Reagent for rapidly distinguishing bacterial virus infection and application

Abstract

The invention discloses a reagent for rapidly distinguishing bacterial and virus infection and application, belongs to the field of molecular biology, relates to medical and biotechnology, and aims to solve the problem that a traditional method cannot clearly distinguish bacterial infection from virus infection. The reagent comprises five antibodies marked with different fluorescence. The five antibodies are all mouse anti-human or rabbit anti-human antibodies, and targets corresponding to the five antibodies are respectively CD45, CD14, CD64, CD169 and HLA-DR (human leukocyte antigen-DR). The tail end of the antibody is connected with different fluorescein through chemical reaction; fluorescence labeling is performed on neutrophils, mononuclear cells and lymphocytes in peripheral blood through an antibody combination of CD molecules, and corresponding infection indexes are calculated through a flow cytometry method, so that the infection type (bacterial / virus infection) of a patient is quickly distinguished and judged.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biomedicine, and in particular relates to a reagent for rapidly distinguishing bacterial and viral infections and its application. Background Art

[0002] Bacteria can invade the human body through the respiratory tract, digestive tract, skin and mucous membranes, and urinary system. Bacterial infection triggers local inflammatory responses (manifested by redness, swelling, heat, pain, and dysfunction at the infected site) and systemic reactions (fever, fatigue, headache, muscle aches, and other discomfort). In severe cases, bacterial infection can lead to life-threatening conditions such as sepsis and septicemia. At this time, large quantities of bacteria and their toxins enter the bloodstream, triggering a systemic inflammatory response syndrome, leading to serious consequences such as shock and multiple organ dysfunction.

[0003] Viral infection refers to the process in which viruses invade the body, proliferate in target organ cells, and interact with the body. Sometimes, although viral infection occurs, no obvious damage or disease is caused. The pathogenic effect of viruses is mainly caused by invading susceptible cells, damaging or changing cell structure and function. And viral infections are contagious to varying degrees. Systemic symptoms include fever, fatigue, muscle aches, cough, sore throat, diarrhea, herpes, headache, etc., and in severe cases, it can lead to impaired consciousness.

[0004] CD molecules, or leukocyte differentiation antigens, are important molecules found on the surface of immune cells, playing a key role in immune cell recognition, activation, proliferation, and differentiation. Detecting the expression of certain CD molecules in peripheral blood plays a crucial role in the diagnosis of certain diseases.

[0005] CD45 is a transmembrane glycoprotein with a large molecular weight. Its extracellular domain is divided into multiple domains with distinct functional regions. It exists in multiple isoforms, resulting from different exon splicing patterns within its gene. Common isoforms include CD45RA, CD45RB, CD45RC, and CD45RO. These isoforms differ primarily in the composition of their extracellular domains, while their transmembrane and intracellular domains are largely identical. CD45 plays a crucial role in the activation of T and B cells. Through its intracellular phosphatase activity, it regulates the phosphorylation status of key proteins in the T cell receptor (TCR) and B cell receptor (BCR) signaling pathways, thereby initiating TCR signaling and promoting T cell activation, proliferation, and differentiation. In B cells, CD45 also plays a crucial role in regulating the initiation and transmission of BCR signaling, influencing processes such as B cell activation and antibody production. In addition, CD45 participates in regulating signal transduction pathways in a variety of immune cells. In immune cells such as natural killer cells (NK cells) and macrophages, CD45 can also regulate cell activation, cytokine secretion, phagocytosis, etc. by acting as a phosphatase on related signal molecules.

[0006] CD14 is a glycosylphosphatidylinositol (GPI)-anchored protein lacking a transmembrane or intracellular domain and is anchored to the cell surface via a GPI anchor. Its molecular structure includes a leucine-rich repeat (LRR) domain, which plays a key role in recognizing ligands such as pathogen-associated molecular patterns (PAMPs). CD14 is a receptor for lipopolysaccharide (LPS). During Gram-negative bacterial infection, CD14 binds to LPS, initiating the cellular immune response to the infection. It forms a complex with LPS and then transmits LPS to signaling receptors such as Toll-like receptor 4 (TLR4), activating intracellular signaling pathways and inducing the production of inflammatory factors such as tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1), thereby initiating the body's immune defense mechanisms. In addition to recognizing LPS, CD14 can also recognize other bacterial components and some endogenous inflammatory mediators, playing a crucial role in the initiation and amplification of inflammatory responses. It can enhance the phagocytosis of pathogens by monocytes, macrophages, and other cells, and promote the release of more inflammatory mediators and cytokines by these cells, further recruiting and activating other immune cells to eliminate pathogens. During the differentiation of monocytes and macrophages into dendritic cells, CD14 expression levels change, affecting cell differentiation and function. Furthermore, CD14 regulates immune cell migration and adhesion, influencing immune cell aggregation at sites of inflammation and the localization of the immune response.

