Slide Quality

Once the slide is prepared, the laboratory scientist must perform an immediate microscopic quality control (QC) check using a Phase Contrast microscope (typically 10x and 20x objectives). This “triage” step determines whether the slide is suitable for staining and analysis or if adjustments must be made to the harvest or dropping conditions. Evaluating slide quality is a subjective skill that balances four distinct parameters: Cell Density, Mitotic Index, Metaphase Spreading, and Chromosome Morphology

Cell Density (The “Crowding” Factor)

Density refers to the total number of cells (interphase nuclei + metaphases) per field of view

• Optimal: Cells are evenly distributed but not touching. There is clear glass space between nuclei. This allows metaphases to spread without colliding with neighboring interphase cells
• Too Sparse: Finding cells requires hunting across the slide. This wastes analyst time
  • Remedy: Re-spin the pellet and remove more fixative to concentrate the suspension
• Too Dense: Cells are piled on top of each other. Metaphases are trapped within clumps of nuclei, preventing proper spreading and banding
  • Remedy: Dilute the suspension by adding more fixative

Mitotic Index (The Yield)

The Mitotic Index (MI) is the percentage of cells in the population that are actively dividing (in metaphase)

• Optimal: A good constitutional blood culture should have dozens of metaphases per low-power field. A bone marrow might have fewer, but should still yield 20+ analyzable cells per slide
• Low MI: Metaphases are rare. This signals a culture failure or biological suppression
  • Remedy: Drop multiple slides (4–6) to ensure enough cells are available for the full analysis. Concentrate the pellet to the absolute minimum volume
• Note: If the MI is zero (no metaphases), no amount of slide dropping will save the case. The failure investigation protocol begins

Metaphase Spreading (The “Explosion”)

This is the most critical variable controlled by the laboratory scientist during dropping. It describes how well the chromosomes have separated from each other

• Optimal (“Textbook Spread”): The chromosomes are dispersed in a circular area, free from the cytoplasm. They are not overlapping, but they are still close enough to be recognized as a single cell unit. No chromosomes have “floated away”
• Under-spread (“Tight”): Chromosomes are bundled in a tight ball, often encased in visible cytoplasm boundaries. Overlaps are frequent
  • Cause: Drying was too fast (low humidity) or hypotonic was insufficient
  • Remedy: Re-drop using “wet” techniques (huffing, cold wet slides) or re-hypotonize the pellet
• Over-spread (“Scattered”): Chromosomes are blasted far apart. It is difficult to tell if a chromosome belongs to Cell A or Cell B. Hypodiploidy (random loss of chromosomes) becomes a major artifact
  • Cause: Drying was too slow (high humidity) or hypotonic was too harsh
  • Remedy: Use heat (hot plate) or airflow to speed up drying. Use dry slides instead of wet ones

Chromosome Morphology (The Appearance)

This refers to the physical structure of the chromatids before staining. Phase contrast microscopy reveals the quality of the chromatin

• Optimal: Chromosomes appear dark grey and crisp against the light grey background (in phase contrast). The chromatids are parallel and distinct. The length is adequate (mid-length, not dots)
• Refractile (The “Glossy” Look): Chromosomes look bright white, shiny, or hollow. They look like glass
  • Cause: Water contamination in the fixative or incomplete drying. These chromosomes will not band. They will stain poorly and look “ghost-like.”
  • Remedy: Wash the pellet in fresh fixative multiple times. Ensure slides are fully dry (bake them)
• Fuzzy / Puffy: Chromosomes look like cotton balls with indistinct edges
  • Cause: Over-hypotonization or drying that was too slow in high humidity
  • Remedy: Speed up drying (heat/air). It is hard to fix the pellet once this damage is done
• Condensed (Short): Chromosomes look like small black dots or “X”s
  • Cause: Colcemid exposure was too long
  • Remedy: None. The chromosomes are physically short. High-resolution analysis is impossible

The QC Loop

The slide check is an iterative process. The laboratory scientist drops one “test slide,” checks it under the microscope, and then adjusts the variable

• Example: “The test slide is too dense and tight.” \(\rightarrow\) Action: “I will add 1 mL of fixative (dilute) and drop the next slide on a cold wet surface (spread).” Only when the test slide is optimal are the final slides for the case prepared