A method for detecting the percentage levels and mean fluorescence intensities of different types of platelet-leukocyte aggregates in a blood sample

Abstract

The present invention relates to the field of biotechnology, and discloses a method for detecting the percentage levels and mean fluorescence intensities of different types of platelet-leukocyte aggregates in a blood sample. An EDTA anticoagulated blood sample collected within 4 hours is used as the sample to be tested, pre-treated at room temperature, and the percentage levels and expression intensities of different platelet-leukocyte aggregates are detected by flow cytometry. The method provided by the present invention can accurately detect different types of platelet-leukocyte aggregates in a blood sample. The method for detecting the percentage levels and mean fluorescence intensities of different types of platelet-leukocyte aggregates in a blood sample has the advantages of convenient operation, easy availability of samples, non-invasive and non-destructive, fast detection speed, low cost, etc. The present invention is of great significance for thrombus risk assessment and prediction and has good clinical application value.

Description

Technical Field

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

[0002] Platelets have traditionally been widely regarded as playing a crucial role in maintaining body homeostasis, as well as in thrombosis and hemostasis. In fact, they can also interact with innate immune cells, activating and regulating immune cells. In the context of an infected and inflamed body environment, platelets can directly bind to circulating leukocytes after expressing surface P-selectin. Leukocytes can recognize platelet P-selectin through PSGL-1 on their surface, ultimately forming platelet-leukocyte aggregates (PLA). Platelet-leukocyte aggregates can be divided into platelet-T cell aggregates (PLyA(T)), platelet-CD4+ T cell aggregates (PLyA(CD4+)), platelet-CD8+ T cell aggregates (PLyA(CD8+)), platelet-B cell aggregates (PLyA(B)), 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 (PMA(intermediate-monocyte)), and platelet-non-classical monocyte aggregates (PMA(non-classical monocyte)) according to the different types of leukocytes. Platelet-leukocyte aggregates are involved in multiple pathological processes including inflammation, thrombosis, and cancer, and the tissues and organs that can be affected include the circulatory system, brain, liver, kidneys, and lungs.

[0003] Currently, there are various methods for detecting platelet-leukocyte aggregates in blood circulation, including flow cytometry (FCM), confocal or electron microscopy imaging, and in vitro microfluidic assays. Currently, there is still a lack of professional guidelines for evaluating platelet-leukocyte aggregate detection internationally. In routine methods, flow cytometry is mostly used to identify circulating PLA. This detection method has high sensitivity in differentiating different subtypes of aggregates and reduces the occurrence of false positives. Currently, there is no recognized standardized FCM detection protocol, nor a detection protocol covering various types of platelet-leukocyte aggregates.

[0004] Using advanced confocal or electron microscopy can provide high-resolution images to visualize platelet-leukocyte interactions. However, this analytical method cannot analyze dynamic behaviors.

[0005] Microfluidic technology is a technique for studying platelet-leukocyte interactions in real time, but its ability to simulate physiological activities in real human blood vessels is still limited. Now, an invention of a method for detecting the percentage levels and average fluorescence intensities of different types of platelet-leukocyte aggregates in a blood sample solves the above problems. Summary of the Invention

[0006] (1) Technical Problems to be Solved

[0007] Aiming at the deficiencies of the prior art, the present invention provides a method for detecting the percentage levels and average fluorescence intensities of different types of platelet-leukocyte aggregates in a blood sample, solving the above problems.

[0008] (2) Technical Solutions

[0009] To achieve the above object, the present invention provides the following technical solutions: A method for detecting the percentage levels and average fluorescence intensities of different types of platelet-leukocyte aggregates in a blood sample, comprising the following steps:

[0010] (A) Place the whole blood sample collected within 4 hours at room temperature, and take 200 μl each into 3 flow cytometry tubes for detecting platelet-leukocyte aggregates of 3 major categories: lymphocyte type (tube (Ⅰ)), granulocyte type (tube (Ⅱ)), and monocyte type (tube (Ⅲ));

[0011] (B) Using the 3 whole blood samples obtained in (A) as the test samples, flow cytometry is used to detect the percentage levels and average fluorescence intensities of different types of platelet-leukocyte aggregates therein;

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

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

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

[0015] (B1) Prepare single-positive tubes with compensation microspheres (Compensation Beads, Biolegend, USA) that can be used for adjustment compensation and each fluorescent antibody. After shaking and mixing evenly, load the samples at a low flow rate; the compensation microspheres include 1 part of positive microspheres and 1 part of negative microspheres. The positive microspheres can bind any mouse or human antibody, and the negative microspheres will never bind any antibody. The purpose of these compensation microspheres is for compensation control in multi-color flow cytometry analysis.

