Improved resolution by mounting of tissue sections for laser microdissection.

BACKGROUND: Laser microbeam microdissection has greatly facilitated the procurement of specific cell populations from tissue sections. However, the fact that a coverslip is not used means that the morphology of the tissue sections is often poor. AIMS: To develop a mounting method that greatly improves the morphological quality of tissue sections for laser microbeam microdissection purposes so that the identification of target cells can be facilitated. METHODS: Fresh frozen tissue and formalin fixed, paraffin wax embedded tissue specimens were used to test the morphological quality of mounted and unmounted tissue. The mounting solution consisted of an adhesive gum and blue ink diluted in water. Interference of the mounting solution with DNA quality was analysed by the polymerase chain reaction using 10-2000 cells isolated by microdissection from mounted and unmounted tissue. RESULTS: The mounting solution greatly improved the morphology of tissue sections for laser microdissection purposes and had no detrimental effects on the isolation and efficiency of amplification of DNA. One disadvantage was that the mounting solution reduced the cutting efficiency of the ultraviolet laser. To minimise this effect, the mounting solution should be diluted as much as possible. Furthermore, the addition of blue ink to the mounting medium restores the cutting efficiency of the laser. CONCLUSIONS: The mounting solution is easy to prepare and apply and can be combined with various staining methods without compromising the quality of the DNA extracted.     

Molecular profiling of cancer

The objective of molecular profiling of cancer is to determine the differential expression of genes and proteins from human tissue in the progression from normal precursor tissue to preneoplastic tissue to cancer in order to discover diagnostic, prognostic, and therapeutic markers. With the development of high-throughput analytical techniques such as microarrays and 2-D PAGE as well as the development of tools for cell procurement from histological sections such as laser capture microdissection (LCM), it is now possible to perform molecular analyses on specific cell populations from tissue. Since recognition of specific cell populations is critical, there is a need to optimize fixation and embedding not only to improve preservation of biomolecules, but also to maintain excellent histology. We have shown that 70% ethanol fixation of prostate tissue improves the recovery of DNA, RNA, and proteins over routine formalin fixation and maintains histological quality comparable to formalin. There is also a need to develop new technologies in order to expand the range of tissue types that can be analyzed. The development and applications of Layered Expression Scanning (LES) for the molecular analysis of whole tissue sections are discussed.     

Laser microdissection and pressure catapulting (LMPC) in paraffin sections mounted on glass slides. A methodological report

The technique of laser microdissection together with laser pressure catapulting (LMPC) is demonstrated in paraffin sections obtained from surgical specimens of brain tumors mounted on glass slides. A sufficient and precise application of microdissection techniques in tissue on glass slides is worthwhile, since it offers the possibility of a retrospective analysis of archived paraffin sections in histopathology. We could demonstrate a precise dissection of areas in tissues of different thicknesses (4 microm and 20 microm). Areas of tissue mounted directly on glass need to be dissected in a scanning mode in order to remove the total region in form of small tissue fragments row by row. This mode provided a precise microdissection of tissue areas of different sizes and shapes. A successful molecular biological analysis of the microdissected regions could be demonstrated. As an example for such an analysis, differential-PCR for detecting an amplification of the gene for the epidermal growth factor receptor (EGFR) was performed.     

A microdissection technique for archival DNA analysis of specific cell populations in lesions < 1 mm in size.

We have developed a microdissection technique that allows for procurement and analysis of specific, minute cell populations from routine, 5-mu, formalin-fixed, paraffin-embedded histological tissue sections. Lesions < 1 mm in size can be specifically examined. Cells of interest are procured under direct microscopic visualization followed by a single-step DNA extraction and subsequent polymerase chain reaction. Amplification of DNA from selected cell populations was demonstrated by detecting a loss of heterozygosity (LOH) at the von Hippel-Lindau disease (VHL) gene in an atypical renal lesion and a renal cell carcinoma in a kidney of a VHL patient. Moreover, previously unrecognized LOH on the short arm of chromosome 3 (3p25-26) was detected in microdissected colorectal carcinoma cells in a non-VHL patient with sporadic colon carcinoma. This technique should prove useful in DNA studies of small lesions and cell populations. Furthermore, microscopic premalignant, in situ, and invasive lesions can be selectively examined.     

