Enteroblastic adenocarcinoma of the ampulla of Vater / Adenocarcinoma enteroblástico de la ampolla de Vater

Adenocarcinoma with enteroblastic differentiation is a rare histologic subtype of adenocarcinoma of the gastrointestinal tract that shows unique histologic and immunohistochemical features that resemble fetal intestinal epithelium. This histological subtype has been widely described in the stomach, where it most frequently appears, but, in other locations, it is misdiagnosed because of the poor experience in routine diagnostic setting. Here we present a case of an 87-year-old male with an adenocarcinoma of the ampulla of Vater with enteroblastic differentiation with a literature review of the cases described of this subtype in this location to date. The anatomical peculiarity of the ampulla, joined with the infrequent nature of this histological subtype, makes this case of great interest to aid to better characterize the biological behavior of these tumors. (AU)

El adenocarcinoma con diferenciación enteroblástica es un subtipo histológico poco frecuente de adenocarcinoma gastrointestinal que muestra características histológicas e inmunohistoquímicas únicas que se asemejan al epitelio intestinal fetal. Este subtipo histológico ha sido ampliamente descrito en el estómago, donde aparece con mayor frecuencia, pero en otras localizaciones es mal diagnosticado debido a la poca experiencia en el diagnóstico de rutina. Presentamos un caso de un varón de 87 años con adenocarcinoma de ampolla de Vater con diferenciación enteroblástica, junto a una revisión bibliográfica de los casos descritos de este subtipo en esta localización hasta el momento. La peculiaridad anatómica de la ampolla, sumada al carácter poco frecuente de este subtipo histológico, dotan a este caso de gran interés para ayudar a caracterizar mejor el comportamiento biológico de estos tumores. (AU)

Super-resolution proximity labeling with enhanced direct identification of biotinylation sites.

Promiscuous labeling enzymes, such as APEX2 or TurboID, are commonly used in in situ biotinylation studies of subcellular proteomes or protein-protein interactions. Although the conventional approach of enriching biotinylated proteins is widely implemented, in-depth identification of specific biotinylation sites remains challenging, and current approaches are technically demanding with low yields. A novel method to systematically identify specific biotinylation sites for LC-MS analysis followed by proximity labeling showed excellent performance compared with that of related approaches in terms of identification depth with high enrichment power. The systematic identification of biotinylation sites enabled a simpler and more efficient experimental design to identify subcellular localized proteins within membranous organelles. Applying this method to the processing body (PB), a non-membranous organelle, successfully allowed unbiased identification of PB core proteins, including novel candidates. We anticipate that our newly developed method will replace the conventional method for identifying biotinylated proteins labeled by promiscuous labeling enzymes.

Coupling imaging mass cytometry with Alcian blue histochemical staining for a single-slide approach.

Imaging mass cytometry (IMC) is a metal mass spectrometry-based method allowing highly multiplex immunophenotyping of cells within tissue samples. However, some limitations of IMC are its 1-µm resolution and its time and costs of analysis limiting respectively the detailed histopathological analysis of IMC-produced images and its application to small selected tissue regions of interest (ROI) of one to few square millimeters. Coupling on a single-tissue section, IMC and histopathological analyses could permit a better selection of the ROI for IMC analysis as well as co-analysis of immunophenotyping and histopathological data until the single-cell level. The development of this method is the aim of the present study in which we point to the feasibility of applying the IMC process to tissue sections previously Alcian blue-stained and digitalized before IMC tissue destructive analyses. This method could help to improve the process of IMC in terms of ROI selection, time of analysis, and the confrontation between histopathological and immunophenotypic data of cells.

Probing cell proliferation: Considerations for dye selection.