[0016] (B2) Set up the blank control tube and each fluorescent single-positive tube according to the following (I).

[0017] (I) Set up as follows:

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

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

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

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

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

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

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

[0025] (B3) In the above (B2), according to the blank control tube, single positive tubes - 1 to - 6 set in the above (I), adjust the voltages of the APC, PE, FITC, APC-Cy7, PE-Cy7, PerCP / 5.5 channels and the compensation between each fluorescence channel;

[0026] According to the blank control tube, single positive tubes - 1 to - 6 set in the above (I), adjust the voltages of the APC, PE, FITC, APC-Cy7, PE-Cy7, PerCP / 5.5 channels and the compensation between each fluorescence channel. The specific parameter settings are as follows: the forward scatter light voltage is 286 V, the side scatter light voltage is 451 V, the voltage of the APC channel is 478 V, the voltage of the PE channel is 500 V, the voltage of the FITC channel is 464 V, the voltage of the APC-Cy7 channel is 507 V, the voltage of the PE-Cy7 channel is 615 V, the voltage of the PerCP / 5.5 channel is 625 V, the compensation of FITC-%APC is 0, the compensation of PE-%APC is 0, the compensation of APC Cy7-%APC is 7.2, the compensation of PE Cy7-%APC is 0, the compensation of PerCP / 5.5-%APC is 1.5, the compensation of FITC-%PE is 0.6, the compensation of APC Cy7-%PE is 0, the compensation of PE Cy7-%PE is 0, the compensation of PercpCy5.5-%PE is 13.5, the compensation of APC Cy7-%FITC is 0, the compensation of PE Cy7-%FITC is 0.1, the compensation of PerCP / 5.5-%FITC is 2.3, the compensation of PE Cy7-%APC Cy7 is 2.6, the compensation of PercpCy5.5-%APC Cy7 is 0.8, the compensation of PerCP / 5.5-%PE Cy7 is 8.9;

[0027] Taking APC Cy7-%APC as an example, its meaning is: the fluorescence intensity leaked from the APC fluorescence channel to the APC Cy7 fluorescence channel. To ensure accurate and true detection, the fluorescence leaked to the APC Cy7 fluorescence channel needs to be subtracted. Therefore, the compensation of APC Cy7-%APC is 7.2;

[0028] (B4) In the above (B1), obtain flow cytometry tubes each containing 200 μl of EDTA whole blood. Add fluorescently labeled antibodies for labeling platelet-lymphocyte aggregates to tube (I), specifically: 2 μl each of FITC CD3 antibody, PE CD61 antibody, APC CD19 antibody, APC Cy7 CD8 antibody, PE Cy7 CD56 antibody, and Percp / 5.5 CD4 antibody; add fluorescently labeled antibodies for labeling platelet-granulocyte aggregates to tube (II), specifically: 2 μl each of FITC CCR3 antibody, PE CD61 antibody, APC FcεRIα antibody, APC Cy7 CD11b antibody, PE Cy7 CD15 antibody, and Percp / 5.5 CD16 antibody; add fluorescently labeled antibodies for labeling platelet-monocyte aggregates to tube (III), specifically: 2 μl each of PE CD61 antibody, APC CD14 antibody, and Percp / 5.5 CD16 antibody; incubate in the dark at 4 °C.

[0029] Preferably, after adding red blood cell lysis buffer (FACSTM Lysing Solution, BD, USA) to fully lyse red blood cells, centrifuge at 1500 rpm / min for 5 min, remove the supernatant, resuspend with PBS buffer, centrifuge again to remove the supernatant, and resuspend with PBS buffer again before performing the upper machine detection.

