FLUORESCENCE IN SITU

HYBRIDIZATION
 OVERVIEW
 Introduction • Trouble shooters and tips
to handle
• Definition • Applications in detail
• History • Quality control
• Principle • Advantages and
• Indications disadvantages
• Types of FISH • FISH vs. other techniques
• Procedure • Summary
• Result and interpretations • References
 INTRODUCTION
• Genome: The entire DNA of an organism
– Humans
• Diploid (chromosome pairs)
• 6 x 109 bp per diploid genome
• Haploid genome is one set of chromosomes

Chromosome: structure found within a cell nucleus consisting of a

continuous length of ds DNA
– Humans
• 22 pairs of autosomal chromosomes
• 2 sex chromosomes
 Numerical abnormalities

 Aneuploidy :- presence of an abnormal number

of chromosomes in a cell (2n+2,2n-2)
 Ex: Human cell having 45 or 47 chromosomes instead of
the usual 46
 Euploidy:- state of a cell or organism having one
or more than one set of the same set of
chromosomes, possibly excluding the sex-
determining chromosomes. i.e 4n
 Ex: A human cell with an extra set out of the 23 normal
ones would be considered euploid.
Structural Abnormalities of Chromosomes

• Deletion refers to loss of a portion of a chromosome

• Ring chromosome is a special form of deletion. It is
produced when a break occurs at both ends of a
chromosome
• Inversion refers to a rearrangement that involves two
breaks within a single chromosome with
reincorporation of the inverted, intervening segment
• Iso chromosome -One arm of a chromosome is lost
and the remaining arm is duplicated, resulting in a
chromosome consisting of two short arms only or of
two long arms
• Duplication-A duplication is the presence of an
additional copy of a chromosome segment
• Translocations - Rearrangements involving two or
more non homologous chromosomes.
• Cytogenetics
 Science that combine methods and findings of
cytology and genetics to allow investigation of
heredity at cellular level.
• Techniques:
1. Karyotyping
2. Fluorescent in situ hybridization
 Karyotype FISH
- All chromosomes - Specific abnormality
- Labour intensive - Relatively rapid
- Requires familiarity - Large numbers of
with chromosome cells can be screened
morphology
 Procedure to examine chromosomes

• Arrest dividing cells in metaphase with mitotic spindle

inhibitors (e.g., N-di acetyl-N-methyl colchicine) and then to
stain the chromosomes
• In a metaphase spread, the individual chromosomes take the
form of two chromatids connected at the centromere
• A karyotype is obtained by arranging each pair of autosomes
according to length, followed by sex chromosomes.
• IN SITU HYBRIDIZATION
 Labelled complementary DNA, RNA with
modified nucleic acids strand (i.e. probe) to
localize a specific DNA or RNA sequence in a
portion or section of tissue
Technique Instrument/ Primary advantage Primary application
visualization
method

Bright-field Ability to view the

CISH microscopy CISH signal and Molecular
tissue morphology pathology
simultaneously diagnostics

Fluorescence Multi plexible: Gene presence,

DNA-FISH microscopy visualize multiple copy number, and
targets in the same location; mutation
sample analysis

Fluorescence Multi plexible: Gene expression,

RNA-FISH microscopy, HCS, visualize multiple RNA temporal and
and flow cytometry targets in the same spatial localization
sample
 DEFINITION
• FISH
 It is the more recent era of molecular cytogenetics,
allows visualization of sequence specific loci using
biochemical technique of in situ hybridization.
HISTORY
• Interphase FISH introduced by cremer et al in 1986 for
chromosomal analysis in hematological malignancies
• Overcome the drawbacks of low mitotic index.
• Permitted the screening of large number of non dividing and
dividing cells in blood and bone marrow.
 DNA/gene mRNA/expression

First in situ detection Bauman et al., 1980 Singer and Ward, 1982

Two-colour detection Hopman et al., 1986 Dirks et al., 1990

Three-color detection Nederlof et al., 1989 Dirks et al., 1991

Combinatorial color-coding Nederlof et al., 1990 Levsky et al., 2002

(M-FISH)

Ratio color-coding Nederlof et al., 1992b

Combinations and ratios Tanke et al., 1999

(COBRA)
 Principles of fluorescence in
situ hybridization
• The basic elements of FISH are a DNA probe and a
target sequence
• Before hybridization, the DNA probe is labelled by
various means, such as nick translation, random
primed labelling, and PCR.
• The labelled probe and the target DNA are denatured
• Combining the denatured probe and target
 Indications for FISH
Diagnosis
1. Culture not successful
2. Expected abnormality not seen
3. To confirm a suspected abnormality
4. To detect a cryptic translocation

