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Journal ArticleDOI

Enhanced flowType/RchyOptimyx: a BioConductor pipeline for discovery in high-dimensional cytometry data.

01 May 2014-Bioinformatics (Oxford University Press)-Vol. 30, Iss: 9, pp 1329-1330

TL;DR: A significantly improved version of the flowType and RchyOptimyx BioConductor-based pipeline is presented that is both 14 times faster and can accommodate multiple levels of biomarker expression for up to 96 markers, positioned to be an integral part of data analysis for high-throughput experiments on high-dimensional single-cell assay platforms.

AbstractWe present a significantly improved version of the flowType and RchyOptimyx BioConductor-based pipeline that is both 14 times faster and can accommodate multiple levels of biomarker expression for up to 96 markers. With these improvements, the pipeline is positioned to be an integral part of data analysis for high-throughput experiments on high-dimensional single-cell assay platforms, including flow cytometry, mass cytometry and single-cell RT-qPCR.

Topics: Bioconductor (60%), Mass cytometry (52%)

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Journal ArticleDOI
Andrea Cossarizza1, Hyun-Dong Chang, Andreas Radbruch, Andreas Acs2  +459 moreInstitutions (160)
TL;DR: These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community providing the theory and key practical aspects offlow cytometry enabling immunologists to avoid the common errors that often undermine immunological data.
Abstract: These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.

483 citations


Journal ArticleDOI
Abstract: The marriage between immunology and cytometry is one of the most stable and productive in the recent history of science. A rapid search in PubMed shows that, as of July 2017, using “flow cytometry immunology” as a search term yields more than 68 000 articles, the first of which, interestingly, is not about lymphocytes. It might be stated that, after a short engagement, the exchange of the wedding rings between immunology and cytometry officially occurred when the idea to link fluorochromes to monoclonal antibodies came about. After this, recognizing different types of cells became relatively easy and feasible not only by using a simple fluorescence microscope, but also by a complex and sometimes esoteric instrument, the flow cytometer that is able to count hundreds of cells in a single second, and can provide repetitive results in a tireless manner. Given this, the possibility to analyse immune phenotypes in a variety of clinical conditions has changed the use of the flow cytometer, which was incidentally invented in the late 1960s to measure cellular DNA by using intercalating dyes, such as ethidium bromide. The epidemics of HIV/AIDS in the 1980s then gave a dramatic impulse to the technology of counting specific cells, since it became clear that the quantification of the number of peripheral blood CD4+ T cells was crucial to follow the course of the infection, and eventually for monitoring the therapy. As a consequence, the development of flow cytometers that had to be easy-to-use in all clinical laboratories helped to widely disseminate this technology. Nowadays, it is rare to find an immunological paper or read a conference abstract in which the authors did not use flow cytometry as the main tool to dissect the immune system and identify its fine and complex functions. Of note, recent developments have created the sophisticated technology of mass cytometry, which is able to simultaneously identify dozens of molecules at the single cell level and allows us to better understand the complexity and beauty of the immune system.

382 citations


01 Jan 2017
TL;DR: It is rare to find an immunological paper or read a conference abstract in which the authors did not use flow cytometry as the main tool to dissect the immune system and identify its fine and complex functions, and recent developments have created the sophisticated technology of mass cytometry.

327 citations


Journal ArticleDOI
TL;DR: This Review provides non-experts with a broad and practical overview of the many recent developments in computational flow cytometry.
Abstract: Recent advances in flow cytometry allow scientists to measure an increasing number of parameters per cell, generating huge and high-dimensional datasets. To analyse, visualize and interpret these data, newly available computational techniques should be adopted, evaluated and improved upon by the immunological community. Computational flow cytometry is emerging as an important new field at the intersection of immunology and computational biology; it allows new biological knowledge to be extracted from high-throughput single-cell data. This Review provides non-experts with a broad and practical overview of the many recent developments in computational flow cytometry.

319 citations


Journal ArticleDOI
TL;DR: The phenotypic signature of hu MG was identified, which was distinct from peripheral myeloid cells but was comparable to fresh huMG, and microglia regional heterogeneity was identified.
Abstract: Microglia, the specialized innate immune cells of the CNS, play crucial roles in neural development and function. Different phenotypes and functions have been ascribed to rodent microglia, but little is known about human microglia (huMG) heterogeneity. Difficulties in procuring huMG and their susceptibility to cryopreservation damage have limited large-scale studies. Here we applied multiplexed mass cytometry for a comprehensive characterization of postmortem huMG (103 - 104 cells). We determined expression levels of 57 markers on huMG isolated from up to five different brain regions of nine donors. We identified the phenotypic signature of huMG, which was distinct from peripheral myeloid cells but was comparable to fresh huMG. We detected microglia regional heterogeneity using a hybrid workflow combining Cytobank and R/Bioconductor for multidimensional data analysis. Together, these methodologies allowed us to perform high-dimensional, large-scale immunophenotyping of huMG at the single-cell level, which facilitates their unambiguous profiling in health and disease.

