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Cytogenetics

Select, Prepare, & Use Reagents

The cytogenetic harvest is a defined sequence of chemical reactions. The laboratory scientist acts as a chemist, using three primary classes of reagents to manipulate the physical state of the cell. The quality of the final chromosome spread - its spreading, contrast, and band resolution - is entirely dependent on the precise preparation and application of these three agents: Mitotic Inhibitors, Hypotonic Solutions, and Fixatives

Mitotic Inhibitors (Spindle Poisons)

These agents are the “stop button” for the cell cycle. Without them, cells would pass through metaphase in minutes, making it statistically impossible to find enough dividing cells on a slide for analysis

  • Selection
    • Colcemid (Demecolcine): A synthetic analog of Colchicine. It is less toxic to cells, allowing for slightly longer exposure times without causing cell death. It is the standard for routine cytogenetics
    • Colchicine: The naturally occurring alkaloid (derived from the Autumn Crocus). It is more potent and acts faster but is more toxic. Often used in plant cytogenetics or specialized rapid harvests
  • Mechanism: It binds to the protein Tubulin, preventing the polymerization of microtubules. This stops the formation of the Mitotic Spindle. The chromosomes align at the metaphase plate but cannot be pulled apart (Anaphase), accumulating cells in Metaphase
  • Preparation & Use
    • Concentration: Typically used at \(10\mu\text{g/mL}\)
    • Exposure Time: 30–60 minutes: (Standard)
      • Too Long (>2 hours): Chromosomes become “super-condensed” (short, dark dots) with no banding resolution. Chromatids may separate (C-anaphase)
      • Too Short (<20 mins): Insufficient accumulation of metaphases (low yield)

Hypotonic Solutions (The “Swell”)

These solutions are the “inflation pump.” They create an osmotic gradient that forces water into the cell, expanding the cell membrane volume while leaving the chromosomes intact. This spatial expansion is vital to prevent chromosomes from overlapping on the slide

  • Selection
    • 0.075M Potassium Chloride (KCl): The industry standard for Blood lymphocytes and fibroblasts. It provides a strong, controlled swell
    • 0.8% Sodium Citrate: A gentler hypotonic. Often preferred for Bone Marrow (leukemic blasts are fragile) because it creates a more dispersed spread without bursting the delicate cell membranes
    • Dilute Serum (1:5): Sometimes used for very fragile cells to provide protein support during swelling
  • Mechanism: The solution has a lower salt concentration (hypotonic) than the intracellular fluid. Osmosis drives water into the cell. The red blood cells (RBCs) in the culture lyse (burst) because they cannot withstand the pressure, effectively “cleaning” the harvest. The white blood cells (WBCs) swell but do not burst
  • Preparation & Use
    • Temperature: Must be pre-warmed to \(37^\circ\text{C}\). Cold hypotonic solution shocks the spindle/chromosomes, causing poor spreading
    • Timing: 15–20 minutes
      • Over-hypotonization: Cells burst in the tube (“soup”) or on the slide (“scattered” metaphases where chromosomes are lost)
      • Under-hypotonization: Cells remain small. Chromosomes are trapped in a tight ball and overlap heavily (unreadable)

Fixatives (The Preservative)

The fixative freezes the biology in time. It hardens the chromatin structure, removes water, and strips proteins to prepare the DNA for banding

  • Selection
    • Modified Carnoy’s Fixative: A 3:1 ratio of Methanol (3 parts) to Glacial Acetic Acid (1 part)
      • Methanol: A dehydrating agent. It replaces the water in the cell, hardening the structure and precipitating proteins
      • Acetic Acid: A swelling/penetrating agent. It prevents the cells from shrinking too much due to the methanol. Crucially, it extracts Histones H1: from the chromatin, which uncoils the DNA slightly to reveal the G-banding pattern
  • Preparation & Use
    • Freshness: Fixative must be prepared fresh: immediately before use (or within 1–2 hours). Over time, Methanol and Acetic Acid react to form Methyl Acetate and water. “Old” fixative contains water, which prevents proper drying and causes “refractile” (shiny/glossy) chromosomes that stain poorly
    • Temperature: Often used Cold (\(4^\circ\text{C}\)): or even frozen (slushy). Cold fixative penetrates rapidly and helps remove lipids
    • The “First Fix”: The initial addition of fixative to the water-filled cell pellet is violent. It generates heat (exothermic). The fixative must be added drop-wise: while vortexing (agitating) the tube. If added too fast, the surface proteins crosslink instantly, trapping the cells in a hard clump that cannot be spread
    • Multiple Washes: A harvest typically requires 3–4 rounds of Fix/Spin/Aspirate
      • Round 1: Turns the pellet brown (hemoglobin destruction)
      • Round 3/4: The pellet should be pure white. Any residual lipids or hemoglobin will ruin the slide (greasy appearance)

Troubleshooting Reagent Failure

  • “Soup” (Broken cells): Hypotonic was too long or too hot
  • “Tight” Metaphases (Overlapping): Hypotonic was too short or Colcemid was too long (short chromosomes)
  • “Greasy” Slides: Fixative was old (contained water) or not enough wash steps were performed
  • No Banding: Fixative lacked Acetic Acid (chromatin didn’t relax) or chromosomes were over-condensed (Colcemid too long)