WO2021012925A1 - 一种人外周血淋巴细胞的检测方法 - Google Patents

一种人外周血淋巴细胞的检测方法 Download PDF

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WO2021012925A1
WO2021012925A1 PCT/CN2020/099875 CN2020099875W WO2021012925A1 WO 2021012925 A1 WO2021012925 A1 WO 2021012925A1 CN 2020099875 W CN2020099875 W CN 2020099875W WO 2021012925 A1 WO2021012925 A1 WO 2021012925A1
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peripheral blood
human peripheral
fluorescent probe
hemolysin
blood lymphocytes
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PCT/CN2020/099875
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French (fr)
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何淼
李国平
张涛
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泛肽生物科技(浙江)有限公司
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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
    • G01N33/53—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

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  • the invention belongs to the technical field of cell detection, and particularly relates to a detection method of human peripheral blood lymphocytes.
  • FCM Flow Cytometry
  • It can not only measure cell size, internal particle properties, but also detect cell surface and cytoplasmic antigens, intracellular DNA, RNA content, etc., and can analyze populations of cells at the single cell level, and detect and analyze a large number of cells in a short time , And collect, store and process data for multi-parameter quantitative analysis; it can sort a certain subgroup of cells with a purity of> 95%. It is widely used in hematology, immunology, oncology, pharmacology, molecular biology and other disciplines.
  • T lymphocytes and NK cells are two types of lymphocytes, which can reflect the immune function of basal cells.
  • Chinese Patent CN107063982A discloses a method for detecting chicken peripheral blood lymphocyte subsets by flow cytometry.
  • the method disclosed in the above patent can only output the positive percentage of each subgroup, and the result is relatively single, and the pure positive percentage is only a relative count. For example, the total cell number of lymphocytes is reduced, that is, the absolute number is reduced, but relative The percentage of each group may remain unchanged.
  • the detection method of the above patent takes a long time to extract lymphocytes, which is not conducive to the absolute count of lymphocytes. At the same time, it will cause damage to the sample cells and a long process of apoptosis, low detection sensitivity, and affect the detection results.
  • the purpose of the present invention is to provide a method for detecting human peripheral blood lymphocytes.
  • the invention has the advantages of simple operation, long sample storage time, high accuracy, multi-index output and the like.
  • a method for detecting human peripheral blood lymphocytes including the following steps:
  • the present invention uses the monoclonal antibody reagent of the cell membrane surface antigen-specific antibody to directly label human peripheral blood.
  • the leukocyte co-antigen molecule CD45 is used to distinguish different lymphocyte populations; the antibodies CD3, CD4, and CD8 specifically label T lymphocytes and Its subgroup molecules; antibodies CD16 and CD56 specifically label NK lymphocytes and their subgroup molecules.
  • the fluorescent probe of the step (1) is a mitochondrial fluorescent probe; preferably, the fluorescent probe is a mitochondrial green fluorescent probe.
  • the fluorescent probe of step (2) is a lysosomal fluorescent probe; preferably, the fluorescent probe is a lysosomal green fluorescent probe.
  • mitochondrial fluorescent probes and lysosomal fluorescent probes are respectively combined with lymphocytes to complete organelle calibration.
  • mitochondrial fluorescent probes are mainly combined based on changes in mitochondrial membrane potential
  • lysosomal fluorescent probes are mainly combined based on changes in lysosomal pH.
  • the volume ratio of the monoclonal antibody to the fluorescent probe is 5:1-10:1.
  • Monoclonal antibodies are mainly protein components, and fluorescent probes are polymer compounds. Interference reactions will occur between the two. By optimizing the appropriate titers of antibodies and probes, the present invention can effectively reduce interference reactions and ensure antigens and antibodies. The precise combination of needle and target.
  • the hemolysin working solution contains ammonium chloride, potassium bicarbonate, disodium EDTA and fetal bovine serum (FBS).
  • each liter of the hemolysin working solution contains 75-85 g of ammonium chloride, 15-25 g of potassium bicarbonate, 300-400 mg of disodium EDTA, and 15-30 ml of fetal bovine serum (FBS).