 To monitor response to treatment

 Engraftment in sex –mismatched BM
transplant
 TYPES OF FISH
TYPES TECHNIQUE USES

Single labelled Individuals RNA molecules

SINGLE MOLECULE oligonucleotides probe
RNA

Fiber - FISH Chromosome combing Small and gene dense

genomes

Flow-FISH Flow cytometry Telomere length

Q-FISH (Cy3 or FITC) Telomere length

• SKY and M-FISH-
1. Visualization and simultaneous analysis of all 24
chromosomes in different colours.
2. Identify multiple cryptic translocations derivative or
marker chromosomes.
• Comparative Genomic Hybridization(CGH)-In
Non HodgkinsLymphoma by identifying the number
of genes
• Matrix CGH-Detection of high level of
amplification or deletion in targets of few kilo bases
is possible.
 Procedure of FISH
• Step I – Denaturation
 Conversion of double stranded DNA in to single stranded DNA
• Step II – Hybridization
 Application of probe DNA to slide & overnight incubation at 37°C
 Binding of probe DNA to target DNA.

• Step III – Post hybridization washing & detection

 Washing of unbound probe DNA.
• Step IV – counter stain
 Application of counter stain.

• Step V – Visualization
 visualization using fluorescence microscopy.
 Nature of specimens
 Formalin-fixed, paraffin-embedded (FFPE) sections
 Confirm or exclude a histologic diagnosis, or as a tool to
determine appropriateness of targeted therapy in cancer
patients.
 Diffuse and solid tissue
 Single-cell suspensions: uncultured cells and cell smears
 Isolated cells from external body fluids: urine, sperm, and
sputum
 Interphase FISH
• Interphase of cell cycle
• Using Formalin Fixed Paraffin Embedded
tissues for solid tumor oncology cases, such as
breast cancer, lung cancer, and brain cancer.
 Procedure
• Make a cell suspension
Apply cells to glass slide.

• Denature cells and probe

• Apply probe to slide

• Hybridize

Wash slides to remove unbound probe

•
Counterstain

• View under epifluorescence microscope

 Metaphase FISH
• Commonly used for hematologic oncology
cases
• Performed in cells that have been arrested in
the metaphase portion of the cell cycle when
DNA is coiled into recognizable chromosomes
 Protocol
• Denaturation
• DNA melting-double-stranded DNA is essentially
unzipped, or opened, using a combination of heat and
chemical treatment.
Hybridization (Day 1 ):-
Anti-Digoxigenin Fluorescein-detection of Digoxigenin labelled
compound
Alexa 594-red fluorescent dye
CAS Block-blocking agent
Pepsin-protease that digest away cytoplasm
Goat Serum –blocking reagent before staining fixed cells and tissue
DAPI Antifade –4,6-diamido-2-phenyindoloefluorescent stain
binds A-T regions
10X PBS-phosphate buffer saline(buffer solution)
Tween 20-nonionic detergent (emulsifying agent)
70% Ethanol
100% Ethanol
37% HCl-permeabilization
20XSSC-saline sodium citrate buffer
• Procedure:
1. Add 25μl pepsin (20mg/ml) to Coplin jar (just prior to putting
slides). Mix well.
2. Incubate slides for 1 min (5 slides max)
3. Rinse slide in PBS
4. Incubate in 70% Ethanol in room temperature for 2 min.
5. Incubate in 100% Ethanol in room temperature for 2 min.
6. Air dry
7. Add probe (usually 20μl)
8. Add Coverslip (22 x 30mm)
9. Seal with rubber cement, air dry for 5 minutes
10.Place slides directly on slide moat (pre-set at 78°C) for 2.5 min.
11.Incubate overnight at 37°C in humidifying box
Detection (Day 2) :
 Preparation
• Set water bath to 42°C.
• Thaw goat serum.
• Place Coplin jars with 0.5X SSC in 42°C bath.
Let warm 10 min.
• Prepare 1x PBS with 1% Tween 20.
• Prepare blocking solution
• Procedure:
 Remove slides from oven, remove rubber
cement and coverslips.
 Place slides in 0.5X SSC at 42°C 5 min.
 Wash in PBS+Tween20 3 times 2 min. each.
 Shake briefly to dry, place on hyb box or tray.
 Add 500μl Blocking solution per slide.
 Block at RT 10 min
 Pour off blocking solution and add 500μl
Blocking solution with antibodies to each slide.
 Incubate in the dark for 1 hour at room
temperature
 Pour off solution; wash 3 times in PBS+ Tween
20 2 min. each.
 Shake briefly, add 20μl DAPI to each slide.
 Coverslip (25x50mm)
 Store in dark
Steps involved in FISH Paraffin embedded tissue Cell preparation and isolated
nuclei

