Flow cytometry has been the premier tool for single cell analysis since its invention in the 1960s. It has maintained this position through steady advances in technology and applications, becoming the main force behind interrogating the complexities of the immune system. Technology development was a three-pronged effort, including the hardware, reagents, and analysis algorithms to allow measurement of as many as 20 independent parameters on each cell, at tens of thousands of cells per second. In the coming years, cytometry technology will integrate with other techniques, such as transcriptomics, metabolomics, and so forth. Ongoing efforts are aimed at algorithms to analyse these aggregated datasaets over large numbers of samples. Here we review the development efforts heralding the next stage of flow cytometry.
"As mentioned above, flow cytometry has been the leading method for measuring this diversity over the past three decades. The latest instruments are remarkably powerful, capable of analyzing and/or sorting cells based on up to ∼18 cellular parameters at >10,000 cells per second4. Such high-dimensional and high-throughput analysis revealed relationships between T-cell phenotype/function and clinical status for a range of diseases. "
[Show abstract][Hide abstract] ABSTRACT: Adaptive immune responses often begin with the formation of a molecular complex between a T-cell receptor (TCR) and a peptide antigen bound to a major histocompatibility complex (MHC) molecule. These complexes are highly variable, however, due to the polymorphism of MHC genes, the random, inexact recombination of TCR gene segments, and the vast array of possible self and pathogen peptide antigens. As a result, it has been very difficult to comprehensively study the TCR repertoire or identify and track more than a few antigen-specific T cells in mice or humans. For mouse studies, this had led to a reliance on model antigens and TCR transgenes. The study of limited human clinical samples, in contrast, requires techniques that can simultaneously survey TCR phenotype and function, and TCR reactivity to many T-cell epitopes. Thanks to recent advances in single-cell and cytometry methodologies, as well as high-throughput sequencing of the TCR repertoire, we now have or will soon have the tools needed to comprehensively analyze T-cell responses in health and disease.
"Building cytometry panels (either user-assembled or with pre-prepared lyophilized reagent cocktails, such as Lyoplates™ from BD Biosciences) should include critical consideration of monoclonal antibody clones and the appropriate conjugate fluorescent detection dyes, as well as maintaining and monitoring consistent reagent performance as has been elaborated elsewhere (Kaminski et al., 2012; Maecker et al., 2012). Also described elsewhere are instrument set-up considerations and the extent of controls needed, which are familiar to many flow cytometry users (Perfetto et al., 2006; Maecker et al., 2010, 2012; Chattopadhyay and Roederer, 2012), but become extremely important to maintain consistency in large studies. These elements include single-color instrument compensation controls, fluorescence-minus-one controls that help determine the lower-limits of a positive stain when gating, and monitoring and documenting instrument set-up and performance on different procedure days of a study. "
[Show abstract][Hide abstract] ABSTRACT: To advance our understanding and treatment of disease, research immunologists have been called-upon to place more centralized emphasis on impactful human studies. Such endeavors will inevitably require large-scale study execution and data management regulation ("Big Biology"), necessitating standardized and reliable metrics of immune status and function. A well-known example setting this large-scale effort in-motion is identifying correlations between eventual disease outcome and T lymphocyte phenotype in large HIV-patient cohorts using multiparameter flow cytometry. However, infection, immunodeficiency, and autoimmunity are also characterized by correlative and functional contributions of B lymphocytes, which to-date have received much less attention in the human Big Biology enterprise. Here, we review progress in human B cell phenotyping, analysis, and bioinformatics tools that constitute valuable resources for the B cell research community to effectively join in this effort.
Frontiers in Immunology 10/2012; 3:302. DOI:10.3389/fimmu.2012.00302
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.