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PubMed1991

Acquired cystic disease of the kidney analyzed by microdissection.

Four cases of acquired cystic disease of the kidney (ACDK) were studied by the microdissection technique (MD) of Darmady and Baert to analyze the cystic transformation. No patient had a history or clinical evidence of the adult polycystic disease of the kidney (APDK). Hypothetical models related the pathogenesis of cystic transformation to either obstructive or degenerative factors. Microdissection was performed in four nephrectomy specimens of hemodialyzed patients and a total of 155 nephrons were isolated. The atrophy of the glomeruli has already been described histopathologically but MD demonstrated the existence of nephrons consisting of sclerotic glomeruli and enlarged segments between the atrophic convoluted proximal and distal tubules. Diverticula and cysts were located, above all along the proximal (mainly dilated) convoluted tubules: they were always in continuity with the tubules. Phase contrast microscopy showed a patent lumen in 80% of the proximal and distal convoluted tubules, and a regular lining of the cysts. These data support the hypothesis that ACDK is the result of hyperplasia and dilation of remaining nephrons, rather than a result of obstruction and/or fibrosis.

Adult↗

PubMed2002

Alumina-alumina artificial hip joints. Part I: a histological analysis and characterisation of wear debris by laser capture microdissection of tissues retrieved at revision.

The aims of this study were to investigate the tissues from uncemented Mittelmeier alumina ceramic-on-ceramic total hip replacements using histological methods and to isolate and characterise the ceramic wear debris using laser capture microdissection and electron microscopy. Tissues from around 10 non-cemented Mittelmeier alumina ceramic on ceramic THRs were obtained from patients undergoing revision surgery. Tissues were also obtained from six patients who were undergoing revisions for aseptic loosening of Charnley, metal-on-polyethylene prostheses. Tissue sections were analysed using light microscopy to determine histological reactions and also the location and content of alumina ceramic wear debris. Tissue samples were extracted from sections using laser capture microdissection and the characteristics of the particles subsequently analysed by TEM and SEM. The tissues from around the ceramic-on-ceramic prostheses all demonstrated the presence of particles, which could be seen as agglomerates inside cells or in distinct channels in the tissues. The tissues from the ceramic-on-ceramic retrievals had a mixed pathology with areas that had no obvious pathology, areas that were relatively rich in macrophages and over half of the tissues had in the region of 60% necrosis/necrobiosis. In comparison, the Charnley tissues showed a granulomatous cellular reaction involving a dense macrophage infiltrate and the presence of giant cells and < 30% necrosis/necrobiosis. The tissues from the ceramic prostheses also showed the presence of neutrophils and lymphocytes, which were not evident in the tissues from the Charnley retrievals. There were significantly more macrophages (p < 0.05), and giant cells (p < 0.01) in the Charnley tissues and significantly more neutrophils (p < 0.01) in the ceramic-on-ceramic tissues. TEM of the laser captured tissue revealed the presence of very small alumina wear debris in the size range 5-90 nm, mean size + SD of 24 +/- 19nm whereas SEM (lower resolution) revealed particles in the 0.05-3.2 microm size range. This is the first description of nanometre sized ceramic wear particles in retrieval tissues. The bi-modal size range of alumina ceramic wear debris overlapped with the size ranges commonly observed with metal particles (10-30 nm) and particles of ultra-high molecular weight polyethylene (0.1-1,000 microm). It is possible that the two size ranges of contributed to the mixed tissue pathology observed. It is speculated that the two types of ceramic wear debris are generated by two different wear mechanisms in vivo, under normal articulating conditions, relief polishing wear and very small wear debris is produced. while under conditions of microseparation of the head and cup and rim contact, intergranular and intragranular fracture and larger wear particles are generated.

Aluminum Oxide↗

PubMed2000

Microdissection, DOP-PCR, and comparative genomic hybridization of paraffin-embedded familial prostate cancers.