Intended application Interphase Interphase or metaphase

Fixative 4-10%formalin Carnoyl fixative or 4%formalin

Aging none Overnight or 2x SSC solution

at 73C for 2 min

Pre-treatment a)Incubation at 52C overnight none

b)Deparaffinization using xylene
c)Incubate sections in 1M sodium
thiocynate at 80Cfor 20-30 min

Protease treatment 0.05% pepsin in 0.01 N HCL 0.05% pepsin in 0.01 N HCL
pH 2 a 37 C for 10-20 min pH 2 a 37 C for 10-20 min

Post fixation 4%buffered formaldehyde 1% buffered formaldehyde

denaturation Target: 70% formamide in Target 70%formamide in 2xSSC PH at
2xSSC,ph7.0 at 72C-74C for 1-2 min 72-74 for 1-2 min
Probe: 50-60 formamide in 2x SSC ph Probe:50-60%formamide in 2XSSC pH7
7.0 at72-74 for 1-2 min at 72-74 for 1-2 min

hybridization Target incubation with probe
prepared in a 2XSSC solution
containing 50-60% formamide and
10%dextran sulfate .PH-7 at 37 C-42C
overnight
Post hybridization a)2XSSC with0.3%NP-40 at 73 C for
wash 2min
b)2XSSC with 0.1%NP-40 at for 1 min
Target incubation with probe prepared
in a 2XSSC solution containing 50-60%
formamide and 10%dextran
sulfate .PH-7 at 37 C-42C overnight
a)2XSSC with0.3%NP-40 at 73 C for
2min
b)2XSSC with 0.1%NP-40 at for 1 min

Detection 1) Incubation with fluorescent labelled 1) Incubation with fluorescent labelled

antibiotin or antidigoxgenin antibody antibiotin or antidigoxgenin antibody
2) Alternative amplification with enzyme 2) Alternative amplification with enzyme –
–fluorophore -tyramide fluorophore -tyramide

Counterstain/ DAPI with antifade DAPI with antifade

mounting

visualization Epifluorescence microscopy with Epifluorescence microscopy with

appropriate filters appropriate filters
 Probes
 Segment of DNA
1. labeled with a marker which allows identification and
quantitation
2. Will hybridize to another nucleic acid on the basis of base
complementarity
• Types of labels
1. Fluorescent - FISH (fluorescence in situ hybridization)
2. Radioactive (32P, 35S, 14C, 3H)
3. Biotinylated (avidin -streptavidin)
Types of probes
• Dual colour break apart probes
 For chromosome segments with multiple
translocation partners
 These are made to span both sides of the breakpoint.
If there is a translocation at the breakpoint, the signals
will split apart
 Ex:-MLL gene on chromosome 11q23
 FILTERS IN FISH
• Types:
1. Excitation filter-selects and transmits the optimal
wavelength of light to excite a FISH probe
2. Emission filter-selects and transmits the optimal
wavelength of fluorescence emitted by a FISH
probe
 Fluorescence microscope
• Uses fluorescence and phosphorescence, or in addition
to, reflection and absorption to study properties of organic
or inorganic substances.
Mitosis is seen under an epifluorescence microscope
using different fluorophores
 Scoring criteria for FISH signals

