Establishing the fluorescent amyloid ligand h-FTAA for studying human tissues with systemic and localized amyloid

Abstract

Rapid and accurate detection of amyloid deposits in routine surgical pathology settings are of great importance. The use of fluorescence microscopy in combination with appropriate amyloid specific dyes is very promising in this regard. Here we report that a luminescent conjugated oligothiophene, h-FTAA, rapidly and with high sensitivity and selectivity detects amyloid deposits in verified clinical samples from systemic amyloidosis patients with AA, AL and ATTR types; as well as in tissues laden with localized amyloidosis of AANF, AIAPP and ASem1 type. The probe h-FTAA emitted yellow red fluorescence on binding to amyloid deposits, whereas no apparent staining was observed in surrounding tissue. The only functional structure stained with h-FTAA showing the amyloidotypic fluorescence spectrum was Paneth cell granules in intestine. Screening of 114 amyloid containing tissues derived from 107 verified (Congo red birefringence and/or immunohistochemistry) amyloidosis patients revealed complete correlation between h-FTAA and Congo red fluorescence (107/107, 100% sensitivity). The majority of Congo red negative control cases (27 of 32, 85% specificity) were negative with h-FTAA. Small Congo red negative aggregates in kidney, liver, pancreas and duodenum were found by h-FTAA fluorescence in five control patients aged 72-83 years suffering from diverse diseases. The clinical significance of these false-positive lesions is currently not known. Because h-FTAA fluorescence is one magnitude brighter than Congo red and as the staining is performed four magnitudes lower than the concentration of dye, we believe that these inclusions are beyond detection by Congo red. We conclude that h-FTAA is a fluorescent hypersensitive, rapid and powerful tool for identifying amyloid deposits in tissue sections. Use of h-FTAA can be exploited as a rapid complementary technique for accurate detection of amyloid in routine surgical pathology settings. Our results also implicate the potential of the technique for detection of prodromal amyloidosis as well as for discovery of new amyloid-like protein aggregates in humans.

ThT 101: a primer on the use of thioflavin T to investigate amyloid formation

“…Polysaccharides such as hyalinous cartilage [8] and certain proteins like acetylcholinesterase [9] also induce the characteristic ThT fluorescence, presumably through the existence of appropriate structural cavities. These properties reduce its usefulness for the identification of amyloid deposits in diagnostic histology compared to Congo red [10] and have spurred the development of ThT derivatives [11][12][13] such as Amytracker TM [14,15] and Proteostat V R [16]. Although commercially available, these are expensive and remain less widely used than ThT, relegating them to a minor role in amyloid science so far.…”

Organic bioelectronics in medicine

“…Of interest, when the LCO backbone consisted of five or more backbone units, prefibrillar amyloid species were detected, which are not identified by any other amyloid staining . Further, based on the spectral shift (Fig.…”

“…). So‐called luminescent conjugated oligothiophenes (LCOs) have been synthesized to visualize and study distinct conformational variants of protein aggregates , components of the bacterial extracellular matrix and carbohydrates .…”

Monoclonal gammopathy of renal significance: Diagnostic workup

“…1 New sensitive method for early detection of amyloidosis in humans[36]. AH: Immunoglobulin heavy chain; AHL: Immunoglobulin heavy and light chain; AL: Immunoglobulin light chain; C3GN: C3 glomerulonephritis; DDD: Dense deposits disease; EM: Electronic microscopy; EP: Electrophoresis; FGN: Fibrillary glomerulonephritis; FLC: Free light chain assay; GN: Glomerulonephritis; GP: Glomerulopathy; IF: Immunofluorescence; Ig: Immunoglobulin; ITG: Immunotactoid glomerulonephritis; LC: Light chain; LM: Light microscopy; MG: Monoclonal gammopathy; MIDD: Monoclonal immunoglobulin deposition disease; MPGN: Membranoproliferative glomerulonephritis; PGNMID: Proliferative glomerulonephritis with monoclonal immunoglobulin deposits.…”