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Cytogenetics

Culture Harvest

The harvest is the critical termination step of the culture process. It involves a precise sequence of chemical reactions designed to arrest cells in metaphase, swell them to disperse the chromosomes, and fix the chromatin structure for staining. Unlike the culture phase, which spans days or weeks, the harvest occurs over minutes. The technique selected depends entirely on whether the cells are growing in suspension (floating) or as a monolayer (attached), and whether the goal is to analyze individual cells (flask method) or distinct colonies (in situ method)

Universal Principles of the Cytogenetic Harvest

Regardless of the specific technique (suspension vs. monolayer), every harvest follows the same three-step chemical logic. The difference lies in the mechanical handling of the cells

  • Step 1: Mitotic Arrest (Colcemid): The addition of Colcemid (demecolcine) or Colchicine. This chemical binds to the protein tubulin, preventing the formation of the spindle fibers (microtubules). Consequently, cells progress through Prophase but arrest at Metaphase because they cannot pull the chromosomes apart into Anaphase
  • Step 2: Hypotonic Treatment (Swelling): The cells are exposed to a solution with a lower salt concentration than the cell cytoplasm (e.g., 0.075M KCl). Water rushes into the cell via osmosis, causing it to swell like a balloon. This internal pressure pushes the chromosomes apart, preventing them from overlapping on the slide
  • Step 3: Fixation (Preservation): The addition of “Carnoy’s Fixative” (3:1 Methanol to Glacial Acetic Acid). This stops the swelling, kills the cell, hardens the chromatin, and removes water. The acetic acid also helps strip histones (proteins) from the DNA, facilitating better banding resolution later

Suspension Harvest Technique

This is the standard technique for Peripheral Blood and Bone Marrow. Since the cells are already floating, no enzymatic detachment is required. The process relies on centrifugation to concentrate the cells into a “pellet” at the bottom of the tube between solution changes

  • The Protocol
    • Arrest: Colcemid is added directly to the culture tube for 30–60 minutes prior to harvest
    • Centrifugation: The tube is spun (typically 1000 rpm for 8–10 minutes) to pellet the cells
    • Hypotonic Shock: The supernatant (culture media) is aspirated and discarded. The hypotonic solution (Potassium Chloride - KCl) is added to the pellet. The cells are resuspended and incubated at \(37^\circ\text{C}\) for 20 minutes
    • Fixation: After another spin/aspirate cycle, cold fixative is added. The tube is often “vortexed” or flicked during the first few drops of fixative addition to prevent the cells from clumping into a hard mass
  • Critical Considerations
    • The Pellet: The pellet is often tiny and white/translucent. The laboratory scientist must be careful not to aspirate the pellet when removing the supernatant
    • Clumping: If fixative is added too fast to a cell pellet that hasn’t been resuspended, the proteins crosslink instantly, creating a “brick” of cells that cannot be analyzed

Flask Method (Monolayer-to-Suspension)

This technique is used for Solid Tissues and Long-term CVS where large numbers of cells are required, or for Amniotic Fluid when the in situ method is not used. The unique challenge here is that cells are stuck to the plastic. They must be detached (trypsinized) to convert them into a suspension before they can be harvested like blood

  • The Protocol
    • Arrest: Colcemid is added to the flask while the cells are still attached
    • Detachment (Trypsinization): The media is removed. Trypsin-EDTA is added to detach the cells from the flask surface. Once detached, they are floating in fluid
    • Neutralization: Serum-containing media is added to stop the trypsin reaction (preventing cell digestion)
    • Transition to Suspension: The contents of the flask are poured into a centrifuge tube. From this point on, the protocol is identical to the Suspension Harvest (Spin \(\rightarrow\) Hypotonic \(\rightarrow\) Fix)
  • Critical Considerations
    • Over-Trypsinization: If cells are left in trypsin too long, the cell membranes weaken. When the hypotonic solution is added later, these weakened cells will burst completely, resulting in “soup” (loose chromosomes) rather than intact metaphases

In Situ Harvest Technique

This technique is the Gold Standard for Amniotic Fluid and CVS grown on coverslips or chamber slides. The fundamental difference is that the cells never leave the glass surface. There is no centrifugation and no trypsinization. The solutions are changed around the stationary cells

  • The Protocol
    • Arrest: Colcemid is added to the petri dish/chamber
    • Hypotonic Shock: The media is carefully aspirated from the dish. Hypotonic solution (usually 0.8% Sodium Citrate or dilute KCl) is gently pipetted onto the coverslip. The cells swell while still attached to the glass
    • Prefixation: A small amount of fixative is added into the hypotonic solution. This “soft fix” stabilizes the swollen cells before the harsh pure fixative is added
    • Fixation: The fluid is aspirated, and pure fixative is added. The coverslip is then dried
  • Advantages
    • Colony Architecture: Because cells are not mixed, distinct colonies remain intact. This allows the cytogeneticist to distinguish between True Mosaicism (abnormality in multiple colonies/founder cells) and Pseudomosaicism (abnormality in a single colony/culture artifact)
    • Minimal Cell Loss: Since there is no centrifugation, there is no risk of aspirating the cell pellet
  • Critical Considerations
    • :** fragility** Hypotonic-swollen cells attached to glass are incredibly fragile. If the fixative is piped directly onto the cell monolayer with force, the cells will be “blown off” the glass, leaving a blank slide. Fluids must be added down the sidewall of the dish

Harvest Variables by Specimen

  • Bone Marrow (Sodium Citrate): While KCl is used for blood, Sodium Citrate is often preferred as the hypotonic agent for bone marrow. It is gentler and provides better spreading for the often-fragile leukemic blasts
  • CVS Direct (Simulated Suspension): In a “Direct” CVS harvest, the villi are incubated in Colcemid and then exposed to hypotonic solution (Acetic Acid) to release the cytotrophoblast layer spontaneously. The remaining mesenchymal core is then removed, leaving a suspension of cells. This is a hybrid physical/chemical harvest