Relating Lipoprotein(a) Concentrations to Cardiovascular Event Risk After Acute Coronary Syndrome: A Comparison of 3 Tests.

BACKGROUND: Lipoprotein(a) is a risk factor for cardiovascular events and modifies the benefit of PCSK9 (proprotein convertase subtilisin/kexin type 9) inhibitors. Lipoprotein(a) concentration can be measured with immunoassays reporting mass or molar concentration or a reference measurement system using mass spectrometry. Whether the relationships between lipoprotein(a) concentrations and cardiovascular events in a high-risk cohort differ across lipoprotein(a) methods is unknown. We compared the prognostic and predictive value of these types of lipoprotein(a) tests for major adverse cardiovascular events (MACE). METHODS: The ODYSSEY OUTCOMES trial (Evaluation of Cardiovascular Outcomes After an Acute Coronary Syndrome During Treatment With Alirocumab) compared the PCSK9 inhibitor alirocumab with placebo in patients with recent acute coronary syndrome. We compared risk of a MACE in the placebo group and MACE risk reduction with alirocumab according to baseline lipoprotein(a) concentration measured by Siemens N-latex nephelometric immunoassay (IA-mass; mg/dL), Roche Tina-Quant turbidimetric immunoassay (IA-molar; nmol/L), and a noncommercial mass spectrometry-based test (MS; nmol/L). Lipoprotein(a) values were transformed into percentiles for comparative modeling. Natural cubic splines estimated continuous relationships between baseline lipoprotein(a) and outcomes in each treatment group. Event rates were also determined across baseline lipoprotein(a) quartiles defined by each assay. RESULTS: Among 11 970 trial participants with results from all 3 tests, baseline median (Q1, Q3) lipoprotein(a) concentrations were 21.8 (6.9, 60.0) mg/dL, 45.0 (13.2, 153.8) nmol/L, and 42.2 (14.3, 143.1) nmol/L for IA-mass, IA-molar, and MS, respectively. The strongest correlation was between IA-molar and MS (r=0.990), with nominally weaker correlations between IA-mass and MS (r=0.967) and IA-mass and IA-molar (r=0.972). Relationships of lipoprotein(a) with MACE risk in the placebo group were nearly identical with each test, with estimated cumulative incidences differing by ≤0.4% across lipoprotein(a) percentiles, and all were incrementally prognostic after accounting for low-density lipoprotein cholesterol levels (all spline P≤0.0003). Predicted alirocumab treatment effects were also nearly identical for each of the 3 tests, with estimated treatment hazard ratios differing by ≤0.07 between tests across percentiles and nominally less relative risk reduction by alirocumab at lower percentiles for all 3 tests. Absolute risk reduction with alirocumab increased with increasing lipoprotein(a) measured by each test, with significant linear trends across quartiles. CONCLUSIONS: In patients with recent acute coronary syndrome, 3 lipoprotein(a) tests were similarly prognostic for MACE in the placebo group and predictive of MACE reductions with alirocumab at the cohort level. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT01663402.

Closing the gaps in patient management of dyslipidemia: stepping into cardiovascular precision diagnostics with apolipoprotein profiling.

In persons with dyslipidemia, a high residual risk of cardiovascular disease remains despite lipid lowering therapy. Current cardiovascular risk prediction mainly focuses on low-density lipoprotein cholesterol (LDL-c) levels, neglecting other contributing risk factors. Moreover, the efficacy of LDL-c lowering by statins resulting in reduced cardiovascular risk is only partially effective. Secondly, from a metrological viewpoint LDL-c falls short as a reliable measurand. Both direct and calculated LDL-c tests produce inaccurate test results at the low end under aggressive lipid lowering therapy. As LDL-c tests underperform both clinically and metrologically, there is an urging need for molecularly defined biomarkers. Over the years, apolipoproteins have emerged as promising biomarkers in the context of cardiovascular disease as they are the functional workhorses in lipid metabolism. Among these, apolipoprotein B (ApoB), present on all atherogenic lipoprotein particles, has demonstrated to clinically outperform LDL-c. Other apolipoproteins, such as Apo(a) - the characteristic apolipoprotein of the emerging risk factor lipoprotein(a) -, and ApoC-III - an inhibitor of triglyceride-rich lipoprotein clearance -, have attracted attention as well. To support personalized medicine, we need to move to molecularly defined risk markers, like the apolipoproteins. Molecularly defined diagnosis and molecularly targeted therapy require molecularly measured biomarkers. This review provides a summary of the scientific validity and (patho)physiological role of nine serum apolipoproteins, Apo(a), ApoB, ApoC-I, ApoC-II, ApoC-III, ApoE and its phenotypes, ApoA-I, ApoA-II, and ApoA-IV, in lipid metabolism, their association with cardiovascular disease, and their potential as cardiovascular risk markers when measured in a multiplex apolipoprotein panel.

