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Cytogenetics

Collection & Transport

The pre-analytical phase is the most critical component of clinical cytogenetics. Unlike molecular diagnostics, which can often utilize DNA extracted from non-viable tissue, conventional cytogenetics (karyotyping) relies entirely on the successful culture of living, dividing cells. The laboratory scientist must rigorously evaluate specimen integrity, understanding that errors in collection, transport, or triage are often irreversible and will result in culture failure

Specimen Requirements

The requirements for cytogenetic specimens are dictated by the need to maintain cell viability and the ability to stimulate mitosis

  • Anticoagulants
    • Sodium Heparin (Green Top): The required anticoagulant for peripheral blood and bone marrow. Heparin prevents clotting without being toxic to cells
    • Contraindications: EDTA (Lavender top) is generally unacceptable for culture because it chelates calcium, a cofactor required for spindle fiber formation during mitosis. Lithium Heparin is historically avoided due to potential cellular toxicity, though modern formulations are often acceptable
    • Fixatives: Specimens must never be placed in formalin. Formalin cross-links proteins and kills cells instantly, rendering karyotyping impossible
  • Volumes and Containers
    • Peripheral Blood: Adults (3–5 mL); Infants (1–2 mL). Collected in Sodium Heparin
    • Bone Marrow: 1–3 mL of aspirate. Ideally the “second pull” to avoid hemodilution. Collected in Sodium Heparin
    • Amniotic Fluid: 15–20 mL. Collected in sterile, non-toxic plastic tubes (glass is avoided). The first 2 mL should be discarded to reduce maternal contamination
    • Solid Tissue (POC/Skin): Sterile biopsy in transport media (Hanks/RPMI) or sterile saline. Never send dry or in water
  • Transport Conditions
    • Temperature: Specimens should generally be maintained at ambient room temperature (20–24°C)
    • Exceptions: Solid tissues may be refrigerated (\(4^\circ\text{C}\)) if there is a significant delay (overnight) to slow bacterial growth and autolysis. Specimens should never be frozen or exposed to heat (\(>40^\circ\text{C}\))

Quality Factors

The laboratory must assess three main quality indicators upon receipt:

  • Viability
    • pH Indicators: Transport media typically contains phenol red. A shift to yellow indicates acidity (bacterial contamination or metabolic waste), while a shift to purple indicates alkalinity (CO2 loss from a loose cap)
    • Clotting: In blood/marrow, clots trap cells in a fibrin mesh, preventing them from accessing nutrients and mitogens, significantly reducing the yield of dividing cells
  • Cellularity
    • Hemodilution: In bone marrow, excessive aspiration volume draws in peripheral blood. This dilutes the leukemic blasts with mature lymphocytes, potentially leading to a False Negative result (normal karyotype in a leukemic patient)
    • Necrosis: In solid tumors, the necrotic center is dead. Viable tissue must be selected from the tumor periphery
  • Contamination
    • Microbial: Bacteria and fungi grow rapidly in the rich culture media, outcompeting human cells and acidifying the culture
    • Maternal Cell Contamination (MCC): In prenatal samples (CVS/Amnio), the presence of maternal blood or decidua can lead to the culture of maternal cells, resulting in a misdiagnosis (e.g., reporting a 46,XX female karyotype for a male fetus)

Compromised or Unacceptable Specimens

A strict distinction is made between specimens that are “unacceptable” (must be rejected) and those that are “compromised” (processed with a disclaimer)

  • Unacceptable (Rejection Criteria)
    • Specimens fixed in formalin or alcohol
    • Frozen specimens (unless in cryoprotectant)
    • Unlabeled or mislabeled specimens (Chain of Custody violation)
    • Amniotic fluid leaking significantly (breach of sterility)
  • Compromised (Salvage Protocols)
    • Hemolysis: Toxic to cells. Must be washed/centrifuged immediately to remove heme-rich supernatant
    • Clotted Samples: Can be treated with mechanical disaggregation or enzymatic digestion (Streptokinase) to release cells, though yield is poor
    • Old Specimens: Samples \(>48\) hours old have reduced viability. May be rescued using enriched media, higher mitogen concentrations, or L-glutamine supplementation

Specimens for Multiple Tests

When a single specimen must serve multiple departments (Hematology, Flow Cytometry, Molecular, Cytogenetics), triage is based on sterility and viability requirements

  • Triage Hierarchy
    1. Sterile Cultures (Microbiology/Cytogenetics): Must be processed first in a sterile hood. Opening the tube elsewhere compromises sterility
    2. Viable Cell Analysis (Flow Cytometry): Requires living cells but not sterile culture
    3. Morphology (Hematology): Smears are made from fresh blood
    4. DNA/RNA Stability (Molecular): DNA is robust and can often be extracted from leftover or fixed material
  • Specific Strategies
    • Amniotic Fluid: Centrifuged to separate the pellet (sent to Cytogenetics for culture) from the supernatant (sent to Chemistry for AFP testing)
    • Bone Marrow: The “First Pull” goes to Hematology for smears (undiluted). The “Second Pull” (Heparinized) is split between Cytogenetics and Flow Cytometry
    • Solid Tissue: Fresh tissue is sent to Surgical Pathology; the Pathologist dissects a portion for Cytogenetics before placing the rest in formalin