FISH

TISH
PRESENTER : DR.ASTHA SRIVASTAVA
MODERATOR : DR ANKITHA PATIL
 OUTLINES
• INTRODUCTION
• RESULT AND
• DEFINITION
INTERPRETATION
• HISTORY
• QUALITY CONTROL
• PRINCIPLE
• ADVANTAGES,
• TYPES
DISADVANTAGES
• PROCEDURE
• TROUBLE SHOOTING
• INDICATIONS
•
 OUTLINES
• FISH VS OTHER TECHNIQUES
• ONE DAY QUICK FISH
• TISH
• SUMMARY
• REFERENCES
INTRODUCTION
 HUMAN SOMATIC CELLS

46 CHROMOSOMES

22 PAIRS AUTOSOMES 2 SEX

CHROMOSOMES

Study of Chromosomes under microscope is CYTOGENETICS

Conventional Molecular
KARYOTYPING FISH
• KARYOTYPING : Study of number and appearance (including abnormal )

chromosome in the cell.

• Arrest the dividing cells in the METAPHASE (routinely ) with mitotic spindle inhibitors

• Eg: Colcemid
• 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
In situ hybridization is simply detection of specific genetic information within a morphologic

context.

This specialized type of solid-support assay involves taking morphologically intact tissue, cells,

or chromosomes affixed to a glass microscope slide through the hybridization process.

Technique Instrument/ Primary advantage Primary application
visualization method

Bright-field microscopy Ability to view the CISH

CISH signal and tissue Molecular pathology
morphology diagnostics
simultaneously

Fluorescence Multi plexible: visualize Gene presence, copy

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

Fluorescence Multi plexible: visualize Gene expression, RNA

RNA-FISH microscopy, HCS, and multiple targets in the temporal and spatial
flow cytometry same sample localization
 OTHERS
• Fluorescence ISH (FISH): direct detection of a fluorescent labeled
probe
• Bright-field ISH (BISH): indirect detection through an enzymatic
reaction
• Silver ISH: detection of silver precipitation (black dots)
• Chromogenic ISH (CISH): detection of chromogen (e.g. red)
• Dual hapten dual probe ISH (DDISH): CISH + SISH
 FISH-
DEFINITION
• Fluorescent in situ hybridization technique (FISH) is also

known as interphase cytogenetics

• In this technique the double-stranded DNA is at first

converted into single-stranded DNA, and then

subsequently a fluorescent-tagged probe is used to

visualize the target DNA part

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
PRINCIPLE
The basic principles of FISH are

• To convert double-stranded DNA into single-stranded DNA.

• DNA probes tagged with fluorescent dyes are also made to single stranded.

• The florescent-tagged single-stranded DNA probes are allowed to bind with the

corresponding single-stranded DNA.

• The hybridized probe-target DNA complexes are visualized by fluorescence

microscope
OPTICAL PRINCIPLE
 FISH PROBES

• Short fragment of synthesized DNA tagged with a fluorochrome

• The nucleotide sequence corresponds to gene of interest
• Commercially available
Categories:

(a) single-copy (locus-specific) genomic probes : Translocations Dual colour and breakapart

probe

(b) centromere-specific probes

(c) whole chromosome (‘painting’) probes

(d) spectral karyotyping (SKY; multiplex metaphase FISH; multicolor FISH)

Repeat sequence probes
● Also called as CENTROMERE-SPECIFIC PROBES
(CENTROMEREENUMERATING PROBES, CEPS), Alphoid Probes

● Repeat sequence probes are usually used in chromosome enumeration (i.e., to

detect the gain or loss of specific chromosomes)
○ Chromosome-specific pericentromeric and subtelomeric probes are typically
used for this purpose
Repeat sequence centromere probe (for chromosome 21)

C, Trisomy 21 seen at left in an interphase nucleus and at right in a metaphase (arrows).

D, Normal complement of two copies of chromosome 21 (arrows) seen in interphase (left and right)
and in a metaphase (center).
 Chromosome painting probes
• Chromosome painting probes
are actually a cocktail of many
unique DNA fragments from along
the entire length of a
chromosome, so that following
hybridization, the entire
chromosome fluoresces

Whole chromosome paint

probe highlighting the two X
chromosomes in a female
cell.
SINGLE-COPY GENOMIC PROBES (SYN: LOCUSSPECIFIC; GENE-SPECIFIC, OR UNIQUE

SEQUENCE PROBES)

•
A unique sequence probe is usually isolated from cloned DNA of a disease-

causing gene or a fragment of DNA of known location associated with a

particular gene.

• This type of probe is used to identify the presence or absence of a gene, gene

region, or chromosomal rearrangement of interest

Multicolor FISH

● With computer assisted system, detection of multiple colors become possible.

● The computer does not see more colors but it can detect subtle differences in

shade better than the human eye.

