Active pulmonary tuberculosis diagnosis and differential diagnosis marker combination and its application

Abstract

The present invention relates to a combination of markers for diagnosis and differential diagnosis of active pulmonary tuberculosis and its application, belonging to the technical field of medical testing. + CD15 ‑ CD14 + CD11b + CD64 high CD16 + New use of monocyte subsets in the diagnosis of ATB by detecting CD45 + CD15 ‑ CD14 + CD11b + CD64 high CD16 + Subpopulation or CD45 + CD15 ‑ CD14 + CD11b + CD64 high CD16 + CD62L + The proportion of ATB in monocytes can realize the diagnosis and differential diagnosis of ATB, which has the advantages of sensitivity, rapidity, safety and simplicity, and is conducive to the early diagnosis and treatment of ATB.

Description

Technical Field

[0001] The invention relates to active pulmonary tuberculosis diagnosis and differential diagnosis marker combination and application thereof, belonging to the technical field of medical testing. Background Art

[0002] Active tuberculosis (ATB) is a chronic infectious disease caused by infection with Mycobacterium tuberculosis (Mtb). According to the "2023 Global Tuberculosis Report" released by WHO, there will be approximately 10.6 million new cases of ATB in the world in 2022, and approximately 1.3 million people will die from the disease. The estimated number of new ATB patients in China in 2022 is 748,000, accounting for 7.1% of the global incidence, second only to India and Indonesia. In recent years, with the emergence of multidrug-resistant Mtb and the increase in cases of secondary tuberculosis infection with AIDS, the incidence of ATB has been on the rise, and ATB has now become a global public health issue.

[0003] The prevention and control of pulmonary tuberculosis depends largely on accurate diagnosis and standard anti-tuberculosis treatment. In clinical practice, the commonly used methods for diagnosing ATB include bacterial culture, imaging examination, molecular biological detection and biopsy. However, to date, clinical diagnostic methods related to tuberculosis have defects, such as low sensitivity of sputum smear microscopy, inability to distinguish between dead and live bacteria, and inapplicability in many environments; sputum tuberculosis culture requires a long period (6-8 weeks) and a low positive rate (30-50%); chest X-ray examinations are difficult to distinguish between pulmonary tuberculosis and other lung lesions; Xpert and other molecular detection technologies have high false positive and false negative rates; tuberculin tests cannot distinguish between natural tuberculosis infection and BCG vaccination; the sensitivity and specificity of serum tuberculosis antibody detection are currently limited. In summary, the current accurate diagnosis of ATB still faces various difficulties, and it is urgent to develop new specific ATB diagnosis and differential diagnosis methods. Summary of the invention

[0004] The invention provides a kit for diagnosing active pulmonary tuberculosis, which contains a reagent for detecting the immune characteristics of human monocytes.

[0005] In one embodiment, the human monocyte immune characteristics include high expression of monocyte CD64 and positive CD16.

[0006] In one embodiment, the human monocyte immunosignature is CD45 + CD15 - CD14 + CD11b + CD64 high CD16 +Monocyte subsets.

[0007] In one embodiment, the diagnosis is to identify and find patients with active pulmonary tuberculosis in a population with unknown health status; the population with unknown health status includes but is not limited to healthy people (HC), latent tuberculosis infected persons (LTBI), and active pulmonary tuberculosis (ATB) patients.

[0008] The invention provides a kit for differential diagnosis of active pulmonary tuberculosis, which contains reagents for detecting the immune characteristics of human monocytes.

[0009] In one embodiment, the human monocyte immune characteristics include high expression of monocyte CD64, CD16 positivity, and CD62L positivity.

[0010] In one embodiment, the human monocyte immunosignature is CD45 + CD15 - CD14 + CD11b + CD64 high CD16 + CD62L + Monocyte subsets.

[0011] In one embodiment, the differential diagnosis is identifying active pulmonary tuberculosis patients among pneumonia (PNA), lung cancer (LC), and pulmonary tuberculosis (TB) patients.

[0012] In one embodiment, the reagents include the following antibodies modified with different fluorescent groups: human CD45 fluorescent antibody, human CD15 fluorescent antibody, human CD14 fluorescent antibody, human CD11b fluorescent antibody, human CD16 fluorescent antibody, human CD64 fluorescent antibody, and human CD62L fluorescent antibody.

