CN104360049A - T lymphocyte immunophenotyping method and kit - Google Patents
T lymphocyte immunophenotyping method and kit Download PDFInfo
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Abstract
本发明属于免疫学技术领域，公开了一种T淋巴细胞免疫分型的方法和试剂盒。本发明提供的T淋巴细胞免疫分型的方法，包括：取不同的荧光标记的抗体，与待检测样品混合，孵育后，经流式细胞术检测，得检测数据，分析所得检测数据；该抗体包括：抗TCRαβ的抗体、抗TCRγδ的抗体、抗CD3的抗体、抗CD4的抗体、抗CD8的抗体、抗CD27的抗体和抗CD45RA抗体。本发明的方法实现了对T淋巴细胞进行更加全面的精细免疫分型，所需待测样品少，操作简单、所需时间短、准确性高、可广泛应用于淋巴细胞亚群免疫分型。The invention belongs to the technical field of immunology, and discloses a method and a test kit for T lymphocyte immune typing. The method for immunophenotyping of T lymphocytes provided by the present invention comprises: taking different fluorescently labeled antibodies, mixing them with the samples to be detected, and after incubation, detecting by flow cytometry to obtain detection data and analyzing the obtained detection data; the antibody Including: anti-TCRαβ antibody, anti-TCRγδ antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody, anti-CD27 antibody and anti-CD45RA antibody. The method of the present invention realizes more comprehensive fine immune typing of T lymphocytes, requires less samples to be tested, is simple to operate, takes a short time, and has high accuracy, and can be widely used in lymphocyte subgroup immune typing.
Description
技术领域technical field
本发明属于免疫学技术领域，特别涉及一种T淋巴细胞免疫分型的方法和试剂盒。The invention belongs to the technical field of immunology, and in particular relates to a method and a kit for immunotyping of T lymphocytes.
背景技术Background technique
淋巴细胞(lymphocyte)也称淋巴球，为白细胞中体积最小的一种，直径6-8微米；在人体约占白细胞数目的20％-30％，圆形细胞核，细胞质很少；由淋巴器官产生，是机体免疫应答功能的重要细胞成分，是一类具有免疫识别功能的细胞系。淋巴细胞是复杂的异质性细胞群体，根据其表型和功能特征可分为不同类别，如T细胞、B细胞、NK细胞等，这些细胞还可以进一步分为若干亚群。淋巴细胞及其亚群在免疫应答过程中相互协作、相互制约，共同完成对抗原物质的识别、应答和清除，从而维持机体内环境稳定。Lymphocytes, also known as lymphocytes, are the smallest type of white blood cells, with a diameter of 6-8 microns; they account for about 20%-30% of the number of white blood cells in the human body, with round nuclei and little cytoplasm; produced by lymphoid organs , is an important cell component of the body's immune response function, and is a type of cell line with immune recognition function. Lymphocytes are complex heterogeneous cell populations, which can be divided into different categories according to their phenotype and functional characteristics, such as T cells, B cells, NK cells, etc. These cells can be further divided into several subgroups. Lymphocytes and their subgroups cooperate and restrict each other in the process of immune response, and jointly complete the recognition, response and clearance of antigenic substances, so as to maintain the stability of the internal environment of the body.
早期研究根据淋巴细胞按其发生迁移、表面分子和功能的不同，大体上将淋巴细胞分为T细胞、B细胞和自然杀伤(NK)细胞。其中T细胞可分为细胞毒性T细胞(cytotoxic T lymphocyte，Tc)、辅助T细胞(helper T lymphocyte，Th)、记忆T细胞(memory T lymphocyte，Tm)和调节/抑制T细胞(regulatory/suppressor T lymphocyte)；B细胞可进一步分为记忆B细胞(memory B cell)、浆细胞(plasma cell)、初始B细胞(B cell)等亚类。Lymphocytes were roughly divided into T cells, B cells, and natural killer (NK) cells according to their migration, surface molecules, and functions in early studies. Among them, T cells can be divided into cytotoxic T cells (cytotoxic T lymphocyte, Tc), helper T cells (helper T lymphocyte, Th), memory T cells (memory T lymphocyte, Tm) and regulatory/suppressor T cells (regulatory/suppressor T cells). lymphocyte); B cells can be further divided into memory B cells (memory B cells), plasma cells (plasma cells), naive B cells ( B cell) and other subcategories.
随着科学研究的不断进步，科学家发现了更多的细胞表面标志，可以将各类淋巴细胞进行更加精细的细胞亚群分类。例如，可以将T细胞进一步划分为：TCRαβ+双阴性T细胞(double negative T lymphocyte，DNT)、γδT细胞、辅助初始T细胞、辅助耗竭T细胞、辅助中心记忆T细胞、辅助效应记忆T细胞、细胞毒性初始T细胞、细胞毒性耗竭T细胞、细胞毒性中心记忆T细胞、细胞毒性效应记忆T细胞。With the continuous progress of scientific research, scientists have discovered more cell surface markers, which can classify various lymphocytes into more refined cell subgroups. For example, T cells can be further divided into: TCRαβ + double negative T cells (double negative T lymphocyte, DNT), γδ T cells, help naive T cells, help exhausted T cells, help center memory T cells, help effector memory T cells, Cytotoxic naive T cells, cytotoxic exhausted T cells, cytotoxic central memory T cells, cytotoxic effector memory T cells.
血液样品中淋巴细胞的种类与机体的免疫系统性能有着重要关系。通过对血液样品中淋巴细胞进行免疫分型和定量分析，能够更加准确地评价机体的免疫功能，也能够更好地研究某些疾病的发生、发展以及治疗效果评价。目前，常用于血液样品中的淋巴细胞免疫分型和定量分析的方法主要有：免疫酶标法、普通淋巴细胞分类、较细淋巴细胞亚群检测。免疫酶标法只是针对某一种或一类特定的抗原或抗体发生特异反应，检测面窄、操作复杂、对待检测样品需求量大，费时费力。普通淋巴细胞分类，普遍应用于淋巴细胞检测，但只能将淋巴细胞分为T细胞、Tc细胞、Th细胞、B细胞和NK细胞，不能进一步对细胞进行分型。较细淋巴细胞亚群检测依赖于流式细胞术，采用多色流式的方法，对淋巴细胞进行较为详细的分型，除传统的T细胞、Tc细胞、Th细胞、B细胞和NK细胞外，还检测Tm细胞及细胞数量。但是，如果想进一步对细胞亚群进行免疫分型和定量分析的话，还需要借助于其他的细胞免疫分型方法，操作复杂，对待测样品的需求量大，并不适合实际应用。所以，还需要对淋巴细胞免疫分型和定量分析的方法进行研究，以求尽可能利用简单的方法将淋巴细胞进行较精细地免疫分型和定量分析。The type of lymphocytes in a blood sample has an important relationship with the performance of the body's immune system. By immunotyping and quantitative analysis of lymphocytes in blood samples, the immune function of the body can be more accurately evaluated, and the occurrence, development, and treatment effect evaluation of certain diseases can be better studied. At present, the methods commonly used for immunophenotyping and quantitative analysis of lymphocytes in blood samples mainly include: immunoenzyme labeling method, common lymphocyte classification, and detection of finer lymphocyte subsets. The immunoenzyme labeling method only reacts specifically to one or a certain type of specific antigen or antibody, which has a narrow detection range, complicated operation, and a large demand for samples to be tested, which is time-consuming and labor-intensive. Ordinary lymphocyte classification is commonly used in lymphocyte detection, but lymphocytes can only be divided into T cells, Tc cells, Th cells, B cells and NK cells, and cells cannot be further classified. The detection of finer lymphocyte subsets relies on flow cytometry. The multi-color flow cytometry method is used to carry out more detailed typing of lymphocytes, except for traditional T cells, Tc cells, Th cells, B cells and NK cells. , Tm cells and cell number are also detected. However, if one wants to further perform immunophenotyping and quantitative analysis of cell subpopulations, it is necessary to resort to other cellular immunotyping methods, which are complicated to operate and require a large number of samples to be tested, which is not suitable for practical application. Therefore, it is still necessary to study the methods of immunophenotyping and quantitative analysis of lymphocytes, in order to use as simple a method as possible to perform more precise immunophenotyping and quantitative analysis of lymphocytes.
发明内容Contents of the invention
有鉴于此，本发明的发明目的在于提供一种T淋巴细胞免疫分型的方法和试剂盒。该T淋巴细胞免疫分型的方法能够对T淋巴细胞进行更为全面的精细免疫分型和定量分析，效率高，且节约了待测样品的用量，更合适T淋巴细胞的免疫分型和定量分析。In view of this, the object of the present invention is to provide a method and kit for T lymphocyte immunophenotyping. The method for immune typing of T lymphocytes can perform more comprehensive fine immune typing and quantitative analysis of T lymphocytes, has high efficiency, saves the amount of samples to be tested, and is more suitable for immune typing and quantification of T lymphocytes analyze.
为了实现本发明的发明目的，本发明采用如下的技术方案：In order to realize the purpose of the invention of the present invention, the present invention adopts following technical scheme:
本发明提供了一种T淋巴细胞免疫分型的方法，包括：The invention provides a method for T lymphocyte immunophenotyping, comprising:
取不同的荧光标记的抗体，与待检测样品混合，孵育后，经流式细胞术检测，得检测数据，分析所述检测数据；Take different fluorescently-labeled antibodies, mix them with the samples to be detected, and after incubation, detect by flow cytometry to obtain detection data, and analyze the detection data;
该抗体包括：This antibody includes:
抗TCRαβ的抗体、抗TCRγδ的抗体、抗CD3的抗体、抗CD4的抗体、抗CD8的抗体、抗CD27的抗体和抗CD45RA抗体；Anti-TCRαβ antibody, anti-TCRγδ antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody, anti-CD27 antibody and anti-CD45RA antibody;
该分析的方法包括：Methods for this analysis include:
细胞表面标志CD3+TCRαβ+CD4-CD8-代表TCRαβ+双阴性T细胞；Cell surface markers CD3 + TCRαβ + CD4 - CD8 - represent TCRαβ + double negative T cells;
细胞表面标志CD3+TCRγδ+代表γδT细胞；Cell surface marker CD3 + TCRγδ + represents γδ T cells;
细胞表面标志CD3+CD4+CD45RA+CD27+代表辅助初始T细胞；Cell surface markers CD3 + CD4 + CD45RA + CD27 + represent helper naive T cells;
细胞表面标志CD3+CD4+CD45RA+CD27-代表辅助耗竭T细胞；Cell surface markers CD3 + CD4 + CD45RA + CD27 - represent helper exhausted T cells;
细胞表面标志CD3+CD4+CD45RA-CD27+代表辅助中心记忆T细胞；Cell surface markers CD3 + CD4 + CD45RA - CD27 + represent helper center memory T cells;
细胞表面标志CD3+CD4+CD45RA-CD27-代表辅助效应记忆T细胞；Cell surface markers CD3 + CD4 + CD45RA - CD27 - represent helper effector memory T cells;
细胞表面标志CD3+CD8+CD45RA+CD27+代表细胞毒性初始T细胞；Cell surface markers CD3 + CD8 + CD45RA + CD27 + represent cytotoxic naive T cells;
细胞表面标志CD3+CD8+CD45RA+CD27-代表细胞毒性耗竭T细胞；Cell surface markers CD3 + CD8 + CD45RA + CD27 - represent cytotoxic exhausted T cells;
细胞表面标志CD8+CD45RA-CD27+代表细胞毒性中心记忆T细胞；Cell surface markers CD8 + CD45RA - CD27 + represent cytotoxic central memory T cells;
细胞表面标志CD3+CD8+CD45RA-CD27-代表细胞毒性效应记忆T细胞。Cell surface markers CD3 + CD8 + CD45RA - CD27 - represent cytotoxic effector memory T cells.
在本发明中，流式细胞术中所用到的抗体可以为与待测样品同源的抗体或也可以为与待测样品非同源的抗体，只要该抗体能够与待测样品中细胞表面标志物产生抗原-抗体特异性结合反应即可。In the present invention, the antibody used in flow cytometry can be an antibody homologous to the sample to be tested or an antibody non-homologous to the sample to be tested, as long as the antibody can bind to the cell surface markers in the sample to be tested The substance can produce antigen-antibody specific binding reaction.
在本发明中，“+”代表阳性，即表示该抗原在细胞表面有表达；In the present invention, "+" represents positive, which means that the antigen is expressed on the cell surface;
“++”代表强阳性，即表示该抗原在细胞表面高表达；"++" stands for strong positive, which means that the antigen is highly expressed on the cell surface;
“-”代表阴性，即表示该抗原在细胞表面不表达。"-" means negative, which means that the antigen is not expressed on the cell surface.
淋巴细胞的表面标志是指存在于淋巴细胞表面的膜分子，它们是淋巴细胞识别抗原、与其他免疫细胞相互作用以及接受信号刺激并产生应答的物质基础，也是鉴别和分离淋巴细胞的重要依据。在淋巴细胞的分化发育过程中，淋巴样干细胞进一步分化成各个细胞亚群，赋予了各个细胞亚群特定的表面标志。有些表面标志是淋巴细胞共有的，有些表面标志是某一类或某几类淋巴细胞特有的，所以在进行淋巴细胞免疫分型时，可以有众多种组合。但是并不是每种组合都能够实现淋巴细胞的免疫分型，本发明通过大量的创造性劳动发现：针对T细胞，细胞表面标志为CD3+TCRαβ+CD4-CD8-时，可对TCRαβ+双阴性T细胞进行免疫分型；细胞表面标志为CD3+TCRγδ+时，可对γδT细胞进行免疫分型；细胞表面标志为CD3+CD4+CD45RA+CD27+时，可对辅助初始T细胞进行免疫分型；细胞表面标志为CD3+CD4+CD45RA+CD27-时，可以对辅助耗竭T细胞进行免疫分型；细胞表面标志为CD3+CD4+CD45RA-CD27+时，可以对辅助中心记忆T细胞进行免疫分型；细胞表面标志为CD3+CD4+CD45RA-CD27-时，可以对辅助效应记忆T细胞进行免疫分型；细胞表面标志为CD3+CD8+CD45RA+CD27+时，可以对细胞毒性初始T细胞进行免疫分型；细胞表面标志为CD3+CD8+CD45RA+CD27-时，可以对细胞毒性耗竭T细胞免疫分型；细胞表面标志为CD8+CD45RA-CD27+时，可以对细胞毒性中心记忆T细胞免疫分型；细胞表面标志为CD3+CD8+CD45RA-CD27-时，可以对细胞毒性效应记忆T细胞免疫分型。本发明根据对各个细胞所对应的细胞表面标志，设计获得了最优的细胞表面标志的抗体组合，通过流式细胞术，对T淋巴细胞进行了更为全面的免疫分型。本发明提供的T淋巴细胞免疫分型的方法所需待测样品少、操作简单、准确性高，能够用于T淋巴细胞的免疫分型。The surface markers of lymphocytes refer to the membrane molecules present on the surface of lymphocytes, which are the material basis for lymphocytes to recognize antigens, interact with other immune cells, receive signal stimulation and respond, and are also an important basis for identifying and separating lymphocytes. During the differentiation and development of lymphocytes, lymphoid stem cells further differentiate into various cell subgroups, endowing each cell subgroup with specific surface markers. Some surface markers are shared by lymphocytes, and some surface markers are unique to a certain type or several types of lymphocytes, so there can be many combinations when performing lymphocyte immunophenotyping. However, not every combination can realize the immunophenotyping of lymphocytes. The present invention has discovered through a lot of creative work: for T cells, when the cell surface marker is CD3 + TCRαβ + CD4 - CD8 - , TCRαβ + double-negative T Immunotyping of cells; when the cell surface markers are CD3 + TCRγδ + , γδT cells can be immunotyped; when the cell surface markers are CD3 + CD4 + CD45RA + CD27 + , helper naive T cells can be immunotyped; When the cell surface markers are CD3 + CD4 + CD45RA + CD27 - , helper exhausted T cells can be immunotyped; when the cell surface markers are CD3 + CD4 + CD45RA - CD27 + , helper central memory T cells can be immunotyped ; When the cell surface markers are CD3 + CD4 + CD45RA - CD27 - , helper effector memory T cells can be immunotyped; when the cell surface markers are CD3 + CD8 + CD45RA + CD27 + , cytotoxic naive T cells can be immunized When the cell surface markers are CD3 + CD8 + CD45RA + CD27 - , cytotoxic exhausted T cells can be immunotyped; when the cell surface markers are CD8 + CD45RA - CD27 + , cytotoxic central memory T cells can be immunotyped type; when the cell surface markers are CD3 + CD8 + CD45RA - CD27 - , memory T cells can be immunotyped for cytotoxic effects. According to the cell surface markers corresponding to each cell, the present invention designs an antibody combination with optimal cell surface markers, and conducts a more comprehensive immune typing of T lymphocytes through flow cytometry. The T lymphocyte immune typing method provided by the invention requires less samples to be tested, is simple to operate and has high accuracy, and can be used for T lymphocyte immune typing.