There is a clear genetic component to prostate cancer susceptibility. Regions reported to be linked to prostate cancer include 1q24-25 (HPC-1), 1q42.2-43, and Xq27-28. There is limited genetic information on familial prostate tumors. We used the Utah Population Database to identify familial prostate cancer cases and selected 35 cases from high-risk families. Tissue blocks containing discernable tumor were available from 19 cases; 13 of these yielded adequate specimens for analysis. Six cases came from families with linkage to HPC-1, 3 were known to have linkage to Xq27-28, and 4 had no linkage to a known locus; 7 cases were analyzed from patients who showed no known linkage (sporadic tumors) as controls. These paraffin-embedded tumors were laser microdissected, degenerate oligonucleotide (DOP)-amplified, and labeled for fluorescence detection by comparative genomic hybridization (CGH). Loss of 7q, 10q, and 16q and gain of 8q were common abnormalities present in both familial and sporadic tumors. Distinctive abnormalities included loss of 3p12-3p22 in 3 of 6 HPC-7-linked cases and in 2 of 3 X-linked cases and gain of 6q11-6q21 in 2 each of HPC-1 and X-linked tumors. In conclusion, laser microdissection, DOP-PCR, and CGH is a feasible method for analysis of paraffin-embedded prostate tumors. This study provides preliminary data suggesting that familial prostate cancer harbors some unique genetic changes when compared with sporadic prostate tumors.

Chromosomes, Human, Pair 1↗

PubMed1998

Characterization of multiple 12p rearrangements in testicular germ cell tumor cell line 833K and its subclone 64CP by chromosome microdissection.

Rearrangements of chromosome arm 12p are known to be common in germ cell tumors (GCT). Previous studies, using fluorescence in situ hybridization (FISH) with a whole chromosome 12 painting probe, showed unusual distributions of chromosome 12-derived chromatin in GCT cell line 833K and its cisplatin-resistant subclone, 64CP, located next to AgNOR (silver staining nucleolus organizer regions), some of which were ectopic. In this study, the ectopic stalk regions were shown by FISH to be composed of 18s and 28s rDNA, but were flanked by beta-satellite DNA, which may form a barrier around the rDNA. In order to determine the specific origins of the rearranged chromosome 12 segments, three different derived chromosome 12 regions were isolated from 64CP, using chromosomal microdissection. The microdissected fragments were labeled and hybridized by FISH to normal human chromosomes. All three segments localized to distal 12p; 12p12-->12pter, but with apparently different breakpoints for each segment. Furthermore, three-color FISH experiments with 12p band-specific probes demonstrated that the derivative chromosome 12 regions in 833K also originate from distal 12p (12p12-->p13). These sequences now can be evaluated for degree of overlap or common breakpoints which may be of significance in the development or progression of GCT.

Chromosome Aberrations↗

PubMed2000

Microdissection and FISH investigations in acute myeloid leukemia: a step forward to full identification of complex karyotypic changes.

Complex chromosomal rearrangements in malignant hemopathies frequently remain unclarified because of paucity of material for further fluorescence in situ hybridization analyses and/or lack of suitable probes. Chromosome microdissection (MD) can be an adequate approach to elucidate chromosome aberrations unrecognizable by conventional karyotyping. We applied MD in two patients with acute myeloid leukemia (AML) and unidentified chromosome changes at karyotype. Microdissection of a ring chromosome in an AML-M5 case revealed 21q polysomy. In an AML-M4 case, MD of an add(15p) disclosed a t(8;15) with over-representation of both 8q22 and 8q24 bands. YAC probes were helpful in showing duplication of the ETO gene at 8q22, and amplification of C-MYC, at 8q24.

Aneuploidy↗

PubMed2002

Laser capture microdissection of Plasmodium falciparum liver stages for mRNA analysis.