● Eg. Two fluorochromes are used and mixed in varying proportions to give a range

of colors.
Fiber FISH
- Interphase chromosomes are attached to a slide in such a
way that they are stretched out in a straight line.
- This is by applying mechanical shear along the length of
the slide, either to cells fixed on slide or to a solution of
purified DNA.
- Extended conformation of the chromosome allows higher
resolution – even down to a few kilobases.
Q-FISH
The technique uses labelled synthetic DNA mimics called peptide
nucleic acid (PNA) oligonucleotides to quantify target sequences
in chromosomal DNA using fluorescent microscopy and analysis software.

Q-FISH is most commonly used to study telomere length, which

in vertebrates are repetitive hexameric sequences (TTAGGG) located at
the distal end of chromosomes.

Telomeres are necessary at chromosome ends to prevent DNA-damage

responses as well as genome instability. To this day, the Q-FISH method
continues to be utilized in the field of telomere research
TECHNIQUE
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
● Nucleated cell sample
○ Heparinized peripheral blood is the preferred
specimen
○ In hematologic disorders, the best results are obtained
from bone marrow samples
○ Fibroblast cultures from skin biopsies
● Prenatal analysis –
○ Amniotic fluid collected by amniocentesis- 16
and 18 weeks of gestation, at which time 20 to 30
mL of amniotic fluid
○ Chorionic villus sampling - The transabdominal
or transvaginal procedure is performed at 10 to 14
weeks’ gestation
○ Cordocentesis, or percutaneous umbilical blood
sampling (PUBS) – Fetal blood specimen, later
gestational ages (≥20 weeks)
PROCEDURE
 Steps to Do FISH
1. Fixation:
• Cytology 4%
paraformaldehyde 10
min.
• Histology 10%
buffered formalin.
5. Hybridization:
• Add 10 μl of
denatured probe
solution over the slide.
• Seal coverslip
• Incubate it at 37 °C
for 1 day (24 h).
2. Deparaffinization
• Bake slide overnight in
56°C.
• Xylene for 10 min
• 70% and 100% ethyl alcohol
twice for 5 min.
6. Post-hybridization
• Remove the coverslip, rinse the
slides in SSC for
5 min twice.
• Slides in a Coplin jar filled with
pre-warmed SSC at 70 °C.
• Keep at room temperature.
3. Pretreatment
• Proteinase K solution (20
μg/ml) for 15 min
• Wash the slide in
deionized water.
• Dehydrate
7. Visualization: Counterstain
the slide by 5 μl DAPI/antifade
solution. Now visualize the
cells by an epifluorescence
fluorescence microscope.
4. Denaturation
• of target DNA- treating the
smear with denaturing
solution (70% formamide) for
2 min time at 72 °C.
• of probe DNA – add 1 μl of
labelled probe with 9 μl of
65% formamide solution.
Now heat the mixture at 75
°C for 5–6 min.
PROCEDURE
INDICATIONS
 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
Mitosis is seen under an epifluorescence microscope using different
fluorophores
INTERPRETATION
 Unaffected individual
↓
2 signals per cell
(one for each chromosome of the pair)

Affected individual(deletion)
↓
single signal per cell
(one normal and one deleted chromosome)
 NOMENCLATURE

In current use is the International system for Human Cytogenetic

Nomenclature from 2009.(ISCN)
nuc ish : interphase in situ hybridization
Abbreviations:
+ present on a specific chromosome
- absent on a specific chromosome
++ duplication on a specific chromosome
amp amplified signal
con connected or adjacent signals
; seperates probes on different derivative chr
. Seperates cytogenetic results from ish
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
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
 DUAL COLOR DUAL FUSION- BCR/ABL1 FOR t(9;22)

NORMAL: 2R2G
Red: ABL1 on chromosome 9
Green: BCR on chromosome 22
POSITIVE for BCR/ABL1 fusion: 2F1R1G
Fusion signals on translocated (derivative)
chromosomes 9 and 22
Green and red signals on normal chromosomes
Trouble shooting

When viewing the results of FISH assay

1)Distorted chromosome morphology
A. Specimen slides dried too
quickly during
preparation
B)Specimen over denatured
 Increase temperature of
water bath
 Decrease temperature
of slide warmer
 Increase drying time at
least overnight
 Prepare denaturation
solution according to
packet insert
 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)Weak or no signals
A)Slides not adequately denatured o Ensure temp of denaturation solution.

o Increase the temperature of denaturation

B)Slides improperly aged after dropping solution to 74°C
specimen o Increase the time the slide is immersed in
denaturation solution by 2-4 minutes

 Warm slides to 45-50°C prior to denaturation/

C)Slides not thoroughly dry prior to dehydrate slides through a series of ethanol
immersion in denaturation solution

 Prepare new probe mixture, allow the probe to

thaw, vortex, centrifuge, pipet probe slowly
D)Probe not added
E)Probe, hybridization buffer, or probe
mixture not mixed well prior to use  Use the volume stated in the assay procedure

