High Dimensional Flow Cytometry In Clinical Trials

By Tia Byer |
04 November 2021
How can the biomarkers industry best implement multi-parameter flow cytometry in clinical drug development? Vilma Decman of GlaxoSmithKline takes us through the advantages of using spectral flow cytometry, the importance of viability dyes, and high-parameter panel design.

Presented by Vilma Decman, Director and Head of the Cellular Biomarkers Group, Bioanalysis, Immunogenicity and Biomarkers at GlaxoSmithKline

Transcribed by Tia Byer

The Cellular Biomarkers Group at GlaxoSmithKline supports clinical biomarker testing through immunohistochemistry, flow cytometry, genomics, imaging mass cytometry, spatial profiling and other emerging technologies. In recent years, multi-parameter technologies have dominated biomarkers research to help streamline the drug development process. As part of Biomarkers Week: Online held in May 2021, this article will examine how utilising viability dyes and implementing a multi-parameter approach to flow cytometry can radically optimise biomarker discovery.

Implementation of Multi-Parameter Flow Cytometry in Clinical Trials:

Implementation of multi-parameter flow cytometry in clinical trials enables simultaneous examination of multiple targets on various cell subsets from a relatively limited sample size. Multi-parameter flow cytometry can identify, enumerate, and track cellular markers (both intracellular and intranuclear) during treatment. The technology provides valuable information on both the target engagement and the mechanism of action of new therapies. Furthermore, it aids in understanding disease pathology, toxicological assessment of new drugs, and their efficacy.

The initial strategy for assay development during clinical sample testing requires understanding the biomarkers needs for a particular drug and its targets. Among the most prevalent biomarkers are the pharmacodynamic biomarkers that show a biological response has occurred in an individual following treatment. Biomarker testing aims to individualise treatment by targeting the right patients with the right therapy at the right time. However, the presence or absence of any single biomarker may not provide a complete understanding of the diverse interactions occurring within the disease microenvironment. Therefore, a toolbox approach is required; evaluating multiple biomarkers in combination by flow cytometry provides a more accurate and comprehensive assessment.

Spectral Flow Cytometry: The Evolution and Advantages

Spectral flow cytometry brought new possibilities of deep profiling of many populations, including more precise tracking of rare events. Conventional flow cytometry uses one detector to capture partial emission of individual fluorochrome that has been excited by one laser. Spectral flow cytometry uses multiple detectors to capture full-spectrum emission of every single fluorochrome extracted from multiple lasers. Spectral flow cytometry, therefore, generates individual fingerprints for each fluorochrome.

There are several advantages of spectral flow cytometry over conventional flow cytometry, such as allowing for more fluorochrome choices and improved flexibility in reagent selection. For instance, fluorochromes with identical peaks of emission but different off-peak spectra can now be efficiently differentiated using unmixing algorithms. This was not possible with conventional flow cytometry approach. Spectral flow cytometry also has a more straightforward set-up process that requires no filter changes.

Figure 1. The advantages of spectral flow cytometry as evidenced by simultaneous staining with APC and eFluor660 showing that fluorochromes with identical peaks of emission but different off-peak spectra can be combined in same panel.

One of the most significant advantages of spectral flow cytometry is that it allows for the removal of autofluorescence which aids in improved and more accurate cell and marker separation. Certain cell types such as monocytes, neutrophils, and different tissue cells can have a very strong autofluorescence signals. These signals can interfere with precise marker detection and separation. Spectral flow cytometry can overcome this issue. In particular, it can detect full spectrum of autofluorescence of unstained cells. By extracting each autofluorescence signature and then separating it from fluorescent data, spectral flow cytometry improves resolution.

Figure 2. Autofluorescence extraction in spectral flow cytometry. Autofluorescence is compromising resolution of dim population. Upon autofluorescence extraction, resolution of the positive population is improved significantly.

The Importance of Viability Dyes:

Clinical flow cytometry assay development process can be divided into three steps; feasibility, assay development and validation. The assay development is perhaps the most critical stage of the process. This step involves both antibodies titration and performing additional panel optimisation. In addition, developing appropriate controls for the panels requires careful consideration. The last step of the process is fit for purpose assay validation tailored to the intended use of data.

An important step in panel development includes the use of viability dyes. Viability dyes for flow cytometry can efficiently and reliably analyse both live and dead cells. Dead cells bind antibodies non-specifically which can result in background staining and false positives. Therefore, excluding dead cells via viability dyes allows for a cleaner separation and detection of cell populations. This approach produces reliable, reproducible, and high-quality data.

The Importance of High-Parameter Panel Design:

GSK recently introduced two Cytek Aurora flow cytometry panels to accelerate flow cytometry biomarker testing, one being a 27-colour panel and the other a 23-colour panel. These panels were created for clinical sample testing and consisted of 8 cross-over backbone markers. Although generating a high-parameter panel can be a tedious and lengthy process, it is important to do careful antibody titrations to ensure high quality data. Indeed, titration of antibodies makes it possible to generate 20+ panels as it allows the determination of the amount of antibody that enables the best separations and the optimal measurement of expression levels of individual markers.

Multi-Parameter Flow Cytometry: Key Take-Aways

High dimensional flow cytometry in clinical trials is essential to improving data quality and optimising drug development. While highly powered designs cannot always predict all possible scenarios and drawbacks associated with a clinical trial setting, recognition of the need for power acceleration can significantly improve efficacies and provide comprehensive biological insights. The utilisation of automated data analysis holds many promises for the biomarker industry. At Oxford Global, we look forward to seeing the next advancements made by this technology over the coming years.

Speaker Biographies

Vilma Decman, Senior Director and Head of Cellular Biomarkers at GSK – As Senior Director and Head of Cellular Biomarkers at GSK, Decman leads several teams that support clinical biomarker testing through immunohistochemistry, flow cytometry, genomics, imaging mass cytometry, spatial profiling, and other emerging technologies. Previously she has worked for companies such as Bristol-Myers Squibb, where she was appointed Associate Director of Clinical Flow Cytometry, and Flow Metric, Inc., and Regeneron. Decman completed her PhD in Immunology at the University of Pittsburgh and holds a Master of Science degree in Molecular Biology from the University of Zagreb.

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