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Cytogenetics

Select Culture Systems

The culture phase transforms the static specimen into a dynamic biological system capable of generating metaphase chromosomes. Success depends on selecting the correct system for the tissue type, preparing the nutrient environment accurately, and establishing redundancy to protect against failure

Prepare Specimens

Preparation involves transitioning cells from transport vessels to culture vessels under sterile conditions (Class II Biological Safety Cabinet) to optimize growth

  • Fluid Specimens (Blood/Marrow)
    • Inoculation: Whole blood is added directly to media. Bone marrow often requires “washing” (centrifugation) to remove toxic plasma/hemolysis or to concentrate cells if the count is low
    • Clots: Must be mechanically minced or enzymatically digested (Streptokinase/Collagenase) to release trapped cells
  • Solid Specimens (Tissue/Amnio)
    • Disaggregation: Solid tissues (biopsies) are minced with scalpels (mechanical) or digested with Trypsin/Collagenase (enzymatic) to create a cell suspension or explants
    • Amniotic Fluid: Centrifuged to pellet the anchorage-dependent amniocytes, which are then resuspended and plated onto coverslips
  • Seeding Density: Critical for growth. Low density causes a “lag phase” (lack of paracrine factors); high density causes nutrient depletion and contact inhibition. Viability stains (Trypan Blue) help calculate the correct inoculum

Optimal Culture for Specimen Type

Cells are biologically categorized as either “suspension” (hematopoietic) or “monolayer” (adherent), dictating the vessel and method

  • Suspension Systems (Blood/Marrow)
    • Cells float freely in tubes or flasks
    • Target: Lymphocytes (Blood) and Leukemic Blasts (Marrow)
    • Advantage: Rapid harvest via centrifugation; no trypsinization needed
  • Monolayer Systems (Amnio/Tissue)
    • Cells must attach to a surface (flask or coverslip) to divide
    • Flask Method (Closed): For solid tissues/tumors. Cells are trypsinized into suspension for harvest
    • In Situ Method (Open): For Amniotic Fluid. Cells grow directly on coverslips. This is the Gold Standard for Prenatal Diagnosis because it allows distinction between true mosaicism (multiple colonies) and in vitro artifact (single colony pseudo-mosaicism)
  • Hybrid Systems
    • CVS Direct: Short-term incubation of villi to capture spontaneously dividing cytotrophoblasts (mimics suspension harvest)

Determine Number of Cultures

Redundancy is the primary safety net against contamination, incubator failure, and artifactual results

  • The Rule of Two: The absolute minimum is two independent cultures (e.g., Tubes A & B). If one fails, the other survives
  • Specimen-Specific Guidelines
    • Bone Marrow: Multiple timepoints (24hr, 48hr, 72hr) are set up to catch leukemic clones with different growth rates
    • Amniotic Fluid: 3 to 4 cultures (dishes) are split between two separate incubators. If one incubator overheats, the backup incubator saves the diagnosis (preventing a repeat amniocentesis)
    • CVS: Both Direct (Short-term) and Culture (Long-term) are required to rule out Confined Placental Mosaicism

Label Cultures

Traceability is a critical patient safety control. A labeling error here results in analyzing the wrong patient

  • Identifiers: Every vessel must have Two Unique Identifiers (Accession Number + Name/DOB). The Accession Number is the primary link to the LIS
  • Sub-Labeling: Must distinguish the culture type (e.g., “72h PHA” vs. “24h Unstim”) to ensure correct processing and harvest timing
  • Durability: Labels must be permanent and resistant to water (water baths), alcohol (sterilization), and fixative (methanol/acetic acid). Petri dishes must be labeled on the Base, not the Lid, to prevent mix-ups

Prepare Media

Media acts as the surrogate physiological environment, requiring precise buffering and supplementation

  • Base Media: RPMI-1640 (Blood/Marrow) or Alpha-MEM/AmnioMAX (Tissue/Amnio)
  • Supplements
    • Serum (FBS): Provides growth factors (10–20%). Must be heat-inactivated
    • L-Glutamine: Essential energy source; unstable at 37°C (add fresh)
    • Antibiotics: Penicillin/Streptomycin to maintain sterility
    • Buffer: Sodium Bicarbonate balances with CO2 to maintain pH 7.2–7.4
  • Mitogens (Stimulants)
    • PHA (Phytohemagglutinin): Stimulates T-Lymphocytes (for constitutional bloods)
    • LPS/DSP-30: Stimulates B-Lymphocytes (for CLL/B-cell disorders)
    • None (Unstimulated): For bone marrow/tumors where spontaneous division is the target
  • Conditions: Incubators maintained at 37.0°C, 5% CO2, and >90% Humidity