Towards an SI-Traceable Reference Measurement System for Seven Serum Apolipoproteins Using Bottom-Up Quantitative Proteomics: Conceptual Approach Enabled by Cross-Disciplinary/Cross-Sector Collaboration.

Current dyslipidemia management in patients with atherosclerotic cardiovascular disease (ASCVD) is based on traditional serum lipids. Yet, there is some indication from basic research that serum apolipoproteins A-I, (a), B, C-I, C-II, C-III, and E may give better pathophysiological insight into the root causes of dyslipidemia. To facilitate the future adoption of clinical serum apolipoprotein (apo) profiling for precision medicine, strategies for accurate testing should be developed in advance. Recent discoveries in basic science and translational medicine set the stage for the IFCC Working Group on Apolipoproteins by Mass Spectrometry. Main drivers were the convergence of unmet clinical needs in cardiovascular disease (CVD) patients with enabling technology and metrology. First, the residual cardiovascular risk after accounting for established risk factors demonstrates that the current lipid panel is too limited to capture the full complexity of lipid metabolism in patients. Second, there is a need for accurate test results in highly polymorphic and atherogenic apolipoproteins such as apo(a). Third, sufficient robustness of mass spectrometry technology allows reproducible protein quantification at the molecular level. Fourth, several calibration hierarchies in the revised ISO 17511:2020 guideline facilitate metrological traceability of test results, the highest achievable standard being traceability to SI. This article outlines the conceptual approach aimed at achieving a novel, multiplexed Reference Measurement System (RMS) for seven apolipoproteins based on isotope dilution mass spectrometry and peptide-based calibration. This RMS should enable standardization of existing and emerging apolipoprotein assays to SI, within allowable limits of measurement uncertainty, through a sustainable network of Reference Laboratories.

Development of a certified reference material for anti-ß2-glycoprotein I IgG - commutability studies.

Objectives: In this paper, we describe the steps followed for the development of a certified reference material for immunoglobulin G antibodies against ß2-glycoprotein I (also known as apolipoprotein H). These steps include processing of the material, commutability, the impact of dilution, the appropriate reconstitution conditions, homogeneity and stability during transport and storage. Methods: We analysed 69 clinical samples from patients suffering from antiphospholipid syndrome with several commercial enzyme-linked immunosorbent assays (ELISA) purchased from in vitro diagnostic manufacturers. Results: Analysis of the results indicated that the candidate reference material can be safely freeze-dried, and that the user should carefully follow the reconstitution instructions as small changes in e.g. temperature may have unwanted effects. The statistical analysis of the commutability studies indicated that the analytical response of the reference material upon dilution is similar to that of clinical samples, and that correlation between results may differ from assay to assay. Finally yet importantly, the presented and developed candidate reference material is commutable for most assays tested, homogeneous and stable. Conclusions: Immunoglobulin G antibodies against ß2-glycoprotein I are associated with a higher risk of thrombosis and pregnancy complications. Their measurement is essential for the diagnosis and monitoring of antiphospholipid syndrome. These antibodies are detected by specific immunoassays, routinely used in clinical diagnostics, but various of these methods show enormous variability, in part due to the lack of a reference material.

First amyloid ß1-42 certified reference material for re-calibrating commercial immunoassays.

INTRODUCTION: Reference materials based on human cerebrospinal fluid were certified for the mass concentration of amyloid beta (Aß)1-42 (Aß42 ). They are intended to be used to calibrate diagnostic assays for Aß42 . METHODS: The three certified reference materials (CRMs), ERM-DA480/IFCC, ERM-DA481/IFCC and ERM-DA482/IFCC, were prepared at three concentration levels and characterized using isotope dilution mass spectrometry methods. Roche, EUROIMMUN, and Fujirebio used the three CRMs to re-calibrate their immunoassays. RESULTS: The certified Aß42 mass concentrations in ERM-DA480/IFCC, ERM-DA481/IFCC, and ERM-DA482/IFCC are 0.45, 0.72, and 1.22 µg/L, respectively, with expanded uncertainties (k = 2) of 0.07, 0.11, and 0.18 µg/L, respectively. Before re-calibration, a good correlation (Pearson's r > 0.97), yet large biases, were observed between results from different commercial assays. After re-calibration the between-assay bias was reduced to < 5%. DISCUSSION: The Aß42 CRMs can ensure the equivalence of results between methods and across platforms for the measurement of Aß42 .