An LC-MS-based designated comparison method with similar performance to the Lp(a) reference measurement procedure to guide molar Lp(a) standardization.

BACKGROUND: The 2022 consensus statement of the European Atherosclerosis Society (EAS) on lipoprotein(a) (Lp(a)) recognizes the role of Lp(a) as a relevant genetically determined risk factor and recommends its measurement at least once in an individual's lifetime. It also strongly urges that Lp(a) test results are expressed as apolipoprotein (a) (apo(a)) amount of substance in molar units and no longer in confounded Lp(a) mass units (mg/dL or mg/L). Therefore, IVD manufacturers should transition to molar units. A prerequisite for this transition is the availability of an Lp(a) Reference Measurement Procedure (RMP) that allows unequivocal molecular detection and quantification of apo(a) in Lp(a). To that end an ISO 17511:2020 compliant LC-MS based and IFCC-endorsed RMP has been established that targets proteotypic peptides of apolipoprotein(a) (apo(a)) in Lp(a). The RMP is laborious and requires highly skilled operators. To guide IVD-manufacturers of immunoassay-based Lp(a) test kits in the transition from mass to molar units, a Designated Comparison Method (DCM) has been developed and evaluated. METHODS: To assess whether the DCM provides equivalent results compared to the RMP, the procedural designs were compared and the analytical performance of DCM and RMP were first evaluated in a head-to-head comparison. Subsequently, apo(a) was quantified in 153 human clinical serum samples. Both DCM and RMP were calibrated using external native calibrators that produce results traceable to SRM2B. Measurement uncertainty (MU) was checked against predefined allowable MU. RESULTS: The major difference in the design of the DCM for apo(a) is the use of only one enzymatic digestion step. The analytical performance of the DCM and RMP for apo(a) is highly similar. In a direct method comparison, equivalent results were obtained with a median regression slope 0.997 of and a median bias of - 0.2 nmol/L (- 0.2%); the intermediate imprecision of the test results was within total allowable error (TEa) (CVa of 10.2% at 90 nmol/L). CONCLUSIONS: The semi-automated, higher throughput, LC-MS-based method for Lp(a) meets the predefined analytical performance specifications and allowable MU and is hence applicable as a higher order Designated Comparison Method, which is ideally suited to guide IVD manufacturers in the transition from Lp(a) mass to molar units.

Blood self-sampling devices: innovation, interpretation and implementation in total lab automation.

The introduction of the vacuum tube in 1949 revolutionized blood collection, significantly improving sample quality and patient comfort. Over the past 75 years, laboratory diagnostics have evolved drastically, from manual to automated processes, reducing required test volumes by over 1,000 times. Despite these advancements, venous blood collection presents logistical challenges, including centralized scheduling and a large volume of biological waste due to the imbalance between the needed blood volume (often very little) and the collected volume (often in excess). The COVID-19 pandemic further emphasized the need for decentralized healthcare solutions and patient empowerment. Capillary blood collection, widely used in point-of-care testing, offers a promising alternative, particularly for patients facing frequently, or difficulties with, venous sampling. The Leiden University Medical Center in the Netherlands experienced a 15 % reduction in volume of laboratory tests during and after the pandemic, attributed to patient preference for local blood collection and testing. To address these challenges, self-sampling devices are emerging, empowering patients and streamlining sample logistics. However, challenges such as cost, transportation regulations, and sample volume adequacy persists. Robust devices tailored for total lab automation and sustainable practices are crucial for widespread adoption. Despite hurdles, the integration of self-sampling into diagnostic processes is inevitable, heralding a shift towards patient-centered, proactive healthcare. Practical recommendations include robust device design, ease of use, affordability, sustainability, sufficient quality and acceptability by seamless integration into laboratory workflows. Although obstacles remain, self-sampling represents the future of laboratory diagnostics, offering convenience, cost-effectiveness, interoperability and patient empowerment.