[0013] In one embodiment, the fluorescent protein includes but is not limited to phycocyanin (APC), Alexa Fluor 647, Alexa 700, APC / Cyanine7, BV421, BV510, BV605, BV650, BV711, BV785, FITC, PE, PE / Dazzle TM 594, one or more of PE / Cyanine5, PE / Cyanine7, Percp, Percp / Cyanine5.5.

[0014] In one embodiment, the detection object of the kit is a body fluid sample, including but not limited to peripheral blood and alveolar lavage fluid.

[0015] In one embodiment, the kit further comprises a lymphocyte separation solution, a red blood cell lysis solution and a washing solution.

[0016] The present invention also provides the use of a reagent for detecting human monocyte immune characteristics in the preparation of a product for diagnosing and / or differentially diagnosing active pulmonary tuberculosis, wherein the human monocyte immune characteristics include CD45 + CD15 - CD14 + CD11b + CD64 high CD16 + Cell subsets or CD45 + CD15 - CD14 + CD11b + CD64 high CD16 + CD62L + Cell subsets.

[0017] In one embodiment, the reagents include the following antibodies modified with different fluorescent groups: human CD45 fluorescent antibody, human CD15 fluorescent antibody, human CD14 fluorescent antibody, human CD11b fluorescent antibody, human CD16 fluorescent antibody, human CD64 fluorescent antibody, and human CD62L fluorescent antibody.

[0018] In one embodiment, the fluorescent markers include, but are not limited to, APC, Alexa Fluor, 647, Alexa 700, APC / Cyanine7, BV421, BV510, BV605, BV650, BV711, BV785, FITC, PE, PE / Dazzle TM 594, PE / Cyanine5, PE / Cyanine7, Percp, Percp / Cyanine5.5.

[0019] In one embodiment, the diagnosis is to screen and find active pulmonary tuberculosis patients among healthy people, latent tuberculosis infected persons, and active pulmonary tuberculosis patients; the active pulmonary tuberculosis patients have high expression of CD64 and positive CD16 in peripheral blood monocytes.

[0020] In one embodiment, the differential diagnosis is to screen and find active pulmonary tuberculosis patients among pneumonia patients, lung cancer patients and active pulmonary tuberculosis patients; the active pulmonary tuberculosis patients have high expression of CD64 in peripheral blood monocytes, and are positive for CD16 and CD62L.

[0021] The present invention also provides a method for screening body fluid samples of active pulmonary tuberculosis patients in an in vitro environment, wherein the body fluid samples to be tested are treated with the reagent or the kit, and the body fluid samples with CD45 + CD15 - CD14 + CD11b + CD64 high CD16 + or CD45 + CD15 - CD14 + CD11b + CD64 high CD16 + CD62L + The monocyte subsets are from body fluid samples of patients with active pulmonary tuberculosis.

[0022] In one embodiment, the body fluid sample includes but is not limited to peripheral blood and bronchoalveolar lavage fluid.

[0023] Beneficial effects:

[0024] (1) The present invention first discovered CD45 + CD15 - CD14 + CD11b + CD64 high CD16 + The proportion of monocyte subsets in peripheral blood mononuclear cells of ATB patients is significantly higher than that of healthy people and patients with latent tuberculosis infection, which can be used as a diagnostic marker for ATB with high specificity and sensitivity. The diagnostic performance of this monocyte subset for ATB was analyzed (ROC curve analysis), and the results showed that CD45 + CD15 - CD14 + CD11b + CD64 high CD16 + Subpopulations of monocytes (CD45 + CD15 - CD14 + CD11b + ) can be used as an ATB diagnostic indicator. When this subgroup is used to distinguish ATB from healthy people and latent tuberculosis infected people, the AUC is 1, the sensitivity is 100.0%, and the specificity is 100.0%. The AUC used to distinguish ATB from healthy people is 1, the sensitivity is 100.0%, and the specificity is 100.0%; the AUC used to distinguish ATB from latent tuberculosis infected people is 1, the sensitivity is 100.0%, and the specificity is 100.0%.

