Method for detecting percentage level and average fluorescence intensity of leukocyte aggregate of different types of platelets in blood sample

Abstract

The invention relates to the technical field of biology, and discloses a method for detecting percentage level and average fluorescence intensity of different types of platelet leukocyte aggregates in a blood sample, which comprises the following steps of: collecting an EDTA (Ethylene Diamine Tetraacetic Acid) anticoagulant blood sample within 4 hours as a sample to be detected, and pretreating at room temperature; the percentage level and the expression intensity of different platelet leukocyte aggregates are subjected to flow detection. By utilizing the method provided by the invention, different types of platelet leukocyte aggregates in a blood sample can be accurately detected. The method for detecting the percentage level and the average fluorescence intensity of the different types of platelet leukocyte aggregates in the blood sample has the advantages of being convenient to operate, easy to obtain the sample, non-invasive, non-destructive, high in detection speed, low in cost and the like, has important significance on thrombus risk assessment and prediction, and has good clinical application value.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for detecting percentage levels and average fluorescence intensities of different types of platelet-leukocyte aggregates in a blood sample. Background Art

[0002] Platelets are traditionally considered to play a vital role in maintaining body homeostasis, thrombosis and hemostasis, but they can also interact with innate immune cells, activate and regulate immune cells. In the context of infection and inflammation, platelets can directly bind to circulating leukocytes by expressing surface P-selectin. Leukocytes can recognize platelet P-selectin through PSGL-1 on their surface, eventually forming platelet-leukocyte aggregates (PLA). Platelet-leukocyte aggregates can be divided into platelet T-cell aggregates (PLyA(T)), platelet CD4+T cell aggregates (platelet T-cell aggregates, PLyA(CD4+)), platelet CD8+T cell aggregates (platelet T-cell aggregates, PLyA(CD8+)), platelet B cell aggregates (platelet B-cell aggregates, PLyA(B)), platelet NK cell aggregates (platelet NK-cell aggregates, PLyA(NK)), platelet-neutrophil aggregates (PNA), platelet-eosinophil aggregates (platelet-eosinophil aggregates), platelet-basophil aggregates (platelet-basophil Platelet-leukocyte aggregates are involved in a variety of pathological processes including inflammation, thrombosis and cancer, and the affected parts include the circulatory system, brain, liver, kidney and lung tissues and organs.

[0003] There are currently a variety of methods for detecting platelet-leukocyte aggregates in the blood circulation, including flow cytometry (FCM), confocal or electron microscopy imaging, and in vitro microfluidic assays. At present, there is still a lack of professional guidelines for evaluating platelet-leukocyte aggregate detection internationally. Flow cytometry is often used in conventional methods to identify circulating PLA. This detection method has high sensitivity in identifying different subclasses of aggregates while reducing the occurrence of false positives. There is currently no recognized standardized FCM detection protocol, and there is also a lack of detection protocols covering multiple types of platelet-leukocyte aggregates.

[0004] The use of advanced confocal or electron microscopy can provide high-resolution images to visualize platelet-leukocyte interactions, however this analytical approach does not allow for analysis of dynamic behavior.

[0005] Microfluidics is a technology for studying the interaction between platelets and leukocytes in real time, but its ability to simulate the physiological activities in real human blood vessels is still limited. A method for detecting the percentage level and mean fluorescence intensity of different types of platelet-leukocyte aggregates in blood samples has been invented to solve the above problem. Summary of the invention

[0006] 1. Technical issues to be resolved

[0007] In view of the deficiencies of the prior art, the present invention provides a method for detecting the percentage levels and mean fluorescence intensity of different types of platelet-leukocyte aggregates in a blood sample, thereby solving the above-mentioned problems.

[0008] (II) Technical solution

[0009] To achieve the above object, the present invention provides the following technical solution: a method for detecting the percentage level and mean fluorescence intensity of different types of platelet-leukocyte aggregates in a blood sample, comprising the following steps:

[0010] (A) The whole blood samples collected within 4 hours were placed at room temperature, and 200ul of each were placed in three flow cytometry tubes for detecting three major types of platelet-leukocyte aggregates: lymphocytes (tube (I)), granulocytes (tube (II)), and monocytes (tube (III));

[0011] (B) The three whole blood samples obtained in (A) were used as test samples, and the percentage levels and mean fluorescence intensities of different types of platelet-leukocyte aggregates were detected by flow cytometry;

[0012] Preferably, in (A), the blood sample may be freshly collected peripheral venous blood, and the anticoagulant used in the blood collection process is EDTA;

[0013] Different types of platelet leukocyte aggregates include platelet T-cell aggregates (PLyA(T)), platelet CD4+T cell aggregates (platelet T-cell aggregates, PLyA(CD4+)), platelet CD8+T cell aggregates (platelet T-cell aggregates, PLyA(CD8+)), platelet B cell aggregates (platelet B-cell aggregates, PLyA(B)), platelet NK cell aggregates (platelet NK-cell aggregates, PLyA(NK)), platelet-neutrophil aggregates (PNA), platelet-eosinophil aggregates, platelet-basophil aggregates, platelet classical-monocyte aggregates (PMA(classical-monocyte)), platelet intermediate monocyte aggregates (platelet intermediate platelet non-classical aggregates, PMA (non-classical monocyte)).Furthermore, the aggregates may specifically be CD3+CD61+, indicating platelet T cell aggregates; CD3+CD4+CD61+, indicating platelet CD4+T cell aggregates; CD3+CD8+CD61+, indicating platelet CD8+T cell aggregates; CD3-CD19+CD61+, indicating platelet B cell aggregates; CD3-CD56+CD61+, indicating platelet NK cell aggregates; CD11b+CD15+CD16+CD61+, indicating platelet neutrophil aggregates; CD11b+CD 15+CD16-CD61+ indicates platelet eosinophil aggregates; CD11b+CD15-CCR3+FcεRiα+CD61+ indicates platelet basophil aggregates; CD14+CD61+ indicates platelet monocyte aggregates; CD14++CD16-CD61+ indicates platelet classical monocyte aggregates; CD14+CD16+CD61+ indicates platelet intermediate monocyte aggregates; CD14-CD16+CD61+ indicates platelet non-classical monocyte aggregates.