优选地，本发明提供T淋巴细胞免疫分型的方法中，还设置对照组和同型对照组。设置对照组的意义在于排除无关变量的影响，增加实验结果的可信度和说服力；设置同型对照组的意义在于区别抗体染色过程中相同亚型所造成的背景信号影响。Preferably, in the method for immunophenotyping of T lymphocytes provided by the present invention, a control group and an isotype control group are also set. The significance of setting the control group is to exclude the influence of irrelevant variables, increasing the credibility and persuasiveness of the experimental results; the significance of setting the isotype control group is to distinguish the background signal effect caused by the same subtype during antibody staining.
在本发明的一些实施例中，本发明提供的T淋巴细胞免疫分型的方法中，对照组所用不同的荧光标记的抗体具体为：In some embodiments of the present invention, in the method for immunophenotyping T lymphocytes provided by the present invention, the different fluorescently-labeled antibodies used in the control group are specifically:
抗CD3的抗体、抗CD4的抗体、抗CD8的抗体。Anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody.
在本发明的一些实施例中，本发明提供的T淋巴细胞免疫分型的方法中，抗TCRαβ的抗体的荧光标记为PE。In some embodiments of the present invention, in the method for immunophenotyping T lymphocytes provided by the present invention, the fluorescent label of the antibody against TCRαβ is PE.
在本发明的另外一些实施例中，本发明提供的T淋巴细胞免疫分型的方法中，抗TCRγδ的抗体的荧光标记为BV421或FITC。在本发明的另外一些实施例中，本发明提供的T淋巴细胞免疫分型的方法中，抗TCRγδ的抗体的荧光标记为FITC。In some other embodiments of the present invention, in the method for immunophenotyping of T lymphocytes provided by the present invention, the fluorescent label of the antibody against TCRγδ is BV421 or FITC. In some other embodiments of the present invention, in the method for immunophenotyping T lymphocytes provided by the present invention, the fluorescent label of the antibody against TCRγδ is FITC.
在本发明的另外一些实施例中，本发明提供的T淋巴细胞免疫分型的方法中，抗CD3的抗体的荧光标记为Percp-cy5.5或Pacific blue。在本发明的另外一些实施例中，本发明提供的T淋巴细胞免疫分型的方法中，抗CD3的抗体的荧光标记为Pacific blue。In some other embodiments of the present invention, in the method for immunophenotyping of T lymphocytes provided by the present invention, the fluorescent label of the anti-CD3 antibody is Percp-cy5.5 or Pacific blue. In some other embodiments of the present invention, in the method for immunophenotyping of T lymphocytes provided by the present invention, the fluorescent label of the anti-CD3 antibody is Pacific blue.
在本发明的另外一些实施例中，本发明提供的T淋巴细胞免疫分型的方法中，抗CD4的抗体的荧光标记为FITC或APC-Cy7。在本发明的另外一些实施例中，本发明提供的T淋巴细胞免疫分型的方法中，抗CD4的抗体的荧光标记为APC-Cy7。In some other embodiments of the present invention, in the method for immunophenotyping T lymphocytes provided by the present invention, the fluorescent label of the anti-CD4 antibody is FITC or APC-Cy7. In some other embodiments of the present invention, in the method for immunophenotyping T lymphocytes provided by the present invention, the fluorescent label of the anti-CD4 antibody is APC-Cy7.
在本发明的另外一些实施例中，本发明提供的T淋巴细胞免疫分型的方法中，抗CD8的抗体的荧光标记为BV510。In some other embodiments of the present invention, in the method for immunophenotyping T lymphocytes provided by the present invention, the fluorescent label of the anti-CD8 antibody is BV510.
在本发明的另外一些实施例中，本发明提供的T淋巴细胞免疫分型的方法中，抗CD27的抗体的荧光标记为APC。In some other embodiments of the present invention, in the method for immunotyping T lymphocytes provided by the present invention, the fluorescent label of the anti-CD27 antibody is APC.
在本发明的另外一些实施例中，本发明提供的T淋巴细胞免疫分型的方法中，抗CD45RA的抗体的荧光标记为PE-Cy7。In some other embodiments of the present invention, in the method for immunophenotyping T lymphocytes provided by the present invention, the fluorescent label of the anti-CD45RA antibody is PE-Cy7.
在本发明的另外一些实施例中，本发明提供的T淋巴细胞免疫分型的方法中，同型对照所用的抗体具体为：PE标记的同型抗体、FITC标记的同型抗体、APC标记的同型抗体、PE-Cy7标记的同型抗体。In some other embodiments of the present invention, in the method for immunotyping of T lymphocytes provided by the present invention, the antibodies used in the isotype control are specifically: PE-labeled isotype antibodies, FITC-labeled isotype antibodies, APC-labeled isotype antibodies, PE-Cy7 labeled isotype antibody.
在本发明的另外一些实施例中，本发明提供的T淋巴细胞免疫分型的方法，具体为：In some other embodiments of the present invention, the method for immunophenotyping of T lymphocytes provided by the present invention is specifically:
取荧光标记的抗TCRαβ的抗体、荧光标记的抗TCRγδ的抗体，与待测样品混合，室温(即20℃～25℃)条件下，孵育25min～35min，得第一产品；Take the fluorescently labeled anti-TCRαβ antibody and the fluorescently labeled anti-TCRγδ antibody, mix with the sample to be tested, and incubate at room temperature (20°C-25°C) for 25min-35min to obtain the first product;
取荧光标记的抗CD3的抗体、荧光标记的抗CD4的抗体、荧光标记的抗CD8的抗体、荧光标记的抗CD27的抗体、荧光标记的抗CD45RA的抗体，与第一产品混合，室温(即20℃～25℃)条件下，孵育25min～35min，得第二产品；Take fluorescently-labeled anti-CD3 antibody, fluorescently-labeled anti-CD4 antibody, fluorescently-labeled anti-CD8 antibody, fluorescently-labeled anti-CD27 antibody, fluorescently-labeled anti-CD45RA antibody, mix with the first product, room temperature (i.e. (20°C～25°C) and incubate for 25min～35min to obtain the second product;
取第二产品与红细胞裂解液混合，并将所得混合液置于36.5℃～37.5℃的中水中水浴9min～11min，洗涤，流式上机检测，得检测数据，分析检测数据；Take the second product and mix it with erythrocyte lysate, place the resulting mixture in a water bath at 36.5°C to 37.5°C for 9 minutes to 11 minutes, wash it, and perform flow cytometry detection on the machine to obtain test data and analyze the test data;
分析的方法包括：Methods of analysis include:
细胞表面标志CD3+TCRαβ+CD4-CD8-代表TCRαβ+双阴性T细胞；Cell surface markers CD3 + TCRαβ + CD4 - CD8 - represent TCRαβ + double negative T cells;
细胞表面标志CD3+TCRγδ+代表γδT细胞；Cell surface marker CD3 + TCRγδ + represents γδ T cells;
细胞表面标志CD3+CD4+CD45RA+CD27+代表辅助初始T细胞；Cell surface markers CD3 + CD4 + CD45RA + CD27 + represent helper naive T cells;
细胞表面标志CD3+CD4+CD45RA+CD27-代表辅助耗竭T细胞；Cell surface markers CD3 + CD4 + CD45RA + CD27 - represent helper exhausted T cells;
细胞表面标志CD3+CD4+CD45RA-CD27+代表辅助中心记忆T细胞；Cell surface markers CD3 + CD4 + CD45RA - CD27 + represent helper center memory T cells;
细胞表面标志CD3+CD4+CD45RA-CD27-代表辅助效应记忆T细胞；Cell surface markers CD3 + CD4 + CD45RA - CD27 - represent helper effector memory T cells;
细胞表面标志CD3+CD8+CD45RA+CD27+代表细胞毒性初始T细胞；Cell surface markers CD3 + CD8 + CD45RA + CD27 + represent cytotoxic naive T cells;
细胞表面标志CD3+CD8+CD45RA+CD27-代表细胞毒性耗竭T细胞；Cell surface markers CD3 + CD8 + CD45RA + CD27 - represent cytotoxic exhausted T cells;
细胞表面标志CD8+CD45RA-CD27+代表细胞毒性中心记忆T细胞；Cell surface markers CD8 + CD45RA - CD27 + represent cytotoxic central memory T cells;
细胞表面标志CD3+CD8+CD45RA-CD27-代表细胞毒性效应记忆T细胞。Cell surface markers CD3 + CD8 + CD45RA - CD27 - represent cytotoxic effector memory T cells.
在本发明中，本发明提供的方法中，各个荧光标记的抗体中的荧光标记物不受本发明的限制，本领域技术人员可以根据实际情况选择合适的荧光标记物，以及对应的同型对照。In the present invention, in the method provided by the present invention, the fluorescent markers in each fluorescently labeled antibody are not limited by the present invention, and those skilled in the art can select appropriate fluorescent markers and corresponding isotype controls according to actual conditions.
优选地，本发明提供的T淋巴细胞免疫分型的方法中，还包括统计待测样品中T淋巴细胞数目的步骤。Preferably, the method for immunophenotyping of T lymphocytes provided by the present invention further includes a step of counting the number of T lymphocytes in the sample to be tested.
在本发明的一些实施例中，本发明提供的T淋巴细胞免疫分型的方法中，检测样品中T淋巴细胞数目的步骤包括：In some embodiments of the present invention, in the method for immunophenotyping of T lymphocytes provided by the present invention, the step of detecting the number of T lymphocytes in the sample includes:
检测待测样品中淋巴细胞的总数；Detect the total number of lymphocytes in the sample to be tested;
检测待测样品中T淋巴细胞占所述淋巴细胞的百分比；Detecting the percentage of T lymphocytes accounting for the lymphocytes in the sample to be tested;
通过计算，即得待测样品中T淋巴细胞的数目。By calculation, the number of T lymphocytes in the sample to be tested can be obtained.
本发明提供的方法中，检测样品中T淋巴细胞数目中，淋巴细胞总数乘以其中T淋巴细胞的百分比，即得T淋巴细胞的数目。在本发明中通过对待测样品各个T细胞亚群进行免疫分型和相对数统计，再结合T淋巴细胞的绝对数，即可获得每个T细胞亚群的细胞绝对数。In the method provided by the present invention, in detecting the number of T lymphocytes in a sample, the total number of lymphocytes is multiplied by the percentage of T lymphocytes therein to obtain the number of T lymphocytes. In the present invention, the absolute number of cells of each T cell subset can be obtained by performing immunophenotyping and relative statistics of each T cell subset in the sample to be tested, combined with the absolute number of T lymphocytes.
在本发明的一些实施例中，本发明提供的方法中，检测样品中T淋巴细胞的百分比的步骤，包括：In some embodiments of the present invention, in the method provided by the present invention, the step of detecting the percentage of T lymphocytes in the sample includes:
取不同的荧光标记的抗体，与新的所述待测样品混合，孵育后，经流式细胞术检测，得检测数据，分析所述检测数据；Take different fluorescently labeled antibodies, mix them with the new sample to be tested, and after incubation, perform flow cytometry detection to obtain detection data, and analyze the detection data;
检测待测样品中T淋巴细胞的百分比中所用的抗体包括：Antibodies used in detecting the percentage of T lymphocytes in the test sample include:
抗CD45的抗体、抗CD3的抗体、抗CD16的抗体、抗CD56的抗体、抗CD19的抗体；Anti-CD45 antibody, anti-CD3 antibody, anti-CD16 antibody, anti-CD56 antibody, anti-CD19 antibody;
检测待测样品中T淋巴细胞的百分比中所用的抗CD3的抗体与T淋巴细胞免疫分型中所述抗CD3的抗体，各自独立地带有荧光标记。The anti-CD3 antibody used in detecting the percentage of T lymphocytes in the sample to be tested and the anti-CD3 antibody described in T lymphocyte immunophenotyping are each independently labeled with a fluorescent light.
在本发明的一些实施例中，本发明提供的方法中，检测样品中T淋巴细胞的百分比的步骤中，对照组所用抗体为：荧光标记的抗CD45抗体、荧光标记的抗CD3抗体、荧光标记的CD4抗体、荧光标记的CD8抗体。In some embodiments of the present invention, in the method provided by the present invention, in the step of detecting the percentage of T lymphocytes in the sample, the antibodies used in the control group are: fluorescently labeled anti-CD45 antibody, fluorescently labeled anti-CD3 antibody, fluorescently labeled CD4 antibody, fluorescently labeled CD8 antibody.
在本发明的一些实施例中，本发明提供的方法中，检测样品中T淋巴细胞的百分比的步骤中，抗CD45的抗体荧光标记为Percp。In some embodiments of the present invention, in the method provided by the present invention, in the step of detecting the percentage of T lymphocytes in the sample, the anti-CD45 antibody is fluorescently labeled as Percp.
在本发明的一些实施例中，本发明提供的方法中，检测样品中T淋巴细胞的百分比的步骤中，抗CD3的抗体的荧光标记为FITC。In some embodiments of the present invention, in the method provided by the present invention, in the step of detecting the percentage of T lymphocytes in the sample, the fluorescent label of the anti-CD3 antibody is FITC.
在本发明的一些实施例中，本发明提供的方法中，检测样品中T淋巴细胞的百分比的步骤中，抗CD16的抗体的荧光标记为PE。In some embodiments of the present invention, in the method provided by the present invention, in the step of detecting the percentage of T lymphocytes in the sample, the fluorescent label of the anti-CD16 antibody is PE.
在本发明的一些实施例中，本发明提供的方法中，检测样品中T淋巴细胞的百分比的步骤中，抗CD56的抗体的荧光标记为PE。In some embodiments of the present invention, in the method provided by the present invention, in the step of detecting the percentage of T lymphocytes in the sample, the fluorescent label of the anti-CD56 antibody is PE.
在本发明的一些实施例中，本发明提供的方法中，检测样品中T淋巴细胞的百分比的步骤中，抗CD19的抗体的荧光标记为APC。In some embodiments of the present invention, in the method provided by the present invention, in the step of detecting the percentage of T lymphocytes in the sample, the fluorescent label of the anti-CD19 antibody is APC.
在本发明的一些实施例中，本发明提供的方法中，检测样品中T淋巴细胞的百分比的步骤中，抗CD4的抗体的荧光标记为APC。In some embodiments of the present invention, in the method provided by the present invention, in the step of detecting the percentage of T lymphocytes in the sample, the fluorescent label of the anti-CD4 antibody is APC.
