Thicker sections, 4–8 μm, are helpful in the detection of small deposits for two reasons: (1) in thicker sections, amyloid deposits are already visible by bright field microscopy as salmon-pink-stained areas, and (2) in thicker sections, sample error is less likely to occur. This is particularly relevant in the early stages of amyloidosis, where small deposits of amyloid may not be present in each section. As discussed earlier in this book, by Linke, the pathognomonic Congo red birefringence seen under polarized light can be demonstrated only in sections that are cut within standard thicknesses. In particular, when sections are cut with a thickness of less than 1 μm, the anisotropy changes from apple green to bluish white. While sections that are too thin may fail to exhibit birefringence, in the author’s own experience, the sections typically used in renal pathology are quite adequate. The thinnest paraffin sections used in renal pathology are at least 2 μm thick, with 3 μm being cut most frequently. In the editor’s own experience, these thicknesses do not interfere with polarization, providing that appropriate equipment is used. Diagnostic birefringence can be seen, even though no salmon-pink color is apparent by bright field illumination (please see also, in this book, Chap. 14, Fig. 1C by Linke). It is also true that small deposits may be easily missed in thinner sections. In order to minimize sampling error, the author routinely stains two slides with Congo red (and more if clinically indicated), preferably obtained from different levels within the block. Again, the importance of proper optics in the evaluation of Congo red-stained slides cannot be overstressed. Please note also that specimens that are too thick may be difficult to interpret; for example, thick fat smears (please see also Chap. 14 by Linke in this book).

Sampling error is one of the most important and also one of the most common pitfalls encountered in the diagnosis of amyloid. It is defined as the apparent absence of amyloid in a tissue section from a patient with amyloidosis. As noted by Linke, elsewhere in this book, a negative diagnosis of amyloid, based on examination of a single section, can never be conclusive. Therefore, in patients with a higher level of suspicion, on both clinical as well as on pathologic grounds, but where initial slides are negative for amyloid, the editor routinely evaluates multiple Congo red-stained sections. Moreover, repeated biopsies may be needed (e.g., repeated abdominal fat biopsies) in order to conclusively establish the diagnosis. Not uncommonly, amyloid deposits may be detected later in the course of the disease, with earlier biopsies being truly negative. Sampling error may also be responsible for reports of the detection of amyloid-like fibrils by electron microscopy but with negative Congo red stain results.

The birefringence with anomalous colors, that is found when Congo red-stained amyloid is examined with polarized light and which is considered to be the most specific finding, is, however, even under optimal circumstances, less sensitive than some other stains. However, these other stains are, in turn, less specific. Despite this lesser specificity of polarization alternatives, the use of fluorochromes is particularly worthwhile due to their markedly increased sensitivity. Interestingly, Congo red itself can be used as a fluorochrome when examined under fluorescent light. This application offers the added benefit of examining the same section using two different techniques: namely, fluorescence and polarization (as shown by Linke elsewhere in this book). Among other fluorochromes, Thioflavin T and S are particularly useful in the diagnosis of amyloid (discussed in this book in Chap. 15, see also Chap. 29) [20].

The use of fluorescence for the detection of amyloid is gaining in popularity. Elimination of the “polarization shadow” phenomenon through the use of fluorescence results in a significant increase in the sensitivity of detection of small deposits. Briefly, using polarization microscopy, at any given time, only a portion of the amyloid deposit shows diagnostic orange–green–yellow birefringence—only by further rotation of the stage, other parts will become visible while, in turn, the formerly visible areas will be obscured by the “polarization shadow.” Thus, at any given time, only a portion of the amyloid is visualized (please see also the Chaps. 13 and 14 in this book). Hence, the enhanced sensitivity of Thioflavin and Congo red stains as fluorochromes for the detection of amyloid is, in part, due to visualization of the entire area containing amyloid, at the same time. The absence of “polarization shadow” makes the detection of small deposits much more sensitive (Fig. 17.3a–c).

