Labile iron concentrations of normal hemoglobin AA erythrocytes in isotonic and hypertonic buffers. Erythrocytes suspended in isotonic or hypertonic buffers were loaded with calcein using calcein acetomethoxy, and fluorescence was measured before and after addition of the 100 μM permeant Fe²⁺ chelator (BIP). 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 7 hemoglobin AA (HbAA) control individuals. The increase in MCHC of erythrocytes suspended in hypertonic buffer was determined using the phthalate ester technique as described in ”Patients, materials, and methods.” The average increase in the labile Fe²⁺ concentration (41%) of cells in hypertonic buffer was greater than the average rise in the MCHC (22%).