December 1, 2014 (Vol. 34, No. 21)

An expert panel sorts through reagent, fluorophore, detection channel, and optical system choices to tell you what counts in different applications.

Flow cytometry has long been used in cell counting and cell sorting. More recent applications include biomarker detection, protein engineering, cell signaling, pathway screening, and systems biology. GEN recently interviewed a number of flow cytometry researchers and specialists to get a better sense of the capabilities of current flow cytometry techniques as well as some of their limitations.

These experts are Robert Balderas, vp of market development at BD Biosciences, Jeannine T. Holden, M.D., director of scientific affairs, flow cytometry at Beckman Coulter Life Sciences, Carol Oxford, staff scientist of cell biology for the business unit at Bio-Rad Laboratories, Tad George, Ph.D., senior director of scientific applications at Fluidigm, Kim Luu, Ph.D., director of assay development at IntelliCyt, Katherine Gillis, applications scientist at EMD Millipore, Mark Dessing, manager of new technologies and scientific applications at Sony Biotechnology, and Jolene Bradford, senior staff scientist cell biology at Thermo Fisher Scientific.

GEN: Are different types of flow cytometry systems and/or reagents used for basic research, drug development, and diagnostics. If yes, why? If no, why not?

Mr. Balderas: Irrespective of the market segment you must focus on what the need is. The research market runs from low-complexity to highest-level multicolor applications. At low complexity, you might be looking at a low-cost, small-footprint, and ease-of-use instrument. High-complexity applications might need an instrument with five or more lasers.

Drug development involves both low- and highly-complex assays depending on the application. The paramount issue, however, is the ability to run many samples quickly.

On the clinical side, applications can range from a low-complexity single-parameter test like a CD4 count for HIV up to an eight-color application for leukemia and lymphoma. You need an FDA-cleared instrument that fits the dynamic of the application.

Dr. Holden: Yes and no, actually. The fundamental technology is the same and most of the platforms, reagents, and analytic targets are the same. But whereas basic researchers want maximum flexibility, diagnosticians need maximum reproducibility and reliability, plus they have to work within regulatory guidelines. Drug development and other translational research require both: very robust and reproducible diagnoses, but flexibility when it comes to investigating potential biomarkers; high throughput and automation are often needs as well. Fortunately, flow cytometry is such an inherently robust technique that it adapts easily to all three types of laboratory settings.

Ms. Oxford: Flow cytometry systems and reagents used for diagnostics are subjected to a rigorous approval process that requires years of validation. In the United States, this is ultimately the responsibility of the FDA, although there are many regulatory agencies involved in the process. This certainly limits the number of parameters and the reagents that can be used for diagnostics. The concern is always for the patient, and for the critical nature of the accuracy required for the correct diagnosis of disease. The stringency of diagnosis of disease requires making sure that the identical results can be reproduced in labs with different instrumentation in different labs all across the country. 

Dr. George: Yes. Basic biomedical research, drug development, and clinical research/diagnostics all share the need to resolve meaningful phenotypes from complex biological systems, consisting of heterogeneous cell types with different functions and in different functional states. A cell’s phenotype and function is conferred primarily by the array of proteins that it expresses.

As a result, cytometry platforms that measure multiple cellular targets (using fluorescent or mass tagged reporter reagents) with single-cell resolution for thousands of cells per sample are ideally suited to resolve biological heterogeneity. These include both imaging (Amnis, now part of EMD Millipore) and nonimaging (traditional flow cytometers by many vendors; mass cytometers by Fluidigm) platforms.

Dr. Luu: Basic researchers are willing to invest more time on assay development and newer reagents that might have untested performance. To fulfill their research missions, we see these researchers looking for more power in a flow system, so that they can assemble increasingly complex experiments.

At the other end of the spectrum, diagnostic experiments rely heavily on standardized, validated reagents that can deliver robust day-to-day performance on less complex systems. Drug discovery seems to be a middle ground where to get to the interesting biology, systems and reagents are being validated so that they can be used for screening.

Ms. Gillis: Similar flow cytometry systems and reagents can generally be used for both basic research and drug discovery depending on the area of interest. Flow cytometry systems that allow for the flexibility to look at multiple colors simultaneously can be used across many research fields. Instruments such as the easyCyte™ HT line allow the researcher the flexibility for both plate- or tube-based analysis in combination with the channel selection, yielding analysis of up to 12 parameters in either a single sample or across a 96-well plate. Diagnostic instruments tend to be much more specialized for the clinical arena.

Mr. Dessing: Over the past years, Sony has developed and commercialized two systems for life science research, which are used in clinical, academic, and pharmaceutical research. The Sony SH800 cell sorter is an easy to use, fully automated “classical” cell sorter with multiple laser options and detectors; capable of bulk cell sorting or single-cell deposition in multiwell plates. The Sony SP6800 is the first commercial flow cytometer using advanced optics, electronics and an array detector to record the full spectral fingerprint of a single cell. Advanced algorithms are used to extract single-cell staining patterns and help analyze complex cellular phenotypes.

Ms. Bradford: Basic researchers need flexibility to meet their changing priorities. The modular, upgradable design of the Attune® NxT Acoustic Focusing Cytometer allows the system to grow with the researcher. As scientists build skills and knowledge, the Attune NxT can grow with them. Drug development applications often require screening using plates. The Attune AutoSampler offers the ability to use 96- and 384-well plates, in standard or deep-well formats. The acoustic focusing of the Attune system allows samples to be processed at extremely high rates, enabling data collection in shorter time without loss of data quality.

