Spectral Flow Cytometry: Understanding Its Advantages

Christy Bucks, Ph.D. October 13, 2025

As an emerging and growing application, spectral flow cytometry offers capabilities beyond what is possible with conventional flow. Its unique capabilities make it a powerful tool for scientists aiming to extract more information from each sample and push the boundaries of discovery. Join us in this post as we explore how cutting-edge advancements in spectral flow cytometry can transform your drug development projects.

What Is Spectral Flow Cytometry?

Building upon the core foundation of conventional flow cytometry, spectral flow utilizes specialized cytometers that enable the detection of 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 capture 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.

How Spectral Flow Cytometry Is Advancing Immunological Investigations

Spectral flow cytometry provides a major advantage in immune studies by allowing scientists to measure many markers on diverse cell types simultaneously with high resolution. Unlike conventional flow cytometry, it captures the full emission spectrum of each fluorochrome, making it easier to distinguish overlapping signals and build larger panels. This enables comprehensive views of the immune system from a single sample, helping to identify rare populations, track dynamic changes, and uncover subtle shifts in immune balance. In immune research, this translates to deeper insights into health and disease, better use of precious samples, and more efficient discovery of meaningful immune signatures.

Advantages of Spectral Flow Cytometry

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.

One way that our experts leverage this advantage is by building customizable backbone panels. By maximizing available channels, a backbone panel is established for core phenotyping markers, and critical channels can be left open for sponsor-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 spectra 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 low 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.

Applications of Spectral Flow Cytometry

In this blog, we highlight several key areas where spectral flow cytometry has a significant impact. Comprehensive reviews, including the work by Czechowska et al.—which includes contributions from KCAS Bio’s Sr. Director of Cellular and Flow Cytometry Services, Adam Cotty—outline these applications and provide additional insights into the advantages of spectral flow cytometry across clinical and translational research settings [1]. The following sections explore how this technology is applied in immunophenotyping, overcoming limited sample volumes, infectious disease research, and drug development.

Immunophenotyping

Spectral flow cytometry enables detailed immunophenotyping by measuring many markers simultaneously, allowing researchers to define immune cell subsets with greater precision and capture rare populations within complex samples.

In pre-clinical study spectral flow is useful for evaluating complex changes in immune cell phenotype, including activation, exhaustion, cytokine production or other functional readouts. In clinical studies, high dimensional analysis is useful for immune monitoring during cancer immunotherapy, rare population detection in leukemic patients or immune tracking of cellular therapies

As one example of the utility of high-parameter analysis, consider clinical trials evaluating cellular therapies. In this setting, the simultaneous assessment of CAR-T product tracking, CAR-T phenotyping, and the characterization of resident immune cells within a single testing tube has yielded critical insights. Denlinger et al utilized spectral flow cytometry to evaluate the phenotype of post-infusion CD8+CAR+ T cells in patients with relapsed/refractory B cell non-Hodkin lymphoma. Here, with a 37-color panel approach, a correlation between PD-1 expression on CAR product and the ability to achieve complete response by 6 months was identified [2]. This is just one example among many, where high dimensional spectral flow cytometry is used to identify key cellular phenotypes which can correlate with cancer treatment outcomes.

Limited Sample Quantity

When flow cytometry options were limited to conventional systems, evaluating either a broad range of cell phenotypes or performing deep interrogation of selected populations required a multi-tube assay design. While technically feasible, limited sample volume often restricts the ability to assess all desired endpoints. Spectral flow cytometry overcomes many of these limitations, enabling comprehensive analysis even from small sample inputs. This approach is particularly advantageous for pediatric samples, bone marrow aspirates, biopsies, and other specimens with limited cellularity, opening the door to deeper cellular evaluation [3,4].

Infectious Disease Research

In the field of infectious disease research, spectral flow cytometry enables high-dimensional monitoring of immune responses to pathogens and vaccines. This approach helps identify protective or functionally relevant cell populations and track dynamic changes in these populations over the course of infection or treatment.

Immunophenotyping of cellular responses can be valuable in both pre-clinical and clinical studies. For example, a 2022 study used spectral flow cytometry to perform deep immunophenotyping of peripheral blood samples from COVID-19 patients, assessing whether specific immune cell subsets correlated with immunotypes that had been identified as predictive of clinical severity and disease outcomes. Using a 40-color spectral flow panel, only minor differences were identified between patient immunotypes, suggesting that the peripheral compartment was not a good predictor for the distinction of patient immunotype [5].

Why Choose KCAS for Spectral Flow Cytometry?

The technology for flow cytometry is evolving rapidly; thus, continuous learning and adaptation are crucial. KCAS Bio is committed to using the latest technologies to ensure your research stays at the forefront of scientific advancements. To meet that commitment, in 2023, we added the Cytek Aurora to our technology platform. With five lasers, 40 colors, and 67 parameter detection, this instrumentation has opened the door to our spectral flow offerings. In 2024, we further expanded our spectral flow offerings to have global impact, with instruments in USA, Europe and Australia.

However, we also recognize that the most significant successes are found when cutting-edge technology is paired with expert scientists. Our team is built with experienced leaders who are well-equipped to develop, optimize, and validate critical assays for you.

While stepping confidently into this new arena, we continue to build upon our firm foundation in conventional flow cytometry. Whether you need assay support using conventional or spectral flow cytometry, KCAS Bio can be your partner of choice.

References

First name *

Last name *

Business Email *

Business phone

Company *

Country/State

Country *

State

Please tell us a little about your needs *