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Flow CytometryB-9 acting Group Leader Babs Marrone authored a feature story on flow cytometry that was published in the June 2009 issue of the Journal of the Association for Laboratory Automation. “Flow Cytometry: A Multipurpose Technology for a Wide Spectrum of Global Biosecurity Applications” examines how flow cytometry evolved over the last 40 years after being first developed at LANL, and how it could be more widely used for biosecurity and public health applications. Flow cytometry, and its offspring - flow sorting, are extremely useful technologies for biosecurity and public health studies related to infectious disease. Applications range from environmental surveillance of pathogens to diagnosis and the development of vaccines and therapeutics for prevention and control of infectious diseases. Flow cytometers have been developed for laboratory analysis and field deployment. In flow cytometry, suspensions of single cells are loaded into the instrument through a sample port. Typically, cells are stained with one or more fluorescent dyes that bind to a specific cell constituent or a protein on the cell surface. The cells are hydrodynamically focused to the center of the fluid stream by a sheath of saline. In the sample stream, cells pass in single file through a detection area where laser light shines on them at a specific wavelength to excite the fluorescent dye(s). Photodetectors record the fluorescence emissions, and specialized software analyzes the fluorescence intensity per each cell event. Light scatter measurements are also taken from each cell as indicators of cell size and shape. If the flow cytometer has cell-sorting capabilities, a charge is put on the specified cells of interest as they pass through the laser beam, and they are deflected into a sort vessel. Marrone describes the features and advantages of flow cytometry such as its sensitivity, throughput, statistics (high-speed sorters have reached sort rates of 40,000 cells per second), reagent availability, and versatility. Using flow cytometry to its full capacity could revolutionize biosecurity research and public health measures. In clinical microbiology laboratories, flow cytometry could be performed without the need for traditional time-consuming culture methods and to identify unculturable or slow-growing organisms. Flow cytometry can be used to study host-cell interactions and for multiplexing assays, dramatically increasing the throughput. Flow cytometry has the capability for direct detection of biological threat agents, and point-of-care devices are in development to make this more accessible. Figure 1 shows the specificity of flow cytometry to discriminate three biothreat pathogens from related strains. Other applications include monitoring host immune response and environmental surveillance of pathogens. The article also addresses the containment issues associated with using flow cytometry with biohazardous agents, and how these risks have been mitigated. Marrone suggests that more flexible designs, additional automation, and the use of fluorescent reporter proteins could help bring sophisticated flow cytometry-based detection to resource-poor area, underdeveloped parts of the world. She states that flow cytometry could play a key role in real-time, global and transparent surveillance of infectious disease. 
Figure 1. Specificity of multiplexed oligonucleotide ligation-PCR (MOL-PCR) microsphere array-based assay of Category A pathogens analyzed on a Luminex flow cytometer platform. A 13-plex bead-based assay developed for the screening of three biothreat pathogens, Bacillus anthracis, Yersinia pestis, and Francisella tularensis is shown. The assay was developed to validate the specificity of the probes to discriminate the three target organisms against phylogenetically related strains (near neighbors) in a biodetection application. The x axis represents genomic DNA from the indicated organisms and the y axis shows the 13 probes used. The z axis is the signal-to-noise ratio, where noise is the fluorescence intensity obtained from the beads in the absence of DNA template. The red-circled strains are the target pathogens. Credit: Alina Deshpande (D-3) and P. Scott White (B-7) The Bioscience Division has been home the National Flow Cytometry Resource (NFCR) for 26 years. As a National Institutes of Health (NIH) Resource, NFCR scientists focus on development of new flow cytometry instrumentation, techniques, and applications and also participate in training and collaborative research. The NFCR has earned 6 R&D 100 Awards, the most recent of which was for the Portable Acoustic Cytometer. This invention also won a 2009 Federal Laboratory Consortium for Technology Transfer Award. In addition, the technique for “Ultrasonic Analyte Concentration and Application in Known Cytometry” received a 2009 LANL Distinguished Patent Award. |
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