[0007] CD64, also known as FcγRI, is a transmembrane glycoprotein. Its extracellular region contains three immunoglobulin-like domains that specifically bind to the Fc region of immunoglobulin G (IgG). This binding ability underlies CD64's immune function. The intracellular region contains an immunoreceptor tyrosine-based activation motif (ITAM). When CD64 binds to a ligand, the ITAM recruits downstream signaling molecules, initiating intracellular signal transduction. CD64 is highly expressed on the surface of cells such as macrophages and monocytes. When infected with pathogens, antigens on the pathogen's surface bind to IgG antibodies to form immune complexes. CD64 specifically recognizes and binds to the IgG Fc region within these immune complexes, mediating phagocytosis of the pathogen by phagocytes and promoting pathogen clearance. After CD64 binds to IgG immune complexes, the ITAM activates intracellular signaling pathways, prompting immune cells to release a variety of cytokines and inflammatory mediators, such as tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1). These substances further recruit and activate other immune cells, enhancing the body's immune defense response.

[0008] CD169 is a member of the sialic acid-binding immunoglobulin-like lectin (Siglec) family of proteins within the immunoglobulin superfamily. CD169 is primarily expressed on macrophages, particularly those in the marginal zone of the spleen and subcapsular sinus macrophages of lymph nodes. However, under certain pathological conditions, such as inflammation and infection, other immune cells, such as monocytes, may also induce CD169 expression. CD169 can recognize and bind to sialic acid residues on the surface of various pathogens, mediating their capture and uptake by macrophages and playing a crucial role in the body's early recognition and clearance of pathogens. For example, during influenza virus infection, CD169 recognizes sialic acid on the viral surface, facilitating viral uptake by macrophages and initiating an immune response. After ingesting antigens, CD169-positive macrophages can present them to T cells via MHC molecules, activating T cell immune responses and playing a crucial role in the initiation and regulation of adaptive immunity. Furthermore, CD169 can interact with molecules on the surface of other immune cells to regulate immune cell activation and cytokine secretion. Under normal physiological conditions, CD169 participates in the clearance of senescent or apoptotic cells and some self-antigens in the body, helping to maintain the homeostasis of the immune system and prevent the occurrence of autoimmune reactions.

[0009] HLA-DR belongs to the MHC class II molecule and is a heterodimer composed of an α chain and a β chain connected by non-covalent bonds. Both α and β chains are polymorphic, with the β chain being more polymorphic and playing a major role in the antigen-binding specificity and immune function diversity of HLA-DR molecules. The primary function of HLA-DR is to present antigens to CD4+ T lymphocytes. Within antigen-presenting cells (such as macrophages, dendritic cells, and B cells), antigens are broken down into peptide fragments, which then bind to HLA-DR molecules to form complexes that are transported to the cell surface for recognition by CD4+ T cells, thereby initiating an immune response. HLA-DR interacts with the TCR and other accessory molecules on the T cell surface, transmitting activation or inhibitory signals, regulating T cell proliferation, differentiation, and cytokine secretion, thereby influencing the intensity and direction of the overall immune response. Under normal physiological conditions, HLA-DR participates in the presentation of self-antigens, helping the immune system recognize self-components and maintain immune tolerance. When autoimmune tolerance is broken, HLA-DR may mistakenly present self-antigens, activate autoreactive T cells, and lead to the occurrence of autoimmune diseases.

[0010] Common clinical diagnosis methods for bacterial and viral infections are as follows:

[0011] 1) White blood cell count and classification: When bacterial infection occurs, the total number of white blood cells increases, but when some viral infections occur, the number of lymphocytes increases;

[0012] 2) C-reactive protein (CRP): CRP levels increase when bacterial infections occur, but the increase in CRP is minimal in patients with viral infections.

