Hemolysis of Frozen-Thawed Red Blood Cells Using a Single Step Deglycerolization Process

Hemolysis of Frozen-Thawed Red Blood Cells Using a Single Step Deglycerolization Process

Jolynn Meza Wynkoop, John Lahmann, Dr. Adam Higgins

Biological Engineering Lab, School of Chemical, Biological and Environmental Engineering

Oregon State University

Corvallis, Oregon 97330

Currently, blood is preserved using cryoprotectants, such as glycerol, which defer the blood from obtaining freezing damage when stored.  The blood is stored in a freezer that is set to -80 degrees Celsius.  Then, when the blood is needed for use, the glycerol is removed from the frozen-thawed red blood cells (RBCs) through a multistep deglycerolization process.  Current deglycerolization techniques are time consuming due to the limitations in how fast the glycerol may be removed from the blood without damaging the cells.  The addition and removal of cryoprotectants, such as glycerol, from the blood, can result in large osmotic gradients in the cells which could eventually lead to damaging cell volumes.  This project involved looking at the effects of different concentrations of saline, a washing solution, on blood.  The saline maximizes the concentration driving force for glycerol removal, allowing the procedure to be completed rapidly.  Dilutions were also performed with different blood to saline ratios to determine the one that would allow for the smallest percent of cells lysed over time.  Human RBCs have shown to undergo hemolysis in two different situations.  The first situation involves the cells swelling beyond their maximum volume.  Our trials showed that extensive exposure of saline to blood over time resulted in hemolysis of RBCs.  Second, the RBCs could also lyse if they exceeded their osmotic tolerance limits and then returned again to isotonic conditions.  Our procedure involved using both a 9 % hypertonic saline solution and a 12 % hypertonic saline solution.  Once the blood was exposed to saline, it was tested for hemolysis at thirty minute intervals over a three hour time frame.  Three hours would be the longest amount of time that the blood would be sitting out for analysis.  One hemolysis assay was run with a dilution of 1:10, blood to 12 % saline.  This dilution, after one hour, had cells lyse up to 24.43±1.43 %.  With a 1:2 dilution, blood to 12 % saline, hemolysis at one hour only reached up to 6.14±1.00 %.  More cells lysed with the 1:10 dilution compared to the 1:2 dilution.  Assays were also held at a 1:4 dilution with 12 % saline and 9 % saline.  After two and a half hours, the 1:10 dilution with 12 % saline had reached a percent hemolysis of 38.71±1.56 %.  The 1:2 dilution with 12 % saline, after two and a half hours, had a hemolysis value of 8.52±1.37 %.  The main focus of this research was to experiment with different concentrations of saline and dilution rations of blood to saline.  Future plans include looking at temperature tolerance and how the RBCs react to the saline under different temperatures.