Plasmodium falciparum liver-stages are important targets for vaccine-induced protective immune responses and prophylactic treatment against malaria. Little is known of the gene expression profile of malaria parasites during their development inside hepatocytes. The sequencing of the P. falciparum genome and the development of DNA microarray technology give new opportunities to identify genes expressed during the development of Plasmodium. However, transcriptome analysis cannot currently be applied to the hepatic stages, due to difficulties in obtaining sufficient amounts of parasite material that lie among the large excess of host cell RNA. Here, we describe the isolation of liver-stages by a modified laser capture microdissection approach applied to human hepatocyte cultures infected with P. falciparum. RT-PCR amplification of several P. falciparum transcripts demonstrated the high quality of the RNA recovered after microdissection. This approach should enable analyses of P. falciparum transcriptome during its hepatic development and substantially assist the identification of new therapeutic and vaccine targets.

Animals↗

PubMed1998

Laser-capture microdissection: opening the microscopic frontier to molecular analysis.

As the list of expressed human genes expands, a major scientific challenge is to understand the molecular events that drive normal tissue morphogenesis and the evolution of pathological lesions in actual tissue. Laser capture microdissection (LCM) has been developed to provide a reliable method to procure pure populations of cells from specific microscopic regions of tissue sections, in one step, under direct visualization. The cells of interest are transferred to a polymer film that is activated by laser pulses. The exact morphology of the procured cells (with intact DNA, RNA and proteins) is retained and held on the transfer film. With the advent of LCM, cDNA libraries can be developed from pure cells obtained directly from stained tissue, and microhybridization arrays of thousands of genes can now be used to examine gene expression in microdissected human tissue biopsies. The fluctuation of expressed genes or alterations in the cellular DNA that correlate with a particular disease stage can ultimately be compared within or between individual patients. Such a fingerprint of gene-expression patterns can provide crucial clues for etiology and might, ultimately, contribute to diagnostic decisions and therapies tailored to the individual patient. Molecules found to be associated with a defined pathological lesion might serve as imaging ot therapeutic targets.

DNA↗

PubMed2003

Application of laser capture microdissection in genetic analysis of neuroblastoma and neuroblastoma precursor cells.

Recently developed quantitative and high-throughput technologies that allow automated and rapid screening of the whole genome, transcriptome and proteome have revolutionized the field of cancer genetics. At the same time, new challenges are met, e.g. the need for improved data analysis and standardization of tumor sample handling. Even if these issues are resolved, an 'old' problem in genetic tumor analysis remains, i.e. contamination of tumor samples by stromal and surrounding normal cells. To overcome this obstacle, laser capture microdissection (LCM) has been developed in order to procure the cells of interest from stained tissue sections with retention of morphology. In this review we describe the possible down-stream applications of LCM in the genetic analysis of neuroblastoma (NB). Special focus is given to MYCN copy number determination using real-time quantitative polymerase chain reaction (Q-PCR), analysis of 1p-, 3p- and 11q-deletions using loss of heterozygosity analysis and Q-PCR expression analysis of microdissected normal neuroblast cells and NB cells.

DNA, Neoplasm↗

PubMed1992

Construction and characterization of a region-specific microdissection library from human chromosome 2q35-q37.

A region-specific genomic library for human chromosome 2q35-q37 has been constructed using the microdissection and polymerase chain reaction-mediated linker-adaptor microcloning method. Twenty fragments from the chromosome region 2q35-q37 were dissected and a library consisting of 20,000 recombinant microclones was obtained. The insert size ranged between 50 and 800 bp, with a mean of approximately 270 bp. About 50-60% of the microclones contained unique sequences. The microdissection library has been demonstrated to derive from the dissected region 2q35-q37 by chromosome painting using the fluorescence in situ hybridization (FISH) technique. Southern blot analysis of the unique sequence microclones from the library showed that 54% (26/48) of the clones are of human origin and chromosome 2 specific. Four of these microclones have been further mapped to the 2q37 region by using a cell hybrid containing only 2q37. The unique sequence microclones have also been characterized for their insert size and the hybridizing genomic fragments cleaved with HindIII. As shown previously, these microclones will be useful in isolating corresponding yeast artificial chromosome (YAC) clones with large inserts for high-resolution physical mapping and also in screening cDNA libraries to isolate expressed gene sequences as candidate genes to facilitate search for the crucial genes underlying genetic diseases and specific forms of cancer assigned to the region.

