Over the last two decades, our ability to interrogate the immune system on a single-cell level has increased dramatically (Chattopadhyay and Roederer, 2012; Bendall et al., 2011), allowing an opportunity to better understand the immunological mechanisms underlying disease. Complex flow cytometry (FCM) data are now surpassing our ability to fully analyze and interpret all information via current standard approaches, such as 2D dot plots and Boolean gates. Indeed, the number of potential cell subpopulations increases exponentially with the number of parameters assessed, making it difficult to decipher all possible combinations included in the raw data (eg, 512 potential subsets with nine markers) via the traditional approaches (Bendall and Nolan, 2012). This could limit the translation of technical advances into new diagnostics or therapies. Newly developed bioinformatics tools that have the potential to bridge this gap are now available. The aim of this letter is to foster the implementation and adoption of these novel computational methodologies for unbiased analysis of complex cytometry data.

In recent years, a host of new dataanalysis tools have emerged, creating workflows for processing and analyzing complex FCM datasets; however, these have gone mostly unnoticed by immunologists. Table S1 provides an overview of many of the currently available tools and their specific applications. They can be assigned to specific categories arranged in a ‘‘FCM data-analysis workflow’’from compensated data as input to biologically interpretable results as output. The vast majority of the listed tools for FCM data processing, analysis, and visualization are made available by the bioinformaticians at no cost and include open source code and unrestrictive software licensing, opening up these computational approaches to broad use by the research community. Many of the tools have been developed to address similar analysis objectives via quite different approaches. They might provide optimal results for different datasets, such that there is no ‘‘right’’or ‘‘best’’tool, and using