[Microdissection of the chick embryo ovocell cultured in vitro].
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Animals↗
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Results for “Tissue microdissection”
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Phenotypic and genotypic characterization of glioblastoma multiforme with epithelial differentiation and adenoid formations.
Rare examples of high-grade gliomas show focal epithelial differentiation, which may require distinction from colliision tumors. These epithelial constituents are not well-characterized immunophenotypically and have rarely been subjected to genotyping. We describe a case of a 54-year-old female with a short history of hemiparesis who was found to have an absolute lymphocytosis and a heterogeneously enhancing frontotemporal tumor. Cytological and histological examination of brain biopsies confirmed the presence of a glioblastoma multiforme also containing CAM5.2/CK7/BerEP4/CEA/EMA-immunopositive and GFAP-immunonegative nests of epithelial cells with a high proliferative index and focal glandular differentiation. Hematological investigations confirmed a diagn of chronic lymphocytic leukemia (CLL) with no demonstrable CNS involvement. Genetic analysis using microsatellite markers and specimens obtained by laser capture microdissection of the CNS tumor and normal brain tissue, showed that both the glial and epithelial components of the brain tumor had identical losses of 2/2 informative markers on chromosome 17p13 and 5/5 informative markers on chromosome 10q22-26. The glial and epithelial components also shared an identical 2 base pair deletion in the TP53 gene at codon 209, exon 6, introducing a stop codon at codon 214. No losses at any of the above loci and no TP53 mutation were detected in the leukemic cells. These results suggest both components of the brain tumor, although differing in phenotype, share the same genetic lineage.
Brain Neoplasms↗
Nodular lymphocyte predominance Hodgkin's disease: a monoclonal or polyclonal B-cell disorder?
Nodular lymphocyte predominance Hodgkin's disease (NLPHD) is characterized by the presence of atypical putatively neoplastic cells (L & H cells) with a B-cell phenotype. A proportion of patients with NLPHD develop a simultaneous or subsequent large cell B lymphoma (LCL) that is thought to evolve directly from the L & H cells of NLPHD. However, the clonal nature of L & H cells remains controversial, and the relationship between NLPHD and complicating LCL has not been fully established. In an attempt to determine the clonality of L & H cells and to clarify the link between NLPHD and complicating LCL, we used polymerase chain reaction (PCR) to analyze 33 cases of NLPHD, including 15 cases with simultaneous or subsequent LCL, for clonal immunoglobulin (lg) heavy chain variable region (VH) gene rearrangements. PCR amplifications with consensus primers covering framework 2 or framework 3 to joining region were performed on paraffin-embedded tissue sections and, in 12 cases, on microdissection-enriched L & H cells. No clonal Ig rearrangements were detected. In eight of the 15 LCL, monoclonal IgVH regions were amplified, four of which were cloned and sequenced. Clone specific primers were designed based on the unique N region sequences. These allowed detection of LCL clones at a sensitivity up to 1,000 times greater than the consensus primers, as determined by dilution assays. However, no LCL clones were detected in the preceding NLPHD, including microdissection-enriched L & H cells. Our results suggest that populations of L & H cells do not carry monoclonal Ig rearrangements and provide no evidence for a clonal link between NLPHD and complicating LCL.
Antibodies, Monoclonal↗
Single sperm cell isolation by laser microdissection.
In 1996, Van Oorschot and Jones firstly reported in scientific correspondence that short tandem repeat (STR) profiles could be obtained from cells left on different objects. Since then, forensic scientists have focused their efforts in isolating single cells as it can be extremely helpful in solving case works where sexual violence was concerned. Laser microdissection is a micromanipulation procedure allowing to cut off precisely the cells of interest from tissue samples or smears by a laser beam fitted with an optical microscope. We have harvested single sperm cells by laser microdissection using a Leica AS LMD (Leica Microsystems, Germany); laser setting, pulse laser intensity and laser alignment as well as recovery of the specimen have been properly fitted to the samples we were dealing with. Different tissue preservation, fixation, histological staining (Papanicolau, Nuclear Fast Red-Picroindigocarmine) methods and number of harvested cells for each sample have been evaluated as well. Finally, the genotype of sperm cells has been determined by STR typing, evaluating the sensibility of this forensic technique according to instrumental and biological above-mentioned variables.
