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Data Standards
Data Standards
Flow Cytometry Standard (FCS)

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.

For more information, see the normative version of the FCS 3.1 specification, and the FCS 3.1 Implementation Guidance

ISAC Classification Results File Format (CLR)

Identifying homogenous sets of cell populations in flow cytometry is an important process for sorting and selecting populations of interests for further data acquisition and analysis. Many computational methods are now available to automate this process, with several algorithms partitioning cells based on high-dimensional separation versus the traditional pairwise two-dimensional visualization approach of manual gating.

ISAC’s Classification Results File Format (CLR) was developed to exchange the results of both manual gating and algorithmic classification approaches in a standardized way based on per event based classifications, including the potential for soft classifications expressed as the probability of an event being a member of a class.

For more information, visit:

Gating-ML 2.0

The lack of software interoperability with respect to gating has traditionally been a bottleneck preventing the use of multiple analytical tools and reproducibility of flow cytometry data analysis by independent parties. To address this issue, ISAC developed Gating-ML, a computer file format to encode and interchange gates.

Gating-ML 1.5 was adopted and published as an ISAC Candidate Recommendation in 2008. Feedback during the probationary period from implementors—including major commercial software companies, instrument vendors and the wider community—has led to a streamlined Gating-ML 2.0. Gating-ML has been significantly simplified and therefore easier to support by software tools.

To aid developers, free open-source reference implementations, compliance tests, and detailed examples are provided to stimulate further commercial adoption. ISAC has approved Gating-ML as a standard ready for deployment in the public domain and encourages its support within the community as it is at a mature stage of development having undergone extensive review and testing, under both theoretical and practical conditions.

For more information, visit:

MIFlowCyt Standard

A fundamental tenet of scientific research is that published results are open to independent validation and refutation. Minimum data standards aid data providers, users, and publishers by providing a specification of what is required to unambiguously interpret experimental findings. The Minimum Information about a Flow Cytometry Experiment (MIFlowCyt) standard officially states the minimum information required to report flow cytometry (FCM) experiments. ISAC developed the standard in 2008, and it was subsequently vetted by ISAC's Data Standards Task Force, Standards Committee, membership, and Council.

Image Cytometry Experiment Format (ICEFormat)

In image cytometry, most instruments produce images and data in proprietary file formats, making researchers dependent on analytical software provided by the specific hardware manufacture, which can hinder the independent validation of experimental results. One of ISAC's projects has been to develop a data format standard for image cytometry in order to promote data sharing and interoperability.

Text of the specification providing a detailed description of the ICEFormat:

Full specification of the ICEFormat including XML schemas and informative parts, such as examples and XML schema documentation:

Calibration Standards

Fluorescence Intensity Calibrations For the calibration of fluorescence measurements, and flow cytometry measurements especially, the use of the Mean Equivalent Soluble Fluorophoes (MESF) as an intensity measurement standard has facilitated the reporting of absolute intensity measurements. Reference particles whose intensities are calibrated in MESF units are available for a few of the most frequently used fluorophores (for example, fluorescein and phycoerythrin), but extension of this approach to highly multiparameter measurements is limited by the availability of reference materials.

An alternative approach is to use a calibration particle with a broad emission spectrum and referenced to a single reference fluorophore to calibrate the response of multiple fluorescence channels over defined spectral ranges. We refer to this as the Equivalent Reference Fluorophore (ERF) unit. The ERF unit has practical advantages with respect to the MESF unit, most notably the possibility to use a single reference particle for calibration across multiple fluorescence channels. The ERF unit will complement the MESF unit by providing practical and usefully accurate calibration of absolute intensities for many fluorescence channels.

Calibration in ERF units is not restricted to multifluorophore beads. Fluorochrome specific beads may also be calibrated in ERF intensity units to reference ERF standards. Cross calibration of fluorochrome-specific beads (e.g. stained with fluorescein or phycoerythrin) to multifluorophore beads will be practical using ERF units.

Reference: L Wang, AK Gaigalas, G Marti, F Abbasi, and RA Hoffman. Toward Quantitative Fluorescence Measurements with Multicolored Flow Cytometry. Cytometry 73A: 279-288 (2008).