The Evolution of Flow Cytometry: Powering Advancements in Research and Drug Discovery

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Flow cytometry enables researchers to examine individual cells based on multiple parameters simultaneously. This flexibility allows for identifying different cell types within a heterogeneous sample. Accordingly, flow cytometry has become an invaluable tool for studying the immune system, characterizing cell populations and investigating various diseases at the cellular level. This technique is widely employed in oncology, hematology and microbiology.

With applications in various fields, flow cytometry has been established as an important tool for understanding therapeutics currently in development. However, it also has a critical role in understanding the complex mechanisms of action (MOA) for future generations of drugs. The pharmaceutical community’s realization of flow cytometry’s power has fueled the proliferation of its use. Pharmaceutical companies and their investors rightly demand hard data when making critical decisions. Flow cytometry provides a unique space for in-depth interrogations.

Leveraging the significant advancements in technology, KCAS Bio researchers support this need and delve deeper into the immune system’s critical role in drug development. Join us in this post as we consider the evolution of flow cytometry and how flow cytometry can transform your research.

Flow Cytometry: A Multi-purpose Analytical Tool

Flow cytometry has been an invaluable tool for scientific advancement in immunology, oncology, stem cell research, infectious disease, vaccines and drug development research. As a versatile and powerful technique, it can be used in various ways and tailored to meet your research needs. Applications include precise evaluation of cell immune profiles, cell cycle, apoptosis or functional responses such as cytokine production, phosphorylation, phagocytosis and more. Importantly, each approach generates data on a single cell level and allows for investigation of cell responses.

Driven by a need for deeper insights, this technology has evolved steadily. From early cell morphology-based characterization to conventional flow cytometers to groundbreaking spectral technology, it continues to evolve in exciting ways.

What is Conventional Flow Cytometry?

Conventional flow cytometry equipment and techniques have a long history of providing reliable and reproducible data from basic research to clinical sample evaluation. Dating back to the mid-20th century, early cytometers allowed the analysis of cells by distinguishing populations based on size and granular complexity. In the following decades, technology evolved to include more complex measurements. This was achieved by using lasers, expanding fluorochrome availability and recognizing commercialization efforts that drove technological advancements. All these efforts led to today’s conventional cytometers that distinguish upwards of 20 unique parameters on a single cell.

So, how do conventional cytometers work? The method begins with sample preparation into a single-cell suspension, followed by staining with fluorochrome-conjugated antibodies directed against specific antigens. During acquisition, the cells are moved through the cytometer by a fluidics system and pass by lasers that excite the cell-bound fluorochrome-labeled antibodies. The emitted light is filtered, separated and captured by detectors to allow simultaneous measurement of multiple parameters.

Following sample acquisition, the data is analyzed using software that allows researchers to gate and identify specific cell populations based on their characteristics. The fluorescent signals are displayed in histograms or dot plots, representing the distribution of fluorescence intensity within the cell population. Quantitative information, such as the percentage of cells expressing a particular marker or the mean fluorescence intensity, can be extracted and provide valuable information about the cellular profile and response.

Conventional flow cytometry’s familiarity, efficiency and speed are advantageous, making it ideal for focused applications and evaluation of routine parameters.

Spectral Flow - Understanding the Advantages

As an emerging and growing application, spectral flow offers capabilities beyond what is possible with conventional flow. Its unique capabilities make it a powerful tool for researchers aiming to extract more information from each sample and push the boundaries of discovery.

Building upon the core foundation of conventional flow cytometry, spectral flow utilizes specialized cytometers that can detect 40 or more antigens per single cell. Detecting a high number of antigens per cell is possible due to prisms and multiple detectors, which split the fluorochrome light and allow capture of the entire emitted light spectrum. As a result, more fluorochromes can be readily distinguished; thus, more can be used per cell. All of this leads to higher dimensional and more data-rich analyses.

