Flow cytometry assessment of apoptotic CD34+cells by annexin V labeling may improve prediction of cord blood potency for engraftment
Flow cytometry assessment of apoptotic CD34+cells by annexin V labeling may improve prediction of cord blood potency for engraftment
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DOI：
10.1111/j.1537-2995.2011.03305.x
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发表时间：
2012-03-01
期刊：
影响因子：
2.9
通讯作者：
Madrigal, J. Alejandro
中科院分区：
文献类型：
--
作者：
Duggleby, Richard C.;Querol, Sergio;Madrigal, J. Alejandro
背景：无法存活的 CD34+ 细胞通常使用核染色剂 7-氨基放线菌素 D (7AAD) 通过标准流式细胞术进行评估。然而，7AAD 只能检测坏死细胞和晚期凋亡细胞，不能检测早期凋亡细胞，而早期凋亡细胞在脐带血（脐带）移植的动物模型中植入效果较差。因此，标准方法可能会高估某些条件下脊髓单元的植入效力。 研究设计和方法：为了检测细胞凋亡事件，使用 7AAD 和膜联蛋白 V (AnnV) 共同染色，同时进行定量、标准计数。使用染色方法和集落形成单位 (CFU) 评估冷冻保存之前和之后的脊髓单位，以确定是否可以使用“功能流式细胞术”方法预测移植物效力。 结果：在 7AAD 门控群体中发现了大量 CD34+ AnnV+ 事件。在小规模 (n = 10) 和大规模库研究 (n = 107) 中，非凋亡细胞剂量 (CD34+ AnnV-) 与 CFU 密切相关。最后，随着时间的推移，解冻后的样品显示凋亡 CD34+ 细胞的数量不断增加，因此与标准计数相比，AnnV 评估剂量能够更好地预测 CFU。结论：定义 CD34+ 细胞凋亡群体改善了 CFU 的预测，使该方法成为临床使用的脊髓单位评估效力的快速测试。
BACKGROUND: Nonviable CD34+ cells are commonly assessed by standard flow cytometry using the nuclear stain 7-aminoactinomycin D (7AAD). 7AAD, however, only detects necrotic and late apoptotic cells, not earlier apoptosis, which engraft poorly in animal models of cord blood (cord) transplantation. The standard method, therefore, may overestimate engraftment potency of cord units under certain conditions.STUDY DESIGN AND METHODS: To detect apoptotic events, costaining with 7AAD and annexin V (AnnV), in parallel with the quantitative, standard enumeration, was used. Cord units were assessed before and after cryopreservation using both staining methods and colony-forming units (CFU) to determine if graft potency can be predicted using a "functional flow cytometry" approach.RESULTS: Significant numbers of CD34+ AnnV+ events were found within the 7AAD-gated population. Nonapoptotic cell dose (CD34+ AnnV-) correlated well with CFUs in both a small-scale (n = 10) and a large-scale banking study (n = 107). Finally, following samples postthaw with time showed increasing numbers of apoptotic CD34+ cells and consequently the AnnV assessed dose was better at predicting the CFU compared with just the standard enumeration.CONCLUSION: Defining the apoptotic population of CD34+ cells improved the prediction of CFU, making this method a rapid test of potency for assessment of cord units for clinical use.