Analyze FISH Slides

The analysis of FISH slides transforms raw fluorescent signals into a clinical diagnosis. This phase demands rigorous adherence to scoring criteria to differentiate true genetic events from artifacts, precise documentation through imaging and nomenclature, and the ability to troubleshoot technical failures

Score & Interpret Signal Patterns

Interpretation depends on the probe strategy employed. Laboratory scientistsscore specific numbers of cells (e.g., 50 for constitutional, 200 for oncology) to ensure statistical validity

• Enumeration (Aneuploidy)
  • Normal: 2 Signals (Disomy)
  • Abnormal: 1 Signal (Monosomy) or 3 Signals (Trisomy)
  • Artifact Alert: Split signals (doublets) in S-phase cells must be counted as one signal, not two
• Translocation (Dual Fusion)
  • Normal: 2 Red, 2 Green
  • Abnormal: 1 Red, 1 Green, 2 Fusions (Yellow). This pattern confirms a reciprocal translocation (e.g., BCR:ABL1)
• Rearrangement (Break-Apart)
  • Normal: 2 Fusions
  • Abnormal: 1 Fusion, 1 Red, 1 Green. The separation of colors indicates the gene (e.g., KMT2A) has broken apart
• Amplification (Copy Number)
  • Normal: Ratio of Target:Control < 2.0
  • Abnormal: Ratio \(\ge\) 2.0 or “Clusters” of signals. This distinguishes true gene amplification (e.g., HER2) from polysomy (extra chromosomes)

Capture Representative Cell Images

The digital image serves as the permanent medical record since fluorescence fades

• Requirements
  • Positive Cases: Must show the specific abnormal pattern reported (e.g., the fusion or deletion)
  • Negative Cases: Must show a clean normal pattern to prove technical success
  • Quality: Images must have a black background, sharp focus (using Z-stacking if needed), and balanced signal intensity
• Archival: Images must be stored securely and unalterably for audit purposes

Document Analyses Using ISCN Nomenclature

Results are reported using the standardized international syntax: [Technique] (Probe) [Pattern]

• Basic Syntax: nuc ish (Interphase) or ish (Metaphase)
• Examples
  • Normal: nuc ish(D21S259x2)[400] (Two copies of Ch21 seen in 400 cells)
  • Trisomy: nuc ish(D21S259x3)[400]
  • Fusion: nuc ish(ABL1,BCR)x3(ABL1 con BCRx2)[200] (Fusion/Connection of ABL1 and BCR)
  • Deletion: nuc ish(D11Z1x2,ATMx1)[200] (One copy of ATM, two copies of Centromere)

Troubleshoot FISH Processing Issues

Diagnosing failures based on microscopic appearance

• No Signal
  • Cause: Under-denaturation: (Refractile nuclei; DNA didn’t melt) or Over-washing (Signal stripped)
  • Fix: Re-denature at higher temp or re-hybridize
• High Background (Green Haze)
  • Cause: Under-washing: (Wash too cold/salty) or dried slide
  • Fix: Re-wash at correct stringency
• Weak Signal
  • Cause: Photobleaching, old slide, or expired probe
  • Fix: Increase hybridization time or use fresh reagents
• Autofluorescence
  • Cause: Cellular debris/RBCs. Identifiable because it glows in all color channels
  • Fix: Use better cleaning/fixation or specific quenching steps