DNA↗
Gene expression patterns in rat dentate granule cells: comparison between fresh and fixed tissue.
RNA from brain tissue (in particular human brain) can often only be extracted from fixed material. As brain tissue is very heterogeneous with regard to cell type, it is important to obtain RNA from small samples of identified cells. The aim of this study was (a) to generate expression profiles from small yet homogeneous samples of fixed brain cells in rat and (b) to verify the reliability of these profiles by comparing them with expression profiles obtained from single fresh neurons of the same cell type. Samples (n=12) of 50 rat dentate granule cells were isolated, using Laser Microdissection and Pressure Catapulting, from paraformaldehyde fixed, paraffin embedded tissue or from frozen, ethanol fixed tissue. In addition, RNA was extracted under visual control from individual dentate granule cells (n=12) in hippocampal slices, after electrophysiological recording with patch clamp electrodes. Our data show that RNA was successfully extracted from ethanol fixed sections yielding expression profiles highly comparable to those from non-fixed, single granule cells. RNA extraction from paraformaldehyde fixed, paraffin embedded tissue was less reliable. The present approach validates expression profiling from small amounts of fixed neurons as a powerful tool to investigate molecular processes if fresh tissue is not available.
Animals↗
A new molecular biology approach in morphology: basic method and application of laser microdissection.
Our understanding of the role played by specific genes in various diseases is advanced as elucidation of the human genome progresses. One important step in this process is profiling gene expression so as to understand the roles of specific genes and the role of the translated protein. Profiling of the gene should also include tissue generation and an explanation of the mechanism of the disease. A useful technique in achieving this goal is the laser microdissection (LMD) method, which can draw genetic information from tissues of individual patients. We describe herein our technique using a special film which we developed and laser microdissection by the Palm Co. With this LMD method, RNA was extracted from carcinoma tissue and genetic analysis was carried out. LMD is equivalent to pathological diagnosis, and it is necessary to do dissection for accurate diagnosis. It seems to make clear gene pathological diagnosis possible in the future.
Gene Expression Profiling↗
Be more specific! Laser-assisted microdissection of plant cells.
Laser-assisted microdissection (LAM) is a powerful tool for isolating specific tissues, cell types and even organelles from sectioned biological specimen in a manner conducive to the extraction of RNA, DNA or protein. LAM, which is an established technique in many areas of biology, has now been successfully adapted for use with plant tissues. Here, we provide an overview of the processes involved in conducting a successful LAM study in plants and review recent developments that have made this technique even more desirable. We also discuss how the technology might be exploited to answer some pertinent questions in plant biology.
Lasers↗
[Progress in molecular medicine: "laser capture microdissection"].
BACKGROUND: With the unravelling of the human genome, we now face the challenge of defining the function and clinical relevance of single genes. To do this, we should be able to isolate normal and diseased cells from complex tissue structures to make them accessible to sensitive molecular analyses. Laser Capture Microdissection (LCM) was developed to meet this challenge. METHOD AND APPLICATION: LCM allows the precise dissection of cells with the help of a laser beam under direct visualization in the microscope, and the sterile transfer of these cells into a DNA or RNA isolation buffer. The technique is ideal for investigating cell-cell interactions, for performing mutation analyses, and for the production of high-quality cDNA libraries. Expression studies of known and unknown genes are currently employed successfully to define tissue- and simple cell-specific patterns which help elucidate the etiology and pathogenesis of colon, lung, breast, prostate, adrenal, ovary, and other organ tumors. The LCM system developed at the NIH is, therefore, an important part of the Cancer-Genome Anatomy Project (CGAP), which sequences and publishes the structures of genes that are expressed in human tumors. In combination with the modern cDNA arrays, it will thus be possible to analyze the expression of several thousands of genes in one step and to develop individual therapeutic strategies in the not too distant future. CONCLUSIONS: The LCM is a major advance in molecular medicine, enabling us to combine highly-sensitive gene analysis techniques with conventional histologic and morphologic methods. Applications range from research to diagnosis, and to monitoring disease progression.
Cell Separation↗
Proteomic characterization of postmortem amyloid plaques isolated by laser capture microdissection.
