Fluorescence In Situ Hybridization

Slide 1 · Fluorescence in situ hybridization

Molecular cytogenetics: principles, interpretation, and diagnostic applications

Pathology teaching lecture

Dr Abu Suraih Sakhri

MBBS, MD (Pathology)

www.abusuraihsakhri.com

Slide 2 · Learning objectives

Explain how a labeled probe identifies a complementary DNA interval in a nucleus

Interpret qualitative patterns for aneuploidy, deletion, amplification, and rearrangement

Select suitable specimens and recognize analytical limitations

Relate FISH results to selected constitutional and neoplastic disorders

Slide 3 · FISH is a targeted DNA-localization assay

Fluorescently labeled DNA probes bind their homologous genomic sequences

Signals are viewed at their chromosomal or nuclear locations

The output is a spatial signal pattern, not a nucleotide-by-nucleotide readout

Probe maps to a defined locus

Target interval

Locus-specific probe

Schematic — relative lengths not scaled

Slide 4 · Why large structural lesions need other methods

Large deletions, duplications, and complex rearrangements may be difficult to characterize by short-target PCR

FISH visualizes a chosen chromosome interval rather than amplifying one short sequence

Its use is strongest when the suspected locus or abnormality is already known

Known locus

Select DNA probe

Spatial readout

Copy / arrangement

Interpret in cells

Slide 5 · The diagnostic question determines the probe

A copy-number question requires a probe at the locus of interest

A deletion question benefits from a separate retained control region

A rearrangement question requires probes whose geometry changes when DNA is reorganized

Probe maps to a defined locus

Target interval

Locus-specific probe

Schematic — relative lengths not scaled

Slide 6 · Probes recognize chromosome intervals

Labeled genomic fragments spanning as much as approximately 200 kb

Each probe hybridizes to a complementary region of the specimen genome

Probe position and span determine what the assay can and cannot interrogate

Probe maps to a defined locus

Target interval

Locus-specific probe

Schematic — relative lengths not scaled

Slide 7 · Complementarity provides specificity

Heat or chemical treatment separates genomic DNA strands

Single-stranded probe DNA binds the homologous specimen sequence

Unbound or nonspecific probe is removed before fluorescence microscopy

Denature DNA

Add labeled probe

Complementary pairing

Wash excess probe

Image fluorescence

Slide 8 · Fluorophores convert binding into signals

The label emits light under suitable excitation

Different dyes permit simultaneous visualization of two or more loci

Signal number, relative position, and intensity are interpreted in assay context

Hybridized probe

Specific fluorescent emission

Slide 9 · FISH workflow: From specimen to interpretation

Prepare cells or fixed tissue while preserving interpretable nuclei

Denature DNA, hybridize probe, wash, and visualize

Score informative cells using a validated probe-specific interpretation scheme

Tissue / cells

DNA pretreatment

Probe hybridization

Wash + stain

Microscopy

Validated interpretation

Slide 10 · Metaphase FISH places signals on chromosomes

Condensed metaphase chromosomes reveal the location of hybridization

Useful when chromosome identity or rearrangement topology matters

Requires an adequate metaphase preparation

Probe binding on homologs

Homolog 1

Homolog 2

Slide 11 · Interphase FISH does not require metaphase

Probes bind loci in nondividing interphase nuclei

Signals can be assessed directly in suitable tissue or cell preparations

Chromosome-wide architecture is less apparent than in a metaphase spread

Two-copy reference pattern

Schematic — illustrative signal pattern

Target A

Target B / control

Slide 12 · Metaphase and interphase answer different questions

Feature

Metaphase

Interphase

Template

Condensed chromosomes

Nuclei without metaphase

Strongest readout

Chromosomal localization

Copy and spatial pattern

Constraint

Needs metaphase cells

Less structural context

Editable comparison table

Slide 13 · The preparation can come from different tissues

Material

Use in FISH

Important context

Prenatal samples

Selected constitutional targets

Appropriate prenatal workflow

Peripheral blood

Constitutional / hematologic cells

Target population present

Biopsy touch imprint

Accessible tumor nuclei

Tumor-cell selection

Fixed tissue sections

Archived histopathology

Truncation artifacts

Editable comparison table

Slide 14 · Targeted resolution is a principal advantage

Conventional chromosome banding requires large alterations to become visibly apparent

