Congo Red Staining of Amyloid: Improvements and Practical Guide for a More Precise Diagnosis of Amyloid and the Different Amyloidoses

Abstract

Congo red (CR) is the most popular dye used as a probe for diagnosing amyloidosis, a very heterogeneous group of diseases with more than 23 chemically different amyloid syndromes of men and animals, leading to more than 400 different individual diseases. Congo red binding increases the natural anisotropy of amyloid, indicating that the elongated and planar CR molecules are aligned parallel to the axis of the amyloid α fi brila and to each other, thereby revealing a structure of amyloid. This structure was established to represent fi brils of similar dimensions, although the amyloid fi brils can be composed of many unrelated proteins. This CR-induced (positive) anisotropy displaying a green color is the hallmark of all amyloids, and is therefore used in the diagnosis of amyloidosis. The specifi city of this criterion, however, is based on very stringent conditions of staining and evaluation. This review will focus on the understanding of the CR staining procedure, its mechanism and, in particular, on its recent practical improvements by increasing the sensitivity of the CR procedure, so that minute and the earliest amyloid deposits in the course of amyloidosis can now be reliably detected in patients. This enables a very early diagnosis in the course of the disease before irreversible organ damage might have occurred, and widens the options for a successful therapy. In addition, the central role of the CR diagnostic procedure and evasion of common pitfalls in arriving at a pathogenetically exact classifi cation must only be based on the chemical nature of the amyloid deposits and not on the soluble precursors of the many different amyloidoses will be highlighted. The proposed bench-tobedside algorithm will enable the physician to arrive at the exact diagnosis for therapeutic considerations. Finally, some possible future applications of CR and analogues will be presented.

Liver transplantation and combined liver-heart transplantation in patients with familial amyloid polyneuropathy: A single-center experience

“…The biopsy samples were formalin‐fixed and paraffin‐embedded. Amyloid was diagnosed in 4‐ to 6‐μm‐thick tissue sections by the specific pinkish coloration in bright light and the pathognomonic green birefringence in polarized light after modified staining with Congo red according to Puchtler et al9 Typing of the amyloid class was performed immunohistochemically with a panel of anti‐amyloid antibodies that have been documented to be reliable 10. A strong reaction to the anti–amyloidosis TTR antibody was taken as proof of the presence of TTR‐derived amyloid, whereas six other hereditary and sporadic amyloids were excluded by much weaker and more inconsistent reactions of specificity.…”

Microbial-generated amyloids and Alzheimer's disease (AD)

“…Amyloid fibrillation is initiated by self-aggregation of protein monomers-into-dimers, oligomers and fibrils, which accumulate over time, and this process is thought to result from the hydrophobic nature of the aromatic amino-acid peptides comprising the primary sequence of the amyloid (O'Brien and Wong, 2011 ; Lukiw, 2012 ). The Congo red dye-based intercalation of β-pleated sheets, induction of a positive anisotropy that is polarized and directionally dependent, and generation of a measureable wavelength shift and apple-green birefringence is the hallmark of all amyloids and is the “gold standard” in the diagnosis of amyloidogenic disease (Linke, 2006 ; Buxbaum and Linke, 2012 ). The polymerization of amyloidogenic proteins is cooperative, and can be accelerated by amyloid aggregates derived from the same protein in a selective “seeding” process.…”

“…Congo red [3,3′-([1,1′-biphenyl]-4,4′-diyl)bis(4-aminonaphthalene-1-sulfonic-acid-disodium salt)], a toxic, water soluble secondary diazo dye, was first synthesized in 1883 and used as a textile dye (Steensma, 2001 ; Linke, 2006 ). Due to its toxicity, Congo red's use in textile dying was discontinued, but because of its important spectroscopic properties gained wide use in investigative microbiology (Linke, 2006 ).…”

“…Congo red [3,3′-([1,1′-biphenyl]-4,4′-diyl)bis(4-aminonaphthalene-1-sulfonic-acid-disodium salt)], a toxic, water soluble secondary diazo dye, was first synthesized in 1883 and used as a textile dye (Steensma, 2001 ; Linke, 2006 ). Due to its toxicity, Congo red's use in textile dying was discontinued, but because of its important spectroscopic properties gained wide use in investigative microbiology (Linke, 2006 ). The proposed Congo red-mediated staining mechanism suggests a substrate-mediated hydrophobic pi-pi orbital stacking interaction between the aromatic rings of the dye molecules and β-pleated sheet structures of both amyloids and textiles (Buxbaum and Linke, 2012 ).…”

A Molecular History of the Amyloidoses

“…12,13 Early studies led to the hypothesis that well-ordered bundles of amyloid fibrils would line up the bound Congo red in a crystal-like configuration with colored anisotropy, with different colors being a function of the thickness of the crystal (in this case a 4-μm tissue section) Fig. 3.…”