Flow cytometry is and always has been a multiparametric technology. Increasingly simultaneous and correlated measurements can be used to provide greater detail about the phenotype of cellular subpopulations. Clinical laboratories now routinely employ 5- or 6-color flow cytometry, while research labs are experimenting with 10- to 20-color capabilities.
Innovations such as image flow cytometry and acoustically focused flow cytometry have added new dimensions and capabilities, and in some cases flow cytometers have begun to replace other instruments like microscopes.
At the same time, in order to reach its full potential, the field of flow cytometry must overcome some significant challenges. Chief among these is the abundance of data generated from increasingly large multiparametric analyses.
At the other end of the spectrum, there are some basic problems that are still being solved, such as how to accurately calculate the sensitivity of the instrument. Potential solutions to a range of problems impeding the efficient use of flow cytometry were presented at the recent “Great Lakes International Imaging and Flow Cytometry Association” conference.
Deconstructing Detection Limits
Many flow cytometer sensitivity measurements do not accurately represent the sensitivity of the instrument, according to Eric Chase, president and CEO of Cytek Development. Typically, detection limits are used to describe instrument sensitivity, equating the noise of the instrument to the mean equivalent fluorochrome (MEFL) value, which is the number of dye molecules the instrument can supposedly detect.
“That number is kind of meaningless,” according to Chase, because the detection limit is not really a function of the performance or optical sensitivity of the cytometer. It’s more a function of the design of the electronics.
“In newer flow cytometers, the way the electronics work, that number should always be zero because the newer digital flow cytometers use area systems,” Chase added. “They give a detection limit of zero.”
That doesn’t mean, however, that the instrument can pick out one or two dye molecules from background noise, which is the kind of information intended to be conveyed by a sensitivity rating.
Chase said that manufacturers continue to use detection limits because the numbers are low and it makes the instrument seem sensitive. A typical detection limit would be 100 dye molecules or less.
Chase’s method is based on values called Q and b that describe flow cytometer performance. Q is related to the ability of the instrument to capture signals, and b is a measurement of the background of the instrument. Although BD Biosciences adopted Chase’s method in its cytometer quality-control package, he says that the Q and b of the instrument do not give information that is directly actionable in the laboratory.
He described how Q and b could be used to calculate a resolution limit, analogous to MEFL, but that describes how many molecules of dye a user needs to be fully resolved from the background noise.
This is one step away from the number of antibody-binding sites on a cell that the instrument can detect, he explained. Users can calculate their own resolution limit based on their instrument’s Q and b values, but more support from instrument and reagent vendors would be necessary for determining the number of antibody sites an instrument could resolve.
Chase foresees a future where resolution limits will be industry standard and every reagent bottle will come labeled with the necessary information for making the calculation. This would represent a major advantage to the user, but first users and vendors alike will have to let go of their attachment to superlow—but not necessarily accurate—detection limits.