École Polytechnique Fédérale de Lausanne, Laboratory of Microsystems, Lausanne, Switzerland.
J Biomed Opt. 2018 Nov;24(2):1-8. doi: 10.1117/1.JBO.24.2.021204.
Immunohistochemistry (IHC) is one of the main clinical techniques for biomarker assessment on tissue biopsies. It consists in chromogenic labeling with specific antibodies, followed by optical imaging, and it is used for diagnosis and therapeutic targeting. A well-known drawback of IHC is its limited robustness, which often precludes quantitative biomarker assessment. We combine microfluidic immunostaining, fluorescence imaging, and image-based cell segmentation to create an ultrafast procedure for accurate biomarker assessment via IHC. The experimental protocol is very simple and based on fast delivery of reagents in a microfluidic chamber created by clamping a half-chamber patterned in a silicon chip on top of a tumor tissue section. Also, the imaging procedure simply requires a standard fluorescence microscope, already widely used in clinical practice. The image processing is based on local-contrast enhancement and thresholding of the obtained fluorescence image, with subsequent Voronoi segmentation. To assess the experimental and analytical procedure on robust biological controls, we apply our method to well-characterized cell lines, which guarantee higher reproducibility than whole-tissue samples and therefore enable to disentangle the technical variability from the biological variability. To increase the potential translationality, we address the detection and quantification of the human epidermal growth factor receptor 2 (HER2) protein, which is a biomarker for HER2-type breast carcinoma diagnosis and therapy. We report both ultrafast immunofluorescence staining (5 min per sample) of two breast cancer biomarkers and ultrafast cell segmentation (1 min per sample = processing of thousands of cells). This provides a quantitative, cell-based immunofluorescent signal, with which we propose a potential diagnostic criterion to separate HER2-positive and HER2-negative breast cancer cells at high sensitivity and specificity.
免疫组织化学（IHC）是组织活检中评估生物标志物的主要临床技术之一。它包括用特异性抗体进行显色标记，然后进行光学成像，用于诊断和治疗靶向。IHC 的一个众所周知的缺点是其有限的稳健性，这通常排除了定量生物标志物评估。我们结合微流控免疫染色、荧光成像和基于图像的细胞分割，创建了一种通过 IHC 进行准确生物标志物评估的超快方法。实验方案非常简单，基于在硅芯片上的半腔图案化顶部夹肿瘤组织切片上创建的微流控室中快速输送试剂。此外，成像过程仅需要标准荧光显微镜，该显微镜已经广泛应用于临床实践。图像处理基于获得的荧光图像的局部对比度增强和阈值处理，随后是 Voronoi 分割。为了在稳健的生物对照上评估实验和分析过程，我们将我们的方法应用于具有良好特征的细胞系，这些细胞系保证了更高的可重复性，因此能够将技术变异性与生物变异性区分开来。为了提高潜在的转化性，我们解决了人表皮生长因子受体 2（HER2）蛋白的检测和定量问题，HER2 蛋白是 HER2 型乳腺癌诊断和治疗的生物标志物。我们报告了两种乳腺癌生物标志物的超快免疫荧光染色（每个样本 5 分钟）和超快细胞分割（每个样本 1 分钟=处理数千个细胞）。这提供了一种定量的、基于细胞的免疫荧光信号，我们提出了一种潜在的诊断标准，以高灵敏度和特异性分离 HER2 阳性和 HER2 阴性乳腺癌细胞。
