(117d) Improving the Post-Thaw Wash Process for Frozen Red Blood Cells: An Integrated Theoretical and Experimental Approach

Transfusion of red blood cells (RBCs) is one of the most commonly practiced medical procedures, with approximately 15 million units transfused per year in the United States alone. Currently, blood banks and hospitals use RBCs refrigerated at 4^oC in their reserve almost exclusively. Depending on the preservative, the FDA allows a storage period of 21 to 42 days for refrigerated blood. The short storage period makes it difficult to maintain a steady supply of refrigerated blood to meet the unpredictable demand for transfusion.  On the contrary, freezing blood in the presence of 40% glycerol extends the FDA approved shelf life of the product to 10 years. The use of frozen blood in blood banking would enable stockpiling strategic reserves to eliminate seasonal shortages, improve availability in the event of a natural disaster, facilitate military use during the time of war, and provide overall stability in the RBC supply chain. Although logistically more advantageous, the use frozen blood in clinical therapy is uncommon due to the time consuming post-thaw wash process required to remove the glycerol before the product can be transfused. The current state of the art removal method requires approximately one hour to process a single blood unit, making short notice use of frozen blood particularly challenging. Until a more rapid method of glycerol removal is discovered, the increased use of frozen blood in blood banking remains unlikely.

 In this study, modern approaches such as mathematical modeling and microfluidics were used to develop a method that expeditiously removes glycerol from thawed frozen RBCs without causing excessive cellular damage. A cell membrane transport model was used in guiding the design of several procedures for varying the extracellular solution concentration with time to theoretically remove glycerol in a rapid manner without causing cell membrane damage. Batch experiments employing a series of dilutions were then conducted to test the theoretical deglycerolization procedures, resulting in the identification of a four-step procedure capable of removing glycerol while achieving a cell recovery that meets transfusion standards in a total process time of three minutes. Direct scaled up implementation of the batch procedure would require excessive amounts of diluting solution and produce an extremely dilute cell suspension. Hence, a membrane microfluidic device was built to practically adapt the batch procedure in a continuous manner. Additionally, a model capable of predicting the glycerol removal in the membrane device was developed to assist in selecting the appropriate operational conditions to match the extracellular concentration changes in the batch procedure. Preliminary experiments using thawed frozen blood and 3.4% saline solution in a single device resulted in a reduction of glycerol mass composition of up to 55.0%±0.3% with a corresponding hemolysis of 15.1%±0.7%. The glycerol removal achieved in this experiment is comparable with the first step of the batch procedure, demonstrating partial implementation in a continuous platform. Matching the step-by-step concentration changes from the remaining steps is possible with multiple membrane devices linked together. The operating conditions necessary to achieve such concentration changes can be predicted using the mathematical model for each device in the assembly. The use of a combination approach of mathematical modeling and microfluidics shows promise in enabling full continuous implementation of the rapid batch deglycerolization procedure. Ultimately, the findings from this study has the potential to dramatically reduce the post processing time of frozen blood, making it a more attractive option for blood banking in the future.