[0030] (B5) In the above (B4), record and save the upper machine detection results, and use Flowjo software for data analysis. Record the percentage levels of various platelet-leukocyte aggregates and the mean 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 levels and mean fluorescence intensity of different types of platelet-leukocyte aggregates in a blood sample, and has the following beneficial effects:

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

[0033] Figure 1 Schematic diagram for establishing the method of flow cytometry for detecting different platelet-lymphocyte aggregates of the present invention;

[0034] (a): Diagram of the five-part white blood cell classification of normal people, with the part where lymphocytes are located circled. (b): Based on the lymphocyte gate, it is divided into CD3+ gate and CD3- gate according to whether the surface molecule CD3 of T lymphocytes is expressed, corresponding to the T lymphocyte population and the non-T lymphocyte population respectively. (c): Based on the CD3+ gate, it is divided into CD3+CD61+ gate according to whether the surface molecule CD61 of platelets is expressed, and CD3+CD61+ is the platelet-T lymphocyte aggregate. (d): Based on the CD3- gate, the CD19+ gate is drawn according to whether the surface molecule CD19 of B lymphocytes is expressed, corresponding to the B lymphocyte population. (e): Based on the CD3- gate, the CD56+ gate is drawn according to whether the surface molecule CD56 of NK cells is expressed, corresponding to the NK cell population. (f): Based on the CD3+ gate, the CD3+CD4+ and CD3+CD8+ gates are drawn according to whether the CD4 or CD8 molecule is expressed, corresponding to the CD4+ T lymphocyte population and the CD8+ T lymphocyte population respectively. (g): Based on the CD19+ gate, the CD19+CD61+ gate is drawn according to whether the surface molecule CD61 of platelets is expressed, and CD19+CD61+ is the platelet-B lymphocyte aggregate. (h): Based on the CD56+ gate, the CD56+CD61+ gate is drawn according to whether the surface molecule CD61 of platelets is expressed, and CD56+CD61+ is the platelet-NK cell aggregate. (i): Based on the CD8+ gate, the CD8+CD61+ gate is drawn according to whether the surface molecule CD61 of platelets is expressed, and CD8+CD61+ is the platelet-CD8+ T lymphocyte aggregate. (j): Based on the CD4+ gate, the CD4+CD61+ gate is drawn according to whether the surface molecule CD61 of platelets is expressed, and CD4+CD61+ is the platelet-CD4+ T lymphocyte aggregate;

[0035] Figure 2 Schematic diagram for establishing the method for detecting different platelet-granulocyte aggregates by flow cytometry of the present invention;

[0036] (a): Normal human white blood cell five-part differential plot, with the part where granulocytes are located circled. (b): Based on the granulocyte gate, the CD11b+CD15+ gate and CD11b+CD15- gate are delineated according to the expression of CD11b and CD15, corresponding to the non-basophil population and basophil population respectively. (c): Based on the CD11b+CD15+ gate, it is divided into the CD11b+CD15+CD16+ gate (N+) and CD11b+CD15+CD16- gate (E+) according to the expression of CD16, corresponding to the neutrophil population and eosinophil population respectively. (d): Based on the CD11b+CD15- gate, the CD11b+CD15-FcεRiα+ / CCR3+ gate (B+) is delineated according to the expression of the basophil surface molecule FcεRiα or CCR3, corresponding to the basophil population. (e): Based on the B+ gate, the B+CD61+ gate is delineated according to the expression of the platelet surface molecule CD61, and B+CD61+ is the platelet-basophil aggregate. (f): Based on the E+ gate, the E+CD61+ gate is delineated according to the expression of the platelet surface molecule CD61, and E+CD61+ is the platelet-eosinophil aggregate. (g): Based on the N+ gate, the N+CD61+ gate is delineated according to the expression of the platelet surface molecule CD61, and N+CD61+ is the platelet-neutrophil aggregate;

[0037] Figure 3 Schematic diagram for the establishment of the method for detecting different platelet-monocyte aggregates by flow cytometry of the present invention;

[0038] (a): Five-part differential white blood cell count of normal people, and the part where monocytes are located is circled. (b): Based on the monocyte gate, it is divided into CD14+CD16- gate (Classical-Mon gate), CD14+CD16+ gate (Intermediate-Mon gate), and CD14-CD16+ gate (Nonclassical-Mon gate) according to the expression levels of CD14 and CD16, corresponding to the classical monocyte population, intermediate monocyte population, and non-classical monocyte population respectively. (c): Based on the monocyte gate, the PMA (all) gate is delineated 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 delineated according to whether the platelet surface molecule CD61 is expressed. PMA (Nonclassical-Mon) is the platelet non-classical monocyte aggregate. (e): Based on the Classical-Mon gate, the PMA (Classical-Mon) gate is delineated 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, it is divided into the PMA (Intermediate-Mon) gate according to whether the platelet surface molecule CD61 is expressed. PMA (Intermediate-Mon) is the platelet intermediate monocyte aggregate;

