Semi-automated approaches for interrogating spatial heterogeneity of tissue samples.

Tissues are spatially orchestrated ecosystems composed of heterogeneous cell populations and non-cellular elements. Tissue components' interactions shape the biological processes that govern homeostasis and disease, thus comprehensive insights into tissues' composition are crucial for understanding their biology. Recently, advancements in the spatial biology field enabled the in-depth analyses of tissue architecture at single-cell resolution, while preserving the structural context.

Saw marks in bone: A preliminary empirical study to inform decision making and best practice.

Forensic toolmark examiners compare marks between those observed on an item/surface and those made by a reference implement, such as a particular tool or weapon, to provide an opinion of the likelihood of common origin. It is widely accepted that such comparison opinions need to be underpinned by empirical research, and this study aimed to add to the knowledge base relied upon when developing and comparing saw marks in bone, a substrate encountered in body dismemberment cases. Porcine bones were used as a human proxy; they were either fresh with residual soft tissue and bodily fluids present ('wet') to replicate dismembered bones shortly post-mortem, or processed to remove soft tissue and moisture content ('dry') to represent cases of dismemberment after an extended period of decomposition and exposure.

Fully automated sequential immunofluorescence (seqIF) for hyperplex spatial proteomics.

Tissues are complex environments where different cell types are in constant interaction with each other and with non-cellular components. Preserving the spatial context during proteomics analyses of tissue samples has become an important objective for different applications, one of the most important being the investigation of the tumor microenvironment. Here, we describe a multiplexed protein biomarker detection method on the COMET instrument, coined sequential ImmunoFluorescence (seqIF).

Acoustofluidic large-scale mixing for enhanced microfluidic immunostaining for tissue diagnostics.

The usage of microfluidics for automated and fast immunoassays has gained a lot of interest in the last decades. This integration comes with certain challenges, like the reconciliation of laminar flow patterns of micro-scale systems with diffusion-limited mass transport. Several methods have been investigated to enhance microfluidic mixing in microsystems, including acoustic-based fluidic streaming.

Efficient AC electrothermal flow (ACET) on-chip for enhanced immunoassays.

Biochemical reaction rates in microfluidic systems are known to be limited by the diffusional transport of reagents, leading often to lowered sensitivity and/or longer detection times in immunoassays. Several methods, including electrically powering electrodes to generate AC electrothermal flow (ACET) on-chip, have been adopted to enhance the mass transport of the reagents and improve microfluidic mixing. Here, we report a novel ACET electrode design concept for generating in-plane microfluidic mixing vortices that act over a large volume close to the reaction surface of interest.

Microfluidics-assisted multiplexed biomarker detection for in situ mapping of immune cells in tumor sections.

Because of the close interaction between tumors and the immune system, immunotherapies are nowadays considered as the most promising treatment against cancer. In order to define the diagnosis and the subsequent therapy, crucial information about the immune cells at the tumor site is needed. Indeed, different types or activation status of cells may be indicative for specific and personalized treatments.

Microfluidic-based immunohistochemistry for breast cancer diagnosis: a comparative clinical study.

Breast cancer is a highly heterogeneous disease. The efficacy of tailored therapeutic strategies relies on the precise detection of diagnostic biomarkers by immunohistochemistry (IHC). Therefore, considering the increasing incidence of breast cancer cases, a concomitantly time-efficient and accurate diagnosis is clinically highly relevant.

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).

Ultra-fast and automated immunohistofluorescent multistaining using a microfluidic tissue processor.

Multistaining of a tissue section targeting multiple markers allows to reveal complex interplays in a tumor environment. However, the resource-intensive and impractically long nature of iterative multiplexed immunostainings prohibits its practical implementation in daily routine, even when using work-flow automation systems. Here, we report a fully automated and ultra-fast multistaining using a microfluidic tissue processor (MTP) in as short as 20 minutes per marker, by immunofluorescent staining employing commercially available tyramide signal amplification polymer precipitation by horse-radish peroxidase (HRP) activation.

