The Utility of Ultrastructural Immunolabeling (Immunogold Electron Microscopy) in the Classification of Systemic Amyloidosis
JC Lee, PT Soo Hoo, LH Connors, CJ O'Hara. Boston Medical Center, Boston, MA
Background: Current management for patients with systemic amyloidosis relies on precise amyloid subtyping. Available modalities for amyloid subtyping include routine immunohistochemistry (IHC), immunofluorescence, immunoblotting, mass spectrometry, and amino acid sequencing. IHC is often hampered by non-specific background staining, due in large part to amyloid stickiness, often rendering an inconclusive result. Ultrastructural immunolabeling or immunogold electron microscopy (EM) has shown promise as an alternate modality. This study presents the utility of immunogold EM in a series of patients in whom IHC was inconclusive.
Design: A total of 26 patients had immunogold EM, of which 11 had IHC performed at our institution. Samples submitted for immunogold EM include heart (6), liver (1), colon (2), and abdominal fat (17). The tissues were fixed in 4% paraformaldehyde and embedded in lowicryl. The sections were incubated overnight with polyclonal rabbit anti-human kappa light chain, anti-human lambda light chain, anti-human transthyretin, and monoclonal rabbit anti-human serum amyloid A. The sections were then incubated for one hour with goat anti-rabbit antibody conjugated to a 20 nm gold particle. The sections were stained with uranyl acetate and lead citrate and examined using a transmission electron microscope.
Results: Amyloid subtyping by immunogold EM was successful in 25 of 26 patients. By immunogold EM, 4 patients had serum amyloid A (AA) amyloidosis, 9 patients had immunoglobulin light chain (AL) lambda amyloidosis, 2 patients had AL kappa amyloidosis, and 10 patients had hereditary transthyretin (TTR) amyloidosis. For 1 patient amyloid subtyping was inconclusive. Eleven of the 26 patients also had amyloid subtyping by IHC. By IHC, 2 patients were diagnosed as AA, 2 as AL lambda, and for 7 amyloid subtyping was inconclusive. Subsequent evaluation by immunogold EM yielded definitive results in all 7 patients with 1 patient diagnosed as AA, 1 as AL lambda, 1 as AL kappa, and 4 as TTR.
Conclusions: Immunogold EM is an effective tool for subtyping amyloid, with a positive identification rate of 96%. This contrasts with IHC which has a rate of 36% in our case series. In addition, immunogold EM can be performed readily on minimally invasive fat pad aspirates. Prior notification for patients with clinically suspicious systemic amyloidosis is required to assure proper specimen fixation and embedding.
Monday, March 9, 2009 1:00 PM
Poster Session II # 248, Monday Afternoon