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FISH tips & troubleshooting

What's wrong with my hematology FISH?

The quality of your FISH results can vary depending on several factors in your protocol. Our hematology troubleshooting tool provides expert guidance on how to identify and resolve common problems to help you get the best results from your FISH probes.

Key
Problem
Identifier
Solution

Insufficient fixation of blood smears Show solutionHide 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

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Insufficient Fixation Of Blood Smears

Incorrect slides used Show solutionHide solution

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

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Incorrect Slides Used

Inadequate signals with CD138+ cells Show solutionHide solution

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

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Inadequate Signals With CD138+ Cells

Baked or aged slides Show solutionHide solution

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 the wash temperatures, concentrations and pH are correct

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Baked Or Aged Slides

Damaged or worn filters Show solutionHide solution

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

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Damaged Or Worn Filters

Light exposure Show solutionHide solution

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

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Under-denaturation Show solutionHide solution

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

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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 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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