immunoassays. In autoimmune thrombocytopenic purpura, free serum antibodies are not found as frequently as platelet-bound antibodies (80–83). In contrast, in cases of alloantibody formation, serum antibodies may be detected without evidence of platelet-associated antibodies (84). Flow cytometry is an excellent method for direct analysis of platelet-bound antibodies, and it has also been shown to be of benefit in detection of free plasma antibodies (81, 85). The use of thiazole orange, a fluorescent dye that binds RNA, allows immature platelets (also referred to as reticulated platelets) to be quantified (86–88). The reticulated platelet count can be used to determine the rate of thrombopoiesis. This measurement can separate unexplained thrombocytopenias into those with increased destruction and those with defects in platelet production. The pathogenesis and molecular defects of many primary thrombocytopathies are well known and relate to defects in structural or functional glycoproteins, such as the abnormal expression of gpIIb/IIIa in Glanzmann thrombasthenia and gpIb in Bernard-Soulier disease (89– 94). Flow cytometry is a rapid and useful method of obtaining a diagnosis. Until recently, functional analysis of platelet activation was used primarily in research. Many immunological markers of platelet activation have been described, and the commercial availability of antibodies permits flow cytometric determination of platelet activation (95–97). Platelet activation may be clinically important in stored blood components, after cardiopulmonary bypass and renal dialysis, and in the treatment of patients with myocardial infarction or thrombotic events. Quantification of Soluble Molecules Soluble antigens or antibodies can be quantified by flow cytometry if standard cells or beads are used. For example, OKT3 is a mouse anti-human antibody useful in treating transplant rejection. Circulating concentrations of OKT3 can be quantified by incubating with normal CD3- positive lymphocytes, followed by a fluorescently labeled anti-mouse antibody (98). Fluorescence values are compared to a calibration curve generated with known amounts of OKT3. Recently, multiplex assays for several antigens have become possible by the use of beads indexed by incorporating two different dyes (99–102). Summary Flow cytometry is a powerful technique for correlating multiple characteristics on single cells. This qualitative and quantitative technique has made the transition from a research tool to standard clinical testing. Applications in hematology include DNA content analysis, leukemia and lymphoma phenotyping, immunologic monitoring of HIV-infected individuals, and assessment of structural and functional properties of erythrocytes, leukocytes, and platelets. Smaller, less expensive instruments and an increasing number of clinically useful antibodies are creating more opportunities for routine clinical laboratories to use flow cytometry in the diagnosis and management of disease. References 1. Keren DF, Hanson CA, Hurtubise PE. Flow cytometry and clinical diagnosis. Chicago: ASCP Press, 1994:664 pp. 2. Shapiro H. Practical flow cytometry, 3rd ed. New York: Wiley-Liss, 1995:542 pp. 3. Orfao A, Ruiz-Arguelles A, Lacombe F, Ault K, Basso G, Danova M. Flow cytometry: its applications in hematology. Haematologica 1995;80:69–81. 4. Recktenwald DJ. Introduction to flow cytometry: principles, fluorochromes, instrument set-up, calibration. J Hematother 1993; 2:387–94. 5. Watson JV. The early fluidic and optical physics of cytom 6.1 INTRODUCTION One of the major advances in laboratory medicine has been the ability to immunologically identify specifie subsets of leukocytes and other cells by their expression of proteins and other markers. This process, termed "immunophenotyping," employs monoclonal antibodies reactive against cell-associated determinants to distinguish specific subsets within a heterogeneous mixture of cells. Quantification of a subset of interest can be readily accomplished by the use of aflow cytometer, an instrument 181 182 Handbook of Human Immunology, Second Eclition that electronically measures the intensity of scattered or emitted