The presence of amyloid plaques in the brain is one of the pathological hallmarks of Alzheimer's disease (AD). We report here a comprehensive proteomic analysis of senile plaques from postmortem AD brain tissues. Senile plaques labeled with thioflavin-S were procured by laser capture microdissection, and their protein components were analyzed by liquid chromatography coupled with tandem mass spectrometry. We identified a total of 488 proteins co-isolated with the plaques, and we found multiple phosphorylation sites on the neurofilament intermediate chain, implicating the complexity and diversity of cellular processes involved in the plaque formation. More significantly, we identified 26 proteins enriched in the plaques of two AD cases by quantitative comparison with surrounding non-plaque tissues. The localization of several proteins in the plaques was further confirmed by the approach of immunohistochemistry. In addition to previously identified plaque constituents, we discovered novel association of dynein heavy chain with the plaques in human postmortem brain and in a double transgenic AD mouse model, suggesting that neuronal transport may play a role in neuritic degeneration. Overall, our results revealed for the first time the sub-proteome of amyloid plaques that is important for further studies on disease biomarker identification and molecular mechanisms of AD pathogenesis.
Aged↗
[Gene expression profiles in different tissues of human nasopharyngeal carcinoma].
OBJECTIVE: To screen the genes that may play an important role in the carcinogenesis of nasopharyngeal carcinoma (NPC). METHODS: Microdissection and cDNA genechip hybridization techniques were used to examine the differentially expressed genes in NPC tissue, the surrounding and adjacent tissues of NPC, and the nasopharyngeal inflammation tissue. The fluorescent signals on cDNA chip were scanned and the results of hybridization analyzed by image processing software. RESULTS: Many differentially expressed genes were identified between the three samples, including many different types of genes, such as those responsible for signal and protein transmission, oncogene and tumor suppression genes, immune-associated genes, apoptosis genes and DNA binding and transcription factor genes. CONCLUSION: The carcinogenesis of NPC involves many genes of a variety of types, suggesting its complex process.
Carcinoma, Squamous Cell↗
[The CHU-CRLCC-UNSA tumour/tissue bank of the Nice area].
The human tissue bank of Nice associates the CHU, the Antoine Lacassagne Center, and the University of Nice Sophia Antipolis. This bank has been labelled by the French Ministery of Health in 2003, and has been open in September 2004. Two main collections have been setting up: the tumoral and non tumoral thyroid pathology and the tumoral and non tumoral pleuro-pulmonary pathology collections. Other collections will be soon incorporated in this Biobank. The main criteria of quality connected with these collections are described. These different criteria of quality have been defined before the beginning of the collect, and thus, they have been initially strictly applied. The quality of the tissue stored at low temperature, the accuracy of the clinical and histopathological data associated with the tissue specimens, and the different procedures, should allow to obtain a labellisation by different appropriate organisms. This is crucial since the different research projects developped from human tissue collections, used expensive and accurate technologies (microarray, CGH array, proteomic, real time PCR, laser capture microdissection, etc.), and the results obtained need to be reproductible. An optimal quality both for the stored tissues and for the procedures applied in a human tissue bank is obliged since some of these tissues specimens are going to be used to try to define new prognosis criteria of tumours and new therapeutic strategies for the patients.
France↗
Transcriptional profiling of enriched populations of stem cells versus transient amplifying cells. A comparison of limbal and corneal epithelial basal cells.
The basal layer of limbal and central corneal epithelium is enriched in stem cells and transient amplifying cells, respectively. This physical separation of stem and transient amplifying cells makes the limbal/corneal epithelium an exceptionally suitable system for isolating basal cells enriched in these two proliferative populations. Prior attempts to isolate epithelial stem cells used methods such as proteolytic tissue dissociation and cell sorting that could potentially alter their gene expression profile. Using laser capture microdissection, we were able to isolate resting limbal and corneal basal cells from frozen sections with minimal tissue processing, thereby improving the yield and quality of RNA. Analyses of RNA isolated from 300 limbal and corneal basal cells from eight mice revealed a set of approximately 100 genes that are differentially expressed in limbal cells versus corneal epithelial basal cells. Semiquantitative reverse transcription-PCR confirmed the up-regulation of three limbal and three corneal genes. LacZ identification of epiregulin from epiregulin-null mice and immunohistochemical staining of wild type mice confirmed that epiregulin, one of the limbal epithelium-enriched genes, was associated with the limbal epithelial basal cells. Within the limbal and corneal basal cells, we detected previously unknown genes that were differentially expressed in these two regions that contribute further to our understanding of the unique heterogeneity of these two closely related basal cell populations. Our findings indicate that we can obtain accurate gene expression profiles of the stem cell-enriched limbal basal cell population in their "natural" quiescent state.
