk assessment and prediction and has good clinical application value. Description of the Drawings

[0032] Figure 1 To establish the EVs gate using the SPHEROTM flow cytometry nano-fluorescent size standard kit for flow cytometry:

[0033] (a) The kit consists of 4 fluorescent beads with different diameters ranging from 0.22μm to 1.33μm;

[0034] (b) The upper limit of the EVs gate is determined according to the 0.88μm calibration microspheres, and the lowest detectable limit of EVs is established according to the 0.22μm calibration microspheres;

[0035] (c) Finally, the EVs gate is determined based on the detectable upper limit and the lowest limit of the beads.

[0036] Figure 2 To detect the results of different amounts of extracellular vesicles promoting thrombin generation using a thrombin generation analyzer, the thrombin generation conditions at concentrations of 10 / μl, 100 / μl, 1000 / μl, 10000 / μl, and 100000 / μl were detected in this experiment:

[0037] (a) Curves of promoting thrombin generation at concentrations of 10 / μl, 100 / μl, 1000 / μl, 10000 / μl, and 100000 / μl;

[0038] Figure 3The results of thrombin generation promoted by extracellular vesicles with different contents detected by a thrombin generation analyzer are bar charts of thrombin generation promoted at concentrations of 10 / μl, 100 / μl, 1000 / μl, 10000 / μl, and 100000 / μl respectively.

[0039] Figure 4 Results of thrombin generation in 26 patients with traumatic lower limb fractures (13 thrombus patients and 13 non - thrombus patients) at an EVs concentration of 100,000 / μl. Detailed implementation mode

[0040] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0041] Embodiment: Ability of a thrombin generation analyzer to detect thrombin generation promoted by extracellular vesicles in peripheral blood samples

[0042] I. Test samples:

[0043] Patients with traumatic lower limb fractures (13 thrombus patients and 13 non - thrombus patients), healthy physical examination subjects, and extracellular vesicles of healthy physical examination subjects at different concentrations.

[0044] 1. Based on previous literature research experience (Gasa, Noluthando, Meiring, Muriel (2021). Microparticles: a link to increased thrombin generation Blood Coagul Fibrinolysis 32(3):204 - 208.), the mean difference in peak thrombin generation between the two groups is Δμ approximately ±246 (nmol / L), the standard deviation σ is approximately 193, the test power is 0.90, and α = 0.05. Calculating the sample size is approximately 7 cases per group. Therefore, the sample size per group is at least 7 cases or more.

[0045] 2. Selection of research subjects:

[0046] A total of 162 consecutive research subjects were selected from May 2024 to December 2024, including 60 male healthy subjects, 60 female healthy subjects, 26 patients with traumatic lower limb fractures (13 in the thrombus group and 13 in the non - thrombus group), and 16 healthy people used to evaluate the experimental performance.

[0047] (1) Healthy people

[0048] Inclusion criteria: Healthy physical examination subjects

[0049] Exclusion criteria: Abnormal test results of blood pressure, routine urine and feces tests, liver and kidney functions, coagulation routine, etc., and internal and surgical diseases such as diabetes, autoimmune diseases, and pancreatitis.

[0050] Grouping criteria: Divided into male and female according to the medical record report

[0051] (2) Trauma patients

[0052] Inclusion criteria: Patients diagnosed by a doctor with lower limb fractures due to trauma

[0053] Exclusion criteria: Active inflammation; cancer; active bleeding; VTE; atrial fibrillation; pregnancy; hemophilia; patients receiving warfarin or other antithrombotic drug treatment before surgery.

[0054] Grouping criteria: The research subjects were divided into a thrombosis group and a non-thrombosis group according to preoperative lower limb venous color Doppler ultrasound.

[0055] 3. Acquisition of relevant information of research subjects

[0056] Information such as the gender, age, underlying diseases, surgical risk factors (thrombosis history, surgical history within half a year before enrollment, smoking history, hypertension history, diabetes history, hemoglobin, platelet count, coagulation routine), and medication history (whether there is a history of antiplatelet drugs) of the research subjects was obtained from their hospital medical record materials.

