Flow Cytometry: It's Not Just For Immunologists Anymore

A low-profile child of the '60s, flow cytometry didn't capture the imagination of most researchers until the early 1980s. The decade saw the birth of the AIDS epidemic, and as attention focused on HIV, researchers needed a method to accurately and reproducibly characterize immune cells. Flow cytometry was suddenly thrust into the spotlight. COMPLETE KITS: Bio-Rad’s KINESIS reagent kits for flow cytometry assays A flow cytometer shines one or more lasers on a sample of cells in suspension, each of which scatters the light and emits fluorescence in response. For each cell that passes, the cytometer measures low-angle forward scatter intensity-which is proportional to the cell's diameter-and the orthogonal- scatter intensity, indicative of the number of granular structures in the cell. The majority of flow cytometers are used in clinical settings for complete cell counts, says Eric Martz, director of the flow cytometry lab at the University of Massachusetts in Amherst. But researchers often take it one step further, adding fluorescently labeled reagents to the cells-such as tagged antibodies, proteins, or oligonucleotide probes-or transfecting cells to make them produce a fluorescent protein. Thus modified, cells yield unique fluorescent patterns under a laser. Flow cytofluorometry, as such techniques are called, can further distinguish a cell based on the receptors on its surface and other molecular characteristics. FOR DNA ANALYSIS: Bio-Rad’s KINESIS-CK kit contains premeasured propidium iodide and FITC conjugated anti-cytokeratin antibody. Through a mechanism that meshes fluidics and electronics -- an exacting description of which is better left to the engineers who designed it -- sorters can also physically separate cells in light of their fluorescence and scattering patterns. That capability has made flow cytometry a favorite in at least one field: "A large percentage of the papers in the Journal of Immunology report flow cytometric data," says Martz, who has set up a Web site that describes flow cytometry and the instruments available at the University of Massachusetts facility (http://www.bio.umass.edu/mcbfacs/flowhome.html). But flow cytometry is pushing beyond the limits of immunology, as researchers in other fields begin to recognize its charms. Close to 1 percent of all biological papers now contain cytometric data, according to Louis A. Kamentsky, chief scientific officer at CompuCyte Corp. of Cambridge, Mass. Kamentsky has been intimately involved with the development of flow cytometry since its inception in the 1960s. He reports that CompuCyte is continuing to pursue cutting-edge improve- ments in the technology. The needs of AIDS researchers helped spur the development of flow cytometry, and a call for automation-the better to rapidly and reproducibly analyze samples-soon followed. Today there are several entries in the automated flow cytometry market. The IMAGN 2000, sold by Atlanta-based Immucor Inc. and manu- factured by Mountain View, Calif.-based Biometric Imaging Inc., is an automated cellular assay system for counting CD4+ and CD8+ T cells in HIV-infected patients. Automation eliminates the need for washing and separating the sample, which can lead to transfer loss and potential error. Unlike other laser analysis cytometry techniques that scan the sample as it flows past a stationary laser, the IMAGN 2000 holds the fluorescently labeled cells in stasis in a volumetric capil- lary while sweeping the laser over the sample. The instrument then stores and analyzes an electronic image of the sample, providing the physician with an absolute CD4 and CD8 cell count and CD4/CD8 ratio. The IMAGN 2000 performs several tests: The 4T8 assay tracks the numbers of CD4+ and CD8+ T cells, providing a measure of disease progression and efficacy of treatment, as these levels are seen by most as an indicator of the severity of disease. These numbers may take several days to determine by normal methods, but the company promises results in less than 45 minutes. The CEQer assay quantifies the number of residual leukocytes in blood and apheresis products after leuko- reduction by filtration. Stray leukocytes can complicate a blood transfusion by transporting cytomegaloviruses or causing graft-vs.-host disease in the recipient. Harvesting stem cells in preparation for stem cell transplan- tation is a tricky business, partly because these undifferen- tiated cells can be nearly impossible to distinguish. The IMAGN 2000 lends a hand here, too. Its STELLer assay, current- ly unavailable in the United States, helps determine the optimal moment to harvest the stem cells after they are stimulated to enter the bloodstream. It also measures the number of stem cells to ensure that enough are harvested for a successful transplant. In 1993, Miami-based Coulter Corp. was the first to introduce a fully automated, four-color cytometer, the EPICS XL and the EPICS XL-MCL, which is fitted with a multicarousel loader. Now in its second generation of software, the unit "was designed from the ground up as a four-color instrument," says Tom Krueger, product manager at Coulter. Other benchtop instruments use two lasers to analyze four color samples, and that complicates the optics, according to Krueger. The single-laser system doesn't have to correlate signals between two separate lasers. The instrument does all of its signal processing on digital signals, avoiding the run-to-run variability that can result from processing analog data. Using the EPICS XL/XL-MCL together with Coulter's tetraONE system, researchers can automate CD4 counts. The system includes a four-color reagent formulated to identify CD4 cells and can typically complete greater than 45 results per hour, according to the company. Similarly, Coulter's reticONE system automates the counting of reticulocytes in whole blood. The four colors of the EPICS system make it state-of-the-art, contends Janis Giorgi, director of the flow cytometry research facility at the University of California, Los Angeles. But she foresees bigger things to come: "In a year to 18 months, five and potentially six colors will likely be commercially avail- able." HIGH-SPEED SORTER: Cytomation’s MoFlow (modular flow) cytometer reportedly can perform multiparameter cell sorting at rates exceeding 25,000 cells per second. Fort Collins, Colo.