预冻温度和冻干机搁板温度对冷冻干燥红细胞的影响

为研究预冻温度和冷冻干燥机 (冻干机 )搁板温度对红细胞冻干保存后回收率的影响 ,采用主要成分为7%二甲亚砜和 4 0 %聚乙烯吡咯烷酮 (PVP)的缓冲液作为保护液 ,在不同预冻温度或搁板温度下进行红细胞的冻干保存。首先将新鲜血液离心、洗涤和平衡以制备浓集红细胞 ,然后将浓集红细胞和保护液按 1∶3混匀制备红细胞悬液 ,在不同温度 (- 2 0 ,- 35 ,- 4 5 ,- 80或 - 196℃ )下预冻后移入冻干机 (搁板温度设为 - 35℃ ,抽真空压力为2 0 0mbar)内进行真空干燥。为研究冻干机搁板温度对冻干红细胞的影响 ,将上述红细胞悬液先在 - 80℃下预冻后移入冻干机 ,在不同的搁板温度 (- 2 0 ,- 2 5 ,- 30 ,- 35 ,- 4 0或 - 4 5℃ )下抽真空干燥 ,冻干结束后用 37℃的再水化液快速水化。结果表明 :在不同预冻温度下 ,冻干后红细胞和血红蛋白的回收率均在 85 %和 75 %以上 ,各组之间差异不显著。但 - 196℃组上清游离血红蛋白浓度显著高于其他各组 (P <0 .0 1) ;当搁板温度等于或高于- 2 5℃时 ,样品无法冻干 ;当搁板温度等于或低于 - 30℃时 ,随着搁板温度的降低 ,冻干时间相应延长。再水化后红细胞和血红蛋白回收率均在 90 %以上 ,各组之间差异不显著 ;但当洗涤至等渗时 ,4组血红蛋白回收率之间差异不显著。结论 :本冻

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.