CYTO 2025 Tutorials: Basics of Quantitative Flow Cytometry - From Instrument Qualification and Calibration to Assay Optimization and Validation

Basics of Quantitative Flow Cytometry: From Instrument Qualification and Calibration to Assay Optimization and Validation

John Nolan, PhD - Professor, Scintillon Institute
Virginia Litwin, PhD - Director, Scientific Affairs Eurofins

The roots of the word “cytometry” mean cell measurement and, in terms of quantitative rigor, flow cytometry is arguably the most cytometry method, providing quantitative measurements of cell number, fluorescence, and light scatter that are essential to its use in clinical and other high impact applications. However, flow cytometry as generally practiced is rarely quantitative or rigorous, and is most often used in a descriptive manner, with cell measurements being relative and semi-quantitative at best or, at worst, difficult to interpret and impossible to reproduce. This situation is not due to a lack of knowledge or understanding, but rather due to a lack of education.

In this tutorial we will review the basic elements of flow cytometer configuration and calibration and assay design, optimization, and validation. Topics to be covered include: qualifying a flow cytometer, calibrating and determining optimal settings for sensitivity, resolution, and dynamic range, estimating limits of detection for particle number and brightness, assay and reagent design, optimization, and validation, with examples from basic, translational, and clinical research. Upon completion, attendees will have the knowledge and resources to effectively configure and calibrate a flow cytometer, and to use it to make a quantitative and reproducible cell measurement.

CMLE Credit: 1.0