Blotting, Southern↗

PubMed2000

Gene expression profiles in squamous cell carcinomas of the oral cavity: use of laser capture microdissection for the construction and analysis of stage-specific cDNA libraries.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer among men in the developed world affecting the oral cavity, salivary glands, larynx and pharynx. Utilizing tissue from patients with HNSCC, we sought to systematically identify and catalog genes expressed in HNSCC progression. Here, we demonstrate the successful use of laser capture microdissection for procuring pure populations of cells from patient tissue sets comprised of oral squamous cell carcinomas (OSCCs) and matching normal tissue. From the estimated 5000 cells procured for each sample, we were able to extract total RNA (14.7-18.6 ng) of sufficient quality to transcribe GAPDH by reverse transcriptase-polymerase chain reaction (RT-PCR). The RNA was used for the synthesis of blunt-ended, double-strand complementary DNAs (cDNAs) by oligo (dT)-mediated reverse transcription, followed by addition of linkers. Primers specific for these linkers with uracil deglycosylase-compatible ends were used to amplify these cDNAs by PCR and the product was subcloned into the pAMP10 cloning vector. Ninety-six clones from each of six libraries were randomly sequenced and results indicated that 76-96% of the inserts represent either anonymous expressed sequence tags (ESTs) (25-48%), known genes (9-29%) or novel sequences (27-51%), respectively, with very little redundancy. These results demonstrate that high quality, representative cDNA libraries can be generated from microdissected OSCC tissue. Furthermore, these finding suggest the existence of at least 132 novel genes expressed in our cDNA libraries, which may have a role in the pathogenesis of HNSCC, and may represent novel markers for early detection as well as targets for pharmacological intervention in this disease.

Aged↗

PubMed2002

Isolating vessels from the mouse brain for gene expression analysis using laser capture microdissection.

Studies of gene expression often examine a pool of RNA extracted from the diverse cell types making up a tissue. We have developed a method for isolating vessels from the brain in order to understand the changes occurring in the vessels during the pathogenesis of cerebral malaria. Vessels were visualised by incubating sections of mouse brain with a substrate for alkaline phosphatase. Vessels were collected by laser capture microdissection and the specificity was monitored by measuring the expression of cell-specific markers. RNA from the captured vessels was highly enriched in mRNA for genes associated with endothelial cells and pericytes. Measurement of indoleamine 2,3-dioxygenase mRNA indicated it was possible to detect changes in gene expression, due to malaria infection, occurring specifically within the vessels. Laser capture microdissection can be used to study changes in gene expression occurring at the blood-brain barrier.

Alkaline Phosphatase↗

PubMedDate not supplied

Laser capture microdissection MALDI for direct analysis of archival tissue.

MALDI mass spectra were obtained from cancer cells isolated by laser capture microdissection (LCM) of archived tissue. Frozen human lung tissue from adenocarcenoma and squamous cell carcenoma cases were cut into 5 to 15 microm thick sections, stained with hematoxylin and dehydrated. Cancer cells were isolated by LCM, mixed with matrix solution, and deposited on a MALDI target for mass spectrometric analysis. For comparison with LCM isolated cells, tissue sections were placed directly on the MALDI target without microdissection. Tissue sections frozen in optimal cutting temperature (OCT) solution and cut into 8 microm thick sections gave the best performance with direct MALDI analysis. Between 15 and 20 peaks were observed in the mass region between 1,000 and 4,000 Da, and roughly half of these peaks were common to either squamous cells or adenocarcenoma. Additional peaks were observed in the non-LCM mass spectra and these may result from biomolecules in the healthy tissue. When compared to fresh tissue, both LCM and non-LCM archived tissue produced fewer peaks, possibly due to degradation of the biomolecules in the archived tissue.

