Christopher T. Wagner

Biochemical stabilization enhances red blood cell recovery and stability following cryopreservation

Glycerolized red blood cells (RBC) are approved for long-term cryopreservation. However, the need to remove the glycerol cryoprotectant prior to transfusion has limited the usefulness of this cryopreservation method. This report describes using non-cryoprotectant biochemical stabilization techniques to substitute for the standard glycerol cryoprotectant. The glycerolized RBC method was compared to a newly developed LC-V method that combines transfusable cryoprotectants (hydroxyethyl starch and dextran) and specific non-cryoprotectant biochemical stabilizers (nicotinamide, nifedipine, and flurbiprofen). Results demonstrate that the biochemical stabilizers significantly reduce cryopreservation-induced hemolysis compared to cryopreservation in their absence and that thaw hemolysis levels approach those of standard 40% (w/v) glycerolized RBC (3.1+/-0.2% for 40% glycerol compared to 8.7+/-0.9% for the LC-V protocol). Furthermore, LC-V cryopreserved RBC exhibit a significantly enhanced post-thaw stability compared to glycerolized RBC as determined by osmotic fragility index (0.557+/-0.034 for 40% glycerol compared to 0.478+/-0.016 for the LC-V protocol). Analysis of biochemically stabilized RBC proteins revealed a transient translocation of carbonic anhydrase to the membrane fraction. However, the enhanced RBC recovery and stability could not be attributed to this event. Finally, DSC analysis demonstrated that the biochemical stabilizers of the LC-V process were not functioning as surrogate cryoprotectants in that they did not affect the quantity or quality of ice formed. Overall, this work demonstrates that cryopreservation-induced RBC damage may be corrected or prevented through specific biochemical stabilization and represents a significant step toward a directly transfusable cryopreserved RBC product.

The Glass Transition Behaviors of Hydroxyethyl Starch Solutions

Despite the increasing use of hydroxyethyl starch (HES) as a cryo- and lyoprotectant, information about its glass transition behavior is scarce. The problem stems from the difficulty in detecting the glass transition of HES samples due to the polydispersity of HES and low sensitivity of calorimetric methods. Using an isothermal desorption (controlled air-drying) method and differential scanning calorimetry (DSC), the present study reports a complete glass state transition diagram of the HES-phosphate-buffered saline (HES-PBS) solution (average molecular weight, 262,600; substitution ratio, 0.46). This state diagram is described by the Gordon-Taylor equation. The glass transition temperature (T g) of amorphous anhydrous solutes and the plasticization constant of water (k) as defined in the model are 406.3 K (133.1°C) and 4.75, respectively. Tg depression of the HES-PBS system is less sensitive to water plasticization than other carbohydrates and biopolymers. Heat capacity change (Δ C p) associated with gla...

Instability of Frozen Human Erythrocytes at Elevated Temperatures

Frozen cells are known to be unstable at elevated subzero temperatures; however, the kinetics of cell damage as a function of storage temperature and time are not well understood. The present study investigated the instability of frozen human erythrocytes during isothermal storage at elevated subzero temperatures. The relationship between the instability of frozen cells and the temperature-dependent state/phase transitions in frozen domains was examined. Human erythrocytes were cryopreserved with 12% (wt/vol) hydroxyethyl starch in phosphate-buffered saline solution by plunging into liquid nitrogen, and were then isothermally stored at elevated subzero temperatures. Hemolysis following thawing and dilution was used as an indicator of cell damage during isothermal storage. The instability of frozen cells was found to conform to a special form of the Johnson–Mehl–Avrami model, H(T, t) = H(T)[1 - exp( -kt)], where H(T, t) represented the percent hemolysis at temperature T after time t, H(T) was the maximal h...