CN114441419B

CN114441419B - Flow type gate looping method and application

Info

Publication number: CN114441419B
Application number: CN202210111689.XA
Authority: CN
Inventors: 范盘生; 严倩蓉; 李正义; 张涛; 陆剑
Original assignee: Hangzhou Xiangyu Medical Laboratory Co ltd
Current assignee: Hangzhou Xiangyu Medical Laboratory Co ltd
Priority date: 2022-01-29
Filing date: 2022-01-29
Publication date: 2022-11-22
Anticipated expiration: 2042-01-29
Also published as: CN114441419A

Abstract

The invention discloses a flow type gate circling method, which comprises the following steps: detecting the blood sample incubated by the CD45 antibody, the CD14 antibody and the CD64 antibody by using side scattered light and CD45 fluorescence, and trapping leukocytes in the sample; among the leukocytes that were trapped, portal neutrophils and lymphocytes were trapped with CD45 fluorescence, respectively; among the gated leukocytes, gated monocytes were fluorescent with CD 14. The invention also discloses application of the mixture of the CD45 antibody, the CD14 antibody and the CD64 antibody in preparing infectious disease diagnosis products, wherein the high expression of CD64 in neutrophils is taken as an indication of the disease. The flow type gate circling method of the invention does not excessively depend on the experience of technical personnel, ensures the consistency of detection and ensures that the gate circling method is more accurate and simpler and clearer. After lymphocytes, monocytes and neutrophils are accurately and respectively circled, whether infectious diseases exist or not can be diagnosed according to the expression of CD64 on the neutrophils.

Description

Flow type gate looping method and application

Technical Field

The invention relates to the technical field of cell flow detection, in particular to a flow type gate circling method and application.

Background

The most common way of circulating lymphocytes in blood is: the sample cells are roughly classified into neutrophils, monocytes and lymphocytes by the skilled person according to experience by performing a gate round using forward light FSC and lateral light SSC of the flow cytometer. The experience of the technician determines the quality of the door of the collar. The drawback of this circle of door mode is that the cells circled out in the door are lymphocytes, and because of the different experience of the detection personnel, the purity of the lymphocytes in the door is different. Particularly, when the abundance of the lymphocytes is low, the lymphocytes are lost, and the result is influenced. Moreover, the round-robin method does not accurately distinguish neutrophils, monocytes and lymphocytes.

Disclosure of Invention

Accordingly, there is a need for a flow gating method and applications that can accurately gate neutrophils, monocytes and lymphocytes.

The invention provides a flow type circle gate method, which comprises the following steps:

detecting the blood sample incubated by the CD45 antibody, the CD14 antibody and the CD64 antibody by using side scattered light and CD45 fluorescence, and trapping leukocytes in the sample;

among the leukocytes that were trapped, portal neutrophils and lymphocytes were trapped with CD45 fluorescence, respectively;

among the gated leukocytes, gated monocytes were fluorescent with CD 14.

In one example, fluorescence intensity peaks expressed by CD64 antibody labeled with fluorescent antibody on neutrophils, lymphocytes and monocytes, respectively, were obtained in the gates of neutrophils, lymphocytes and monocytes, respectively.

In one embodiment, the volume ratio of the CD45 antibody, the CD14 antibody or the CD64 antibody to the blood is 1 (8-12) based on the concentration of the CD45 antibody, the CD14 antibody and the CD64 antibody being 1ug/ul respectively.

In one embodiment, the incubation is performed in the dark, and the incubation time is 15min to 30min.

In one embodiment, the flow gating method further comprises removing red blood cells from the blood after the incubation, followed by flow detection.

In one embodiment, the method for removing red blood cells from blood is incubation with a hemolytic agent.

In one embodiment, the volume ratio of the hemolytic agent to the blood is 1 (0.8-1.2); preferably, the incubation with the hemolytic agent is carried out for a period of 14min to 16min.

