|Ahmet Dogan, Mayo Clinic, Rochester, MN|
Julie A. Vrana, Mayo Clinic, Rochester, MN
Steven R. Zeldenrust, Mayo Clinic, Rochester, MN
Jason D. Theis, Mayo Clinic, Rochester, MN
Jeffrey D. Gamez, Mayo Clinic, Rochester, MN
Paul J. Kurtin, Mayo Clinic, Rochester, MN
|Abdominal subcutaneous fat aspiration is one of the most practical, sensitive and specific methods for the diagnosis of systemic amyloidosis. One limitation of this method, compared to more invasive tissue biopsy based approaches, remains the technical difficulties in further classification of the amyloidosis as commonly used methods, such as immunohistochemistry, are not readily applicable to fat aspiration specimens. To overcome these difficulties we developed a method using liquid chromatography tandem mass spectrometry (LC-MS/MS) that could identify amyloid subtypes in freshly obtained Congo Red positive fat aspirate specimens with great accuracy.|
The study used two independent groups of fat aspirate specimens. The first group of cases was used as a training set to establish diagnostic criteria. The second group was used as the validation set. The training set contained fat aspirate specimens from 73 patients with clinical suspicion for systemic amyloidosis. One half of each specimen was stained with Congo red and used for diagnosis of amyloidosis and the other half was processed and enzyme digested for LC-MS/MS analysis. The resulting LC-MS/MS data was correlated to theoretical fragmentation patterns of tryptic peptide sequences from the Swissprot database using Mascot, Sequest, and X!Tandem search algorithms. Peptide identifications were accepted according to previously established criteria. The identified proteins were subsequently examined for the presence or absence of amyloid related peptides. Of the 73 cases studied, 41 were positive for Congo red consistent with systemic amyloidosis. In Congo red positive cases, LC-MS/MS peptide profiles consistent with AL-lambda (28/31), AL-kappa (6/7), and ATTR (2/3) were observed. Only one case in the Congo red negative control group (31/32) gave a kappa light chain profile which was attributed to a high level of kappa in the serum (285 mg/dL). Of the 35 out of 41 cases of systemic amyloidosis successfully classified by LC-MS/MS, additional clinical and pathology data validating the amyloid type was available. In each of these cases the MS/MS results accurately predicted the amyloid type. The validation set contained Congo red positive fat aspirate specimens from 69 patients. In the validation set the amyloid type was accurately identified in 64/69 (93%) of the specimens.
In conclusion, LC-MS/MS proteomic analysis of subcutaneous fat aspiration specimens involved by amyloidosis provides a highly specific (97% specificity) and sensitive (>93% sensitivity) method for diagnosis and classification of amyloidosis. The method is rapid and readily applicable in a clinical setting and will greatly improve the clinical management of amyloidosis patients.