Clinical Biodetection And Research Tool

The term cytometry refers to the measurement of physical and/or chemical characteristics of cells or, in general, of any biological assemblies (Shapiro, 1995; Givan, 2001; Stewart et al., 2002). In flow cytometry, such measurements are made while the cells, the biological assemblies, or microbeads (as calibration standards) flow in suspension, preferably in a single file, one by one, past a sensing point. The sensing is conducted by using an optical technique where the beam from a light source interacting with each individual cell, a bioassembly, or a microbead produces scattering or fluorescence. The optical response is used to determine cellular features and organelles, providing counts and ability to distinguish different types of cells in a heterogeneous population. Though the fluorescence detected can be autofluorescence, generally the cells or the intra-cellular products are tagged with special fluorescently labeled antibodies or dyes that bind to cellular components and are capable of producing fluorescence. Flow cytometry yields measurement of various optical responses that provide a set of properties, also called parameters, that provide unique characteristics of a specific type of cell. The identification and quantification of a particular type of cells can be then used to correlate with a specific pathological condition that can be used to identify a specific disease or microbial invasion.

Another name used for flow cytometry and thus applied interchangeably is fluorescence-activated cell sorting (FACS), which emphasizes the utilization of fluorescence detection and the ability of the instrument to sort cells that meet specific measured criteria. In a sense, both flow cytometry and optical microscopy perform similar functions—that is, to look at microscopic objects. Like a microscope, a flow cytometer incorporates a light source, an illumination optics, and a light collection optics. However, while in a regular microscope with point detection (such as in a confocal microscope), the light (laser) beam moves (scanned) to detect and image cells, the cells are moving (flowing in a single file) in a flow cytometer.

Furthermore, incorporation of the cell sorting feature in a flow cytometer by using electrical or mechanical methods allows one to collect cells with one or more specific characteristics. This feature thus allows one to isolate pure population of viable cells with more homogeneous characteristics from within a mixed heterogeneous population of cells. The process of diverting a particular type of cell, after measuring its identifying features, will be described later. In addition to isolating a pure population, the cell sorting capability also allows for further biochemical analysis of the selected cells, or other desired processing such as cell culture.

Although a flow cytometer measures optical response from one individual cell at a time, progress in fluidics and detection, together with rapid data acqui sition and processing software, can now readily provide the ability to detect and analyze up to 75,000 cells per second. Thus, the two significant advantages offered by a flow cytometer over a traditional microscope are its rapid throughput rate at which each cell is interrogated and the ability to sort selected populations while maintaining viability. Thus real-time monitoring of many biological events can be achieved.

The current market for flow cytometry is near one billion dollars worldwide, showing the wide usage of this instrument, even through primary application has been on eukaryotic cells. The usage of flow cytometry for the study of prokaryotic cells is only beginning to emerge. An instrument capable of accurately measuring the properties of microbes, whether bacterial, fungal, or viral, can be expected to significantly expand the current market. The impact will be felt in many diverse areas, such as the pharmaceutical industry, microbiology applications, and agriculture. As pharmaceutical research moves more toward target-directed intervention and biological intervention and away from drug screening with intact animals, flow cytometry offers a cost-effective method for the development and testing of agents by the pharmaceutical industry.

Biological organisms are now being recognized as offensive weapons against population centers. Flow cytometry will likely be a method of choice for the rapid and sensitive screening of potential sources of deliberate contamination. New generation flow cytometers can significantly impact on the ability of physicians and clinicians to quickly determine microorganism infection in humans, animals, food supplies, and water supplies with a compact highperformance cytometer.

Flow cytometry is also beginning to impact agricultural research and livestock development. Selection of sex type in feed animals, or the ability to selectively sort male and female sperm, will have an enormous economic impact on food supply. The ability to select desired animal gender, with flow cyto-metric techniques, and to use artificial insemination ensures desired selection of offspring gender and physical traits. This capability dramatically reduces breeding and maintenance costs. This application is expected to grow substantially in coming decades.

