CYTO 2025 Tutorials: Debunking Myths About Data Spread

Debunking Myths About Data Spread |

Diana L. Bonilla Escobar, PhD - Scientific Director, Cytek Biosciences
Dave Novo-Lake, PhD - De Novo Research

Spread assessment is critical to understand and troubleshoot data resolution in multicolor flow cytometry, being essential in panel design and assay optimization, as well as instrument optical performance evaluation. Spread manifest as dispersion in the distribution of a population and is caused by the stochastic nature of fluorescence emission and the extent of the spectral overlap between fluorochromes. A large amount of spread can severely impact resolution, especially for dimly expressed markers. In a multiparametric assay, the amount of spread can be influenced by multiple factors including the cytometer optical performance, the instrument setup, the choice of fluorochromes, the biology of the samples, the staining conditions, the extraction of autofluorescence, and the mathematical algorithm used to calculate the abundance of each fluorochrome in fully stained samples. In this tutorial, we will summarize the fundamentals of data spreading and evaluate in detail the impact of multiple factors in the extent of the spread. In addition, we will provide recommendations on how to mitigate spread to generate high quality multicolor flow cytometry assays and accurate result interpretation.

CMLE Credit: 1.0