Spectral compensation for flow cytometry: Visualization artifacts, limitations, and caveats

Background: In multicolor flow cytometric analysis, compensation for spectr al overlap is nearly always necessary. For the most part, such compensation has been relatively simple, producing the desired rectilinear distribution s. However, in the realm of multicolor analysis, visualization of compensat ed often results in unexpected distributions, principally the appearance of a large number of events on the axis, and even more disconcerting, an inab ility to bring the extent of compensated data down to "autofluorescence" le vels. Materials and Methods: A mathematical model of detector measurements with v ariable photon intensities, spillover parameters, measurement errors, and d ata storage characteristics was used to illustrate sources of apparent erro r in compensated data. Immunofluorescently stained cells were collected und er conditions of limiting light collection and high spillover between detec tors to confirm aspects of the model. Results: Photon-counting statistics contribute a nonlinear error to compens ated parameters. Measurement errors and log-scale binning error contribute linear errors to compensated parameters. These errors are most apparent wit h the use of red or far-red fluorochromes (where the emitted light is at lo w intensity) and with large spillover between detectors. Such errors can le ad to data visualization artifacts that can easily lead to incorrect conclu sions about data, and account for the apparent "undercompensation" previous ly described for multicolor staining. Conclusions: There are inescapable errors arising from imperfect measuremen ts, photon-counting statistics, and even data storage methods that contribu te both linearly and nonlinearly to a "spreading" of a properly compensated autofluorescence distribution. This phenomenon precludes the use of "quadr ant" statistics or gates to analyze affected data; it also precludes visual adjustment of compensation. Most importantly, it is impossible to properly compensate data Using standard visual graphical interfaces (histograms or dot plots). Computer-assisted compensation is required, as well as careful gating and experimental design to determine the distinction between positiv e and negative events. Finally, the use of special staining controls that e mploy all reagents except for the one of interest (termed fluorescence minu s one, or "FMO" controls) becomes necessary to accurately identify expressi ng cells in the fully stained sample, Cytometry 45: 194 - 205, 2001. (C) 20 01 Wiley-Liss, Inc.