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  • Key
  • Problem
  • Identifier
  • Solution
Likely causes
  • High levels of red background caused by presence of erythrocytes and cellular debris on the slide.
  • The fixation time is critical and depends on the size and erythrocyte density of the smear.
Solution
  • 10-30 minutes fixation time with 3:1 methanol/glacial acetic acid is recommended.
  • Replace with fresh fixative (3:1 methanol/glacial acetic acid fix) after 10 minutes.
  • Hypotonic solutions, such as potassium chloride, can be used with smear slides to improve FISH results.
Insufficient fixation on blood smears  Image
Likely causes
  • Positively-charged slides are designed for tissue sections to ensure good adhesions; however, these can produce higher levels of green background on other cell samples.
Solution
  • Review the type of slides used and try using non-adhesive slides.
High levels of green background Image
Likely causes
  • CD138+ separated samples can produce a high volume of cytoplasm and debris which can be seen as bright green fluorescence.
Solution
  • Use <3µl cell suspension and treat with a hypotonic solution, such as potassium chloride, followed by an enzymatic digestion step using a protease.
Bright, green signals on cell separated samples Image
Likely causes
  • Caused by baking or ageing slides.
  • Could also be a result of high stringency in the post-hybridization wash.
Solution
  • Avoid baking (>45°C) or ageing slides in heated SSC solutions. This reduces signal intensity and can be time consuming.
  • Re-make 0.4xSSC ensuring that the pH=7 and the wash temperature is 72+/-1°C.
Small, weak signals Image
Likely causes
  • Bacteria may fluoresce very brightly under both the FITC (Fluorescein isothiocyanate) and Texas Red® filters, depending on the type of bacteria.
  • The level of contamination can vary depending on the length of culture (i.e 24 or 72 hours).
Solution
  • Always use fresh cell samples and reset the culture if required.
  • Ensure aseptic techniques are correctly followed when culturing cells to minimize risk of contamination.
  • Regularly refresh your solutions, including reagent bottles and coplin jars.
Bacterial contamination Image
Identifier

 

Example of normal breakapart signal pattern compared to abnormal signal pattern:

In some breakapart probes, the red and green signals may not be tightly co-localized in all cells, especially when chromatin is extended in interphase. It is therefore important to determine the cut-off levels for a normal result1.

Solution

 

Scoring recommendations1:

  • Analyse only intact nuclei, not overlapped or crowded nuclei or nuclei covered by cytoplasmic debris or high degree of auto-fluorescence.
  • Signal intensity may vary, even within a single nucleus. In such cases, use single filters and/or adjust the focal plane.

 

1. Analysis and scoring guidelines recommended by The AGT Cytogenetics Laboratory Manual (fourth edition).

Identifier

 

Example of normal translocation signal pattern compared to abnormal signal pattern:

The dual-fusion translocation probes span the breakpoints of interest. The abnormal pattern is rarely seen as an artefact of chromosome overlap in interphase1. In an abnormal cell, this produces two fusion signals (co-localized red and green signals).

Solution

 

Scoring recommendations1:

  • Analyse only intact nuclei, not overlapped or crowded nuclei or nuclei covered by cytoplasmic debris or high degree of auto-fluorescence.
  • Signal intensity may vary, even within a single nucleus. In such cases, use single filters and/or adjust the focal plane.

 

1. Analysis and scoring guidelines recommended by The AGT Cytogenetics Laboratory Manual (fourth edition).

FISH tips & troubleshooting
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