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BRIGITTE SCHLEGELBERGER et al.
red (Cy3 mter), yellow (FITC mter) or white (AMCA filter), whereas true
Cy3 fluorescence is not visible if the AMCA filter set is used.
For the evaluation of FICTION slides the same rules have to be observed as for conventional FISH. Controls have to be performed for
each immunofluorescence as this may interfere with probe binding
and signal evaluation.
Evaluation criteria and cut-offlevels: For inter-observer comparability
the evaluation criteria have to be defmed exactly. This is particularly necessary for translocation probes, in which a colocalization of two signals
indicates a translocation. As to the organization of the nucleus in interphase cells, a spatial separation of the two colocalized signals is possible.
Thus, it has to be defined up to which distance between the signals, (calculated in relative signal size, i.e. once or twice the signal diameter) a real
colocalization is assumed.
In each laboratory, diagnostic thresholds have to be defined for each
probe. This is done by analyzing at least five control cases with a minimum
of 200 interphase nuclei each. For each slide preparation process, these
controls have to be performed independently as there are significant differences in the false positive and negative rates according to the protocols
used. The cut-off levels are usually defined as mean percentage of false
positives in these controls plus three standard deviations. Positive controls
on tumor specimens with a known karyotype have to be performed to
verify the specificity of each assay.
Evaluation of tumor specimens: For the evaluation of tumor specimens the same rules have to be applied as for the controls. At least
100 interphase nuclei have to be evaluated by the observer. Only good
quality slides and clearly analyzable cells can be counted. In cases with
low percentages of aberrant cells (in the range of the thresholds) at least
200 nuclei have to been counted. Each result should be confirmed independently by a second observer.
Troubleshooting
In situ hybridization
Too much background
- Unspecific cross-hybridization: Add human Cot-l DNA to probe and!
or coprecipitate.
- Too low hybridization stringency: Increase hybridization and!or wash
temperature. Increase formamide concentration. Decrease salt concentration. Prepare new wash solutions.
BRIGITTE SCHLEGELBERGER et al.
red (Cy3 mter), yellow (FITC mter) or white (AMCA filter), whereas true
Cy3 fluorescence is not visible if the AMCA filter set is used.
For the evaluation of FICTION slides the same rules have to be observed as for conventional FISH. Controls have to be performed for
each immunofluorescence as this may interfere with probe binding
and signal evaluation.
Evaluation criteria and cut-offlevels: For inter-observer comparability
the evaluation criteria have to be defmed exactly. This is particularly necessary for translocation probes, in which a colocalization of two signals
indicates a translocation. As to the organization of the nucleus in interphase cells, a spatial separation of the two colocalized signals is possible.
Thus, it has to be defined up to which distance between the signals, (calculated in relative signal size, i.e. once or twice the signal diameter) a real
colocalization is assumed.
In each laboratory, diagnostic thresholds have to be defined for each
probe. This is done by analyzing at least five control cases with a minimum
of 200 interphase nuclei each. For each slide preparation process, these
controls have to be performed independently as there are significant differences in the false positive and negative rates according to the protocols
used. The cut-off levels are usually defined as mean percentage of false
positives in these controls plus three standard deviations. Positive controls
on tumor specimens with a known karyotype have to be performed to
verify the specificity of each assay.
Evaluation of tumor specimens: For the evaluation of tumor specimens the same rules have to be applied as for the controls. At least
100 interphase nuclei have to be evaluated by the observer. Only good
quality slides and clearly analyzable cells can be counted. In cases with
low percentages of aberrant cells (in the range of the thresholds) at least
200 nuclei have to been counted. Each result should be confirmed independently by a second observer.
Troubleshooting
In situ hybridization
Too much background
- Unspecific cross-hybridization: Add human Cot-l DNA to probe and!
or coprecipitate.
- Too low hybridization stringency: Increase hybridization and!or wash
temperature. Increase formamide concentration. Decrease salt concentration. Prepare new wash solutions.