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Cytogenetics

Troubleshoot Analysis

The analysis phase is where the limitations of the specimen meet the demands of the diagnosis. “Troubleshooting” here refers to the intellectual and technical steps taken when the microscopic data is ambiguous, incomplete, or contradictory. The goal is to resolve uncertainty so a definitive ISCN diagnosis can be reported

Problem 1: Poor Banding Quality (The “Fuzzy” Case)

  • Symptom: Chromosomes are grey, puffy, or lack distinct light/dark bands. It is impossible to identify breakpoints or distinguish Chromosome 4 from 5
  • Cause: Specimen quality, poor spreading, or over-trypsinization
  • Troubleshooting Steps
    • Re-Stain: If the slide was under-stained, de-stain and re-stain with a longer Trypsin/Giemsa time
    • Drop Fresh Slides: If the original slide was poor, go back to the fixed cell pellet. Adjust drying conditions (humidity/temp) to improve spreading
    • Different Stain: Attempt R-Banding (Reverse banding). Some abnormalities (like telomeric deletions) are clearer in R-banding than G-banding
    • Report Limitation: If the sample cannot be improved (e.g., poor bone marrow), the report must state “Resolution limited to 300 bands,” implying that subtle defects may be missed

Problem 2: Ambiguous Rearrangement (The “Marker” Chromosome)

  • Symptom: There is extra chromosomal material (Add) or a mysterious extra chromosome (Marker), but the banding pattern is unrecognizable. You know it’s abnormal, but you don’t know what it is
  • Troubleshooting Steps
    • FISH (Painting): Use Whole Chromosome Paints (WCP) or Spectral Karyotyping (SKY). Painting the marker will reveal its origin (e.g., “It paints with Chromosome 12, so it’s a derivative 12”)
    • Microarray (CMA): If the marker represents extra material (gain), microarray will identify the specific genetic content and size
    • Parental Studies: If the marker is in a child, checking the parents can prove if it is a harmless familial variant (e.g., a bisatellited marker)

Problem 3: Single Cell Abnormality (The “Artifact” Question)

  • Symptom: 19 cells are normal (46,XY). 1 cell has Trisomy 8 (47,XY,+8)
  • Dilemma: Is this early leukemia (Clone) or a culture error (Artifact)?
  • Troubleshooting Steps
    • Extend Count: Analyze 10–30 more cells. If a second +8 cell is found, it is a clone. If not, it remains a singleton
    • Check FISH: Perform Interphase FISH on 200 nuclei. If FISH shows 5% +8 cells, the clone is real (low level). If FISH is 0%, the metaphase was likely an artifact
    • Check Culture: Did the single cell come from a separate flask? (See “Appropriate Number of Cultures”)

Problem 4: Overlapping/Clumped Chromosomes

  • Symptom: Critical chromosomes (e.g., 9 and 22) are lying on top of each other, obscuring the translocation check
  • Troubleshooting Steps
    • Find Better Cells: Do not rely on “digital surgery” (software separation) for critical diagnoses. Scan more slides to find an open metaphase
    • Different Culture: Sometimes the synchronized culture is clumped, but the unsynchronized culture spreads better

Problem 5: Inconsistent Counts (Random Loss)

  • Symptom: Cell 1 is 45,X,-7. Cell 2 is 45,X,-14. Cell 3 is 45,X,-22
  • Diagnosis: This is “Random Loss” due to fragile cell membranes rupturing during dropping (scattering chromosomes)
  • Action
    • Ignore the random losses
    • However, if Composite Karyotype: is required (e.g., complex cancer), these might be noted, but generally, valid diagnosis requires finding the same loss in 3 cells
    • Caution: In older men, loss of Y (-Y) is a common age-related phenomenon, not necessarily cancer. It is reported but noted as “age-related.”