Background Texture
  • Key
  • Problem
  • Identifier
  • Solution
Identifier
  • High levels of red background.
Solution
  • Check under phase microscope for bright round lymphocyte cells prior to applying FISH probe.
  • Hypotonic solutions, e.g. potassium chloride can be used with smear slides to improve FISH results.
  • 10-30 minutes fixation (in the freezer) - 3:1 methanol/glacial acetic acid recommended.
  • Replace with fresh fixative after 10 minutes.
Insufficient fixation of blood smears Image
Identifier
  • High levels of green background.
Solution
  • Review the type of slides used – positively charged slides (intended for use with tissue sections) can cause issues with other cell samples. Non-adhesive/charge neutral slides are recommended.
Incorrect slides used Image
Identifier
  • Absent signals.
  • Weak signals.
  • High background levels.
Solution
  • Use <3μl cell suspension and treat with a hypotonic solution, e.g. potassium chloride during harvest of the cells.
  • Check correct and adequate fixative method used, i.e. 3:1 methanol/acetic acid
  • Follow with an enzymatic digestion step using Pepsin or protease K to remove cytoplasm and debris – 37°C for 15 mins.
Inadequate signals with CD138+ cells Image
Identifier
  • Small signals.
  • Weak signals.
Solution
  • Always make slides fresh on same day you intend to FISH, do not age or bake.
  • Check post hybridization wash solutions - remake all SSC solutions ensuring that pH=7, wash temperature is 72 +/- 1°C and concentrations are correct (2x and 0.4x).
Baked or aged slides Image
Identifier
  • Weak signals.
  • High background.
Solution
  • Check filters for damage.
  • Treat filters as consumables and replace in line with manufacturer’s guidelines – typically every 2-4 years.
Damaged or worn filters Image
Identifier
  • Non-specific binding.
  • Auto-fluorescence.
Solution
  • Reduce the level of exposure to light.
  • Cover probe and store in -20°C freezer when not in use.
  • Store washed slides in dark until analysis.
  • Aliquot probe into single use shots on arrival.
  • Consider type of lighting in lab - some are more damaging than others, e.g. LED lights with high K values.
Light exposure Image
Identifier
  • Absent signals.
  • Very weak signals.
Solution
  • Calibrate hotplate/hybridizer to ensure that the correct denaturation temperature is achieved.
  • 75°C for 2 mins is recommended to ensure sufficient hybridization.
Under-denaturation Image
FISH tips & troubleshooting

All laboratories should validate their own protocol and analysis guidelines according to their regional guidelines. This webpage is for general hematology FISH troubleshooting, following standard cytogenetic practices such as those found in the AGT Cytogenetics Laboratory Manual (2017), and is not suggesting intended uses for any CytoCell product nor should any be inferred by the reader.

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