[0057] II. Sample collection and preparation

[0058] A nurse drew a tube of peripheral venous blood from the research subject (3 mL in total, and the volume ratio of 3.2% sodium citrate to peripheral blood was 1:9) using a blue-capped anticoagulant tube containing 3.2% sodium citrate anticoagulant, and gently inverted and mixed well. The fresh sodium citrate anticoagulated blood was centrifuged at 2500 g for 15 minutes at room temperature, and the upper layer of platelet-poor plasma (PPP) was aspirated until 1 cm away from the white blood cell layer. The aspirated plasma was placed into a 1.5 mL Eppendorf tube and centrifuged again at 2500 g for 15 minutes at room temperature, and the upper layer of platelet-free plasma (PFP) was aspirated and transferred into a 1.5 mL Eppendorf tube. The aspirated platelet-free plasma was centrifuged at 20000 g for 1 h at 4°C, and the upper layer of extracellular vesicle-free plasma was aspirated and aliquoted for later use. The lower layer of extracellular vesicle precipitate and the aliquoted extracellular vesicle-free plasma were stored in an -80°C refrigerator for later use. All the above operations were completed within 2 hours after fresh whole blood collection.

[0059] III. Instruments

[0060] Flow cytometry instrument Canto II, Thrombin Generation Analyzer

[0061] IV. Reagents

[0062] SPHEROTM Flow Cytometry Nanofluorescent Size Standard Kit, TruCount Absolute Count Tubes, Thrombin Calibrator, Fluca-Kit, Phosphate Buffered Saline

[0063] V. Reagent Preparation

[0064] Before the experiment, reconstitute the thrombin calibrator with sterile water. Add 1 ml of sterile water to each vial of calibrator. Place the Fluo-Buffer in the Fluca-Kit in a 37°C water bath and add the Fluo-Substrate and mix well by pipetting immediately before loading onto the instrument. Reagents should be prepared immediately before use.

[0065] VI. Experimental Procedures

[0066] 1. Establish a method for detecting the content of circulating extracellular vesicles

[0067] (1) Pipette one drop of fluorescently labeled microspheres capable of localizing the 0.1 μm and 1 μm detection gates, each with four diameter sizes (0.22 μm, 0.45 μm, 0.88 μm, 1.34 μm), into a flow tube, add 500 μl of PBS, and load at low flow rate.

[0068] (2) Adjust the threshold of the side scatter channel of the flow cytometer, the voltages of the side scatter, forward scatter, and FITC channels to determine the upper and lower limits of the extracellular vesicle detection gate;

[0069] In step (2), continuously debug to obtain the specific parameters of the voltages of each channel and the thresholds of the FSC and SSC channels in this process, as shown in Tables 1 and 2

[0070] Table 1. Voltages of FSC, SSC, and FITC Channels

[0071] Parameter Voltage (V) FSC 780 SSC 566 FITC 500

[0072] Table 2. Thresholds of FSC and SSC Channels

[0073] Parameter Threshold FSC 200 SSC 200

[0074] 2. Standardization of the concentration of extracellular vesicles

[0075] Add normal human mixed plasma to the obtained extracellular vesicle precipitate to prepare an extracellular vesicle resuspension. After oscillation, pipette 10 μl and place it in a TruCount absolute counting tube. Then add 300 μl of PBS solution and load the sample at a low flow rate for flow cytometry detection. Calculate the content of the obtained extracellular vesicle resuspension according to the known number of counting fluorescent microspheres in the TruCount absolute counting tube and the flow cytometry results. The calculation formula for the extracellular vesicle resuspension is: EVs / μL = total counting microspheres in the absolute counting tube × number of collected extracellular vesicles / (number of collected counting microspheres × sample volume in μL). Adjust the extracellular vesicle suspensions of different samples to the same concentration using extracellular vesicle-free plasma. The EVs concentration set in this experiment is 100,000 / μl.