Broadly speaking, cell tracking dyes are fluorescent compounds that bind stably to components on or within the cells so the fate of the labeled cells can be followed. Their staining should be bright and homogeneous without affecting cell function. For purposes of monitoring cell proliferation, each time a cell divides the intensity of cell tracking dye should diminish equally between daughter cells. These dyes can be grouped into two different classes. Protein reactive dyes label cells by reacting covalently but non-selectively with intracellular proteins. Carboxyfluorescein diacetate succinimidyl ester (CFSE) is the prototypic general protein label. Membrane intercalating dyes label cells by partitioning non-selectively and non-covalently within the plasma membrane. The PKH membrane dyes are examples of lipophilic compounds whose chemistry allows for their retention within biological membranes without affecting cellular growth, viability, or proliferation when used properly. Here we provide considerations based for labeling cell lines and peripheral blood mononuclear cells using both classes of dyes. Examples from optimization experiments are presented along with critical aspects of the staining procedures to help mitigate common risks. Of note, we present data where a logarithmically growing cell line is labeled with both a protein dye and a membrane tracking dye to compare dye loss rates over 6days. We found that dual stained cells paralleled dye loss of the corresponding single stained cells. The decrease in fluorescence intensity by protein reactive dyes, however, was more rapid than that with the membrane reactive dyes, indicating the presence of additional division-independent dye loss.

Immunophenotyping challenging tissue types using high-dimensional full spectrum flow cytometry.

Technological advancements in fluorescence flow cytometry and an ever-expanding understanding of the complexity of the immune system, have led to the development of large flow cytometry panels, reaching up to 40 markers at the single-cell level. Full spectrum flow cytometry, that measures the full emission range of all the fluorophores present in the panel instead of only the emission peaks is now routinely used in many laboratories internationally, and the demand for this technology is rapidly increasing. With the capacity to use larger and more complex staining panels, optimized protocols are required for the best panel design, panel validation and high-dimensional data analysis outcomes. In addition, for ex vivo experiments, tissue preparation methods for single-cell analysis should also be optimized to ensure that samples are of the highest quality and are truly representative of tissues in situ. Here we provide optimized step-by-step protocols for full spectrum flow cytometry panel design, tissue digestion and panel optimization to facilitate the analysis of challenging tissue types.

Chondrosarcoma evaluation using hematein-based x-ray staining and high-resolution 3D micro-CT: a feasibility study.

BACKGROUND: Chondrosarcomas are rare malignant bone tumors diagnosed by analyzing radiological images and histology of tissue biopsies and evaluating features such as matrix calcification, cortical destruction, trabecular penetration, and tumor cell entrapment. METHODS: We retrospectively analyzed 16 cartilaginous tumor tissue samples from three patients (51-, 54-, and 70-year-old) diagnosed with a dedifferentiated chondrosarcoma at the femur, a moderately differentiated chondrosarcoma in the pelvis, and a predominantly moderately differentiated chondrosarcoma at the scapula, respectively. We combined a hematein-based x-ray staining with high-resolution three-dimensional (3D) microscopic x-ray computed tomography (micro-CT) for nondestructive 3D tumor assessment and tumor margin evaluation. RESULTS: We detected trabecular entrapment on 3D micro-CT images and followed bone destruction throughout the volume. In addition to staining cell nuclei, hematein-based staining also improved the visualization of the tumor matrix, allowing for the distinction between the tumor and the bone marrow cavity. The hematein-based staining did not interfere with further conventional histology. There was a 5.97 ± 7.17% difference between the relative tumor area measured using micro-CT and histopathology (p = 0.806) (Pearson correlation coefficient r = 0.92, p = 0.009). Signal intensity in the tumor matrix (4.85 ± 2.94) was significantly higher in the stained samples compared to the unstained counterparts (1.92 ± 0.11, p = 0.002). CONCLUSIONS: Using nondestructive 3D micro-CT, the simultaneous visualization of radiological and histopathological features is feasible. RELEVANCE STATEMENT: 3D micro-CT data supports modern radiological and histopathological investigations of human bone tumor specimens. It has the potential for being an integrative part of clinical preoperative diagnostics. KEY POINTS: • Matrix calcifications are a relevant diagnostic feature of bone tumors. • Micro-CT detects all clinically diagnostic relevant features of x-ray-stained chondrosarcoma. • Micro-CT has the potential to be an integrative part of clinical diagnostics.

Double Chromogen-based Immunohistochemical Staining: An Efficient Approach for Utilizing Long-term Formalin-fixed Tissue in Biobanks.

