Fractionation and analysis of normal erythrocytes, sickle cells and reticulocytes; removal of erythrocytes from blood and bone marrow

A number of density gradient strategies have been developed for the fractionation of human erythrocytes according to their age. 

As the cells age, so their density tends to increase; reticulocytes therefore have the lowest densities. Reticulocytes have frequently been partially purified on discontinuous gradients of arabinogalactan; the actual density range being quite variable, sometimes broad, e.g. 1.066-1.220 g/ml 

[1], sometimes narrow, e.g. 1.083-1.124 g/ml [2]. These gradients have entailed the use of unusually high RCFs of 27,000g [1] 74,000g [2] for approx 1 h.

 Such high RCFs are rarely used for other types of cell and are often detrimental to cell function; the viscosity of arabinogalactan gradients maybe a contributory factor. Percoll® gradients have also been used to fractionate erythrocytes on the basis of density in selfgenerated gradients, again RCFs more associated with the banding of smaller particles (33,000g) were used [3].

 Moreover importantly, the properties of reticulocytes, which control their release from bone marrow, i.e. their deformability and rigidity, may be compromised by the use of Percoll®. 

There is evidence that the colloidal silica particles of Percoll® adhere to the surface of erythrocytes (even after washing) and cause progressive hemolysis; the elongation index of erythrocytes is thus difficult to measure and their deformability is affected [4]