Spatial resolution of renal amyloid deposits through MALDI-MSI: a combined digital and molecular approach to monoclonal gammopathies

Abstract

AimsIdentification and characterisation of monoclonal gammopathies of renal significance (MGRS) is critical for therapeutic purposes. Amyloidosis represents one of the most common forms of MGRS, and renal biopsy remains the gold standard for their classification, although mass spectrometry has shown greater sensitivity in this area.MethodsIn the present study, a new in situ proteomic technique, matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI), is investigated as an alternative to conventional laser capture microdissection MS for the characterisation of amyloids. MALDI-MSI was performed on 16 cases (3 lambda light chain amyloidosis (AL), 3 AL kappa, 3 serum amyloid A amyloidosis (SAA), 2 lambda light chain deposition disease (LCDD), 2 challenging amyloid cases and 3 controls). Analysis began with regions of interest labelled by the pathologist, and then automatic segmentation was performed.ResultsMALDI-MSI correctly identified and typed cases with known amyloid type (AL kappa, AL lambda and SAA). A ‘restricted fingerprint’ for amyloid detection composed of apolipoprotein E, serum amyloid protein and apolipoprotein A1 showed the best automatic segmentation performance (area under the curve >0.7).ConclusionsMALDI-MSI correctly assigned minimal/challenging cases of amyloidosis to the correct type (AL lambda) and identified lambda light chains in LCDD cases, highlighting the promising role of MALDI-MSI for amyloid typing.

Feasibility of MALDI-MSI-Based Proteomics Using Bouin-Fixed Pathology Samples: Untapping the Goldmine of Nephropathology Archives

“…Finally, once the feasibility of performing MALDI-MSI spatial proteomics analysis in BFPE tissue was demonstrated, cases of renal amyloidosis were selected to document the “real-world” feasibility of performing MALDI-MSI on a BFPE kidney bioptic sample obtained from routine clinical archives. Here, the comparable detection of fingerprint proteins in both BFPE and FFPE cases of renal amyloidosis, facilitating the MSI-based detection of renal deposits, confirms the applicability of this approach in BFPE cases of renal amyloidosis with results comparable to those previously obtained . As a consequence, these findings suggest that renal pathology archives containing vast numbers of BFPE biopsies from cases of renal amyloidosis can also be used to further optimize this MALDI-MSI-based approach for detecting and subtyping renal amyloidosis.…”

“…Here, the comparable detection of fingerprint proteins in both BFPE and FFPE cases of renal amyloidosis, facilitating the MSI-based detection of renal deposits, confirms the applicability of this approach in BFPE cases of renal amyloidosis with results comparable to those previously obtained. 10 As a consequence, these findings suggest that renal pathology archives containing vast numbers of BFPE biopsies from cases of renal amyloidosis can also be used to further optimize this MALDI-MSI-based approach for detecting and subtyping renal amyloidosis. Additionally, they can amplify the statistical significance of the case series, particularly for cases that arise from rarer subsets of precursors among the over 30 proteins known to have amyloidogenic potential.…”

“…While studies have presented the application of MALDI-MSI on BFPE tissues (e.g., human testis) with promising results, none have preceded to assess the technical reliability of this method through the direct parallel analysis of coupled FFPE cases . Thus, starting from our previous results on FFPE renal biopsies in the context of different kidney diseases, ,,− here we sought to investigate whether BFPE samples are equally suitable for spatial proteomics analysis with MALDI-MSI in order to further enrich the capabilities of this tool and, subsequently, unlock a wide retrospective series of rare renal diseases that can be exploited to increase our knowledge in this field.…”

“…FFPE and BFPE samples were subjected to deparaffinization, rehydration, antigen retrieval, and tryptic digestion, according to our previously published method. 10 α-Cyano-4-hydroxycinnamic acid matrix (10 mg/μL in 70% acetonitrile and 1% TFA) was sprayed onto the slides using an HTX TM-Sprayer (HTX Technologies, Chapel Hill, NC) with the following parameters: temperature 75 °C; number of passes 4; flow rate 0.12 mL/min; speed 1200 mm/min; and pressure 10 psi. A mixture of known masses (PepMix I) was applied directly onto the ITO slide to be used as an external calibrant within the m/ z range of 750−3150.…”

“…The ability to detect and localize amyloid-associated proteins in BFPE tissue, as an additional stress test, was also evaluated using cases of renal amyloidosis. Using a recently described protocol for amyloid fingerprint protein detection on FFPE CNBs, 10 the pipeline was adopted on a BFPE renal biopsy. This comparison also demonstrated the feasibility to detect the presence of amyloid fingerprint proteins and underline their colocalization within amyloid deposits in BFPE tissue, corresponding to the annotations of the renal pathologist on the Congo Red stained tissue and presented the same reliability as the FFPE counterpart (Figure 5).…”