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Acquisition and Preservation of Tissue for Microarray Analysis
Published in Brian Leyland-Jones, Pharmacogenetics of Breast Cancer, 2020
Recently, focus has shifted away from whole-tumor microarray analysis to analysis of individual cell types within a tumor. A tumor is composed of a heterogeneous group of cells, malignant cells, premalignant cells, normal parenchymal cells, and supportive cells. This cellular heterogeneity must be taken into account when applying array-based techniques (25). Numerous successful expression-profiling studies have been performed using whole-tumor macromolecular extraction (26–28). These analyses thus incorporate a variety of different cell types. Laser capture microdissection allows one to isolate histologically distinct cell types and perform subsequent analysis. The technique employs an infrared laser in conjunction with light microscopy. Prepared histologic slides are covered with a transfer film. When cells of interest are identified, the laser is activated over the cells. This process causes the cells to attach to the transfer film. The remainder of the sample remains, and the isolated cells can undergo extraction procedures. Notably, this procedure can result in lower yields of cells requiring amplification of nucleic acids for use in microarray experiments (29,30). Laser capture microdissection has the potential to not only improve our understanding of cellular deregulation but also to help determine how other cells within the tumor contribute to the overall tumor phenotype. Immune cells, vascular structures, and normal parenchymal cells within a tumor may contribute significantly to the growth and metastatic potential. Expression analysis of these individual cell types may help elucidate their roles.
Mechanisms of Fibril Formation and Cellular Response
Published in Martha Skinner, John L. Berk, Lawreen H. Connors, David C. Seldin, XIth International Symposium on Amyloidosis, 2007
Martha Skinner, John L. Berk, Lawreen H. Connors, David C. Seldin
Human amyloidogenic LC can be expressed in vivo by injecting stably transfected plasmacytoma cells into mice. These LC enter the circulation and induce functional and biochemical changes in the heart. No congophilic amyloid fibrils were observed in this 4-6 week time frame, however, amorphous deposits and casts were deposited in the kidneys and LC and albumin were present in the urine. Injection of amyloidogenic LC-secreting plasmacytomas in mice is a useful model for testing the acute effects of multiple LC in vivo. We have generated three transgenic lines using the CMV early intermediate promoter which express the A6 LC in many tissues and at lower levels in the serum. Lambda-staining, congophilic fibrillar amyloid was present in the lumens of the gastric glands, demonstrating that AL amyloid can form in mice, but suggesting that the increased local concentration in the gastric glands and the acidic environment of the stomach may promote amyloid formation in this in vivo model. These deposits are being extracted by laser capture microdissection to analyze the composition of the proteins in the deposits in more detail, and the tissue responses to them. Additional transgenic models that express exclusively in plasma cells are planned, which may more faithfully replicate the human disease. Such models will be useful for testing peptide based immunotherapy.
Molecular Diagnostics of Pulmonary Neoplasms
Published in Philip T. Cagle, Timothy C. Allen, Mary Beth Beasley, Diagnostic Pulmonary Pathology, 2008
Translational research focused on potential diagnostic and particularly prognostic markers of lung carcinoma using techniques of molecular biology at different levels of resolution from the whole chromosome down to the specific nucleotide sequence, has resulted in a large number of studies that have significantly improved our understanding of pulmonary carcinogenesis. However, only a few DNA or RNA based diagnostic tests have been implemented in clinical practice. RNA based tests usually require fresh or frozen tissue, while DNA based tests can be successfully performed on formalin-fixed paraffin-embedded tissue (FFPE). Fixatives that have a low pH (e.g., picric acid containing Bouin’s fixative or decalcifying solutions) or that contain heavy metals (e.g., B5 with mercury) should be avoided when molecular testing is considered as they may interfere with testing (1). The majority of molecular tests are very sensitive and can detect abnormalities from a very small amount of tissue. Therefore, the first requirement for most molecular tests is to obtain a relatively pure cell population by tissue microdissection, which is an excellent method that leads to more accurate test results. Microdissection can be performed in a variety of ways, all of which have different advantages and disadvantages that have been reviewed elsewhere (1–6). Basically, these methods range from simple and inexpensive manual methods to laser-capture microdissection (LCM) methods that require expensive and complex equipment (7,8). Microdissection of target tissue, either frozen or paraffin embedded, is followed by DNA or RNA extraction. In molecular anatomic pathology laboratories, genetic material is often analyzed at the nucleic acid level with polymerase chain reaction (PCR). Other methods commonly used include in situ hybridization (ISH) or fluorescence in situ hybridization (FISH). This chapter provides a short general overview of major areas of molecular testing in lung cancer, which are employed by clinical diagnostic laboratories.
