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Cytogenetics

Ambient Conditions

The process of dropping fixed cells onto a glass slide, often viewed as the “art” of cytogenetics, is in reality a complex interplay of physics and chemistry governed by the surrounding environment. The quality of the metaphase spread - specifically the separation of chromosomes and the removal of cytoplasm - is dictated by the evaporation rate of the fixative. This evaporation rate is directly controlled by three environmental variables: Temperature, Relative Humidity, and Airflow. Because the laboratory’s HVAC system can fluctuate with the seasons, the laboratory scientist must understand how to manipulate these conditions to ensure consistent slide quality

The Physics of Spreading

To understand ambient conditions, one must understand the mechanism of spreading. When a drop of cell suspension hits the slide, it spreads out. As the fixative (Methanol/Acetic Acid) evaporates, the surface of the slide cools (evaporative cooling). This drop in temperature causes moisture from the ambient air to condense onto the slide surface, mixing with the remaining Acetic Acid. This microscopic influx of atmospheric water increases the cell volume momentarily and changes the surface tension, causing the cell membrane to rupture and the chromosomes to “explode” outward. Therefore, the drying time is the critical control point

Relative Humidity (The Critical Variable)

Humidity is the most influential factor in slide making. It provides the “wedge” of water that breaks the cell open. Labs generally aim for a “Goldilocks” zone, typically between 40% and 55% Relative Humidity (RH)

  • Low Humidity (<35% - “Dry”)
    • The Phenomenon: In dry air (e.g., winter), the fixative evaporates extremely rapidly. There is insufficient moisture in the air to condense onto the slide
    • The Result (Under-spreading): The cell membrane dries and hardens before it can rupture. The chromosomes remain trapped inside the cytoplasm, encased in a tight ball. These metaphases are often heavily overlapped and unreadable
    • Laboratory Scientist Intervention: To counteract this, the laboratory scientist must slow the drying or add moisture. Techniques include breathing on the slide (“huffing”) to add warm water vapor, using a warm water bath under the slide, or dropping onto cold wet slides to extend the drying time
  • High Humidity (>60% - “Wet”)
    • The Phenomenon: In humid air (e.g., summer), evaporation is slow, and excessive water condenses onto the slide
    • The Result (Over-spreading): The cells burst violently. Chromosomes are scattered across the slide (“chromosome soup”), making it impossible to determine which chromosomes belong to which cell. Additionally, excess water remaining during the final drying phase leads to “refractile” (shiny) chromosomes that resist banding (staining)
    • Laboratory Scientist Intervention: The drying process must be accelerated. This is done by placing the slide immediately on a hot plate (\(50^\circ\text{C}\) to \(60^\circ\text{C}\)) or using an electric fan

Temperature (The Energy Source)

Temperature dictates the speed of evaporation and the energy available for membrane rupture. While the room temperature is usually static (\(20^\circ\text{C}\) to \(24^\circ\text{C}\)), the temperature of the slide and the fixative can be manipulated

  • The Slide Temperature
    • Cold Slides (Wet or Dry): Dropping cells onto a chilled slide (kept in the freezer) slows down the evaporation of the methanol. This increases the spreading time and is useful in low-humidity environments to prevent “tight” metaphases
    • Room Temperature Slides: The standard for most routine preparations. Often dipped in water (“sheeting water”) just before dropping to ensure an even spread of the fixative
  • The Drying Surface (Hot Plate)
    • Many protocols involve placing the slide on a hot plate immediately after dropping. The heat accelerates evaporation (counteracting high humidity) and helps flatten the cells against the glass, which improves the geometric plane of focus for microscopy

Airflow

Airflow removes the boundary layer of saturated air directly above the drying slide, accelerating evaporation

  • Static Air: If the air is still, a localized cloud of methanol vapor forms over the slide, slowing drying. This can lead to “grey” or “grainy” chromosomes
  • Active Airflow: Laboratory scientists may gently blow on the slide or wave it in the air. This removes the vapor cloud and speeds up drying. This is a crucial technique when humidity is high or when the chromosomes appear “puffy” and indistinct

Environmental Chambers (Standardization)

Because relying on the weather is risky for clinical diagnostics, many high-volume laboratories utilize Environmental Chambers (e.g., the Hanabi Harvester or Thermotron units). These are enclosed boxes where the slide dropping occurs. The machine maintains a precise, user-defined environment (e.g., exactly \(25^\circ\text{C}\) and 50% RH) regardless of the weather outside. This standardization removes the variability from slide making, ensuring that a slide dropped in January looks identical to a slide dropped in July