myeloperoxidase and terminal deoxynucleotidyl transferase are also commercially available and permit analysis of an increasing number of intracellular markers. erythrocyte analysis The use of flow cytometry for the detection and quantification of fetal red cells in maternal blood has increased in recent years. Currently in the United States, rhesus Dnegative women receive prophylactic Rh-immune globulin at 28 weeks and also within 72 h of delivery (17). The standard single dose is enough to prevent alloimmunization from ;15 mL of fetal rhesus D1 red cells. If fetomaternal hemorrhage is suspected, the mother’s blood is tested for the presence and quantity of fetal red cells, and an appropriate amount of Rh-immune globulin is administered. The quantitative test most frequently used in clinical laboratories is the Kleihauer-Betke acid-elution test. This test is fraught with interobserver and interlaboratory variability, and is tedious and time-consuming (18). The use of flow cytometry for the detection of fetal cells is much more objective, reproducible, and sensitive than the Kleihauer-Betke test (19–21). Fluorescently labeled antibodies to the rhesus (D) antigen can be used, or more recently, antibodies directed against hemoglobin F (19–27). This intracellular approach, which uses permeabilization of the red cell membrane and an antibody to the g chain of human hemoglobin, is precise and sensitive (21). This method has the ability to distinguish fetal cells from F-cells (adult red cells with small amounts of hemoglobin F). Fig. 2 is a histogram of a positive test for feto-maternal hemorrhage. Although the flow cytometry method is technically superior to the Kleihauer-Betke test, cost, instrument availability, and stat access may limit its practical utility. Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal stem cell disorder that leads to intravascular hemolysis with associated thrombotic and infectious complications. PNH can arise in the setting of aplastic anemia and may be followed by acute leukemia (28). The disease is caused by deficient biosynthesis of a glycosylphosphatidylinositol linker that anchors several complement and immunoregulatory surface proteins on erythrocytes, monocytes, neutrophils, lymphocytes, and platelets (28–31). On erythrocytes, deficiencies of decayaccelerating factor and membrane-inhibitor of reactive lysis render red cells susceptible to complement-mediated lysis (30, 31). Conventional laboratory tests for the diagnosis of PNH include the sugar water test and the Ham’s acid hemolysis test (32). Problems associated with these tests include stringent specimen requirements and limited specificity. Antibodies to CD55 and CD59 are specific for decay-accelerating factor and membrane-inhibitor of reactive lysis, respectively, and can be analyzed by flow cytometry to make a definitive diagnosis of PNH (29, 33– 35). In affected patients, two or more populations of erythrocytes can be readily identified, with different degrees of expression of CD55 and CD59 (Fig. 3) Reticulocyte counts are based on identification of residual ribosomes and RNA in immature nonnucleated red blood cells (RBCs). Traditionally, a blood smear is stained with a dye that precipitates the nucleic acid, and the cells are counted manually (36). This method is subjective, imprecise, labor-intensive, and tedious. The flow cytometric enumeration of reticulocytes uses fluorescent dyes that bind the residual RNA, such as thiazole orange (37, 38). This method provides excellent discrimination between reticulocytes and mature RBCs, with greater precision, sensitivity, and reproducibility than the traditional method (37, 38). However, Howell-Jolley bodies (a remnant of nuclear DNA) are not distinguished from reticulocytes (39). Because the fluorescence intensity is directly proportional to the amount of RNA and related to the immaturity of the RBC, a reticulocyte maturity index has been used clinically to assess bone marrow engraftment and erythropoietic activity and to help classify anemias (34, 38, 40, 41). Some current automated cell counters use similar technology to estimate reticulocyte counts (42). In the blood bank, flow cytometry can be used as a complementary or replacement test for red cell immunology, including