  • FBS fetal bovine serum
  • the hemolysin working solution used in the present invention is a gentle red blood cell lysate, which can lyse human peripheral blood samples so that the samples retain only white blood cells, ensure the integrity of the molecular structure of the white blood cell membrane surface, and maintain good cell activity, which can ensure The stained sample maintains stability within 48 hours.
  • the volume concentration of the fluorescent probe in the hemolysin working solution is 2% to 5%.
  • the volume ratio of human anticoagulant peripheral blood to hemolysin working solution is 1:10 to 1:5.
  • the light-proof incubation time is 15-60 minutes.
  • the invention innovatively uses organelle-specific fluorescent probes to bind the lymphocytes therein to complete the organelle calibration.
  • the signal strength after the fluorescent probes are combined with the organelles can accurately analyze cell populations with stable quality.
  • T lymphocytes can improve the sensitivity of lymphocyte detection;
  • NK lymphocyte subgroups as innate immune cells, are extremely sensitive to the content environment where the cells are located. By analyzing the changes in their subgroups, the sensitivity can be improved. The sensitivity of cell detection.
  • the detection steps of the present invention are simple and easy to operate.
  • the detection steps are only two steps.
  • the sample is combined with the reagents and the red blood cells are lysed.
  • the machine can be tested, which reduces manual operations and reduces operational risks.
  • the samples are stored for a stable time after preparation. Longer
  • the present invention has high accuracy and sensitivity.
  • the detection of human peripheral blood lymphocytes is achieved through two indicators: the positive percentage of lymphocyte subpopulations and the absolute count. It can be applied to assess cell damage in the future, even for Determine the risk of illness.
  • Figure 1 is a scatter plot of CD45/SSC, used to circle lymphocyte population.
  • Figure 2 is a scatter plot of reading CD45+Lymph, Mito/CD3, marking Mito+CD3+ (ie Q3-2).
  • Figure 3 is a scatter plot of reading Mito+CD3+, CD4/CD8, marking CD3+CD8+, CD3+CD4+.
  • Figure 4 is a scatter plot of CD45/SSC, used to circle lymphocyte population.
  • Figure 5 is a scatter plot of reading CD45+Lymph, Lyso/CD3, marking Lyso+CD3- (ie Q2-1).
  • Figure 6 is a scatter plot of reading Lyso+CD3-, CD16/CD56, marking CD3-CD56+CD16+, CD3-CD56dimCD16-, CD3-CD56brightCD16-.
  • Example 1 Detection of T lymphocyte subsets expressed by human peripheral blood mitochondria
  • the reagent for detecting T lymphocyte subgroups expressed by human peripheral blood mitochondria of the present invention includes: hemolysin (10 ⁇ ), mitochondrial fluorescent dye, CD3APC, CD4PE-Cy7, CD8PE, CD45APC-Cy7 monoclonal antibody.
  • Mitochondrial fluorescent dye preparation (1 serving): Add 1 ⁇ L of mitochondrial fluorescent dye to 499 ⁇ L of hemolysin working solution, vortex and mix at low speed, and use at 2 ⁇ 8°C for standby.
  • the concentration of CD3APC monoclonal antibody is 0.05 ⁇ g/test (5 ⁇ L)
  • the concentration of CD4PE-Cy7 monoclonal antibody is 0.1 ⁇ g/test (5 ⁇ L)
  • the concentration of CD8PE monoclonal antibody is 0.02 ⁇ g/test (5 ⁇ L)
  • the CD45APC-Cy7 monoclonal antibody The concentration is 0.01 ⁇ g/test (5 ⁇ L).
  • On-machine testing (testing will be completed within 6 hours, if not testing immediately, please place the sample at 2 ⁇ 8°C in the dark).
  • Figure 1 is a CD45/SSC scatter diagram.
