Ex situ and in situ demonstration of amyloid fibrils for confirmation of amyloidosis using transmission electron microscopy.

Confirmation of extracellular amyloid deposition across various animal species and tissue types has been a long-standing challenge in veterinary diagnostic pathology. Transmission electron microscopy (TEM) has historically been used to advance the understanding of amyloid fibril morphology and confirm amyloid fibril deposition when histologic methods provide unclear results. We assessed the feasibility of utilizing TEM for routine confirmation of amyloidosis as an addition to histology. We analyzed ex situ amyloid fibrils with direct, negative-contrast TEM and in situ amyloid fibrils with aldehyde-fixed, plastic-embedding TEM to confirm amyloidosis in a variety of cases in which amorphous extracellular amyloid deposits had been identified by H&E and Congo red staining. We compared the 2 TEM methods and documented amyloid fibril morphology and morphometry in 7 species (goat, guinea pig, kudu, fox, sheep, flamingo, and duck). Ex situ fibrils had helical morphology and widths of 15-18 nm across all species. Fibril crossover distances had more interspecies variation of 60-130 nm, and species could be grouped based on pitch (twist size). Twisting patterns of in situ fibrils could not be visualized, but in situ widths of 10 nm were measured across all species. In 4 different chicken cases, fibrils differing morphologically from amyloid were consistently detected via both TEM methods, suggesting the possibility of a non-amyloid deposit that is commonly diagnosed as amyloidosis based on its histologic appearance. When available, we recommend routine confirmation of amyloid fibril deposition by TEM.