How to Generate More Reliable Immunophenotyping Data

Although surface CD molecules are still the most common markers used to immunophenotype cells, the number of CD molecules that are routinely detected in parallel has increased. It has also become standard practice for researchers to include other cell surface proteins and intracellular markers in immunophenotyping panels. Because these developments have inevitably made study design more challenging, we asked a group of experts for their advice to help you generate more reliable immunophenotyping data.

Immunophenotyping studies vary in complexity

There are many different reasons for researchers to immunophenotype cells, ranging from basic research all the way through to clinical monitoring. According to Mark Carter, manager of assay development, cell analytics at Sartorius, immunophenotyping studies range from identifying the presence of a single phenotype, such as identifying CD4+ T cells in blood samples taken from HIV patients, to intricate multiparameter applications. “Advances in reagents, instrumentation, and software have allowed immunophenotyping studies to become more complex,” he says. “For instance, it is now possible to perform quality control of differentiated cells; profile cancers and their response to treatment; investigate the effect of a disease on a patient’s immune system; or to immuno-match donors and recipients for organ donation.”

The aim of an immunophenotyping study dictates the number and types of markers that will be used. “The minimum number of readouts possible for immunophenotyping by flow cytometry is two, since you will always want to include a stain for cell viability with your marker of choice,” notes Sarah Klein, Ph.D., senior development scientist at Cell Signaling Technology. “Some labs perform 3–5 marker immunophenotyping when they have a specific question to ask, while others routinely use panels of 10–15 markers on multiple platforms. The maximum number of markers is constrained only by the technology used for detection, the antibodies available, and the bioinformatic platforms required to analyze the enormous quantities of data delivered by larger panels.”

Mike Blundell, field marketing specialist for flow cytometry at Bio-Rad, agrees, noting that while just one marker can be used to identify some cell types (e.g. CD3 for T cells or CD19 for B cells), determining general lineages in blood may require four markers and identifying specific lineage subsets might increase this number to eight. “Monitoring the activation state, memory status, and subsets of multiple lineages often necessitates >20 markers,” he says. “Even if you’re just looking at T cell subsets, for example, you’ll generally want to identify other cell types within the sample to ensure they’re not affected by a treatment or to eliminate them from your analysis.”

“The more markers that can be included in a panel, the less sample is needed to collect more data,” explains Michelle M. Poulin, Ph.D. senior manager for mass cytometry field applications at Fluidigm. “Researchers who use mass cytometry for immunophenotyping typically combine 30 or even 40-plus markers in their panels, often adding in functional markers as well, because mass cytometry (unlike flow or spectral cytometry) benefits from minimal signal overlap. This makes it a valuable technique to study conditions where the underlying biology is unknown—COVID-19 being a prime example—since performing as broad a screen as possible is the fastest way to reveal key determinants of disease.”

Dr. Michael Fiebig, VP product portfolio and innovation at Absolute Antibody, reports that in addition to the widely studied CD markers, there are many other important markers that (without another reporter system) require intracellular staining. “FoxP3 and RORγt are widely used intracellular markers for T cell biology,” he says. “Other kinds of intracellular ‘marker’ that are perhaps of particular importance right now are intracellular disease agents such as viruses, intracellular bacteria, or intracellular parasites; a lot of information can be obtained from staining for infected versus non-infected cells.”

Lauren Jachimowicz, application development scientist at Agilent, adds that many of the common markers used for immunotherapies (e.g. PD-1 and CTLA-4) are also intracellular. “With the recent surge in immunotherapy research, intracellular markers are being used more than ever,” she says, “however a caveat of using intracellular markers is that they extend staining protocols because cells must be fixed and permeabilized, and surface markers must often be stained first.”

Tips and tricks for panel design

“It might sound obvious, but before you even begin designing an immunophenotyping panel, it is critically important that you choose the right markers to identify your cell type or phenotype,” says Urmi Roy, Ph.D., product manager at Miltenyi. “Marker selection can vary widely based on factors such as species, tissue distribution, and more, but there are many publicly available resources that can help. Scientific publications, the OMIP panels curated by the journal Cytometry Part A, and vendor-validated panels are all good places to start, and fellow researchers can provide useful information.” Reagent and instrumentation vendors also offer a wealth of knowledge.

