Rapid tissue processing using a temperature-controlled collection device to preserve tumor biomarkers.

Precision tissue diagnostics rely on high quality input specimens so that assay results are not affected by artifact, but advances in collection and processing of tissue specimens have lagged behind innovations in diagnostic assay development. Therefore, we have designed and evaluated a novel surgical tissue collection device that maintains and monitors sample temperature and motion throughout transport so that the major preanalytical variable of tissue temperature can be controlled and measured. This device, in combination with an improved cold-hot tissue fixation protocol affords optimal biomarker preservation in less overall time, thereby simultaneously improving diagnostic quality and turnaround time. We collected 50 primary and metastatic liver tumors using a novel transport device. Tissue was fixed using a rapid cold-hot fixation protocol and immunohistochemical assays were used to assess the performance of the device, in comparison to control tissue preserved using standard clinical fixation protocol. Two pathologists evaluated the IHC studies in a blinded fashion to determine the immunophenotype of each tumor. The observed IHC staining intensities and the clinical impressions of the immunophenotypes did not differ between tissue collected with the novel device and control tissue, while improvements in processing time were achieved. The novel cold transport device and rapid fixation protocol can be successfully and safely combined and used to monitor specimen conditions, thus preserving the diagnostic utility of specimens and improving the overall turn-around time of the diagnostic process.

Q&A: How can advances in tissue clearing and optogenetics contribute to our understanding of normal and diseased biology?

Mammalian organs comprise a variety of cells that interact with each other and have distinct biological roles. Access to evaluate and perturb intact biological systems at the cellular and molecular levels is essential to fully understand their functioning in normal and diseased conditions, yet technical limitations have constrained most research to small pieces of tissue. Tissue clearing and optogenetics can help overcome this hurdle: tissue clearing affords optical interrogation of whole organs at the molecular level, and optogenetics enables the scalable control and measurement of cellular activity with light. In this Q&A, we delineate recent advances and practical challenges associated with these two techniques when applied body-wide.

[A DEVICE FOR THE VACUUM TREATMENT OF HISTOLOGICAL MATERIAL].

A device to carry out all types of vacuum infiltration in a wide temperature range. The device is easy to use and allows you to simultaneously conduct the infiltration of a large number of tissue samples. The main element of the device is a temperature-controlled cuvette, the temperature of which is set in the range of 23-65 °C. The design of the cell allows pumping air from the cell to the 50-100 mm Hg. In practice, histological methods are used to remove the vacuum infiltration of air from the tissue fixation (plant tissue), to accelerate the penetration of fasteners into the tissue. To carry out such a procedure, syringes, water pump and other hand-held devicesare used. Only vacuum infiltration allows a fill lyophilized tissue in paraffin. Efficiency of the described device is checked during the experiments at various fixing plant tissue and nervous tissue filling lyophilized in paraffin.

The museum maze in oral pathology demystified-part I.

Museum technologies provide a wide array of choice of museums to those who wish to exploit technology to attract, excite and ensure an unrivalled visitor experience, as well as capture and sustain share of mind and heart. Museum being a combination of both art and science requires skilled workmanship, meticulous planning and execution to exhibit a specimen to its optimal elegance due to its relatively smaller size and fragile nature. A well established oral pathology museum is rarely seen due to negligence of oral specimens, dearth of knowledge in this field and also available data on it. An insight on oral pathology museum, including its establishment, importance and advanced technologies to make it more simple and accessible are discussed in two parts. Part I emphasizes on basics in oral pathology museum, whereas part II highlights the specialized techniques and recent advances in museum technology. Our effort is to present this article as hands on experience for the pathologists, student population and the technicians.

High-quality lung fixation by controlled closed loop perfusion for stereological analysis in a large animal model.

