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WO2005124350A1 - Utilisation de cytometrie de flux multiparametrique pour diagnostic, pronostique et validation d'immunotherapies pour malades autoimmunes, hematologiques et lymphoproliferatives - Google Patents

Utilisation de cytometrie de flux multiparametrique pour diagnostic, pronostique et validation d'immunotherapies pour malades autoimmunes, hematologiques et lymphoproliferatives Download PDF

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Publication number
WO2005124350A1
WO2005124350A1 PCT/IT2004/000352 IT2004000352W WO2005124350A1 WO 2005124350 A1 WO2005124350 A1 WO 2005124350A1 IT 2004000352 W IT2004000352 W IT 2004000352W WO 2005124350 A1 WO2005124350 A1 WO 2005124350A1
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Prior art keywords
cells
apc
fitc
autoimmune
prognosis
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Ceased
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PCT/IT2004/000352
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English (en)
Inventor
Gionvanna Borsellino
Adamo Diamantini
Luca Battistini
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Fondazione Santa Lucia Irccs
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Fondazione Santa Lucia Irccs
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Priority to EP04745177A priority Critical patent/EP1842068A1/fr
Priority to PCT/IT2004/000352 priority patent/WO2005124350A1/fr
Publication of WO2005124350A1 publication Critical patent/WO2005124350A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1456—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
    • G01N15/1459—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals the analysis being performed on a sample stream
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/42—Low-temperature sample treatment, e.g. cryofixation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N2015/1477—Multiparameters
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants

