Knapp Michael, Kadić Samir, Lux Astrid, Paust Nils, Zengerle Roland, Hoffmann Jochen
Department of Microsystems Engineering - IMTEK, University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany.
Robert Bosch GmbH, Corporate Sector Research and Advance Engineering, Robert-Bosch-Campus 1, 71272 Renningen, Germany.
Lab Chip. 2025 Apr 8;25(8):1938-1946. doi: 10.1039/d4lc00759j.
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 = 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.
我们提出了一种基于从全血中无选择地分离有核细胞来改进肿瘤细胞分析的微流控解决方案。它由一个高密度硅微腔阵列与微流控倾析的新型流体策略相结合组成。这使得能够进行多步芯片上染色方案，包括样品加载-封闭-细胞外染色-固定-通透和细胞内染色，以量化肿瘤细胞。通过将结肠癌细胞系加入全血中以检测上皮肿瘤标志物EpCAM和细胞角蛋白，对该工作流程的性能进行了研究和验证。对于不同的样品类型，总细胞回收率达到了≥95%。与参考离心处理中约50%的细胞损失相比，该方法几乎能在每次10秒内快速进行试剂交换且几乎没有细胞损失。有核细胞的分离导致了较高的分析内精度，变异系数为2%，每孔单细胞分布率为90%，这与使用泊松统计的理论估计一致。该方法的线性在三个数量级上得到了证明，相关系数为0.9998。这些结果证明了一种从全血中量化肿瘤细胞的高效方法，未来可集成到自动化即时检测设备中。
