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Pathobiology : Journal of... 2023Histopathology has historically been the critical technique for the diagnosis and treatment of human disease. Today, genomics, transcriptomics, and proteomics from... (Review)
Review
Histopathology has historically been the critical technique for the diagnosis and treatment of human disease. Today, genomics, transcriptomics, and proteomics from specific cells, rather than bulk tissue, have become key to understanding underlying disease mechanisms and rendering useful diagnostic information. Extraction of desired analytes, i.e., nucleic acids or proteins, from easily accessible formalin-fixed paraffin-embedded tissues allows for clinically relevant activities, such as sequencing biomarker mutations or typing amyloidogenic proteins. Genetic profiling has become routine for cancers as varied as non-small cell lung cancer and prostatic carcinoma. The five main tissue dissection techniques that have been developed thus far include: bulk scraping, manual macrodissection, manual microdissection, laser-capture microdissection, and expression microdissection. In this review, we discuss the importance of tissue dissection in clinical practice and research, the basic methods, applications, as well as some advantages and disadvantages for each modality.
Topics: Humans; Carcinoma, Non-Small-Cell Lung; Prognosis; Lung Neoplasms; Dissection; Microdissection; Tissue Fixation; Paraffin Embedding
PubMed: 35952628
DOI: 10.1159/000525979 -
The Journal of Clinical Endocrinology... Mar 2021Spermatogenesis is strictly regulated by the intratesticular hormonal milieu, in which testosterone (T) and estradiol (E2) play pivotal roles. However, the optimal...
CONTEXT
Spermatogenesis is strictly regulated by the intratesticular hormonal milieu, in which testosterone (T) and estradiol (E2) play pivotal roles. However, the optimal expression of aromatase and intratesticular T (ITT) and E2 (ITE2) levels are unknown.
OBJECTIVE
To investigate ITT/ITE2 and aromatase expression in men with nonobstructive azoospermia (NOA) and to elucidate the roles of aromatase in spermatogenesis, as determined based on sperm retrieval by microdissection testicular sperm extraction (micro-TESE).
DESIGN AND SETTING
A retrospective study at a reproductive center using serum, testicular specimens, and intratesticular fluid.
PATIENTS
Seventy-six men with NOA, including 4 men who received 3 months of anastrozole administration prior to micro-TESE, and 18 men with obstructive azoospermia.
INTERVENTIONS
Testicular aromatase expression was evaluated using immunohistochemistry and quantitative reverse transcription-polymerase chain reaction (RT-PCR). Intratesticular T and ITE2 levels were determined using liquid chromatography-tandem mass spectrometry.
RESULTS
Aromatase was mainly located in Leydig cells, and the levels of its transcript and protein expression levels were increased in men with NOA. No correlation was observed between serum T/E2 and ITT/ITE2 levels, whereas significant associations were observed between decreased ITT and increased ITE2, aromatase expression, and sperm retrieval. Treatment with anastrozole increased the ITT/ITE2 ratio and decreased aromatase expression.
CONCLUSIONS
A close association between the expression of aromatase in Leydig cells and ITT/ITE2 was shown. Leydig cell aromatase is a factor that is independently correlated with spermatogenesis, and aromatase inhibitors may open a therapeutic window by increasing ITT/ITE2 in selected patients.
Topics: Adult; Anastrozole; Aromatase; Azoospermia; Estradiol; Humans; Male; Microdissection; Retrospective Studies; Sperm Retrieval; Spermatogenesis; Testis; Testosterone
PubMed: 33236081
DOI: 10.1210/clinem/dgaa860 -
Journal of Visualized Experiments : JoVE Apr 2020Gliomas are primary brain tumors characterized by their invasiveness and heterogeneity. Specific histological patterns such as pseudopalisades, microvascular...
Gliomas are primary brain tumors characterized by their invasiveness and heterogeneity. Specific histological patterns such as pseudopalisades, microvascular proliferation, mesenchymal transformation and necrosis characterize the histological heterogeneity of high-grade gliomas. Our laboratory has demonstrated that the presence of high densities of mesenchymal cells, named oncostreams, correlate with tumor malignancy. We have developed a unique approach to understand the mechanisms that underlie glioma's growth and invasion. Here, we describe a comprehensive protocol that utilizes laser capture microdissection (LMD) and RNA sequencing to analyze differential mRNA expression of intra-tumoral heterogeneous multicellular structures (i.e., mesenchymal areas or areas of tumor invasion). This method maintains good tissue histology and RNA integrity. Perfusion, freezing, embedding, sectioning, and staining were optimized to preserve morphology and obtain high-quality laser microdissection samples. The results indicate that perfusion of glioma bearing mice using 30% sucrose provides good morphology and RNA quality. In addition, staining tumor sections with 4% Cresyl violet and 0.5% eosin results in good nuclear and cellular staining, while preserving RNA integrity. The method described is sensitive and highly reproducible and it can be utilized to study tumor morphology in various tumor models. In summary, we describe a complete method to perform LMD that preserves morphology and RNA quality for sequencing to study the molecular features of heterogeneous multicellular structures within solid tumors.