-based Cytomation Inc. touts its MoFlo flow cytometer as the first and only true high-speed cell sorter. Developed at Lawrence Livermore National Laboratory in Livermore, Calif., the MoFlo has proved its mettle in sorting human chromosomes as part of the Human Genome Project, the company claims. The MoFlo also has devoted optical paths so that the four lasers it employs are not compromised by sharing optics. In contrast, when two lasers are focused through the same lens, the lasers' accuracy is compromised. Focusing on the optical axis gives the best result, but two lasers can't share the same axis, so one or both must be shifted away from it. The four lasers can separate four components simultaneously, outstripping less versatile competitors that at most can separate two at a time, maintains Peter A. Lopez, applications scientist at Cytomation. And its optics are solid as a rock, he claims: "It's built on an industry-standard laser optical bench and bolted on to it-you could dance on the thing." Also stressing excellent optical stability, Aber Instruments Ltd. of Aberystwyth, U.K., offers its Microcyte flow cytome- ter. The patented design features a solid aluminum block that contains all of the optical components, reportedly removing the need for any optical adjustments. Its small dimensions and light weight are said to make it a good fit for both laborato- ry and field applications. ARC LAMP FLOW CYTOMETER: Bio-Rad’s BRYTE HS has applications in microbiology, DNA analysis, and immunophenotyping. Bio-Rad Laboratories Inc. of Hercules, Calif., is targeting microbiologists with its BRYTE HS system. Bio-Rad touts the BRYTE HS-with a detection sensitivity of 0.2 micron and size resolution of 20 nanometers-as the only flow cytometer that can reliably visualize and discriminate among different populations of bacteria-even spores-using only the light- scatter signatures of these populations. That frees up the fluorescence parameters to measure other characteristics of the microbes. The company also sells reagent kits to supplement the BRYTE HS that provide one-step protocols for sample preparation, technique standardization, and streamlining efficiency. Kits are available for labeling of cellular DNA (the KINESIS-50), enhanced identification of tumor cell populations (KINESIS-C- K), proliferative cell identification (KINESIS-PCNA), and analysis of the p53 oncoprotein and cellular DNA for improved tumor characterization (KINESIS-P53). Alone, the instruments only measure the scattering of light from cells and cell components. That's of no small importance, of course, but to do really interesting stuff, many research- ers contend, fluorescently labeled tags are the key. These reagents can close in on receptors, proteins, and carbohy- drates on the surface of the cells-or chromosomes and other structures on the interior. Once situated, they lend their fluorescent signature to the cell, further distinguishing it from its mates. Two cell types with different levels of expression of a given protein may go unsorted by light scattering alone, but a fluorescently tagged antibody may broadcast the difference to the cell sorter. The use of one or two fluorophore reagents-typically FITC, or fluorescein isothiocyanate (530 nm, green), or RPE, which is short for R-phycoerythrin (570 nm, orange)-is common in many laboratories, but DAKO Corp. of Carpinteria, Calif., offers a third line of fluorophores that allow the researcher to study three populations of cells simultaneously. The RPE-Cy5 conju- gates emit at 670 nm (red) and are available as reagent sets, as individual conjugated antibodies, and as streptavidin conjugates and control reagents. REAGENT KITS: R&D Systems' Fluorokine kits and Apoptosis Detection Kit are designed with specialized uses in mind. A wide variety of reagents are sold with specialized uses in mind. For instance, the Fluorokine reagents offered by R&D Systems Inc. of Minneapolis are useful in studying cells with poorly characterized cytokine receptors, says Frank Mortari, assistant director of the flow cytometry department at R&D. The line includes a wide range of human and nonhuman cytokine conjugates, and any cell that expresses a given cytokine's receptor can be pinpointed with the appropriate conjugate, according to Mortari. ANTI-HUMAN CYTOKINES: Becton Dickinson's FastImmune anti-human cytokine conjugates Becton Dickinson Immunocytometry Systems Corp. of San Jose, Calif., takes a different approach to the study of cytokine function, selling labeled anti-cytokine antibodies in the form of its FastImmune Cytokine System (IL-2, IL-4, and IFN-g) for Type 1 and Type 2 determination of lymphocytes, and the new TNF-a, IL-1a, IL-1b, IL-8, and IL-13 for monokine studies. "Unlike ELISA techniques-which measure a bulk response on a global level-these conjugates allow a researcher to determine which cells are producing what cytokines in an autologous environment," says Tony Ward, product manager at Becton Dickinson. There is also growing interest in the platelet activation area, as researchers attempt to come up with a gpIIb/IIIa