CD64 infection index detection kit and method for quality control by using same

Abstract

The invention discloses a CD64 infection index detection kit and a method for quality control by using the kit, the kit comprises an antibody reagent, a buffer solution and a blocking agent, the antibody reagent comprises four antibodies marked by different fluorescence markers, namely CD64, CD14, CD45 and HLA-DR, and the buffer solution is PBS; the blocking agent is a mixed blocking agent composed of mouse IgG, a HAMA blocking agent and heparin sodium; the addition concentration of the mouse IgG, the HAMA blocking agent and the heparin sodium mixed blocking agent is not less than 1.34 mu g / mL and not more than 10 mu g / mL. A blocking agent is added into the kit to reduce background fluorescence of negative control lymphocytes, so that the accuracy of a detection result is improved, meanwhile, compensation microspheres, calibration microspheres and a quality control product are introduced to be used for standardized establishment and verification of a detection template, and the consistency of the detection result is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of marker detection, and specifically relates to a CD64 infection index detection kit and a quality control method using the kit. Background Art

[0002] The T lymphocyte surface antigen CD64 is a high-affinity receptor for the Fcγ segment of the immunoglobulin (IgG) heavy chain. It can bind to IgG1 and IgG3 and is a bridge connecting humoral immunity and cellular immunity. The CD64 molecule on the surface of neutrophils is low in normal state, and the expression level increases rapidly when suffering from infectious diseases. It has high sensitivity and specificity for infectious diseases. Therefore, CD64 can be used as a biomarker for early diagnosis of pathogenic microbial infection in patients. The reference range of the CD64 index is generally used to guide clinical judgment of infection: CD64 index = (neutrophil CD64MFI × neutrophil CD64MFI) / (monocyte CD64MFI × lymphocyte CD64MFI). CD64 is a highly sensitive and specific early marker for the diagnosis of infectious diseases. It can distinguish the active stage of autoimmune diseases or concurrent infections, and can monitor the efficacy of antimicrobial drugs and whether there are concurrent infections after surgery. It has good clinical application prospects. Leukocyte differentiation antigen 14 (CD14) is a key molecule in the body's immune response process. Recent studies have shown that the expression of HLA-DR on the surface of CD14-positive cells is a sensitive indicator of the body's immune function in patients with infectious diseases. Too low HLA-DR levels in CD14+ cells may lead to low immune function and even immune paralysis. Therefore, observing the HLA-DR expression rate of CD14+ cells in patients is helpful to understand the changes in the disease and guide clinical treatment.

[0003] The method for determining neutrophil CD64 disclosed in patent publication number CN110823789A includes the following steps: sample collection; flow cytometry; using only CD64-PE monoclonal fluorescent antibody to circle lymphocytes, monocytes and neutrophils respectively in the FSC / SSC scatter plot; then establishing a histogram to obtain the geometric median fluorescence intensity data of the three groups of cells, and calculating according to the infection index formula. This method has simple reagent components, but ignores the differences in the test samples. Most samples cannot accurately circle lymphocytes, monocytes and neutrophils in the FSC / SSC scatter plot. The boundaries between these three groups of cells in the FSC / SSC scatter plot are not very clear, which can easily lead to inaccurate final infection index results.

[0004] The method for determining the neutrophil infection index for clinical diagnosis of infectious diseases disclosed in the patent publication number CN114942217A, which jointly detects CD64 and CD35 on the surface of neutrophils, can not only judge infection early, but also distinguish and diagnose pathogen types, which has important guiding significance for the diagnosis of clinical diseases. CD35 is a transmembrane protein that binds to complement components C3b and C4b. CD35 mediates phagocytosis by coating C3b and C4b on neutrophil and monocyte particles. The CD35 antigen inhibits complement activation through both classical and bypass pathways. The CD35 antigen is found to be expressed in red blood cells, B cells, T cell subsets, monocytes, macrophages, neutrophils, eosinophils, etc. CD35 on the surface of neutrophils is highly expressed during bacterial infection; but during viral infection, CD35 on the surface of neutrophils is less expressed. The invention introduces the combined diagnosis of CD64 and CD35, and its sensitivity and specificity are better than the conventional detection of CD64 infection index alone.

[0005] The patent publication number CN115327135A discloses a reagent and method for determining the neutrophil CD64 infection index. The CD64 expression of neutrophils in blood samples is detected by flow cytometry, and four antibodies, CD14, CD45, CD64, and HLA-DR, are used to perform gating. Not only can the CD45 channel be used to distinguish the white blood cell population from the debris, lymphocytes and neutrophils can be circled out, but also the CD14 channel can be used to circle out monocytes. The three circles of lymphocytes, monocytes, and neutrophils are more independent, with less mutual influence and overlap, and the gating results are more accurate, and the calculation accuracy is improved. At the same time, the expression of HLA-DR on the surface of CD14-positive cells can also be measured to assist in reflecting the patient's immune status.

[0006] In actual applications, the above three kits or methods for CD64 infection index detection have not taken into account the problem of excessively high background fluorescence of lymphocytes as negative controls; nor have they taken into account the independence of the three cell groups of lymphocytes, monocytes and neutrophils, which has introduced the problem of multicolor compensation faced by multicolor antibodies; nor have they considered the standardization and quality control methods of the detection template, which has easily led to the problem of large differences in detection results between different models of flow cytometers or different personnel.

[0007] Therefore, there is an urgent need in the art to develop a simple, efficient, low-cost kit and quality control method thereof that can detect the CD64 infection index on different models of flow cytometers. Summary of the invention

[0008] In order to solve at least one of the above problems, the present invention provides a CD64 infection index detection kit and a quality control method using the kit.

[0009] In order to achieve the above-mentioned purpose, the present invention adopts the following technical means: the first aspect of the present invention provides a kit for detecting the CD64 infection index, including an antibody reagent, a buffer and a blocker, wherein the antibody reagent includes antibodies labeled with different fluorescent markers: CD64, CD14, CD45, HLA-DR, and the buffer is PBS; the blocker is selected from FC receptor blocker; RF blocker; mouse serum and EDTA-2Na mixed blocker; one of mouse IgG, HAMA blocker and heparin sodium mixed blocker. In some embodiments of the present invention, the four antibodies CD64, CD14, CD45, and HLA-DR in the antibody reagent are mixed in equal volumes; in some embodiments, the labeling of the antibody reagent can be a single labeling or a combined labeling, such as in some embodiments, the antibody reagent is CD64-PE, CD14-FITC, CD45-Percp cy5.5 or CD45-PE-cy7, HLA-DR-APC.