1. Score non overlapping individuals with intense

bright signals using 10x100x magnifications
2. Do not count cells with overlapping signals
3. Cell with diffuse or split signal is considered as one
signals
4. Use optimum combination of filter set according to
fluorochromes excitation of signal and counterstain.
 Interpretation of FISH
Guidelines as per the international society of
cytogenetics nomenclature (ISCN) as follows:
1. Number of total chromosomes (autosomes and sex
chromosomes).
2. Methods of ish used.
3. Is there any structural chromosome abnormality if present
mention
4. Probe used (fluorochromes used)
5. Number of cells counted and the cell cycle phase (interphase
and metaphase)
• CLINICAL DETAILS: Global development delay, syndrome obesity. Height -129
cm, weight-40.3 kg
• INDICATION: Suspected Prader Willi syndrome
• TEST RESULTS:INTERPHASE/ NUCLEAR IN SITU HYBRIDIZATION
• Nue ish (D15Z1X2),(SNRPNX2),(PMLX2)
• FISH ANALYSIS
• PROBE USED :LSISNRPN/LSIPML/CEP 15
• 15p11.2(D15Z1)-spectrum aqua
• 15 q11-13(SNPRN)-spectrum orange
• 15 q22-24(PML)-spectrum green
• Number of INTERPHASE CELLS COUNTED-100
• NUMBER OF CELLS NEGATIVE FOR DELETIONOF THESNPRN LOCUS-
100%
• COMMENT:
• FISH analysis of 100 interphase cells is negative for deletion of the SNPRN locus
on chromosome 15 band q11-13 in 100% of cells
• 46,XX.ish 22q11.2(TUPLE1x2)(ARSAx2)

• 46 -The total number of chromosomes present

• XX -The sex chromosomes present (XX for girls, XY for boys)
• ish -The analysis was by fluorescent in situ hybridisation
(FISH)
• 22- The FISH test was performed on chromosome 22
• q11.2- The chromosome has two breakpoints, both in band
22q11.2 and material between these two breakpoints is missing
• (TUPLE1x2)- The DNA segment (probe) called TUPLE is
present in two copies (as expected)
• (ARSAx2)- The DNA segment (probe) called ARSA2 is
present in two copies (as expected)
If a deletion is present, the karyotype is written
slightly differently, as shown below:
46,XX.ish del(22)(q11.2q11.2)(TUPLE1-)

• 46 -The total number of chromosomes present

• XX -The sex chromosomes present (XX for girls, XY for boys)
• ish -The analysis was by fluorescent in situ hybridisation (FISH)
• del -A deletion, or material is missing
• (22)- The deletion is from chromosome 22
• (q11.2q11.2)- The chromosome has two breakpoints, both in band
22q11.2 and material between these two breakpoints is missing
• (TUPLE1-)- One copy of the DNA segment (marker) called
TUPLE is missing
 Locus specific probe BCR / ABL1
Negative for t(9;22) – two red, two green signals
Cryptic abnormalities such as the t(12;21) in
ALL -TEL/ AML1 fusion – favourable impact
on outcome
EXTRA SIGNAL DUAL COLOR
TRANSLOCATION PROBE
• TEL/AML1 fusion pattern :
1F1R1r1G
• One fused orange and green
signal which may sometimes
appear yellow (TEL/AML1)
• One green signal (native TEL),
• One large, orange signal (native
AML1),
• One smaller orange signal
(residual AML1)

(The green native TEL signal may be

absent in some instances due to
deletion
 DUAL COLOR DUAL FUSION- BCR/ABL1 FOR t(9;22)

NORMAL: 2R2G POSITIVE for BCR/ABL1 fusion: 2F1R1G

Red: ABL1 on chromosome 9 Fusion signals on translocated (derivative)
chromosomes 9 and 22
Green: BCR on chromosome 22 Green and red signals on normal chromosomes
 Trouble shooting
When viewing the results of FISH
assay
 1)Distorted chromosome morphology
 Increase temperature of
A. Specimen slides dried too water bath (dropping slides)
quickly during  Decrease temperature of
preparation slide warmer (sample
preparation)
 Increase drying time at least
overnight
 Don't bake slides at high
temperature

 Prepare denaturation
B)Specimen over denatured solution according to packet
insert
 Ensure temperature of
denaturation solution is
73+/-1°C
 Decrease time for
denaturation
C)Slides not thoroughly  Warm slides to 45-
dry prior to immersion 50°C prior to
in denaturation solution denaturation/
dehydrate slides
through series of
ethanol

D)Slides to fresh prior to

denaturation  Age slide at least 24
hours at ambient
temperature
 2)High slide background
• Immersed glass slide in ethanol
• Glass slide not sufficiently and wipe dry using lint free
cleaned prior to sample paper prior to dropping slides
preparation