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.     

Persistent p53 mutations in single cells from normal human skin

Epidermal clones of p53-mutated keratinocytes are abundant in chronically sun-exposed skin and may play an important role in early development of skin cancer. Advanced laser capture microdissection enables genetic analysis of targeted cells from tissue sections without contamination from neighboring cells. In this study p53 gene mutations were characterized in single cells from normal, chronically sun-exposed skin. Biopsies were obtained from skin subjected to daily summer sun and skin totally protected from the sun by blue denim fabric. Using laser capture microdissection, 172 single-cell samples were retrieved from four biopsies and analyzed using single-cell polymerase chain reaction and direct DNA sequencing. A total of 14 different mutations were identified in 26 of 99 keratinocytes from which the p53 gene could be amplified. Mutations displayed a typical UV signature and were detected in both scattered keratinocytes and in a small cluster of p53-immunoreactive keratinocytes. This minute epidermal p53 clone had a diameter of 10 to 15 basal cells. Two missense mutations were found in all layers of epidermis within the p53 clone. The presented data show that p53 mutations are common in normal skin and that a clone of keratinocytes with a mutated p53 gene prevailed despite 2 months of total protection from ultraviolet light.     

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.     

Prospects for tissue-specific analysis of gene expression in Xenopus embryos through laser-mediated microdissection of histological sections

Analysis of spatiotemporal patterns of gene expression is an important prerequisite for understanding the molecular basis of embryogenesis. Tissue-specific resolution is desirable, but often not achieved owing to methodical limitations. We used a common model system for embryonic development--the South African clawed frog Xenopus laevis--to demonstrate that laser microdissection and laser-mediated catapulting of tissue samples from histologic sections are feasible even for yolk-rich, fragile embryonic tissue. A combination with RT-PCR provides the possibility of detecting tissue-specific gene expression with high resolution and fidelity. We show that specimens of various sizes and shapes can easily be procured by laser microdissection and pressure catapulting (LMPC). Subsequent RNA-isolation and nested RT-PCR for marker genes revealed that the combination of these methods allows for analysis of specific gene expression in micro-areas. We report on the efficiency and reliability of detection of marker genes in dissected tissue. We further discuss the question of whether such a combination can be applied to certain issues raised in developmental biology with regard to other techniques.     

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.     

Pitfalls in diagnostic molecular pathology – significance of sampling error

Today, molecular diagnostic tests are widely used in clinical medicine with polymerase chain reaction (PCR)-based techniques being of particular interest. In tissue specimens, however, false-positive and false-negative results can be obtained if pathomorphological and processing aspects are not considered. We therefore studied the impact of tissue sampling in three widely used diagnostic tests: (1) assessment of clonality in B-cell non-Hodgkin's lymphoma, (2) analysis of microsatellite instability (MSI) in colorectal neoplasia, and (3) demonstration of mycobacterium tuberculosis. Tissue sections of routinely formalin-fixed and paraffin-embedded diagnostic specimens were taken, and the significance of sampling was systematically investigated using laser microdissection or processing of the entire section. PCR analyses were done according to standard protocols. False-positive pseudo-monoclonality was obtained in small gastrointestinal biopsies and in laser microdissected lymph follicles of non-neoplastic tonsils. False negativity (pseudo-stability) could be demonstrated in a case with hereditary rectal adenoma if whole tissue sections were taken without microdissection of the most dysplastic subareas. Demonstration of mycobacteria failed in tissue sections of a formalin-fixed lymph node that was positive after complete digestion or in fresh frozen material of the same patient. In diagnostic molecular pathology, sampling error is an important source of false-positive and false-negative results, particularly if disease- and tissue-specific morphological features, such as sample size, type of fixation, and intralesional heterogeneity, are ignored.     

