Since chromosomes are visible as individual
structures under the light microscope only
during metaphase (or under special conditions
and for certain purposes, during prometaphase),
every chromosome analysis requires
dividing cells. In vivo, only bone marrow cells
contain a sufficient proportion of cells in mitosis.
Thus, in-vivo chromosome analysis of cells
is limited to bone marrow. All other procedures
for analyzing chromosomes in mitosis require
culturing of suitable cells (cell culture). Most
commonly, lymphocytes from blood are cultured
for chromosome preparations.
Peripheral blood lymphocytes stimulated by
phytohemagglutinin grow in a suspension culture.
Their life span is limited to a few cell divisions.
However, by exposing the culture to Epstein-
Barr virus they can be transformed into a
lymphoblastoid cell line with permanent
growth potential. Such cultures arewidely used
because they are much easier to handle than adhesion
cultures (p. 122).
In addition, fibroblasts from a piece of skin can
be propagated in cell culture for analysis (see
p. 122). However, since this procedure is somewhat
elaborate and time-consuming, it is used
only for certain purposes.
Sunday, April 12, 2009
Chromosome analysis from blood
For the cell culture, either peripheral blood is
used directly or lymphocytes are isolated from
peripheral blood (T lymphocytes). A sample of
about 2ml of peripheral blood is needed. The
blood is prevented from clotting by use of a heparinized
syringe, since clumping of the blood
cells precludes culturing (the proportion of heparin
to blood is about 1: 20). Peripheral blood
or isolated lymphocytes are placed in a vessel
with culture medium. The cells are generally
stimulated with phytohemagglutinin, a protein
from plants that unspecifically stimulates
lymphocytes to divide. The culture requires
about 72 hours at 37!C for cells to divide.
Lymphocyte cultures are suspension cultures;
i. e., the cells divide in culture medium without
attaching to the culture vessel. Cell division is
arrested and the culture is terminated by
adding a suitable concentration of a colchicine
derivative (colcemid) two hours prior to
harvest. Colcemid interrupts mitosis during
metaphase, so that a relative enrichment of
cells in metaphase results.
used directly or lymphocytes are isolated from
peripheral blood (T lymphocytes). A sample of
about 2ml of peripheral blood is needed. The
blood is prevented from clotting by use of a heparinized
syringe, since clumping of the blood
cells precludes culturing (the proportion of heparin
to blood is about 1: 20). Peripheral blood
or isolated lymphocytes are placed in a vessel
with culture medium. The cells are generally
stimulated with phytohemagglutinin, a protein
from plants that unspecifically stimulates
lymphocytes to divide. The culture requires
about 72 hours at 37!C for cells to divide.
Lymphocyte cultures are suspension cultures;
i. e., the cells divide in culture medium without
attaching to the culture vessel. Cell division is
arrested and the culture is terminated by
adding a suitable concentration of a colchicine
derivative (colcemid) two hours prior to
harvest. Colcemid interrupts mitosis during
metaphase, so that a relative enrichment of
cells in metaphase results.
Cell preparation is carried out as follows:
the
culture solution is centrifuged; the cell sediment
is placed in a hypo-osmolar KCI solution
(0.075 molar), incubated for about 20 minutes,
and centrifuged again. The resulting cell sediment
is placed in fixative. The fixing solution is
a mixture of methyl alcohol and glacial acetic
acid in a ratio of 3:1. Usually the fixative is
changed two to three times with subsequent
centrifugation. After that, the fixed cells are
taken up in a pipette and dropped onto a slide.
The preparation is stained, and the slide is
covered with a cover glass.
culture solution is centrifuged; the cell sediment
is placed in a hypo-osmolar KCI solution
(0.075 molar), incubated for about 20 minutes,
and centrifuged again. The resulting cell sediment
is placed in fixative. The fixing solution is
a mixture of methyl alcohol and glacial acetic
acid in a ratio of 3:1. Usually the fixative is
changed two to three times with subsequent
centrifugation. After that, the fixed cells are
taken up in a pipette and dropped onto a slide.
The preparation is stained, and the slide is
covered with a cover glass.
At this point the cells are ready for analysis.
Suitable metaphases are located under the microscope
with about 100x magnification and
are subsequently examined at about 1250x
magnification. During direct analysis with the
microscope, the number of chromosomes and
the presence or absence of all chromosomes
and recognizable chromosome segments are
noted. Since the preparation procedure itself
may induce deviations from the normal chromosome
number or structure in some cells,
more than one cell must be analyzed. Depending
on the purpose of the analysis, between 5
and 100 metaphases (usually 10–15) are examined.