• Cellular debris in sample • Wash cell pellet with fresh

preparation fixative three times and repeat
the slide dropping procedure

• Metaphase spreads were aged by

baking/ contain cytoplasm • Increase time the slides
immersed in denaturation
solution to 10 minutes
• Slide inadequately o Ensure the wash solution are made
washed following according to the package insert
hybridization
o Ensure pH and temperature of
wash solution are correct

o Remove coverslip and repeat the

wash procedure

 Wash solutions containing

formamide can be stored at 4°C ;
discard all other wash solutions
after one day
• Wash solution used for
too long or stored  Ensure pH of the formamide wash
improperly solution are pH7.0-8.0
 3)Weak or no signals
A)Slides not adequately denatured
B)Slides improperly aged after
dropping specimen
C)Slides not thoroughly dry prior to
immersion in denaturation solution
D)Probe not added
o Ensure temp of denaturation solution is
73+/-1°C prior to immersion of slides
o Increase the temperature of denaturation
solution to 74°C
o Increase the time the slide is immersed in
denaturation solution by 2-4 minutes
 Age for 24 hours at ambient temp before
performing FISH on them*
 Warm slides to 45-50°C prior to
denaturation/ dehydrate slides through a
series of ethanol
 Prepare new probe mixture, allow the
probe to thaw, vortex, centrifuge, pipet
probe slowly
E)Probe, hybridization buffer, or probe
mixture not mixed well prior to use
F)Probes improperly diluted for
hybridization
G)Probe not adequately denatured
H)Probe not applied to the target
sample immediately after probe
denaturation
 Vortex /pipette reagent to mix,
centrifuge
 Use the volume stated in the assay
procedure
• Ensure the pipette is calibrated
• Allow hybridization buffer to thaw
completely, pipet slowly
• Ensure the temp of water bath to
denature the probe mix is 73+/-1°C
 Denature the probe mixture for 5
minutes
 Plan work flow so probe is
applied immediately after slides are
removed from 100% ethanol
 Remove tube containing probe mix
from 73+/-1°C water bath and
place it at 45-50°C slide warmer
I)Probe mix dried too much on the
specimen slide
J)Air bubbles trapped under the cover
slip during hybridization
K)Hybridization condition
inappropriate
• Immediately place the cover slip
over the target area after applying
probe mix
• For washing post hybridization,
remove the cover slip from one
slide at a time and immediately
immerse the slide into the wash
solution before removing the
cover slip from the next slide.
 Apply cover slip by first touching
the surface of the probe mixture.
 Place the slide with cover slip
down on the blotter and very
gently press out visible bubbles
 Ensure the stated time and
temperature for hybridization
were followed
 Seal the cover slip well with
rubber cement, leaving no gaps
 Low signal specificity
A)Wrong counterstain used
&Counterstain too bright
B)Viewed hybridization using
inappropriate filter set
C)Microscope configuration/
objectives not adequate for
viewing FISH results
o Remove cover slip, immerse slides
for 5 minutes in 2XSSC / 0.1% NP-
40 at ambient temp; dehydrate slide
thorough a series of ethanol, air dry
and reapply counterstain
 Use correct filter for viewing
fluorophore
 Multi-band pass filter sets provide
less light than single band pass filter
sets, so probe may appear fainter
when viewed through the multi-
band pass sets
 Contact microscope manufacture

D)Probe diluted inappropriately
E)Inappropriate hybridization
condition
F)Wash temp too low
G)Wash solution stringency too low
• Ensure the probe mixture was
made according to the package
insert
 Ensure temp of incubator is 37°C
 Ensure that the hybridization
buffer was added to the probe
mixture and in proper amount
 Maintain the wash temp of the
wash solutions by placing no
more than four slide in one
Coplin jar at a time
 Ensure the wash solution are
made according to the package
insert
 Bright or weak counterstain
A)Counter stain appear weak: slides
not dehydrated prior to applying
counterstain or oil droplets in
counterstain
B)Wrong concentration of
counterstain
C)Counterstain too old or exposed to
light for extended periods
• Remove cover slip. Immerse slides
for 5 minutes in 2XSSC/ 0.1%NP-
40 at ambient temp; dehydrate
slide through a series of ethanol.
Air dry and reapply counterstain
 If it appears too bright, dilute the
counterstain in antifade solution
before applying
 Store counter stain at – 20°C
protected from light
 Ensure the counterstain is not used
past the expiration date
Trouble shooting results from
denaturation assay
 Cross hybridization
• Repeat the assay on a new specimen using one
of the following:
1. Increase the temp of 0.4xSSC/0.3%NP-40 by 2C
2. Decrease the melting temperature by 2C
 Probe appears dim
• Repeat the assay on a new specimen using one
of the following:
1. Increase the hybridization time
2. Increase the melting temperature
3. Wash the slides using 0.4 SSC/0.3 NP-40 at 70-73C
 Diffuse signals (speckling)
• Repeat the hybridization using one of the
following:
1. Decrease the melt temp by 2C
2. Decrease the melt time
3. Wash the slide using 0.4x SSC/0.3%NP-40 at 70-
73C
Inadequate signal quality in non-small-cell lung cancer sections for
the epidermal growth factor receptor fluorescence in situ
hybridisation assay that direct to subjective judgment.
 CLINICAL APPLICATIONS
• Classifications
 Haematolymphoid neoplasms