[0014] Preferably, in said (B), it can be carried out according to a method comprising the following steps:

[0015] (B1) Compensation beads (Compensation Beads, Biolegend, USA) that can be used to adjust compensation are made into single positive tubes with each fluorescent antibody, shaken and mixed, and loaded at a low flow rate; the compensation beads include 1 positive microsphere and 1 negative microsphere. The positive microsphere can bind to any mouse or human antibody, and the negative microsphere will never bind to any antibody. The purpose of this compensation microsphere is to be used as a compensation control for multi-color flow analysis.

[0016] (B2) Set up the blank control tube and each fluorescent single positive tube as follows (I)

[0017] (I) The following settings are made:

[0018] Blank control tube: add the whole blood sample obtained in (A) and positive microspheres and negative microspheres, incubate at room temperature in the dark, and load the sample at a low flow rate;

[0019] Single positive tube-1: Add positive microspheres, negative microspheres, and APC CD14 antibody, incubate at room temperature in the dark, and load at a low flow rate;

[0020] Single positive tube-2: Add positive microspheres, negative microspheres, and PE CD61 antibody, incubate at room temperature in the dark, and load at a low flow rate;

[0021] Single positive tube-3: Add positive microspheres, negative microspheres, and FITC CD3 antibody, incubate at room temperature in the dark, and load at a low flow rate;

[0022] Single positive tube-4: Add positive microspheres, negative microspheres, and APC-Cy7 CD11b antibody, incubate at room temperature in the dark, and load at a low flow rate;

[0023] Single positive tube-5: Add positive microspheres, negative microspheres, and PE-Cy7 CD15 antibody, incubate at room temperature in the dark, and load at a low flow rate;

[0024] Single positive tube-6: Add positive microspheres, negative microspheres, and Percp / 5.5CD16 antibody, incubate at room temperature in the dark, and load at a low flow rate;

[0025] (B3) In (B2), the voltages of the APC, PE, FITC, APC-Cy7, PE-Cy7, and Percp / 5.5 channels and the compensation between the fluorescence channels can be adjusted according to the blank control tube and single positive tube-1 to single positive tube-6 set in (I);

[0026] According to the blank control tube and single positive tube-1 to single positive tube-6 set up in (I), the voltages of the APC, PE, FITC, APC-Cy7, PE-Cy7, and Percp / 5.5 channels and the compensation between the fluorescence channels are adjusted. The specific parameters were set as 286 V for forward scattering, 451 V for side scattering, 478 V for APC channel, 500 V for PE channel, 464 V for FITC channel, 507 V for APC-Cy7 channel, 615 V for PE-Cy7 channel, 625 V for Percp / 5.5 channel, compensation for FITC-%APC was 0, compensation for PE-%APC was 0, compensation for APC Cy7-%APC was 7.2, compensation for PE Cy7-%APC was 0, compensation for Percp / 5.5-%APC was 1.5, compensation for FITC-%PE was 0.6, compensation for APC Cy7-%PE was 0, compensation for PE Cy7-%PE was 0, compensation for PercpCy5.5-%PE was 13.5, compensation for APC Cy7-%FITC was 0, and compensation for PE The compensation for Cy7-%FITC was 0.1, the compensation for Percp / 5.5-%FITC was 2.3, the compensation for PE Cy7-%APC Cy7 was 2.6, the compensation for PercpCy5.5-%APC Cy7 was 0.8, and the compensation for Percp / 5.5-%PE Cy7 was 8.9;

[0027] Taking APC Cy7-%APC as an example, its meaning is: the fluorescence intensity of the APC fluorescence channel leaking into the APC Cy7 fluorescence channel. In order to ensure the accuracy and authenticity of the detection, it is necessary to subtract the fluorescence intensity leaking into the APC Cy7 fluorescence channel. Therefore, the compensation of APC Cy7-%APC is 7.2;

[0028] (B4) In the above (B1), three flow tubes each containing 200ul EDTA whole blood are obtained, and fluorescently labeled antibodies for labeling platelet lymphocyte aggregates are added to tube (I), specifically 2ul each of FITC CD3 antibody, PE CD61 antibody, APCCD19 antibody, APC Cy7 CD8 antibody, PE Cy7 CD56 antibody, and Percp / 5.5CD4 antibody; fluorescently labeled antibodies for labeling platelet granulocyte aggregates are added to tube (II), specifically 2ul each of FITC CCR3 antibody, PE CD61 antibody, APCFcεRIα antibody, APC Cy7 CD11b antibody, PE Cy7 CD15 antibody, and Percp / 5.5CD16 antibody; fluorescently labeled antibodies for labeling platelet monocyte aggregates are added to tube (III), specifically 2ul each of PE CD61 antibody, APC CD14 antibody, and Percp / 5.5CD16 antibody; incubate at 4°C in the dark;

[0029] Preferably, after adding red blood cell lysis solution (FACSTM Lysing Solution, BD, USA) to fully lyse red blood cells, centrifuge at 1500 rpm / min for 5 min, remove the supernatant, add PBS buffer to resuspend, centrifuge to remove the supernatant, add PBS buffer to resuspend again, and detect on the machine;

[0030] (B5) In the step (B4), the on-machine test results are recorded and saved, and data analysis is performed using Flowjo software to record the percentage level of each platelet-leukocyte aggregate and the average fluorescence intensity of platelets on the platelet-leukocyte aggregates.

[0031] Compared with the prior art, the present invention provides a method for detecting the percentage level and mean fluorescence intensity of different types of platelet-leukocyte aggregates in a blood sample, which has the following beneficial effects:

[0032] 1. The method for detecting the percentage level and average fluorescence intensity of different types of platelet-leukocyte aggregates in blood samples has the advantages of convenient operation, easy sample acquisition, non-invasive and non-destructive, fast detection speed and low cost. The present invention is of great significance for thrombosis risk assessment and prediction and has good clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 A schematic diagram is established for the method of detecting different platelet-lymphocyte aggregates by flow cytometry of the present invention;

