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Cytogenetics

Prepare Slides

The slide preparation phase, often termed “dropping,” is the pivotal moment where the suspended cells are physically transferred to a solid support for analysis. The quality of the final cytogenetic result is heavily dependent on the interaction between the fixed cells, the glass surface, and the ambient environment. Mastery of this phase requires balancing physical variables to achieve the optimal “spread” of chromosomes

Ambient Conditions

The evaporation rate of the fixative dictates the spreading force. Laboratory scientists manipulate the environment to control this rate

  • Relative Humidity (RH): The most critical factor. The “Goldilocks Zone” is 40–55%
    • Low Humidity (<35%): Causes rapid drying, leading to “Tight” (under-spread) metaphases encased in cytoplasm. Correction: Add moisture (“huffing” on the slide), use cold wet slides, or decrease airflow
    • High Humidity (>60%): Causes slow drying and excessive water uptake, leading to “Scattered” (over-spread) metaphases or “refractile” chromosomes. Correction: Use heat (hot plate) or airflow to accelerate drying
  • Temperature
    • Cold Slides: Slow evaporation (good for dry days)
    • Hot Plate: Accelerates evaporation (good for humid days) and flattens cells
  • Airflow: Moving air removes methanol vapor, speeding up drying. Used to prevent “grey” morphology

Slide Quality (The QC Check)

Every slide is triaged under a phase-contrast microscope immediately after dropping to assess four parameters

  1. Cell Density: Cells should be evenly distributed but not touching. If too sparse, concentrate the pellet. If too dense, dilute with fixative
  2. Mitotic Index: The yield of dividing cells. If low, multiple slides are dropped to ensure sufficient analysis material
  3. Metaphase Spreading: The separation of chromosomes
    • Optimal: Circular spread with no overlaps
    • Tight: Ball of chromosomes (needs more humidity/spreading)
    • Scattered: Chromosomes lost from the group (needs faster drying)
  4. Morphology
    • Crisp/Dark: Good fixation
    • Refractile/Shiny: Water contamination (needs fresh fixative)
    • Fuzzy: Poor drying or over-hypotonization

Evaluate Harvest

A biological assessment of the culture’s success, guiding the downstream workflow

  • Grading Scale
    • Grade 4 (Excellent): High index, long chromosomes (prometaphase), no cytoplasm. Suitable for High-Resolution Banding
    • Grade 3 (Standard): Good index, mid-length chromosomes. Suitable for Routine Banding
    • Grade 2 (Suboptimal): Low index, short/fuzzy chromosomes. Requires Rescue Banding (solid stain or short trypsin)
    • Grade 1 (Failure): No metaphases. Do not band; investigate failure
  • Biological Artifacts: Evaluating for C-Anaphase (colcemid toxicity) or Pulverization (virus/premature condensation) helps diagnose culture issues

Troubleshoot (Reagents, Equipment, Specimens)

Corrective actions for suboptimal slides

  • Reagents
    • Refractile Chromosomes: Caused by old/watery fixative. Fix: Wash pellet with fresh 3:1 fixative
    • Greasy/Dirty: Caused by lipids/RBCs. Fix: Perform additional washes until pellet is white
  • Equipment
    • Tight Spreads: Caused by dry air. Fix: Drop on wet slides or use a water bath
    • Scattered Spreads: Caused by humid air. Fix: Drop on dry warm slides
  • Suboptimal Specimens
    • Low Cellularity: Fix: Concentrate the entire pellet into a single drop and spot onto a small area
    • Clumping: Fix: Allow large debris to settle, then carefully pipette the single-cell supernatant from the top