RED BLOOD CELL STABILIZATION: EFFECT OF HYDROXYETHYL STARCH ON RBC VIABILITY, FUNCTIONALITY AND OXIDATIVE STATE DURING DIFFERENT FREEZE THAW CONDITIONS. A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF Master of Technology Biotechnology By DEEPANWITA DAS

R. P. Goodrich and co-workers, 1989 have reported that red blood cells (RBCs) can be preserved in the dry state by addition of mixtures of hydroxyethyl starch (HES) and glucose (59,60). In this thesis work we tried to investigate the effect of HES alone on the viability, functionality and oxidative state of red blood cells after freeze thaw stress. Here we prepared eight formulations having varying concentrations of HES along with Adenine glucose mannitol sodium chloride (ADSOL) and Phosphate buffer saline (PBS) which were ADSOL, 5% HES in ADSOL, 10% HES in ADSOL, 15% HES in ADSOL, PBS, 5% HES in PBS, 10% HES in PBS, 10% HES in PBS and finally 15% HES in PBS as protective solutions during freeze thaw. Control sets used in this study did not contain HES. Red blood cell suspensions were prepared using the formulations at around 25% hematocrit and were frozen in liquid nitrogen for 10 mins. After thawing at different temperatures (4°C, 37°C and 60°C) the percentage hemolysis, percent methemoglobin oxidation, Thiobarbituric acid reactive species (TBARS) and catalase activity were determined using spectrophotometric assays. In all formulations percentage hemolysis observed was found to be more than 30% which has been reported in normal freeze thaw experiments on RBCs. In case of red blood cells in 15% HES in ADSOL which were thawed at 4°C showed moderate amount of hemolysis, lowest amount of methemoglobin, lipid peroxidation and highest Catalase activity. Therefore, it was found out to be the best formulation to preserve cells against freeze thaw stress among all the formulations used in this work. Thus, it can be concluded that freeze thaw experiments using HES alone in ADSOL or PBS showed a trend of protective effect in 15% concentration of HES but it is not sufficient alone for providing protection against different stresses during freeze thaw experiments due to significant amount of percent hemolysis. Further combination of disaccharide along with HES needs to be investigated and also the cooling rate during freeze thaw experiments needs to be controlled.