Courtesy of DakoCytomation
Conventional wisdom holds that flow cytometers are expensive, massive, high-maintenance instruments that require trained operators. They are plumbed into centralized facilities of large institutions, where investigators can pay to have their cells sorted, or perform the analyses themselves (provided they have the requisite skills) under the watchful eye of the center's personnel.
But as so often happens, the conventional wisdom is wrong. Nowadays, flow cytometers are small enough to be found on the benchtops of individual labs. While not as sophisticated as the colossal high-end sorters, these new instruments can easily hold their own for a wide range of common cytometry tasks.
Becton Dickinson (now BD Biosciences-Immunocytometry Systems of San Jose, Calif.), developed one of the first commercial flow cytometers in the early 1970s, but didn't introduce a benchtop version (the FACScan cytometers) until the mid-1980s. That release was followed closely by systems from Coulter (now Beckman Coulter of Fullerton, Calif.); Ortho Diagnostics in Raritan, NJ; and others. These smaller instruments, which at around $100,000 (US) cost one-third to one-fifth what the larger sorters cost, were within the reach of many larger labs and clinics, says Karen Hagen, director of the flow cytometry service at the University of Illinois, Chicago. A steady stream of improvements in lasers, optics, data processing, fluidics, and detection, coupled with commensurate improvements in and availability of the necessary antibody reagents, have driven prices down and put high-performance analyzers within the reach of even small to midsize individual labs.
WHAT IS FLOW CYTOMETRY, ANYWAY? Many investigators use the term FACS generically to mean flow cytometry and the instruments that perform it, just as people say "Xeroxing" instead of photocopying. But FACS stands for Fluorescence-Activated Cell Sorter and is a trademark of BD Biosciences. In the broadest sense, flow cytometers move particles in a liquid stream past a light source and record what happens to the light. The ensuing data provide information about the particles, such as shape and color.
In HIV research, for example, a researcher might want to examine a patient's blood cells for expression of the CD4 and CCR7 cell-surface antigens. First the cells are coated with specific antibodies that are tagged directly or indirectly with fluorescent moieties. The cells are then drawn into the cytometer, where a laser excites the fluorophores, causing each dye to emit light (fluoresce) at its distinct wavelength. The cytometer quantifies this light, using filters to distinguish the different colors. It may also collect data on how the incident light interacts with the cells; known as forward scatter and side scatter, FSC describes a particle's diameter, and SSC relates to its granularity. Operators then determine, based on a graph of the raw data, which cells are of interest--a process called gating. They might, for instance, gate on cells bearing a characteristic FSC versus SSC profile and subject those cells to greater scrutiny, measuring the surface expression of CD4 and CCR7.
Other cytometers supplement these features with sorting capabilities. Users can delineate specific cell populations, such as lymphocytes with intermediate CD4 expression but no discernable CCR7, and collect them for further analysis. Researchers might harvest RNA at this point, measuring gene expression in this narrow cellular subset, or, if the sorting process was done under sterile conditions, they can culture the cells. Cell- sorting functionality adds considerably to the instrument's cost.
Courtesy of Union Biometrica
LASER LIGHT SHOW At the heart of every cytometer is at least one light source and detector; the number and type of these components directly affects the system price. Gas lasers, the standard through the late 1990s, "gave great flexibility in their usable excitation wavelengths, particularly krypton sources," says Bill Telford, the director of the National Cancer Institute's Experimental Transplantation and Immunology Branch Flow Cytometry Core Laboratory. In other words, they were tunable. But these were also very large, powerful, water-cooled units that required substantial warm-up time. Smaller, air-cooled gas lasers have since become more commonplace.
Smaller still are the solid-state lasers, which, though not tunable, are now becoming available in many colors commonly used for flow cytometry. Telford's facility, for example, often uses violet laser light to excite certain fluorochromes. "The only source of that until about five or six years ago was a $30,000 krypton gas laser, which was expensive and a bit fussy, maintenance-wise," he points out. "Now there are smaller violet diode lasers that provide useful violet excitation in a package the size of a flashlight [and are] air-cooled, [and] switch-on/switch-off."
Some cytometers possess multiple lasers to stimulate different excitation wavelengths. The CyAn™ LX, for example, from DakoCytomation of Denmark, can carry three solid-state lasers and detect nine colors. But for a lower-cost option, a single laser can often excite several different fluorophores, so long as their absorption spectra overlap; the cytometer differentiates these signals by their distinct emission spectra. A cytometer with only a single light source can therefore simultaneously examine several different spectral parameters. Beckman Coulter's Epics XL, for instance, can detect up to four colors excited by its 488-nm argon ion air-cooled laser. (For a sampling of spectral options, BD Biosciences offers a Java-based Web tool for viewing the excitation and emission spectra of various fluorophores at www.bdbiosciences.com/spectra.)
Excitation is not limited to light in the visible spectrum; several dyes, such as the Indo series (for measuring intracellular calcium) and the Hoesch series (for cell-cycle analyses) are excitable in the ultraviolet range. Thus, some manufacturers offer instruments configured with ultraviolet-emitting sources, such as the four-laser, 10-color BD LSR II. And light sources needn't be lasers; German company Partec offers models with a high-power mercury arc lamp capable of exciting in the UV, blue, and green ranges.