● Ensure the pipette is calibrated

F)Probes improperly diluted for hybridization
● Allow hybridization buffer to thaw completely, pipet

slowly
● Ensure the temp of water bath to denature the
G)Probe not adequately denatured
probe mix is 73+/-1°C
 Denature the probe mixture for 5 minutes

H)Probe not applied to the target sample  Remove tube containing probe mix from 73+/-1°C
immediately after probe denaturation
water bath and place it at 45-50°C slide warmer
 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 . 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
● 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
CLINICAL APPLICATIONS
Haematolymphoid neoplasms
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
 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
  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)
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.
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 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 [...]
Micro deletion & 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
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
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. Probes are expensive – 20 µL can cost Rs 50,000 to 70,000
4. Recommended - 20 tests from this amount; in practice, about 40- 50 tests
from this volume
5. 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 hybridization Complementary amplification Complementary 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 -amplification Possibility-amplification

False negative possible negligible negligible

quantitation Possible Semiquantitative Semi quantitative

characteristics FISH PCR RT-PCR

Single cell details and tissue Comprehensible Unavailable Unavailable

architecture

Screening of genetic possible in metaphase Not possible Not possible

abnormalities

Coverage of multiple breakpoint Easy Cumbersome and requires multiple primer Cumbersome and requires
application multiple primer application

Detection of single nucleotide Not possible Possible with additional nucleotide Possible with additional
sequencing nucleotide sequencing
One-Day Quick FISH
• The classical FISH requires overnight incubation for proper hybridization result.

• Tissue morphological features are varying due to aggressive pretreatment and high

temperatures applied.

• To increase the speed of the molecular cytogenetic finding and eliminate some of the

technical limitations, an alternative oneday FISH method was recently introduced.

• Allows completion and evaluation of a FISH reaction within one day by the
reduction of the hybridization time to 60–120 min.

• Low denaturation temperature  better tissue and cell morphology.

• The utility of the instant quality FISH (IQFISH) was carefully evaluated for the
determination of HER2 copy number status in breast carcinoma and ALK
translocation status in lung adenocarcinoma samples.

• Summary, the one-day IQFISH diagnostic kits point with fast and stable
hybridization reaction in the diagnostic practice without any major loss compared to
the conventional (overnight) FISH
FISH CENTRES
● Karnataka
 Genelon institute of life science-Bengaluru
● Tamil Nadu
 CMC Vellore
● Delhi
 Dr Lal PathLabs
Third-strand in situ
hybridization (TISH) to non-
denatured metaphase spreads
and interphase nuclei
• A methodology developed for binding oligodeoxyribonucleotide ’third

strands’ to duplex DNA targets in fixed but not additionally denatured

metaphase spreads and interphase nuclei under conditions found to be

optimal in solution.

• Third-strand in situ hybridization (TISH) at pH 6.0 of a psoralen- and biotin-

modified 16-nucleotide homopyrimidine third strand to a unique multicopy

target sequence in human chromosome 17 α-satellite (D17Z1 locus) is

described.
• UVA-photofixed third strands, rendered fluorescent by fluorescein
isothiocyanate-labeled avidin, are reproducibly centromere-specific
for chromosome 17, and visible without amplification of the signal
in lymphocyte and somatic cell hybrid spreads and interphase
nuclei.
• Two third-strand-specific D17Z1 haplotypes were identified. TISH
has potential diagnostic, biochemical, and flow cytometric
applicability to native metaphase and interphase chromatin.
SUMMARY
• Fluorescence in Situ Hybridization (FISH) is a molecular
cytogenetic technique that uses fluorescent probes which
bind to specific chromosomal locations within the nucleus to
search and detect chromosomal abnormality.

• In the last decade, tumor-specific chromosomal

translocations, deletions, gains, amplifications, and novel
oncogenes have become increasingly important in the field
of diagnostic, therapeutic, and prognostic concepts in
medicine.
• FISH technique provides a quick analysis of formalin-fixed paraffin-

embedded cells which can be used in daily practice of pathology for

several tumor types.

• Fluorescence in Situ Hybridization (FISH) technique is based on

hybridiza-tion of tagged DNA probes which are fluorescent reporter

molecules that affirm the presence or absence of particular genetic

anomaly under fluorescence microscopy.

THANKYOU
REFERENCES

• Robbins & Cotran, Pathologic Basis of Disease 9th edition

• Henry’s, Clinical Diagnosis and Management by Laboratory Methods, 22nd edition.

• Anderson’s Pathology 10th edition.

• The Washington Manual of Surgical Pathology 3Rd edition.

• Thomas liehr, Anja Wise; Fluorescence In Situ Hybridization (FISH), Springer 2017

• Susan Mahler, Chromosal , FISH and Microarray based Reporting, Cytogenetic

abnormalities; 1st edition ; 2014

• Cytogenetic Nomenclature: Changes in the ISCN 2013 Compared to the 2009 Edition