The New South Wales Brain Tissue Resource Centre is a human brain bank that provides top-quality brain tissue for cutting-edge neuroscience research spanning various conditions from alcohol use disorder to neurodegenerative diseases. However, the conventional practice of preserving brain tissue in formalin poses challenges for immunofluorescent staining primarily due to the formalin's tendency, over time, to create cross-links between antigens, which can obscure epitopes of interest. In addition, researchers can encounter issues such as spectral bleeding, limitations in using multiple colors, autofluorescence, and cross-reactivity when working with long-term formalin-fixed brain tissue. The purpose of the study was to test chromogen-based double immunolabeling to negate the issues with immunofluorescent staining. Colocalization of antigens was explored using chromogens 3-amino-9-ethylcarbazole (AEC) and 3,3,-diaminobenzidine in a sequential staining procedure where the AEC signal was eliminated by alcohol treatment. Combinations of 2 or 3 primary antibodies from the same or different species were trialed successfully with this protocol. The colocalization of antigens was also demonstrated with pseudocoloring that mimicked immunofluorescence staining. This staining technique increases the utility of archival formalin-fixed tissue samples.

A fast, easy, cost-free method to remove excess dye or drug from small extracellular vesicle solution.

Tracking small extracellular vesicles (sEVs), such as exosomes, requires staining them with dyes that penetrate their lipid bilayer, a process that leaves excess dye that needs to be mopped up to achieve high specificity. Current methods to remove superfluous dye have limitations, among them that they are time-intensive, carry the risk of losing sample and can require specialized equipment and materials. Here we present a fast, easy-to-use, and cost-free protocol for cleaning excess dye from stained sEV samples by adding their parental cells to the mixture to absorb the extra dye much like sponges do. Since sEVs are considered a next-generation drug delivery system, we further show the success of our approach at removing excess chemotherapeutic drug, daunorubicin, from the sEV solution.

Array tomography of in vivo labeled synaptic receptors.

Array tomography (AT) allows one to localize sub-cellular components within the structural context of cells in 3D through the imaging of serial sections. Using this technique, the z-resolution can be improved physically by cutting ultra-thin sections. Nevertheless, conventional immunofluorescence staining of those sections is time consuming and requires relatively large amounts of costly antibody solutions. Moreover, epitopes are only readily accessible at the section's surface, leaving the volume of the serial sections unlabeled. Localization of receptors at neuronal synapses in 3D in their native cellular ultrastructural context is important for understanding signaling processes. Here, we present in vivo labeling of receptors via fluorophore-coupled tags in combination with super-resolution AT. We present two workflows where we label receptors at the plasma membrane: first, in vivo labeling via microinjection with a setup consisting of readily available components and self-manufactured microscope table equipment and second, live receptor labeling by using a cell-permeable tag. To take advantage of a near-to-native preservation of tissues for subsequent scanning electron microscopy (SEM), we also apply high-pressure freezing and freeze substitution. The advantages and disadvantages of our workflows are discussed.

Microwave-assisted and methanol/acetic acid-free method for rapid staining of proteins in SDS-PAGE gels.

We describe a microwave-assisted, methanol and acetic acid-free, inexpensive method for rapid staining of SDS-PAGE proteins. Only citric acid, benzoic acid, and Coomassie brilliant blue G-250 (CBG) were used. Microwave irradiation reduced the detection duration, and proteins in a clear background were visualized within 30 min of destaining, after 2 min of fixing and 12 min of staining. By using this protocol, comparable band intensities were obtained to the conventional methanol/acetic acid method.

One-Dimensional Acrylamide Gel Electrophoresis for Analysis of Plant Samples.

Polyacrylamide gel electrophoresis (PAGE) is a widely used technique for separating proteins from complex plant samples. Prior to the analysis, proteins must be extracted from plant tissues, which are rather complex than other types of biological material. Different protocols have been applied depending on the protein source, such as seeds, pollen, leaves, roots, and flowers. Total protein amounts must also be determined before conducting gel electrophoresis. The most common methodologies include PAGE under native or denaturing conditions. Both procedures are used consequently for protein identification and characterization via mass spectrometry. Additionally, various staining procedures are available to visualize protein bands in the gel, facilitating the software-based digital evaluation of the gel through image acquisition.

