RBC-bound immunoglobulins and red cell antigens (43). In multiply transfused patients, determining the recipient’s blood type can be very difficult. Flow cytometry has been used to accurately identify and pheFig. 2. Hemoglobin F test for feto-maternal hemorrhage. Most adult RBCs do not have any hemoglobin F and are included in the large peak on the left. A few adult red cells have a small amount of hemoglobin F and are called F cells. Higher quantities of hemoglobin F in fetal cells yield a higher fluorescence signal and allow discrimination between fetal cells and adult F cells. Clinical Chemistry 46, No. 8(B), 2000 1223 notype the recipient’s red cells (44). Flow cytometry is being used increasingly in the blood bank to assess leukocyte contamination in leukocyte-reduced blood products (45, 46). leukocyte analysis Immunologic monitoring of HIV-infected patients is a mainstay of the clinical flow cytometry laboratory. HIV infects helper/inducer T lymphocytes via the CD4 antigen. Infected lymphocytes may be lysed when new virions are released or may be removed by the cellular immune system. As HIV disease progresses, CD4-positive T lymphocytes decrease in total number. The absolute CD4 count provides a powerful laboratory measurement for predicting, staging, and monitoring disease progression and response to treatment in HIV-infected individuals. Quantitative viral load testing is a complementary test for clinical monitoring of disease and is correlated inversely to CD4 counts (47, 48). However, CD4 counts directly assess the patient’s immune status and not just the amount of virus. It is likely that both CD4 T-cell enumeration and HIV viral load will continue to be used for diagnosis, prognosis, and therapeutic management of HIV-infected persons. Perhaps the best example of simultaneous analysis of multiple characteristics by flow cytometry involves the immunophenotyping of leukemias and lymphomas. Immunophenotyping as part of the diagnostic work-up of hematologic malignancies offers a rapid and effective means of providing a diagnosis. The ability to analyze multiple cellular characteristics, along with new antibodies and gating strategies, has substantially enhanced the utility of flow cytometry in the diagnosis of leukemias and lymphomas. Different leukemias and lymphomas often have subtle differences in their antigen profiles that make them ideal for analysis by flow cytometry. Diagnostic interpretations depend on a combination of antigen patterns and fluorescence intensity. Several recent review articles are available (49–60). Flow cytometry is very effective in distinguishing myeloid and lymphoid lineages in acute leukemias and minimally differentiated leukemias. Additionally, CD45/side scatter gating often can better isolate the blast population for more definitive phenotyping than is possible with forward scatter/side scatter gating. Fig. 4 is an example of CD45/side scatter gating for an acute myeloid leukemia. Although most acute myeloid leukemias are difficult to classify by phenotype alone, flow cytometry can be useful in distinguishing certain acute myeloid leukemias, such as acute promyelocytic leukemia (61, 62). Flow cytometry can also be used to identify leukemias that may be resistant to therapy (63). In ALL, phenotype has been shown to correlate strongly with outcome (64, 65). The B-cell lymphoproliferative disorders often have specific antigen patterns. The use of a wide range of antibodies allows clinicians to make specific diagnoses based on patterns of antigen expression. Table 2 lists some of the common phenotypes expressed by various B-cell lymphoproliferative disorders. Not only is the presence or absence of antigens useful in making specific diagnoses, the strength of antigen expression can also aid in diagnosis. One example is the weak expression of CD20 and Fig. 3. Diagnosis of PNH. Control individuals (A) show high expression of CD55 and CD59 on all red cells. In PNH (B), some stem cell clones produce RBCs with decreased expression of CD55 and CD59. In the PNH patient (B), two distinct populations are present: normal red cells with high CD55 and CD59 expression and a second population with low CD55 and CD59 expression. 1224 Brown and Wittwer: Flow Cytometry