Abstract
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. The current state of the art could enjoy more widespread use if instruments and data analysis were made simpler and had more automated functions, and if technology was modified to reduce biosafety concerns related to analysis and sorting of infectious organisms. The full spectrum of possible applications of flow cytometry technology to global biosecurity challenges has not yet been realized.
Introduction
Flow cytometry is a powerful platform for fluorescence measurement of multiple characteristics of single biological particles, cells, or subcellular components, flowing in a sample stream. The past 40 years has seen significant changes in flow cytometry technology that have enabled important advances in the biomedical sciences. Primary examples are what multicolor flow cytometry and the ability to do immunophenotyping by flow cytometry did to revolutionize the field of immunology, and what chromosome sorting and chromosome-specific library construction did to help create the field of genomics. Today, the global burden of infectious disease is at the highest level ever and itself creates a source of political strife and economic instability that can lead to international security issues. AIDS and tuberculosis are two infectious diseases that account for 4.5 million deaths per year, mainly in resource-poor settings where there is limited access to clean water and inadequate sanitation, nutrition, and health care. 22 New microbial pathogens are emerging and reemerging at an alarming rate; from 1940 to 2004, 335 new infectious diseases have emerged. 30 Flow cytometry is now poised to enable scientific breakthroughs in biosecurity and global public health related to the control of infectious disease. Specifically, flow cytometry and cell sorting can play a critical role in the diagnosis of infectious disease, the development of treatments and evaluation of their effectiveness, and in the detection and identification of pathogenic microbes in the environment whether naturally present or intentionally introduced in an act of terrorism or war.
Many of the applications of flow cytometry and sorting have been automated, or are amenable to automation and high-sample throughput technologies. This article reviews the use of flow cytometry in biosecurity and related public health applications to date, addresses limitations and areas for improvement, and makes recommendations for future improvements that would help to expand new uses of flow cytometry and sorting to detect pathogens and develop treatments for infectious disease worldwide.
Flow cytometry and fluorescence-activated cells sorting technologies have been around since the early 1970s. In flow cytometry, suspensions of single cells are loaded into the instrument through a sample port. Typically, cells will be stained with one or more fluorescent dyes that bind to a specific cell constituent such as DNA, 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 a laser light is shined on them at a specific wavelength to excite the fluorescent dye(s). Photodetectors record the fluorescence emissions, and specialized software is used to analyze 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. The International Society for Advancement of Cytometry (ISAC at http://www.isac-net.org) is a useful starting point for linking to a wealth of information concerning the principles of flow cytometry and cell sorting, and current application areas.
Features of flow cytometry
Modern flow cytometry has many attributes that make it a desirable technology for new applications in biosecurity and control of infectious disease. Some of the more unique features are described below.
Sensitivity
By definition, flow cytometry is a single-cell analysis method. Measurements made at the level of individual cells, whether microbial or mammalian, provide a discrete observation that is not achievable by bulk analysis methods, which average results across all the cells in the sample. This feature of flow cytometry has long been exploited by cell biologists, but only recently has been appreciated by microbiologists. The high sensitivity is achieved by the inherent ability of flow cytometry to resolve free from bound fluorescence by virtue of the small analysis volume used. This unique feature of flow cytometry makes possible assays that do not require a wash step. In terms of fluorescence sensitivity, custom flow cytometers are capable of measurement down to the single fluorescent molecule level. 39
Throughput
Flow cytometry sometimes arrives “in the disguise of multiarray technology.” 28 An entire industry has sprung up around the use of optically encoded microspheres as solid supports for large-scale, high-throughput molecular analysis and screening on a flow cytometry platform. This technology is referred to as bead-based microarrays, liquid suspension arrays, or microsphere arrays. 47 48 The Luminex technology (Luminex Corp., Austin, TX) uses 100 distinct bead sets encoded with varying ratios of two fluorescent dyes, which can be coated with different reagents. This allows multiplexing of up to 100 unique assays within a single sample tube. A 100-plex assay can be performed approximately every 30 s, thus enabling hundreds of thousands of assays to be performed in a day. The high-throughput capability of bead-based microarrays has been used for clone library screening 3 71 and single-nucleotide polymorphism analysis. 9 The broad range of high-throughput, microsphere-based, nucleic acid detection approaches and applications has been reviewed recently. 13
Statistics
Flow cytometry has been referred to in the community as “moving microscopy,” but in contrast to traditional microscopy methods, flow cytometry can measure and sort many single cells in a short amount of time. Analysis rates are typically 10,000–20,000 cells per second, and sorting rates for conventional sorters can range from 5000 to 20,000 cells per second. Recently, high-speed sorters have reached sort rates of 40,000 cells per second and sorters with even higher rates of 1 million cells per second are being developed. 34 The high statistics achieved by flow cytometers make then ideally suited for quantitative measurements.
