Manual exfoliation of fresh tissue obviates the need for frozen sections for molecular profiling.
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.
Mojica
Wilfrido D
WD
Department of Pathology, University at Buffalo, State University of New York, Buffalo, New York 14203, USA. mojica@buffalo.edu
Rapkiewicz
Amy V
AV
Liotta
Lance A
LA
Espina
Virginia
V
D002469:Cell Separation; D004247:DNA / Q000032:analysis*; D005629:Frozen Sections; D006801:Humans; D007834:Lasers; D042282:Microdissection; D016133:Polymerase Chain Reaction; D011309:Preservation, Biological; D020543:Proteome / Q000032:analysis*; D012313:RNA / Q000032:analysis*; D013048:Specimen Handling; D046888:Tissue Array Analysis; D016707:Tissue Fixation
D020543:Proteome; D012313:RNA; D004247:DNA