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Table 1 Guidelines for Microscope Analysis of NSCLC tissue sections hybridized with the SpectrumOrange LSI EGFR/SpectrumGreen CEP 7 FISH probe set (Vysis/Abbott Molecular) at UCCC.

From: Stratification of non-small cell lung cancer patients for therapy with epidermal growth factor receptor inhibitors: the EGFR fluorescence in situ hybridization assay

• Examine the parallel H&E stained section to locate areas rich in tumor cells. Recognize the tumor pattern, verify the cell density in the tumor areas and the size of the tumor nuclei.
• Identify 4–5 distinct tumor areas and define tissue landmarks for them. Perform this selection with the assistance of a lung pathologist.
• Use low power objective (20× or 40×) and DAPI filter to re-find the selected tumor areas in the FISH section based on the landmarks recognized in the H&E slide. Record the location of these areas.
• Move to a high power objective (100×), change to red, green, double red/green and/or triple blue/red/green band pass filters to inspect quality of the hybridization.
   • The normal green signals (CEP 7) signals should be bright, compact (occasionally slightly stringy or diffuse) oval shapes. The red (EGFR) signals should be bright, small round shapes, commonly adjacent to a green CEP 7 signal. The green CEP7 green signal should be larger and brighter than the EGFR red signal.
   • Background should appear dark and free of fluorescence particles or haziness.
   • At least 75% of cells in the selected tumor areas should display hybridization signals not hampered by background noise for the specimen to qualify for analysis.
• Select approximately 10–20 representative nuclei for analysis in 2–3 microscope fields in each selected tumor area. Record the number of red and green signals for each individual nucleus in the FISH analysis worksheet. Select nuclei should have:
   • Not less than median diameter compared with overall tumor nuclei to reduce the effect of the nuclear truncation.
   • Unambiguous borders and objectively interpretable signals.
   • At least one signal for each target.
• Scan the focus through the entire depth of the section to ensure that all signals are identified within each nucleus.
• Score a minimum of 50 representative nuclei per specimen (or 30 cells when gene amplification is present).
• Document results capturing images of representative fields (two if the specimen is homogeneous or more if the specimen is heterogeneous).