在本发明的一些实施例中，本发明提供的方法中，检测样品中T淋巴细胞的百分比的步骤中，抗CD8的抗体的荧光标记为PE。In some embodiments of the present invention, in the method provided by the present invention, in the step of detecting the percentage of T lymphocytes in the sample, the fluorescent label of the anti-CD8 antibody is PE.
在本发明中，本发明提供的方法中，检测样品中T淋巴细胞的百分比的步骤为常规的淋巴细胞免疫分型和定量分析方法，该方法不受本发明的限制，本领域技术人员可以根据实际情况选择测定待测样品中T淋巴细胞的百分比的方法。In the present invention, in the method provided by the present invention, the step of detecting the percentage of T lymphocytes in the sample is a routine lymphocyte immunophenotyping and quantitative analysis method, which is not limited by the present invention, and those skilled in the art can according to The actual situation chooses the method of determining the percentage of T lymphocytes in the sample to be tested.
在本发明的另外一些实施例中，本发明提供的方法中，检测待测样品中淋巴细胞的总数的方法为采用血细胞计数仪进行计数。在本发明中，本发明提供的方法中，检测待测样品中淋巴细胞的总数的方法为常规的方法，该方法不受本发明的限制，本领域技术人员可以根据实际情况选择检测待测样品中淋巴细胞的总数的方法。In some other embodiments of the present invention, in the method provided by the present invention, the method of detecting the total number of lymphocytes in the sample to be tested is counting by using a blood cell counter. In the present invention, in the method provided by the present invention, the method for detecting the total number of lymphocytes in the sample to be tested is a conventional method, which is not limited by the present invention, and those skilled in the art can choose to detect the sample to be tested according to the actual situation. The total number of lymphocytes in the method.
本发明还提供了一种淋巴细胞免疫分型的方法，其包括本发明提供的T淋巴细胞免疫分型的步骤；The present invention also provides a method for immunophenotyping of lymphocytes, which includes the step of immunophenotyping of T lymphocytes provided by the present invention;
该T淋巴细胞免疫分型的方法包括：The method of immunophenotyping of T lymphocytes includes:
取不同的荧光标记的抗体，与待检测样品混合，孵育后，经流式细胞术检测，得检测数据，分析所述检测数据；Take different fluorescently-labeled antibodies, mix them with the samples to be detected, and after incubation, detect by flow cytometry to obtain detection data, and analyze the detection data;
该抗体包括：This antibody includes:
抗TCRαβ的抗体、抗TCRγδ的抗体、抗CD3的抗体、抗CD4的抗体、抗CD8的抗体、抗CD27的抗体和抗CD45RA抗体；Anti-TCRαβ antibody, anti-TCRγδ antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody, anti-CD27 antibody and anti-CD45RA antibody;
该分析的方法包括：Methods for this analysis include:
细胞表面标志CD3+TCRαβ+CD4-CD8-代表TCRαβ+双阴性T细胞；Cell surface markers CD3 + TCRαβ + CD4 - CD8 - represent TCRαβ + double negative T cells;
细胞表面标志CD3+TCRγδ+代表γδT细胞；Cell surface marker CD3 + TCRγδ + represents γδ T cells;
细胞表面标志CD3+CD4+CD45RA+CD27+代表辅助初始T细胞；Cell surface markers CD3 + CD4 + CD45RA + CD27 + represent helper naive T cells;
细胞表面标志CD3+CD4+CD45RA+CD27-代表辅助耗竭T细胞；Cell surface markers CD3 + CD4 + CD45RA + CD27 - represent helper exhausted T cells;
细胞表面标志CD3+CD4+CD45RA-CD27+代表辅助中心记忆T细胞；Cell surface markers CD3 + CD4 + CD45RA - CD27 + represent helper center memory T cells;
细胞表面标志CD3+CD4+CD45RA-CD27-代表辅助效应记忆T细胞；Cell surface markers CD3 + CD4 + CD45RA - CD27 - represent helper effector memory T cells;
细胞表面标志CD3+CD8+CD45RA+CD27+代表细胞毒性初始T细胞；Cell surface markers CD3 + CD8 + CD45RA + CD27 + represent cytotoxic naive T cells;
细胞表面标志CD3+CD8+CD45RA+CD27-代表细胞毒性耗竭T细胞；Cell surface markers CD3 + CD8 + CD45RA + CD27 - represent cytotoxic exhausted T cells;
细胞表面标志CD8+CD45RA-CD27+代表细胞毒性中心记忆T细胞；Cell surface markers CD8 + CD45RA - CD27 + represent cytotoxic central memory T cells;
细胞表面标志CD3+CD8+CD45RA-CD27-代表细胞毒性效应记忆T细胞。Cell surface markers CD3 + CD8 + CD45RA - CD27 - represent cytotoxic effector memory T cells.
本发明还提供了一种用于T淋巴细胞免疫分型的试剂盒，包括如下不同的荧光标记的抗体：The present invention also provides a kit for immunophenotyping of T lymphocytes, including the following different fluorescently labeled antibodies:
抗TCRαβ的抗体、抗TCRγδ的抗体、抗CD3的抗体、抗CD4的抗体、抗CD8的抗体、抗CD27的抗体和抗CD45RA抗体。Anti-TCRαβ antibody, anti-TCRγδ antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody, anti-CD27 antibody and anti-CD45RA antibody.
在本发明中，本发明提供的用于T淋巴细胞免疫分型的试剂盒中，荧光标记的抗体，可以是荧光标记物与抗体单独放置，在使用时将两者偶联获得荧光标记的抗体；也可以是荧光标记的抗体，在使用时，直接使用即可。In the present invention, in the kit for immunophenotyping of T lymphocytes provided by the present invention, the fluorescently labeled antibody can be placed separately with the fluorescent label and the antibody, and the two are coupled to obtain the fluorescently labeled antibody during use. ; It can also be a fluorescently labeled antibody, which can be used directly when used.
在本发明的一些实施例中，本发明提供的用于T淋巴细胞免疫分型的试剂盒包括荧光标记物和抗体；In some embodiments of the present invention, the kit for immunophenotyping of T lymphocytes provided by the present invention includes fluorescent markers and antibodies;
该抗体包括：This antibody includes:
抗TCRαβ的抗体、抗TCRγδ的抗体、抗CD3的抗体、抗CD4的抗体、抗CD8的抗体、抗CD27的抗体和抗CD45RA抗体。Anti-TCRαβ antibody, anti-TCRγδ antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody, anti-CD27 antibody and anti-CD45RA antibody.
在本发明一些实施例中，本发明提供的用于T淋巴细胞免疫分型的试剂盒中，抗TCRαβ的抗体的荧光标记为PE。In some embodiments of the present invention, in the kit for immunophenotyping of T lymphocytes provided by the present invention, the fluorescent label of the antibody against TCRαβ is PE.
在本发明的另外一些实施例中，本发明提供的用于T淋巴细胞免疫分型的试剂盒中，抗TCRγδ的抗体的荧光标记为BV421或FITC。在本发明的另外一些实施例中，本发明提供的用于T淋巴细胞免疫分型的试剂盒中，抗TCRγδ的抗体的荧光标记为FITC。In some other embodiments of the present invention, in the kit for immunophenotyping of T lymphocytes provided by the present invention, the fluorescent label of the anti-TCRγδ antibody is BV421 or FITC. In some other embodiments of the present invention, in the kit for immunophenotyping of T lymphocytes provided by the present invention, the fluorescent label of the anti-TCRγδ antibody is FITC.
在本发明的另外一些实施例中，本发明提供的用于T淋巴细胞免疫分型的试剂盒中，抗CD3的抗体的荧光标记为Percp-cy5.5或Pacific blue。在本发明的另外一些实施例中，本发明提供的用于T淋巴细胞免疫分型的试剂盒中，抗CD3的抗体的荧光标记为Pacific blue。In some other embodiments of the present invention, in the kit for immunophenotyping of T lymphocytes provided by the present invention, the fluorescent label of the anti-CD3 antibody is Percp-cy5.5 or Pacific blue. In some other embodiments of the present invention, in the kit for immunophenotyping of T lymphocytes provided by the present invention, the fluorescent label of the anti-CD3 antibody is Pacific blue.
在本发明的另外一些实施例中，本发明提供的用于T淋巴细胞免疫分型的试剂盒中，抗CD4的抗体的荧光标记为FITC或APC-Cy7。在本发明的另外一些实施例中，本发明提供的用于T淋巴细胞免疫分型的试剂盒中，抗CD4的抗体的荧光标记为APC-Cy7。In some other embodiments of the present invention, in the kit for immunophenotyping of T lymphocytes provided by the present invention, the fluorescent label of the anti-CD4 antibody is FITC or APC-Cy7. In some other embodiments of the present invention, in the kit for immunophenotyping of T lymphocytes provided by the present invention, the fluorescent label of the anti-CD4 antibody is APC-Cy7.
在本发明的另外一些实施例中，本发明提供的用于T淋巴细胞免疫分型的试剂盒中，抗CD8的抗体的荧光标记为BV510。In some other embodiments of the present invention, in the kit for immunophenotyping of T lymphocytes provided by the present invention, the fluorescent label of the anti-CD8 antibody is BV510.
在本发明的另外一些实施例中，本发明提供的用于T淋巴细胞免疫分型的试剂盒中，抗CD27的抗体的荧光标记为APC。In some other embodiments of the present invention, in the kit for immunophenotyping of T lymphocytes provided by the present invention, the fluorescent label of the anti-CD27 antibody is APC.
在本发明的另外一些实施例中，本发明提供的用于T淋巴细胞免疫分型的试剂盒中，抗CD45RA的抗体的荧光标记为PE-Cy7。In some other embodiments of the present invention, in the kit for immunophenotyping of T lymphocytes provided by the present invention, the fluorescent label of the anti-CD45RA antibody is PE-Cy7.
在本发明的另外一些实施例中，本发明提供的用于T淋巴细胞免疫分型的试剂盒中的抗体，还包括同型对照抗体，具体为：PE标记的同型抗体、BV421标记的同型抗体、APC标记的同型抗体、PE-Cy7标记的同型抗体。In some other embodiments of the present invention, the antibodies in the kit for immunophenotyping of T lymphocytes provided by the present invention also include isotype control antibodies, specifically: PE-labeled isotype antibodies, BV421-labeled isotype antibodies, APC-labeled isotype antibody, PE-Cy7-labeled isotype antibody.
在本发明中，本发明提供的试剂盒中，各个荧光标记的抗体中的荧光标记物不受本发明的限制，本领域技术人员可以根据实际情况选择合适的荧光标记物，以及对应的同型对照。In the present invention, in the kit provided by the present invention, the fluorescent markers in each fluorescently labeled antibody are not limited by the present invention, and those skilled in the art can select appropriate fluorescent markers and corresponding isotype controls according to actual conditions. .
本发明还提供了一种用于淋巴细胞免疫分型的试剂盒，其包括如下不同的荧光标记的抗体：The present invention also provides a kit for immunophenotyping of lymphocytes, which includes the following different fluorescently labeled antibodies:
抗TCRαβ的抗体、抗TCRγδ的抗体、抗CD3的抗体、抗CD4的抗体、抗CD8的抗体、抗CD27的抗体和抗CD45RA抗体。Anti-TCRαβ antibody, anti-TCRγδ antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody, anti-CD27 antibody and anti-CD45RA antibody.
在本发明一些实施例中，本发明提供的用于淋巴细胞免疫分型的试剂盒中，抗TCRαβ的抗体的荧光标记为PE。In some embodiments of the present invention, in the kit for immunophenotyping of lymphocytes provided by the present invention, the fluorescent label of the antibody against TCRαβ is PE.
在本发明的另外一些实施例中，本发明提供的用于淋巴细胞免疫分型的试剂盒中，抗TCRγδ的抗体的荧光标记为BV421或FITC。在本发明的另外一些实施例中，本发明提供的用于淋巴细胞免疫分型的试剂盒中，抗TCRγδ的抗体的荧光标记为FITC。In some other embodiments of the present invention, in the kit for immunophenotyping of lymphocytes provided by the present invention, the fluorescent label of the anti-TCRγδ antibody is BV421 or FITC. In some other embodiments of the present invention, in the kit for immunophenotyping of lymphocytes provided by the present invention, the fluorescent label of the antibody against TCRγδ is FITC.
在本发明的另外一些实施例中，本发明提供的用于淋巴细胞免疫分型的试剂盒中，抗CD3的抗体的荧光标记为Percp-cy5.5或Pacific blue。在本发明的另外一些实施例中，本发明提供的用于淋巴细胞免疫分型的试剂盒中，抗CD3的抗体的荧光标记为Pacific blue。In some other embodiments of the present invention, in the kit for immunophenotyping of lymphocytes provided by the present invention, the fluorescent label of the anti-CD3 antibody is Percp-cy5.5 or Pacific blue. In some other embodiments of the present invention, in the kit for immunophenotyping of lymphocytes provided by the present invention, the fluorescent label of the anti-CD3 antibody is Pacific blue.
在本发明的另外一些实施例中，本发明提供的用于淋巴细胞免疫分型的试剂盒中，抗CD4的抗体的荧光标记为FITC或APC-Cy7。在本发明的另外一些实施例中，本发明提供的用于淋巴细胞免疫分型的试剂盒中，抗CD4的抗体的荧光标记为APC-Cy7。In some other embodiments of the present invention, in the kit for immunophenotyping of lymphocytes provided by the present invention, the fluorescent label of the anti-CD4 antibody is FITC or APC-Cy7. In some other embodiments of the present invention, in the kit for immunophenotyping of lymphocytes provided by the present invention, the fluorescent label of the anti-CD4 antibody is APC-Cy7.
在本发明的另外一些实施例中，本发明提供的用于淋巴细胞免疫分型的试剂盒中，抗CD8的抗体的荧光标记为BV510。In some other embodiments of the present invention, in the kit for immunophenotyping of lymphocytes provided by the present invention, the fluorescent label of the anti-CD8 antibody is BV510.
在本发明的另外一些实施例中，本发明提供的用于淋巴细胞免疫分型的试剂盒中，抗CD27的抗体的荧光标记为APC。In some other embodiments of the present invention, in the kit for immunophenotyping of lymphocytes provided by the present invention, the fluorescent label of the anti-CD27 antibody is APC.
在本发明的另外一些实施例中，本发明提供的用于淋巴细胞免疫分型的试剂盒中，抗CD45RA的抗体的荧光标记为PE-Cy7。In some other embodiments of the present invention, in the kit for immunophenotyping of lymphocytes provided by the present invention, the fluorescent label of the anti-CD45RA antibody is PE-Cy7.
在本发明中，流式细胞术检测过程中，通过目标细胞分群是否清楚、目标细胞荧光强度等表示准确性的高低；目标细胞分群清楚、目标细胞荧光强度准确高，则表示准确性高。In the present invention, during the detection process of flow cytometry, whether the target cell grouping is clear or not, and the fluorescence intensity of the target cells are used to indicate the accuracy; if the target cell grouping is clear, and the target cell fluorescence intensity is high, it indicates high accuracy.
