Identification of fetal aneuploidy with dual-probe fluorescence in situ hybridization analysis in circulating trophoblasts after enrichment using a high-sensitivity microfluidic platform.

OBJECTIVE: To evaluate a microfluidics-based positive selection technology for isolating circulating trophoblasts (CTs) from peripheral blood of women whose pregnancies are affected by aneuploidy and to evaluate fetal karyotype using fluorescence in situ hybridization (FISH). METHOD: Ten 18-ml samples of peripheral blood were collected consecutively from pregnant women whose fetus was affected by aneuploidy. A preservation buffer was added, and the specimens were shipped overnight to the testing laboratory at ambient temperature. The specimen was infused into the fully automated microfluidics-based LiquidScan® instrument without pre-processing. This instrument contains microfluidic chips, which are coated with antibodies (anti-huEpCAM and a proprietary antibody mixture) specific to CT surface epitopes. FISH analysis was performed on the enriched cells. RESULTS: Fetal aneuploidy evaluated included trisomy 21 (n = 3), trisomy 18 (n = 1), trisomy 13 (n = 1), monosomy X (n = 3), and triploidy (n = 1). CTs for analysis by FISH were identified in all samples. The average number of mononucleate cells per 1 ml of whole blood was 2.11 (range 0.38-4.63) overall and was 2.67 (range 1.13-4.63) using the proprietary combination of antibodies. FISH results were concordant with the aneuploidy based on other testing in all cases. Multinucleate cells were searched for and identified in the last seven samples (average number: 0.84/1 ml). CONCLUSIONS: Our study demonstrates that the LiquidScan® , a high-sensitivity microfluidic platform, can enrich circulating trophoblasts (mononucleate and multinucleate). FISH can then be used to detect fetal aneuploidy.

Microfluidic Device for On-Chip Immunophenotyping and Cytogenetic Analysis of Rare Biological Cells.

The role of circulating plasma cells (CPCs) and circulating leukemic cells (CLCs) as biomarkers for several blood cancers, such as multiple myeloma and leukemia, respectively, have recently been reported. These markers can be attractive due to the minimally invasive nature of their acquisition through a blood draw (i.e., liquid biopsy), negating the need for painful bone marrow biopsies. CPCs or CLCs can be used for cellular/molecular analyses as well, such as immunophenotyping or fluorescence in situ hybridization (FISH). FISH, which is typically carried out on slides involving complex workflows, becomes problematic when operating on CLCs or CPCs due to their relatively modest numbers. Here, we present a microfluidic device for characterizing CPCs and CLCs using immunofluorescence or FISH that have been enriched from peripheral blood using a different microfluidic device. The microfluidic possessed an array of cross-channels (2-4 µm in depth and width) that interconnected a series of input and output fluidic channels. Placing a cover plate over the device formed microtraps, the size of which was defined by the width and depth of the cross-channels. This microfluidic chip allowed for automation of immunofluorescence and FISH, requiring the use of small volumes of reagents, such as antibodies and probes, as compared to slide-based immunophenotyping and FISH. In addition, the device could secure FISH results in <4 h compared to 2-3 days for conventional FISH.

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

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.

Fluorescence In Situ Hybridization and Rehybridization Using Bacterial Artificial Chromosome Probes.

Fluorescence in situ hybridization (FISH) method enables in situ genetic analysis of both metaphase and interphase cells from different types of material, including cell lines, cell smears, and fresh and paraffin-embedded tissue. Despite the growing number of commercially available FISH probes, still for large number of gene loci or chromosomal regions commercial probes are not available. Here we describe a simple method for generating FISH probes using bacterial artificial chromosomes (BAC). Due to genome-wide coverage of BAC clones, there are almost unlimited possibilities for the analysis of any genomic regions using BAC FISH probes.

Analysis of Common Abnormalities Seen in Chronic Lymphocytic Leukemia Using Fluorescence In Situ Hybridization.

Since fluorescence in situ hybridization (FISH) was used to define a prognostic heierarchy for chronic lymphocytic leukemia (CLL) in 2000, the method has been employed widely in cytogenetics laboratories worldwide. This chapter describes techniques and trouble-shooting to maximize the efficiency of microscope slide preparation for FISH analysis in CLL.