 Genetic disorders

 Other indications
 Malignancy

Leukemias

Acute lymphoblastic leukemia(ALL)

Acute myelogenous leukemia(AML)

Chronic lymphocytic leukemia(CLL)

Chronic myelogenous leukemia(CML)

Acute monocytic leukemia (AMoL)

lymphomas

Hodgkin's lymphomas (all four subtypes)

Non-Hodgkin's lymphomas(all subtypes)

Follicular lymphoma
 Genetic disorders
• Classified as:-
1. Chromosomal abnormalities
2. Single gene disease
3. Multifactorial disorders
CHROMOSOMAL ABNORMALITIES

NUMERICAL STRUCTURAL
• Aneuploidy • Deletion
Types- Hyper and hypo • Inversion
1. Trisomy • Duplication
2. Monosomy • Ring
3. Tetrasomy • Translocation
• These abnormalities are diagnosed at three
stages of life:
1. Prenatal
2. Postnatal
3. Childhood and adult
• Prenatal Cytogenetics
• Studies have shown that 1 in 13 conceptuses has a
chromosomal abnormality, but, of these, only 6 in 1000 are
live born, indicating that most errors are recognized and
biologically eliminated
• Methods of prenatal diagnosis:
 Maternal serum screening
1. Alpha fetoprotein estimation
2. Estriol and human chorionic gonadotrophin estimation
 Amniocentesis
1. Fetoprotein and acetyl cholinesterase
2. Chromosomal analysis
3. Biochemical analysis
 Chorionic villus sampling
1. DNA analysis
2. • Chromosomal analysis
3. • Biochemical analysis
 Fetal blood sampling
1. Chromosomal analysis
2. • DNA analysis
• Examples:-
1. Trisomy (13,18,21)
2. Cystic fibrosis.
3. Duchenne muscular dystrophy.
4. Haemophilia A.
5. Thalassemia.
6. Sickle cell anemia.
7. Polycystic kidney disease.
8. Tay-Sachs disease
 Postnatal
• Approximately 0.6% of newborns have a chromosome
abnormality
• Methods to collect postnatal samples:
1. Umbilical cord testing-after child is born sample is collected
from umbilical cord
2. Venipucture-obtain from vein via syringe or vacuum
3. Buccal swab-rubbed on inside cheek to obtain DNA sample
• Types of disorders-
1. Metabolic disorders (e.g., phenylketonuria (PKU),
galactosemia)
2. Endocrine disorders (e.g., congenital
hypothyroidism and congenital adrenal hyperplasia )
3. Haemoglobin disorders (e.g., sickle cell anemia)
4. Other disorders (e.g., cystic fibrosis)
 Childhood and Adult
• Most difficult diagnostic problems occur in
children and adults.
• Molecular and biochemical options must also
be taken into account.
• Examples:-
 Nonbrainstem glioblastomas of childhood
 Large B-cell lymphoma
 Autosomal Aneuploidies

• Trisomy 21(Down syndrome)

• The most common cause of mental retardation.
• This disorder has a birth incidence of 1 in 700.
• Approximately 92.5% of all Down syndrome individuals
have 47 chromosomes including three copies of
chromosome 21 resulting from a nondisjunction error in
parental meiosis.
 Trisomy 18
• .
 FISH (fluorescence in situ hybridization) probes for chromosomes 21 (red signal), 18
(yellow or fused red/green signal), and 12 (green signal) were used to identify cells with
trisomy 21 (left) and both trisomy 18 and trisomy 21 (right).
 Sex Chromosome Aneuploidies
• Common(overall frequency of 1 in 500 births)
• Phenotypically milder than autosomal aneuploidies because of
the effect of X inactivation and the limited number of genes
present on the Y chromosome
• Types:
1. Klinefelter syndrome (47,XXY)
2. Turner syndrome
 FISH in Klinefelter syndrome
FISH analyses using sex chromosome-specific probes to the α-satellite of the X
centromere region (red signal) and satellite III of Yq12 (green signal) on a buccal
smear specimen revealed 86% XXY and 11.5% XY. The remaining 2.5% of cells had
an XX signal pattern, probably due to the artificial loss of 1 Y chromosome in the
FISH procedure or true mosaicism with 3 cell lines
Turner syndrome
FISH analysis with centromeric X (DXZ1; spectrum green) and centromeric Y (DYZ3; spectrum
red) probes showing one 46,XY metaphase and five X interphases with one green signal
indicating monosomy X.
 Structural Chromosome Anomalies

