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CHAPTER 4: Immunohistochemistry
a novel hapten (3-hydroxy-2-quinoxaline; HQ) with tyramide amplification, was developed (Nitta 2013). With this HQ-tyramide IHC detection system, all tumor cells were positive, despite variations of intensity, suggesting that heterogeneous staining with some methods is a matter of a detection threshold. Furthermore, the hypothesis was confirmed using Regular Polymer Method Linker-Polymer Method the gene-protein assay that combined the highly sensitive IHC with a bright-field breakapart ISH.
Evaluation of Staining
The postanalytic phase starts with microscopic evaluation of the stained slide. In NSCLC, ALK Polymer with Primary antibody staining is cytoplasmic; it may secondary antibodies !"#$%"&'%()*+,&' Antigens have a granular character and, Linker in some cases, there may be membrane accentuation. The Figure 5. Schemes of regular polymer and linker-polymer methods. assessment of staining intensity is subjective, but the use of successive microscope objective lenses with inherent related spatial resolution is a physical aid in establishing the intensity level, as first applied to HER2 testing (Ruschoff 2012). The use of this approach may lead to more uniformity in intensity scoring. Strong staining (3+) is clearly visible with use of a x2 or x4 microscope objective lens; moderate staining (2+) requires a x10 or x20 objective lens to be seen clearly; and weak staining (1+) can be seen only with a x40 objective lens. The classic H-score is derived by multiplying the percentage of tumors that stain positively by the intensity (0, 1, 2, or 3), giving a range of 0 to 300. This approach takes greater account of the heterogeneity of the staining. Different criteria for ALK-positive and ALKnegative results on IHC have been applied in different studies. Some 2+ authors have scored the 3+ intensity from 1+ to 3+ (Figure 6), with an ambiguous threshold around 1+ or 2+ ; this scoring approach seems to be mainly related to the amplification system used and the background 1+ 0 observed with some antibodies (Mino-Kenudson Figure 6. An example of ALK IHC scores ranging from 0 to 3+.