Highly efficient isolation and multistep analysis of tumor cells from whole blood†

IF 6.1 2区 工程技术 Q1 BIOCHEMICAL RESEARCH METHODS
Lab on a Chip Pub Date : 2025-03-10 DOI:10.1039/D4LC00759J
Michael Knapp, Samir Kadić, Astrid Lux, Nils Paust, Roland Zengerle and Jochen Hoffmann
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引用次数: 0

Abstract

We present a microfluidic solution for improved tumor cell analysis based on selection-free isolation of nucleated cells from whole blood. It consists of a high-density silicon microcavity array combined with the novel fluidic strategy of microfluidic decanting. This enables multistep on-chip staining protocols comprising sample loading–blocking–extracellular staining–fixation–permeabilization and intracellular staining to quantify tumor cells. The performance of the workflow was investigated and proven by spiking colon cancer cell lines into whole blood for the detection of the epithelial tumor markers EpCAM and cytokeratin. Total cell recovery rates of ≥95% were achieved for different sample species. The method allows for rapid reagent exchange within 10 s each almost without cell loss compared to approximately 50% cell loss in reference centrifugal processing. The isolation of nucleated cells resulted in a high intra-assay precision with a CV of 2% and a single cell per well distribution of 90%, which is consistent with the theoretical estimate using Poisson statistics. The linearity of the method was demonstrated over three orders of magnitude with r2 = 0.9998. These results demonstrate a highly efficient approach for the quantification of tumor cells from whole blood that could be integrated into automated point-of-care devices in the future.

Abstract Image

全血肿瘤细胞的高效分离和多步骤分析。
我们提出了一种基于全血无选择分离有核细胞的改进肿瘤细胞分析的微流体解决方案。它由高密度硅微腔阵列和新型的微流控倒液策略相结合而成。这使得包括样品装载-阻断-细胞外染色-固定-渗透和细胞内染色在内的多步骤芯片染色方案能够量化肿瘤细胞。通过将结肠癌细胞系注入全血中检测上皮肿瘤标志物EpCAM和细胞角蛋白,研究并证实了该工作流程的性能。不同样品种类的细胞总回收率均≥95%。该方法允许在10秒内快速交换试剂,几乎没有细胞损失,而参考离心处理的细胞损失约为50%。有核细胞的分离导致了较高的测定内精度,CV为2%,每孔分布的单个细胞为90%,这与使用泊松统计的理论估计一致。该方法的线性关系在3个数量级以上,r2 = 0.9998。这些结果证明了一种高效的全血肿瘤细胞定量方法,可以集成到未来的自动化点护理设备中。
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来源期刊
Lab on a Chip
Lab on a Chip 工程技术-化学综合
CiteScore
11.10
自引率
8.20%
发文量
434
审稿时长
2.6 months
期刊介绍: Lab on a Chip is the premiere journal that publishes cutting-edge research in the field of miniaturization. By their very nature, microfluidic/nanofluidic/miniaturized systems are at the intersection of disciplines, spanning fundamental research to high-end application, which is reflected by the broad readership of the journal. Lab on a Chip publishes two types of papers on original research: full-length research papers and communications. Papers should demonstrate innovations, which can come from technical advancements or applications addressing pressing needs in globally important areas. The journal also publishes Comments, Reviews, and Perspectives.
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