In one embodiment, the molar ratio of the CD45 antibody, the CD14 antibody, and the CD64 antibody used in the co-incubation is (0.8-1.2): (0.8-1.2).

The second objective of the invention is to provide application of a mixture of a CD45 antibody, a CD14 antibody and a CD64 antibody in preparation of infectious disease diagnosis products, wherein high expression of CD64 in neutrophils is taken as an indication of the disease.

In one embodiment, the infectious disease comprises upper respiratory infection, viral hepatitis, sepsis, epstein-barr virus infection, infectious mononucleosis, encephalitis b, and sepsis.

The invention combines three antibodies of CD14, CD45 and CD64 to perform circle gating, integrates all the advantages of single antibody and double antibody, not only distinguishes leucocyte groups from fragments by using a CD45 channel and can circle lymphocytes and neutrophils, but also circles monocytes by using the CD14 channel, three circles of the lymphocytes, the monocytes and the neutrophils are more independent, the influence and overlapping parts among the three circles are less, and the circle gating result is more accurate. The flow type gate circling method of the invention does not excessively depend on the experience of technical personnel, ensures the consistency of detection and ensures that the gate circling method is more accurate and simpler and clearer.

In addition, high expression of CD64 in neutrophils is an indicator of infectious disease. After lymphocytes, monocytes and neutrophils are accurately and respectively circled, whether infectious diseases exist or not can be diagnosed according to the expression of CD64 on the neutrophils.

Drawings

FIG. 1 is a result of comparing example 1, front side to circled gate, which is seen by the percentage of White Blood Cells (WBC) by cell size and complexity, circled gate name P1;

FIG. 2 is a schematic representation of the CD64 fluorescence channel in the WBC encircled in FIG. 1 to depict gated granulocytes, monocytes and lymphocytes, respectively encircled by gated lymphocytes P9, granulocytes P8, monocytes P10;

FIG. 3 is a graph of the fluorescence intensity peaks expressed on lymph, granulocytes and monocytes by the CD64 antibody labeled with a fluorescent antibody and an average value obtained in the same circle as FIG. 2;

FIG. 4 is a graph showing the results of the front and rear circling of comparative example 1;

FIG. 5 shows the CD64 fluorescence channel used in comparative example 1 to enclose portal granulocytes, monocytes and lymphocytes, portal lymphocytes P16, granulocytes P15, monocytes P14, respectively;

FIG. 6 is a graph of the fluorescence intensity peaks expressed on lymph, granulocytes and monocytes by the CD64 antibody labeled with a fluorescent antibody and an average value obtained by the same, circled in FIG. 5;

FIG. 7 is another sectional view of the CD64 fluorescent channel surrounding portal granulocytes, monocytes and lymphocytes in comparative example 1;

FIG. 8 is the results of the anterior lateral circled gate of comparative example 2, which is shown by the percentage of White Blood Cells (WBC), circled gate name P1, in terms of cell size and complexity;

FIG. 9 is a schematic representation of the CD14 fluorescence channel delineating gated granulocytes, monocytes and lymphocytes in the WBCs encircled in FIG. 8;

FIG. 10 is a graph of the fluorescence intensity peaks expressed on lymph, granulocytes and monocytes by the CD64 antibody labeled with a fluorescent antibody and an average value obtained by the same, circled in FIG. 9;

FIG. 11 is a photograph of the WBCs encircled with CD64 fluorescence channel to circumscribe portal granulocytes, monocytes and lymphocytes in FIG. 8;

FIG. 12 is the result of comparing example 3 from the front side to the circled gate, which shows the percentage of White Blood Cells (WBC) by cell size and complexity, the name of the circled gate P1;

FIG. 13 is a photograph of the WBCs encircled with CD45 fluorescence channels to enclose the portal granulocytes, monocytes and lymphocytes in FIG. 12;