[0034] (a): A diagram of the five groups of normal white blood cells, from which the lymphocytes are circled. (b): Based on the lymphocyte gate, the CD3+ gate and CD3- gate are divided according to whether the T lymphocyte surface molecule CD3 is expressed, corresponding to the T lymphocyte group and the non-T lymphocyte group, respectively. (c): Based on the CD3+ gate, the CD3+CD61+ gate is divided according to whether the platelet surface molecule CD61 is expressed, and CD3+CD61+ is the platelet T lymphocyte aggregate. (d): Based on the CD3- gate, the CD19+ gate is divided according to whether the B lymphocyte surface molecule CD19 is expressed, corresponding to the B lymphocyte group. (e): Based on the CD3- gate, the CD56+ gate is divided according to whether the NK cell surface molecule CD56 is expressed, corresponding to the NK cell group. (f): Based on the CD3+ gate, CD3+CD4+ and CD3+CD8+ gates are drawn according to whether CD4 or CD8 molecules are expressed, corresponding to CD4+T lymphocyte population and CD8+T lymphocyte population, respectively. (g): Based on the CD19+ gate, CD19+CD61+ gate is drawn according to whether platelet surface molecule CD61 is expressed, and CD19+CD61+ is platelet B lymphocyte aggregates. (h): Based on the CD56+ gate, CD56+CD61+ gate is drawn according to whether platelet surface molecule CD61 is expressed, and CD56+CD61+ is platelet NK cell aggregates. (i): Based on the CD8+ gate, CD8+CD61+ gate is drawn according to whether platelet surface molecule CD61 is expressed, and CD8+CD61+ is platelet CD8+T lymphocyte aggregates. (j): Based on the CD4+ gate, the CD4+CD61+ gate is drawn according to whether the platelet surface molecule CD61 is expressed. CD4+CD61+ is the platelet CD4+T lymphocyte aggregate;

[0035] Figure 2 A schematic diagram is established for the method of detecting different platelet-granulocyte aggregates by flow cytometry of the present invention;

[0036] (a): A diagram of the five groups of normal white blood cells, from which the granulocytes are circled. (b): Based on the granulocyte gate, the CD11b+CD15+ gate and CD11b+CD15- gate are drawn according to whether CD11b and CD15 are expressed, corresponding to the non-basophil group and the basophil group, respectively. (c): Based on the CD11b+CD15+ gate, the CD11b+CD15+CD16+ gate (N+) and CD11b+CD15+CD16- gate (E+) are drawn according to whether CD16 is expressed, corresponding to the neutrophil group and the eosinophil group, respectively. (d): Based on the CD11b+CD15- gate, the CD11b+CD15-FcεRiα+ / CCR3+ gate (B+) is drawn according to whether the basophil surface molecules FcεRiα or CCR3 are expressed, corresponding to the basophil group. (e): Based on the B+ gate, the B+CD61+ gate is drawn according to whether the platelet surface molecule CD61 is expressed. B+CD61+ is the platelet basophil aggregate. (f): Based on the E+ gate, the E+CD61+ gate is drawn according to whether the platelet surface molecule CD61 is expressed. E+CD61+ is the platelet eosinophil aggregate. (g): Based on the N+ gate, the N+CD61+ gate is drawn according to whether the platelet surface molecule CD61 is expressed. N+CD61+ is the platelet neutrophil aggregate.

[0037] Figure 3 A schematic diagram is established for the method of detecting different platelet-monocyte aggregates by flow cytometry of the present invention;

[0038] (a): A diagram of the five groups of normal white blood cells, from which the monocytes are circled. (b): Based on the monocyte gate, the expression levels of CD14 and CD16 are divided into CD14+CD16- gate (Classical-Mon gate), CD14+CD16+ gate (Intermediate-Mon gate), and CD14-CD16+ gate (Nonclassical-Mon gate), which correspond to the classical monocyte population, intermediate monocyte population, and nonclassical monocyte population, respectively. (c): Based on the monocyte gate, the PMA(all) gate is drawn according to whether the platelet surface molecule CD61 is expressed. PMA(all) is the total platelet monocyte aggregate. (d): Based on the Nonclassical-Mon gate, the PMA(Nonclassical-Mon) gate is drawn according to whether the platelet surface molecule CD61 is expressed. PMA(Nonclassical-Mon) is the nonclassical platelet monocyte aggregate. (e): Based on the Classical-Mon gate, the PMA (Classical-Mon) gate is drawn according to whether the platelet surface molecule CD61 is expressed. PMA (Classical-Mon) is the platelet classical monocyte aggregate. (f): Based on the Intermediate-Mon gate, the PMA (Intermediate-Mon) gate is drawn according to whether the platelet surface molecule CD61 is expressed. PMA (Intermediate-Mon) is the platelet intermediate monocyte aggregate;

[0039] Figure 4 The figure is a schematic diagram of the results of detecting the percentage level and mean fluorescence intensity (MFI) of platelet-lymphocyte aggregates by flow cytometry of the present invention;

[0040] The circulating percentage levels of (a) platelet T lymphocyte aggregates, (b) platelet CD4+ T lymphocyte aggregates, (c) platelet CD8+ T lymphocyte aggregates, (d) platelet B lymphocyte aggregates, and (e) platelet NK lymphocyte aggregates were evaluated, and the mean fluorescence intensity (MFI) of (f) platelet T lymphocyte aggregates, (g) platelet CD4+ T lymphocyte aggregates, (h) platelet CD8+ T lymphocyte aggregates, (i) platelet B lymphocyte aggregates, and (j) platelet NK lymphocyte aggregates were detected.

[0041] Figure 5The figure is a schematic diagram of the results of detecting the percentage level and mean fluorescence intensity (MFI) of platelet-granulocyte aggregates by flow cytometry of the present invention;

[0042] The circulating percentage levels of (a) platelet-neutrophil aggregates, (b) platelet-eosinophil aggregates, and (c) platelet-basophil aggregates were evaluated, and the mean fluorescence intensity of (d) platelet-neutrophil aggregates, (e) platelet-eosinophil aggregates, and (f) platelet-basophil aggregates were measured.

