[Evaluation of an automated assay of thyrotropin receptor antibody (TRAb) on Elecsys Analyser 2010]. / Evaluation du dosage automatisé des auto-anticorps anti-récepteur de la TSH sur l'analyseur Elecsys 2010.

2nd generation assays are currently available to determine the concentration of thyrotropin receptor antibody (TRAb) present in Graves' disease. The aim of this study was to evaluate the analytical performance of TRAb assay on Elecsys 2010 and Cobas(R)e of Roche Diagnostics, a new test using electrochemiluminescence immunoassay (ECLIA). The analytical performance observed with this assay is accurate (repetability and reproductibility, analytical and functional sensitivities). Comparing this method with Brahms' assay (TRAK human RIA), the correlation observed can be put in the equation: y = 1.013 x + 1.381; r = 0.92. With a positive value in ECLIA at 0.9 UI/L and at 1 UI/L in RIA (functional sensitivity fixed by manufacturers), concordance study shows a false negative and two false positive. With a positive value at 0.8 UI/L with ECLIA (functional sensitivity calculated from precision's profile for a CV inferior to 10%), there is no false negative and two false-positive results, this value seems to give a better sensitivity.

Tag-mass: specific molecular imaging of transcriptome and proteome by mass spectrometry based on photocleavable tag.

MALDI tissue imaging of tissues has become a promising technique for tracking biomarkers while determining their location and structural characterization. We have now developed specific targeting probes (oligonucleotides, antibodies), named Tag-Mass. This approach is based on probes modified with a photocleavable linker coupled with a tag cleaved and detected using mass spectrometry. Tag-Mass development is the key for a rapid, sensitive, and accurate approach to correlate levels of expression of different mRNA or proteins in diseases.

Direct analysis and MALDI imaging of formalin-fixed, paraffin-embedded tissue sections.

Formalin fixation, generally followed by paraffin embedding, is the standard and well-established processing method employed by pathologist. This treatment conserves and stabilizes biopsy samples for years. Analysis of FFPE tissues from biopsy libraries has been, so far, a challenge for proteomics biomarker studies. Herein, we present two methods for the direct analysis of formalin-fixed, paraffin-embedded (FFPE) tissues by MALDI-MS. The first is based on the use of a reactive matrix, 2,4-dinitrophenylhydrazine, useful for FFPE tissues stored less than 1 year. The second approach is applicable for all FFPE tissues regardless of conservation time. The strategy is based on in situ enzymatic digestion of the tissue section after paraffin removal. In situ digestion can be performed on a specific area of the tissue as well as on a very small area (microdigestion). Combining automated microdigestion of a predefined tissue array with either in situ extraction prior to classical nanoLC/MS-MS analysis or automated microspotting of MALDI matrix according to the same array allows the identification of both proteins by nanoLC-nanoESI and MALDI imaging. When adjacent tissue sections are used, it is, thus, possible to correlate protein identification and molecular imaging. These combined approaches, along with FFPE tissue analysis provide access to massive amounts of archived samples in the clinical pathology setting.

MALDI-MS direct tissue analysis of proteins: Improving signal sensitivity using organic treatments.

Direct tissue analysis using MALDI-MS allows the generation of profiles while maintaining the integrity of the tissue, displaying cellular localizations and avoiding tedious extraction and purification steps. However, lower spectral quality can result from direct tissue analysis due to variations in section thickness, the nature of the tissue, and the limited access to peptides/proteins due to high lipid content. To improve signal sensitivity, we have developed a tissue-washing procedure using organic solvents traditionally used for lipid extraction, i.e., CHCl3, hexane, toluene, acetone, and xylene. The increased detection for peptides/proteins (m/z 5000-30,000) is close to 40% with chloroform or xylene, and 25% with hexane, while also improving sample reproducibility for each solvent used in the present study. This strategy improved matrix cocrystallization with tissue peptides/proteins and more importantly with cytoplasmic proteins without delocalization. The extracted lipids were characterized by nanoESI-QqTOF/MS/MS using the precursor ion mode, lithium adducts, or both and were identified as phospholipids including phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and lysophosphatidylinositol, confirming membrane lipid extraction from the tissues.