Reagent Availability
A vast and ever-growing selection of fluorescent molecules is available for studying microorganisms and host-cell immune responses by flow cytometry. The reagents assist in the measurement of cell constituents such as DNA, RNA, proteins, lipids, and Gram-staining characteristics; cell function and metabolism (viability, redox potential, ion flux, membrane potential, enzyme activity); and gene expression using reporter systems. The use of fluorescently labeled antibodies that are specific to the pathogen of interest, or a component of the host cell, has also expanded the application of flow cytometry methods in clinical microbiology and biodetection.
Versatility
Last but not least, one of the most important features of flow cytometry is that the same instrument can be used for multiple purposes: for example, classifying and identifying bacteria, and characterizing a host-cell immune response or susceptibility. The ability of flow cytometry to collect information-rich data sets drawn from analyses of multiple features on thousands of single cells makes this technology unique among other single-cell measurement approaches. 8 Specific cells can be isolated (sorted) from the rest of the cells in the population for further analysis on the basis of one or more measurement parameters. In addition, flow cytometry data analysis software has been developed to be readily adapted to different measurements. Measurements can be standardized and calibrated to aid in the interpretation of results and quality control. Table 1 shows a list of possible applications of flow cytometry to biosecurity.
Possible applications of flow cytometry technology in biosecurity-related areas
Biosecurity applications of flow cytometry
In this article, biosecurity is broadly defined as a flow cytometry application area that encompasses direct measurement of traditional biological threat agents and also other pathogens in the laboratory or in the field, and measurement of pathogen effects in human hosts. The following sections focus on four major areas of flow cytometry applications related to biosecurity and public health: flow cytometry in the clinical microbiology laboratory; flow cytometry for direct detection of biological threat agents; monitoring host immune response to infection using flow cytometry; and environmental surveillance of pathogens by flow cytometry.
Flow Cytometry Applied to Clinical Microbiology
Flow cytometry is an attractive technology for clinical microbiology labs because in concept it can be performed without the use of traditional time-consuming culture methods and can be used for the identification of unculturable or slow-growing organisms. Eliminating the culture step would significantly speed up time to diagnosis of life-threatening diseases. However, the challenge to overcome is the small size of most microbes, resulting in small signals. In addition, the propensity of some microbes to aggregate can be problematic for flow cytometry and sorting. Despite these challenges, there is now a substantial literature on the application of flow cytometry technology to detection and quantification of virus- or bacteria-infected cells, and on the direct measurement of viruses and bacteria by flow cytometry. 2 43 45
In terms of measuring pathogen and host-cell interactions, analyses can include measurement of apoptosis, regulation of antigen expression, measurement of immune response of infected cells, identification of cell surface receptors, and identification and measurement of cell surface or intracellular antigens, and induction of cytoplasmic function by infection. Direct detection of microbes (viruses, bacterial, fungi, and parasites) in blood or other clinical specimens is accomplished by fluorescent dyes that label nucleic acids and/or by labeling with antibodies to specific microbial antigens. Sensitive and specific detection has been enabled by amplification of microbial DNA or RNA and detection by fluorescence in situ hybridization methods. Sample preparation technologies, such as gel microdrop encapsulation, have also helped to facilitate flow cytometry analysis and sorting of microbes (e.g., Ref. 55).
There is a large body of literature on the application of flow cytometry to studies of antimicrobial effects and susceptibility testing to guide the development of improved therapeutics and medical countermeasures (reviewed by McSharry, 44 and by Alvarez-Barrientos et al. and Pils et al. 52 ). Flow cytometric analysis of the effects of various treatments can provide significantly more information that is more useful for screening than standard culture and count testing. Alvarez-Barrientos et al. suggests that to expand the use of flow cytometry in clinical microbiology, particularly to optimize the cost–benefit ratio, automation is necessary for both sample analysis and for analysis of results.