[0043] Figure 6 The figure is a schematic diagram of the results of detecting the percentage level and mean fluorescence intensity (MFI) of platelet-monocyte aggregates by flow cytometry of the present invention;

[0044] The circulating percentage levels of (a) total platelet-monocyte aggregates, (b) classical platelet-monocyte aggregates, (c) nonclassical platelet-monocyte aggregates, and (d) intermediate platelet-monocyte aggregates were evaluated, and the mean fluorescence intensity of (e) total platelet-monocyte aggregates, (f) classical platelet-monocyte aggregates, (g) nonclassical platelet-monocyte aggregates, and (h) intermediate platelet-monocyte aggregates was detected. DETAILED DESCRIPTION

[0045] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0046] Embodiment 1:

[0047] A method for detecting the percentage level and mean fluorescence intensity of different types of platelet-leukocyte aggregates in a blood sample, characterized in that it comprises the following steps:

[0048] 1) (A) The whole blood sample collected within 4 hours is transported to the laboratory smoothly and placed at room temperature; the blood sample (A) is freshly collected peripheral venous blood, and the anticoagulant used is EDTA;

[0049] 2) (B) Using the blood sample obtained in (A) as the test sample, flow cytometry is performed to detect different types of platelet-leukocyte aggregates therein; the different types of platelet-leukocyte aggregates may be platelet T-cell aggregates (platelet T-cell aggregates, PLyA(T)), platelet CD4+T cell aggregates (platelet T-cell aggregates, PLyA(CD4+)), platelet CD8+T cell aggregates (platelet T-cell aggregates, PLyA(CD8+)), platelet B cell aggregates (platelet B-cell aggregates, PLyA(B)), platelet NK cell aggregates (platelet NK-cell aggregates, PLyA(NK)), platelet-neutrophil aggregates (platelet-neutrophil aggregates, PNA), platelet-eosinophil aggregates (platelet-eosinophil aggregates), platelet-basophil aggregates (platelet-basophil aggregates), platelet-classical monocyte aggregates (platelet classical-monocyte aggregates, PMA (classical-monocyte)), platelet intermediate aggregates (PMA (intermediate-monocyte)), platelet non-classical aggregates (PMA (non-classical monocyte));

[0050] 3) (B1) using compensation microspheres and specific antibodies to prepare each fluorescent single positive tube, and setting up a blank control tube; adjusting the forward angle, lateral angle and voltage of the corresponding fluorescent type channel of the flow cytometer and the compensation between each fluorescent channel;

[0051] 4) (B2) Take out 200ul of the blood sample obtained in (A) and place it in a flow cytometer, add fluorescently labeled antibodies targeting surface molecules of different platelet-leukocyte aggregates, and add red blood cell lysis solution to fully lyse the red blood cells. After incubation at 4°C in the dark, centrifuge to remove the supernatant and add PBS buffer. Centrifuge again to remove the supernatant and load the sample for flow cytometry detection. Analyze with Flowjo software to obtain the percentage level of different subtypes of platelet-leukocyte aggregates and the average fluorescence intensity;

[0052] 5) In the (B1), a blank control tube and a single positive tube are set as follows (I), and (I) is set as follows:

[0053] Blank control tube: add the whole blood sample obtained in (A) and positive microspheres and negative microspheres, incubate at room temperature in the dark, and load the sample at a low flow rate;

[0054] Single positive tube-1: Add positive microspheres, negative microspheres, and APC CD14 antibody, incubate at room temperature in the dark, and load at a low flow rate;

[0055] Single positive tube-2: Add positive microspheres, negative microspheres, and PE CD61 antibody, incubate at room temperature in the dark, and load at a low flow rate;

[0056] Single positive tube-3: Add positive microspheres, negative microspheres, and FITC CD3 antibody, incubate at room temperature in the dark, and load at a low flow rate;

[0057] Single positive tube-4: Add positive microspheres, negative microspheres, and APC-Cy7 CD11b antibody, incubate at room temperature in the dark, and load at a low flow rate;

[0058] Single positive tube-5: Add positive microspheres, negative microspheres, and PE-Cy7 CD15 antibody, incubate at room temperature in the dark, and load at a low flow rate;

[0059] Single positive tube-6: Add positive microspheres, negative microspheres, and Percp / 5.5CD16 antibody, incubate at room temperature in the dark, and load at a low flow rate;

[0060] 6) In (B1), according to the blank control tube and single positive tubes -1 to -6 set in (I), adjust the voltages of the APC, PE, FITC, APC-Cy7, PE-Cy7, and Percp / 5.5 channels and the compensation between the fluorescence channels;

[0061] 7) According to the blank control tube and single positive tubes -1 to -5 set in (I), the voltages of the APC, PE, FITC, APC-Cy7, PE-Cy7, and Percp / 5.5 channels and the compensation between the fluorescence channels are adjusted, and the parameters are set as follows: the forward scattered light voltage is 286 volts, the side scattered light voltage is 451 volts, the APC channel voltage is 478 volts, the PE channel voltage is 500 volts, the FITC channel voltage is 464 volts, the APC-Cy7 channel voltage is 507 volts, the PE-Cy7 channel voltage is 615 volts, the Percp / 5.5 channel voltage is 625 volts, the FITC-%APC compensation is 0, the PE-%APC compensation is 0, the APC Cy7-%APC compensation is 7.2, and the PE The compensation for Cy7-%APC was 0, the compensation for Percp / 5.5-%APC was 1.5, the compensation for FITC-%PE was 0.6, the compensation for APC Cy7-%PE was 0, the compensation for PE Cy7-%PE was 0, the compensation for PercpCy5.5-%PE was 13.5, the compensation for APC Cy7-%FITC was 0, the compensation for PE Cy7-%FITC was 0.1, the compensation for Percp / 5.5-%FITC was 2.3, the compensation for PE Cy7-%APC Cy7 was 2.6, the compensation for PercpCy5.5-%APC Cy7 was 0.8, and the compensation for Percp / 5.5-%PE Cy7 was 8.9;

[0062] 8) In the above (B2), 200ul of the blood sample obtained in (A) is taken out and placed in a flow tube, which is divided into three tubes. According to the following detections, (II) different platelet-lymphocyte aggregates, (III) different platelet-granulocyte aggregates, and (IV) different platelet-monocyte aggregates, fluorescently labeled antibodies against specific surface molecules are added;

[0063] (II), as follows: 200ul of the blood sample obtained in (A) was taken out and placed in a flow tube, and FITC CD3 antibody, PECD61 antibody, APC CD19 antibody, APC Cy7 CD8 antibody, PE Cy7 CD56 antibody, and Percp / 5.5CD4 antibody were added;

[0064] (III), as follows: 200ul of the blood sample obtained in (A) was taken out and placed in a flow tube, and FITC CCR3 antibody, PECD61 antibody, APC FcεRIα antibody, APC Cy7 CD11b antibody, PE Cy7 CD15 antibody, and Percp / 5.5CD16 antibody were added;