Animals↗
Telomerase activity analysis of esophageal carcinoma using microdissection-TRAP assay.
OBJECTIVES: To investigate telomerase activity in esophageal squamous cell carcinoma (SCC) and its preneoplasia lesions, and to study the relationships between telomerase activity and cancer differentiation, cancer invasiveness, and lymphatic metastasis. METHODS: Telomerase activity in esophageal SCC tissues, adjacent dysplasia tissues and normal epithelia from the surgical edge were assessed by microdissection-TRAP (telomeric repeat amplification protocol)-silver staining assay. RESULTS: Telomerase activity was detected in 37 (82.2%) of 45 esophageal tumors, 23 (79.3%) of 29 dysplasias, and 2 (5%) of 40 normal epithelia. There was a significant difference in activity between dysplasia and normal epithelium, as well as between tumor and normal epithelium. Twenty-six (92.9%) of 28 tumors with lymphatic metastasis had detectable telomerase activity compared to 11 (64.7%) of 17 non-lymphatic metastasis tumors. These relationships were statistically significant (P < 0.05), but the one between telomerase activity and tumor grade was not. CONCLUSION: Telomerase activity was high both in esophageal SCC and their preneoplasia lesions. The telomerase activity in SCC tissue was related to lymphatic metastasis, but not to cancer differentiation.
Adult↗
Immunohistoselective sequencing (IHSS) of p53 tumor suppressor gene in human oesophageal precancerous lesions.
Accumulation of p53 protein occurs in human oesophageal precancerous lesions and even in near-normal oesophageal epithelium. In some instances, p53 gene mutations have been detected. In many of the cases of p53 protein accumulation in early lesions, however, p53 mutations were not detected due to either the lack of mutation or the low abundance of cells with a mutation. In order to enrich p53 immunostain-positive cells for single strand conformation polymorphism (SSCP) analysis and DNA sequencing, an immunohisto-selective sequencing (IHSS) method was developed. Anti-p53 antibody-peroxidase stained oesophageal tissue sections were subjected to ultraviolet (UV) irradiation to damage the DNA in p53 immunostain-negative cells. The immunostain protected p53 immunostain-positive cells from the UV light and thus preserved the DNA in those cells for PCR amplification. Comparison of the SSCP results from sections with and without UV treatment showed that the IHSS method selectively enriched p53 immunostain-positive cells. With this method, we could analyse mutations in samples with as few as 30 p53 immunostain-positive cells per tissue section. Analysis was carried out on tissues with precancerous lesions from six surgically-resected oesophageal specimens and 13 oesophageal biopsies from symptom-free subjects. The results of mutation analysis for some of the samples were confirmed by microdissection to enrich the p53-positive cells. The mutations in tissues with precancerous lesions were compared with those in the corresponding squamous cell carcinomas. The IHSS method is shown to be a simple and effective way to analyse mutations in p53 immunostain-positive cells. IHSS may also be a general method for molecular analysis of biological specimens after immunohistochemical staining.
DNA↗
Detection of plasminogen activators in oral cancer by laser capture microdissection combined with zymography.
Plasminogen activation is believed to be critical to the progression of oral squamous cell carcinoma by facilitating matrix degradation during invasion and metastasis, and high levels of urokinase plasminogen activator (uPA) and plasminogen activator (PA) inhibitor-1 (PAI-1) in tumors predict poor disease outcome. We describe the development of a novel method for studying PA in oral cancer that combines the sensitivity and specificity of zymography with the spatial resolution of immunohistochemistry. Laser capture microdissection (LCM) was combined with plasminogen-casein zymography to analyze uPA, tissue PA (tPA), uPA-PAI-1 complexes, and tPA-PAI-1 complexes in 11 tumors and adjacent non-malignant epithelium from squamous cell carcinomas of the tongue, floor of mouth, larynx, and vocal cord. uPA was detectable in all tumor samples analyzed, uPA-PAI-1 complexes in three samples, and tPA in nine. PA was detectable in as little as 0.5 microg protein lysate from microdissected tumors. In all specimens, uPA expression was highly increased in tumor tissue compared to adjacent non-malignant tissue. In conclusion, LCM combined with zymography may be excellently suited for analyzing the prognostic significance and causal involvement of the plasminogen activation system in oral cancer.