[0025] (2) The present invention also detected the peripheral blood mononuclear cells of ATB patients compared with those of patients with other common lung diseases such as pneumonia (PNA) and lung cancer (LC). + CD15 - CD14 + CD11b + CD64 high CD16 + The expression level of CD62L in the monocyte subset was significantly increased, while the expression level of CD62L in the peripheral blood of patients with other common lung diseases PNA or LC was low, suggesting that CD45 + CD15 - CD14 + CD11b + CD64 high CD16 + CD62L + Monocyte subsets can be used as clinical ATB and PNA / LC differential diagnostic markers. Further, we analyzed the performance of this monocyte subset in the differential diagnosis of ATB (ROC curve analysis), and the results showed that CD45 + CD15 - CD14 + CD11b + CD64 high CD16 + CD62L + Subpopulations of monocytes (CD45 + CD15 - CD14 + CD11b + ) significantly increased (greater than 15%), which can be used as a marker for the differential diagnosis of ATB and other common lung diseases (pneumonia, lung cancer). Among them: the AUC for the differential diagnosis of ATB and PNA was 1, the sensitivity was 100.0%, and the specificity was 100.0%; the AUC for the differential diagnosis of ATB and LC was 0.988, the sensitivity was 100.0%, and the specificity was 98.2%.

[0026] The present invention provides a combination of ATB diagnosis and differential diagnosis markers, which can simply and efficiently identify and diagnose ATB, and has the advantages of being sensitive, rapid, safe and simple. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 :CD45 in peripheral blood of healthy volunteers (Healthy control, HC), latent tuberculosis infection (Latent tuberculosis infection, LTBI), and active tuberculosis patients (ATB)+ CD15 - CD14 + CD11b + CD64 high CD16 + Typical flow cytometry analysis and statistical graphs of monocyte subset content.

[0028] Figure 2 :Peripheral blood CD45 + CD15 - CD14 + CD11b + CD64 high CD16 + ROC curve analysis of monocyte subsets for ATB diagnosis.

[0029] Figure 3 :CD45 in peripheral blood of patients with ATB and PNA + CD15 - CD14 + CD11b + CD64 high CD16 + CD62L + Typical flow cytometry analysis and statistical graphs of monocyte subset content.

[0030] Figure 4 :Peripheral blood CD45 + CD15 - CD14 + CD11b + CD64 high CD16 + CD62L + ROC curve analysis of monocyte subsets for differential diagnosis of ATB and PNA.

[0031] Figure 5 :CD45 in peripheral blood of patients with ATB and LC + CD15 - CD14 + CD11b + CD64 high CD16 + CD62L + Typical flow cytometry analysis and statistical graphs of monocyte subset content.

[0032] Figure 6 :Peripheral blood CD45 + CD15 - CD14 + CD11b + CD64 highCD16 + CD62L + ROC curve analysis of monocyte subsets for differential diagnosis of ATB and LC. DETAILED DESCRIPTION

[0033] The experimental methods in the following examples of the present invention, where no specific conditions are specified, are usually carried out under conventional conditions or under conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0034] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0035] The terms "including" and "having" and any variations thereof of the present invention are intended to cover non-exclusive inclusions. For example, a process, method, device, product or equipment comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps not listed, or may optionally include other steps inherent to these processes, methods, products or equipment.

[0036] In the present invention, the term "and / or" is used to describe the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B may represent three situations: A exists alone, A and B exist at the same time, and B exists alone. The character " / " generally indicates that the associated objects before and after are in an "or" relationship.

[0037] The term "active tuberculosis" (ATB) of the present invention refers to an infectious disease caused by Mycobacterium tuberculosis, characterized by positive sputum tuberculosis test, accompanied by symptoms such as low fever, cough, weight loss, fatigue, poor appetite, etc. Active tuberculosis exists in the body of patients with active tuberculosis and may be contagious.

[0038] The term "latent tuberculosis infection" (LTBI) of the present invention refers to people who have a persistent immune response to Mycobacterium tuberculosis antigens in their bodies but do not have active tuberculosis infection. They usually have no symptoms and are not contagious.

[0039] Example 1 ATB diagnosis based on flow cytometry

[0040] This example is used to describe the method of diagnosing TB patients among healthy persons (HC), latent tuberculosis infected persons (LTBI) and active tuberculosis (ATB) patients.