  • CD45 divides human peripheral blood leukocytes into three groups of lymphocytes, monocytes and granulocytes;
  • Figure 2 reads the CD45+Lymph group, shown as A scatter plot of mitochondrial probe expression and T lymphocyte count (CD3), in which a blank tube with unstained mitochondrial probe is superimposed;
  • Figure 3 reads the Mito+/CD3+ group (ie Q3-2) , Shows the CD4/CD8 scatter plot, the figure respectively identifies the helper-induced T lymphocyte population (CD3+CD4+) and the inhibitory T lymphocyte population (CD3+CD8+).
  • Use ACEA NovoCyte flow cytometer volume method absolute count Method the absolute counts and positive percentages of the three groups are obtained, as shown in Table 1.
  • Table 1 Absolute counts and positive percentages of the three groups Q3-2, CD3+CD4+ and CD3+CD8+
  • Example 2 Detection of NK lymphocyte subsets expressed by lysosomes in human peripheral blood
  • the reagent for detecting NK lymphocyte subsets expressed by human peripheral blood lysosomes of the present invention includes: hemolysin (10 ⁇ ), lysosomal fluorescent dye, CD3APC, CD56PE, CD16-PE/Cy7, CD45-APC-Cy7 monoclonal antibody.
  • Lysosomal Fluorescent Dye (1 serving): Add 1 ⁇ L of Lysosomal Fluorescent Dye to 499 ⁇ L of Hemolysin working solution, vortex and mix at low speed, and use at 2 ⁇ 8°C for later use.
  • the concentration of CD3APC monoclonal antibody is 0.05 ⁇ g/test (5 ⁇ L)
  • the concentration of CD16PE-Cy7 monoclonal antibody is 0.05 ⁇ g/test (5 ⁇ L)
  • the concentration of CD56PE monoclonal antibody is 0.1 ⁇ g/test (5 ⁇ L)
  • the CD45APC-Cy7 monoclonal antibody The concentration is 0.01 ⁇ g/test (5 ⁇ L).
  • On-machine testing (testing will be completed within 6 hours, if not testing immediately, please place the sample at 2 ⁇ 8°C in the dark).
  • Figure 4 is a CD45/SSC scatter plot.
  • CD45 divides human peripheral blood leukocytes into three groups of lymphocytes, monocytes and granulocytes;
  • Figure 5 reads the CD45+Lymph group, showing This is a scatter plot of lysosomal probe expression and T lymphocyte count (CD3), in which a blank tube with unstained lysosomal probe is superimposed;
  • Figure 6 is reading Lyso+/CD3-group (I.e. Q2-1), showing the CD16/CD56 scatter plot.
  • the figure shows the NK1 lymphocyte population (CD3-CD56+CD16+), NK2 lymphocyte population (CD3-CD56dimCD16-) and NK3 lymphocyte population (CD3- CD56brightCD16-), using ACEA NovoCyte flow cytometer's volumetric absolute counting method to obtain the absolute counts and positive percentages of the four groups, as shown in Table 2.
  • the quality of the organelles can be analyzed by flow cytometry, and the cell population with stable quality can be accurately analyzed based on the signal strength of the fluorescent probe combined with the organelle.
  • the percentage and absolute count value of the two subgroups are read, so as to realize the detection of human peripheral blood lymphocytes.
  • the method of the invention effectively improves the accuracy and sensitivity of lymphocyte detection.

