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At least 271 records · Page 15Linked to original sources

PubMed2006

Microdissection genotyping analysis of the effect of intraarterial cytoreductive chemotherapy in the treatment of lacrimal gland adenoid cystic carcinoma.

PURPOSE: To investigate the feasibility of integrating molecular analysis into standard histopathology for lacrimal gland adenoid cystic carcinoma (ACC), and to gain insights into the molecular pathogenesis of this tumor and its response to intraarterial cytoreductive chemotherapy (IACC) that is of clinical use. DESIGN: A retrospective, comparative case series. METHODS: setting: Institutional. patient population: Nine consecutive patients with lacrimal gland ACC were treated with IACC, followed by orbital exenteration and chemoradiotherapy. This case series was compared with a series of seven patients treated by conventional local therapies. intervention procedure: Gene analysis was performed on microdissected tissue samples. Mutational allelotyping targeting nine genomic loci was performed with 15 polymorphic microsatellite markers situated in proximity to known tumor suppressor genes serving as markers for the presence of gene deletion. main outcome measure: A fractional mutation index was used to compare the acquired mutational load between different tumors having nonidentical patterns of microsatellite informativeness. RESULTS: Allelic imbalance (loss of heterozygosity [LOH]) for microsatellite markers at 1p36 was the single most common site affected by imbalance in this series, followed by LOH in temporal sequence involving 9p21, 22q12, 10q23, and 9q22. CONCLUSIONS: Microdissection genotyping holds promise as a clinical tool in integrating molecular analysis into standard histopathology to advance the understanding of lacrimal gland ACC tumorigenesis. A unique time course for temporal mutation acquisition in ACC is proposed, consisting of 1p36 loss first. Allelic loss for microsatellite markers at 1p36 may be a common as well as an early event in ACC formation and progression.

Adult↗

PubMed2006

Evaluation of laser microdissection as a tool in cancer glycomic studies.

Laser microdissection (LMD) is a recent development that enables the isolation of specific cell populations from tissue sections. This study focuses on the potential of LMD as a tool in cancer glycomics using colon cancer as a model. LMD was performed on hematoxylin and eosin stained frozen tissue sections. Tumor cells and normal epithelial cells were selectively microdissected. N-Glycans from the LMD- and the bulk tissue-derived samples were liberated by hydrazinolysis and then labeled with 2-aminopyridine. After sialidase digestion, the resulting asialo-N-glycans were analyzed by normal and reversed phase HPLC combined with mass spectrometry. Comparison of the various N-glycan profiles with the aid of LMD identified seven characteristic N-glycans with significantly different expression profiles between normal and cancerous cells that could not be detected by conventional analysis. Thus, LMD is a potent and useful tool for analyzing variations in the expression of N-glycans by overcoming the problem of tissue sample heterogeneity.

Carbohydrate Sequence↗

PubMed2006

Isolation of erythroid cells from the mouse embryonic yolk sac by laser capture microdissection and subsequent microarray hybridization.

Erythropoietic tissues are complex, containing both erythroid and other cells. The embryonic yolk sac in particular contains primitive erythroid cells in low abundance. Laser capture microdissection (LCM) was performed to isolate erythroid cells, and epithelial cells, from mouse embryonic day 10 (E10) yolk sac. Quantitative RT-PCR was performed to confirm that enriched cell populations were obtained. epsilony- and betaH1-globin mRNAs were enriched in the erythroid compared to the epithelial fraction, and villin mRNA was enriched in the epithelial compared to the erythroid fraction. RNA isolated from the microdissected erythroid cells was of high quality as indicated by capillary electrophoresis. The RNA from the LCM erythroid fraction was linearly amplified with T7 RNA polymerase and hybridized to a Mouse 430A 2.0 Affymetrix array. Forty-eight percent of genes were present in the microarray assays, including low abundance transcripts such as erythroid transcription factors and enzymes involved in heme synthesis. With the LCM/microarray strategy, it will be possible to identify genes that are differentially regulated in native primitive and definitive erythroid cells.

Animals↗

PubMed2005

Births using sperm retrieved via immediate microdissection of a solitary testis with cancer.

OBJECTIVE: To determine the feasibility of achieving births using sperm retrieved from a solitary testis with cancer. DESIGN: Prospective clinical study of azoospermic men with testis cancer in a solitary testis. SETTING: Infertility patients in an academic environment. PATIENT(S): Azoospermic men with previous history of orchiectomy and testis cancer in a remaining solitary testis. INTERVENTION(S): Viable sperm were retrieved by immediate microdissection of paratumor testicular tissue from orchiectomy specimen. MAIN OUTCOME MEASURE(S): Live births were achieved using sperm from immediate microdissection of orchiectomy specimen with testis cancer. CONCLUSION(S): Azoospermic men with cancer in a solitary testis have potential for fertility.

