Effects of preparative procedures on the volume and content of resealed red cell ghosts

The effects of variations in preparative procedures on the volume and content of resealed red cell ghosts have been investigated. Following hypotonic lysis at 0 degrees C, and after a variable delay time (td), concentrated buffer was added to restore isotonicity; resealing was then induced by incubation at 37 degrees C for one hour. Using this procedure, both the resealed ghost volume and the residual hemoglobin (Hb) content decreased for increasing td. If ghosts were maintained at 0 degree C (i.e., no 37 degrees C incubation), they remained nearly spherical until isotonicity was restored. Their volume then fell abruptly, but subsequently increased toward an intermediate level. The fall in volume was greater and the final level achieved was smaller for longer delay times. At 0 degree C, return to isotonicity also halted the otherwise gradual loss of residual Hb from unsealed ghosts. In addition, ghosts with internal osmolality of 40 to 300 mosmol/kg were prepared by adding different amounts of concentrated buffer before resealing for one hour at 37 degrees C. Under these conditions, the final ghost volume was inversely related to the resealing osmolality (i.e., lower osmolality yielded a larger volume). Ghost volume also increased, along with Hb content, if the quantity or concentration of the red cell suspension added to the lysing medium was increased. We conclude that resealed ghost volume is influenced by the ratio of lysate to resealing medium osmolality and by the colloid osmotic pressure of the residual ghost Hb. These data indicate methods by which ghosts with desired characteristics can be prepared, and have potential application for studies of ghost mechanical and biophysical behavior.