• Deletion
1. Wolf-Hirschhorn syndrome-4p– syndrome, is due to a
terminal deletion of the short arm of chromosome 4, del(4)
(p16).
2. Cri-du-chat, or 5p– syndrome, del(5)(p15), is characterized
by a distinctive high-pitched, catlike cry in infancy
3. Micro deletion Syndromes and contiguous gene syndrome
4. Breakage syndrome
 Wolf-Hirschhorn syndrome (WHS)
FISH result on metaphase spreads for case 2 shows the presence of only
one red signal (WHSCR), demonstrating the deletion of WHSCR on
chromos [...]
 Fluorescent in situ hybridization (FISH) study of a patient with cri-
du-chat syndrome.
Spectrum orange colour represents chromosome 5–specific signal and
spectrum green is cri-du-chat locus signal. Absence of a green signal indicates
monosomy for that region (left, interphase cell; right, metaphase
chromosome spread)
Micro deletion Syndromes and
Contiguous Gene Syndromes
 Breakage Syndromes
• Set of autosomal recessive disorders that were originally
grouped together because of the common finding of
chromosome instability or fragility
• Examples:
1. Fanconi anemia
2. Cockayne syndrome
3. Ataxia telangiectasia
4. Bloom syndrome
RING CHROMOSOMES
Amplification of MDM2 (red), CDK4 (green), and chromosome 9 sequences (blue) in a
low-grade malignant fibrous histio cytoma (a). Rings from six different cells from the
same tumour showing extensive variability in size and structure (b). Anaphase bridges
containing amplified MDM2 sequences (red) in a well-differentiated lipo sarcoma.
(A) BCR/ABL rearrangement and inversion of chromosome 9 involved in the translocation
with chromosome 22 were detected using the LSI BCR/ABL+9q34 Tricolor dual fusion
translocation probe. (B) The application of MCB 9 shows the chromosomal breakpoints
of inversion, inv (9)(q22q34). (C) M-FISH confirms the complexity of the karyotype
46,XX,t(9;22),t(10;17). (D) The deletion of TP53 on der(17)(p13.1) was identified using
17p13(p53)/Alpha-satellite 17, dual color. #, chromosome; der, derivative chromosome;
Ph, Philadelphia chromosome.
 Conventional cytogenetic and FISH findings in BM of patients with HSTCL. A,
Conventional karyotype analysis showing both iso chromosome 7q(10) (blue) and
trisomy 8 (red) (case 8). B, FISH shows iso chromosome 7q (red: D7S522; green:
CEP7)
Chromosomal duplication
FISH analysis on metaphase spread and interphase nuclei showed
duplication of chromosome 8p22 region
Chromosomal translocation
A, Banded chromosomes showing the 9;22 translocation. The derivative
chromosome 22 is known as the Ph′, or Philadelphia chromosome
C, No translocation is present as seen by two copies each of the chromosome 9 (red) and chromosome 22 (green) probes
in both interphase (left) and metaphase (right) cells.
Cells with a M-BCR translocation
as detected by the single yellow fusion signal (arrow) plus one green, one large
red, and one small red signal (metaphase on the left and interphase on the right).
 QUALITY CONTROL
• It includes:
1. Internal quality control
2. External quality assessment
• Internal quality control:
 Document of the internal process of a laboratory such
as reagent batch and expiry dates, specificity and
sensitivity of FISH probes, probe validation, standard
operating procedure (SOP) for technical staff and
analytical aspect of laboratory service.
• External quality assessment:
1. After obtaining result from the patient sample
2. The same sample is been sent to different laboratory along
with the results so that there is no biased
3. Assure to patients and referring clinicians
 Advantages