FIG. 14 is a graph of the fluorescence intensity peaks expressed on lymph, granulocytes and monocytes by the CD64 antibody labeled with a fluorescent antibody and an average value obtained in the same circle as FIG. 13;

FIG. 15 is a photograph of the WBCs encircled with CD64 fluorescence channel to circumscribe portal granulocytes, monocytes and lymphocytes in FIG. 12;

FIG. 16 shows the results of the anterior lateral circumvallate of example 1, where the percent White Blood Cells (WBC) is shown by the cell size and complexity, and the circumvallate designation P1;

FIG. 17 depicts the gated White Blood Cells (WBC) enclosed by the CD45 fluorescent channel of example 1;

FIG. 18 is a photograph of the WBCs encircled with CD45 fluorescence channels to circumscribe portal granulocytes and lymphocytes in FIG. 17;

FIG. 19 is a photograph of a monocyte gated with a CD14 fluorescence channel in the WBC circled in FIG. 17;

FIG. 20 is a graph of fluorescence intensity peaks expressed on lymphocytes, granulocytes and monocytes by CD64 antibody labeled with fluorescent antibody with granulocytes and lymphocytes of the circle gate of FIG. 18 and monocytes of the circle gate of FIG. 19 and an average value obtained.

Detailed Description

To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

As used herein, the terms "comprising," "including," and "comprising" are synonymous, inclusive or open-ended, and do not exclude additional, unrecited members, elements, or method steps.

The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within that range and the recited endpoints.

As used herein, the term "about" when describing a measurable value, such as a parameter, amount, time period, etc., is intended to encompass variations of +/-20% or less, preferably +/-10% or less, more preferably +/-5% or less, more preferably +/-1% or less, more preferably +/-0.1% or less, from the specified value, such variations being suitable for use in the disclosed invention.

CD14: i.e., LPS (Lipopolysaccharide) receptor, is originally a leukocyte differentiation antigen present on the cell surface of monocytes, macrophages, and the like. The chemical structure of CD14 (including mCD14 and sCD 14) is glycoprotein, the biological function of the glycoprotein is mainly to recognize and combine LPS or LPS/LBP complex, mediate cell reaction caused by LPS, and play an important role in pathological reactions such as LPS inflammatory reaction, endotoxin shock and the like.

CD45: the molecule is expressed on all leukocytes and is called Leukocyte Common Antigen (LCA). CD45 is composed of a kind of transmembrane protein with similar structure and larger molecular weight, is widely existed on the surface of leucocytes, and a cytoplasmic segment of the protein has the function of protein tyrosine phosphatase, can lead tyrosine on substrates P56lck and P59fyn to be dephosphorylated and activated, and plays an important role in the information conduction of cells, CD45 is a key molecule of signal conduction on cell membranes, and has important significance in the developmental maturation, function regulation and signal transmission of lymphocytes, and the distribution of CD45 can be used as a classification mark of certain T cell subsets.

CD64: igG Fc fragment receptor 1 (Fc gamma RI) which can recognize immunoglobulin, has high affinity to IgG monomer, mediates humoral immunity and cellular immunity and has early diagnosis value to infectious diseases. Normally, CD64 is mainly expressed on the surfaces of macrophages, monocytes and dendritic cells, and CD64 is hardly expressed on the surface of neutrophils. When the body suffers from infectious diseases (such as the vast majority of upper respiratory tract infections, viral hepatitis caused by hepatitis B and hepatitis C virus infections, sepsis, EB virus infections, infectious mononucleosis, japanese encephalitis, septicemia and the like), the CD64 expression on the surface of neutrophils is rapidly increased. CD64 has wide application prospect in clinic as a good diagnostic index of infectious diseases.

Therefore, neutrophils are separately screened, and CD64 expression on the neutrophils is detected, so that the neutrophils can be used as a good diagnostic index for infectious diseases.

