Immunohistochemical typing of amyloid in fixed paraffin-embedded samples by an automatic procedure: Comparison with immunofluorescence data on fresh-frozen tissue.

Antonella Barreca,Mauro G Papotti,Luigi Biancone,Angelo Attanasio,Paola Cassoni,Manuela Giarin,Alberto Nocifora,Emanuel Bottasso,Francesca Veneziano,Dario Roccatello,Nadia Martinetti,Giulia Benevolo,Vincenzo L’Imperio

doi:10.1371/journal.pone.0256306

Abstract

Amyloidosis comprises a spectrum of disorders characterized by the extracellular deposition of amorphous material, originating from an abnormal serum protein. The typing of amyloid into its many variants represents a pivotal step for a correct patient management. Several methods are currently used, including mass spectrometry, immunofluorescence, immunohistochemistry, and immunogold labeling. The aim of the present study was to investigate the accuracy and reliability of immunohistochemistry by means of a recently developed amyloid antibody panel applicable on fixed paraffin-embedded tissues in an automated platform. Patients with clinically and pathologically proven amyloidosis were divided into two cohorts: a pilot one, which included selected amyloidosis cases from 2009 to 2018, and a retrospective one (comprising all consecutive amyloidosis cases analyzed between November 2018 and May 2020). The above-referred panel of antibodies for amyloid classification was tested in all cases using an automated immunohistochemistry platform. When fresh-frozen material was available, immunofluorescence was also performed. Among 130 patients, a total of 143 samples from different organs was investigated. They corresponded to 51 patients from the pilot cohort and 79 ones from the retrospective cohort. In 82 cases (63%), fresh-frozen tissue was tested by immunofluorescence, serving to define amyloid subtype only in 30 of them (36.6%). On the contrary, the automated immunohistochemistry procedure using the above-referred new antibodies allowed to establish the amyloid type in all 130 cases (100%). These included: ALλ (n = 60, 46.2%), ATTR (n = 29, 22.3%), AA (n = 19, 14.6%), ALκ (n = 18, 13.8%), ALys (n = 2, 1.5%), and Aβ2M amyloidosis (n = 2, 1.5%). The present immunohistochemistry antibody panel represents a sensitive, reliable, fast, and low-cost method for amyloid typing. Since immunohistochemistry is available in most pathology laboratories, it may become the new gold standard for amyloidosis classification, either used alone or combined with mass spectrometry in selected cases.

Highlights

Amyloidosis encompasses a wide range of systemic or localized disorders in humans caused by the extracellular deposition of insoluble fibrils under the form of “amyloid”, in some cases leading to organ dysfunction
Other amyloidosis variants are related to chronic inflammatory diseases, like serum amyloid A protein amyloidosis (AA), or have a genetic background, being caused by hereditary or sporadic mutations in different genes encoding for soluble proteins, such as transthyretin amyloidosis (ATTR), fibrinogen amyloidosis (AFib), apolipoprotein A1 amyloidosis (AApo A1), gelsolin amyloidosis (AGel), cystatin C amyloidosis (ACys), and lysozyme amyloidosis (ALys), among others
Thirty-five patients were males and 16 were females. This cohort was constituted by cases that still had an unclassified form of amyloidosis (40 patients, 78.4%), whereas in the remaining 11 patients (21.6%), amyloid typing had already been performed by means of IF alone and such results were compared to those obtained by IHC

Summary

Introduction

Amyloidosis encompasses a wide range of systemic or localized disorders in humans caused by the extracellular deposition of insoluble fibrils under the form of “amyloid”, in some cases leading to organ dysfunction. These deposits form homogeneous eosinophilic agglomerates staining positive for Congo red dye and showing apple-green birefringence under polarized light because of their characteristic β-pleated sheet conformation [1]. Congo red staining on amyloid deposits shows bright red appearance under ultraviolet light on fluorescent microscopy (the so-called “Congo red fluorescence”, CRF) [2]. Other amyloidosis variants are related to chronic inflammatory diseases, like serum amyloid A protein amyloidosis (AA), or have a genetic background, being caused by hereditary or sporadic mutations in different genes encoding for soluble proteins, such as transthyretin amyloidosis (ATTR), fibrinogen amyloidosis (AFib), apolipoprotein A1 amyloidosis (AApo A1), gelsolin amyloidosis (AGel), cystatin C amyloidosis (ACys), and lysozyme amyloidosis (ALys), among others

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