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Quantification of T-cell-mediated apoptosis in heterogeneous leukemia populations using four-color multiparameter flow cytometry. 2005

Theresia M Westers, and Ilse Houtenbos, and Gerrit Jan Schuurhuis, and Gert J Ossenkoppele, and Arjan A van de Loosdrecht
Department of Hematology, VU University Medical Center, Amsterdam, The Netherlands.

BACKGROUND The unique capacity of dendritic cells to present antigens to naive T cells is being increasingly utilized in cancer therapy. The efficacy of cell-based immunotherapy can be analyzed by determination of cytotoxic activity of T cells toward tumor cells in vitro. This study supplies a flow cytometric method to analyze T-cell-mediated cytotoxic activity toward heterogeneous leukemic cell populations at a single-cell level. METHODS The fluorescent probe SYTO16 and the dead-cell dye 7-aminoactinomycine-D (7-AAD) were used to identify early and late stages of apoptosis in combination with leukemia cell-identifying markers. Determination of viable, apoptotic, and necrotic cells was performed by inclusion of fluorescent beads. RESULTS In nine acute myeloid leukemia samples and three leukemic cell lines the use of SYTO16 next to the dead-cell marker 7-AAD significantly increased (P = 0.001) the sensitivity of the cytotoxicity assay as compared with single use of 7-AAD. Analysis of several effector-to-target ratios revealed the ability to determine dose-response effects. Enumeration of absolute numbers resulted in coefficients of variation of 4.1% and 8.4% for cell lines and leukemic samples, respectively. CONCLUSIONS The presented flow cytometric cytotoxicity assay enables the study of T-cell-mediated apoptosis in a heterogenous leukemia population.