[0039] Figure 4 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 respectively, and the mean fluorescence intensities (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 5Schematic diagram of the results of detecting the percentage level and mean fluorescence intensity (MFI) of platelet-granulocyte aggregates by flow cytometry according to the present invention;

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

[0043] Figure 6 Schematic diagram of the results of detecting the percentage level and mean fluorescence intensity (MFI) of platelet-monocyte aggregates by flow cytometry according to the present invention;

[0044] The circulating percentage levels of (a) total platelet-monocyte aggregates, (b) classical platelet-monocyte aggregates, (c) non-classical platelet-monocyte aggregates, and (d) intermediate platelet-monocyte aggregates were evaluated respectively, and the mean fluorescence intensities of (e) total platelet-monocyte aggregates, (f) classical platelet-monocyte aggregates, (g) non-classical platelet-monocyte aggregates, and (h) intermediate platelet-monocyte aggregates were detected. Detailed implementation manners

[0045] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0046] Example 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 by comprising the following steps

[0048] 1) (A) Transport the whole blood sample collected within 4 hours to the laboratory smoothly and place it 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 used 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 (PNA), platelet-eosinophil aggregates, platelet-basophil 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) Prepare each single-fluorescence positive tube using compensation microspheres and specific antibodies, and set up a blank control tube at the same time; adjust the forward angle, side angle of the flow cytometer, the voltage of the corresponding fluorescence type channel, and the compensation between each fluorescence channel;

[0051] 4) (B2) Take out 200 μl of the blood sample obtained in (A) and place it in a flow tube, add a fluorescence-labeled antibody against the surface molecules of different platelet-leukocyte aggregates thereto, and add a red blood cell lysate to fully lyse the red blood cells. After incubation in the dark at 4°C, centrifuge to remove the supernatant, add PBS buffer, centrifuge again to remove the supernatant, and then load the sample for flow cytometry detection. Analyze using Flowjo software to obtain the percentage levels and mean fluorescence intensities of different subclasses of platelet-leukocyte aggregates;

[0052] 5) In (B1), set up blank control tubes and single-positive tubes according to the following (I), and the setting of (I) is as follows:

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

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

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

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

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

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

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

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

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

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

[0063] (II) is as follows: 200 μl of the blood sample obtained in (A) is taken 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 / 5.5 CD4 antibody are added;

[0064] (III) is as follows: 200 μl of the blood sample obtained in (A) is taken 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 / 5.5 CD16 antibody are added;

[0065] (IV) is as follows: Take 200 μl of the blood sample obtained in (A) and place it in a flow cytometry tube. Add FITC CD62P antibody, PEC CD61 antibody, APC CD14 antibody, and Percp / 5.5 CD16 antibody.

[0066] 9) In (B2), after adding the fluorescently labeled antibodies against specific surface molecules in (II), (III), and (IV), incubate in the dark for 20 minutes. Then add 2 ml of red blood cell lysis buffer to each and continue to incubate in the dark for 8 minutes. Centrifuge at 1500 rpm / min for a total of 5 minutes. After centrifuging to remove the supernatant, add 2 ml of PBS buffer to each, and continue to centrifuge at the same speed for 5 minutes. Then remove the supernatant. After resuspending with 200 μl of PBS buffer, perform on-machine detection.

[0067] 10) In (B2), record and save the on-machine detection results, and use Flowjo software for data analysis. Record the percentage levels of platelet-leukocyte aggregates and the mean fluorescence intensity of platelets on platelet-leukocyte aggregates.

[0068] I. Test Samples

[0069] Patients with coagulopathy after sepsis and patients without coagulopathy.

[0070] 1. Based on the experience of previous literature research (Li, N., Goodall, A. H., & 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 average number of platelet-leukocyte aggregates between the two groups is Δμ approximately ±15.3, the standard deviation σ is approximately 8.5, the test power is 0.90, and α = 0.05. Calculate that the sample size is approximately 8 cases per group. Therefore, the sample size per group is at least 8 cases or more.

[0071] 2. Selection of research subjects

[0072] From May 2024 to December 2024, 71 sepsis patients (41 in the non-coagulopathy group and 30 in the coagulopathy group) were continuously enrolled as research subjects at the Fourth Clinical Medical College of Peking University. This study has been approved by the hospital ethics committee.