Click Chemistry with Cell-Permeable Fluorophores Expands the Choice of Bioorthogonal Markers for Two-Color Live-Cell STED Nanoscopy.

STED nanoscopy allows for the direct observation of dynamic processes in living cells and tissues with diffraction-unlimited resolution. Although fluorescent proteins can be used for STED imaging, these labels are often outperformed in photostability by organic fluorescent dyes. This feature is especially crucial for time-lapse imaging. Unlike fluorescent proteins, organic fluorophores cannot be genetically fused to a target protein but require different labeling strategies. To achieve simultaneous imaging of more than one protein in the interior of the cell with organic fluorophores, bioorthogonal labeling techniques and cell-permeable dyes are needed. In addition, the fluorophores should preferentially emit in the red spectral range to reduce the potential phototoxic effects that can be induced by the STED light, which further restricts the choice of suitable markers. In this work, we selected five different cell-permeable organic dyes that fulfill all of the above requirements and applied them for SPIEDAC click labeling inside living cells. By combining click-chemistry-based protein labeling with other orthogonal and highly specific labeling methods, we demonstrate two-color STED imaging of different target structures in living specimens using different dye pairs. The excellent photostability of the dyes enables STED imaging for up to 60 frames, allowing the observation of dynamic processes in living cells over extended time periods at super-resolution.

Fluorogenic cell surface glycan labelling with fluorescence molecular rotor dyes and nucleic acid stains.

We show that covalent labelling of sialic acids on live cell surfaces or mucin increases the fluorescence of the fluorescence molecular rotors (FMRs) CCVJ, Cy3 and thioazole orange, enabling wash-free imaging of cell surfaces. Dual labelling with an FMR and an environmentally insensitive dye allows detection of changes that occur, for example, when cross-linking is altered.

Detection of pit and fissure sealant microleakage using quantitative light-induced fluorescence technology: an in vitro study.

This in vitro study aimed to evaluate the feasibility of quantitative light-induced fluorescence (QLF) technology for detecting the presence and severity of microleakage of pit and fissure sealants. The areas of interest (AOIs) were 160 pits and fissures of 40 extracted permanent teeth. Fluorescent images were acquired using a QLF device, and the maximum fluorescence loss ΔFmax of each AOI was analyzed. After staining and cross-sectioning of the teeth, histological dye penetration was scored on a scale of 0 to 3. The relationship between ΔFmax and microleakage depth was analyzed, and the areas under the curve (AUCs) were calculated. The │ΔFmax│ increased as microleakage depth increased. The ΔFmax values of microleakage areas showed a strong significant correlation with the histological scores of dye penetration (r = - 0.72, P = 0.001). AUC analysis showed a high diagnostic accuracy for microleakage depth (AUC = 0.83-0.91). The highest AUC of 0.91 was found when differentiating the outer half microleakage of the sealant (histological score 0 vs. 1-3). QLF technology is effective in assessing the presence and severity of microleakage, suggesting its potential for noninvasive detection and monitoring of sealant microleakage in clinical settings.

Multi-domain stain normalization for digital pathology: A cycle-consistent adversarial network for whole slide images.

The variation in histologic staining between different medical centers is one of the most profound challenges in the field of computer-aided diagnosis. The appearance disparity of pathological whole slide images causes algorithms to become less reliable, which in turn impedes the wide-spread applicability of downstream tasks like cancer diagnosis. Furthermore, different stainings lead to biases in the training which in case of domain shifts negatively affect the test performance. Therefore, in this paper we propose MultiStain-CycleGAN, a multi-domain approach to stain normalization based on CycleGAN. Our modifications to CycleGAN allow us to normalize images of different origins without retraining or using different models. We perform an extensive evaluation of our method using various metrics and compare it to commonly used methods that are multi-domain capable. First, we evaluate how well our method fools a domain classifier that tries to assign a medical center to an image. Then, we test our normalization on the tumor classification performance of a downstream classifier. Furthermore, we evaluate the image quality of the normalized images using the Structural similarity index and the ability to reduce the domain shift using the Fréchet inception distance. We show that our method proves to be multi-domain capable, provides a very high image quality among the compared methods, and can most reliably fool the domain classifier while keeping the tumor classifier performance high. By reducing the domain influence, biases in the data can be removed on the one hand and the origin of the whole slide image can be disguised on the other, thus enhancing patient data privacy.

