Highly Efficient Assay of Circulating Tumor Cells by Selective Sedimentation with a Density Gradient Medium and Microfiltration from Whole Blood
Highly Efficient Assay of Circulating Tumor Cells by Selective Sedimentation with a Density Gradient Medium and Microfiltration from Whole Blood
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DOI：
10.1021/ac3011704
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发表时间：
2012-09-04
影响因子：
7.4
通讯作者：
Huh, Nam
中科院分区：
文献类型：
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作者：
Park, Jong-Myeon;Lee, June-Young;Huh, Nam
密度大小排除法分离循环肿瘤细胞（CTCs）纯度低，回收率低，因为CTCs大小变化大，白细胞扩增，可能与9个白细胞重叠，使基于大小的过滤方法不可靠。本报告提出了一种灵敏、选择性强、快速、新颖的ctc分离检测方法。我们的检测平台由三个步骤组成：(i)用抗epcam偶联微珠捕获ctc， （ii）通过密度梯度介质中ctc的选择性沉淀去除不需要的血液细胞（例如，白细胞，红细胞等），（iii）简单的微滤收集这些细胞。为了证明该方法的有效性，我们使用MCF-7乳腺癌细胞（平均直径，μ pm）和DMS-79小细胞肺癌细胞（平均直径，10 μ m）来模拟ctc。我们研究了不同细胞和/或颗粒的相对沉降速率，例如与不同类型微珠、白细胞和红细胞结合的ctc，以最大限度地提高物理性质的差异。我们观察到，由于其沉降速率的差异，在最佳离心力下，全血中超过99%的白细胞被有效去除，与其他基于过滤器的方法相比，产生了更纯净的样品。我们还研究了过滤条件对回收率和样品纯度的影响，以及我们的检测平台的灵敏度。我们的结果显示MCF-7细胞的近乎完美的回收率（类似于99%）和DMS-79细胞的非常高的回收率（类似于89%），存在少量的白细胞。
Isolation of circulating tumor cells (CTCs) by Size Density size exclusion can yield poor purity and low recovery rates, due Leukocytes amplification to large variations in size of CTCs, which may overlap with 9 leukocytes and render size-based filtration methods unreliable. This report presents a very sensitive, selective, fast, and novel method for isolation and detection of CTCs. Our assay platform consists of three steps: (i) capturing CTCs with anti-EpCAM conjugated microbeads, (ii) removal of unwanted hematologic cells (e.g., leukocytes, erythrocytes, etc.) by selective sedimentation of CTCs within a density gradient medium, and (iii) simple microfiltration to collect these cells. To demonstrate the efficacy of this assay, MCF-7 breast cancer cells (average diameter, mu pm) and DMS-79 small cell lung cancer cells (average diameter, 10 mu m) were used to model CTCs. We investigated the relative sedimentation rates for various cells and/or particles, such as CTCs conjugated with different types of microbeads, leukocytes, and erythrocytes, in order to maximize differences in the physical properties. We observed that greater than 99% of leukocytes in whole blood were effectively removed at an optimal centrifugal force, due to differences in their sedimentation rates, yielding a much purer sample compared to other filter-based methods. We also investigated not only the effect of filtration conditions on recovery rates and sample purity but also the sensitivity of our assay platform. Our results showed a near perfect recovery rate (similar to 99%) for MCF-7 cells and very high recovery rate (similar to 89%) for DMS-79 cells, with minimal amounts of leukocytes present.