With the emergence of fetal hemoglobin (HbF)-stimulating agents as potential treatments for sickle-cell disease, procedures to monitor the effect of these agents on HbF levels in patients are needed. Flow cytometric assay was developed as a rapid procedure to detect fetal hemoglobin in erythrocytes (F cells) and reticulocytes (F reticulocytes) using a Tricolor-conjugated monoclonal antibody against HbF. In order to detect fetal hemoglobin in reticulocytes, these same erythrocytes are subsequently incubated with a staining solution of Thiazole Orange. This method permits rapid distinction and semi-quantitation of F cells and F reticulocytes in EDTA anticoagulated peripheral blood. In collaboration with Dr. Mark Gladwin group, we are following patients with sickle syndromes that are treated with hydroxyurea (HU) and/or erythropoietin. Flow cytometric analysis is used to establish baseline levels of HbF and F reticulocytes in SCD patients and to assess their responses to treatment regimens. Fetal hemoglobin (HbF) induction involves NO-cGMP signaling pathways. The NO substrate L-arginine and the phosphodiesterase (PDE) 5 inhibitor sildenafil, which potentiates cGMP, are studied in adults with sickle cell disease (SCD) who are stably on HU. 24 courses of L-arginine or sildenafil, assigned based on gender, were successfully completed. L-arginine increased plasma arginine and ornithine, but not citrulline, suggesting diversion by plasma arginase from NO, and citrulline, generation. Sildenafil increased plasma cGMP and citrulline, but not other amino acids. Glutathione (GSH) increased in patients on L-arginine. Pulmonary pressures and 6-minute walk distances improved in patients on sildenafil. In 14 subjects with stable baseline HbF levels, HbF changed little on L-arginine, down from baseline by 2.916.1%, n=6; p=n.s., but increased on sildenafil, up from baseline by 7.511.7%, n=8, p<.05. Reticulocyte counts decreased in patients on sildenafil. L-arginine at doses that increase plasma arginine levels, alters redox potential in red cells. The lack of clinical changes after L-arginine supplementation may be due to increased arginine metabolism in SCD patients, caused by plasma arginase. In vivo augmentation of the cyclic nucleotide pathway by PDE inhibition minimally induces HbF, but strikingly improves hemodynamic and functional status in SCD.