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Cytogenetics

Chromosome Banding & Staining Techniques

Once metaphase slides are prepared, the chromosomes are uniformly transparent. To identify individual chromosomes and detect structural rearrangements, they must be processed to produce a characteristic banding pattern. The standard method in clinical cytogenetics is G-banding (Giemsa banding), which creates a unique barcode of light and dark bands along the length of each chromosome

G-banding (Mechanism & Protocol)

  • Mechanism: G-banding relies on the differential sensitivity of chromatin to enzymatic digestion
    • Heterochromatin (AT-rich): Tightly packed and gene-poor. It is resistant to digestion and stains Dark (G-Positive)
    • Euchromatin (GC-rich): Loosely packed and gene-rich. It is susceptible to digestion and stains Light (G-Negative)
  • Protocol Steps
    1. Aging: Slides are baked (\(60^\circ\text{C}\)–\(90^\circ\text{C}\)) to harden the chromatin. Fresh slides are too sensitive and melt
    2. Trypsinization: Slides are dipped in the proteolytic enzyme Trypsin. This relaxes the chromatin structure
    3. Staining: Slides are immersed in Giemsa (buffered to pH 6.8). The dye binds to the resistant (heterochromatic) regions
    4. Rinsing: Excess stain is removed with water
  • Resolution: The quality is defined by the number of visible bands per haploid set (e.g., 400, 550, or 850 bands). Higher resolution requires longer chromosomes (prometaphase) and gentler trypsin treatment

Evaluate & Troubleshoot Staining/Banding

Banding is an “art” dependent on variables like slide age, trypsin activity, and pH. Laboratory scientiststypically run a “test slide” to calibrate the protocol for each batch

  • Evaluation Criteria
    • Contrast: Sharp distinction between light and dark bands
    • Definition: Crisp edges (not fuzzy)
    • Completeness: Banding visible from centromere to telomere
  • Troubleshooting Matrix
    • Under-Trypsinized (Solid Stain): Chromosomes look uniformly dark with no bands
      • Cause: Trypsin time too short, enzyme inactive/old, or slide baked too long
      • Fix: Increase trypsin time or temperature. Can be re-treated
    • Over-Trypsinized (Ghosting): Chromosomes look puffy, fuzzy, or “melted” (pale outlines)
      • Cause: Trypsin time too long, enzyme too strong, or slide too fresh
      • Fix: Decrease trypsin time/concentration or age slides longer. Cannot be saved (must drop new slide)
    • Poor Color (pH Issues)
      • Red/Pink: Buffer too Acidic (<6.4)
      • Blue/Black: Buffer too Basic (>7.2)
    • Precipitate: Black dirt on slide. Caused by oxidized stain scum. Fix: Filter stain or skim surface before dipping