Aged↗
The role of the pathologist as tissue refiner and data miner: the impact of functional genomics on the modern pathology laboratory and the critical roles of pathology informatics and bioinformatics.
This article provides an overview of how functional genomics is likely to impact on the pathology laboratory and highlights how informatics and tissue banking will greatly facilitate the molecular age of medicine. Important aspects of functional genomics in the post-genome era, including the roles of laser capture microdissection, DNA- and complementary DNA-based microarrays, proteomic methods, collaborative human tissue banking, tissue microarrays, and pathobioinformatics in the modern pathology laboratory are discussed. The role of mass spectroscopy in the analysis of RNA, DNA, and protein and its impact on the clinical laboratory, particularly in cost-effectiveness and time savings, are evaluated. This article explores how laboratory information systems (LISs) and the devices that feed them information may need to be modified to adapt to greater volumes of data for the new testing modalities that require understanding sophisticated fluorescence detection methods and image processing. Emerging genomic testing methods and their impact on pathology laboratory testing, especially in the area of molecular classification of neoplasms, are examined. The role of the tissue bank in the modern pathology laboratory as an archive of control normal tissues, as well as subsamples of the spectrum of progressive neoplastic states, is discussed in light of its critical importance to the molecular classification of cancer. Establishing a database that combines structured reports in pathology LISs and construction of tissue banking information systems will provide a rich resource for pathology departments. The article discusses a hypothetical resource, such as the Shared Tumor Expression Profiler, that would provide access to well-characterized tissue-based research resources for clinicians and researchers. Last, the article emphasizes how LISs can prepare for these changes, and how training pathologists in pathology informatics and bioinformatics (pathobioinformatics) is critical to ensure pathology's overall leadership role in the post-genome era.
Clinical Laboratory Techniques↗
Application of laser capture microdissection to phage display peptide library screening.
OBJECTIVE: When identifying important regulatory genes using methods such as phage display peptide library screening it is critical to select such peptides from cells and tissues in their native state. Here, we report a novel approach to screen tumors using phage display and laser capture microdissection (LCM). STUDY DESIGN: A phage peptide library was screened directly on fresh oral tumor tissue, such that specifically bound peptides were selected from fresh tumor cells in the native tissue state. Tissue processing conditions were modified to ensure the survival of the bacteriophage. RESULTS: Our results demonstrate that live phage-peptide conjugates can be recovered from laser capture microdissected cells in a form suitable for additional cycles of amplification. CONCLUSION: Thus, LCM will be a valuable adjunct to phage display studies.
Animals↗
Epstein-Barr virus (EBV) genome and expression in breast cancer tissue: effect of EBV infection of breast cancer cells on resistance to paclitaxel (Taxol).
The Epstein-Barr virus (EBV) has been detected in subsets of breast cancers. In order to elaborate on these observations, we quantified by real-time PCR (Q-PCR) the EBV genome in biopsy specimens of breast cancer tissue as well as in tumor cells isolated by microdissection. Our findings show that EBV genomes can be detected by Q-PCR in about half of tumor specimens, usually in low copy numbers. However, we also found that the viral load is highly variable from tumor to tumor. Moreover, EBV genomes are heterogeneously distributed in morphologically identical tumor cells, with some clusters of isolated tumor cells containing relatively high genome numbers while other tumor cells isolated from the same specimen may be negative for EBV DNA. Using reverse transcription-PCR, we detected EBV gene transcripts: EBNA-1 in almost all of the EBV-positive tumors and RNA of the EBV oncoprotein LMP-1 in a smaller subset of the tissues analyzed. Moreover, BARF-1 RNA was detected in half of the cases studied. Furthermore, we observed that in vitro EBV infection of breast carcinoma cells confers resistance to paclitaxel (taxol) and provokes overexpression of a multidrug resistance gene (MDR1). Consequently, even if a small number of breast cancer cells are EBV infected, the impact of EBV infection on the efficiency of anticancer treatment might be of importance.
Adenocarcinoma↗