Microfluidic staining technology and automated image processing for fast and accurate tissue-based diagnostics

Abstract

The development of microfluidics has led to significant improvements in many areas of in vitro diagnostics. This thesis proposes the use of microfluidic device technology in the field of anatomic pathology, leading to faster and more accurate tissue-based diagnosis than was provided by standard diagnostic tools until now. First, we characterize the fluorescence of the intermediate Parylene C bonding layer used in the fabrication of silicon/Pyrex microfluidic chips. Subsequently, we show how long-term illumination of Parylene C under bonding conditions can deliberately modify the autofluorescence of this material. We then use these programming capabilities to demonstrate several microfluidic applications of interest. In a first study, we show data storage on silicon/Pyrex microfluidic devices, where dynamic programming can be achieved by alternating the exposure of Parylene C to UV and green light. In a second study, we show how modifying the fluorescence of the intermediate Parylene C bonding layer we can create an on-chip reference, which can be used to estimate concentrations and flow-rates of fluorescent molecules in a microfluidic channel. Subsequently, we focus our research on the use of microfluidic technologies to answer clinically relevant questions in the field of anatomic pathology. We propose a microfluidic precision immunofluorescence method, which accurately quantifies antigen expression levels in a continuous scale based on microfluidic staining of breast carcinoma tissue sections and automated image analysis. We show that the level of human epidermal growth factor receptor 2 (HER2) protein expression, as continuously quantified using microfluidic precision immunofluorescence in 25 breast cancer cases, can predict the number of HER2 gene copies as assessed by fluorescence in situ hybridization. This method has the potential of providing automated, fast and high-quality quantitative in situ biomarker data using low-cost immunofluorescence assays, as increasingly required in the era of individually tailored cancer therapy. Next, we propose two solutions for intraoperative staining using microfluidics: (1) a rapid immunohistochemical (IHC) staining of frozen sections using a polymer microfluidic chip, and (2) an automated fluorescent staining of the surface of thick (> 2 mm) fresh tumoral specimens. Frozen sections of tumor samples play an important role in the microscopic analysis of specimens during surgery. IHC stainings on frozen sections would be of great use during intraoperative consultations, if only the turn-around time was not a limitation. In a first intraoperative application, we show a complete pan-cytokeratin chromogenic staining protocol on frozen sections using a polymer microfluidic chip. We demonstrate an optimized cytokeratin IHC staining protocol that takes less than 12 minutes on several autopsy and tumor biopsy tissues. In the second application, we propose a new microfluidic tool that allows automated staining and imaging of thick (> 2 mm) tissue samples. Sections of breast mastectomies taken from the proximity of the tumor location are inserted in a chamber and fluorescently stained for cytokeratin using the reagent delivery of a microfluidic chip that is custom-fabricated for this application. The dimensions of the microfluidic system used in these studies are compatible with the space constraints of an intraoperative pathology laboratory.