Silencing cuticular pigmentation genes enables RNA FISH in intact insect appendages.

Optical imaging of gene expression by fluorescence in situ hybridisation (FISH) in insects is often impeded by their pigmented cuticle. As most chemical bleaching agents are incompatible with FISH, we developed an RNA interference (RNAi)-based method for clearing cuticular pigmentation which enables the use of whole-mount body appendages for RNA FISH (termed RNA-i-FISH). Silencing laccase2 or tyrosine hydroxylase in two leaf beetles species (Chrysomela populi and Phaedon cochleariae) cleared their pigmented cuticle and decreased light absorbance. Subsequently, intact appendages (palps, antennae, legs) from RNAi-cleared individuals were used to image the expression and spatial distribution of antisense mRNA of two chemosensory genes encoding gustatory receptor and odorant-binding protein. Imaging did not work for RNAi controls because the pigmentation was retained, or for FISH controls (sense mRNA). Several bleaching agents were incompatible with FISH, because of degradation of RNA, lack of clearing efficacy or long incubation times. Overall, silencing pigmentation genes is a significant improvement over bleaching agents, enabling FISH in intact insect appendages.

3D FISH to analyse gene domain-specific chromatin re-modeling in human cancer cell lines.

Fluorescence in situ hybridization (FISH) is a common technique used to label DNA and/or RNA for detection of a genomic region of interest. However, the technique can be challenging, in particular when applied to single genes in human cancer cells. Here, we provide a step-by-step protocol for analysis of short (35 kb-300 kb) genomic regions in three dimensions (3D). We discuss the experimental design and provide practical considerations for 3D imaging and data analysis to determine chromatin folding. We demonstrate that 3D FISH using BACs (Bacterial Artificial Chromosomes) or fosmids can provide detailed information of the architecture of gene domains. More specifically, we show that mapping of specific chromatin landscapes informs on changes associated with estrogen stimulated gene activity in human breast cancer cell lines.

Standardization and optimization of fluorescence in situ hybridization (FISH) for HER-2 assessment in breast cancer: A single center experience.

Accurate assessment of human epidermal growth factor receptor 2 (HER-2) is crucial in selecting patients for targeted therapy. Commonly used methods for HER-2 testing are immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Here we presented the implementation, optimization and standardization of two FISH protocols using breast cancer samples and assessed the impact of pre-analytical and analytical factors on HER-2 testing. Formalin fixed paraffin embedded (FFPE) tissue samples from 70 breast cancer patients were tested for HER-2 using PathVysion™ HER-2 DNA Probe Kit and two different paraffin pretreatment kits, Vysis/Abbott Paraffin Pretreatment Reagent Kit (40 samples) and DAKO Histology FISH Accessory Kit (30 samples). The concordance between FISH and IHC results was determined. Pre-analytical and analytical factors (i.e., fixation, baking, digestion, and post-hybridization washing) affected the efficiency and quality of hybridization. The overall hybridization success in our study was 98.6% (69/70); the failure rate was 1.4%. The DAKO pretreatment kit was more time-efficient and resulted in more uniform signals that were easier to interpret, compared to the Vysis/Abbott kit. The overall concordance between IHC and FISH was 84.06%, kappa coefficient 0.5976 (p < 0.0001). The greatest discordance (82%) between IHC and FISH was observed in IHC 2+ group. A standardized FISH protocol for HER-2 assessment, with high hybridization efficiency, is necessary due to variability in tissue processing and individual tissue characteristics. Differences in the pre-analytical and analytical steps can affect the hybridization quality and efficiency. The use of DAKO pretreatment kit is time-saving and cost-effective.

HiCTMap: Detection and analysis of chromosome territory structure and position by high-throughput imaging.

