Laser Capture Microdissection–Guided Fluorescence In Situ Hybridization and Flow Cytometric Cell Cycle Analysis of Purified Nuclei from Paraffin Sections

Abstract

Laser capture microdissection (LCM) has recently been identified as a quick, simple, and effective method by which microdissection of complex tissue specimens for molecular analysis can be routinely performed. Assessment of gene copy number by fluorescence in situ hybridization (FISH) is useful for the analysis of molecular genetic alterations in cancer. Unfortunately, the application of FISH to paraffin sections of tumor specimens is fraught with technical difficulty and potential artifacts. Our results demonstrate that LCM-microdissected nuclei are suitable for FISH gene copy analysis. Amplification of genes in cancer specimens can be detected as easily in LCM-prepared nuclei as in fresh nuclei from cancer tissue specimens. Furthermore, contamination of tumor specimens by normal cells can make interpretation of flow cytometric cell cycle analysis difficult. Our results show that LCMmicrodissected nuclei can also be used for flow cytometric cell cycle and ploidy analysis. LCM/FISH offers the advantages of multicolor FISH in a morphologically defined cell population, without the technical problems of FISH performed on paraffin sections. This technique should further simplify the methodology required to perform copy number analysis of tumor suppressor or protooncogenes in archived cancer specimens. The use of LCM specimens will also improve the specificity and simplify the interpretation of flow cytometric cell cycle and ploidy analysis of breast cancer specimens.

Loss of Heterozygosity Associated with Uniparental Disomy in Breast Carcinoma

“…Nuclei for FISH were either prepared by touches from fresh-frozen tumor samples when available (25), or by laser capture microdissection of pure cancer nuclei from paraffin sections (26). FISH was performed according to our established methods (27).…”

Improvement in the detection rate of t(14;18) translocation on paraffin-embedded tissue: a combination approach using PCR and FISH

“…33,54 Moreover, the possibility of performing FISH analysis on laser microdissected tumour cells and on tissue microarray slides will enable studies to be conducted on a very large scale. 58,59 In conclusion, we suggest application of PCR as the first screening tool on retrospective archival materials to identify the translocated cases within these frequently reported breakpoints, followed by FISH on PCR-negative cases. This combination of approaches improves the detection rate of t(14;18) translocation in archival tissues.…”

Histological microdissection in diagnostic and investigative pathology

“…DNA in situ hybridisation has been used with laser microdissection to select cells by genotype 45,46 and microdissection of living cells has been used for molecular analysis and sub-culture, as microdissection does not appear to kill cells.…”