Culture Harvest

The harvest phase marks the critical transition from biological culture to chemical preservation. It acts as the “point of no return” in the cytogenetic workflow. Once the harvest begins, the living process is terminated, and the quality of the final analysis depends entirely on the precise execution of chemical reactions and physical manipulation. The objective is to arrest cells in metaphase, disperse the chromosomes to prevent overlapping, and fix the chromatin structure for staining. This process is divided into two distinct technical stages: the chemical harvest (generating a cell suspension) and slide preparation (generating the physical spread)

Select Harvest Techniques

The harvest protocol allows the laboratory scientist to manipulate the cell cycle and physical state of the cell. The specific technique is chosen based on the culture type (suspension vs. monolayer) and the diagnostic resolution required

• Harvest Methodologies
  • Suspension Harvest (Blood/Marrow)
    • Since cells are naturally floating, the process relies on centrifugation: to pellet the cells between solution changes. The supernatant is aspirated, and reagents are added to the pellet
    • Critical Step: Careful aspiration is required to avoid removing the loose cell pellet along with the supernatant
  • Flask Harvest (Monolayer-to-Suspension)
    • Used for solid tissues or mass-culture amniocytes. Cells must be Trypsinized: (enzymatically detached) to convert the monolayer into a suspension. Once floating, the protocol mirrors the suspension harvest (Spin/Hypotonic/Fix)
    • Critical Step: Serum must be added to neutralize the trypsin before the hypotonic step, or the weakened cell membranes will rupture
  • In Situ Harvest (Amnio/CVS)
    • Cells are harvested while attached: to the coverslip or slide surface. There is no centrifugation. Solutions are gently exchanged in the dish
    • Clinical Value: This method preserves the original colony architecture, allowing the cytogeneticist to distinguish between True Mosaicism: (abnormality present in multiple independent colonies) and Pseudomosaicism (culture artifact limited to a single colony)
• Chromosome Elongation (High-Resolution)
  • Standard harvests yield 400–550 bands. To achieve 850+ bands (Prometaphase), special techniques are used
  • Synchronization (MTX): Methotrexate blocks DNA synthesis (S-phase). Cells are released with Thymidine, creating a synchronized wave of division harvested in Prophase
  • Intercalating Agents (EtBr): Chemicals like Ethidium Bromide insert between DNA base pairs, physically preventing the condensation proteins from shortening the chromosomes
• Reagent Selection & Use
  • Mitotic Inhibitors (Colcemid): The “Stop” button. Binds tubulin to prevent spindle formation, arresting cells in metaphase. Over-exposure leads to short, condensed chromosomes; under-exposure yields low mitotic indices
  • Hypotonic Solutions (KCl / NaCitrate): The “Swell.” Osmosis drives water into the cell, expanding the membrane to separate chromosomes and lyse RBCs. Must be used at 37°C; cold hypotonic shocks the cells and causes poor spreading
  • Fixatives (Carnoy’s): The “Preservative.” A 3:1 ratio of Methanol to Acetic Acid. Hardens chromatin, removes water, and strips histones. Must be prepared Fresh; old fixative contains water, leading to “refractile” (shiny) chromosomes that stain poorly
• Storage: Fixed pellets are stored at -20°C to prevent evaporation. Old pellets must be washed with fresh fixative before use to remove water absorbed from the atmosphere

Prepare Slides

Slide preparation, or “dropping,” is the physical transfer of fixed cells onto glass. The quality of the metaphase spread - whether chromosomes are distinct or overlapped - is governed by the evaporation rate of the fixative, which is controlled by ambient environmental conditions

• Ambient Conditions
  • Relative Humidity (RH): The dominant variable. The optimal range is 40–55%
    • Low Humidity (<35%): Fixative evaporates too fast. Cells dry before bursting, resulting in “Tight” spreads (chromosomes encased in cytoplasm). Correction: Add moisture (“huffing”), use wet slides, or decrease airflow
    • High Humidity (>60%): Fixative evaporates too slowly. Cells absorb excess water, resulting in “Scattered” spreads (chromosomes broken apart) or refractile morphology. Correction: Use heat (hot plate) or airflow to accelerate drying
  • Temperature: Cold slides slow evaporation (for dry days); hot plates accelerate evaporation (for humid days)
• Slide Quality Assessment (QC)
  • Every slide is checked under phase-contrast microscopy immediately after dropping
  • Cell Density: Cells should be evenly distributed. If too sparse, the pellet is concentrated; if too dense, it is diluted
  • Mitotic Index: Ensures enough metaphases are present for analysis. Low index requires dropping more slides
  • Spreading: Chromosomes should be distinct and non-overlapping. “Tight” vs. “Scattered” indicates humidity issues
  • Morphology: Chromosomes should be dark and crisp. “Refractile” (shiny/hollow) appearance indicates water contamination in the fixative
• Evaluate Harvest
  • The harvest is graded (e.g., Grade 1–4) to determine the downstream workflow. Grade 4: (long, clean chromosomes) qualifies for High-Resolution Banding. Grade 2 (short, fuzzy) may require “Rescue Banding” (solid stain). Grade 1 (failure) triggers an investigation
  • Biological artifacts like C-Anaphase: (separated chromatids) indicate Colcemid toxicity; Pulverization indicates premature condensation or viral issues
• Troubleshooting
  • Reagent Issues: Refractile or greasy slides are fixed by washing the pellet 3–4 times in fresh fixative
  • Environmental Issues: Tight spreads are fixed by increasing humidity/wetness; scattered spreads are fixed by increasing heat/dryness
  • Specimen Issues: Low cellularity samples are managed by concentrating the entire pellet into a single drop and spotting it in a small area on the slide