The 10 µl volume of blood is important. Less than 10 µl makes it difficult to find cells. More than 10 µl causes background noise that makes it difficult to read the result.
It is important to add only one drop of fix 1. Fix 1 has to be added before two drops of fix 2. If fix 2 is added before fix 1 the result gets very unclear.
Fix 2 is not a critical step. 1-3 drops of fix 2 is ok.
Fixation longer than 5 min at 55 C can cause false positive results due to non-specific binding. True positive samples can lose signal.
If more than one drop of “blue” or “yellow” is added to the cover slip, the cover slip has to be discarded.
It doesn’t make a difference if “yellow” is added before “blue” to the cover slip.
Foam or air bubbles (>3) changes the ratio between the reagents. In case of foam or air bubbles the cover slip should be discarded.
The cover slip has to be flipped into the marked area on the slide. If the cover slip is placed on the white writing area it will not be in proper contact with the smear. That can cause floating cells under the microscope. It can also cause false negative results due to insufficient hybridisation.
Hybridisation > 20 min can cause non-specific binding and false positive results.
As QuickFISH is designed to provide rapid diagnostics it is always best to read the slides when they are freshly prepared.
However, the slides can be stored in the dark for several hours should this be required. If the slides are not kept in the dark and the results aren’t read within 2 hours there is a risk of false negative results or weak results as the controls will lose signal.