Flow Cytometer Performance Characterization, Standardization, and Control

Flow cytometry is a widely used technique for the analysis of single cells and particles. It is an essential tool for immunological research, drug and device development, clinical trials, disease diagnosis, and therapy monitoring. However, measurements made on different instrument platforms are often inconsistent, leading to variable results for the same sample on different instruments and impeding advances in biomedical research. This chapter describes methodologies to obtain key parameters for characterizing flow cytometer performance, including precision, sensitivity, background, electronic noise, and linearity. Further, various fluorescent beads, hard dyed and surface labeled, are illustrated for use in quality control, calibration, and standardization of flow cytometers. To compare instrument characteristics, fluorescence intensity units have to be standardized to mean equivalent soluble fluorochrome (MESF) or equivalent reference fluorophore (ERF) units that are traceable to the existing primary fluorophore solution standards. With suitable biological controls or orthogonal method, users will be able to quantitatively measure DNA and RNA content per cell or biomarker expression in antibodies bound per cell. Comparable, reproducible, and quantitative measurements using flow cytometers can be accomplished only upon instrument standardization through performance characterization and calibration, and use of proper biological controls.