Production of reactive oxygen species is elevated in SS RBC. (A) Comparison of ROS levels in total populations of AA and SS RBC. SS RBC manifest a 1.5- to 2.5-fold higher signal than AA RBC by flow cytometric measurement of CM-H2-DCFDA-derived signal (P < .05). The left panel shows fluorescence histograms of representative AA and SS samples while the right panel shows normalized MFI data of AA (n = 3) and SS samples (n = 5). (B) Elevated ROS production in SS RBC is not confined to the reticulocyte subpopulation. Total AA and SS RBC samples were simultaneously stained for ROS levels with CM-H2-DCFDA and for the reticulocyte marker CD71. While SS reticulocytes have a higher average signal than non-reticulocyte SS RBC (right panel), the whole population of non-reticulocyte SS RBC demonstrates a right shift relative to AA non-reticulocyte RBC (left panel). Flow cytograms are representative of 3 AA and 3 SS samples. (C) Elevated ROS production in SS RBC relative to AA RBC is preserved across all fractions (F1 - F6) of density-fractionated erythrocytes. Fractions 5 and 6 cells represent sickle “dense cells” and are not typically found in AA samples. n = 3 AA and 3 SS samples and P < .05 where designated by asterisks.