A microfluidic platform towards automated multiplexed in situ sequencing.

Advancements in multiplexed in situ RNA profiling techniques have given unprecedented insight into spatial organization of tissues by enabling single-molecule quantification and sub-micron localization of dozens to thousands of RNA species simultaneously in cells and entire tissue sections. However, the lack of automation of the associated complex experimental procedures represents a potential hurdle towards their routine use in laboratories. Here, we demonstrate an approach towards automated generation and sequencing of barcoded mRNA amplicons in situ, directly in fixed cells.

Microfluidics-based immunofluorescence for fast staining of ALK in lung adenocarcinoma.

Background: Anaplastic lymphoma kinase (ALK) is a key oncogenic driver in lung adenocarcinoma patients and its fusion proteins are routinely assessed. The microfluidic tissue processor (MTP) device is based on a chip-confined low-volume technology allowing for rapid immunohistochemistry/immunofluorescence (IHC/IF) stainings of formalin-fixed paraffin-embedded (FFPE) or frozen tissue samples.

Methods: A novel ALK IF protocol was developed for the MTP device using the primary mouse anti-human ALK antibody clone 5A4.

Microfluidics for rapid cytokeratin immunohistochemical staining in frozen sections.

Frozen sections (FS) of tumor samples represent a cornerstone of pathological intraoperative consultation and have an important role in the microscopic analysis of specimens during surgery. So far, immunohistochemical (IHC) stainings on FS have been demonstrated for a few markers using manual methods. Microfluidic technologies have proven to bring substantial improvement in many fields of diagnostics, though only a few microfluidic devices have been designed to improve the performance of IHC assays.

Continuous quantification of HER2 expression by microfluidic precision immunofluorescence estimates HER2 gene amplification in breast cancer.

Chromogenic immunohistochemistry (IHC) is omnipresent in cancer diagnosis, but has also been criticized for its technical limit in quantifying the level of protein expression on tissue sections, thus potentially masking clinically relevant data. Shifting from qualitative to quantitative, immunofluorescence (IF) has recently gained attention, yet the question of how precisely IF can quantify antigen expression remains unanswered, regarding in particular its technical limitations and applicability to multiple markers. Here we introduce microfluidic precision IF, which accurately quantifies the target expression level in a continuous scale based on microfluidic IF staining of standard tissue sections and low-complexity automated image analysis.

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.

Microparticles from apoptotic vascular smooth muscle cells induce endothelial dysfunction, a phenomenon prevented by beta3-integrin antagonists.

Fragile atherosclerotic plaques are rich in apoptotic smooth muscle cells (SMCs) and macrophages, generating microparticules (MPs) which accumulate locally and may be released in blood in case of mechanical or spontaneous plaque disruption. Besides being highly procoagulant, this material may interact with downstream endothelium. Using a model of mouse aorta vaso-reactivity, we have investigated the effects of apoptotic MPs prepared in vitro from Fas-ligand sensitive SMCs.

Population pharmacokinetics of pyrimethamine and sulfadoxine in children with congenital toxoplasmosis.

Aims: To develop a population pharmacokinetic model for pyrimethamine (PYR) and sulfadoxine (SDX) in children with congenital toxoplasmosis.

Methods: Children were treated with PYR (1.25 mg kg(-1)) and SDX (25 mg kg(-1)) (Fansidar) plus folinic acid (Lederfoline) 5 mg).

Shedding of active tissue factor by aortic smooth muscle cells (SMCs) undergoing apoptosis.

As apoptosis of neo-intimal SMCs is a feature of advanced atherosclerotic plaques, the procoagulant properties of SMCs of synthetic phenotype undergoing apoptosis were investigated. SMCs isolated from rat aorta obtained 10 days after balloon injury, previously found to up-regulate Tissue Factor (TF) and Tissue Factor Inhibitor (TFPI) and to release large amounts of TFPI (Ghrib et al. Thromb Haemost 2002;87:1043-50), were sensitive to the apoptosis induced by Fas-ligand.