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Abstract

一种人外周血淋巴细胞的检测方法，取人抗凝外周血，分别加入抗人CD3、CD4、CD8、CD45单克隆抗体，以及抗人CD3、CD56、CD16、CD45单克隆抗体，然后再加入包含荧光探针的溶血素工作液，从而分别获得人外周血T淋巴细胞亚群和NK淋巴细胞亚群百分比和绝对计数值，实现对人外周血淋巴细胞的检测。该方法具有操作简便，样本稳定保存时间长，精确度高，多指标输出等优点。

Description

一种人外周血淋巴细胞的检测方法 技术领域
本发明属于细胞检测技术领域，特别是涉及一种人外周血淋巴细胞的检测方法。
背景技术
流式细胞术(Flow Cytometry,FCM)是七十年代发展起来的高科学技术，它集计算机技术、激光技术、流体力学、细胞化学、细胞免疫学于一体，同时具有分析和分选细胞功能。检测原理是荧光标记待测试细胞并制成单细胞悬液，根据接收激光照射后液流内细胞的散色光信号和荧光信号分析细胞的物理化学特征，如细胞大小、被测细胞内部颗粒的信息等。它不仅可测量细胞大小、内部颗粒的性状，还可检测细胞表面和细胞浆抗原、细胞内DNA、RNA含量等，可对群体细胞在单细胞水平上进行分析，在短时间内检测分析大量细胞，并收集、储存和处理数据,进行多参数定量分析；能够分类分选某一亚群细胞，分选纯度＞95％。在血液学、免疫学、肿瘤学、药物学、分子生物学等学科广泛应用。
T淋巴细胞和NK细胞是淋巴细胞的两种类群，能够体现基体细胞的免疫功能。
中国专利CN107063982A公开了一种流式细胞术检测鸡外周血 淋巴细胞亚群的方法。但是上述专利公开的方法仅能输出各亚群的阳性百分比，结果较为单一，而单纯的阳性百分比仅为相对计数，如淋巴细胞整体细胞数都减少了，即绝对个数减少了，但相对的各群百分比可能维持不变。此外，上述专利的检测方法对淋巴细胞的提取操作时间较长，不利于淋巴细胞的绝对计数，同时会导致样本细胞的损伤和凋亡过程长，检测灵敏度偏低，影响检测结果。
发明内容
基于现有技术存在的缺陷，本发明的目的在于提供一种人外周血淋巴细胞的检测方法。本发明具有操作简便，样本稳定保存时间长，精确度高，多指标输出等优点。
为了达到上述的目的，本发明采取以下技术方案：
一种人外周血淋巴细胞的检测方法，包括以下步骤：
(1)T淋巴细胞亚群检测：
取人抗凝外周血加入流式管底部，加入抗人CD3、CD4、CD8、CD45单克隆抗体，涡旋混匀，室温下避光孵育；然后进一步加入包含荧光探针的溶血素工作液，涡旋混匀，室温下避光孵育；最后使用流式细胞仪进行检测，获得人外周血T淋巴细胞亚群百分比和绝对计数值；
(2)NK淋巴亚群检测：
取人抗凝外周血加入流式管底部，加入抗人CD3、CD56、CD16、 CD45单克隆抗体，涡旋混匀，室温下避光孵育；然后进一步加入包含荧光探针的溶血素工作液，涡旋混匀，室温下避光孵育；最后使用流式细胞仪进行检测，获得人外周血NK淋巴细胞亚群百分比和绝对计数值。
本发明使用细胞膜表面抗原特异性抗体的单克隆抗体试剂直接标记人外周血，其中：白细胞共抗原分子CD45，用于区分不同的淋巴细胞群；抗体CD3、CD4、CD8特异性标记T淋巴细胞及其亚群分子；抗体CD16和CD56特异性标记NK淋巴细胞及其亚群分子。
进一步地，在上述方法中，所述步骤(1)的荧光探针为线粒体荧光探针；优选的，所述荧光探针为线粒体绿色荧光探针。
进一步地，在上述方法中，所述步骤(2)的荧光探针为溶酶体荧光探针；优选的，所述荧光探针为溶酶体绿色荧光探针。
本发明步骤(1)和步骤(2)分别采用线粒体荧光探针和溶酶体荧光探针结合淋巴细胞，完成细胞器标定。其中，线粒体荧光探针主要依据线粒体膜电位变化进行结合，溶酶体荧光探针主要依据溶酶体pH值变化进行结合。
进一步地，在上述方法中，所述单克隆抗体与荧光探针的体积比为5:1～10:1。
单克隆抗体主要为蛋白质成分，荧光探针为高分子化合物，两者之间会出现干扰反应，本发明通过优化抗体和探针的合适滴度， 可有效降低干扰反应，保证抗原和抗体，探针和标靶的精准结合。
