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Cytogenetics

Prepare FISH Slides

Fluorescence In Situ Hybridization (FISH) bridges the gap between traditional cytogenetics and molecular genetics. It utilizes fluorescently labeled DNA probes to detect specific chromosomal sequences with high resolution. Unlike karyotyping, which requires dividing cells, FISH can be applied to both metaphase chromosomes and interphase nuclei. The preparation of FISH slides is a multi-step process involving specimen evaluation, strategic probe selection, and rigorous chemical processing to ensure the probe successfully binds to its target

Evaluate Specimen Quality

The quality of the hybridization signal is directly proportional to the quality of the specimen on the slide. Evaluation is performed at two distinct stages: before hybridization (to assess accessibility) and after hybridization (to assess DNA integrity)

  • Pre-Hybridization (Phase Contrast)
    • Cellularity: Slides must have optimal density. If too sparse, selection bias occurs (scoring only the few cells found). If too dense, nuclei overlap, making it impossible to assign signals to specific cells (false polysomy)
    • Cytoplasm: Probes must penetrate the cell membrane. Heavy cytoplasmic debris acts as a barrier (“mask”). Ideally, nuclei should appear dark and distinct under phase contrast. Refractile or “haloed” nuclei indicate cytoplasm that may require pre-treatment (Pepsin digestion)
  • Post-Hybridization (DAPI Check)
    • Morphology: DAPI counterstain reveals the status of the chromatin. “Ghostly” or hollow nuclei indicate over-denaturation (heat damage). Shiny, refractile nuclei indicate under-denaturation (DNA strands didn’t melt)
    • Background: The space between cells should be dark. High background noise (haze) indicates insufficient washing or non-specific binding
  • FFPE Considerations: For solid tumors, the pathologist must verify Tumor Burden. If the tumor percentage is low (<10%), the results may be falsely normal. Additionally, section thickness is critical; sections cut too thin (\(<3\mu\text{m}\)) cause nuclear truncation (false deletions), while thick sections cause overlapping (false gains)

Determine Analysis Type

The choice between analyzing non-dividing cells (Interphase) or dividing chromosomes (Metaphase) depends on the clinical question and urgency

  • Interphase FISH (nuc ish)
    • Application: Used for rapid results (STAT aneuploidy, APL), non-dividing tissues (solid tumors, plasma cells), and quantifying clone size (% abnormal)
    • Pros/Cons: Extremely fast (no culture required) and sensitive for counting signals. However, it lacks structural context (cannot see where a gene has translocated) and is prone to signal overlap artifacts
  • Metaphase FISH (met ish)
    • Application: Used for mapping “Marker” chromosomes, characterizing complex rearrangements, and validating new probes
    • Pros/Cons: Provides definitive structural localization. However, it requires a successful culture (dividing cells) and cannot be performed on fixed tissue (FFPE) or samples with a mitotic index of zero

Identify Appropriate Probe Strategy

Probe selection is dictated by the mechanism of the genetic defect. The laboratory scientist must match the probe design to the pathology

  • Enumeration (Centromere Probes - CEP)
    • Targets repetitive alpha-satellite DNA at the centromere. Used to count chromosomes (Aneuploidy)
    • Pattern: 2 signals = Normal; 3 signals = Trisomy; 1 signal = Monosomy
  • Fusion (Dual-Color Dual-Fusion)
    • Targets specific translocations where both partners are known (e.g., BCR:ABL1 in CML)
    • Pattern: The juxtaposition of Red and Green signals creates a Yellow (Fusion): signal
  • Break-Apart (Separation Probes)
    • Targets “promiscuous” genes that have multiple partners (e.g., KMT2A, EWSR1). Flanking probes (Red/Green) bind to the 5’ and 3’ ends of the gene
    • Pattern: A translocation splits the gene, resulting in separated Red and Green signals
  • Amplification/Deletion (Locus Specific - LSI)
    • Targets unique gene sequences to determine copy number. Must be paired with a Control Probe (CEP) to distinguish gene amplification from whole-chromosome gain (polysomy)
    • Pattern: Deletion = 1 Target / 2 Controls. Amplification = Cluster of signals or Ratio \(\ge 2.0\)

Processing (The Workflow)

The physical processing of the slide involves thermodynamic manipulation to allow the probe to find the target

  • Denaturation (The Melt)
    • High heat and Formamide: are used to separate double-stranded DNA into single strands without destroying cell morphology
    • Control: Typical temp is \(\sim 72^\circ\text{C}\). Under-denaturation = No signal; Over-denaturation = DNA destruction
  • Hybridization (The Annealing)
    • Incubation at \(37^\circ\text{C}\): allows the probe to bind. Cot-1 DNA is added to block repetitive sequences, ensuring specificity
  • Postwash (Stringency)
    • A critical cleanup step to remove non-specific binding. Stringency: (strictness) is controlled by Heat and Salt
    • High Stringency: High Temp / Low Salt (0.4x SSC). Removes weak bonds (clean signal)
    • Low Stringency: Low Temp / High Salt. Preserves weak bonds (high background)
  • Specialized Processing
    • Plasma Cell Enrichment: Magnetic bead sorting (CD138+) is required for Multiple Myeloma to isolate rare, non-dividing plasma cells from bone marrow
    • FFPE Pre-treatment: Solid tissues require Deparaffinization (Xylene), Heat Pre-treatment (to break formalin crosslinks), and Pepsin digestion (to strip proteins). This is essential to allow probe entry into the fixed tissue matrix