Fluorescence In Situ Hybridization

1. FISH- Fluorescence In Situ Hybridization
Dr.C.Lingasamy.C
II Year PG
26/3/2018

2. FISH Preview
• What is FISH?
• Historic perspectives.
• Basics of cytogenetics .
• Basic methodology.
• Basic requirements.
• Samples .
• Probes .
• FISH protocol.
• Applications of FISH.
• Factors affecting FISH.
• Latest about FISH.

3. What is FISH?
• Cytogenetic technique to detect and localize the presence or
absence of specific DNA sequences in chromosomes.
• Performed in order to detect inherited or disease related
genotypes, mutations, phenotypes or karyotypes for clinical
purposes.
• Gain or loss of chromosomes.
• Fusion of genes.
• Altered position of genes.

4. HISTORIC PERSPECTIVES
• 1953 – Watson and Crick - DNA double helix.
Pre banding era
• 1956 – Tijo and Levan - 46 chromosomes.
• 1960 – Denver Conference – 23 human chromosome pairs divided into 7
groups(A-G)
Banding era
• 1970 – Chaudhuri & Clasperson – G and Q banding.
• Smith & Wilcox – Birth of molecular genetics- specific restriction endonuclease.
FISH era
• 1981 - Harper & Saunders – Localization of single copy genes by ISH.
• 1986 – Saiki – PCR technology, Pinkel – FISH
• 1990 – Nederlof – Multicolor FISH

5. BASICS OF CYTOGENETICS

7. TERMINOLOGY IN CYTOGENETICS
• Amplification – production of additional copies of gene sequences.
• Anneal – to join 2 strands of complementary nucleic acids.
• Complementary sequence- nucleic acid sequence of bases that can
form a double stranded structure by matching base pairs.eg.
CATG= GTAC
• Denature – to dissociate a double stranded region of nucleic acid
into homologous single strands.

8. TERMINOLOGY IN CYTOGENETICS
• Hybridisation – 2 complementary single stranded pieces of nucleic
acids are joined to form a double stranded segment.
• Nick translation – incorporating a labelled deoxyribonucleotide
triphosphate into DNA.
• Probe – single stranded piece of labeled DNA or RNA that will
bind to a complementary sequence.

9. BASIC METHODOLOGY
Fluorescence microscopy
Probe binding to target DNA
Labelled with fluorochromes
Hybridised to specific DNA probe sequences
DNA denatured – single stranded
Fixation of DNA
Metaphase chromosomes Interphase chromosomes

11. BASIC REQUIREMENTS
• Biological specimen with preservation of sufficient morphological
detail to determine the localization of the labelled probe after
hybridisation.
• Probe that is specific for the sequence of interest.
• Fluorescent labelling of this probe.

12. SAMPLES

13. SAMPLES
 Blood
 Bone marrow
 Epithelial cells
 Hair root cells
 Amniotic fluid cells
 Human sperm cells
 Formalin fixed and paraffin embedded tissue sections
 Cryofixed tissue
 Yeast and bacteria

14. • Methanol/ acetic acid fixative for metaphase spreads.
• 10% neutral buffered formalin.
• 4% formaldehyde – cyrostat.
• Bouin’s fixative
• Alcohol and mercuric fixatives – not recommended.

15. PROBES

16. PROBES ?????
TYPES:
 Repetitive sequences (centromeres or alpha satellite regions of
chromosomes)
 Whole chromosome sequences (short arm, long arm of chromosome)
 Locus specific - Unique sequences (1kb to >1 Mb of DNA)
Commonly used probes –
• Cloned DNA fragments like plasmids, cosmids
• Polypeptide nucleic acid(PNA)
• Locked nucleic acid (LNA)
• Padlock probes

17. PROBES

18. LABELLING
• Direct (fluorochrome)
• Indirect (incorporation of hapten biotin/digoxygenin into DNA via
nick translation or PCR.
• Detected by fluorescently labelled antibody
strepavidin/antidigoxygenin

19. METHODS OF LABELLING
• Enzymatic methods.
• Nick translation or randomly primed extension.
• Chemical crosslinking.
• PCR based methods.

20. FISH INSTRUMENT

21. FISH PROTOCOL
• Sample preparation (source, fixation & unmasking)
• Probe preparation and labelling.
• Denaturation of probe and sample.
• Hybridisation of probe to sample.
• Post hybridisation wash.
• Detection.

22. CLINICAL APPLICATIONS

23. Diagnostic Research
Identification of specific chromosome
abnormalities
Identification of new non-random
abnormalities (by M-FISH or SKY)
Characterization of marker chromosomes Gene mapping
Interphase FISH for specific abnormalities in
cases of failed cytogenetics
Identification of regions of amplification or
deletion by CGH
Monitoring disease progression Identification of translocation breakpoints
Monitoring the success of bone marrow
transplantation
Study of 3D chromosome organization in
interphase nuclei
APPLICATIONS OF FISH

24. CLINICAL APPLICATIONS OF FISH
• CONSTITUTIONAL ABNORMALITIES
- Microdeletion syndromes
- Subtelomeric rearrangements
- Prenatal studies
• ACQUIRED ABNORMALITIES
- Hematological malignancies
- Solid tumors
- Breast cancer
- Bladder cancer
- Diagnosis of infections

25. CLINICAL APPLICATIONS
• CONSTITUTIONAL ABNORMALITIES
Microdeletion syndromes
- Small deletion of genetic material results in loss of
several genes from one chromosomal region.
- <2Mb

26. MICRODELETION SYNDROMES

28.  Subtelomeric rearrangements
- Located immediately proximal to the terminal telomeric repeated DNA
sequences.
- Unique sequence and highly gene rich FISH probes designed for
subtelomeric regions of every chromosome except
1)acrocentric short arms( chr 13,14,15,21&22)
2)short arms of X & Y chromosomes which share sequence homology
3)long arms of X & Y chromosomes which also share sequence
homology
• Growth delay and mental retardation.

