The light scatter signals are usually used for screening. However, the characterization of cells is mainly based on their surface, cytoplasmic, and nuclear antigens, which are detected through a special optical signal: the fluorescent signal. Fluorescent signals are generated through fluorochrome-labeled antibodies that react specifically with various cell antigens, thus facilitating the identification of cell lineage, developmental stage, and special groups of tumor cells. Cellular DNA and RNA can be directly stained by intercalating agents, such as propidium iodide, which can bind DNA and RNA with the resultant generation of fluorescent signals.

Fluorochrome-labeled cells absorb incident light from the laser, resulting in emission of a longer wavelength, which is called fluorescence. The color of light emitted is a function of its wavelength (Table 2.1). As long as the wave-lengths emitted from the various fluorochromes do not overlap, the same specimen can be treated with 2 to as many as 10 fluorochrome-conjugated antibodies. The selection of fluorochrome is determined by the compatibility between the wavelength spectrum of the light source and the excitation ranges of the fluorochromes (Table 2.2). The most frequently used fluorochrome pair is fluorescein isothiocyanate (FITC) and phycoerythrin (PE).

A series of optical devices is used to direct the incident light, reflected light, and fluorescence to detectors. A beam-shaping lens is used to focus the laser down to a narrow beam waist of 20 to 100 µm in the sensing area. In multicolor analysis, separation of multicolored light is accomplished by the combined use of filters and dichroic mirrors. A short-pass filter allows only light of short wavelengths to pass and blocks that of the longer wavelengths. A long-pass filter acts the opposite way. Dichroic mirrors, in contrast, allow light of certain wavelengths to pass and reflect the light that is not allowed to pass.

This is a graphic display of dots as determined by two related parameters along the x and y axes. For instance, one can plot forward-angle light scatter against right-angle light scatter; each dot represents a single cell or event. On the basis of cell size (forward angle) and cytoplasmic granularity (right angle), the scattergram usually shows three distinct groups of cells in peripheral blood samples. The lymphocytes are the smallest (with no cytoplasmic granules); the monocytes are the largest; the granulocytes have the most cytoplasmic granules (Fig. 2.5). In lymph node specimens, the distinction between a group of large cells and a group of small cells frequently helps to separate lymphoma cells from reactive lymphocytes or a small cell lymphoma from a mixed small and large cell lymphoma.

This graph is usually used to display the number of cells (y axis) versus fluorescence intensity (x axis). When cells are stained with a fluorochrome-labeled antibody, a single histogram provides the percentages of positive and negative populations (Fig. 2.6). An isotopic-negative control should be used to determine the cutoff point between these two populations. For instance, if the monoclonal antibody is mouse immunoglobulin G (IgG), the mouse IgG with no specific antibody function should be used. When DNA stain is used, the single histogram can demonstrate the percentage of cells in different stages of the cell cycle and determine the ploidy of chromosomes with a normal control.

The contourgram computes the percentage of cell groups as determined by two parameters (e.g., two monoclonal antibodies or DNA and RNA contents) (Fig. 2.7). In contrast to the dot plot, the contour plot is composed of isocontour lines representing the cross-sections of the peaks and valleys of the data. An isocontour line connects all elements with a similar frequency of events. When the cutoff points of these two parameters are determined, the contourgram can be divided into four quadrants, and the percentages of subpopulations are readily computed. This is a very useful means for further characterization of subpopulations. Dot plots can also be used for this two-dimensional analysis.

The isometric plot is most frequently used for simultaneous DNA and/or RNA analysis, but it can be used to display the correlation of any three parameters (Fig. 2.8). An isometric plot is often used to present multifactor analysis in research projects and is seldom used in clinical laboratories for routine phenotypic analysis.

Although cell sorting can be based on the principle of electroacoustic or electromechanical fluid switching, most instruments use droplet sorting. For droplet sorting, the stream of cell suspension first passes through an ultrasonically vibrating nozzle so that the stream can be broken up into evenly spaced droplets (e.g., 30,000 to 40,000 droplets per second). At the discretion of the operator, the droplets containing the cells of interest, which have been identified by a monoclonal antibody, are selectively charged electronically, either positive or negative, and then deflected as
they fall through an electromagnetic field formed by two deflection plates. The uncharged droplets fall vertically into a waste container. The cells separated in this way are usually viable and 95% pure. The cell sorter is undoubtedly a useful tool in research laboratories; however, it is not an absolute necessity for a clinical laboratory, because different groups of cells can be separated by electronic gating, and the cell group of interest can thus be selected.