[0065] (IV), as follows: 200ul of the blood sample obtained in (A) was taken out and placed in a flow tube, and FITC CD62P antibody, PECD61 antibody, APC CD14 antibody, and Percp / 5.5CD16 antibody were added;

[0066] 9) In the above (B2), after adding fluorescent labeled antibodies against specific surface molecules to (II), (III), and (IV), incubate in dark for 20 minutes, add 2 ml of red blood cell lysis buffer and continue incubating in dark for 8 minutes, centrifuge at 1500 rpm / min for 5 minutes, remove the supernatant after centrifugation, add 2 ml of PBS buffer, continue centrifugation at the same speed for 5 minutes, remove the supernatant, add 200 ml of PBS buffer to resuspend, and then detect on the machine;

[0067] 10) In (B2), the on-machine test results are recorded and saved, and data analysis is performed using Flowjo software to record the percentage level of each platelet-leukocyte aggregate and the average fluorescence intensity of platelets on the platelet-leukocyte aggregates.

[0068] 1. Test samples

[0069] Patients who developed coagulopathy following sepsis and those who did not.

[0070] 1. Based on previous research experience (Li, N., Goodall, AH, & Hjemdahl, P. (1997). A sensitive flow cytometric assay for circulating platelet-leucocyte aggregates. British journal of haematology, 99 (4), 808–816.), the difference in the mean number of platelet-leucocyte aggregates between the two groups was Δμ approximately ±15.3, the standard deviation σ was approximately 8.5, the test power was 0.90, α = 0.05, and the calculated sample size was approximately 8 cases per group. Therefore, the sample size of each group was at least 8 cases.

[0071] 2. Selection of research subjects

[0072] A total of 71 patients with sepsis (41 in the non-coagulopathy group and 30 in the coagulopathy group) were consecutively enrolled in the Peking University Fourth Clinical Medical College from May to December 2024. This study was approved by the hospital ethics committee.

[0073] Inclusion criteria: ICU patients with sepsis, evaluated by SOFA score according to the Sepsis 3.0 guidelines; age ≥ 18 years

[0074] Exclusion criteria: pregnant or breastfeeding patients; patients with malignant tumors or malignant tumors related to coagulation or platelets who are undergoing chemotherapy; patients with immunosuppressive diseases such as HIV infection; patients who have used immunosuppressants in the past three months; patients aged less than 18 years

[0075] Grouping criteria: According to the ISTH SIC score diagnostic criteria, sepsis patients were judged to have sepsis-induced coagulopathy (SIC) and divided into SIC group and non-SIC group.

[0076] 3. Obtaining relevant information about the research subjects

[0077] The gender, age, underlying diseases, and surgical risk factors (history of thrombosis, surgery within six months before enrollment) of the subjects were analyzed.

[0078] Information such as medical history, smoking history, hypertension history, diabetes history, blood type, C-reactive protein, hemoglobin, platelet count), medication history (whether there is a history of antiplatelet drugs), etc. was obtained from their hospital medical records.

[0079] 2. Sample Collection and Preparation

[0080] The nurse draws a tube of peripheral venous blood from the research subject using a purple-capped anticoagulant tube containing EDTA, and gently inverts it to mix it. The tube is transported to the laboratory smoothly and fixed within 4 hours (the red blood cell lysis solution contains a fixative).

[0081] 3. Instruments

[0082] Flow cytometer Canto II (BD, USA)

[0083] IV. Reagents

[0084] Compensation Beads (Biolegend, USA), PE CD61 antibody (Biolegend, USA), FITCCD3 antibody (Biolegend, USA), APC CD19 antibody (Biolegend, USA), APC Cy7 CD8 antibody (Biolegend, USA), PE Cy7 CD56 antibody (Biolegend, USA), Percp / 5.5CD4 antibody (Biolegend, USA), FITC CCR3 antibody (Biolegend, USA), APC FcεRIα antibody (Biolegend, USA), APC Cy7CD11b antibody (Biolegend, USA), PE Cy7 CD15 antibody (Biolegend, USA), Percp / 5.5CD16 antibody (Biolegend, USA), APC CD14 antibody (Biolegend, USA), FACSTM Lysing Solution, (BD, USA).

[0085] 5. Reagent Preparation

[0086] Preparation of 1X red blood cell lysis buffer: 10X FACSTM Lysing Solution buffer, add deionized water to dilute to 1X FACSTM Lysing Solution binding buffer. That is 1X red blood cell lysis buffer, which needs to be prepared before use.

[0087] VI. Experimental steps

[0088] Establishment of a flow cytometric method for detecting different platelet-leukocyte aggregates

[0089] (1) Use compensation microspheres (including positive microspheres and negative microspheres) to adjust the voltage and compensation between each channel, and set up a blank control tube and a single positive tube for each fluorescent channel.

[0090] (2) Blank control tube: add the whole blood sample obtained in (A) and positive microspheres and negative microspheres, incubate at room temperature in the dark, and load the sample at a low flow rate;

[0091] (3) Single positive tube-1: Add positive microspheres, negative microspheres, and APC CD14 antibody, incubate at room temperature in the dark, and load the sample at a low flow rate;

[0092] (4) Single positive tube-2: Add positive microspheres, negative microspheres, and PE CD61 antibody, incubate at room temperature in the dark, and load the sample at a low flow rate;

[0093] (5) Single positive tube-3: Add positive microspheres, negative microspheres, and FITC CD3 antibody, incubate at room temperature in the dark, and load the sample at a low flow rate;

[0094] (6) Single positive tube-4: Add positive microspheres, negative microspheres, and APC-Cy7 CD11b antibody, incubate at room temperature in the dark, and load the sample at a low flow rate;

[0095] (7) Single positive tube-5: Add positive microspheres, negative microspheres, and PE-Cy7 CD15 antibody, incubate at room temperature in the dark, and load the sample at a low flow rate;

[0096] (8) Single positive tube-6: Add positive microspheres, negative microspheres, and Percp / 5.5CD16 antibody, incubate at room temperature in the dark, and load the sample at a low flow rate;

[0097] Steps (2) to (8) are continuously debugged with each other to jointly obtain the voltage and compensation value of each channel in this process. The specific parameters are shown in Table 1 and Table 2.

