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Cytogenetics

Positive & Negative Controls

Quality Control (QC) in Fluorescence In Situ Hybridization (FISH) is the daily mechanism that ensures the validity of every test run. Unlike Validation (a one-time setup), QC is performed concurrently with every batch of patient samples. The principle is simple: if the controls fail, the patient results cannot be trusted, regardless of how good they look. The laboratory utilizes both Internal and External controls to monitor assay performance

Negative Controls (Specificity Check)

A negative control demonstrates that the probe does not produce a signal in the absence of the target. However, in FISH, “negative” has a nuanced meaning because the probe always binds to the chromosome (it’s DNA)

  • Internal Control Probe (within the patient cell)
    • Most diagnostic FISH probes (e.g., HER2) are dual-color, containing a Target: probe and a Control (CEP) probe
    • The Negative Check: The Control probe (e.g., CEP17) serves as the internal negative control for the hybridization conditions. If the HER2 signal is absent (deleted), the presence of the bright CEP17 signal proves that the hybridization worked and the lack of HER2 is a true deletion, not a technical failure (e.g., failed denaturation)
  • Biological Negative Control (Normal Samples)
    • A sample known to be normal for the target locus (e.g., a normal male blood sample run alongside a batch of leukemia marrows)
    • Purpose: To monitor the False Positive Rate (Background) for that specific day’s run
    • Scenario: If the laboratory scientist accidentally washes the slides at too low a temperature (low stringency), the background noise will spike. The patient sample might look abnormal, but the Normal Control will also look abnormal (high background). This alerts the laboratory scientist to reject the run

Positive Controls (Sensitivity Check)

A positive control demonstrates that the probe is capable of detecting the target when it is present

  • External Positive Control (Known Abnormal)
    • A sample (cell line or previously tested patient) known to carry the specific abnormality (e.g., a cell line with Trisomy 8)
    • Purpose: To verify that the probe can detect the abnormality and that the signal intensity is sufficient
    • Usage: While ideal, running a positive control for every specific probe (e.g., rare translocations) in every run is logistically impossible and cost-prohibitive
    • Alternative: Labs often rotate positive controls or rely on the Internal Normal cells (see below)
  • Internal Positive Control (Normal Cells within Patient)
    • In almost every patient sample, there are normal cells mixed in with the tumor cells (e.g., T-lymphocytes in a Myeloma sample, or stromal cells in a Breast Cancer section)
    • The Check: These normal cells should show the expected normal signal pattern (e.g., 2 Red, 2 Green)
    • Significance: If the normal internal cells show no signal, the assay failed. If the normal cells show 2 signals, the assay worked. This serves as a built-in positive control for the hybridization efficiency

Batch Controls (Process Control)

For every batch of FISH slides processed (e.g., a tray of 12 slides run on the ThermoBrite), at least one control slide must be included to verify the physical conditions (Temperature, Reagents, Wash)

  • The “Batch Normal”: A normal male peripheral blood slide is commonly included in every run
    • Verification: The laboratory scientist checks this slide first
      • Are the signals bright? (Probe quality)
      • Is the background low? (Wash quality)
      • Is the specific pattern (XY) correct? (Sex verification)
    • Failure Rule: If the Batch Control fails (e.g., no signal), the entire batch of patient slides is invalid and must be repeated. You cannot “cherry pick” readable slides from a failed run

Control of Reagents (Probe Checks)

  • Probe Testing: Every new shipment or lot number of probe is tested on a control slide before being released for patient use
  • Daily Temperature Checks: The temperature of the water baths, incubators, and hybridizers must be recorded daily. A drift of \(1^\circ\text{C}\) in the stringency wash can cause a run to fail (False Positives from low temp, False Negatives from high temp)

Troubleshooting Control Failures

  • Scenario: Patient shows “Deletion of TP53,” but the Internal Control (CEP17) on the same cell is also invisible
    • Conclusion: Invalid.: This is not a deletion; this is a hybridization failure (no signal)
  • Scenario: Patient shows “Amplification,” but the Normal Batch Control also shows high background/extra signals
    • Conclusion: Invalid.: The run had low stringency (under-washed). The “amplification” is likely just background noise. Repeat the run