[0073] Inclusion criteria: According to the sepsis 3.0 guidelines, evaluate the SOFA score, and ICU patients meeting the criteria for sepsis; age ≥ 18 years

[0074] Exclusion criteria: Pregnant or lactating patients; patients with malignant tumors undergoing chemotherapy or malignant tumors related to coagulation or platelets; patients with immunosuppressive diseases such as HIV infection; those who have used immunosuppressants within the past three months; age < 18 years old

[0075] Grouping criteria: According to the diagnostic criteria of ISTH SIC score, it is determined whether sepsis patients belong to sepsis-induced coagulopathy (SIC), and they are divided into the SIC group and the non-SIC group.

[0076] 3. Acquisition of relevant information of the research subjects

[0077] Information such as the gender, age, underlying diseases, surgical risk factors (history of thrombosis, surgery within half a year before enrollment

[0078] history, smoking history, hypertension history, diabetes history, blood type, C-reactive protein, hemoglobin, platelet count), and medication history (whether there is a history of antiplatelet drugs) of the research subjects are obtained from their hospital medical records.

[0079] II. Collection and preparation of samples

[0080] A tube of peripheral venous blood of the research subject is drawn by a nurse using a purple-capped anticoagulant tube containing EDTA, and gently inverted and mixed evenly. It is transported to the laboratory smoothly and fixed within 4 hours (the red blood cell lysate contains a fixative)

[0081] III. Instruments

[0082] Flow cytometry instrument Canto II (BD Company, USA)

[0083] IV. Reagents

[0084] Compensation Beads (Biolegend, USA), PE CD61 antibody (Biolegend, USA), FITC CD3 antibody (Biolegend, USA), APC CD19 antibody (Biolegend, USA), APC Cy7 CD8 antibody (Biolegend, USA), PE Cy7 CD56 antibody (Biolegend, USA), Percp / 5.5 CD4 antibody (Biolegend, USA), FITC CCR3 antibody (Biolegend, USA), APC FcεRIα antibody (Biolegend, USA), APC Cy7 CD11b antibody (Biolegend, USA), PE Cy7 CD15 antibody (Biolegend, USA), Percp / 5.5 CD16 antibody (Biolegend, USA), APC CD14 antibody (Biolegend, USA), FACSTM Lysing Solution, (BD, USA).

[0085] V. Reagent Preparation

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

[0087] VI. Experimental Procedures

[0088] Establish a flow cytometry detection method for different platelet-leukocyte aggregates

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

[0090] (2) Blank control tube: Add the whole blood sample obtained in (A), 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, APC-Cy7 CD11b antibody, incubate at room temperature in the dark, and load the sample at low flow rate;

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

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

[0097] Steps (2) to (8) are continuously adjusted to each other to jointly obtain the channel voltages and compensation values 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, PerCP / 5.5 channels:

[0099]

[0100]

[0101] Table 2. Compensation of FITC, PE, APC, APC Cy7, PE Cy7, PerCP / 5.5 fluorescence channels:

[0102]

[0103]

[0104] (9) Place the whole blood sample collected within 4 hours at room temperature, and take 200 μl each and put them into 3 flow cytometry tubes for detecting platelet-leukocyte aggregates of 3 major categories, namely lymphocyte category (tube (Ⅰ)), granulocyte category (tube (Ⅱ)), and monocyte category (tube (Ⅲ)).

[0105] Tube (I): Add 2 μl of FITC CD3 antibody, PE CD61 antibody, APC CD19 antibody, APC Cy7CD8 antibody, PECy7 CD56 antibody, PerCP / 5.5CD4 antibody, and incubate at 4°C in the dark for 20 min

[0106] Tube (Ⅱ): Add 2 μl of FITC CCR3 antibody, PE CD61 antibody, APC FcεRIα antibody, APC Cy7 CD11b antibody, PECy7 CD15 antibody, PerCP / 5.5CD16 antibody, and incubate at 4°C in the dark for 20 min

[0107] Tube (Ⅲ): Add 2 μl of PE CD61 antibody, APC CD14 antibody, and PerCP / 5.5 CD16 antibody, and incubate in the dark at 4°C for 20 min

[0108] (10) After incubation of Tube (Ⅰ), Tube (Ⅱ), and Tube (Ⅲ), add the prepared 1X red blood cell lysate to each tube to fully lyse the red blood cells, and simultaneously fix the platelets (the lysate contains 5% paraformaldehyde). After shaking, incubate in the dark at 4°C for 8 min.

