Identification & Karyogram Review

The final stage of the cytogenetic process is the interpretation of the visual data. This requires the laboratory scientist to possess a deep knowledge of chromosomal morphology, the rules of standardized arrangement, and the clinical significance of specific genomic changes

Metaphase Chromosomes

• Identification (Groups A–G)
  • Group A (1–3): Large metacentric/submetacentric. Chromosome 1 is the largest with a heterochromatic block at 1q12. Chromosome 3 is metacentric (“bow-tie”)
  • Group B (4–5): Large submetacentric. Differentiated by banding (Ch 4 is lighter; Ch 5 has a distinct dark band at 5q12)
  • Group C (6–12, X): Medium submetacentric. The most difficult group. Includes Ch 9 (secondary constriction) and Ch 10 (three distinct bands on q-arm)
  • Group D (13–15): Medium acrocentric with satellites
  • Group E (16–18): Small metacentric/submetacentric. Ch 16 has a heterochromatic block (16q12). Ch 18 is notably dark
  • Group F (19–20): Small metacentric. Ch 19 is very light (GC-rich); Ch 20 is dark
  • Group G (21–22, Y): Small acrocentric. Ch 21 is the smallest autosome. Y has a heterochromatic distal q-arm
• Abnormalities
  • Numerical: Recognition of aneuploidy (Trisomy 21, Monosomy X) or Polyploidy (Triploidy 69,XXY)
  • Structural: Identification of Asymmetry between homologs. Includes Translocations (reciprocal exchange), Deletions (terminal or interstitial loss), Inversions (flipped banding sequence), and Isochromosomes (mirror image arms)

Karyogram

• Placement and Ordering
  • Chromosomes are arranged in numerical order (1–22) followed by Sex Chromosomes (XY)
  • Homologs: Normal pairs are placed side-by-side
  • Abnormalities: Derivative/Abnormal chromosomes are placed to the right of their normal homolog to facilitate comparison (e.g., Normal 9 | Derivative 9). Unidentified markers are placed at the very end
• Orientation (ISCN Rules)
  • p-arm Up: The short arm must always face the top
  • q-arm Down: The long arm must always face the bottom
  • Acrocentrics: The satellite stalks represent the p-arm; therefore, satellites must point up
  • Alignment: Centromeres should be aligned horizontally across the row to allow for easy length comparison

Assess Band Resolution

• Definition: Resolution is determined by the degree of chromosomal condensation. Longer chromosomes show more split bands (higher resolution)
• Sentinel Chromosomes (Visual Checks)
  • Chromosome 10: In low resolution (400 bands), the q-arm has distinct but solid bands. In high resolution (550 bands), the proximal and middle dark bands split into sub-bands
  • Chromosome 16: In low resolution, the q-arm is a solid block. In high resolution, the dark band at 16q22 separates clearly from the centromeric heterochromatin
• Diagnostic Adequacy
  • 400 Bands: Sufficient for routine aneuploidy (Down syndrome) and large rearrangements (CML)
  • 550+ Bands: Required for ruling out Microdeletion Syndromes (e.g., Prader-Willi, DiGeorge). Analyzing these cases at low resolution poses a risk of false negatives

Clinical Implications

• Constitutional (Germline)
  • Abnormalities affect development and reproductive health
  • Trisomy 13/18/21: Developmental delay and congenital anomalies
  • Sex Chromosomes (Turner/Klinefelter): Infertility and endocrine issues
  • Balanced Translocations: Phenotypically normal carriers but high risk of recurrent miscarriage
• Acquired (Oncology)
  • Abnormalities are specific to the tumor and drive malignancy
  • Diagnostic: t(15;17) confirms Acute Promyelocytic Leukemia (APL)
  • Prognostic: In MDS, del(5q) indicates a favorable prognosis, while Monosomy 7 indicates a poor prognosis
• Normal Variants
  • Benign polymorphisms such as inv(9), 1qh+, or prominent satellites: must be recognized as normal to avoid misdiagnosis