[0010] In some embodiments of the present invention, the FC receptor blocker; RF blocker; mouse serum and EDTA-2Na mixed blocker are added at a concentration of less than 200 μg / mL. In some preferred embodiments of the present invention, the blocking effect is better when the mouse serum and EDTA-2Na mixed blocker is added at a concentration of 20 μg / mL.

[0011] In some embodiments of the present invention, the added concentration of the mixed blocker of mouse IgG, HAMA blocker and heparin sodium is not less than 1.34 μg / mL, and does not exceed the recommended concentration of 10 μg / mL. In some embodiments of the present invention, the blocking effect of the mixed blocker of mouse IgG, HAMA blocker and heparin sodium when the added concentration is less than 1.34 μg / mL is not as good as the blocking effect when the added concentration is 1.34 μg / mL.

[0012] In some embodiments of the present invention, in the mixed blocking agent of mouse serum and EDTA-2Na, the ratio of mouse serum to EDTA-2Na is (8-10): 1. In some preferred embodiments of the present invention, the ratio of mouse serum to EDTA-2Na is 10:1.

[0013] In some embodiments of the present invention, the ratio of mouse ploy IgG: HAMA blocker: heparin sodium in the mixed blocker of mouse IgG, HAMA blocker and heparin sodium is (1-5): (0.1-2): (5-10). In some preferred embodiments of the present invention, the ratio of mouse ploy IgG: HAMA blocker: heparin sodium is 1: 0.2: 8.

[0014] In some embodiments of the present invention, the volume ratio of the four antibodies in the antibody reagent to the buffer is CD64:CD14:CD45:HLA-DR:buffer = (3-12):(3-12):(3-12):(3-12):(52-88).

[0015] In some embodiments of the present invention, the kit further comprises hemolysin, freeze-dried protective solution, and phosphate buffer containing 1% BSA. In some preferred embodiments, the hemolysin can be selected from staphylococcal hemolysin, red blood cell lysis solution containing 1-2% ammonium chloride, organic hemolysin containing 30-37% formaldehyde, 1-3.5% methanol, and 9-10% diethylene glycol, and the freeze-dried protective solution comprises ultrapure water, 10% water-soluble starch, 1% trehalose, 2% inulin, 5% sorbitol, 2% polyglutamic acid, 1% glycerol, and 2% vitamin C.

[0016] In some embodiments of the present invention, the kit further includes compensation microspheres, calibration microspheres, and quality control products, and the quality control products include high-value quality control products and low-value quality control products.

[0017] In some embodiments of the present invention, the preparation method of the quality control product is as follows: 1 mL of fresh blood samples with low and high CD64 values ​​that meet the requirements of the quality control product is taken from each blood sample, and the samples are mixed separately according to the high value group and the low value group, and hemolyzed by adding hemolysin at a volume ratio of 1:20 between the blood sample and the hemolysin. After hemolysis for 15 minutes, the sample is centrifuged at 400 g for 5 minutes, the supernatant is discarded, and then mixed with 1 mL of PBS, and the supernatant is discarded by centrifugation at 400 g for 5 minutes; the leukocytes in the precipitated part are added with freeze-drying protection solution at a ratio of 1:10, and the sample is dispensed into each vial at a volume of 100 μL / bottle for freeze-drying. After freeze-drying for 48 hours, the CD64 quality control product can be obtained.

[0018] In some embodiments of the present invention, the step of reconstituted freeze-dried quality control products is also included: the freeze-dried quality control products are reconstituted with 100 μL 1×PBS reconstitution solution, gently shaken and allowed to stand for 15 minutes; the products can be used after being fully dissolved, and the dissolved freeze-dried quality control products must be used within 30 minutes.

[0019] In some embodiments of the present invention, the step of using the reconstituted CD64 quality control product for sample preparation is also included: 50 μL of the reconstituted quality control sample is aspirated, diluted CD45-Percp-cy5.5, CD14-FITC, HLA-DR-APC, and CD64-PE antibodies are added for incubation in the dark for 15 min, 1 mL of PBS is added after incubation to mix, the supernatant is discarded after centrifugation at 400 g for 5 min, and finally 300 μL of PBS is added for resuspending.

[0020] The second aspect of the present invention provides a method for quality control of CD64 infection index detection using the kit described in the first aspect, comprising the following steps: (1) preparing a whole blood calibration microsphere tube; a compensation microsphere blank tube; a CD64 single positive tube; a CD14 single positive tube; a CD45 single positive tube; an HLA-DR single positive tube; a low-value quality control tube containing an antibody reagent; and a high-value quality control tube containing an antibody reagent; the CD64 single positive tube, CD14 single positive tube, CD45 single positive tube, and HLA-DR single positive tube are prepared using four antibodies, CD64, CD14, CD45, and HLA-DR, respectively, a phosphate buffer solution containing 1% BSA, PBS, and compensation microspheres; the antibody reagent contains four antibodies, CD64, CD14, CD45, and HLA-DR; further, the antibody reagent contains equal volumes of the four antibodies CD64, CD14, CD45, and HLA-DR; (2) Test the whole blood calibration microsphere tube on the machine, adjust the voltage of FSC-H\SSC-H, so that the sample cells and calibration microspheres are in the center of the FSC-H\SSC-H scatter plot; adjust the voltage of each fluorescent channel so that the negative cell population is located in each fluorescent channel -H 10 3 Nearby, the calibration microsphere is located within the coordinate axis; (3) Based on the voltage adjusted for the whole blood calibration microsphere tube, the compensation microsphere blank tube is tested on the machine and repeatedly debugged to ensure that the sample cells, calibration microspheres, and compensation microspheres are all in the center of the FSC-H\SSC-H scatter plot at the same voltage, and at the same time, the negative cell population / compensation microspheres are located in each fluorescent channel-H 10 3 The calibration microspheres are located in the coordinate axis; the final voltage and the median fluorescence intensity of the calibration microspheres in each channel are recorded; (4) Test the CD64 single-positive tube, CD14 single-positive tube, CD45 single-positive tube, and HLA-DR single-positive tube on the machine in turn, and make compensation adjustments in turn to ensure that the median fluorescence intensity of the negative and positive microspheres at the same level in the vertical and horizontal directions is the same, record the final compensation matrix, and save the detection template; (5) Two different levels of quality control test templates, a low-value quality control tube containing antibody reagents and a high-value quality control tube containing antibody reagents, are used to test whether the infection index results are within the target value range of the quality control products, and to verify whether the template establishment is qualified and accurate.

