Breast Cancer Diagnosis Using a Microfluidic Multiplexed Immunohistochemistry Platform

Abstract

BackgroundBiomarkers play a key role in risk assessment, assessing treatment response, and detecting recurrence and the investigation of multiple biomarkers may also prove useful in accurate prediction and prognosis of cancers. Immunohistochemistry (IHC) has been a major diagnostic tool to identify therapeutic biomarkers and to subclassify breast cancer patients. However, there is no suitable IHC platform for multiplex assay toward personalized cancer therapy. Here, we report a microfluidics-based multiplexed IHC (MMIHC) platform that significantly improves IHC performance in reduction of time and tissue consumption, quantification, consistency, sensitivity, specificity and cost-effectiveness.Methodology/Principal FindingsBy creating a simple and robust interface between the device and human breast tissue samples, we not only applied conventional thin-section tissues into on-chip without any additional modification process, but also attained perfect fluid control for various solutions, without any leakage, bubble formation, or cross-contamination. Four biomarkers, estrogen receptor (ER), human epidermal growth factor receptor 2 (HER2), progesterone receptor (PR) and Ki-67, were examined simultaneously on breast cancer cells and human breast cancer tissues. The MMIHC method improved immunoreaction, reducing time and reagent consumption. Moreover, it showed the availability of semi-quantitative analysis by comparing Western blot. Concordance study proved strong consensus between conventional whole-section analysis and MMIHC (n = 105, lowest Kendall's coefficient of concordance, 0.90). To demonstrate the suitability of MMIHC for scarce samples, it was also applied successfully to tissues from needle biopsies.Conclusions/SignificanceThe microfluidic system, for the first time, was successfully applied to human clinical tissue samples and histopathological diagnosis was realized for breast cancers. Our results showing substantial agreement indicate that several cancer-related proteins can be simultaneously investigated on a single tumor section, giving clear advantages and technical advances over standard immunohistochemical method. This novel concept will enable histopathological diagnosis using numerous specific biomarkers at a time even for small-sized specimens, thus facilitating the individualization of cancer therapy.

Microfluidic processor allows rapid HER2 immunohistochemistry of breast carcinomas and significantly reduces ambiguous (2+) read-outs

“…The system could work mostly in the diffusion-limited mode, and the signal was detected using a special sophisticated time-resolved fluorescent detection method. Other researchers opted for multiplex IHC (16,17) with multiple parallel small channels targeting different biomarkers at spatially displaced locations using higher-antibody dilutions than needed for conventional IHC analysis. However, none of these microfluidic approaches resulted in an increase in accuracy of quantitative biomarker expression analysis and hence a reduction of ambiguous diagnostic results.…”

Quantitative proteomic profiling of breast cancers using a multiplexed microfluidic platform for immunohistochemistry and immunocytochemistry

“…Thus, rounded channels were fabricated for microvalves, and rectangular channels were created to form even fluid profiles in terms of the width of the reaction channels [21]. The fluidic channel mold of the device was fabricated by two-step multilayer soft lithography.…”

“…The preparation of cell blocks and tissues was described in our previous report [21]. Briefly, HCC70, MCF-7, and AU-565 were maintained in RPMI-1640 medium, and SK-BR-3 cells were maintained in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 100 IU mL À1 penicillin, and 100 mg mL À1 streptomycin.…”

“…The three parts were classified by a Bayesian classification. The Gaussian mixture model and expectation-maximization method were used for the color distribution of each part in RGB color space [21].…”

Passive removal of immiscible spacers from segmented flows in a microfluidic probe

“…Techniques to localize (bio)chemicals on surfaces at the micrometer length scale enable numerous applications, such as local staining of tissue samples, 1 targeted stimulation of cells, 2,3 and patterning of biochemicals on surfaces. 4 To this end, we developed the microfluidic probe (MFP), a noncontact scanning probe technology that leverages hydrodynamic flow confinement (HFC).…”