General Principles Of Flow Cytometry

Flow cytometry is a technique in which single cells in a fluid suspension are analyzed with respect to their intrinsic light-scattering properties and are simultaneously evaluated for one or more extrinsic properties (i.e., the presence of specific molecules) using fluorescent probes. The fluorescent probe might bind directly to the targeted molecule (e.g., propidium iodide in DNA content analysis) or a fluorescent dye might be coupled to an antibody probe, to enable detection of a specific protein. By using several different fluorochromes with substantially nonoverlapping emission spectra, the laboratorian can simultaneously evaluate the expression of multiple extrinsic cellular properties. Moreover, because the flow rate of cells within the flow cytometer is rapid, thousands of cells can be analyzed in seconds. Despite the rapidity with which data can be acquired, however, because each cell is analyzed individually, multiple intrinsic and extrinsic parameters are retained for each cell; that is, flow cytometric analysis is multiparametric. In this section, we review some general principles of flow cytometry (1-6).

1.1. FLUIDICS A requirement of flow cytometry is that the sample to be analyzed must comprise single cells in a fluid suspension. Therefore, hematopoietic precursors and lymphocytes are ideally suited for flow cytometric analysis, because they normally exist as single-cells (i.e., without intercellular attachments) in vivo. The single-cell suspension is aspirated into the flow cytometer, whereupon it encounters an isotonic fluid referred to as sheath fluid. The sheath fluid surrounds the sample fluid and produces conditions of laminar flow within the stream of sample fluid. This process of hydrodynamic focusing results in a single-file arrangement of cells within the sample stream.

1.2. LIGHT SOURCE AND LIGHT SCATTER Next, each cell is interrogated by a light source. Typically, clinical flow cytometers use small air-cooled lasers as a light source. The wavelength of monochromatic light emitted by the laser (e.g., 488 nm for argon) in turn dictates the fluorochromes suitable for use with a given instrument; that is, a useful fluorochrome must absorb significantly at that wavelength. As each cell passes through the interrogation point, it scatters the incident laser light in all directions. Light scattered at two specific angles is measured by the flow cytometer: forward-angle light scatter (FSC), and orthogonal or right-angle light scatter (SSC). FSC refers to light deflected by the cell at a small angle relative to the vector of the incident light, whereas SSC refers to light deflected at a right angle to the vector of the incident light. Appropriately arrayed photodetectors capture the light scattered by the cell at low (FSC) or right (SSC) angles and convert its energy into an electrical signal proportional to the light's intensity. This electrical signal is ultimately converted into a digital signal, which is stored by a dedicated computer system. To a rough approximation, the FSC signal of a cell correlates with its size, whereas the SSC signal correlates with granularity or other intra-cellular "complexity" (e.g., vacuoles) (Fig. 1).

1.3. FLUORESCENCE AND EXTRINSIC PROPERTIES

Unlike FSC and SSC, which represent light-scattering properties intrinsic to the cell, extrinsic properties require the addition of a fluorescent probe for their measurement. A fluorescent molecule is one that absorbs light across a spectrum of wavelengths and emits light of lower energy across a spectrum of longer wavelengths. The difference between the peak absorption and emission wavelengths of a fluorochrome is referred to as its Stokes' shift. By using a series of fluorochromes with progressively larger Stokes' shifts, each of which absorbs reasonably well at the wavelength of the laser used in the flow cytometer, the laboratorian can simultaneously evaluate the cell for several extrinsic properties. The clinical utility of such multicolor analysis is enhanced when the fluorescent data are analyzed in conjunction with FSC and SSC.

1.4. MULTICOLOR ANALYIS The number of fluoro-chromes capable of being used simultaneously is limited by the number of photodetectors in the flow cytometer. The specificity of each photodetector for a given band of wavelengths results from the arrangement of a series of mirrors and filters, which permits only light of certain wavelengths to reach each detector. Most clinical flow cytometers in use today are capable of three-color or four-color analysis. However, the range of suitable fluorochromes can be expanded by adding additional light sources whose emission differs from that of the primary laser and by using tandem dyes. The latter consist of two coupled fluorescent molecules; the first is excited by the light source

From: Molecular Diagnostics: For the Clinical Laboratorian, Second Edition Edited by: W. B. Coleman and G. J. Tsongalis © Humana Press Inc., Totowa, NJ

Fig. 1. Normal peripheral blood specimen illustrating the intrinsic light-scatter properties of lymphocytes (R2), monocytes (R3), and granulocytes (R4).

and the second is excited by the emission of the first. In this way, a larger net Stokes' shift is produced compared with individual fluorochromes, and more extrinsic properties can be measured using a single light source. By using multiple light sources and judicious combinations of fluorochromes, including tandem dyes, investigators have developed research instruments capable of 11-color analysis (7).

1.5. SPECTRAL OVERLAP AND COMPENSATION One pitfall of multicolor analysis is spectral overlap. Because the emission spectra of individual fluorochromes are often broad and overlapping, a portion of the fluorescence signal arriving at a specific photodetector could have originated from a different fluorochrome. The contribution of such extraneous signals can be measured by omitting individual fluorochromes in multicolor experiments and subtracting the fraction of signal originating from inappropriate fluorochromes. For example, the "compensated" signal for photodetector number 2 might be expressed as the total signal recorded in photodetector number 2, minus 20% of the signal recorded in photodetector number 1 (i.e., compensated FL2 = [FL2] - 0.2[FL1]), whereas the "compensated" signal for photodetector number 1 might be expressed as the total signal recorded in photodetector number 1, minus 5% of the signal recorded in photodetector number 2 (i.e., compensated FL1 = [FL1] - 0.05[FL2]) (Fig. 2). Compensation is commonly performed prior to acquisition of the actual experimental data, but software has more recently become available that enables postacquisition compensation of raw (uncompensated) data.

1.6. DATA ANALYSIS AND GATING Once the intrinsic and extrinsic cellular properties of many cells (typically 5000-20,000 in routine clinical studies) have been measured and recorded in the form of a list (so-called "list-mode" data), the laboratorian must analyze the data. Such multiparametric analysis invariably involves electronic gating. When primary data are displayed (typically as one-dimensional histograms, or two-dimensional dot plots, with each dot, or event, representing a single cell), specific populations can be selected for analysis to the exclusion of other (irrelevant) populations. For example, a polygonal or amorphous gate might be drawn around events positive for the B-cell antigen CD20 in the case of a B-cell non-Hodgkin's lymphoma (Fig. 3). Gating enables precise characterization of the extrinsic properties of specific subsets of cells, without contamination from fluorescence signals originating from cells not relevant to a particular analysis.

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