[0021] When establishing the template again, the voltage should be adjusted based on the median fluorescence intensity of the calibration microspheres in each fluorescence channel recorded in step (3) as a reference to control the CV between the median fluorescence intensity of each fluorescence channel and the median fluorescence intensity of each fluorescence channel to less than 2%. Refer to steps (4) and (5) to complete template establishment and quality control.

[0022] The third aspect of the present invention provides a method for establishing a standard detection template for a detection machine and detecting a CD64 infection index, which specifically includes the following steps: (1) Prepare the tube to be tested Tube ①-Whole blood calibration microsphere tube: prepared from whole blood sample, hemolysin, calibration microspheres and PBS; preparation method: 50μL whole blood sample is mixed with 1mL hemolysin for hemolysis; after 15min, centrifuge at 400g for 5min and discard the supernatant; add 1mL PBS and centrifuge at 400g for 5min and discard the supernatant; add 3μL calibration microspheres and 500μL PBS into the tube for later use; Tube ②-Compensation microsphere blank tube: prepared from compensation microspheres and PBS, the preparation method is: 50μL compensation microspheres are mixed with 500μL PBS and set aside; Tube ③-CD64 single positive tube: prepared from compensation microspheres, CD64, phosphate buffer containing 1% BSA and PBS; the preparation method is: 50μL compensation microspheres plus 5μL CD64 plus 15μL phosphate buffer containing 1% BSA, mixed and incubated for 15 minutes; add 500μL PBS and set aside; Tube ④-CD14 single positive tube: prepared from compensation microspheres, CD14, phosphate buffer containing 1% BSA and PBS; the preparation method is: 50 μL compensation microspheres plus 5 μL CD14 plus 15 μL phosphate buffer containing 1% BSA, mixed and incubated for 15 minutes; add 500 μL PBS and set aside; Tube ⑤-CD45 single positive tube: prepared from compensation microspheres, CD45, phosphate buffer containing 1% BSA and PBS; the preparation method is: 50μL compensation microspheres plus 5μL CD45-percp cy5.5 plus 15μL phosphate buffer containing 1% BSA, mixed and incubated for 15 minutes; add 500μL PBS and set aside; Tube ⑥-HLA-DR single positive tube: prepared from compensation microspheres, HLA-DR, phosphate buffer containing 1% BSA and PBS; the preparation method is: 50 μL compensation microspheres plus 5 μL HLA-DR plus 15 μL phosphate buffer containing 1% BSA, mixed and incubated for 15 minutes; add 500 μL PBS and set aside; Tube ⑦ - Low value quality control tube: prepared from low value quality control, antibody reagent and PBS; preparation method: 50μL low value quality control plus 20μL antibody reagent incubation; add 1mL PBS 400g centrifugation for 5min, discard the supernatant; add 300μL PBS and set aside; Tube ⑧-High value quality control tube: prepared from high value quality control, antibody reagent and PBS; preparation method: 50 μL high value quality control plus 20 μL antibody reagent incubation; add 1 mL PBS 400g centrifugation for 5 min, discard the supernatant; add 300 μL PBS and set aside; Tube ⑨ - Sample test tube: prepared from the whole blood sample to be tested, antibody reagent, hemolysin and PBS, the preparation method is: 50μL of the whole blood sample to be tested is added with 20μL of antibody reagent for mixed incubation; after 15 minutes, 1mL of hemolysin is added for hemolysis; after 15 minutes, centrifuge at 400g for 5 minutes and discard the supernatant; add 1mL of PBS and centrifuge at 400g for 5 minutes and discard the supernatant; add 500μL of PBS and set aside; In the preparation of the test tubes on the above machine, the antibody reagent is a mixture of 5 μL CD64-PE, 5 μL CD14-FITC, 5 μL CD45-percp cy5.5, and 5 μL HLA-DR APC.

[0023] In some embodiments, a blocking agent may be added to tube ⑨-sample test tube, and the blocking agent is selected from one of FC receptor blocker; RF blocker; mouse serum and EDTA-2Na mixed blocker; mouse IgG, HAMA blocker and heparin sodium mixed blocker; the method of adding the blocking agent is: adding the blocking agent to the buffer, and diluting the antibody reagent to the working concentration with the buffer containing different blocking agents for standby use. In the sample test tube, the volume ratio of the four antibodies of the antibody reagent to the buffer is CD64:CD14:CD45:HLA-DR:buffer = (3-12): (3-12): (3-12): (3-12): (52-88).

[0024] In some embodiments, the FC receptor blocker; RF blocker; mouse serum and EDTA-2Na mixed blocker are added at a concentration of less than 200 μg / mL. In some preferred embodiments of the present invention, in the mouse serum and EDTA-2Na mixed blocker, the ratio of mouse serum to EDTA-2Na is (8-10):1. In some preferred embodiments, the ratio of mouse serum to EDTA-2Na is 10:1; the added concentration of 20 μg / mL has a better blocking effect.

[0025] In some embodiments, in the mixed blocker of mouse IgG, HAMA blocker and heparin sodium, the ratio of mouse ployIgG: HAMA blocker: heparin sodium is (1-5): (0.1-2): (5-10); the added concentration is not less than 1.34 μg / mL and not more than 10ug / mL. In some embodiments, when the added concentration of the mixed blocker of mouse IgG, HAMA blocker and heparin sodium is less than 1.34 μg / mL, its blocking effect is not as good as the blocking effect of the added concentration of 1.34 μg / mL. When the concentration is too high, the mixed blocker has poor stability.

[0026] (2) Establishment of detection template for detection machine S1. Test tube ① on the machine, adjust the voltage of FSC-H\SSC-H, make the sample cells and calibration microspheres in the center of the FSC-H\SSC-H scatter plot, avoid cell particles flying out of the interface, and ensure that the cell population is in the scatter plot. Figure 2 3D interface for cell population analysis; adjust the voltage of each fluorescent channel so that the negative cell population is located in each fluorescent channel -H 10 3 Nearby, the calibration microsphere is located within the coordinate axis; S2. Based on the voltage adjusted by tube ①, test tube ② on the machine and adjust it repeatedly to ensure that the sample cells, calibration microspheres, and compensation microspheres can be in the center of the FSC-H\SSC-H scatter plot at the same voltage, and at the same time, the negative cell population / compensation microspheres are located in each fluorescent channel -H 10 3 The calibration microspheres are located in the coordinate axis; the final voltage and the median fluorescence intensity of the calibration microspheres in each channel are recorded; S3. Test tubes ③ to ⑥ sequentially on the machine, and make compensation adjustments in sequence to ensure that the median fluorescence intensity of the negative and positive microspheres at the same level in the longitudinal and transverse directions is the same. Record the final compensation matrix and save the detection template.

