119 Osmotic tolerance limits of fetal and adult red blood cells

Abstract

Fetal and neonatal anemias are serious complications that may occur during pregnancy and post-natal development. The most common treatment is intrauterine or neonatal transfusion of adult red blood cells (RBCs). However, fetal RBCs that are otherwise discarded after cord blood stem cell isolation may be a superior treatment alternative. Proper cryopreservation techniques for these cells would assist in the future development of a fetal RBC transfusion product. Currently, adult RBCs are routinely cryopreserved in the presence of 40% w/v glycerol. However, because fetal RBCs possess a unique membrane composition and biophysical properties, this cryopreservation technique may not be appropriate for use with fetal cells. In this study, the osmotic tolerance limits of fetal and adult RBCs were investigated under several conditions by exposing the cells to anisotonic conditions and measuring the resulting hemolysis. In the first experiment the osmotic tolerance limits of untreated adult and fetal RBCs were evaluated to assess the osmotic sensitivity of the cell membrane in its natural state. Hemolysis was measured at anisotonic conditions as well as after the cells had been returned to isotonic conditions. This experiment revealed that untreated fetal cells are significantly more susceptible to both hypertonic and hypotonic damage compared to adult cells. Furthermore, both cell types exhibited some hypertonic damage that was not apparent while the cells were in hypertonic solution and only manifested once the cells were brought back to isotonic conditions. The second experiment investigated the osmotic sensitivity of RBCs after the glycerolization process. Fetal and adult RBCs were glycerolized to 40% w/v and induced to swell by exposure to relatively hypotonic glycerol solution. The effect of shrinkage in the presence of glycerol was not examined due to the extremely concentrated glycerol solutions that would have been required to shrink the cells. Both fetal and adult RBCs survived the glycerolization process, but underwent hemolysis when they were induced to swell. The glycerolized fetal RBCs were more resistant to swelling than adult RBCs. Additionally, the results suggest that glycerolization has a negative effect on hemolysis for adult cells, lowering the upper osmotic tolerance limit when compared to untreated cells. The same effect was not observed for fetal cells. The third experiment evaluated the osmotic sensitivity of glycerolized blood after it had been frozen and thawed. Fetal and adult cells were glycerolized to 40% w/v, frozen at −80 °C for at least 48 h, thawed in a 37 °C bath, and induced to swell using various anisotonic glycerol solutions. Both fetal and adult cells survived the freezing and thawing process, but subsequent swelling induced hemolysis. The fetal cells that had been frozen and thawed were more resistant to swelling than adult cells. However, the results indicated that the freeze-thaw process has a negative effect on hemolysis for both fetal and adult cells, lowering the upper osmotic tolerance limit in comparison to untreated cells, as well as unfrozen glycerolized cells. The information obtained from this study will facilitate the design of an optimized cryoprotectant loading and removal procedure for fetal RBCs. Source of funding: Canadian Institute for Health Research and Canadian Blood Services and National Science Foundation Grant #1150861. Conflict of interest: None declared. [email protected]