本发明提供了一种T淋巴细胞免疫分型的方法和试剂盒。本发明提供的T淋巴细胞免疫分型的方法，包括：取不同的荧光标记的抗体，与待检测样品混合，孵育后，经流式细胞术检测，得检测数据，分析所得检测数据；该抗体包括：抗TCRαβ的抗体、抗TCRγδ的抗体、抗CD3的抗体、抗CD4的抗体、抗CD8的抗体、抗CD27的抗体和抗CD45RA抗体；该分析的方法包括：细胞表面标志CD3+TCRαβ+CD4-CD8-代表TCRαβ+双阴性T细胞；细胞表面标志CD3+TCRγδ+代表γδT细胞；细胞表面标志CD3+CD4+CD45RA+CD27+代表辅助初始T细胞；细胞表面标志CD3+CD4+CD45RA+CD27-代表辅助耗竭T细胞；细胞表面标志CD3+CD4+CD45RA-CD27+代表辅助中心记忆T细胞；细胞表面标志CD3+CD4+CD45RA-CD27-代表辅助效应记忆T细胞；细胞表面标志CD3+CD8+CD45RA+CD27+代表细胞毒性初始T细胞；细胞表面标志CD3+CD8+CD45RA+CD27-代表细胞毒性耗竭T细胞；细胞表面标志CD8+CD45RA-CD27+代表细胞毒性中心记忆T细胞；细胞表面标志CD3+CD8+CD45RA-CD27-代表细胞毒性效应记忆T细胞。实验结果证实，本发明设计获得了最优的细胞表面标志的抗体组合，通过流式细胞术，实现了对T淋巴细胞进行更加全面的免疫分型和定量分析。在本发明的另外一些实施例中，本发明提供的方法，所需待测样品少、操作简单、所需时间短。在本发明的另外一些实施例中，本发明提供的方法重复性好、准确性高、可广泛应用于T淋巴细胞亚群免疫分型和定量分析。The invention provides a method and kit for T lymphocyte immunophenotyping. The method for immunophenotyping of T lymphocytes provided by the present invention comprises: taking different fluorescently labeled antibodies, mixing them with the samples to be detected, and after incubation, detecting by flow cytometry to obtain detection data and analyzing the obtained detection data; the antibody Including: anti-TCRαβ antibody, anti-TCRγδ antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody, anti-CD27 antibody and anti-CD45RA antibody; the analysis method includes: cell surface marker CD3 + TCRαβ + CD4 - CD8 - represents TCRαβ + double-negative T cells; cell surface markers CD3 + TCRγδ + represents γδ T cells; cell surface markers CD3 + CD4 + CD45RA + CD27 + represents helper naive T cells; cell surface markers CD3 + CD4 + CD45RA + CD27 - Represents helper-exhausted T cells; cell surface markers CD3 + CD4 + CD45RA - CD27 + represent helper central memory T cells; cell surface markers CD3 + CD4 + CD45RA - CD27 - represent helper effector memory T cells; cell surface markers CD3 + CD8 + CD45RA + CD27 + represents cytotoxic naive T cells; cell surface markers CD3 + CD8 + CD45RA + CD27 - represent cytotoxic exhausted T cells; cell surface markers CD8 + CD45RA - CD27 + represent cytotoxic central memory T cells; cell surface markers CD3 + CD8 + CD45RA - CD27 - represent cytotoxic effector memory T cells. Experimental results confirm that the present invention designs and obtains the optimal antibody combination of cell surface markers, and realizes more comprehensive immune typing and quantitative analysis of T lymphocytes through flow cytometry. In some other embodiments of the present invention, the method provided by the present invention requires few samples to be tested, simple operation and short time. In some other embodiments of the present invention, the method provided by the present invention has good repeatability and high accuracy, and can be widely used in immunophenotyping and quantitative analysis of T lymphocyte subsets.
附图说明Description of drawings
图1示实施例1中淋巴细胞分类结果；Fig. 1 shows lymphocyte classification result in embodiment 1;
图2示实施例1中T淋巴细胞分型结果；其中，图2-A、图2-B示TCRαβ+双阴性T细胞的细胞分型结果；图2-C示γδT细胞的细胞分型结果；图2-D示辅助T细胞亚类的细胞分型结果；图2-E示细胞毒性T细胞亚类的细胞分型结果；Figure 2 shows the results of T lymphocyte typing in Example 1; wherein, Figure 2-A and Figure 2-B show the results of cell typing of TCRαβ + double-negative T cells; Figure 2-C shows the results of cell typing of γδT cells ; Figure 2-D shows the cell typing results of helper T cell subsets; Figure 2-E shows the cell typing results of cytotoxic T cell subsets;
图3示实施例1中B淋巴细胞分型结果；Fig. 3 shows B lymphocyte typing result among the embodiment 1;
图4示实施例2中淋巴细胞分类结果；Fig. 4 shows lymphocyte classification result in embodiment 2;
图5示实施例2中T淋巴细胞分型结果；其中，图5-A、图5-B示TCRαβ+双阴性T细胞的细胞分型结果；图5-C示γδT细胞的细胞分型结果；图5-D示辅助T细胞亚类的细胞分型结果；图5-E示细胞毒性T细胞亚类的细胞分型结果；Figure 5 shows the results of T lymphocyte typing in Example 2; wherein, Figure 5-A and Figure 5-B show the results of cell typing of TCRαβ + double-negative T cells; Figure 5-C shows the results of cell typing of γδT cells ; Figure 5-D shows the cell typing results of helper T cell subsets; Figure 5-E shows the cell typing results of cytotoxic T cell subsets;
图6示实施例2中B淋巴细胞分型结果；Fig. 6 shows the B lymphocyte typing result in embodiment 2;
图7示实施例3中淋巴细胞分类结果；Fig. 7 shows lymphocyte classification result in embodiment 3;
图8示实施例3中T淋巴细胞分型结果；其中，图8-A、图8-B示TCRαβ+双阴性T细胞的细胞分型结果；图8-C示γδT细胞的细胞分型结果；图8-D示辅助T细胞亚类的细胞分型结果；图8-E示细胞毒性T细胞亚类的细胞分型结果；Figure 8 shows the results of T lymphocyte typing in Example 3; among them, Figure 8-A and Figure 8-B show the results of cell typing of TCRαβ + double-negative T cells; Figure 8-C shows the results of cell typing of γδT cells ; Figure 8-D shows the cell typing results of helper T cell subsets; Figure 8-E shows the cell typing results of cytotoxic T cell subsets;
图9示实施例3中B淋巴细胞分型结果；Fig. 9 shows the B lymphocyte typing result in embodiment 3;
图10示实施例4中淋巴细胞分类结果；Figure 10 shows the lymphocyte classification result in embodiment 4;
图11示实施例4中T淋巴细胞分型结果；其中，图11-A、图11-B示TCRαβ+双阴性T细胞的细胞分型结果；图11-C示γδT细胞的细胞分型结果；图11-D示辅助T细胞亚类的细胞分型结果；图11-E示细胞毒性T细胞亚类的细胞分型结果；Figure 11 shows the results of T lymphocyte typing in Example 4; wherein, Figure 11-A and Figure 11-B show the results of cell typing of TCRαβ + double-negative T cells; Figure 11-C shows the results of cell typing of γδT cells ; Figure 11-D shows the cell typing results of helper T cell subsets; Figure 11-E shows the cell typing results of cytotoxic T cell subsets;
图12示对比例中CD45RO和CCR7相组合时，所得的流式图。Fig. 12 shows the flow chart obtained when CD45RO and CCR7 are combined in the comparative example.
具体实施方式Detailed ways
本发明公开了一种T淋巴细胞免疫分型的方法和试剂盒。本领域技术人员可以参考本文内容，实施该方法，实现其应用，特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明内。本发明的方法及应用已经通过较佳的实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文制备方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。The invention discloses a method and a kit for T lymphocyte immunophenotyping. Those skilled in the art can refer to the content herein to implement the method and realize its application. In particular, it should be noted that all similar replacements and modifications are obvious to those skilled in the art, and they are all deemed to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and the relevant personnel can obviously make changes or appropriate changes and combinations to the preparation method and application herein without departing from the content, spirit and scope of the present invention to realize and apply The technology of the present invention.
本发明提供的一种T淋巴细胞免疫分型的方法和试剂盒中所用到的试剂和原料均可由市场购得。The reagents and raw materials used in the T lymphocyte immunophenotyping method and kit provided by the present invention can be purchased from the market.
本发明中用到的荧光标记Percp、FITC、PE、APC、BV421、Percp-cy5.5、BV510、PE-Cy7、BV450均为常见的荧光标记可以由市场购得，各个荧光标记的抗体也可以由市场购得。The fluorescent markers Percp, FITC, PE, APC, BV421, Percp-cy5.5, BV510, PE-Cy7, and BV450 used in the present invention are common fluorescent markers that can be purchased from the market, and each fluorescently labeled antibody can also be purchased from the market.
为了使本技术领域的技术人员能够更好地理解本发明的技术方案，下面结合实施例，进一步阐述本发明：In order to enable those skilled in the art to better understand the technical scheme of the present invention, the present invention will be further set forth below in conjunction with the examples:
实施例1 淋巴细胞精细免疫分型和定量分析Example 1 Fine Immunotyping and Quantitative Analysis of Lymphocytes
实验材料：Experimental Materials:
待测样品：抗凝外周血样品，来源于健康志愿者，为正常人的外周血。Sample to be tested: anticoagulated peripheral blood sample, derived from healthy volunteers, which is the peripheral blood of normal people.
BD Biosciences淋巴细胞分类试剂盒(cat340503)，购买于BD biosciences，其中混合抗体1包括：荧光标记Percp的抗CD45抗体、抗CD3(FITC)的抗体、抗CD4(APC)的抗体、抗CD8(PE)的抗体；混合抗体2包括：荧光标记Percp的抗CD45抗体、抗CD19(APC)的抗体、抗CD56和CD16(PE)的抗体、抗CD3(FITC)的抗体。BD临床试剂盒自带的红细胞裂解液。BD Biosciences lymphocyte classification kit (cat340503), purchased from BD biosciences, wherein mixed antibody 1 includes: fluorescently labeled Percp anti-CD45 antibody, anti-CD3 (FITC) antibody, anti-CD4 (APC) antibody, anti-CD8 (PE ) antibody; mixed antibody 2 includes: fluorescently labeled Percp anti-CD45 antibody, anti-CD19 (APC) antibody, anti-CD56 and CD16 (PE) antibody, anti-CD3 (FITC) antibody. The red blood cell lysate that comes with the BD clinical kit.
自配的红细胞裂解液：每1L红细胞裂解液中含NH4Cl8.29g、KHCO3 1g,EDTA0.37g。Self-prepared erythrocyte lysate: Each 1L of erythrocyte lysate contains 8.29g of NH 4 Cl, 1g of KHCO 3 , and 0.37g of EDTA.
实验方法：experimental method:
取500μL抗凝外周血样品，取200μL待测样品，通过血细胞计数仪测得淋巴细胞绝对数，得淋巴细胞的绝对数为4.22×109个/L。Take 500 μL of anticoagulated peripheral blood sample, take 200 μL of the sample to be tested, and measure the absolute number of lymphocytes by a blood cell counter, and the absolute number of lymphocytes is 4.22×10 9 /L.
剩下300μL用于检测淋巴细胞各亚群，进行淋巴细胞免疫分型和定量分析。The remaining 300 μL was used to detect each subset of lymphocytes for lymphocyte immunotype and quantitative analysis.
淋巴细胞分类Lymphocyte classification
1、取两根流式管，分别标记为L-1、L-2，将BD Biosciences淋巴细胞分类试剂盒(cat340503)中两种混合抗体分别加入到对应的流式管中，其中混合抗体1加入到L-1号流式管中；混合抗体2分别加入对L-2号流式管中。1. Take two flow tubes, marked as L-1 and L-2 respectively, and add the two mixed antibodies in the BD Biosciences Lymphocyte Sorting Kit (cat340503) into the corresponding flow tubes respectively, among which the mixed antibody 1 Add to the L-1 flow tube; add the mixed antibody 2 to the L-2 flow tube respectively.
2、向L-1、L-2号流式管中，各加入50μL待测样品，充分涡旋，室温(25℃)避光孵育30min；2. Add 50 μL of the sample to be tested to flow tubes L-1 and L-2, vortex fully, and incubate at room temperature (25°C) in the dark for 30 minutes;
3、用试剂盒中1×BD红细胞裂解液裂解红细胞，5min；3. Lyse red blood cells with 1×BD red blood cell lysate in the kit for 5 minutes;
4、加入1mL PBS洗涤一次后(3500rpm/min，2min)，加入200μL PBS流式上机，并分析结果。4. Add 1mL PBS to wash once (3500rpm/min, 2min), add 200μL PBS to the flow cytometer, and analyze the results.
T淋巴细胞精细免疫分型Fine T lymphocyte immunophenotyping
1、取两根流式管，分别标记为T-1、T-2，其中T-1为对照组和同型对照组，T-2为待检测组；按照表1向各个流式管中加入以下抗体：1. Take two flow tubes and mark them as T-1 and T-2 respectively, where T-1 is the control group and the same type control group, and T-2 is the group to be tested; add to each flow tube according to Table 1 The following antibodies:
表1 各个流式管中加入的抗体的类别和加入的体积Table 1 Types and volumes of antibodies added to each flow tube
2、向T-1、T-2号流式管中，各加入50μL待测样品，充分涡旋后，室温(25℃)避光孵育30min；2. Add 50 μL of the sample to be tested to flow tubes T-1 and T-2, vortex fully, and incubate at room temperature (25°C) in the dark for 30 minutes;
3、按照表2向各个流式管中加入以下抗体：3. Add the following antibodies to each flow tube according to Table 2:
表2 各个流式管中加入的抗体的类别和加入的体积Table 2 Types and volumes of antibodies added to each flow tube
4、用自配的红细胞裂解液裂解红细胞后，37℃水浴10min；4. After lysing red blood cells with self-prepared red blood cell lysate, bathe in water at 37°C for 10 minutes;
5、各加入1mL PBS洗一次后，流式上机并分析结果。5. After adding 1mL PBS to wash once, run the flow cytometry machine and analyze the results.
B淋巴细胞精细免疫分型Refined immunophenotyping of B lymphocytes
1、取两个流式管，分别编号为B-1、B-2，其中，B-1为对照组和同型对照组，B-2为待检测组；按照表3向各个流式管中加入以下抗体：1. Take two flow tubes, numbered B-1 and B-2 respectively. Among them, B-1 is the control group and the same type control group, and B-2 is the test group; Add the following antibodies:
表3 各个流式管中加入的抗体的类别和加入的体积Table 3 Types and volumes of antibodies added to each flow tube
2、向B-1、B-2号流式管中，各加入混匀的50μL待测样品，混匀后室温(25℃)避光孵育30min；2. Add 50 μL of the sample to be tested into the flow tubes B-1 and B-2, and incubate at room temperature (25°C) for 30 minutes in the dark after mixing;
3、用自配的红细胞裂解液裂解红细胞；3. Use self-prepared red blood cell lysate to lyse red blood cells;
4、加入1mL PBS洗一次后，流式上机并分析结果。4. After adding 1mL PBS to wash once, run the flow cytometry machine and analyze the results.
结果分析：Result analysis:
通过以上三步，得到各淋巴细胞亚群相对数(百分数)和淋巴细胞绝对数。各淋巴细胞亚群相对数(百分数)×淋巴细胞绝对数，即得到各淋巴细胞亚群绝对数。这里的各淋巴细胞亚群相对数是指：针对淋巴细胞分类时为分别为T、B、NK等细胞占淋巴细胞的百分比；针对T细胞精细免疫分型时为各细胞亚群占T细胞的百分比；针对B细胞精细免疫分型时为各细胞亚群占B细胞的百分比。Through the above three steps, the relative number (percentage) and the absolute number of lymphocytes of each lymphocyte subgroup are obtained. The relative number (percentage) of each lymphocyte subgroup × the absolute number of lymphocytes, that is, the absolute number of each lymphocyte subgroup is obtained. The relative number of each lymphocyte subset here refers to the percentage of T, B, NK and other cells in lymphocytes for lymphocyte classification; the percentage of each cell subset in T cells for T cell fine immune typing Percentage; for fine immunophenotyping of B cells, it is the percentage of each cell subgroup in B cells.