[0041] 1. Volunteer inclusion criteria

[0042] In this example, 15 HC volunteers, 15 LTBI volunteers, and 15 ATB patients were randomly selected. Gender and body mass index (BMI) were not limited. After obtaining the consent of the above subjects, an informed consent form was signed.

[0043] Inclusion criteria for HC volunteers: no discomfort symptoms, good mental state, no known history of exposure to Mycobacterium tuberculosis, and negative Mtb infection test using a commercially available interferon-γ release assay (IGRA) kit.

[0044] Inclusion criteria for LTBI volunteers: history of exposure to Mycobacterium tuberculosis, no clinical ATB indications, but positive test results for Mtb infection using a commercially available interferon-γ release assay (IGRA) kit.

[0045] Inclusion criteria for ATB patients: a clear clinical diagnosis of pulmonary tuberculosis, a positive Mtb infection test using a commercially available interferon-γ release assay (IGRA) kit, and a positive Mycobacterium tuberculosis culture in respiratory specimens. The above-mentioned pulmonary tuberculosis includes primary pulmonary tuberculosis, hematogenous disseminated pulmonary tuberculosis, and secondary pulmonary tuberculosis (including infiltrative, fibrocavitary, and caseous pneumonia), but does not include tuberculous meningitis.

[0046] All the above participants were aged ≥16 years and had no mental illness.

[0047] 2. Isolation of human peripheral blood mononuclear cells

[0048] Blood was collected after the patients signed informed consent forms. Peripheral blood was collected by fasting intravenous EDTA anticoagulation in the early morning. Mononuclear lymphocytes (PBMCs) in all peripheral blood samples were separated using lymphocyte separation solution (TBD). The operation steps were strictly carried out according to the instructions of the kit.

[0049] (1) adding an equal volume of PBS solution to the collected peripheral blood sample and mixing well to obtain a diluted blood sample;

[0050] (2) Add an equal volume of lymphocyte separation solution to the diluted blood sample into a centrifuge tube, carefully pipette the diluted blood sample onto the surface of the separation solution, and centrifuge at 25°C and 650 g (3 for 1, 3 for 2).

[0051] (3) After centrifugation, a ring-shaped milky white lymphocyte layer can be observed. Carefully pipette the cell layer into a clean centrifuge tube and add 10 mL of washing solution (TBD) to mix the cells. Centrifuge at room temperature at 2,000 rpm / min (9 for up and 9 for down) for 10 min, discard the supernatant, and collect the cell pellet.

[0052] (4) Add 1 mL of red blood cell lysis buffer (Solebol) to the cell pellet, lyse at room temperature for 5 min, centrifuge at 1,000 rpm / min for 5 min, discard the supernatant, and collect the cell pellet;

[0053] (5) Resuspend the obtained cells with 5 mL of washing solution, centrifuge at room temperature at 2,000 rpm / min (9 for up and 9 for down) for 10 min, discard the supernatant, and collect the cell pellet.

[0054] (III) Antibody staining method based on flow cytometry:

[0055] (1) Peripheral blood mononuclear cells from healthy subjects (HC), latent tuberculosis infected persons (LTBI) and ATB patients were isolated according to the method of step (ii), and 200 μL of flow cytometry antibody diluent was added to the cell pellet, mixed thoroughly and incubated at room temperature in the dark for 20 min. The flow cytometry antibody diluent contained human anti-CD45-BV421 fluorescent antibody (Biolegend, 368522), human anti-CD14-BV785 fluorescent antibody (Biolegend, 301840), human anti-C D15-BV510 fluorescent antibody (Biolegend, 323028), human anti-CD11b-BV605 fluorescent antibody (Biolegend, 301332), human anti-CD16-FITC fluorescent antibody (Biolegend, 302006), human anti-CD64-PE fluorescent antibody (Biolegend, 305008), all kinds of antibodies were added into PBS buffer for dilution at a ratio of 1:200 to obtain the flow antibody dilution solution;

[0056] (2) After incubation, centrifuge at 2,000 rpm / min for 5 min and discard the supernatant. Add 1 mL of PBS solution and mix well. Centrifuge at 2,000 rpm / min for 5 min and discard the supernatant.

