| The following companies offer flow cytometry instruments, equipment, and/or reagents for clinical and laboratory purposes. Please contact the companies directly for more information concerning specific products. |
Bangs Laboratories Inc.
Becton Dickinson Immunocytometry Systems
The Binding Site Inc
Biological Detection Systems
BioSource International Inc.
Boehringer Mannheim Biochemicals Inc.
Duke Scientific Corp.
Frederick Research Center
Gen Trak Inc.
Jackson Immuno Research Labs
Life Technologies Inc.
Molecular Probes Inc.
New Brunswick Scientific (UK) Ltd. PharMingen R & D Systems Inc. Research Organics Inc. Riese Enterprises Inc. Sigma Chemical Co. Vector Laboratories Inc. VWR Scientific Inc. Zymed Laboratories Inc.
R & D Systems Inc.
Research Organics Inc.
Riese Enterprises Inc.
Sigma Chemical Co.
Vector Laboratories Inc.
VWR Scientific Inc.
Zymed Laboratories Inc.
These sophisticated instruments use lasers, fluorochrome dyes, and photomultiplier tubes to analyze large numbers of individual cells or subcellular structures. A significant strength of the technology lies in its ability to rapidly characterize individual cells by recording and correlating several pieces of data about each one. Cells or cell particles can be counted and even physically sorted according to multiple parameters.
As the name implies, flow cytometry (or simply "flow," as users call it) analyzes cells or cell particles flowing in a stream of liquid. First, the cells are stained with fluorochromes, dyes that emit light in various colors when illuminated by a laser beam.
Some flow cytometers are equipped with up to four lasers, although instruments with one or two are more common. The most widely used lasers in flow cytometry are argon lasers, but helium-neon lasers are also used.
The choice of fluorochrome used depends somewhat on the laser that will be illuminating it. Fluorescein, a commonly used dye, fluoresces green when hit by an argon laser, while phycoerythrin fluoresces orange. Additional laser types broaden the range of fluorochromes that can be used in flow cytometry.
One by one, the fluorescently stained particles, in a fluid suspension, pass through a laser beam. As each particle fluoresces, the light is picked up by a photodetector, which converts the light signal to an electrical impulse. In addition to fluorescence, laser light scattered by the particles can also be measured.
A flow cytometer usually includes several photodetectors. Through the use of filters and mirrors, different-colored light signals can be sent to specific photodetectors, which then send the information to a computer, where it is stored for analysis. For any one particle, the data for each parameter are stored discretely, so that statistical correlations are possible between parameters.
If the particles are to be sorted, the machine puts an electrical charge, either positive or negative, on the droplet of fluid containing the cell, and the charged droplet is deflected into a test tube. By using both positive and negative charges, a researcher can sort two distinct populations of cells.
Although the technology involved in flow cytometry is itself not new, it took a while to catch on, according to Alice L. Givan, codi-rector of the cell analysis laboratory at Dartmouth-Hitchcock Medical Center's Norris Cotton Cancer Center, in Lebanon, N.H. Flow cytometry was developed during the late 1960s by researchers in several locations, but much of the work that went into present-day flow cytometry took place in the 1970s at Stanford University in California, and at Los Alamos and Lawrence Liver-more national laboratories, in New Mexico and California, respectively. At that time, says Givan, "it was a fairly rarefied technique with a few experts building their own instrumentation." By the early 1980s, commercial flow cytometers were available. However, "these were pretty difficult instruments to use. They required a lot of care and feeding and constant alignment and things of that sort."
It was not until the late 1980s that flow cytometry really caught on. "The field really exploded because `black box' flow cytometry became available," says Givan. Flow cytom-eters "became much more foolproof, much easier to use. They didn't require quite the high-tech technical support." At this point, she says, "they moved fairly rapidly into hospital labs, and into research labs," more or less on a lab bench, where people could use them fairly easily. Givan points out that flow cytometry instrumentation has grown in two different directions. One is that the simpler, more accessible "black box" machines have made flow cytometry a routine technique in the clinical lab. In addition, increasingly sophisticated machines are being developed with faster sorting capabilities, multiple color analysis capabilities (determined by the number of photodetectors), and multiple lasers, machines that can be configured for many different types of experiments.
Paul Ginouves, United States product manager for cytometry research and instrumentation at Miami-based Coulter Corp., one of the leaders in flow-cytometry instrumentation, says that Coulter's current crop of flow cytometers extends these advances in the technique. For example, he says, the Coulter EPICS XL, introduced in 1992, is the first four-color analyzer available in a benchtop system. The EPICS XL has many automated features, useful in the clinical laboratory. Its four-color capability makes it an important research tool, says Ginouves, as long as multiple lasers and sorting are not required. The XL sells for $89,500 to $122,500, depending on the configuration.
Coulter's other flow cytometer on the market is the EPICS Elite ESP. The ESP also has some automated functions, and can sort between 10,000 and 20,000 cells per second, and use up to four lasers. The cost of the ESP ranges from $153,000 to $300,000. Another major player in flow-cytometry instrumentation is Becton Dickinson Immunocytometry Systems of San Jose, Calif. Becton Dickinson offers an assortment of instruments, with laser capabilities ranging from one to three, and color capabilities from two to four.
Fluorescent reagents for use in flow cytometry are available from many suppliers, including Coulter and Becton Dickinson. Others include Sigma Chemical Co. in St. Louis; Indianapolis-based Calbio-chem-Novabiochem Corp.; and Molecular Probes Inc. in Eugene, Ore.