In one aspect, an embodiment of the present invention provides a flow type circle gate method, including the following steps:

detecting the blood sample incubated by the CD45 antibody, the CD14 antibody and the CD64 antibody by using side scattered light and CD45 fluorescence, and enclosing white blood cells in the sample;

of the leukocytes that were circled, portal neutrophils and lymphocytes were circled with CD45 fluorescence, respectively;

among the gated leukocytes, gated monocytes were fluorescent with CD 14.

In some embodiments, a plot of the fluorescence intensity peaks expressed by CD64 antibody labeled with fluorescent antibody on neutrophils, lymphocytes, and monocytes, respectively, is obtained in the gates of neutrophils, lymphocytes, and monocytes, respectively.

Flow cytometry: (Flow Cytometry, FCM) is a technique for multiparameter, rapid quantitative analysis and sorting of cells or biological particles in a rapid linear Flow state. The method is widely applied to immunophenotyping analysis, DNA content and cell cycle analysis, quantitative analysis, cell function analysis, apoptosis research and other aspects.

Setting a door (a ring door) (gating): refers to the selection of a particular cell population in a cell distribution map in which analysis is desired based on the cell population distribution of the map. The different regions (regions) are obtained by "gating" to allow single or multiparameter analysis of the cells therein.

The purpose of flow cytometry data analysis is to identify the organelles of interest to be studied, which involves gating. Gating is the definition of a population of cells in a region that is individually analyzed or sorted. The shape of the door can be arbitrary, and the following methods are available:

(1) And setting a gate on the threshold value. FSC (forward scattered light) is the most commonly used threshold parameter. FSCs are positively correlated with cell size. By setting the threshold value by FSC, signals of other impurities such as cell debris below the threshold value can be prevented from being processed.

(2) The scattered light is provided with a gate. It is common to gate with a combination of FSC and SSC (side scatter light). The greatest advantage is that interference from debris or noise can be excluded. The cell population of interest can be designed based on the different cell distributions on the FSCvsSSC scattergram.

In addition, a fluorescent door, a reverse door and a combined door are arranged. The data analysis process of flow cytometry is actually the process of gating and setting gates. It should be noted that gating is a subjective act, and is a decision made by a human, and different people can have great difference in gating, which is the most difficult technique to master in flow cytometry. Thus, there are 2 points to grasp that, first, gating is as objective as possible, and, second, it should be recognized that gating requires subjective decisions. To minimize errors in subjective determinations, it is desirable to sort cells and observe them under a microscope to further confirm the objectivity and accuracy of the portal.

Gating considerations for flow cytometry:

when analyzing a cell sample by flow cytometry, it is very important to apply an optimized gating strategy in order to obtain a more accurate analysis result. Factors to be considered include:

1) Removing cell debris;

2) Applying a suitable fluorescent negative control;

3) Removing dead cells;

4) Staining with a shared marker (such as the CD45 leukocyte marker or a pan-cell population marker equivalent thereto), if appropriate;

5) Set up the necessary fluorescence analysis dot map.

Generally speaking, gating is an important technique in flow cytometry analysis, and accurate acquisition and analysis can only be achieved by optimal gating. In order to correctly identify the target cells, it is necessary to combine biological knowledge and flow manipulation experience to correctly circle the final target region. Gating is an all or nothing data reduction process. Cells inside the gate are moved to the next point of analysis, while cells outside the gate are excluded.

The invention combines three antibodies of CD14, CD45 and CD64 to perform circle gating, integrates all the advantages of single antibody and double antibody, not only distinguishes leucocyte groups from fragments by using a CD45 channel and can circle lymphocytes and neutrophils, but also circles monocytes by using the CD14 channel, three circles of the lymphocytes, the monocytes and the neutrophils are more independent, the influence and overlapping parts among the three circles are less, and the circle gating result is more accurate. The flow type gate circling method of the invention does not depend on the experience of technical personnel excessively, ensures the consistency of detection and ensures that the gate circling method is more accurate and simpler and more clear.