Note particularly that different filters may be used in the Congo red fluorescence technique, including a filter for detecting fluorescein isothiocyanate (FITC) with an absorption maximum of 495 (blue) and an emission maximum of 525 nm (green), and tetramethylrhodamine isothiocyanate (TRITC) with an absorption maximum of 555 nm (green) and an emission maximum of 580 nm (red). With the former filter, amyloid deposits appear orange while, with the latter filter, deposits are red. In the editor’s own experience, the red fluorescence (TRITC) filter gives a cleaner and more-easily discernible visualization of amyloid (Fig. 17.3c, see also Chap. 28, Fig. 28.​6d). For detecting amyloid in laser microdissection techniques, a newer generation of filters is being used, i.e., the BGR filter cube; here also, red fluorescence is used preferentially. A practical approach would be to try different filters and see which gives the best results with the microscope/optics available in a given laboratory.

As noted in the chapter devoted to the Thioflavin stain, this is easy to perform, the outcome is predictable and interpretation is much easier than with the Congo red stain (see Chap. 15). The Thioflavin T stain is used extensively in the research setting and, given the advantages listed above, it should be considered as a desirable screening test for use in the clinical setting as well. As can be seen from other chapters in this book, several laboratories have used Thioflavin stains (T or S) successfully in amyloid diagnosis (please see also Chap. 29, Figs. 29.​1c, 29.​2h, i, and 29.​8c, g). For the detection of amyloid with the Thioflavin stain, different filters may also be used as shown in Chap. 29. Other issues, namely the need for a fluorescence microscope, or the fading of sections, are relatively minor. While such equipment may not be readily available in general surgical pathology laboratories, it is standard equipment in renal pathology/dermatopathology laboratories, and renal pathologists, in particular, also have the necessary experience for handling such microscopes. Also, faded sections can be restained. In sum, the thioflavin stain is a desirable option for screening purposes and is worthy of inclusion in most laboratory protocols, in particular, those that also have renal/dermathopathologists on the staff.

The combination of Congo red and immunohistochemistry on a single slide (the “overlay technique”) is discussed extensively by Linke in Chap. 14 in this book. However, while the combination of Congo red stain with immunohistochemistry results in enhanced sensitivity for amyloid detection, prior knowledge of the amyloid type is necessary. This is possible in certain clinical situations; for example, when patients with rheumatoid arthritis, or periodic fevers, are monitored for the development of AA amyloidosis, or when patients with a known mutation associated with one of the hereditary amyloidoses are monitored for development of the corresponding amyloidosis (see chapter by Linke, Chap. 14, and Refs. [15, 17]). Another approach to consider is the use of immunostain for amyloid P component, which, being invariably associated with deposits of amyloid, may allow its early detection and localization [21, 22] (see also discussion regarding amyloid P component in chapters on amyloid typing).

The concept of “amyloid signature” has been introduced in amyloid proteomics. Since several compounds, most notably amyloid P component and Apolipoprotein E, are invariably associated with all deposits of amyloid, regardless of their amyloid protein precursor type, their presence in abundant amounts, above the “baseline,” is considered indicative of the presence of amyloid deposits (please see chapters on mass spectrometry) in this part of the book.

Other stains: sulfated alcian blue (SAB), crystal violet, methyl violet, various cotton dyes (e.g., pagoda red, Sirius red), other fluorescent dyes (Phorwhite BBU), periodic acid-Schiff (PAS), and even other fabric dyes (RIT Scarlet No. 5, and RIT Cardinal Red No. 9 [yielding a bright orange color]) have been used to detect amyloid, with variable results. The presence of carbohydrate moieties in amyloid fibrils has also encouraged some investigators to use carbohydrate stains, such as alcian blue or even periodic acid-Schiff, to demonstrate amyloid deposits; however, dye uptake is variable and generally poor with these methods. SAB identifies glycosaminoglycans (GAGs) that are present in all types of amyloid, similar to amyloid P component. Likewise, metachromatic stains, such as crystal violet or methyl violet, capitalize on the carbohydrate content of amyloid fibrils, but their staining is not very specific, and the low sensitivity of these methods has caused them to fall into disfavor; these stains also fade with time. Sirius red F3B (but not Sirius red F4B) stains amyloid rose-red while the background is clear to pale pink. However, the Sirius red stain is not specific for amyloid as it also binds to all types of collagen and, for this reason, has been used as a stain for collagen.