Laser capture microscopy.

Human tissues are composed of complex admixtures of different cell types and their biologically meaningful analysis necessitates the procurement of pure samples of the cells of interest. Many approaches have been used in attempts to overcome this difficulty, including a variety of microdissection methods. This review concerns a recent advance in microdissection techniques, namely laser capture microdissection (LCM). The principle underlying this technique is outlined, and practical issues pertaining to LCM are considered. In addition, the literature relating to LCM is reviewed, with examples of research applications of this technique being outlined.     

Selective genetic analysis of p53 immunostain positive cells.

The isolation of p53 immunostain positive cells from histological sections for molecular genetic studies is a difficult task, especially if there are few positive cells. To eliminate contaminating DNA from p53 negative cells, which can obscure the results of molecular assays, a variation on the technique of immunohistoselective sequencing was developed. This is a highly selective approach, whereby immunostained sections of formalin fixed, paraffin wax embedded tissue are exposed to ultraviolet irradiation to damage the DNA in p53 negative cells. The DNA in positive cells remains unaffected because the dark immunostain protects their nuclei from ultraviolet light. Polymerase chain reaction single strand conformation polymorphism of samples enriched with p53 immunostain positive cells has shown that this method can produce pure samples of mutated DNA. The isolation of DNA from minority immunostain positive cells allows a wide range of molecular analyses to be carried out on these samples, which would otherwise be hampered by the problem of contaminating background cells.     

Vascular endothelial growth factor-A is expressed both on lymphoma cells and endothelial cells in angioimmunoblastic T-cell lymphoma and related to lymphoma progression

Vascular endothelial growth factor-A (VEGF-A), a main stimulator of endothelial cell proliferation, plays an important role on tumor angiogenesis. Angioimmunoblastic T-cell lymphoma (AITL) show the most prominent vascular component among lymphomas and their prognosis is difficult to predict. To assess the clinical significance of VEGF-A in AITL, VEGF-A gene expression was studied in the tumoral lymph nodes of 24 patients using laser microdissection and quantitative polymerase chain reaction. VEGF-A gene was overexpressed in both microdissected lymphoma and endothelial cells. Increased levels of VEGF-A gene expression in lymphoma cells, as in endothelial cells, were related to extranodal involvement and to short survival time. Accordingly, VEGF-A protein expression was also found in both types of cells in lymph nodes and bone marrows with lymphomatous involvement. Triple immunofluorescent labeling on lymph node sections showed that VEGF-A protein and its receptor VEGF-R1 were coexpressed on endothelial cells of microvessels in the areas of lymphoma invasion. In these areas, ultrastructural study showed dystrophic microvessels. Taken together, the value of VEGF-A gene expression as an adverse prognostic marker in AITL should thus be considered. In addition to lymphoma cells themselves, the vascular component, a critical pathologic characteristic in AITL, also contributes to lymphoma progression.     

An exfoliation and enrichment strategy results in improved transcriptional profiles when compared to matched formalin fixed samples

Background  

Identifying the influence formalin fixation has on RNA integrity and recovery from clinical tissue specimens is integral to determining the utility of using archival tissue blocks in future molecular studies. For clinical material, the current gold standard is unfixed tissue that has been snap frozen. Fixed and frozen tissue however, both require laser capture microdissection to select for a specific cell population to study. The recent development of a sampling method capable of obtaining a viable, enriched cell population represents an alternative option in procuring cells from clinical material for molecular research purposes. The expression profiles of cells obtained by using this procurement approach, in conjunction with the profiles from cells laser capture microdissected from frozen tissue sections, were compared to the expression profiles from formalin fixed cells to determine the influence fixation has on expression profiles in clinical material.