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PubMed2002

A microdissected thin tensor fasciae latae perforator flap.

A new method, named "microdissection," has been introduced to create a thin flap by elevating the tensor fasciae latae perforator flap to serve as microdissected thin tensor fasciae latae perforator flap. In microdissection, perforators that run in the posterolateral direction in the adipose tissue after penetrating the deep fascia are dissected meticulously using an operative microscope, and a thin flap is elevated in a single process. The caliber of the perforator artery and vein in the tensor fasciae latae muscle measures approximately 0.7 mm and 0.9 mm, respectively. When transplanting the flap, an end-to-side anastomosis to the main artery measuring 1 to 2 mm is preferable to avoid the risk of arterial thrombosis. In contrast, an end-to-end anastomosis of the perforator vein to the comitans vein of the main artery can be performed safely. In the present study, 11 flaps were transplanted to the sites of skin defects of the neck, hand, axilla, knee, and foot. The author considers that the first clinical indication of this flap is reconstruction of hand skin defects.

Adult↗

PubMed1999

Clonality of cutaneous B-cell infiltrates determined by microdissection and immunoglobulin gene rearrangement.

Diagnosis of primary cutaneous B-cell lymphoma (PCBCL) is supported by the demonstration of a monoclonal B-cell population. Immunoglobulin heavy chain (IgH) gene-rearrangement analysis by polymerase chain reaction (PCR) is a reliable technique to detect B-cell monoclonality in paraffin-embedded tissue, but the presence of numerous reactive B lymphocytes in PCBCL may complicate the interpretation of clonality test results. To test this hypothesis, IgH gene-rearrangement analysis by PCR was performed on paraffin-embedded whole tissue sections of 19 cutaneous B-cell infiltrates diagnosed either as consistent with PCBCL (10 specimens) or unclassified lymphoid infiltrates (ULI) (9 specimens). In specimens that did not show monoclonal bands by IgH gene-rearrangement on DNA extracted from whole tissue sections, clonality assays were repeated on microdissected B-cell subpopulations suspicious for neoplastic cells. In the analysis of whole tissue sections, 4 (40%) of 10 specimens consistent with PCBCL showed one or two monoclonal bands, whereas 9 of 9 ULI specimens showed either a ladder or a smear. Clonality analysis of microdissected B-cell subpopulations showed 3 additional PCBCL specimens (total, 7 of 10) and 1 ULI specimen (total, 1 of 9) with unequivocal and reproducible monoclonal bands. Addition of microdissection increases the sensitivity of PCR-based B-cell clonality assay in PCBCL compared with analysis performed on the whole section (70% versus 40% monoclonal cases) and allows the recognition of a dominant clone in ULI specimens, possibly representing early PCBCL.

B-Lymphocyte Subsets↗

PubMed2002

Construction of cDNA libraries from microdissected benign and malignant thyroid tissue.

cDNA libraries were constructed from thyroid epithelial cells gained by laser capture microdissection for gene expression analysis of the progression of thyroid cancer. Six histologically diverse thyroid tissue specimens were used. A mean of 93 ng of total RNA was gained per tissue sample from a mean estimated number of 25,000 microdissected cells per sample. Analysis of randomly selected clones from six libraries showed an average insert size of 600 (range, 300-1500) bp. Preliminary sequencing of clones selected from the six libraries indicates a range of 46% to 62% known genes per library, 4% to 25% anonymous expressed sequence tags per library, and 15% to 43% novel expressed sequence tags per library. Thyroglobulin was found in normal thyroid epithelium and follicular thyroid adenoma, whereas calcitonin precursor transcripts were found in medullary thyroid carcinoma. We demonstrate production of high-quality cDNA libraries of microdissected tissue of the thyroid, which should prove useful for gene expression analysis of human thyroid tumors.

Adenoma↗

PubMed2002

An efficient method for the assessment of DNA quality of archival microdissected specimens.

