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chapter 3: Fluorescence in situ Hybridization
choose any of these protocols, as long as conditions are properly adjusted to the characteristics of the specimen.
Quality Assessment of the Hybridized Specimen and Selection of Scorable Cells
It is essential that the quality of tissue morphology and signal intensity be rigorously assessed before a specimen is accepted for analysis. Specimens are optimal for analyses when they exhibit excellent morphology and signal intensity with very low background noise (Figures 2 and 3). Specimens with evidence of chromatin overdigestion or poor probe penetration are not acceptable and must be retested after troubleshooting technical conditions. For example, specimens are not acceptable when the pretreatment of tissue is insufficient or excessive (Figure 4) or when technical sectioning artifacts that generate overlapped nuclei with stringy signals make it impossible to measure the separation between red and green (Figure 5). ALK rearrangement appears homoA geneously distributed in the tumor, reflecting its critical oncogenic role (Camidge 2010). Therefore, it is not necessary to select a specific tumor area based on morphology or immunoprofile, and scoring in several different tumor areas is recommended. Scoring must be done on the well-preserved C B nonoverlapping tumor cells that have
Figure 2. Microscopic fields of ALK-negative lung tumors.
D
A
B
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Figure 3. Microscopic fields of ALK-positive lung tumors, showing predominantly the split 3'-5' pattern (3A-3C) and the isolated 3' pattern (3D, 3E).