Select Harvest Techniques

The harvest phase is a precise biochemical procedure that terminates the living culture to produce analyzable metaphase chromosomes. The goal is to arrest cells in division, swell them to disperse chromosomes, and fix the structure for staining. The technique varies based on the culture type (suspension vs. monolayer) and the required resolution (standard vs. high-resolution), but the fundamental chemical logic remains constant

Culture Harvest Techniques

• Suspension Harvest (Blood/Marrow)
  • Method: Cells are naturally floating. The process relies on centrifugation to pellet cells between solution changes
  • Protocol: Colcemid \(\rightarrow\) Spin \(\rightarrow\) Hypotonic (KCl) \(\rightarrow\) Spin \(\rightarrow\) Fixative
  • Critical: Careful aspiration of supernatant to avoid sucking up the loose cell pellet
• Flask Harvest (Monolayer-to-Suspension)
  • Method: Used for solid tissues (fibroblasts) requiring large biomass
  • Protocol: Cells must be Trypsinized (detached) first to create a suspension. Once floating, they are treated identically to blood (Spin/Hypotonic/Fix)
  • Critical: Neutralizing trypsin with serum is vital before starting the hypotonic step to prevent cell membrane lysis
• In Situ Harvest (Amnio/CVS)
  • Method: Cells are harvested while attached to the coverslip. There is no centrifugation
  • Protocol: Solutions are gently added/removed from the dish. Hypotonic causes swelling on the glass. Fixative is added carefully to avoid “blowing” cells off the surface
  • Advantage: Preserves colony architecture, allowing distinction between True Mosaicism (multiple colonies) and Pseudomosaicism (single colony artifact)

Chromosome Elongation Techniques

Standard harvests yield 400–550 bands. High-resolution analysis (850+ bands) requires capturing chromosomes in Prophase/Prometaphase (long/uncondensed)

• Synchronization (MTX Block)
  • Mechanism: Methotrexate (MTX) blocks DNA synthesis (S-phase) by inhibiting thymidine availability. Cells arrest
  • Release: Thymidine is added to release the block. Cells rush through the cycle in a synchronized wave. Harvest is timed (4–5 hours later) to catch the wave in prophase
• Intercalating Agents (Ethidium Bromide/Actinomycin D)
  • Mechanism: These chemicals insert between DNA base pairs, physically preventing the condensation proteins from packing the chromatin tightly
  • Use: Added 45mins–2hrs before harvest. Easier than synchronization but involves handling toxic mutagens (EtBr)

Select, Prepare, & Use Reagents

Three chemical classes define the harvest quality

1. Mitotic Inhibitors (The Stop)
   • Colcemid: Binds tubulin, preventing spindle formation. Arrests cells in Metaphase
   • Control: 30–60 mins. Too long = short, condensed chromosomes (poor banding). Too short = few metaphases
2. Hypotonic Solutions (The Swell)
   • 0.075M KCl: Standard for blood. Strong swell
   • Sodium Citrate: Gentler, used for fragile Bone Marrow
   • Mechanism: Osmosis drives water into the cell, expanding the membrane to prevent chromosome overlap and lysing RBCs
   • Control: Must be \(37^\circ\text{C}\). Cold hypotonic shocks cells (poor spreading)
3. Fixatives (The Preserve)
   • Carnoy’s (3:1 Methanol:Acetic Acid): Hardens chromatin, removes water, strips histones for banding
   • Control: Must be prepared Fresh. Old fixative contains water (refractile chromosomes). Added drop-wise initially to prevent cell clumping

Store Fixed-Cell Pellets

• Conditions: stored at \(-20^\circ\text{C}\) (Freezer) to prevent evaporation and chemical degradation. Never room temperature overnight
• Maintenance: Over time, fixative degrades (absorbs water). Old pellets must be Washed (spun and resuspended in fresh fixative) before making new slides for analysis or FISH
• Retention: Kept for years (especially leukemias) to allow for retrospective FISH testing if the patient relapses or new markers are discovered