Certified reference material against PR3 ANCA IgG autoantibodies. From development to certification.

Background The importance of the standardisation of immunoassays for autoantibodies has been widely discussed. The appropriate use of certified reference materials (CRM) could contribute to a more accurate diagnosis and follow-up of a series of diseases such as small vessel-associated vasculitis. This is a systemic autoimmune disorder during which two autoantibodies can be present, MPO ANCA IgG and PR3 ANCA IgG. Results from different commercially available immunoassays used for PR3 ANCA IgG measurement can vary significantly. Therefore the potential for improvement using a suitable certified reference material was assessed and led to the development of a CRM. Methods Thirty clinical samples were evaluated using 10 immunoassays. The correlation between results from these assays was assessed in a pairwise manner. Feasibility studies were conducted in order to find a reference material format most suitable for the preparation of a CRM. Results The evaluation of two sets of 30 clinical samples with 10 assays showed that differences between assays can result in different interpretations for individual clinical samples. Most of the samples had the same result classification in all assays. However, six of the samples tested led to inconsistent results. Conclusions The correlation between results from clinical samples was systematically good for combinations of eight of those assays. Therefore, it should be possible to improve the comparability of results using a commutable CRM for calibration. Based on these studies, a final format for the CRM was selected and eventually produced and certified for its PR3 ANCA IgG content.

Commutability Assessment of Candidate Reference Materials for Pancreatic α-Amylase.

BACKGROUND: Measurement standardization of the catalytic concentration of α-amylase in serum is based on 3 pillars: the primary reference measurement procedure (PRMP), reference laboratories, and suitable certified reference materials (CRMs). Commutability is a prerequisite when using a CRM for calibration and trueness control of routine methods or for value transfer from the PRMP to end-user calibrators of routine methods through a calibration hierarchy. METHODS: We performed a commutability study with 30 serum pools and 5 candidate reference materials (RMs) for pancreatic α-amylase using an automated version of the PRMP and 5 different routine methods. Four candidate RMs had an artificial matrix, each with a different composition, and 1 candidate RM was based on human serum. Data were analyzed according to a linear regression analysis with prediction interval as described in the Clinical and Laboratory Standards Institute guideline EP30-A and a difference in bias analysis as described in the recommendations of the IFCC Working Group on Commutability. RESULTS: The commutability profile of the 4 candidate RMs with an artificial matrix was variable. Only 1 candidate RM, with human serum albumin in the matrix, showed a good profile like that of the candidate RM based on serum. The comparison of both commutability assessment approaches indicated some differences because of inconclusive results for the difference in bias approach, suggesting a large uncertainty on the commutability assessment. CONCLUSIONS: A CRM for pancreatic amylase in an artificial matrix can be commutable for routine methods using the same substrate as the PRMP, but the matrix composition is crucial.

IFCC Working Group Recommendations for Assessing Commutability Part 1: General Experimental Design.

Commutability is a property of a reference material (RM) that relates to the closeness of agreement between results for an RM and results for clinical samples (CSs) when measured by ≥2 measurement procedures (MPs). Commutability of RMs used in a calibration traceability scheme is an essential property for them to be fit for purpose. Similarly, commutability of trueness controls or external quality assessment samples is essential when those materials are used to assess trueness of results for CSs. This report is part 1 of a 3-part series describing how to assess commutability of RMs. Part 1 defines commutability and addresses critical components of the experimental design for commutability assessment, including selection of individual CSs, use of pooled CSs, qualification of MPs for inclusion, establishing criteria for the determination that an RM is commutable, generalization of commutability conclusions to future measurements made with the MPs included in the assessment, and information regarding commutability to be included in the certificate for an RM. Parts 2 and 3 in the series present 2 different statistical approaches to commutability assessment that use fixed criteria related to the medical decisions that will be made using the laboratory test results.