Definitions

  • This invention concerns a method which allows to perform flow cytometric analysis using determined antibody combinations; these antibodies are conjugated with different fluorochromes, and the antibody combinations include several panels which allow to perform an analysis of the immunological status of the individual (in case of screening for autoimmune disorders or chronic infections) or a detailed study of neoplastic cell populations (when screening for haematological diseases).
  • the increased knowledge on how the immune system works has brought, in recent years, to a better understanding of the pathogenesis of many diseases, and, consequently, to new therapeutic approaches.
  • the interactions between the different components of the immune system are indeed very complex, and new technologies have revealed that cellular populations Which apparently seem homogeneous are in fact composed of several subpopulations with different phenotypes and functions.
  • the cells of the immune system circulate in the blood and the lymph which can also migrate to tissue to protect the organism efficiently. Lymphocytes specifically recognise and respond to foreign antigens, while phagocytes and granulocytes are classified as "inflammatory" cells, and they play a major role in the innate immune response.
  • lymphocytes appear to be macroscopically homogeneous, they can actually be divided in distinct subpopulations: in the first place one must distinguish between B-lymphocytes, which produce antibodies, and T lymphocytes. Based on expression of surface molecules, the latter can be further divided in CD4+ "helper” cells and CD8+ cytotoxic cells. Both CD4+ and CD8+ cells comprise subsets, which have not yet encountered a foreign antigen, so called “na ⁇ ve" cells, and memory cells, which represent the expanded pool of antigen experienced cells. But these subsets are still heterogeneous: indeed some memory cells produce proinflammatory proteins, while other tend to regulate and to switch off the immune response.
  • the complexity of the immune system is such that the identification of cellular subsets with unique functional characteristics requires the simultaneous measurement of multiple parameters on each single cell.
  • multiparametric flow cytometry it is possible to measure the presence of surface markers, which permit the identification of distinct cellular subsets: for instance, during a viral infection, it is possible to identify virus-specific cells activated by the infection and to follow them during the anti-viral response.
  • virus-specific cells activated by the infection and to follow them during the anti-viral response.
  • the cells of the immune system become activated and acquire the functions which permit the elimination of the offending agent.
  • adhesion molecules which enable the cells of the immune system to interact with the endothelial cells lining the capillaries: this interaction represents the initial step of the migration process by which the cells of the immune system gain access to inflamed tissues.
  • the study of the membrane receptors expressed by the different subsets of lymphocytes represents a powerful tool for the evaluation of the "immunological status" of an individual.
  • autoimmune diseases are the results of the dysregulation of the complex interactions between cells of the immune system, so these pathologies represent a good target for studies involving the definition of fine cellular subsets.
  • Flow cytometry represents the ideal tool for the study of non adherent cells (in suspension), and can be used also for the study of lymphoid tissues, from which single cell suspensions can be easily obtained. Data obtained with flow cytometry - both concerning the distribution of the different cellular subsets and the expression of multiple cellular markers are then evaluated with powerful software.
  • Fig. 1 show the multiparametric analysis of CD3+CD8+ lymphocytes obtained from the peripheral blood of a healthy subject.
  • Fig.2. show the multiparametric analysis of CD3+CD8+ lymphocytes in an MS patient
  • Fig.3. show the analysis of the ability of CD3+CD8+ lymphocytes to produce the inflammatory cytokine interferon gamma (IFN ⁇ ).
  • This invention concerns a method which allows to perform flow cytometric analysis using determined antibody combinations.
  • These antibodies are conjugated with different fluorochromes, and the antibody combinations include several panels which allow to perform an analysis of the immunological status of the individual (in case of screening for autoimmune disorders or chronic infections) or a detailed study of neoplastic cell populations (when screening for haematological diseases).
  • Antibodies are aliquoted in small tubes at predetermined concentrations, and then lyophilised.
  • Antibodies which we have tested are: CD3 PE-Cy7, CD8 PE-Cy7, CD8 PE-TxRed, CD45RA PE-TxRed, CD62L PE-TxRed, CD45RO PE-TxRed, CD8 beta PE, CD49dFlTC, NKRP1A PE, CD3 FITC, CD3 PE, CD8 FITC, CD8 PE, CD8 CyChrome, CD8 APC, CD4 FITC, CD4 APC, CD62L PE-Cy5, CD62L APC, CD162 PE, CCR7 PE, CD45RO FITC, CD11a PE, CD11a APC, CD49d PE, CD8 APC-Cy7, CDHa ' FITC, CD45RA PE-Cy5, CD5 PE-Cy5, CD23 PE-TxRed, CD19 PE-Cy7, CD19 APC- Cy7, CD79b APC, CD38 PE, kappa FITC, lambda PE, F
  • Antibodies are used are optimal predetermined concentrations, (usually 0,25 ⁇ g/ml). Each new antibody lot is accurately tested in serial dilutions, and antibodies are aliquoted in 1 ,5 ml tubes in different combinations. Tubes are then put in the speedvac, until completely lyophilised (20 minutes). Before use each antibody combinations is reconstituted with saline solution, and added to the tube containing cells to be analyzed. For each antibody combination, 2x10 6 cells are stained. This method can be used for staining of whole blood or of peripheral blood lymphocytes (PBLs) obtained from centrifugation on a density gradient.
  • PBLs peripheral blood lymphocytes
  • heparinized blood will be diluted with 1 volume of RPMI media and gently layered over the Fycoll-Hypaque (Pharmacia). Following centrifugation at 660g for 30 minutes, cells at the interface of the gradient will be collected and washed three times in medium. After the final wash cells will be resuspended in PBS with 1% human serum. For each labelling cells will be resuspended in a volume of 100 ⁇ l. All antibodies will have been tested at saturating conditions, to exclude differences in staining.
  • Samples will then be acquired at the flow cytometer using constant instrument settings, obtained by a careful daily calibration of the instruments with beads and by the use of appropriate compensation controls (beads coated with the antibodies used in the stainings). For each sample 1x106 events will be acquired, in order to guarantee an appropriate representation of all cellular subsets, and allowing significant statistical analysis.
  • To study cytokine production we will follow the procedure of intracellular staining, as established in our laboratory. Briefly, cells are plated at a density of 2x10 6 /ml in 96 well plates and triggered with either mitogens (PHA, PMA+ionomycin) or antibodies directed against cell surface molecules (such as CD3), for 6 hours at 37°C, in the presence of monensin (10 mM).
  • Flow cytometric analysis permits not only the phenotypic analysis of cellular subpopulations r but also the ability of each subpopulation to produce soluble factors which modulate the immune response (Fig. 3).
  • the panel of cytokine produced by each cell type provides important information on the immunological status of the patient, and can be used as a "marker" of the efficacy of immunomodulatory treatments.
  • this invention may be used for the diagnosis of hematologic and lymphoproliferative disorders.
  • flow cytometry is a powerful tool used for the characterization of neoplastic cells present in these diseases.
  • the ability to simultaneously measure multiple markers on each ' single cell enables to carry out a detailed analysis of tumoral cells, and to perform an accurate diagnosis.
  • the diagnosis of hematologic tumours requires an accurate characterization of the neoplastic clone, since treatment and prognosis depend closely on the diagnosis.
  • a standard panel of antibodies specific for membrane or cytoplasmic markers is used in order to define the lineage and differentiation stage of the neoplastic clone (Tab.1).

