Mass Spectrometry Amyloid Typing Is Reproducible across Multiple Organ Sites

Abstract

We have determined patient's amyloid subtype through immunohistochemical and proteomic analyses of formalin-fixed, paraffin-embedded (FFPE) tissue samples from two affected organs per patient. Amyloid typing, via immunohistochemistry (IHC) and laser microdissection followed by the combination of liquid chromatography with mass spectrometry (LMD-LC-MS), was performed using tissue samples of the human heart, liver, kidney, tongue, and small intestine from 11 patients, and the results were compared with clinical data. LMD-LC-MS correctly typed AL amyloidosis in all 22 FFPE tissue samples despite tissue origin. In contrast, IHC was successful only in the analysis of eight FFPE tissue samples with differences between the examined organs. In the majority of LMD-LC-MS typed samples, the level of IHC staining intensity for transthyretin and serum amyloid A was the same as that for Ig κ and Ig λ antibodies, suggesting low Ig κ or Ig λ antibodies reactivity and the additional antibody clones were essential for correct typing. Both methods used in the study were found to be suitable for amyloid typing, although LMD-LC-MS yielded more promising results than IHC.

Figure 1

Immunohistochemical typing of amyloid in myocardial tissue by a basic panel of antibodies. The confirmation of amyloid deposition was done via Congo red and Sirius red staining, the amyloid typing via IHC analysis (right panels). The tissue sample of case 1 failed during IHC staining with AL κ, AL λ, SAA, and TTR antibodies. The examined tissues of cases 2 and 3 had a false positive reaction with SAA and/or TTR antibodies, weak and/or negative reaction with AL κ antibody, respectively. In cases 4, 7, 10, and 11 examined tissue had a positive reaction with more than one antibody which is classified as no immunospecific staining (NS). Amyloid fibril protein (AL κ) was typed correctly in case 5. Amyloid fibril protein (AL λ) was typed correctly in cases 6, 8, and 9. IHC staining intensity was classified as negative (-), weak (+), moderate (++), and strong (+++). The amyloid subtype was determined based on the strongest IHC reaction.