Commutability of the certified reference materials for the standardization of ß-amyloid 1-42 assay in human cerebrospinal fluid: lessons for tau and ß-amyloid 1-40 measurements.

BACKGROUND: The core Alzheimer's disease cerebrospinal fluid (CSF) biomarkers total tau (T-tau), phosphorylated tau (P-tau), ß-amyloid 1-42 (Aß42) and ß-amyloid 1-40 (Aß40) are increasing in importance and are now part of the research criteria for the diagnosis of the disease. The main aim of this study is to evaluate whether a set of certified reference materials (CRMs) are commutable for Aß42 and to serve as a feasibility study for the other markers. This property is a prerequisite for the establishment of CRMs which will then be used by manufacturers to calibrate their assays against. Once the preanalytical factors have been standardized and proper selection criteria are available for subject cohorts this harmonization between methods will allow for universal cut-offs to be determined. METHODS: Thirty-four individual CSF samples and three different CRMs where analyzed for T-tau, P-tau, Aß42 and Aß40, using up to seven different commercially available methods. For Aß40 and Aß42 a mass spectrometry-based procedure was also employed. RESULTS: There were strong pairwise correlations between the different methods (Spearman's ρ>0.92) for all investigated analytes and the CRMs were not distinguishable from the individual samples. CONCLUSIONS: This study shows that the CRMs are commutable for the different assays for Aß42. For the other analytes the results show that it would be feasible to also produce CRMs for these. However, additional studies are needed as the concentration interval for the CRMs were selected based on Aß42 concentrations only and did in general not cover satisfactory large concentration intervals for the other analytes.

Assessing the commutability of reference material formats for the harmonization of amyloid-ß measurements.

BACKGROUND: The cerebrospinal fluid (CSF) amyloid-ß (Aß42) peptide is an important biomarker for Alzheimer's disease (AD). Variability in measured Aß42 concentrations at different laboratories may be overcome by standardization and establishing traceability to a reference system. Candidate certified reference materials (CRMs) are validated herein for this purpose. METHODS: Commutability of 16 candidate CRM formats was assessed across five CSF Aß42 immunoassays and one mass spectrometry (MS) method in a set of 48 individual clinical CSF samples. Promising candidate CRM formats (neat CSF and CSF spiked with Aß42) were identified and subjected to validation across eight (Elecsys, EUROIMMUN, IBL, INNO-BIA AlzBio3, INNOTEST, MSD, Simoa, and Saladax) immunoassays and the MS method in 32 individual CSF samples. Commutability was evaluated by Passing-Bablok regression and the candidate CRM termed commutable when found within the prediction interval (PI). The relative distance to the regression line was assessed. RESULTS: The neat CSF candidate CRM format was commutable for almost all method comparisons, except for the Simoa/MSD, Simoa/MS and MS/IBL where it was found just outside the 95% PI. However, the neat CSF was found within 5% relative distance to the regression line for MS/IBL, between 5% and 10% for Simoa/MS and between 10% and 15% for Simoa/MSD comparisons. CONCLUSIONS: The neat CSF candidate CRM format was commutable for 33 of 36 method comparisons, only one comparison more than expected given the 95% PI acceptance limit. We conclude that the neat CSF candidate CRM can be used for value assignment of the kit calibrators for the different Aß42 methods.

Round robin test on quantification of amyloid-ß 1-42 in cerebrospinal fluid by mass spectrometry.