Fluorescence detection is accomplished using photomultiplier tubes; the number of detectors (channels) determines the number of optical parameters the instrument can simultaneously examine while bandpass filters ensure that only the intended wavelengths are collected.
DATA STORAGE AND RETRIEVAL Cytometry data are collected at a furious pace (the BD LSR II, for example, can sample 10 million times per second), producing mammoth files that are generally stored on dedicated servers. The data, says Alan Saluk, director of the flow core facility at Scripps Research Institute, La Jolla, Calif., are saved in a standardized file format, called the flow cytometry standard, or FCS. Data can be read not only by the instrument that generated them, but also by other cytometers or computers.
Each instrument sports its own data-acquisition and analysis software. But other, aftermarket, postacquisition analysis software, such as San Carlos, Calif.-based Tree Star's FlowJo, promise advantages over their bundled cousins. Postacquisition freeware programs also exist, such as WinMDI, which converts data between Macintosh format (which many BD instruments use) and PC format (the basis of most other cytometers).
Courtesy of BD Biosciences
WHAT SORT OF SORTER? Some sorters, such as BD Biosciences' FACSCalibur system, employ a mechanical sorting process, but this method has drawbacks. Most notably, it's slow: The FACSCalibur sorts around 300 particles per second, while a specially equipped FACSVantage system, using an electrical sorting method, can sort 25,000 events per second.
Another disadvantage: Mechanical sorters "dilute the cells tremendously into saline with no protein," says Hagen. "Your 50-ml tube might have only a few thousand cells in it."
In March, BD began shipping the first benchtop cytometer, the FACSAria system, with electrical sorting capabilities; it can sift through 100,000 droplets per second. According to Ken Murchison, BD product manager, the instrument costs between $325,000 and $400,000 (US).
Courtesy of Union Biometrica
PERSONAL CYTOMETERS "Not every lab has to have their own expensive piece of equipment," says Telford, who has a vested interest in researchers utilizing core facilities. He explains that sophisticated flow cytometers are expensive to maintain, with service contracts costing perhaps tens of thousands of dollars per year. And that does not even take into account the salary costs of the trained personnel who are often required to operate them.
Philippe Goix, founder and chief technology officer of Guava Technologies of Hayward, Calif., echoes this opinion. His company and another startup, NPE Systems, of Pinebroke, Fla., recently introduced small, inexpensive (less than $50,000, US) cytometers designed to carry out a "limited set of applications," Telford says, adding that they "tend to do those particular applications quite well."
For example, Telford notes that the Guava PCA™ (personal cell analysis system) handles simple cell proliferation and apoptosis "rather nicely. It's built for that. And the NPE was originally built to do cell-cycle analysis, which it does better than any other commercial instrument I've seen so far." (Telford has worked with the designers of some of these instruments.)
Each offers a series of software-driven, factory-set protocols that allow users to do routine analyses with very little training or calibration: "It's really made to be very intuitive for the user," Goix says. Ernie Thomas, executive vice president of NPE Systems describes that company's Analyzer™ as turnkey: The user can "push a button and walk away."
Most cytometers focus cells through an outer, hollow stream of sheath fluid. Goix notes that Guava's innovative capillary flow cell does not use sheath fluid to draw the cell past the laser. This allows the instrument to analyze small numbers of cells in small volumes, and to keep the per-sample costs down to a minimum. "The instrument is a sipper," says Goix. Susan Almquist of Vertex Pharmaceuticals, Cambridge, Mass., who has used the instrument for just under a year, points out that "you generate a 50-ml tube [of waste] in a week, instead of having to dump once or twice for a run."
But that same capillary flow cell, Telford points out, makes the Guava lack the resolution of some other instruments. "For many applications that's not very important," he says, but it is critical for DNA cell-cycle analysis. "I would not do cell cycle on the Guava, for example," he says. Goix concurs that the current instrument "does not have the resolution to do ploidy analysis" (the technique used to determine cell cycle) but adds that Guava will shortly introduce one that does.
For his part, Thomas says that the NPE Analyzer is the only system capable of simultaneously measuring both particle volume and fluorescence. But because it uses a mercury arc lamp as its light source, it is incapable of exciting FITC (fluorescein isothio-cyanate) and many other common fluorochromes. The company plans to release a 488-nm laser option later this year, Thomas notes.
Other manufacturers offer specialty products as well. Partec, for example, markets portable cytometers, including one for field analysis that is battery-powered and mounted on a sport utility vehicle. There are several cytometers dedicated to analyzing milk for the dairy industry, or yeast for brewing and winemaking. And one company, Union Biometrica of Somerville, Mass., makes flow cytometers that can handle objects as large as 40-1,200 microns, and are used for such applications as sorting Drosophila and zebrafish embryos, remarks Telford.
With the advent of smaller, less-expensive lasers, faster computers, and improved optics, the benchtop flow-cytometry analyzer has become a standard laboratory tool, regularly used for research, clinical diagnostics, quality control, and drug discovery. And though trained operators will most likely continue to handle high-speed sorting in centralized facilities for some time to come, that application is coming within the reach of individual laboratories.
Josh P. Roberts (email@example.com) is a freelance writer in Minneapolis, Minn.