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

[0110] (12) Centrifuge at 1500 rpm / min for 5 min, then remove the supernatant. Resuspend each tube with 400 μl of PBS buffer and detect on the machine

[0111] (13) Import the flow cytometry results into Flowjo software to obtain the percentage levels and mean fluorescence intensities of different platelet-leukocyte aggregates

[0112] VII. Statistical Analysis

[0113] Statistical analysis was performed using SPSS software (version 23.0, IBM Corporation, USA) and Graphpad Prism software (version 7.0, Graphpad Company, USA). Normality tests were performed on the measurement data. For data that conform to a normal distribution, they are expressed as mean ± standard deviation. Measurement data that do not conform to a normal distribution are expressed as median (2.5th percentile, 97.5th percentile). The Mann-Whitney test was used to compare the percentage levels and mean fluorescence intensities of each platelet-leukocyte aggregate between the SIC group and the non-SIC group

[0114] VIII. Experimental Results

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

[0116] From May 2024 to December 2024, 71 sepsis patients (41 in the non-coagulopathy group and 30 in the coagulopathy group) were continuously enrolled as research subjects at the Fourth Clinical Medical College of Peking University. The demographic characteristics and clinical data of the research subjects

[0117] As shown in Table 3

[0118]

[0119]

[0120]

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

[0122] 2. Detection of the percentage levels and mean fluorescence intensities of various platelet-leukocyte aggregates by flow cytometry

[0123] The gating strategy for platelet-lymphocyte aggregates is as Figure 1 shown. (a): Five-part white blood cell diagram of normal people, from which the part where lymphocytes are located is circled. (b): Based on the lymphocyte gate, it is divided into CD3 + gate and CD3 - gate according to whether the surface molecule CD3 of T lymphocytes is expressed, corresponding to the T lymphocyte population and the non-T lymphocyte population respectively. (c): Based on the CD3 + gate, it is divided into CD3 + CD61 + gate and CD3 + CD61 + gate according to whether the platelet surface molecule CD61 is expressed. CD3 - CD61 + gate is the platelet-T lymphocyte aggregate. (d): Based on the CD3 - gate, the CD19 + gate is drawn according to whether the surface molecule CD19 of B lymphocytes is expressed, corresponding to the B lymphocyte population. (e): Based on the CD3 + gate, the CD56 + CD4 + gate and CD3 + CD8 + gate are drawn according to whether the CD4 or CD8 molecule is expressed, corresponding to the CD4 + T lymphocyte population and the CD8 + T lymphocyte population respectively. (f): Based on the CD19 + gate, the CD19 + CD61 + gate and CD19 + CD61 + gate are drawn according to whether the platelet surface molecule CD61 is expressed. CD19 + CD61 + CD61 + gate is the platelet-B lymphocyte aggregate. (h): Based on the CD56 + CD61 + gate and CD56 +Based on the gate, CD8 was delineated according to whether the platelet surface molecule CD61 was expressed. + CD61 + gate, CD8 + CD61 + That is platelet CD8 + T lymphocyte aggregates. (j): Based on the CD4 + gate, CD4 was delineated according to whether the platelet surface molecule CD61 was expressed. + CD61 + gate, CD4 + CD61 + That is platelet CD4 + T lymphocyte aggregates.

[0124] The gating strategy for platelet granulocyte aggregates is as Figure 2 shown. (a): The five-part differential white blood cell count diagram of normal people, from which the part where granulocytes are located was circled. (b): Based on the granulocyte gate, the CD11b+CD15+ gate and the CD11b+CD15- gate were delineated according to whether CD11b and CD15 were expressed, corresponding to the non-basophil population and the basophil population respectively. (c): Based on the CD11b+CD15+ gate, it was divided into the CD11b+CD15+CD16+ gate (N+) and the CD11b+CD15+CD16- gate (E+) according to whether CD16 was expressed, corresponding to the neutrophil population and the eosinophil population respectively. (d): Based on the CD11b+CD15- gate, the CD11b+CD15-FcεRiα+ / CCR3+ gate (B+) was delineated according to whether the basophil surface molecule FcεRiα or CCR3 was expressed, corresponding to the basophil population. (e): Based on the B+ gate, the B+CD61+ gate was delineated according to whether the platelet surface molecule CD61 was expressed, and B+CD61+ is the platelet basophil aggregate. (f): Based on the E+ gate, the E+CD61+ gate was delineated according to whether the platelet surface molecule CD61 was expressed, and E+CD61+ is the platelet eosinophil aggregate. (g): Based on the N+ gate, the N+CD61+ gate was delineated according to whether the platelet surface molecule CD61 was expressed, and N+CD61+ is the platelet neutrophil aggregate.

