Evaluate Harvest

The evaluation of the harvest is the final quality gate before the slide moves to the banding and analysis stages. While the “Slide Quality” check focuses on the physical dropping conditions (spreading/density), the “Harvest Evaluation” assesses the underlying biological success of the entire culture process. It answers the question: “Did the chemical manipulation of the cell cycle work?” This evaluation is often performed by a senior laboratory scientist or supervisor because the decision made here determines the downstream workflow (e.g., standard banding vs. high-resolution banding vs. failure reporting)

Evaluation Criteria: The “Harvest Index”

Laboratories often use a semi-quantitative scoring system (e.g., 1 to 4 scale) to grade harvests. This score dictates the optimal banding technique

• Grade 4 (Excellent)
  • Characteristics: High Mitotic Index (\(>50\) spreads/slide). Chromosomes are long, straight, and non-overlapping (prometaphase). Cytoplasm is completely absent (clean background)
  • Action: Suitable for High-Resolution G-Banding (850 band level). Prioritize for complex cases or microdeletion queries
• Grade 3 (Good/Standard)
  • Characteristics: Adequate Mitotic Index (20–50 spreads). Chromosomes are mid-length (metaphase). Some minor overlaps or slight cytoplasm visible
  • Action: Suitable for Routine G-Banding (400–550 band level). Standard workflow
• Grade 2 (Suboptimal)
  • Characteristics: Low Mitotic Index (<20 spreads). Chromosomes are short/condensed or “fuzzy.” Heavy cytoplasmic background (background noise)
  • Action: Requires “Rescue Banding.” The lab may use Giemsa-only staining (solid stain) first to find the few spreads, or use shorter trypsin times to avoid destroying the poor morphology
• Grade 1 (Failure/Unanalyzable)
  • Characteristics: No metaphases found, or chromosomes are shattered (“pulverized”)
  • Action: Do not band. Review backup cultures or request re-draw

Troubleshooting Biological Issues

The evaluation identifies specific biological errors that occurred during the harvest steps

• “C-Anaphase” (Colcemid Artifact)
  • Observation: The sister chromatids have separated completely at the centromere and are lying parallel to each other (looking like “ski tracks” or “railroad tracks”) rather than forming an “X”
  • Diagnosis: Over-exposure to Colcemid. The cells were arrested for too long, and the centromeric cohesion degraded. These cells cannot be karyotyped accurately because the chromatids may drift apart
• “Chromatid Breakage” vs. “Pulverization”
  • Observation: Chromosomes appear fragmented or shattered
  • Diagnosis:
    • Pulverization: Often seen in S-phase cells that were forced to condense prematurely (e.g., by virus infection or cell fusion)
    • Breakage: May indicate Clastogen exposure: (radiation/chemicals) or a breakage syndrome (Fanconi Anemia). However, it can also be a sign of over-trypsinization during the harvest
• “Soup” (Ruptured Membranes)
  • Observation: Loose chromosomes are scattered randomly across the field with no defined cell borders
  • Diagnosis: Hypotonic Overdose. The hypotonic solution was too warm, applied for too long, or the wrong concentration was used. The cells exploded before hitting the slide

The Decision to Re-Harvest

If the evaluation reveals a systemic error (e.g., Grade 2 or 1), the laboratory scientist checks if there is a remaining fixed pellet

• Re-Fixation: If the slide is “greasy” or refractile, the pellet is washed 3–4 times in fresh fixative to remove lipids/water
• Re-Hypotonic (The “Hypo-Fix” Trick): If the cells are stubbornly “tight” (under-spread) despite good dropping technique, some labs attempt a “Hypo-Fix” maneuver on the fixed pellet (adding a drop of water/hypotonic to the fixed cells briefly) to re-soften the membranes, though this is risky
• The evaluation saves the lab from wasting expensive banding reagents and analyst time on slides that are destined to fail