| The conservation of human red blood cells has important implications for blood transfusion in clinical medicine. The lyophilization is one of the methods for long-term preservation of human red blood cells. As early as 1960s, some scholars tried to perform lyophilization of red blood cells. Until the 1990s, the lyophilized preservation of red blood cells has become a hot topic in cryobiology. Scientists both in China and abroad did many researches on screening freeze-dried protective agents, freezing-dry procedures and restoring procedures. In the dry state, there is still some water left in red blood cells, also known as "residual moisture". Issues related to residual moisture constitute a major problem. In recent years, we find that the overall effect is better from using glycerol as a permeability protectant combining with PVP. However, glycerol has excellent capacity to moisturize, thus it seems contradictory to the effects of lyophilization. Therefore, more research is needed to investigate on this problem. Based on the prior accomplishments in our research group, we conducted a research on residual moisture that resulted from lyophilization of red blood cells.Objective:To investigate the effect of different concentrations of glycerol on protective agent, the effect of different lyophilization methods and retention time on the recovery of RBCs and hemolysis rate, the effect of residual moisture on the lyophilization of red blood cells, and to compare and study the methods of red cell recovery and moisture content determination.Methods:RBCs were subjected to different concentrations of glycerol, and then freeze-dried by freeze dryer at different shelf temperature and secondary drying time. Corresponding parameters were cell morphology, number of blood cells measured by automatic blood cell counter and blood cell counting chamber, and the concentration of free hemoglobin in supernatant measured by HiCN. The residual moisture was measured by thermogravimetric analyzer, Karl-Fischer gauge and modified method. Different methods were compared and analyzed statistically, followed by an analysis on the change of water content in different protective agent groups.Results:1. The influence of the concentrations of glycerol (3%,6%,9%,12%,15%, 18%,21%) (w/v) on RBCs freeze-drying was as followed:The cell morphology was normal under light microscopes after rehydration. The parameters showed that the content of residual water was (11.533±0.327)%～(35.315±1.529)%. The recovery rate of RBCs ranged from (51.04±6.14)% to (84.37±8.62)%. The hemolysis rate was (51.04±6.14)%～(84.37±8.62)%. Meanwhile, residual moisture increased with the concentration of glycerol. The fitting correlation coefficient is 0.9963, which showed a relatively good correlation between residual moisture and concentration of glycerol. Furthermore, the quality of freeze-drying RBCs in 9%,12%,15% concentration of glycerol condition was better than others. However, the recovery rate of cells and hemolysis difference after rehydration has no statistical significance (p>0.076). On the contrary, there were significance differences among other experimental groups.2. Four experimental groups under the shelf temperature from -60℃,-50℃,-40℃ to-30℃ showed that the cell recovery rates after the removal of protective agent were 58.50±7.29,22.67±2.10,21.52±1.70 and 29.73±1.35, respectively. The hemolysis rates were 77.55±3.26,82.31±1.16,85.30±1.35 and 84.37±0.62, respectively. The concentrations of residual moisture were 25.18±0.19,22.12±1.46,23.02±1.25 and 26.19±0.58, respectively. The experimental group at -60℃ and dried for 2 hours showed better effect on freeze-drying RBCs. Under this condition, the cell recovery rate and the rate of hemolysis after removal of protective agent showed significance differences (P<0.01) compared with the other groups. For example, the result quality of RBCs freeze-drying for 26 hours was worse than that with 2 hours drying time, though the differences in residual moisture were negligibly small (P=0.273). Furthermore, cell recovery declined with the increase in preservation time after the process of freeze-drying. The hemolysis rate had no statistical difference in different reservation time (P>0.05).3. After rehydration and removal of protective agent, automatic blood cell counter and blood cell counting chamber were used to estimate the number of RBCs. The results were (2.29±0.08)×1012 and (0.71±0.11)×1012, showing significant difference (P<0.01). The results of residual moisture by different methods were significantly different (P<0.01), ranging from (3.20±0.52)% to (25.18±0.42)%.Conclusion:1. When the concentrations of glycerol were between 9% and 15%, and the shelf temperature was set at -60℃ without prolonging secondary drying time, the damage on RBCs was minimal.2. The proportion of residual moisture increased with the concentration of glycerol in the protective regents. The concentration of glycerol can be used to control the concentration of residual moisture. Prolonging secondary drying time had less impact on residual moisture.3. The results of thermogravimetric method reported to be higher than the results of Karl Fischer chemical method or its modified methods.4. Blood cell counting chamber should be used then to measure the recovery rate of lyophilized RBCs. |
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