Erythrocyte labile iron concentrations using calcein and Fe²⁺ chelator versus calcein and Fe³⁺ chelator. Erythrocytes were loaded with calcein using acetomethoxyl-calcein, and fluorescence was measured before and after addition of 100 μM permeant Fe²⁺ chelator (BIP) or Fe³⁺ chelator (SIH) to separate identical cell suspensions from the same subject. Erythrocyte labile iron was then calculated based on the fractional increase in fluorescence, the intracellular calcein concentration, and the dissociation constant, K_d, of calcein and iron, as described in “Patients, materials, and methods.” The bar graphs show mean ± SD of erythrocyte labile iron concentration of 10 hemoglobin AA control individuals and 12 patients with hemoglobin SS.