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PubMed2001

Allelic loss detection in inflammatory breast cancer: improvement with laser microdissection.

Solid tumors are composed not only of tumor cells but also of stromal nonneoplastic cells. In whole tumor samples, stromal cells retaining their alleles may therefore obscure detection of loss of heterozygosity (LOH) in tumor cells. An increasing number of studies have used laser-assisted tissue microdissection to improve LOH detection, but the real gain in sensitivity has been poorly quantified. We studied a group of 16 inflammatory breast carcinomas that were submitted to both standard DNA extraction from frozen whole tumor samples and laser microdissection performed on paraffin-embedded tumor samples. Using PCR with fluorescence-labeled primers, we comparatively analyzed ten polymorphic markers with both sources of DNA. With the LOH detection threshold set at -25%, we showed that 25 LOHs could not be diagnosed with whole tumor samples out of 73 LOHs positively diagnosed in microdissected samples (34%). With the LOH detection threshold set at -50%, the respective figures were 39 LOHs not diagnosed out of 55 LOHs (71%). Measuring the intensity of the allelic decrease, we showed that the mean decrease of the lost allele is -34% with whole tumor samples and -67% with microdissected samples. The increase in sensitivity of LOH detection with microdissection is associated with the density of stromal cells. This strong improvement in LOH detection in this aggressive type of breast cancer indicates that many other molecular studies performed on heterogeneous solid tumors may benefit from a first step of laser microdissection.

Breast Neoplasms↗

PubMed1999

Testicular sperm extraction: microdissection improves sperm yield with minimal tissue excision.

Testicular sperm extraction (TESE) is often an effective method for sperm retrieval from men with non-obstructive azoospermia. However, TESE has been a blind procedure that does not identify the focal sperm-producing areas of the testicle until after tissue has been excised from the patient. Experience with a new technique of microdissection of testicular tubules is presented here that identifies sperm-containing regions before their removal. Identification of spermatogenically active regions of the testicle is possible by direct examination of the individual seminiferous tubules. The underlying concept for this technique is simple: seminiferous tubules containing many developing germ cells, rather than Sertoli cells alone, are likely to be larger and more opaque than tubules without sperm production. In a sequential series of TESE cases for men with non-obstructive azoospermia, the ability to find spermatozoa increased from 45% (10/22) to 63% (17/27) after introduction of the microdissection technique. Microdissected samples yielded an average of 160,000 spermatozoa per sample in only 9.4 mg of tissue, whereas only 64,000 spermatozoa were found in standard biopsy samples that averaged 720 mg in weight (P < 0.05 for all comparisons). For men where microdissection was attempted, successful identification of enlarged tubules was possible in 56% (15/27) of cases. However, spermatozoa were retrieved with microdissection TESE for six men in whom sperm retrieval was unsuccessful with standard TESE approaches (35% of all men with spermatozoa retrieved). These findings suggest that microdissection TESE can improve sperm retrieval for men with non-obstructive azoospermia over that achieved with previously described biopsy techniques.

Dissection↗

PubMed2000

Laser capture microdissection in pathology.

The molecular examination of pathologically altered cells and tissues at the DNA, RNA, and protein level has revolutionised research and diagnostics in pathology. However, the inherent heterogeneity of primary tissues with an admixture of various reactive cell populations can affect the outcome and interpretation of molecular studies. Recently, microdissection of tissue sections and cytological preparations has been used increasingly for the isolation of homogeneous, morphologically identified cell populations, thus overcoming the obstacle of tissue complexity. In conjunction with sensitive analytical techniques, such as the polymerase chain reaction, microdissection allows precise in vivo examination of cell populations, such as carcinoma in situ or the malignant cells of Hodgkin's disease, which are otherwise inaccessible for conventional molecular studies. However, most microdissection techniques are very time consuming and require a high degree of manual dexterity, which limits their practical use. Laser capture microdissection (LCM), a novel technique developed at the National Cancer Institute, is an important advance in terms of speed, ease of use, and versatility of microdissection. LCM is based on the adherence of visually selected cells to a thermoplastic membrane, which overlies the dehydrated tissue section and is focally melted by triggering of a low energy infrared laser pulse. The melted membrane forms a composite with the selected tissue area, which can be removed by simple lifting of the membrane. LCM can be applied to a wide range of cell and tissue preparations including paraffin wax embedded material. The use of immunohistochemical stains allows the selection of cells according to phenotypic and functional characteristics. Depending on the starting material, DNA, good quality mRNA, and proteins can be extracted successfully from captured tissue fragments, down to the single cell level. In combination with techniques like expression library construction, cDNA array hybridisation and differential display, LCM will allow the establishment of "genetic fingerprints" of specific pathological lesions, especially malignant neoplasms. In addition to the identification of new diagnostic and prognostic markers, this approach could help in establishing individualised treatments tailored to the molecular profile of a tumour. This review provides an overview of the technique of LCM, summarises current applications and new methodical approaches, and tries to give a perspective on future developments. In addition, LCM is compared with other recently developed laser microdissection techniques.