29. Prenatal studies
• Rapid detection of aneuploidy numerical abnormalities in
uncultured cells.
• 24 to 48 hrs – turnaround time.
• Advanced maternal age >35yrs.
• Abnormal USG findings.
• Abnormal maternal screening results.

31. ACQUIRED ABNORMALITIES
• HEMATOLOGICAL FISH PROBES (Vysis; Abbott labs)
1) Dual color/single fusion probes
2) Extrasignal probes
3) Dual color/ break apart probes
4) Dual color/ dual fusion probes

32. SOLID TUMORS
 Ewing’s sarcoma – EWS gene chr 22 and FL1 gene chr 11.
- Break apart / dual color FISH - telomeric and centromeric to EWS
gene.
- Normal 2 yellow signals.
-Abnormal – one yellow, one red and one green – represents
disruption and translocation of EWS gene.
 Synovial sarcoma – t (X ;18) SSX1 or SSX2, SYT
 Myxoid round cell liposarcoma – chromosomal rearrangements
involving DDIT 3 gene on chr 12.
 Alveolar rhabdomyosarcoma – abnormal fusion gene FOXO1
chr13 and PAX3chr 2 or PAX7 on chr 1.

34. BREAST CANCER
 Her 2 gene chr 17
• Overexpressed or amplified in 25% of breast cancers.
• Poor prognosis
• Increased recurrence
• Shortened survival time
1)IHC – level of expression of gene.
2)FISH – number of copies of gene.
 Chemotherapy responsiveness
 Selection of targeted monoclonal antibody therapy - Herceptin

35. • 4 µm
• Tumor areas scored.
• FISH analysis with Her2 gene in combination with alpha
satellite probe for the centromere of chr 17.
• Number of Her2 and chr 17 signals is scored.
• Ratio of >2.2 - amplification

37. BLADDER CANCER
• Aneuploidy for centromeric region of chr 3,7 ,17 –
histological progression.
• Homozygous loss of short arm of chr 9 – recurrence.
• FISH-intial diagnosis with hematuria
• Voided urine may used

39. SCORING CRITERIA
• METAPHASE CRITERIA
 Only complete metaphases should be scored
 Analysis of 10 metaphase cells
 Atleast 2 images captured- abnormal
 Atleast 1 image – normal
 Mosaicism – additional metaphase cells counted
• INTERPHASE CRITERIA
 Atleast 200 individual cells
 Only cells in monolayer are countable
 Signals should be discrete

40. TROUBLESHOOTING
• Slide background
 Inadequate post hybridisation wash
 Inadequate cleaning of glass
• Weak or no signal
 Specimen or probe inadequately denatured
 Probe not added
 Counterstain too bright
• Distorted chromosome morphology
 Specimen over- natured

41. FISH REPORT
• Metaphase (ish) or interphase (nuc ish) cells
• Chromosomal location
• Probe name
• Number of signals observed
• Eg. ish del(15)(q11.2q11.2)(SNRPN- )
Prader Willi syndrome

42. FACTORS AFFECTING FISH

43. FACTORS AFFECTING FISH
• Size of target sequences.
• Target sequence unique or multiple copies at the site of location.
• Size of probe sequence. (300 to 500 base pairs)
• Indirect labelling techniques.

44. ADVANTAGES OF FISH
 Rapid (AML M3)
 Analyze large numbers of cells
 Both metaphase and interphase cells. (CLL)
 High sensitivity and specificity
 Samples with low mitotic index and terminally differentiated cells
 To detect residual disease and early relapse
 To assess therapeutic efficacy

45. DISADVANTAGES OF FISH
× Inability to interrogate a more than
a few abnormalities.
× Specific abnormality sought is only
identified.
× Limited probes.
× Cost .

46. LATEST IN FISH

47. • Spectral karyotyping analysis/multiplex FISH
• Microarray

48. AML M3 t(15;17)

49. CML bcr;abl t (9;22)

50. REFERENCES
• S. Kim suvarna, Christopher layton, John D. Bancroft,Theory and Practices of
Histological Techniques ,6 th edition.
• Douglas C. Tkachuk, Jan V. Hirschmann,Wintrobe’s hematology 12 th edition
• Sir John V. Dacie, S. Mitchell Lewis,Dacie and Lewis Practical Hematology 11th edition
• Liang Cheng, Shaobo Zhang, Fluorescence in situ hybridization in surgical
pathology:principles and applications:J Path: Clin Res April 2017; 3: 73–99.
• Ratan Z, Zaman S, Mehta V, et al. (June 09, 2017) Application of
Fluorescence In Situ Hybridization(FISH) Technique for the Detection of
Genetic Aberration in Medical Science. Cureus 9(6): e1325.
DOI10.7759/cureus.1325

51. THANK YOU