Adult↗

PubMed2004

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↗

PubMed2004

Laser microdissection and DNA typing of cells from single hair follicles.

Isolation and identification of single cells from tissue samples or smears assume a great relevance in pathological and forensic applications; in this latter field, the possibility to identify a specific genetic profile can be obtained by short tandem repeat (STR) typing, allowing to achieve a scientific proof important in law courts. It is well known that DNA extraction may be performed from several tissue fragments, blood traces, spermatozoa as well as telogen hair. However, in the last case, few follicle cells are coupled to a great amount of keratin reducing the efficiency of DNA amplification. Recently, the introduction of laser microdissection technique has greatly improved the capability to select single cells without any cross-contamination. In the present report, we have performed a laser microdissection using a Leica AS LMD (Leica Microsystems, Germany), utilized on cutting the telogen hair in order to exclusively collect the lower part of the follicle and reduce keratin contamination. In this way we can accurately extract an adequate amount of DNA, successfully typed by STR profile.

DNA↗

PubMed2005

Detection of mutations in whole genome-amplified DNA from laser-microdissected neurons.

Laser microdissection allows removal of individual cells for DNA extraction, but obtaining reliably amplified DNA from the small numbers of cells that survive neurodegenerative diseases can be difficult. We therefore tested a recently-available technique to amplify genomic DNA to see if it could reliably detect a mutation in nervous system cells. Fifty cortical motor neurons were removed by laser microdissection from cases of motor neuron disease both with and without known mutations in the gene for superoxide dismutase 1 (SOD1). DNA was extracted and amplified with a linear genomic amplification kit (GenomiPhitrade mark). The PCR product of exon 4 of SOD1 from this DNA was then sequenced. The GenomiPhi kit amplified the extracted DNA approximately 70 times. When exon 4 of SOD1 from this DNA was amplified by PCR the E100G SOD1 mutation could be identified on sequencing. No errors in replication were found. In conclusion, the GenomiPhi method of genomic DNA amplification appears to result in reliable replication of mutations in neurons. This technique can be used to obtain increased amounts of genomic DNA to study mutations within cells of the nervous system.

DNA↗

PubMed2005

Combining laser capture microdissection with quantitative real-time PCR: effects of tissue manipulation on RNA quality and gene expression.

Laser capture microdissection (LCM) is increasingly being used in quantitative gene expression studies of the nervous system. The current study aimed at determining the impact of various tissue manipulations on the integrity of extracted RNA in LCM studies. Our data indicate that various tissue preparation strategies prior to microdissection may decrease RNA quality by as much as 25%, thus affecting expression profiles of some genes. To circumvent this problem, we developed a strategy for reverse transcriptase real-time PCR that has considerable sensitivity and can be used to calculate relative changes in gene expression. This approach was validated in subregions of the rat cerebellum. Accordingly, expression of glial gene markers - myelin-associated glycoprotein and proteolipid protein 1 - was found 70-160-fold higher in the white matter layer of the cerebellar cortex as compared to the neuron-enriched granular layer. In contrast, expression of a specific neuronal maker, neuron-specific enolase, was found seven-fold higher in the granular layer, as compared to the white matter layer. Furthermore, this approach had high sensitivity and specificity as we were able to detect a 38% decrease in the expression of neuron-specific enolase without a change in the expression of glial markers following administration of the neurotoxin, ibotenic acid. These results demonstrate feasibility of performing accurate semi-quantitative gene expression analyses in LCM samples.

Animals↗

PubMed2006

Laser-assisted microdissection for real-time PCR sample preparation.

Laser-assisted microdissection (LMD) has been developed to procure precisely the cells of interest in a tissue specimen, in a rapid and practical manner. Together with real-time PCR and RT-PCR techniques, it is now feasible to study genetic alterations, gene expression features and proteins in defined cell populations from complex normal and diseased tissues. The process that brings from sample collection to the final quantitative results is articulated in several steps, each of which requires optimal choices in order to end up with high-quality nucleic acid or protein that allows successful application of the final quantitative assays. This review will describe shortly the development of LMD technologies and the principles they are based on. Trying to highlight the advantages and disadvantages of LMD, the main problems related to specimens collection and processing, section preparation and extraction of bio-molecules from microdissected tissue samples have been analysed.