Cell Adhesion↗

PubMed2001

Effect of tissue processing on the ability to recover nucleic acid from specific renal tissue compartments by laser capture microdissection.

The anatomic heterogeneity of the nephron poses obstacles to microdissection of individual renal compartments for analysis of gene expression. We have systematically analyzed the effects of fixation time and nuclear staining on the ability to recover nucleic acid from individual renal compartments by laser capture microdissection (LCM). Formalin-fixed kidney sections from Wistar rats and archival human renal biopsies were used for DNA analysis. From 1 to 10 individual glomeruli and from 1 to 10 individual proximal tubules were captured sequentially onto polymer films. DNA for beta-globin could be amplified by PCR from even a single glomerulus or tubule. Optimal conditions for DNA amplification were brief (1- or 2-day) formalin fixation. Use of nuclear counterstains, including Weigert's hematoxylin, Harris's hematoxylin, Mayer's hematoxylin, or methyl green, did not adversely affect the ability to extract and amplify DNA. For RNA extraction, glomeruli and tubules were microdissected from renal cryostat sections stored for up to 6 months. By RT-PCR, mRNA expression of the glomerulus-specific gene, Wilms' tumor-1, was identified in as few as 5 microdissected glomeruli and of the tubule-specific gene, aminopeptidase N, in as few as 5 microdissected tubules, with no cross-contamination between renal compartments. Our findings indicate that the LCM method can successfully microdissect pure glomerular and tubular tissue compartments and that the optimal fixation and staining conditions are those employed routinely for renal biopsies, namely overnight formalin fixation and hematoxylin counterstain for DNA extraction, and cryostat sectioning with hematoxylin counterstain for RNA extraction. The specificity of LCM coupled with the sensitivity of RT-PCR should prove a powerful tool for the analysis of gene expression in specific renal compartments from archival human renal biopsies.

Animals↗

PubMed2000

Laser microdissection and microsatellite analyses of breast cancer reveal a high degree of tumor heterogeneity.

Carcinomas with productive fibrosis are the most common forms of breast cancer. Analysis of tumor-specific genomic alterations can be compromised by the presence of normal cells, demanding microdissection of small tumor areas to detect loss of heterozygosity (LOH) and microsatellite instability (MSI). The aim of this study was to evaluate the importance of precise laser microdissection for microsatellite analyses and investigation of tumor heterogeneity in breast cancer. 39 primary breast tumor samples were analyzed for MSI and LOH by PCR followed by polyacrylamide gel electrophoresis and silver staining using 15 microsatellite markers. Different tumor areas were processed separately in 30 patients. Both intraductal and invasive breast cancer regions were investigated in 11 patients. The following results were obtained: (1) accurate microdissection revealed MSI in 3 or more of the investigated markers (> or =20%) in 33% of the patients, a higher frequency than reported previously; (2) laser microdissection was 43% more sensitive in detection of LOH compared to manual microdissection due to a reduction of contamination by normal cells, and (3) 29 of 30 investigated tumors showed heterogeneity of genetic alterations in different tumor regions. Laser-based microdissection is a valuable tool in genetic analysis of desmoplastic tumors and allows an accurate determination of genetic alterations in histologically different tumor regions.

Adult↗

PubMed2002

Microdissection techniques for cancer analysis.

