I am the Director of the Northwest Lipid Metabolism and Diabetes Research Laboratories (NWRL) at the University of Washington in Seattle WA, USA NWRL/UW has received grant and research funding from: • National Institutes of Health • Amgen Inc • Ionic Pharmaceuticals • Kaiser Permanente I am consultant to: • Denka Seiken, Japan • Roche Diagnostics, Germany • Medtest DX Inc, USA EAS 2019
Santica M Marcovina PhD ScD FAHA Research Professor of Medicine, UW Director, Northwest Lipid Metabolism and Diabetes Research Laboratories EAS 2019
Aim : To obtain values in patient samples that are accurate and comparable independently of the analytical method used for the measurement. Accuracy is obtained if calibrator values are traceable to a high order primary reference material directly or via a suitable secondary reference material and the methods are certified to guarantee that accuracy of the calibrator results in accurate values in samples. Aim : To obtain values in patient samples that are comparable even though not necessarily accurate . EAS 2019
• Antibody specificity • Immunoreactivity of the antibody per particle should be the same for the assay calibrator and for the samples • An accuracy-based target value should be assigned to the assay calibrators EAS 2019
Kringle 4 kDa Kringle 4 Type V PD Type 2 repeats 1 3-10 2 3 187 10 274 20 399 30 524 40 649 EAS 2019 SM Marcovina, University of Washington
EAS 2019
• For Lp(a) the “signal” does not reflect the number of particles • In samples with apo(a) sizes smaller than the apo(a) size of the assay calibrator, Lp(a) levels will be UNDERESTIMATED • In samples with apo(a) sized larger than the apo(a) size of the assay calibrator, Lp(a) levels will be OVERESTIMATED EAS 2019
• To circumvent this problem, we produced a variety of monoclonal antibodies against Lp(a) and we were able to obtain and characterize a high-affinity monoclonal antibody directed to an epitome only present in the KIV Type 9 region of apo(a) • Using this unique monoclonal antibody (a-40), we developed and fully validated an ELISA method that can measure Lp(a) protein on an equimolar basis • We demonstrated that Lp(a) contains one molecule of apo(a) per Lp(a) particle and therefore, the Lp(a) protein values expressed in nmol/L reflect the number of circulating Lp(a) particles. EAS 2019
Calibrated with the same sample containing apo(s) with 21 Kingle 4 EAS 2019
% B i a s 5 0 2 5 0 y = 4 . 0 5 x - 8 4 . 7 - 2 5 r = 0 . 9 6 7 - 5 0 1 4 1 6 1 8 2 0 2 2 2 4 2 6 2 8 3 0 3 2 K r i n g l e 4 n u m b e r EAS 2019
• In the mid 1970’s, John Albers developed the first, high sensitive RIA to measure Lp(a) • Lp(a) was isolated from an individual with high Lp(a) levels and measurements of the lipid and the protein components of Lp(a) were performed • This Lp(a) preparation was used as the RIA primary calibrator with the assigned value being the sum of lipid and protein values • Results of Lp(a) were therefore expressed in mg/dL of TOTAL MASS of Lp(a), not knowing that the Lp(a) mass is highly variable within and between individuals EAS 2019
International Federation of Clinical Chemistry 1997: IFCC Working Group Members: A. Steinmetz (Chair) F. Dati K. Berg R. Couderc G. Kostner N. Rifai I. Sakurabayashi J. Tate Phase 1: Assessment of analytical performance of 40 test systems by testing serum samples for precision, linearity, and parallelism. Results: ❖ A significant number of assays were not optimized and failed to meet the criteria for precision, linearity, and parallelism. ❖ The among method CV of all systems on reference samples ranged from 22% to 60%. ❖ The among method CV of optimized systems ranged from 16% to 35% Clin Chem 1998; 44:1629-40 EAS 2019
International Federation of Clinical Chemistry 1998: Phase 2 Aims: Selection of a secondary reference material for Lp(a) ❖ Four proposed materials were compared in 27 optimized analytical systems ❖ Results of precision and linearity were comparable ❖ Among-method CV on each of the four materials ranged from 11% to 22% ❖ Based on the overall results, the IFCC Working Group decided to select the material 2B as a proposed reference material (PRM-2B) for further evaluation and for final target value assignment. Decision was made to express Lp(a) values in nmol/L. Clin Chem Lab Med 1999; 37:949-958 EAS 2019