Topics: Animals; Brain Neoplasms; Glioma; Humans; Laser Capture Microdissection; Mice; Neoplasm Invasiveness; Sequence Analysis, RNA; Staining and Labeling
PubMed: 32338655
DOI: 10.3791/60939 -
Molecular Neurodegeneration Aug 2021Alzheimer's disease (AD) is pathologically defined by the presence of fibrillar amyloid β (Aβ) peptide in extracellular senile plaques and tau filaments in... (Review)
Review
Alzheimer's disease (AD) is pathologically defined by the presence of fibrillar amyloid β (Aβ) peptide in extracellular senile plaques and tau filaments in intracellular neurofibrillary tangles. Extensive research has focused on understanding the assembly mechanisms and neurotoxic effects of Aβ during the last decades but still we only have a brief understanding of the disease associated biological processes. This review highlights the many other constituents that, beside Aβ, are accumulated in the plaques, with the focus on extracellular proteins. All living organisms rely on a delicate network of protein functionality. Deposition of significant amounts of certain proteins in insoluble inclusions will unquestionably lead to disturbances in the network, which may contribute to AD and copathology. This paper provide a comprehensive overview of extracellular proteins that have been shown to interact with Aβ and a discussion of their potential roles in AD pathology. Methods that can expand the knowledge about how the proteins are incorporated in plaques are described. Top-down methods to analyze post-mortem tissue and bottom-up approaches with the potential to provide molecular insights on the organization of plaque-like particles are compared. Finally, a network analysis of Aβ-interacting partners with enriched functional and structural key words is presented.
Topics: Alzheimer Disease; Amyloid beta-Peptides; Apolipoproteins; Autopsy; Blood Coagulation Factors; Carrier Proteins; Cell Adhesion Molecules; Complement System Proteins; Extracellular Fluid; Extracellular Matrix Proteins; Humans; Immunoglobulins; Laser Capture Microdissection; Lipid Metabolism; Microscopy, Confocal; Nerve Tissue Proteins; Plaque, Amyloid; Protein Interaction Maps; Protein Isoforms; Proteoglycans; Tandem Mass Spectrometry
PubMed: 34454574
DOI: 10.1186/s13024-021-00465-0 -
Analytica Chimica Acta Apr 2022Developments in quantitative proteomics and data-independent acquisition (DIA) methodology is enabling quantification of proteins in biological samples. Currently, there...
Developments in quantitative proteomics and data-independent acquisition (DIA) methodology is enabling quantification of proteins in biological samples. Currently, there are a few reports on DIA mass spectrometry (MS) approaches for proteome analysis of formalin-fixed paraffin-embedded (FFPE) tissues. Therefore, to facilitate detection and quantification of immune- and glioblastoma (GBM)-relevant proteins from FFPE patient materials, we established a simple and precise DIA-MS workflow. We first evaluated different lysis buffers for their efficiency in protein extractions from FFPE GBM tissues. Our results showed that more than 1700 proteins were detected and over 1400 proteins were quantified from GBM FFPE tissue microdissections. GBM-relevant proteins (e.g., GFAP, FN1, VIM, and MBP) were quantified with high precision (median coefficient of variation <12%). In addition, immune-related proteins (e.g., ILF2, MIF, and CD38) were consistently detected and quantified. The strategy holds great potential for routinizing protein quantification in FFPE tissue samples.