[0076] Sample addition to standard wells and sample wells

[0077] The sample addition settings for the sample wells are as follows:

[0078] To avoid experimental errors, set 3 replicate wells for the sample wells;

[0079] Sample addition to the sample wells: Add 80 μl of the obtained extracellular vesicle resuspension with the same concentration to each sample, and then add 20 μl of PBS solution respectively. At this time, the total volume of the solution in each well is 100 μl;

[0080] The sample addition settings for the standard wells are as follows:

[0081] To avoid experimental errors, set 3 replicate wells for the standard wells;

[0082] Sample addition to the standard wells: Add 80 μl of the obtained extracellular vesicle-free plasma to each well, and then add 20 μl of thrombin calibrator respectively. At this time, the total volume of the solution in each well is 100 μl;

[0083] For the 96-well plate: The 96-well plate is required to be transparent round-bottomed and cannot be capped for on-machine detection. After sample addition, it can be directly loaded onto the machine for detection.

[0084] The operations after loading onto the machine are as follows:

[0085] After loading onto the machine, incubate at 37 °C for 10 min. Place the fluorescence buffer in a 37 °C water bath. After incubation, take out the fluorescence buffer from the 37 °C water bath, add 40 μl of fluorescence substrate, and pipette to mix evenly to form fluca. Then add 20 μl of fluca to each of the standard wells and sample wells by the machine. At this time, the total volume of the solution in the sample wells and standard wells is 120 μl.

[0086] 3. Precision determination:

[0087] (1) Take 10 μl of the EVs resuspension of healthy human mixture in a TruCount absolute counting tube, add 300 μl of PBS, mix well, and then load onto the machine to detect the EVs concentration;

[0088] (2) Adjust the concentration of the mixed EVs suspension to 100,000 / μl, aliquot, and repeat the measurement using the established method for detecting the promotion of thrombin generation by extracellular vesicles three times a day for three consecutive days.

[0089] (3) Analyze the precision of Lagtime, ETP, Peak, and ttPeak.

[0090] 4. Result reading

[0091] Evaluate the ability of extracellular vesicles to promote thrombin production according to the following four indicators: Lagtime: The time to peak, which is the time required from the start of the reaction to the start of thrombin generation. The shorter the time, the stronger the ability to promote thrombin generation; ETP: Thrombin generation potential, which is the amount of thrombin generated per minute. The higher the content, the stronger the ability to promote thrombin generation; Peak: The maximum peak of thrombin generation, which is the maximum amount of thrombin generated. The higher the peak, the stronger the ability to promote thrombin generation; ttPeak: The time required from the start of thrombin generation to the highest point of thrombin generation. The shorter the time, the stronger the ability to promote thrombin generation. Record the results of Lagtime, ETP, Peak, and ttPeak of extracellular vesicles in different wells for data analysis.

[0092] VII. Statistical analysis

[0093] Statistical analysis was performed using SPSS software and Graphpad Prism software. Normality tests were performed on measurement data. For data that conformed to a normal distribution, they were expressed as mean ± standard deviation, and the t-test was used to test the statistical differences between the two groups. Measurement data that did not conform to a normal distribution were expressed as median (2.5th percentile, 97.5th percentile), and the Mann-Whitney test was used to test the statistical differences between the two groups.

[0094] VIII. Experimental results

[0095] 1. Demographic characteristics and clinical data of the research subjects

[0096] A total of 120 research subjects (60 healthy males and 60 healthy females) were continuously enrolled from May 2024 to December 2024. The demographic characteristics and clinical data of the research subjects are shown in Table 3.

[0097] Table 3. Demographic characteristics and clinical data of the research subjects

[0098] Characteristic Male (n = 60) Female (n = 60) P value Age 44(36-56) 49(38-62) 0.3 WBC 6.51(5.46,7.585) 6.17(5.06,7.23) 0.09 RBC 5.14(4.823,5.333) 4.44(4.2,4.63) <0.001 PLT 235.5(212.5,271) 238(204,279) 0.76 Prothrombin time 11.4(11.03,11.7) 11.4(11.1,11.7) 0.368 Prothrombin activity 121.5(113.5,126.6) 120.5(113,126.6) 0.431 INR 0.885(0.87,0.927) 0.89(0.87,0.92) 0.591 Activated partial thromboplastin time 27.1(26.2,28.08) 26.75(25.4,28.2) 0.176 Fibrinogen quantification 247.9(214.2,280.2) 268.3(226.8,311.4) 0.02 D-dimer 0.19(0.16,0.25) 0.23(0.19,0.31) 0.007 Fibrin degradation product 1.4(1.3,1.7) 1.6(1.4,1.8) 0.04 Thrombin time 18.5(17.9,18.95) 18.15(17.7,18.5) 0.01

