Joint Research Centre Institute for Reference Materials and Measurements (IRMM) http://irmm.jrc.ec.europa.eu Abeta42 standardisation Definition of the SI unit “Pure” peptide calibrator CRM matrix material Manufacturer’s working calibrator Manufacturer’s product calibrator e.g. AAA, refractive index Reference procedure, immunoassay ring trial Value transfer 1: LC-MS, immunoassay Value transfer 2: immunoassay Immunoassay Routine sample Aim: Setting up a reliable traceability chain linking results on routine samples to a common reference, the international system of units (SI) 2 ✔ Purity assessment amino acids Commutability studies Processing Feasibility studies processing Definition of the SI unit ✔ Purity assessment calibrant Pure amino acids Abeta42 calibrant CRM matrix material Amino acid analysis validation Amino acid analysis LC-MS Reference procedure Correlation studies LC-MS ring trial Calibrator batch: - Intended use: Calibration of LC-MS measurements for the value assignment of the CRM - Format: recombinant Abeta42 in 20 % acetonitrile, 1 % ammoniumhydroxide - Concentration: determined by Amino acid analysis and purity assessment 4 Calibrator batch: Amino Acid analysis: - Problem in-house with repeatability and between AA variation, indicating a problem. 5 Possible explanations: - Purity: very high purity (Q-TOF, ion-trap, sequencing) - Digestion conditions optimised - Different sequences present? Unlikely as recombinant, confirmed by MS sequencing - Problematic interaction between solvent and HCl → freezedrying of samples before measurements: good conditions found on a different test batch To be repeated on actual calibrant (ongoing) 6 ✔ Purity assessment amino acids Commutability studies Processing Feasibility studies processing Definition of the SI unit ✔ ✔ Pure amino acids Abeta42 calibrant CRM matrix material ✔ ✔ ✔ Purity assessment calibrant Amino acid analysis validation Amino acid analysis LC-MS Reference procedure Correlation studies LC-MS ring trial LC-MS reference method Must give results that correlate with routine assays → correlation studies: OK Pannee et al (2013) J Alzheim Dis Leinenbach et al (2014) Clin Chem Korecka et al (2014) J Alzheim Dis Bjerke et al (2015) CCLM submitted 8 Ring trial with common protocol for calibration 9% 6 datasets: Uncertainty for value assignment: Acceptable for value assignment of the CRM ! 9 ✔ Purity assessment amino acids Commutability studies Processing Feasibility studies processing Definition of the SI unit ✔ ✔ Pure amino acids Abeta42 calibrant CRM matrix material ✔ ✔ ✔ ✔ ✔ ✔ Purity assessment calibrant Amino acid analysis validation Amino acid analysis LC-MS Reference procedure Correlation studies LC-MS ring trial Commutability: a reference material must behave in the same way as a patient sample (2 studies, 10 methods) Bjerke et al, CCLM submitted 11 Processing tests: - Manual filling/ machine filling - Stirring/ not stirring (no difference) - Abeta42 concentration over time while stirring (no slope) - Flash freezing versus slow freezing (better slow freezing) Drift according to filling sequence (aCSF) problem at beginning and end of filling (aCSF) 12 Conditions chosen: - Machine filling - Stirring - slow freezing Statistical evaluation of data: Soverall = 3.8 % Srepeatability = 3.2 % (method variability) Sbetween vial = 1.9 % (between vial variability) Results from last test (CSF) 13 → Decision on format of the matrix reference material: - CSF pool - No spiking - 0.5 mL/vial - 3 levels: low, medium and high - Stored at -70°C 14 1st level filled 15 ✔ ✔ ✔ ✔ Purity assessment amino acids Commutability studies Processing Feasibility studies processing Definition of the SI unit ✔ ✔ ✔ Pure amino acids Abeta42 calibrant CRM matrix material ✔ ✔ ✔ ✔ ✔ ✔ Purity assessment calibrant Amino acid analysis validation Amino acid analysis LC-MS Reference procedure Correlation studies LC-MS ring trial Planning Homogeneity study Feasibility study (collection, processing, characterisation, commutability) Correlation studies Storage Short-term stability study Processing Value assignment Long-term stability study Follow-up stability monitoring of CRM Interim transport and storage Assessment by experts CRM Distribution and Sales Material selection Commutability study Documentation 17 → Next step 1: homogeneity study first material → Next step 2: processing 2 other levels → Next step 3: Value assignment: - LC-MS methods validated - 6 laboratories or methods 3 levels At least 2 days Common protocol for calibration 18 Thanks to a large collaboratve network: GBSC IFCC Participating laboratories Manufacturers Some people devoting a majority of their time: Josef Pannee, Julia Kuhlmann, Maria Bjerke, Magdalena Korecka, Andreas Leinenbach… 19
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