Locus-specific FISH may disclose lesions below standard karyotype resolution

Resolution depends on probe span, spacing, and the structural abnormality

Large lesion

Banding may detect

Smaller locus loss

FISH may resolve

Unknown locus

Broader assay

Slide 15 · Aneuploidy changes chromosome copy number

Normal diploid cells generally contain two copies of an autosomal locus

A trisomic cell may show an additional locus signal

Signal enumeration must consider nuclear truncation, overlap, and assay controls

Two-copy reference pattern

Schematic — illustrative signal pattern

Target A

Target B / control

Slide 16 · Normal two-color enumeration pattern

A disomic reference nucleus is expected to show two signals per autosomal target in an idealized preparation

Color identifies which chromosome or locus each signal represents

An individual nucleus is illustrative; Diagnostic scoring evaluates a validated collection of cells

Two-copy reference pattern

Schematic — illustrative signal pattern

Target A

Target B / control

Slide 17 · Trisomy 21: An extra locus signal

Chromosome 21–specific probes identify an additional chromosome-21 target copy

Robbins Figure 5.21 depicts three chromosome-21 signals with two chromosome-13 signals

This demonstrates numeric chromosome imbalance at the interrogated loci

Three target / two reference signals

Schematic — illustrative signal pattern

Target A

Target B / control

Slide 18 · Read the trisomy signal pattern precisely

Three target signals are consistent with an extra chromosome-21 locus

A two-signal control supports adequate detection at the reference locus

The pattern should be integrated with specimen identity and other cytogenetic information

Three target / two reference signals

Schematic — illustrative signal pattern

Target A

Target B / control

Slide 19 · Mosaicism creates more than one cell population

Different nuclei may display different locus counts within one specimen

A mixture of two-signal and three-signal populations suggests possible mosaicism

Biologic mosaicism must be distinguished from technical signal loss or scoring artifacts

Two cell populations in one specimen

Disomic example

Trisomic example

Interpret the proportions across multiple nuclei

Slide 20 · Signal enumeration requires multiple nuclei

Single-cell patterns can be distorted by overlap, splitting, or incomplete nuclei

Assess a sufficient number of interpretable nuclei under the laboratory protocol

Report the observed distribution rather than extrapolating from one striking field

Score interpretable nuclei, not one field

Cell A

Cell B

Validated cell counts + exclusion rules

Slide 21 · A deletion removes a targeted DNA interval

A locus-specific probe fails to bind when its complementary interval is absent

A separate chromosome control helps show that the chromosome or nucleus is present

A normal control does not exclude loss of the interrogated target

Single-locus copy loss

Schematic — illustrative signal pattern

Target A

Target B / control

Slide 22 · Microdeletions may escape routine banding

Some pathogenic chromosomal deletions are too small for routine karyotypic visualization

Targeted FISH can test a suspected microdeletion directly

A negative FISH result does not survey unprobed regions of the chromosome

Single-locus copy loss

Schematic — illustrative signal pattern

Target A

Target B / control

Slide 23 · The 22q11.2 example uses an internal control

Robbins Figure 5.23 uses a red probe at 22q11.2

A green probe targets another chromosome-22 region for control

Loss of one red signal with preservation of green signals supports a 22q11.2 deletion

22q11.2 deletion pattern

Schematic — illustrative signal pattern

One 22q11.2 target

Two chr22 controls

Slide 24 · Deletion interpreted in an interphase nucleus

An idealized deletion pattern shows one target signal and two control signals

The control indicates that both homologous chromosomes contribute detectable signal

Apparent loss still requires exclusion of truncation and hybridization failure

22q11.2 deletion pattern

Schematic — illustrative signal pattern

One 22q11.2 target

Two chr22 controls

Slide 25 · Metaphase FISH localizes the missing interval

A normal homolog carries both target and control signals

The homolog with a deletion retains control but lacks the target signal

Matching interphase and metaphase patterns can support a coherent interpretation

Chromosome 22 homolog comparison

Homolog 1

Deletion homolog

Slide 26 · A control probe addresses alternative explanations

Without a control, a missing target may reflect chromosome loss or technical failure

