Data File Standard for Flow Cytometry, version FCS 3.1

Abstract

The flow cytometry data file standard provides the specifications needed to completely describe flow cytometry data sets within the confines of the file containing the experimental data. In 1984, the first Flow Cytometry Standard format for data files was adopted as FCS 1.0. This standard was modified in 1990 as FCS 2.0 and again in 1997 as FCS 3.0. We report here on the next generation flow cytometry standard data file format. FCS 3.1 is a minor revision based on suggested improvements from the community. The unchanged goal of the standard is to provide a uniform file format that allows files created by one type of acquisition hardware and software to be analyzed by any other type. The FCS 3.1 standard retains the basic FCS file structure and most features of previous versions of the standard. Changes included in FCS 3.1 address potential ambiguities in the previous versions and provide a more robust standard. The major changes include simplified support for international characters and improved support for storing compensation. The major additions are support for preferred display scale, a standardized way of capturing the sample volume, information about originality of the data file, and support for plate and well identification in high throughput, plate based experiments. Please see the normative version of the FCS 3.1 specification in Supporting Information for this manuscript (or at http://www.isac-net.org/ in the Current standards section) for a complete list of changes. ' 2009 International Society for Advancement of Cytometry

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“…Nevertheless, there is a great need for both sharing of raw data, as well as analysis workflows to enable reproducible research in immunology. Computational approaches towards flow cytometry depend on highly standardized data formats and workflows, and since 1984 the Flow Cytometry Standard (FCS) file format has emerged as a standard for the exchange of raw data 15 . Since 2010, the International Society for Advancement of Cytometry (ISAC) has developed the Archival Cytometry Standard (ACS) file format as a container that stores all files relevant to an experiment, including support for audit trails, versioning and digital signatures 16 .…”

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“…These reporter ions are quantified by time of flight mass spectrometry [34] and recorded in an IMD format file. These data are typically parsed into single cell events and converted to a flow cytometry standard (FCS) file for analysis [35]. Many software programs can handle FCS files, including Cytobank (www.cytobank.org), FlowJo (www.FlowJo.com), R/Bioconductor, MATLAB, Cytoscape, and GenePattern (http://genepattern.broadinstitute.org/) [36].…”

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“…The raw data acquired from the flow cytometer needs to be processed to obtain data for independent evaluation [30]. To ensure that the data can be evaluated, list mode data files, preferably in '.fcs' format, are processed by using software from the vendor of the flow cytometer or by using software from a third party, for example, FlowJo™ (Ashland, OR, USA), FACSDiva™ (Becton Dickinson, SJ, USA), Infinicyt™ (Becton Dickinson), CellQuest Pro™ (Becton Dickinson), Winlist™ (Verity Software House, ME, USA), FCS Express™ (De Novo Software, CA, USA), Kaluza, Guava™ (B eckman Coulter, IN, USA) and many others [30,31].…”