INTRODUCTION: Cerebrospinal fluid (CSF) amyloid-ß 1-42 (Aß42) is an important biomarker for Alzheimer's disease, both in diagnostics and to monitor disease-modifying therapies. However, there is a great need for standardization of methods used for quantification. To overcome problems associated with immunoassays, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as a critical orthogonal alternative. METHODS: We compared results for CSF Aß42 quantification in a round robin study performed in four laboratories using similar sample preparation methods and LC-MS instrumentation. RESULTS: The LC-MS results showed excellent correlation between laboratories (r(2) >0.98), high analytical precision, and good correlation with enzyme-linked immunosorbent assay (r(2) >0.85). The use of a common reference sample further decreased interlaboratory variation. DISCUSSION: Our results indicate that LC-MS is suitable for absolute quantification of Aß42 in CSF and highlight the importance of developing a certified reference material.

Performance criteria for reference measurement procedures and reference materials.

The concept of metrological traceability of measurement results to property values assigned to measurement standards of higher metrological order or to the International System of Units (SI) through sequential calibrations, using reference materials and reference measurement procedures, plays a key role in ensuring that end user measurement procedures perform at an acceptable level in the clinical context. The aim is that measurement results produced over time or by different end users or with different end user measurement procedures for the same measurand will be equivalent within their corresponding uncertainties. These goals can only be reached under certain conditions and if requirements laid down in international standards on calibration concepts, reference measurement procedures and reference materials are fulfilled. Calibration hierarchies have to be implemented correctly and parameters contributing to measurement uncertainty and systematic bias need to be controlled and eliminated, respectively, by technically improving methods and reference materials and intermediate calibrators used for effectively achieving equivalence of measurement results and for meeting analytical performance requirements for in vitro diagnostic devices.

Generation of a new cystatin C-based estimating equation for glomerular filtration rate by use of 7 assays standardized to the international calibrator.

BACKGROUND: Many different cystatin C-based equations exist for estimating glomerular filtration rate. Major reasons for this are the previous lack of an international cystatin C calibrator and the nonequivalence of results from different cystatin C assays. METHODS: Use of the recently introduced certified reference material, ERM-DA471/IFCC, and further work to achieve high agreement and equivalence of 7 commercially available cystatin C assays allowed a substantial decrease of the CV of the assays, as defined by their performance in an external quality assessment for clinical laboratory investigations. By use of 2 of these assays and a population of 4690 subjects, with large subpopulations of children and Asian and Caucasian adults, with their GFR determined by either renal or plasma inulin clearance or plasma iohexol clearance, we attempted to produce a virtually assay-independent simple cystatin C-based equation for estimation of GFR. RESULTS: We developed a simple cystatin C-based equation for estimation of GFR comprising only 2 variables, cystatin C concentration and age. No terms for race and sex are required for optimal diagnostic performance. The equation, [Formula: see text] is also biologically oriented, with 1 term for the theoretical renal clearance of small molecules and 1 constant for extrarenal clearance of cystatin C. CONCLUSIONS: A virtually assay-independent simple cystatin C-based and biologically oriented equation for estimation of GFR, without terms for sex and race, was produced.

Ruptured Baker Cyst on 18 F-FDG PET/CT.

ABSTRACT: A 78-year-old woman with diffuse large B-cell lymphoma was referred for an 18 F-FDG PET/CT to evaluate therapy response after 6 cycles of R-mini-CHOP. A new 18 F-FDG accumulation was noticed medial in the upper part of the right lower leg, spreading along the medial head of the gastrocnemius muscle. The shaft-bow-looking curvature, arch sign, of 18 F-FDG revealed a fluid collection on CT. This typical pattern and its specific location are indicative of a ruptured Baker's cyst. It can manifest in 1 or both legs.

Gaps in the traceability chain of human growth hormone measurements.