[0027] (3) Quality control of the detection template of the detection machine Two different levels of quality control product testing templates, tube ⑦ and tube ⑧, were used to test whether the infection index results were consistent with the target value range of the quality control product, and to verify whether the template establishment was qualified and accurate.

[0028] (4) Testing machine sample testing Tube ⑨ was tested using the established and quality-controlled test template: lymphocytes were circled using CD45 fluorescence and SSC gating, monocytes and neutrophils were circled using CD14 fluorescence and SSC gating, and PE histogram gates were set for the three groups of cells, respectively. The PE fluorescence signal values ​​corresponding to the three types of cells, namely, lymphocytes, monocytes and neutrophils, were recorded for high-value and low-value quality control products, respectively. The infection index was calculated and the test results were recorded. The infection index calculation formula was: CD64 infection index = (neutrophil CD64 MFI × neutrophil CD64 MFI) / (monocyte CD64 MFI × lymphocyte CD64 MFI).

[0029] When the detector establishes the template again, it is necessary to adjust the voltage based on the median fluorescence intensity of the calibration microspheres in each fluorescence channel recorded in step (2) as a reference, and control the CV between the median fluorescence intensity of each fluorescence channel and the median fluorescence intensity to be less than 2%. Refer to step (3) to complete the quality control of the established template.

[0030] The method for establishing the CD64 infection index template on different models using the kit is as follows: (1) Basic voltage adjustment of each fluorescence channel of different models Select fresh blood samples, compensation microspheres and calibration microspheres with no disease symptoms and normal blood routine as the standard, and debug the voltage template of CD64 infection index detection on flow cytometers of different brands and models. Repeated debugging ensures that sample cells, calibration microspheres and compensation microspheres can be in the center of the FSC-H\SSC-H scatter plot at the same voltage to avoid cell particles flying out of the interface and ensure that the cell population is in the scatter plot. Figure 2 dimensional interface for cell population analysis, and at the same time satisfy the negative cell population / compensation microspheres located in each fluorescent channel-H 10 3 The calibration microspheres are located within the coordinate axis; the final voltage and the median fluorescence intensity of the calibration microspheres in each channel are recorded. For reagent preparation, refer to the previous article.

[0031] (2) Confirmation of primary CD64 infection index template for different models Compensation microspheres were selected and incubated with CD64-PE, CD14-FITC, CD45-Percp, and HLA-DR-APC as blank control tubes, single-positive tubes, and all-positive tubes, and tested on the basic voltage template. Compensation adjustment was performed according to the horizontal and vertical principles of compensation adjustment to confirm the detection template of the primary CD64 infection index on different models.

[0032] (3) Confirmation of the final CD64 infection index template for different models Two freeze-dried quality control products with different levels were selected for testing on different models. The templates for each model were established using the standard template establishment method, while ensuring that the quality control products were within the target value range.

[0033] The fourth aspect of the present invention provides an application of a CD64 infection index detection kit in CD64 infection index detection, wherein the kit reduces the background fluorescence of negative control lymphocytes during the detection process by adding a blocker, and improves the accuracy of the detection results and reduces errors by establishing a standardized detection model.

[0034] Beneficial effects of the present invention Compared with the prior art, the present invention has the following beneficial effects: ① The kit of the present invention has a simple composition and is easy to use. It can simultaneously detect two indicators, CD64 infection index and HLA-DR. Compared with the prior art, blocking agent components are added to the antibody reagent composition, which greatly reduces the background fluorescence of negative control lymphocytes, improves the accuracy of the test results, and can better reflect the infection degree of clinical infectious diseases.

[0035] ② Compensation microspheres, calibration microspheres and quality control products are introduced into the kit for the standardized establishment of the detection template. The quality control products are used to verify whether the template establishment meets the requirements. The method for quality control of CD64 infection index detection of different models is introduced by using the kit of the present invention, which can solve the problem of inaccurate results caused by the independence of the three groups of lymphocytes, monocytes and neutrophils, and the fluorescence leakage caused by multi-color antibodies; at the same time, the establishment and quality control of the on-machine detection template solves the problem of large differences in detection results of different models of flow cytometers or different personnel, greatly reduces the differences in detection results of different models or different personnel, improves the accuracy of detection, and can better assist in the diagnosis of clinical infectious diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 A comparison of the median fluorescence intensity of lymphocytes of blocker C and blocker D in Example 3 is shown; Figure 2 A comparison of the median fluorescence intensity of lymphocytes of blockers E, F, and G in Example 4 is shown; Figure 3 A comparison of the median fluorescence intensity of lymphocytes at different concentrations of blocker C in Example 5 is shown; Figure 4 A comparison of the median fluorescence intensity of lymphocytes at different concentrations of blocker D in Example 5 is shown; Figure 5 A comparison of the median fluorescence intensity of lymphocytes at different concentrations of blocker E in Example 5 is shown; Figure 6 A comparison chart of the infection index at different concentrations of blocker E in Example 5 is shown; Figure 7 A comparison of the median fluorescence intensity of lymphocytes at different concentrations of the blocker Z in Example 6 is shown; Figure 8 A comparison chart of the infection index at different concentrations of the blocking agent Z in Example 6 is shown; Fig. 9 A comparison of the median fluorescence intensity of lymphocytes of blockers C, E, CE, and Z in Example 7 is shown; Fig.10 A comparison of the infection indexes of blockers C, E, CE, and Z in Example 7 is shown; Fig.11 A comparison of the median fluorescence intensity of lymphocytes under the conditions of blockers CE and Z amplification of samples in Example 8 is shown; Fig.12 A comparison of infection index under the conditions of sample amplification by blockers CE and Z in Example 8 is shown; Fig.13A comparison chart of the infection index detected by different experimenters on the quality-controlled standard detection template in Example 9 is shown; Fig.14 A comparison chart of the infection index detected by different experimenters on the random template of the conventional method in Example 9 is shown. DETAILED DESCRIPTION

[0037] The following examples are used to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to implement the present invention and therefore can be considered as preferred embodiments of the present invention. However, it will be appreciated by those skilled in the art based on this specification that many modifications may be made to the specific embodiments disclosed herein and still achieve the same or similar results without departing from the spirit or scope of the present invention.

