High-Throughput Analysis of Microdissected Tissue Samples - High-Throughput Image Reconstruction and Analysis - page 242

that has been fixed and embedded. Both experimental design and subsequent data
analysis must take into account this possibility [11].
In order to analyze pure cell populations, tissue microdissection techniques
have become critical to the field of molecular pathology. In the present chapter we
review the molecular approaches that have been applied to dissected cells in order
to generate high-throughput molecular profiling data. Typically, a multidisciplinary
approach is required for these efforts, as knowledge of tissue handling and pro-
cessing, histopathology, imaging and signal quantitation, physics and engineering
for the development of microscopy and dissection instruments, and bioinformatics
need to be integrated together in order to efficiently perform the profiling experi-
ments and to analyze the large datasets that are produced.
11.2 Microdissection Techniques and Molecular Analysis of Tissues
11.2.1 General Considerations
Tissue specimens from patients offer the possibility of analyzing both the morpho-
logical and molecular basis of diseases, which can then be matched with clinical
records. An extraordinary example of the value of integrating high throughput
molecular techniques with histopathological analysis of samples is the sequencing
of the 1918 Spanish Influenza Virus genome. Archival material from autopsy cases
almost 90 years old allowed for the characterization and reconstruction of this pan-
demic virus and its subsequent study in the laboratory. This remarkable feat is lead-
ing to identification of new prognostic indicators for the illness and potentially to
the development of novel antiviral therapies [12, 13]. Similar molecular pathology-
based investigations of disease processes such as cancer are also revealing novel
etiological insights and generating new targets for clinical intervention [14--16].
11.2.2 Fixation---- A M a j o r C o n s i d e r a tion When Working with Tissue Samples
Formalin fixation followed by paraffin embedding (FFPE) is the usual practice for
processing clinical samples and is the gold standard for histopathological diagnosis.
However, this method limits the feasibility of molecular analysis to some extent
[7, 11, 17, 18]. The main drawback of FFPE is the damage to biomolecules that
ensues, particularly to RNA and proteins, due to the cross-linking of biomolecules
induced by the fixation process [7, 17, 18]. DNA and to some extent RNA can
still be retrieved from the samples and analyzed, although RNA extraction from
FFPE samples and subsequent analysis is challenging [7, 17, 18]. Alternative
fixatives, including ethanol-based ones, are under development and assessment
as a potential means to preserve biomolecules as well as morphological detail for
diagnosis [19, 20].
Snap-freezing of tissues is the optimal procedure for molecular analysis in
terms of the quality and quantity of biomolecules that can be recovered; however,
these samples are not as readily available for study as most pathology laboratories
have only recently started collecting snap-frozen tissue in biorepositories [21, 22].
Moreover, the histological detail in these specimens is inferior to that in FFPE
samples, and the resource needs are increased when using frozen tissue with respect
to freezer space and cryostat sectioning.