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• W2101565335 abstract "From its origins in the 16th century, microscopy has allowed the cell, as the basic unit of eukaryotic life and disease, to be identified and analyzed. Today, quantitative cytometric technologies, either microscope based or flow cytometric, are the most powerful tools to analyze the proliferation, physiology and differentiation of cells generally, and are particularly useful in immunopathology. In combination with monoclonal antibodies (which recognize specific gene products) conjugated to sensitive fluorescent dyes, cell types can be identified according to the genes they express. They can also be isolated using either fluorescence-activated cell sorting (FACS) or magnetic cell sorting (MACS). In the past 20 years, immunofluorescence-based cytometry and cell sorting have become 'state of the art' technologies, mostly serving to identify subsets of lymphocytes and systemic changes in the immune system. Although it is certainly of value for diagnosis and analysis of immunopathology, cytometry did have one major limitation; except in a few experimental situations, it was not possible to focus analysis on those lymphocytes that specifically recognize the relevant antigens in a normal or pathological immune reaction. This drawback has recently been overcome both for B and T lymphocytes, using antigen to identify the cells. Today, a number of exciting new technologies make it possible to analyze and isolate specifically those lymphocytes that are directly involved in the immune reaction to given antigens. These advances will spur research in arthritis considerably.Why did this take so long? The problem is twofold. First, the diversity of the immune system means that lymphocytes recognizing a particular antigen are rare. Estimations of the frequencies of cells specific for one antigen used to range from 10-5 to 10-6, based for example on limiting dilution analyses. For a number of biological and physical reasons immunofluorescence, either with antigens or antibodies, shows considerable variation in intensity. This makes it technically difficult to identify accurately rare cells of interest at frequencies below 10-3 to 10-4. Apart from that basic limitation, it is extremely time consuming to analyze a sufficient number of rare cells to obtain a reliable result.Nevertheless, experimental work [1,2,3,4] has shown that it is possible cytometrically to identify and analyze B memory lymphocytes and plasma cells that occur at very low frequencies and that recognize a particular antigen with high affinity, using native antigens conjugated to haptens or fluorochromes. The decisive technological advance in those experiments was the use of a 'parallel' cell-sorting technology (MACS), providing a nonoptical (in this case magnetic) label to enrich antigen-binding cells to make them detectable by flow cytometry and to isolate them for proof of specificity. Today, the cytometry of B lymphocytes according to antigen specificity is not so much a problem of technology as of biology, because B cells that bind to one particular antigen often occur at frequencies of 1-10/ml blood, thus making the availability of sufficient blood for analysis a limiting factor.A second challenge for antigen-specific cytometry has been the fact that the antigen receptors of T lymphocytes recognize fragments of antigen only in the context of either major histocompatibility complex (MHC) class I or class II molecules. Initial attemps to use recombinant and labelled MHC molecules, and to load them with peptides of interest to stain peptide-specific T cells failed in the late 1980s. Recently, not only has the direct labelling of T cells with MHC-peptide complexes finally been achieved, but also alternative technologies have been developed that identify T cells that react to particular antigens by DNA synthesis, proliferation or cytokine expression. In combination or alone, those technologies now offer unique options to analyze antigen-specific T lymphocytes directly ex vivo, and to isolate them for molecular and functional studies. Innovative diagnostic and therapeutic strategies based on the identification and isolation of antigen-reactive lymphocytes can now be developed, targeted at the smallest functional unit of immunological disease: the cell. These technologies will have a profound impact not only in arthritis research, but also on research of numerous other diseases." @default.
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• W2101565335 doi "https://doi.org/10.1186/ar7" @default.
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