name but a few are available. In particular, the detection of protein activation states with antibodies directed against specific phosphoepitopes, has opened up a new area in which flow cytometry can be used to probe cellular function [18,19]. Antibodies recognize and react against small peptide sequences of complex antigenic molecules. Thus, each antigenic protein contains numerous small peptide sequences, termed epitopes, to which monoclonal antibodies may be raised. Antibodies to any epitope of the parent molecule can be used to identify the entire molecule. Initially, this led to a nomenclature dilemma; each laboratory and commercial source had unique designations for their monoclonal antibodies. To facilitate communication, an international workshop established a consensus nomenclature (see historical review of consensus nomenclature in Chapter 11) [20]. Cell surface antigens were assigned a cluster ofdifferentiation (CD) number based on their recognition by monoclonal antibodies. All antibodies to the same antigen, regardless of their epitope specificity, were grouped together. Thus, two or more antibodies, each reactive with a different epitope on the same antigen are considered as directed against the same CD. In general, the antigens themselves are identified by a CD number (e.g., CD3 on T cells) and the monoclonal antibodies recognizing them are grouped as anti-CD number (e.g., anti-CD3). At a workshop held in Vienna in 2006, 3S0 CD clusters were recognized. These are listed and annotated as Tables 11.2 and 11.3 in Chapter II. It is important to note that few of the CD antigens are truly restricted to a single cellular lineage. With rare exceptions (e.g., CD3 on T cells), lineage assignments cannot be made on the basis of a single CD antigen. Rather, they are determined by the presence and absence of a combination of markers. 6.3 FlUOROCHROMES AND flUORESCENCE DETECTION To detect the presence of a cell-bound monoclonal antibody by flow cytometry, the antibody must be coupled either directly or indirectly to a fluorescent dye (fluorochrome). These are the dyes that, when excited by light of a specific wavelength, emit light at a longer wavelength (this is known as the Stokes shift). Because different dyes have different Stokes shifts, they can be distinguished by passing emitted light through a series of optical filters that permit only specific bandwidths of light to reach the photomultiplier tubes (PMTs) (Figure 6.1). The PMTs generate a signal proportional to the intensity of the incident light. Through spectral compensation, which will be discussed in detail later, the processed signals can be quantitatively related to the amount of dye bound to a particular cell. With appropriate standards, this in turn, can be used to estimate the number of molecules targeted by the fluorescence-tagged antibody. 184 Handbook of Human Immunology, Second I::ditiol1 r FIGURE 6.1 Schematic operation of a three-color single-laser flow cytometer. The fluorochrome-labeled cells are pumped through the flow cell, where they enter the light path of a 488-nm argon laser (arrow). A photodiode aligned with the laser path captures light scattered between 1.5° and 19° (FSc). Before encountering this FSc detector, the scattered laser light can be attenuated by a neutral density (ND) filter if needed; the laser beam itself is blocked by an obscuration bar. A collection lens is positioned 90° from the incident laser light. This light (which consists of both laser light scattered by the cell and light emitted by the fluorochromes associated with the cell) passes through a series of dichroic mirrors and filters that separates the light sources on the basis of their wavelengths. In the instrument depicted here, light from the argon laser (488 nm) is deflected to the side scatter photomultiplier tube (SSc PMT) by a 488 dichroic long-pass (DL) mirror. Before reaching the SSc PMT, this light passes through a 488-nm band-pass (BP) filter, which blocks all other wavelengths. The use of these filters ensures that the SSc PMT measures only light emitted by the argon laser and scattered at 90° by the cell. The light transmitted through the 488 DL mirror then passes through blocking filters that