[0057] (3) Add 100 μL of 4% paraformaldehyde, mix well, and incubate in the dark for 20 min. After incubation, centrifuge at 2,000 rpm / min for 5 min, discard the supernatant, add 1 mL of PBS solution, mix well, centrifuge at 2,000 rpm / min for 5 min, and discard the supernatant;

[0058] (4) The cells were resuspended in 200 μL PBS solution, passed through a membrane and added to a flow cytometer. The cells were tested using a flow cytometer (Fortessa, USA). Data were acquired and analyzed using FlowJo v10 software.

[0059] (IV) Flow cytometry results analysis:

[0060] FlowJo v10 software was used to analyze and compare the CD45 in peripheral blood of healthy subjects (HC), latent tuberculosis infected persons (LTBI) and active pulmonary tuberculosis patients (ATB). + CD15 CD14 + CD11b + CD64 high 、CD45 + CD15 CD14 + CD11b + CD16 + and CD45 + CD15 CD14 + CD11b + CD64 high CD16 + Subpopulations of monocytes (CD45 + CD15 - CD14 + CD11b + The results showed that compared with the HC and LTBI groups, the percentage of CD45 + CD15 - CD14 + CD11b + CD64 high CD16 + Monocyte subsets as diagnostic markers for ATB patients can more significantly distinguish ATB from HC / LTBI. + CD15 - CD14 + CD11b + CD64 high CD16 + The proportion of monocyte subsets in monocytes increased significantly ( Figure 1 ), suggesting that it can be used as a diagnostic marker for ATB patients.

[0061] ROC curve was further used to analyze the expression of CD45 + CD15 - CD14 + CD11b + CD64 high CD16 + The diagnostic performance of monocyte subsets in ATB patients was shown by the proportion of monocyte subsets in monocytes (CD45 + CD15 - CD14 + CD11b +) was significantly increased (greater than 30%) as a diagnostic indicator for ATB patients. When used to distinguish ATB from HC / LTBI, the AUC was 1, the sensitivity was 100.0%, and the specificity was 100.0%. Among them, the AUC for distinguishing ATB from healthy people was 1, the sensitivity was 100%, and the specificity was 100.0%; the AUC for distinguishing ATB from latent tuberculosis infection was 1, the sensitivity was 100%, and the specificity was 100.0%. The above results show that this monocyte subset marker is a highly specific diagnostic indicator for ATB ( Figure 2 ).

[0062] Example 2 Differential diagnosis of ATB based on flow cytometry

[0063] This example is used to describe a method for differentially diagnosing active tuberculosis (ATB), pneumonia (PNA), and lung cancer (LC) patients.

[0064] 1. Volunteer inclusion criteria

[0065] In this example, 15 ATB patients, 15 PNA patients, and 57 LC patients were randomly selected. Gender and body mass index (BMI) were not limited. After obtaining the consent of the above subjects, an informed consent form was signed.

[0066] Inclusion criteria for ATB patients: a clear clinical diagnosis of pulmonary tuberculosis, a positive Mtb infection test using a commercially available interferon-γ release assay (IGRA) kit, and a positive Mycobacterium tuberculosis culture in respiratory specimens. The above-mentioned pulmonary tuberculosis includes primary pulmonary tuberculosis (primary syndrome and intrathoracic lymph node tuberculosis), hematogenous disseminated pulmonary tuberculosis, and secondary pulmonary tuberculosis (including infiltrative, fibrocavitary, and caseous pneumonia), but does not include tuberculous meningitis.

[0067] Inclusion criteria for PNA patients: T-spot, sputum smear, X-pert, and bacterial culture results are all negative, and NGS detects pathogenic pathogens such as Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Gram-negative bacteria, fungi, etc. Including bacterial pneumonia, fungal pneumonia, obstructive pneumonia, etc.

[0068] Inclusion criteria for LC patients: pathological diagnosis combined with immunohistochemistry results clearly confirmed lung malignancy, chest X-ray showed that the lung cancer mass was lobed, with burrs and notches, multiple sputum exfoliated cells were positive, no history of tuberculosis, and no tuberculosis-related symptoms. This includes adenocarcinoma, squamous cell carcinoma, small cell carcinoma, etc.

