Manual exfoliation of fresh tissue obviates the need for frozen sections for molecular profiling.

Cancer 2005 Dec;105(6):483-91

Department of Pathology, University at Buffalo, State University of New York, Buffalo, New York 14203, USA.

Background: Simple, rapid tissue processing that preserves macromolecules will enhance translational research capabilities. Traditional fixative-based approaches for specimen preservation are ideal for histologic evaluation but are not conducive to molecular studies of nucleic acids and protein. Tissue cryosections preserve macromolecule integrity, but the process is labor intensive and technically challenging. To the authors' knowledge to date, an alternative method capable of retrieving cells while providing adequate histologic detail yet preserving macromolecule integrity has been lacking. In the current study, the authors evaluated the utility of using manual exfoliation of clinical tissue samples as a means of obtaining cells for molecular analysis. This technique possesses the advantages of fixed and frozen tissue sections without their drawbacks. This simple, rapid, nonfixative based technique is capable of preparing cells from human clinical material for further isolation without compromising the preservation of macromolecules in the tissue.

Methods: Cells from a variety of clinical resection specimens from solid tumors were directly scraped from the tissue samples using the edge of a glass microscope slide and smeared onto another slide for cytologic evaluation. The manually exfoliated cells were evaluated microscopically for cytologic quality and cellular quantity. Pure cell populations were procured by laser capture microdissection (LCM) with subsequent extraction of nucleic acids and proteins. The integrity and suitability of the recovered nucleic acids and proteins for molecular analysis were evaluated using the polymerase chain reaction (PCR), reverse transcriptase-PCR, and reverse-phase protein microarray, respectively.

Results: Manual exfoliation permits the selection of homogeneous cell populations by LCM based on well established cytologic characteristics. DNA and mRNA, of comparable quality to frozen sections, can be amplified from the manual exfoliation cells. Proteins of similar quality can be recovered using this technique and quantitated via reverse-phase protein microarray.

Conclusions: Molecular macromolecules of high quality and sufficient quantity can be retrieved from human clinical samples using manual exfoliation and LCM to procure specific cell populations. The manual exfoliation technique does not destroy the original tissue source, thereby allowing subsequent formalin tissue fixation. The technique of manual exfoliation in conjunction with LCM can enable the molecular profiling of a sampled selected cell population. Because it does not destroy the original tissue, histologic correlation can be combined with molecular profiling.

Download full-text PDF

Source
http://dx.doi.org/10.1002/cncr.21347DOI Listing
December 2005
9 Reads

Publication Analysis

Top Keywords

manual exfoliation
28
nucleic acids
12
cell populations
12
molecular profiling
12
tissue
9
frozen sections
8
simple rapid
8
macromolecule integrity
8
original tissue
8
molecular analysis
8
destroy original
8
reverse-phase protein
8
tissue samples
8
acids proteins
8
human clinical
8
molecular
7
manual
7
exfoliation
6
cells
6
technique
5

References

(Supplied by CrossRef)

Stanley et al.
1993

DeMay et al.
1995
Reliable mutation analysis of archival tissue: critical issues and procedures
Roemen et al.
Proc Amer Assoc Cancer Res. 2003
Immunohistochemical (IHC) expression of phosphorylated proteins (PP) in breast cancer is significantly affected by formalin fixation
Mohsin et al.
Mod Pathol. 2004
Deterioration of phosphor-epitopes prior to and during specimen fixation
Sharifi et al.
Proc Amer Assoc Cancer Res. 2005

Similar Publications