FCS Format

FCS Files

Example compensated FCS data from Human FFPE

Flow Cytometry Standard (FCS) is a file standard specified by the International Society for the Advancement of Cytometry Data Standards Task Force, last updated to FCS 3.1 in 2011. Publication is available here. CODEX® outputs FCS files in addition to CSV files for compatibility with many flow cytometry analysis platforms.

CODEX® FCS files contain integrated expression intensity of all markers for all cells. For each region, there are two FCS files, compensated and uncompensated. Compensated FCS file contains expressions compensated for spatial fluorescence spillover, while uncompensated FCS file contains the raw integrated expressions. In addition to marker expression, FCS files contain cell identifiers and metadata, such as cell size and location.

FCS files can be exported from CODEX® MAV.

Third-party FCS files requirements

All FCS files provided for the 'Import clustering' operation in CodexMAV must meet the following requirements:

1. All files for a single clustering run shall be stored in one folder.

2. Clustering run folder shall have a name, that has not yet been used by another clustering run folder in experiment.

3. There shall be one FCS file for each region in the experiment.

4. The name of each file shall start with the 'regXXX_' prefix.

5. There shall be no duplicate files for any region.

6. 'X', 'Y' and 'Z'' (case insensitive) columns shall be presented in each file.

7. Each file shall contain a column which will be used as the 'ClusterID'. User will be able to select this column through the combo box.

8. If all cells from the experiment are presented in clustering FCS files, then these cells shall be arranged in the same order as in the original FCS files.

9. If clustering FCS files don't contain all cells from the experiment, then the cells can be arranged in any order.

10. All marker names must meet the following requirements:

    a. Long and short names must be separated by ':' character (e.g. 'CollagenIV:cyc001_ch002').

    b. Short name must be written using one of two naming conventions:

    1. Cyc_X_Ch_X (case insensitive)

    2. cycXXX_chXXX (case insensitive)