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Cytogenetics

Determine Number of Cultures

The determination of “how many cultures to set up” is a strategic decision that balances laboratory economics (cost of media/reagents/time) against the risk of culture failure. In clinical cytogenetics, the specimen is often irreplaceable. Therefore, the primary rule of culture initiation is Redundancy. A single culture vessel is a single point of failure; if it becomes contaminated, breaks, or fails to grow, the diagnosis is lost. The standard of care dictates establishing multiple independent cultures to safeguard against these risks and to provide the necessary diagnostic resolution (e.g., distinguishing true mosaicism from artifact)

The Rule of Two (Minimum Requirement)

For almost every specimen type, the absolute minimum standard is two independent cultures. This redundancy serves as a safety net

  • Sterility Protection: If one tube is contaminated by bacteria during setup or handling, the second tube (handled separately) remains sterile
  • Safety Net: If an incubator fails (overheats or loses CO2), or a flask is accidentally dropped, the backup culture survives
  • Mosaicism Confirmation: Finding a chromosomal abnormality in a single culture vessel might represent an in vitro artifact (an error that happened in the lab dish). Finding the same abnormality in two separate cultures confirms it is present in the patient (constitutional/clonal)

Specific Guidelines by Specimen Type

While “two” is the minimum, specific guidelines exist for different sample types based on cell fragility, clinical urgency, and the biology of the target tissue

Peripheral Blood (Constitutional)

  • Standard Setup: Two cultures
    • Typically designated “A” and “B” (e.g., 72-hour A and 72-hour B)
    • Both are usually stimulated with PHA
  • Rationale: T-lymphocytes are robust. Two tubes provide enough metaphases for a standard 20-cell count. If one tube fails, the other is usually sufficient. Some labs set up a third culture stored in the refrigerator as a “backup inoculum” in case both fail, but active culture of two is standard

Bone Marrow (Leukemic)

  • Standard Setup: Two to Four cultures
  • The Problem: Leukemic blasts are unpredictable. We do not know a priori which culture condition will make them divide. Some divide spontaneously; some need time to recover from the stress of aspiration
  • The Strategy: The lab sets up multiple cultures with different incubation times to cast a “wide net”
    • 24-hour Unstimulated: Captures cells already in the cell cycle
    • 48-hour Unstimulated: Captures cells that needed recovery time (often the highest yield)
    • Optional: 72-hour or Mitogen-stimulated (e.g., LPS for B-cell disorders)
    • Rationale: If the lab only sets up a 24-hour culture and the patient’s cells were slow-growing, the result will be a False Negative (Normal). Setting up multiple timepoints maximizes the detection of the abnormal clone

Amniotic Fluid (Prenatal)

  • Standard Setup: Three to Four cultures (distributed across multiple vessels)
  • The “In Situ” Method: Typically 15 to 20 colonies are needed for diagnosis. This usually requires:
    • 3 or 4 Petri dishes (each with a coverslip)
    • OR 2 Chamber Slides (each with 2 chambers)
  • The “Split Incubator” Rule: This is critical for prenatal samples. The cultures must be physically separated into two different incubators (e.g., Dishes A and B in Incubator 1; Dishes C and D in Incubator 2)
    • Rationale: If Incubator 1 malfunctions (thermostat failure “cooks” the cells), the cultures in Incubator 2 survive, preventing the need for a repeat amniocentesis (which carries a risk of miscarriage)

Solid Tissues (Biopsies/POC)

  • Standard Setup: Two to Three Flasks
  • The Strategy: Because contamination (fungal/bacterial) is common in skin and POC samples, and because solid tissue takes weeks to grow, redundancy is vital
    • Flask A: Fed with standard media
    • Flask B: Fed with media + heavy antibiotics/antifungals
    • Flask C: (Optional) Backup frozen early or kept in a different incubator
  • Rationale: If the primary flask is overtaken by mold (common in POCs), the backup flask with antifungals might be salvaged

Chorionic Villi (CVS)

  • Standard Setup: Two Culture Types
    • Direct/Short-Term: (24-48 hours) Targets the Cytotrophoblast (placental origin). Provides rapid results
    • Long-Term Culture: (7-10 days) Targets the Mesenchymal Core (fetal origin). Provides confirmatory results
  • Rationale: Comparing the two cultures allows for the detection of “Confined Placental Mosaicism” (CPM) - where the placenta has an abnormality (e.g., Trisomy) but the fetus is normal. Both must be set up to ensure accurate diagnosis

Resource Management

While “more is better,” labs are constrained by incubator space and reagent costs. The number of cultures is determined by the Standard Operating Procedure (SOP) which is validated to ensure a \(>95\text{--}99\%\) success rate

  • Low Volume Samples: If a sample arrives with insufficient volume (e.g., 0.5 mL of blood), the lab reduces the volume per culture rather than the number of cultures. It is better to have two “micro-cultures” (0.25 mL each) than one standard culture (0.5 mL) to maintain the safety of redundancy