[0069] All the above participants were aged ≥16 years and had no mental illness.

[0070] Special note: Patients with severe pneumonia, silicosis, and moderate to severe anemia do not meet the inclusion criteria.

[0071] (II) Antibody staining method based on flow cytometry:

[0072] (1) Peripheral blood mononuclear cells from clinical ATB patients, PNA patients, and LC patients were isolated according to the method of step (ii) of Example 1, and 200 μL of flow cytometry antibody diluent was added to the cell precipitate, mixed thoroughly, and incubated at room temperature in the dark for 20 min. The flow cytometry antibody diluent contained human anti-CD45-BV421 fluorescent antibody (Biolegend, 368522), human anti-CD14-BV785 fluorescent antibody (Biolegend, 301840), and human anti-CD15-BV510 fluorescent antibody (Biolegend, 323028), human anti-CD11b-BV605 fluorescent antibody (Biolegend, 301332), human anti-CD16-FITC fluorescent antibody (Biolegend, 302006), human anti-CD64-PE fluorescent antibody (Biolegend, 305008), human anti-CD62L-PerCP / Cy5.5 fluorescent antibody (Biolegend, 304823), all kinds of antibodies were added into PBS buffer for dilution at a ratio of 1:200 to obtain the flow antibody dilution solution;

[0073] (2) After incubation, centrifuge at 2,000 rpm / min for 5 min and discard the supernatant. Add 1 mL of PBS solution and mix well. Centrifuge at 2,000 rpm / min for 5 min and discard the supernatant.

[0074] (3) Add 100 μL of 4% paraformaldehyde, mix well, and incubate in the dark for 20 min. After incubation, centrifuge at 2,000 rpm / min for 5 min, discard the supernatant, add 1 mL of PBS solution, mix well, centrifuge at 2,000 rpm / min for 5 min, and discard the supernatant;

[0075] (4) The cells were resuspended in 200 μL PBS solution, passed through a membrane and added to a flow cytometer. The cells were tested using a flow cytometer (Fortessa, USA). Data were acquired and analyzed using FlowJo v10 software.

[0076] FlowJo v10 software was used to analyze and compare the peripheral blood CD45 of different populations + CD15 - CD14 + CD11b + CD64 high CD16 + CD62L + The proportion of subpopulations in monocytes varies in different populations.

[0077] (III) Flow cytometry results analysis:

[0078] FlowJo v10 software was used to analyze and compare the CD45 in peripheral blood of patients with active pulmonary tuberculosis (ATB) and pneumonia (PNA). + CD15 - CD14 + CD11b + CD64 high CD16 + CD62L + The results showed that compared with the PNA patient group, the proportion of this monocyte subset in the peripheral blood of ATB patients was significantly increased ( Figure 3 ), suggesting that it can be used as a differential diagnostic marker for ATB and PNA. + CD15 - CD14 + CD11b + CD64 high CD16 + CD62L + The performance of the proportion of monocyte subsets in the differential diagnosis of ATB patients was evaluated. The results showed that a significantly increased proportion of this group in monocytes (greater than 15%) can be used as a differential diagnostic marker for ATB. The AUC for the differential diagnosis of ATB and pneumonia (PNA) was 1, the sensitivity was 100.0%, and the specificity was 100.0% ( Figure 4 ).

[0079] FlowJo v10 software was used to analyze and compare the CD45 expression in the peripheral blood of patients with active ATB (ATB) and lung cancer (LC). + CD15 - CD14 + CD11b + CD64 high CD16 + CD62L + The results showed that compared with LC patients, the proportion of this monocyte subset in the peripheral blood of ATB patients was significantly increased ( Figure 5 ), suggesting that this subgroup can be used as a differential diagnostic marker for ATB and LC. + CD15 - CD14 + CD11b + CD64 high CD16 + CD62L +The performance of the proportion of monocyte subsets in the differential diagnosis of ATB patients was studied. The results showed that the proportion of this subset in monocytes was significantly increased (greater than 15%) and could be used as a differential diagnostic marker for ATB. The AUC for the differential diagnosis of ATB and lung cancer (LC) was 0.988, the sensitivity was 100.0%, and the specificity was 98.2% ( Figure 6 ).

