Light and electron microscopy immunohistochemical characterization of amyloid deposits

Abstract

The present study reports our optimized fixation and processing methods for the light and electron microscopic immunohistochemical characterization of tissue amyloid. The study involved a series of 25 abdominal fat aspirates and of 11 tissue biopsy samples from different organs from 36 patients. We tested a short fixation and processing method and a series of enzymatic, acidic and physical (microwave oven) pretreatments on paraffin slides for light microscopy, Congo red stain and immunohistochemistry. Microwave antigen retrieval provided the highest enhancement for both Congo red stain and immunohistochemical reactions with all tested antibodies; formic acid also gave good results, but tissue morphology may have been altered. Immunoelectron microscopy provided evidence of fibril accumulation and immunoreactivity in both mixed (double immunoreactivity) and non-mixed forms, along with information on the relative amount of immunoreaction for the two components in mixed amyloid deposits; the technique also yielded details on spatial fibril arrangement, which differs, at least for β2-microglobulin and TTR, from that of AL amyloid deposits.In our experience, light and electron microscopic immunocharacterization of amyloid reliably identifies the nature of amyloid proteins, which may not necessarily reflect laboratory (serum or urine), and clinical data. In these last conditions and in mixed amyloidosis, ultrastructural immunocytochemical study is an essential tool for complete and definite amyloid fibril characterization.