Preserved reference signals provide a check on the imaged nucleus

Control strategy depends on the assay and suspected lesion

Target loss with retained reference

Schematic — illustrative signal pattern

Target A

Target B / control

Slide 27 · A focused probe is not a genome-wide screen

FISH directly interrogates only intervals represented by the selected probes

Unanticipated alterations elsewhere may remain invisible

A broader assay is appropriate when the abnormal locus is unknown

Whole genome

Only selected segment probed

Slide 28 · Copy-number gain and amplification are distinct ideas

A modest increase in a locus signal can reflect increased chromosome content

Focal gene amplification generates disproportionate gain of a genomic interval

The signal pattern and a suitable comparator determine the interpretation

Whole-chromosome gain vs focal gain

Co-gain concept

Locus-enriched concept

Slide 29 · Gene amplification produces extra probe signals

An amplified target can form multiple discrete signals or clustered signal groups

Signal intensity and clustering are assay- and preparation-dependent

A visual impression alone is not a validated amplification call

Disproportionate target signal gain

Schematic — illustrative signal pattern

Target A

Target B / control

Slide 30 · HER2 amplification is an actionable example

Breast cancer testing can detect ERBB2 (HER2) copy-number amplification by FISH

Robbins Figure 23.19 illustrates HER2 probes together with chromosome-17 control probes

Gene amplification provides molecular context for HER2-positive disease

ERBB2/HER2 target and chr17 control

Schematic — illustrative signal pattern

Red = HER2 target

Green = chr17 control

Slide 31 · Why the reference chromosome probe matters

A locus count must be interpreted relative to overall chromosome content where appropriate

A chromosome-17 centromeric probe supplies comparative information in HER2 FISH

Clinical classification requires the applicable assay standard; No universal cut-off is shown here

ERBB2/HER2 target and chr17 control

Schematic — illustrative signal pattern

Red = HER2 target

Green = chr17 control

Slide 32 · Do not equate all signal gain with amplification

Pattern

Interpretive concept

Qualification

Target and reference co-gain

Broader copy-number increase

Interpret assay context

Target-enriched gain

Possible focal amplification

Use validated criteria

Mixed cells

Tumor heterogeneity

Representative scoring

Editable comparison table

Slide 33 · Tumor heterogeneity changes what is sampled

Genomic copy-number patterns may differ between tumor regions

A selected field may not represent the entire lesion

Morphologic selection and validated sampling procedures are essential

Intermingled tumor-cell populations

Lower-copy cell

Higher-copy cell

Cell identity and selection matter

Slide 34 · Rearrangements alter chromosome architecture

Translocations and other rearrangements reorganize genomic intervals

A probe arrangement can be designed to change its spatial signal pattern

FISH identifies the chosen rearrangement signature without sequencing its breakpoint

5′ side

3′ side

Two probes straddle one locus

Slide 35 · Two common rearrangement strategies

Design

Question

Interpretation

Locus-specific

Target present / how many?

Signal enumeration

Deletion + control

Is one locus lost?

Relative target loss

Break-apart

Is this locus rearranged?

Probe separation

Fusion-oriented

Are two loci juxtaposed?

Co-localized probe pattern

Editable comparison table

Slide 36 · Break-apart probes flank a single locus

Different fluorophores label sequences on opposite sides of the target gene

An intact locus produces adjacent or overlapping colors

A rearrangement involving the locus can separate the colors

5′ side

3′ side

Two probes straddle one locus

Slide 37 · An unrearranged locus shows paired colors

Two intact homologs generate two paired red/green configurations in an idealized diploid cell

Close red and green signals may appear fused at optical resolution

Pairing alone does not identify exact nucleotide structure

Both alleles retain paired flanking probes

Schematic — illustrative signal pattern

Paired red/green on both homologs

Slide 38 · A rearranged locus can split paired colors

A locus-spanning break can separate the flanking probes

One homolog may show a split while the other remains paired

The validated assay determines what separation is considered abnormal

One paired allele and one split allele

Schematic — illustrative signal pattern

Partner cannot be identified from split alone

Slide 39 · MYC break-apart FISH detects partner-variable events

MYC break-apart probes for gene rearrangements

A split pattern can flag a MYC locus rearrangement without naming its partner

Further work is necessary when the translocation partner changes diagnosis or management