Validation and verification framework and data integration of biosensors and in vitro diagnostic devices: a position statement of the IFCC Committee on Mobile Health and Bioengineering in Laboratory Medicine (C-MBHLM) and the IFCC Scientific Division.

Advances in technology have transformed healthcare and laboratory medicine. Biosensors have emerged as a promising technology in healthcare, providing a way to monitor human physiological parameters in a continuous, real-time, and non-intrusive manner and offering value and benefits in a wide range of applications. This position statement aims to present the current situation around biosensors, their perspectives and importantly the need to set the framework for their validation and safe use. The development of a qualification framework for biosensors should be conceptually adopted and extended to cover digitally measured biomarkers from biosensors for advancing healthcare and achieving more individualized patient management and better patient outcome.

Current Pyuria Cutoffs Promote Inappropriate Urinary Tract Infection Diagnosis in Older Women.

BACKGROUND: Pre-existing lower urinary tract symptoms (LUTS), cognitive impairment, and the high prevalence of asymptomatic bacteriuria (ASB) complicate the diagnosis of urinary tract infection (UTI) in older women. The presence of pyuria remains the cornerstone of UTI diagnosis. However, >90% of ASB patients have pyuria, prompting unnecessary treatment. We quantified pyuria by automated microscopy and flowcytometry to determine the diagnostic accuracy for UTI and to derive pyuria thresholds for UTI in older women. METHODS: Women ≥65 years with ≥2 new-onset LUTS and 1 uropathogen ≥104 colony-forming units (CFU)/mL were included in the UTI group. Controls were asymptomatic and classified as ASB (1 uropathogen ≥105 CFU/mL), negative culture, or mixed flora. Patients with an indwelling catheter or antimicrobial pretreatment were excluded. Leukocyte medians were compared and sensitivity-specificity pairs were derived from a receiver operating characteristic curve. RESULTS: We included 164 participants. UTI patients had higher median urinary leukocytes compared with control patients (microscopy: 900 vs 26 leukocytes/µL; flowcytometry: 1575 vs 23 leukocytes/µL; P < .001). Area under the curve was 0.93 for both methods. At a cutoff of 264 leukocytes/µL, sensitivity and specificity of microscopy were 88% (positive and negative likelihood ratio: 7.2 and 0.1, respectively). The commonly used cutoff of 10 leukocytes/µL had a poor specificity (36%) and a sensitivity of 100%. CONCLUSIONS: The degree of pyuria can help to distinguish UTI in older women from ASB and asymptomatic controls with pyuria. Current pyuria cutoffs are too low and promote inappropriate UTI diagnosis in older women. Clinical Trials Registration. International Clinical Trials Registry Platform: NL9477 (https://trialsearch.who.int/Trial2.aspx?TrialID=NL9477).

Developing an SI-traceable Lp(a) reference measurement system: a pilgrimage to selective and accurate apo(a) quantification.

In the past decade a remarkable rebirth of serum/plasma lipoprotein(a) (Lp(a)) as an independent risk factor of cardiovascular disease (CVD) occurred. Updated evidence for a causal continuous association in different ethnic groups between Lp(a) concentrations and cardiovascular outcomes has been published in the latest European Atherosclerosis Society (EAS) Lp(a) consensus statement. Interest in measuring Lp(a) at least once in a person's lifetime moreover originates from the development of promising new Lp(a) lowering drugs. Accurate and clinically effective Lp(a) tests are of key importance for the timely detection of high-risk individuals and for future evaluation of the therapeutic effects of Lp(a) lowering medication. To this end, it is necessary to improve the performance and standardization of existing Lp(a) tests, as is also noted in the Lp(a) consensus statement. Consequently, a state-of-the-art internationally endorsed reference measurement system (RMS) must be in place that allows for performance evaluation of Lp(a) field tests in order to certify their validity and accuracy. An ELISA-based RMS from Northwest Lipid Research Laboratory (University of Washington, Seattle, USA) has been available since the 1990s. A next-generation apo(a)/Lp(a) RMS is now being developed by a working group from the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC). The envisioned apo(a) RMS is based on the direct measurement of selected proteotypic fragments generated after proteolytic digestion using quantitative protein mass spectrometry (MS). The choice for an MS-based RMS enables selective measurement of the proteotypic peptides and is by design apo(a) isoform insensitive. Clearly, the equimolar conversion of apo(a) into the surrogate peptide measurands is required to obtain accurate Lp(a) results. The completeness of proteolysis under reaction conditions from the candidate reference measurement procedure (RMP) has been demonstrated for the quantifying apo(a) peptides. Currently, the candidate apo(a) RMP is endorsed by the IFCC and recommendations for suitable secondary reference materials have been made in a recent commutability study paper. Ongoing efforts toward a complete apo(a) RMS that is listed by the Joint Committee on Traceability in Laboratory Medicine (JCTLM) are focused on the peptide-based calibration and the establishment of a network of calibration laboratories running the apo(a) RMS in a harmonized way. Once completed, it will be the holy grail for evaluation and certification of Lp(a) field methods.