进一步地，在上述方法中，所述溶血素工作液包含氯化铵、碳酸氢钾、EDTA二钠和胎牛血清(FBS)。
进一步地，在上述方法中，每升所述溶血素工作液包含氯化铵75～85g、碳酸氢钾15～25g、EDTA二钠300～400mg、胎牛血清(FBS)15～30ml。
本发明采用的溶血素工作液配方是一种温和型红细胞裂解液，能够裂解人外周血样本，使样本仅保留白细胞，保证白细胞膜表面分子结构的完整性，并维持细胞良好的活性，可以保证染色后的样本维持48小时内的稳定性。
进一步地，在上述方法中，所述溶血素工作液中荧光探针的体积浓度为2％～5％。
进一步地，在上述方法中，人抗凝外周血与溶血素工作液的体积比为1:10～1:5。
进一步地，在上述方法中，所述避光孵育时间为15-60分钟。
本发明具有以下技术特点：
1)本发明创新的使用细胞器特异性荧光探针结合其中的淋巴细胞，完成细胞器标定，荧光探针结合细胞器后的信号强弱能够精确分析出质量稳定的细胞群。
2)本发明同时检测T淋巴细胞和NK淋巴细胞。T淋巴细胞作为淋巴细胞群中的主要表达，能够提高对淋巴细胞检测的灵敏度； NK淋巴细胞亚群作为固有免疫细胞，对细胞所在的内容环境极为敏感，通过分析其亚群的变化，可提高细胞检测的敏感度。
3)本发明检测步骤简单，操作简便，检测步骤仅为两步，样本与试剂结合，红细胞裂解，即可上机检测，减少了人工操作，降低了操作风险，同时制备后样本的稳定保存时间更长
4)本发明具有较高的精确度和灵敏度，通过淋巴细胞亚群的阳性百分比与绝对计数两个指标来实现对人外周血淋巴细胞的检测，将来可应用于评估细胞损伤情况，甚至用于判断患病风险。
附图说明
图1是CD45/SSC散点图，用于圈淋巴细胞群.
图2是读取CD45+Lymph，Mito/CD3散点图，标识Mito+CD3+(即Q3-2).
图3是读取Mito+CD3+，CD4/CD8散点图，标识CD3+CD8+，CD3+CD4+.
图4是CD45/SSC散点图，用于圈淋巴细胞群.
图5是读取CD45+Lymph，Lyso/CD3散点图，标识Lyso+CD3-(即Q2-1).
图6是读取Lyso+CD3-，CD16/CD56散点图，标识CD3-CD56+CD16+，CD3-CD56dimCD16-，CD3-CD56brightCD16-。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将对本发明实施例的技术方案进行清楚、完整的描述。显然，所描述的实施例是本发明的一部分实施例，而不是全部的实施例。基于所描述的本发明的实施例，本领域普通技术人员在无需创造性劳动的前提下所获得的所有其他实施例，都属于本发明的保护范围。
除非另作定义，本公开所使用的技术术语或者科学术语应当为本发明所属领域内有一般技能的人士所理解的通常意义。
本发明具体实施方式中使用的试剂来源如表1所示。
表1试剂名称、厂家和货号
Figure PCTCN2020099875-appb-000001
Figure PCTCN2020099875-appb-000002
实施例1：人外周血线粒体表达的T淋巴细胞亚群检测
本发明的人外周血线粒体表达的T淋巴细胞亚群的检测试剂包括：溶血素(10×)、线粒体荧光染料、CD3APC、CD4PE-Cy7、CD8PE、CD45APC-Cy7单克隆抗体。
(一)人外周血线粒体表达的T淋巴细胞亚群试剂准备
1.溶血素
将10×溶血素用去离子水稀释成1×工作液(1×溶血素)，按照50μL人外周血加入0.5mL工作液的量计算量配制。
配制1L 10×溶血素需要使用82.9g氯化铵，10g碳酸氢钾，372mg EDTA二钠，20mL胎牛血清(Fetal calf serum，FBS)。
2.线粒体荧光染料和单克隆抗体
线粒体荧光染料配制(1人份用量)：将1μL线粒体荧光染料加入至499μL溶血素工作液中，低速涡旋混匀，2～8℃备用。
CD3APC单克隆抗体浓度为0.05μg/test(5μL)，CD4PE-Cy7单克隆抗体浓度为0.1μg/test(5μL)，CD8PE单克隆抗体浓度为0.02μg/test(5μL)，CD45APC-Cy7单克隆抗体浓度为0.01μg/test(5μL)。