[0013] 3) Procalcitonin (PCT): PCT levels increase when bacterial infection occurs, but the increase in PCT is minimal in patients with viral infection.

[0014] 4) Antibody testing: When the body has a bacterial infection, the antibody level increases, but there may be false positives or false negatives. However, in patients with viral infections, some antibodies increase, but they can only be detected 2 days after the onset of symptoms. Summary of the Invention

[0015] In order to be able to quickly detect bacterial and viral infections at the clinical end. The present invention proposes a detection kit for quickly distinguishing bacterial and viral infections based on a flow cytometry method, comprising 5 antibodies labeled with different fluorescence. The 5 antibodies are all mouse anti-human or rabbit anti-human antibodies, and the targets corresponding to the 5 antibodies are CD45, CD14, CD64, CD169, and HLA-DR, respectively. The ends of the antibodies are connected to different fluorescein through a chemical reaction; the present application uses an antibody combination of CD molecules to fluorescently label neutrophils, monocytes, and lymphocytes in the peripheral blood, and calculates the corresponding infection index by a flow cytometry method, thereby quickly distinguishing and judging the patient's infection type (bacterial / viral infection). This solves the problem that traditional methods cannot clearly distinguish between bacterial and viral infections, and greatly facilitates the diagnosis of bacterial and viral infections at the hospital end.

[0016] In a first aspect, the present invention provides a reagent for rapidly distinguishing bacterial and viral infections, wherein the reagent comprises a plurality of antibodies, the ends of the plurality of antibodies being connected to different fluoresceins, and the fluorescein concentration is 2 mg / mL.

[0017] In some preferred examples of this aspect, there are 5 antibodies, and all of the 5 antibodies are mouse anti-human or rabbit anti-human antibodies.

[0018] In some preferred examples of this aspect, the targets corresponding to the five antibodies are CD45, CD14, CD64, CD169, and HLA-DR, respectively.

[0019] In some preferred examples of this aspect, the fluorescein labeling concentration is 2 mg / mL, and the fluorescein is allophycocyanin dye.

[0020] In some preferred examples of this aspect, the capture antibodies included in the four antibody pairs are CD70 monoclonal antibody, CD83 monoclonal antibody, CCL3 monoclonal antibody and CCL4 monoclonal antibody respectively.

[0021] In a second aspect, the present invention provides a kit for rapidly distinguishing bacterial and viral infections, the kit comprising the reagents described above.

[0022] In a third aspect, the present invention provides a method for fluorescently labeling peripheral blood. Using the above-mentioned practical implementation, the labeling method comprises the following steps:

[0023] Step 1: Collect peripheral blood samples;

[0024] Step 2: Assess the quality and quantity of peripheral blood samples;

[0025] Step 3: Use reagents to perform fluorescent staining on peripheral blood samples;

[0026] Step 4: The fluorescently stained cells were detected by flow cytometry and the results were analyzed.

[0027] In some preferred examples of this aspect, an anticoagulant is added to the peripheral blood sample in step 1, and the anticoagulant is any one or a combination of EDTA, sodium heparin, and sodium citrate.

[0028] In some preferred examples of this aspect, step 2 specifically includes:

[0029] (1) Collection time: Peripheral blood samples collected and submitted for testing within 3 hours are considered fresh samples;

[0030] (2) Sample collection volume: Peripheral blood samples with a collection volume greater than 3 mL are considered fresh samples.

[0031] In some preferred examples in this aspect, the specific process of step 3 is as follows:

[0032] Add vortexed CD45, CD14, CD64, CD169, and HLA-DR antibodies to each labeled flow tube; then add anticoagulated peripheral blood sample to the bottom of each flow tube, gently shake the flow tube by hand to mix the peripheral blood sample and reagent evenly, incubate in the dark for 10 minutes, add red blood cell lysis buffer, shake gently by hand, incubate in the dark for 10 minutes, centrifuge for 5 minutes, use a pipette to remove the supernatant, add 300 μL PBS solution, vortex and shake to resuspend the cells for use on the machine.