Pulsed field gel electrophoresis (PFGE) is a mainstay of clinical microbiology for molecular subtyping of bacterial strains, as exemplified by the Centers for Disease Control and Prevention (CDC)'s PulseNet system for typing of bacteria involved in outbreaks of foodborne disease. 65 Marrone et al. at the National Flow Cytometry Resource at Los Alamos National Laboratory pioneered the application of high sensitivity, DNA fragment sizing flow cytometry for rapid DNA fingerprinting of pathogens (e.g., Refs. 20, 32). In this method, a macrorestriction digest of a bacterial genome is prepared and the resulting DNA fragments are stained with a fluorescent dye and analyzed by a custom flow cytometer with single molecule detection sensitivity. The fluorescence intensity of each event is directly proportional to the length of the DNA fragment. The restriction digest profile that results from this method is equivalent in precision and accuracy to the PFGE method, but each analysis by flow cytometry takes minutes instead of days. Large-scale analysis of DNA fragments by this flow cytometry approach would require much software and hardware development and automation for it to replace PFGE for large DNA fragment analysis. However, this example illustrates the potential power of flow cytometry approaches to streamline laborious, reagent-intensive laboratory methods.
The small sizes of viruses, typically 10–300 nm in diameter, have made direct measurement by flow cytometry difficult. Using a flow cytometer that was adapted for single molecule detection, Ferris et al. 18 19 demonstrated the rapid enumeration of adenovirus, respiratory syncytial virus, and influenza A virus stained with a fluorescent nucleotide-binding dye that is selective for single-stranded DNA. In a more recent study, Agrawal et al. 1 showed that by using two colors of fluorescent nanoparticles conjugated to adjacent nucleic acid sequences or antibody probes, that target viral particles could be specifically measured in a flow channel by coincidence detection of the two-color signals.
Combining molecular biology and immuno-detection approaches with flow cytometry has been particularly successful for detection of viruses to determine viral load and for detecting virus-infected cells. Kao et al. 31 used indirect immunofluorescence staining with antibodies to dengue virus to detect the virus in mosquito cells earlier than possible with conventional methods. Likewise, microsphere-based technologies are particularly suitable for enhancing the detection of small fluorescent signals from pathogens. The Luminex LabMAP system is a flow cytometry platform that has been demonstrated for its ability to detect many different pathogenic organisms through the presence of antigen or DNA in a multiplexed format. For DNA-based detection, specific pathogen DNA sequence targets are amplified, denatured, then hybridized to synthetic oligonucleotide capture probes on the surface of microspheres and fluorescently labeled for detection. For example, multiplexed assays for sequence-specific targets have been developed using microsphere-based flow cytometry to enable detection and discrimination among common respiratory viruses. 40 49 For antigen-based detection, capture-sandwich immunoassays were developed using pathogen-specific antibodies coupled to microspheres and fluorescently labeled for detection. 15 Multiplexed, microsphere-based flow cytometry immunoassays have also been developed for influenza virus detection and differentiation of A and B types. 69 This format was optimized and shown to be about 10 times more sensitive compared to conventional enzyme-linked immuno-absorbent assay (ELISA) methods. 70
Direct Detection of Biological Threat Agents by Flow Cytometry
In the event of a bioterrorist attack, rapid, accurate detection of biological threat agents is important for protection of public health. As soon as a biological threat agent is detected in the environment, presumed exposed individuals can be treated appropriately to minimize infection. Flow cytometry has been shown to be an effective platform for direct biological threat agent detection and identification. Immunoassays for detection of Bacillus anthracis spores by multiparameter flow cytometry have been demonstrated. 59 63 Immunoassays using bead-based, suspension arrays and the Luminex system or other flow cytometer platforms have been developed for the detection of botulinum toxin 17 and for B. anthracis and Yersinia pestis. 42 A Luminex flow cytometer is at the heart of the autonomous pathogen detection system (APDS), developed at Lawrence Livermore National Laboratory for the integrated continuous, unattended collection, sample preparation, and detection of aerosol samples for the presence of multiple biological threat pathogens. 26 27 41 The initial version of the APDS coupled Luminex microsphere immunoassays with a flow-through PCR module for confirmation of a positive immunoassay detection event by DNA amplification of the sample using pathogen-specific DNA primers. In the subsequent version of APDS, the Luminex system will be used both for the initial immunoassay detection of multiple biological threat agents and for confirmation of a positive detection event by multiplexed PCR-liquid bead detection. A multiplexed PCR-based microsphere microarray assay was developed for integration into the APDS and was demonstrated for the simultaneous detection of B. anthracis, Y. pestis, Francisella tularensis, and Brucella melitensis. 67 Their approach was based on hybridization of a biotinlabeled, pathogen-specific PCR product to a complementary capture sequence on a color-coded microsphere, followed by reaction with streptavidin–phycoerythrin.
