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Dec 1, 2013 (Vol. 33, No. 21)

Spectral Flow Cytometry Makes Debut

  • Click Image To Enlarge +
    Immunophenotyping using the La Jolla Bioengineering Institute’s approach to spectral flow cytometry: Peripheral blood mononuclear cells were stained with a cocktail of fluorescently labeled antibodies, and the complete emission spectrum of each cell was measured. Singly stained antibody capture beads provide reference spectra for spectral unmixing, which is used to calculate the abundance of each marker on each cell, quantities that can be displayed in familiar histogram plots.

    Although flow cytometry was invented more than 45 years ago, it continues to grow and expand into new areas that marry it with other modern cutting-edge technologies.

    Harnessing the power of the full spectrum, coupling it with mass spectrometry, and utilizing it for expression profiling are a few of the new and intriguing applications heralding the next generation of flow cytometry.

    Recent advances in optics and detectors are allowing a new technology, spectral flow cytometry, to make full spectral measurements on the sub-millisecond scale. John Nolan, Ph.D., principal investigator at La Jolla Bioengineering Institute, describes his approach: “There has been a longstanding interest in measuring the complete emission spectra from individual cells. We’ve developed the instrumentation to do this in a robust and routine way.”

    Dr. Nolan says the new technology differs in several important aspects from conventional flow cytometry. “Instead of utilizing photomultiplier tubes, dichroic mirrors, and band-pass filters to measure cell-derived fluorescence at specific wavelengths, spectral flow cytometry uses prisms or gratings to disperse light over a detector array for high-speed, wavelength-resolved detection. Spectral unmixing, a data-analysis approach that estimates the amount of each fluorescent probe in the mixture spectrum, replaces compensation, which is required in conventional flow cytometry to account for fluorescence spillover between channels.”

  • Spectral Flow

    Although autofluorescence can be a significant problem in typical flow cytometry, spectral flow easily resolves the issue. “With spectral flow, autofluorescence is just treated as another ‘color’ and can be resolved from other colors using spectral unmixing, effectively reducing a major source of background,” notes Dr. Nolan.

    Dr. Nolan and colleagues demonstrated proof of principle by using calibrated beads stained with six different quantum dots to demonstrate the analytical performance of the instrument. To evaluate performance in a typical immunophenotyping application, they analyzed peripheral blood mononuclear cells stained with canonical surface markers used to discriminate lymphocyte subsets.

    “Spectral flow was clearly able to resolve CD14+ monocytes, CD3+ T cells, and CD4+ and CD8+ subsets,” says Dr. Nolan. “This shows that spectral flow cytometry has a dynamic range suitable for such conventional applications.”

    Dr. Nolan and colleagues, aware that the emergence of spectral flow cytometry opens the doors to using many different types of fluorescent and other optical probes, are tackling other challenges for this evolving technology. “At present, commercial options are designed for the conventional flow cytometry paradigm of one color per detector,” remarks Dr. Nolan. “Within 5 to 10 years, we will likely see development of new probes, improved instrumentation, new analytical software, and new applications that take advantage of the spectral flow cytometry approach.”

  • Mass Cytometry

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    The CyTOF 2, from DVS Sciences, analyzes cells labeled with stable heavy metal isotopes using time-of-flight atomic mass cytometry technology.

    A new technology adapts the analytical capabilities of atomic mass spectrometry to address the challenges of multiparametric flow cytometry. Advancing this technology is CyTOF®, a mass cytometry system developed and produced by DVS Sciences. Emulating flow cytometry, this technology involves tagging antibodies with isotopically pure rare-earth metals rather than fluorophores.

    After incubation with a panel of metal-conjugated antibodies, hundreds of cells per second are passed through an argon plasma that atomizes and ionizes the metal tags that are subsequently analyzed by a time-of-flight mass spectrometer. In a typical cell analysis experiment, the data derived in less than four minutes of raw collection provides analysis of more than 100,000 cells.

    “The CyTOF system has the capability to measure more than 100 different parameters with negligible signal overlap,” notes Scott Tanner, Ph.D., CTO and co-founder. “Currently, we offer 34 distinct metal tags and continue to develop more,” adds Nicole Ellis-Ovadia, head of marketing. Researchers can label their own antibodies or choose from a catalog of over 200 pre-labeled antibodies or application-specific panel kits.

    With the huge amount of data generated by mass cytometry, one key challenge is high-dimensional data analysis. According to Dr. Tanner, “Mass cytometry requires novel multivariate analytical solutions. While our data files (standard .fcs format) are compatible with all flow analytical platforms, our preferred solution is DVS Cytobank, a new platform that provides conventional flow data workflow as well as validated and emerging unsupervised protocols designed for high dimensional data.”

    Applications for mass cytometry continue to grow and include basic research on cell genesis and functionality, drug discovery (including screening samples with limited availability), and translational applications such as in the clinical diagnostics arena. Dr. Tanner says, “All of these areas share a huge demand for high dimensional, single-cell analysis and will also benefit from application-specific hardware, reagents, and software solutions.”

  • Single-Cell Transcriptomics

    In the last five years, highly multiplexed, single-cell gene expression studies have revealed a great degree of heterogeneity in cell populations thought to be relatively uniform. However, little work has been done in immunological systems, where heterogeneity is known and expected, according to Mario Roederer, Ph.D., a senior investigator at the NIH. Dr. Roederer’s team performed a series of studies to define the gene signatures and identify the coordinate expression patterns of multiple genes in unique cell subsets.

    “Our goal is to marry the power of protein expression analysis via flow cytometry with gene expression profiling of single cells to interrogate peripheral blood mononuclear cells,” says Dr. Roederer. “Combining these technologies also provides information as to the post-transcriptional regulation in these cells, something that neither technology alone can do.”

    Dr. Roederer’s team utilizes a multicolor, fluorescence-activated, cell-sorting system along with Fluidigm’s BioMark™ system to dissect single-cell gene expression. The latter is performed on 96 samples simultaneously and can measure 96 or more genes in each sample. “Although the technology has been used in several disparate biological settings, methodological details for optimal and quantitative application were lacking,” comments Dr. Roederer.

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