inhibitor to help treat chronic cardiovascular disease and promote healing of burns and vascular injuries. Becton Dickinson's PAC-1 platelet activation marker recognizes the fibrinogen binding site in the gpIIb/IIIa complex on activated platelets. It is the only commercially available antibody to date that blocks that binding site, maintains Ward. As inter- est in the area grows, "this could be a big market opportunity for us." R&D's Apoptosis Detection Kit uses annexin V, a member of the calcium and phospholipid binding proteins, to detect pro- grammed cell death. Under defined calcium concentrations, annexin V has a high affinity for phosphatidylserine. Cells proceeding toward programmed cell death expose phosphatidylse- rine on the outer leaflet of the cell membrane, making annexin binding a useful red flag for apoptosis. Traditionally, researchers have awaited signs of DNA cleavage patterns to confirm an apoptotic event, but the surface presentation of phosphatidylserine occurs "orders of magnitude" faster, according to Mortari. DAKO's Lambda antibodies react with the human lambda light chains of antibodies exposed on the surface of B cells, or to determine which antibodies leukemic B cells are expressing. They are available conjugated to either the FITC or RPE fluorophore. Detecting the production of molecules within a cell-as its enzymes churn them out-has eluded researchers. Enzyme cross- reactivity, the need to maintain cell viability, and the sensitive nature of kinetic assays and problems with cell permeability have kept this field largely untapped, according to Coulter. The company's CellProbe Reagent line offers a selection of tagged enzyme substrates, specially designed to enter cells. That allows the researcher to perform an assay without pretreating the cell to make it more permeable, often a necessary step. Once processed, the reagents release a fluorescent molecule, allowing the cell to be analyzed using flow cytometry. The line includes more than 30 enzyme sub- strates. For those who wish to label their own reagents, Research Organics Inc. of Cleveland offers the Cy3, Cy5, Fluorolink, and labeling agents. Each has a distinct absorption and emission spectra, and can be used to label proteins, oligonuc- leotides, or any compound with an amine group. These cyanine dyes produce conjugates that are much brighter, are more photostable, and emit less background than other fluorophores, says the company. The dyes have relatively long fluorescence lifetimes of greater than 300 msec, making it possible to conduct short-term excitation followed by a time-delayed signal detection. That helps eliminate interference from background components that have relatively short lifetime fluorescence, the company points out. One of the oft-cited limitations of flow cytometry is that the cells must be in suspension. Solid-tissue analysis can be difficult because the procedures used to put tissue cells in suspension may causes sample degradation and loss. ENABLES SAMPLE PROFILES: CompuCyte’s LSC measures four-color fluorescence and light scatter and records the position and time of measurement of each cell. "For solid tissue, and for a lot of new and interesting things in molecular biology, flow cytometry isn't adequate," notes CompuCyte's Kamentsky. "A lot of the results are artifactual, and it would be very nice to be able go back and look" at images of individual cells, he says. Flow cytometry also isn't very conducive to small samples, since small amounts can easily get lost during specimen preparation. CompuCyte has developed a system that makes the same measure- ments as flow cytometers, but reverses the paradigm: Rather than have a sample flowing by the laser, the cells are placed on a conventional microscope slide placed on a computerized stage. The laser then sweeps over the sample, yielding an image of individual cells. What's more, Kamentsky points out, the researcher can go back and investigate a suspicious-look- ing cell. "Things you thought were artifacts may turn out to be unusual events." Introduced last year, CompuCyte's Laser Scanning Cytometer (LSC) allows users to do sample profiles. One user is doing 12 simultaneous immunophenotyping tests on one patient's speci- men, the company reports. The blood is placed in a device with 12 chambers-each with three antibodies-and then the device is placed on the LSC stage, yielding a 12-test profile on one specimen. "That turns out to be a fair advantage-with flow you'd have to do 12 separate assays and then put the data together," says Kamentsky. The technology is a conceptual throwback. Kamentsky and his colleagues envisioned a stationary sample for flow cytometry in the 1960s, but the lasers and computer systems available at the time didn't permit it. The system is extremely calculation -intensive, and computers powerful enough to handle it didn't become widely available until around 1990, he states. Flow cytometry remains a maturing technology, with a wide variety of applications designed for specific clinical needs and new designs that are forging into new research fields. As novel applications materialize, flow cytometry should take a prominent place in the toolbox of 21st-century biological scientists. Selected Vendors of Equipment and Supplies For Flow Cytometry: Aber Instruments Ltd. Becton Dickinson Immunocytometry Systems Corp. Biometric Imaging Inc. Bio-Rad Laboratories Inc. CompuCyte Corp. Coulter Corp. Cytomation Inc. DAKO Corporation Immucor Inc. Research Organics Inc. R&D Systems Inc. James Kling, a science writer based in Bellingham, Wash., can be reached online at jkling@nasw.org.

Flow Cytometry: It's Not Just For Immunologists Anymore

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