Establishment and optimization of a flow cytometric method for evaluation of viability of CD34+cells after cryopreservation and comparison with trypan blue exclusion staining
Establishment and optimization of a flow cytometric method for evaluation of viability of CD34+cells after cryopreservation and comparison with trypan blue exclusion staining
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DOI：
10.1111/j.1537-2995.2005.00174.x
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发表时间：
2005-07-01
期刊：
影响因子：
2.9
通讯作者：
Horst, HA
中科院分区：
文献类型：
--
作者：
Humpe, A;Beck, C;Horst, HA
背景技术背景：台盼蓝拒染法可能是最常用的方法（方法1），用于评估冷冻保存后外周血祖细胞移植物的活力。或者，流式细胞术为基础的方法（方法11）的建立和optimized.Study设计和方法：在第一系列的22个自体单采产品，抗体染色和红细胞（RBC）裂解的持续时间对生存能力的影响进行了研究。在第二个系列的21个自体和1个同种异体单采产品中，评价了省略RBC裂解的影响。在前两个系列的结果的基础上，155自体和57异基因单采产品进行了分析与方法I和现在优化的方法II。结果：减半的孵育时间不影响CD 45+或CD 34+细胞的活力。忽略RBC裂解导致CD 45+细胞的中位活力显著（p = 0.022）增加（75.8% vs. 71.0%），而对CD 34+细胞无任何影响。在第三个系列中，CD 34+细胞的中位活力（96.9%）显著（p < 0.0001）高于CD 45+细胞的活力（76.2%）和方法1测定的活力（86.5%）。结论：CD 34+细胞的活力显著高于所有白色血细胞的活力。所提出的基于细胞计数的方法上级标准台盼蓝方法，关于可分析细胞和文件的数量，关于观察者的独立性和标准化;它允许在最小的样品操作后分析用于移植的感兴趣的细胞。
BACKGROUND: Trypan blue exclusion staining is probably the most frequently applied method (Method 1) for assessment of viability in peripheral blood progenitor cell grafts after cryopreservation. Alternatively, a flow cytometry-based method (Method 11) was established and optimized.STUDY DESIGN AND METHODS: In a first series of 22 autologous apheresis products, the influence of duration of antibody staining and red cell (RBC) lysis on viability was investigated. In a second series of 21 autologous and 1 allogeneic apheresis products, the effect of omitting the RBC lysis was evaluated. On the basis of the results of the first two series, 155 autologous and 57 allogeneic apheresis products were analyzed with Method I and the now optimized Method II.RESULTS: Halving the incubation times did not influence the viability of CD45+ or CD34+ cells. Omission of RBC lysis resulted in a significantly (p = 0.022) increased median viability of CD45+ cells (75.8% vs. 71.0%) without any influence on CD34+ cells. In the third series, the median viability of CD34+ cells (96.9%) was significantly (p < 0.0001) higher compared with the viability of CD45+ cells (76.2%) and the viability determined by Method 1 (86.5%).CONCLUSION: The viability of CD34+ cells was significantly higher compared with the viability of all white blood cells. The presented cytometry-based method is superior to the standard trypan blue method regarding the number of analyzable cells and documentation, regarding observer independence and standardization; it allows the analysis of the cells of interest for transplantation after minimal sample manipulation.