Some of the metaphases are photographed
with the microscope and can subsequently
be cut out of the photograph (karyotyping).
In thisway a karyotype can be obtained
from the photograph of a metaphase. The time
needed for a chromosome analysis varies depending
on the problem, but is usually 3–4
hours. Analysis and karyotyping time can be
shortened by computer procedures.
with about 100x magnification and
are subsequently examined at about 1250x
magnification. During direct analysis with the
microscope, the number of chromosomes and
the presence or absence of all chromosomes
and recognizable chromosome segments are
noted. Since the preparation procedure itself
may induce deviations from the normal chromosome
number or structure in some cells,
more than one cell must be analyzed. Depending
on the purpose of the analysis, between 5
and 100 metaphases (usually 10–15) are examined.
Some of the metaphases are photographed
with the microscope and can subsequently
be cut out of the photograph (karyotyping).
In thisway a karyotype can be obtained
from the photograph of a metaphase. The time
needed for a chromosome analysis varies depending
on the problem, but is usually 3–4
hours. Analysis and karyotyping time can be
shortened by computer procedures.
In Situ Hybridization in Metaphase and Interphase
In situ hybridization refers to procedures that
demonstrate DNA sequences directly on chromosome
preparations (in situ). Since resolution
is relatively good (about 12 x 107 base pairs), the
exact regional localization of a sequence on its
corresponding chromosome can be determined.
demonstrate DNA sequences directly on chromosome
preparations (in situ). Since resolution
is relatively good (about 12 x 107 base pairs), the
exact regional localization of a sequence on its
corresponding chromosome can be determined.
Principle of in situ hybridization
Cells in metaphase or interphase are fixed on a
slide and denatured to change the doublestranded
DNA (1 a) into single-stranded DNA
(2). The metaphase or interphase preparation is
then hybridized (3) with DNA sequences that
are complementary to the region of interest and
that have been labeledwith biotin (1 b). The hybridization
site is made visible by means of a
primary antibody against biotin; this antibody
is bound to a fluorochrome (4), e. g., fluorescein
isothiocyanate (FITC). Since the primary signal
is quite weak, a secondary antibody (e. g.,
avidin) bound to biotin is attached (5). A further
primary antibody can then be attached to the
secondary antibody (6). This amplifies the signal,
which can then be demonstrated by bright
fluorescence under the light microscope.
slide and denatured to change the doublestranded
DNA (1 a) into single-stranded DNA
(2). The metaphase or interphase preparation is
then hybridized (3) with DNA sequences that
are complementary to the region of interest and
that have been labeledwith biotin (1 b). The hybridization
site is made visible by means of a
primary antibody against biotin; this antibody
is bound to a fluorochrome (4), e. g., fluorescein
isothiocyanate (FITC). Since the primary signal
is quite weak, a secondary antibody (e. g.,
avidin) bound to biotin is attached (5). A further
primary antibody can then be attached to the
secondary antibody (6). This amplifies the signal,
which can then be demonstrated by bright
fluorescence under the light microscope.
Demonstration of the Philadelphia translocation in chronic myeloid leukemia (CML)
The Philadelphia translocation (1) in chronic
myeloid leukemia (CML, see p. 332) can be demonstrate
in metaphase (2) and in interphase (3)
by means of in situ hybridization. When a probe
for the BCR gene is used in interphase, the normal
signal consists of two fluorescing dots, one
dot on each chromosome 22. (On good preparations
of metaphase chromosomes, one dot is
seen over each chromatid and appears as a
double dot on a chromosome.) When the probe
includes the breakpoint of the translocation,
three signals are visible: the largest over the
normal chromosome 22, a small one over the
BCR sequences remaining in the distal long arm
of a chromosome 22 (22q), and another small
one over the sequences translocated to the distal
long arm of chromosome 9.
myeloid leukemia (CML, see p. 332) can be demonstrate
in metaphase (2) and in interphase (3)
by means of in situ hybridization. When a probe
for the BCR gene is used in interphase, the normal
signal consists of two fluorescing dots, one
dot on each chromosome 22. (On good preparations
of metaphase chromosomes, one dot is
seen over each chromatid and appears as a
double dot on a chromosome.) When the probe
includes the breakpoint of the translocation,
three signals are visible: the largest over the
normal chromosome 22, a small one over the
BCR sequences remaining in the distal long arm
of a chromosome 22 (22q), and another small
one over the sequences translocated to the distal
long arm of chromosome 9.
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