WO2020107496A1 - 一种免疫细胞中淋巴细胞的流式细胞仪检测方法 - Google Patents

一种免疫细胞中淋巴细胞的流式细胞仪检测方法 Download PDF

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WO2020107496A1
WO2020107496A1 PCT/CN2018/118802 CN2018118802W WO2020107496A1 WO 2020107496 A1 WO2020107496 A1 WO 2020107496A1 CN 2018118802 W CN2018118802 W CN 2018118802W WO 2020107496 A1 WO2020107496 A1 WO 2020107496A1
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姚伟丽
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铭道创新（北京）医疗技术有限公司
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Priority to PCT/CN2018/118802 priority Critical patent/WO2020107496A1/zh
Priority to US17/295,655 priority patent/US20210389225A1/en
Priority to EP18941524.3A priority patent/EP3889576A4/en
Priority to CN201880002857.3A priority patent/CN111527395B/zh
Publication of WO2020107496A1 publication Critical patent/WO2020107496A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/38—Diluting, dispersing or mixing samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/4077—Concentrating samples by other techniques involving separation of suspended solids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1429—Signal processing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1456—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
    • G01N15/1459—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals the analysis being performed on a sample stream
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G01N2001/302—Stain compositions
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells
    • G01N2015/016—White blood cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/1006—Investigating individual particles for cytology
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N2015/1402—Data analysis by thresholding or gating operations performed on the acquired signals or stored data
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N2015/1488—Methods for deciding

Definitions

  • the invention relates to the field of medicine, and specifically provides a method for detecting lymphocyte flow cytometry in immune cells.
  • the human immune system is usually divided into: immune organs and immune tissues, immune cells (phagocytes and lymphocytes), immune active substances (antibodies, lysozyme, complement, immunoglobulins, interferons, interleukins, tumor necrosis factor and other cells factor).
  • immune lymphocytes usually 100 microliters of peripheral blood, adding fluorescent antibodies, incubating for 15-30 minutes, and then lysing red blood cells with hemolysin, adding washing buffer after washing, and using flow cytometry for detection.
  • CN201680006060.1 discloses an immune state inspection method and inspection device, which can classify immune cells: T, B, NK, NKT, and T cells are subdivided into CD4+T cells and CD8+T cells.
  • the invention has the following technical deficiencies: 1. Waste of antibodies and reagent materials; 2. The subtype of DC cells that do not define innate immunity; 3. Individual lymphocyte subsets are not fully defined, and there is no systematic consideration of the function of the lymphatic immune system, and then Subgroup typing is not comprehensive and balanced; 4. After erythrocytes are lysed with red blood cell lysate after staining with immunofluorescent antibodies, the background is not clean, there are many fragments, and the data is prone to errors; 5. For a small number of lymphocyte subgroups The method cannot be detected or the detection result is inaccurate; 6. The detection method is prone to excessive staining or insufficient staining, resulting in inaccurate data.
  • a lymphocyte immunotyping method and kit divide T cells into finer classifications of the same categories T, B, NK, NKT, and T cells are further divided into CD4+T cells and CD8+T cells.
  • T cells were jointly defined using CD45RA and CD27, and four types of B cells were defined using CD19, CD24, CD38, and CD27.
  • the invention has the following technical deficiencies: 1. Waste of antibodies and reagent materials; 2. The subtype of DC cells that do not define innate immunity; 3. Individual lymphocyte subsets are not fully defined, and there is no systematic consideration of the function of the lymphatic immune system, and then Subgroup typing is not comprehensive and balanced; 4.
  • the present invention provides a method for detecting lymphocytes in immune cells.
  • the method includes the method including:
  • step c) Add the mixed cell sample obtained in step a) to the sample tube and then test.
  • the pre-cooling temperature in step a) is a temperature commonly used in flow cytometry detection in the art.
  • the present invention includes but is not limited to 2-8°C.
  • the PBS-EDTA solution in step a) or step b) is pH 7.2-7.4 containing 0.005-0.05M PBS and a final concentration of 2-3mM EDTA mixture;
  • the 0.005-0.05M PBS as an exemplary description may be 0.005M PBS, 0.01M PBS, 0.02M PBS, 0.03M PBS, 0.04M PBS, or 0.05M PBS.
  • the step a) further includes: the concentration of cells in the lymphocyte sample is 1 ⁇ 10 6 to 10 ⁇ 10 6 cells/ml; the preferred concentration range is 1.5 ⁇ 10 6 Pieces/ml to 5 ⁇ 10 6 pieces/ml, more preferably 1.5 to 3.0 ⁇ 10 6 pieces/ml.
  • the step a) further includes: the volume of the lymphocyte sample is a volume suitable for detection, and the present invention includes but is not limited to 50-200 ⁇ l.
  • the adjustment liquid flow rate is in the range of 30-44 ⁇ l/min; as one of further embodiments, the adjustment liquid flow rate is 35-40 ⁇ l/min
  • the medium speed range can be 35 ⁇ l/min, 36 ⁇ l/min, 37 ⁇ l/min, 38 ⁇ l/min, 39 ⁇ l/min or 40 ⁇ l/min as an exemplary illustration.
  • the preheating time in the step b) may be a time suitable for detection and may be adjusted by a person skilled in the art, and the present invention is preferably 5-10 minutes.
  • the amount of the PBS-EDTA solution added to the sample tube in step b) can be determined by those skilled in the art according to the detection equipment and conditions, including but not limited to 3 ml, for example.
  • the step c) further includes setting a condition that 3000 to 20000 cells in the gate, preferably 15000 cells in the gate, and collecting a sample.
  • the flow lymphocyte sample is prepared by the following method:
  • step 4) Take the cells in step 3) into the centrifuge tube, add PBS solution and mix to obtain a suspension, centrifuge and remove the supernatant, then add PBS solution, adjust the cell concentration to 1 ⁇ 10 5 cells/ ml ⁇ 1 ⁇ 10 8 pcs/ml, the preferred concentration range is 1.5 ⁇ 10 6 pcs/ml ⁇ 5 ⁇ 10 6 pcs/ml, further preferably 1.5 ⁇ 3.0 ⁇ 10 6 pcs/ml; as an exemplary description, it can be of 1.5 ⁇ 10 6 th /ml,2.0 ⁇ 10 6 th /ml,2.5 ⁇ 10 6 th /ml,3.0 ⁇ 10 6 cells / ml.
