Sensitive Method Detects Early Amyloidosis in Humans

These deposits, termed “amyloid,” result from misfolding and/or partial unfolding of proteins followed by their ordered aggregation into amyloid fibrils with specific β-sheet conformation.

Diagnosis of amyloidosis requires a tissue biopsy and the biopsy is assessed for evidence of characteristic amyloid deposits. The most useful stain in the diagnosis of amyloid is Congo red, which, combined with polarized light, makes the amyloid proteins appear apple-green on microscopy.

Scientists at the Linköping University (Sweden) and their colleagues identified patients diagnosed with amyloidosis in the amyloid collection of Uppsala Biobank (Sweden). They chose 53 paraffin-embedded tissue blocks with noticeable amyloid deposits from 50 patients. A second set of amyloid specimens was retrieved and included 61 paraffin-embedded tissue blocks with conspicuous amyloid deposits from 61 patients, including 24 women and 37 men. A control set of eight tissue samples were retrieved from five patients aged 69 to 78 years, comprising heart, liver and kidney. Tissues from a control group of 27, included kidney, stomach, heart, lung, ovary, pancreas and duodenum, which showed no interstitial amyloid deposits as assessed by Congo red staining and polarization microscopy.

Archived paraffin-embedded formalin fixed sections were processed and stained with a luminescent conjugated oligothiophene, h-FTAA, that stains 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. For the archived paraffin-embedded formalin fixed tissue specimens as well as dried fine needle aspired (FNA) abdominal fat biopsies it was found that a 1:5,000 staining solution corresponding to 0.2 mg/L of h-FTAA was optimal. Fluorescence images and spectra were recorded with a Leica DM6000 B fluorescence microscope (Leica Microsystems; Wetzlar, Germany).

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. 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 authors concluded that 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. The 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. The study was published on March 17, 2016, in Amyloid: The Journal of Protein Folding Disorders.

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Linköping University
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