In some embodiments, the volume ratio of the CD45 antibody, the CD14 antibody or the CD64 antibody to the blood is 1 (8-12) at a concentration of 1ug/ul of each of the CD45 antibody, the CD14 antibody and the CD64 antibody.

In some embodiments, the co-incubation of the CD45 antibody, the CD14 antibody, and the CD64 antibody is incubation protected from light for 15min to 30min.

In some embodiments, the flow gating method further comprises removing red blood cells from the blood after the incubation, followed by flow detection.

In some embodiments, the method of removing red blood cells from blood is incubation with a hemolytic agent.

In some embodiments, the volume ratio of the hemolytic agent to the blood is 1 (0.8-1.2). The hemolytic agent is mainly prepared by adding ultrapure water into magnesium chloride, calcium chloride, sodium citrate, glycerol and formaldehyde according to different proportions.

In some embodiments, the incubation with the hemolytic agent is for a time period of 14min to 16min.

In some embodiments, the molar ratio of the CD45 antibody, the CD14 antibody, and the CD64 antibody used in the co-incubation is (0.8-1.2): (0.8-1.2).

In a second aspect, embodiments of the invention provide the use of a mixture of CD45, CD14 and CD64 antibodies in the preparation of a product for the diagnosis of an infectious disease, wherein high expression of CD64 in neutrophils is indicative of the disease.

Optionally, the infectious disease comprises upper respiratory infection, viral hepatitis, sepsis, epstein barr virus infection, infectious mononucleosis, japanese encephalitis, and sepsis.

The following are specific examples.

The method comprises the following steps:

collecting a specimen: performing fasting blood sampling by adopting an EDTA-K2 or heparin anticoagulation tube to obtain not less than 2mL;

sample requirements: 1. collecting peripheral blood sample of not less than 200 μ L, anticoagulating with EDTA-K2 or heparin, and storing at room temperature.

2. The samples were stored at room temperature and were protected from shaking and used within 24 hours after collection.

3. After the sample is dyed, the sample is kept at 2-8 ℃ in a dark place, and is required to be matched with a hemolytic agent for blood cell analysis for use, red blood cells in the sample are cracked, and the sample is subjected to on-machine detection within 24 hours.

4. Samples with microbial contamination, lipemia, clotting and poor cell viability should be avoided unless the sample is irreplaceable, as noted in the results report. In the case of chronic liver disease or hyperlipemia and other patient's specimen, hemolytic agent can not completely lyse erythrocyte, and density gradient centrifugation may be used.

And (3) checking the principle: when the reagent is added into the experimental detection sample, the specificity of the fluorescence labeling antibody in the reagent is combined with the surface antigen of the white blood cells, and then the hemocyte analysis is used for treating the stained sample with hemolytic agent to crack the red blood cells. During the acquisition, the cells pass through the laser beam and scatter the laser, while the stained cells fluoresce. From these instrumentally detected scattered fluorescent signals we can learn information about the size of the cells, the internal complexity and the strength of expression of the corresponding antigen.

The operation steps are as follows:

1. taking the fully sterilized flow tubes for numbering, wherein the number of the flow tubes is consistent with the number of the sample;

2. taking three antibodies as an example, respectively taking 5 mu L of monoclonal fluorescent antibodies (labeled fluorescence is as follows: CD14-FITC, CD45-PerCP, CD 45-PE) of CD45, CD14 and CD64, fully and uniformly mixing, adding into the step 1, sucking 50ul of reversely and uniformly mixed peripheral blood samples by adopting a reverse pipetting technology, adding into the bottom of a tube, and avoiding the samples from touching the upper part of the tube wall; if the antibody is a single antibody or double antibody, 5 mu L of each corresponding antibody is mixed with 50ul of reversely-mixed peripheral blood samples;

3. incubating for 20min at room temperature in dark place;

4. adding 500 μ L of 1 x hemolysin into the flow tube, mixing well, incubating at room temperature in dark for 15min, and taking out;

5. fully mixing and waiting for loading.