[0080] Example 3 Preparation of ATB diagnostic kit

[0081] The ATB diagnostic kit includes: the following antibodies modified with different fluorescent groups: human CD45 fluorescent antibody, human CD15 fluorescent antibody, human CD14 fluorescent antibody, human CD11b fluorescent antibody, human CD16 fluorescent antibody, human CD64 fluorescent antibody, human CD62L fluorescent antibody; the fluorescent group is selected from: phycocyanin (APC), Alexa Fluor 647, Alexa 700, APC / Cyanine7, BV421, BV510, BV605, BV650, BV711, BV785, FITC, PE, PE / Dazzle TM 594, PE / Cyanine5, PE / Cyanine7, Percp, Percp / Cyanine5.5.

[0082] Optionally, the kit further contains a lymphocyte separation solution, a red blood cell lysis solution and a washing solution.

[0083] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Use of a reagent for detecting the immune characteristics of human monocytes in the preparation of a product for diagnosing active pulmonary tuberculosis, characterized in that: The product contains a reagent for detecting the immune characteristics of human monocytes; the immune characteristics of human monocytes are CD45 + CD15 - CD14 + CD11b + CD64 high CD16 + Cell subsets; the diagnosis is to identify and discover active pulmonary tuberculosis patients among a population with unknown health status; the population with unknown health status includes healthy people, latent tuberculosis infected people, and active pulmonary tuberculosis patients.

2. The use according to claim 1, characterized in that: The reagents include the following antibodies modified with different fluorescent groups: human CD45 fluorescent antibody, human CD15 fluorescent antibody, human CD14 fluorescent antibody, human CD11b fluorescent antibody, human CD16 fluorescent antibody, and human CD64 fluorescent antibody.

3. The use according to claim 2, characterized in that: The fluorescent agent includes one or more of phycocyanin, Alexa Fluor® 647, Alexa Fluor® 700, APC / Cyanine7, BV421, BV510, BV605, BV650, BV711, BV785, fluorescein isothiocyanate, glucosidase aldehyde hemoglobin, PE / Dazzle™ 594, PE / Cyanine5, PE / Cyanine7, Percp, and Percp / Cyanine5.

5.

4. The use according to any one of claims 1 to 3, characterized in that: The product also includes lymphocyte separation solution, red blood cell lysis solution and washing solution.

5. The use according to any one of claims 1 to 3, characterized in that: The detection object of the product is a human body fluid sample, including one or more of a blood sample or a bronchoalveolar lavage fluid.

6. The use according to claim 4, characterized in that: The product is a detection kit.

7. Use of a reagent for detecting the immune characteristics of human monocytes in the preparation of a product for differential diagnosis of active pulmonary tuberculosis, characterized in that: The human monocyte immune characteristics are CD45 + CD15 - CD14 + CD11b + CD64 high CD16 + CD62L + Cell subsets; the differential diagnosis is to identify active tuberculosis patients among patients with pneumonia, lung cancer and tuberculosis.

8. The use according to claim 7, characterized in that: The reagents include the following antibodies modified with different fluorescent groups: human CD45 fluorescent antibody, human CD15 fluorescent antibody, human CD14 fluorescent antibody, human CD11b fluorescent antibody, human CD16 fluorescent antibody, human CD64 fluorescent antibody, and human CD62L fluorescent antibody.

9. The use according to claim 8, characterized in that The fluorescent agent includes one or more of phycocyanin, Alexa Fluor® 647, Alexa Fluor® 700, APC / Cyanine7, BV421, BV510, BV605, BV650, BV711, BV785, fluorescein isothiocyanate, glucosidase aldehyde hemoglobin, PE / Dazzle™ 594, PE / Cyanine5, PE / Cyanine7, Percp, and Percp / Cyanine5.

5.

10. The use according to any one of claims 7 to 9, characterized in that: The product also includes lymphocyte separation solution, red blood cell lysis solution and washing solution.

11. The use according to any one of claims 7 to 9, characterized in that: The detection object of the product is a human body fluid sample, including one or more of a blood sample or a bronchoalveolar lavage fluid.

12. The use according to claim 10, characterized in that: The product is a detection kit.

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