[0038] The numerical ranges in this application are approximate values, so unless otherwise specified, they may include values ​​outside the range. Numerical ranges include all values ​​from the lower limit to the upper limit increased by 1 unit, provided that there is an interval of at least 2 units between any lower value and any higher value. For example, if the recorded component, physical or other properties (such as molecular weight, melt index, etc.) are 100 to 1000, it means that all single values ​​are clearly listed, such as 100, 101, 102, etc., and all sub-ranges, such as 100 to 166, 155 to 170, 198 to 200, etc. For a range containing a value less than 1 or containing a fraction greater than 1 (such as 1.1, 1.5, etc.), 1 unit is appropriately regarded as 0.0001, 0.001, 0.01 or 0.1. For a range containing a single digit less than 10 (such as 1 to 5), 1 unit is usually regarded as 0.1. These are merely specific examples of what is intended to be expressed, and all possible combinations of numerical values ​​between the lowest value and the highest value listed are considered to be clearly recorded in this application. The numerical values ​​in the following examples are only for specific schemes and do not serve to limit the numerical range.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs, and the materials cited herein and those cited by them are incorporated by reference. Those skilled in the art will recognize or will learn through routine experimentation that there are many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0040] The technical solution of this patent is further described in detail below in conjunction with specific implementation methods.

[0041] Example 1: Establishment of a standard test template for on-machine testing 1. Establishment of standard test template 1. Prepare the tube to be tested Tube ①- Whole blood microsphere tube: 50μL whole blood sample + 1mL staphylolysin hemolysis; 15min later, centrifuge at 400g for 5min, discard the supernatant; add 1mL PBS, centrifuge at 400g for 5min, discard the supernatant; add 3μL calibration microspheres and 500μL PBS to the tube for later use; Tube ②-Beads blank tube: 50 μL compensation microspheres plus 500 μL PBS mixed for later use; Tube ③-CD64-PE single positive tube: 50 μL compensation microspheres plus 5 μL CD64-PE plus 15 μL phosphate buffer containing 1% BSA, incubate for 15 min; add 500 μL PBS and set aside; Tube ④ - CD14-FITC single positive tube: 50 μL compensation microspheres plus 5 μL CD14-FITC plus 15 μL phosphate buffer containing 1% BSA, mix and incubate for 15 min; add 500 μL PBS and set aside; Tube ⑤ - CD45-percp cy5.5 single positive tube: 50 μL compensation microspheres plus 5 μL CD45-percp cy5.5 plus 15 μL phosphate buffer containing 1% BSA, mix and incubate for 15 minutes; add 500 μL PBS and set aside; Tube ⑥ - HLA-DR APC single positive tube: 50 μL compensation microspheres plus 5 μL HLA-DR APC plus 15 μL phosphate buffer containing 1% BSA, mix and incubate for 15 min; add 500 μL PBS and set aside; Tube ⑦- Whole blood all positive tube: 50μL whole blood sample is mixed with 20μL antibody reagent for incubation; after 15 minutes, 1mL staphylolysin is added for hemolysis; after 15 minutes, centrifuge at 400g for 5 minutes and discard the supernatant; add 1mL PBS and centrifuge at 400g for 5 minutes and discard the supernatant; add 500μL PBS and set aside; Tube ⑧ - Quality control tube: 50 μL quality control product plus 20 μL antibody reagent were mixed and incubated; 1 mL PBS was added and centrifuged at 400 g for 5 min, and the supernatant was discarded; 300 μL PBS was added and set aside; among them, 20 μL antibody reagent was a mixture of 5 μL CD64-PE, 5 μL CD14-FITC, 5 μL CD45-percp cy5.5, and 5 μL HLA-DR APC.

[0042] 2. Adjust the voltage and record it (1) Place tube ① on the test machine and adjust the voltage of FSC-H\SSC-H at the same time, so that the sample cells and calibration microspheres are in the center of the FSC-H\SSC-H scatter plot to prevent cell particles from flying out of the interface and ensure that the cell population is in the scatter plot. Figure 2 3D interface for cell population analysis; adjust the voltage of each fluorescent channel so that the negative cell population is located in each fluorescent channel -H 10 3 Near the left and right, the calibration microsphere is located within the coordinate axis; (2) Based on the voltage adjusted for tube ①, test tube ② on the machine to observe whether the compensation microspheres are centered in the FSC-H\SSC-H scatter plot, which is convenient for cell population analysis; (3) If not, fine-tune. Repeatedly test tubes ① and ② at the fine-tuned voltage to ensure that the sample cells, calibration microspheres, and compensation microspheres are all centered in the FSC-H\SSC-H scatter plot at the same voltage, which is convenient for cell population analysis, and that the negative cell population / compensation microspheres are located in each fluorescent channel -H 10 3 Nearby positions, the calibration microspheres are located within the coordinate axis; (4) Record the final voltage and the median fluorescence intensity of the calibration microspheres in each channel.

[0043] 3. Compensation adjustment Test tubes ③, ④, ⑤, and ⑥ on the machine in sequence, and make compensation adjustments in sequence to ensure that the MFI of the negative and positive microspheres at the same level is the same, that is, follow the principle of "horizontal and vertical". Record the final compensation matrix and save the detection template.

[0044] 4. Sample testing Perform all-positive tube and quality control tube tests on the template saved in the previous step, use CD45 fluorescence and SSC gating to circle lymphocytes, use CD14 fluorescence and SSC gating to circle monocytes and neutrophils, and set PE histogram gates for the three cell clusters, record high and low quality control lymphocyte, monocyte, and neutrophil-PE fluorescence signal values, calculate the infection index, and record the test results.

[0045] 5. Notes When this model establishes the template again, it is necessary to adjust the voltage based on the MFI value of the calibration microspheres in each fluorescent channel recorded in step 2 as the reference, control the median fluorescence intensity MFI of each fluorescent channel and the CV between them < 2%, and then refer to the subsequent steps to complete the template establishment. Whether the established template meets the requirements can be determined by detecting whether the value of the quality control tube is within the target value range.