[0099] Note: Measurement data are expressed as median (2.5th percentile, 97.5th percentile), and count data are expressed as absolute values.

[0100] 2. Establishment of the gate for detecting extracellular vesicles content by flow cytometry

[0101] Use the SPHEROTM Flow Cytometry Nanofluorescent Size Standard Kit (containing fluorescently labeled microspheres with diameters of 0.22 μm, 0.45 μm, 0.88 μm, and 1.34 μm) to establish the gate for extracellular vesicles. As shown in Figure 1 (a), this kit consists of 4 types of fluorescent beads with different diameters, ranging from 0.22 μm to 1.33 μm. The upper limit of the EVs gate is determined based on the 0.88 μm labeled microspheres, and the lowest detectable limit of EVs is established based on the 0.22 μm labeled microspheres. As shown in Figure 1 (b). Finally, the EVs gate is determined according to the upper detectable limit and the lowest limit of the beads, as shown in Figure 1 (c).

[0102] 3. Precision of the thrombin generation ability of extracellular vesicles

[0103] Use the established method for detecting the ability of extracellular vesicles to promote thrombin generation to detect the thrombin generation of 100,000 / μl EVs concentration mixed from 16 healthy individuals. Detect 6 times a day for 5 consecutive days. The CV values are shown in Table 4. This result indicates that the method for detecting the ability of extracellular vesicles in blood samples to promote thrombin generation is relatively standardized and has good precision.

[0104] Table 4. Precision of the method for detecting the ability of extracellular vesicles to promote thrombin generation by thrombin generation analyzer (n = 16)

[0105] Test result Lagtime (min) ETP (nM / min) Peak (nM) ttPeak (min) Intra-assay CV 8.225％ 7.322％ 6.118％ 7.635％ Inter-assay CV 12.03％ 11.81％ 10.02％ 9.210％

[0106] 4. Comparison of the thrombin generation ability of extracellular vesicles from the study subjects

[0107] Use the established method for detecting the ability of extracellular vesicles in blood samples to promote thrombin generation by thrombin generation analyzer to conduct thrombin generation ability tests on 120 healthy physical examination subjects, 26 patients with traumatic lower limb fractures, and 16 healthy physical examination subjects used for detecting experimental performance who were enrolled.

[0108] (1) Mix the EVs of 16 healthy physical examination subjects, and use EVs - free plasma to dilute the mixed EVs to 10 / μl, 100 / μl, 1000 / μl, 10000 / μl, and 100000 / μl respectively to detect the thrombin generation status. The thrombin generation gradually increases between 10 / μl and 100000 / μl EVs. As shown in Figure 2(a). Among them, for the statistical comparison between different concentration groups, except that there was no statistical difference in the peak time of the EVs suspension at the concentrations of 10,000 / μl and 100,000 / μl, there were statistical differences between the remaining concentration groups (p < 0.001, p < 0.01, p < 0.05), as Figure 3 (b). Among the ETPs of the four groups, the differences in ETP between different concentration groups were statistically significant (p < 0.001, p < 0.05), as Figure 3 (c). Among the Peaks of the four groups, the Peak value increased with the increase in the EVs concentration, and the differences in Peak between different concentration groups were statistically significant (p < 0.001, p < 0.05), as Figure 3 (d). Among the ttPeaks of the four groups, the ttPeak value decreased with the increase in the EVs concentration, and there were statistical differences between each concentration group (p < 0.001, p < 0.01, p < 0.05), as Figure 3 (e).

