by Caitlin Smith
If you’ve ever had your blood count assessed, you probably weren’t thinking at the time about how the results would be produced. In the old days of cytometry, the test involved counting cells with a microscope—tedious, slow, and more error-prone when compared to today’s alternative. Using flow cytometry, the number of different types of leukocytes can be counted quickly and easily, allowing accurate assessments of blood health. Each type of cell has a set of unique characteristics—such as size, shape, volume, surface area, DNA content, and proteins—that allow it to be read like a bar code by the computer that receives the flow cytometry data.
Basically, flow cytometry consists of injecting a cell suspension into an increasingly narrow flow of solution, such that the cells are forced to move one at a time through a beam of laser light. The scattered light is picked up by detectors, and the unique characteristics of the scatter are analyzed by computer. Many properties can be read from each cell at rates of up to 1000 cells per second. This high rate enables researchers to study rare cells, for example, or a small population of cells that shows a fleeting or inducible effect. To facilitate this, the cells of interest in the starting suspension may be tagged with fluorescent dyes, an especially useful tool when combined with a certain type of flow cytometer known as a cell sorter, which sorts different cell types into designated collection tubes.
Research and clinical applications abound
All of these useful attributes are responsible for flow cytometers finding their way into both the clinic and the research lab, where they continue to advance in technology and convenience. Turnkey systems that minimize the chance of human error are available, as are highly automated high-throughput systems that measure, sort, analyze, and more. “In clinical settings, BD Biosciences’ cytometers help pathologists and physicians provide care by enabling them to monitor disease progression—most notably CD-4 testing for HIV/AIDS,” says Tony Ward, director of strategic marketing and cell analysis at BD Biosciences. “Many lab-developed assays in the areas of leukemia and lymphoma can be validated and performed by users as well.” He notes that BD Biosciences’ products are also used by clinical research scientists, “to study how differences in patient phenotypes could be used to guide therapy decisions and improve clinical outcomes within patient populations.” According to Ward, flow cytometry is also used in basic research on immune function, infectious disease, oncology, drug discovery and vaccine development, clinical trials, in vitro diagnostic testing, and disease management.
Sticking with specificity: the more colors, the better
No matter what the ultimate application, antibodies are vital to most researchers using flow cytometry, especially those using multiple colors of fluorophores to differentiate cell types. For example, antibodies are a key tool in the detection of newly defined, very small subsets of cells in mixed populations. There are many choices available in fluorescent dyes now, so finding the right choice for your experiment should not be difficult. Primary antibodies conjugated to the more traditional FITC, RPE, and APC dyes are available, in addition to the Alexa Fluor series and Pacific Blue dyes, among others. “The full range of fluorescent dyes is completed by a wide range of tandem conjugates, including RPE-Cy5, RPE-AlexaFluor647, and RPE-AlexaFluor750,” says Andrew Lane, scientific affairs and licensing manager at AbD Serotec. “These dyes offer options to customers working with new generation three laser flow cytometers.”
With more and more fluorophores available, researchers’ options are expanding with respect to applications. “Multi-color techniques now make it possible to define cell subsets by their expression of four or more antigens, visualizing functional cell populations that have not been recognized previously,” says Lane. Using cell sorting and multiple fluorophore colors, a flow cytometer can produce a highly pure sample of even a rare cell type. Some companies offer custom design services for fluorophores: “AbD Serotec offers customers the possibility of preparing our antibodies conjugated to any of the dyes within our range, making available conjugates that are not currently commercially available,” says Lane. “This option is increasingly popular with the rapid growth of multi-color flow cytometry, with customers looking to maximize their options for staining.”
Along with the right antibodies and fluorophores, one needs the most appropriate instrumentation to address the challenge of small subsets of cells. Cindy Coughenour, head of the flow cytometry unit at Dako, notes the importance of using “instruments capable of fast high multi-color acquisition and sorting,” such as Dako’s cytometers that can handle up to 12 colors. Another option is BD Biosciences’ flow cytometry systems that can use up to 13 colors.
Detecting low level expression
“An increasingly common challenge in flow cytometry stems from the fact that many newly identified antigens are expressed at very low levels,” says Lane. “To overcome this, customers need to utilize high affinity antibodies combined with highly sensitive dyes.” He gives an example of this in the detection of FOXP3 positive regulatory T cells. “The weak intracellular expression of FOXP3 demands high quality conjugated antibodies that can be combined in multi-color assays with cell surface markers.” Coughenour agrees that “detection of very rare events, down to one in a thousand” is a challenge for Dako’s customers as well. She remarks that their customers address this challenge using flow cytometers that are “capable of high multi-color, high event acquisition and sorting, [that give] low [coefficients of variation] of one to three percent.”
Anticipated developments
Lane expects still more fluorescent dyes to become available, with more color options, and also further technical improvements. These include “dyes with much higher Stokes shifts and alternatives for tandem dyes, which often suffer from inherent problems in multi-laser applications. There are also improvements to some existing dyes in the pipeline, which will offer increased brightness for conjugated antibodies.” He also predicts that new dyes will be offered together with new laser options from flow cytometer manufacturers, with an emphasis on accessibility: “New solid state lasers in digital instruments are providing options in bench top machines that were previously restricted to top-end instruments.” Take advantage of the latest technological developments in flow cytometry, and your next cell selection task may prove to be easier than you think.