The spatial organization of chromosomes in the nuclear space is an extensively studied field that relies on measurements of structural features and 3D positions of chromosomes with high precision and robustness. However, no tools are currently available to image and analyze chromosome territories in a high-throughput format. Here, we have developed High-throughput Chromosome Territory Mapping (HiCTMap), a method for the robust and rapid analysis of 2D and 3D chromosome territory positioning in mammalian cells. HiCTMap is a high-throughput imaging-based chromosome detection method which enables routine analysis of chromosome structure and nuclear position. Using an optimized FISH staining protocol in a 384-well plate format in conjunction with a bespoke automated image analysis workflow, HiCTMap faithfully detects chromosome territories and their position in 2D and 3D in a large population of cells per experimental condition. We apply this novel technique to visualize chromosomes 18, X, and Y in male and female primary human skin fibroblasts, and show accurate detection of the correct number of chromosomes in the respective genotypes. Given the ability to visualize and quantitatively analyze large numbers of nuclei, we use HiCTMap to measure chromosome territory area and volume with high precision and determine the radial position of chromosome territories using either centroid or equidistant-shell analysis. The HiCTMap protocol is also compatible with RNA FISH as demonstrated by simultaneous labeling of X chromosomes and Xist RNA in female cells. We suggest HiCTMap will be a useful tool for routine precision mapping of chromosome territories in a wide range of cell types and tissues.

Canine Hepatitis Associated with Intrahepatic Bacteria in Three Dogs.

This case report describes the detection of intrahepatic bacteria in formalin-fixed paraffin-embedded histopathological sections from three dogs with neutrophilic, pyogranulomatous, or lymphoplasmacytic hepatitis and cholangiohepatitis. In each of these cases, eubacterial fluorescence in situ hybridization enabled colocalization of intrahepatic bacteria with neutrophilic and granulomatous inflammation in samples that were negative for bacteria when evaluated by routine hematoxylin and eosin histopathology augmented with histochemical stains. Positive responses to antimicrobial therapy were observed in of 2 out of 2 patients that were treated with antimicrobials. These findings suggest that eubacterial fluorescence in situ hybridization analysis of formalin-fixed paraffin-embedded histopathological sections is more sensitive than conventional histochemical stains for the diagnosis of bacteria-associated canine hepatitis.

Automated Image Analysis of HER2 Fluorescence In Situ Hybridization to Refine Definitions of Genetic Heterogeneity in Breast Cancer Tissue.

Human epidermal growth factor receptor 2 gene- (HER2-) targeted therapy for breast cancer relies primarily on HER2 overexpression established by immunohistochemistry (IHC) with borderline cases being further tested for amplification by fluorescence in situ hybridization (FISH). Manual interpretation of HER2 FISH is based on a limited number of cells and rather complex definitions of equivocal, polysomic, and genetically heterogeneous (GH) cases. Image analysis (IA) can extract high-capacity data and potentially improve HER2 testing in borderline cases. We investigated statistically derived indicators of HER2 heterogeneity in HER2 FISH data obtained by automated IA of 50 IHC borderline (2+) cases of invasive ductal breast carcinoma. Overall, IA significantly underestimated the conventional HER2, CEP17 counts, and HER2/CEP17 ratio; however, it collected more amplified cells in some cases below the lower limit of GH definition by manual procedure. Indicators for amplification, polysomy, and bimodality were extracted by factor analysis and allowed clustering of the tumors into amplified, nonamplified, and equivocal/polysomy categories. The bimodality indicator provided independent cell diversity characteristics for all clusters. Tumors classified as bimodal only partially coincided with the conventional GH heterogeneity category. We conclude that automated high-capacity nonselective tumor cell assay can generate evidence-based HER2 intratumor heterogeneity indicators to refine GH definitions.

Detection of Gene Rearrangements in Circulating Tumor Cells: Examples of ALK-, ROS1-, RET-Rearrangements in Non-Small-Cell Lung Cancer and ERG-Rearrangements in Prostate Cancer.

Circulating tumor cells (CTCs) hold promise as biomarkers to aid in patient treatment stratification and disease monitoring. Because the number of cells is a critical parameter for exploiting CTCs for predictive biomarker's detection, we developed a FISH (fluorescent in situ hybridization) method for CTCs enriched on filters (filter-adapted FISH [FA-FISH]) that was optimized for high cell recovery. To increase the feasibility and reliability of the analyses, we combined fluorescent staining and FA-FISH and developed a semi-automated microscopy method for optimal FISH signal identification in filtration-enriched CTCs . Here we present these methods and their use for the detection and characterization of ALK-, ROS1-, RET-rearrangement in CTCs from non-small-cell lung cancer and ERG-rearrangements in CTCs from prostate cancer patients.

