Microdissection Techniques for Molecular Testing in Surgical Pathology

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

MANUAL MICRODISSECTION

The simplest method of microdissection is the manual method, which involves use of a standard or inverted microscope. A standard microscope has the advantage of universal availability, but because of the optics, the operator's movements are inverted in the visual field. Thus, the operator must learn to identify a target and microdissect while visualizing the opposite movement. Also when using a standard microscope, if the lowest power is used the distance between the stage and the objective may be very small, making it difficult to manipulate the microdissecting implement under direct visualization. A stereomicroscope may be available in many laboratories, and the advantage of its use is that slides can be placed on the flat stage with backlight and movements under the objectives are not inverted. Relatively simple microdissection can take place while the target is visualized directly through the objectives. The larger distance between the slide and the objectives gives the operator the space necessary to manipulate the tissue on the slide under direct visualization.

Tissue sections for manual microdissection can be prepared as paraffin-embedded or frozen sections. Staining of the slides is a matter of personal preference, and many investigators are adept at interpreting unstained sections. Stains that should not be used, however, include some types of hematoxylin, especially Delafield and Mayer alum or Weigert iron hematoxylin, because these stains can interfere with DNA and RNA extraction after microdissection.16 The stains that are reported to aid in visualization and preserve nucleic acids are methyl green and nuclear fast red.17 Microdissection tissue sections that are unstained should be coupled with a hematoxylin-eosinstained scout section. First, the operator should examine the stained section and carefully mark areas for microdissection with a marker to easily compare the unstained levels for microdissection.

Microdissection from paraffin-embedded tissues will be easiest when the tissue sections are deparaffinized before beginning. To deparaffinize, a standard series of washes in xylenes and alcohol can be utilized (Table 3).18 When sections are not deparaffinized first, the tissue fragments or tissue scrolls must be deparaffinized in the tube using xylenes and alcohol before nucleic acid extraction proceeds. Care must be taken because the xylenes can degrade some plastic tubes.

Different implements have been recommended for performing microdissection, including any sharp and precise instrument such as a 30-gauge needle, a pointed surgical blade, or specialized oscillating needles with various attachments. The implement should be easy to hold in one hand, because the prosector's second hand is used to stabilize the slide, focus the microscope, and hold the tube for the microdissected tissue. Regardless of the implement used, the prosector will perform the best microdissection while looking through the eyepieces of the microscope.