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Standardization, Calibration, and Control in Flow Cytometry

Published

Author(s)

Lili Wang, Robert A. Hoffman

Abstract

Standardization, control, and calibration provide different degrees of certainty about the data acquired with an instrument. Each process is aimed at assuring that results from the instrument have the quality required for the intended purpose. The purpose may be an individual research experiment or a clinical result that determines the course of patient treatment. In the terminology used in this commentary, an instrument is standardized at certain time points and subsequently operated under quality control conditions. These processes maintain the instrument within predetermined bounds and ensure that results will vary only within certain limits. If results are also calibrated when instruments are standardized, then future results can be objectively and quantitatively compared. Results are frequently expressed either in terms of “percent positive” or in qualitative terms such as “dim” or “bright.” These terms are relative: what is considered “dim,” and “bright” in one laboratory may be quite different in another laboratory. Flow cytometers measure the amount of fluorescence and provide objective criteria for expressing results. After providing extensive background on particle types and cautions, this unit describes practical aspects of methods to standardize and calibrate flow cytometers (e.g., in terms of optical alignment, resolution, and sensitivity). Finally, suggestions are given for analyzing particles used as calibrators, including how to assign to fluorescent beads a value for molecules of equivalent soluble fluorochrome (MESF) and equivalent reference fluorophores (ERF) and how to determine the inherent fluorescence coefficient of variation (CV) of a dim bead sample.
Citation
Current Protocols in Cytometry
Created January 6, 2017, Updated February 19, 2017