In addition to clinical and biodetection applications of flow cytometry, its ability as a valuable research tool is also rapidly expanding. It has proved to be a powerful technique for cell cycle analysis where measurements of DNA content can be used to provide a great deal of information about the cell cycle. Cell division, apoptosis, and necrosis (Darzynkiewicz et al., 1997; also discussed in Chapter 3) can be studied. Furthermore, metabolic characteristics such as calcium flux, mitochondrial activity, cellular pH, and free radical production in live cell populations can be probed and quantified in real time. Flow cytome-try using fluorescent tagged protein or a reporter gene can readily be used to measure gene expression in cells transfected with recombinant DNA. Thus using flow cytometry one can readily measure the following (source: (i) expression of proteins and (ii)

transfection efficiency. In addition, transfection assays can be combined with staining and sorting for other markers. Also, flow cytometry can be used to purify transfected cells for further analysis or use. Other cellular functions studied by flow cytometry are (source: (i) phagocycosis, (ii) intracellular cytokines, (iii) oxidative burst, and (iv) membrane potential.

Molecular cytometry is a relatively new area of application of cytometry which is attracting a great deal of attention from the research community (Nunez, 2001). It utilizes flow cytometry to obtain information on cell-to-cell variations in molecular parameters being investigated. Fluorescence resonance energy transfer (FRET), discussed in Chapter 7, can also be used in flow cytometry to determine if two protein markers are closely associated on the cell surface or inside the cell. Another technique being used in combination with flow cytometry to obtain molecular (or submolecular) information is fluorescence in situ hybridization (also abbreviated as FISH and discussed in Chapter 8).

Another active area of research is detecting or assessing the activities of microorganisms in a wide variety of samples such as milk, bean, river water, biosolids, and biofilms.

The various applications of flow cytometry are further summarized in Tables 11.1,11.2, and 11.3. The field of flow cytometry bridges many disciplines involving biologists, physicians, organic chemists, laser physicists, and optical

TABLE 11.1. Clinical Applications

• HIV monitoring

• Leukemia or lymphoma immunophenotyping

• Organ transplant monitoring

• DNA analysis for tumor ploidy and SPF

• Primary and secondary immunodeficiency

• Hematopoietic reconstitution

• Paroxysmal nocturnal hemoglobinuria

TABLE 11.2. Research Applications

• Multiplexing immunoassays

• Multiparameter immunophenotyping

• Measurement of intracellular cytokines

• Signal transduction pathways

• Cell cycle analysis

• Measuring cellular function

TABLE 11.3. Molecular Flow Cytometry

• Multiplexing oligonucleotide assays

• Measuring gene expression

• In situ hybridization

• Drug discovery and fluidic engineers, as well as software and hardware engineers for data acquisition systems. It holds tremendous opportunities for future development, ranging from basic research at the molecular and cell biology level, to functional genomics and proteomics, to new applications, to new designs of flow cytometers with considerably expanded capabilities. Areas of future development are detailed in Section 11.6. This wide diversity of flow cytome-try is clearly reflected at various flow cytometry centers around the world as well as at numerous conferences which attract a truly multidisciplinary participation. The rapidly growing interest of the scientific and clinical community is also evident from the popularity of a number of courses offered on cytometry which cover principles, applications, and hands-on training. Detailed information about these courses can readily by obtained from various websites, some of which are:

National Flow Cytometry Resource:

The International Society for Analytical Cytometry:

Clinical Cytometry Society:

Purdue University Cytometry Laboratories:

Roswell Park Cancer Institute Laboratory of Flow Cytometry:

Cancer Research UK FACS Laboratory:

Major journals specializing in research reports using cytometry are:

Cytometry : The Journal of the International Society for Analytical Cytology Clinical Cytometry: A Publication of the Clinical Cytometry Society and The

International Society for Analytical Cytology The Journal of Immunological Method

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