GEN: What are the practical limits for parameters that can be tested in a population of cells? In a single cell?

Mr. Balderas: The most important parameter in any flow application is to match the appropriate color with the appropriate number of receptors on the cell surface. Some cells, e.g., CD4 and CD8, have tens of thousands of cell-surface receptors while others, IL2-secreting cells such B-cells, leukemia cells, LAK cells, have 100 or fewer cell-surface receptors. Thus, the limits range from a high number of receptors on a per-cell basis to a very low number.

The unique aspect about flow cytometry is that you do not need to separate out and look at a single cell. Flow allows one to see the dimensions of a single cell in a heterogeneous population of cells. Based on the sensitivity of the instrument, we can find a single cell—the needle in the haystack—and determine its size and number of surface receptors in a population of millions of cells.

Dr. Holden: The better the technology the more difficult it becomes to define practical limits. The conventional lower limit for particle size that could be assessed using benchtop flow cytometry analyzers, for example, was thought to be about one micron, but the improved optics on Beckman Coulter’s new CytoFLEX benchtop analyzer enable detection of particles as small as 200 nanometers using light scatter only. Feasibility for single-cell analysis is similarly a moving target. Depending on targets and technology, ballpark numbers of 30 to 50 parameters are attainable. Beckman’s Astrios EQ can sort cell populations according to 32 different parameters and deposit single cells of interest into microtiter plates for functional studies, further stretching the practical limits.

Ms. Oxford: The theoretical limits of cytometry technology are advancing at an incredible pace. At the cutting edge, we can detect between 27 and 50 parameters on a single cell, or a population of cells. This technology is complicated and expensive, requiring an advanced level of understanding, and highly sophisticated and expensive equipment. Choosing the right antibodies and fluorochromes, and the understanding of the evaluation of the data are challenges that not many labs have the expertise or instrumentation to complete. In most labs, the practical limits are constrained by cost, and many research questions can be answered with simpler experiments that require only four to six parameters. It really depends on the nature of the question the scientist is asking, and in many cases, there are many simple questions left to answer.  At Bio-Rad, we’ve designed the S3e Cell Sorter to sort either specific populations of cells or single cells for further analysis by real-time PCR using the CFX96Touch real-time PCR system, or digital PCR using the QX200 droplet digital PCR system.

Dr. George: Mass cytometry uses stable high-purity metal isotopes as probes that are separated into separate channels with minimal signal overlap. Endogenous cellular ions are filtered out and, therefore, do not contribute any background. While Fluidigm’s CyTOF2 has 120 separate detection channels, the principal limitation to panel size in mass cytometry is the number of metal tags available (~40).
For flow cytometry platforms, the physical properties of fluorescence provide the greatest obstacle to high dimensional panel design. Fluorescence tags emit unique but overlapping spectra. High-end flow cytometers use a sophisticated array of lasers and filters to direct the composite emission to separate detection channels

Dr. Luu: What is considered a practical limit is difficult to say, as it varies according to a lot of variables. The limit is really theoretical at this point, and is the same whether you’re looking at an entire population or within a single cell. Systems can certainly boast the ability to excite from six different lasers with five to six different detection channels per laser, but at some point you run out of available reagents to make use of the detection capability. Alternative technologies likewise advertise 100 detection channels, but the quantity of reagents available for multiplexing put the number in the range of 30 parameters per sample. The power of these technologies lies in the promise for greater multiplexing with each successive generation of tools.

Ms. Gillis: The number of parameters you can test in a population or single cell is limited by the parameters available on a flow cytometer, availability of dyes and/or antibody conjugates for these detection channels, as well as the complexity of setting up multicolor experiments. One of the challenges with multiparametric assays is the cross-channel spillover for fluorophores utilized. While the emergence of new dyes with long stoke shift emissions has improved multicolor flow cytometry, channel selection has become much more feasible giving the user the capability to perform multiparameter experiments with little to no spillover compensation. There are constant new efforts being developed to increase capabilities for multiparametric analysis while reducing difficulties for the user to acquire, analyze, and interpret data.

Mr. Dessing: In classical flow cytometry, the number of parameters a particular instrument can measure is dependent on the number of detectors. Sony’s spectral analyzer acquires the complete spectrum of a single cell from multiple lasers in the range from 420 to 800 nm. In essence, the instrument records 66 different fluorescent measurements of a single cell. Using this “spectral fingerprint,” together with the appropriate spectral references and advanced un-mixing algorithms, allows the user to extract staining patterns from many different dyes simultaneously. Even strongly overlapping dyes can be resolved, making it possible to measure more dyes from a single laser. The practical limit on the number of parameters is not really known and is depending on available and yet to be developed dyes with appropriate emission spectra.

Ms. Bradford: Recently we have seen development of new reagents that expand the choice of fluorophores. Flow cytometry instrumentation has progressed by expanding laser and detector choices. Combining these advances to maximize use of the spectra allows expanded multiplexing ability. As our systems detect more parameters, multicolor panel design becomes more challenging. Correction for spectral overlap of fluorophores using automated compensation is standard on most instruments and facilitates multiplexing, as does careful reagent selection paired with the ability to easily change instrument detection filters. The Attune NxT Acoustic Focusing Cytometer offers a modular system of lasers, easy filter switching, and intuitive software with automated compensation to enable multiplexing for up to 16 parameters.