(二)利用流式细胞仪检测人外周血线粒体表达的T淋巴细胞亚群
1.用合适的微量移液器移取50μL抗凝外周血，缓缓加入流式管底部，如管壁有血液沾染，需要使用棉签擦净，以避免后续红细胞裂解不完全，引起检测结果碎片增多现象；
2.使用合适的微量移液器移取各抗体试剂加入至步骤1中的样本，中速涡旋混匀，室温下避光孵育15分钟；
3.使用合适的微量移液器移取0.5mL溶血素工作液(含线粒体荧光染料)，缓缓加入步骤2中的流式管中，中速涡旋混匀，室温下避光孵育60分钟；
4.上机检测(6小时内完成检测，如不立即检测，请将样本放置在2～8℃避光下)。
5.流式图形数据结果分析，检测结果如图1-图3所示。其中，图1是以CD45/SSC散点图，CD45将人外周血白细胞分成了三群淋巴细胞群，单核细胞群和粒细胞群；图2是读取的是CD45+Lymph群，显示为线粒体探针表达和T淋巴细胞数(CD3)散点图，在该图中叠加上了以未染色线粒体探针的空白管；图3是读取的是Mito+/CD3+群(即Q3-2)，显示CD4/CD8散点图，图中分别标识出辅助诱导T淋巴细胞群(CD3+CD4+)，抑制毒T淋巴细胞群(CD3+CD8+)，使用ACEA NovoCyte流式细胞仪的体积法绝对计数方法，得到三个群的绝对计数和阳性百分比，如表1所示。
表1 Q3-2、CD3+CD4+和CD3+CD8+三个群的绝对计数和阳性百分比
Gate Count Abs.Count ％Q3-2
Q3-2 9,919 1,475 100.00％
CD3+CD8+ 4,036 599 40.69％
CD3+CD4+ 4,701 699 47.39％
实施例二：人外周血溶酶体表达的NK淋巴细胞亚群检测
本发明的人外周血溶酶体表达的NK淋巴细胞亚群检测试剂包括：溶血素(10×)、溶酶体荧光染料、CD3APC、CD56PE、CD16-PE/Cy7、CD45-APC-Cy7单克隆抗体。
(一)人外周血溶酶体表达的NK淋巴细胞亚群试剂准备
1.溶血素
将10×溶血素用去离子水稀释成1×工作液(1×溶血素)，按照100μL人外周血加入2mL工作液的量计算量配制。
配制1L 10×溶血素需要使用82.9g氯化铵，10g碳酸氢钾，372mgEDTA二钠。
2.溶酶体荧光染料和单克隆抗体
溶酶体荧光染料配制(1人份用量)：将1μL溶酶体荧光染料加入至499μL溶血素工作液中，低速涡旋混匀，2～8℃备用。
CD3APC单克隆抗体浓度为0.05μg/test(5μL)，CD16PE-Cy7单克隆抗体浓度为0.05μg/test(5μL)，CD56PE单克隆抗体浓度为0.1μg/test(5μL)，CD45APC-Cy7单克隆抗体浓度为0.01μg/test(5μL)。
(二)人外周血溶酶体表达的NK淋巴细胞亚群
1.用合适的微量移液器移取50μL抗凝外周血，缓缓加入流式管底部，如管壁有血液沾染，需要使用棉签擦净，以避免后续红细胞裂解不完全，引起检测结果碎片增多现象；
2.使用合适的微量移液器移取各抗体试剂加入至步骤1中的样本，中速涡旋混匀，室温下避光孵育15分钟；
3.使用合适的微量移液器移取0.5mL的溶血素(含溶酶体荧光染料)加入步骤2中的流式管中，中速涡旋混匀，室温下避光孵育60分钟；
4.上机检测(6小时内完成检测，如不立即检测，请将样本放置在2～8℃避光下)。
5.流式图形数据结果分析，检测结果如图4-图6所示。其中，图4是是以CD45/SSC散点图，CD45将人外周血白细胞分成了三群淋巴细胞群，单核细胞群和粒细胞群；图5是读取的是CD45+Lymph群，显示为溶酶体探针表达和T淋巴细胞数(CD3)散点图，在该图中叠加上了以未染色溶酶体探针的空白管；图6是读取的是Lyso+/CD3-群(即Q2-1)，显示CD16/CD56散点图，图中分别标识出NK1淋巴细胞群(CD3-CD56+CD16+)，NK2淋巴细胞群(CD3-CD56dimCD16-)和NK3淋巴细胞群(CD3-CD56brightCD16-)，使用ACEA NovoCyte流式细胞仪的体积法绝对计数方法，得到四个群的绝对计数和阳性百分比，如表2 所示。
