Select Representative Images

A karyogram is the definitive visual representation of a patient’s chromosomal constitution. It serves as the primary medical record supporting the cytogenetic diagnosis. In a standard clinical case, dozens of metaphase cells are examined microscopically, but only a select few (typically 2 to 5) are captured, processed, and arranged into formal karyograms. The selection of these specific images is a critical decision. They must not only be technically superior but also accurately represent the biological reality of the patient’s condition, including any clonal abnormalities or variations

Technical Criteria for Selection

The primary filter for selecting an image is technical quality. A “representative” image that is blurry or poorly spread is useless for documentation

• Resolution (Banding Level)
  • The selected metaphase should demonstrate the highest banding resolution achieved in the case
  • Why: High-resolution chromosomes (long, with distinct sub-bands) provide the visual proof that the analyst could indeed see subtle defects (e.g., verifying that a 15q11.2 deletion is not present). Using a short, condensed metaphase implies a limited analysis
• Spreading (Geometry)
  • Minimal Overlaps: The ideal image has zero overlapping chromosomes. Overlaps hide bands and require “digital surgery” (separation) which introduces artifacts
  • Intactness: The spread should be circular and complete. Selecting a cell that is “broken” (scattered over two fields of view) increases the risk that chromosomes are missing or belong to a neighbor cell
• Staining Contrast
  • The image must have crisp, high-contrast G-bands. Cells that are “puffy” (over-trypsinized) or solid black (under-trypsinized) should not be selected for the final report, even if they were countable
• Straightness
  • While software can straighten chromosomes, it is preferable to select cells where the chromosomes are naturally straight. Excessive digital straightening distorts the banding pattern

Diagnostic Criteria for Selection

Once technically adequate cells are identified, the specific cells chosen for the karyogram must reflect the diagnosis reported in the ISCN string

• Documenting the Abnormal Clone
  • If the patient has a clonal abnormality (e.g., t(8;21)), at least one (and usually two) karyograms must: display this abnormality
  • Clarity: The selected cell should show the abnormality clearly. For example, in a subtle translocation, choose the cell where the derivative chromosomes are lying flat and the breakpoints are most obvious, rather than a cell where the translocation is obscured by a cross-over
• Documenting Normal Cells (in Mosaicism)
  • If the patient is Mosaic (e.g., 45,X[10]/46,XX[10]), the report usually includes two karyograms: one representing the abnormal cell line (45,X) and one representing the normal cell line (46,XX). This visually proves the existence of both populations
• Documenting Clonal Evolution
  • In complex cancer cases (e.g., CML progressing to Blast Crisis), the patient may have multiple sub-clones:
    • Clone 1: t(9;22) only
    • Clone 2: t(9;22) + Isochromosome 17q
  • Selection: The karyograms should ideally capture the most complex: clone (Evolution) to document the full extent of disease progression
• Documenting Variations (Polymorphisms)
  • If a notable polymorphism is seen (e.g., a very large heterochromatic block on Chromosome 9qh+ or a Pericentric Inversion of Chromosome 9), a karyogram should be prepared to document it, even if it is benign. This serves as a reference for future family studies

The “Representative” Rule

The image must be representative of the count

• Scenario: In a 20-cell count, 19 cells were 46,XY and 1 cell was 45,X,-Y (random loss)
• Selection: The karyogram must be prepared from one of the 46,XY: cells
• Error: Preparing the karyogram from the single 45,X cell would mislead the physician into thinking the patient has a clone, whereas the report states the patient is normal. The image must match the conclusion