  • step 5) Take the adjusted concentration of lymphocytes from step 4) into the flow tube; add the flow antibody for labeling lymphocyte subsets, mix well, let stand at 2 ⁇ 8°C and incubate in the dark;
  • the PBS solution is a 0.005-0.05 M PBS solution with a pH of 7.2 to 7.4; as one of the embodiments, the PBS buffer solution may include calf serum and/or EDTA, The concentration of the calf serum or EDTA may be a concentration commonly used in the art.
  • the PBS-EDTA solution is a mixed solution containing 0.005-0.05M PBS and a final concentration of 2-3mM EDTA at pH 7.2-7.4.
  • the step 1) further includes centrifugation conditions of 1500-3500 rpm for 5-30 minutes; preferably 1500-2900 rpm for 15-20 minutes.
  • the step 2) further includes diluting the PBS solution for blood cell precipitation at a volume ratio of 1:0.5 to 1:2, preferably 1:1.
  • the lymphocyte separation liquid in the step 3 may be prepared by a person skilled in the art according to the existing technology, or may be commercially available.
  • the present invention is preferably from the American reagent company GE Lymphocyte separation fluid.
  • the step 3) further includes a centrifugal condition of 1500-3500rpm, 10-20min, 4°C; preferably 1500-2900rpm, 15-20min, 4°C; in the method of the present invention, as In one embodiment, if necessary in step 4), an appropriate amount of suspension is optionally taken before centrifugation and counted by a blood analyzer, so as to adjust the cell concentration later.
  • the step 4) further includes a centrifugal condition of 1500-3500rpm, 5-20min, 4°C; as a further embodiment, the centrifugal condition is 1500-2900rpm, 5- 10min, 4°C.
  • step 4 a PBS solution is added to the centrifuge tube until the volume is 10 ml to 15 ml.
  • the amount of the diluted lymphocytes added to the step 5) in the flow tube in step 5) may be the amount commonly used in the art, and exemplary illustrations include but are not limited to 100 ⁇ l;
  • the incubation time may include but not limited to 10-30 minutes.
  • the step 6) further includes centrifugation conditions of 1500-2900 rpm and centrifugation for 5-10 min.
  • the amount of the PBS solution added in step 6) is 2 ml.
  • the PBS-EDTA solution in step 7) is a mixed solution of EDTA containing 0.005-0.05M PBS and a final concentration of 2-3mM at pH 7.2-7.4;
  • the PBS solution is a 0.005-0.05M PBS solution with a pH of 7.2 to 7.4; alternatively, the PBS buffer solution may contain calf serum and/or EDTA, and the concentration of the calf serum or EDTA may be commonly used in the art concentration.
  • the centrifugal conditions in step 7) are 1500-2900 rpm and 5-10 min.
  • the method includes:
  • it may be 1.5 ⁇ 10 6 th /ml,1.6 ⁇ 10 6 th /ml,1.7 ⁇ 10 6 th /ml,1.8 ⁇ 10 6 th /ml,1.9 ⁇ 10 6 cells / ml or 2.0 ⁇ 10 6 /ml concentration.
  • the method of the invention can realize that the lymphocyte sample is easier to obtain and can be quantified, and only a small amount of isolated peripheral blood sample can be used to detect all the detection indicators; at the same time, it can include but not limited to DC cells (dendritic cells) and the like.
  • the specific experimental steps of the detection method of the present invention are divided into four steps: obtaining an isolated blood sample, separating cells, incubating antibodies, and testing on the computer.
  • the present invention uses antibodies that bind to different fluorescence to bind to specific antigens on the surface of cells, thereby identifying such subgroups.
  • Obtaining blood samples in vitro Collect blood from blood collection tubes in vitro.
  • the blood sample separation method of the present invention saves more material costs and time costs, making the experimental results faster and more accurate.
  • the peripheral blood was separated, washed, diluted to the specified concentration range using lymphocyte separation solution, and set at 4°C for use.
  • Incubation of antibodies select fluorescent antibodies corresponding to the specific antigens on the surface of each type of cells, dilute the isolated cells to an appropriate concentration, incubate according to the verified fluorescent antibody concentration and time, and then add washing solution to wash off excess antibodies. Add machine fluid and mix well.
  • On-board testing use flow cytometry for testing and analysis.
  • the present invention highly enriches lymphocytes and removes red blood cells, granulocytes and other cells that affect the detection result, so that many subpopulations of cells that are not highly expressed and traces are clearly presented The grouping is obvious, which is convenient for analysis and research. From another perspective, the high-purity lymphocytes isolated by the present invention will have much higher efficiency of binding antibody per unit volume, while saving more than half of the antibody cost and antibody incubation time.
  • the present invention has the following advantages: 1. Lymphocytes are separated and enriched by the lymphocyte separation solution to ensure reliable and accurate detection results, saving antibodies and reagents; 2. Through the detection of enriched lymphocyte subsets, It can effectively analyze a small number of lymphocyte subpopulations, and also ensure effective and reasonable staining of defined lymphocyte subpopulations; 3.
  • the lymphocyte subpopulations that can be detected by the method of the present invention include immune recognition and early warning system, cellular immune system, and humoral immunity System and non-specific immune cell system, so the test results can fully reflect the status of the lymphocyte system; 4.
  • the present invention can clearly define the two subtypes of DC cells; 5. Through the present invention, the lymphocyte subsets can be comprehensively analyzed Analysis and detection provide a scientific basis for a comprehensive understanding of the immune status.
  • Figure 4 Detection chart of different lymphocyte subsets in Example 3.
  • Figure 6 Comparison of the cell population of the sample obtained by the method of Example 1 in Example 4 and the sample obtained by the prior art lysis method; (The top three figures are the flow cytograms obtained by the lysis method, and the bottom three figures are the top three figures (Flow cytogram obtained by the corresponding sample separation method)
  • Figure 7 Comparison of the cell enrichment of the sample obtained in Example 4 according to the method of Example 1 and the sample obtained by the prior art lysis method;
  • Figure 8 Comparison of the enrichment of different types of cells between the sample obtained in Example 4 according to the method of Example 1 and the sample obtained in the prior art lysis method.