Adenocarcinoma↗

PubMed2007

Identification of specific protein markers in microdissected hepatocellular carcinoma.

At present, the molecular mechanisms of hepatocellular carcinogenesis are not well-understood, and hepatocellular carcinoma (HCC) stays one of the most frequent and high-risk metastatic visceral neoplasms worldwide. For the identification of tumor-relevant proteins, we analyzed microdissected cells from nontumorous liver tissue (n = 28) and tissue derived from hepatic tumor center (n = 25), as well as tumor margin (n = 23). We unequivocally identified 53 proteins from hepatic tumor tissues by peptide fingerprint mapping and SELDI mass spectrometry that were separated using two-dimensional gel electrophoresis. Among a number of signals that were detected as significantly different in the protein profiling analysis, we identified for the first time ferritin light subunit (FLS) and adenylate kinase 3 alpha-like 1 (AK3), showing decreased expressions in hepatic tumor, as well as biliverdin reductase B (BVRB) that was upregulated in HCC. The use of ProteinChip technology in combination with tissue microdissection gives insight of the complex changes occurring at the protein level in hepatocellular cancer associated with tumor development and progression and resulted in three new potential diagnostically useful markers.

Biomarkers↗

PubMed1989

Cloning defined regions of the human genome by microdissection of banded chromosomes and enzymatic amplification.

The molecular analysis of many genetic diseases requires the isolation of probes for defined human chromosome regions. Existing techniques such as the screening of chromosome-specific libraries, subtractive DNA cloning and chromosome jumping are either tedious or not generally applicable. Microdissection and microcloning has successfully been applied to various chromosome regions in Drosophila and mouse, but conventional microtechniques are too coarse and inefficient for analysis of the human genome. Because microdissection has previously been used on unbanded chromosomes only, cell lines in which the chromosome of interest could be identified without banding had to be used. At least one hundred chromosomes were needed for dissection and lambda vectors used to achieve maximum cloning efficiency. Recombinant phage clones are, however, more difficult to characterize than plasmid clones. Here we describe the dissection of the Langer-Giedion syndrome region on chromosome 8 from GTG-banded metaphase chromosomes (G-banding with trypsin-Giemsa) and the universal enzymatic amplification of the dissected DNA. Eighty per cent of clones from this library (total yield 20,000) identify single-copy DNA sequences. Fifty per cent of clones detect deletions in two patients with Langer-Giedion syndrome. Although the other clones have not yet been mapped, this result demonstrates that thousands of region-specific probes can be isolated within ten days.

Base Sequence↗

PubMed1995

A microdissection approach to detect molecular markers during progression of prostate cancer.

To investigate the underlying mechanisms of carcinogenesis, we have developed a technique to determine the frequency of genetic changes in prostatic carcinoma tissue. We have demonstrated that at a ratio of between 1:4 and 1:9 mutant-normal alleles, the signal from a mutant TP53 allele is not apparent after polymerase chain reaction (PCR) amplification and further direct sequencing or single-strand conformation polymorphism (SSCP) analysis. To bypass this problem, which is inherent in the heterogeneity of the prostate tissue and of the tumour, we selected areas of graded prostate tumours (Gleason score) from cryosectioned preparations and microdissected these cells (20-100 cells). After anionic resin removal of proteins, PCR amplification of TP53 gene exons 5/6 and SSCP analysis, an abnormal SSCP band shift was observed in suspected tumour cells, compared with microdissected stromal cells used as an internal control, while (1) a crude preparation of tissue DNA carrying the tumour did not show any abnormality and (2) immunostaining by a set of monoclonal antibodies against TP53 protein remained negative. Nucleotide sequence analysis of the different bands confirmed the presence of a mutation in the TP53 gene exon 6 position 13,336 in an abnormal band for one specimen, while no mutation was detected in the normal SSCP band. By targeting recognised tumour cells we can find DNA mutations which are undetectable using the standard technique of whole-tissue DNA extraction, particularly in a heterogeneous tumour such as carcinoma of the prostate.

