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CHAPTER 4: Immunohistochemistry
of 10 times more than the volume 20 of the specimen) of 10% neutral buffered formalin and embedded in paraffin (? FPE). Fixation F must be done as soon as possi10 ble to avoid cold ischemia effects. Fixation times of less than 6 hours are not recommended because conventional staining as well as IHC can be adversely affected. 0 1 10 80 Antigen preservation for IHC is Epitope Concentration epitope dependent, and some epitopes may not be hampered Figure 3. The effect of a low (blue circles) and a high (red triangles) signal by fixation times of as long as 120 enhancement system in IHC. Note that a low epitope concentration may hours. For practical purposes, a become positive with a high signal enhancement system, (thin arrow), but fixation interval of 6 to 48 hours is negative with a low signal enhancement system. In addition, a higher intensity plateau is reached; once positive, a higher epitope concentration will not lead to recommended for all specimens. darker staining. With ALK IHC, the epitope concentration is higher (thick arrow) After paraffin embedding, the in lymphomas than in NSCLC (thin arrow), and a low signal enhancement system may suffice. In NSCLC, a high-affinity antibody with high concentration and tumor tissue is stable and pre- high enhancement is necessary. Epitope concentration has a logarithmic scale served against oxidative damages and a linear scale for intensity (A.U. = arbitrary units). Adapted from Prinsen CF, or other degenerative effects. Klaassen CH, Thunnissen FB. Microarray as a model for quantitative visualization chemistry. Appl Immunohistochem Mol Morphol. 2003;11:168-173. However, once 3 to 4-µm thick slides are cut from the FFPE block, the storage time of these sections mounted on glass microscope slides at room temperature is limited to a maximum of 6 weeks. For storage at colder temperatures, the slides remain adequate for a longer period of time. However, slides of tissue sections that were prepared more than 6 weeks earlier should be interpreted very carefully, as they may present false-negative results.
Immunostaining
For the analytic procedure, i.e., the actual ALK IHC testing, several issues need to be controlled and optimized: epitope retrieval, type and concentration of the antibody, incubation time, incubation temperature, and amplification. A single uniform technique, or comparator, has not been evaluated in the studies on ALK IHC in NSCLC. Instead, the type or source of antibodies, the process of antigen retrieval and antibody detection, and the amplification techniques have varied substantially (Table 2). When different antibodies were compared head-to-head, D5F3 (Cell Signaling Technology) and 5A4 (Novocastra) with the ADVANCE system (Dako) appeared to be both more sensitive and more specific than the ALK1 antibody (Dako) (Figure 4) (Conklin 2013). (See Chapter 6 for a discussion of various platforms.) Commercial ALK IHC kits are currently in development and validation. The sensitivity for detecting the ALK fusion protein has been enhanced by using several signal amplification steps (Figure 5) (Rodig 2009, Sakairi 2010, McLeer-Florin 2012). Standardization may also be obtained with automation of IHC using a commercially available IHC kit. Standardization with automated stainers may lead to more consistent staining, occasionally at the expense of a higher concentration of the primary antibody. Recently, a highly sensitive detection method that combined
Intensity (A.U.)