One difficulty in studying molecular changes of tumours has been the inability to isolate DNA and RNA from a homogeneous cell population. The combination of several new technologies should help overcome these hurdles. Microdissection is a technique for rapid and easy procurement of a pure cellular subpopulation away from its complex tissue milieu. Laser-assisted microdissection has recently been identified as a quick, simple and effective method by which microdissection of complex tissue specimens can be routinely performed for molecular analysis. With the advent of laser microdissection, cDNA libraries can be developed from pure cells obtained directly from stained neoplastic tissue, and microarrays of thousands of genes can now be used to examine gene expression in microdissected tumour tissue samples. This review will concentrate on the application of different microdissection techniques in the area of cancer research.

Cell Separation↗

PubMed2002

[Application of microdissection combined with RNA linear amplification].

BACKGROUND & OBJECTIVE: Microdissection has become indispensable for the selective analysis of stroma-free tumor cell. However, To obtain sufficient RNA from microdissected cells is difficult. The study was designed to seek a specific way to separate nasopharyngeal carcinoma (NPC) cells from stromal cells and to amplify the RNA from microdissected NPC cells. METHODS: NPC cells were obtained using microdissection from frozen NPC tissue sections, then RNA was extracted from the microdissected NPC cells and reverse transcribed in vitro. The expression levels of beta-actin and GADPH in amplified RNA were detected using RT-PCR. RESULTS: About 20,000-40,000 NPC cells were obtained, RNA was extracted from the cells, about 0.5-2.5 kb RNA fragments were obtained after RNA linear amplification and beta-actin and GADPH levels were integral. CONCLUSION: Microdissection combined with RNA linear amplification can be used to successfully obtain pure NPC cells, the integrity of amplified RNA is good and can be used in further research.

Dissection↗

PubMed2004

Serial follow-up study of serum testosterone and antisperm antibodies in patients with non-obstructive azoospermia after conventional or microdissection testicular sperm extraction.

Testicular sperm extraction (TESE) combined with intracytoplasmic sperm injection is becoming a first-line treatment even for non-obstructive azoospermia. The current focus of TESE is the identification of seminiferous tubules that contain spermatozoa and minimization of testicular damage. Although microdissection TESE has been introduced as a preferred procedure for sperm retrieval, no serial follow-up studies of testicular damage have been reported. In the present study, we assayed serum testosterone concentrations and for the presence of antisperm antibodies (ASA) for 1 year after conventional multiple TESE or microdissection TESE and compared postoperative testicular damage between procedures. Thirteen patients who underwent conventional multiple TESE and 12 patients who underwent microdissection TESE were included in this study. Serum total and free testosterone concentrations were evaluated before operation and 1, 6 and 12 months after TESE. Serum ASA was also evaluated before and 12 months after TESE. Serum total and free testosterone concentrations in all patients in both groups showed no significant postoperative decrease. A comparison between the two groups of serum total and free testosterone concentrations showed no significant difference (total testosterone, p = 0.2477; free testosterone, p = 0.3098). No incidence of new ASA formation was identified in the present study. In conclusion, TESE procedures cause neither a decrease of serum testosterone nor formation of ASA. Serum testosterone concentration are similar between patients in the conventional multiple TESE and microdissection groups. Therefore, microdissection TESE is safe with respect to testicular damage, particularly for patients with hypogonadism.

Adult↗

PubMed2004

The influence of immunohistochemistry on mRNA recovery from microdissected frozen and formalin-fixed, paraffin-embedded sections.

Laser-assisted microdissection (LAM) is now widely used to obtain specific cell populations from heterogeneous tissues. A major disadvantage of LAM is poor tissue morphology during microscopy, in part because coverslips are not used. Immunohistochemical labeling can improve identification of target cells but may affect the subsequent analysis of the microdissected tissue. We studied the effect of immunohistochemistry (IHC) on mRNA recovery from labeled cells after microdissection from both frozen and formalin-fixed, paraffin-embedded (FFPE) sections, using Melan-A and Ki-67 staining in lymph nodes with metastatic melanoma as a model. We developed rapid protocols for immunostaining in an attempt to limit loss of mRNA during procedures. A sensitive real-time quantitative reverse transcription-PCR was used to measure mRNA. We found a marked decrease in the mRNA yield from 500 microdissected cells from frozen and paraffin sections after immunostaining for both markers. Recovery of mRNA decreased by up to 89%, comparing the immunostained with the routinely stained sections. Interestingly, the ratio between mRNA for the two markers was similar in all stains, indicating that immunostained sections may be used for mRNA analysis. We also investigated the effect of storing membrane-mounted sections for microdissection under different conditions. Slides mounted with paraffin sections could be stored at room temperature for up to 90 days with no significant decrease in mRNA recovery.

