Cytology.
Fibroblast cells derived from Huntington's Disease patients fixed
and immunostained for actin (red-Phalloidin conjugated with Texas
red (Molecular
Probes) and a-tubulin (green anti-a-tubulin (Sigma) for a-tubulin
and secondary antibody conjugated with Alexa 488 (Molecular Probes)).
Captured by SmartCapture software using a Zeiss Axio Plan II microscope.
Image provided by Kirupa Sathasivam and Prof. Gill Bates, Division
of Medical and Molecular Genetics, Guy's, King's and St Thomas' School
of Medicine, London. |
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| 7
Colour Fish. SmartCapture 2 can visualise 7 colour chromosome
paints from a palette from a palette of 3 fluorochromes and a DAPI
counterstain for a variety of applications such as Leukaemia / lymphoma
monitoring or early indicative diagnosis. |
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| Ratio
Imaging. Example of 'ratio painting' on B / NHL cell line G452.
wcp9 Sp Orange; wcp14 Sp Green; wcp 22 Sp Orange/Cy5; wcp 8 Sp Orange/Sp
Green; wcp13 Cy5. |
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| CGH
& 24 Colour FISH. SmartCapture 2 is an ideal platform for
the acquisition of images derived from CGH (Comparative Genomic Hybridisation)
and multiplex FISH experiments. |
|
| Z-Stacks.
A series of images captured at intervals through the Z (Vertical)
plane of a 3 dimensional object may be produced onto a single plane
to visualise the structure. Conterstained outlined in yellow. |
|
Time
Lapse. Multiplane images may be automatically captured at time
intervals and saved as filmstrips or converted into a movie.
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Colour
karyotype of K562.
Resolution of multiple chromosome abnormalities. Note the presence
of an abnormal chromosome 22 shown to contain material from chromosome
9.
(Gribble
et al.,Cancer Genet Cytogenet,2000)
|
|
The
principle of M-FISH analysis.
A labelling scheme (on the left) using 5 fluorochromes to achieve
24 combinations was used to visualise the chromosomes from a normal
cell. Images are presented as seen under the microscope (raw image
) and after digital processing using Smartcapture (processed image).
(Gribble
et al.,Cancer Genet Cytogenet,2000)
|
|
A
G banding karyotype
of the B type NH Lymphoma cell line (Karpas 1106).
Note the presence of structural abnormalities of chromosomes 3, 9,15,12,18,
20, and X as well the loss of chromosome 5.
(Nacheva
et al., Blood, 1994)
|
|
| Advantages
of M-FISH analysis.
Conventional banding analysis failed due to the typically poor morphology
for ALL (reversed DAPI counterstain image on the right). Whilst a
dic (9;20) chromosome was identified by M-FISH analysis.
|
|
Seven
chromosome painting.
A labelling scheme (on the left), of an image of a normal cell after
hybridisation as seen by eye under the microscope (raw image) and
after digital manipulation (processed images).
(Nacheva
et al., Cancer Genet Cytogenet,2000)
|
|
| Chromosome
painting with a mixture of seven chromosomes selected to screen for
specific chromosome aberrations
associated with disease progression in CML. A raw image of a cell
from a CML cell line (CM3) demonstrates that the painting probe can
detect all chromosome aberrations including changes affecting very
small chromosome segments (arrows). |
|
Seven
chromosome painting in CML.
Painting analysis can detect subtle chromosome translocations such
as (1;8)(p32;q24.1), which are difficult to identify by conventional
analysis (see arrow). This example illustrates the high degree of
resolution possible.
|
|
Seven
colour chromosome painting
with CML panel using hypermetaphase cell preparations (magnification
40x). Many condensed mitoses can be produced per slide.
|
|
| Reverse
chromosome painting
identifies the origin of the G(+) band as 20q13.2 in the structure
of a del(20)q marker thus defining the deletion as del(20)(q11.2q13.1)
(Nacheva
et al.,Cancer Genet Cytogenet, 1995)
|
|
FISH
mapping.
A dual color approach used to identify the CDR in cases with del(20q)
using two probes - one from the centromere 20 (in red) and one from
a specific locus (green).
In
the cell on the left both markers are retained, whilst in the cell
on the right only one of the chromosome 20 homologues shows both
signals. The other, for the locus specific marker, is ‘deleted’
|
|
Schematic
presentation of FISH:protocols
|
|
Examples
of FISH probes
|
|
| The
three colour-triple probe system.
A signal pattern in a Ph positive cell.
|
|
| The
three colour-triple probe system.
A normal, Ph positive cell and a cell with a false positive signal
pattern.
(Sinclair
et al.,Blood,1997)
|
|
| A
simple variant Ph translocation.
