Skip to main content

Cytogenetics

Probe Strategy

The selection of a Fluorescence In Situ Hybridization (FISH) probe is not a “one size fits all” decision. It requires a precise understanding of the underlying genetic mechanism being investigated. Different chromosomal abnormalities - gains, losses, specific translocations, or indefinite rearrangements - require distinct probe designs to visualize the defect accurately. The laboratory scientist must select a strategy that maximizes sensitivity and specificity for the suspected pathology

Enumeration Strategy (Centromere Probes)

When the clinical question involves Aneuploidy (the gain or loss of whole chromosomes), the most appropriate tool is a Chromosome Enumeration Probe (CEP). These probes target the highly repetitive alpha-satellite DNA sequences found at the centromere of specific chromosomes. Because centromeric DNA is abundant and condensed, these probes produce bright, distinct signals that are easy to count in both interphase nuclei and metaphase spreads

  • Mechanism: The probe binds to the centromere
  • Signal Pattern
    • Normal (Disomy): Two signals (2 dots)
    • Trisomy (Gain): Three signals
    • Monosomy (Loss): One signal
  • Clinical Applications
    • Prenatal Screening: Rapid detection of Trisomy 13, 18, 21, X, and Y in uncultured amniocytes
    • Post-Bone Marrow Transplant: Using X and Y probes to monitor chimerism in sex-mismatched transplants (e.g., female donor cells in a male recipient)
    • Control Probes: CEPs are often paired with gene-specific probes to act as a mathematical denominator (see Amplification below)

Fusion Strategy (Dual-Color Dual-Fusion)

When the clinical question involves a Specific Translocation where both partner genes are known, a Dual-Color Dual-Fusion (DCDF) probe is selected. This strategy is designed to detect the juxtaposition of two genes that are normally on different chromosomes. It is the gold standard for diagnosing leukemias driven by specific chimeric proteins

  • Mechanism
    • Probe A (e.g., BCR) is labeled Red
    • Probe B (e.g., ABL1) is labeled Green
    • In a normal cell, the red and green signals are on separate chromosomes. In a translocation, the breakage and reunion bring the red and green dyes physically close together. Due to the optical physics of the microscope, overlapping red and green light appears Yellow (or “Fusion”)
  • Signal Pattern
    • Normal: 2 Red, 2 Green (2R, 2G)
    • Abnormal (Translocation): 1 Red, 1 Green, 2 Yellow Fusions (1R, 1G, 2F). This represents the two derivative chromosomes [der(9) and der(22)] created by the reciprocal exchange
  • Clinical Applications
    • CML: BCR:ABL1 t(9;22)
    • APL: PML:RARA t(15;17)
    • Burkitt Lymphoma: IGH:MYC t(8;14)

Break-Apart Strategy (Separation Probes)

When the clinical question involves a “Promiscuous Gene” - a gene that can translocate with many different partners - the Fusion strategy is impractical because you would need a different probe for every possible partner. Instead, the Break-Apart Probe (BAP) strategy is used. This design does not look for what the gene connected to; it simply asks, “Did this specific gene break?”

  • Mechanism
    • The probe consists of two differently colored sequences (Red and Green) that bind to the flanking regions of the target gene (one at the 5’ end, one at the 3’ end)
    • In a normal intact gene, the 5’ and 3’ ends are adjacent. The red and green signals merge to form a Yellow/Fusion signal
  • Signal Pattern
    • Normal: 2 Fusions (2F)
    • Abnormal (Rearrangement): The translocation breaks the gene in half. The 5’ end moves to one chromosome, and the 3’ end moves to another. The red and green signals separate physically. The pattern shows 1 Normal Fusion and 1 Red/Green split (1F, 1R, 1G)
  • Clinical Applications
    • KMT2A (MLL) Rearrangements: This gene on 11q23 has over 50 fusion partners in leukemia. A break-apart probe detects the disruption of KMT2A regardless of the partner
    • Lymphoma: BCL6 or ALK rearrangements
    • Sarcoma: EWSR1 rearrangements in Ewing Sarcoma

Amplification/Deletion Strategy (Locus-Specific)

When the clinical question involves the Copy Number of a specific gene (Gene Amplification or Gene Deletion), Locus-Specific Identifiers (LSI) are used. These probes bind to unique DNA sequences found only within the gene of interest. Because hybridization efficiency is never 100%, these probes are almost always paired with a Control Probe (usually a CEP for the same chromosome) to distinguish true genetic changes from technical artifacts

  • Deletion Strategy
    • Goal: To detect the loss of a critical region (e.g., Tumor Suppressor gene or Microdeletion syndrome)
    • Pattern
      • Normal: 2 Target Signals, 2 Control Signals
      • Deletion: 1 Target Signal, 2 Control Signals
    • Why the Control matters: If a cell has 1 Target and 1 Control (Monosomy), the entire chromosome is missing, not just the gene. The deletion strategy specifically looks for 1 Target / 2 Controls
    • Example: DiGeorge Syndrome (22q11.2 deletion) or TP53 deletion in CLL
  • Amplification Strategy
    • Goal: To detect Oncogene overexpression (Multiple copies)
    • Pattern
      • Normal: 2 Target signals
      • Amplification: Clusters of target signals or >6 discrete dots
    • The Ratio (Target:CEP): In solid tumors, cells often have extra whole chromosomes (polysomy). If a cell has 4 copies of Chromosome 17, it will naturally have 4 copies of the HER2 gene. This is not amplification; it is polysomy
    • True Amplification: Defined by a mathematical ratio. For HER2, if the ratio of HER2 signals to CEP17 signals is \(\ge 2.0\), it confirms the gene is amplified relative to the chromosome count
    • Example: ERBB2 (HER2) in Breast Cancer; MYCN in Neuroblastoma