  • the present invention further illustrates the present invention through the following examples and test examples, but does not limit the effective scope of the present invention in any way.
  • PBMC samples also referred to herein as “samples”.
  • a relatively complete antigen or receptor remains on the surface of the living cell, and the antibody labeled with fluorescence binds to the corresponding antigen on the cell surface, and the density and distribution of the corresponding antigen can be known based on the measured fluorescence intensity and positive percentage.
  • PBMC peripheral blood samples
  • the antibodies and cells should be kept at a low temperature (4°C) as much as possible throughout the operation.
  • the antibody and the cells should be mixed thoroughly.
  • the isolated blood sample is prepared according to the method of Example 1;
  • circle gate 1 lymphocyte group, you can clearly separate the lymphocyte subgroup, and the main analysis of the cell group, the data will be more reliable and accurate.
  • Sample to be tested 5ml of isolated blood sample is prepared according to the method of Example 1;
  • Figure 3 shows the analysis of lymphocyte subsets obtained by the lymphocyte separation method in the peripheral blood of this healthy person.
  • the P1 gate in the circle is the lymphocyte population.
  • the background is clean, the lymphocyte population and cells
  • the debris cluster is very open and ideal.
  • the lymphocytes are separated and enriched by the lymphocyte separation liquid, and the clustering is obvious, which guarantees the reliable and accurate detection results, and at the same time saves antibodies and reagents.
  • Figure 4 shows that through the detection of enriched lymphocyte subpopulations, a small number of lymphocyte subpopulations are effectively analyzed, and the lymphocyte subpopulations defined by effective staining are defined effectively;
  • Figure 4 left sample 4, Q3-2 is CD8+ T cells are separated and enriched, and the grouping is obvious, which can analyze the subgroups very well.
  • the right picture of Figure 4 shows the cell subsets with a small proportion of lymphocyte subsets, and the DC cell subsets can also be accurately analyzed by enrichment.
  • Figure 5 clearly defines two subtypes of DC cells, which is not available in the analysis of lymphocyte subsets in the past. Two subtype cells were enriched by the separation method. As shown in Figure 1, right 1, the groups are obvious and the boundaries are clear.
  • Example 4 Comparison between the sample prepared in the method of the present invention and the sample obtained by the hemolysin lysis method
  • the lymphocyte sample of the present invention the isolated peripheral blood sample is prepared according to the method of Example 1;
