ther a material is amyloid or something else. Drury and Wallington describe amyloid as being eosinophil, moderately PAS positive, variable with a Van Gieson and blue or green with a Masson type trichrome, disagreeing with Wolman, who said that amyloid is not stained like collagen (i.e. blue or green) in a trichrome. Others require different staining results for a positive identification, such as a positive staining with congo red in conjunction with the DMAB nitrite test for tryptophane, or crystal violet metachromasia in conjunction with green birefringence after congo red staining.
These differences of opinion as to what constitutes definitive staining results for amyloid are rooted in the inherent variability of the material itself. Many examples of amyloid will meet the stated criteria, but it is not by any means unusual for particular examples to stain according to a different pattern. For example, during the author's career he has had at least three examples in which the staining results caused confusion. In one, the congo red stain (Highman's) was positive, but sirius red (Llewellyn's) failed to stain it. In another the staining was reversed, with the congo red failing to stain with a positive sirius red. In the third, both congo red and sirius red were negative, as was crystal violet metachromasia, but Vassar and Culling's thioflavine T was weakly positive. The material was later confirmed to be amyloid by electron microscopy.
The point is that amyloid is a variable material and the staining may be variable as well. Particular examples of amyloid may stain with a particular method or not, and failure to stain may not mean that a particular material is definitively not amyloid. In practice, each laboratory should make available several staining methods, so that negative results which were expected to demonstrate amyloid can be checked with alternate procedures, or, as a last resort, sent for electron microscopy. I suggest either congo red or sirius red as the initial staining method, followed by whichever 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 