[0109] (2) Establishment of the reference intervals for each parameter of the thrombin generation ability of 120 healthy people.

[0110] Using the established method for detecting the ability of blood extracellular vesicles to promote thrombin generation, 120 enrolled healthy people were detected, and the EVs concentration of each healthy person was adjusted to 100,000 / μl. The reference ranges for each parameter are shown in Table 5

[0111] Table 5. Reference intervals of blood extracellular vesicles of 120 healthy people at a concentration of 100,000 / μl

[0112]

[0113] (3) Comparison of each parameter of the thrombin generation ability between 60 healthy male people and 60 healthy female people.

[0114] Table 6. Comparison of the thrombin generation ability between 60 healthy male people and 60 healthy female people

[0115] Thrombin generation-related parameter Total (n = 120) Male (n = 60) Female (n = 60) P value Lagtime (min) 7.33(6.33,10) 8.335(6.753,12) 7(6.33,8.248) P＝0.01 ETP (nM / min) 933.3(837.8,1010) 909.7(753.8,994.8) 958.8(847.4,1018) P＝0.11 Peak (nM) 91.37(72.3,121.9) 84.48(51.07,115.1) 97.84(77.47,126.2) P＝0.06 ttPeak (min) 11(10,15.92) 12.34(10.33,18.59) 10.67(9.67,12) P＝0.008

[0116] (4) Comparison of the thrombin generation ability between 13 thrombus patients and 13 non - thrombus patients at a concentration of 100,000 / μl

[0117] Thrombin generation was as Figure 4 shown. Patients with thrombus had a stronger thrombin generation ability than those without thrombus, and the difference in ETP between the two groups was statistically significant (p < 0.05), as Figure 4(as shown in (b)). The peak of thrombin generation in the thrombus group was significantly higher than that in the non-thrombus group, and the difference was statistically significant (p < 0.05). As Figure 4 (as shown in (c)).

[0118] The beneficial effects of the present invention are as follows: A relatively standardized method with good precision for detecting extracellular vesicles in plasma was established. On this basis, the present invention detected the thrombin generation ability of 120 healthy people at a concentration of 100,000 / μl EVs, and established a reference interval. At the same time, the thrombin generation potential of patients with thrombosis and those without thrombosis was compared at a concentration of 100,000 / μl EVs. Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for detecting the ability of extracellular vesicles in a blood sample to promote thrombin generation, characterized in that, It includes the following steps: Step 1: Centrifuge the blood sample collected within 2 hours at 2500g for 15 minutes at room temperature. Aspirate the upper platelet-poor plasma and place it in a centrifuge tube, then centrifuge it again at 2500g for 15 minutes at room temperature. Aspirate the upper platelet-free plasma, and centrifuge the obtained platelet-free plasma at 20000g for 1 hour at 4°C. Aspirate the upper extracellular vesicle-free plasma for standby; Step 2: Place a mixture containing four kinds of fluorescent localization microspheres with diameters that can be used to locate the detection gates of 0.1μm and 1μm in a flow tube, load the sample, adjust the threshold of the side scatter channel of the flow cytometer, the voltages of the side scatter, forward scatter, and FITC channels, and determine the upper and lower limits of the extracellular vesicle detection gate; Step 3: Add normal human mixed plasma to the extracellular vesicle precipitate obtained in Step 1 to prepare an extracellular vesicle resuspension. Oscillate and mix well, place it in a TruCount absolute counting tube, load the sample for flow cytometry detection, and calculate the content of the obtained extracellular vesicle resuspension according to the known number of counting fluorescent microspheres in the TruCount absolute counting tube and the flow cytometry results; Step 4: According to the content of the extracellular vesicles obtained in Step 3, adjust the test samples with different extracellular vesicle contents to the same concentration with normal human mixed extracellular vesicle-free plasma; Step 5: Add the test samples with the same concentration to a 96-well plate and load the machine to detect the ability of extracellular vesicles to promote thrombin production. This well is the sample well; Step 6: Add the extracellular vesicle-free plasma obtained in Step 1 to a 96-well plate as the standard well; Step 7: After completing the sample addition in the sample well and the standard well, load the machine to detect the ability of extracellular vesicles to promote thrombin production.

