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Cytogenetics

Prepare Specimens

The preparation of specimens for culture is the transitional phase where biological material is moved from the transport vessel into the culture system. The objective is to isolate the specific cell type of interest, remove inhibitory substances (such as red blood cells, necrotic tissue, or toxic transport media), and introduce the cells into a nutrient-rich environment at the optimal concentration to support logarithmic growth. The specific preparation technique varies significantly based on the physical state of the specimen (fluid vs. solid) and the growth requirements of the target cells (anchorage-dependent vs. suspension)

General Principles of Sterile Preparation

All specimen preparation must occur within a certified Class II Biological Safety Cabinet (Laminar Flow Hood) to protect both the specimen from environmental contamination and the laboratory scientist from potential pathogens. Before the culture is initiated, the laboratory scientist must perform a gross examination of the sample to determine the processing path

  • Aseptic Technique: All pipettes, tubes, and culture vessels must be sterile. The workspace should be wiped with 70% ethanol before and after use. Open flame (Bunsen burners) is generally avoided in modern tissue culture hoods due to airflow disruption
  • Media Equilibration: Culture media should be warmed to \(37^\circ\text{C}\) and adjusted to the correct pH (7.2–7.4) prior to inoculation. Cold media can shock the cells, causing a lag in the cell cycle recovery

Preparation of Peripheral Blood (Suspension Culture)

Peripheral blood preparation is relatively straightforward because it is a “whole blood” culture technique. The target cells (T-lymphocytes) are suspended in plasma. Separation of the white blood cells (buffy coat) is generally not required for routine constitutional studies unless the white blood cell (WBC) count is exceptionally high or low

  • Inoculation: The specimen tube is inverted gently to mix the cells. Using a sterile pipette, a specific volume of whole blood is added directly to the culture tube or flask containing media and mitogen
  • Seeding Density: The standard ratio is approximately 0.3 mL to 0.5 mL of whole blood per 5 mL to 10 mL of culture media
    • High Counts: If the WBC count is elevated (e.g., leukocytosis), the inoculum volume is reduced to prevent rapid acidification of the media
    • Low Counts: If the patient is leukopenic, a larger volume (up to 1.0 mL) may be used, or the buffy coat may be concentrated via centrifugation to discard excess red blood cells (RBCs), as high RBC concentrations can inhibit lymphocyte proliferation

Preparation of Bone Marrow (Suspension Culture)

Bone marrow aspirates differ from blood because the target cells (leukemic blasts) are often fragile and the sample may contain fat, bone chips, or clots. Additionally, the plasma may contain antibodies or drugs (chemotherapy) inhibitory to growth, often necessitating a “washing” step

  • Washing Protocol: If the sample is hemolyzed or the volume is large, the specimen is centrifuged (typically 800–1000 rpm for 8–10 minutes). The supernatant (plasma/transport media) is aspirated and discarded. The cell pellet is resuspended in fresh, sterile culture media or Hanks Balanced Salt Solution (HBSS)
  • Cell Counting: Unlike peripheral blood, bone marrow cellularity is highly variable. A manual or automated cell count is often performed. The target seeding density is generally \(1\times 10^6\) cells per mL of culture media
  • Handling Clots: If the marrow is clotted, the clot should be physically transferred to a sterile dish. It can be minced with sterile scalpels or washed vigorously with media to dislodge trapped cells. In severe cases, enzymatic digestion (Collagenase) may be used to release the cells

Preparation of Amniotic Fluid (Monolayer Culture)

Amniotic fluid contains very few viable cells (amniocytes) relative to the total volume, and these cells are anchorage-dependent (they must attach to a surface to divide). Therefore, the preparation focuses on concentration and attachment

  • Centrifugation: The fluid (typically 15–20 mL) is centrifuged to pellet the cells. The supernatant (amniotic fluid) is removed, leaving approximately 0.5 mL of fluid with the pellet. The supernatant is often saved for biochemical testing (AFP/AChE)
  • Resuspension: The cell pellet is gently resuspended in the remaining fluid
  • Plating: The suspension is inoculated onto coverslips (for in situ culture) or into T-flasks. Because amniocytes settle by gravity, the culture vessels are usually left undisturbed for 48–72 hours to allow the cells to adhere to the plastic or glass surface before fresh media is added

Preparation of Solid Tissues (Monolayer Culture)

Solid tissues (skin biopsies, products of conception, chorionic villi, solid tumors) require physical or chemical breakdown to release individual cells capable of attachment and division. This process is known as disaggregation

  • Mechanical Disaggregation (Mincing): The tissue is placed in a sterile petri dish with a small amount of media. Using sterile scalpels or scissor-forceps, the tissue is minced into pieces smaller than 1 mm cubed. These “explants” are then placed directly onto the culture surface. Fibroblasts will migrate out of the tissue chunk and form a monolayer around it
  • Enzymatic Disaggregation (Digestion): For tougher tissues or to obtain a single-cell suspension, the minced tissue is incubated with enzymes
    • Collagenase: Breaks down the collagen matrix of the connective tissue (often used for chorionic villi and tumors)
    • Trypsin: A proteolytic enzyme used to separate cells from each other
  • Cleaning and Dissection: Before disaggregation, the tissue must be cleaned
    • Chorionic Villi (CVS): Must be microscopically dissected to separate the branching fetal villi from the flat, membranous maternal decidua to prevent maternal cell contamination (MCC)
    • Products of Conception (POC): Necrotic tissue does not grow and releases toxins. The laboratory scientist must identify and select firm, white/pink fetal tissue and discard mushy, dark/necrotic areas

Seeding Density & Viability Assessment

For all specimen types, the success of the culture depends on the “Goldilocks” principle of seeding density

  • Cell Viability (Trypan Blue): When cellularity is questionable, a drop of cell suspension is mixed with Trypan Blue dye. Viable cells have intact membranes and exclude the dye (appearing clear/shiny). Dead cells take up the dye (appearing blue). Only viable cells contribute to the seeding calculation
  • The Density Impact
    • Too Low: If cells are plated too sparsely, they experience a “lag phase” or fail to divide entirely because they lack the autocrine and paracrine growth factors secreted by neighboring cells (the “feeder” effect)
    • Too High: If cells are overcrowded, they rapidly deplete the nutrients in the media and produce excessive lactic acid (pH drop). In monolayer cultures, overcrowding leads to contact inhibition, where cells stop dividing once they touch each other, preventing the formation of mitotic figures