There will be an increasing need of methods for assessing the suitability of specimens for genetic-based assays as DNA markers become an integral part of molecular diagnosis. The targeting of specimens for specific analyses will require the ability to rapidly screen for DNA quality. Conventional methods such as Southern analysis and gene specific-polymerase chain reaction (PCR) often require quantities of material that represent a significant portion of the specimen, especially in microdissected samples. Here we describe a novel application of a commonly used PCR-based DNA-fingerprinting technology that requires minimal quantities of DNA to simultaneously assess multiple regions throughout the genome for DNA quality. Randomly amplified polymorphic DNA (RAPD) PCR generates DNA fragments of a broad size range with the product size reflecting the degree of sample fragmentation. Fourteen DNA samples extracted from cells microdissected from seven formalin-fixed, paraffin-embedded oral cancer biopsies were assessed for DNA quality using gene-specific PCR and RAPD-PCR. Although the more conventional assay required 2-ng DNA (or 300-cell equivalents) to examine DNA quality at a single locus, RAPD-PCR provided a more informative profile of DNA quality from the same microdissected archival specimens.

Carcinoma, Squamous Cell↗

PubMed2004

Expression microdissection: operator-independent retrieval of cells for molecular profiling.

Tissue microdissection is an important method for the study of disease states. However, it is difficult to perform high-throughput molecular analysis with current techniques. We describe here a prototype version of a novel technique (expression microdissection) that allows for the procurement of desired cells via molecular targeting. Expression microdissection (xMD) offers significant advantages over available methods, including an increase in dissection speed of several orders of magnitude. xMD may become a valuable tool for investigators studying cancer or other disease states in patient specimens and animal models.

Animals↗

PubMed1997

Isolation of chromosome-specific ESTs by microdissection-mediated cDNA capture.

Despite dramatic advances in the identification of human expressed sequence tags (ESTs), techniques that facilitate isolation of chromosome or chromosome band-specific ESTs would be of considerable value. This report demonstrates the feasibility of identifying chromosome-specific ESTs following microdissection of a single-copy chromosome region. For this study, a reduced complexity cDNA library was linkered and hybridized to normal human metaphase chromosomes. After stringency washes, the entire long arm of chromosome 6 (6q) was microdissected. Following PCR amplification using linker-specific primers, captured cDNAs were subcloned and 187 individual clones picked at random. These 187 clones were then sorted by filter cross-hybridization into 34 unique groups. Of these 34 groups, 19 (56%) mapped to chromosome 6 by Southern blot. We identified three previously known genes, human cytovillin (ezrin) mapped previously to 6q25-26, human cardiac gap junction protein (connexin 43) mapped previously to 6q21-23.2 and prolyloligopeptidase, which had not been mapped previously. BLASTN identified three clone groups with homology to known ESTs and 12 representing novel cDNA sequences. Six of the groups were sublocalized to specific band regions of 6q using a chromosome 6 hybrid mapping panel, five representative clones were tested on Northern analysis to verify their expression, and finally, nine clones were mapped against the Gene bridge 4 reduction hybrid panel to confirm their genetic map location on 6q. These results demonstrate that microdissection of single-copy sequences has sufficient specificity for isolation of chromosome-specific cDNAs.

Animals↗

PubMed1994

Clonality analysis of defined B-cell populations in archival tissue sections using microdissection and the polymerase chain reaction.

A simple microdissection technique involving the use of a drawn-out glass pipette was developed for isolation of defined cell subsets from tissue sections. Using this technique and the polymerase chain reaction (PCR), clonally rearranged immunoglobulin (Ig) heavy chain genes were reliably amplified in single neoplastic follicles or few hundreds of tumour cells isolated from archival haematoxylin and eosin or immunostained sections of B-cell lymphomas. A polyclonal nature was consistently demonstrated in reactive lymphoid follicles or interfollicular reactive B-cells within the same lymphoma sections. Microdissection of lymphoma cells from within foci of chronic inflammation improved the resolution of tumour-specific PCR products by reducing amplification of background polyclonal B-cell sequences. The combination of microdissection and PCR techniques, therefore, provides an important tool for the investigation of B-cell lymphomas and also allows simple and specific access for other molecular genetic analyses of different cell subsets on tissue sections.

B-Lymphocytes↗

PubMed1998

Microdissection of pig chromosomes: dissection of whole chromosomes, arms and bands for construction of paints and libraries.