One paired allele and one split allele

Schematic — illustrative signal pattern

Partner cannot be identified from split alone

Slide 40 · What a break-apart result does not establish

Signal separation does not directly determine the partner chromosome

It also does not sequence the breakpoint or prove fusion-transcript expression

An orthogonal or partner-specific assay may be required

Target gene

Split FISH signal

Rearranged locus

Partner unknown

Further assay if needed

Slide 41 · MYC signal separation differs from MYC gain

Break-apart detects changes in spatial relationship between probe colors

Amplification is a copy-number phenomenon involving increased signal abundance

An assay may reveal both types of abnormalities; Classify the visual pattern correctly

One paired allele and one split allele

Schematic — illustrative signal pattern

Partner cannot be identified from split alone

Slide 42 · Fusion-oriented probes test a partner hypothesis

Two selected genomic loci are assigned distinct labels

A relevant chromosomal rearrangement may juxtapose the labels

Colocalization must be assessed against expected background and assay geometry

Juxtaposition of two named loci

Schematic — illustrative signal pattern

Proximity is interpreted using a validated assay

Slide 43 · PML::RARA is an important disease-associated fusion

Acute promyelocytic leukemia typically carries t(15;17), generating PML::RARA

The altered retinoic-acid receptor biology contributes to impaired myeloid maturation

Identification of this lesion has urgent clinical implications

T(15;17)

PML::RARA fusion

Altered retinoid signaling

Maturation block

Targeted diagnosis

Slide 44 · Suspected APL demands a rapid diagnostic pathway

Suspected acute promyelocytic leukemia as a setting for rapid FISH

Detection of the relevant retinoic-acid receptor rearrangement supports lesion-directed care

Clinical urgency is not a justification to treat an unvalidated signal pattern as definitive

Suspected APL

Rapid molecular test

Identify lesion

Integrated diagnosis

Urgent care pathway

Slide 45 · The clinical decision requires the correct target

A generic structural abnormality is not equivalent to identifying PML::RARA

Choose a probe configuration appropriate for the suspected leukemia

Correlate the molecular result with morphology and the clinical presentation

Specific suspicion

Choose matching probe

Evaluate pattern

Check quality

Correlate clinically

Slide 46 · FISH complements other diagnostic modalities

Morphologic examination selects the relevant cell population

Cytogenetics can provide chromosome-wide structural context

Sequencing or transcript assays may provide base-level or expression information

Morphology

Choose tumor cells

FISH cytogenomics

PCR / sequencing

Integrated report

Slide 47 · Prenatal material can support targeted FISH

Interphase or metaphase preparations can assess selected chromosomal loci

Targeted FISH can be useful when a specific aneuploidy or deletion is suspected

Results must be interpreted within a validated prenatal-testing workflow

Specimen context

Prenatal cells

Targeted locus evaluation

Slide 48 · Peripheral blood is suitable in selected settings

Circulating cells may provide material for constitutional cytogenetic assessment

Hematologic malignancies may be investigated in blood when abnormal cells are present

Specimen appropriateness depends on the clinical and biologic compartment

Specimen context

Appropriate circulating cells

Targeted locus evaluation

Slide 49 · Touch preparations retain accessible tumor nuclei

Fresh biopsy touch imprints can preserve cells for hybridization

Direct nuclear evaluation can be tied to material sampled from a tumor

Poor cellularity or wrong target-cell selection can compromise the result

Specimen context

Tumor-cell imprint

Targeted locus evaluation

Slide 50 · Archival tissue expands diagnostic use

FISH on fixed archival tissue sections

Sectioned nuclei allow molecular assessment alongside histopathologic context

Incomplete nuclear profiles can underestimate visible copy number

Specimen context

Interpretable section nuclei

Targeted locus evaluation

Slide 51 · Sectioning may remove part of the nucleus

A thin section may not contain every locus copy from a three-dimensional nucleus

A missing signal can therefore be artifactual

Scoring criteria and adequate control patterns reduce overinterpretation

Partial nucleus intersects a tissue section

Schematic — illustrative signal pattern

Section plane (schematic)

Slide 52 · Overlapping cells can simulate extra signals

Superposed nuclei may appear to contain more signals than either cell truly carries

