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Cytogenetics

Select & Analyze Suitable Metaphases

The generation of a karyotype is a multi-step process requiring rigorous selection criteria and adherence to established statistical guidelines. The cytogeneticist transforms a biological sample into a structured dataset (the ISCN report) through careful observation and documentation

Selection Criteria

  • Spreading: Chromosomes must be well-separated (no clumps) but not scattered (risk of loss)
  • Banding: Quality is graded (Routine 400 bands vs. High-Res 550+ bands). Fuzzy or over-digested cells are rejected
  • Process: Count chromosomes to detect aneuploidy (e.g., 47,+21). Analyze (karyotype) band-for-band to detect structural changes (e.g., deletions, translocations)
  • Clonality: A diagnosis requires finding the same structural defect/gain in 2 cells or the same loss in 3 cells

Review Previous/Related Results

  • History: Comparison with prior karyotypes is essential to distinguish new disease from old clones (Disease Progression vs. Remission)
  • Family: In prenatal cases, parental studies distinguish de novo mutations (pathogenic) from inherited familial variants
  • Correlation: FISH and Microarray results guide the analyst on where to look (e.g., “FISH says MLL is rearranged; check 11q23”)

Analyze Appropriate Number of Cells

  • Constitutional: Count 20, Analyze 5. (Target: Homogeneous germline defects)
  • Oncology: Count 20, Analyze 20. (Target: Mosaic clones and subtle structural changes)
  • Mosaicism: If a single abnormal cell is found, extend the count to 30 or 50 cells to rule out artifact

Analyze Appropriate Number of Cultures

  • Rule of Two: Always analyze cells from at least 2 independent cultures (flasks/dishes)
  • Reason: To detect Pseudomosaicism (artifacts arising in vitro in a single dish). A true clone will be present in multiple cultures
  • Amnio: Requires analysis of cells from multiple distinct colonies

Document Analysis

  • Worksheet: Records coordinates (X/Y) and counts for every cell
  • Karyogram: Digital arrangement of homologous pairs for the analyzed cells. Raw images must be preserved
  • ISCN: Final report string (e.g., 46,XY,t(9;22)(q34;q11.2)[20])

Troubleshoot Analysis

  • Poor Banding: Re-stain or drop fresh slides. If unfixable, report “Limited Resolution.”
  • Unknown Marker: Use FISH (WCP/SKY) or Microarray to identify the origin of extra material
  • Single Cell: Extend count or use Interphase FISH to determine if it is a real clone or a culture artifact
  • Random Loss: Scattered missing chromosomes (different in each cell) are attributed to technical breakage (cell rupture) and ignored