淋巴细胞免疫分型和定量分析结果(采用BD自动分析软件分析)Lymphocyte immunotype and quantitative analysis results (analyzed by BD automatic analysis software)
淋巴细胞分型结果见图1，从图中可知，T细胞、Tc细胞、Th细胞、B细胞、NK细胞的相对数(百分数)；根据淋巴细胞的绝对数、和各个细胞亚群的相对数(百分数)，计算获得各个细胞亚群的绝对数，具体实验结果见表4。The results of lymphocyte typing are shown in Figure 1. From the figure, it can be seen that the relative numbers (percentages) of T cells, Tc cells, Th cells, B cells, and NK cells; according to the absolute number of lymphocytes and the relative numbers of each cell subgroup (percentage), the absolute number of each cell subpopulation was calculated, and the specific experimental results are shown in Table 4.
表4 各个细胞亚群免疫分型所用的细胞表面标志、各个细胞亚群的相对数和绝对数Table 4 The cell surface markers used for immunophenotyping of each cell subgroup, the relative number and absolute number of each cell subgroup
T细胞精细免疫分型和定量分析结果(采用BD Diva软件分析)Fine T cell immunophenotyping and quantitative analysis results (analyzed by BD Diva software)
T淋巴细胞分型结果见图2-A、2-B、2-C、2-D和2-E，从图中可知，各类T细胞亚群的相对数(百分数)，该相对数为相对于T细胞数目的百分数；根据淋巴细胞的绝对数、和各个细胞亚群的相对数(百分数)，计算获得各个细胞亚群的绝对数。The results of T lymphocyte typing are shown in Figures 2-A, 2-B, 2-C, 2-D and 2-E. It can be seen from the figure that the relative numbers (percentages) of various T cell subsets are Percentage relative to the number of T cells; according to the absolute number of lymphocytes and the relative number (percentage) of each cell subgroup, the absolute number of each cell subgroup was calculated.
1、TCRαβ+双阴性T细胞(TCRαβ+DNT细胞)1. TCRαβ + double-negative T cells (TCRαβ + DNT cells)
由图2-A，图2-B可知，TCRαβ+DNT(即，CD3+TCRαβ+CD4-CD8-)即为目标细胞。由流式细胞仪可测得目标细胞占T细胞的1.82％，可由血细胞计数仪测得的淋巴细胞绝对数算出，TCRαβ+双阴性T细胞的绝对数为50个/μL。It can be known from Fig. 2-A and Fig. 2-B that TCRαβ + DNT (ie, CD3 + TCRαβ + CD4 - CD8 - ) is the target cell. The target cells accounted for 1.82% of the T cells measured by the flow cytometer, which can be calculated from the absolute number of lymphocytes measured by the hemocytometer, and the absolute number of TCRαβ + double-negative T cells was 50/μL.
2、γδT细胞2. γδT cells
由图2-C可知，CD3+TCRγδ+即为目标细胞。由流式细胞仪可测得目标细胞占T细胞的3.4％，可由血细胞计数仪测得的淋巴细胞绝对数算出，γδT细胞的绝对数为93个/μL。It can be seen from Figure 2-C that CD3 + TCRγδ + is the target cell. The target cells accounted for 3.4% of the T cells as measured by the flow cytometer, which can be calculated from the absolute number of lymphocytes measured by the hemocytometer, and the absolute number of γδT cells was 93/μL.
3、辅助T细胞亚类3. Subtypes of helper T cells
从图2-D可得，以下四个细胞亚群：①辅助初始T细胞(即CD4+ CD3+CD4+CD45RA+CD27+)，相对数34.36％，绝对数936个/μL；②辅助耗竭T细胞(即图中Q4-2，CD3+CD4+CD45RA+CD27-)相对数0.12％，绝对数3个/μL；③辅助中心记忆T细胞(即CD4+CM，CD3+CD4+CD45RA-CD27+)相对数6.34％，绝对数173个/μL；④辅助效应记忆T细胞(即CD4+EM，CD3+CD4+CD45RA-CD27-)相对数0.37％，绝对数10个/μL。From Figure 2-D, the following four cell subsets can be seen: ① helper naive T cells (ie, CD4 + CD3 + CD4 + CD45RA + CD27 + ), the relative number was 34.36%, and the absolute number was 936 cells/μL; ②The relative number of helper-exhausted T cells (that is, Q4-2 in the figure, CD3 + CD4 + CD45RA + CD27 - ) was 0.12%, the absolute number 3/μL; ③The relative number of helper center memory T cells (ie CD4 + CM, CD3 + CD4 + CD45RA - CD27 + ) was 6.34%, and the absolute number was 173/μL; ④ Helper effector memory T cells (ie CD4 + EM , CD3 + CD4 + CD45RA - CD27 - ) relative number 0.37%, absolute number 10/μL.
4、细胞毒性T细胞亚类4. Subtypes of cytotoxic T cells
由图2-E可得，以下四个细胞亚群：①细胞毒性初始T细胞(即CD8+ CD3+CD8+CD45RA+CD27+)，相对数17.68％，绝对数482个/μL；②细胞毒性耗竭T细胞(即CD8+TEMRA，CD3+CD8+CD45RA+CD27-)相对数2.69％，绝对数73个/μL；③细胞毒性中心记忆T细胞(即CD8+CM，CD8+CD45RA-CD27+)相对数1.09％，绝对数30个/μL；④细胞毒性效应记忆T细胞(即CD8+EM，CD3+CD8+CD45RA-CD27-)相对数0.28％，绝对数8个/μL。From Figure 2-E, the following four cell subgroups can be obtained: ① Cytotoxic initial T cells (namely CD8 + CD3 + CD8 + CD45RA + CD27 + ), the relative number was 17.68%, and the absolute number was 482 cells/μL; ② The relative number of cytotoxic exhausted T cells (CD8 + TEMRA, CD3 + CD8 + CD45RA + CD27 - ) was 2.69%, the absolute number 73 cells/μL; ③The relative number of cytotoxic central memory T cells (ie CD8 + CM, CD8 + CD45RA - CD27 + ) was 1.09%, and the absolute number was 30 cells/μL; ④ Cytotoxic effector memory T cells (ie CD8 + EM, CD3 + CD8 + CD45RA - CD27 - ) relative number 0.28%, absolute number 8/μL.
B细胞精细免疫分型和定量分析结果(采用BD Diva软件分析)B cell fine immunophenotyping and quantitative analysis results (analyzed by BD Diva software)
B淋巴细胞分型结果见图3-A和图3-B，从图中可知，各类B细胞亚群的相对数(百分数)，该相对数为相对于B细胞数目的百分数；根据淋巴细胞的绝对数、和各个细胞亚群的相对数(百分数)，计算获得各个细胞亚群的绝对数。The results of B lymphocyte typing are shown in Figure 3-A and Figure 3-B. It can be seen from the figure that the relative number (percentage) of various B cell subsets is the percentage relative to the number of B cells; The absolute number of and the relative number (percentage) of each cell subgroup were calculated to obtain the absolute number of each cell subgroup.
由图3可知，待测样品中以下四个细胞亚群和该亚群的细胞数目：①记忆B细胞(CD19+D27+)，相对数12.0％，绝对数127个/μL；②初始B细胞(CD19+D27-IgD+)，相对数69.5％，绝对数605个/μL；③过渡B细胞(CD19+CD24++CD38++)，相对数9.0％，绝对数95个/μL；④浆母细胞(CD19+CD24-CD38++)，相对数3.4％，绝对数37个/μL。It can be seen from Figure 3 that the following four cell subgroups in the sample to be tested and the number of cells in this subgroup: ① Memory B cells (CD19 + D27 + ), the relative number is 12.0%, and the absolute number is 127/μL; ② Naive B cells (CD19 + D27 - IgD + ), relative number 69.5%, absolute number 605 cells/μL; ③ transitional B cells (CD19 + CD24 ++ CD38 ++ ), relative number 9.0%, absolute number 95 cells/μL; ④ slurry Blast cells (CD19 + CD24 - CD38 ++ ), relative number 3.4%, absolute number 37 cells/μL.
根据文献报道，正常人的淋巴细胞中的各个细胞亚群的相对数的参考范围见表5所示。According to literature reports, the reference ranges of the relative numbers of each cell subpopulation in normal human lymphocytes are shown in Table 5.
表5 正常人的淋巴细胞中各个细胞亚群相对数Table 5 The relative number of each cell subgroup in lymphocytes of normal people
其中，※代表该数据并没有通用的参考范围，仅是目前研究报道的实验结果；Among them, ※ means that there is no general reference range for the data, and it is only the experimental results reported in the current research;
根据图1可知，本实施例所用的方法能够成功将淋巴细胞分为T淋巴细胞、B淋巴细胞和NK细胞，并且进行定量分析，获得了各个细胞的相对数和绝对数。从表5中结果可知，各个细胞亚群的相对数完全落入正常人的参考范围，与待检测样品的实际情况相一致，说明本发明实验结果稳定、准确。According to Fig. 1, it can be seen that the method used in this example can successfully divide lymphocytes into T lymphocytes, B lymphocytes and NK cells, and quantitative analysis was performed to obtain the relative and absolute numbers of each cell. From the results in Table 5, it can be seen that the relative numbers of each cell subgroup completely fall within the reference range of normal people, which is consistent with the actual situation of the sample to be tested, indicating that the experimental results of the present invention are stable and accurate.
根据图2-A、图2-B、图2-C、图2-D、图2-E可得，本实施例所用的方法能够准确地将T淋巴细胞分为TCRαβ+双阴性T细胞、γδT细胞、辅助初始T细胞、辅助耗竭T细胞、辅助中心记忆T细胞、辅助效应记忆T细胞、细胞毒性初始T细胞、细胞毒性耗竭T细胞、细胞毒性中心记忆T细胞、细胞毒性效应记忆T细胞，并且进行定量分析，获得了各个细胞亚群的相对数和绝对数。从表5中结果可知，各个细胞亚群的相对数与正常人的参考范围基本一致，与待检测样品的实际情况相一致，说明本发明实验结果稳定、准确。According to Figure 2-A, Figure 2-B, Figure 2-C, Figure 2-D, and Figure 2-E, the method used in this example can accurately divide T lymphocytes into TCRαβ + double-negative T cells, γδ T cells, helper naive T cells, helper exhausted T cells, helper central memory T cells, helper effector memory T cells, cytotoxic naive T cells, cytotoxic exhausted T cells, cytotoxic central memory T cells, cytotoxic effector memory T cells , and quantitative analysis was carried out to obtain the relative and absolute numbers of each cell subgroup. From the results in Table 5, it can be seen that the relative numbers of each cell subgroup are basically consistent with the reference range of normal people, consistent with the actual situation of the sample to be tested, indicating that the experimental results of the present invention are stable and accurate.
根据图3可知，本实施例所用的方法能够准确地将B淋巴细胞分为记忆B细胞、初始B细胞、过渡B细胞、浆母细胞，并且进行定量分析，获得了各个细胞亚群的相对数和绝对数。从表5中结果可知，各个细胞亚群的相对数与正常人的参考范围基本一致，与待检测样品的实际情况相一致，说明本发明实验结果稳定、准确。According to Figure 3, it can be seen that the method used in this example can accurately divide B lymphocytes into memory B cells, naive B cells, transitional B cells, and plasmablasts, and quantitative analysis was performed to obtain the relative numbers of each cell subgroup. and absolute numbers. From the results in Table 5, it can be seen that the relative numbers of each cell subgroup are basically consistent with the reference range of normal people, consistent with the actual situation of the sample to be tested, indicating that the experimental results of the present invention are stable and accurate.
综上所述，本发明提供的方法采用较少的待测样品就实现了对待测样品的淋巴细胞免疫分型，并统计出了每个细胞亚群的含量；且本发明提供的方法能够准确将各个细胞亚群进行免疫分型。In summary, the method provided by the present invention uses fewer samples to be tested to achieve lymphocyte immunophenotyping of the sample to be tested, and counts the content of each cell subgroup; and the method provided by the present invention can accurately Individual cell subsets were immunotyped.
实施例2 淋巴细胞精细免疫分型和定量分析Example 2 Fine Immunotyping and Quantitative Analysis of Lymphocytes
实验材料：Experimental Materials:
待测样品：抗凝外周血样品，来源于来我院就诊患儿静脉血样品(已签知情同意书)，为拟诊系统性红斑狼疮(SLE)患儿的外周血。Samples to be tested: anticoagulated peripheral blood samples, derived from venous blood samples from children who came to our hospital (with signed informed consent), and peripheral blood from children with suspected systemic lupus erythematosus (SLE).
BD Biosciences淋巴细胞分类试剂盒(cat340503)，购买于BD biosciences，其中混合抗体1包括：荧光标记Percp的抗CD45抗体、抗CD3(FITC)的抗体、抗CD4(APC)的抗体、抗CD8(PE)的抗体；混合抗体2包括：荧光标记Percp的抗CD45抗体、抗CD19(APC)的抗体、抗CD56和CD16(PE)的抗体、抗CD3(FITC)的抗体。BD临床试剂盒自带的红细胞裂解液。BD Biosciences lymphocyte classification kit (cat340503), purchased from BD biosciences, wherein mixed antibody 1 includes: fluorescently labeled Percp anti-CD45 antibody, anti-CD3 (FITC) antibody, anti-CD4 (APC) antibody, anti-CD8 (PE ) antibody; mixed antibody 2 includes: fluorescently labeled Percp anti-CD45 antibody, anti-CD19 (APC) antibody, anti-CD56 and CD16 (PE) antibody, anti-CD3 (FITC) antibody. The red blood cell lysate that comes with the BD clinical kit.
自配的红细胞裂解液：每1L红细胞裂解液中含NH4Cl8.29g、KHCO3 1g,EDTA0.37g。Self-prepared erythrocyte lysate: Each 1L of erythrocyte lysate contains 8.29g of NH 4 Cl, 1g of KHCO 3 , and 0.37g of EDTA.
实验方法：experimental method:
取500μL抗凝外周血样品，取200μL待测样品，通过血细胞计数仪测得淋巴细胞绝对数，得淋巴细胞的绝对数为0.68×109个/L。Take 500 μL of anticoagulated peripheral blood sample, take 200 μL of the sample to be tested, and measure the absolute number of lymphocytes by a blood cell counter, and the absolute number of lymphocytes is 0.68×10 9 /L.
剩下300μL用于检测淋巴细胞各亚群，进行淋巴细胞免疫分型和定量分析。The remaining 300 μL was used to detect each subset of lymphocytes for lymphocyte immunotype and quantitative analysis.
淋巴细胞分类Lymphocyte classification
与实施例1中记载的淋巴细胞分类方法相同，其中孵育时的温度为室温(20℃)，孵育时间为35min。The lymphocyte classification method described in Example 1 is the same, wherein the temperature during incubation is room temperature (20° C.), and the incubation time is 35 min.
T淋巴细胞精细免疫分型Fine T lymphocyte immunophenotyping
与实施例1中记载的T淋巴细胞分类方法相同，其中加入PE标记的抗TCRαβ抗体、BV421标记的抗TCRγδ抗体后，孵育的温度为室温(20℃)，孵育时间为35min；加入剩余抗体后，孵育的温度为室温(20℃)，孵育时间为35min；用自配的红细胞裂解液裂解红细胞后，36.5℃水浴11min。The T lymphocyte classification method described in Example 1 was the same, wherein after adding PE-labeled anti-TCRαβ antibody and BV421-labeled anti-TCRγδ antibody, the incubation temperature was room temperature (20°C) and the incubation time was 35min; after adding the remaining antibody, , the incubation temperature is room temperature (20° C.), and the incubation time is 35 minutes; after the erythrocytes are lysed with self-prepared erythrocyte lysate, they are placed in a water bath at 36.5° C. for 11 minutes.