[0033] In some preferred examples in this aspect, the specific process of step 4 is as follows:

[0034] Different horizontal and vertical coordinate channel markers were set to obtain the relationship diagram between two antibodies. First, the coordinates were set to the CD45 / SSC two-coordinate relationship diagram and circled the all-leukocyte gate P1. Then, the coordinates were set to the CD14 / SSC two-coordinate relationship diagram in the P1 gate and circled the granulocyte gate P2, the monocyte gate P3, and the lymphocyte gate P4. Then, the horizontal coordinates were set to the CD64 and CD169 histograms in the P2 and P4 gates and the mean fluorescence intensity (MFI) of CD64 and CD169 in neutrophils and lymphocytes were calculated. Similarly, the horizontal coordinates were set to the CD64, CD169, and HLA-DR histograms in the P3 gate and the mean fluorescence intensity of CD64 and CD169 in monocytes and the percentage of HLA-DR in monocytes were calculated. The specific gating strategy is shown in the figure.

[0035] Compared with the prior art, the present invention has the following advantages:

[0036] 1. The reagent or kit for rapidly distinguishing bacterial and viral infections described in this application is a reagent that detects cell surface proteins. It can quickly, accurately and specifically detect the type of infection in patients through the mean fluorescence intensity (MFI) of each target, and can perform repeated tests multiple times, greatly improving the detection throughput and reducing the difficulty of distinguishing bacterial and viral infections on the hospital side.

[0037] 2. The detection kit for rapid differentiation of bacterial and viral infections described in this application can be adapted to most 2-light 6-color flow cytometers on the market and has high versatility.

[0038] 3. Compared with traditional CRP or PCT methods, the kit has small inter-well differences and small CV values.

[0039] 4. The detection kit for rapid differentiation of bacterial and viral infections described in this application can detect and evaluate peripheral blood cells from multiple angles, greatly improving the sensitivity and accuracy of detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Gating step for flow cytometry data analysis. DETAILED DESCRIPTION

[0041] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments and accompanying drawings. It should be understood that the specific embodiments described are only for explaining the specific application of the present invention and are not intended to limit the scope of the present invention. Through these embodiments, it is intended to help understand the principles, operating methods and advantages of the present invention, but it is not excluded that appropriate adjustments and modifications can be made during the implementation of the present invention.

[0042] Example 1

[0043] Flow cytometry (FCM) is a technique for rapid, multi-parameter, quantitative analysis and sorting of single cells or other biological particles, such as bacteria and microspheres, in suspension. This technique uses fluorescent signals from labeled cells to perform high-speed quantitative analysis of single cells or other biological particles. It is widely used in immunology, hematology, oncology, cell biology, and biochemistry.

[0044] This embodiment proposes a reagent for quickly distinguishing bacterial and viral infections based on a flow cytometry method, which comprises 5 antibodies labeled with different fluorescence. The 5 antibodies are all mouse anti-human or rabbit anti-human antibodies, and the targets corresponding to the 5 antibodies are CD45, CD14, CD64, CD169, and HLA-DR, respectively. The ends of the antibodies are connected to different fluorescein through a chemical reaction, and the fluorescein concentration is 2 mg / mL; the present application uses a combination of CD molecule antibodies to fluorescently label neutrophils, monocytes, and lymphocytes in peripheral blood, and calculates the corresponding infection index by flow cytometry, thereby quickly distinguishing and judging the patient's infection type (bacterial / viral infection).

[0045] The above reagents can be used to prepare a test kit for quickly distinguishing between bacterial and viral infections. The test kit is based on a flow cytometry method for quickly distinguishing between bacterial and viral infections. The detection instrument is a flow cytometer with 488nm and 638nm lasers and 6 fluorescence channels.

[0046] Example 2

[0047] The method for fluorescently labeling peripheral blood using the above reagent or kit comprises the following steps:

[0048] S1. Collect a peripheral blood sample, where the anticoagulant for the peripheral blood sample includes but is not limited to EDTA, sodium heparin, and sodium citrate;

[0049] S2. Evaluate the quality and quantity of collected samples. The specific steps are as follows:

[0050] 1) Collection time: fresh samples must be submitted for testing within 3 hours after collection to meet the requirements.

[0051] 2) Sample collection volume: Samples with a collection volume greater than 3 mL are considered fresh samples that meet the requirements.

