The Cancer Research Institute of West Tennessee

Background of Flow Cytometry and Examples of Clinical Applications.

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Flow cytometry permits the measurement of single cells as they flow single file (>100 cells/sec) through a small orifice (flow chamber). During the 1960's, Kametsky and co-workers and the Los Alamos group led by Fulwyler, developed for Leonard Herzenberg and his colleagues at Stanford the first fluorescence activated cell sorter (FACS) as a means to sort lymphocytes into various subcategories, and provide cell counts in the sorted fractions. Flow cytometry provided unprecedented speed and specificity for cell analysis. The Stanford concept formed the basis for the first commercial research flow cytometer, the Becton-Dickinson's FACS. A typical analysis instrument consists of an injector which propels a stream of cells into a flow chamber (orifice or transducer). The flow chamber passes the cells, one at time, through a laser light beam. Light scattered forward or at a right angle to the incident beam and fluorescence from fluorochromes attached to each cell are each quantitated by a light detecting system (photomultiplier tube or photo diode) placed appropriately in the optical pathway. In a typical clinical application, the fluorescent dye is attached chemically to a protein, often a monoclonal antibody. The antibody is capable of binding to a particular surface component of one or more classes of cells in a cell suspension. For example, if the sample is a mixed population of lymphocytes, the operator might add a mixture, such as that sold by Becton Dickinson,, of Anti-Leu4 labeled with fluorescein isothiocyanate (FITC) which emits green light,. and Anti-Leu-12 labeled with phycoerythrin (PE) which emits orange-red light. These antigens are found on T and B cells, respectively. The antibodies bind to these cells, and when the cells pass through the laser light beam which excites them at 488mn (blue light), they emit green and orange-red light, respectively. It is possible on this basis to separate cells into three groups, T cells, B cells, and all others of which analytical data, such as percentages, can be obtained on the different populations.

Current flow cytometers can measure two additional parameters other than fluorescence emission. Typically, these are forward light scattering, which gives an index of cell size, and side scattering, which gives an index of cell granularity. Plots of forward scattering versus side scattering for blood white cells provide graphs containing clusters of points; each cluster consists of a distinct cell grouping, e.g., lymphocytes, monocytes, and granulocytes. Some non-optical flow cytometers, also called hematology analyzers, measure electronic cell volume (ECV). This measure is more sensitive to cell volume than the light scatter measurement and has the capability to give added information on cells, such as cell volume, capacitance and membrane conformational changes. Furthermore, ECV can be used as a non-optical trigger for detection of a cell entering the flow chamber. None of the commercial flow cytometers can simultaneously measure the combination of fluorescence light scatter, and electronic cell volume. Using flow cytometric analysis, it is possible to enumerate members of the various classes of lymphocytes. For example, B-D sells a Simultest Immune Monitoring Kit featuring four vials of mixed antibodies labeled with FITC, PE and PE-Texas Red (the mixed labels give the third emission wavelength via a proprietary energy transfer process). One sample pinpoints monocytes, a second flags total T cells together with activated T and B cells, a third gives the ratio of cytotoxic to suppressor T cells together with the ratio of helper to inducer T cells, and a fourth shows specific antigenic subsets of T cells and NK (natural killer) cells. This panel provides clinically vital information on AIDS and other immunodeficiency diseases, on autoimmune diseases, viral infections, and transplant rejection. These dye mixtures are used to perform three color analysis but are far from ideal for reasons to be stated later. However, the attempts to extract 3-color fluorescence from excitation at one wavelength show the great clinical need in this area. Simply stated, the more accurate parameters which can be detected on a single cell, the more accurate the diagnosis, prognosis, and treatment monitoring. Individually labeled monoclonal antibodies to a wide variety of blood cell antigens are sold and often provide useful diagnostic information of a highly specialized nature. For example, anti-leu-M5 (which binds to monocytes and macrophages) is useful in the immunophenotyping of hairy cell leukemia.

Another clinical area for which Dr. Thornthwaite has the best reagents, is DNA analysis, also known as ploidy measurement. Depending on what stage of the growth cycle a cell is in, it may be diploid, tetraploid, or haploid; i.e. the cell may contain a normal amount of DNA (2C) twice that amount (4C), or half of it (I C) as in sperm. Under certain pathological conditions, abnormal amounts of DNA are present (aneuploidy) due to an abnormal number and/or type of chromosomes. Aneuploidy has been found in leukemia, lymphoma, myeloma, and all solid tumors. Evidence for clinical significance of ploidy measurements has been found in several carcinomas including colorectal, melanoma, breast, urothelia and bladder, among others. Tumors that contain cells with abnormal amounts of DNA offer a significantly worse prognostic picture than those that contain the normal diploid quantity (Thornthwaite,, 1980-1990). Solid tumors must be separated into single nuclei for flow cytometry to give useful information about them. This has been difficult, but Dr. Thornthwaite's Nuclear Isolation Medium (NIM) has solved this problem.

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