Crowded regions also obscure nuclear boundaries

Select well-preserved, separable, representative nuclei

Overlapping nuclei create false aggregates

Schematic cell 1

Schematic cell 2

Slide 53 · Hybridization failure can mimic true loss

Inadequate DNA access or probe binding can reduce signals

Weak or absent labeling must be evaluated with controls and quality criteria

Failed technical runs are not interpretable negative molecular findings

Loss of probe signal need not mean deletion

Schematic — illustrative signal pattern

Interpret only if controls and assay QC pass

Slide 54 · Signal patterns may vary within a tumor

Biologic heterogeneity and specimen sampling influence apparent fractions

Neoplastic and non-neoplastic nuclei may coexist in the field

A valid interpretation identifies the population actually evaluated

Intermingled tumor-cell populations

Lower-copy cell

Higher-copy cell

Cell identity and selection matter

Slide 55 · Laboratories need assay-specific scoring criteria

Signals have finite optical size and spatial variability

Cutoffs, minimum nuclei, and acceptance criteria depend on the validated probe/application

Do not transfer an arbitrary numeric threshold between FISH assays

Nuclear quality

Probe controls

Signal pattern

Validated cutoff

Clinical report

Slide 56 · A normal targeted FISH assay has limits

Unprobed loci and unrelated rearrangements remain outside its interrogation range

Single-nucleotide substitutions cannot be characterized by routine locus-level FISH

Some cryptic rearrangements may not produce detectable probe separation

Whole genome

Only selected segment probed

Slide 57 · PCR and FISH interrogate different features

Characteristic

FISH

PCR

Principal readout

Position / count of locus signals

Amplified target sequence

Resolution

Probe / optical context

Short defined nucleic acid interval

Cell-by-cell

Yes

Typically bulk extraction

Best question

Copy / rearrangement at locus

Defined sequence-level target

Editable comparison table

Slide 58 · Arrays answer the genome-survey question

Characteristic

FISH

Array strategy

Coverage

Selected locus/loci

Many loci genome-wide

Readout

Spatial signals in cells

Aggregate copy-number data

Balanced event

Can detect with suitable probe

Not ordinarily by copy number alone

Best context

Specific alteration suspected

Locus not known

Editable comparison table

Slide 59 · Selecting a test starts with the abnormality

Suspected known locus deletion: Targeted locus/control FISH

Partner-variable rearrangement: Gene-specific break-apart design

Unexplained genome-wide copy-number changes: Consider an array-based strategy

Specific suspicion

Choose matching probe

Evaluate pattern

Check quality

Correlate clinically

Slide 60 · Case 1 — a possible chromosome-number abnormality

Clinical reasoning

A child is being evaluated for a suspected chromosome-21 gain

Illustrative nuclei show three chromosome-21 locus signals and two reference signals

Interpret as a pattern consistent with trisomy 21; Confirm within the full diagnostic context

Three target / two reference signals

Schematic — illustrative signal pattern

Target A

Target B / control

Slide 61 · Case 2 — an occult chromosome-22 deletion

Clinical reasoning

A child has a phenotype prompting investigation of 22q11.2 loss

One target 22q11.2 signal is present, whereas both control signals remain

The pattern supports deletion of one interrogated 22q11.2 locus

22q11.2 deletion pattern

Schematic — illustrative signal pattern

One 22q11.2 target

Two chr22 controls

Slide 62 · Case 3 — a rearrangement with unknown partner

Clinical reasoning

Tumor nuclei show a reproducible separation of a MYC break-apart probe pair

The arrangement indicates involvement of the MYC locus

Do not assign a specific partner or transcript from break-apart signals alone

One paired allele and one split allele

Schematic — illustrative signal pattern

Partner cannot be identified from split alone

Slide 63 · Case 4 — rapid testing in suspected APL

Clinical reasoning

Morphology raises concern for acute promyelocytic leukemia

A rapid targeted genetic assay is requested to address the relevant rearrangement

Interpret the assay alongside urgent specialist clinical and pathologic assessment

Suspected APL

Rapid molecular test

Identify lesion

Integrated diagnosis

Urgent care pathway

Slide 64 · Core principles to retain

Targeted hybridization: A probe must match the suspected genomic interval

Pattern-based reading: Count, proximity, and internal controls all matter

Clinical integration: Target selection, sample quality, and validated interpretation govern utility

A negative result excludes only abnormalities detectable by the selected probe design

Slide 65 · Principal reference

Robbins, Cotran & Kumar: Pathologic Basis of Disease, 11th edition (2026)

Chapter 5: Genes and Human Diseases — molecular genetic diagnosis and FISH

Related illustrations and context: Figures 5.21, 5.23, and 23.19; APL molecular genetics

The schematic signal patterns in this lecture are original teaching diagrams, not specimen images