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| List of contributors | |
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| List of abbreviations | |
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| Principles of flow cytometry | |
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| History and development of flow cytometry | |
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| Principles of flow cytometry | |
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| Fluorescence analysis | |
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| Light scatter and fluorescence detection | |
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| Acquisition | |
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| Amplification | |
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| Histograms | |
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| Coefficients of variation | |
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| Spectral overlap and compensation | |
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| Safety aspects of lasers | |
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| Cell sorting | |
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| Commercial flow cytometers | |
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| References | |
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| Introduction to the general principles of sample preparation | |
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| Introduction | |
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| Factors affecting the choice of preparation procedure | |
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| Blood and bone marrow | |
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| Preparation of cell suspensions from solid tissues and cell cultures | |
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| Live samples | |
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| Fixed samples | |
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| Permeabilisation techniques for immunolabelling intracellular components | |
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| Immunolabelling | |
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| Determining absolute cell counts | |
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| Safety | |
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| Further reading | |
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| Internet resources | |
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| References | |
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| Fluorescence and fluorochromes | |
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| Introduction | |
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| The interaction of light and matter | |
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| Light absorption and fluorescence | |
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| Energy transfer by resonance between fluorochromes | |
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| Fluorochrome options for cytometric applications | |
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| The characteristics of fluorochromes used for conjugating to antibodies and other proteins | |
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| Coupling fluorochromes to antibodies or other proteins | |
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| Linking antibodies to fluorochromes through protein A or G, avidin--or streptavidin--biotin links | |
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| Fluorescent dyes for labelling nucleic acids | |
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| Probes for cell viability and death: necrosis and apoptosis | |
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| Fluorescent dyes for the detection of intracellular ions | |
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| Probes for phagocytosis and oxidative metabolism | |
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| Fluorochrome-labelled substrate analogues for measuring enzyme activity | |
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| Fluorescent ligands | |
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| Fluorescent dyes for measuring total protein | |
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| Autofluorescence | |
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| Further reading | |
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| Internet sites | |
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| References | |
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| Quality control in flow cytometry | |
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| Introduction | |
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| Internal quality control | |
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| Instrument quality control | |
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| Quality control issues and pitfalls | |
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| External quality assessment | |
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| Reagent selection | |
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| Definition of positive values | |
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| Absolute count enumeration | |
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| Conclusion | |
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| References | |
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| Data analysis in flow cytometry | |
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| Introduction | |
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| Experiment design | |
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| Analysis of flow cytometric cell phenotyping data | |
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| Cluster analysis | |
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| Measurement of fluorescent intensity | |
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| Standardisation | |
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| Logarithmic and linear amplification | |
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| Fluorescence calibration and quantification | |
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| Conclusions | |
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| References | |
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| Laser scanning cytometry: application to the immunophenotyping of hematological malignancies | |
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| General considerations for the immunophenotyping of leukaemias and lymphomas | |
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| The laser scanning cytometer | |
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| Immunophenotyping by laser scanning cytometry | |
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| Extensions to the basic method | |
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| Conclusions | |
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| Internet sites | |
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| References | |
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| Leukocyte immunobiology | |
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| Introduction to human leukocytes and leukocyte antigens | |
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| Monoclonal antibodies and clusters of differentiation | |
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| Surface antigen changes during hematopoiesis | |
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| Progenitor cell differentiation | |
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| Myeloid maturation | |
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| Blymphocyte maturation | |
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| Tlymphocyte maturation | |
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| Natural killer cell maturation | |
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| Dendritic cell maturation | |
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| Peripheral blood lymphocyte subpopulations: normal ranges | |
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| The identification of leukocyte subclasses by flow cytometry | |
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| Analysis of surface and intracellular molecules | |
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| Characteristic changes in lymphocyte subsets in disease | |
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| The identification and quantification of lymphocyte functional subsets | |
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| Measurement of leukocyte activation | |
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| Human hemopoietic cell lines | |
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| References | |
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| Immunophenotypic analysis of leukocytes in disease | |
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| Introduction | |
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| Classification of leukaemias | |
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| Acute myeloid leukaemia | |
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| The classification of B-cell acute lymphoblastic leukaemia | |
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| T-lineage acute lymphoblastic leukaemia | |
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| Biphenotypic and undifferentiated acute leukaemia | |
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| B-cell lymphomas and chronic leukaemias | |
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| Chronic T-cell leukaemias | |
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| Myeloproliferative disorders | |
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| Flow cytometric analysis of leukaemic samples | |
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| Therapeutic monitoring | |
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| Diagnosis of other hematological disorders | |
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| Analysis of genetic variation in polymorphic markers | |
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| Transplantation crossmatching | |
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| References | |