Base Sequence↗

PubMed1978

Hereditary polycystic kidney disease (adult form): a microdissection study of two cases at an early stage of the disease.

Kidney fragments from two cases of hereditary polycystic kidney disease (adult form) at an early stage were examined by microdissection. Localized cystic cystic dilatations were found in proximal and distal tubules, loops of Henle, and collecting tubules. Entirely normal nephrons and collecting tubules were also observed. Abnormal branching of collecting tubules or the abnormal attachment of nephrons, as described in other microdissection studies, were not found. Our observations do not confirm the hypothesis that the adult form of hereditary polycystic kidney disease is the consequence of ampullary dysfunction during early development.

Adult↗

PubMed1982

Localization of active and inactive kallikrein (kininogenase activity) in the microdissected rabbit nephron.

Active and total (trypsin-activated) kallikrein were measured in discrete segments of the nephron by their kininogenase activity. The kinins generated were measured by radioimmunoassay and the amount of inactive kallikrein was calculated as the difference between total and active kallikrein. Single nephrons from collagenase-treated rabbit kidneys (N = 12) were microdissected and divided into eight segments: (1) glomerulus; (2) proximal convoluted tubule; (3) cortical thick ascending limb; (4) bright portion of distal convoluted tubule; (5) granular portion of distal convoluted tubule; (6) granular portion of cortical collecting tubule; (7) light portion of cortical collecting tubule; and (8) medullary collecting tubules. After pooled segments (approximately 50 nephron segments in each assay) were homogenized and treated with deoxycholic acid, active and total kallikrein were measured and inactive kallikrein was calculated. Active and inactive kallikrein were localized in granular portions of the distal and cortical collecting tubule, which contains more than 85% of the active and inactive kallikrein found in the total microdissected nephron. Little or no kallikrein was detected in other segments, including the bright portion of the distal segment which has the macula densa. The discrete localization of active and inactive kallikrein suggests a specific role for renal kallikrein in these nephron segments.

Animals↗

PubMed1993

Viral DNA in microdissected renal biopsy tissue from HIV infected patients with nephrotic syndrome.

Focal glomerulosclerosis (FGS) has been considered as HIV-associated nephropathy, a specific renal complication of infection. To determine whether renal disease in HIV infected patients has one highly prevalent pathologic expression, and whether renal parenchymal viral genomic incorporation affects pathologic outcome, we reviewed renal biopsies performed at our center. Twenty-eight HIV infected patients with nephrotic range proteinuria underwent renal biopsy for diagnosis of renal disease: 85.7% led homosexual or bisexual lifestyles; 10.7% admitted to intravenous drug use; and 85.7% were Black. Only 53.6% had FGS; 28.6% had glomerulonephritis. Two patients had diabetic renal disease; 93.3% of patients with FGS and 87.5% of patients with glomerulonephritis were Black. Paraffin slides of twenty-two of the patients' renal biopsies were evaluated by polymerase chain reaction (PCR) for the presence of HIV DNA, using primers and probes to the gag gene, detected by liquid hybridization and polyacrylamide gel electrophoresis. Twenty-one of the twenty-two evaluated tissue specimens showed the presence of HIV DNA. Microdissection studies of glomeruli, tubules, interstitial cells and infiltrating inflammatory cells showed the presence of HIV genome in all but interstitial cells. HIV infected patients without renal disease also had positive PCR evaluations of microdissected tissue, while non-infected patients were all negative. We conclude that although focal glomerulosclerosis is the most common renal pathologic lesion in patients with HIV infection and nephrotic range proteinuria, glomerulonephritis is a relatively frequent finding. HIV genome is present in renal tissue in HIV infected subjects with nephrotic range proteinuria, but is also found in HIV infected subjects without nephropathy.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