Assembling a Panel for Spectral Flow Cytometry

The growing field of spectral flow cytometry allows researchers to use more fluorochromes, or fluorophores, simultaneously to detect more targets expressed by cells, compared to traditional flow cytometry. Unlike the latter, spectral flow cytometry detects the entire emission spectrum of each fluorochrome, and is better able to separate those with overlapping emission spectra, which enhances resolution.

“Spectral flow cytometry’s high-dimensional capabilities make it ideal for emerging fields like single-cell multi-omics, immuno-oncology, and infectious disease research, where understanding complex cellular landscapes is essential,” says Fanuel Messaggio, Senior Application Scientist of the Flow Cytometry Business Unit at Beckman Coulter Life Sciences. “The field of clinical diagnostics is starting to leverage spectral flow cytometry for complex disease monitoring and immune profiling in areas such as cancer.” Here are the important factors to consider when choosing a set of fluorochromes for spectral flow cytometry, and some expert troubleshooting advice.

Design considerations

To find the most favorable set of fluorophores, define the biology of the assay by researching the literature and consulting colleagues. “The more detailed and accurate the information about the biology, the higher the chances of designing an optimal panel,” says Maria Jaimes, VP Scientific Commercialization at Cytek Biosciences. Information is also available from reagent vendors; for example, Cytek^® Cloud offers spectral signatures for a majority of commercially available fluorochromes and can compare them to help researchers find unique options. “Metrics that quantify spread between fluorochromes, along with information on marker co-expression, are key for a successful panel design,” adds Jaimes.

Choosing the right fluorophore to label the antibody for each target is very important, because the spectral characteristics of different fluorophores must be carefully balanced. “It is recommended to use relatively bright fluorophores for more rare targets, or for targets that are not highly expressed in or on the cell,” says Annika Dalheim, Senior Scientist at Proteintech. “Whereas relatively dimmer fluorophores should be reserved for targets that are abundant and highly expressed on cells.” The experts agreed this is an important principle for successful spectral flow cytometry. “When designing a panel of fluorophores for spectral flow cytometry, begin with matching the antigen density to relative fluor brightness,” says Seddon Thomas, Staff Scientist, Cell Biology at Thermo Fisher Scientific. “This ensures that spectral space is not wasted in off-target channels by using excessively bright fluorophores.”

Depending on the biological question, define your markers or targets of interest, assess their relative expression levels, and determine whether they are co-expressed with other targets. Then select fluorophores with unique spectral signatures to label them. “The result of the selection process should be a combination of fluorochromes that have unique spectral signatures, varying levels of brightness, introduce the least amount of spread, and have optimal performance characteristics,” says Jaimes.

Remember that more is not necessarily better. “Avoid the temptation to use more fluorochromes than necessary to answer your biological questions,” says Rodrigo Pestana Lopes, Senior Manager in Scientific Marketing at BD. “Fluorochromes can optically interfere with each other and create more spillover spread.” Similarly, try to avoid using very bright fluorochromes. “A good panel design includes a balanced combination of dyes at all levels of brightness,” says Jaimes. Thomas agrees that “brighter is not always better—prioritizing the brightest fluorophores at the expense of other important characteristics can make panel optimization harder than it needs to be,” she says.

Also consider buffer compositions for optimal staining. “Indeed, depending on the target localization (extracellular or intracellular), a specific permeabilization buffer might be needed and users should evaluate the impact of this buffer on other marker expression or on fluorochrome brightness,” says Laurissa Ouaguia, Scientist, Reagents and Application Development
 at Agilent Technologies.

Watching for potential pitfalls

Self-education is key, because potential pitfalls lurk in lack of knowledge regarding the sample and its interaction with fluorophores. “High autofluorescent samples, like tumor specimens, are complex to work with, and building a high-parameter panel around those types of samples will require careful considerations for fluorophore combinations,” says Messaggio. Using proper controls during sample preparation can also prevent pitfalls. “An example is the appropriate use of blocking reagents, like the monocyte blocker, to avoid non-specific binding of some antibodies and fluorophores, or different staining buffer to avoid specific fluorescent dye interactions,” she says.

