A new approach for rapid and reliable enumeration of circulating endothelial cells in patients
A new approach for rapid and reliable enumeration of circulating endothelial cells in patients
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DOI：
10.1111/j.1538-7836.2012.04681.x
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发表时间：
2012-05-01
影响因子：
10.4
通讯作者：
Gratama, J. W.
中科院分区：
文献类型：
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作者：
Kraan, J.;Strijbos, M. H.;Gratama, J. W.
。背景：破碎的垂直条成熟循环内皮细胞(CECs)是内皮损伤/功能障碍的替代标志物。由于缺乏关于CEC表型的标准化分析和共识，导致报告的CEC数量存在很大差异(每毫升41300个)。目的考虑到快速、可靠且价格合理的CEC检测方法的必要性，我们提出了一种新的方法，该方法使用多参数流式细胞仪(FCM)来计数CEC，而无需免疫学预富集。方法根据静脉壁内皮细胞的免疫表型，将破裂的垂直条形CEC定义为CD34+、CD45neg、CD146+和DNA+事件。由于CEC表达高水平的CD34，我们在分析了总血容量中所有CD34阳性事件后，基于CD34的绝对计数，以1个细胞Mu L-1的频率进行CEC的精确计数。结果形态、免疫组织化学和基因表达均证实CECs来源于内皮细胞。新的FCM检测与经过验证的检测(即CellSearch(R))同时进行。健康人CEC水平为4~79CECmL-1，在晚期实体恶性肿瘤患者(P=0.0008)和恶性血液病患者(P<；0.0001)中，CEC水平显著升高。结论流式细胞术是一种快速、经济的CEC计数和鉴定方法。
. Background broken vertical bar Mature circulating endothelial cells (CECs) are surrogate markers of endothelial damage/dysfunction. A lack of standardized assays and consensus on CEC phenotype has resulted in a wide variation of reported CEC numbers (41300 per mL). Objectives broken vertical bar Given the need for a quick, reliable, robust and validated CEC assay at an affordable price, we present a novel approach to enumerate CECs using a multi-parameter flow cytometric (FCM) method without immunological pre-enrichment. Methods broken vertical bar CECs were defined as CD34+, CD45neg, CD146+ and DNA+ events based on the immunophenotype of endothelial cells from vein-wall dissections. As CECs express high levels of CD34, we based our assay on absolute CD34 counts after analyzing all CD34 positive events in a total blood volume of 4 mL needed for a precise enumeration of CECs at a frequency of < 1 cell mu L-1. Results broken vertical bar The endothelial origin of CECs was confirmed by morphology, immunohistochemistry and gene expression. The new FCM assay was tested in parallel with a validated assay (i.e. CellSearch (R)). CEC levels ranged from 4 to 79 CEC mL-1 in healthy individuals and were significantly higher in patients with advanced solid malignancies (P = 0.0008) and in patients with hematological malignancies (P < 0.0001). Conclusions broken vertical bar This flow cytometric method should be useful as a fast and economical assay to enumerate and characterize CECs.