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
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  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Rheumatology (AREA)
  • Rehabilitation Therapy (AREA)
  • Dispersion Chemistry (AREA)
  • Zoology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

Cette invention concerne un procédé permettant d'effectuer une analyse cytométrique de flux à l'aide de combinaisons d'anticorps déterminées. Ces anticorps sont conjugués à différents fluorochromes, et les combinaisons d'anticorps comprennent plusieurs panels qui permettent effectuer une analyse du statut immunologique de l'individu (en cas de dépistage de troubles autoimmuns ou d'infections chroniques) ou une étude détaillée de populations de cellules néoplasiques (lors du dépistage de maladies hématologiques). Les anticorps sont répartis dans de petits tubes selon des concentrations prédéterminées, puis lyophilisés.
PCT/IT2004/000352 2004-06-21 2004-06-21 Utilisation de cytometrie de flux multiparametrique pour diagnostic, pronostique et validation d'immunotherapies pour malades autoimmunes, hematologiques et lymphoproliferatives Ceased WO2005124350A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP04745177A EP1842068A1 (fr) 2004-06-21 2004-06-21 Utilisation de cytometrie de flux multiparametrique pour diagnostic, pronostique et validation d'immunotherapies pour malades autoimmunes, hematologiques et lymphoproliferatives
PCT/IT2004/000352 WO2005124350A1 (fr) 2004-06-21 2004-06-21 Utilisation de cytometrie de flux multiparametrique pour diagnostic, pronostique et validation d'immunotherapies pour malades autoimmunes, hematologiques et lymphoproliferatives

Applications Claiming Priority (1)

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PCT/IT2004/000352 WO2005124350A1 (fr) 2004-06-21 2004-06-21 Utilisation de cytometrie de flux multiparametrique pour diagnostic, pronostique et validation d'immunotherapies pour malades autoimmunes, hematologiques et lymphoproliferatives

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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102062774A (zh) * 2009-11-13 2011-05-18 程小星 检测结核病治疗效果的试剂盒
US8148101B2 (en) 2008-07-22 2012-04-03 Abbott Laboratories Method for classifying and counting bacteria in body fluids
WO2014135924A1 (fr) * 2013-03-08 2014-09-12 Cells For Cells Cellules souches de sang menstruel pour le soutien et le développement efficaces des cellules souches hématopoïétiques cd34+ et cd133+ in vitro
CN109030322A (zh) * 2018-08-17 2018-12-18 成都赋智健康科技有限公司 一种节约成本的流式细胞术检测方法
CN110412286A (zh) * 2019-07-11 2019-11-05 上海宸安生物科技有限公司 一种利用质谱流式系统对肿瘤样本进行单细胞检测的方法
CN110488009A (zh) * 2019-04-12 2019-11-22 北京市理化分析测试中心 一种非疾病诊断目的的人体免疫功能的检测方法及应用
CN111316098A (zh) * 2017-09-15 2020-06-19 拜克门寇尔特公司 用于治疗的基于流动的测定
CN111527395A (zh) * 2018-12-01 2020-08-11 铭道创新（北京）医疗技术有限公司 一种免疫细胞中淋巴细胞的流式细胞仪检测方法
CN114112868A (zh) * 2020-08-26 2022-03-01 上海睿昂基因科技股份有限公司 一种用于监测肿瘤相关免疫微环境的流式细胞术试剂盒及监测方法
WO2023115702A1 (fr) * 2021-12-22 2023-06-29 重庆医科大学附属儿童医院 Procédé et kit pour l'immunotypage de sous-groupes de développement des lymphocytes t
CN116741281A (zh) * 2023-06-12 2023-09-12 广东省人民医院 一种采用多参数流式细胞术检测外周血免疫细胞的方法
CN119178711A (zh) * 2024-11-25 2024-12-24 国家卫生健康委科学技术研究所 在流式细胞仪上检测自然杀伤细胞的方法及其试剂盒
CN119290541A (zh) * 2024-12-13 2025-01-10 天津医科大学总医院空港医院 一种淋巴组织中b细胞亚群的检测方法
CN119530150A (zh) * 2024-11-29 2025-02-28 北京肿瘤医院(北京大学肿瘤医院) 一种肿瘤反应性非耗竭t细胞亚型及其分离、鉴定方法

