The Evolution from Conventional to High-Parameter Cytometry

The earliest flow cytometers, developed in the late 1960s, measured just a single fluorescent signal and light scatter. Since then, the technology has evolved, and it is now possible to simultaneously detect more than 50 different markers. This article explores the advantages and challenges of conventional and high-parameter cytometry and comments on future directions.

High-parameter analysis is possible with conventional flow cytometry

Conventional flow cytometers have come a long way in the last 50 plus years. “Today, instruments featuring the classic architecture (one fluorescent marker per detector) are capable of high-parameter analysis, as demonstrated by the publication of a 30-color panel in 2024,” says Rodrigo Pestana Lopes, Ph.D., Sr. Global Scientific Marketing Manager at BD. Yet this level of plex is far from being the norm. “Most users typically measure between 8 to 12 markers simultaneously with conventional flow cytometry,” reports Kate Alford, Product Manager at Thermo Fisher Scientific. “Limiting factors include the number of detectors available in the instrument, as well as spectral overlap, fluorochrome availability, and compensation complexity.”

According to Matthew Goff, Flow Cytometry Product Manager at Beckman Coulter Life Sciences, one way in which conventional flow cytometers have become more powerful is by replacing photomultiplier tubes with avalanche photodiode detectors, which possess higher quantum efficiency, especially for wavelengths greater than 800 nm. Another is through the use of integrated optics, rather than a series of lenses and filters, to focus light onto the flow cell. “Our CytoFLEX platform incorporates both of these technologies, as well as the wavelength division multiplexer (WDM), which deconstructs and detects multiple wavelengths of light,” he says. “In combination, these measures ensure efficient light management for optimal excitation and emission of fluorochrome-tagged cells, providing exceptional sensitivity.”

Limiting factors for conventional flow cytometry

• Instrument configuration—the number of lasers and detectors available on the cytometer restricts the number of markers that can be simultaneously analyzed
• Spectral overlap—fluorescent markers often have overlapping emission spectra, making it difficult to distinguish between closely related markers
• Fluorochrome availability—conventional flow cytometry requires distinct fluorochromes that can be excited and detected without significant spillover
• Compensation complexity—as the number of markers increases, the compensation for spectral overlap becomes more difficult to manage accurately

Spectral flow cytometers capture distinct fluorochrome signatures

Spectral flow cytometry was first described by the Robinson group at Purdue University in 2004. “The main difference between a spectral flow cytometer and a conventional instrument is that the spectral system is engineered with more detectors, which are filtered so that their combination covers the entire optical spectrum,” explains Pestana Lopes. “Since there are more detectors, and all of them are used irrespective of the fluorochrome, the spectral signature of each fluorochrome is captured.” Ultimately, spectral flow cytometry allows for using more fluorochromes simultaneously, and a 50-color spectral flow cytometry panel has recently been published.

Advantages of spectral flow cytometry over conventional flow cytometry

• Increased marker detection—allows for using a higher number of fluorochromes simultaneously by capturing the entire emission spectrum of each fluorochrome
• Greater flexibility in panel design—enables fluorochromes with overlapping spectra to be more easily combined in the same experiment
• Enhanced signal resolution—measures autofluorescence as a separate parameter, allowing for its extraction
• Potential for improved detection of less abundant markers—collects more signal from each fluorochrome, which may help with detecting lower-expressed markers

Mass cytometry replaces fluorochromes with heavy metal tags

Mass cytometry, also known as Cytometry by Time-of-Flight (on which CyTOF™ systems are based), is a variation of flow cytometry that uses antibodies labeled with metal tags instead of fluorochromes. It has the potential to detect over 100 different markers per sample, more than any other cytometric technology, and offers unique advantages.

“The high-parameter capabilities of CyTOF systems enable the comprehensive measurement of functional profiles for immune cells, including intracellular markers such as cytokines, phosphorylation events, and transcription factors,” says Jennifer Ellis, MS, Director of Scientific Content at Standard BioTools. “Immunophenotyping using surface markers is just the tip of the iceberg when it comes to translational immunology. One must measure immune function, commonly indicated by intracellular markers, yet many such proteins can be difficult to detect. CyTOF technology enables optimal detection of a multitude of functional markers at an improved signal resolution to allow for the detailed determination of cellular functional diversity and to more clearly understand distinct immune signatures.”

Advantages of mass cytometry

• Simple panel design—stable and discrete metal tags with over 50 commercially available markers, kit-based custom conjugations, and modular panels to enable easy panel customization
• No reference controls required—no spectral overlap to manage or unmix helps save on cells and time, as single cell CyTOF data is ready to analyze
• Streamlined workflows—batch sample processing with multiplex barcoding capabilities and ability to freeze stained samples for multi-site and longitudinal studies
• Exceptional signal resolution—high signal to noise, especially with intracellular readouts

Data analysis challenges

All of the methods described come with their own data analysis challenges. “For conventional flow cytometry, managing compensation for spectral overlap can be complex and error prone, while limited marker detection forces the need for multiple experiments to gather comprehensive data, increasing data complexity,” notes Alford. “Furthermore, variability in instrument performance and setup can impact reproducibility and standardization across experiments.” And, for high-parameter spectral flow cytometry, handling the vast quantity of data generated is not the only problem.

“As panels get bigger, data sets are collected over time, and studies become longitudinal, curating and analyzing data sets becomes complex beyond the challenges created by volume,” says Goff. “Dimensionality reduction and drawing meaningful qualitative and quantitative relationships between cellular populations in the context of any treated sample or disease state has become harder for legacy methods. Tools like Cytobank allow users to make meaningful insights into the data sets and create analytical conclusions not previously possible in two dimensional analysis methods.”

As the first high-parameter cytometry platform, CyTOF data led to the development of high-dimensional cytometry software, such as Citrus, SPADE, viSNE, FlowSOM, UMAP, PhenoGraph, and opt-SNE. These software tools are commonly used for analysis of high-parameter datasets. “Clustering and visualization tools also enable an important shift away from manual cytometry analysis to a more scalable and comprehensive approach of automated identification of the many cell populations and functional states enabled by high-parameter cytometry, like CyTOF,” reports Ellis.

Future directions

While conventional flow cytometry meets the needs of most users, researchers are increasingly turning to high-parameter technologies for the greater flexibility and more detailed cellular analysis they can provide. To address growing demand, new tools and technologies are being developed.

Recently launched products include the BD FACSDiscover™ S8 and the Attune Xenith™, which are respectively the latest spectral instruments from BD and Thermo Fisher Scientific, while in March the CytoFLEX mosaic Spectral Detection Module from Beckman Coulter Life Sciences is set to launch, which lets users extend certain CytoFLEX Flow Cytometer models to include spectral flow cytometry acquisition.

Additionally, technologies are being combined to answer more complex questions. “Mass cytometry and fluorescence flow cytometry are often used together, the former for high-parameter analysis of marker expression in a single cell and the latter for lower parameter panel validations,” says Ellis. “Mass cytometry also supports spatial biology initiatives with the use of an attached imager to add in spatial context to phenotypic and functional characterization of diverse cell populations in a single sample.”

“Whichever approach you decide to take, careful experimental design is key to advance your research,” cautions Pestana Lopes. “Rigorous experimental planning, considering the advantages and limitations of each method, and adopting proper experimental controls are critical to obtain meaningful information from your samples.”