In fact, in this high-parameter analysis, the relative amount of data per sample is increased not linearly but exponentially. Accordingly, data analysis and management are multifaceted and involve careful pre-processing, appropriate data storage and sophisticated analysis techniques. Expert data analysis requires a robust knowledge of flow cytometry and a solid understanding of topic-specific biology. In short, it is a dynamic and often collaborative process requiring technical and biological expertise.

Advantages of Spectral Flow

Harnessing the power of full-spectrum analysis means performing highly multiplexed experiments and gaining a more nuanced understanding of cell populations. However, the benefits of spectral flow extend beyond just multiplexing capabilities.

Flexibility in Panel Design: Spectral flow cytometry is less constrained by spectral overlap, resulting in greater flexibility in panel design. This flexibility enables the inclusion of additional markers without the limitations imposed by conventional flow cytometry.

At KCAS Bio, our experts leverage this advantage by preparing customizable backbone panels. By maximizing available channels, a backbone panel can be built for core marker, and critical channels can be left open for client-driven customization. Within one panel, there can be broad research applications; this approach maximizes efficiency in both time and budget for our sponsors.

Improved Resolution and Sensitivity: Spectral flow cytometry offers enhanced resolution, allowing better discrimination between closely spaced fluorochromes. This can be especially beneficial when working with dimly expressed markers or populations with subtle differences. The improved sensitivity of spectral flow cytometry makes it well-suited for detecting rare events.

Reduced Compensation Issues: Traditional flow cytometry often requires compensation for spectral overlap and can complicate the analysis process. Because spectral flow cytometry uses the entire emission spectrum for each fluorochrome, the need for compensation is minimized. In spectral flow, reference controls are used for spectral unmixing to separate overlapping fluorescent spectrums and discriminate individual fluorescent signals.

Extraction of Autofluorescence: Cell structure or metabolic components can have autofluorescence that interferes with the proper signal detection of antibody-bound fluorochromes. This is particularly impactful when detecting a lowly expressed antigen or using dim fluorochromes. In conventional flow cytometry, this can be difficult to correct. However, in spectral flow cytometry, the autofluorescence of the unstained cells is included in the spectral unmixing and can be appropriately subtracted.

At KCAS Bio, we’ve leveraged this benefit to improve the data resolution in studies that require a sample matrix or a target cell type known to have high autofluorescence. Where compensation could be challenging with conventional flow, spectral flow cytometry provides an advantageous solution.

Partnership and Collaboration

Flow cytometry is not just a technique; it’s more accurately considered a growing community. When everything functions as it should, a symbiotic relationship develops between pharmaceutical companies and contract research organizations (CROs) like KCAS Bio. Intentionally taking a collaborative approach enhances the robustness and reproducibility of flow cytometry applications.

Our partnerships go beyond meeting timelines; partnership involves communication, standardization and continuous improvement that benefits both players. Throughout its history, KCAS Bio has consistently emphasized the value of interactions within this community. Both new faces and returning clients contribute to a collaborative learning environment. The ongoing exchange of feedback and the validation that KCAS Bio’s data aids in therapeutic development underscore the shared goal of advancing science.

A History of Innovation That Speaks for Itself

KCAS Bio has a long-standing presence in the field of flow cytometry. It stands as a clear testament to the ongoing KCAS Bio commitment to advancing the science underlying flow cytometry. With over 11 years in Philadelphia and 40 years in the Kansas City area, KCAS Bio has actively participated in the evolution of this technique and remains at the forefront with cutting-edge instrumentation. Moreover, KCAS Bio continues to expand its global reach and has selected Crux Biolabs (Melbourne, Australia) as its partner in providing harmonized spectral flow cytometry across the US, Europe and Australia. This alliance enables KCAS Bio to further meet customer demand and accelerate harmonized processes to support advanced flow cytometry capabilities using Cytek Aurora flow cytometers. KCAS Bio invites researchers, pharmaceutical companies and industry professionals to explore the possibilities of this transformative technique.

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