Actins↗

PubMed1996

Chromosome microdissection in leukemia: a powerful tool for the analysis of complex chromosomal rearrangements.

In many human cancers the presence of marker chromosomes or unbalanced translocations prevents complete karyotypic analysis. Chromosome microdissection has become an increasingly important method for assessing chromosome rearrangements. However, most studies using chromosome microdissection have been carried out on established cancer cell lines that provide an unlimited supply of abnormal metaphase cells. We have routinely performed microdissection of as few as three marker chromosome copies from short-term cultures of acute myeloid leukemias, followed by in vitro DNA amplification and fluorescence in situ hybridization (FISH) to normal metaphase spreads. Using this type of "reverse chromosome painting," we were able to characterize precisely the chromosomal constitution of each marker chromosome in the samples, confirming the diagnostic usefulness of microdissection in cancer cytogenetics. In addition, in one leukemia with atypical cytological features, microdissection enabled us to detect a novel retinoic acid receptor alpha gene rearrangement.

Acute Disease↗

PubMed2003

Differential gene expression analysis using paraffin-embedded tissues after laser microdissection.

Recent advances in laser microdissection allow for precise removal of pure cell populations from morphologically preserved tissue sections. However, RNA from paraffin-embedded samples is usually degraded during microdissection. The purpose of this study is to determine the optimal fixative for RNA extractions from laser microdissected paraffin-embedded samples. The integrity of RNA was evaluated with the intactness of 18S and 28S ribosomal RNA by electrophoresis and by the length of individual gene transcripts using RT-PCR. The various fixatives were methacarn (a combination of methanol, chloroform, and acetic acid) and several concentrations of ethanol and isopropanol. Methacarn was the optimal fixative for RNA preservation in paraffin-embedded tissues, which included liver, lung, kidney, muscle, and limb. Based on RT-PCR analysis, methacarn fixed samples exhibited the expected RNA sizes for individual genes such as glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and bone-related genes (e.g., alkaline phosphatase and osteonectin). The laser microdissection technique with methacarn fixation was then applied to analyze the differential gene expression between hypertrophic and proliferative chondrocytes in the growth plate of long bone. The expression of type X collagen, a specific gene for hypertrophic chondrocytes, was only observed in hypertrophic chondrocytes, while type II collagen was observed more broadly in the growth plate as anticipated. Thus, combining laser microdissection with methacarn fixation facilitates the examination of differentially expressed genes from various tissues.

Acetic Acid↗

PubMed1999

Microassay of 5'-nucleotidase and adenosine deaminase activity in microdissected nephron segments.

The present study describes a new method for microassay of the activity of 5'-nucleotidase (5'-ND) and adenosine deaminase (ADA) in the microdissected nephron segments. The nephron segments including glomeruli, proximal convoluted and straight tubules (PCT and PST), cortical and medullary thick ascending limbs, and cortical and medullary collecting ducts were microdissected. 5'-ND and ADA in the nondenatured lysate of 20-mm microdissected tubules and 20 glomeruli were separated by agarose gel electrophoresis and by isoelectric focusing, respectively. The gels were incubated with specific substrates and staining dyes to exhibit the dephosphorylation by 5'-ND or deamination by ADA. The enzyme activity was estimated by measuring the intensity of the reaction bands on the gels. The 5'-ND activity was detected in all microdissected tubular segments and glomeruli. Among these nephron segments, PCT and PST exhibited the greatest enzyme activity, averaging 1142 and 939 mU/mg tissue protein, respectively. The activity of ADA was also detected in all tubular segments and glomeruli. However, the greatest activity of this enzyme was found in the glomeruli (649.8 mU/mg protein). Using reverse transcriptase-polymerase chain reaction technique, we verified the presence of mRNA of 5'-ND and ADA in all microdissected tubular segments and glomeruli. Based on these results, we conclude that 5'-ND and ADA are present in all nephron segments studied, but the activity of these enzymes is nonuniformly expressed along the nephron. This microassay is a highly specific, sensitive, and reliable method for the segmental analysis of adenosine metabolism in the kidney.