  • Comparative lymphocyte sample prepared by following the method in section 4.2.1;
  • PBS solution namely 1 ⁇ PBS
  • PBS-EDTA solution add 0.5M EDTA to 1 ⁇ PBS until the final concentration of EDTA is 2.5mM;
  • Antibodies for routine flow cytometry anti-CD3 antibody, lin1 [Lineage Cocktail 1], anti-CD123 antibody, anti-CD11 antibody, CD3FITC, CD8PerCP, CD4PerCP.
  • Sample staining add 100 ⁇ l of anticoagulated blood sample to each flow tube, and add flow cytometry antibody to label lymphocyte subgroups respectively, so that the antibody and cells are mixed well. Incubate at 2 ⁇ 8°C for 20 minutes in the dark.
  • the gates in the circle of the figure in the analysis of the lymphocyte subgroup analysis obtained by the lysis method are lymphocytes, and it can be seen from the figure that the division of lymphocyte groups and cell debris groups is very unsatisfactory (see three pictures in the first row of Figure 6) .
  • the gates in the circle in the figure for the analysis of the lymphocyte subsets obtained by the present invention are the lymphocyte groups, and it can be seen from the figure that the lymphocyte groups and the cell debris groups are very open and ideal (see the third figure in the second row of Figure 6) .
  • the separation method has been significantly enriched.

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Abstract

提供一种免疫细胞中淋巴细胞的流式细胞仪检测方法，该方法包括：a)取免疫荧光抗体染色的淋巴细胞样品，加入预冷的PBS-EDTA溶液，混均细胞，备流式细胞仪检测待用；b)对流式细胞仪系统进行开机预热，调节液流速度，然后向样品管中加入PBS-EDTA溶液，冲洗液流的喷嘴系统；c)将步骤a)所得混均细胞样品加入样品管然后进行检测。该检测方法在检测时可使亚群分群明显，分析检测更加准确，有利于检测数量较少的细胞亚群，并节省抗体试剂。

Description

一种免疫细胞中淋巴细胞的流式细胞仪检测方法 技术领域
本发明涉及医药领域，具体提供一种免疫细胞中的淋巴细胞流式细胞仪检测方法。
背景技术
人体免疫系统通常分为：免疫器官和免疫组织、免疫细胞(吞噬细胞和淋巴细胞)、免疫活性物质(抗体、溶菌酶、补体、免疫球蛋白、干扰素、白细胞介素、肿瘤坏死因子等细胞因子)。其中传统的免疫淋巴细胞的检测通常是外周血100微升，加入荧光抗体，孵育15-30分钟，然后用溶血素进行裂解红细胞，洗涤后加入上机缓冲液，并用流式细胞仪进行检测。
CN201680006060.1公开了一种免疫状态的检查方法以及检查装置，该方法可以免疫细胞进行分型：T、B、NK、NKT、T细胞再分为CD4+T细胞，CD8+T细胞。该发明存在以下技术不足：1、浪费抗体和试剂材料；2、没有定义固有免疫的DC细胞这个亚型；3、个别淋巴细胞亚群定义不全面，而没有系统的考虑淋巴免疫系统功能，进而亚群分型不够全面、不够平衡；4、染完免疫荧光抗体后运用红细胞裂解液进行裂解红细胞，导致背景不干净，碎片多，数据容易出现误差；5、对于数量较少的淋巴细胞亚群，该方法检测不出来或者检测结果不准确；6、该检测方法容易出现过度染色或染色不足的情况，导致数据不准确。
CN201410486089.7一种淋巴细胞免疫分型的方法和试剂盒将T细胞更细的分为同样的分类T、B、NK、NKT，T细胞再分为CD4+T细胞，CD8+T细胞，在此基础上，运用CD45RA和CD27联合定义了几种T细胞，运用CD19、CD24、CD38、CD27，定义了4种B细胞。该发明存在以下技术不足：1、浪费抗体和试剂材料；2、没有定义固有免疫的DC细胞这个亚型；3、个别淋巴细胞亚群定义不全面，而没有系统的考虑淋巴免疫系统功能，进而亚群分型不够全面、不够平衡；4、染完免疫荧光 抗体后运用红细胞裂解液进行裂解红细胞，导致背景不干净，碎片多，数据容易出现误差；5、对于数量较少的淋巴细胞亚群，该方法检测不出来或者检测结果不准确；6、该检测方法容易出现过度染色或染色不足的情况，导致数据不准确。
因此、如何更全面的平衡的淋巴细胞亚群的检测方法依然是目前研究的热点，目前依然没有相关方面的报道。
发明内容
针对以上技术现状，本发明提供一种免疫细胞中淋巴细胞的检测方法，所述方法包括所述方法包括：
a)取免疫荧光抗体染色的淋巴细胞样品，加入预冷的PBS-EDTA溶液，混均细胞，备流式细胞仪检测待用；
b)对流式细胞仪系统进行开机预热，调节的流速度为10-29μl/min的低速范围或30～44μl/min的中速范围，然后向样品管中加入PBS-EDTA溶液，冲洗液流的喷嘴系统；
c)将步骤a)所得混均细胞样品加入样品管然后进行检测。
本发明方法中，作为实施方案之一，所述步骤a)中预冷的温度为本领域进行流式细胞仪检测时常用的温度，本发明包括但不限于2～8℃。