BACKGROUND: Human growth hormone (hGH) is measured for the diagnosis of secretion disorders. These measurements fall under the EU Directive 98/79/EC on in vitro diagnostic medical devices requiring traceability of commercial calibrator values to higher-order reference materials or procedures (Off J Eur Communities 1998 Dec 7;L 331:1-37). External quality assessment schemes show large discrepancies between results from different methods, even though most methods provide results traceable to the recommended International Standard (IS 98/574). The aim of this study was to investigate possible causes for these discrepancies. METHODS: We investigated the commutability and recovery of hGH in reconstituted IS 98/574. We tested different reconstitution protocols and used 4 different serum matrices for spiking. These IS preparations were measured together with serum samples. We quantified hGH by 5 different methods in 4 different laboratories. RESULTS: Results from the different methods correlated well for the serum samples. Mean discrepancies between results from different methods were ≤20%. None of the IS preparations was commutable for all the method comparisons. The recovery of hGH in preparations of IS 98/574 depended on the reconstitution protocol (>10-fold differences) and background matrix (relative differences ≤17% for different serum matrices). CONCLUSIONS: The use of different protocols for reconstitution and spiking of hGH reference preparations affects quantification by immunoassays, potentially leading to a bias between commercial methods, despite the use of calibrators with values claimed to be traceable to the same higher-order reference material.

The importance of commutability of reference materials used as calibrators: the example of ceruloplasmin.

BACKGROUND: Different methods for ceruloplasmin tend to give different results in external quality assessment schemes. During the production of the certified reference material ERM-DA470k/IFCC discrepant measurement results were also found for ceruloplasmin measured with different methods, and consequently the protein could not be certified in the material. METHODS: We performed a commutability study with 30 serum samples and the reference materials ERM-DA470, ERM-DA470k/IFCC, and ERM-DA472/IFCC, using 6 different methods. Data were analyzed according to the CLSI Guideline C53-A to assess whether the reference materials had the same behavior as the serum samples with respect to measurement results obtained with combinations of the methods used. RESULTS: Measurement results from different methods showed a good linear correlation for the serum samples. ERM-DA470 showed marked noncommutability for certain combinations of methods. ERM-DA470k/IFCC and ERM-DA472/IFCC were commutable for more combinations of methods. The lack of commutability of ERM-DA470 for certain combinations of methods correlates with results from the UK National External Quality Assessment Service showing discrepancies between results from these methods. For serum stored in the presence of sodium azide the results from different methods are essentially equivalent. CONCLUSIONS: Ceruloplasmin in ERM-DA470 is a fully documented example of a situation in which, due to lack of commutability, the use of a common material for calibration did not lead to harmonization .

A reference system for urinary albumin: current status.

BACKGROUND: Increased urinary excretion of albumin reflects kidney damage and is a recognized risk factor for progression of renal and cardiovascular disease. Considerable inter-method differences have been reported for both albumin and creatinine measurement results, and therefore the albumin-to-creatinine ratio. Measurement accuracy is unknown and there are no independent reference measurement procedures for albumin and no reference materials for either measurand in urine. METHODS: The National Kidney Disease Education Program (NKDEP) Laboratory Working Group and the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) have initiated joint projects to facilitate standardization of urinary albumin and creatinine measurement. RESULTS: A candidate LC-MS/MS reference measurement procedure for urinary albumin and candidate reference materials for urinary albumin and creatinine has been developed. The status of validations of these reference system components is reported. CONCLUSIONS: The development of certified reference materials and reference measurement procedures for urinary albumin will enable standardization of this important measurand.

Analysis of the physicochemical state of C-reactive protein in different preparations including 2 certified reference materials.