Dunbar and Carrano 14 reviewed the many studies that have used the Luminex flow cytometry system for detection of infectious agents using immunoassay or nucleic acid detection approaches. Although the immunoassay approaches are fairly similar antigen capture-sandwich formats, the detection of nucleic acid sequences have used a variety of approaches including simple direct hybridization of a labeled PCR product, to more complex methods for detecting genotype and single-nucleotide polymorphisms that could enable discrimination of strains of organisms. One such method is multiplexed oligonucleotide ligation–PCR (MOL-PCR), and readout using the Luminex system. 38 MOL-PCR facilitates multiplexing because it uses a pre-PCR ligation step that accomplishes the initial, specific detection of the target nucleic acid sequence. The detection step is then followed by a singleplex PCR that serves to amplify and label the products of the ligation step. After PCR, the ligated, amplified, and labeled products are hybridized to addressable microspheres and analyzed by flow cytometry. MOL-PCR has been demonstrated in a 50-plex assay to detect multiple plant pathogens, and a multiple-marker assay for detection of Category A pathogens B. anthracis, Y. pestis, and F. tularensis and their close relatives (Fig. 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 uses the MOL-PCR method, and the Luminex microsphere array was analyzed on a Luminex 100 flow cytometer platform. 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: four for Y. pestis, five for B. anthracis, and four for F. tularensis. 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.
The HyperCyt system developed at the University of New Mexico is a high-throughput system that interfaces a flow cytometer and autosampler and allows for the sampling and analysis of a 96-well plate within less than 3 min. Designed for high-content measurements in a drug discovery environment, the HyperCyt system surpasses the high-throughput capacity of commercial flow cytometry autosamplers by 30-fold. 16 HyperCyt technology was used with a multiplexed microsphere-based suspension array designed for protease screening and demonstrated using the lethal factor protease toxin of B. anthracis. 56 The screening assay is applicable for high-throughput screening of samples for protease activity, and for the screening and characterization of protease inhibitors.
Mark et al. 38 point out that the need for rapid information in the event of a bioterrorism attack has forced a trade-off between the high information content desired and the high cost of deployed biodetection systems. The liquid suspension microsphere-based array as implemented in the Luminex system, for example, is a rapid, cost-effective approach that can be highly multiplexed so that information content is not sacrificed. Moreover, the potential for flow cytometry platforms to be automated and integrated with other devices, as demonstrated by the APDS and HyperCyt systems, makes the flow cytometry platform an attractive one for super high-throughput, routine biological threat agent surveillance without sacrificing high information content.
Monitoring Host Immune Response to Infection
Our ability to strengthen biodefense internationally has an “inherent synergy” with our ability and willingness to promote global health, in part by sharing technology advancement with less fortunate countries to combat naturally occurring infectious disease. 50 The approach that the flow cytometry community of practitioners, instrument developers, and reagent suppliers are taking to deliver critical technology to resource-poor areas of the world is instructive to the future of flow cytometry applications in biosecurity and related public health matters.
Measurement and typing of peripheral blood lymphocytes (immunophenotyping) is probably the most widespread use of flow cytometry, and measurement of the CD4 T lymphocyte subset in particular has emerged as one of the most important laboratory tests for evaluating the immune system, because it is critical for determining the immune status of individuals with HIV infections and monitoring the response to antiretroviral therapies in HIV/AIDS. Although CD4 T-cell enumeration by flow cytometry is part of routine HIV monitoring in the developed world, in resource-limited regions of the world, where there is the greatest need for large-scale monitoring of HIV infection and AIDS progression, the needed technology is too costly, too difficult to operate, and not robust enough for the harsh environmental conditions often encountered. There is a movement within the flow cytometry community to develop affordable flow cytometers for CD4 T-cell counts that can be robust and simple to use so that technology for CD4 T-cell counting can reach resource-poor regions. The status of this effort was recently reviewed. 36 Beginning in 2004, with the introduction of the Apogee MiniFCM based on a ruggedized flow cytometer prototype developed originally at Los Alamos National Laboratory under contract with the U.S. Army for application to detection of biowarfare agents, Mandy et al. compares several different varieties of affordable flow cytometers for dedicated CD4 T-cell counting.