Cell Line↗

PubMed2006

Fusobacterium necrophorum determined as abortifacient in sheep by laser capture microdissection and fluorescence in situ hybridization.

Fluorescent in situ hybridization (FISH) has been extensively used for identification of individual microbial cells within their natural environment. The present work describes the identification of Fusobacterium necrophorum in formalin-fixed tissue samples from three sets of ovine twins aborted at late pregnancy by a technique that combines laser capture microdissection (LCM) and fluorescent in situ hybridization (LCM-FISH). Cultural bacteriological examination had failed to identify an infectious agent but by histological examination, large colonies of bacteria associated with tissue inflammation were seen. In situ hybridization visualized the bacteria in the tissue samples and micro-colonies closely associated with lesions were isolated by LCM. PCR-amplification and sequencing of 16S rRNA gene from the microdissected bacteria identified the organisms as Fusobacterium necrophorum. A rRNA-targeting oligonucleotide probe specific for F. necrophorum was used in a FISH assay. In situ hybridization showed a high density of F. necrophorum in all examined tissue sections. Simultaneous probing with a general bacterial probe EUB338 and the specific probe for F. necrophorum showed that no other bacteria could be detected in the tissue sections. We therefore conclude that F. necrophorum was the likely cause of abortion in these sheep.

Abortion, Spontaneous↗

PubMed2004

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↗

PubMed2004

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↗

PubMed2005

Microsatellite analysis of hereditary nonpolyposis colorectal cancer-associated colorectal adenomas by laser-assisted microdissection: correlation with mismatch repair protein expression provides new insights in early steps of tumorigenesis.

Although microsatellite instability (MSI) testing is a useful tool for molecular screening of hereditary nonpolyposis colorectal cancer (HNPCC) carcinomas, conflicting results have been obtained in colorectal adenomas. This might result from different techniques of tissue sampling and MSI analysis. Alternatively, some HNPCC-associated adenomas may follow a molecular route that differs from the MSI pathway. In the present study we examined the MSI status of 18 adenomas from 17 HNPCC patients by comparing manual adenoma dissection under gross visual control with laser microdissection of single adenoma crypts. After manual gross dissection, 50% (9 of 18) and 11.1% (2 of 18) of the adenomas displayed high-level (MSI-H) and low-level (MSI-L) MSI, respectively. The same set of adenomas split into 83.3% (15 of 18) MSI-H and 5.6% (1 of 18) MSI-L after laser microdissection. The expression pattern of mismatch repair (MMR) proteins showed a higher concordance rate with the MSI status in laser-dissected (94%) than gross-dissected (47%) adenomas. Whereas two adenomas remained microsatellite stable (MSS) and MMR proficient even after laser-assisted dissection, two MSI-H cases showed either rare instabilities at coding microsatellites or intratumoral heterogeneity of MSI with and without MSH2 expression. This suggests that in some adenomas development of MMR dysfunction occurs stepwise with MSI, arising before complete loss of MMR gene expression, whereas other HNPCC-associated adenomas might develop independently of MMR deficiency.

Adenoma↗

PubMed2006

Analysis of gene expression in mineralized skeletal tissues by laser capture microdissection and RT-PCR.

The analysis of gene expression by growth plate chondrocytes in vivo has been hampered by the inherent difficulty in performing in situ hybridization on mineralized tissues. The combination of laser capture microdissection and reverse transcription-polymerase chain reaction (RT-PCR) allows analysis of gene expression by cells selectively removed from histologic sections by laser ablation. In order to apply this method to mineralized tissues, a decalcification process is required. The object of this study was to determine the optimal method for tissue decalcification prior to laser capture microdissection RT-PCR that will preserve integrity of the mRNA population. Acetone, 10% formalin, and methacarn were evaluated as fixatives, while Surgipath Decalicifier I, 10% ethylenediaminetetraacetic acid (EDTA), and 20% EDTA were evaluated as decalcifying reagents. Our results demonstrate that the optimal RNA quality was preserved by a decalcification protocol consisting of 20% EDTA for decalcification followed by fixation in methacarn, although this method is also associated with a reduction in RNA quantity.

Animals↗

PubMed2003

A novel technique for the examination of skin biopsies by laser capture microdissection.

