Microdissection Techniques for Molecular Testing in Surgical Pathology

Archives of Pathology & Laboratory Medicine, Dec 2004 by Hunt, Jennifer L, Finkelstein, Sydney D

Objective.-To describe the techniques for microdissection of paraffin-embedded and frozen tissue sections for the use in molecular applications.

Data Sources.-Original research papers and review papers and the authors' personal experiences.

Data Synthesis.-Manual and laser-capture microdissection are described in detail, with specific protocols for sample preparation and instructions for performing the microdissection. A section addressing frequently asked questions is also included.

Conclusions.-Microdissection is a technique that is very useful both in the research setting and for clinical molecular testing in paraffin-embedded tissue samples. The available techniques range from simple and inexpensive (manual microdissection) to complex and expensive (laser-capture microdissection). All of the techniques, however, require the user to be familiar with microscopy and histology.

(Arch Pathol Lab Med. 2004;128:1372-1378)

Molecular testing in anatomic pathology is becoming more prevalent, in both diagnostic and prognostic clinical applications and in translational research on tissue samples. It is critical, however, to overcome the innate difficulties of tissue heterogeneity when designing assays that use molecular techniques. Techniques for addressing cellular and tissue heterogeneity have included tissue homogenization (single cell suspension), gradient controlled cell separation (eg, ficoll), and expression-based cell separation (flow cytometry). These techniques may allow gross separation of tumor cells from lymphocytes in a lymph node, for example, or isolation of cellular components with specific phenotypes. These assays are ideal when substantial amounts of fresh tissue are available and morphologic discrimination is not necessary.

Archived paraffin-embedded material, however, is an attractive resource for investigators in pathology and basic sciences. These quality-controlled, histologically verified tissues are available in many surgical pathology laboratory archives. When using them for research purposes, it may be possible to include subjects with long-term clinical follow-up data available with appropriate authorization. To separate the cellular components in these samples, tissue microdissection may be necessary to obtain relatively pure cellular samples of morphologically confirmed cell types. Various methods for microdissection have been developed for use in frozen or paraffin-embedded tissue sections.1 These techniques allow the investigator to visually select the cells or areas of interest and to separate them cleanly from the background tissue. The techniques that are most commonly used for tissue microdissection are reviewed here.

Our understanding of the molecular events associated with disease processes is growing exponentially, and the applications of molecular biological testing to anatomic pathology tissues are becoming richer and more diverse every year. Although many of the tests and techniques are experimental, translation from basic science to clinical applicability is occurring regularly. The best examples of transition from bench to bedside can be seen in hematopathology, where diagnostic molecular testing is now performed regularly as part of the clinical workup and management of lymphomas and leukemias.2 Molecular tests are being used both for initial diagnosis and in following patients for minimal residual disease over the course of their treatment.3 Other applications that are used diagnostically include testing for genetic translocations in sarcomas,4 gene loss in gliomas,5,6 and gene amplifications in breast cancer and other types of tumors.7

Currently, many of the polymerase chain reaction (PCR)-based diagnostic molecular tests yield positive or negative results to identify tumor-specific genetic changes. Because any amplification is diagnostic of a disease process, gross tissue samples can be used with little need to isolate cell populations or to start testing with pure samples. However, as molecular testing becomes more focused on quantitation of molecular targets, there will be a need to begin testing with relatively pure cell populations. Tissue samples without microdissection may not be ideal for these types of applications because they may be contaminated with normal stromal and lymphoid tissues. The pathologist, therefore, is likely to be faced with requests for tissue microdissection to enhance the clinical value of molecular testing on tissue samples.8

One of the major benefits of microdissection is the proauction of relatively pure samples of morphologically confirmed cellular populations.L) This relative purity may be essential for assessing a genetic change or for quantification of genetic changes. For example, in assessing possible loss of heterozygosity of tumor suppressor genes, normal contaminating cells will artifactually equalize the allelic balance, and it may appear that the tumor cells contain normal DNA. In molecular identity testing for tissue floaters (tissue carryover artifacts), microdissection is essential for separating the fragments of the suspected floater from the rest of the tissue sample.10,11 When examining expression of some gene targets in tumor cells at the mRNA level, results may be confounded by the expression found in normal contaminating stromal and lymphoid cells. In some diseases it may be important to measure events in rare single neoplastic cells. Perhaps the most notable example is in Hodgkin lymphoma, where molecular experiments require detailed microdissection to isolate the Reed-Sternberg cells from the surrounding lymphoid infiltrates.12,13