HOYADOCS
Block 5 · Heme · HEM24 · Rev 1.0 · Updated Oct 8, 2026
HEM24 · Genetics · Study Guide

Clinical Molecular Cytogenetics

Updated Oct 9, 2026 Revision 1.0 2 learning objectives

Learning objectives

  1. LO 1Explain how molecular cytogenetic tests (FISH and chromosomal microarray) work, and what each can and can't detect.
  2. LO 2Choose the right cytogenetic test for constitutional and acquired chromosomal abnormalities.
◆ In-house exam ★ Step 1 ▲ High yield Memory tool Analogy Trap

MAPThe test ladder: each step sees smaller, but loses something

G-banded karyotypemetaphase · culture · 10–14 d FISHtargeted probe · any phase · 1–3 d Chromosomal microarraygenome-wide · DNA only Genome sequencingsequence + structure SEES Number, big changesBalanced translocations~5–10 Mb resolution SEES Only the probed targetMicrodeletions, fusionsMosaicism, non-dividing cells SEES Gains/losses of any sizeDown to exon levelAneuploidy · ROH (SNP) SEES Point mutations+ copy number Microarray's blind spots balanced translocations/inversions · where extra material sits · low-level mosaicism · point mutations

LO 1Principles of molecular cytogenetics

Q1. What can a conventional karyotype do, and where does it fall short?

Pros

  • Detects changes in chromosome number
  • Detects large structural changes (deletions, duplications, insertions, translocations)
  • Sees balanced rearrangements
  • Cheap

Cons

  • Low resolution (~5–10 Mb): small deletions missed
  • Needs cells in metaphase
  • Needs culture; slow (10–14 days)
Q2. How does FISH work?
  1. Design a single-stranded DNA probe complementary to the region, tagged with a fluorophore
  2. Denature (heat) the patient's DNA into single strands
  3. Cool → probe hybridizes to its complement
  4. Wash away unbound probe
  5. Read under a fluorescence microscope
FISH schematic: a labeled probe base-pairs with its complementary sequence on a chromosome, viewed by fluorescence microscope

FISH principle

  • Labeled probe finds its complementary target

Pros

  • High sensitivity for the target
  • Detects large and micro changes
  • Works in any cell-cycle stage (interphase too)
  • Detects mosaicism

Cons

  • Only finds what you probe for
  • An unexpected abnormality elsewhere is missed
High yield

FISH extended cytogenetics from metaphase chromosomes to all stages of the cell cycle: interphase analysis shows morphology and genetic change in the same cell.

Analogy — glowing magnets

Each probe is a glowing magnet for one address. Count the dots (copy number), look for a missing dot (deletion), a merged dot (fusion) or a cluster (amplification).

Q3. What are the FISH probe types?
Probe types on a chromosome: centromeric at the centromere, telomeric at the tip, locus-specific at one band, and whole-chromosome paint covering the whole chromosome

Four probe types

  • Centromeric · telomeric · locus-specific · paint
ProbeBindsUse
CentromericChromosome-specific centromere repeatsCount chromosomes (aneuploidy), even in interphase
TelomericChromosome endsSubtle terminal deletions/rearrangements
Locus-specificOne gene/regionMicrodeletions (22q11.2), fusions (BCR-ABL), amplification (HER2)
Whole-chromosome paintPool of fragments covering a chromosomeTranslocations; multicolor FISH / SKY (all 24)
Normal2 control + 2 test Deletion2 control + 1 test Trisomy3 centromere signals Fusionred + green → yellow Amplificationcluster of test signals
In-house exam

Match the probe to the question: centromeric → aneuploidy; locus-specific → microdeletion, fusion, amplification; paint → translocation.

Q4. How does chromosomal microarray (array CGH) work?
  1. Label test DNA one color (green) and reference DNA another (red); mix 1:1
  2. Hybridize to an array of DNA sequences spanning the genome
  3. Scanner reads the ratio at each spot
RatioColorMeaning
EqualYellowNormal copy number (plot at 0)
Test > referenceGreenGain (plot rises)
Test < referenceRedLoss (plot drops)
Array CGH: control and test genomic DNA labeled in two colors hybridize to an array; a computer plots the ratio along the chromosome

Array CGH method

  • Two colors, one chip, ratio plot
Microarray plot with the line raised across the entire length of chromosome 13

Trisomy 13

  • Gain across the whole chromosome
Microarray plot with a dip at 15q11.2 to q13.1

15q11.2–q13.1 deletion

  • Line drops = loss

Can detect

  • Deletions/duplications of any size, down to exon level
  • Aneuploidy
  • Microdeletions (15q11.2, 22q11.2)
  • Regions of homozygosity (SNP arrays)
  • Needs DNA only: no metaphase, no culture

Cannot detect

  • Balanced rearrangements (translocation, inversion)
  • Location of extra material (free vs Robertsonian trisomy)
  • Low-level mosaicism
  • Point mutations
High yield — why microarray misses balanced changes

The DNA is chopped up. Total copy number is unchanged in a balanced translocation, so it looks normal, and you can't tell where extra material sits.