1. Can be used on any tissue; dividing cells not

required
2. Rapid screening of large numbers of cells
3. Detects specific abnormalities
 Disadvantages
1. Very specific- will detect abnormalities only of the region that
is under study and not any abnormalities on other
chromosomes
2. Therefore will not detect any associated abnormality that
could represent disease progression
3. Interphase FISH may not detect variant translocations
4. Probes are expensive – 20 µL can cost Rs 50,000 to 70,000
5. Recommended - 20 tests from this amount; in practice, about
40- 50 tests from this volume
6. Mercury vapour lamp expensive – about Rs 15,000, needs to be
changed every 200 hours of use; filters also need replacement
FISH vs. other techniques
characteristics FISH PCR RT-PCR
target DNA DNA M-RNA
Detection principle Complementary Complementary Complementary
hybridization amplification reverse transcription
and amplification
sensitivity 1 in 1000 cells 1 in 100000 cells 1 in 100000 cells
specificity Very high Relatively high Relatively high
Assay duration 1-2 d 1d 1d
False positive rate negligible Possibility - Possibility-
amplification amplification
False negative possible negligible negligible
Chance cross unlikely likely likely
examination
quantitation Possible Semiquantitative Semi quantitative
Single cell details Comprehensi Unavailable Unavailable
and tissue ble
architecture

Screening of possible in Not possible Not possible

genetic metaphase
abnormalities

Coverage of Easy Cumbersome and Cumbersome and

multiple breakpoint requires multiple primer requires multiple
application primer application

Detection of single Not possible Possible with additional Possible with

nucleotide nucleotide sequencing additional
nucleotide
sequencing
 Targeted Gene Sequencing

• Useful tools for analysing specific mutations in a given sample.

• Produces a smaller, more manageable data set compared to broader
approaches such as whole-genome sequencing, making analysis
easier.
• ADVANTAGES:-
1. Sequences key genes or regions of interest to high depth (500–
1000× or higher), allowing identification of rare variants
2. Provides cost-effective findings for studies of disease-related genes

3. Delivers accurate, easy-to-interpret results, identifying variants at

low allele frequencies (down to 5%)
4. Enables confident identification of causative novel or inherited
mutations in a single assay
 FLOW CYTOMETRY
• Methods of studying cells and chromosomes
• ADVANTAGES:
1. Used to study heterogeneous population of cells
2. Analyse the populations in few minutes
3. Detail data is produced
• Disadvantages
1. It is expensive
 DNA MICRORRAY
• Solid supports upon which DNA is attached in an organized
grid fashion
• Advantages:
1. Provides data for thousands of genes
2. One experiment instead of many
3. Fast and easy to obtain results
• Disadvantages:
1. Expensive to create
2. Long time for analysis
3. DNA chips do not have long shelf life
 Summary
• Expensive and performed in few higher centres- helps in
diagnosing specific chromosomal disorders and mutations
which will help to establish diagnosis.
• Guiding the clinicians to decide treatment options.
• Helps in monitoring the response to treatment and to modify
treatment protocol if required.
• The other family members can be investigated on similar lines
if they is strong suspicion. The family members include
parents and siblings
• Genetic counselling of parents of the patients(infant
or child or adolescence) if they are planning for
another baby.
• Though they are many higher molecular diagnostic
methods available, the integrated diagnosis has to be
followed i.e. consideration of overall clinical features,
routine investigations complemented by higher
molecular diagnostic techniques.
 FISH CENTRES
• Karnataka
 Genelon institute of life science-Bengaluru
• Tamil Nadu
 CMC Vellore
• Delhi
 Dr Lal PathLabs
videoplayback.mp4
 Reference
• Constance K Stein. Applications of cytogenetics in modern pathology. In; Henry
clinical diagnosis and management by laboratory methods. Elsevier 21 st edition.
• Suneel D Mundle and Robert J. Kosea. Fluorescence in situ hybridization. In;
Molecular diagnostic: For the clinical laboratorian. Humana press INC 2 nd edition.
• Neoplasia. In; Robin and Cotran Pathological basis of disease. Elsevier 9 th edition.
• Amare PS. The practical aspects of cytogenetics in haematlogical Malignancies. In
laboratory techniques in heamatology.Jaypee
• Journal of Cell Science 116, 2833-2838 © 2003 The Company of Biologists Ltd
doi:10.1242/jcs.00633
• Cytogenetics of acute leukemias Dr Vivi M Srivastava Cytogenetics Unit Christian
Medical College, Vellore
• Journal of Cell Science 116, 2833-2838 © 2003 The Company of Biologists
Ltddoi:10.1242/jcs.00633