An extensive data analysis has been conducted using the Swedish cardiac registry SWEDEHEART to identify the subgroups that will most profit from determining lipoproteins as additional biomarkers for CVD risk. Based on the outcome of the analysis a clinical study has been devised and submitted to ethical approval. Currently, 63 000 patients are included in the analyses. Baseline data and definitions have been performed. Long-term prognosis in these patients with up to 11 years of follow-up is being assessed. Further, UPP is conducting two large-scale studies aiming at assessing low density lipoprotein cholesterin (LDL-C) levels during MCI and prognosis: One analysis is investigating the level of LDL-C at the time of an MI in 60 000 patients and its association to long-term prognosis. In order to document the state of the art regarding CVD risk prediction on the basis of diagnostic tests currently used in day to day clinical practice and determine what is the measurement uncertainty needed for routine methods to accurately stratify patients based on concentration of conventional biomarkers (e.g. LDL-C, non-HDL-C, TG, …), preparation of an EQA scheme has been initiated.  Clinical specimens that are intended to be used in an EQA scheme later in 2021 have been sourced. An initial commutability study has been organised, comparing the results of fresh single patient samples, pooled native serum pools and conventional EQA material using the major measurement techniques. In addition, LNE sourced additional samples from the Cholesterol Reference Method Laboratory Network (CRMLN) that were already value assigned for LDL-C by the Center for Disease Control and Prevention’s (CDC) reference method by beta quantification. 25 pools of human frozen serum were received by LNE and will be distributed to clinical laboratories running the most popular assays for LDL-C. In order to establish recommendations for analytical performance criteria for assays relying on conventional biomarkers currently used to estimate long-term CVD risk, LNE joined CDC’s working group on lipids analytical performance criteria that brings together world class expert of this field. Recommendations which are currently discussed with assay manufacturers will be published soon.
Substantial progress was made in the development of a candidate reference measurement procedures for a panel of apolipoproteins (ApoA-I, B, C-I, C-II, C-III, E and Apo(a)) with a target uncertainty of below 15 %. The procedure for sample preparation is almost in place: a list of target peptides was established, digestion conditions were optimised, a protocol for isolation of proteotypic peptides was established and LC-MS conditions were optimised. The developed preliminary standard operating procedure (SOP) was tested among three clinical reference laboratories using serum-based calibrators and the needs for further improvement have been identified. Sourcing and evaluation of calibration materials for absolute quantification of a panel of apolipoproteins by LC-IDMS has been started. Preliminary purity assessment for four peptides showed sufficient purity to proceed. Equimolarity of digestion is a key step to demonstrate the suitability of the bottom-up approach and specially to establish the traceability of the results to the International System of Units (SI). Ongoing experiments will result in validating the final list of peptides that will be used to quantify the whole protein(s) and, therefore, to decide which primary calibrators are needed. Meanwhile, a preliminary purity assessment of 5 candidate peptides for apo(a) was conducted to ensure that preliminary concentrations determined by amino acid analysis are sufficiently accurate to conduct the equimolarity experiments. After this work has been completed, additional purity evaluation and amino acid analysis will be conducted with the objective to perform formal certification of the primary calibrators that will be used to calibrate the candidate reference method.