September 6, 2023

Why Labs Should Use Spectral Flow Cytometry

Flow cytometry has shifted the goalposts of what biomedical research can achieve, enabling simple, rapid, and reliable analysis and characterization of several biomarkers simultaneously using fluorescent signals. However, emission spectra of different fluorochromes within a cell can interfere with each other as their peaks are broad, causing spectral overlap and subsequently complicating analysis. Hence, the current limitation of conventional flow cytometry.

By empowering the spectral overlap instead of attempting to eliminate it, spectral flow cytometry has emerged as a powerful alternative to conventional approaches.

But what is spectral flow cytometry, and why should labs embrace it?

What is spectral flow cytometry?

Both conventional and spectral flow cytometry use fluorescently labeled antibodies to detect specific markers or molecules on individual cells. Unlike conventional, which detects broad overlapping peaks, spectral flow cytometry analyzes the entire spectrum of each fluorochrome used during an assay. Thus, analysts can identify and quantify over 40 multiple fluorochromes simultaneously (compared to less than 20 markers for conventional cytometry) for more detailed information about cell populations and biomarkers in a single sample.

Cytek® is the current market leader in the field of spectral flow cytometry with their Aurora instrument.

How is spectral flow cytometry used?

Spectral flow cytometry continues to gain traction within the flow cytometry community, as both instruments and services become increasingly accessible. Talks at CYTO 2023 highlighted three emerging areas for this approach:

1.    Lung analysis

Lung is a very challenging tissue to analyze, owing to cellular complexity and high autofluorescence. Kewal Asosingh, Cleveland Clinic, highlighted how spectral flow cytometry handled autofluorescence and helped identify cellular subsets associated with impaired lung function in asthma.[i] The study also showed a strong correlation between a decreased lung function and eosinophil — something more commonly thought to be associated with neutrophils.

2.    Global clinical trials

Spectral flow cytometry provides detailed insights into immune cell identification, drug kinetics and biomarker characterization. Global standardization across laboratories ensuring consistency and high-quality data generation is a current need for this growing market. Standardized workflow achieved by several groups have shown spectral flow cytometry’s utility in global clinical trials[ii], offering deeper immune profiling.

3. High parameter analysis

A combination of an increasing number of dyes and advances in spectral technology opens new avenues for panel development. . As an example, Sandrine Schmutz, Institut Pasteur, reported a panel of 42 fluorescent markers, enabling deep phenotyping of most subsets of hematopoietic cells.[ii]

Deeper insights into complex samples

Spectral flow cytometry has several advantages over traditional analytical methods, offering a higher number of parameters and autofluorescence handling. Even small panels usually reserved for conventional flow cytometry could see their resolution increased with spectral flow cytometry.

CellCarta offers a broad range of services and biomarker expertise for deeper insights into your studies. Contact the team to speak to an expert about how to leverage spectral flow cytometry into your clinical programs.



About the author:

author photo

Martin Turcotte (PhD) is a Senior Scientist at CellCarta, specializing in cytometry assay development. He led the implementation of the CYTEK Aurora spectral flow cytometry platform at the Montreal site. Martin has over 5 years of experience in development and validation of flow-based assays. He studied biopharmaceutical sciences and obtained a PhD in immuno-oncology.



[i] Asosingh, K. Four-dimensional functional pulmonary imaging in combination with high-dimensional flow cytometry identifies cellular subsets associated with impaired lung function in asthma (talk), CYTO 2023 (2023).

[ii] Schmutz, S. Full Spectrum Spectral Cytometry: evolution & new features for high parameter analysis (talk), CYTO 2023 (2023).