Diagnosis of Amyloidosis Subtype by Laser-Capture Microdissection (LCM) and Tandem Mass Spectrometry (MS/MS) Proteomic Analysis — ASN Events
  1. Schedule
  2. Lunch/Poster Session Two
  3. Diagnosis of Amyloidosis Subtype by Laser-Capture Microdissection (LCM) and Tandem Mass Spectrometry (MS/MS) Proteomic Analysis

Diagnosis of Amyloidosis Subtype by Laser-Capture Microdissection (LCM) and Tandem Mass Spectrometry (MS/MS) Proteomic Analysis (#224)

Peter Mollee 1 2 , Patricia Renaut 3 , Samuel Boros 3 , Dorothy Loo 1 4 , Michelle M Hill 1 4
  1. The University of Queensland Diamantina Institute, The University of Queensland, Brisbane, QLD, Australia
  2. Princess Alexandra Hospital, Brisbane, QLD, Australia
  3. Pathology Queensland, Brisbane, QLD, Australia
  4. Translational Research Institute, Brisbane, Australia

Background & Aims

Correct identification of the protein that is causing amyloidosis is crucial for clinical management. Current standard laboratory methods have limited ability to detect the full range of amyloid forming proteins. We assessed the diagnostic value of LCM-MS/MS, which combines specific sampling of amyloid deposits by LCM with protein identification by MS/MS.

Methods

Biopsy specimens were referred to the Princess Alexandra Hospital Amyloidosis Centre. 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 extracted with FFPE Protein Extraction Solution, digested with trypsin and peptides were analysed by nano-liquid chromatography-coupled MS/MS using an Agilent Chip CUBE-QTOF.

Results

Biopsies were received for 136 patients from 35 different organ sites. There was insufficient tissue in the block for 7 cases, repeat LCM was required in 15 cases. An amyloid forming protein was identified in 121 out of 129 attempted cases (94%). Of the 121 successfully cases, the Mayo Clinic (at least two of SAP, ApoE and ApoA4) was detected in 92 (76%), and was associated with higher Amyloid protein score and % coverage. We found that vitronectin provided complementary information to the existing 3-protein amyloid signature. In terms of clinical impact, amyloid typing by immunohistochemical stains had been attempted in 87 cases and reported as diagnostic in 39. Five of these were subsequently revealed by proteomic analysis to be incorrect. Overall, the clinical diagnosis of amyloid subtype was altered by proteomic analysis in 24% of cases.

Conclusions

LCM with LC-MS/MS successfully identified an amyloid forming protein in 94% of clinical biopsy samples. Amyloid deposits often contain small amounts of other amyloid forming proteins. Because of this, results need to be interpreted in the context of full clinical information to enable correct diagnosis of amyloid subtype.