[0046] Example 2 Quality Control of Standard Test Templates for Machine Testing 1. Preparation method of quality control products ① Screen out low-value and high-value CD64 fresh blood samples that meet the requirements of quality control products. Among them, fresh blood samples with no disease symptoms and normal blood routine as the standard are low-value CD64 quality control products, and fresh blood samples from patients with bacterial infection, viral infection, sepsis, high CD64 infection index, etc. are used as high-value PD-1 quality control products; 1 mL of each blood sample was taken and mixed separately according to the high value group and the low value group. Staphylococcus hemolysin was added to the blood sample at a volume ratio of 1:20 for hemolysis. After hemolysis for 15 minutes, the sample was centrifuged at 400 g for 5 minutes, the supernatant was discarded, and then mixed with 1 mL of PBS, centrifuged at 400 g for 5 minutes, and the supernatant was discarded; ② Add freeze-drying protection solution to the precipitated white blood cells at a ratio of 1:10, and dispense into each vial at a volume of 100 μL / vial for freeze-drying. After freeze-drying for 48 hours, take out and obtain the CD64 quality control product.

[0047] 2. Testing steps for quality control products: ① Take one bottle of freeze-dried high-value and low-value quality control products, and reconstitute them with 100-200 μL 1×PBS solution for 15 minutes respectively; the fully dissolved freeze-dried quality control products must be used within 30 minutes; ②After complete dissolution, aspirate 50 μL of CD64 quality control sample, add 20 μL of CD64 detection reagent, and incubate in the dark for 15 minutes; ③Add 1 mL of PBS and mix well, centrifuge at 400 g for 5 minutes, and discard the supernatant; ④ Add 300 μL PBS and resuspend for testing; ⑤ According to the method of Example 1, the parameters of the instrument were calibrated with fluorescent calibration microspheres. After all the parameters passed the test, the quality control products were tested: lymphocytes were circled by CD45 fluorescence and SSC gating, monocytes and neutrophils were circled by CD14 fluorescence and SSC gating, and PE histogram gates were set for the three groups of cells respectively. The PE fluorescence signal values ​​of the three types of cells, namely, lymphocytes, monocytes and neutrophils, of the quality control products with high and low values ​​were recorded respectively, and the infection index was calculated by the formula: CD64 infection index = (neutrophil CD64 MFI × neutrophil CD64 MFI) / (monocyte CD64 MFI × lymphocyte CD64 MFI).

[0048] 3. Qualification of the quality control product verification template Based on the high-value and low-value quality control products, test whether the infection index results are within the target value range of the quality control products of this model on the newly established template, so as to verify whether the template establishment is qualified and accurate.

[0049] Example 3 Blocker C, D Experiment In this example, blocker C and blocker D were added to the antibody reagent to conduct experiments to reduce the background fluorescence of negative control lymphocytes and compare the response of the test results to the actual infection degree of clinical infectious diseases.

[0050] The experimental plan is as follows: Blocker C: FC receptor blocker; Blocker D: RF blocker.

[0051] The experimental process is as follows: Add 1% volume concentration of blocking agent C and blocking agent D to the buffer solution: PBS. Dilute the antibody reagent to the working concentration with the buffer solution added with blocking agent and set aside; the antibody reagent is a mixture of 5μL CD64-PE, 5μL CD14-FITC, 5μL CD45-percp cy5.5, and 5μL HLA-DR APC.

[0052] The cells without adding blocking agents were used as the normal control group.

[0053] Sample preparation: Take 50 μL of whole blood sample and incubate it with 20 μL of antibody reagent. After incubation for 15 minutes, add 1 mL of staphylolysin for hemolysis; after 15 minutes, centrifuge at 400g for 5 minutes and discard the supernatant; add 1 mL of PBS and centrifuge at 400g for 5 minutes and discard the supernatant; add 500 μL of PBS and set aside.

[0054] The relevant methods of Example 1 and Example 2 are used to establish a standard test template, and quality control is performed to meet the qualified requirements.

[0055] In this experimental group, we used 10 blood samples for testing each blocker.

[0056] The above 10 samples were tested on the machine: CD45 fluorescence and SSC gating were used to circle lymphocytes, CD14 fluorescence and SSC gating were used to circle monocytes and neutrophils, and PE histogram gating was performed on the three groups of cells respectively. The high and low value quality control lymphocyte, monocyte, and neutrophil-PE fluorescence signal values ​​were recorded respectively, and the infection index was calculated. The test results are recorded as shown in Table 1. The median fluorescence intensity of lymphocytes is compared as shown in Figure 1 shown.

[0057] Table 1 Test results of blocker CD and control group

[0058] The results showed that the C and D blockers at a volume concentration of 1% reduced the overall fluorescence intensity of CD64 to a certain extent, thereby improving its infection index results.

[0059] Example 4 Blocker E, F, G Experiment In this example, blockers E, F, and G were added to the antibody reagent to conduct experiments to reduce the background fluorescence of negative control lymphocytes and compare the response of the test results to the actual infection degree of clinical infectious diseases.

[0060] The experimental plan is as follows: Blocker E: mouse serum and EDTA-2Na mixed in a ratio of 10:1; Blocker F: fetal bovine serum, mouse poly IgG, and sodium oxalate mixed in a ratio of 1:10:1; Blocker G: mouse IgG.

[0061] The experimental process is as follows: Add 1% volume concentration of blocker E, blocker F and blocker G to buffer: PBS. Dilute the antibody reagents to working concentrations with the buffer added with blockers for later use. The antibody reagents are prepared by mixing 5μL CD64-PE, 5μL CD14-FITC, 5μL CD45-percp cy5.5 and 5μL HLA-DR APC.

[0062] The cells without adding blocking agents were used as the normal control group.

[0063] Sample preparation: Take 50 μL of whole blood sample and incubate it with 20 μL of antibody reagent. After incubation for 15 minutes, add 1 mL of staphylolysin for hemolysis; after 15 minutes, centrifuge at 400g for 5 minutes and discard the supernatant; add 1 mL of PBS and centrifuge at 400g for 5 minutes and discard the supernatant; add 500 μL of PBS and set aside.

[0064] The relevant methods of Example 1 and Example 2 are used to establish a standard test template that meets quality control requirements.

[0065] In this experimental group, 10 blood samples were used to test each blocker.

[0066] The above 10 samples were tested on the machine: CD45 fluorescence and SSC gating were used to circle lymphocytes, CD14 fluorescence and SSC gating were used to circle monocytes and neutrophils, and PE histogram gating was performed on the three groups of cells respectively. The high and low value quality control lymphocyte, monocyte, and neutrophil-PE fluorescence signal values ​​were recorded respectively, and the infection index was calculated. The test results are recorded as shown in Table 2. The median fluorescence intensity of lymphocytes is compared as shown in Figure 2 shown.