B淋巴细胞精细免疫分型Refined immunophenotyping of B lymphocytes
1、取两个流式管，分别编号为B-1、B-2，其中，B-1为对照组和同型对照组，B-2为待检测组；按照表6向各个流式管中加入以下抗体：1. Take two flow tubes, numbered B-1 and B-2 respectively. Among them, B-1 is the control group and the same type control group, and B-2 is the group to be tested; put them into each flow tube according to Table 6. Add the following antibodies:
表6 各个流式管中加入的抗体的类别和加入的体积Table 6 Types and volumes of antibodies added to each flow tube
2、向B-1、B-2号流式管中，各加入混匀的50μL待测样品，混匀后室温(20℃)避光孵育30min；2. Add 50 μL of the sample to be tested into the flow tubes B-1 and B-2, and incubate at room temperature (20°C) for 30 minutes in the dark after mixing;
3、用自配的红细胞裂解液裂解红细胞；3. Use self-prepared red blood cell lysate to lyse red blood cells;
4、加入1mL PBS洗一次后，流式上机并分析结果。4. After adding 1mL PBS to wash once, run the flow cytometry machine and analyze the results.
结果分析：Result analysis:
通过以上三步，得到各淋巴细胞亚群相对数(百分数)和淋巴细胞绝对数。具体分析方法与实施例1提供的方法相同。Through the above three steps, the relative number (percentage) and the absolute number of lymphocytes of each lymphocyte subgroup are obtained. The specific analysis method is the same as the method provided in Example 1.
淋巴细胞免疫分型和定量分析结果(采用BD自动分析软件分析)Lymphocyte immunotype and quantitative analysis results (analyzed by BD automatic analysis software)
淋巴细胞分型结果图4，从图中可知，T细胞、Tc细胞、Th细胞、B细胞、NK细胞的相对(百分数)；根据淋巴细胞的绝对数、和各个细胞亚群的相对数(百分数)，计算获得各个细胞亚群的绝对数，具体实验结果见表7。Lymphocyte typing results Fig. 4, as can be seen from the figure, the relative (percentage) of T cell, Tc cell, Th cell, B cell, NK cell; ), the absolute number of each cell subpopulation was calculated, and the specific experimental results are shown in Table 7.
表7 各个细胞亚群免疫分型所用的细胞表面标志、各个细胞亚群的相对数和绝对数Table 7 The cell surface markers used for immunophenotyping of each cell subgroup, the relative number and absolute number of each cell subgroup
T细胞精细免疫分型和定量分析结果(采用BD Diva软件分析)Fine T cell immunophenotyping and quantitative analysis results (analyzed by BD Diva software)
T淋巴细胞分类结果见图5-A、5-B、5-C、5-D和图5-E，从图中可知，各类T细胞亚群的相对数(百分数)，该相对数为相对于T细胞数目的百分数；根据淋巴细胞的绝对数、和各个细胞亚群的相对数(百分数)，计算获得各个细胞亚群的绝对数。The results of T lymphocyte classification are shown in Figure 5-A, 5-B, 5-C, 5-D and Figure 5-E. It can be seen from the figure that the relative numbers (percentages) of various T cell subsets are Percentage relative to the number of T cells; according to the absolute number of lymphocytes and the relative number (percentage) of each cell subgroup, the absolute number of each cell subgroup was calculated.
1、TCRαβ+双阴性T细胞(TCRαβ+DNT细胞)1. TCRαβ + double-negative T cells (TCRαβ + DNT cells)
由图5-A，图5-B可知，TCRαβ+DNT(即CD3+TCRαβ+CD4-CD8-)即为目标细胞。由流式细胞仪可测得目标细胞占T细胞的3.04％，可由血细胞计数仪测得的淋巴细胞绝对数算出，TCRαβ+双阴性T细胞的绝对数为14个/μL。It can be known from Fig. 5-A and Fig. 5-B that TCRαβ + DNT (ie CD3 + TCRαβ + CD4 - CD8 - ) is the target cell. The target cells accounted for 3.04% of the T cells as measured by the flow cytometer, which can be calculated from the absolute number of lymphocytes measured by the hemocytometer, and the absolute number of TCRαβ + double-negative T cells was 14/μL.
2、γδT细胞2. γδT cells
由图5-C可知，CD3+TCRγδ+即为目标细胞。由流式细胞仪可测得目标细胞占T细胞的1.32％，可由血细胞计数仪测得的淋巴细胞绝对数算出，γδT细胞的绝对数为6个/μL。It can be seen from Figure 5-C that CD3 + TCRγδ + is the target cell. The target cells accounted for 1.32% of the T cells as measured by the flow cytometer, which can be calculated from the absolute number of lymphocytes measured by the hemocytometer, and the absolute number of γδT cells was 6/μL.
3、辅助T细胞亚类3. Subtypes of helper T cells
从图5-D可得，以下四个细胞亚群：①辅助初始T细胞(即CD4+ CD3+CD4+CD45RA+CD27+)，相对数16.87％，绝对数77个/μL；②辅助耗竭T细胞(即图中Q4-2，CD3+CD4+CD45RA+CD27-)相对数0.09％，绝对数0.3个/μL；③辅助中心记忆T细胞(即CD4+CM，CD3+CD4+CD45RA-CD27+)相对数22.16％，绝对数101个/μL；④辅助效应记忆T细胞(即CD4+EM，CD3+CD4+CD45RA-CD27-)相对数6.43％，绝对数29个/μL。From Figure 5-D, the following four cell subsets can be seen: ① helper naive T cells (namely CD4 + CD3 + CD4 + CD45RA + CD27 + ), the relative number was 16.87%, and the absolute number was 77 cells/μL; ②The relative number of helper-exhausted T cells (that is, Q4-2 in the figure, CD3 + CD4 + CD45RA + CD27 - ) was 0.09%, the absolute number 0.3 cells/μL; ③The relative number of helper center memory T cells (ie CD4 + CM, CD3 + CD4 + CD45RA - CD27 + ) was 22.16%, and the absolute number was 101 cells/μL; ④ Helper effector memory T cells (ie CD4 + EM , CD3 + CD4 + CD45RA - CD27 - ) relative number 6.43%, absolute number 29/μL.
4、细胞毒性T细胞亚类4. Subtypes of cytotoxic T cells
由图5-E可得，以下四个细胞亚群：①细胞毒性初始T细胞(即CD8+ CD3+CD8+CD45RA+CD27+)，相对数12.33％，绝对数56个/μL；②细胞毒性耗竭T细胞(即CD8+TEMRA，CD3+CD8+CD45RA+CD27-)相对数1.97％，绝对数9个/μL；③细胞毒性中心记忆T细胞(即CD8+CM，CD8+CD45RA-CD27+)相对数3.78％，绝对数17个/μL；④细胞毒性效应记忆T细胞(即CD8+EM，CD3+CD8+CD45RA-CD27-)相对数2.24％，绝对数10个/μL。From Figure 5-E, the following four cell subgroups: ① Cytotoxic initial T cells (namely CD8 + CD3 + CD8 + CD45RA + CD27 + ), relative number 12.33%, absolute number 56 cells/μL; ②cytotoxic exhausted T cells (ie CD8 + TEMRA, CD3 + CD8 + CD45RA + CD27 - ) relative number 1.97%, absolute number 9 cells/μL; ③The relative number of cytotoxic central memory T cells (ie CD8 + CM, CD8 + CD45RA - CD27 + ) was 3.78%, and the absolute number was 17 cells/μL; ④ Cytotoxic effector memory T cells (ie CD8 + EM, CD3 + CD8 + CD45RA - CD27 - ) relative number 2.24%, absolute number 10/μL.
B细胞精细免疫分型和定量分析结果(采用BD Diva软件分析)B cell fine immunophenotyping and quantitative analysis results (analyzed by BD Diva software)
B淋巴细胞分型结果见图6-A和图6-B，从图中可知，各类B细胞亚群的相对数(百分数)，该相对数为相对于B细胞数目的百分数；根据淋巴细胞的绝对数、和各个细胞亚群的相对数(百分数)，计算获得各个细胞亚群的绝对数。The results of B lymphocyte typing are shown in Figure 6-A and Figure 6-B. It can be seen from the figure that the relative number (percentage) of various B cell subsets is the percentage relative to the number of B cells; The absolute number of and the relative number (percentage) of each cell subgroup were calculated to obtain the absolute number of each cell subgroup.
由图6可知，待测样品中以下四个细胞亚群和该亚群的细胞数目：①记忆B细胞(CD19+D27+)，相对数28.0％，绝对数59个/μL；②初始B细胞(CD19+D27-IgD+)，相对数48.5％，绝对数102个/μL；③过渡B细胞(CD19+CD24++CD38++)，相对数0.5％，绝对数1个/μL；④浆母细胞(CD19+CD24-CD38++)，相对数13.8％，绝对数29个/μL。It can be seen from Figure 6 that the following four cell subgroups and the cell numbers of the subgroups in the sample to be tested: ① memory B cells (CD19 + D27 + ), the relative number is 28.0%, and the absolute number is 59/μL; ② naive B cells (CD19 + D27 - IgD + ), relative number 48.5%, absolute number 102/μL; ③ transitional B cells (CD19 + CD24 ++ CD38 ++ ), relative number 0.5%, absolute number 1/μL; ④ plasma Blast cells (CD19 + CD24 - CD38 ++ ), the relative number was 13.8%, and the absolute number was 29 cells/μL.
根据文献报道，正常人的淋巴细胞中的各个细胞亚群的相对数的参考范围见表5所示。According to literature reports, the reference ranges of the relative numbers of each cell subpopulation in normal human lymphocytes are shown in Table 5.
根据图4可知，本实施例所用的方法能够成功将淋巴细胞分为T淋巴细胞、B淋巴细胞和NK细胞，并且进行定量分析，获得了各个细胞的相对数和绝对数。从结果可知，待测样品中的NK细胞(CD16+CD56+)含量明显低于正常参考范围下限(7％-40％)，而B细胞(CD19+)高于正常参考范围上限(5-18％)，其余无明显异常，提示该待测样品中B细胞异常活化或增殖。According to Fig. 4, it can be seen that the method used in this example can successfully divide lymphocytes into T lymphocytes, B lymphocytes and NK cells, and quantitative analysis was performed to obtain the relative and absolute numbers of each cell. From the results, it can be seen that the content of NK cells (CD16 + CD56 + ) in the test sample is significantly lower than the lower limit of the normal reference range (7%-40%), while the content of B cells (CD19 + ) is higher than the upper limit of the normal reference range (5-18 %), and the rest had no obvious abnormalities, suggesting abnormal activation or proliferation of B cells in the sample to be tested.
根据图5可知，本实施例所用的方法能够准确地将T淋巴细胞分为TCRαβ+双阴性T细胞、γδT细胞、辅助初始T细胞、辅助耗竭T细胞、辅助中心记忆T细胞、辅助效应记忆T细胞、细胞毒性初始T细胞、细胞毒性耗竭T细胞、细胞毒性中心记忆T细胞、细胞毒性效应记忆T细胞，并且进行定量分析，获得了各个细胞亚群的相对数和绝对数。从结果可知，各个细胞亚群的相对数与正常人的参考范围基本一致，无明显异常。According to Figure 5, it can be seen that the method used in this example can accurately divide T lymphocytes into TCRαβ + double-negative T cells, γδT cells, helper naive T cells, helper exhausted T cells, helper central memory T cells, and helper effector memory T cells. Cells, cytotoxic naive T cells, cytotoxic exhausted T cells, cytotoxic central memory T cells, and cytotoxic effector memory T cells were quantitatively analyzed to obtain relative and absolute numbers of each cell subset. From the results, it can be known that the relative number of each cell subgroup is basically consistent with the reference range of normal people, and there is no obvious abnormality.
根据图6可知，本实施例所用的方法能够准确地将B淋巴细胞分为记忆B细胞、初始B细胞、过渡B细胞、浆母细胞，并且进行定量分析，获得了各个细胞亚群的相对数和绝对数。从结果可知，待测样品中过渡B细胞(TransitionalB cell)仅占B细胞的0.05％，几乎没有，提示其体液免疫过渡活化或失调。According to Figure 6, it can be seen that the method used in this embodiment can accurately divide B lymphocytes into memory B cells, naive B cells, transitional B cells, and plasmablasts, and quantitative analysis was performed to obtain the relative numbers of each cell subgroup. and absolute numbers. From the results, it can be seen that the transitional B cells (Transitional B cells) in the samples to be tested account for only 0.05% of the B cells, almost none, suggesting that the humoral immunity is excessively activated or disordered.
综上所述，本发明提供的方法采用较少的待测样品就实现了对待测样品的淋巴细胞免疫分型，并统计出了每个细胞亚群的含量；且本发明提供的方法能够准确将各个细胞亚群进行免疫分型。In summary, the method provided by the present invention uses fewer samples to be tested to achieve lymphocyte immunophenotyping of the sample to be tested, and counts the content of each cell subgroup; and the method provided by the present invention can accurately Individual cell subsets were immunotyped.
实施例3 淋巴细胞精细免疫分型和定量分析Example 3 Fine Immunotyping and Quantitative Analysis of Lymphocytes
实验材料：Experimental Materials:
待测样品：抗凝外周血样品，来源于来我院就诊患儿静脉血样品(已签知情同意书)，为拟诊高IgM综合征患儿的外周血。Samples to be tested: anticoagulant peripheral blood samples, derived from venous blood samples of children who came to our hospital (with signed informed consent), peripheral blood of children with suspected hyper-IgM syndrome.
BD Biosciences淋巴细胞分类试剂盒(cat340503)，购买于BD biosciences，其中混合抗体1包括：荧光标记Percp的抗CD45抗体、抗CD3(FITC)的抗体、抗CD4(APC)的抗体、抗CD8(PE)的抗体；混合抗体2包括：荧光标记Percp的抗CD45抗体、抗CD19(APC)的抗体、抗CD56和CD16(PE)的抗体、抗CD3(FITC)的抗体。BD临床试剂盒自带的红细胞裂解液。BD Biosciences lymphocyte classification kit (cat340503), purchased from BD biosciences, wherein mixed antibody 1 includes: fluorescently labeled Percp anti-CD45 antibody, anti-CD3 (FITC) antibody, anti-CD4 (APC) antibody, anti-CD8 (PE ) antibody; mixed antibody 2 includes: fluorescently labeled Percp anti-CD45 antibody, anti-CD19 (APC) antibody, anti-CD56 and CD16 (PE) antibody, anti-CD3 (FITC) antibody. The red blood cell lysate that comes with the BD clinical kit.
自配的红细胞裂解液：每1L红细胞裂解液中含NH4Cl8.29g、KHCO3 1g,EDTA0.37g。Self-prepared erythrocyte lysate: Each 1L of erythrocyte lysate contains 8.29g of NH 4 Cl, 1g of KHCO 3 , and 0.37g of EDTA.
实验方法：experimental method:
取500μL抗凝外周血样品，取200μL待测样品，通过血细胞计数仪测得淋巴细胞绝对数，得淋巴细胞的绝对数为0.89×109个/L。Take 500 μL of anticoagulated peripheral blood sample, take 200 μL of the sample to be tested, and measure the absolute number of lymphocytes by a blood cell counter, and the absolute number of lymphocytes is 0.89×10 9 /L.
剩下300μL用于检测淋巴细胞各亚群，进行淋巴细胞免疫分型和定量分析。The remaining 300 μL was used to detect each subset of lymphocytes for lymphocyte immunotype and quantitative analysis.
淋巴细胞分类Lymphocyte classification
与实施例1中记载的淋巴细胞分类方法相同，其中孵育时的温度为室温(22℃)，孵育时间为30min。The lymphocyte classification method described in Example 1 is the same, wherein the temperature during incubation is room temperature (22° C.), and the incubation time is 30 min.