[0098] Table 1. Voltages of FSC, SSC, FITC, PE, APC, APC Cy7, PE Cy7, and Percp / 5.5 channels:

[0099]

[0100]

[0101] Table 2. Compensation of each fluorescence channel of FITC, PE, APC, APC Cy7, PE Cy7, and Percp / 5.5:

[0102]

[0103]

[0104] (9) The whole blood samples collected within 4 hours were placed at room temperature, and 200 μl of each sample was placed in three flow cytometry tubes for detecting three major types of platelet-leukocyte aggregates: lymphocytes (tube (I)), granulocytes (tube (II)), and monocytes (tube (III)).

[0105] Tube (I): Add 2ul FITC CD3 antibody, PE CD61 antibody, APC CD19 antibody, APC Cy7CD8 antibody, PECy7 CD56 antibody, Percp / 5.5CD4 antibody, and incubate at 4℃ in dark for 20min

[0106] Tube (II): Add 2ul FITC CCR3 antibody, PE CD61 antibody, APC FcεRIα antibody, APC Cy7 CD11b antibody, PE Cy7 CD15 antibody, Percp / 5.5CD16 antibody, and incubate at 4℃ in the dark for 20min

[0107] Tube (III): Add 2ul PE CD61 antibody, APC CD14 antibody, Percp / 5.5CD16 antibody, and incubate at 4℃ in dark for 20min

[0108] (10) After incubation, add the prepared 1X red blood cell lysis buffer to fully lyse the red blood cells and fix the platelets (the lysis buffer contains 5% paraformaldehyde). After shaking, incubate at 4°C in the dark for 8 minutes.

[0109] (11) Centrifuge at 1500 rpm / min for 5 min, remove the supernatant, add 2 ml of PBS buffer to each tube, and shake thoroughly.

[0110] (12) After centrifugation at 1500 rpm / min for 5 min, remove the supernatant, add 400 μl PBS buffer to each tube for resuspending, and then load onto the analyzer for detection.

[0111] (13) The flow cytometry results were imported into Flowjo software to obtain the percentage levels and mean fluorescence intensity of different platelet-leukocyte aggregates.

[0112] 7. Statistical Analysis

[0113] Statistical analysis was performed using SPSS software (version 23.0, IBM, USA) and Graphpad Prism software (version 7.0, Graphpad, USA). The normality test was performed on the measurement data. The mean ± standard deviation was used to represent the data that conformed to the normal distribution. The median (2.5 percentile, 97.5 percentile) was used to represent the measurement data that did not conform to the normal distribution. The Mann-Whitney test was used to compare the percentage level and mean fluorescence intensity of each platelet-leukocyte aggregate between the SIC group and the non-SIC group.

[0114] 8. Experimental Results

[0115] 1. Demographic characteristics and clinical data of the study subjects

[0116] A total of 71 patients with sepsis (41 in the non-coagulopathy group and 30 in the coagulopathy group) were consecutively enrolled in the Peking University Fourth Clinical Medical College from May to December 2024. Demographic characteristics and clinical data of the subjects.

[0117] As shown in Table 3:

[0118]

[0119]

[0120]

[0121] Note: Measurement data are expressed as median (2.5 percentile, 97.5 percentile), and count data are expressed as absolute value.

[0122] 2. Flow cytometry detection of various platelet-leukocyte aggregate percentage levels and mean fluorescence intensity

[0123] The gating strategy for platelet-lymphocyte aggregates is as follows Figure 1 As shown in the figure, (a): A diagram of the five groups of normal human leukocytes, from which the lymphocytes are circled. (b): Based on the lymphocyte gate, the T lymphocytes are divided into CD3 + Door, CD3 - The gates correspond to the T lymphocyte population and the non-T lymphocyte population, respectively. (c): In CD3 + Based on the expression of the platelet surface molecule CD61, CD3 + CD61 + Door, CD3 + CD61 + It is a platelet T lymphocyte aggregate. (d): In CD3 - Based on the gate, CD19 cells were selected according to whether they expressed CD19 on the surface of B lymphocytes. + Gate, corresponding to the B lymphocyte population. (e): In CD3 - Based on the gate, CD56 cells were selected according to whether they expressed the surface molecule CD56 of NK cells. + Gate, corresponding to the NK cell population. (f): In CD3 + Based on the gate, CD3 is divided according to whether it expresses CD4 or CD8 molecules + CD4 + 、CD3 + CD8 + gate, corresponding to CD4 + T lymphocyte population, CD8 + T lymphocyte population. (g): CD19 + Based on the gate, CD19 was selected based on the expression of platelet surface molecule CD61. + CD61 + Door,CD19 + CD61 + It is a platelet-B lymphocyte aggregate. (h): In CD56 + Based on the gate, CD56 + CD61 + Door,CD56 + CD61 + That is, platelet NK cell aggregates. (i): In CD8 +Based on the gate, CD8 + CD61 + Door, CD8 + CD61 + Platelet CD8 + T lymphocyte aggregates. (j): in CD4 + Based on the gate, CD4 + CD61 + Door, CD4 + CD61 + Platelet CD4 + T lymphocyte aggregates.

[0124] The gating strategy for platelet-granulocyte aggregates is as follows Figure 2 As shown, (a): A diagram of the five groups of normal human leukocytes, from which the part where the granulocytes are located is circled. (b): Based on the granulocyte gate, the CD11b+CD15+ gate and CD11b+CD15- gate are drawn according to whether CD11b and CD15 are expressed, corresponding to the non-basophil group and the basophil group, respectively. (c): Based on the CD11b+CD15+ gate, the CD11b+CD15+CD16+ gate (N+) and CD11b+CD15+CD16- gate (E+) are divided according to whether CD16 is expressed, corresponding to the neutrophil group and the eosinophil group, respectively. (d): Based on the CD11b+CD15- gate, the CD11b+CD15-FcεRiα+ / CCR3+ gate (B+) is drawn according to whether the basophil surface molecules FcεRiα or CCR3 are expressed, corresponding to the basophil group. (e): Based on the B+ gate, the B+CD61+ gate is drawn according to whether the platelet surface molecule CD61 is expressed. B+CD61+ is the platelet basophil aggregate. (f): Based on the E+ gate, the E+CD61+ gate is drawn according to whether the platelet surface molecule CD61 is expressed. E+CD61+ is the platelet eosinophil aggregate. (g): Based on the N+ gate, the N+CD61+ gate is drawn according to whether the platelet surface molecule CD61 is expressed. N+CD61+ is the platelet neutrophil aggregate.