Chromosome painting reveals a reciprocal t(11;22)(q13.1q11.2) and
no involvement of chromosome 9 (cell on the left). Chromosome 11 paint
and BCR/ABL probes, demonstrate the presence of a fusion BCR/ABl gene
at the der(22)t(11;22) immediately proximal to the breakpoint (cell
on the right).
(Gribble
et al.,Cancer Genet Cytogenet,1999)
|
|
Chromosome
painting analysis of a variant Ph translocation t(9;17;22).
Diagrammatic
presentation (on the left) and G banded partial karyotypes (on the
right) of the chromosome pairs 9, 19 and 22. This demonstrates the
presence of 22q material in the structure of the der(9) chromosome
shown here from three different cells painted with the relevant
chromosome paints. This finding illustrates the higher sensitivity
of chromosome painting compared to conventional banding analysis.
|
|
Interphase
cell FISH analysis
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|
FISH
detection of the BCR/ABL fusion
in metaphase and interphase cells.
|
|
Schematic
representation of the BCR/ABL detection with FISH
|
|
| Triple
probe-three colour system.
Signal pattern in a case with classical Ph translocation.
(Sinclair
et al.,Blood,2000)
|
|
A
triple probe-three colour BCR/ABL detection system.
A typical signal pattern due to the deletion of the ASS contig in
a der(9) chromosome.
|
|
| Chromosome
profile of CML blast crisis.
Amplification of the 8q24 qter region identified by colour karyotyping
(cell on the left), locus specific probes (cell in the middle) and
CGH (a graph to the bottom right) in the cell line BV173 with 4 abnormal
chromosomes (partial G banded karyotype top, far right). |
|
Hypermetaphase
cell preparations
can be used as a tool for following disease progression using multicolour
FISH analysis with a panel of seven chromosomes.
|
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| Top
of page |
| |
| References: |
Gribble
S, Grace C, Sinclair PS, Green AR, Nacheva EP
COMPARATIVE ANALYSIS OF G BANDING, CHROMOSOME PAINTING, LOCUS SPECIFIC
FLUORESCENCE IN SITU HYBRIDISATION, AND COMPARATIVE GENOMIC HYBRIDISATION
IN CHRONIC MYELOID LEUKAEMIA BLAST CRYSIS.
Cancer
Genet Cytogenet.1999,111:7-17 |
Gribble
S, Roberts I, Grace C, Andrews K, Green AR, Nacheva EP.
CYTOGENETICS OF THE CML DERIVED CELL LINE K562 REVIEWED: COMPLETE
KARYOTYPE CLARIFICATION BY 24 COLOUR M-FISH, CGH AND FISH
Cancer
Genet Cytogenet.,2000, In press. |
Nacheva
E, Dyer MJS, Metivier C, Jadayel D, Stranks G, Morilla R, Howard JM,
Holloway T, O'Connor S, Bevan PC, Larsen CJ, Karpas A.
B-CELL NON-HODGKIN'S LYMPHOMA CELL LINE (KARPAS 1106) WITH COMPLEX
TRANSLOCATION INVOLVING 18q21.3 BUT LACKING BCL2 REARRANGEMENT AND
EXPRESSION. Blood
1994 Nov
15 84(10): 3422-8. |
Nacheva
EP, White NJ, Asimakopoulos FA, Green AR.
CHARACTERIZATION OF 20q DELETIONS IN PATIENTS WITH MYELOPROLIFERATIVE
DISORDERS OR MYELODYSPLASTIC SYNDROMES. Cancer
Genet Cytogenet. 1995
Apr 80(2): 87 -94 |
| Nacheva
EP, Gribble S, Andrews K, Wienberg J, Grace CD (2000). SCREENING FOR
SPECIFIC CHROMOSOME INVOLVEMENT IN HAEMATOLOGICAL MALIGNANCIES USING
A SET OF SEVEN CHROMOSOME PAINTING PROBES - AN ALTERNATIVE APPROACH
FOR CHROMOSOME ANALYSIS USING STANDARD FISH INSTRUMENTATION. Cancer
Genetics & Cytogenetics, 2000, In press |
| Sinclair
P, Grace CD, Green AR, Nacheva EP. IMPROVED SENSITIVITY OF BCR-ABL
DETECTION: A TRIPLE-PROBE THREE-COLOUR FLUORESCENCE IN SITU HYBRIDIZATION
SYSTEM. Blood
1997
Aug 15;90(4):1395-402. |
| Sinclair
PS, Nacheva EP, Laversha M, Telford N, Champion K, Bench A, Huntley
B, Green AR. LARGE DELETIONS AT THE t(9;22) BREAK POINT ARE COMMON
AND MAY IDENTIFY A POOR PROGNOSTIC SUB GROUP OF PATIENTS WITH CHRONIC
MYELOID LEUKEMIA. Blood
2000,
995:738-743 |
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