Molecular Identification of Transthyretin Amyloid in Fixed Skin Biopsies: A Simplified Sample Preparation Method

Hereditary transthyretin (TTR) amyloidosis (ATTRv) is associated with peripheral neuropathy resulting from the accumulation of fibrillar TTR amyloid deposits in tissues including peripheral nerves and skin. Accurate diagnosis requires molecular identification of amyloid deposits in tissue specimens. Skin punch biopsy is a minimally invasive, well-established diagnostic tool used to confirm small-fiber neuropathy. Congo-red (CR) staining confirms amyloid deposition but does not determine amyloid type. Identification of TTR proteins within deposits commonly employs immunohistochemistry, which is limited by non-specific binding to blood-derived proteins and can lead to misdiagnosis. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) of laser micro-dissected amyloid fibrils precisely identifies deposited proteins but requires specialized equipment available only in dedicated medical centers. This study describes a streamlined protocol for extracting insoluble amyloid proteins from Zamboni-fixed skin biopsies for subsequent Western blotting (WB) and LC-MS/MS. The protocol includes: (a) tissue collection by cryo-sectioning of biopsy samples; (b) removal of soluble background proteins by repeated phosphate buffer rinsing; (c) extraction of insoluble proteins with formic acid; (d) removal of the volatile solvent by vacuum evaporation and lyophilization. In a proof-of-concept cohort of 24 biopsies, 15-kDa and 30-kDa immunoreactive bands consistent with TTR monomer and dimer forms were detected in 10 of 12 CR-positive, genetically confirmed ATTRv samples and in none of 12 CR-negative neurological controls. This corresponded to an estimated sensitivity of 83.3% (95% confidence interval [CI], 55.2%-97.0%) and specificity of 100% (95% CI, 75.8%-100%) within this selected cohort. An in-gel LC-MS/MS analysis of excised 15 kDa and 30 kDa bands confirmed TTR identity in WB-positive samples tested. The findings were also in concordance with an LC-MS/MS analysis of micro-dissected CR-positive fibril deposits in three tested biopsies. This protocol expands the analytical use of routinely collected skin biopsies and provides an accessible proof-of-concept approach for TTR detection.