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Cytogenetics

Assess Cultures for Harvest

The decision to harvest is the culmination of the entire culture process. It is a “point of no return.” Once the harvest chemicals (Colcemid/Hypotonic) are added, the living process stops. If the harvest is initiated too early, there will be insufficient cells (low mitotic index), resulting in analysis failure. If initiated too late, the culture becomes overgrown (confluent), cells arrest in G0, and the chromosomes become short and condensed, leading to poor banding resolution. The laboratory scientist must assess the “readiness” of the culture based on cell density, mitotic activity, and incubation time

The “Goldilocks” Zone: Mitotic Index vs. Confluence

The goal of the harvest is to capture the maximum number of cells in Metaphase

  • Mitotic Index (MI): The percentage of the cell population actively dividing at any given moment. A good harvest requires a high MI
    • Visual Cues: In monolayer cultures, cells in mitosis change shape. They retract their attachment fibers, “round up,” and become highly refractile (bright/shiny) under the phase-contrast microscope. A laboratory scientist looks for a field of view sprinkled with these bright, round cells floating slightly above the dark, flat monolayer
  • Confluence Limit
    • Optimal: 60% to 80% Confluence. At this density, cells are signaling each other to grow (paracrine factors) but still have physical space to divide
    • Overgrown: >90% Confluence. When cells touch on all sides, “Contact Inhibition” triggers cell cycle arrest. Even if the flask is packed with millions of cells, if none are dividing, the karyotype yield is zero. An overgrown flask must be subcultured (split), not harvested

Assessment by Specimen Type

Peripheral Blood (Suspension)

  • Time-Dependent: Unlike adherent cultures, blood cultures are harvested based on a strict biological clock, not visual inspection of density
  • The Clock: T-lymphocytes stimulated by PHA undergo their first division around 48 hours and reach peak mitotic activity at 72 hours
    • Standard Harvest: 70–72 hours post-setup
    • Visual Check: While density is hard to judge in a red suspension, a “good” culture will show agglutination (clumping) due to the PHA and a visible “buffy coat” layer of white cells settling on top of the red cells. The media should turn slightly orange/yellow (acidic) due to metabolic activity

Bone Marrow (Suspension)

  • Unpredictable Kinetics: Leukemic blasts do not follow a set clock
  • The Strategy: Because we cannot “see” the blasts dividing in the suspension, we rely on the multi-tube strategy
    • 24-hour harvest: Captures spontaneous divisions (fast clones)
    • 48-hour harvest: Captures clones that needed recovery time
  • Visual Check: The laboratory scientist checks for turbidity and clumping. If a 24-hour culture looks completely dormant (clear supernatant, no pellet), the lab might decide to extend it to 48 hours or add growth factors (like Giant Cell Tumor Conditioned Media) rather than harvesting a likely failure

Amniotic Fluid (In Situ Monolayer)

  • Colony Counting: The assessment is purely visual
    • Criteria: A minimum of 15 colonies: distributed across the coverslips/dishes is standard. Alternatively, if colonies are large (\(>10,000\) cells), fewer may suffice
    • Morphology: Ideally, the colonies should be actively expanding at the periphery. “Starburst” colonies (dense center, spreading rays) are ideal. If colonies look static or granular, they are not ready
  • Sequential Harvest: Not all dishes are harvested at once. The laboratory scientist identifies the “best” dishes for the first harvest (e.g., Day 7) and leaves the slower dishes (“backups”) to grow for another 2–3 days. This staggered approach ensures a result even if the first harvest is suboptimal

Solid Tissues (Flask Monolayer)

  • The “Explosion”: Tissue explants grow slowly for weeks, then suddenly accelerate
  • Assessment: The laboratory scientist looks for the halo of fibroblasts migrating out from the tissue chunk. Once the flask floor is 50–70% covered, it is ready
  • Super-Confluence Warning: Fibroblasts are very prone to contact inhibition. If a flask looks “full” on Friday afternoon, it must be harvested immediately or subcultured. Leaving it until Monday will result in a “shut down” culture

Preparation for Harvest

Once the assessment is “Go,” the laboratory scientist prepares the logistics:

  • Colcemid Addition: The timing of Colcemid (the spindle poison) determines the chromosome length
    • Standard: 30–60 minutes incubation. Longer exposure yields more metaphases but shorter, condensed chromosomes (lower band resolution)
    • Synchronized: For high-resolution banding (prophase), chemicals like Methotrexate or Thymidine are added the day before harvest to block cells, followed by a release step. The assessment for this starts 24 hours in advance