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Cytogenetics

Document Analysis

In clinical cytogenetics, the analysis is not complete until it is meticulously documented. The physical or digital record of the microscopic examination serves as the primary data source for the final diagnosis, the legal record of the work performed, and the basis for Quality Control (QC) review. “If it is not written down, it did not happen.” Documentation encompasses recording microscope coordinates, chromosome counts, sex chromosome composition, band level resolution, and the standardized nomenclature (ISCN)

The Bench Worksheet (The Primary Record)

The bench worksheet (whether paper or electronic) is the official log of the laboratory scientist ’s observation. It must be created during the analysis, not transcribed from memory later. This document ensures traceability and reproducibility

  • Mandatory Identifiers
    • Patient Name and Medical Record Number (MRN)
    • Laboratory Accession Number
    • Date of Analysis
    • Laboratory scientist Initials
    • Slide Identification: Which specific slide (e.g., Slide A or B) was used
    • Microscope ID: The specific identifier of the microscope or workstation used (critical for relocating cells)
  • Recording Conventions
    • Data must be recorded in permanent ink (if paper)
    • Errors must be corrected with a single strikethrough, initialed, and dated (no whiteout)
    • In electronic systems, an audit trail automatically tracks changes

Recording Microscope Coordinates (Relocation)

A fundamental requirement of cytogenetic documentation is the ability to relocate every analyzed metaphase. This allows a supervisor or director to verify the finding (QC) or to re-examine a cell if a subtle abnormality is suspected later

  • Vernier Scale Coordinates
    • Most manual microscopes use a stage with a Vernier scale (X and Y axes). The laboratory scientist must record the X (horizontal) and Y (vertical) numbers for every cell counted
    • Example: \(114.5 / 32.1\)
  • England Finder
    • To allow a cell found on “Microscope A” to be found on “Microscope B,” labs may use an England Finder (a master slide with a gridded coordinate system) to convert the stage coordinates
  • Automated Scanning Systems
    • Modern labs use automated slide scanners (e.g., Metafer, GenASIs). In these systems, the “documentation” consists of a digital gallery. The system records the precise stage coordinates automatically. The laboratory scientist “documents” the analysis by clicking to accept or reject a cell in the software gallery

Documenting the Count & Sex

For every case, a specific number of metaphases must be counted to determine the modal number and constitution. Standard protocol typically requires counting 20 cells for constitutional cases and 20 to 30 cells for oncology cases

  • The Count
    • The total number of chromosomes in the cell is recorded (e.g., 45, 46, 47)
    • Laboratory scientistsoften use a shorthand “tic sheet” or grid on the worksheet to tally counts as they go
  • Sex Chromosome Composition
    • For every counted cell, the sex chromosomes must be identified and recorded (e.g., XX, XY, X, XXY)
    • This serves as an internal check against the patient’s stated gender and indicates potential mix-ups or sex-chromosome aneuploidy (e.g., Turner Syndrome)
  • Discrepancy Documentation
    • If a cell has a non-modal count (e.g., one cell has 45 chromosomes while the others have 46), it must be documented
    • Random Loss: If the missing chromosome is different in each hypodiploid cell (e.g., -4 in one cell, -21 in another), it is documented as random loss (often technical artifact)
    • Clonal Loss: If the same chromosome is missing in at least 3 cells, it represents a clone and must be fully documented

Documenting Structural Analysis (The Karyotype)

While 20 cells are counted, a subset (typically 5 cells) undergoes “Full Analysis.” This involves a band-for-band verification of every chromosome pair to detect structural abnormalities (deletions, inversions, translocations)

  • Slide/Cell Selection
    • The worksheet must indicate which of the 20 counted cells were fully analyzed. These should be the cells with the best spreading and highest band resolution
  • Band Level Estimation
    • The resolution of the preparation must be estimated and recorded for the analyzed cells
    • Standard: Constitutional studies usually require 550 band level. Bone marrow studies may accept 400 band level
    • The laboratory scientist compares the observed chromosomes to an idiogram (standardized drawing) to assign the level (e.g., “Analyzed at 550 bphs”)
  • Sketching/Annotation
    • On paper worksheets, laboratory scientist s may sketch a specific abnormal chromosome (e.g., a derivative 9) next to the cell coordinates to illustrate the breakpoint
    • On digital systems, digital annotations (arrows, circles) are placed directly on the image file

Digital Imaging & Karyograms

The Karyogram (the aligned image of the chromosomes) is the final visual document of the analysis

  • Capture Requirement
    • Regulations (CAP/CLIA) usually require a minimum of 2 karyograms: to be prepared and stored for normal cases, and more for abnormal cases (to demonstrate the abnormality in multiple cells)
  • Image Integrity
    • The raw image file and the processed karyogram must be saved
    • Software tools used to straighten or overlap chromosomes must be used carefully so as not to create artificial deletions or hide real ones. The documentation system preserves the “original” vs. “enhanced” image

Reporting via ISCN

The final step of documenting the analysis is translating the visual findings into the text-based International System for Human Cytogenomic Nomenclature (ISCN). This string summarizes the entire analysis

  • String Components
    • Modal Number: (e.g., 46)
    • Sex Chromosomes: (e.g., XX)
    • Abnormalities: Listed in alphanumeric order (e.g., t(9;22)(q34;q11.2))
  • Contextual Documentation
    • The ISCN string on the worksheet must match the findings in the cell count log
    • Example: If the worksheet shows 10 cells with 46,XY and 10 cells with 47,XY,+8, the ISCN must document the mosaicism: mos 47,XY,+8[10]/46,XY[10]

Managing Abnormal Results (Protocol for Documentation)

When an abnormality is found, the documentation requirements increase to prove the validity of the finding

  • Expansion of Count
    • If a suspected abnormality is seen, the protocol usually requires analyzing additional cells (e.g., extending the count from 20 to 30 or 50) to determine the size of the clone. This extension must be logged
  • Confirmation
    • Documenting that a second laboratory scientist or supervisor reviewed the abnormal cell (double-check) is standard practice before the report is released