Skip to main content

Cytogenetics

Analysis Type

One of the defining advantages of Fluorescence In Situ Hybridization (FISH) over standard karyotyping is its versatility: it can be performed on cells at different stages of the cell cycle. The decision to perform Interphase FISH (on non-dividing nuclei) or Metaphase FISH (on cultured, dividing chromosomes) is not arbitrary. It is determined by the specific clinical question, the urgency of the result (STAT status), the type of specimen available, and the nature of the genetic abnormality being investigated

Interphase FISH (nuc ish)

Interphase FISH involves hybridizing probes to the uncoiled chromatin of the nucleus in non-dividing cells. Because the DNA is not condensed into distinct chromosomes, the signals appear as distinct colored “dots” within the nucleus. This is the most common form of FISH in routine clinical oncology and prenatal screening

  • Primary Indications & Advantages
    • Speed (Rapid Results): Interphase analysis bypasses the need for cell culture. A specimen (e.g., blood, amniotic fluid) can be processed immediately (uncultured), allowing for results within 24–48 hours. This is critical for STAT indications, such as identifying PML:RARA fusion in Acute Promyelocytic Leukemia (a medical emergency) or rapid aneuploidy screening (Trisomy 13, 18, 21) in prenatal diagnosis
    • Non-Mitotic Samples: It is the only option for tissues that do not divide well or at all in culture, such as Paraffin-Embedded Tissue (FFPE) from solid tumors (e.g., Breast Cancer HER2 testing) or terminally differentiated cells (e.g., mature granulocytes or plasma cells in Multiple Myeloma)
    • Quantitative Analysis (Counting): Interphase FISH is superior for determining the percentage of abnormal cells (clone size). Because thousands of nuclei can be scored rapidly by a laboratory scientist or automated scanner, it provides high statistical power to detect low-level mosaicism or monitor Residual Disease (MRD) after treatment
  • Limitations
    • Lack of Structural Context: While you can count signals (e.g., 3 dots = trisomy), you cannot see where those sequences are located. A signal might be amplified, but you cannot determine if it is on the correct chromosome or part of a complex “Double Minute” structure
    • Signal Overlap: Because the nucleus is a 3D sphere flattened onto a 2D slide, signals may overlap, potentially masking a trisomy or creating a false fusion signal

Metaphase FISH (met ish)

Metaphase FISH involves hybridizing probes to chromosomes that have been cultured, arrested in metaphase, and spread on a slide. The signals appear as “double dots” (one on each chromatid) on the specific chromosome structures. This method essentially paints a specific region of the standard G-banded karyotype

  • Primary Indications & Advantages
    • Structural Localization (Mapping): This is the definitive method for determining the physical location of a DNA sequence. If a standard karyotype shows an unidentified piece of genetic material (a “Marker Chromosome” or “Add”), Metaphase FISH with a Whole Chromosome Paint or specific probe can identify the origin of that material
    • Microdeletion Confirmation: While interphase FISH can detect a deletion (loss of signal), Metaphase FISH confirms the deletion is on the correct chromosome homolog. For example, in diagnosing DiGeorge Syndrome (22q11.2 deletion), Metaphase FISH confirms the signal is missing from one Chromosome 22 while the control probe is present
    • Complex Rearrangements: In cases of complex translocations involving three or more chromosomes, Metaphase FISH allows the analyst to trace the exchange of segments between the chromosomes visually
  • Limitations
    • Culture Dependence: This method requires dividing cells. If the culture fails or the Mitotic Index is low (common in some lymphoid malignancies), Metaphase FISH cannot be performed
    • Turnaround Time: It requires the full culture duration (24–72 hours) plus harvest time, delaying results compared to uncultured interphase analysis
    • Resolution: It cannot detect micro-deletions smaller than the probe size (typically ~100kb), though this limitation applies to both methods

Decision Matrix: Selecting the Approach

The laboratory scientist or director selects the method based on the following hierarchy of needs:

  • Scenario A: “Does the patient have Leukemia/Aneuploidy NOW?” (Urgency)
    • Selection: Interphase FISH.
    • Reason: The priority is speed. The physician needs to know if the patient has Trisomy 21 or t(15;17) immediately to start treatment. Culture is too slow
  • Scenario B: “What is this mystery extra material found on the Karyotype?” (Characterization)
    • Selection: Metaphase FISH.
    • Reason: The question is structural. Interphase FISH would just show extra signals floating in the nucleus; Metaphase FISH will show exactly which chromosome the extra material is attached to
  • Scenario C: “Is the probe valid?” (Quality Control)
    • Selection: Metaphase FISH.
    • Reason: When validating a new probe lot, the lab must prove the probe binds to the correct chromosomal locus (specificity). Only metaphase spreads allow for this visual confirmation (mapping)
  • Scenario D: “Solid Tumor Analysis” (Sample Constraints)
    • Selection: Interphase FISH (Tissue Section).
    • Reason: Solid tumor cells (lung, breast, colon) are difficult to culture for cytogenetics. The analysis is almost exclusively performed on thin slices of fixed tissue (FFPE) where the nuclei are sliced (sectioned), necessitating interphase scoring