Wherein LYM is a lymphocyte, PMN is a granulocyte, and MONO is a monocyte.

Comparative example 1 Single antibody (CD 64) circle-peripheral blood samples were incubated with CD64 monoclonal fluorescent antibody.

The results are shown in fig. 1 to 3 for the front side to the hoop door.

The results of looping the door back and forth are shown in fig. 4.

The results obtained with the CD64 fluorescence channel circle gate are shown in FIGS. 5-7.

In comparative example 1, the CD64 single antibody circle was used. The result shows that when the cells are circled in the front direction, no clustering can be found, the three cell populations of neutrophile, mononuclear and lymph can not be effectively distinguished, even the specific circled position of the mononuclear cell population can not be determined, so that the mononuclear cell fluorescent antibody can not express strongly, the obtained data is low, the calculation result is increased, and false positive is easy to appear.

All WBCs are circled as an integral image from front to back, impurities and fragments at the lower left corner are many, and the WBCs cannot be completely removed. Moreover, the lymphocyte population is mostly overlapped with the fragment at the lower left corner, so that no way is provided for separately closing the lymph, and the fragments have great influence on the data after closing.

After the CD64 fluorescence channel is changed into the round gate, the three gates can be effectively distinguished, the problem that the strong expression of the monocytes is low is solved, the overall data is more stable, but the color difference shows that the part circled at the lower left corner is the impurity and fragment part which cannot be removed from the overall image, and is still overlapped with the lymphocytes and cannot be adjusted.

Therefore, the advantages and disadvantages of a single antibody are as follows:

the advantages are that: the single CD64 antibody has the advantages of simple operation, convenient map adjustment, reduced cost and strong universality;

the disadvantages are that: the ineffective fragments are numerous and cannot be effectively distinguished from the lymphocyte population.

Comparative example 2 diabody (CD 64+ CD 14) -peripheral blood samples were incubated with CD64 antibody + CD14 monoclonal fluorescent antibody.

The results are shown in FIGS. 8-10, which are a general graph of the CD14 fluorescence channel cycle. It can be seen that the three-cycle gate of the diabody (CD 64+ CD 14) is more clear than the mab of comparative example 1. However, when the leukocyte general diagram in fig. 8 is obtained, it is found that many impurities and fragments cannot be removed, so that lymphocytes and fragments cannot be distinguished.

The result is shown in FIG. 11, comparing the CD14 channel gate with the CD64 fluorescence channel gate, the three cell populations are more concentrated and have unclear boundaries, and the gate is more complex.

Diabody CD64+ CD14 has advantages and disadvantages:

the advantages are that: the three groups of cells are relatively aggregated and relatively dispersed, so that the cells are easy to circle, and the result is relatively stable.

The disadvantages are as follows: the ineffective fragments are numerous and cannot be effectively distinguished from the lymphocyte population.

Comparative example 3 diabody (CD 64+ CD 45) -peripheral blood samples were incubated with CD64+ CD45 monoclonal fluorescent antibody.

The results are shown in FIGS. 12-14, which are summarized in the CD45 fluorescence channel circles. It can be seen that the three clusters are found to be relatively concentrated and the impact of debris on the gate of the trap is relatively small. However, compared with the CD14 circle gate of comparative example 2, the three groups are compact, the edges of the three groups coincide with each other, the circle pattern is irregular, and the data is not as stable as CD 14.

As shown in fig. 15, the WBC population could not be well stripped from the debris and lymph could not be distinguished from debris-trash coincidence with the CD64 fluorescence channel circle overall.

EXAMPLE 1 Tri-antibody (CD 64+ CD14+ CD 45) -incubation of peripheral blood samples with CD64+ CD14+ CD45 monoclonal fluorescent antibody

The result is shown in FIG. 16.

