Diagnosis Of Amyloidosis Subtype By Laser-Capture Microdissection (LCM) and Tandem Mass Spectrometry (MS) Proteomic Analysis

Correct identification of the protein that is causing amyloidosis is crucial for clinical management. Current standard diagnostic methods have limited ability to detect the full range of amyloid forming proteins. We assessed combining specific sampling of amyloid deposits by LCM and analysis of tryptic digests by tandem MS proteomic analysis to determine whether the majority of amyloidosis clinical samples can be correctly classified as reported by the Mayo Clinic pathology department.

We studied 58 cases of well characterised amyloid deposition and 10 cases in which the amyloid subtype was unable to be diagnosed with confidence. For all specimens, 10µm sections of formalin-fixed paraffin embedded tissue were stained with Congo Red using a standard technique. LCM was performed using an Arcturus XT instrument with an infrared capture laser. Proteins were digested with trypsin and peptides were analysed by nano-liquid chromatography-coupled tandem mass spectrometry using a Chip CUBE-QTOF. Database searching was performed using Spectrum Mill (Agilent) with the NCBInr human protein database. Protein identification cut-offs were protein score > 11, peptide score > 10 and % scored peak intensity >60.

Biopsy sites included: GIT (n=16), cardiac (n=12), soft tissue (n=8), renal (n=9), liver (n=3) and other (n=20). The amyloid subtype was able to be determined in 64 cases analysed. In 7 of these cases a second sample or second LCM was required as the first analysis was non-diagnostic. In 4 cases the amyloidogenic protein was not identified mostly due to the amyloid deposits being very small. Proteins identified included immunoglobulin light chain (localised amyloid n=6, systemic AL n=32), transthyretin (senile amyloid n=17, hereditary ATTR n=2), serum amyloid A (AA n=4), fibrinogen (AFib n=1), TGFb (corneal lattice amyloid n=1) and semenogelin (seminal vesicle amyloid n=1).

Three diagnostically challenging cases are detailed as examples of the utility of LCM and tandem MS. The first case had extensive gastrointestinal amyloidosis and no evidence of clonal light chain disease; negative kappa, lambda, SAA and transthyretin immunohistochemistry; and negative genetic studies. Tandem MS revealed immunoglobulin lambda light chain type. The second diagnostically challenging case had: isolated renal amyloidosis with a positive AA stain and kappa restricted serum free light chains. Tandem MS revealed serum amyloid A2 protein. The third case had: cardiac, neurological and gastrointestinal involvement; and equivocal immunohistochemistry. Tandem MS demonstrated transthyretin and genetic studies showed a A97S ATTR mutation.

Various other proteins were identified by tandem MS in amyloid extracts. Of particular interest is the presence of proteins typically known to be co-located in amyloid deposits which helps confirm that the microdissected tissue is amyloid. Typical amyloid-associated proteins were identified in the following number of cases: SAP (n=36), apolipoprotein A4 (n=42), vitronectin (n=44), apolipoprotein E (n=40) and clusterin (n=21). Various types of collagen were frequently present (n=29) and various, presumably contaminating, keratins were identified (n=24).

LCM and tandem MS allows correct typing of amyloid deposits in the majority of clinical biopsy samples.

No relevant conflicts of interest to declare.