UI MeSH Term Description Entries
D003601 Cytotoxicity Tests, Immunologic The demonstration of the cytotoxic effect on a target cell of a lymphocyte, a mediator released by a sensitized lymphocyte, an antibody, or complement. AHG-CDC Tests,Anti-Human Globulin Complement-Dependent Cytotoxicity Tests,Microcytotoxicity Tests,Anti Human Globulin Complement Dependent Cytotoxicity Tests,Anti-Human Globulin Complement-Dependent Cytotoxicity Test,Antiglobulin-Augmented Lymphocytotoxicity Test,Antiglobulin-Augmented Lymphocytotoxicity Tests,Cytotoxicity Test, Immunologic,Cytotoxicity Tests, Anti-Human Globulin Complement-Dependent,Cytotoxicity Tests, Immunological,Immunologic Cytotoxicity Test,Immunologic Cytotoxicity Tests,Lymphocytotoxicity Test, Antiglobulin-Augmented,Lymphocytotoxicity Tests, Antiglobulin-Augmented,Microcytotoxicity Test,AHG CDC Tests,AHG-CDC Test,Anti Human Globulin Complement Dependent Cytotoxicity Test,Antiglobulin Augmented Lymphocytotoxicity Test,Antiglobulin Augmented Lymphocytotoxicity Tests,Cytotoxicity Test, Immunological,Cytotoxicity Tests, Anti Human Globulin Complement Dependent,Immunological Cytotoxicity Test,Immunological Cytotoxicity Tests,Lymphocytotoxicity Test, Antiglobulin Augmented,Lymphocytotoxicity Tests, Antiglobulin Augmented
D003602 Cytotoxicity, Immunologic The phenomenon of target cell destruction by immunologically active effector cells. It may be brought about directly by sensitized T-lymphocytes or by lymphoid or myeloid "killer" cells, or it may be mediated by cytotoxic antibody, cytotoxic factor released by lymphoid cells, or complement. Tumoricidal Activity, Immunologic,Immunologic Cytotoxicity,Immunologic Tumoricidal Activities,Immunologic Tumoricidal Activity,Tumoricidal Activities, Immunologic
D003609 Dactinomycin A compound composed of a two CYCLIC PEPTIDES attached to a phenoxazine that is derived from STREPTOMYCES parvullus. It binds to DNA and inhibits RNA synthesis (transcription), with chain elongation more sensitive than initiation, termination, or release. As a result of impaired mRNA production, protein synthesis also declines after dactinomycin therapy. (From AMA Drug Evaluations Annual, 1993, p2015) Actinomycin,Actinomycin D,Meractinomycin,Cosmegen,Cosmegen Lyovac,Lyovac-Cosmegen,Lyovac Cosmegen,Lyovac, Cosmegen,LyovacCosmegen
D005434 Flow Cytometry Technique using an instrument system for making, processing, and displaying one or more measurements on individual cells obtained from a cell suspension. Cells are usually stained with one or more fluorescent dyes specific to cell components of interest, e.g., DNA, and fluorescence of each cell is measured as it rapidly transverses the excitation beam (laser or mercury arc lamp). Fluorescence provides a quantitative measure of various biochemical and biophysical properties of the cell, as well as a basis for cell sorting. Other measurable optical parameters include light absorption and light scattering, the latter being applicable to the measurement of cell size, shape, density, granularity, and stain uptake. Cytofluorometry, Flow,Cytometry, Flow,Flow Microfluorimetry,Fluorescence-Activated Cell Sorting,Microfluorometry, Flow,Cell Sorting, Fluorescence-Activated,Cell Sortings, Fluorescence-Activated,Cytofluorometries, Flow,Cytometries, Flow,Flow Cytofluorometries,Flow Cytofluorometry,Flow Cytometries,Flow Microfluorometries,Flow Microfluorometry,Fluorescence Activated Cell Sorting,Fluorescence-Activated Cell Sortings,Microfluorimetry, Flow,Microfluorometries, Flow,Sorting, Fluorescence-Activated Cell,Sortings, Fluorescence-Activated Cell
D005456 Fluorescent Dyes Chemicals that emit light after excitation by light. The wave length of the emitted light is usually longer than that of the incident light. Fluorochromes are substances that cause fluorescence in other substances, i.e., dyes used to mark or label other compounds with fluorescent tags. Flourescent Agent,Fluorescent Dye,Fluorescent Probe,Fluorescent Probes,Fluorochrome,Fluorochromes,Fluorogenic Substrates,Fluorescence Agents,Fluorescent Agents,Fluorogenic Substrate,Agents, Fluorescence,Agents, Fluorescent,Dyes, Fluorescent,Probes, Fluorescent,Substrates, Fluorogenic
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D013601 T-Lymphocytes Lymphocytes responsible for cell-mediated immunity. Two types have been identified - cytotoxic (T-LYMPHOCYTES, CYTOTOXIC) and helper T-lymphocytes (T-LYMPHOCYTES, HELPER-INDUCER). They are formed when lymphocytes circulate through the THYMUS GLAND and differentiate to thymocytes. When exposed to an antigen, they divide rapidly and produce large numbers of new T cells sensitized to that antigen. T Cell,T Lymphocyte,T-Cells,Thymus-Dependent Lymphocytes,Cell, T,Cells, T,Lymphocyte, T,Lymphocyte, Thymus-Dependent,Lymphocytes, T,Lymphocytes, Thymus-Dependent,T Cells,T Lymphocytes,T-Cell,T-Lymphocyte,Thymus Dependent Lymphocytes,Thymus-Dependent Lymphocyte
D014408 Biomarkers, Tumor Molecular products metabolized and secreted by neoplastic tissue and characterized biochemically in cells or BODY FLUIDS. They are indicators of tumor stage and grade as well as useful for monitoring responses to treatment and predicting recurrence. Many chemical groups are represented including HORMONES; ANTIGENS; amino and NUCLEIC ACIDS; ENZYMES; POLYAMINES; and specific CELL MEMBRANE PROTEINS and LIPIDS. Biochemical Tumor Marker,Cancer Biomarker,Carcinogen Markers,Markers, Tumor,Metabolite Markers, Neoplasm,Tumor Biomarker,Tumor Marker,Tumor Markers, Biochemical,Tumor Markers, Biological,Biochemical Tumor Markers,Biological Tumor Marker,Biological Tumor Markers,Biomarkers, Cancer,Marker, Biochemical Tumor,Marker, Biologic Tumor,Marker, Biological Tumor,Marker, Neoplasm Metabolite,Marker, Tumor Metabolite,Markers, Biochemical Tumor,Markers, Biological Tumor,Markers, Neoplasm Metabolite,Markers, Tumor Metabolite,Metabolite Markers, Tumor,Neoplasm Metabolite Markers,Tumor Markers, Biologic,Tumor Metabolite Marker,Biologic Tumor Marker,Biologic Tumor Markers,Biomarker, Cancer,Biomarker, Tumor,Cancer Biomarkers,Marker, Tumor,Markers, Biologic Tumor,Markers, Carcinogen,Metabolite Marker, Neoplasm,Metabolite Marker, Tumor,Neoplasm Metabolite Marker,Tumor Biomarkers,Tumor Marker, Biochemical,Tumor Marker, Biologic,Tumor Marker, Biological,Tumor Markers,Tumor Metabolite Markers
D015470 Leukemia, Myeloid, Acute Clonal expansion of myeloid blasts in bone marrow, blood, and other tissue. Myeloid leukemias develop from changes in cells that normally produce NEUTROPHILS; BASOPHILS; EOSINOPHILS; and MONOCYTES. Leukemia, Myelogenous, Acute,Leukemia, Nonlymphocytic, Acute,Myeloid Leukemia, Acute,Nonlymphocytic Leukemia, Acute,ANLL,Acute Myelogenous Leukemia,Acute Myeloid Leukemia,Acute Myeloid Leukemia with Maturation,Acute Myeloid Leukemia without Maturation,Leukemia, Acute Myelogenous,Leukemia, Acute Myeloid,Leukemia, Myeloblastic, Acute,Leukemia, Myelocytic, Acute,Leukemia, Myeloid, Acute, M1,Leukemia, Myeloid, Acute, M2,Leukemia, Nonlymphoblastic, Acute,Myeloblastic Leukemia, Acute,Myelocytic Leukemia, Acute,Myelogenous Leukemia, Acute,Myeloid Leukemia, Acute, M1,Myeloid Leukemia, Acute, M2,Nonlymphoblastic Leukemia, Acute,Acute Myeloblastic Leukemia,Acute Myeloblastic Leukemias,Acute Myelocytic Leukemia,Acute Myelocytic Leukemias,Acute Myelogenous Leukemias,Acute Myeloid Leukemias,Acute Nonlymphoblastic Leukemia,Acute Nonlymphoblastic Leukemias,Acute Nonlymphocytic Leukemia,Acute Nonlymphocytic Leukemias,Leukemia, Acute Myeloblastic,Leukemia, Acute Myelocytic,Leukemia, Acute Nonlymphoblastic,Leukemia, Acute Nonlymphocytic,Leukemias, Acute Myeloblastic,Leukemias, Acute Myelocytic,Leukemias, Acute Myelogenous,Leukemias, Acute Myeloid,Leukemias, Acute Nonlymphoblastic,Leukemias, Acute Nonlymphocytic,Myeloblastic Leukemias, Acute,Myelocytic Leukemias, Acute,Myelogenous Leukemias, Acute,Myeloid Leukemias, Acute,Nonlymphoblastic Leukemias, Acute,Nonlymphocytic Leukemias, Acute
D016923 Cell Death The termination of the cell's ability to carry out vital functions such as metabolism, growth, reproduction, responsiveness, and adaptability. Death, Cell

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