本发明方法中，作为实施方案之一，所述步骤a)或步骤b)中的所述PBS-EDTA溶液为pH7.2～7.4的含有0.005-0.05M PBS和终浓度为2-3mM EDTA的混合溶液；
本发明中，作为实施方案之一，所述0.005-0.05M PBS作为示例性的说明，可以为0.005M PBS、0.01M PBS、0.02M PBS、0.03M PBS、0.04M PBS或0.05M PBS。
本发明方法中，作为实施方案之一，所述步骤a)中进一步包括：淋巴细胞样品中细胞的浓度为1×10 6～10×10 6个/ml；优选的浓度范围是1.5×10 6个/ml～5×10 6个/ml，进一步优选1.5～3.0×10 6个/ml。
本发明方法中，作为实施方案之一，所述步骤a)中进一步包括：淋巴细胞样品的体积为适于检测的体积，本发明包括但不限于为50～200μl。
本发明方法中，作为实施方案之一，所述步骤b)中调节液流速度为 30-44μl/min的中速范围；作为进一步实施方案之一，所述调节液流速为35～40μl/min的中速范围，作为示例性的说明可以为35μl/min、36μl/min、37μl/min、38μl/min、39μl/min或40μl/min。
本发明方法中，作为实施方案之一，所述步骤b)中预热时间可为适于检测的时间，可以由本领域技术人员进行调整，本发明优选为5～10分钟。
本发明方法中，作为实施方案之一，所述步骤b)中加入样品管中的PBS-EDTA溶液的量可以由本领域技术人员根据检测设备和条件进行确定，例如包括但不限于3ml。
本发明方法中，作为实施方案之一，所述步骤c)进一步包括设置条件为：门内3000～20000个细胞，优选门内15000个细胞，采集样品。
本发明方法中，作为实施方案之一，所述流式淋巴细胞样品采用如下方法制备：
1)取离体抗凝血液样品进行离心，分离血浆和血液细胞沉淀，得血液细胞沉淀；
2)将血液细胞沉淀用PBS溶液进行稀释，得血液细胞稀释液；
3)将血液细胞稀释液缓慢加入到含等体积的淋巴细胞分离液的离心管中，然后离心，去掉血浆，取中间白膜层细胞；
4)取步骤3)所得白膜层细胞加入到离心管中，加入PBS溶液混合均匀得混悬液，离心并去除上清液，然后再加入PBS溶液，调整细胞浓度在1×10 5个/ml～1×10 8个/ml，优选的浓度范围是1.5×10 6个/ml～5×10 6个/ml，进一步优选1.5～3.0×10 6个/ml；作为示例性的说明，可以为1.5×10 6个/ml、2.0×10 6个/ml、2.5×10 6个/ml、3.0×10 6个/ml。
5)取步骤4)所得调整浓度后的淋巴细胞加入流式管内；并加入用于标记淋巴细胞亚群的流式抗体，混匀，2～8℃静置并避光孵育；
6)加入PBS溶液、混匀，离心洗掉未结合抗体；
7)样品的完成
①如果立即上机检测，则弃去上清，加入预冷至2～8℃的PBS-EDTA溶液，混匀细胞，用于流式检测；
②如不能立即检测，不需PBS溶液稀释，直接用0.05-5％福尔马林 500μl悬起细胞，混匀，静置于2～8℃避光保存；检测前各管加入PBS溶液，离心弃掉上清液，用2～8℃的PBS-EDTA溶液、混匀细胞，用于流式检测；
本发明方法中，作为实施方案之一，所述PBS溶液为pH7.2～7.4的0.005～0.05M PBS溶液；作为实施方案之一，所述PBS缓冲溶液可以有小牛血清和/或EDTA，所述小牛血清或EDTA的浓度可以为本领域常用的浓度。
本发明方法中，作为实施方案之一，所述PBS-EDTA溶液为pH7.2～7.4的含有0.005-0.05M PBS和终浓度为2-3mM EDTA的混合溶液。
本发明方法中，作为实施方案之一，所述步骤1)进一步包括离心条件为1500～3500rpm离心5～30min；优选1500-2900rpm离心15-20分钟。
本发明方法中，作为实施方案之一，所述步骤2)进一步包括血液细胞沉淀用PBS溶液以1:0.5～1:2，优选1:1按体积比例进行稀释。
本发明方法中，作为实施方案之一，所述步骤3)中淋巴细胞分离液可以由本领域技术人员根据现有技术进行制备，还可以来自商业购得，本发明优选来自美国试剂公司GE公司的淋巴细胞分离液。
本发明方法中，作为实施方案之一，所述步骤3)进一步包括离心条件为1500-3500rpm，10-20min，4℃；优选1500-2900rpm，15-20min，4℃；本发明方法中，作为实施方案之一，所述步骤4)中如果必要时，可选择地在离心前取适量混悬液采用血液分析仪计数，以便后续调整细胞浓度。
本发明方法中，作为实施方案之一，所述步骤4)进一步包括离心条件为1500-3500rpm，5-20min，4℃；作为进一步实施方案之一，所述离心条件为1500-2900rpm，5-10min，4℃。
本发明方法中，作为实施方案之一，所述步骤4)中向离心管中加入PBS溶液直至体积为10ml～15ml。
本发明方法中，作为实施方案之一，所述步骤5)中加入流式管内骤4)所得稀释后淋巴细胞的量可以为本领域常用的量，作为示例性的说明包括但不限于100μl；
本发明方法中，作为实施方案之一，所述步骤5)中；孵育时间作为示例性的说明，可以包括但不限于10-30分钟。
本发明方法中，作为实施方案之一，所述步骤6)进一步包括离心条件为1500-2900rpm，离心5-10min。
本发明方法中，作为实施方案之一，所述步骤6)中加入PBS溶液的量为2ml。
本发明方法中，作为实施方案之一，所述步骤7)中所述PBS-EDTA溶液为pH7.2～7.4的含有0.005-0.05M PBS和终浓度为2-3mM的EDTA混合溶液；所述PBS溶液为pH7.2～7.4的0.005-0.05M PBS溶液；可选择地，所述PBS缓冲溶液可以含有小牛血清和/或EDTA，所述小牛血清或EDTA的浓度可以为本领域常用的浓度。
本发明方法中，作为实施方案之一，所述步骤7)中所述离心条件为1500-2900rpm，5-10min。
本发明方法中，作为实施方案之一，所述方法包括：
(一)外周血中分离人外周血单核细胞
(1)取离体原血血样0.2ml计数，留样1ml，标记信息，于4℃下保存；
(2)将血液加入到离心管中，1500-2900rpm离心15-20min；取上层血浆加入到2ml冻存管中于-80℃保存；
(3)剩余血液细胞沉淀与PBS溶液按1:1的体积比进行稀释。
(4)准备1支15ml离心管，用移液管加入与血液细胞稀释液等量的淋巴细胞分离液。
(5)将血液细胞稀释液缓慢加入到步骤(4)的离心管中，保持分离液分层清晰。
(6)缓慢放入低速离心机内，配平后1500-2900rpm，离心15-20min。
