Age-dependent elution of human red blood cells in gravitational field-flow fractionation

The technique of field-flow fractionation (FFF) which combines the earth gravitational force with a carrier liquid flow in a horizontal, ribbon-like channel is well suited for the separation of micron-sized particulate species such as cells. We investigated the selective elution, in a phosphate buffer, of human red blood cells (RBC) which migrate along the FFF channel more slowly than the carrier. Fractions of the channel effluent were collected and the activities of various intracellular enzymes, which either reveal the presence of white cells or are known to be related to cell age, were evaluated for each fraction. The hemoglobin sub-fraction composition was also determined. From analysis of these biochemical determinations, nucleated cells and reticulocytes appeared to be eluted as unretained species while mature RBC form a well defined, retained peak. The steady variation of the activity of age-related enzymes within this peak demonstrates that RBC are separated according to age. These observations, linked to the fact that reticulocytes have a drastically different FFF behavior from RBC, reveal that particle shape and stiffness are, when combined with size and density. key biophysical factors controlling the retention of biological micron-sized particles in FFF.