are detected by photomultiplier tubes and digitized for computer analysis. Fig. 1 is a schematic diagram of the fluidic and optical components of a flow cytometer. The resulting information usually is displayed in histogram or twodimensional dot-plot formats. Department of Pathology, University of Utah, ARUP Laboratories, Inc., Salt Lake City, UT 84132. *Address correspondence to this author at: Department of Pathology, University of Utah, 50 North Medical Dr., Salt Lake City, UT 84132. E-mail carl_wittwer@hlthsci.med.utah.edu. Received February 15, 2000; accepted April 10, 2000. Clinical Chemistry 46:8(B) 1221–1229 (2000) Beckman Conference 1221 DNA Content Analysis The measurement of cellular DNA content by flow cytometry uses fluorescent dyes, such as propidium iodide, that intercalate into the DNA helical structure. The fluorescent signal is directly proportional to the amount of DNA in the nucleus and can identify gross gains or losses in DNA. Abnormal DNA content, also known as “DNA content aneuploidy”, can be determined in a tumor cell population. DNA aneuploidy generally is associated with malignancy; however, certain benign conditions may appear aneuploid (7–12). DNA aneuploidy correlates with a worse prognosis in many types of cancer but is associated with improved survival in rhabdomyosarcoma, neuroblastoma, multiple myeloma, and childhood acute lymphoblastic leukemia (ALL)1 (11, 13–16). In multiple myeloma, ALL, and myelodysplastic syndromes, hypodiploid tumors cells portend a poor prognosis. In contrast, hyperdiploid cells in ALL have a better prognosis (11, 13). For many hematologic malignancies, there are conflicting reports regarding the independent prognostic value of DNA content analysis. Although con1 Nonstandard abbreviations: ALL, acute lymphoblastic leukemia; PNH, paroxysmal nocturnal hemoglobinuria; and RBC, red blood cell. Table 1. Common clinical uses of flow cytometry. Field Clinical application Common characteristic measured Immunology Histocompatibility cross-matching IgG, IgM Transplantation rejection CD3, circulating OKT3 HLA-B27 detection HLA-B27 Immunodeficiency studies CD4, CD8 Oncology DNA content and S phase of tumors DNA Measurement of proliferation markers Ki-67, PCNAa Hematology Leukemia and lymphoma phenotyping Leukocyte surface antigens Identification of prognostically important subgroups TdT, MPO Hematopoietic progenitor cell enumeration CD34 Diagnosis of systemic mastocytosis CD25, CD69 Reticulocyte enumeration RNA Autoimmune and alloimmune disorders Anti-platelet antibodies IgG, IgM Anti-neutrophil antibodies IgG Immune complexes Complement, IgG Feto-maternal hemorrhage quantification Hemoglobin F, rhesus D Blood banking Immunohematology Erythrocyte surface antigens Assessment of leukocyte contamination of blood products Forward and side scatter, leukocyte surface antigens Genetic disorders PNH CD55, CD59 Leukocyte adhesion deficiency CD11/CD18 complex a PCNA, proliferating cell nuclear antigen; TdT, terminal deoxynucleotidyltransferase; MPO, myeloperoxidase. Fig. 1. Schematic of a flow cytometer. A single cell suspension is hydrodynamically focused with sheath fluid to intersect an argon-ion laser. Signals are collected by a forward angle light scatter detector, a sidescatter detector (1), and multiple fluorescence emission detectors (2–4). The signals are amplified and converted to digital form for analysis and display on a computer screen. 1222 Brown and Wittwer: Flow Cytometry ventional cytogenetics can detect smaller DNA content differences, flow cytometry allows more rapid analysis of a larger number of cells. Immunophenotyping Applications in Hematology The distributed nature of the hematopoietic system makes it amenable to flow cytometric analysis. Many surface proteins and glycoproteins on erythrocytes, leukocytes, and platelets have been studied in great detail. The availability of monoclonal antibodies directed against these surface proteins permits flow cytometric analysis of erythrocytes, leukocytes, and platelets. Antibodies against intracellular proteins such as