Chromosome microdissection is an important means to efficiently generate a large number of markers from a desired region of a genome. The present study was designed to initiate microdissection and amplification of DNA from whole chromosomes, arms, or bands of porcine chromosomes. The following pig (SSC) chromosomes/segments were scraped: SSC1p, SSC1q26-q2.13, SSC2q11-q14, SSC4q12-q25, SSC13, SSC13q12-q31, SSC13q32-q43, SSC13q32-q43, SSC15, and SSC16q21-q23. After amplification and PCR-labelling, the DNA from the dissected segments were painted back to normal metaphase chromosomes to test their identity. Microdissection of some of the segments (on SSC4, 13 and 15) coincides with the mapping of economically important traits. As a first step towards generation of markers, microcloning of amplified product from SSC1p and SSC15 was carried out. The libraries were screened with a (GT)15 oligonucleotide probe. Future prospects of such a work in farm animals are discussed.

Animals↗

PubMed1996

Disclosure of five breakpoints in a complex chromosome rearrangement by microdissection and FISH.

Microdissection and fluorescence in situ hybridisation (FISH) were used to elucidate the nature of a complex chromosome translocation, after GTG banding failed in the complete characterisation of the structural rearrangement between chromosomes 6 and 12. These chromosomes were painted with chromosome specific paints and one of the chromosome regions involved in the translocation was isolated by microdissection. Ten copies of the microdissected region were collected with microneedles from GTG banded metaphases, transferred to a collecting drop, and amplified by means of DOP-PCR. The PCR product was labelled with biotin-14-dATP and used as a FISH probe for hybridisation to normal metaphase chromosomes and metaphase chromosomes of the patients (microFISH). FISH with this chromosome region specific painting probe and with chromosome band specific probes enabled the characterisation of a complex chromosome rearrangement with five breakpoints in two chromosomes. This resulted in the following karyotype: 46,XY,t(6;12)(6pter--> 6q12::12q24.1-->12qter;12qter-->12q13.3:: 6q16.2-->6q26::12q13.3-->12q24.1::6q12--> 6q16.2::6q26-->6qter).

Adolescent↗

PubMed1998

A simple combined microdissection and aspiration device for the rapid procurement of single cells from clinical peripheral blood smears.

Molecular analysis of cells from cytology specimens can help to establish a diagnosis in ambiguous cases. However, mutations in heterogeneous samples might not be detected because of the diluting effect of DNA from normal background cells. Even if a mutation were detected, it could not be traced back to a specific cell type. Molecular analysis of single cells circumvents this problem. Both mechanical and laser assisted methods have been described for the selective procurement of cells from histology slides; however, they have the drawback of either being technically demanding or expensive. Furthermore, it is nuclear whether they can be applied to cytology specimens. Finally, few of these techniques are able to procure single cells. Therefore, we developed a simplified combined microdissection and aspiration device for the rapid procurement of single cells from clinical cytology specimens. The principle of this device, called the cytopicker, is the combination of the microdissection tool, a steel cannula, with the aspiration tool, a glass capillary connected to a vacuum, into one device. Steel cannulae are optimal for microdissection of cells from the hard matrix of cytology specimens but aspirate poorly. On the other hand, glass capillaries are suboptimal for dissecting but aspirate very well. Combining both tools into one by inserting the capillary into the cannula allows optimal dissection using the cannula (with the glass capillary with-drawn and thus protected), followed by optimal aspiration using the capillary (after being advanced through the cannula). All movements of the device are controlled by just one micromanipulator, making the cytopicker inexpensive to manufacture. The cytopicker can rapidly and simply procure single cells, such as lymphoblasts, from cytology specimens, such as peripheral blood smears. DNA from these cells can be amplified by PCR. However, precautions have to be taken to avoid contamination. Once improved further, the cytopicker might facilitate molecular analysis in the routine cytology laboratory.

Blood Specimen Collection↗

PubMed1999

A simple method for PCR based analyses of immunohistochemically stained, microdissected, formalin fixed, paraffin wax embedded material.

Microdissection was performed on sections cut from formalin fixed, paraffin wax embedded archival material, which had been subjected to conventional immunohistochemistry. Crude DNA extracts, which were obtained from these microdissected samples by a simple microwave step, were then added directly to amplification reactions. Analyses using a range of polymerase chain reaction (PCR) based techniques, including microsatellite repeat polymorphism analysis at the NM23-H1 locus and sequencing of exons 5, 7, and 8 of the p53 gene, were performed successfully. Universal PCR amplification was also carried out on the microdissected material and probes suitable for use in comparative genomic hybridisation (CGH) were obtained in all cases. This technique will enable a range of effective genetic analyses to be carried out on specific subsets of cells that have been characterised previously by immunohistochemistry.