BACKGROUND: Stereology is an essential method for quantitative analysis of lung structure. Adequate fixation is a prerequisite for stereological analysis to avoid bias in pulmonary tissue, dimensions and structural details. We present a technique for in situ fixation of large animal lungs for stereological analysis, based on closed loop perfusion fixation. MATERIALS AND METHODS: Twenty anesthetized ventilated pigs (30 ± 3 kg) underwent cannulation of the pulmonary artery and ligation of the right hilus. Following circulatory arrest a continuous positive pressure of 12 mbar was applied to the airways and lung perfusion started with the fixative solution (1.5% paraformaldehyde; 1.5% glutaraldehyde in 0.15 M HEPES). In five animals, a single-pass perfusion technique was performed, in 15 subsequent animals, the closed-loop technique was applied. Afterwards, lungs were removed, externally postfixed in the recycled fixative solution, and stored at 4 °C. Fifteen lung specimens underwent stereological analysis with volume estimation and subsequent systematic uniform random sampling for light and electron microscopic analysis. RESULTS: Singlepass perfusion did not result in satisfactory fixation. Left lung closed loop perfusion rate was 0.5-0.7 L/min with total median [min-max] perfusion time of 15 min (11-19). Perfusion pressure was 15 mm Hg (9-33). Subsequent lung analysis revealed well-preserved cell and tissue ultrastructure. CONCLUSION: The closed loop perfusion technique represents a valuable and reproducible fixation method in large animal models. Pressure controlled fixation perfusion results in high-quality preservation of in situ parenchymal architecture of lungs with or without injury, which is ideally suited for quantitative assessment of lung structure by stereology.

Preservation and Processing of Intestinal Tissue for the Assessment of Histopathology.

The intestine is often examined histologically in connection with allergies and in search for pathological changes. To be able to examine the intestine histologically with a microscope, it must be sampled and processed correctly. For microscopic analysis, the samples have to be cut into thin sections, stained, and mounted on slides. Since it is not possible to cut fresh samples without damaging them, they must first be fixed. The most common method, which is described herein, is the fixation in formalin with subsequent embedding in paraffin and staining of the slides with hematoxylin and eosin (H&E). Hematoxylin solutions (in this case Mayer's hemalum solution) stain the acidic components of the cell, i.e., cell nuclei, blue. The staining with eosin gives a pink staining of cytoplasm. This chapter describes the method of processing intestinal tissue for paraffin-embedding, sectioning, and staining with H&E. Tissue processing can be done in tissue processing machines or manually. We describe the manual processing that is often used for smaller batches of samples.

Preparing Cell Smears for Staining.

Increasing use is being made of cell smears for cell-staining studies. Suspension cells can be attached to slides by drying, and cell smears can also be prepared from biopsy samples, such as needle aspirates, tissue scrapings, or freshly dissected tissues. In these procedures, a thin layer of cells is deposited on a dry slide by physical methods. The most important factor in obtaining good staining patterns is that the smear be only a single cell thick. Tissue smears do not preserve tissue architecture, but are useful for identifying pathological changes and infectious organisms in tissue samples. Cell smears are easily prepared and can be fixed readily by any of the methods used for attached cells.

Effect of immediate cold formalin fixation on phosphoprotein IHC tumor biomarker signal in liver tumors using a cold transport device.

Phosphoproteins are the key indicators of signaling network pathway activation. Many disease treatment therapies are designed to inhibit these pathways and effective diagnostics are required to evaluate the efficacy of these treatments. Phosphoprotein IHC have been impractical for diagnostics due to inconsistent results occurring from technical limitations. We have designed and tested a novel cold transport device and rapid cold plus warm formalin fixation protocol using phosphoproteins IHC. We collected 50 liver tumors that were split into two experimental conditions: 2 + 2 rapid fixation (2 hours cold then 2 hour warm formalin) or 4 hour room-temperature formalin. We analyzed primary hepatocellular carcinoma (n = 10) and metastatic gastrointestinal tumors (n = 28) for phosphoprotein IHC markers pAKT, pERK, pSRC, pSTAT3, and pSMAD2 and compared them to slides obtained from the clinical blocks. Expression of pERK and pSRC, present in the metastatic colorectal carcinoma, were better preserved with the rapid processing protocol while pSTAT3 expression was detected in hepatocellular carcinoma. Differences in pSMAD2 expression were difficult to detect due to the ubiquitous nature of protein expression. There were only 3 cases expressing pAKT and all exhibited a dramatic loss of signal for the standard clinical workflow. The rapid cold preservation shows improvement in phosphoprotein preservation.

Optimizing the reaction temperature to facilitate an efficient osmium maceration procedure.

