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Cytogenetics

Analyze Number of Cells

The statistical power of a cytogenetic study depends entirely on the sample size - the number of cells examined. Because analyzing chromosomes is labor-intensive, laboratories must balance diagnostic confidence with practical efficiency. The number of cells scored (counted) and analyzed (karyotyped) is dictated by guidelines from the American College of Medical Genetics (ACMG) and the specific clinical indication

Routine Constitutional Analysis (Blood/Amnio)

For congenital diagnosis (e.g., Down syndrome, Infertility), the goal is to define the patient’s constitutional karyotype. Since every cell in the body theoretically shares the same genetics, a smaller number of cells is usually sufficient

  • Standard Protocol
    • Count: 20 cells
    • Analyze (Karyotype): 5 cells
  • Rationale: Detecting a uniform (non-mosaic) abnormality like Trisomy 21 is statistically virtually guaranteed within 20 cells. Analyzing 5 cells band-for-band ensures that structural rearrangements (like a balanced translocation) are not missed due to a single bad spread

Routine Oncology Analysis (Bone Marrow/Leukemia)

In cancer, the patient is a Mosaic by definition: they have a population of normal host cells and a population of tumor cells (the clone). The goal is to detect the abnormal clone, which might be small

  • Standard Protocol
    • Count: 20 cells
    • Analyze (Karyotype): 20 cells
  • Why Analyze All 20?: In leukemia, structural changes can be subtle. Counting alone (just checking for 46 chromosomes) would miss a balanced translocation like t(9;22) or t(15;17). Therefore, every cell counted in an oncology case is typically fully analyzed for structural defects

Mosaicism Workup (The Extended Count)

If mosaicism is suspected (either clinically or because a single abnormal cell was found during the routine count), the sample size must be increased to rule out artifact

  • Scenario: During a routine 20-cell count, 1 cell is found with Trisomy 8 (+8). Is this a clone or a random error?
  • Protocol: Extend the count to 30 or 50 cells
  • Statistical Power
    • Counting 20 cells: can rule out mosaicism of 14% or higher with 95% confidence. (Meaning, if you see 20 normal cells, you are 95% sure the patient doesn’t have a clone larger than 14%)
    • Counting 30 cells: rules out 10% mosaicism
    • Counting 50 cells: rules out 6% mosaicism
  • Diagnosis: If a second +8 cell is found during the extension, the “2-cell rule” for clonality is met, and the patient is diagnosed with mosaic Trisomy 8. If no other cells are found, the single cell is reported as a probable cultural artifact (pseudomosaicism)

Single-Colony vs. Multiple-Colony (Amniocytes)

For amniotic fluid cultures (which grow in colonies), analysis must prove that an abnormality is present in the fetus, not just an artifact arising in one culture dish (in vitro error)

  • The Rule: Cells must be selected from multiple independent colonies (usually at least 2 or 3 different culture vessels)
  • Rationale
    • True Mosaicism: Present in multiple colonies/dishes. Indicates the fetus is mosaic
    • Pseudomosaicism: All abnormal cells are confined to a single colony. This indicates the mutation happened in the lab dish during culture growth, not in the baby. This result is generally considered artifactual and clinically insignificant

Abbreviated Analysis (Follow-Up)

In known positive oncology cases (e.g., a known CML patient on therapy), fewer cells might be fully analyzed if the specific abnormality is easily identifiable

  • Protocol: Count 20 cells. Karyotype only 2–5 cells to confirm it’s the same clone, but scan the rest for the specific known defect (e.g., Ph chromosome). However, most labs still default to full analysis to catch clonal evolution