表2 Q2-1、NK1、NK2和NK3四个群的绝对计数和阳性百分比
Gate Count Abs.Count ％Q2-1
Q2-1 3,414 596 100.00％
NK1 1,841 321 53.93％
NK2 57 9.9 1.67％
NK3 8 1.39 0.23％
综上，从本发明的实施例1和实施例2可以通过流式图分析细胞器的质量分群，依据荧光探针结合细胞器后的信号强弱精准分析出质量稳定的细胞群。通过分析稳定细胞群的T淋巴细胞亚群及NK淋巴细胞亚群，读取两种亚群的百分比和绝对计数值，从而实现人外周血淋巴细胞的检测。本发明的方法有效的提高了淋巴细胞检测的精确度和灵敏度。
以上实施例的说明只是用于帮助理解本发明方法及其核心思想。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求保护范围内。

Claims (9)

  1. 一种人外周血淋巴细胞的检测方法，其特征在于，包括以下步骤：
    (1)T淋巴细胞亚群检测：
    取人抗凝外周血加入流式管底部，加入抗人CD3、CD4、CD8、CD45单克隆抗体，涡旋混匀，室温下避光孵育；然后进一步加入包含荧光探针的溶血素工作液，涡旋混匀，室温下避光孵育；最后使用流式细胞仪进行检测，获得人外周血T淋巴细胞亚群百分比和绝对计数值；
    (2)NK淋巴细胞亚群检测：
    取人抗凝外周血加入流式管底部，加入抗人CD3、CD56、CD16、CD45单克隆抗体，涡旋混匀，室温下避光孵育；然后进一步加入包含荧光探针的溶血素工作液，涡旋混匀，室温下避光孵育；最后使用流式细胞仪进行检测，获得人外周血NK淋巴细胞亚群百分比和绝对计数值。
  2. 根据权利要求1的一种人外周血淋巴细胞的检测方法，其特征在于，所述步骤(1)的荧光探针为线粒体荧光探针；优选的，所述荧光探针为线粒体绿色荧光探针。
  3. 根据权利要求1的一种人外周血淋巴细胞的检测方法，其特征在于，所述步骤(2)的荧光探针为溶酶体荧光探针；优选的，所述荧光探针为溶酶体绿色荧光探针。
  4. 根据权利要求1的一种人外周血淋巴细胞的检测方法，其特 征在于，所述单克隆抗体与荧光探针的体积比为5:1～10:1。
  5. 根据权利要求1的一种人外周血淋巴细胞的检测方法，其特征在于，所述溶血素工作液包含氯化铵、碳酸氢钾、EDTA二钠和胎牛血清(FBS)。
  6. 根据权利要求1的一种人外周血淋巴细胞的检测方法，其特征在于，每升所述溶血素工作液包含氯化铵75～85g、碳酸氢钾15～25g、EDTA二钠300～400mg、胎牛血清(FBS)15～30ml。
  7. 根据权利要求1的一种人外周血淋巴细胞的检测方法，其特征在于，所述溶血素工作液中荧光探针的体积浓度为2％～5％。
  8. 根据权利要求1的一种人外周血淋巴细胞的检测方法，其特征在于，人抗凝外周血与溶血素工作液的体积比为1:10～1:5。
  9. 根据权利要求1的一种人外周血淋巴细胞的检测方法，其特征在于，所述避光孵育时间为15-60分钟。
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CN113049815A (zh) * 2019-12-26 2021-06-29 上海益诺思生物技术股份有限公司 一种食蟹猴淋巴细胞的流式圈门方法
CN111504887B (zh) * 2020-05-09 2023-05-09 苏州四正柏生物科技有限公司 一种溶血素及其制备方法
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CN113218847A (zh) * 2021-04-12 2021-08-06 武汉凯普瑞生物技术有限公司 一种流式细胞分析用溶血剂及其制备方法及其应用方法
