Light scatter and fluorescence calibration allow standard comparisons of small particle data between different instruments.

Flow cytometry analysis has been utilized since the late 1970s for the analysis of small particles, initially viruses before extracellular vesicles (EVs) in the 1990s. Despite its long-established use, a lack of standardization has resulted in a growing body of literature that is cannot be easily interpreted, validated, and reproduced between instrumentation. This inconsistency in data reporting greatly impacts the progress of the small particle field by making it difficult for researchers to make objective assessments on assays and instrumentation for small particle analysis. The methods and reagents for fluorescence and light scatter standardization are not novel. In this pilot study, we demonstrate calibration of small particle data with commercially available materials, published methods, and freely available software tools. We show that light scatter, fluorescence, and concentration can be calibrated and result in highly concordant data between flow cytometry platforms independent of instrument collection angle or gain/voltage; providing a means of cross-comparison in standard units.