P169-S Rapid and Sensitive Identification of Amyloid Proteins from Formalin-Fixed Paraffin-Embedded Sections and Fat Aspirates: Potential for Aiding in Clinical Diagnosis of Systemic Amyoidosis

摘要

Human amyloidosis describes a condition where one of some 23 known proteins form non-soluble pathological fibrils in various organs and tissues throughout the body. A common feature of these amyloid deposits is distinctive green birefringence when stained with Congo Red. Staining is performed on formalin fixed paraffin embedded (FFPE) tissue sections of tissue biopsies or on subcutaneous abdominal fat aspirates. This method is combined with clinical, immunohistochemical, and genetic information to diagnose the condition and in some cases, identify the amyloid involved. Definitive identification of the amyloid protein is essential for proper therapies in treating patients. Identification in the cases where the other methods give ambiguous results, involve solubilizing and extracting the amyloid proteins from the fixed sections or from fat aspirates. In an attempt to reduce the processing time of these types of clinical samples and still provide definitive identification, we explored in-situ enzymatic digestion methods that exploit the speed and sensitivity of nanoRPLC-ESI-tandem mass spectrometry. Using previously diagnosed clinical samples, we tested a variety of conditions to perform trypsin digestion directly on laser capture microdisected (LCM) generated samples and on fat aspirate samples with the goal of having a robust method suitable for mass spectrometry based identification. Trypsin digestions were performed using, ammonioum bicarbonate, Tris, Tris EDTA/ ± zwittergent 3–16, with and without solubilization steps of hexafluoroisopropanol, acetonitrile, or DMSO. A commercially available FFPE solubilization kit, Liquid Tissue, was also tested. The efficacies of these treatments are judged by performing Sequest or Mascot searches of LTQ generated MS/MS spectra, noting the presence of peptides from the expected amyloid proteins. This method has been successfully utilized to correctly identify amyloids comprised of transthyretin (TTR), serum amyloid A (SAA), lambda and kappa light chains. Controls taken from regions adjacent to the amyloid plaques were consistently devoid of the amyloid proteins.