165 citations


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TL;DR: K shortest paths are given for finding the k shortest paths connecting a pair of vertices in a digraph, and applications to dynamic programming problems including the knapsack problem, sequence alignment, and maximum inscribed polygons are described.
Abstract: We give algorithms for finding the k shortest paths (not required to be simple) connecting a pair of vertices in a digraph. Our algorithms output an implicit representation of these paths in a digraph with n vertices and m edges, in time O(m+n log n+k). We can also find the k shortest paths from a given source s to each vertex in the graph, in total time O(m+n log n+kn). We describe applications to dynamic programming problems including the knapsack problem, sequence alignment, and maximum inscribed polygons. >

708 citations


"Enhanced flowType/RchyOptimyx: a Bi..." refers methods in this paper

  • ...RchyOptimyx uses a dynamic programing algorithm for efficiently constructing k-shortest paths (Eppstein, 1998)....

    [...]


Journal ArticleDOI
TL;DR: These cytometric technologies, capable of high-content, high-throughput single-cell assays, and a new technology that promises to extend these capabilities significantly are reviewed.
Abstract: In recent years, advances in technology have provided us with tools to quantify the expression of multiple genes in individual cells. The ability to measure simultaneously multiple genes in the same cell is necessary to resolve the great diversity of cell subsets, as well as to define their function in the host. Fluorescence-based flow cytometry is the benchmark for this; with it, we can quantify 18 proteins per cell, at >10 000 cells/s. Mass cytometry is a new technology that promises to extend these capabilities significantly. Immunophenotyping by mass spectrometry provides the ability to measure >36 proteins at a rate of 1000 cells/s. We review these cytometric technologies, capable of high-content, high-throughput single-cell assays.

538 citations


"Enhanced flowType/RchyOptimyx: a Bi..." refers background in this paper

  • ...Since then, mass cytometry has enabled measurement of 30–45markers/cell (Bendall et al., 2012), whereas single-cellmultiplexed RT-qPCR can measure 50–96 messenger RNAs/cell (White et al., 2011)....

    [...]


Journal ArticleDOI
TL;DR: Several methods performed well as compared to manual gating or external variables using statistical performance measures, which suggests that automated methods have reached a sufficient level of maturity and accuracy for reliable use in FCM data analysis.
Abstract: Traditional methods for flow cytometry (FCM) data processing rely on subjective manual gating. Recently, several groups have developed computational methods for identifying cell populations in multidimensional FCM data. The Flow Cytometry: Critical Assessment of Population Identification Methods (FlowCAP) challenges were established to compare the performance of these methods on two tasks: (i) mammalian cell population identification, to determine whether automated algorithms can reproduce expert manual gating and (ii) sample classification, to determine whether analysis pipelines can identify characteristics that correlate with external variables (such as clinical outcome). This analysis presents the results of the first FlowCAP challenges. Several methods performed well as compared to manual gating or external variables using statistical performance measures, which suggests that automated methods have reached a sufficient level of maturity and accuracy for reliable use in FCM data analysis.

503 citations


"Enhanced flowType/RchyOptimyx: a Bi..." refers methods in this paper

  • ...FlowType uses partitioning of cells, either manually or by clustering, into positive or negative for each marker to enumerate all cell types in a sample, e.g. Aghaeepour et al. (2013)....

    [...]


Journal ArticleDOI
TL;DR: This work presents a fully integrated microfluidic device capable of performing high-precision RT-qPCR measurements of gene expression from hundreds of single cells per run, and shows that nanoliter volume processing reduced measurement noise, increased sensitivity, and provided single nucleotide specificity.
Abstract: A long-sought milestone in microfluidics research has been the development of integrated technology for scalable analysis of transcription in single cells Here we present a fully integrated microfluidic device capable of performing high-precision RT-qPCR measurements of gene expression from hundreds of single cells per run Our device executes all steps of single-cell processing, including cell capture, cell lysis, reverse transcription, and quantitative PCR In addition to higher throughput and reduced cost, we show that nanoliter volume processing reduced measurement noise, increased sensitivity, and provided single nucleotide specificity We apply this technology to 3,300 single-cell measurements of (i) miRNA expression in K562 cells, (ii) coregulation of a miRNA and one of its target transcripts during differentiation in embryonic stem cells, and (iii) single nucleotide variant detection in primary lobular breast cancer cells The core functionality established here provides the foundation from which a variety of on-chip single-cell transcription analyses will be developed

459 citations