AGE-DEPENDENT ELUTION OF HUMAN RED-BLOOD-CELLS IN GRAVITATIONAL FIELD-FLOW FRACTIONATION

The technique of field-flow fractionation (FFF) which combines the ear th gravitational force with a carrier liquid flow in a horizontal, rib bon-like channel is well suited for the separation of micron-sized par ticulate species such as cells. We investigated the selective elution, in a phosphate buffer, of human red blood cells (RBC) which migrate a long the FFF channel more slowly than the carrier. Fractions of the ch annel effluent were collected and the activities of various intracellu lar enzymes, which either reveal the presence of white cells or are kn own to be related to cell age, were evaluated for each fraction. The h emoglobin 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 w ell defined, retained peak. The steady variation of the activity of ag e-related enzymes within this peak demonstrates that RBC are separated according to age. These observations, linked to the fact that reticul ocytes 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 micr on-sized particles in FFF.