National Institutes of Health National Heart, Lung, and Blood Institute Principal Investigator: ❖ Santica M Marcovina, University of Washington Co-Investigators: ❖ John J Albers, University of Washington ❖ Angelo Scanu, University of Chicago ❖ Celina Edelstein, University of Chicago EAS 2019
National Institutes of Health National Heart, Lung, and Blood Institute Specific Aims: • Evaluation of Lp(a) assays with particular emphasis on antibody specificity and sensitivity to apo(a) size polymorphism • Feasibility of standardization of Lp(a) assays • Preparation and characterization of primary reference material • Assignment of target value to the IFCC Reference Material EAS 2019
Study performed in collaboration with the IFCC Working • Group on Lp(a) standardization NWRL ELISA served as the reference method • 21 commercially available methods were evaluated by using • 30 fresh-frozen samples spanning a large range of Lp(a) levels and apo(a) isoforms The reference material, PRM-2B, with an assigned value of • 107 nmol/L, was used as a common assay calibrator EAS 2019
• Using PRM-2B to calibrate all the systems, the among-method CVs for each of the 30 samples ranged from 6% to 31% • Even though the CVs were lower than those obtained using individual calibration (CV = 16% to 35%), no harmonization in Lp(a) values measured by different methods was achieved • The impact of apo(a) isoform size on Lp(a) concentrations varied among the different methods as a function of the apo(a) size of the assay calibrators • Other factors, like difference in instrumentation, also contributed to the lack of comparability of results • In 2003, the WHO Expert Committee on Biological Standardization accepted PRM- 2B, with an assigned value of 107 nmol/L, as the “First WHO/IFCC International Reference Reagent for Lipoprotein(a) Immunoassay” EAS 2019
Bias (%) 100 50 0 -50 -100 10 15 20 25 30 35 Major Kringle 4 Repeat EAS 2019
Bias (%) 100 50 0 -50 -100 10 15 20 25 30 35 Major Kringle 4 Repeat EAS 2019
Bias (%) 50 25 0 -25 -50 10 15 20 25 30 35 Major Kringle 4 Repeat EAS 2019
• Polyclonal antibody-based. Antibodies strongly reacting with apo(a) KIV Type 2 repeats. • Antibodies are coated on the surface of latex particles (diameter 120 nm) • Lp(a) particles in plasma are then bound to the antibodies on the latex particles to form immunocomplexes • Five independent calibrators with Lp(a) levels from low to high and apo(a) size from large to small EAS 2019
Signal Very Small Medium Small Medium Large Very Large Lp(a) nmol/L EAS 2019
Values expressed in mg/dL of total Lp(a) mass Single calibrator: no traceability to a common ➔ reference material • Effect of apo(a) size variation is inevitable Values traceable to the WHO/IFCC Reference Material and expressed in nmol/L of Lp(a) protein 5-point calibrators ➔ • Effect of apo(a) size variation is minimized EAS 2019
ITA-DENKA ITA apo(a) Kringle number accounts for 2.5% of the bias variation apo(a) Kringle number accounts for 61.1% of the bias variation EAS 2019
Denka Method – Instrument 1 Denka Method – Instrument 2 apo(a) Kringle number accounts for 1.0% of the bias variation apo(a) Kringle number accounts for 0.7% of the bias variation EAS 2019
Step 1 – Assignment of target values to assay calibrators • Target values traceable to the WHO-IFCC Reference Material Step 2 – Validation of the accuracy of value transfer • Six fresh-frozen samples from individual donors were prepared at the NWRL and were selected to have a suitable range of Lp(a) levels and apo(a) isoforms. EAS 2019
Step 3 • 80 fresh-frozen samples are analyzed, collected from apparently healthy donors and selected to represent a large range of Lp(a) levels and apo(a) isoforms • Detailed certification criteria have been established including analytical CV, correlation and absolute bias limits between obtained and assigned values, and the correlation between the % bias and apo(a) isoform size EAS 2019
System 1 System 2 System 3 System 4 EAS 2019
Recommend
More recommend
