Improved preservation of human red blood cells by lyophilization.

The lyophilization of human red blood cells has important implications for blood transfusion in clinical medicine. In this study, sugars, human serum albumin, polyvinylpyrrolidone, and dimethyl sulfoxide were used as protective reagents for the lyophilization of red blood cells. Freezing temperature, shelf temperature, and the rehydration conditions were optimized. The results showed that extracellular disaccharides, especially trehalose, did not increase the recovery of hemoglobin. However, when the concentration of human serum albumin was higher than 25%, it had a considerable protective effect on the recovery of lyophilized red blood cells; the cellular hemoglobin recovery was over 70%, which was significantly higher than that in the group without human serum albumin (P<0.01). As the concentration of polyvinylpyrrolidone was increased, the extent of vitrification also increased. But when the concentration of polyvinylpyrrolidone was over 40%, the resulting concentration of free hemoglobin was over 1g/L, which was significantly higher than that with 40% (P<0.01). When lyophilization was carried out after freezing at different temperatures, the recovery of cells and hemoglobin was 70-80% and there were no significant differences among the five groups. When the shelf temperature was higher than -30 degrees C, the samples were partly collapsed, but when the shelf temperature was lower than -30 degrees C, the recovery of cells in the -40 and -45 degrees C groups was significantly higher than in the -30 and -35 degrees C groups (P<0.05). The recovery of cells and hemoglobin after lyophilization and rehydration in solutions containing low concentrations of polymers was over 80%, which is significantly higher than the other groups (P<0.01). In addition, when the temperature was higher than 25 degrees C, the concentration of free hemoglobin was significantly lower than it was at 4 degrees C (P<0.01). In conclusion, our study showed the lyophilization of red blood cells is feasible. Disaccharides have no protective effect on lyophilized cells when they are only extracellular and extensive vitrification may be not beneficial. Although the recovery of cells after lyophilization and rehydration by our method was over 70%, the ultrastructure of the cells may be compromised and some hemolysis does still exist. Further research is required.

[A simple and convenient method for assaying human RBCs glycolysis rate without oxygen].

AIM: To convenience of the methods for assaying red blood cell glycolysis without oxygen condition in the studies. METHODS: Reagent kit of glucose, perchloric acid, visible light prismatic photometer, battle of nitrogen and rocking bed are used in the studies. The process includes 4 steps prepare Tris- HCI solution and so on, assay of red blood cell glycolysis without oxygen condition and account of glycolysis rate. RESULTS: Human red blood cells stored at 4 degrees C for 75 d, in SOD solution, the glycolysis rate is 86.2% +/- 5.0%, distinctly better than GMA solution (39.2% +/- 8.9%). CONCLUSION: The methods of assaying glycolysis without oxygen condition not use Habea's apparatus. The operation is convenient and simple and its determinations can be performed in ordinary laboratory and is is accurate.

[Effect of pre-freezing temperature and lyophilizer shelf temperature on recovery of red blood cells after lyophilization].

To study effect of pre-freezing temperature and lyophilizer shelf temperature on recovery of human red blood cells after lyophilization and determine solidifying temperature of this lyophilization system, the protective solution composed of 7% DMSO, 40% polyvinylpyrrolidone (PVP) and isotonic buffer were adopted to lyophilize red blood cells at different pre-freezing temperatures or shelf temperatures. At first, fresh whole blood was centrifugated, washed and equilibrized to prepare concentrated red blood cells. Then concentrated red blood cells were mixed with the protective solution at 1:3 and pre-freezed at different temperature (-20, -35, -45, -80 or -196 degrees C) before lyophilization in lyophilizer. To study effect of shelf temperature on lyophilization of red blood cells, red blood cells were lyophilized at different shelf temperature after pre-freeze at -80 degrees C. After lyophilization, the samples were quickly rehydrated by 37 degrees C rehydration solution. The results showed the recovery rate of red blood cells and hemoglobin after pre-freeze at different temperature and lyophilization were > 85% and > 75%, there was not significant difference among these groups, but the concentration of free hemoglobin in -196 degrees C group was significantly higher than that in other groups (P < 0.01). With decreasing of shelf temperature, the lyophilizing time was also prolonged. When shelf temperature was > or = -25 degrees C, samples were not fully lyophilized; when shelf temperature was < or = -30 degrees C, the recovery rate of red blood cells and hemoglobin after lyophilization and rehydration were above 90%; after washed to isotonic state, the recovery rate of hemoglobin of the four groups was similar to each other. In conclusion, only when pre-freezing temperature is between -20 and -80 degrees C and the lyophilizer shelf temperature is < or = -30 degrees C, the effect of lyophilization is better, but the effect of excessively low pre-freezing temperature may even be worse.

[Analysis on ultrastructure of lyophilized red blood cells].