Establishing Conditions for Blood Smear Drying and Staining on Sysmex SP-50 for Leukocyte Differential Count.

BACKGROUND: The purpose of this study was to determine the staining conditions and appropriate fan1 start time (FAN1ST) for Sysmex SP-50 to produce blood smears (BS) that reflect the true lymphocyte morphology of patient samples. METHODS: Using different start times of fan1, we obtained a set of 84 blood smear slides from 21 blood samples and measured 10,920 lymphocyte areas, which were then converted to compare lymphocyte sizes. We also performed a leukocyte differential count using Sysmex DI-60 on 202 blood smear slides prepared before and after the change in staining conditions and compared the results. RESULTS: The mean lymphocyte sizes at FAN1ST 0 second, 5 seconds, 10 seconds, and 30 seconds were 12.55 µm, 12.14 µm, 11.27 µm, and 10.50 µm, respectively. The mean differences in the preclassification of neutrophils, lymphocytes, monocytes, eosinophils, and basophils in DI-60, according to the SP-50 staining conditions, were 0.88, -1.58, -0.24, 0.37, and 0.07, respectively. CONCLUSIONS: Wright-Giemsa staining of blood smears prepared on the SP-50 showed that changing the pH of the concentrated phosphate buffer to 6.6 and adjusting the staining time did not affect the results of the leukocyte differential count. However, since fan1 was used to dry the blood smear on the SP-50 and the lymphocyte size gradually decreased as the start time was delayed, it was necessary to set a start time for fan1 that did not affect the lymphocyte size.

Effect of Meconium Staining Amniotic Fluid on Fetal Outcome.

Meconium-stained amniotic fluid is the passage of meconium by a fetus in utero during the antenatal period or in labour. It has for long been considered to be a bad predictor of fetal distress and meconium aspiration syndrome (MAS). The objective of this study was to find out the fetal outcome of MSAF and clear amniotic fluid. This cross- sectional comparative study was carried out in Upazilla Health Complex, Palash, Narshingdi from July 2016 to June 2017. A total of 100 pregnant women among them 50 women with MSAF and 50 women with clear liquor were studied to see the record of ANC, mode of delivery and fetal outcome by APGAR score. Study showed that among MSAF group 76.0% (n=38) had irregular ANC and 24.0% (n=12) had regular ANC whereas in clear liquor 86.0% (n=43) had regular ANC 14.0% had irregular ANC. Among MSAF (50 cases) thick meconium was in 20 cases (40.0%) and thin meconium was in 30 cases (60.0%). Regarding mode of delivery 52.0% (n=26) MSAF cases had instrumental delivery and Caesarean section compared to 24.0% (n=12) in clear liquor group. Regarding thick MSAF among 40.0% (n=20), (n=14) had low APGAR score and (n=6) had normal score at one minute and (n=9) low APGAR score and (n=11) normal score at five minutes. In clear liquor, among 100.0% (n=50), 20.0% (n=10) had low APGAR score and 80.0% (n=40) had normal score at one-minute and at five minutes 8.0% (n=4) had low APGAR score and 92.0% (n=46) had normal score. Among MSAF 26.0% (n=13) were admitted to SCBU compare to 12.0% (n=6) in clear liquor group. The mean SCBU stay was 3.1 days in MSAF whereas 1.2 days in clear liquor. Among MSAF babies 4.0% (n=2) had MAS compared to no MAS in clear liquor group. Regarding Survivalist 92.0% (n=46) were alive in MSAF whereas 100.0% all (n=50) were alive in clear liquor group.

The virtual staining method by quantitative phase imaging for label free lymphocytes based on self-supervised iteration cycle-consistent adversarial networks.

