PART ONE
General Cytology
Fig. 2.3 G banded karyotype from
a normal male
showing 22 pairs of autosomal
chromosomes and two X and Y sexual
chromosomes
uncoiled chromatin. The dense Q and G bands are G+C-rich
and contain repetitive inactive DNA. Active genes are supposed
to be in clear bands; constitutive heterochromatin is located
in the pericentromeric regions as revealed by C banding and
appears as chromocenters in the nondividing nucleus. Chromo-
some Y has a unique strong fluorescent appearance visible in the
interphase nucleus as a bright dot also visible as a dark C band.
With these staining methods, the chromosomes 21, already rec-
ognized in the prebanding era because of their known involve-
ment in Down syndrome, remained classified as such, and the
minute marker of CML was consequently considered as belong-
ing to pair 22.
The Standardized Reporting
In 1971 at the International Conference in Paris, a nomencla-
ture for banded chromosomes was adopted and extensively
explained in a later revised version (ISCN 1985), codifying the
way in which all possible numerical and structural chromosome
anomalies should be reported.6
On each banded chromosome pair, the upper arms are des-
ignated as p arms (petit, meaning "short" in French); the longer
arms below are designated as q arms. Regions and bands are
numbered starting from the centromere.
According to this nomenclature, the Philadelphia (Ph)
chromosome was revealed by J. Rowley as being a t(9;22)
translocation, hence the result of a reciprocal translocation
between the q arms of chromosomes 9 and 22, with break-
points positioned in q34 and q11 chromosomal regions,
respectively7 (Fig. 2.4). Rapidly the complexity of neoplasia-
associated chromosome
aberrations
appeared
difficult to
adequate with the current nomenclature. Two standing com-
mittees proposed in 1991 and 1995 new consensus guidelines
to suit the description of tumor karyotypes, including fluo-
rescent in situ hybridization (FISH) methodology. However,
the new abnormalities reported with an increasing variety of
FISH probes and the new confusing subtleties of ISCN 1995
accumulated a greater rate of syntax errors. The heterogene-
ity of the observations and the variability of the banding
resolution made these ISCN nomenclatures not very practical
to use for the description of cancer-associated chromosomal
abnormalities, and still favors the use of personal simplified
nomenclatures.8
Karyotyping
Mitoses suitable for karyotyping are obtained more eas-
ily from lymphocyte cultures stimulated to grow by phyto-
hemagglutinin. Cells cultures from amniotic fluid or biopsy
of chorionic villi allow antenatal diagnoses. Skin fibroblasts
or fetal blood samples from the umbilical cord of stillborns
are also suitable. Direct examination of bone marrow or
short-term cultures are the techniques of choice for hemato-
logical disorders. Short-term tissue cultures took advantage
of technical improvements such as methotrexate synchroni-
zation or collagenase digestion in the analysis of lymphomas
and solid tumors.
Chromosomes are counted and analyzed on slides. For
decades, the better metaphase spreads were photographed,
and each chromosome was manually cut out before being clas-
sified on a sheet of paper. Nowadays, on-screen karyotyping is
the commonly used method for routine metaphase analysis.
Once acquired by the automated capture device, metaphases
can be quickly and accurately presented for chromosome
assignment. The CytoVision system (Applied Imaging) used
in our laboratory provides classifiers for standard banding
methods (Fig. 2.5).
Fluorescent in Situ Hybridization
The principle of in situ hybridization (ISH) is an uncoiling of
the double DNA strand by heat denaturation followed by subse-
quent specific hybridization of the targeted DNA molecules with
the complementary labeled DNA probe. By this procedure, ISH
detects the precise location of unique DNA sequences directly
on the chromosomes. The hybridized sequence is then revealed
by two layers of fluorescent or chromogenic-labeled antibod-
ies. FISH is preferred to chromogenic ISH (CISH) because of
its higher sensitivity and the greater palette of artificial colors
available.
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