The expression of tissue factor and tissue factor pathway inhibitor in aortic smooth muscle cells is up-regulated in synthetic compared to contractile phenotype.

Tissue factor (TF) and its specific inhibitor TF pathway inhibitor (TFPI) are produced by vascular smooth muscle cells (SMCs) in vitro and are increased in vivo in atherosclerotic compared to normal vessels. Besides local regulation of the hemostatic balance, this may be related to non-hemostatic TF/protease dependent functions such as SMC proliferation, adhesion and migration. The aim of the study was to compare the expression of both proteins between the contractile (normal adult) and synthetic (neo-intimal) SMC phenotypes.

Preconception seroconversion and maternal seronegativity at delivery do not rule out the risk of congenital toxoplasmosis.

We describe two unusual cases of congenital toxoplasmosis, one occurring after preconception maternal infection with cervical adenopathies and the other occurring after maternal infection at the very end of pregnancy with maternal seronegativity at delivery. These documented cases of congenital toxoplasmosis demonstrate the value of extending the serologic monitoring period during pregnancy, according to the individual clinical context.

Polymorphonuclear leukocytes modulate tissue factor production by mononuclear cells: role of reactive oxygen species.

To determine whether polymorphonuclear leukocytes (PMN) modulate the production of tissue factor (TF) by monocytes, PBMC were incubated with increasing concentrations of PMN. PMN did not express any procoagulant activity. After 20-h cocultures, PMN enhanced or inhibited the TF production of PBMC, and this effect depended on the PMN/PBMC ratio.

Detection of specific immunoglobulin E during maternal, fetal, and congenital toxoplasmosis.

Toxoplasma immunoglobulin E (IgE) antibodies in 664 serum samples were evaluated by using an immunocapture method with a suspension of tachyzoites prepared in the laboratory in order to evaluate its usefulness in the diagnosis of acute Toxoplasma gondii infection during pregnancy, congenital infection, and progressive toxoplasmosis. IgE antibodies were never detected in sera from seronegative women, from patients with chronic toxoplasma infection, or from infants without congenital toxoplasmosis. In contrast, they were detected in 86.

Further studies on the mechanism for the antithrombotic effects of naroparcil, an orally active thiozyloside compound.

The antithrombotic beta-D-xyloside, naroparcil, has previously been shown to induce a dose-related increase of circulating glycosaminoglycans (GAGs) together with an antithrombin activity (anti-IIa) via heparin cofactor II (HCII) in the rabbit. In order to go further in the mechanisms, the relationship between the antithrombotic activity, the HCII-mediated anti-IIa activity and the plasma GAG content was investigated. We showed that the in vitro specific activity on the inhibition of thrombin by HCII of the plasma GAG extract from naroparcil-treated rabbits was increased by a factor of 60 when compared to controls.

Prenatal diagnosis of congenital toxoplasmosis transmitted by an immunocompetent woman infected before conception. Reims Toxoplasmosis Group.

We report a rare case of congenital toxoplasmosis transmitted by an immunocompetent woman infected before conception. Active toxoplasmosis was suspected due to persistent lymphadenitis with specific IgM, IgA, IgE antibodies. Prenatal diagnosis based on amniocentesis and fetal blood sampling at 24 weeks' amenorrhoea was positive on amniotic fluid, and fetal infection was confirmed after termination.

Pyrimethamine-sulfadoxine treatment of congenital toxoplasmosis: follow-up of 78 cases between 1980 and 1997. Reims Toxoplasmosis Group.

Unlabelled: The purpose of this study was to determine the clinical and immunological outcome of 78 children with congenital toxoplasmosis treated with the pyrimethamine-sulfadoxine combination between 1980 and 1997.

Methods: Children were divided into 3 groups according to the initial duration of treatment (always including folinic acid, 5 mg/week by mouth), as follows: pyrimethamine (1.25 mg/kg every 15 d) + sulfadoxine (25 mg/kg every 15 d) for 12 months (Group 1, 47 children), or for 24 months, with or without prenatal therapy (respectively, Group 2, 19 children, and Group 3, 12 children).