light from individual cells as they pass before a laser. In clinical laboratories, immunophenotyping by flow cytometry has been invaluable for defining the cell of origin of specific neoplasms, particularly in patients with acute leukemia or non-Hodgkin's lymphoma (see Chapter 7). A second major application is to provide information critical for the staging and management of human immunodeficiency virus (HIV)-l infected patients and patients with other immune deficiency syndromes (see Chapters 8, 12 respectively); the data derived from analysis of T-cell subsets play an important role in guiding therapeutic decisions. A host of additional applications, including quantification of hematopoietic stem cells [I] and reticulocytes [2], diagnosis of acquired and congenital immune deficiency syndromes (see Chapter 9), paroxysmal nocturnal hemoglobinuria [3], detection of minimal residualleukemia after therapy [4], and many others are either approved as in vitro diagnostic (IVD) tests, or serve as diagnostic adjuncts to licensed tests. In addition to clinical considerations, these analytic techniques provide essential information concerning the structure and function of the cells comprising normal hematopoietic and lymphoid cells. In addition to the analysis of cells that occur naturally as single-cell suspensions (blood, lymph, bone marrow, cerebrospinal fluid, ascites, and effusions), flow cytometry can also be used to analyze marker expression and function in disaggregated tissue specimens. This review will focus on the factors that make cytometry as robust as it is, and also on avoiding the pitfalls inherent in such a flexible technique. Flow cytometry is a mature clinical technique at present. When it was first introduced as a research tool a little more than 30 years ago, both instrumentation and analytical reagents were of limited usefulness [5]. The first instruments measured fluorescence on a linear scale at a single wavelength and displayed the values on an oscilloscope. The only means of recording and analyzing data was to make Polaroid photographs of the oscilloscope screen. Major advances in hardware and software spurred by the digital revolution, which together with the development of monoclonal antibodies and new dye technologies, have resulted in the emergence of instruments and procedures that are readily adaptable to clinical laboratories. Several publications are available that detail the principles of cytometry and the operation of flow cytometers [6-12]. 6.2 MONOCLONAL ANTIBODIES The invention of monoclonal antibodies by Milstein and colleagues in 1977 [13] changed the field of flow cytometry, providing an inexhaustible supply of reagents of exquisite specificity. By definition, a monoclonal antibody is an immunoglobulin produced by a clonal population of immortalized hybridoma cells. Accordingly, the antigen-binding sites are completely homogeneous, a situation very different from naturally occurring (polyclonal) antibodies. Because of their homogeneity, hybridomas can be selected which produce high-affinity antibodies with great specificity for particular antigenic determinants. Within a year of Milstein's invention, monoclonal antibodies specific for rat [14] and murine [15] helper T cells and murine MHC antigens [16] were described. Shortly thereafter, the description of three murine monoclonal antibodies specific for human T-cell surface determinants, designated OKTl, Understanding Clinical Flow Cytomelry HI3 OKT3, and OKT-l-lI7 J paved the way for human studies. Today, thesc hybrid()ma~ are rccognized as producing antibodies against the CDS, CD3, and CD4 determinants, respectively. Most of the monoclonal antibodies currently in use for the detection of determinants on human cells are of murine origin. The antibodies are generated to proteins, glycoproteins, glycolipids, and carbohydrates. The biological functions of these molecules are varied and include serving as membrane receptors or ligands for soluble and contact-dependent signaling, acting as enzymes, membrane pumps, or adhesion molecules. Antibodies that recognize intracellular components such as histones, lysosomes, signal transducing molecules, and cytokines, to