T淋巴细胞精细免疫分型Fine T lymphocyte immunophenotyping
与实施例1中记载的T淋巴细胞分类方法相同，其中加入PE标记的抗TCRαβ抗体、BV421标记的抗TCRγδ抗体后，孵育的温度为室温(22℃)，孵育时间为25min；加入剩余抗体后，孵育的温度为室温(22℃)，孵育时间为25min；用自配的红细胞裂解液裂解红细胞后，37.5℃水浴9min。The T lymphocyte classification method described in Example 1 was the same, wherein after adding PE-labeled anti-TCRαβ antibody and BV421-labeled anti-TCRγδ antibody, the incubation temperature was room temperature (22°C) and the incubation time was 25min; after adding the remaining antibody, , the incubation temperature is room temperature (22° C.), and the incubation time is 25 minutes; after the erythrocytes are lysed with self-prepared erythrocyte lysate, they are placed in a water bath at 37.5° C. for 9 minutes.
B淋巴细胞精细免疫分型Refined immunophenotyping of B lymphocytes
与实施例1中记载的B淋巴细胞分类方法相同，其中其中孵育时的温度为室温(22℃)，孵育时间为25min。The method for classifying B lymphocytes described in Example 1 is the same, wherein the temperature during incubation is room temperature (22° C.), and the incubation time is 25 minutes.
结果分析：Result analysis:
通过以上三步，得到各淋巴细胞亚群相对数(百分数)和淋巴细胞绝对数。具体分析方法与实施例1提供的方法相同。Through the above three steps, the relative number (percentage) and the absolute number of lymphocytes of each lymphocyte subgroup are obtained. The specific analysis method is the same as the method provided in Example 1.
淋巴细胞免疫分型和定量分析结果(采用BD自动分析软件分析)Lymphocyte immunotype and quantitative analysis results (analyzed by BD automatic analysis software)
淋巴细胞分型结果见图7，从图中可知，T细胞、Tc细胞、Th细胞、B细胞、NK细胞的相对(百分数)；根据淋巴细胞的绝对数、和各个细胞亚群的相对数(百分数)，计算获得各个细胞亚群的绝对数，具体实验结果见表8。Lymphocyte typing results are shown in Figure 7, as can be seen from the figure, the relative (percentage) of T cells, Tc cells, Th cells, B cells, NK cells; according to the absolute number of lymphocytes and the relative number of each cell subgroup ( Percentage), the absolute number of each cell subpopulation was calculated, and the specific experimental results are shown in Table 8.
表8 各个细胞亚群免疫分型所用的细胞表面标志、各个细胞亚群的相对数和绝对数Table 8 The cell surface markers used for immunophenotyping of each cell subgroup, the relative number and absolute number of each cell subgroup
T细胞精细免疫分型和定量分析结果(采用BD Diva软件分析)Fine T cell immunophenotyping and quantitative analysis results (analyzed by BD Diva software)
T淋巴细胞分类结果见图8-A、8-B、8-C、8-D和图8-E，从图中可知，各类T细胞亚群的相对数(百分数)，该相对数为相对于T细胞数目的百分数；根据淋巴细胞的绝对数、和各个细胞亚群的相对数(百分数)，计算获得各个细胞亚群的绝对数。The results of T lymphocyte classification are shown in Fig. 8-A, 8-B, 8-C, 8-D and Fig. 8-E. It can be seen from the figure that the relative numbers (percentages) of various T cell subsets are Percentage relative to the number of T cells; according to the absolute number of lymphocytes and the relative number (percentage) of each cell subgroup, the absolute number of each cell subgroup was calculated.
1、TCRαβ+双阴性T细胞(TCRαβ+DNT细胞)1. TCRαβ + double-negative T cells (TCRαβ + DNT cells)
由图8-A，图8-B可知，TCRαβ+DNT(即CD3+TCRαβ+CD4-CD8-)即为目标细胞。由流式细胞仪可测得目标细胞占T细胞的1.24％，可由血细胞计数仪测得的淋巴细胞绝对数算出，TCRαβ+双阴性T细胞的绝对数为8个/μL。It can be seen from Fig. 8-A and Fig. 8-B that TCRαβ + DNT (ie CD3 + TCRαβ + CD4 - CD8 - ) is the target cell. The target cells accounted for 1.24% of the T cells measured by the flow cytometer, which can be calculated from the absolute number of lymphocytes measured by the hemocytometer, and the absolute number of TCRαβ + double-negative T cells was 8/μL.
2、γδT细胞2. γδT cells
由图8-C可知，CD3+TCRγδ+即为目标细胞。由流式细胞仪可测得目标细胞占T细胞的18.1％，可由血细胞计数仪测得的淋巴细胞绝对数算出，γδT细胞的绝对数为113个/μL。It can be seen from Figure 8-C that CD3 + TCRγδ + is the target cell. The target cells accounted for 18.1% of the T cells as measured by the flow cytometer, which can be calculated from the absolute number of lymphocytes measured by the hemocytometer, and the absolute number of γδT cells was 113/μL.
3、辅助T细胞亚类3. Subtypes of helper T cells
从图8-D可得，以下四个细胞亚群：①辅助初始T细胞(即CD4+ CD3+CD4+CD45RA+CD27+)，相对数23.36％，绝对数146个/μL；②辅助耗竭T细胞(即图中Q4-2，CD3+CD4+CD45RA+CD27-)相对数0.17％，绝对数1个/μL；③辅助中心记忆T细胞(即CD4+CM，CD3+CD4+CD45RA-CD27+)相对数9.47％，绝对数59个/μL；④辅助效应记忆T细胞(即CD4+EM，CD3+CD4+CD45RA-CD27-)相对数0.71％，绝对数4个/μL。From Figure 8-D, it can be seen that the following four cell subsets: ① helper naive T cells (ie CD4 + CD3 + CD4 + CD45RA + CD27 + ), the relative number was 23.36%, and the absolute number was 146 cells/μL; ②The relative number of helper-exhausted T cells (that is, Q4-2 in the figure, CD3 + CD4 + CD45RA + CD27 - ) was 0.17%, the absolute number Count 1/μL; ③The relative number of helper center memory T cells (ie CD4 + CM, CD3 + CD4 + CD45RA - CD27 + ) was 9.47%, and the absolute number was 59/μL; ④ Helper effector memory T cells (ie CD4 + EM , CD3 + CD4 + CD45RA - CD27 - ) relative number 0.71%, absolute number 4/μL.
4、细胞毒性T细胞亚类4. Subtypes of cytotoxic T cells
由图8-E可得，以下四个细胞亚群：①细胞毒性初始T细胞(即CD8+ CD3+CD8+CD45RA+CD27+)，相对数15.66％，绝对数98个/μL；②细胞毒性耗竭T细胞(即CD8+TEMRA，CD3+CD8+CD45RA+CD27-)相对数10.15％，绝对数63个/μL；③细胞毒性中心记忆T细胞(即CD8+CM，CD8+CD45RA-CD27+)相对数1.20％，绝对数7个/μL；④细胞毒性效应记忆T细胞(即CD8+EM，CD3+CD8+CD45RA-CD27-)相对数0.81％，绝对数5个/μL。From Figure 8-E, the following four cell subgroups: ① Cytotoxic initial T cells (namely CD8 + CD3 + CD8 + CD45RA + CD27 + ), the relative number was 15.66%, and the absolute number was 98 cells/μL; ②The relative number of cytotoxic exhausted T cells (namely CD8 + TEMRA, CD3 + CD8 + CD45RA + CD27 - ) was 10.15%, the absolute number 63 cells/μL; ③The relative number of cytotoxic central memory T cells (ie CD8 + CM, CD8 + CD45RA - CD27 + ) was 1.20%, and the absolute number was 7 cells/μL; ④ Cytotoxic effector memory T cells (ie CD8 + EM, CD3 + CD8 + CD45RA - CD27 - ) relative number 0.81%, absolute number 5/μL.
B细胞精细免疫分型和定量分析结果(采用BD Diva软件分析)B cell fine immunophenotyping and quantitative analysis results (analyzed by BD Diva software)
B淋巴细胞分型结果见图9-A和图9-B，从图中可知，各类B细胞亚群的相对数(百分数)，该相对数为相对于B细胞数目的百分数；根据淋巴细胞的绝对数、和各个细胞亚群的相对数(百分数)，计算获得各个细胞亚群的绝对数。The results of B lymphocyte typing are shown in Figure 9-A and Figure 9-B. It can be seen from the figure that the relative number (percentage) of various B cell subsets is the percentage relative to the number of B cells; The absolute number of and the relative number (percentage) of each cell subgroup were calculated to obtain the absolute number of each cell subgroup.
由图9可知，待测样品中以下四个细胞亚群和该亚群的细胞数目：①记忆B细胞(CD19+D27+)，相对数0.5％，绝对数1个/μL；②初始B细胞(CD19+D27-IgD+)，相对数95.3％，绝对数216个/μL；③过渡B细胞(CD19+CD24++CD38++)，相对数28.3％，绝对数64个/μL；④浆母细胞(CD19+CD24-CD38++)，相对数0.9％，绝对数2个/μL。It can be seen from Figure 9 that the following four cell subgroups in the sample to be tested and the number of cells in this subgroup: ① memory B cells (CD19 + D27 + ), the relative number is 0.5%, and the absolute number is 1/μL; ② naive B cells (CD19 + D27 - IgD + ), relative number 95.3%, absolute number 216 cells/μL; ③ transitional B cells (CD19 + CD24 ++ CD38 ++ ), relative number 28.3%, absolute number 64 cells/μL; ④ slurry Blast cells (CD19 + CD24 - CD38 ++ ), relative number 0.9%, absolute number 2 cells/μL.
根据文献报道，正常人的淋巴细胞中的各个细胞亚群的相对数的参考范围见表5所示。According to literature reports, the reference ranges of the relative numbers of each cell subpopulation in normal human lymphocytes are shown in Table 5.
根据图7可知，本实施例所用的方法能够成功将淋巴细胞分为T淋巴细胞、B淋巴细胞和NK细胞，并且进行定量分析，获得了各个细胞的相对数和绝对数。从结果可知，待测样品中的NK细胞(CD16+CD56+)为2.22％，明显低于正常参考范围下限(7-40％)，而B细胞(CD19+)为25.42％，高于正常参考范围上限(5-18％)，其余无明显异常，提示B细胞异常活化或增殖。According to Fig. 7, it can be seen that the method used in this embodiment can successfully divide lymphocytes into T lymphocytes, B lymphocytes and NK cells, and quantitative analysis was performed to obtain the relative and absolute numbers of each cell. From the results, it can be seen that the NK cells (CD16 + CD56 + ) in the test sample were 2.22%, which was significantly lower than the lower limit of the normal reference range (7-40%), while the B cells (CD19 + ) were 25.42%, which was higher than the normal reference range The upper limit of the range (5-18%), and the rest have no obvious abnormalities, suggesting abnormal activation or proliferation of B cells.
根据图8可知，本实施例所用的方法能够准确地将T淋巴细胞分为TCRαβ+双阴性T细胞、γδT细胞、辅助初始T细胞、辅助耗竭T细胞、辅助中心记忆T细胞、辅助效应记忆T细胞、细胞毒性初始T细胞、细胞毒性耗竭T细胞、细胞毒性中心记忆T细胞、细胞毒性效应记忆T细胞，并且进行定量分析，获得了各个细胞亚群的相对数和绝对数。从结果可知，待测样品中细胞毒性耗竭T细胞(CD8+TEMRA)的含量占T细胞的36.5％，显著高于正常同龄儿童，提示患儿可能存在长期慢性病毒感染；待测样品中记忆T细胞(各类中心/效应记忆T)均低于正常参考范围，提示该患儿可能存在细胞免疫异常；待测样品中γδT细胞的含量占T细胞的18.1％，远高于正常参考范围(低于5％)，提示患儿病毒感染、T细胞异常活化或增值。According to Figure 8, it can be seen that the method used in this example can accurately divide T lymphocytes into TCRαβ + double negative T cells, γδ T cells, helper naive T cells, helper exhausted T cells, helper central memory T cells, helper effector memory T cells Cells, cytotoxic naive T cells, cytotoxic exhausted T cells, cytotoxic central memory T cells, and cytotoxic effector memory T cells were quantitatively analyzed to obtain relative and absolute numbers of each cell subset. It can be seen from the results that the content of cytotoxic exhausted T cells (CD8 + TEMRA) in the tested samples accounted for 36.5% of the T cells, which was significantly higher than that of normal children of the same age, suggesting that the children may have long-term chronic viral infections; memory T cells in the tested samples Cells (all types of center/effect memory T) were lower than the normal reference range, suggesting that the child may have abnormal cellular immunity; the content of γδT cells in the sample to be tested accounted for 18.1% of T cells, which was much higher than the normal reference range (low less than 5%), suggesting viral infection, abnormal activation or proliferation of T cells in children.
根据图9可知，本实施例所用的方法能够准确地将B淋巴细胞分为记忆B细胞、初始B细胞、过渡B细胞、浆母细胞，并且进行定量分析，获得了各个细胞亚群的相对数和绝对数。从结果可知，初始B细胞(B)过多，而记忆B细胞(memory B)仅占B细胞的0.5％，远低于正常下限，提示患儿体液免疫功能障碍。According to Figure 9, it can be seen that the method used in this embodiment can accurately divide B lymphocytes into memory B cells, naive B cells, transitional B cells, and plasmablasts, and quantitative analysis was performed to obtain the relative numbers of each cell subgroup. and absolute numbers. From the results, it can be seen that naive B cells ( B) Excessive, and memory B cells (memory B) accounted for only 0.5% of B cells, far below the lower limit of normal, suggesting humoral immune dysfunction in children.
综上所述，本发明提供的方法采用较少的待测样品就实现了对待测样品的淋巴细胞免疫分型，并统计出了每个细胞亚群的含量；且本发明提供的方法能够准确将各个细胞亚群进行免疫分型。In summary, the method provided by the present invention uses fewer samples to be tested to achieve lymphocyte immunophenotyping of the sample to be tested, and counts the content of each cell subgroup; and the method provided by the present invention can accurately Individual cell subsets were immunotyped.
实施例4 T淋巴细胞精细免疫分型和定量分析Example 4 T Lymphocyte Fine Immunotyping and Quantitative Analysis
实验材料：Experimental Materials:
待测样品：抗凝外周血样品，来源于健康志愿者静脉血标本，为多次患“感冒”健康成人的外周血。Samples to be tested: anticoagulated peripheral blood samples, derived from venous blood samples of healthy volunteers, which are peripheral blood of healthy adults who have suffered from "cold" many times.
BD Biosciences淋巴细胞分类试剂盒(cat340503)，购买于BD biosciences，其中混合抗体1包括：荧光标记Percp的抗CD45抗体、抗CD3(FITC)的抗体、抗CD4(APC)的抗体、抗CD8(PE)的抗体；混合抗体2包括：荧光标记Percp的抗CD45抗体、抗CD19(APC)的抗体、抗CD56和CD16(PE)的抗体、抗CD3(FITC)的抗体。BD临床试剂盒自带的红细胞裂解液。BD Biosciences lymphocyte classification kit (cat340503), purchased from BD biosciences, wherein mixed antibody 1 includes: fluorescently labeled Percp anti-CD45 antibody, anti-CD3 (FITC) antibody, anti-CD4 (APC) antibody, anti-CD8 (PE ) antibody; mixed antibody 2 includes: fluorescently labeled Percp anti-CD45 antibody, anti-CD19 (APC) antibody, anti-CD56 and CD16 (PE) antibody, anti-CD3 (FITC) antibody. The red blood cell lysate that comes with the BD clinical kit.
自配的红细胞裂解液：。Self-prepared erythrocyte lysate:.
实验方法：experimental method:
取500μL抗凝外周血样品，取200μL待测样品，通过血细胞计数仪测得淋巴细胞绝对数，得淋巴细胞的绝对数为1.38×109个/L。Take 500 μL of anticoagulated peripheral blood sample, take 200 μL of the sample to be tested, and measure the absolute number of lymphocytes by a blood cell counter, and the absolute number of lymphocytes is 1.38×10 9 /L.
