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Cytogenetics

Specimen Culture

The culture phase represents the “black box” of cytogenetics. Once the specimen is collected and processed, it enters a period of biological incubation where the laboratory scientist acts as a surrogate for the human body. The objective is to transform a static sample into a dynamic, exponentially dividing population of cells. This phase requires a mastery of cell biology, strict aseptic discipline, and the ability to visually assess microscopic growth kinetics. Success relies on selecting the correct culture system, preventing contamination, and actively managing cell health up to the moment of harvest

Select Appropriate Culture Systems

The selection of the culture system is dictated by the tissue of origin. Cells are broadly categorized as either hematopoietic (suspension-adapted) or mesenchymal/epithelial (anchorage-dependent). Using the wrong system results in immediate failure (e.g., fibroblasts will not divide if floating; lymphocytes will not attach)

  • Prepare Specimens & Optimal Culture Types
    • Suspension Cultures (Blood/Marrow)
      • Target: T-Lymphocytes (Blood) and Leukemic Blasts (Marrow)
      • Method: Whole blood is inoculated directly into tubes. Bone marrow is often washed to remove toxic plasma or concentrated to improve cellularity. These cells float freely in the media, allowing for rapid harvest via centrifugation without enzymatic detachment
    • Monolayer Cultures (Amnio/Tissue)
      • Target: Amniocytes, Fibroblasts, Trophoblasts
      • Method: Solid tissues are disaggregated (minced/digested) to release cells. Amniotic fluid is centrifuged to pellet cells. These cells must attach to a plastic or glass surface to form a cytoskeleton and divide
      • In Situ vs. Flask: In Situ (growing on coverslips) is the gold standard for prenatal diagnosis as it allows for the analysis of distinct colonies to rule out pseudomosaicism. Flasks are used for solid tissues requiring large biomass expansion
  • Redundancy (Number of Cultures)
    • The Rule of Two: A minimum of two independent cultures is established for every patient. This protects against random contamination or physical accidents (dropping a tube)
    • Strategic Split: For prenatal samples, cultures are split between two separate incubators to protect against equipment failure (e.g., thermostat malfunction). For bone marrow, multiple timepoints (24hr, 48hr, 72hr) are set up to capture clones with different growth rates
  • Media Preparation & Labeling
    • Media Components: Base media (RPMI-1640 or Alpha-MEM) is supplemented with Serum (10–20% FBS) for growth factors, L-Glutamine for energy, Antibiotics for sterility, and Sodium Bicarbonate to buffer pH against CO2
    • Mitogens: PHA is added to bloods to stimulate T-cells. LPS is added to B-cell disorders. No mitogen is used for spontaneous tumors/marrow
    • Labeling Every vessel must have two unique identifiers (Accession # and Name) and sub-labels indicating culture type (e.g., “72h PHA”). Labels must be resistant to water, alcohol, and fixative

Aseptic Culture Technique

The culture vessel is a nutrient-rich environment (\(37^\circ\text{C}\) with glucose and protein) that supports microbial growth as well as, or better than, human cells. Aseptic technique is the primary barrier against failure

  • Prevent Microbial Contamination
    • Engineering Controls: All work is performed in a Class II Biological Safety Cabinet (BSC). The laminar airflow curtain protects the open specimen. Scientists must avoid blocking grilles or overcrowding the hood, which disrupts airflow
    • The “No-Pass” Rule: A critical motor skill where the laboratory scientist never passes a non-sterile object (hand/arm) over an open sterile container. This prevents skin flakes and dust from falling into the culture
    • Equipment Hygiene: Incubator water pans (humidity source) are frequent sources of fungal spores and must be cleaned regularly. Water baths must be disinfected weekly
  • Prevent Cross-Contamination
    • Definition: The accidental transfer of cells from “Patient A” to “Patient B,” leading to a chimeric or false diagnosis
    • One Patient at a Time: Only one patient’s tubes are allowed on the rack at any given moment. The workspace is cleared between cases
    • Liquid Handling: Pipette tips are never reused between vessels. Media is aliquoted into secondary containers rather than pipetted directly from the stock bottle to patient tubes
    • Maternal Cell Contamination (MCC): In prenatal work, maternal decidua must be dissected away from fetal villi (CVS), and the first draw of amniotic fluid is discarded to prevent culturing the mother’s cells

Monitor & Document Cell Growth

Once established, cultures require active surveillance (“feeding and reading”) to maintain cells in the exponential growth phase and determine the precise moment for harvest

  • Detect, Identify, and Control Contamination
    • Signs: Turbidity (cloudiness), rapid Yellowing of media (acidic pH from bacterial metabolism), or “cotton ball” colonies (fungi)
    • Identification: Bacterial contamination is usually rapid (24h) and often due to skin flora (technique breach). Fungal contamination is slower and often environmental. Mycoplasma is invisible and requires PCR testing
    • Control: Contaminated vessels are immediately isolated/discarded. “Rescue” with heavy antibiotics is only attempted for irreplaceable specimens (e.g., marrow/amnio)
  • Culture Maintenance & Evaluation
    • Feeding: Replacing used media with fresh nutrients every 2–3 days. Monolayer cultures often receive a “partial feed” (50% change) to retain beneficial paracrine factors
    • Confluence Assessment: Using an inverted microscope to judge density
      • Log Phase (50–80%): The optimal state for harvest. Cells are dividing rapidly
      • Contact Inhibition (100%): Overgrown cultures stop dividing. These must be Subcultured (trypsinized and split) to restart the cell cycle; harvesting a 100% confluent flask yields zero metaphases
  • Assess Cultures for Harvest
    • Suspension: Harvested based on time (e.g., 72 hours for PHA-stimulated blood) to match the peak mitotic wave of lymphocytes
    • Monolayer: Harvested based on visual cues: 60–80% confluence and the presence of rounded, refractile cells (mitotic figures). In situ amnio requires a minimum of 15 independent colonies
  • Investigate Failures
    • Biological vs. Technical: Failures are categorized. Biological failures (low viability, necrosis) are reported with a description of the specimen. Technical failures (incubator malfunction, missed PHA addition) trigger a Quality Assurance audit. All failures are documented to explain the lack of results to the clinician