Colorectal Neoplasms↗

PubMed2006

Laser microdissection and pressure-catapulting technique to study gene expression in the reoxygenated myocardium.

For focal events such as myocardial infarction, it is important to dissect infarction-induced biological responses as a function of space with respect to the infarct core. Laser microdissection pressure catapulting (LMPC) represents a recent variant of laser capture microdissection that enables robot-assisted rapid capture of catapulted tissue without direct user contact. This work represents the maiden effort to apply laser capture microdissection to study spatially resolved biological responses in myocardial infarction. Infarcted areas of the surviving ischemic-reperfused murine heart were identified using a standardized hematoxylin QS staining procedure. Standard staining techniques fail to preserve tissue RNA. Exposure of the tissue to an aqueous medium (typically used during standard immunohistochemical staining), with or without RNase inhibitors, resulted in a rapid degradation of genes, with approximately 80% loss in the 1st h. Tissue elements (1 x 10(4)-4 x 10(6) microm(2)) captured from infarcted and noninfarcted sites with micrometer-level surgical precision were collected in a chaotropic RNA lysis solution. Isolated RNA was analyzed for quality by microfluidics technology and reverse transcribed to generate high-quality cDNA. Real-time PCR analysis of the cDNA showed marked (200- and 400-fold, respectively) induction of collagen Ia and IIIa at the infarcted site compared with the noninfarcted site. This work reports a sophisticated yet rapid approach to measurement of relative gene expressions from tissue elements captured from spatially resolved microscopic regions in the heart with micrometer-level precision.

Animals↗

PubMed1985

ATP and cAMP system in the in vitro response of microdissected cortical tubules to PTH.

Responsiveness of proximal convoluted tubule (PCT) and distal convoluted tubule (DCT) microdissected from mouse kidney to PTH, in terms of cAMP accumulation and stimulation of adenylate cyclase, was examined. In both PCT and DCT, the cell-free adenylate cyclase was stimulated at least 10-fold by the same dose (10 U/ml) of PTH, and activity of cAMP phosphodiesterase was about 80% higher in DCT than in PCT. In intact tubules, while the incubation with PTH increased cAMP content in DCT more than 10-fold, it failed to increase the cAMP levels in PCT. To explain discrepancies between cell-free and intact cell incubations, ATP content in microdissected tubules was determined with use of a microbioluminescence assay. ATP content in PCT (4.0 +/- 1.3 fmol/mm, n = 30) was dramatically lower than ATP content of DCT (376.8 +/- 54.3 fmol/mm, n = 25). Incubation with 1 microM rotenone reduced markedly (delta -98%) the ATP content in DCT. In DCT, with ATP depleted by 1 microM rotenone, PTH failed to increase the cAMP, although 1 microM rotenone did not inhibit the adenylate cyclase activity. When 0.1 mM of 1-methyl-3-isobutylxanthine (MIX) was added to the incubation medium, PTH caused a marked elevation in tubular cAMP in PCT and to even a greater degree in DCT. Present results show that various segments of microdissected tubules differ greatly in their ability to maintain adequate ATP levels for cAMP generation in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗

PubMed2000

Quantitative molecular analysis of laser-microdissected paraffin-embedded human tissues.

Laser microdissection enables the contamination-free isolation of morphologically defined pure cell populations from archival formalin-fixed paraffin-embedded tissue specimens. Cells isolated by this method have been characterized by a wide variety of qualitative molecular assays, e.g. loss of heterozygosity, point mutations, clonality and lineage origin. The recently introduced real-time PCR technology renders the reliable quantification of very small amounts of nucleic acids possible. Several groups including our own showed that this technique can be successfully applied for the quantification of DNA and RNA isolated from microdissected archival tissue sections, even after immunohistochemical staining. The exact analysis of quantitative changes of nucleic acids during the course of pathological alterations has thus become possible. In many situations these quantitative changes can be expected to be more important than qualitative changes. The new technology for the quantification of structural genomic alterations and changes in the gene expression pattern in conjunction with microdissection have equipped morphologists with a powerful tool to study reactive and neoplastic changes of tissues.

Cell Separation↗

PubMed2004

Chromosomal anchoring of linkage groups and identification of wing size QTL using markers and FISH probes derived from microdissected chromosomes in Nasonia (Pteromalidae: Hymenoptera).

