Pattern illustration of DNA helix fish with a blue background for the CytoCell FISH product range.
  • 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
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
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
Identifier
  • Small signals
  • Weak signals
Solution
  • Always make slides fresh on same day you intend to FISH, do not age or bake
  • Check post hybridisation wash solutions — remake all SSC solutions ensuring that the wash temperatures, concentrations and pH are correct
Baked Or Aged Slides
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
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
Identifier
  • Absent signals
  • Very weak signals
Solution
  • Calibrate hotplate/hybridiser to ensure that the correct denaturation temperature is achieved
  • 75°C for 2 mins is recommended to ensure sufficient hybridisation
Under Denaturation
FISH tips & troubleshooting

All laboratories should validate their own protocol and analysis guidelines according to their regional guidelines. This webpage is for general haematology 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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