[0125] The gating strategy for platelet-monocyte aggregates is as follows Figure 3As shown, (a): A diagram of the five groups of normal human leukocytes, from which the monocytes are circled. (b): Based on the monocyte gate, according to the expression levels of CD14 and CD16, it is divided into CD14+CD16- gate (Classical-Mon gate), CD14+CD16+ gate (Intermediate-Mon gate), and CD14-CD16+ gate (Nonclassical-Mon gate), which correspond to the classical monocyte population, intermediate monocyte population, and nonclassical monocyte population, respectively. (c): Based on the monocyte gate, the PMA(all) gate is drawn according to whether the platelet surface molecule CD61 is expressed. PMA(all) is the total platelet monocyte aggregate. (d): Based on the Nonclassical-Mon gate, the PMA(Nonclassical-Mon) gate is drawn according to whether the platelet surface molecule CD61 is expressed. PMA(Nonclassical-Mon) is the nonclassical platelet monocyte aggregate. (e): Based on the Classical-Mon gate, the PMA (Classical-Mon) gate is drawn according to whether the platelet surface molecule CD61 is expressed. PMA (Classical-Mon) is the platelet classical monocyte aggregate. (f): Based on the Intermediate-Mon gate, the PMA (Intermediate-Mon) gate is drawn according to whether the platelet surface molecule CD61 is expressed. PMA (Intermediate-Mon) is the platelet intermediate monocyte aggregate. The specific voltage and compensation parameters of each fluorescence channel are shown in Table 1 and Table 2.

[0126] 3. Comparison of platelet-leukocyte aggregate percentage and mean fluorescence intensity among subjects

[0127] Using the established flow cytometric method for detecting different platelet-leukocyte aggregates, platelet T lymphocyte aggregates, platelet CD4+T lymphocyte aggregates, platelet CD8+T lymphocyte aggregates, platelet B lymphocyte aggregates, platelet NK cell aggregates, platelet neutrophil aggregates, platelet eosinophil aggregates, platelet basophil aggregates, platelet total monocyte aggregates, platelet classical monocyte aggregates, platelet intermediate monocyte aggregates, and platelet non-classical monocyte aggregates were detected in 71 patients with sepsis.

[0128] In the detection of platelet lymphocyte aggregates, platelet CD4 +There were significant differences in the percentage levels of T lymphocyte aggregates and platelet B lymphocyte aggregates. Compared with non-SIC patients with sepsis, SIC patients with sepsis expressed lower platelet CD4 + The percentage levels of platelet T lymphocyte aggregates (P=0.045) and platelet B lymphocyte aggregates (P=0.037). In addition, the mean fluorescence intensity of all types of platelet lymphocyte aggregates between the two types of patients was statistically significant. Patients with sepsis SIC had higher mean fluorescence intensity of platelet T lymphocyte aggregates (P<0.001), platelet CD4+T lymphocyte aggregates (P=0.005), platelet CD8+T lymphocyte aggregates (P=0.007), platelet B lymphocyte aggregates (P=0.017), and platelet NK cell aggregates (P=0.031). Figure 4 Shown

[0129] In the detection of platelet-granulocyte aggregates, the differences in the percentage levels of platelet-neutrophil aggregates and platelet-eosinophil aggregates between the two types of patients were statistically significant. Compared with non-SIC patients with sepsis, patients with SIC with sepsis expressed lower levels of platelet-neutrophil aggregates (P=0.005) and platelet-eosinophil aggregates (P<0.001). In addition, patients with SIC with sepsis had higher mean fluorescence intensity of platelet-neutrophil aggregates (P<0.001). Figure 5 Shown

[0130] In the detection of platelet-monocyte aggregates, there was a statistically significant difference in the percentage level of total platelet-monocyte aggregates between the two types of patients. Compared with non-SIC patients with sepsis, patients with SIC had lower total platelet-monocyte aggregates (P=0.038). In addition, there was a statistically significant difference in the mean fluorescence intensity of total platelet-monocyte aggregates and classic platelet-monocyte aggregates between the two types of patients. Patients with SIC had higher mean fluorescence intensity of total platelet-monocyte aggregates (P<0.001) and classic platelet-monocyte aggregates (P<0.001). Figure 6 Shown

[0131] It can be seen that the present invention has established a relatively standardized and relatively extensive method that can simultaneously detect the percentage level and mean fluorescence intensity of platelet-leukocyte aggregation of 12 cell sources. On this basis, the present invention has carried out the detection of different percentage levels and mean fluorescence intensity of platelet-leukocyte aggregation in patients with sepsis coagulopathy and patients with sepsis without coagulopathy.

[0132] The beneficial effects of the present invention are: taking an EDTA anticoagulated blood sample collected within 4 hours as the test sample, pre-treating it at room temperature, and flow cytometry detecting the percentage level and expression intensity of different platelet-leukocyte aggregates therein. The method provided by the present invention can accurately detect different types of platelet-leukocyte aggregates in blood samples. The present invention has the advantages of convenient operation, easy sample acquisition, non-invasive and non-destructive, fast detection speed, and low cost. The present invention is of great significance for thrombosis risk assessment and prediction, and has good clinical application value.