Topics: Formaldehyde; Glioblastoma; Humans; Paraffin Embedding; Proteome; Tandem Mass Spectrometry; Tissue Fixation
PubMed: 35397901
DOI: 10.1016/j.aca.2022.339695 -
Medecine Sciences : M/S Nov 2019One of the most fascinating aspects of the use of a laser beam in the field of biology has emerged with the development of devices able to perform fine dissections of... (Review)
Review
One of the most fascinating aspects of the use of a laser beam in the field of biology has emerged with the development of devices able to perform fine dissections of biological tissues. Laser microdissection can collect phenotypically identical cells from tissue regions laid on a microscope slide in order to make differential molecular analyses on these microdissected cells. Laser microdissection can be used many areas including oncology to specify molecular mechanisms that enable to adapt a treatment related to diagnosis and research in biology, but also forensic science for tissue selection, neurology for post-mortem studies on patients with Alzheimer's disease, for clonality studies from cell cultures and cytogenetics to decipher chromosomal rearrangements. This technology represents the missing link between clinical observations and the intrinsic physiological mechanisms of biological tissues and its major applications will be addressed here.
Topics: Histological Techniques; Humans; Laser Capture Microdissection; Molecular Diagnostic Techniques
PubMed: 31845879
DOI: 10.1051/medsci/2019166 -
Journal of Clinical Pathology Feb 2022Here we explore the presence of mediator complex subunit 12 () exon 2 and telomerase reverse transcriptase () promoter hotspot mutations in complex fibroadenomas (CFAs)...
AIMS
Here we explore the presence of mediator complex subunit 12 () exon 2 and telomerase reverse transcriptase () promoter hotspot mutations in complex fibroadenomas (CFAs) of the breast.
METHODS
The stromal components from 18 CFAs were subjected to Sanger sequencing of exon 2 and the promoter hotspot loci. The epithelial and stromal components of two mutated CFAs were subjected to laser capture microdissection, and Sanger sequencing of exon 2, promoter and exons 9 and 20, separately.
RESULTS
exon 2 mutations were identified in the stroma of 17% of CFAs. The analyses of epithelial and stromal components, microdissected separately, revealed that mutations were restricted to the stroma. No promoter or mutations in exons 9 and 20 were detected in analysed CFAs.
CONCLUSIONS
Like conventional fibroadenomas, exon 2 mutations appear to be restricted to the stromal component of CFAs, supporting the notion that CFAs are stromal neoplasms.
Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Biopsy; Breast Neoplasms; Class I Phosphatidylinositol 3-Kinases; DNA Mutational Analysis; Exons; Female; Fibroadenoma; Genetic Predisposition to Disease; Humans; Mediator Complex; Middle Aged; Mutation; Phenotype; Stromal Cells; Telomerase
PubMed: 33376197
DOI: 10.1136/jclinpath-2020-207062 -
Journal of Visualized Experiments : JoVE Nov 2021Mouse model systems are unmatched for the analysis of disease processes because of their genetic manipulability and the low cost of experimental treatments. However,...
Mouse model systems are unmatched for the analysis of disease processes because of their genetic manipulability and the low cost of experimental treatments. However, because of their small body size, some structures, such as the oviduct with a diameter of 200-400 μm, have proven to be relatively difficult to study except by immunohistochemistry. Recently, immunohistochemical studies have uncovered more complex differences in oviduct segments than were previously recognized; thus, the oviduct is divided into four functional segments with different ratios of seven distinct epithelial cell types. The different embryological origins and ratios of the epithelial cell types likely make the four functional regions differentially susceptible to disease. For example, precursor lesions to serous intraepithelial carcinomas arise from the infundibulum in mouse models and from the corresponding fimbrial region in the human fallopian tube. The protocol described here details a method for microdissection to subdivide the oviduct in such a way to yield a sufficient amount and purity of RNA necessary for downstream analysis such as reverse transcription-quantitative PCR (RT-qPCR) and RNA sequencing (RNAseq). Also described is a mostly non-enzymatic tissue dissociation method appropriate for flow cytometry or single cell RNAseq analysis of fully differentiated oviductal cells. The methods described will facilitate further research utilizing the murine oviduct in the field of reproduction, fertility, cancer, and immunology.
Topics: Animals; Cell Separation; Fallopian Tubes; Female; Humans; Immunohistochemistry; Mice; Microdissection; Oviducts
PubMed: 34806701
DOI: 10.3791/63168 -
Acta Neuropathologica May 2022Sudden unexplained death in childhood (SUDC) is death of a child over 1 year of age that is unexplained after review of clinical history, circumstances of death, and...