Trap

A 15q11.2–q13.1 deletion on array can't distinguish Prader-Willi vs Angelman: that needs parent-of-origin (methylation) studies.

Step 1

SNP arrays show long regions of homozygosity: uniparental isodisomy or consanguinity.

ApplyLO 1 application questions

A1A couple has had three miscarriages; both parents' microarrays are normal. Is a chromosomal cause excluded?
No: microarray misses balanced translocations. Order parental karyotypes.
A2A microarray shows a gain of all of chromosome 21. Is it free or Robertsonian trisomy?
Microarray can't tell (no location). Order a karyotype; it matters for recurrence risk.

LO 2Diagnostic applications

Q1. How is FISH used to find a microdeletion?
  • Microdeletions are usually <5 Mb → missed by G-banding; seen by FISH, array CGH or sometimes high-resolution karyotype
  • Probe cocktail: 22q11.2 test probe (orange) + 22q13 control (green)
  • 2 control + 2 test = normal; 2 control + 1 test = del(22)(q11.2) → DiGeorge / velocardiofacial syndrome
Probe map: 22q11.2 TUPLE1 test probe in orange and 22q13 ARSA control probe in green

22q11.2 probe cocktail

  • Test (orange) + control (green)
Metaphase FISH: one chromosome 22 shows both signals, the other (arrow) only the control signal

22q11.2 deletion

  • Arrow: chromosome 22 missing the test signal
Step 1

22q11.2 deletion = CATCH-22: Cardiac (conotruncal), Abnormal facies, Thymic aplasia (T-cell deficiency), Cleft palate, Hypocalcemia (parathyroid aplasia).

Trap

FISH is targeted: a normal 22q11.2 FISH doesn't rule out a deletion elsewhere. When the cause is unknown, microarray is preferred (and it sizes the deletion).

Q2. How is FISH used for translocations and aneuploidy?

Translocations (paints)

  • A paint color on another chromosome = translocation
  • Shows approximate size, not the exact sequence
  • Multicolor FISH / SKY: 24 paints at once → markers, complex rearrangements

Aneuploidy (centromeric, interphase)

  • 3 signals = trisomic; 2 = disomic
  • Works on uncultured cells (amniotic fluid), fixed tumor sections, low-mitotic samples (chemo, post-BMT)
  • Fast: ~48–72 h vs 10–14 days
Chromosome 1 paint in green and chromosome 3 paint in red, each showing material exchanged with the other chromosome

Whole-chromosome paints

  • Chromosomes 1 and 3 reveal an exchange
G-banded metaphase beside a spectral karyotype in which each chromosome has its own color

Spectral karyotype

  • All 24 chromosomes in different colors
Interphase nuclei: three chromosome 18 signals with one X and one Y; normal 13 and 21; and three chromosome 21 signals

Interphase centromeric FISH

  • Trisomy 18 male · normal 13/21 · trisomy 21
High yield

Rapid prenatal FISH on amniocytes should be confirmed by karyotype or microarray before irreversible decisions.

Q3. When is microarray the first-tier test?

Postnatal (ACMG 2010)

  • Multiple anomalies not specific to a known syndrome
  • Apparently nonsyndromic developmental delay / ID
  • Autism spectrum disorder
  • Follow-up of an imbalance: FISH/cytogenetics, parental studies, counseling

Prenatal (ACOG)

  • ≥1 major structural anomaly + invasive testing → microarray (replaces karyotype)
  • Structurally normal fetus → karyotype or microarray
  • Not restricted to women ≥35 (findings mostly not age-related)
  • Stillbirth → microarray on fetal tissue
  • Pre/post-test counseling + consent (uncertain findings, non-paternity, consanguinity)
Memory tool — stillbirth

Karyotype needs living cells to culture; dead tissue often won't grow. Microarray needs DNA only.

Q4. How is FISH used in cancer?

