Simple Protocol for Amyloid Extraction from FFPE Sections using Organic Solvent

Background: Recently, we have established a simple method of amyloid extraction from Formalin-Fixed, Paraffin-Embedded (FFPE) sections using organic solvent. This method provides an amyloid-rich extract that can be analyzed by electrophoresis and mass spectrometry. This study aimed to show the utility of the established method and present a simple protocol of amyloid extraction for electrophoresis and mass spectrometry. Methods: Amyloid deposition was confirmed pathologically in two dogs diagnosed with medullary carcinoma and plasmacytoma. Amyloid deposition was classified by immunohistochemistry. Amyloid proteins were extracted from FFPE sections of these cases of plasmacytoma and medullary carcinoma using Dimethyl Sulfoxide (DMSO). Amyloid extracts were analyzed by electrophoresis and mass spectrometry. Results: Amyloid deposition was confirmed pathologically in both cases. Immunohistochemistry revealed that the proteins forming amyloid deposits were calcitonin in medullary carcinoma and the lambda chain of immunoglobulin in plasmacytoma. DMSO extracts showed case-specific isolation patterns of proteins in electrophoresis. Mass spectrometry identified the specific peptides of calcitonin and the lambda chain of immunoglobulin in DMSO extracts from the cases. Conclusion: We demonstrated the utility of the amyloid extraction method using DMSO for calcitonin and AL amyloid in canine cases. This simple method might contribute to the diagnosis and study of amyloidosis.