Considerations for Immunophenotyping Panel Design

Immunophenotyping—the process of identifying and quantifying different cell types within a mixed sample using labeled antibodies—is a core technique for many different applications. Depending on the cellular population of interest, immunophenotyping can involve detecting upwards of 40 markers simultaneously, making panel design critical to the accuracy of results.

A technique with broad utility

According to Richard J. Cuthbert, Ph.D., Global Commercialization Product Manager for Flow and Antibody Business at Bio-Rad Laboratories, immunophenotyping first gained widespread recognition during the HIV/AIDS epidemic of the 1980s. “AIDS diagnosis is based on the proportion of T-helper cells in whole blood, identified by their expression of CD4 on the cell surface—an application of immunophenotyping that is still used today,” he explains. Other clinical applications of immunophenotyping include its use to diagnose various cancers, immune disorders, and infectious diseases; monitor response to treatment; track disease progression; and even personalize disease management. “In a research setting, immunophenotyping is used to understand the immune system, including what types of cells are present, how they react to different stimuli, how they differentiate, and how they interact with each other,” says Cuthbert. “Here, immunophenotyping can be employed as a filter to allow deeper investigation of a specific cell type, often in combination with assays for measuring cellular processes such as activation, proliferation, apoptosis, phosphorylation, or cytokine expression.”

It's not all about traditional flow cytometry

While early immunophenotyping experiments were based on traditional flow cytometry, this is no longer the only method available to researchers. Spectral flow cytometry and cytometry by time-of-flight (the latter technology is used in CyTOF^® instruments) are also popular for immunophenotyping, especially when detecting large numbers of markers. Julie Hill, Technical Application Specialist at Cytek^® Biosciences, notes that a major advantage of spectral flow cytometry over traditional flow is that it allows fluorochromes with highly overlapping emission maxima to be combined in the same experiment. “Unlike traditional flow cytometry, which captures a distinct range of wavelengths using one detector per fluorochrome, our Full Spectrum Profiling™ spectral flow cytometry measures the entire emission spectrum of each fluorochrome across a detector array,” she says. “This allows researchers to better discriminate fluorochromes from one another, opening the door to more reagent options. Ultimately, this means researchers can investigate greater numbers of markers per sample for a more complete snapshot of the immune system—something for which there is growing demand in immunology.”

CyTOF has been delivering on the need for higher-dimensional immunophenotyping for over a decade, and researchers have published hundreds of 30-plus marker panels to date. “By using antibodies labeled with metal tags for detection, CyTOF addresses the inherent challenges associated with fluorescence detection,” comments Thiru Selvanantham, Ph.D., Senior Product Manager for Mass Cytometry at Standard BioTools™ (formerly Fluidigm^®). “Specifically, metal tags allow for the easy modification of panels and deep analysis of different cell types without worrying about co-expressed markers, signal overlap, or the need to run multiple control tubes to create complex compensation or unmixing algorithms,” she says. “In addition, metal-labeled antibodies are very stable, and the tags are not affected by different fixation or permeabilization methods the way fluorochromes can be, and they can even be frozen as cocktails. Currently, CyTOF instruments can readily detect 40 to 60 markers in high parameter panels, and this has the potential to increase as more metals are added.”

How many markers do you need?

The number of markers detected in an immunophenotyping experiment will vary according to the research question being asked. While some cell populations can potentially be identified using just a single marker, others may necessitate detecting as many as 40 or more markers simultaneously. “By and large, most researchers are not routinely using huge numbers of markers—within the region of 10–15 markers is probably more common,” observes Cuthbert. “Ultimately, the number of markers you need to detect comes down to the subpopulation you are interested in and how specific you want to be.” As soon as you start using two or more markers, panel design comes into play.

Initiating panel design

So, where should you begin when designing an immunophenotyping panel? Although there will be some differences depending on your chosen technology, knowing as much as possible about the biology of your experimental system is always step one. “Making a list of the antigens you want to measure, along with information about antigen co-expression and antigen density, will help guide panel design,” advises Hill. “If antigen density is unknown, or if you are designing a panel for the first time, a terrific resource is the set of Optimized Multicolor Immunophenotyping Panels, or OMIPs, published in Cytometry Part A. These describe panels for various species and cell types, including successful clones, fluorochrome choices, and gating strategies, and show titrations for every antibody in a panel. For example, OMIP-069  explains in detail how a 40-color immunophenotyping panel was designed and optimized.”

Cuthbert also recommends knowing what the instrument you will be using is capable of. “If you have an instrument that can handle a maximum of 10 fluorescent parameters, then that’s the limit for the number of markers you can use,” he says. “You should also consider the excitation and emission profile of each fluorochrome and how those fit with the available lasers and filters. Bio-Rad’s ZE5 Cell Analyzer simplifies this process by allowing users to select fluorochromes from the instrument’s built-in library, which are then automatically assigned to the most appropriate laser and filter combinations.”

For CyTOF-based immunophenotyping studies, Selvanantham suggests starting with the Standard BioTools Maxpar® Direct™ Immune Profiling Assay™, a dry-format, single-tube assay measuring 37 cell populations from a 30-marker antibody panel. Notably, this is designed to improve immunophenotyping standardization by allowing either PBMC or whole blood samples to be added directly to the tube, with no assay-specific optimization required, and using an automated data analysis solution (Maxpar Pathsetter™). “The immune cell coverage provided by the Maxpar Direct Immune Profiling Assay can be enhanced by adding at least 18 further antibodies for deeper insights into specific immune cell populations or to achieve functional analysis study goals,” she says. “In fact, Standard BioTools has 9 preconfigured ready-to-go drop-in panels for deeper characterization of T, B, NK, and myeloid subsets including cytokine expression to explore antigen-specific T cell responses.”

Considerations for flow cytometry

When assigning fluorochromes to markers for flow cytometry-based immunophenotyping, it is essential to consider brightness and spread. Hill explains that the relative brightness of fluorochromes is described by the stain index, which is calculated using the formula (MFI positive population—MFI negative population) / (2 x SD negative population), where MFI is the median fluorescence intensity. “For those using the Cytek® Northern Lights™ or Cytek® Aurora, we suggest referring to our fluorochrome guides, which contain a stain index chart of over 110 commercially available fluorochromes,” she says. “In general, spread can be minimized by matching low-density antigens with bright fluorochromes, and vice versa. Another way to manage spread—and potentially save assay costs—is by titrating antibodies during assay development.”

Echoing Hill’s comments, Cuthbert stresses that fluorochromes with narrow profiles are often preferred for immunophenotyping studies. “Narrow profiles are best, as broad profiles increase the degree of spillover,” he says. “Bio-Rad’s StarBright™ Dyes have narrow excitation and emission profiles, and are exceptionally bright, making them useful tools for building immunophenotyping panels. They are also highly stable, resistant to photobleaching, and can be pre-mixed in any antibody diluent buffer, making them much easier to use than some other dyes.”

Panel design tools

No immunophenotyping editorial would be complete without mentioning the extensive range of tools and services that have been developed to streamline panel design. These include marker selection tools, spectra viewers, and online panel builders, with some companies even offering to design immunophenotyping panels free-of-charge. For those performing CyTOF experiments, the Maxpar Panel Designer from Standard BioTools allows researchers to build a panel of metal-conjugated antibodies using an algorithm that assists in metal tag selection for each target. Panels can then be saved for future use, either by collaborators or through import into any CyTOF instrument control software.