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WP3: Biomarkers for acute and chronic heart failure
This WP aims to enhance BNP measurements and ultimately improve patient outcomes through provision of reference measurement procedures and standardisation of EQA schemes achieved by:
1. the development of a reference measurement procedure for NT-proBNP and its application to EQA schemes and clinical samples (Task 3.1).
2. data interrogation from Task 3.1 to define procedures for the calculation of measurement uncertainty within EQA schemes and inform about the requirements of commutable NT-proBNP EQA materials. This will facilitate the comparison of the results and provide additional information on sample requirements (Task 3.2).
3. the advancement of the published reference measurement procedure for 1-32 BNP to monitor BNP circulating forms and its application to the EQA schemes and clinical samples (Task 3.3).
The new MS-based methods developed will enable clinicians and measurement laboratories to understand the discrepancies between different commercially available devices for routine measurement of BNP and its derivatives.
Different BNP materials (recombinant, synthetic and glycosylated) were sourced and intact protein analysis was carried out to confirm their identity and preliminary assess their purity. Recombinant NT-proBNP was selected as primary calibrator of choice and quantified traceably to the SI. Hydrogen-deuterium-exchange experiments were carried out to define higher order structural differences between recombinant and glycosylated NT-proBNP that may cause discrepancies in immunoassay binding. No structural differences were observed and immunoassay measurements are currently on-going on the samples analysed. A targeted LC-MS method for the quantification of NT-proBNP in plasma was preliminary developed. The NT-proBNP signature peptides were selected and the optimisation of a tryptic digestion and sample clean-up protocol is on-going. Preliminary experiments were carried out to increase the sensitivity of the published method on quantification of 1-32 BNP in plasma.