The osmium maceration method is a powerful technique for observing the three-dimensional ultrastructure of cellular organelles by scanning electron microscopy. In the conventional osmium maceration method, tissues are immersed in a diluted osmium tetroxide solution for several days at 20°C to remove soluble cytosolic proteins from the freeze-cracked surface of cells, and the optimal duration of this process is dependent on the cell type. To improve the efficiency of the osmium maceration procedure, we have examined systematically the relationship between the reaction temperature and time of the osmium maceration procedure. Treatment at temperatures higher than 20°C drastically shortened the time required to remove cytosolic proteins from the freeze-cracked surface of specimens with optimal durations for the osmium maceration of hepatocytes at 30, 40, 50 and 60°C being 30, 15, 5 and 1 h, respectively. Considering the stability and reproducibility of the macerated specimens, we concluded that the most appropriate temperature was 30 to 40°C. This rapid osmium maceration procedure was used successfully to observe the 3D ultrastructure of Purkinje cells in the cerebellum and proximal convoluted tubule cells in the kidney. This simple and reproducible rapid osmium maceration protocol should find wide appeal for the 3D analysis of cellular organelles in various cell types.

State of the art in antigen retrieval for immunohistochemistry.

The masking effects of antigens by chemical fixation, processing, embedding media interactions, represent a serious problem for immunohistochemical purposes. Fortunately, different approaches in antigen retrieval exist. These techniques are relatively recent and continuously expanding. This review focuses on the present state of the art in antigen retrieval methods for immunohistochemistry in light and electron microscopy. Moreover, a brief discussion on the chemical aspects of fixation, mechanism of retrieval, as well as its efficacy, is given.

Immunostaining of Skeletal Tissues.

Immunohistochemistry (IHC) is a routinely used technique in clinical diagnosis of pathological conditions and in basic and translational research. It combines anatomical, immunological, and biochemical methods and relies on the specific binding of an antibody to an antigen. Using the technique with mineralized tissues is more challenging than with soft tissues. Demineralizing the samples allows for embedding in paraffin wax, and also facilitates cryosectioning. This chapter describes methods for IHC on formaldehyde-fixed, demineralized, paraffin-embedded, or frozen sections to detect antigens in skeletal tissues.

Double Immunohistochemistry and Digital Image Analysis.

Immunohistochemistry (IHC) is a commonly used technique for protein detection in tissue sections. The method requires high-affinity antibodies that are specific for the target proteins of interest. More advanced IHC techniques have been developed to meet the need for simultaneous detection of more than one target protein in the same tissue section. This chapter provides general guidelines for double IHC staining of formalin-fixed, paraffin-embedded tissue sections. Chromogenic substrates are chosen based on their excellent contrast and compatibility with the subsequent digital image analysis.

Single-Cell Mass Cytometry of Archived Human Epithelial Tissue for Decoding Cancer Signaling Pathways.

The emerging phenomenon of cellular heterogeneity in tissue requires single-cell resolution studies. A specific challenge for suspension-based single-cell analysis is the preservation of intact cell states when single cells are isolated from tissue contexts, in order to enable downstream analyses to extract accurate, native information. We have developed DISSECT (Disaggregation for Intracellular Signaling in Single Epithelial Cells from Tissue) coupled to mass cytometry (CyTOF: Cytometry by Time-of-Flight), an experimental approach for profiling intact signaling states of single cells from epithelial tissue specimens. We have previously applied DISSECT-CyTOF to fresh mouse intestinal samples and to Formalin-Fixed, Paraffin-Embedded (FFPE) human colorectal cancer specimens. Here, we present detailed protocols for each of these procedures, as well as a new method for applying DISSECT to cryopreserved tissue slices. We present example data for using DISSECT on a cryopreserved specimen of the human colon to profile its immune and epithelial composition. These techniques can be used for high-resolution studies for monitoring disease-related alternations in different cellular compartments using specimens stored in cryopreserved or FFPE tissue banks.

Multispectral Fluorescence Imaging Allows for Distinctive Topographic Assessment and Subclassification of Tumor-Infiltrating and Surrounding Immune Cells.

