Microdissection Techniques for Molecular Testing in Surgical Pathology

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

In major laboratories or in core facilities, it is practical and reasonable to have both an LCM and a manual microdissection setup available and ready for use at all times. In smaller laboratories that may not have access to LCM, the manual methods can provide a sufficiently pure population of cells for most molecular applications in anatomic pathology.

FREQUENTLY ASKED QUESTIONS

1. Does It Matter How Much Tissue Is Microdissected?

The amount of tissue that should be included in a microdissection target is dependent on the type of testing to be performed on the tissue.26 Because PCR assays are highly sensitive, most PCR applications will require a very small volume of DNA as the starting template. Samples can potentially contain as few as 10 cells or can be very cellular if there is a need for a large amount of DNA.27 However, the reagents utilized should be scaled for the amount of raw DNA included in the reaction mixture, and the amount used should be consistent throughout an experiment.

One major concern with very low DNA concentrations is allelic dropout or PCR infidelity due to inadequate DNA content in the initial starting mixture.28 In our experience, care should be taken in samples that have very low DNA concentrations as measured by ultraviolet spectrophotometry. In these samples, performing the reactions in triplicate or using touchdown or hotstart PCR assays can help to alleviate problems of interpretation when samples are small.

2. Is It Better to Take Multiple Different Targets From the Same Slide or the Same Area From Multiple Serial sections?

In most cases, the purpose of microdissection is to obtain a relatively pure population of clonal tumor cells, and it is preferable that these cells be adjacent to one another. Therefore, we prefer to obtain 3-dimensional clusters of cells from sequential tissue sections rather than cells from many different areas across a tissue section. This approach will lead to a more pure and homogeneous tissue sample than the alternative of taking different areas from the same unstained slide and combining them into 1 tube.

3. Can Microdissection Be Performed on Previously Stained Slides?

Microdissection can certainly be performed on any type of tissue section. However, the molecular use of the microdissected tissue may be limited based on the type of stain that was originally used because certain stains can inhibit PCRs. Furthermore, some pretreatments for immunohistochemistry or even histochemistry may substantially degrade the nucleic acids or may inhibit PCRs. Nevertheless, in selected circumstances it is possible to analyze previously stained material using PCR primer sets that are especially robust with respect to nucleic acid amplication.

4. Do I Need to Take Special Care in Designing a PCR Assay if I Am Going to Use Microdissected Tissue?

If the tissue sections are frozen, PCR primer design can be standard. However, when the PCR assays are planned from tissue microdissected from fixed, paraffin-embedded tissues, the DNA from these samples will most certainly have undergone some degree of degeneration. Fragments of DNA in paraffin-embedded tissues are still relatively intact but are much shorter than those obtained from fresh or snap-frozen tissue samples. Therefore, in designing PCR primers for microdissected paraffin-embedded tissues, it is optimal to keep PCR products small to optimize the amplification (ie,