Anthony Nolan Research Institute, Royal Free Hospital, London, UK.
Transfusion. 2012 Mar;52(3):549-59. doi: 10.1111/j.1537-2995.2011.03305.x. Epub 2011 Aug 24.
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.
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.
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.
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.
通常使用核染料 7-氨基放线菌素 D（7AAD）通过标准流式细胞术评估非存活 CD34+细胞。然而，7AAD 仅检测坏死和晚期凋亡细胞，而不能检测早期凋亡细胞，这些细胞在脐带血（cord）移植的动物模型中植入效果较差。因此，在某些情况下，标准方法可能会高估脐带单位的植入潜力。
使用 7AAD 和膜联蛋白 V（AnnV）的双重染色来平行检测凋亡事件，同时进行定量标准计数，以评估冷冻保存前后的脐带单位，以确定是否可以使用“功能流式细胞术”方法预测移植物的效力。
在 7AAD 门控群体中发现了大量的 CD34+ AnnV+事件。非凋亡细胞剂量（CD34+ AnnV-）与小样本量（n=10）和大规模银行研究（n=107）中的 CFU 相关性良好。最后，随着时间的推移，对解冻后的样本进行检测，发现凋亡的 CD34+细胞数量增加，因此，与仅进行标准计数相比，AnnV 评估的剂量更能预测 CFU。
定义 CD34+细胞的凋亡群体可提高 CFU 的预测能力，使该方法成为评估临床使用脐带单位效力的快速测试。
