of those two was not used initially, along with thioflavine T. In addition, crystal violet metachromasia, standard toluidine blue, alcian blue and the DMAB nitrite test should also be available for those occasions when these initial methods are unexpectedly unsuccessful.
Both amyloid and fibrin have elevated levels of tryptophan, so both materials will be stained by the DMAB nitrite test. This means that the technique cannot be used as a primary staining method for amyloid, since positively stained hyaline could be either amyloid or fibrin, but it can be used as a confirmatory stain when other staining methods are positive.
Amyloid often stains with alcian blue, thus indicating the presence of acid mucopolysaccharide. Both sulphated and non-sulphated types have been suggested, and it is quite possible that both may be present. Although amyloid is metachromatic with toluidine blue and crystal violet, the metachromasia may be dependent on a feature of the material other than the mucopolysaccharide content (i.e. its structure), and the metachromasia is therefore an unreliable indicator of sulphated acid mucopolysaccharide content in this particular case. On the other hand, the assumption sometimes made that alcian blue staining is merely a reflection of the mucopolysaccharides in the ground substance is unlikely, since amyloid is stained more darkly than the ground substance, indicating that it has a higher content.
Lendrum, Slidders and Fraser noted that alcian blue staining is more intense in fresh deposits of amyloid, suggesting that the alcian blue staining material may be being blocked or removed as the amyloid ages. In the same paper, these authors also comment on the variably of amyloid staining with a PAS. They observed that amyloid is initially PAS negative, then becomes positive, then once again becomes negative as the deposit ages. They used this progression and the variability with alcian blue to support their contention that amyloid undergoes changes in chemical composition and hence in staining as the deposit ages. However, this variability of alcian blue and PAS staining once again emphasises the inherent difficulty in recommending a single staining method to identify the material.
As previously noted, the only definitive way to identify amyloid is by its appearance with an electron microscope. Cohen & Calkins and Gueft & Ghidoni described it as extracellular fibres, often in small bundles. Each fibril is 75 Angstroms wide and composed of three linear bands, each 25 Angstroms wide. The fibrils are beaded at 100 Angstrom intervals.
The selectivity of many methods for amyloid appears to depend on two things: hydrogen bonding and the way that its amino acids fold. In most proteins amino acids are folded in a helical manner, rotating around a central point, then deforming the coil as various exposed groups come in contact and influence each other, whether by ionic or non-ionic forces. Amyloid has a different structure, called a beta pleated sheet (β-pleated sheet), which is composed of regular folds like a concertina. This is similar to a sheet of paper folded longitudinally in alternate directions making a zig-zag pattern when viewed on end (see to the right).The dye is often positioned within the creases of the folds, producing a regular alignment of dye molecules (shown in red), much like a crystal would have. This arrangement is referred to as a pseudocrystal, because it causes amyloid to exhibit some of the optical properties of a crystal, such as dichroism and dichroic birefringence. It may also explain the metachromasia with crystal violet.
What causes amyloid
It used to be thought that amyloid was caused by some plasma constituent extruding into the tissues, much like fibrin is formed from fibrinogen in the plasma. Then it was considered to be the results of the reactions between antigens and antibodies which, for some reason, were deposited in the tissues rather than being removed. From the links in the chart below, it appears there may be several causes for the deposition of abnormal proteins as amyloid