Microfluidics-assisted fluorescence in situ hybridization for advantageous human epidermal growth factor receptor 2 assessment in breast cancer

Nguyen, Huu Tuan; Trouillon, Raphaël; Matsuoka, Seiya; Fiche, Maryse; Leval, Laurence de; Bisig, Bettina; Gijs, Martin A. M.

doi:10.1038/labinvest.2016.121

Cited by 24 publications

(21 citation statements)

References 39 publications

(50 reference statements)

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“…For FISH-based microfluidic analysis of FFPE tissue sections, only a few implementations have been reported; Kao et al as an example focused on automation of the assay 25 and reduced the FISH probe consumption from 10 μ l to 2 μ l with 16 h static hybridization resulting in a turnaround assay time of 20 h. Using flow-based FISH probe incubation for 4 h, Nguyen et al 26 reduced the FISH probe consumption to 2 μ l and the turnaround assay time from >24 h to 8 h. They use a PDMS device, which is contacted with the tissue section and FISH probes are loaded into the microchannels for hybridization. In a recent study, 27 they presented extra short incubation microfluidic assisted (ESIMA) FISH for HER2 testing of tissue sections with an incubation time of 35 min and a probe consumption of 3 μ l per test using the recently reported Instant Quality (IQ) FISH buffer 28 …”

Section: Introductionmentioning

confidence: 99%

Rapid micro fluorescence in situ hybridization in tissue sections

Huber

Kaigala

2018

Biomicrofluidics

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This paper describes a micro fluorescence in situ hybridization (μFISH)-based rapid detection of cytogenetic biomarkers on formalin-fixed paraffin embedded (FFPE) tissue sections. We demonstrated this method in the context of detecting human epidermal growth factor 2 (HER2) in breast tissue sections. This method uses a non-contact microfluidic scanning probe (MFP), which localizes FISH probes at the micrometer length-scale to selected cells of the tissue section. The scanning ability of the MFP allows for a versatile implementation of FISH on tissue sections. We demonstrated the use of oligonucleotide FISH probes in ethylene carbonate-based buffer enabling rapid hybridization within <1 min for chromosome enumeration and 10–15 min for assessment of the HER2 status in FFPE sections. We further demonstrated recycling of FISH probes for multiple sequential tests using a defined volume of probes by forming hierarchical hydrodynamic flow confinements. This microscale method is compatible with the standard FISH protocols and with the Instant Quality FISH assay and reduces the FISH probe consumption ∼100-fold and the hybridization time 4-fold, resulting in an assay turnaround time of <3 h. We believe that rapid μFISH has the potential of being used in pathology workflows as a standalone method or in combination with other molecular methods for diagnostic and prognostic analysis of FFPE sections.

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Section: Introductionmentioning

confidence: 99%

Rapid micro fluorescence in situ hybridization in tissue sections

Huber

Kaigala

2018

Biomicrofluidics

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“…The Gijs group developed a flow cell for tissue perfusion to study HER2 expression [69] termed microfluidics-assisted FISH (MA-FISH). Here, the tissue slice was mounted on a conventional microscope slide and placed into a custommade copper holder.…”

Section: Tissue Slices In Fluidic Chambersmentioning

confidence: 99%

“…Reproduced with permission from Ref. [69]. d Concept of vertical microfluidic device with inlets and outlets for confined delivery and withdrawal of nL volumes of (i) hybridization probes and (ii) wash buffer.…”

Section: Comparison Of Microfluidic Fish Platformsmentioning

confidence: 99%

FISH and chips: a review of microfluidic platforms for FISH analysis

Rodriguez-Mateos

Azevedo

Almeida

et al. 2020

Med Microbiol Immunol

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Fluorescence in situ hybridization (FISH) allows visualization of specific nucleic acid sequences within an intact cell or a tissue section. It is based on molecular recognition between a fluorescently labeled probe that penetrates the cell membrane of a fixed but intact sample and hybridizes to a nucleic acid sequence of interest within the cell, rendering a measurable signal. FISH has been applied to, for example, gene mapping, diagnosis of chromosomal aberrations and identification of pathogens in complex samples as well as detailed studies of cellular structure and function. However, FISH protocols are complex, they comprise of many fixation, incubation and washing steps involving a range of solvents and temperatures and are, thus, generally time consuming and labor intensive. The complexity of the process, the relatively high-priced fluorescent probes and the fairly high-end microscopy needed for readout render the whole process costly and have limited wider uptake of this powerful technique. In recent years, there have been attempts to transfer FISH assay protocols onto microfluidic lab-on-a-chip platforms, which reduces the required amount of sample and reagents, shortens incubation times and, thus, time to complete the protocol, and finally has the potential for automating the process. Here, we review the wide variety of approaches for lab-on-chip-based FISH that have been demonstrated at proof-of-concept stage, ranging from FISH analysis of immobilized cell layers, and cells trapped in arrays, to FISH on tissue slices. Some researchers have aimed to develop simple devices that interface with existing equipment and workflows, whilst others have aimed to integrate the entire FISH protocol into a fully autonomous FISH on-chip system. Whilst the technical possibilities for FISH on-chip are clearly demonstrated, only a small number of approaches have so far been converted into off-the-shelf products for wider use beyond the research laboratory. Keywords Microfluidics-assisted FISH • µFISH • Microfluidics • Lab-on-a-Chip (LOC) • Fluorescence in situ hybridization (FISH) Edited by Volkhard A. J. Kempf.

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“…The structure and fabrication of the chip were reported previously. 7,10,14 First, a 4-in. silicon wafer with a 2.5-μm wet-oxide layer was taken, on which 5-μm AZ9260 photoresist was spun, exposed with the channel mask, and developed to form the channels.…”

Section: Fabrication Of the Microfluidic Chipmentioning

confidence: 99%

Combining fluorescence-based image segmentation and automated microfluidics for ultrafast cell-by-cell assessment of biomarkers for HER2-type breast carcinoma

2018

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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.

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Microfluidics-assisted fluorescence in situ hybridization for advantageous human epidermal growth factor receptor 2 assessment in breast cancer

Cited by 24 publications

References 39 publications

Rapid micro fluorescence in situ hybridization in tissue sections

Rapid micro fluorescence in situ hybridization in tissue sections

FISH and chips: a review of microfluidic platforms for FISH analysis

Combining fluorescence-based image segmentation and automated microfluidics for ultrafast cell-by-cell assessment of biomarkers for HER2-type breast carcinoma

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