Skip to main content

Cytogenetics

Store Fixed-Cell Pellets

Once the harvest is complete, the final product is a suspension of fixed cells in 3:1 Methanol:Acetic Acid. Unlike living cultures, which are dynamic and fragile, fixed cells are biologically inert and stable. However, they are chemically sensitive. Proper storage is essential to preserve the nuclear architecture for future slide making, re-analysis, or additional testing (like FISH). A poorly stored pellet will degrade, resulting in “grey” chromosomes with poor morphology or complete evaporation of the sample

The Storage Environment (Temperature)

The defining enemy of the fixed pellet is heat and hydrolysis

  • Standard Storage: \(-20^\circ\text{C}\) (The Freezer)
    • This is the optimal long-term storage condition
    • Mechanism: The freezing temperature significantly slows chemical degradation. More importantly, it minimizes the evaporation of the volatile fixative
  • Short-Term Storage: \(4^\circ\text{C}\) (The Refrigerator)
    • Acceptable for pellets that will be used within a few days (e.g., active clinical cases)
    • Risk: At \(4^\circ\text{C}\), the reaction between Methanol and Acetic Acid (esterification) proceeds faster than at \(-20^\circ\text{C}\), leading to “stale” fixative more quickly
  • Prohibition: Never store pellets at Room Temperature (\(20^\circ\text{C}\)) overnight. The fixative will degrade rapidly, and the acetic acid will continue to macerate the chromosomes, causing them to look “fuzzy” or “exploded” when dropped

Evaporation Control (The Vessel)

Methanol and Acetic Acid are highly volatile organic solvents. They will evaporate through loose caps or seemingly sealed plastic, leaving a dried-out “crust” of cells that cannot be salvaged

  • Seal Integrity
    • Caps must be screwed on tightly
    • Parafilm: For long-term storage (archiving), the cap junction should be wrapped in Parafilm to create a gas-tight seal
  • Tube Material: Polypropylene tubes are standard. Polystyrene (clear plastic) can sometimes crack or degrade after months of exposure to organic solvents at freezing temperatures

“Refreshing” the Pellet (Maintenance)

Even in the freezer, the fixative chemistry changes. Over time (weeks/months), Methanol and Acetic Acid react to form Methyl Acetate and Water. Water is the enemy of good spreading

  • The “Wash” Protocol
    • Before making new slides from an archived pellet (e.g., a case from 2 years ago pulled for a family study), the pellet must be washed
    • Procedure: Centrifuge the tube \(\rightarrow\) Aspirate the old yellowed/evaporated fixative \(\rightarrow\) Resuspend in freshly prepared: 3:1 fixative
    • Rationale: This removes the water and re-acidifies the solution. Without this step, the chromosomes will appear “refractile” (shiny/glassy) under the microscope and will not accept Giemsa stain or FISH probes
  • Top-Up
    • Pellets should be checked periodically (e.g., annually). If the volume has dropped due to slow evaporation, fresh fixative should be added to prevent the cells from drying out completely

Retention Policy (How long to keep?)

  • Regulatory Requirement: Most accreditation bodies (CAP/CLIA) require retaining fixed pellets for a minimum period (typically 2 weeks after the final report is released) to allow for repeat testing if the initial analysis fails or if the physician adds a FISH order
  • Best Practice: Many labs retain leukemic bone marrow pellets for 10+ years
    • Why? Leukemia is a relapsing disease. If a patient relapses 5 years later, the doctor may want to compare the new cytogenetics to the original diagnostic clone. The archived pellet allows for retrospective FISH testing of new markers that weren’t discovered at the time of the original diagnosis

FISH Considerations

  • Fixed pellets are the primary substrate for Fluorescence In Situ Hybridization (FISH)
  • Note: Pellets stored for years may become “hard.” The cytoplasm becomes resistant to digestion. When making slides from old pellets for FISH, the laboratory scientist may need to use a pepsin digestion step or slightly higher denaturation temperatures to get the probe to penetrate the aged chromatin