(7)离心结束后用10ml移液管或者3ml巴氏吸管缓慢吸取上层血浆弃掉。
(8)转圈吸取中间白膜层细胞至一个新的15ml离心管内，加PBS溶液至10ml，取200μl细胞悬液用全血细胞计数仪计数，以备后续调整细胞浓度用；剩余细胞悬液1500-2900rpm，离心5-10min。
(9)离心后，弃上清。根据计数结果，加入适量2～8℃预冷的PBS溶液，调整细胞浓度在1.5-2.0×10 6个/ml。
作为示例性的说明，可以为1.5×10 6个/ml、1.6×10 6个/ml、1.7×10 6个/ml、1.8×10 6个/ml、1.9×10 6个/ml或2.0×10 6个/ml的浓度。
(二)制作流式细胞样品
(10)样品染色
每个流式管内加入100μl细胞悬液，加入用于标注淋巴细胞的抗体(注意设阴性细胞管，ISO管，单阳管)。并用涡旋振荡器轻缓震荡，使抗体与细胞充分混均，2～8℃，避光孵育10-30分钟，优选15～20分钟，最佳20分钟。
(11)样品洗涤、加液
孵育结束后，加入2ml 2～8℃的PBS溶液重悬细胞，1500-2900rpm，离心5-10min，离心后，倒掉上清。
①如果立即上机检测，用预冷的PBS-EDTA溶液400μl混匀细胞，用于流式检测。
②如不能立即检测，则用0.05-5％福尔马林500μl悬起细胞，涡旋振荡器，充分混匀，4℃避光保存。检测前各管加入2ml的PBS溶液，1500-2900rpm，离心5-10min，弃掉上清。用预冷的PBS-EDTA溶液400μl，混匀细胞，上机检测。
本发明方法可以实现淋巴细胞样品较容易获取且可以量化，只需少量离体外周血液样品，就能实现所有检测指标的检测；同时可以实现包含但不限于DC细胞(树突状细胞)等。
本发明检测方法具体实验步骤分为四个步骤离体血样的获取、细胞分离、孵育抗体、上机检测。本发明通过抗原抗体结合原理，采用结合着不同荧光的抗体，与细胞表面的特异性抗原结合，从而将此类亚群进行标识。
在流式细胞仪检测时，可以进行分析此类细胞的比例和数量。
离体血液样品的获取：取自离体的采血管采集外周血。
细胞分离：本发明血液样品的分离方法更加节约物质成本和时间成本，使得实验结果更快更准。
按照验证过的实验方法，利用淋巴细胞分离液，对外周血进行分离，洗涤，稀释到指定浓度范围，置4℃备用。
孵育抗体：选取每类细胞表面特异性的抗原对应的荧光抗体，分离后的细胞稀释到合适浓度，按照验证的荧光抗体浓度和时间进行孵育，然后加入洗涤液洗掉多余的抗体。加上机液后混匀。
上机检测：利用流式细胞仪进行检测、分析。
本实验方法与传统的流式检测方法不同，本发明将淋巴细胞高度富集，去除了红细胞、粒细胞等影响检测结果的细胞，使得很多表达不高和表达微量的细胞亚群得以清晰呈现，分群明显，便于分析研究。从另一个角度讲，本发明分离出的高纯度的淋巴细胞，单位体积结合抗体的效率也会高很多，同时节省一半以上的抗体成本和抗体孵育的时间。
相对于现有技术，本发明利用具有如下优势：1、通过淋巴细胞分离液分离富集了淋巴细胞，保证检测结果可靠准确，节省抗体和试剂；2、通过富集的淋巴细胞亚群检测，能有效分析出数量较少的淋巴细胞亚群，也保证有效合理染色定义的淋巴细胞亚群；3、本发明方法能够检测的淋巴细胞亚群涵盖了免疫识别预警系统、细胞免疫系统、体液免疫系统和非特异免疫细胞系统，因此检测结果能够全面的反应淋巴细胞系统的状态；4、本发明可清楚定义出DC细胞的两种亚型；5、通过本发明可全面地对淋巴细胞亚群分析和检测，对于全面了解免疫状态提供了科学依据。
附图说明
图1：实施例2中淋巴细胞亚群检测图；
图2：实施例2中淋巴细胞亚群检测图；
图3：实施例3中淋巴细胞亚群检测图；
图4：实施例3中不同淋巴细胞亚群检测图；
图5：实施例3中DC细胞亚型检测图；
图6：实施例4中按实施例1方法所得样品与现有技术裂解法所得样品的细胞群对比图；(上边3个图为裂解法所得流式细胞图，下边3个图是上边3图对应样品的分离法所得的流式细胞图)
图7：实施例4中按实施例1方法所得样品与现有技术裂解法所得样品的细胞富集对比图；
图8：实施例4中按实施例1方法所得样品与现有技术裂解法所得样品的不同类型细胞富集对比图。
具体实施方式
本发明通过以下实施例和试验例进一步阐述本发明，但不以任何的方式限制本发明的有效范围。
实施例1流式检测所需的PBMC样品的制备
1、目的
制备流式检测所需的PBMC样品(文中也称为“样本”)。
2、原理
活细胞表面保留有较完整的抗原或受体，用荧光标记的抗体与细胞表面相应抗原结合，根据所测定的荧光强度和阳性百分率即可知相应抗原的密度和分布。
3、仪器耗材准备
3.1仪器
苏净超净工作台，时代北利低速离心机DT5-4，涡旋振荡器，各量程移液器，血细胞计数仪。
3.2耗材
15ml离心管，10ml移液管，1.5ml离心管，流式管，3ml巴氏吸管，各量程枪头
3.3试剂配制
Figure PCTCN2018118802-appb-000001
常规流式检测抗体；
4、操作步骤(以离体原血样5ml为例进行说明)
4.1离体外周血样(PBMC)的分离
(1)取离体原血样0.2ml计数。
(2)将5ml血液加入到15ml离心管中，1500-2900rpm，离心15-20min，去除上层血浆。
(3)剩余血液细胞沉淀与PBS溶液稀释。
(4)准备1支离心管，用移液管加入与血液细胞稀释液等量的淋巴细胞分离液。
(5)将血液细胞稀释液缓慢加入到步骤(4)的离心管中，保持分离液分层清晰。
(6)缓慢放入离心机内，配平后1500-2900rpm，离心15-20min。
(7)离心结束后用移液管缓慢吸取上层血浆弃掉。
(8)吸出中间白膜层细胞至一个新的离心管内，加PBS溶液至10ml，取200μl细胞悬液，用血细胞计数仪计数。剩余细胞悬液1500-2900rpm，离心5-10min。
(9)离心后，弃上清，用移液器吸尽残余液体。根据计数结果，加入适量4℃预冷的PBS溶液，调整细胞浓度在1.5-3.0×10 6个/ml的范围；作为示例性的说明，调整至2×10 6个/ml。
4.2制作流式细胞样品(本操作控制温度，全程样品及抗体在冰盒内操作)
(1)样品染色
每个流式管内加入100μl细胞悬液，加入流式检测抗体。并用涡旋振荡器轻缓震荡，使抗体与细胞充分混均，2～8℃，避光孵育20分钟。
(3)样品洗涤
孵育结束后，弃掉上清，加入2ml预冷的PBS溶液重悬细胞，1500-2900rpm，离心5-10min。
(4)样品完成
①如果立即上机检测，则弃去上清，用预冷的PBS-EDTA溶液400μl混匀细胞，用于流式检测。
