After five decades of use, flow cytometry is entrenched in biomedical science. Besides enabling the quick processing of cells in suspension, flow cytometry provides quantitative results across multiple information parameters. Over time, having gained remarkable versatility and precision, flow cytometry has evolved into a highly sophisticated analytical tool, becoming one of the most powerful platforms for rapid analysis of single cells and cell populations. Today, upwards of 50 parameters can be measured at a rate of tens of thousands of cells per second.
Recent flow cytometry enhancements include spectral and imaging proficiencies and the advent of more laser-specific dyes that facilitate a deeper dive into cellular behavior by increasing the number of experimental parameters. The ongoing challenge is to balance that complexity with accessibility to broaden the technology’s uptake in the research, clinical, and diagnostic markets.
Broader palettes
In a recent paper, scientists from the Fred Hutchinson Cancer Center, ETH Zurich, and BD Biosciences described an experiment in which a flow cytometry system was used to analyze blood samples and enable the 50-color phenotyping of the human immune system with in-depth assessment of T cells and dendritic cells (Konecny et al. Cytometry A 2024; 105: 430–436). The experiment’s optimized multicolor immunophenotyping panel was acquired on a BD Biosciences instrument, the FACSDiscover S8 Cell Sorter.
“The new FACSDiscover S8 Cell Sorter combined with new bright and spectrally narrow laser-specific dyes enabled this landmark experiment,” says Eric Diebold, PhD, worldwide vice president of R&D, BD Biosciences. “We now offer a palette of RealYellow and RealBlue dyes that are specific to the blue and yellow-green lasers in flow cytometers.” Conventional dyes, such as phycoerythrin and phycoerythrin-
tandem dyes that can be excited by both the blue and yellow-green lasers, can cause issues while multiplexing.
Redesigned from the ground up, the spectral FACSDiscover S8 Cell Sorter contains subsystems leveraging new technologies, including 78 detectors and up to five lasers. The optical system and dye palette provide certain advantages for flexible panel design and scaling the number of experimental parameters. “These advantages,” Diebold asserts, “are particularly powerful when studying complex biology within a single sample.”
The instrument also incorporates an imaging cell sorter that allows the user to visualize the spatial distribution of fluorescent, light-scatter, and brightfield signals. Users can generate spatial information from cells in addition to conventional flow data, and then sort this information and quantitatively segment populations for downstream use. Users can also take advantage of the company’s FlowJo software, which allows advanced data manipulation, analysis, and editing.
“From a single-cell deposition perspective, the FACSDiscover S8 is one of our highest performing cell sorters for use in applications such as single-cell genomics or antibody development,” Diebold emphasizes. “It allows a deep hierarchical dive into immune populations to look at rare cells with high resolution. The imaging capabilities offer the ability, with high precision and quality control, to visualize and sort out doublets or other rare events for further downstream analysis.”
Three- and four-laser configuration instruments were introduced in 2024. Diebold adds that BD Biosciences is “advancing the core technology of flow cytometry while also striving to make it easier to use.”
Deeper dives
Flow cytometry has been applied to immunology, cell therapy, genetic engineering, microbiology, and food and environmental testing. And because flow cytometry is so adaptable and amenable to automation, it is particularly useful in diagnostic testing and drug development.
“We recently launched the most extensive offering of fluorophores for flow antibodies,” says Trisha Dowling, vice president of flow and imaging technologies, Thermo Fisher Scientific. “[It reflects the power of] NovaFluor Phiton technology and a growing line of Brilliant Ultra Violet and Brilliant Violet dye conjugates.” The portfolio, designed with narrow excitation for minimal cross-laser excitation, facilitates an increase in panel size and customization.
In addition, acoustic-focusing technology has supported improvements in instrument speed, sample type flexibility, and data fidelity. “Acoustic focusing allows cells to be positioned with sound waves, enabling higher flow rates, the analysis of larger cells, and the dilution of samples,” Dowling points out.
The imaging capability of Thermo Fisher’s Attune CytPix Flow Cytometer adds another dimensional measure that is coupled with recent software advancements using artificial intelligence and machine learning (AI/ML). Improved statistical analysis, advanced data processing, and image analysis tools allow the pairing of traditional fluorescence and morphological image data.
For spectral flow cytometry applications, the combination of jet-in-air cell sorting with multiple configurations of lasers and detectors in the Invitrogen Bigfoot Spectral Cell Sorter delivers high speed, cell viability, and high-throughput fluorescence-activated cell sorting.
“Our recent developments have focused on immunology and improving the ability to isolate, characterize, and manipulate single-cell populations,” Dowling adds. “Our broad preconjugated antibody portfolio enables experimental design.” The NovaFluor Phiton technology has powered the development of 31 new fluorophores with many novel spectra for high-plex immunophenotyping workflows.
Software and instrument updates provide researchers with unique opportunities to capture multiple cell states, including exceedingly rare populations. The combination of acoustic focusing and imaging, coupled with AI/ML analyses of images, provides multidimensional analysis and throughput to power a broad set of applications.
Accessibility issues
Flow cytometry’s evolution has also impacted its complexity and cost, making it less accessible to the casual user and hindering large-scale adoption in the diagnostic market. “Our goal is to maintain the technology’s enhancement while removing the impediments to its widespread adoption and growth,” says Shervin Javadi, CEO, Stratedigm. To realize the technology’s potential in the research and clinical markets, instruments need to be scalable, adaptable, “future proofed,” and easy to adopt.
