Flow Cytometry: Principles and Clinical Applications in Hematology Michael Brown and Carl Wittwer* The use of flow cytometry in the clinical laboratory has grown substantially in the past decade. This is attributable in part to the development of smaller, userfriendly, less-expensive instruments and a continuous increase in the number of clinical applications. Flow cytometry measures multiple characteristics of individual particles flowing in single file in a stream of fluid. Light scattering at different angles can distinguish differences in size and internal complexity, whereas light emitted from fluorescently labeled antibodies can identify a wide array of cell surface and cytoplasmic antigens. This approach makes flow cytometry a powerful tool for detailed analysis of complex populations in a short period of time. This report reviews the general principles in flow cytometry and selected applications of flow cytometry in the clinical hematology laboratory. © 2000 American Association for Clinical Chemistry Flow cytometry provides rapid analysis of multiple characteristics of single cells. The information obtained is both qualitative and quantitative. Whereas in the past flow cytometers were found only in larger academic centers, advances in technology now make it possible for community hospitals to use this methodology. Contemporary flow cytometers are much smaller, less expensive, more user-friendly, and well suited for high-volume operation. Flow cytometry is used for immunophenotyping of a variety of specimens, including whole blood, bone marrow, serous cavity fluids, cerebrospinal fluid, urine, and solid tissues. Characteristics that can be measured include cell size, cytoplasmic complexity, DNA or RNA content, and a wide range of membrane-bound and intracellular proteins. This review will describe the basic principles of flow cytometry and provide an overview of some applications to hematology. General Principles Flow cytometry measures optical and fluorescence characteristics of single cells (or any other particle, including nuclei, microorganisms, chromosome preparations, and latex beads). Physical properties, such as size (represented by forward angle light scatter) and internal complexity (represented by right-angle scatter) can resolve certain cell populations. Fluorescent dyes may bind or intercalate with different cellular components such as DNA or RNA. Additionally, antibodies conjugated to fluorescent dyes can bind specific proteins on cell membranes or inside cells. When labeled cells are passed by a light source, the fluorescent molecules are excited to a higher energy state. Upon returning to their resting states, the fluorochromes emit light energy at higher wavelengths. The use of multiple fluorochromes, each with similar excitation wavelengths and different emission wavelengths (or “colors”), allows several cell properties to be measured simultaneously. Commonly used dyes include propidium iodide, phycoerythrin, and fluorescein, although many other dyes are available. Tandem dyes with internal fluorescence resonance energy transfer can create even longer wavelengths and more colors. Table 1 lists clinical applications and cellular characteristics that are commonly measured. Several excellent texts and reviews are available (1–6). Inside a flow cytometer, cells in suspension are drawn into a stream created by a surrounding sheath of isotonic fluid that creates laminar flow, allowing the cells to pass individually through an interrogation point. At the interrogation point, a beam of monochromatic light, usually from a laser, intersects the cells. Emitted light is given off in all directions and is collected via optics that direct the light to a series of filters and dichroic mirrors that isolate particular wavelength bands. The light signals