Sudden unexplained death in childhood (SUDC) is death of a child over 1 year of age that is unexplained after review of clinical history, circumstances of death, and complete autopsy with ancillary testing. Multiple etiologies may cause SUDC. SUDC and sudden unexpected death in epilepsy (SUDEP) share clinical and pathological features, suggesting some similarities in mechanism of death and possible abnormalities in hippocampus and cortex. To identify molecular signaling pathways, we performed label-free quantitative mass spectrometry on microdissected frontal cortex, hippocampal dentate gyrus (DG), and cornu ammonis (CA1-3) in SUDC (n = 19) and pediatric control cases (n = 19) with an explained cause of death. At a 5% false discovery rate (FDR), we found differential expression of 660 proteins in frontal cortex, 170 in DG, and 57 in CA1-3. Pathway analysis of altered proteins identified top signaling pathways associated with activated oxidative phosphorylation (p = 6.3 × 10, z = 4.08) and inhibited EIF2 signaling (p = 2.0 × 10, z = - 2.56) in frontal cortex, and activated acute phase response in DG (p = 8.5 × 10, z = 2.65) and CA1-3 (p = 4.7 × 10, z = 2.00). Weighted gene correlation network analysis (WGCNA) of clinical history indicated that SUDC-positive post-mortem virology (n = 4/17) had the most significant module in each brain region, with the top most significant associated with decreased mRNA metabolic processes (p = 2.8 × 10) in frontal cortex. Additional modules were associated with clinical history, including fever within 24 h of death (top: increased mitochondrial fission in DG, p = 1.8 × 10) and febrile seizure history (top: decreased small molecule metabolic processes in frontal cortex, p = 8.8 × 10) in all brain regions, neuropathological hippocampal findings in the DG (top: decreased focal adhesion, p = 1.9 × 10). Overall, cortical and hippocampal protein changes were present in SUDC cases and some correlated with clinical features. Our studies support that proteomic studies of SUDC cohorts can advance our understanding of the pathogenesis of these tragedies and may inform the development of preventive strategies.
Topics: Autopsy; Child; Death, Sudden; Hippocampus; Humans; Proteomics; Seizures, Febrile
PubMed: 35333953
DOI: 10.1007/s00401-022-02414-7 -
Annals of Diagnostic Pathology Aug 2020p16 hypermethylation in Barrett's carcinogenesis has been evaluated in studies which did not take into account sample heterogeneity and yielded qualitative... (Comparative Study)
Comparative Study
UNLABELLED
p16 hypermethylation in Barrett's carcinogenesis has been evaluated in studies which did not take into account sample heterogeneity and yielded qualitative (methylated/unmethylated) instead of accurate quantitative (percentage of CpG methylation) data. We aimed to measure the degree of p16 methylation in pure samples representing all the steps of Barrett's tumorogenesis and to evaluate the influence of sample heterogeneity in methylation analysis.
METHODS
77 paraffin-embedded human esophageal samples were analyzed. Histological grading was established by two pathologists in: negative for dysplasia, indefinite for dysplasia, low-grade dysplasia, high-grade dysplasia and adenocarcinoma. Areas of interest were selected by laser-capture microdissection. p16 methylation was quantified by pyrosequencing. An adjacent section of the whole sample was also analyzed to compare methylation data.
RESULTS
After microdissection, we obtained 15 samples of squamous epithelium, 36 non-dysplastic Barrett's esophagus, 3 indefinite for dysplasia, 24 low-grade dysplasia, 4 high-grade dysplasia and 12 adenocarcinoma. Squamous epithelium showed the lowest methylation rates: 6% (IQR 5-11) vs. 11%(7-39.50) in negative/indefinite for dysplasia, p<0.01; 10.60%(6-24) in low-grade dysplasia, p<0.05; and 44.50%(9-66.75) in high-grade dysplasia/adenocarcinoma, p<0.01. This latter group also exhibited higher methylation rates than Barrett's epithelium with and without low-grade dysplasia (p<0.05). p16 methylation rates of microdissected and non-microdissected samples did not correlate unless the considered histological alteration comprised >71% of the sample.
CONCLUSIONS
p16 methylation is an early event in Barrett's carcinogenesis which increases with the severity of histological alteration. p16 methylation rates are profoundly influenced by sample heterogeneity, so selection of samples is crucial in order to detect differences.
Topics: Adenocarcinoma; Barrett Esophagus; Carcinogenesis; Cyclin-Dependent Kinase Inhibitor p16; DNA Methylation; Disease Progression; Esophageal Neoplasms; Evaluation Studies as Topic; Female; High-Throughput Nucleotide Sequencing; Humans; Laser Capture Microdissection; Male; Middle Aged; Neoplasm Staging; Severity of Illness Index
PubMed: 32570024
DOI: 10.1016/j.anndiagpath.2020.151554