Diagnosis and Typing of Cardiac Amyloidosis in Routine Clinical Specimens by Mass Spectrometry Based Proteomic Analysis
JA Vrana, JD Theis, JD Gamez, CF Bouse, DV Miller, WD Edwards, A Dogan. Mayo Clinic, Rochester, MN
Background: Cardiac amyloidosis is a frequent cause of restrictive cardiomyopathy and, if untreated, leads to cardiac failure and death. Treatment strategies target the underlying pathogenesis and often involve high risk approaches such as organ transplantation. Therefore accurate typing of amyloid is of great clinical significance. Unfortunately, immunohistochemistry (IHC), currently used for typing amyloidosis, is problematic due to non-specific staining in approximately half of the cases. Here, we describe a novel mass spectrometry based proteomic approach which can type amyloidosis with high sensitivity and specificity and overcome many of the problems associated with IHC.
Design: We studied 56 cases of paraffin embedded cardiac biopsies involved by amyloidosis and 4 cases of normal cardiac biopsies. Congo red positive amyloid plaques were laser microdissected, trypsin digested, and analyzed by nano-flow liquid chromatography electrospray tandem MS (LC-MS/MS). The resulting LC-MS/MS data was correlated to theoretical fragmentation patterns of tryptic peptide sequences from the Swissprot database using Scaffold. Peptide identifications were accepted if established at greater than 90.0% probability and protein identifications were accepted if established at greater than 90.0% probability and contained at least 2 identified spectra. The identified proteins were examined for the presence or absence of amyloid related peptides. IHC for immunoglobulin kappa (IGK) and lambda (IGL) light chains, transthyretin (TTR), serum amyloid A (SAA) was performed in 52 cases.
Results: In 53/56 cases studied, LC MS/MS identified the presence of a single amyloidogenic protein. 35 cases showed a peptide profile consistent with TTR, 15 cases with IGL, 2 cases IGK and 1 case with SAA. No amyloidogenic peptides were identified in normal cardiac stroma or muscle. Of the cases where IHC was performed, staining was considered to be diagnostic in 19 cases and inconclusive in 33 cases. In each case, the IHC confirmed LC MS/MS findings. In those cases where the IHC was non-contributory, additional clinical and pathological information supported the amyloid type assigned by mass spectrometry.
Conclusions: LC-MS/MS proteomic analysis provides a highly specific and sensitive method for diagnosis and classification of amyloidosis in cardiac biopsy specimens. The method is rapid and readily applicable in a clinical setting to paraffin embedded tissues and will greatly improve the diagnosis and clinical management of cardiac amyloidosis.
Tuesday, March 10, 2009 11:45 AM
Platform Session: Section H2, Tuesday Morning