Some of the most powerful applications of flow cytometry involve combining it with molecular biological techniques, such as the polymerase chain reaction (PCR). Last May, researchers at Northwestern University Medical School in Chicago published a paper describing a methodology they developed to discern very small sequences of DNA and messenger RNA within a cell using PCR and flow cytometry (B.K. Patterson, et al., Science, 260:976-9, 1993). By detecting single copies of DNA, the researchers pushed the lower limits of detection possible in flow cytometry from repetitive sequences of DNA several hundred thousand bases long to single-copy genes of less than a thousand bases. This work had important ramifications in the understanding of the HIV infection, says study coauthor Chuck Goolsby, an assistant professor of pathology at Northwestern.
First, explains Goolsby, the Northwestern team performed PCR, using primers directed against the desired sequences, keeping the PCR product within the cell membrane. They were looking for HIV proviral DNA, which indicates that the cell is infected, and HIV messenger RNA, found in cells that are not only infected, but also actively expressing the HIV viral genes. In addition to using cell samples from individuals, which may have contained multiple copies of the DNA, they used cultured cell lines that contained only one copy of the HIV DNA. They then stained the cells using fluorescent DNA probes directed against the sequence of interest, and analyzed them via flow cytometry.
The researchers successfully detected the desired nucleic acids. In addition, they showed that, in HIV-positive people, while only a small percentage of cells actively express the gene, a significant percentage of cells are latently carrying the HIV genome, acting as a reservoir for long-term maintenance of the infection.
Also working with HIV is Philip McCoy, an associate professor of pediatrics and director of flow cytometry at Cooper Hospital/University Medical Center in Camden, N.J. McCoy is using flow to analyze helper T lymphocytes in HIV infection.
The HIV virus, McCoy explains, infects only a small proportion of the helper T (CD4+) cells at any given time. McCoy is trying to determine upon what basis the virus selects CD4+ cells to infect. He believes that selection may be based on one part of the T-cell receptor, the beta chain. To test this, he is surveying cells from a large number of HIV-positive and HIV-negative individuals, staining the cells with fluorescently conjugated antibodies to CD4, CD8, and T-cell receptor variable beta chains. The cells are then being analyzed through molecular biological techniques for presence of the HIV virus. If McCoy and his colleagues do find a correlation between HIV-infected helper T cells and variable beta chain expression, they hope to eventually develop a clinical assay based on this.
One of flow cytometry's innovative nonbiomedical applications is in oceanography. Researchers are performing flow-cytometric analyses of sea water to study phytoplankton. Similar to leukocytes swimming in blood, plankton easily lend themselves to the fluid nature of flow-cytometry measurements.
Phytoplankton contain pigments that fluoresce on their own, so there is no need to dye them, says Sallie Chisholm, a professor of civil and environmental engineering at Massachusetts Institute of Technology, Cambridge. The chlorophyll in photosynthetic plankton fluoresces red, while the phycoerythrin that naturally occurs in cyanobacteria fluoresces orange. By combining fluorescence data with light scatter data, Chisholm can identify and sort various populations of plankton. In addition to studying the distribution of phytoplankton populations, Chisholm uses fluorescence signal intensity to measure the amount of pigment per cell, which corresponds to the amount of light reaching the organism. She and other researchers are currently using these data to make deductions about surface ocean water mixing.
Chisholm, who directs MIT's part of a joint educational program with Woods Hole Oceanographic Institution in Woods Hole, Mass., has been doing flow cytometry at sea for more than 10 years. In 1988, she and colleague Robert Olson, an associate scientist at Woods Hole, discovered a new species of phytoplankton using the technology.
"We had a flow cytometer on a ship, and we were actually studying the little orange-fluorescing cyanobacteria," recalls Chisholm. "We noticed some signals that were coming from things smaller than them, but had red fluorescence. At that time, there was no known organism that would have those qualities."
They were living organisms, however, and were dubbed Prochlorococcus marinus. It turns out that Prochlorococcus marinus are more abundant than any other plankton known, and, in the equatorial region, the species accounts for 40 percent to 50 percent of total chlorophyll in the sea. "They're just so small that people couldn't see them with a microscope," says Chisholm.
Los Alamos and Lawrence Livermore national laboratories, where many flow-cytometry techniques were first developed, are still focal points for the advancement of flow technology. Researchers at both locations are pushing the lowest limits of detection possible in flow systems, as well as increasing sorting speeds, and are applying this work to molecular biological research, including chromosome sorting for the Human Genome Project.
The laboratory is working on the development of a "fast kinetic" flow-cytometry system, which would allow the measurement of enzyme kinetics within the cell. "With the kinetic system," says James Jett, a program manager at Los Alamos, "we can measure enzyme levels by looking at how fast products are produced." Other applications include the possibility of looking at assembly of macromolecular structures in cells, processes that happen on sub-second time frames.
Some of the most promising work taking place at Los Alamos involves bringing the limit of detection in flow cytometry down to the single-molecule level, says Jett. At present, single-molecule detection has been accomplished using molecules of fluorescent dyes in solution, without cells or other biological material.
Based on this, says Jett, researchers have developed a technique for measuring the length of individual DNA fragments using flow cytometry. While this is traditionally done by electrophoresis, a method that requires several hours and significant quantities of DNA, Jett and his colleagues use very little DNA and finish the job in about three minutes.
In ongoing work based on single-molecule detection, researchers at Los Alamos, in collaboration with Life Technologies Inc. in Gaithersburg, Md., are developing a new approach to DNA sequencing. The plan, says Jett, is to label DNA so that each of the four constituent bases has a different, base-specific tag. The labeled bases will then be cleaved individually into the flow, and they will be identified as they pass through the laser beam.
Rebecca Krumm is a freelance science writer based in Audubon, Pa.