Histomorphology has significantly changed over the last decades due to technological achievements in immunohistochemistry (IHC) for the visualization of specific proteins and in molecular pathology, particularly in the field of in situ hybridization of small oligonucleotides and amplification of DNA and RNA amplicons. With an increased availability of suitable methods, the demands regarding the observer of histomorphological slides were the supply of complex quantitative data as well as more information about protein expression and cell-cell interactions in tissue sections. Advances in fluorescence-based multiplexed IHC techniques, such as multispectral imaging (MSI), allow the quantification of multiple proteins at the same tissue section. In histopathology, it is a well-known technique for over a decade yet harboring serious problems concerning quantitative preciseness and tissue autofluorescence of multicolor staining when using formalin-fixed, paraffin-embedded (FFPE) tissue specimen. In recent years, milestones in tissue preparation, fluorescent dyes, hardware imaging, and software analysis were achieved including automated tissue segmentation (e.g., tumor vs. stroma) as well as in cellular and subcellular multiparameter analysis.This chapter covers the role that MSI plays in anatomic pathology for the analysis of FFPE tissue sections, discusses the technical aspects of MSI, and provides a review of its application in the characterization of immune cell infiltrates and beyond regarding its prognostic and predictive value and its use for guidance of clinical decisions for immunotherapeutic strategies.

The biochemistry and immunohistochemistry of versican.

Versican is a chondroitin sulfate proteoglycan found in the extracellular matrix that is important for changes in cell phenotype associated with development and disease. Versican has been shown to be involved in cardiovascular disorders, as well as lung disease and fibrosis, inflammatory bowel disease, cancer, and several other diseases that have an inflammatory component. Versican was first identified as a fibroblast proteoglycan and forms large multimolecular complexes with hyaluronan and other components of the provisional matrix during wound healing and inflammation. The biology of versican has been well studied. Versican plays a major role in embryogenesis, particularly heart formation, where versican deletion proves lethal. The ability to purify versican to characterize and to use in experimental systems is vital to defining its role in development and disease. Protein expression systems have proven challenging to obtain milligram quantities of full-length versican. Here, we describe proteoglycan biochemical purification techniques that have been developed by others, but which we have adapted to use with our source tissues and cells. We also include methods for immunohistochemical localization and quantitation of versican in tissue sections.

Predictive Marker: HER2 in Esophageal Adenocarcinoma.

HER2 positivity is based on the fundamental principle of amplification of the human epidermal growth factor receptor 2 (HER2) gene resulting in overexpression of the protein products . Arising from that a "HER2-positive cancer" is one that shows HER2 gene amplification and resultant protein expression as demonstrated by in situ hybridization and immunohistochemistry, respectively. Testing of the HER2 status is crucial to ensure selection of the correct patient who may benefit from target therapy for esophageal adenocarcinoma. Accurate testing is dependent on several pre-analytical and analytical factors including sample selection, laboratory techniques, and accurate interpretation of HER2 test results.

Comprehensive periorbital rejuvenation with resorbable endotine implants for trans-lid brow and midface elevation.

Periorbital rejuvenation can enhance a patient's appearance, with changes of only a few millimeters making a significant impact. Many patients undergoing blepharoplasty often have mild brow or midfacial changes for which they are unwilling to undergo additional concurrent forehead or midface procedures, however, because these procedures may be associated with an unacceptable postoperative recovery and may not provide adequate tissue fixation. This article describes limited incision procedures capable of providing excellent elevation and support to the brow and midface that can be incorporated easily into a comprehensive periorbital rejuvenation treatment plan using semi-permanent subperiosteal fixation devices.

Cancer Detection in Human Tissue Samples Using a Fiber-Tip pH Probe.

Intraoperative detection of tumorous tissue is an important unresolved issue for cancer surgery. Difficulty in differentiating between tissue types commonly results in the requirement for additional surgeries to excise unremoved cancer tissue or alternatively in the removal of excess amounts of healthy tissue. Although pathologic methods exist to determine tissue type during surgery, these methods can compromise postoperative pathology, have a lag of minutes to hours before the surgeon receives the results of the tissue analysis, and are restricted to excised tissue. In this work, we report the development of an optical fiber probe that could potentially find use as an aid for margin detection during surgery. A fluorophore-doped polymer coating is deposited on the tip of an optical fiber, which can then be used to record the pH by monitoring the emission spectra from this dye. By measuring the tissue pH and comparing with the values from regular tissue, the tissue type can be determined quickly and accurately. The use of a novel lift-and-measure technique allows for these measurements to be performed without influence from the inherent autofluorescence that commonly affects fluorescence-based measurements on biological samples. The probe developed here shows strong potential for use during surgery, as the probe design can be readily adapted to a low-cost portable configuration, which could find use in the operating theater. Use of this probe in surgery either on excised or in vivo tissue has the potential to improve success rates for complete removal of cancers. Cancer Res; 76(23); 6795-801. ©2016 AACR.