脐带血从采集到制备最佳保存条件及保存时间的研究

The Optimal Storage Condition and Storage Time of Umbilical Cord Blood from Collection to Preparation

目的:探讨脐带血从采集到制备的最佳保存条件和保存时间。方法:选择30例健康新生儿的脐带血标本,将每例脐带血平均分为两份,一份置于冷藏(4℃)保存,一份置于室温(20-24℃)保存,分别在24、36、48、60和72 h取样,分析有核细胞计数、有核细胞活率,流式细胞术检测活的CD34^(+)细胞占活的CD45+细胞的百分比及CD34^(+)细胞活率,并通过造血祖细胞集落培养进行CFU-GM计数。观察随时间延长各指标的变化趋势,比较相同保存时间不同条件(冷藏和室温)下各指标的差异。结果:冷藏(4℃)、室温(20-24℃)条件下保存的脐带血随保存时间延长,有核细胞计数(r_(冷藏)=-0.9588,r_(室温)=-0.9790)、有核细胞活率(r_(冷藏)=-0.9941,r_(室温)=-0.9970)、CD34^(+)细胞活率(r_(冷藏)=-0.9932,r_(室温)=-0.9828)均呈现出一致性下降趋势,活的CD34^(+)细胞百分率(r_(冷藏)=0.9169,r_(室温)=0.7470)和CFU-GM计数(r_(冷藏)=-0.2537,r_(室温)=-0.8098)未呈现出一致性变化趋势。当保存时间相同时,冷藏保存较室温保存的脐带血有核细胞计数、有核细胞活率、CD34^(+)细胞活率和CFU-GM计数均更高,且当保存时间同为24、36、48、60和72 h时,室温保存较冷藏保存有核细胞活率均显著降低(P<0.001),有核细胞计数、活的CD34^(+)细胞百分率和CFU-GM计数均无显著差异;当保存时间同为24和36 h时,室温保存较冷藏保存CD34^(+)细胞活率显著降低(P<0.001、P<0.01),保存时间同为48、60、72 h时,室温保存较冷藏保存CD34^(+)细胞活率无显著差异。结论:脐带血从采集到制备建议冷藏(4℃)保存,并尽量缩短采集到制备的时间。

Objective:To explore the optimal storage condition and time of umbilical cord blood from collection to preparation.Methods:Collect cord blood samples from 30 healthy newborns,with each new born′s umbilical cord blood was divided into two parts on average.One part was stored in cold storage(4℃)and the other was stored at room temperature(20-24℃).Samples were taken at 24,36,48,60 and 72 h,respectively,total nucleated cells(TNC)count and TNC viability was analyzed.Flow cytometry was used to detect the ratio of viable CD34^(+)cells to viable CD45+cells and viability of CD34^(+)cells,and colony-forming unit-granulocyte-macrophage(CFU-GM)count was performed by hematopoietic progenitor cell colony culture.The change trend of each index over time was observed,and the differences in each index was compared between cold storage and room temperature storage under the same storage time.Results:The TNC count(r_(4℃)=-0.9588,r_(20-24℃)=-0.9790),TNC viability(r_(4℃)=-0.9941,r 20-24℃=-0.9970),CD34^(+)cells viability(r_(4℃)=-0.9932,r_(20-24℃)=-0.9828)of cord blood stored in cold storage(4℃)and room temperature storage(20-24℃)showed a consistent downward trend with the prolongation of storage time.The percentage of viable CD34^(+)cells(r_(4℃)=0.9169,r_(20-24℃)=0.7470)and CFU-GM count(r_(4℃)=-0.2537,r_(20-24℃)=-0.8098)did not show consistent trends.When the storage time was the same,the TNC count,TNC viability,CD34^(+)cells viability and CFU-GM count of cord blood stored in cold storage were higher than those stored at room temperature.Under the same storage time(24,36,48,60 or 72 h),TNC viability in room temperature storage was significantly lower than that in cold storage(P<0.001),but TNC count,percentage of viable CD34^(+)cells and CFU-GM count were not significantly different between room temperature storage and cold storage.When stored at room temperature for 24 h and 36 h,the viability of CD34^(+)cells was significantly lower than that in cold storage(P<0.001,P<0.01),when the storage time for 48,60 and 72 h,there was no significant difference in the CD34^(+)cells viability between room temperature storage and cold storage.Conclusion:It is recommended that cord blood be stored in cold storage(4℃)from collection to preparation,and processed as soon as possible.