②如不能立即检测，则用0.05-5％福尔马林500μl悬起细胞，涡旋振荡器，充分混匀，4℃避光保存。检测前各管加入2ml的PBS溶液，1500-2900rpm，离心5-10min，弃掉上清。用预冷的PBS-EDTA溶液400μl，混匀细胞，上机检测。
5、注意事项
5.1、整个操作中抗体、细胞尽量保持在低温(4℃)下进行。
5.2、加入抗体后要使抗体与细胞充分混匀。
实施例2 PBMC样品的检测
2.1仪器、试剂和样品：
样品：取离体血液样品按实施例1方法制备即得；
流式细胞仪：ACEA NovoCyte
2.2检测方法：
a)开机前检查仪器状态；
b)对流式细胞仪系统进行开机预热5～10分钟，进行灌注(约15分钟)；
c)进行质控程序，通过以后进行下一步；
d)设置仪器各个参数，调节各个检测通道的电压，调节液流速度为优选35～40μl/min；
e)将制备好的流式样品涡旋振荡3秒，进行检测。设置条件：门内15000个细胞；采集样品。
2.3检测结果
由图1可以看出：在FSC、SSC界面里，圈门1：淋巴细胞群，可以清晰地把淋巴细胞亚群分离出来，并且是主要分析细胞群，数据会更可靠准确。
由图2可以看出：(1)选取门1，在门1内在显示FL1和FL3界面里，(2)门1内圈十字门2：选取的是CD3+CD4+细胞群(即Q2区域)。待分析淋巴细胞亚群会分离比较开，门非常容易设定，数据更加准确。
实施例3 PBMC样品的检测
3.1仪器、试剂和样品：
样品：
待测样品：取5ml离体血液样品按实施例1方法制备即得；
流式细胞仪：ACEA NovoCyte。
3.2检测方法：
a)开机前检查仪器状态；
b)对流式细胞仪系统进行开机，进行灌注(约15分钟)；
c)进行质控程序，通过以后进行下一步；
d)设置仪器各个参数，调节各个检测通道的电压，调节液流速度为优选35～40μl/min；
e)将制备好的流式样品涡旋振荡，进行检测。设置条件：门内15000个细胞；采集样品。
3.3检测结果
1)图3为这一个健康人的外周血液用淋巴细胞分离法得到的淋巴细胞亚群分析的图，圈的P1门内为淋巴细胞群，由图3可见，背景干净，淋巴细胞群和细胞碎片群分的很开，很理想。
可以看出本发明通过淋巴细胞分离液分离富集了淋巴细胞，分群明显，保证检测结果可靠准确，同时节省抗体和试剂。
2)图4为通过富集的淋巴细胞亚群检测，有效地分析出了数量较少的淋巴细胞亚群，有效合理染色定义的淋巴细胞亚群；图4左图样品4，Q3-2为CD8+T细胞，通过分离富集，分群明显，可以很好的分析亚群。图4右图为淋巴细胞亚群比例较小的细胞亚群，DC细胞亚群，通过富集，也可以准确分析。
3)图5清楚定义出了DC细胞两种亚型，这是以往淋巴细胞亚群分析所不具备的。通过分离法富集了两个亚型细胞。如图5右1，分群明显，界限清晰。
实施例4本发明方法中制备样品与血溶素裂解法所得样品的对比
4.1样品和试剂：
本发明淋巴细胞样品：取离体外周血液样本按实施例1方法制备即得；
对比淋巴细胞样品：如下4.2.1部分方法制备即得；
5ml抗凝血样；
1×PBS：10×PBS溶液：纯水按照1:9体积比配制；
PBS溶液：即1×PBS；
PBS-EDTA溶液：向1×PBS中添加0.5M EDTA直至EDTA的终浓度为2.5mM；
常规流式检测用抗体：抗CD3抗体、lin1[Lineage Cocktail 1]、抗CD123的抗体、抗CD11的抗体、CD3FITC、CD8PerCP、CD4PerCP。
4.2试验方法：
4.2.1血溶素裂解法处理方法：
(1)样本染色：每个流式管内加入100μl抗凝血液样本，分别加入要标记淋巴细胞亚群的流式抗体，使抗体与细胞充分混均，2～8℃，避光孵育20分钟。
(2)裂解：向流式管中加入2ml 1×BD溶血素(即红细胞裂解液)，裂解5分钟。
(3)样本洗涤：5分钟后，离心，1500-2900rpm，10分钟，离心结束后，弃掉上清，加入2ml预冷的PBS溶液重悬细胞，1500-2000rpm，离心5-10min。倒掉上清后加入400μl 2～8℃PBS-EDTA溶液，混匀即得。
3.2.2上流式细胞仪检测方法：
a)开机前检查鞘液桶、废液通、洗涤剂、冲洗剂体积，添加足够的鞘液，倒掉废液；
b)对流式细胞仪系统进行开机预热5～10分钟，进行灌注(约15分钟)；
c)进行质控程序，通过以后进行下一步；
d)设置仪器各个参数，调节各个检测通道的电压，调节液流速度为35～40μl/min；
e)分别取制备好的本发明淋巴细胞样品和对比淋巴细胞样品、涡旋振荡3秒，分别进行检测。设置条件：门内15000个细胞；采集样品。
4.3实验结果：
4.3.1通过对比可以看出裂解得到样品碎片多，背景不干净，影响流式细胞仪数据的收集和分析，对淋巴细胞补偿调整可能引起偏倚现象；本方法(淋巴细胞分离液分离方法)使得背景干净、分群清晰明显(参见图6)。
其中，通过观察裂解法得到的淋巴细胞亚群分析的图中圈的门内为淋巴细胞，由图可见淋巴细胞群和细胞碎片群分的很不理想(参见图6第一排三幅图)。
本发明所得到的淋巴细胞亚群分析的图中圈的门内为淋巴细胞群，由图可见淋巴细胞群和细胞碎片群分的很开，很理想(参见图6第二排三幅图)。
4.3.2图7可以看出：本发明在使用荧光抗体时，由于是检测了淋巴细胞数量，根据比例添加荧光抗体，节约了70％以上的抗体，本发明检测时组合抗体的使用量为15μl，而裂解法的使用量为60μl。而本发明法不会造成抗体过量或不足。由图7可知：1组标记的均是CD3FITC、CD8PerCP；2组标记的均是CD3FITC、CD4PerCP。由于处理方法所得样品的不同，对于同一份离体外周血样品，其结果为裂解法得到的样品中的双阳性群比例明显减少，且抗体用量无法进行定量；而本发明方法得到的样品可以根据细胞的量进行抗体定量添加，除此之外，很明显，分离法细胞得到了显著的富集。
4.3.3图8可以看出：本发明制得的样品对于细胞亚群比例较小的样本，具有富集作用。同一份离体外周血样品，细胞亚群mDC和pDC所占比例比较小，在裂解法和本发明方法下，其所得样品的观察结果是不同的，本发明所得到样品中的细胞亚群都得到了富集。从细胞数目和百分比上看都可证明。

Claims (19)

  1. 一种免疫细胞中淋巴细胞的检测方法，其特征在于，所述方法包括：
    a)取免疫荧光抗体染色的淋巴细胞样品，加入预冷的PBS-EDTA溶液，混均细胞，备流式细胞仪检测待用；
    b)对流式细胞仪系统进行开机预热，调节液流速度为10-29μl/min的低速范围或30～44μl/min的中速范围，向样品管中加入PBS-EDTA溶液，冲洗液流的喷嘴系统；
    c)将步骤a)所得混均细胞样品加入样品管然后进行检测。