Mass Spectrometry Imaging of Fibroblasts: Promise and Challenge
Published in Expert Review of Proteomics, 2021
Peggi M. Angel, Denys Rujchanarong, Sarah Pippin, Laura Spruill, Richard Drake
A schematic of approaches that can be used toward imaging the single cell fibroblast niche is shown in Figure 3. In a general imaging approach, the entire tissue is scanned by systematically stepping through x, y coordinates. This is currently the general approach to tissue imaging MS that is done and molecular signatures that can be compared within one tissue section based on histological features, e.g. tumor, tumor margins, and normal adjacent. Understanding the comparative pathology involves working with a pathologist and combining the imaging data with pathology stains such as hematoxylin and eosin (H&E). Here, a pathologist marks the pathology either on the same tissue section that will be used for imaging MS [91,187] or on a serial section. This strategy allows relative quantitative analysis of patient-specific tissue features that may also be combined in large patient cohorts. The higher speed, higher spatial resolution MS instruments allow investigation of gradient patterns from different cellular regions. In contrast to laser capture microdissection where regionalized cells are removed, gradient patterns from different cellular regions can be added based on user selection of a cell types and compared at any point once the data is collected. In this strategy, one should consider that acquiring high spatial resolution data over an entire data sets requires significantly more computational resources to analyze, particularly when studies involve large clinical cohorts.
A large-scale histological investigation gives insight into the structure of ischemic stroke thrombi
Published in Platelets, 2021
Natalie J. Jooss, Natalie S. Poulter
Whilst this is a well-executed study, the conclusions are based upon observations and further experimental work is required. For example, laser capture microdissection could be utilized to not only visualize areas of interest but also enable incorporation of other experimental measures such as proteomics, transcriptomics, or genomics to investigate regions/cells [33]. This type of approach would complement existing data obtained from mass spectrometry on stroke thrombi where Munoz et al. [34]. identified 339 proteins commonly detected in the four patients investigated [34]. Further, as there is evidence that the composition of the thrombus plays an important role in the efficacy of thrombolytic treatment, it would be of interest to know what the underlying mechanism is for the formation of the two thrombi subpopulations (RBC- or platelet-rich). For example, different shear rates may influence the composition of the thrombus [35] and this could be something to further interrogate. Also, changes in the vessel wall, which can be picked up by noninvasive imaging techniques such as CT or MRI, have been correlated to an increased amount of RBC in the thrombus [36].
Protein biomarkers for subtyping breast cancer and implications for future research
Published in Expert Review of Proteomics, 2018
Claudius Mueller, Amanda Haymond, Justin B. Davis, Alexa Williams, Virginia Espina
Normal breast or tumor epithelial cells were procured by laser capture microdissection to reduce interference from stromal and immune cells. Two hundred and ninety eight differentially expressed proteins were applied to PSEA to render functional protein maps of normal and luminal breast epithelium [114]. Based on PSEA, tumors exhibited decreased levels of cytoskeletal proteins, collagens, fibrinogen, laminins, hemopexin, 14-3-3σ, lumican, TGF-β, and serpin peptidase inhibitor. Enrichment of proteins related to transcription-binding factor motifs, including p53, SMAD, NF-κB, were also found. Using spectral index abundance for the tumor cohort, elevated levels of fibronectin and mitochondrial isoleucyl-tRNA synthetase were discovered [114]. Exploiting the wealth of IHC information in the Human Protein Atlas (HPA) [140,141], Cha el al. visually quantified IHC breast images in the HPA to verify expression levels and subcellular location for 25 of their differentially expressed proteins. Seventy-five percent concordance was noted with the spectral index abundance. The lack of complete concordance was surmised to be due to the limited dynamic range of IHC immunoperoxidase staining intensity or lack of breast cancer subtype annotation in version 5.0 of the HPA [114].