Molecular Analyses in Endocrine Pathology
Dr. George Kontogeorgos
Dr. Robert Yoshiyuki Osamura
Dr. Jennifer Hunt
Section 1 -
Fluorescent In Situ Hybridization Techniques: Principles and Limitations
Department of Pathology,
G. Gennimatas General Hospital,
Conventional cytogenetics or karyotyping is a well-established method to study chromosomes based on
cells capable of in vitro growth and division. Different types of samples,
such as amniotic fluid and peripheral blood, can be used and several banding methods can be applied for
karyotyping analysis in clinical cytogenetic laboratories. However, karyotyping is a time consuming
technique that requires tissue culture conditions, equipment and expert technical qualification. For
these reasons, application of conventional cytogenetics has been limited to some specific laboratories.
In addition, karyotyping requires fresh tissue material in order to obtain metaphases, which are quite
difficult to achieve from solid tumors, especially from those having low proliferation rate. As a
result, analysis of a small number of metaphases from solid tumors may lead to underestimation of
cytogenetic abnormalities and obscure the heterogeneity and complexity of cytogenetic changes.
Furthermore, overgrowth of nontumorous cells such as lymphocytes and fibroblasts may result in loss of
overall genetic information and thus, lead to erroneous results .
Fluorescence in situ hybridization (FISH) or molecular cytogenetics is a modern technique alternative
to karyotyping . FISH combines molecular genetics with classic cytogenetics and allows simultaneous
morphologic evaluation on a single slide . It is currently recognized as a reliable, sensitive and
reproducible technique for the identification of numerical and structural chromosomal aberrations. FISH
is been applied with increasing frequency in diagnostic and research laboratories. The clinical utility
of FISH is of particular interest; it covers a wide spectrum of diagnostic applications including
autosomal and sex chromosome disorders, and cancer cytogenetics
FISH allows cytogenetic investigation of metaphase spreads and interphase nuclei
represents the main advantage of FISH application for practicing and research pathologist, for it permits
cytogenetic analysis of solid tumors without the need of metaphases, particularly of those with low
proliferation rate. Interphase FISH can determine diagnosis, by identifying the presence of known
abnormalities, and in some tumors, it can predict responses to targeted therapy. Several types of fresh
and frozen samples or archival material can be used.
FISH is based on the formation of a hybridization product between a selected DNA probe and the target
specimen DNA. Fluorescent labeling of the probe enables the detection and study of the specimen under an
epifluorescent microscope. A variety of DNA probe types are available and can be used for specific
purposes. Centromeric probes are used to detect specific chromosomes and telomeric probes to demonstrate
all chromosomes. Sequence-specific probes can localize a single gene copy on a specific chromosome locus
Comparative genomic hybridization (CGH) is a novel cytogenetic technique, which combines FISH with
automatic digital image analysis. Comparative analysis of the hybridization products of tumor-DNA and
reference-DNA with normal metaphase chromosomes, each labeled with different color fluorochrome, can
detect non-balanced chromosomal aberrations of the entire genome in a single experiment .
Tissue sampling and fixation
For interphase FISH nuclei from touch imprints, fine needle aspiration biopsies, biological fluids
preparations and nuclei isolated from frozen or paraffin-embedded tissues can be used.
Fixation in methanol/acetic acid or chilled acetone gives excellent results, although other fixatives
including formalin can also be used . Sections from formalin-fixed and paraffin-embedded tissues
require careful digestion with proteinase K before application of the technique; this step is often
crucial to obtain optimal results. Tissue overdigestion leads to loss of nuclear borders and the cells
appear "ghosts". In such case, depending on the extent of overdigestion, the experiment should be
repeated reducing either concentration or incubation time of proteinase K. In contrast, underdigested
tissue sections generate persistent auto-fluorescence of the background, often associated with poor
propidium iodide staining. To restore this problem, additional digestion, depending on the intensity of
background fluorescence is required.
Utility of Fluorescent Microscope
A high quality fluorescent microscope is necessary to enable optimal visualization of the
results.A 100-watt high-pressure mercury lamp is required, when dual or
triple band filters are used for multicolor FISH analysis, particularly for detecting the weak
fluorescent signals of sequence specific probes. Evaluation and recording of fluorescent signals also
requires high power, dry or oil objective lenses. For the latter, only non-fluorescing immersion oil
should be used. Filters are specifically designed for certain fluorochromes used for probe labeling and
counterstaining. Therefore, selection of filters should be carefully planed. A wide range of filters
and combinations, that allow only certain wavelengths of light pass through are used for single or
simultaneous dual signal fluorescence detection. Optimal use of filters can subject the fluorescent
signal to the lowest amount of excitation light to retain fluorescence as much as possible. Modern
fluorescent microscopes are equipped with digital photographic camera integrated with computer systems
and provide the user with many facilities to overcome problems in detecting weak or multicolor signals.
Using different types of single bandpass filters, multiple pictures are captured form the same filed and
then, with the aid of specially designed software, all pictures are merged together. Automatic FISH
systems can provide accurate scoring by counting a large number of interphase nuclei. In addition, they
can analyze image information from different focal planes and detect co-localizations via the 3-D
distances between fluorescent signals.
Interphase FISH is based on nuclear DNA content analysis. Only intact nuclei from touch preparations
and FNA biopsies or nuclei extracted from frozen or paraffin-embedded tissues retain the whole DNA mass
. Thus, the use of intact nuclei permits accurate estimation of the results. In contrast, tissue
sectioning leads to partial loss of nuclear DNA mass. Therefore, the pathologist cannot rely with
accuracy on this material; the results require cautious interpretation, particularly in the assessment of
aberrant chromosomes or gene copy number. In that case, due to partial analysis of the DNA material,
monosomies or polysomies can be overestimated or underestimated respectively.
FISH and CGH are powerful morphologic tools in understanding physiologic mechanisms and in resolving
problems of the pathogenesis of several diseases. These techniques shed light on the cytogenetic
background in many pathological disorders providing a better understanding of the activities and
alterations of cell function.
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