  2. 根据权利要求1所述的方法，其特征在于，所述PBS-EDTA溶液为pH7.2～7.4的含有0.005-0.05M PBS和终浓度为2-3mM EDTA的混合溶液。
  3. 根据权利要求1所述的方法，其特征在于，所述步骤a)中进一步包括：淋巴细胞样品中细胞的浓度为1×10 6～10×10 6个/ml。
  4. 根据权利要求1所述的方法，其特征在于，所述步骤a)中进一步包括：淋巴细胞样品的体积为50-200μl。
  5. 根据权利要求1所述的方法，其特征在于，所述步骤b)中调节液流速为30～44μl/min的中速范围；优选35～40μl/min的中速范围。
  6. 根据权利要求1所述的方法，其特征在于，所述步骤b)中进一步包括预热时间为5～10分钟。
  7. 根据权利要求1所述的方法，其特征在于，所述步骤c)进一步包括设置条件为：门内3000～20000个细胞，优选门内15000个细胞；采集样品。
  8. 根据权利要求1～7任一所述的方法，其特征在于，所述流式淋巴细胞样品采用如下方法制备：
    1)取离体抗凝血液样品进行离心，分离血浆和血液细胞沉淀，得血液细胞沉淀；
    2)将血液细胞沉淀用PBS溶液进行稀释，得血液细胞稀释液；
    3)将血液细胞稀释液缓慢加入到含等体积的淋巴细胞分离液的离心 管中，然后离心，去掉血浆，取白膜层细胞；
    4)取步骤3)所得白膜层细胞加入到离心管中，加入PBS溶液混合均匀，离心并去除上清液，然后再加入PBS溶液，调整细胞浓度在1×10 5个/ml～1×10 8个/ml，优选的浓度范围是1.5×10 6个/ml～5×10 6个/ml，进一步优选1.5～3.0×10 6个/ml；
    5)取步骤4)所得调整浓度后的淋巴细胞加入流式管内；并加入用于标记淋巴细胞亚群的流式抗体，混匀，2～8℃静置并避光孵育；
    6)加入PBS溶液、混匀，离心洗掉未结合抗体；
    7)样品的完成
    ①如果立即上机检测，则弃去上清，加入预冷至2～8℃的PBS-EDTA溶液，混匀细胞，用于流式检测；
    ②如不能立即检测，直接用0.05-5％福尔马林悬起细胞，混匀，静置于2～8℃避光保存；检测前各管加入PBS溶液，离心弃掉上清液，用2～8℃的PBS-EDTA溶液、混匀细胞，用于流式检测。
  9. 根据权利要求8所述的方法，其特征在于，所述PBS溶液为pH7.2～7.4的0.005-0.05M PBS溶液；所述PBS-EDTA溶液为pH7.2～7.4的含有0.005-0.05M PBS和终浓度为2-3mM EDTA的混合溶液。
  10. 根据权利要求8所述的方法，其特征在于，所述步骤1)进一步包括离心条件为1500～3500rpm离心5～30min；优选1500-2900rpm离心15-20分钟。
  11. 根据权利要求8所述的方法，其特征在于，所述步骤2)进一步包括血液细胞沉淀用PBS溶液以1:0.5～1:2体积比例；优选1:1体积比例进行稀释。
  12. 根据权利要求8所述的方法，其特征在于，所述步骤3)进一步包括离心条件为1500-3500rpm，10-20min，4℃；优选1500-2900rpm，15-20min，4℃。
  13. 根据权利要求8所述的方法，其特征在于，所述步骤4)进一步包括离心条件为1500-3500rpm，5-20分钟，4℃；优选离心条件为1500-2900rpm，离心5-10分钟，4℃。
  14. 根据权利要求8所述的方法，其特征在于，所述步骤4)中PBS 溶液的量为加至10ml～15ml。
  15. 根据权利要求8所述的方法，其特征在于，所述步骤6)进一步包括离心条件为1500-2900rpm，离心5-10分钟。
  16. 根据权利要求8所述的方法，其特征在于，所述步骤6)中PBS溶液的量为2ml。
  17. 根据权利要求8所述的方法，其特征在于，所述步骤7)中所述PBS-EDTA溶液为pH7.2～7.4的含有0.005-0.05M PBS和终浓度为2-3mM EDTA的混合溶液；所述PBS溶液为pH7.2～7.4的0.005-0.05M PBS。
  18. 根据权利要求8所述的方法，其特征在于，所述步骤7)中所述离心条件为：1500-2900rpm，离心5-10min。
  19. 根据权利要求8所述的方法，其特征在于，所述方法包括：
    (一)外周血中分离人外周血单核细胞
    (1)取离体原血血样0.2ml计数，留样1ml，标记信息，于4℃下保存；
    (2)将5ml血液加入到离心管中，1500-2900rpm离心15-20min；取上层血浆加入到2ml冻存管中于-80℃保存；
    (3)剩余血液细胞沉淀与PBS溶液按1:1的体积比进行稀释；
    (4)准备1支15ml离心管，用移液管加入与血液细胞稀释液等量的淋巴细胞分离液；
    (5)将血液细胞稀释液缓慢加入到步骤(4)的离心管中，保持分离液分层清晰；
    (6)放入离心机内，配平后1500-2900rpm，离心15-20min；
    (7)离心结束后用10ml移液管或者3ml巴氏吸管缓慢吸取上层血浆弃掉；
    (8)吸取中间白膜层细胞至一个新的离心管内，加PBS溶液至10ml，取200μl细胞液，用全血细胞计数仪计数，以备后续调整细胞浓度用，剩余细胞悬液1500-2900rpm，离心5-10min；
    (9)离心后，弃上清，用1ml移液器吸尽残余液体，根据计数结果，加入适量4℃度预冷的PBS溶液，调整细胞数在1.5-3.0×10 6个/ml的浓度；优选2×10 6个/ml的浓度；
    (二)制作流式细胞样品
    (10)样品染色
    每个流式管内加入100μl细胞悬液，加入相应抗体，使抗体与细胞充分混均，4℃，避光孵育10-30分钟，优选15～20分钟，最佳20分钟；
    (11)样品洗涤、加液
    离心结束后，弃掉上清，加入2ml 2～8℃度的PBS溶液重悬细胞，1500-2900rpm，离心5-10min，离心后，倒掉上清；
    ①如果立即上机检测，用预冷的PBS-EDTA溶液400μl混匀细胞，用于流式检测；
    ②如不能立即检测，则用0.05-5％福尔马林500μl悬起细胞，涡旋振荡器，充分混匀，4℃避光保存；检测前各管加入2ml PBS溶液，1500-2900rpm，离心5-10min，弃掉上清；用预冷的PBS-EDTA溶液400μl，混匀细胞，上机检测。
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