plasma-torchMass cytometry has been hailed as a revolutionary new addition to the immunologist’s tool kit. Developed at Dr. Scott Tanner’s lab in the Department of Chemistry at the University of Toronto in 2011, this homegrown technology allows scientists to explore cell populations, tissues, and biologic systems with unprecedented depth. Mass cytometry has been met with great excitement, as it allows for the simultaneous interrogation of several times the number of parameters previously thought possible.

Over the past 40 years, fluorescence-based flow cytometry uncovered the great diversity of cells in various tissues and networks at a single-cell level. This multiparametric analysis tool uses fluorescent dyes to identify individual proteins within cells, and has led to the discovery and understanding of many complex immunological processes. But as we expand our understanding of cellular networks, it becomes desirable to simultaneously analyze an increased number of cellular markers to paint a more global view of how individual cells are positioned within systems. While state-of-the-art flow cytometers can simultaneously detect up to 20 different protein markers within a single cell, expansion of a flow cytometry panel to 10-18 parameters is limited due to significant challenges presented in increasing the number of fluorochromes used per experiment.  This has left scientists in need of a new way to analyze cells in a highly multiparametric manner without the roadblocks that multicolor flow cytometry experiments face.

That need has arguably been met with the invention of mass cytometry. A combination of mass spectrometry and flow cytometry, this technique uses rare earth metals (found at the bottom of the periodic table) to identify individual antigens, and can detect upwards of 38 parameters, with over 100 available detection channels. New reagents are in development, which will soon allow the detection of more than 50 parameters. CyTOF (cytometry time-of-flight) is the only commercially available mass cytometer, and was developed alongside tagging technology at the Tanner lab in 2011. The technology was commercialized by DVS Sciences, which was acquired by Fluidigm for $207.5 million in 2014.

The SickKids-UHN flow cytometry facility in Toronto, Canada houses the CyTOF2, the second generation of DVS’s mass spectrometer. IMMpress got in touch with Dr. Cynthia Guidos, Scientific Director of the facility, to ask some commonly raised questions from students in the Immunology Department about CyTOF’s potential to revolutionize our research.

CyTOF is the first technology to provide a “systems-level”
view of the development and function of the immune
system at a single cell level.

cindy-guidosMany labs already have large, expensive repertoires of fluorescently conjugated antibodies. Why move from established flow cytometry panels to CyTOF?
There are several potential advantages, especially for projects aiming to characterize phenotypic and functional differences in cellular subsets from healthy vs. disease patients, from wild-type vs. mutant mice, or from naive vs. immunized mice. With CyTOF you can use ~35 markers/panel in one tube, thus casting a wider net that can allow you to discover novel subsets, functional states or unusual patterns of marker expression that you would miss using 10-12 markers across multiple tubes. Thus, CyTOF is ideal for “screening” a small number of patients or mice. You can then select a subset of markers to test using traditional flow on a large number of samples for validation.

Another clear advantage is that there is essentially no background signal from auto-fluorescence or “spill-over” from other channels in CyTOF, both considerable sources of background in traditional flow. Although the maximum signals generated by CyTOF are a bit lower, overall resolution sensitivity is typically comparable or better. Improvements in metal conjugation methods will increase sensitivity even further in the near future.
Importantly, a CyTOF experiment doesn’t necessarily cost more, since you might need to run five 12-color panels (with some overlap for cross-correlation) to cover all markers included in one CyTOF panel. The cost of metal-conjugated vs. fluorochrome-conjugated Abs is similar, so the reagent cost for 5×12-color panels would likely be higher, and the CyTOF experiment would require far fewer cells.

What services does the SickKids flow facility offer to ease a transition to CyTOF experiments?
We sell pre-validated and pre-optimized metal-conjugated Abs, to make it easy for labs to do pilot studies. We will also provide a metal conjugation service for markers not included in our menu. Our CyTOF service manager (Tina Chen) and Research Associate (Dr. Tatiana Perova) can help potential clients get started. Dr. Perova has optimized CyTOF protocols for intracellular staining with antibodies specific for cytokines, transcription factors and phosphorylated signaling molecules, and can help clients optimize their own CyTOF applications. Finally, we can provide clients with a preliminary “SPADE” analysis of their CyTOF data – see below!

How does analysis of CyTOF data differ from traditional flow data?
Analyzing CyTOF data and highly dimensional traditional flow experiments present similar challenges. For a 12-color flow experiment, 66 biaxial plots are needed to analyze all possible 2-marker combinations! I’m guessing most of you never do this? Thus, the burgeoning field of “flow informatics” seeks to develop unsupervised methods to summarize and visualize the data. There is not yet a “one-size-fits-all” algorithm, but there are a few good options to get started. We typically use “SPADE”, an agglomerative clustering method that generates a branched tree structure with closely related cell clusters (based on marker expression levels) connected by lines. Importantly, SPADE does not require you to make arbitrary decisions about whether cells are positive or negative for each marker. A “plug and play” version of SPADE is provided in Cytobank, a cloud-based single cell analysis and data storage platform used by the SickKids-TMDT flow facility. Clients also have the option to purchase their own Cytobank license and experiment with SPADE (which is quite user friendly). Cytobank also has some nice features for storing and visualizing traditional flow data.

CyTOF has been called a “game-changer” for single-cell analysis. How do you foresee this technology impacting the field of immunology?
CyTOF is the first technology to provide a “systems-level” view of the development and function of the immune system at a single cell level, and so will undoubtedly transform our understanding of immune system functions in health and disease. As for any cutting edge technology, there will be growing pains but there is gold to be mined for those willing to take the plunge! But CyTOF does not spell the end for fluorescence-based flow cytometry, because there are new instruments on the horizon that allow fluorescence-based detection of the full emission spectrum for each fluorochrome, minimizing or eliminating the need for fluorescence compensation between channels. So stay tuned – 30-color fluorescence flow cytometry may be just around the corner!

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Nyrie Israelian

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