2D and 3D FISH of expanded repeat RNAs in human lymphoblasts.

The first methods for visualizing RNAs within cells were designed for simple imaging of specific transcripts in cells or tissues and since then significant technical advances have been made in this field. Today, high-resolution images can be obtained, enabling visualization of single transcript molecules, quantitative analyses of images, and precise localization of RNAs within cells as well as co-localization of transcripts with specific proteins or other molecules. In addition, tracking of RNA dynamics within single cell has become possible. RNA imaging techniques have been utilized for investigating the role of mutant RNAs in a number of human disorders caused by simple microsatellite expansions. These diseases include myotonic dystrophy type 1 and 2, amyotrophic lateral sclerosis/frontotemporal dementia, fragile X-associated tremor/ataxia syndrome, and Huntington's disease. Mutant RNAs with expanded repeats tend to aggregate predominantly within cell nuclei, forming structures called RNA foci. In this study, we demonstrate methods for fluorescent visualization of RNAs in both fixed and living cells using the example of RNAs containing various expanded repeat tracts (CUG, CCUG, GGGGCC, CGG, and CAG) from experiment design to image analysis. We describe in detail 2D and 3D fluorescence in situ hybridization (FISH) protocols for imaging expanded repeats RNAs, and we review briefly live imaging techniques used to characterize RNA foci formed by mutant RNAs. These methods could be used to image the entire cellular pathway of RNAs, from transcription to degradation.

Genome organization in the nucleus: From dynamic measurements to a functional model.

A biological system is by definition a dynamic environment encompassing kinetic processes that occur at different length scales and time ranges. To explore this type of system, spatial information needs to be acquired at different time scales. This means overcoming significant hurdles, including the need for stable and precise labeling of the required probes and the use of state of the art optical methods. However, to interpret the acquired data, biophysical models that can account for these biological mechanisms need to be developed. The structure and function of a biological system are closely related to its dynamic properties, thus further emphasizing the importance of identifying the rules governing the dynamics that cannot be directly deduced from information on the structure itself. In eukaryotic cells, tens of thousands of genes are packed in the small volume of the nucleus. The genome itself is organized in chromosomes that occupy specific volumes referred to as chromosome territories. This organization is preserved throughout the cell cycle, even though there are no sub-compartments in the nucleus itself. This organization, which is still not fully understood, is crucial for a large number of cellular functions such as gene regulation, DNA breakage repair and error-free cell division. Various techniques are in use today, including imaging, live cell imaging and molecular methods such as chromosome conformation capture (3C) methods to better understand these mechanisms. Live cell imaging methods are becoming well established. These include methods such as Single Particle Tracking (SPT), Continuous Photobleaching (CP), Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Correlation Spectroscopy (FCS) that are currently used for studying proteins, RNA, DNA, gene loci and nuclear bodies. They provide crucial information on its mobility, reorganization, interactions and binding properties. Here we describe how these dynamic methods can be used to gather information on genome organization, its stabilization mechanisms and the proteins that take part in it.

Yeasts identification in microfluidic devices using peptide nucleic acid fluorescence in situ hybridization (PNA-FISH).

Peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) is a highly specific molecular method widely used for microbial identification. Nonetheless, and due to the detection limit of this technique, a time-consuming pre-enrichment step is typically required before identification. In here we have developed a lab-on-a-chip device to concentrate cell suspensions and speed up the identification process in yeasts. The PNA-FISH protocol was optimized to target Saccharomyces cerevisiae, a common yeast that is very relevant for several types of food industries. Then, several coin-sized microfluidic devices with different geometries were developed. Using Computational fluid dynamics (CFD), we modeled the hydrodynamics inside the microchannels and selected the most promising options. SU-8 structures were fabricated based on the selected designs and used to produce polydimethylsiloxane-based microchips by soft lithography. As a result, an integrated approach combining microfluidics and PNA-FISH for the rapid identification of S. cerevisiae was achieved. To improve fluid flow inside microchannels and the PNA-FISH labeling, oxygen plasma treatment was applied to the microfluidic devices and a new methodology to introduce the cell suspension and solutions into the microchannels was devised. A strong PNA-FISH signal was observed in cells trapped inside the microchannels, proving that the proposed methodology works as intended. The microfluidic designs and PNA-FISH procedure described in here should be easily adaptable for detection of other microorganisms of similar size.