The objective of the present study was designed to evaluate lyophilized red blood cells of the ultrastructure. Blood was drawn from healthy adult. In group 1, sample was fresh blood; in group 2, sample was added 35% glycerine, stored at -80 degrees C for 24 hours; in group 3, red blood cells stored at 4 degrees C for 5 hours, then were lyophilized for 16 hour. The sample was resuspended for measurements of count and electron microscopy study. The result showed that lyophilized red blood cells possessed relative integrated structure, red blood cell recovery was 53%. The mean diameter, optical density and integral optical density of red blood cell were 4.7 +/- 0.4, 0.14 +/- 0.03 and 1.58 +/- 0.46 in group 1; 4.6 +/- 0.7, 0.14 +/- 0.02 and 2.35 +/- 0.64 in group 2; 4.4 +/- 0.4, 0.17 +/- 0.05 and 2.35 +/- 0.46 in group 3, respectively. There was no significant difference in lyophilized and frozen group, but there was significant difference in lyophilized group and normal group. In conclusion, human red blood cells could be successfully lyophilized and possess relative integrated structure. The mean diameter, optical density and integral optical density of lyophilized red blood cells were similar to that of cryopreservation red cells.

[Dynamic changes of ultrastructure of erythrocytes in prolonged preservation at 4 degrees C].

To study the dynamic changes of ultrastructure of erythrocytes in prolonged preservation of blood with preservative fluid containing superoxide dismutase (SOD), the whole blood samples were preserved at 4 degrees C in SOD-containing solution, the morphologic changes of erythrocyte were dynamically ob served by transmission microscopy after preservation for 42, 75 and 85 days, an d the blood samples preserved in GMA solution served as control. Three variance was applied to analyze the data with SAS software. The results showed that the metamorphotic rates of erythrocyte preserved in SOD-containing solution for 42, 75 and 85 days were lower than those of erythrocytes preserved in GMA solution. Most of metamorphotic rates of erythrocyte preserved in SOD-containing solution for 42, 75 and 85 days were correspond to those of erythrocytes preserved in GMA solution for 42 days, or even lower. It is concluded that SOD-containing preservative fluid might help to maintain the normal morphology of erythrocytes in prolonged preservation at 4 degrees C.

[Labeling and survival studies on rabbit RBC].

AIM: To establish a simple and effective method for RBCs labeling and survival assays, and the qualities of rabbit RBCs preserved in GMA solution at 4 degrees C were verified. METHODS: The bloods were taken through the ear arteries of the rabbits. The RBCs were labeled by fluorescein isothiocyanate (FITC), and were reinjected to the same rabbit through ear veins. The percentage of FITC labeled RBCs was assayed by FACS at a series of times after injection. The SAS software was employed to analyze the data and establish the regression equations. The 24-hour recovery and the half-life span of the labeled RBCs were calculated according to the equations. RESULTS: The 24-hour recovery and the half-life span of the labeled RBCs in the control group were 93.76% +/- 5.40% and 22.50% +/- 4.37 days respectively, which was in agreement with the previous papers. The 24-hour recovery and the half-life span of the labeled RBCs in the GMA group were 89.13% +/- 7.10% and 11.41% +/- 1.63 days respectively, which was coincident with the infusion conditions. CONCLUSION: Compared with other methods of RBCs labeling in vivo, FITC labeling was thought to be easier and cheaper to use, which could facilitate the analysis of the biological character of the labeled cells, and could be used to trace the fate of labeled cells.

[New tactics of human red blood cells stored at 4 degrees C-protective effect of antioxidant solution on red blood cells damage].

Aliquots of venous blood from healthy donor were collected in plastic blood storage bags with ACD, GMA or antioxidant solution (superoxide dismutase, SOD), respectively, and stored at 4 degrees C. After storage for varying periods, the parameters of the blood were detected in the blood samples. Results showed that the parameters of the blood stored at 4 degrees C for 75 days in SOD group were following: the recovery of RBC-Hb was 87.2%, plasma-Hb (mg/L) was 193.2, P50 (mmHg) was 34.0 (normal value was 33.1); deformability (DImax) was 0.2413 (74.3% of normal value). There was no evident hemolysis, color change, air bubble and clots. It was concluded that human RBC stored at 4 degrees C for 75 days with SOD solution, recovery of levels of RBC-Hb and plasma-Hb were accorded with the requirements of "Basic Demands of Blood Station" in China.

[Studies of protective effect of SOD on damage of human RBCs stored at 4 degrees C].

AIM: To investigate the mechanism of protective effect of SOD (superoxide dismutase) on damage of RBCs stored at 4 degrees C, the studies of erythrocyte glucose and energy metabolism were performed. METHODS: whole blood collected from healthy donors and stored at 4 degrees C in ACD, GMA and SOD solutions. Before and post storage, some parameters were assayed. Standard methods were used for the in vitro tests. The 24-hour in vivo recoveries were measured by FTTC (Fluorescein 5-isothiocyanate) from SIGMA Company. RESULTS: All parameters of red blood cell glucolysis rate without oxygen condition, ATP, PK (pyruvic kinase) and 24 h recoveries level were 86.2%, 56.4%, 64.3% and 86.2% of normal respectively stored in SOD solution at 4 degrees C for 75 days, distinctly more than in ACD and GMA groups at 75 days stored. The 24 h recovery at 75d in group SOD was near the recovery at 42d in group GMA. CONCLUSION: Whole blood in SOD solution can be stored satisfactorily for 75 days at 4 degrees C, and furnished theoretical evidence for RBCs survival.