Abstract: Objective To study the correlation between the changes of RBCs morphology and injury in the process of freezing-thawing-deglycerolizing. Methods The matching design method was employed to prepare frozen RBCs and thawed deglycerolized RBCs. Method1 : without supernatant glycerol reduction before being frozen. Method2 : supernatant glycerol reduction by centrifugation before being frozen. Take samples for blood routine test and free hemoglobin (FHb) content detection in the process of freezing-thawing-deglycerolizing, analysis the shape variation of red blood cells and its correlation with injury. Result (1) In the process of freezing-thawing-deglycerolizing, volume of red blood cell increase due to glycerolize ,and then recovery due to deglycerolize. Compare with the method 2 and method 1, mean corpuscular volume (MCV) of glycerolized RBCs (before being frozen) and thawed deglycerolized RBCs increased (P<0.01, P<0.05);(2) The destruction of red blood cells is distributed in three processes, deglycerolizing process (63.20±10.99) %, glycerolizing process (30.78±10.47) %, freezing-thawing process (6.02±4.11) %, no statistical difference between two kinds of preparation methods.(3) the red blood cell damage is moderate negative correlation with volume change rate of glycerolized RBCs ; (4) with the extension of storage time, free hemoglobin content of thawed deglycerolized RBCs gradually increased. In which the 0.9% NaCl suspension thawed deglycerolized RBCs, the content of FHb showed significantly different 12 h and 24 h after preparation as well (P<0.05).In which the MAP suspension thawed deglycerolized RBCs, the content of FHb showed significantly different 21d and 28d after preparation as well (P<0.05). Conclusion In the process of freezing-thawing-deglycerolizing, red blood cell damage is negatively related to its deformation ability, and the damage mainly happens on the deglycerolizing process. The removal of supernatant glycerol by centrifugation caused the red cell membrane skeleton to be damaged by the shear stress, thereby adversely affecting the storage quality of the thawed deglycerolized RBCs.

Key words: Freezing, Thawing, Deglycerolizing, Red blood cells, MCV, Injury