The result of the CD45 fluorescence channel cycle is shown in FIGS. 17-18, and the granulocytes and lymphocytes can be circled.

Monocytes were circled in the white blood cells circled in fig. 17 with the CD14 fluorescence channel as shown in fig. 19.

The fluorescence intensity peaks expressed by CD64 antibody labeled with fluorescent antibody on lymph, granulocytes, and monocytes are plotted in fig. 18, granulocyte and lymphocyte, monocyte circle gate, fig. 19, to obtain an average value, as shown in fig. 20.

From the overall graph of the three antibodies, the three antibody images combine all the advantages of the monoclonal antibody and the double antibody, not only the WBC group is distinguished from the fragments by the CD45 channel, but also the mononuclear cells are separated by the CD14 channel, the three groups are more independent, the mutual influence is less, the overlapping part is less, and the result is more accurate and reliable.

The three antibodies CD64+ CD45+ CD14 have the following advantages: the three groups are independent, the influence of fragments is small, and the result is more accurate and reliable.

Therefore, no clustering was found in the monoclonal antibody CD64 anterior lateral channel gate, and neutrophils, monocytes, and lymphocytes could not be distinguished effectively. The key on-machine parameter is fluorescence intensity, CD64 belongs to strong expression on the surface of a monocyte, the fluorescence intensity is naturally high, but the strong expression of the CD64 cannot be embodied by 64 circles of gates, so the fluorescence intensity is low, the monocyte expression is reduced as an important member of a calculation formula, and the calculation result is high. All leukocyte impurities and ineffective figures are circled by the front side and the rear side as an overall graph, cannot be completely eliminated, and has large influence on data after the circled. Lymphocytes cannot be extracted by the CD14 round gate, and the fragments and impurities are more. The CD14 and the CD64 are used for looping the gate, the looping gate is irregular, the data has no CD14 and has high stability, the CD14, the CD45 and the CD64 are used for looping the gate, all the advantages of the monoclonal antibody and the double antibody are integrated, the CD45 channel is used for distinguishing the leucocyte groups from fragments, the CD14 can be used for looping out mononuclear cells, the three loops are more independent, the mutual influence and the overlapping part are less, and the result is more accurate.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, so as to understand the technical solutions of the present invention specifically and in detail, but not to be understood as the limitation of the patent protection scope of the invention. It should be noted that various changes and modifications can be made by those skilled in the art without departing from the spirit of the invention, and these changes and modifications are all within the scope of the invention. Therefore, the protection scope of the patent of the invention is subject to the appended claims, and the description can be used for explaining the contents of the claims.

Claims

1. A flow-around gate method, comprising the steps of:

detecting the blood sample incubated by the CD45 antibody, the CD14 antibody and the CD64 antibody by using side scattered light and CD45 fluorescence, and trapping leukocytes in the sample; (ii) the volume ratio of said CD45 antibody, said CD14 antibody or said CD64 antibody to said blood sample is 1; the CD45, CD14, and CD64 antibodies used in the co-incubation are at a molar ratio of 1;

the incubation is dark incubation, and the incubation time is 15-30 min;

among the gated leukocytes, gated monocytes were gated with CD14 fluorescence;

fluorescence intensity peaks expressed by the fluorescence-labeled CD64 antibody on neutrophils, lymphocytes and monocytes, respectively, were obtained in the gates of neutrophils, lymphocytes and monocytes, respectively.

2. The flow gate method of claim 1, wherein the incubation is protected from light and the incubation time is 20min.

3. The flow gating method of claim 1, further comprising:

after the incubation, the red blood cells in the blood sample are removed and then subjected to flow detection.

4. A flow gating method according to claim 3, wherein the removal of red blood cells from the blood is by incubation with a haemolysing agent.

5. The flow gating method of claim 4, wherein the volume ratio of the hemolytic agent to the blood sample is 1 (0.8-1.2); the incubation time with the hemolytic agent is 14-16 min.