Automated Measurement of Multiple Cancer Biomarkers Using Quantum-Dot-Based Microfluidic Immunohistochemistry

Abstract

We report an automated multiple biomarker measurement method for tissue from cancer patients using quantum dot (QD)-based protein detection combined with reference-based protein quantification and autofluorescence (AF) removal. For multiplexed detection of biomarkers in tissue samples, visualization of QDs on cytokeratin was performed to create a multichannel microfluidic device on sites with dense populations of tumor cells. Three major breast cancer biomarkers (i.e., estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2) were labeled using QDs successively on cancer cells in tissue sections. For the automated measurement of biomarkers, a cytokeratin-based biomarker normalization method was used to measure the averaged expression of proteins. A novel AF-removal algorithm was developed, which normalizes the reference AF spectra reconstructed from unknown AF spectra based on random sampling. For accurate quantification of QDs, we automatically and accurately removed the AF signal from 344 spots of QD-labeled tissue samples using 240 reference AF spectra. Using analytical data with 10 tissue samples from breast cancer patients, the measured biomarker intensities were in good agreement with the results of conventional analyses.

Continuous quantification of HER2 expression by microfluidic precision immunofluorescence estimates HER2 gene amplification in breast cancer

“…CK constitutes a marker for epithelial cells and it has been widely used in carcinomas to distinguish epithelia from stroma 19 20 21 . Furthermore, CK-based normalization has proven to be a useful technique to increase accuracy in the quantification of HER2, by compensating for variations in the staining quality of the samples 22 . In a second step, two fluorescently labeled secondary antibodies were sequentially delivered into the chamber, first for HER2 and then for CK detection.…”

A Microfluidic Immunostaining System Enables Quality Assured and Standardized Immunohistochemical Biomarker Analysis

“…To facilitate the finding of the same locations in the tissue, a double color QD staining was performed to stain cytokeratin and HER2, to mark the epithelial region of a tissue and simultaneously target the biomarker (Fig. 3D)33. Due to the cell heterogeneity of the breast cancer tissue34, the difference in the average QD intensities among the HER2-labeled tissue sections was larger than that among the cell sections (Fig.…”

Finding the perfect match between nanoparticles and microfluidics to respond to cancer challenges

“…This time they used cytokeratin as protein reference, which allowed not only to normalize the values but also to remove the autofluorescence. 73 They used breast cancer tissue' sections, to compare with conventional scoring methods and labelled cytokeratin, to detect cancer area. First, they aligned a microfluidic chip on top of the cancer tissue samples.…”