BACKGROUND: Standardization of clinical measurements of C-reactive protein (CRP) is based on the availability of reference materials. The serum protein reference material ERM-DA470 was used as a calibrant for various commercially available immunoassays but has now been exhausted. The recently released ERM-DA470k/IFCC was intended to fully replace ERM-DA470. However, the new material was not suited for the certification of CRP because of a bias introduced by the lyophilization process that caused loss of about 20% of CRP measurable by routine immunoassays, compared with the nonlyophilized material that was stored in a liquid frozen state. METHODS: We investigated the physicochemical state of CRP in a set of 4 lyophilized and 2 nonlyophilized serum-based CRP-containing materials by semi-native gel electrophoresis, Western blotting, and gel filtration. RESULTS: We detected a monomeric form of CRP (mCRP) in lyophilized materials at a concentration significantly higher than seen in the materials not subjected to lyophilization. Different reconstitution protocols led to variations of the monomeric CRP fraction found in reconstituted, previously lyophilized material. CONCLUSIONS: Most of the 20% loss in measured CRP after lyophilization of the material can be accounted for by the dissociation of natively pentameric CRP into subunits. The observed dissociation results from lyophilization and subsequent reconstitution of the material at insufficient concentration levels of calcium ions. In the presence of various protein forms, differences in antibody specificity and reactivity between immunoassays and alterations of stoichiometry of antigen-antibody interactions can contribute to the divergence of the measured values.

Characterization of the new serum protein reference material ERM-DA470k/IFCC: value assignment by immunoassay.

BACKGROUND: The availability of a suitable matrix reference material is essential for standardization of the immunoassays used to measure serum proteins. The earlier serum protein reference material ERM-DA470 (previously called CRM470), certified in 1993, has led to a high degree of harmonization of the measurement results. A new serum protein material has now been prepared and its suitability in term of homogeneity and stability has been verified; after characterization, the material has been certified as ERM-DA470k/IFCC. METHODS: We characterized the candidate reference material for 14 proteins by applying a protocol that is considered to be a reference measurement procedure, by use of optimized immunoassays. ERM-DA470 was used as a calibrant. RESULTS: For 12 proteins [α(2) macroglobulin (A2M), α(1) acid glycoprotein (orosomucoid, AAG), α(1) antitrypsin (α(1)-protease inhibitor, AAT), albumin (ALB), complement 3c (C3c), complement 4 (C4), haptoglobin (HPT), IgA, IgG, IgM, transferrin (TRF), and transthyretin (TTR)], the results allowed assignment of certified values in ERM-DA470k/IFCC. For CRP, we observed a bias between the lyophilized and liquid frozen materials, and for CER, the distribution of values was too broad. Therefore, these 2 proteins were not certified in the ERM-DA470k/IFCC. Different value transfer procedures were tested (open and closed procedures) and found to provide equivalent results. CONCLUSIONS: A new serum protein reference material has been produced, and values have been successfully assigned for 12 proteins.

First certified reference material for cystatin C in human serum ERM-DA471/IFCC.

The IFCC Working Group for the Standardisation of Cystatin C (WG-SCC), in collaboration with the Institute for Reference Materials and Measurements (IRMM), announces the availability of the new certified reference material ERM-DA471/IFCC. The material was characterised using a pure protein primary reference preparation (PRP) as calibrant. The PRP was prepared from recombinant cystatin C, and its concentration measured using dry mass determination. The characterisation of ERM-DA471/IFCC was performed by particle enhanced immuno-nephelometry, particle enhanced immuno-turbidimetry, and enzyme amplified single radial immuno-diffusion. The certified cystatin C mass concentration in ERM-DA471/IFCC, if reconstituted according to the specified procedure, is 5.48 mg/L, the expanded uncertainty (k=2) being 0.15 mg/L.

Standardizing plasma protein measurements worldwide: a challenging enterprise.

The need for harmonizing laboratory results is particularly intense in the field of quantitative protein assays in consideration of the clinical impact of specific protein measurements and their relevance in monitoring disease. We report the efforts made by the Committee on Plasma Proteins of the IFCC Scientific Division to achieve worldwide comparability in plasma protein results. We focus on the production of reference materials and the methods applied throughout their production process. Particularly, the recent characterization of ERM-DA470k/IFCC and ERM-DA472/IFCC has demonstrated that it is possible to reproduce the earlier established procedures and thereby maintain standardization. Plasma protein reference materials have had a substantial impact in improving the harmonization of patient protein results that should translate into better patient care.