[0052] S3. Perform fluorescent staining on the blood sample. The specific steps are as follows:

[0053] Take the flow tubes, mark them, and add vortexed CD45, CD14, CD64, CD169, and HLA-DR antibodies to each tube. Add 100 μL of anticoagulant blood to the bottom of the flow tube to prevent the sample from touching the tube wall; gently shake the flow tube by hand to mix the sample and reagent evenly; incubate in the dark for 10 minutes, add 1 mL of red blood cell lysis buffer, shake gently by hand, and incubate in the dark for 10 minutes; centrifuge at 500g for 5 minutes, use a pipette to remove the supernatant, be careful not to suck up the cell pellet; add 300 μL of PBS solution and vortex to resuspend the cells for use on the machine.

[0054] S4. Perform detection and analysis on a flow cytometer. The analysis steps are as follows:

[0055] Flow cytometry was performed using a Beckman Dxflex flow cytometer, and data were analyzed using Beckman Cytexpert flow cytometry software. Different horizontal and vertical coordinate channel markers were set to obtain relationship diagrams between each marker. First, the coordinates were set to the CD45 / SSC two-coordinate relationship diagram and the all-leukocyte gate P1 was circled. Then, the coordinates were set to the CD14 / SSC two-coordinate relationship diagram in gate P1, and the granulocyte gate P2, the monocyte gate P3, and the lymphocyte gate P4 were circled. Then, the horizontal coordinates of gates P2 and P4 were set to the CD64 and CD169 histograms, and the mean fluorescence intensity (MFI) of CD64 and CD169 in neutrophils and lymphocytes was calculated. Similarly, the horizontal coordinates of gate P3 were set to the CD64, CD169, and HLA-DR histograms, and the mean fluorescence intensity of CD64 and CD169 in monocytes and the percentage of HLA-DR in monocytes were calculated.

[0056] The calculated MFI data were substituted into the following formula to obtain the bacterial infection index and viral infection index:

[0057] Bacterial infection index was calculated as Index = (neutrophil CD64 MFI / lymphocyte CD64 MFI) / (monocyte CD64 MFI / neutrophil CD64 MFI); viral infection index was calculated as Index = monocyte CD169 MFI / lymphocyte CD169 MFI.

[0058] Reference interval setting: for bacterial infection, index>1 indicates bacterial infection; for viral infection, index>1 indicates viral infection.

[0059] Example 3

[0060] Performance evaluation: The specific performance of the detection kit for rapid differentiation of bacterial and viral infections according to the above embodiment was confirmed by measuring real infected samples.

[0061] The experimental steps are as follows:

[0062] Take the flow tubes, mark them, and add vortexed CD45, CD14, CD64, CD169, and HLA-DR antibodies to each tube. Add 100 μL of anticoagulant blood to the bottom of the flow tube to prevent the sample from touching the tube wall; gently shake the flow tube by hand to mix the sample and reagent evenly; incubate in the dark for 10 minutes, add 1 mL of red blood cell lysis buffer, shake gently by hand, and incubate in the dark for 10 minutes; centrifuge at 500g for 5 minutes, use a pipette to remove the supernatant, be careful not to suck up the cell pellet; add 300 μL of PBS solution and vortex to resuspend the cells for use on the machine.

[0063] Flow cytometry was performed using a Beckman Dxflex flow cytometer, and data were analyzed using Beckman Cytexpert flow cytometry software. Different horizontal and vertical coordinate channel markers were set to obtain relationship diagrams between each marker. The data analysis gating strategy is shown in the figure. First, the coordinates were set to the CD45 / SSC two-coordinate relationship diagram and the all-leukocyte gate P1 was circled. Then, the coordinates were set to the CD14 / SSC two-coordinate relationship diagram in gate P1, and the granulocyte gate P2, the monocyte gate P3, and the lymphocyte gate P4 were circled. Then, the horizontal coordinates of gates P2 and P4 were set to the CD64 and CD169 histograms, and the mean fluorescence intensity (MFI) of CD64 and CD169 in neutrophils and lymphocytes was calculated. Similarly, the horizontal coordinates of gate P3 were set to the CD64, CD169, and HLA-DR histograms, and the mean fluorescence intensity of CD64 and CD169 in monocytes and the percentage of HLA-DR in monocytes were calculated. The calculated MFI data were substituted into the formula to obtain the bacterial infection index and viral infection index.