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Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080194508A1 (en) * 2005-02-25 2008-08-14 Christensen Nanna K Cell Counting
CN101424692A (zh) * 2008-12-12 2009-05-06 南京医科大学第一附属医院 一种定量检测乙型肝炎病毒特异性细胞毒性t淋巴细胞的方法
CN104360050A (zh) * 2014-09-22 2015-02-18 重庆医科大学附属儿童医院 一种淋巴细胞免疫分型的方法和试剂盒
CN104677810A (zh) * 2015-01-30 2015-06-03 广东医学院附属医院 嗜碱性粒细胞活化的检测试剂盒及其使用方法
CN106290812A (zh) * 2016-09-08 2017-01-04 北京海思特临床检验所有限公司 B细胞恶性肿瘤相关抗原表达量检测试剂盒及检测方法
CN107576789A (zh) * 2017-11-03 2018-01-12 深圳市巴德生物科技有限公司 一种冻干nk细胞表面抗原质控品及其制备方法
CN108375675A (zh) * 2018-01-29 2018-08-07 李小峰 外周血淋巴细胞亚群细胞浓度检测试剂盒及其检测方法
CN110487706A (zh) * 2019-07-24 2019-11-22 泛肽生物科技(浙江)有限公司 一种人外周血淋巴细胞的检测方法

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103424540A (zh) * 2012-05-18 2013-12-04 嘉善加斯戴克医疗器械有限公司 一种白细胞分类试剂盒及其分类方法
CN105223361B (zh) * 2015-08-28 2017-05-17 北京大学人民医院 一种检测急性t淋巴细胞白血病幼稚t细胞的试剂盒、应用及方法
CN106525699A (zh) * 2016-10-21 2017-03-22 深圳市职业病防治院 一种外周血淋巴细胞微核的检测试剂盒及其检测方法
CN108732082A (zh) * 2018-06-07 2018-11-02 北京唯公医疗技术有限公司 细胞分群方法

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080194508A1 (en) * 2005-02-25 2008-08-14 Christensen Nanna K Cell Counting
CN101424692A (zh) * 2008-12-12 2009-05-06 南京医科大学第一附属医院 一种定量检测乙型肝炎病毒特异性细胞毒性t淋巴细胞的方法
CN104360050A (zh) * 2014-09-22 2015-02-18 重庆医科大学附属儿童医院 一种淋巴细胞免疫分型的方法和试剂盒
CN104677810A (zh) * 2015-01-30 2015-06-03 广东医学院附属医院 嗜碱性粒细胞活化的检测试剂盒及其使用方法
CN106290812A (zh) * 2016-09-08 2017-01-04 北京海思特临床检验所有限公司 B细胞恶性肿瘤相关抗原表达量检测试剂盒及检测方法
CN107576789A (zh) * 2017-11-03 2018-01-12 深圳市巴德生物科技有限公司 一种冻干nk细胞表面抗原质控品及其制备方法
CN108375675A (zh) * 2018-01-29 2018-08-07 李小峰 外周血淋巴细胞亚群细胞浓度检测试剂盒及其检测方法
CN110487706A (zh) * 2019-07-24 2019-11-22 泛肽生物科技(浙江)有限公司 一种人外周血淋巴细胞的检测方法

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