5'-Nucleotidase↗

PubMed1994

Rapid generation of whole chromosome painting probes (WCPs) by chromosome microdissection.

A strategy for rapid construction of whole chromosome painting probes (WCPs) by chromosome microdissection has recently been developed. WCPs were prepared from 20 copies of each target chromosome microdissected from normal human metaphase chromosomes and then directly amplified by PCR using a universal primer. Fifteen WCPs, including chromosomes 1, 3, 6, 7, 9, 12, 13, 14, 15, 17, 19, 20, 21, 22, and X, have been generated using this strategy. The probe complexity and hybridization specificity of these WCPs have been characterized by gel electrophoresis and fluorescence in situ hybridization. Analysis of WCPs constructed by chromosome microdissection indicated that microdissected WCPs invariably provide strong and uniform signal intensity with no cytologically apparent cross-hybridization. To demonstrate the application of WCPs generated from microdissection, we have used these probes to detect complex chromosome rearrangements in a melanoma cell line, UM93-007. Two different translocations involving three chromosomes [t(1;3;13) and t(1;7;13)] have been identified, both of which were undetectable by conventional banding analysis. Further application of these WCPs (including generation of WCPs from mouse and other species) should greatly facilitate the cytogenetic analysis of complex chromosome rearrangements.

Animals↗

PubMed1994

Use of chromosome microdissection, the polymerase chain reaction, and dot blot hybridization to analyze double minute chromosomes.

The potential usefulness of chromosome microdissection, the polymerase chain reaction (PCR), and dot blot hybridization as a quick screening method for determining the genetic composition of double minute chromosomes (DMs) was evaluated. DMs or abnormally banding regions (ABRs) were microdissected from multidrug-resistant hamster cell lines and amplified with PCR using primers specific for the hamster multidrug-resistance (MDR) gene, pgp 1. The microdissected-PCR-amplified products were shown to (a) hybridize to a 32P-labeled pCHP1 probe for the hamster MDR gene by using dot blot or Southern blot analysis and also (b) hybridize back to the chromosome region from which they were originally dissected by using fluorescent in situ hybridization. Microdissected/PCR-amplified DMs were also shown to hybridize to ABRs. When microdissected DMs and ABRs were amplified using hamster specific Alu primers, the resulting material was shown to hybridize with probes for hamster MDR and Alu. These results suggest that the DMs contained in these MDR hamster cell lines contain Alu-like sequences and the chromosome microdissection-PCR-hybridization approach might be used as a quick screening method for identifying genes amplified in DMs and ABRs in cell lines and human tumor samples.

Animals↗

PubMed2001

A new method for histological microdissection utilizing an ultrasonically oscillating needle: demonstrated by differential mRNA expression in human lung carcinoma tissue.

Molecular analysis of microdissected tissue samples is used for analyzing tissue heterogeneity of histological specimens. We have developed a rapid one-step microdissection technique, which was applied for the selective procurement of tissue areas down to a minimum of 10 cell profiles. The special features of our microdissection system consist of an ultrasonically oscillating needle and a piezo-driven micropipette. The validity of this technique is demonstrated in human lung large-cell carcinoma by real-time quantitative reverse transcriptase-polymerase chain reaction assays of vimentin, cyclin D1, and carcinoembryonic antigen after linear RNA amplification. mRNA expression values of microdissected samples scattered around those of bulk tumor tissue and showed differential mRNA expression between samples of tumor parenchyma and supportive stromal cells for vimentin and carcinoembryonic antigen as confirmed by immunohistochemistry. In conclusion, this procedure requires simple equipment, is easily performed, and delivers microdissected tissue samples of oligocellular clusters suitable for further molecular analysis.