剩下300μL用于检测淋巴细胞各亚群，进行淋巴细胞免疫分型和定量分析。The remaining 300 μL was used to detect each subset of lymphocytes for lymphocyte immunotype and quantitative analysis.
淋巴细胞分类Lymphocyte classification
与实施例1中记载的淋巴细胞分类方法相同，其中孵育时的温度为室温(20℃)，孵育时间为30min。The lymphocyte classification method described in Example 1 is the same, wherein the temperature during incubation is room temperature (20° C.), and the incubation time is 30 min.
淋巴细胞免疫分型和定量分析结果(采用BD自动分析软件分析)Lymphocyte immunotype and quantitative analysis results (analyzed by BD automatic analysis software)
淋巴细胞分型结果见图10，从图中可知，T细胞、Tc细胞、Th细胞、B细胞、NK细胞的相对(百分数)；根据淋巴细胞的绝对数、和各个细胞亚群的相对数(百分数)，计算获得各个细胞亚群的绝对数，具体实验结果见表9。Lymphocyte typing results are shown in Figure 10, as can be seen from the figure, the relative (percentage) of T cells, Tc cells, Th cells, B cells, NK cells; according to the absolute number of lymphocytes and the relative number of each cell subgroup ( Percentage), the absolute number of each cell subpopulation was calculated, and the specific experimental results are shown in Table 9.
表9 各个细胞亚群免疫分型所用的细胞表面标志、各个细胞亚群的相对数和绝对数Table 9 The cell surface markers used for immunophenotyping of each cell subgroup, the relative number and absolute number of each cell subgroup
T淋巴细胞精细免疫分型Fine T lymphocyte immunophenotyping
与实施例1中记载的T淋巴细胞分类方法相同，其中加入PE标记的抗TCRαβ抗体、BV421标记的抗TCRγδ抗体后，孵育的温度为室温(20℃)，孵育时间为30min；加入剩余抗体后，孵育的温度为室温(20℃)，孵育时间为30min。The T lymphocyte classification method described in Example 1 was the same, wherein after adding PE-labeled anti-TCRαβ antibody and BV421-labeled anti-TCRγδ antibody, the incubation temperature was room temperature (20°C) and the incubation time was 30min; after adding the remaining antibody, , the incubation temperature is room temperature (20° C.), and the incubation time is 30 min.
T细胞精细免疫分型和定量分析结果(采用BD Diva软件分析)Fine T cell immunophenotyping and quantitative analysis results (analyzed by BD Diva software)
T淋巴细胞分类结果见图11-A、11-B、11-C、11-D和图11-E，从图中可知，各类T细胞亚群的相对数(百分数)，该相对数为相对于T细胞数目的百分数；根据淋巴细胞的绝对数、和各个细胞亚群的相对数(百分数)，计算获得各个细胞亚群的绝对数。The results of T lymphocyte classification are shown in Figures 11-A, 11-B, 11-C, 11-D and Figure 11-E. From the figure, it can be seen that the relative numbers (percentages) of various T cell subsets are Percentage relative to the number of T cells; according to the absolute number of lymphocytes and the relative number (percentage) of each cell subgroup, the absolute number of each cell subgroup was calculated.
1、TCRαβ+双阴性T细胞(TCRαβ+DNT细胞)1. TCRαβ + double-negative T cells (TCRαβ + DNT cells)
由图11-A，图11-B可知，TCRαβ+DNT(即CD3+TCRαβ+CD4-CD8-)即为目标细胞。由流式细胞仪可测得目标细胞占T细胞的0.73％，可由血细胞计数仪测得的淋巴细胞绝对数算出，TCRαβ+双阴性T细胞的绝对数为8个/μL。It can be known from Fig. 11-A and Fig. 11-B that TCRαβ + DNT (ie CD3 + TCRαβ + CD4 - CD8 - ) is the target cell. The target cells accounted for 0.73% of the T cells measured by the flow cytometer, which can be calculated from the absolute number of lymphocytes measured by the hemocytometer, and the absolute number of TCRαβ + double-negative T cells was 8/μL.
2、γδT细胞2. γδT cells
由图11-C可知，CD3+TCRγδ+即为目标细胞。由流式细胞仪可测得目标细胞占T细胞的30.2％，可由血细胞计数仪测得的淋巴细胞绝对数算出，γδT细胞的绝对数为347个/μL。It can be seen from Figure 11-C that CD3 + TCRγδ + is the target cell. The target cells accounted for 30.2% of the T cells measured by the flow cytometer, which can be calculated from the absolute number of lymphocytes measured by the hemocytometer, and the absolute number of γδT cells was 347/μL.
3、辅助T细胞亚类3. Subtypes of helper T cells
从图11-D可得，以下四个细胞亚群：①辅助初始T细胞(即CD4+ CD3+CD4+CD45RA+CD27+)，相对数13.64％，绝对数157个/μL；②辅助耗竭T细胞(即图中Q4-2，CD3+CD4+CD45RA+CD27-)相对数0.9％，绝对数4个/μL；③辅助中心记忆T细胞(即CD4+CM，CD3+CD4+CD45RA-CD27+)相对数16.01％，绝对数184个/μL；④辅助效应记忆T细胞(CD4+EM，CD3+CD4+CD45RA-CD27-)相对数11.6％，绝对数45个/μL。From Figure 11-D, it can be seen that the following four cell subsets: ① helper naive T cells (namely CD4 + CD3 + CD4 + CD45RA + CD27 + ), the relative number was 13.64%, and the absolute number was 157 cells/μL; ②The relative number of helper-exhausted T cells (that is, Q4-2 in the figure, CD3 + CD4 + CD45RA + CD27 - ) was 0.9%, the absolute number 4/μL; ③The relative number of helper center memory T cells (CD4 + CM, CD3 + CD4 + CD45RA - CD27 + ) was 16.01%, and the absolute number was 184/μL; ④ Helper effector memory T cells (CD4 + EM, CD3 + CD4 + CD45RA - CD27 - ) relative number 11.6%, absolute number 45/μL.
4、细胞毒性T细胞亚类4. Subtypes of cytotoxic T cells
由图11-E可得，以下四个细胞亚群：①细胞毒性初始T细胞(即CD8+ CD3+CD8+CD45RA+CD27+)，相对数12.98％，绝对数149个/μL；②细胞毒性耗竭T细胞(即CD8+TEMRA，CD3+CD8+CD45RA+CD27-)相对数22.05％，绝对数253个/μL；③细胞毒性中心记忆T细胞(即即CD8+CM，CD8+CD45RA-CD27+)相对数4.99％，绝对数57个/μL；④细胞毒性效应记忆T细胞(即CD8+EM，CD3+CD8+CD45RA-CD27-)相对数1.58％，绝对数18个/μL。From Figure 11-E, the following four cell subgroups: ① Cytotoxic naive T cells (namely CD8 + CD3 + CD8 + CD45RA + CD27 + ), the relative number was 12.98%, and the absolute number was 149 cells/μL; ② The relative number of cytotoxic exhausted T cells (ie CD8 + TEMRA, CD3 + CD8 + CD45RA + CD27 - ) was 22.05%, the absolute number 253 cells/μL; ③The relative number of cytotoxic central memory T cells (that is, CD8 + CM, CD8 + CD45RA - CD27 + ) was 4.99%, and the absolute number was 57 cells/μL; ④ Cytotoxic effector memory T cells (that is, CD8 + EM , CD3 + CD8 + CD45RA - CD27 - ) relative number 1.58%, absolute number 18/μL.
根据文献报道，正常人的淋巴细胞中的各个细胞亚群的相对数的参考范围见表5所示。According to literature reports, the reference ranges of the relative numbers of each cell subpopulation in normal human lymphocytes are shown in Table 5.
根据图10可知，本实施例所用的方法能够成功将淋巴细胞分为T淋巴细胞、B淋巴细胞和NK细胞，并且进行定量分析，获得了各个细胞的相对数和绝对数。从结果可知，该患者这集群细胞相对数在正常参考范围之内。According to Fig. 10, it can be seen that the method used in this embodiment can successfully divide lymphocytes into T lymphocytes, B lymphocytes and NK cells, and perform quantitative analysis to obtain the relative and absolute numbers of each cell. From the results, it can be seen that the relative number of cells in this patient group is within the normal reference range.
根据图11可知，本实施例所用的方法能够准确地将T淋巴细胞分为TCRαβ+双阴性T细胞、γδT细胞、辅助初始T细胞、辅助耗竭T细胞、辅助中心记忆T细胞、辅助效应记忆T细胞、细胞毒性初始T细胞、细胞毒性耗竭T细胞、细胞毒性中心记忆T细胞、细胞毒性效应记忆T细胞，并且进行定量分析，获得了各个细胞亚群的相对数和绝对数。从结果可知，待测样品中细胞毒性耗竭T细胞(CD8+TEMRA)的含量占T细胞的22.05％，显著高于正常参考范围，提示该患者可能存在长期慢性病毒感染；待测样品中γδT细胞的含量占T细胞的30.2％，远高于正常参考范围，提示该患者由于常患“感冒”，可能存在慢性病毒感染的情况。According to Figure 11, it can be seen that the method used in this example can accurately divide T lymphocytes into TCRαβ + double negative T cells, γδ T cells, helper naive T cells, helper exhausted T cells, helper central memory T cells, helper effector memory T cells Cells, cytotoxic naive T cells, cytotoxic exhausted T cells, cytotoxic central memory T cells, and cytotoxic effector memory T cells were quantitatively analyzed to obtain relative and absolute numbers of each cell subset. It can be seen from the results that the content of cytotoxic exhausted T cells (CD8 + TEMRA) in the test sample accounts for 22.05% of the T cells, which is significantly higher than the normal reference range, suggesting that the patient may have long-term chronic viral infection; the γδT cells in the test sample The content of T cells accounts for 30.2%, which is much higher than the normal reference range, suggesting that the patient may have chronic viral infection due to frequent "colds".
对比例 不同的T淋巴细胞表面标记对T淋巴细胞精细免疫分型和定量分析的影响Comparative example Effects of different T lymphocyte surface markers on fine immunophenotyping and quantitative analysis of T lymphocytes
不同的淋巴细胞亚群具有不同的细胞表面标志，但是，并不是任何细胞表面标志的抗体都可以用于淋巴细胞的免疫分型和定量分析。在T细胞精细免疫分型中，以辅助中心/效应记忆T细胞为例，可用CD45RA和CD45RO表面标志(即抗原)区分辅助初始T细胞和辅助记忆T细胞，CCR7/CD62L/CD27任意一个标记区分辅助中心T细胞、辅助效应记忆T细胞。但CD62L易受PBMC提取过程中梯度离心和环境温度的影响；CCR7和CD45RA/CD45RO均为连续表达，用同型对照或者FMO对照划门很可能影响结果准确性和可操作性，导致无法准确区分各个细胞亚群。Different lymphocyte subsets have different cell surface markers, however, not all antibodies to cell surface markers can be used for immunophenotyping and quantitative analysis of lymphocytes. In fine immunophenotyping of T cells, taking helper center/effector memory T cells as an example, CD45RA and CD45RO surface markers (i.e. antigens) can be used to distinguish helper naive T cells from helper memory T cells, and any marker of CCR7/CD62L/CD27 can be used to distinguish Helper center T cells, helper effector memory T cells. However, CD62L is easily affected by gradient centrifugation and ambient temperature during the PBMC extraction process; CCR7 and CD45RA/CD45RO are both continuously expressed, and gating with isotype control or FMO control is likely to affect the accuracy and operability of the results, resulting in the inability to accurately distinguish between each cell subgroups.
图12为以CD45RO和CCR7相组合，以抗CD45RO的抗体和抗CCR7的抗体作为检测抗体，对待测样品进行淋巴细胞免疫分型，所得的流式图，从图中可知，相比本发明用CD45RA和CD27划分上述四群细胞，用CD45RO和CCR7组合划分的方式分群不清晰，对同型对照抗体滴度要求高，划门难度大。Figure 12 is a combination of CD45RO and CCR7, using anti-CD45RO antibodies and anti-CCR7 antibodies as detection antibodies, and performing lymphocyte immunophenotyping on the sample to be tested. CD45RA and CD27 divide the above four groups of cells, and the combination of CD45RO and CCR7 is not clear, and the requirement for isotype control antibody titers is high, and it is difficult to draw gates.
以上仅是本发明的优选实施方式，应当指出的是，上述优选实施方式不应视为对本发明的限制，本发明的保护范围应当以权利要求所限定的范围为准。对于本技术领域的普通技术人员来说，在不脱离本发明的精神和范围内，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。The above are only preferred implementations of the present invention, and it should be noted that the above preferred implementations should not be regarded as limiting the present invention, and the scope of protection of the present invention should be based on the scope defined in the claims. For those skilled in the art, without departing from the spirit and scope of the present invention, some improvements and modifications can also be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.
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| WO2019023148A1 (en) * | 2017-07-24 | 2019-01-31 | Regeneron Pharmaceuticals, Inc. | Anti-cd8 antibodies and uses thereof |
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| CN114578048A (en) * | 2021-12-22 | 2022-06-03 | 重庆医科大学附属儿童医院 | T lymphocyte development subgroup immunophenotyping method and kit |
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| CN106947835A (en) * | 2017-04-24 | 2017-07-14 | 首都医科大学附属北京友谊医院 | The authentication method of ebv infection lymphocyte subgroup and its application |
| CN107144687A (en) * | 2017-05-03 | 2017-09-08 | 首都医科大学附属北京妇产医院 | Detection method and kit for decidua lymphocyte subtype and application thereof |
| US12077587B2 (en) | 2017-07-24 | 2024-09-03 | Regeneron Pharmaceuticals, Inc. | Anti-CD8 antibodies and uses thereof |
| WO2019023148A1 (en) * | 2017-07-24 | 2019-01-31 | Regeneron Pharmaceuticals, Inc. | Anti-cd8 antibodies and uses thereof |
| US10730944B2 (en) | 2017-07-24 | 2020-08-04 | Regeneron Pharmaceuticals, Inc. | Anti-CD8 antibodies and uses thereof |
| US11525001B2 (en) | 2017-07-24 | 2022-12-13 | Regeneron Pharmaceuticals, Inc. | Anti-CD8 antibodies and uses thereof |
| CN108226016A (en) * | 2018-01-12 | 2018-06-29 | 浙江普罗亭健康科技有限公司 | The mass spectrum flow cytometer detection kit of the accurate parting of tumor vaccine cells subgroup |
| CN112557283A (en) * | 2020-11-25 | 2021-03-26 | 华中科技大学 | Diagnosis and treatment immune marker for recurrent pregnancy loss diseases and application thereof |
| CN114578048A (en) * | 2021-12-22 | 2022-06-03 | 重庆医科大学附属儿童医院 | T lymphocyte development subgroup immunophenotyping method and kit |
| CN114578048B (en) * | 2021-12-22 | 2023-08-08 | 重庆医科大学附属儿童医院 | T lymphocyte development subgroup immunophenotyping method and kit |
| WO2023115702A1 (en) * | 2021-12-22 | 2023-06-29 | 重庆医科大学附属儿童医院 | Method and kit for immunotyping of t lymphocyte development subgroups |
| US20240418718A1 (en) * | 2021-12-22 | 2024-12-19 | Children’s Hospital Of Chongqing Medical University | Method and kit for immunotyping of t lymphocyte development subgroups |
| CN117169517A (en) * | 2023-11-03 | 2023-12-05 | 赛德特(北京)生物工程有限公司 | Method and kit for detecting CD28 antibody residues in T lymphocyte preparation |
| CN117169517B (en) * | 2023-11-03 | 2024-01-19 | 赛德特(北京)生物工程有限公司 | Method and kit for detecting CD28 antibody residues in T lymphocyte preparation |
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