I. BASIC PRINCIPLE. Flow cytometry simultaneously measures and analyzes multiple physical and/or chemical characteristics of single particles, usually cells, as they flow in a fluid stream through a beam of light. With this technique, any suspended microparticle, ranging in size from 0.2 to 150 µm, can be analyzed. Peripheral blood or bone marrow aspirate specimens already represent a suspension of single cells but they must be prevented from clotting by using collection tubes containing disodium ethylenediaminetetraacetic acid (EDTA), sodium citrate, or heparin. Enrichment of leukocytes can be achieved by lysis of accompanying red blood cells with ammonium chloride buffer or use of density-gradient separation. Many protocols also exist for producing cell suspensions from solid tissue suspensions.
II. FLOW CYTOMETER. The flow cytometer is composed of three main systems (Fig. 57.1).
A. The flow system. The sample is injected into a stream of sheath fluid within the flow chamber. Through the principle of hydrodynamic focusing, the particles are forced into the center of the stream and transported through a laser beam for analysis, one particle or cell at a time. A higher flow rate is generally used for the immunophenotyping of cells. A lower flow rate is important in applications where greater resolution is needed, such as DNA analysis.
B. The optical system. Lasers illuminate the particles in the sample stream and optical mirrors and filters route the different wavelengths of the generated light scatter and fluorescent signals to the appropriate photodetectors.
1. Light scatter. Light scattering occurs when a particle or cell deflects laser light. Forward-scattered (FSC) light is in line with the laser light beam and represents a measurement of the cell surface size. Side-scattered (SSC) light is collected perpendicular to the laser light beam and analyzes the granularity or internal complexity of a cell. Leukocytes can be separated into different subpopulations using FSC and SSC. For example, lymphocytes will show both a low forward scatter and a low side scatter due to the small size and lack of cytoplasmic granulation. In contrast, neutrophils are larger in size and show granular cytoplasm as well as a complex nucleus, and therefore will show both a high forward and a side scatter (e-Fig. 57.1).*
2. Fluorescence. Another way to identify particular subpopulations is to conjugate fluorescent dyes to monoclonal antibodies directed toward antigens on a particular cell subset. The staining procedure can be carried out in a direct or indirect staining process. The direct staining procedure involves a single staining incubation, followed by several washes to remove nonspecifically bound antibodies. The indirect staining procedure involves the incubation of cells with a nonfluorescent monoclonal antibody directed toward the specific antigen. After washing to remove nonspecifically bound antibody, there is a second incubation with a fluorescent antibody directed against the monoclonal antibody. Although more time-consuming, the indirect staining procedure is less expensive.
Argon ion lasers are the most common lasers used in flow cytometry because the 488-nm light emitted can be absorbed by more than one fluorochrome. Examples of fluorochromes that are conjugated to antibodies are fluorescein isothiocyanate (FITC) and phycoerythrin (PE). FITC absorbs light in the range of 460 to 510 nm and then fluoresces in the range of 510 to 560 nm, with a peak at ˜525 nm, giving a green fluorescent color. PE absorbs light in the range of 480 to 565 nm and fluoresces at ˜570 nm, giving a red fluorescent color. Although both fluorochromes absorb light at ˜488 nm, the resulting different peak emission wavelengths can be detected by different detectors, which make it possible to use more than one fluorochrome in one sample to simultaneously collect information on the expression of several markers in a specific cell. Combined with the FSC and SSC data, the staining pattern of each subpopulation will aid in delineating which cells are present in a sample and in what percentage they are present (e-Fig. 57.2).
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