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Cytogenetics

Capture Cell Images

In modern cytogenetics, the microscope is only half the tool; the imaging system is the other. Because fluorescent signals fade (photobleach) rapidly and the slides themselves degrade over time, the Digital Image becomes the permanent medical record of the analysis. Capturing a representative image is not merely “taking a picture”; it is a documentation step that serves as the proof of diagnosis, the basis for peer review, and the primary artifact for future audits

The Anatomy of a FISH Image

A final FISH image is a composite (overlay) constructed from multiple separate monochromatic exposures. The camera does not “see” color; it sees intensity (greyscale)

  1. The Channels (Filters): The system takes a series of black-and-white photos through different optical filters
    • Blue Channel (DAPI): Captures the nucleus/chromosomes
    • Green Channel (FITC/SpectrumGreen): Captures the green probe signals
    • Red Channel (Texas Red/SpectrumOrange): Captures the red probe signals
  2. The Merge: The software assigns artificial colors (pseudocolors) to these greyscale images (Blue to DAPI, Green to FITC, Red to Texas Red) and stacks them on top of each other
  3. The Result: A single composite image showing a blue nucleus with distinct red and green dots
    • Significance: If the alignment (registration) of these layers is slightly off (pixel shift), a Red and Green signal that are actually physically separated might look like they are overlapping (False Fusion). Precise optical alignment is critical

Criteria for a “Representative” Image

The laboratory scientist cannot simply photograph the first cell they see. The image must accurately reflect the final reported result and demonstrate that the technical quality was sufficient for diagnosis

  • Documentation of Abnormality
    • If the case is Positive: (Abnormal), the image must show the specific abnormal pattern reported (e.g., a cell clearly showing 1 Red, 1 Green, and 2 Fusions for BCR:ABL1)
    • Ideally, the image should capture a field with multiple cells: showing the abnormality to prove it is a clonal event, not a single-cell artifact
  • Documentation of Normality
    • If the case is Negative: (Normal), the image must show a clean, unambiguous normal pattern (e.g., 2 Red, 2 Green) to prove the negative result was due to normal genetics, not technical failure (e.g., “no signal”)
  • Technical Quality Indicators
    • Focus: Both the DAPI (nucleus) and the probe signals must be in sharp focus. Since signals sit on different focal planes (Z-axis) within the 3D nucleus, this often requires “Z-stacking” (taking images at different depths and flattening them) to get everything sharp
    • Background: The background should be black. An image with high green haze or glowing debris suggests a “noisy” slide, undermining confidence in the result
    • Signal Balance: The Red and Green signals should be of roughly equal brightness. If the Red is blindingly bright and the Green is barely visible, the image (and the assay) is unbalanced

Archival & Liability

  • The “Permanent Record”: FISH slides are temporary. The fluorescence fades within weeks. The captured image is the only thing that remains for review 5 or 10 years later
  • Audit Trail: Accreditation bodies (CAP/CLIA) require that the image be stored in a way that is retrievable and unalterable
    • Annotation: The image must be labeled with the Patient ID, Probe Name, and Date
    • No “Photoshop”: While adjusting brightness/contrast is acceptable to improve visibility, removing or adding signals: digitally is fraudulent. Most clinical software tracks all edits in a hidden log

Automated Imaging Systems

Many labs now use automated scanners (e.g., BioView, MetaSystems) that scan the entire slide and present a gallery of cells to the laboratory scientist on a screen

  • Selection: The laboratory scientist selects the “best” cells from this gallery for the final report
  • Verification: The laboratory scientist must verify that the software didn’t make a mistake (e.g., identifying a piece of dirt as a signal). The human eye is the final filter
  • Relocation: If the digital image is ambiguous (e.g., “Is that a fusion or just overlap?”), the system allows the laboratory scientist to click the cell and the motorized microscope stage will drive back to that exact physical location on the glass slide for live manual inspection through the eyepieces. This “Relocation” capability is a mandatory QC step for ambiguous signals