Although one approach to making flow cytometers more simple and affordable is to target a specific measurement, such as CD4 T-cell counts, another parallel approach is to use less expensive components in the instrument. 60 Combining these approaches enables development of a simpler device, with fewer options for the operator, and potentially more sustainable operation. A lost-cost, portable flow cytometer is being developed currently at Los Alamos National Laboratory using innovative approaches such as acoustic energy, rather than large volumes of sheath fluid, to focus particles to the center of a flow stream 21 ; low-cost light sources such as laser pointer modules 24 ; and open, reconfigurable digital data acquisition systems. 46 The availability of a “minimalist” flow cytometer 29 would immediately improve the quality of medical care that is available to diagnose and treat infectious diseases in poor regions of the world, which could in turn have a positive impact on international security. Such devices could also be used to enhance military medical support to troops deployed in some of these same, politically unstable regions around the world, or as portable biological agent detection devices.
Environmental Surveillance of Pathogens Using Flow Cytometry
Flow cytometry is an extremely useful tool for analysis of microbial populations in nature 11 53 whether from collected aerosol samples 62 or from water samples. 6 25 37 64 66 When coupled with molecular methods for specific cell identification, the capability of flow cytometers to rapidly analyze thousands of cells is highly advantageous for characterizing heterogeneous populations of environmental microbes, particularly in aqueous environments such as drinking water systems. A popular application of flow cytometry is the analysis of Cryptosporidium parvum cysts in drinking water samples, and recently a protocol for fluorescent antibody-based detection by flow cytometry was published. 4
Monitoring water for target cells by flow cytometry, as for many other methods, typically requires preconcentration of large volumes of water (500–100 mL) to enrich the sample for the target cell. Enumeration of microbes, analysis of target microbes, and assessment of viability can all be achieved on a conventional flow cytometry platform. Gruden et al. 23 have advocated for the use of flow cytometry for sensing of microbial contaminants in water systems. They compared six other technology platforms for microbial sensing and found that flow cytometry provided the best balance of sensitivity, speed, adaptability, and robustness in complex matrices. This last characteristic is particularly important for detecting specific microbes in water samples, because the complex chemical and physical characteristics of natural environmental samples pose a challenge to optical detection methods, and can also inhibit immunological and nucleic acids-based detection methods used to generate detection signals. The rich microbial diversity inherent in environmental samples may also result in cross-reactivity with even the most carefully designed immunological or nucleic acids technologies. High-throughput, large-scale screening, suspension arrays have also been applied to monitoring for the presence of fecal indicating bacteria in environmental samples of water and nearshore sand using molecular technologies and the Luminex system. 5 This study noted the poor efficiency of nucleic acid extraction methods from environmental samples as a challenge for molecular technologies applied to detection of pathogenic microbes in water. However, the growing literature on the use of flow cytometry for water quality assessment suggests that flow cytometry has a promising future in water biosecurity applications.
Biosafety considerations
When analysis and especially droplet sorting of potentially infectious material is being conducted, a biohazardous environment develops. The increased analysis and sort rates of modern flow cytometers and cell sorters are achieved by increasing the operating pressure of the system, and this high pressure naturally creates aerosols. This problem is compounded by the nonenclosed, jet-in-air design that is used on sorters. Likewise, droplet splashing and clogs during analysis and sorting can also create biohazardous conditions for the operator and bystanders. Because so many flow cytometry laboratories use their instruments for sorting cells from potentially infectious human material (blood, primary cell lines, etc.) the ISAC took a lead in developing biosafety guidelines for sorting of unfixed cells 57 and updated the guidelines a decade later. 58 The current guidelines reflect advances in cell sorting, improvements to biosafety equipment and techniques for the operator, and new cell-sorting applications. The 2007 guideline publication is a rich source of information on the biosafety concerns for a variety of cell-sorting applications, recommendations for personnel training, instrument set-up, maintenance and decontamination, and a detailed appendix containing protocols for testing the flow cytometer/sorter for efficiency of aerosol containment.
To deal with the problems of biohazardous aerosols created during cell sorting, various engineering adaptations have been made such as integrating a commercial flow cytometer into a Class II biosafety cabinet. 35 However, most labs that sort highly biohazardous samples resort to putting their cell sorters into biocontainment facilities, such as a Biosafety Level-3 laboratory. Leary 34 suggests that the droplet-based design of high-speed cell sorters, which reach incredible speeds of 40,000 cells/s and higher, should be replaced with more biosafe designs, such as fluidic switching in closed systems.