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| Analysis and isolation of minor cell populations | |
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| Introduction | |
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| Application of statistical equations to conventional situations | |
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| Application of statistical equations to small subpopulations (rare events) | |
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| Practical examples illustrating different features of rare event analysis | |
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| The statistics of sorting using a flow cytometer | |
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| Conclusions | |
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| References | |
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| Cell cycle, DNA and DNA ploidy analysis | |
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| Introduction | |
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| Cell cycling | |
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| Cell cycle regulation | |
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| Flow cytometric analysis of the cell cycle | |
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| DNA content to determine cell cycle distribution | |
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| S phase analysis using bromodeoxyuridine uptake | |
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| Numerical chromosomal aberrations | |
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| Conclusions | |
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| References | |
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| Cell viability, necrosis and apoptosis | |
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| Introduction | |
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| Apoptosis | |
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| Genetic control of apoptosis | |
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| Bcl-2 expression and apoptosis | |
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| Necrosis | |
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| Mitochondrial membrane potential, cytochrome c and cell death | |
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| Flow cytometry | |
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| Protocols | |
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| References | |
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| Phagocyte biology and function | |
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| Introduction to phagocyte biology | |
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| Maturational states of neutrophils and monocytes | |
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| Expression of functional antigens and receptors on the cell surface | |
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| Receptor signalling | |
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| Priming and activation | |
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| Prolonged responses to cytokines and/or hormones | |
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| Chemoattractant binding and rapid responses to chemotaxins/activators | |
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| Shape changes | |
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| Membrane potential and changes in ion permeability | |
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| Phagocytosis, endocytosis and oxidative burst | |
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| Nitric oxide release | |
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| Multiparameter techniques to assess function and phenotype | |
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| Clinical applications of phagocyte function assays | |
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| Neutrophil antibodies | |
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| Clinical applications of flow cytometry to the study of autoimmune neutropenia | |
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| Neutrophil IgG Fc receptor expression and function | |
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| Conclusions | |
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| References | |
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| Intracellular measures of signalling pathways | |
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| Introduction | |
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| Regulatory pathways | |
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| Detection of intracellular protein epitopes | |
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| Determination of DNA and protein content | |
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| Assessing the physiological status of cells | |
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| Monitoring physiological changes during cell death | |
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| Immunodetection of target molecules and pathways | |
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| Protocols | |
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| Authors' comments | |
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| References | |
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| Cell-cell interactions | |
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| Adhesion molecules in cell-cell interactions | |
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| Analysis of cell-cell interactions | |
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| Platelet-platelet interactions | |
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| Platelet activation in clinical disorders | |
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| Flow cytometric analysis of platelet activation | |
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| Methods for the analysis of platelet adhesion and activation molecules | |
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| Platelet-leukocyte interactions | |
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| Leukocyte-leukocyte interactions | |
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| Leukocyte-endothelial cell interactions | |
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| Dysregulated cell-cell interactions and adhesion molecule deficiency states | |
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| Defects in human integrins | |
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| Defects in other human adhesion molecules | |
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| Pharmacologically induced deficiency states | |
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| Analysis of platelet-directed antibodies | |
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| Fluorescence resonance energy transfer | |
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| Clinical relevance of platelet-directed antibodies | |
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| References | |
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| Nucleic acids | |
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| Introduction | |
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| Flow karyotyping | |
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| Fluorescence in situ hybridisation analyses with cells in suspension | |
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| Approaches using in situ polymerase chain reaction or reverse transcriptase polymerase chain reaction with cells in suspension | |
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| Conclusions | |
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| References | |
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| Microbial infections | |
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| Introduction | |
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| Viruses | |
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| Bacteria | |
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| Yeasts | |
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| Protozoa | |
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| The immune response to infection | |
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| Conclusions | |
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| Internet sites | |
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| References | |
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| Leucocyte cell surface antigens | |
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| Introduction | |
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| Modules and domains | |
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| Clusters of differentiation | |
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| References | |
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| Recent and future developments: conclusions | |
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| Introduction | |
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| Developments in fluorescent labels | |
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| Gaining additional information from light scatter and fluorescence signals | |
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| Portable flow cytometers | |
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| Microscale analysis and sorting using miniature fluidic circuits | |
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| New static cytometers | |
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| New applications of existing technologies | |
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| Current and future clinical needs | |
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| Conclusions | |
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| Internet sites | |
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| References | |
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| Appendix | |
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| Useful Internet sites | |
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| Sites of general interest for cytometry | |
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| Suppliers of instruments, reagents, antibodies etc. | |
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| Index | |