34 Simultaneous Fluorescence Immunophenotyping and FISH (FICTION)
459
- Signal amplification too strong: Omit some amplification steps.
No hybridization signals
- Defective DNA probe: Test probe on freshly prepared slides. If unsuccessful, try a new probe.
- Paraformaldehyde solution too old (pHI): Replace.
- Defective detection reagents: Use another detection cascade as control.
AMCA fluorescence intensity consistently too low
- AMCA unstable: Buy new AMCA reagents. Store AMCA reagents in
50% glycerol at -20°e.
Immunophenotyping
No staining/high background staining
- Inappropriate slide preservation: Use freshly prepared slides. Preserve
slides correctly.
- Defective monoclonal primary antibody: Test antibody on different
freshly prepared slides.
- Defective secondary antibodies: If unsatisfactory immunostaining occurs on different freshly prepared slides, all antibodies have to be
tested separately. Exchange all antibodies for new ones (only one
per test).
Immunostaining is sufficient prior to FISH but less intensive after FISH
- Paraformaldehyde solution is too old. Replace.
References
Haferlach T, Winkemann M. Loffler H, et al. 1996 Blood, 87:2459-2463
Haferlach T, Winkemann M, Nickenig C. et al. 1997 Br J Haematol, 97:99-106
ScWegelberger B, Weber-Matthiesen K, Eumann S, et al 1992 Cytogenet Cell Genet.
59:10-11
Siebert R, and Weber-Matthiesen K 1997 Histochem Cell BioI, 108:391-402
Siebert R. and Schlegelberger B 1997 Rev Clin Exp Hematol, 4:41-55
Teerenhovi L. Knnutila S. Ekblom M et al. 1984 Blood, 64:1116-1122
Weber-Matthiesen K, Winkemann M, Miiller-Hermelink A. Schlegelberger B, and
Grote W 1992 J Histochem Cytochem. 40:171-175