Summary and conclusions
In recent years, with the growing concern about bioterrorism and emerging infectious diseases, flow cytometry and cell sorting have been successfully applied to address a diverse set of challenges in biosecurity and related public health areas. Flow cytometry technology is now poised to become a mainstay measurement approach in biosecurity applications. To realize this possibility, there are improvements to be made to instrumentation to increase automation and to provide safer design to control biohazards that are present when sampling and sorting infectious materials.
A recent trend in software development has been “open source,” which allows users to adapt a software package to their own specifications. The open source approach has been applied at Los Alamos National Laboratory, by researchers in the National Flow Cytometry Resource, to the creation of the Open Reconfigurable Acquisition System (ORCAS). ORCAS is a digital data acquisition system that can be used to collect data from a variety of commercial and custombuilt flow cytometers. 46 A similar approach could be adopted for flow cytometry instrumentation, following along the lines of the Polonator DNA sequencing system developed by Dover Systems in collaboration with the George Church laboratory at Harvard Medical School, which was built using an open platform, “plug and play” approach (Polinator.org). The open platform, configurable approach has the potential advantage that it may stimulate the needed innovation in instrument components that the commercial flow cytometry manufacturers have not been able to provide. Implementing such an approach to flow cytometer instrumentation might clear the apparent technology bottleneck that has stalled the introduction of point-of-care flow cytometers in resource-poor settings and might help to make low-cost, portable flow cytometry a reality. The BD Influx (Becan, Dickinson and Company, Franklin Lakes, NJ) cell sorter appears to be the first commercial flow cytometry instrument to feature this type of flexible design.
Likewise for biosafety concerns when analyzing infectious material by flow cytometry, automated sampling features will reduce the opportunity for operator exposure to biohazards. The design of cell sorters, in particular, could be improved to integrate containment features, such as disposable flow cells and tubing for lengthy sorting procedures, or disposable and enclosed sort modules to contain aerosols. The future of flow cytometry applied to an expanded array of applications in biosecurity lies in the further addition of automated features and integrated biocontainment, and is within our technology reach.
Although we have seen many exciting applications of flow cytometry in biosecurity over the past decade, flow cytometry and cell sorting have still not been used to their full capabilities in biosecurity science and applications. The multicolor capability of cutting-edge flow cytometry (6–12 colors in one sample) opens the door for multiplexed interrogations of mixed microbial cell populations using immunologic or molecular detection methods. 12 51 The wide spectral range of fluorescent reporter proteins now available begs for application in the area of biosecurity for the insight that it can give to gene expression and pathogenesis of some of our most dangerous and least understood pathogens. 61 Likewise, the growing field of MHC tetramer technology 10 has yet to be applied extensively to the study of host immune response to select agent pathogens. The ability to fully characterize and understand the host immune response to pathogens is critical information for vaccine development and drug screening for biosecurity, and flow cytometry can be an important technology for providing that foundational knowledge. Likewise, the coupling of advanced molecular technology for amplification of whole genomes from single bacteria 54 has not been exploited in cell-sorting studies applied to environmental surveillance for pathogens, for example, in water biosecurity to characterize microbial constituents. Yet, the ability of flow cytometers to rapidly analyze individual particles in a mixed sample and selectively sort rare events of interest is one of the hallmark features of flow cytometry and can uniquely be exploited to advance biosecurity science and applications.
There are many technology challenges involved in bringing a highly sophisticated diagnostic test optimized in the developed world, to use for diagnostics in resource-poor, undeveloped parts of the world. Understandably, there has been a question raised even by the flow cytometry community as to whether flow cytometry or lab-on-a-chip/image-based technology will win the day for providing the affordable and sustainable solution for CD4 T-cell counts to resource-limited parts of the world dealing with the HIV/AIDS epidemic 36 60 68 . Advances in this area should be watched closely for their potential application to biodetection, environmental surveillance, and military medicine.
It has been suggested that an essential next step for global biosecurity is near real-time, global and transparent surveillance of infectious disease. 7 33 50 As evidenced by the wide spectrum of possible applications presented here, flow cytometry technology can play a key role in reducing human suffering from infectious diseases and can assist the nation with providing peaceful solutions to the challenge of global biosecurity.
Acknowledgments
Figure 1 was generously provided by Drs. Alina Deshpande and P. Scott White, Los Alamos National Laboratory. The author gratefully acknowledges the support of the National Flow Cytometry Resource (National Institutes of Health, National Center for Research Resources RR-01315). This is Los Alamos Unclassified Report Number: LA-UR 08–07652.