Biomedical Engineering Reference
In-Depth Information
put expression methodologies such as cDNA microarrays or two-dimensional
polyacrylamide gel electrophoresis (2D-PAGE). Therefore, a crucial need exists for
processing methodologies that produce good histologic detail and also allow recov-
ery of mRNA and protein of sufficient quantity and quality for molecular profiling
studies. Currently the best tissue preservation method that fulfills this requirement is
OCT embedding, followed by rapid freezing. OCT (Optimal Cutting Temperature
compound; Tissue-Tek) is a cryosolidifiable compound used commonly in pathol-
ogy laboratories. A tissue sample selected for research is placed in a plastic cryomold
and topped with OCT; the entire apparatus is allowed to rapidly freeze on dry ice or
in a liquid medium such as cooled isopentane or liquid nitrogen. Tissue sections are
then cut in a cryostat and mounted onto special slides. This approach not only
retains tissue morphology, but allows investigators to perform high-throughput
molecular analyses on all of the various biomolecules in a sample.
As previously discussed, breast tissue is heterogeneous. A single section of tissue
can contain multiple cell types, including invasive carcinoma cells, preinvasive
intraductal carcinoma cells, benign ductal epithelial cells, myoepithelial cells, leuko-
cytes, fat cells, endothelial cells, and connective tissue fibroblasts. Laser
microdissection is a relatively new technology that allows cells of interest to be iso-
lated from a tissue-mounted slide, thus creating a pure cell population. Molecular
studies can then be performed, reducing contamination by bystander cells and pro-
ducing more reliable results [14]. OCT-embedded tissue is especially well adapted to
this technique because it allows the tissue to be sectioned directly on a microtome
and provides good histologic detail.
At our institution stock tissue is created by freezing excess tissue directly in liq-
uid nitrogen. These flash frozen specimens can be converted into OCT sections if
required or can be utilized for studies that do not require histologic analysis (e.g.,
measurement of exogenous chemicals).
Although not well suited for molecular profiling studies, formalin fixed paraffin
embedded tissue represents a valuable and vast resource. FFPE tissue is useful for a
variety of applications including the targeting of proteins, RNA, and DNA by means
of immunohistochemistry, in situ hybridization, and fluorescence in situ hybridiza-
tion (FISH), respectively. A specific application of FFPE tissue is the analysis of
allelic imbalance/loss of heterozygosity (AI/LOH) in tumors. Also, because of the
excellent tissue morphology, FFPE tissue can be utilized for laser microdissection to
analyze selected populations of cells.
Utilization of paraffin blocks for research depends on various factors such as
storage guidelines issued by regulatory agencies, medicolegal issues, and database
management. Clinically acquired FFPE tissue is usually stored for 10 years as man-
dated by the College of American Pathologists (CAP). Many pathology groups dis-
card the blocks after that time. To prevent this loss, many repositories are now
archiving and utilizing these discarded blocks for research. With approval by an
institutional review board (IRB), it is also possible to utilize FFPE tissue that is
within that 10-year period providing the diagnostic integrity of the tissue block is
not adversely affected by the research process.
With regard to storage, there is no consensus on the optimal storage condition.
Storage for frozen specimens (OCT-embedded or snap frozen) ranges from
−
80°C in
mechanical freezers to
−
190°C in the vapor phase of liquid nitrogen. Storage at
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