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Cytogenetics

Quality Factors

In clinical cytogenetics, the diagnostic result is only as good as the starting material. Unlike molecular techniques (like PCR) that can amplify minute amounts of DNA from dead cells, conventional karyotyping requires living, dividing cells. Therefore, the “Quality Assurance” phase begins the moment the specimen arrives in the laboratory. The laboratory scientist must assess three critical pillars of specimen quality: Viability (are the cells alive?), Cellularity (are there enough cells?), and Contamination (are these the correct cells?)

Viability

Viability refers to the physiological health of the cells and their ability to undergo mitosis. If cells cannot divide, they cannot reach metaphase, and no karyotype can be produced

  • Transport Time and Temperature: These are the primary external determinants of viability
    • The “Goldilocks” Zone: Most mammalian cells thrive at body temperature (\(37^\circ\text{C}\)) but are stable for transport at room temperature (\(20\text{--}24^\circ\text{C}\))
    • Hyperthermia: Temperatures above \(40^\circ\text{C}\) denature proteins and kill cells rapidly. Specimens left in courier lockboxes during summer often arrive with “cooked” blood (dark, brown/black appearance) and zero viability
    • Hypothermia: Freezing causes ice crystals to form, puncturing cell membranes. While refrigeration (\(4^\circ\text{C}\)) is acceptable for solid tissues to slow metabolism during delays, it is detrimental to amniotic fluid and bone marrow, which should remain at room temperature
  • Media pH: Transport media contains a pH indicator (usually Phenol Red)
    • Normal: Red/Pink (pH 7.2–7.4)
    • Acidic (Yellow/Orange): Indicates bacterial overgrowth or excessive cellular metabolism releasing \(CO_2\). Viability is likely compromised
    • Alkaline (Purple): Indicates the container was not tightly sealed, allowing \(CO_2\) to escape
  • Clotting (in Blood/Marrow)
    • Clots trap the viable cells within a fibrin mesh. While mechanical disaggregation (cutting/mashing) or enzymatic digestion (Streptokinase) can sometimes salvage a clotted sample, the yield of viable cells is significantly reduced

Cellularity

Cellularity refers to the concentration of the specific cell type of interest. High volume does not always equal high cellularity

  • Peripheral Blood
    • WBC Count: The lab needs T-lymphocytes. A patient with severe leukopenia (low white count) may require a larger volume of blood or the seeding of more culture vessels to ensure enough metaphases are captured. Conversely, a patient with extremely high counts (e.g., CLL) requires dilution to prevent nutrient depletion in the media
  • Bone Marrow
    • Hemodilution: This is the most common quality issue in bone marrow aspirates. If the physician draws too much volume, peripheral blood rushes in to fill the vacuum. The resulting sample mimics peripheral blood, containing mature lymphocytes rather than the spontaneous leukemic blasts needed for diagnosis. A “dilute” marrow often yields a normal karyotype (\(46,XX\) or \(46,XY\)) even in a leukemic patient, leading to a false-negative result
  • Amniotic Fluid
    • Gestational Age: Fluid from early gestation (\(<15\) weeks) has very low cellularity. Fluid from late gestation (\(>20\) weeks) has high cellularity, but many cells are dead/terminally differentiated squames that will not divide
    • Bloody Taps: Large numbers of red blood cells can smother the adherent amniocytes on the coverslip, preventing their attachment and growth
  • Solid Tissue
    • Necrosis vs. Viable Tissue: Large tumors often have necrotic (dead) centers due to outgrowing their blood supply. Sampling the necrotic core yields low cellularity. The laboratory scientist must identify and culture the firm, white/tan viable tissue from the periphery rather than the mushy, dark necrotic tissue

Contamination

Contamination involves the introduction of unwanted microorganisms or cells from a different individual/tissue source

Microbial Contamination

Because cytogenetic cultures use rich media (serum, glucose, amino acids) and incubate at \(37^\circ\text{C}\), they are perfect breeding grounds for bacteria and fungi

  • Bacterial: Usually results in a cloudy (turbid) culture that turns yellow (acidic) rapidly. Bacteria grow faster than human cells and deplete the nutrients/oxygen, killing the specimen
  • Fungal: Often appears as “cotton balls” floating in the media. Fungi are resistant to many standard antibiotics (Penicillin/Streptomycin) used in culture, often requiring stronger antifungals (Nystatin) if detected early
  • Mycoplasma: A “silent” contaminant. It does not cause turbidity but alters cell metabolism and chromosome morphology (causing breakage or fuzziness), leading to diagnostic errors

Maternal Cell Contamination (MCC)

In prenatal samples (Amnio, CVS, POC), the greatest risk is culturing the mother’s cells instead of the fetus’s

  • Amniotic Fluid: Maternal blood from the needle stick can introduce maternal lymphocytes. Because amniocytes grow as adherent colonies and lymphocytes float, this is usually managed by changing the media (washing away the blood) 24 hours after setup
  • Chorionic Villus Sampling (CVS): The biopsy contains fetal villi entangled with maternal decidua. The laboratory scientist must microscopically dissect the sample
    • Fetal Villi: Look like branching coral/seaweed with a budding surface and visible central blood vessels
    • Maternal Decidua: Looks like flat, amorphous sheets or membranous tissue
    • Risk: If decidua is not removed, the fast-growing maternal fibroblasts will overtake the culture, leading to a \(46,XX\) result that misdiagnoses the fetus
  • Products of Conception (POC): In miscarriage samples, identifying fetal tissue is difficult. If only maternal decidua is cultured, the result will be a normal female karyotype (\(46,XX\)), masking the chromosomal cause of the miscarriage (e.g., Trisomy 16)

Evaluation & Triage

Upon receipt, the specimen undergoes a gross examination to determine processing modifications:

  • Bloody Amniotic Fluids: Require “clean-up” procedures (ammonium chloride lysis of RBCs) or increased seeding density
  • Low Volume: May require “micro-culture” techniques (using less media to keep cell concentration high)
  • Clotted Marrow: May be treated with enzymes or mechanical dissociation
  • Transport Delay: Specimen may be set up in “enriched” media (extra serum/growth factors) to rescue struggling cells