Pitfalls can also stem from simply not understanding how fluorochrome properties relate to instrument performance. “Since spectral flow cytometers utilize all lasers and fluorescent detectors to create a spectral signature profile of each fluorochrome used, aspects such as cross-laser excitation, dye-to-dye interactions, and potential changes in the spectral profile based on experimental conditions can introduce greater variances than in polychromatic flow cytometry,” says Lopes.

Spillover or spread from overlapping emission spectra, also called spectral overlap, and the resultant inability to fully resolve individual fluorochromes, is another common pitfall. This can make data interpretation unnecessarily complicated or introduce uncertainty into results. “The primary contributors to spreading errors are the cleanliness of the fluorophores’ spectral signatures and their brightness,” says Thomas. “Therefore, looking for fluorophores with optimized signatures, those with low cross-excitation and narrow emission spectra and the right brightness level, is a key starting point in panel design.”

A related pitfall is using fluorophores that are too spectrally similar when labeling the same cell type, which can yield ambiguous results or false positives. Whenever possible, choose fluorophores that are easily unmixed. “However, if spectrally similar fluorophores are necessary for the panel, use them on targets that are not co-expressed on the same cell type, and that are on cell populations that can be clearly distinguished from one another,” says Dalheim. She also recommends using smaller groups of antibodies first, to verify that each target is detectable without interference, before using the entire spectral flow panel.

Lopes recommends a new panel design tool called the spectral hotspot matrix, created from a metric defined as unmixing spreading error, and recently introduced in a publication that optimized a 50-fluorochrome spectral flow cytometry panel in an analysis of human immune system cells. “This tool takes the predictive evaluation of how multiple fluorochromes would interfere with each other in a panel one step further, by assessing the unmixing-dependent spreading for each fluorochrome and highlighting which combinations are problematic (the hotspot) for the spectral unmixing calculation applied, to identify the signal that pertains to each individual fluorochrome,” he says.

Troubleshooting

If you find yourself troubleshooting, understanding the limits and specifications of the spectral cytometer you’re using may help you to understand (or hopefully avoid) your conundrum. For example, “the spectral signature of a specific dye excited by a 5-laser instrument can appear different when excited by a 3-laser instrument and as such, the similarity index of two fluorochromes might not always be the same depending on the number of excitation lasers used on the spectral cytometer,” says Ouaguia.

Running the correct controls is always advisable. In particular, Dalheim recommends a control called fluorescence minus one (FMO). “For each target, there should be a control sample that is stained with all other antibodies in the panel,” says Dalheim. “This provides confidence that the signal in the fully stained sample is specific for that target.” Indeed, Ouaguia also recommends using FMO controls, single-stain controls, a reference control, and viability dyes, and making sure to account for autofluorescence. “Using appropriate reference controls or single-stained controls and autofluorescence controls, researchers should always evaluate the unmixing, as error in the unmixing calculation could impact data interpretation,” she says.

As the number of fluorochromes on a panel increases, their potential interference can lead to loss of resolution. “Running smaller panels with optimal resolution can serve as valuable additional controls to understand if a newly designed panel is performing properly, in terms of its capacity to resolve cell populations and represent true biological expression patterns of the targeted cells being evaluated,” says Lopes.

Indeed, bigger panels are not always better, especially if a larger panel results in loss of biological information. Run small and large versions of your panel to evaluate whether you get the same cell population percentages. “If you are losing populations or percentages in the larger panel, then the number of colors or dye positioning may be affecting resolution,” says Thomas. “It’s important to remember that the resolution of the biology is more critical in many cases than building the largest panel.”

After spending the time and effort designing a panel for spectral flow cytometry, remember that this is a theoretical exercise that requires testing to ensure that each target can be optimally resolved. “The process of testing the panel is straightforward but requires a methodical approach that includes titration of each reagent, identifying the best unmixing controls, and determining the ideal staining conditions, which all lead to ensuring the optimal resolution of each marker,” says Jaimes.

If your nascent panel doesn’t meet your expectations at first, despite your careful planning, don’t despair—and do learn from the experience. “At times, a panel that appears ideal in theory simply doesn’t perform as expected when you get to the experimentation phase, but these findings can be used to create a more developed panel,” says Thomas. “Thermo Fisher Scientific’s free Panel Design Service works with researchers to develop panels until they are satisfactory to the researcher, understanding that the first attempt might need further optimization.”