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Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8148101B2 (en) 2008-07-22 2012-04-03 Abbott Laboratories Method for classifying and counting bacteria in body fluids
CN102062774A (zh) * 2009-11-13 2011-05-18 程小星 检测结核病治疗效果的试剂盒
WO2014135924A1 (fr) * 2013-03-08 2014-09-12 Cells For Cells Cellules souches de sang menstruel pour le soutien et le développement efficaces des cellules souches hématopoïétiques cd34+ et cd133+ in vitro
CN111316098A (zh) * 2017-09-15 2020-06-19 拜克门寇尔特公司 用于治疗的基于流动的测定
CN111316098B (zh) * 2017-09-15 2024-05-14 拜克门寇尔特公司 用于治疗的基于流动的测定
CN109030322A (zh) * 2018-08-17 2018-12-18 成都赋智健康科技有限公司 一种节约成本的流式细胞术检测方法
CN111527395A (zh) * 2018-12-01 2020-08-11 铭道创新（北京）医疗技术有限公司 一种免疫细胞中淋巴细胞的流式细胞仪检测方法
CN111527395B (zh) * 2018-12-01 2024-01-26 铭道创新（北京）医疗技术有限公司 一种免疫细胞中淋巴细胞的流式细胞仪检测方法
CN110488009B (zh) * 2019-04-12 2023-09-05 北京市理化分析测试中心 一种非疾病诊断目的的人体免疫功能的检测方法及应用
CN110488009A (zh) * 2019-04-12 2019-11-22 北京市理化分析测试中心 一种非疾病诊断目的的人体免疫功能的检测方法及应用
CN110412286A (zh) * 2019-07-11 2019-11-05 上海宸安生物科技有限公司 一种利用质谱流式系统对肿瘤样本进行单细胞检测的方法
CN114112868A (zh) * 2020-08-26 2022-03-01 上海睿昂基因科技股份有限公司 一种用于监测肿瘤相关免疫微环境的流式细胞术试剂盒及监测方法
WO2023115702A1 (fr) * 2021-12-22 2023-06-29 重庆医科大学附属儿童医院 Procédé et kit pour l'immunotypage de sous-groupes de développement des lymphocytes t
CN116741281A (zh) * 2023-06-12 2023-09-12 广东省人民医院 一种采用多参数流式细胞术检测外周血免疫细胞的方法
CN119178711A (zh) * 2024-11-25 2024-12-24 国家卫生健康委科学技术研究所 在流式细胞仪上检测自然杀伤细胞的方法及其试剂盒
CN119530150A (zh) * 2024-11-29 2025-02-28 北京肿瘤医院(北京大学肿瘤医院) 一种肿瘤反应性非耗竭t细胞亚型及其分离、鉴定方法
CN119290541A (zh) * 2024-12-13 2025-01-10 天津医科大学总医院空港医院 一种淋巴组织中b细胞亚群的检测方法

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