BACKGROUND: Skin is an inherently heterogeneous tissue, thus the procurement of pure cell populations is critical for the accurate correlation of a molecular profile to a particular cell type or histological location. Laser Capture Microdissection (LCM) permits the efficient procurement of cells and mapping of genetic changes from histologically prepared samples. METHODS: This paper describes a robust LCM protocol established in our laboratory for the extraction of high quality DNA which sequenced from 100% of microdissected samples without the need for cloning. The unique properties of skin, in particular its strong intercellular adhesive forces, have dictated a significant modification to the normal procedure of tissue preparation to ensure reliable cell procurement. RESULTS: Using the methods outlined below we were able to precisely map the pattern of genomic mutations in our target gene of interest in normal skin, actinic keratosis and squamous cell carcinoma. CONCLUSIONS: The capability to select pure cell populations from the skin will revolutionise our ability to understand the processes involved in cutaneous tumourigenesis.

Biopsy↗

PubMed2004

Detection and quantitation of HCV core protein in single hepatocytes by means of laser capture microdissection and enzyme-linked immunosorbent assay.

Immunohistochemistry provides valuable information concerning the localization and distribution of hepatitis C virus (HCV)-related proteins in histological sections of liver tissue, but does not readily permit their quantitation in individual cells and the staining intensity of cell immunodeposits cannot be calibrated with the current number of antigen molecules. We specifically detected and quantitated HCV core protein in single hepatocytes by coupling laser capture microdissection (LCM) with a sensitive enzyme-linked immunosorbent assay (ELISA). Quantitation of HCV core protein per cell was carried out on liver tissue cells obtained by LCM from fixed and stained frozen sections of 10 HCV-positive patients with chronic active hepatitis (CAH). Macromolecules from captured cells were solubilized in an extraction buffer and directly assayed for core protein using a sandwich ELISA. Calibration was achieved by developing a standard curve based on known concentrations of HCV core protein. Precision, linearity and sensitivity were verified for known numbers of microdissected tissue cells. In this study, the concentration of HCV core protein in single hepatocytes ranged from 7 to 56 pg/cell. Specificity was verified on 10 replicates of 10 HCV-negative liver tissues. Immunohistochemical staining of HCV core protein was compared with the results of the soluble immunoassay for the adjacent liver tissue sections. Independent scoring of HCV immunostaining failed to parallel the LCM quantitative immunoassay. LCM-based immunoassay significantly expands our ability to investigate function-related antigens in apparently pure cell populations in HCV infection.

Aged↗

PubMed2004

Accurate qualitative and quantitative proteomic analysis of clinical hepatocellular carcinoma using laser capture microdissection coupled with isotope-coded affinity tag and two-dimensional liquid chromatography mass spectrometry.

Laser capture microdissection (LCM) is a powerful tool that enables the isolation of specific cell types from tissue sections, overcoming the problem of tissue heterogeneity and contamination. This study combined the LCM with isotope-coded affinity tag (ICAT) technology and two-dimensional liquid chromatography to investigate the qualitative and quantitative proteomes of hepatocellular carcinoma (HCC). The effects of three different histochemical stains on tissue sections have been compared, and toluidine blue stain was proved as the most suitable stain for LCM followed by proteomic analysis. The solubilized proteins from microdissected HCC and non-HCC hepatocytes were qualitatively and quantitatively analyzed with two-dimensional liquid chromatography tandem mass spectrometry (2D-LC-MS/MS) alone or coupled with cleavable ICAT labeling technology. A total of 644 proteins were qualitative identified, and 261 proteins were unambiguously quantitated. These results show that the clinical proteomic method using LCM coupled with ICAT and 2D-LC-MS/MS can carry out not only large-scale but also accurate qualitative and quantitative analysis.

Affinity Labels↗

PubMed2005

Detection of mitochondrial DNA from human inner ear using real-time polymerase chain reaction and laser microdissection.

CONCLUSIONS: In this study we were able to amplify and analyze extremely small amounts of template DNA from only a few individually dissected cells. We anticipate that this approach will facilitate the detection and analysis of mitochondrial (mt) DNA mutations in specific cell types in the inner ear, which should shed new light on genetic disorders leading to hearing loss. OBJECTIVE: To isolate mtDNA from selected tissues in the inner ear. Although several methods for extracting DNA from formalin-fixed, celloidin-embedded, archival human temporal bones have been reported, the isolation of DNA from the inner ear by means of laser microdissection has not been previously demonstrated. MATERIAL AND METHODS: This was a retrospective study. Temporal bones were obtained from subjects with no known otological history at autopsy. The combined method of laser microdissection and real-time polymerase chain reaction was used to isolate mtDNA from selected tissues in the inner ear. RESULTS: mtDNA could be isolated from the stria vascularis, spiral ligament, spiral ganglion cells and organ of Corti.

DNA Mutational Analysis↗