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

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

Association of the PHACTR1/EDN1 genetic locus with spontaneous coronary artery dissection

Background: Spontaneous coronary artery dissection (SCAD) is an increasingly recognized cause of acute coronary syndromes (ACS) afflicting predominantly younger to middle-aged women. Observational studies have reported a high prevalence of extracoronary vascular anomalies, especially fibromuscular dysplasia (FMD) and a low prevalence of coincidental cases of atherosclerosis. PHACTR1/EDN1 is a genetic risk locus for several vascular diseases, including FMD and coronary artery disease, with the putative causal noncoding variant at the rs9349379 locus acting as a potential enhancer for the endothelin-1 (EDN1) gene. Objectives: This study sought to test the association between the rs9349379 genotype and SCAD. Methods: Results from case control studies from France, United Kingdom, United States, and Australia were analyzed to test the association with SCAD risk, including age at first event, pregnancy-associated SCAD (P-SCAD), and recurrent SCAD. Results: The previously reported risk allele for FMD (rs9349379-A) was associated with a higher risk of SCAD in all studies. In a meta-analysis of 1,055 SCAD patients and 7,190 controls, the odds ratio (OR) was 1.67 (95% confidence interval [CI]: 1.50 to 1.86) per copy of rs9349379-A. In a subset of 491 SCAD patients, the OR estimate was found to be higher for the association with SCAD in patients without FMD (OR: 1.89; 95% CI: 1.53 to 2.33) than in SCAD cases with FMD (OR: 1.60; 95% CI: 1.28 to 1.99). There was no effect of genotype on age at first event, P-SCAD, or recurrence. Conclusions: The first genetic risk factor for SCAD was identified in the largest study conducted to date for this condition. This genetic link may contribute to the clinical overlap between SCAD and FMD

Programmable parylene-C bonding layer fluorescence for storing information on microfluidic chips

We demonstrate data storage on glass/silicon microfluidic devices fabricated using parylene-C as a bonding layer. In particular, we report intermediate parylene-C bonding layer fluorescence (iPBLF) and its use as an on-chip medium for data storage by dynamic programming of iPBLF intensity, using alternating exposure of parylene-C to UV and Green light. This technique allows data on the microfluidic chip to be read, written and erased by a common fluorescent microscope. Until now, no studies have focused on storing data like expiry date, protocol or operational parameters on a chip. However, this can be useful to overcome certain automation challenges in industrial applications for which communication of information is required, like needed during operation of remote microfluidic platforms. Finally, we also demonstrate the application of iPBLF for detecting channel dimensions and positions, and for marking on-chip zones of particular interest

Method for quantitative measurement of a biomarker by in situ immunofluorescence and uses thereof

The invention relates to a method for quantitative measurement of a biomarker by in situ immunofluorescence and uses thereof. In particular, the invention relates to a method which is a useful tool for use in the field of diagnosis, prevention and/or treatment of disease or disorders, in particular in the field of cancer management and therapy

Microfluidic network device

Microfluidic network device (2) configured to supply reagents to a biological tissue sampling device (1), comprising a plurality of microfluidic inlet channels (12) connected to respective sources of said reagents, at least one common outlet channel (22), and a plurality of valves (36) interconnecting an outlet end (14) of each of said plurality of inlet channels to said at least one common outlet channel

Programming and use of Parylene C fluorescence as a quantitative on-chip reference

A large number of lab-on-a-chip applications use fluorescence for quantifying biological entities. In such applications, incorporation of a stable on-chip fluorescent reference source would be highly desirable in order to compensate for instrumental parameter variations, like for example non-constant illumination intensity over time. In this study, we exploit Parylene C that is used as a bonding layer material in a microfluidic chip. We first show programming of intermediate Parylene C bonding layer fluorescence (iPBLF) and its characterization as a function of the ultraviolet (UV) dose and Parylene C thickness. This technique requires no additional steps in the fabrication of the microfluidic chip and the fluorescence reference zones can simply be incorporated by local exposure to UV light after fabrication. Next, we demonstrate a fluorescence-based analyte concentration and flow-rate measurement in a microfluidic channel, under changing experimental conditions of illumination intensity and taking different microscope objectives. Sensing is realized by analyzing programmed reference and channel images by straightforward data handling using an open-source image processing tool. We anticipate that the demonstrated method will be a key technique allowing low complexity and reliable quantitative fluorescent measurements, for example in point-of-care and mobile diagnostic applications, where intensity calibration can present a major challenge

Microfluidic-Based Immunohistochemistry Combined With Next-Generation Sequencing on Diagnostic Tissue Sections for Detection of Tumoral BRAF V600E Mutation

OBJECTIVES Tailored diagnostics requires immunohistochemistry (IHC) and next generation sequencing (NGS). Here we combined on a single paraffin-embedded slide microfluidic-based IHC (micro-IHC) and NGS for BRAF V600E mutation detection in BRAFomas. METHODS For micro-IHC, we performed the primary antibody incubation step of conventional chromogenic IHC in a LabSat device (Lunaphore Technologies SA). Tumor areas immunoreactive for pan-cytokeratin, pan-melanoma, and BRAF V600E mutation-specific antibody were H-scored, microdissected, and analyzed by NGS. RESULTS After 2 minutes, pan-cytokeratin and BRAF micro-IHC increased exponentially (half-time values: 1.7 and 3.2 minutes). Pan-melanoma displayed a higher half-time value of 15 minutes. There was no significant difference in H-score and staining quality, respectively, between conventional and micro-IHC. BRAF V600E mutation was detected in all pan-cytokeratin and pan-melanoma stained samples without amplification but in only 40% of BRAF V600E stained samples with amplification. CONCLUSIONS Micro-IHC enables short antibody incubation times and subsequent NGS. Preprocessing is critical for preservation of DNA quality