Microdissection Techniques for Molecular Testing in Surgical Pathology

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

Tissue fragments must adhere to the microdissecting implement to be deposited into the collection tube. This adherence can be either through electrostatic forces (if the tissue is dry) or through hydrostatic forces (if the tissue is slightly damp or wet). Damp tissue fragments are somewhat easier to manipulate because the fragments will roll up into a compact, easy-to-manipulate ball, which can be deposited into the collection tube. If microdissection is performed immediately after deparaffinization, the tissue will still be damp from the final immersion in 3% glycerol. If the slides have been stored, the tissue will be brittle and dried out. Slides can be immersed in 3% glycerol for 1 to 2 minutes to rehydrate, with removal of the fluid layer before microdissection. Alternatively, a nonreactive liquid (3% glycerol, Tris-borate-ethylenediaminetetraacetic acid buffer, or deionized water) can be used to moisten the tissue sections by dipping the microdissecting implement into the fluid and allowing the tip to gently touch the surface of the tissue. The liquid will wick across a small area of the tissue, causing the microdissected tissue to form a small sticky ball of cells, which can be deposited into the collection tube.

LASER-CAPTURE MICRODISSECTION

LCM was first described in 1996, and commercial machines became available soon after.19 Preparation of slides for microdissection will depend on the machine being used and the molecular application. For standard PCRbased assays, formalin-fixed, paraffin-embedded tissue samples can be prepared with routine 5- to 10-�m-thick sections. Frozen sections are ideal for RNA applications and will probably yield the most optimal PCR template because the nucleic acids are relatively intact20,21 (see Table 2 for protocol).

Slides for LCM must be deparaffinized before microdissection is attempted. The sample protocol for deparaffinization (Table 3) can be used, but modifications of the times and numbers of washes may be necessary if microdissection is difficult. If the tissue is dehydrated too much, it may fall off in large fragments during the microdissection procedure rather than as single isolated cells. If the tissue is incompletely dehydrated, however, it may adhere too tightly to the slide and will not be removed during microdissection.22

Several commercially available microdissection systems use laser technology: PixCell, AutoPix, and Veritas LCM Systems (Arcturus Bioscience Inc, Mountainview, Calif), Leica AS LMD system (Leica Microsystems, Bannockburn, 111), and the PALM MicroBeam System (Mikrolaser Technologie, Bernried, Germany). The basic system components for LCM include an inverted microscope, an infrared laser, control unit for the laser, a control mechanism for the microscope stage, a digital camera, and a monitor for target visualization.22 The systems involve different technologies for separating selected target areas from tissue sections. In the Arcturus system, the laser is used to melt a specialized thermoplastic film onto the tissue, thereby enabling the lifting of the target embedded in polymer for deposition into a microfuge tube. The Leica system is a no-contact system in which the tissue enters the tube by gravity alone after laser dissection. The PALM and Leica systems require that sections be mounted on membranes or specially coated slides instead of glass slides, and the laser is used to cut around selected target areas. The target and the membrane are then directly deposited into a tube for molecular analysis. All of these systems allow the operator to visualize and photograph the microdissected fragments and to photograph the sections after removal for quality assurance.