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Effects of Glycerol Pretreatment on Recovery and Antioxidant
Enzyme Activities of Lyophilized Red Blood Cells
1
Xinli Zhou
1
,Hui He
1
,Baolin Liu
1
,TseChao Hua
1*
,Ying Chen
2
1
Institute of Biothermal Sciences,University of Shanghai for Science and Technology,Shanghai
(200093)
2
Institute of Blood test,Shanghai Antu Hospital,Shanghai (200093)
∗
E-mail:tchua@sh163.net
Abstract
Successful storage of red blood cells (RBCs) by freeze-drying technique has important implications in
blood transfusion and clinical medicine. We presented a method of preservation RBCs by pretreating
them with glycerol solution and then freeze-drying. The effects of glycerol pretreatment on recovery
and antioxidant enzyme activities of lyophilized RBCs were investigated. RBCs pretreated in 40%
glycerol resulted in 55.3±4.26% numerical recovery and 53.5±3.85% hemoglobin recovery, which were
significantly different from control group (P<0.01). Superoxide dismutase(SOD), catalase (CAT) and
glutathione peroxidase (GSH-PX) activities of 40% glycerol pretreated, freeze-dried RBCs almost
remained fully active after freeze-drying. After 180 days storage, they decreased by 25.6
,
% 21.5% and
18.4%, respectively, which are significantly lower than those of no glycerol pretreated, freeze-dried
RBCs (P<0.1). These data demonstrated that glycerol pretreatment had beneficial effects on the
recovery and antioxidant enzyme activities of lyophilized RBCs.
Keywords:Glycerol;pretreatment;Freeze-drying;Red Blood Cells
1 Introduction
Two methods for storing red blood cells (RBCs)—liquid storage at 4 ºC in an additive solution and
frozen storage at -80 ºC with 40% (w/v) glycerol as a cryoprotectant—are widely used in blood banks.
However, the shelf life at 4 ºC is short, because of rapid loss of cell function and risk of bacterial
contamination during storage. Cryopreservation of RBCs using glycerol has led to some successes in
addressing this problem, but it requires careful maintenance of low temperature and thawed RBCs must be
washed to remove the potentially harmful glycerol.
Preservation of RBCs by freeze-drying has received a lot of attention over the years, and several
approaches for lyophilization have been reported. Goodrich et al.(1) and Sowemimo-Coker et al.(2)
incubated erythrocytes in a buffer containing glucose and biopolymers, such as polyvinylpyrrolidone (PVP),
dextran or hydroxyethyl starch (HES) before freeze-drying. Samples prepared in this manner exhibits
moisture contents of 25 to 30% and only allows for storage at freezing temperatures. Bakaltcheva et al.(3)
proposed a method for red cell preservation based on reversible cross-linking reagent DTBP in combination
with CO treatment before freeze-drying. This technique has questions regarding possible toxicity of
cross-linker and reducing agents in application. Török et al.(4) presented a method for loading human
erythrocytes with trehalose and freeze-dried trehalose-loaded human erythrocytes in a formulation of HES,
albumin and trehalose, resulted in 55% survival based on percent hemolysis. The high level of hemolysis is
unacceptable, although they showed normal metabolic properties. Kheirolomoom et al.(5) improved that
method, they discovered that addition of phospholipid vesicle to the lyophilization buffer substantially
reduces hemolysis of freeze-dried RBCs after rehyration to 30%. This is an important advance in
1Support by Specialized research fund for the doctoral program of higher education (20050252002), Science
Foundation for training excellent young teacher of Shanghai (21035) and Science Foundation of Dawn plan of
Shanghai (06SG47).