FISH-in-CHIPS: A Microfluidic Platform for Molecular Typing of Cancer Cells.

Microfluidics offer powerful tools for the control, manipulation, and analysis of cells, in particular for the assessment of cell malignancy or the study of cell subpopulations. However, implementing complex biological protocols on chip remains a challenge. Sample preparation is often performed off chip using multiple manually performed steps, and protocols usually include different dehydration and drying steps that are not always compatible with a microfluidic format.Here, we report the implementation of a Fluorescence in situ Hybridization (FISH) protocol for the molecular typing of cancer cells in a simple and low-cost device. The geometry of the chip allows integrating the sample preparation steps to efficiently assess the genomic content of individual cells using a minute amount of sample. The FISH protocol can be fully automated, thus enabling its use in routine clinical practice.

Binding induced colocalization activated hybridization chain reaction on the surface of magnetic nanobead for sensitive detection of adenosine.

Herein, a sensitive and enzyme-free assay for adenosine detection has been developed on the basis of binding induced colocalization activated hybridization chain reaction (HCR) strategy on the surface of magnetic nanobead. First, the recognition probe was fabricated and divided into two parts: the Apt-1 that composed a part of adenosine aptamer and toehold domain, and the Apt-2 that consisted of another part of adenosine aptamer and branch migration domain. The Apt-1 was immobilized on a streptavidin-magnetic nanobead (streptavidin-MNBs) that played the roles of enrichment and separation. Then the recognition event of adenosine could bring the two parts of aptamer together and induce the colocalization of toehold domain and branch migration domain, which could serve as an integrated initiator to trigger the HCR, producing a long nicked double-stranded polymer. Finally, the intercalating dye SYBR Green I was inserted into the polymer, generating an enhanced fluorescence signal. In this strategy, the initiator was divided into two parts and could be suppressed effectively in the absence of adenosine. Utilizing the separated function, the spontaneous hybridization of H1 and H2 could be avoided, and a low background could be acquired. Moreover, through the double amplification of HCR and multimolecules binding of SYBR Green I, highly sensitive and enzyme-free detection were achieved. The detection limit for adenosine detection was 2.0×10(-7)mol/L, which was comparable or superior to the previous aptasensors. Importantly, adenosine analysis in human urines has been performed, and this strategy could significantly distinguish the adenosine content in normal human urines and cancer patient urines, suggesting that this proposed assay will become a reliable and sensitive adenosine detection method in early clinical diagnosis and medical research.

DNA-based hybridization chain reaction and biotin-streptavidin signal amplification for sensitive detection of Escherichia coli O157:H7 through ELISA.

This study reported on a novel sandwich enzyme linked immunosorbent assay (ELISA) for the sensitive determination of Escherichia coli O157:H7 (E. coli O157:H7) by using DNA-based hybridization chain reaction (HCR) and biotin-streptavidin signal amplification. The anti-E. coli O157:H7 polyclonal antibody (pAb) was immobilized in the ELISA wells. The anti-E. coli O157:H7 monoclonal antibody (mAb) and initiator strand (DNA1) were labeled on gold nanoparticle (AuNP) to form a mAb-AuNP-DNA1 complex. In the presence of the target E. coli O157:H7, the sandwiched immunocomplex, which is pAb-E. coli O157:H7-mAb-AuNP-DNA1, could be formed. Two types of biotinylated hairpin were subsequently added in the ELISA well. A nicked double-stranded DNA (dsDNA) that contained abundant biotins was formed after HCR. Detection was performed after adding horseradish peroxidase-streptavidin and substrate/chromogen solution. Under optimal conditions, E. coli O157:H7 could be detected in the range of 5×10(2) CFU/mL to 1×10(7) CFU/mL; the limit of detection was 1.08×10(2) CFU/mL in pure culture. The LOD of the novel ELISA was 185 times lower than that of traditional ELISA. The proposed method is considerably specific and can be applied in the detection of whole milk samples inoculated with E. coli O157:H7. The coefficient of variation of in pure culture and in whole milk was 0.99-5.88% and 0.76-5.38%, respectively. This method offers a promising application in the detection of low concentrations of food-borne pathogens.