Data File Standard for Flow Cytometry, version FCS 3.1

FCS 3.2 is a revision of the flow cytometry data standard based on a decade of suggested improvements from the community as well as industry needs to capture instrument conditions and measurement features more precisely. 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 standard retains the overall FCS file structure and most features of previous versions, but also contains a few changes that were required to support new types of data and use cases efficiently. These changes are incompatible with existing FCS file readers. Notably, FCS 3.2 supports mixed data types to, for example, allow FCS measurements that are intrinsically integers (e.g., indices or class assignments) or measurements that are commonly captured as integers (e.g., time ticks) to be more represented as integer values, while capturing other measurements as floating-point values in the same FCS data set. In addition, keywords explicitly specifying dyes, detectors, and analytes were added to avoid having to extract those heuristically and unreliably from measurement names. Types of measurements were formalized, several keywords added, others removed, or deprecated, and various aspects of the specification were clarified. A reference implementation of the cyclic redundancy check (CRC) calculation is provided in two programming languages since a correct CRC implementation was problematic for many vendors. © 2020 International Society for Advancement of Cytometry.