Quantitative phase imaging (QPI) provides 3D structural and morphological information for label free living cells. Unfortunately, this quantitative phase information cannot meet doctors' diagnostic requirements of the clinical "gold standard," which displays stained cells' pathological states based on 2D color features. To make QPI results satisfy the clinical "gold standard," the virtual staining method by QPI for label free lymphocytes based on self-supervised iteration Cycle-Consistent Adversarial Networks (CycleGANs) is proposed herein. The 3D phase information of QPI is, therefore, trained and transferred to a kind of 2D "virtual staining" image that is well in agreement with "gold standard" results. To solve the problem that unstained QPI and stained "gold standard" results cannot be obtained for the same label free living cell, the self-supervised iteration for the CycleGAN deep learning algorithm is designed to obtain a trained stained result as the ground truth for error evaluation. The structural similarity index of our virtual staining experimental results for 8756 lymphocytes is 0.86. Lymphocytes' area errors after converting to 2D virtual stained results from 3D phase information are less than 3.59%. The mean error of the nuclear to cytoplasmic ratio is 2.69%, and the color deviation from the "gold standard" is less than 6.67%.

An algorithm-based technique for counting mitochondria in cells using immunohistochemical staining of formalin-fixed and paraffin-embedded sections.

PURPOSE: Visualizing mitochondria in cancer cells from human pathological specimens may improve our understanding of cancer biology. However, using immunohistochemistry to evaluate mitochondria remains difficult because almost all cells contain mitochondria and the number of mitochondria per cell may have important effects on mitochondrial function. Herein, we established an objective system (Mito-score) for evaluating mitochondria using machine-based processing of hue, saturation, and value color spaces. METHODS: The Mito-score was defined as the number of COX4 (mitochondrial inner membrane) immunohistochemistry-positive pixels divided by the number of nuclei per cell. The system was validated using four lung cancer cell lines, normal tissues, and lung cancer tissues (199 cases). RESULTS: The Mito-score correlated with MitoTracker, a fluorescent dye used to selectively label and visualize mitochondria within cells under a microscope (R2 = 0.68) and with the number of mitochondria counted using electron microscopy (R2 = 0.79). Histologically, the Mito-score of small cell carcinoma (57.25) was significantly lower than that of adenocarcinoma (147.5, p < 0.0001), squamous cell carcinoma (120.6, p = 0.0004), and large cell neuroendocrine carcinoma (111.8, p = 0.002). CONCLUSION: The Mito-score method enables the analysis of the mitochondrial status of human formalin-fixed paraffin-embedded specimens and may provide insights into the metabolic status of cancer.

Prediction of cell cycle distribution after drug exposure by high content imaging analysis using low-toxic DNA staining dye.

Interference in cell cycle progression has been noted as one of the important properties of anticancer drugs. In this study, we developed the cell cycle prediction model using high-content imaging data of recipient cells after drug exposure and DNA-staining with a low-toxic DNA dye, SiR-DNA. For this purpose, we exploited HeLa and MCF7 cells introduced with a fluorescent ubiquitination-based cell cycle indicator (Fucci). Fucci-expressing cancer cells were subjected to high-content imaging analysis using OperettaCLS after 36-h exposure to anticancer drugs; the nuclei were segmented, and the morphological and intensity properties of each nucleus characterized by SiR-DNA staining were calculated using imaging analysis software, Harmony. For the use of training, we classified cells into each phase of the cell cycle using the Fucci system. Training data (n = 7500) and validation data (n = 2500) were randomly sampled and the binary classification prediction models for G1, early S, and S/G2/M phases of the cell cycle were developed using four supervised machine learning algorithms. We selected random forest as the model with the best performance through 10-fold cross-validation; the accuracy rate was approximately 75%-87%. Regarding feature importance, variables expected to be biologically related to the cell cycle, for example, signal intensity and nuclear size, were highly ranked, suggesting the validity of the model. These results showed that the cell cycle can be predicted in cancer cells by simply exploiting the current prediction model using fluorescent images of DNA-staining dye, and the model could be applied for the use of future ex vivo drug sensitivity diagnosis.