IN-VITRO STUDY OF THE PROTECTIVE EFFECT OF TREHALOSE AND DEXTRAN DURING FREEZING OF HUMAN RED-BLOOD-CELLS IN LIQUID-NITROGEN

Two nonpermeant cryoprotectants, the disaccharide trehalose and the po lymeric carbohydrate (dextran, 40 kDa), were assessed as substitutes f or glycerol in the cryopreservation of human red blood cells (RBC). Th e agents were evaluated by measuring the percentage of RBC recovery (t otal of free hemoglobin after freezing) and by evaluating the erythroc yte state after freezing. Ninety percent of the red cells were recover ed after freezing in 30% (w/v) dextran in liquid nitrogen, which is ve ry close to the recovery obtained in 35.5% (w/v) glycerol (92%). The a ctivities of pyruvate kinase and glucose-6-phosphate dehydrogenase of RBCs frozen and thawed with dextran were not modified, and the 2,3-dip hosphoglycerate was reduced by 26%, but remained within normal. values . ATP was reduced by 56%. The erythrocyte membrane integrity, evaluate d by its osmotic fragility, was not altered, and the RBCs protected by dextran retained their normal discoid shape without the formation of microvesicles. The 24-h hemolysis of the washed red cells after storag e at 4 degrees C was 7%. These results suggest that dextran protects r ed blood cells during freezing in liquid nitrogen, but that some effor t is still needed to limit the drop of ATP concentration. One of the m ain advantages of dextran is that it does not penetrate the RBCs and r equires less washing than glycerol. (C) 1997 Academic Press.