An improved method of harvesting haemoglobin-free human erythrocyte ghosts

The ghosts (stromas) of human erythrocytes bearing Hb of different phenotypes were separately isolated by incubating the erythrocytes in hypotonic (20 imOsm) phosphate buffer saline (PBS) for 1 h at pH 7.4. All of these washed ghosts were found to react with benzidine indicating the presence of Hb. In the preparation of Hb free ghosts, the pH 8.2 of the phosphate buffer, incubation time (4 h) and extra oxygen supply to the medium were noticed to be very effective. The results shows that certain ghosts bound to SS, SF, and S-Thal type of Hb were more resistant to the clearing procedure than the ghosts bound to AA, AF, ASF and AC types of Hb. The combined effect of above variables for the optimum isolation of clear ghosts with minimum or no loss of membrane components has been described.