[0133] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for detecting the percentage level and mean fluorescence intensity of different types of platelet-leukocyte aggregates in a blood sample, characterized in that: The following steps are involved: 1) (A) Transport the whole blood sample collected within 4 hours to the laboratory and place it at room temperature; 2) (B) using the blood sample obtained in (A) as the test sample, and detecting different types of platelet-leukocyte aggregates therein by flow cytometry; 3) (B1) using compensation microspheres and specific antibodies to prepare each fluorescent single positive tube, and setting up a blank control tube; adjusting the forward angle, lateral angle and voltage of the corresponding fluorescent type channel of the flow cytometer and the compensation between each fluorescent channel; 4) (B2) Take out 200ul of the blood sample obtained in (A) and place it in a flow cytometer, add fluorescently labeled antibodies targeting surface molecules of different platelet-leukocyte aggregates, and add red blood cell lysis solution to fully lyse the red blood cells. After incubation at 4°C in the dark, centrifuge to remove the supernatant and add PBS buffer. Centrifuge again to remove the supernatant and load the sample for flow cytometry detection. Analyze with Flowjo software to obtain the percentage level of different subtypes of platelet-leukocyte aggregates and the average fluorescence intensity; 5) In the (B1), a blank control tube and a single positive tube are set as follows (I), and (I) is set as follows: Blank control tube: add the whole blood sample obtained in (A) and positive microspheres and negative microspheres, incubate at room temperature in the dark, and load the sample at a low flow rate; Single positive tube-1: Add positive microspheres, negative microspheres, and APC CD14 antibody, incubate at room temperature in the dark, and load at a low flow rate; Single positive tube-2: Add positive microspheres, negative microspheres, and PE CD61 antibody, incubate at room temperature in the dark, and load at a low flow rate; Single positive tube-3: Add positive microspheres, negative microspheres, and FITC CD3 antibody, incubate at room temperature in the dark, and load at a low flow rate; Single positive tube-4: Add positive microspheres, negative microspheres, and APC-Cy7 CD11b antibody, incubate at room temperature in the dark, and load at a low flow rate; Single positive tube-5: Add positive microspheres, negative microspheres, and PE-Cy7 CD15 antibody, incubate at room temperature in the dark, and load at a low flow rate; Single positive tube-6: Add positive microspheres, negative microspheres, and Percp / 5.5CD16 antibody, incubate at room temperature in the dark, and load at a low flow rate; 6) In (B1), according to the blank control tube and single positive tubes -1 to -6 set in (I), adjust the voltages of the APC, PE, FITC, APC-Cy7, PE-Cy7, and Percp / 5.5 channels and the compensation between the fluorescence channels; 7) According to the blank control tube and single positive tubes -1 to -5 set in (I), the voltages of the APC, PE, FITC, APC-Cy7, PE-Cy7, and Percp / 5.5 channels and the compensation between the fluorescence channels are adjusted, and the parameters are set as follows: the forward scattered light voltage is 286 volts, the side scattered light voltage is 451 volts, the APC channel voltage is 478 volts, the PE channel voltage is 500 volts, the FITC channel voltage is 464 volts, the APC-Cy7 channel voltage is 507 volts, the PE-Cy7 channel voltage is 615 volts, the Percp / 5.5 channel voltage is 625 volts, the FITC-%APC compensation is 0, the PE-%APC compensation is 0, the APC Cy7-%APC compensation is 7.2, and the PE The compensation for Cy7-%APC was 0, the compensation for Percp / 5.5-%APC was 1.5, the compensation for FITC-%PE was 0.6, the compensation for APC Cy7-%PE was 0, the compensation for PE Cy7-%PE was 0, the compensation for PercpCy5.5-%PE was 13.5, the compensation for APC Cy7-%FITC was 0, the compensation for PE Cy7-%FITC was 0.1, the compensation for Percp / 5.5-%FITC was 2.3, the compensation for PE Cy7-%APC Cy7 was 2.6, the compensation for PercpCy5.5-%APC Cy7 was 0.8, and the compensation for Percp / 5.5-%PE Cy7 was 8.9; 8) In the above (B2), 200ul of the blood sample obtained in (A) is taken out and placed in a flow tube, which is divided into three tubes. According to the following detections, (II) different platelet-lymphocyte aggregates, (III) different platelet-granulocyte aggregates, and (IV) different platelet-monocyte aggregates, fluorescently labeled antibodies against specific surface molecules are added; (II), as follows: 200ul of the blood sample obtained in (A) was taken out and placed in a flow tube, and FITC CD3 antibody, PE CD61 antibody, APC CD19 antibody, APC Cy7 CD8 antibody, PE Cy7 CD56 antibody, and Percp / 5.5CD4 antibody were added; (III), as follows: 200ul of the blood sample obtained in (A) was taken out and placed in a flow tube, and FITC CCR3 antibody, PE CD61 antibody, APC FcεRIα antibody, APC Cy7 CD11b antibody, PE Cy7 CD15 antibody, and Percp / 5.5CD16 antibody were added; (IV), as follows: 200ul of the blood sample obtained in (A) was taken out and placed in a flow tube, and FITC CD62P antibody, PE CD61 antibody, APC CD14 antibody, and Percp / 5.5CD16 antibody were added; 9) In the above (B2), after adding fluorescent labeled antibodies against specific surface molecules to (II), (III), and (IV), incubate in dark for 20 minutes, add 2 ml of red blood cell lysis buffer and continue incubating in dark for 8 minutes, centrifuge at 1500 rpm / min for 5 minutes, remove the supernatant after centrifugation, add 2 ml of PBS buffer, continue centrifugation at the same speed for 5 minutes, remove the supernatant, add 200 ml of PBS buffer to resuspend, and then detect on the machine; 10) In (B2), the on-machine test results are recorded and saved, and data analysis is performed using Flowjo software to record the percentage level of each platelet-leukocyte aggregate and the average fluorescence intensity of platelets on the platelet-leukocyte aggregates.

2. A method for detecting percentage levels and mean fluorescence intensities of different types of platelet-leukocyte aggregates in a blood sample according to claim 1, characterized in that: The blood sample (A) is freshly collected peripheral venous blood, and the anticoagulant used is EDTA.

3. A method for detecting percentage levels and mean fluorescence intensities of different types of platelet-leukocyte aggregates in a blood sample according to claim 1, characterized in that: The different types of platelet leukocyte aggregates can be platelet T cell aggregates (platelet T-cell aggregates, PLyA (T)), platelet CD4+T cell aggregates (platelet T-cell aggregates, PLyA (CD4+)), platelet CD8+T cell aggregates (platelet T-cell aggregates, PLyA (CD8+)), platelet B cell aggregates (platelet B-cell aggregates, PLyA (B)), platelet NK cell aggregates (platelet NK-cell aggregates, PLyA (NK)), platelet neutrophil aggregates (platelet-neutrophil aggregates, PNA), platelet eosinophil aggregates (platelet-eosinophil aggregates), platelet basophil aggregates (platelet-basophil aggregates), platelet classical-monocyte aggregates (platelet classical-monocyte aggregates, PMA (classical-monocyte)), platelet intermediate monocyte aggregates (platelet intermediate aggregates, PMA (intermediate-monocyte)), platelet non-classical aggregates (PMA (non-classical monocyte)).

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