[0125] The gating strategy for platelet monocyte aggregates is as Figure 3As shown in the figure, (a): Five-part differential white blood cell diagram of normal people, and the part where monocytes are located is 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), CD14-CD16+ gate (Nonclassical-Mon gate), corresponding to the classical monocyte population, intermediate monocyte population, and non-classical monocyte population respectively. (c): Based on the monocyte gate, the PMA(all) gate is delineated 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 delineated according to whether the platelet surface molecule CD61 is expressed. PMA(Nonclassical-Mon) is the platelet non-classical monocyte aggregate. (e): Based on the Classical-Mon gate, the PMA(Classical-Mon) gate is delineated 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 delineated according to whether the platelet surface molecule CD61 is expressed. PMA(Intermediate-Mon) is the platelet intermediate monocyte aggregate. The specific voltages and compensation parameters of each fluorescence channel are shown in Table 1 and Table 2.

[0126] 3. Comparison of the percentage levels and mean fluorescence intensities of different platelet-leukocyte aggregates in the research subjects

[0127] Using the established flow cytometry method for detecting different platelet-leukocyte aggregates, 71 septic patients included in the study were tested for 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.

[0128] In the detection of platelet-lymphocyte aggregates, platelet CD4 in the two groups of patients +There was a statistically significant difference in the percentage levels between T lymphocyte aggregates and platelet B lymphocyte aggregates. Compared with non-SIC patients with sepsis, septic SIC patients had lower platelet CD4 + T lymphocyte aggregates (P = 0.045) and the percentage level of platelet B lymphocyte aggregates (P = 0.037). In addition, there were statistically significant differences in the mean fluorescence intensities of all types of platelet lymphocyte aggregates between the two groups of patients. Septic SIC patients had higher mean fluorescence intensities 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). As Figure 4 shown

[0129] In the detection of platelet granulocyte aggregates, there was a statistically significant difference in the percentage levels between platelet neutrophil aggregates and platelet eosinophil aggregates in the two groups of patients. Compared with non-SIC patients with sepsis, septic SIC patients had lower platelet neutrophil aggregates (P = 0.005) and the percentage level of platelet eosinophil aggregates (P < 0.001). In addition, septic SIC patients had a higher mean fluorescence intensity of platelet neutrophil aggregates (P < 0.001). As 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 groups of patients. Compared with non-SIC patients with sepsis, septic SIC patients had lower total platelet monocyte aggregates (P = 0.038). In addition, there was a statistically significant difference in the mean fluorescence intensities between total platelet monocyte aggregates and classical platelet monocyte aggregates in the two groups of patients. Septic SIC patients had higher mean fluorescence intensities of total platelet monocyte aggregates (P < 0.001) and classical platelet monocyte aggregates (P < 0.001). As Figure 6 shown

[0131] It can be seen that the present invention has established a relatively standardized, relatively extensive method for simultaneously detecting the percentage levels and mean fluorescence intensities of platelet leukocyte aggregates from 12 cell sources. On this basis, the present invention has detected the percentage levels and mean fluorescence intensities of different platelet leukocyte aggregates in patients with septic coagulopathy and non-septic coagulopathy patients with sepsis

[0132] The beneficial effects of the present invention are as follows: Using an EDTA anticoagulated blood sample collected within 4 hours as the sample to be tested, pretreating it at room temperature, and flow detecting the percentage levels and expression intensities of different platelet-leukocyte aggregates therein. The method provided by the present invention can accurately detect different types of platelet-leukocyte aggregates in a blood sample. The present invention has the advantages of convenient operation, easy availability of samples, non-invasive and non-destructive, fast detection speed, low cost, etc. The present invention is of great significance for thrombus risk assessment and prediction and has good clinical application value.

[0133] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and 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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