[0067] Table 2 Test results of blocker EG and control group

[0068] The results showed that compared with the normal control, the addition of 1% volume fraction of blocker E, blocker F, and blocker G had no blocking effect on CD64 fluorescence. The possible reason was that the high concentration of blocker had a hook effect on the blocking effect.

[0069] Example 5 Experiments with different concentration gradients of blockers C, D, and E In this embodiment, different concentration gradients of the blocker are set for experiments, and the concentration gradient of the blocker is set as follows: Blocker C: 200 μg / mL, 100 μg / mL, 50 μg / mL; Blocker D: 200 μg / mL, 40 μg / mL, 20 μg / mL; Blocker E: 200 μg / mL, 100 μg / mL, 50 μg / mL The remaining operations were the same as those in Example 3. In this example, a total of 6 blood samples were used for testing. The results of lymphocytes, monocytes, neutrophils and infection index at different concentrations are shown in Table 3. The comparison of the median fluorescence intensity of lymphocytes at different concentrations of blocker C is shown in Table 3. Figure 3 As shown in Figure 2, the median fluorescence intensity of lymphocytes under different concentrations of blocker D is compared as shown in Figure 2. Figure 4 As shown in Figure 2, the median fluorescence intensity of lymphocytes under different concentrations of blocker E is compared as shown in Figure 2. Figure 5 shown.

[0070] Table 3 Test results of different concentrations of blockers C, D, and E

[0071] The results showed that after reducing the concentration of blockers C, D, and E, the blocking effect of each blocker on the median fluorescence intensity of lymphocytes increased. Among them, blocker E at a concentration of -50μg / mL had the best blocking effect, the strongest reduction in the median fluorescence intensity of lymphocytes, and the highest test infection index result.

[0072] To further explore the blocking effect of blocker E at a lower concentration, the blocker concentration gradient was set as follows: Blocker E: 25 μg / mL, 20 μg / mL, 10 μg / mL; The remaining operations were the same as those in Example 3. In this example, a total of 8 blood samples were used for testing. The results of lymphocytes, monocytes, neutrophils and infection index at different concentrations are shown in Table 4. The results of infection index at different concentrations of blocker E are shown in Table 4. Figure 6 shown.

[0073] Table 4 Test results of different concentrations of blocker E

[0074] The results showed that, under the same conditions, when blocker E was added at 20 μg / mL, the blocking effect was better and the infection index result was the best.

[0075] Example 6 Experiment of different concentration gradients of blocker Z In this embodiment, different concentration gradients of the blocker Z were set for experiments, and the composition and concentration gradient of the blocker Z were set as follows: Blocker Z: Mouse poly IgG, HAMA blocker and heparin sodium were mixed in a ratio of 1:0.2:8.

[0076] Blocker Z concentrations: 1.34 μg / mL, 0.536 μg / mL, 0.268 μg / mL, 0.134 μg / mL, 0.0268 μg / mL.

[0077] The remaining operations were the same as those in Example 3. In this example, a total of 5 blood samples were used for testing. The results of lymphocytes, monocytes, neutrophils and infection index at different concentrations are shown in Table 5. The results of lymphocyte median fluorescence intensity and infection index at different concentrations of blocker Z are shown in Table 5. Figure 7 , Figure 8 shown.

[0078] Table 5 Test results of different concentrations of blocker Z

[0079] The results showed that blocker Z was a combined blocker, and the stability of the reagent deteriorated at higher concentrations. When blocker Z did not exceed the recommended concentration of 10 μg / mL, the lymphocyte fluorescence intensity was the lowest, the infection index was the highest, and the blocking effect was the best when blocker Z was added at a concentration of 1.34 μg / mL.

[0080] Example 7 Blocker Screening Experiment to Determine Concentration According to the screening of the types and concentrations of the blockers in Examples 3 to 6, a further comparison scheme was designed: Blocker C: 20 μg / mL; Blocker E: 20 μg / mL; Blocker CE: 20 μg / mL; Blocker Z: 1.34 μg / mL; Wherein, blocker CE is obtained by mixing blocker C and blocker E in a volume ratio of 1:1; The blank control sample was prepared without adding any blocking agent.

[0081] The remaining operations are the same as those in Example 3. In this example, a total of 10 blood samples are used for testing, wherein the blood samples are samples with different levels of CD64 infection index.

[0082] The results of lymphocytes, monocytes, neutrophils and infection index under different blocking agent addition conditions are shown in Table 6. The median fluorescence intensity and infection index of lymphocytes under different conditions are compared as shown in Table 6. Fig. 9 , Fig.10 shown.

[0083] Table 6 Test results of different blockers

[0084] The results showed that in the comparison and verification of sample blockers with different levels of CD64 infection index, the group with blocker Z had the lowest lymphocyte fluorescence intensity, the highest infection index and the best blocking effect.

[0085] Example 8 Blocker expansion sample verification experiment In this example, blocker CE: 20 μg / mL and blocker Z: 1.34 μg / mL were used to expand the sample to compare the blocking effect. The blank control sample was not added with any blocker.

[0086] The remaining operations were the same as those in Example 3. In this example, a total of 30 blood samples were used for testing, wherein the blood samples were samples with different levels of CD64 infection index. The results of lymphocytes, monocytes, neutrophils and infection index under different conditions of adding blockers are shown in Table 7. The comparison of lymphocytes and infection index results under different conditions is shown in Table 7. Fig.11 , Fig.12 shown.

[0087] Table 7 Blocker CE and Blocker Z expanded sample test results

[0088] The results showed that when the samples were expanded for blocking experiment comparison, the blocking effect of adding blocker Z was better than that of adding mixed blocker CE.

[0089] Compared with the control group, the PE fluorescence signal of the lymphocyte cluster was significantly reduced after the addition of the blocker. During normal cell expression, the CD64 antigen surface binding sites are rarely expressed on the lymphocyte surface, so the real PE signal value is low. The addition of the blocker can significantly reduce the nonspecific binding of antibodies on the lymphocyte surface, thereby making the data results closer to the real infection index results of the antibody and the real clinical immune status of the patient.

[0090] Example 9: Influence of the on-machine testing template on the test results Solution 1: First, the various parameters of the instrument are calibrated with fluorescent calibration microspheres, and then used for standby; the experimenter prepares the detection reagents and test samples for 15 samples. The experimenter establishes the respective standard templates for the detection model A and the detection model B according to the relevant methods of Example 1 and Example 2, and uses the quality control product to detect whether it is within the target value range.