CML: BCR-ABL

  • Every CML has a BCR (22q11)–ABL (9q34) fusion: t(9;22), Philadelphia chromosome
  • 95% visible on karyotype (simple or complex)
  • 5% submicroscopic ("masked"): normal karyotype, FISH or RT-PCR positive
  • FISH: ABL green, BCR red → fusion = yellow

Breast cancer: HER2/NEU

  • Locus-specific HER2 (17q12) probe + chromosome 17 control on fixed tumor
  • Normal: 2 HER2 + 2 control; amplified: many HER2 signals
  • More aggressive; guides trastuzumab
Karyotype 46,XY,t(9;22)(q34;q11.2) with arrows on chromosomes 9 and 22

Philadelphia karyotype

  • 46,XY,t(9;22)(q34;q11.2)
Interphase nucleus with separate ABL and BCR signals plus one yellow BCR-ABL fusion signal

BCR-ABL FISH

  • Separate ABL + BCR, plus one yellow fusion
Diagram: normal interphase and metaphase show separate signals; Philadelphia interphase and metaphase show a fusion signal on the derivative 22

CML detection

  • Fusion signal on der(22) = Ph chromosome
Tumor nuclei with clusters of red HER2 signals beside nuclei with few signals

HER2 FISH

  • Amplified = clusters of red HER2 signals
In-house exam

Classic CML with a normal karyotype → order BCR-ABL FISH (or RT-PCR): ~5% have a masked fusion. Don't call it "Ph-negative CML" first.

Step 1

HER2 FISH is used when IHC is equivocal (2+); HER2/CEP17 ratio ≥2.0 = amplified. BCR-ABL FISH/PCR also monitors TKI (imatinib) response.

Q5. Which test do I pick?
QuestionBest test
Balanced translocation / recurrent miscarriageKaryotype
Free vs Robertsonian trisomyKaryotype
Suspected specific microdeletionFISH or microarray (microarray sizes it)
DD/ID, autism, multiple anomaliesMicroarray first-tier
Fetal structural anomalyMicroarray (replaces karyotype)
StillbirthMicroarray on fetal tissue
Rapid aneuploidy on amniocytes; non-dividing marrowInterphase FISH
CML with normal karyotypeBCR-ABL FISH / RT-PCR
HER2 status, equivocal IHCHER2 FISH
Point mutation (CFTR)Sequencing
PWS vs AngelmanMethylation studies

ApplyLO 2 application questions

A1A 32-week stillborn fetus's skin culture failed to grow. Best test for a chromosomal cause?
Microarray on fetal tissue or placenta (DNA only, no culture).
A2A patient on chemotherapy needs a chromosome count, but the marrow cells won't divide. Which test?
Interphase FISH with centromeric probes.

IDImage ID

Image 1 — metaphase FISHUnknown image 1
One chromosome 22 lacks the test signal → 22q11.2 deletion (DiGeorge/VCFS).
Image 2 — interphase nucleiUnknown image 2
Centromeric FISH: 3 signals = trisomy (18; 21).
Image 3 — plotUnknown image 3
Microarray gain across chromosome 13 → trisomy 13.
Image 4 — plotUnknown image 4
Microarray loss at 15q11.2–q13.1 → needs methylation for PWS vs AS.
Image 5 — nucleusUnknown image 5
Yellow fusion signal → BCR-ABL (CML).
Image 6 — tumor nucleiUnknown image 6
HER2 amplification → trastuzumab.
Image 7 — colored chromosomesUnknown image 7
Spectral karyotype (multicolor FISH, 24 paints).

MCQExam-style questions

Score: 0 / 0

1An infant has a conotruncal heart defect, cleft palate and hypocalcemia. FISH with a 22q11.2 test probe and a 22q13 control probe shows two control signals and one test signal. Interpretation?

2Which abnormality will a chromosomal microarray miss?

3A 4-year-old has nonsyndromic developmental delay and autism. Recommended first-tier genetic test?

4A couple has had three first-trimester losses. Which parental test best looks for a chromosomal cause?

5An amniocentesis is done at 18 weeks; the family wants a rapid answer about trisomy 21. Which test gives the fastest result on uncultured cells?

6A fetus has a major cardiac anomaly on ultrasound, and the mother chooses amniocentesis. Per ACOG, which test is recommended?

7A 45-year-old has splenomegaly, WBC 150,000/µL with all stages of granulocytes, and basophilia. Karyotype is 46,XY with no visible t(9;22). Next step?

8A breast cancer is HER2 2+ (equivocal) by IHC. FISH shows many HER2 signals per nucleus relative to the chromosome 17 control. Implication?

9Why is microarray preferred over karyotype after a stillbirth?

10A microarray shows a deletion at 15q11.2–q13.1 in a hypotonic infant. Which test distinguishes Prader-Willi from Angelman?

11Which FISH probe type is best for identifying an unknown marker chromosome or a complex translocation?

12A microarray shows a gain of the whole of chromosome 21. What additional information requires a karyotype?

!Source notes

ItemThis lectureOther lectures / standard
FISH turnaround24–72 hPrenatal lecture: 48–72 h
MosaicismFISH detects it (targeted region)Microarray misses low-level mosaicism
HER2 cutoff"Many" HER2 signalsHER2/CEP17 ratio ≥2.0 = amplified

✓Rapid review