[0064] Table 1. Test results of real samples

[0065]

[0066]

[0067] The above test data demonstrates that the reagent effectively discriminates between viral and bacterial infections and can sensitively detect bacterial and viral co-infections. Conventional detection methods are less able to distinguish co-infections, and the results for normal samples are relatively stable. Furthermore, the kit's high sensitivity allows for effective detection of samples with varying degrees of infection.

[0068] The above descriptions are merely a few embodiments of the present application and do not constitute any form of limitation to the present application. Although the present application discloses the preferred embodiments as above, they are not intended to limit the present application. Any technical personnel familiar with the present profession, without departing from the scope of the technical solution of the present application, using the technical content disclosed above to make slight changes or modifications are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

Claims

1. A reagent for rapidly distinguishing bacterial and viral infections, characterized in that: The reagent comprises a plurality of antibodies, and the ends of the plurality of antibodies are respectively connected to different fluorescent substances.

2. A reagent for rapidly distinguishing bacterial and viral infections according to claim 1, characterized in that: The antibodies are set to 5, and all of the 5 antibodies are mouse anti-human and rabbit anti-human antibodies.

3. A reagent for rapidly distinguishing bacterial and viral infections according to claim 2, characterized in that: The targets corresponding to the five antibodies are CD45, CD14, CD64, CD169, and HLA-DR, respectively.

4. A reagent for rapidly distinguishing bacterial and viral infections according to claim 2, characterized in that: The concentration of the fluorescent labeling agent is 2 mg / mL.

5. A kit for rapidly distinguishing bacterial and viral infections, characterized in that: The kit comprises the reagent according to any one of claims 1 to 4.

6. A method for fluorescent labeling of peripheral blood, characterized in that: The labeling method is implemented using the reagent according to any one of claims 1 to 4, comprising the following steps: Step 1: Collect peripheral blood samples; Step 2: Assess the quality and quantity of peripheral blood samples; Step 3: Use reagents to perform fluorescent staining on peripheral blood samples; Step 4: The fluorescently stained cells were detected by flow cytometry and the results were analyzed.

7. A method for fluorescent labeling of peripheral blood according to claim 6, characterized in that: In step 1, an anticoagulant is added to the peripheral blood sample, wherein the anticoagulant is any one or a combination of EDTA, sodium heparin, and sodium citrate.

8. The method for fluorescent labeling of peripheral blood according to claim 6, characterized in that: Step 2 specifically includes: (1) Collection time: Peripheral blood samples collected and submitted for testing within 3 hours are considered fresh samples; (2) Sample collection volume: Peripheral blood samples with a collection volume greater than 3 mL are considered fresh samples.

9. The method for fluorescent labeling of peripheral blood according to claim 6, characterized in that: The specific process of step 3 is as follows: Add vortexed CD45, CD14, CD64, CD169, and HLA-DR antibodies to each labeled flow tube; then add anticoagulated peripheral blood sample to the bottom of each flow tube, gently shake the flow tube by hand to mix the peripheral blood sample and reagent evenly, incubate in the dark for 10 minutes, add red blood cell lysis buffer, shake gently by hand, incubate in the dark for 10 minutes, centrifuge for 5 minutes, use a pipette to remove the supernatant, add 300 μL PBS solution, vortex and shake to resuspend the cells for use on the machine.

10. The method for fluorescent labeling of peripheral blood according to claim 6, characterized in that: The specific process of step 4 is as follows: Set different horizontal and vertical coordinate channel labels to obtain the relationship diagram between two antibodies. First, set the coordinates to the CD45 / SSC two-coordinate relationship diagram, circle all leukocyte gates P1, then set the coordinates to the CD14 / SSC two-coordinate relationship diagram in the P1 gate, circle the granulocyte gate P2, the monocyte gate P3, and the lymphocyte gate P4; then set the horizontal coordinates in the P2 and P4 gates to the histograms of CD64 and CD169, and calculate the mean fluorescence intensity (MFI) of CD64 and CD169 in neutrophils and lymphocytes. Similarly, set the horizontal coordinates in the P3 gate to the histogram of CD64, CD169, and HLA-DR, and calculate the mean fluorescence intensity of CD64 and CD169 in monocytes and the percentage of HLA-DR in monocytes.

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