Carcinoembryonic Antigen↗

PubMed1999

Immuno-LCM: laser capture microdissection of immunostained frozen sections for mRNA analysis.

Microdissection of routinely stained or unstained frozen sections has been used successfully to obtain purified cell populations for the analysis of cell-specific gene expression patterns in primary tissues with a complex mixture of cell types. However, the precision and usefulness of microdissection is frequently limited by the difficulty to identify different cell types and structures by morphology alone. We therefore developed a rapid immunostaining procedure for frozen sections followed by laser capture microdissection (LCM) and RNA extraction, which allows targeted mRNA analysis of immunophenotypically defined cell populations. After fixation, frozen sections are immunostained under RNAse-free conditions using a rapid three-step streptavidin-biotin technique, dehydrated and immediately subjected to LCM. RNA is extracted from captured tissue, DNAse I treated, and reverse transcribed. Acetone-, methanol-, or ethanol/acetone-fixed sections give excellent immunostaining after 12 to 25 minutes total processing time. Specificity, precision, and speed of microdissection is markedly increased due to improved identification of desired (or undesired) cell types. The mRNA recovered from immunostained tissue is of high quality. Single-step PCR is able to amplify fragments of more than 600 bp from both housekeeping genes such as beta-actin as well as cell-specific messages such as CD4 or CD19, using cDNA derived from less than 500 immunostained, microdissected cells. Immuno-LCM allows specific mRNA analysis of cell populations isolated according to their immunophenotype or expression of function-related antigens and significantly expands our ability to investigate gene expression in heterogeneous tissues.

Cells↗

PubMed1998

Laser-assisted microdissection of the zona pellucida facilitates polar body biopsy.

OBJECTIVE: To investigate whether polar body biopsy can be performed after laser microdissection of the zona pellucida (ZP). DESIGN: Mouse zygotes were allocated randomly to three groups. The zygotes were subjected to laser microdissection of the ZP and polar body biopsy (group 1), laser microdissection alone (group 2), or no treatment (group 3). SETTING: University-based IVF program. PATIENT(S): Animal study. INTERVENTION(S): A hole was drilled in the ZP of mouse zygotes using a 1.48-micron noncontact diode laser. A microneedle was inserted and the polar body was aspirated. MAIN OUTCOME MEASURE(S): The efficacy of polar body biopsy after laser microdissection of the ZP was evaluated. RESULT(S): The laser diode beam allowed for precise drilling of a 14- to 18-micron hole in the ZP. Polar bodies could be aspirated without damaging the zygote and did not disintegrate during the biopsy. Zygotes developed to blastocysts and underwent the same hatching as control zygotes. Lower hatching rates were observed in untreated zygotes. CONCLUSION(S): Laser microdissection of the ZP with a noncontact laser system facilitates subsequent polar body biopsy. The use of blunt-ended micropipettes greatly reduces the risk of damage to the zygote or the polar body. This procedure makes polar body biopsy more accurate and effective for preimplantation genetic diagnosis.

Animals↗

PubMed2002

Use of laser microdissection in complex tissue.

Concomitant with the rapid development in biomedical knowledge, including the methods of molecular biology and proteomics, and the manufacture of ever more precise optical instruments, powerful lasers, and sophisticated microcomputing hardware and software, laser microdissection systems have emerged which are now entering the field of routine research. Today, several devices are commercially available, congresses devoted to the latest advances in laser microdissection are now held on regular occasions, and the number of publications based on the use of these techniques has risen to over 250. With laser microdissection, histological treatment, such as chemical or immunological fixation and staining, can readily be combined with methods suitable for molecular biology or proteomics. As the optical, technical, and methodological resolution of polymerase chain reaction (PCR) and microdissection increases, genetic and phenotypic studies of biological material are possible even at the level of single cells and subcellular elements. Moreover, questions such as the paracrine interaction of cells within complex tissues, the development of cancer, and the role of single cells in tissue remodeling or development on the microscopic and molecular level can now be addressed precisely at the molecular level. This chapter reviewed the development of laser microdissection platforms, its potential impact on the future of research, and how, in particular, these technologies can be successfully integrated into modern research and routine histopathological studies of complex tissue.

Animals↗