2. The method for detecting the ability of extracellular vesicles in a blood sample to promote thrombin generation according to claim 1, wherein: In the said Step 1, the blood sample is freshly collected peripheral venous blood, and the anticoagulant used is sodium citrate.

3. The method for detecting the ability of extracellular vesicles in a blood sample to promote thrombin generation according to claim 1, wherein: In the said Step 2, the four diameters are 0.22μm, 0.45μm, 0.88μm, and 1.34μm respectively.

4. The method for detecting the ability of extracellular vesicles in a blood sample to promote thrombin generation according to claim 1, wherein: In the said Step 3, the calculation formula for the extracellular vesicle resuspension is: EVs / μL = total counting microspheres in the absolute counting tube × number of collected extracellular vesicles / (number of collected counting microspheres × sample volume in μL).

5. The method for detecting the ability of extracellular vesicles in a blood sample to promote thrombin generation according to claim 1, characterized in that: In the said Step 2 and Step 3, continuously adjust each other to jointly obtain the voltage and threshold of this process, specifically as follows: the threshold of the FSC and SSC channels is 200 volts, the voltage of the SSC channel is 566 volts, the voltage of the FSC channel is 780 volts, and the voltage of the FITC channel is 500 volts.

6. The method for detecting the ability of extracellular vesicles in a blood sample to promote thrombin generation according to claim 1, wherein: In the said Step 4, try to use serial dilution to adjust the concentration of extracellular vesicles as much as possible. A maximum dilution of 10 times is acceptable. Dilution exceeding 10 times will be regarded as having a large error and not meeting the experimental requirements.

7. The method for detecting the ability of extracellular vesicles in a blood sample to promote thrombin generation according to claim 1, characterized in that: In the said Step 5 and Step 6, 3 replicate wells are set for the sample well to avoid experimental errors. Add 80μl of the extracellular vesicle resuspension with the same concentration obtained in Step 4 to each sample well, and then add 20μl of PBS solution respectively. At this time, the total volume of the solution in each well is 100μl; 3 replicate wells are set for the standard well to avoid experimental errors; Add 80 μl of the acellular extracellular vesicle plasma obtained in Step 1 to the standard wells, and then add 20 μl of thrombin calibrator to each well. At this time, the total volume of the solution in each well is 100 μl; the 96-well plate is required to be a transparent round bottom and cannot be covered when loaded onto the machine. After sample addition, it can be loaded onto the machine for detection.

8. The method for detecting the ability of extracellular vesicles in a blood sample to promote thrombin generation according to claim 1, characterized in that: In Step 7, after the sample wells and standard wells are loaded onto the machine, they are first incubated at 37 °C for 10 min. The fluorescence buffer is placed in a 37 °C water bath. After incubation, the fluorescence buffer is taken out of the 37 °C water bath, and 40 μl of fluorescence substrate is added and mixed by pipetting to form fluca. The machine adds 20 μl of fluca to each of the standard wells and sample wells. At this time, the total volume of the solution in the sample wells and standard wells is 120 μl.

9. The method for detecting the ability of extracellular vesicles in a blood sample to promote thrombin generation according to claim 1, wherein: In Step 7, the ability of extracellular vesicles to promote thrombin production is evaluated according to the following four indicators: Indicator 1: Lagtime, the time from the start of the reaction to the start of thrombin generation. The shorter the time, the stronger the ability to promote thrombin generation; Indicator 2: ETP, thrombin generation potential, the amount of thrombin generated per minute. The higher the amount, the stronger the ability to promote thrombin generation; Indicator 3: Peak, the maximum peak of thrombin generation, the maximum amount of thrombin generated. The higher the peak, the stronger the ability to promote thrombin generation; Indicator 4: ttPeak, the time required from the start of thrombin generation to the highest point of thrombin generation. The shorter the time, the stronger the ability to promote thrombin generation; Record the results of Lagtime, ETP, Peak, and ttPeak of extracellular vesicles in different wells for data analysis.

Images