Specimen Quality

Fluorescence In Situ Hybridization (FISH) is a molecular cytogenetic technique that uses fluorescent DNA probes to target specific chromosomal sequences. Unlike standard karyotyping, FISH can be performed on both metaphase (dividing) and interphase (non-dividing) cells. However, the success of the hybridization - the binding of the probe to the target DNA - is entirely dependent on the quality of the specimen on the slide. If the probe cannot penetrate the cell membrane or the chromatin structure, the assay will fail. Therefore, specimen evaluation is a mandatory quality control step performed before the expensive probe is applied and after the hybridization process (using DAPI counterstain)

Pre-Hybridization Evaluation (Phase Contrast Microscopy)

For suspension cultures (Peripheral Blood, Bone Marrow, Amniotic Fluid) prepared from fixed pellets, the slide is evaluated using a phase-contrast microscope immediately after dropping. The goal is to ensure the biological material is accessible to the probe and present in sufficient quantity

• Cell Density (Cellularity)
  • Optimal: Interphase nuclei should be evenly distributed but distinct (not touching). A standard FISH analysis requires scoring 50 to 200 nuclei; the slide should contain at least double this amount to allow for random selection
  • Too Dense: If nuclei overlap, their fluorescent signals will merge. It becomes impossible to distinguish whether a signal belongs to Nucleus A or Nucleus B, leading to inaccurate counts (e.g., false trisomy or fusion calls)
  • Too Sparse: Evaluating a slide with extremely low cellularity increases the risk of “selection bias,” where the laboratory scientist scores only the few cells they can find, which may not represent the patient’s true clonal population
• Cytoplasmic Debris (The Barrier)
  • FISH probes must physically penetrate the cell membrane and nuclear envelope to reach the DNA. Residual cytoplasm acts as a physical barrier (“mask”) that blocks the probe
  • Evaluation: Under phase contrast, nuclei should appear dark grey with distinct edges. If the nuclei appear “refractile” (shiny, glowing, or haloed) or are surrounded by a visible ring of cytoplasm, the probe will not penetrate
  • Correction: Slides with heavy cytoplasm require Pretreatment (chemical aging or pepsin digestion) to strip the proteins before hybridization
• Morphology and Integrity
  • Nuclei should be intact and round/oval
  • “Ghost” nuclei (pale outlines with no substance) or “smudged” chromatin indicate cellular degradation. These cells will likely result in non-specific background binding or zero signal

Evaluation of FFPE Specimens (Solid Tumor FISH)

Formalin-Fixed Paraffin-Embedded (FFPE) tissues present unique challenges compared to suspension cells. The evaluation is typically performed on a hematoxylin and eosin (H&E) stained guide slide by a pathologist before the FISH laboratory scientist processes the unstained sections

• Tumor Enrichment (Burden)
  • FISH is often used to detect amplification (e.g., HER2) or rearrangement in cancer cells. The tissue section must contain a sufficient percentage of tumor cells versus normal stromal cells
  • If the tumor burden is low (<10%), the pathologist may circle the tumor area on the slide, instructing the laboratory scientist to score signals only within that specific boundary
• Section Thickness (Truncation Artifacts)
  • Tissue sections are typically cut at 4–5 microns
  • Too Thick: Nuclei overlap vertically (3D stacking). This creates false “gains” (trisomy) because the analyst counts signals from two nuclei thinking it is one
  • Too Thin: The cutting blade slices through the nucleus (nuclear truncation). This creates false “losses” (monosomy) because part of the nucleus containing the probe target was physically cut away and discarded

Post-Hybridization Evaluation (DAPI Counterstain)

Once the hybridization wash is complete, the slide is stained with DAPI (4’,6-diamidino-2-phenylindole), a blue fluorescent dye that binds to DNA. The laboratory scientist evaluates the slide under a fluorescence microscope to determine if the hybridization conditions were successful

• Nuclear Morphology (DAPI Intensity)
  • Nuclei should stain a moderate, even blue
  • Over-denaturation: If the DAPI signal is weak or the nuclei look “hollow” or “puffy,” the denaturation step (using heat and Formamide to melt the DNA strands) was too harsh. This destroys the DNA target, leading to signal loss
  • Under-denaturation: If the DAPI is intensely bright and the nuclei look shiny/refractile, the DNA strands did not open up. The probe could not bind
• Background Noise
  • The background (space between cells) should be black
  • Hazy green or red background fluorescence indicates “non-specific binding,” where the probe stuck to cellular debris or glass rather than the target DNA. This reduces the signal-to-noise ratio, making it hard to see small deletions
• Autofluorescence
  • Certain biological materials naturally fluoresce
  • Red Blood Cells: Often fluoresce green, mimicking a FITC signal
  • Necrotic Tissue: Dead tissue fluoresces broadly across all color channels
  • The laboratory scientist must distinguish between true probe signals (sharp, distinct dots) and autofluorescence (diffuse, fuzzy glows)

The Decision to Score vs. Fail

Based on these evaluations, the laboratory scientist makes a critical decision:

• Proceed to Score: Signals are bright, background is low, and nuclear boundaries are distinct
• Reprocess (Rescue): If no signals are visible but cells are intact, the slide might be re-hybridized with a different denaturation temperature
• Fail/Reject: If the specimen is necrotic, crushed, or autofluorescent beyond interpretation, the slide is deemed “Unanalyzable.” Scoring poor-quality slides is a primary cause of false-positive and false-negative reporting in molecular cytogenetics