“Our S1000 cytometer is the only instrument that has been consistently scalable and configurable at the time of order to enable complex, high-parameter experiments or simple two- or three-color assays,” Javadi asserts. “It can also be field upgraded after purchase to allow for more colors and avoid obsolescence.” The latest evolution, the S1000 Spectra, scheduled to launch in the fourth quarter of 2024, will have spectral capabilities with up to six lasers.
Service is an integral component of cytometry. Stratedigm’s service program, called Flowraissance, offers no-charge upgrades to its legacy systems, provided they are at least 10 years old, have been maintained under an uninterrupted service contract, and will be signed up for three additional years of service.
“For flow cytometry to gain momentum in diagnostics, products need to focus on automation and one-click operation,” Javadi emphasizes. The A810 cell incubator as been designed to be affordable, compact, and automated; to integrate seamlessly into the company’s cytometry automation suite; and to keep samples protected at the proper ambient temperature, enabling the automatic running of thousands of samples.
To allow optimization of complex protocols across any number of samples, the Multi-Plate Conductor software further expands the capabilities of the automation suite by leveraging machine learning, thereby maximizing speed and operator-free throughput. The latest automation suite expansion will be the A900 intelligent centrifuge (scheduled for release in the fourth quarter). The A900 is capable of accommodating hundreds of samples in two plates without the need for balancing.
Clinical approvals
Data acquired on single cells and populations can be paired to their environmental data and, more recently, their genomic data in an indexed manner. “Flow cytometry does many things with a very high value,” says Matthew Goff, senior commercial product manager, Beckman Coulter Life Sciences. “The data generated versus the time, cost, and effort is enviable compared to other methods.”
This year, the company released the DxFLEX Flow Cytometer for the U.S. clinical market and the RUO CytoFLEX nano Flow Cytometer, built specifically for submicron particle detection. The company’s software also continues to evolve. For example, enhancements in the Cytobank platform’s machine learning engine have been paired with additional analysis tools.
The DxFLEX Flow Cytometer gives U.S. clinical labs access to easier-to-use, higher complexity flow cytometry with more information per tube and up to 13 colors. The clinical instrument, with advanced sensitivity and resolution, facilitates separation of less visible populations, and allows easier and more robust gating of leukemia and lymphoma assays in a space-saving compact design. In addition, FDA clearance was secured for the ClearLLab 10C Reagent System on the DxFLEX Flow Cytometer, providing U.S. clinical labs with a unique integrated leukemia and lymphoma testing solution.
In Europe, more than 20 new reagents were released and certified under the new In Vitro Diagnostic Medical Devices regulatory framework, expanding the availability of compliant reagents and reducing the validation burden. According to Goff, Beckman Coulter was the first to market breakthrough antibodies such as anti-TRBC1 and TRBC2, which the company believes will lead to a paradigm shift in the way T-cell malignancies are assessed.
“The most rapidly developing facets of flow cytometry are in imaging cytometry, dye chemistry, and software tools,” Goff states. “Label-free flow cytometry and novel labeling methods are also generating a lot of interest. We are interested in finding technologies that will offer our users a robust ecosystem of analytical tools to make discoveries or improve diagnostics.”
But as technologies progress, there remains an inextricable need to improve methods of cellular and particle interrogation between aggregate methods and more detailed genomic and proteomic tools.
Labeling advances
With the possible exception of microscopy, it is difficult to name another technology with broader utility in biological research than flow cytometry. “It isn’t surprising,” says Richard Cuthbert, PhD, flow cytometry product manager, Bio-Rad Laboratories, “since flow cytometry is essentially an adaptation of microscopy.” Indeed, when flow cytometry was introduced, roughly 50 years ago, the key innovation, other than a flow cell, was the swapping out of image magnification for the measurement of optical parameters. Then, as now, flow cytometry facilitates the quantification of cells in suspension. Moreover, throughout its evolution, flow cytometry has been improved to allow the analysis of additional parameters.
StarBright Dyes are Bio-Rad’s latest contribution to the field. Their unique chemistry makes them exceptionally bright but with narrow excitation and emission spectra, meaning they can deliver more signal with better signal resolution compared to traditional dyes. They are particularly useful when they allow high-parameter spectral instruments to realize their full potential. According to Bio-Rad, StarBright Dyes are fixable, usable without special buffers, and exceptionally stable over time.
Flow cytometry is an extremely dynamic field, and manufacturers are quick to adopt and integrate new technology. “Laser technology has had a profound impact, allowing ever more parameters to be analyzed simultaneously,” Cuthbert says. He anticipates that manufacturers will continue making greater use of wavelengths outside the visible spectrum. Furthermore, screening analyzers such as the ZE5 Cell Analyzer also process samples faster. “With more parameters being collected from more samples faster than ever before, widespread adoption of computational tools is needed to make efficient use of advances in instrument and dye technology,” Cuthbert advises.
Although flow cytometry can provide vast amounts of data across a vast number of applications, the evolving technology can daunt new users. To that end, Bio-Rad offers a range of resources and tools to help new users get started. In fact, the company was recently awarded the CiteAb Educational Initiative of the Year 2024 for their free-to-attend Flow Cytometry Academy.