[0091] The experimenter used the templates established by each tester A and B to test 15 samples with the same conditions. The method is as follows: The CD45 fluorescence and SSC gating method was used to circle lymphocytes, and the CD14 fluorescence and SSC gating method was used to circle monocytes and neutrophils. The PE histogram gates were set for the three groups of cells, and the PE fluorescence signal values ​​of the high-value and low-value quality control lymphocytes, monocytes, and neutrophils were recorded respectively. The infection index was calculated and the test results were recorded as shown in Table 8. The infection index of different operators was compared as shown in Table 8. Fig.13 shown.

[0092] Table 8 Test results of the same sample by the experimenter under the standard test template of different models

[0093] Solution 2: The laboratory technician prepares the test reagents and test samples for 10 samples. The laboratory technician directly tests the samples on the test machine A and the test machine B according to the conventional test operation method (that is, the quality control step of establishing a standard template is not performed, and the test templates on the two devices are random).

[0094] Here’s how: The CD45 fluorescence and SSC gating method was used to circle lymphocytes, and the CD14 fluorescence and SSC gating method was used to circle monocytes and neutrophils. The PE histogram gates were set for the three groups of cells, and the PE fluorescence signal values ​​of the high-value and low-value quality control lymphocytes, monocytes, and neutrophils were recorded respectively. The infection index was calculated and the test results were recorded as shown in Table 9. The infection index of different operators was compared as shown in Table 9. Fig.14 shown.

[0095] Table 9 Test results of the same sample by the experimenter under the random test templates of different models

[0096] The results showed that before the quality control method was adjusted, the CV of the random template test results on different models ranged from 2.3% to 51.92%, and the result deviation was large; after the quality control method was adjusted, the overall CV of the standard detection template test results on different models was <10%, which significantly reduced the difference in test results on different models. It can be seen that the quality control method of the present invention plays a great role in improving the authenticity and accuracy of the test results.

[0097] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as references separately. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined in the present application.

Claims

1. A CD64 infection index detection kit, characterized in that: The invention comprises an antibody reagent, a buffer and a blocking agent, wherein the antibody reagent comprises antibodies marked with different fluorescent markers: CD64, CD14, CD45 and HLA-DR, and the buffer is PBS; the blocking agent is a mixed blocking agent composed of mouse IgG, HAMA blocker and sodium heparin; in the mixed blocking agent composed of mouse IgG, HAMA blocker and sodium heparin, the ratio of mouse IgG: HAMA blocker: sodium heparin is (1-5): (0.1-2): (5-10).

2. A CD64 infection index detection kit according to claim 1, characterized in that: The added concentration of the mouse IgG, HAMA blocker and heparin sodium mixed blocker is not less than 1.34 μg / mL and not more than 10 μg / mL.

3. A CD64 infection index detection kit according to claim 1, characterized in that: The blocker is one of FC receptor blocker, RF blocker, and mixed blocker of mouse serum and EDTA-2Na; the added concentration of the FC receptor blocker, RF blocker, and mixed blocker of mouse serum and EDTA-2Na is less than 200 μg / mL.

4. A CD64 infection index detection kit according to claim 3, characterized in that: In the mixed blocking agent of mouse serum and EDTA-2Na, the ratio of mouse serum to EDTA-2Na is (8-10):

1.

5. A CD64 infection index detection kit according to claim 1, characterized in that: The ratio of the four antibodies in the antibody reagent to the buffer is CD64:CD14:CD45:HLA-DR:buffer = (3-12):(3-12):(3-12):(3-12):(52-88).

6. A CD64 infection index detection kit according to claim 1, characterized in that: The kit also includes hemolysin, freeze-dried protective solution, and phosphate buffer containing 1% BSA.

7. A CD64 infection index detection kit according to claim 6, characterized in that: The kit also includes compensation microspheres, calibration microspheres, and quality control products, and the quality control products include high-value quality control products and low-value quality control products.

8. A method for quality control of CD64 infection index detection using the kit according to claim 7, characterized in that: The steps include: (1) Prepare whole blood calibration microsphere tubes; compensation microsphere blank tubes; CD64 single positive tubes; CD14 single positive tubes; CD45 single positive tubes; HLA-DR single positive tubes; low-value quality control tubes containing antibody reagents; high-value quality control tubes containing antibody reagents; the CD64 single positive tubes, CD14 single positive tubes, CD45 single positive tubes, and HLA-DR single positive tubes are prepared using four antibodies, CD64, CD14, CD45, and HLA-DR, respectively, phosphate buffer containing 1% BSA, PBS, and compensation microspheres; the antibody reagents contain four antibodies, CD64, CD14, CD45, and HLA-DR; (2) Test the whole blood calibration microsphere tube on the machine, adjust the voltage of FSC-H\SSC-H, so that the sample cells and calibration microspheres are in the center of the FSC-H\SSC-H scatter plot; adjust the voltage of each fluorescent channel so that the negative cell population is located in each fluorescent channel -H 10 3 Nearby, the calibration microsphere is located within the coordinate axis; (3) Based on the voltage adjusted for the whole blood calibration microsphere tube, the compensation microsphere blank tube is tested on the machine and repeatedly adjusted to ensure that the sample cells, calibration microspheres, and compensation microspheres are all in the center of the FSC-H\SSC-H scatter plot at the same voltage, and at the same time, the negative cell population / compensation microspheres are located in each fluorescent channel-H 10 3 The calibration microspheres are located in the coordinate axis; the final voltage and the median fluorescence intensity of the calibration microspheres in each channel are recorded; (4) Test the CD64 single-positive tube, CD14 single-positive tube, CD45 single-positive tube, and HLA-DR single-positive tube on the machine in turn, and make compensation adjustments in turn to ensure that the median fluorescence intensity of the negative and positive microspheres at the same level in the vertical and horizontal directions is the same, record the final compensation matrix, and save the detection template; (5) Use two different levels of quality control test templates, a low-value quality control tube containing antibody reagents and a high-value quality control tube containing antibody reagents, to test whether the infection index results are within the target value range of the quality control products, and to verify whether the template establishment is qualified and accurate.

9. The method according to claim 8, characterized in that: When the template is established again for the same model, first use the median fluorescence intensity of the calibration microspheres in each fluorescence channel recorded in step (3) as a reference to adjust the voltage, control the median fluorescence intensity of each fluorescence channel and the CV between them to be less than 2%, and then refer to steps (4) and (5) to complete the detection template establishment and quality control.

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