Nasonia vitripennis is a small parasitic hymenopteran with a 50-year history of genetic work including linkage mapping with mutant and molecular markers. For the first time we are now able to anchor linkage groups to specific chromosomes. Two linkage maps based on a hybrid cross (N. vitripennis x N. longicornis) were constructed using STS, RAPD and microsatellite markers, where 17 of the linked STS markers were developed from single microdissected banded chromosomes. Based on these microdissections we anchored all linkage groups to the five chromosomes of N. vitripennis. We also verified the chromosomal specificity of the microdissection through in situ hybridization and linkage analyses. This information and technique will allow us in the future to locate genes or QTL detected in different mapping populations efficiently and fast on homologous chromosomes or even chromosomal regions. To test this approach we asked whether QTL responsible for the wing size in two different hybrid crosses (N. vitripennis x N. longicornis and N. vitripennis x N.giraulti) map to the same location. One QTL with a major effect was found to map to the centromere region of chromosome 3 in both crosses. This could indicate that indeed the same gene/s is involved in the reduction of wing in N. vitripennis and N. longicornis.

Animals↗

PubMed1995

Seeding of YACs over regions 1q41-q42.3 and 11q14.3-q23 with microdissection clones.

We describe the use of pooled, region-specific hybridisation probes to screen high-density replica filters of a human genome YAC library. The probes were derived by microdissection of an approximately 30-Mbp region subtending the translocation breakpoint on a der(1)(1;11)(q42.1;q14.3) chromosome. Of 70 microdissection clones used in pools of 4-10, 47 identified a total of 77 YAC recombinants, representing over 50% of the microdissected region. This strategy can easily be adapted to other poorly mapped subchromosomal regions of the human or other mammalian genomes and will provide a solid framework for detailed contig map constructions.

Chromosomes, Artificial, Yeast↗

PubMed1987

Effects of atrial natriuretic factor on cyclic guanosine monophosphate and cyclic adenosine monophosphate accumulation in microdissected nephron segments from rats.

Atrial natriuretic factor (ANF) (1 microM) markedly increased cyclic guanosine monophosphate (cGMP) content in microdissected glomeruli (35-fold) and in microdissected inner medullary collecting ducts (IMCD) (20-fold). ANF caused little or no increase in cGMP content in other nephron segments. The threshold concentration for increased cGMP accumulation by ANF was 0.1-1 nM in IMCD, which is in the range reported for rat plasma. Sodium nitroprusside (1 mM), which selectively stimulates soluble guanylate cyclase, increased cGMP content in glomeruli but not in IMCD. ANF did not alter cAMP accumulation in the absence or presence of vasopressin (AVP) or parathyroid hormone (PTH) in outer and inner medullary tubule suspensions, or in microdissected proximal convoluted tubules (PCT), medullary thick ascending limbs (MAL) or IMCD. These data are compatible with the hypothesis that cGMP is a second messenger for a physiologic action of ANF in the inner medullary collecting duct. ANF apparently activates membrane-bound guanylate cyclase in this segment.

Animals↗

PubMed2002

Methacarn fixation for genomic DNA analysis in microdissected, paraffin-embedded tissue specimens.

We recently found methacarn to be a versatile fixative for analysis of RNA and protein applicable for microdissected specimens from paraffin-embedded tissue (PET). In this study we investigated the performance of methacarn for genomic DNA analysis using microdissected rat tissues. We found that extensive portions of DNA up to 2.8 kb could be amplified by nested PCR using DNA templates extracted by a simple and rapid extraction procedure from a 1 x 1-mm area of cerebral cortex of a 10-microm-thick section. By nested PCR, a 522-bp fragment from a single cell could be amplified in 20% of cresyl violet-stained Purkinje cells, and the minimal number of cells required, as estimated using hippocampal neurons, was on the order of 10-20. Although tissue staining with hematoxylin and eosin affected the PCR, amplification of a 522-bp fragment was successful, with 150-270 cells by 35 cycles of single-step PCR. Immunostaining resulted in a substantial decrease of yield and degradation of extracted DNA. However, even after immunostaining, a 184-bp DNA fragment could be amplified with 150-270 cells by 35 cycles of PCR. The results thus demonstrate the superior performance of methacarn to that reported with formalin in genomic DNA analysis using microdissected PET specimens.

Acetic Acid↗