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Cytogenetics

Select, Count, & Analyze

The core diagnostic activity in cytogenetics is the microscopic evaluation of metaphase chromosomes. Because the cell culture and harvest processes produce thousands of mitotic cells of varying quality, the laboratory scientist must exercise professional judgment to select the most representative and technically adequate cells for analysis. This process follows a hierarchy of rigor: Selection (finding the cell), Counting (determining the modal number), and Analysis (evaluating banding patterns for structural defects)

Selection Criteria: Identifying Suitable Metaphases

Not all metaphases are analyzable. The laboratory scientist scans the slide using low power (10x) to identify candidates and then switches to high power (100x Oil) to verify quality. The selection process must balance the need for high-quality banding with the need for a random, unbiased sample of the patient’s cell population

  • Spreading (dispersion)
    • Optimal: Chromosomes should be well-separated with no overlaps, arranged in a circular area. The “metaphase plate” should appear intact
    • Under-spread: Chromosomes are tightly clumped or overlapping (the “ball of yarn”). These are unanalyzable because bands are obscured
    • Over-spread: Chromosomes are scattered too widely. This creates a risk that chromosomes from this cell have drifted away (hypodiploidy) or chromosomes from a neighbor cell have drifted in (hyperdiploidy)
  • Banding Quality (Resolution)
    • The degree of chromosomal condensation determines resolution. Longer chromosomes have more visible bands
    • Routine (400 bands): Sufficient for numerical abnormalities (aneuploidy) and large translocations
    • High Resolution (550+ bands): Required for detecting subtle microdeletions or complex rearrangements. Cells with short, “stubby,” and dark chromosomes are rejected for structural analysis
  • Staining Quality
    • Chromosomes should show crisp, high-contrast G-banding (dark grey bands vs. white bands)
    • Over-trypsinized: Chromosomes look “fuzzy,” “ghost-like,” or melted
    • Under-trypsinized: Chromosomes are solid black with no distinct banding pattern

The Counting Protocol

Counting is the process of determining the total number of chromosomes per cell to detect Aneuploidy (Gains or Losses). In a standard assay (e.g., peripheral blood or bone marrow), the standard protocol typically requires counting 20 cells

  • Methodology
    • The laboratory scientist counts the chromosomes, usually grouping them mentally or physically on a printed image/screen to avoid double-counting
    • If the count is normal (46), the cell is recorded as such
    • If the count is abnormal (e.g., 45 or 47), the specific missing or extra chromosome is identified roughly (e.g., “47, +C group”)
  • Handling Discrepancies
    • Random Loss: During the dropping process, cell membranes rupture. It is common for a single chromosome to “roll away,” creating a technically broken cell with 45 chromosomes. If the missing chromosome varies from cell to cell (e.g., -C in one cell, -E in another), this is attributed to Random Technical Loss and generally ignored
    • Clonal Loss: If the same chromosome is missing in multiple cells (e.g., Monosomy 7), it represents a true biological clone
  • Mosaicism Checks
    • If mosaicism (a mix of normal and abnormal cells) is suspected, the count is typically extended from 20 cells to 30 or 50 cells: to increase the statistical power of detecting a low-level population

The Analysis Protocol

Analysis is the rigorous, band-for-band evaluation of chromosomal structure. It goes beyond simple counting. In a standard 20-cell count, typically 5 cells are fully analyzed (karyotyped)

  • Band-for-Band Comparison
    • The laboratory scientist compares homologous pairs (e.g., Chromosome 1 Left vs. Chromosome 1 Right)
    • They look for Asymmetry. If one homolog is longer than the other, or if a band is in a different position, it suggests a structural rearrangement (deletion, duplication, or translocation)
  • Landmark Identification
    • Analysis relies on ISCN landmarks (centromeres and major bands). The analyst verifies that every landmark is present in its proper sequence
    • Example: In a suspected inversion, the analyst checks if the banding sequence is reversed between two landmarks
  • Resolution Requirements
    • If a subtle abnormality is suspected (e.g., Prader-Willi 15q11.2 deletion), the laboratory scientist must specifically select pro-metaphase cells (longer chromosomes) for the analysis portion, as standard metaphases may hide the defect

Defining a Clone (ISCN Criteria)

In oncology and constitutional mosaicism, a single abnormal cell is often considered a cultural artifact/technical error unless it meets the criteria for a Clone. Diagnosing a patient as “Abnormal” requires satisfying the ISCN definition of clonality:

  • Structural Abnormalities and Gains: At least two cells must show the same aberration (e.g., two cells with Trisomy 8 or two cells with t(9;22))
  • Losses (Monosomy): At least three cells must show the loss of the same chromosome (e.g., three cells with Monosomy 7)
    • Reasoning: Because random technical loss (broken cells) is common, the threshold for diagnosing a biological loss is higher to rule out artifacts

Documentation

Every cell selected for counting or analysis must be traceable

  • Coordinates: The Vernier scale coordinates (X and Y axis) of every scored cell represent the “address” of that cell. These are recorded on the worksheet so that a supervisor or director can relocate and verify the cell if needed
  • Karyogram: For the fully analyzed cells, digital images are captured, and the chromosomes are electronically cut and arranged into a Karyogram (homologous pairs aligned by size and centromere position) to serve as the permanent medical record