The following tips are collated from all contributors to this editorial:

The sample

• Choose and prepare your sample carefully; factors such as cell concentration, buffer, temperature, and time from harvest to acquisition can all impact cell health
• Be aware of any potential effects of cell/tissue dissociation methods; for instance, enzymatic methods may lead to cleavage of surface proteins
• Note that fixation and permeabilization can alter surface marker staining; be sure to validate reagents for your panel
• Fix cells quickly if looking for transient changes (e.g. transcription factors or phosphorylation states)
• Consider the cell frequency of your population of interest; this will indicate how many events you should collect to obtain meaningful data

The antibodies

• Ensure your chosen antibodies have been validated for flow cytometry; always perform your own validation to optimize for your system
• Consider using recombinant antibodies where available to ensure optimal batch-to-batch reproducibility
• Think about using antibodies with an engineered Fc region to eliminate the need for Fc receptor blocking
• Capitalize on the ability to streamline antibody and isotype control panels by using engineered antibodies with the same species, isotype, or subtype; these types of controls are not necessary in mass cytometry
• Don’t be afraid to perform indirect staining; through antibody engineering you can choose to switch the species of primary antibodies to create diversity in your panel
• Consider conjugating antibodies yourself; easy-to-use antibody labeling kits enable you to tailor reagents to your experiment

The fluorophores

• Check that the fluorophores you are using are compatible with each other in your chosen buffers and staining conditions
• Pair bright fluorophores (e.g. PE, Alexa Fluor^® 674) with less abundant markers such as Granzyme B or certain phosphoproteins, and dim fluorophores (e.g. Pacific Blue™, FITC) with highly expressed targets like CD4, CD8, or CD19
• Choose fluorophores with as little spectral overlap as possible; dyes with narrow excitation and emission (e.g. StarBright dyes) can be useful to minimize compensation
• Use the resources on vendor websites; panel builders are useful tools to choose markers and dyes
• Place overlapping fluorophores on markers that are not on the same cells (e.g. CD3 and CD19)
• If the antigen density is unknown, has a broad range, or may change, use a bright fluorophore that is influenced as little as possible by other fluorophores

The flow cytometer

• Consider the configuration of your flow cytometer during panel design; differences in detection channels and collection optics can affect your data
• For larger panels, use a flow cytometer with more lasers and filters to spread out your fluorophores and reduce spillover
• Understand how much excitation there is from additional lasers than the primary excitation laser; for instance, with tandem dyes such as PE-Cy5, the Cy5 acceptor may be excited by the 640 nm laser to produce some signal at 670 nm that will require compensation

The analysis

• Don’t skimp on controls and compensation; single stained compensation controls are essential in multicolor panels to remove fluorescence from neighboring channels, while fluorescence-minus-one (FMO) controls are critical to see the effect of all the fluorophores in your panel in the missing channel; these types of controls are not necessary in mass cytometry
• Include a viability marker for removal of dead cells from your analysis; dead cells will bind antibodies non-specifically and have higher autofluorescence, reducing the resolution of detection
• Remove doublets and aggregates during analysis to avoid false positives
• Try different gates to identify the right population—but keep these consistent once a gating strategy has been chosen
• Although traditional two-color dot plots and sequential gating may be suitable for smaller panels, more complex analysis tools such as tSNE plots are a better choice for larger panels

Mass cytometry as an alternative to flow

• Remember that mass cytometry panel design is significantly easier than panel design for high-dimensional flow or spectral cytometry since low abundance targets are protected from signal overlap, and the only necessary controls are biologic controls
• Mass cytometry allows assay multiplexing where individual samples are barcoded; this removes many sources of experimental variability and can reveal subtle changes in cellular behavior

Finally, don’t be deterred if you discover new cell subtypes, functional states, or signaling pathways, says Klein. “With the right, strong controls and confidence in your antibodies, protocols, and technology, you can be confident in your immunophenotyping data.”