Urinary Kidney Injury Biomarkers Are Associated with Ischemia-Reperfusion Injury Severity in Kidney Allograft Recipients.

BACKGROUND: We explored the potential of emerging and conventional urinary kidney injury biomarkers in recipients of living donor (LD) or donation after circulatory death (DCD) kidney transplantation, patients with chronic kidney disease (CKD), and individuals from the general population. METHODS: Urine samples from kidney allograft recipients with mild (LD; n = 199) or severe (DCD; n = 71) ischemia-reperfusion injury (IRI) were analyzed for neutrophil gelatinase-associated lipocalin (NGAL), insulin-like growth factor-binding protein 7 (IGFBP7), tissue inhibitor of metalloproteinases 2 (TIMP2), kidney injury molecule-1 (KIM-1), chemokine C-X-C motif (CXCL9), solute carrier family 22 member 2 (SLC22A2), nephrin, and uromodulin (UMOD) by quantitative multiplex LC-MS/MS analysis. The fold-change in biomarker levels was determined in mild and severe IRI and in patients with CKD stage 1-2 (n = 127) or stage ≥3 (n = 132) in comparison to the general population (n = 1438). Relationships between the biomarkers and total protein, ß2-microglobulin (B2M), creatinine, and osmolality were assessed. RESULTS: NGAL, IGFBP7, TIMP2, KIM-1, CXCL9, and UMOD were quantifiable, whereas nephrin and SLC22A2 were below the limit of detection. Kidney injury biomarkers were increased up to 6.2-fold in allograft recipients with mild IRI and 8.3-fold in recipients with severe IRI, compared to the reference population, with the strongest response observed for NGAL and B2M. In CKD stage 1-2, B2M, NGAL, IGFBP7, TIMP2, KIM-1, UMOD, and CXCL9 were not altered, but in individuals with CKD stage ≥3, B2M, NGAL, and KIM-1 were increased up to 1.3-fold. IGFBP7, TIMP2, NGAL, and CXCL9 were strongly correlated (all r ≥ 0.8); correlations with B2M and TP were smaller (all r ≤ 0.6). CONCLUSIONS: IRI, but not stable CKD, was associated with increased urinary levels of kidney injury biomarkers determined by LC-MS/MS. Absolute and multiplexed protein quantitation by LC-MS/MS is an effective strategy for biomarker panel evaluation for translation toward the clinical laboratory.

Development of an LC-MRM-MS-Based Candidate Reference Measurement Procedure for Standardization of Serum Apolipoprotein (a) Tests.

BACKGROUND: Medical results generated by European CE Marking for In Vitro Diagnostic or in-house tests should be traceable to higher order reference measurement systems (RMS), such as International Federation of Clinical Chemistry and Laboratory Medicine (IFCC)-endorsed reference measurement procedures (RMPs) and reference materials. Currently, serum apolipoprotein (a) [apo(a)] is recognized as a novel risk factor for cardiovascular risk assessment and patient management. The former RMS for serum apo(a) is no longer available; consequently, an International System of Units (SI)-traceable, ideally multiplexed, and sustainable RMS for apo(a) is needed. METHODS: A mass spectrometry (MS)-based candidate RMP (cRMP) for apo(a) was developed using quantitative bottom-up proteomics targeting 3 proteotypic peptides. The method was provisionally validated according to ISO 15193 using a single human serum based calibrator traceable to the former WHO-IFCC RMS. RESULTS: The quantitation of serum apo(a) was by design independent of its size polymorphism, was linear from 3.8 to 456 nmol/L, and had a lower limit of quantitation for apo(a) of 3.8 nmol/L using peptide LFLEPTQADIALLK. Interpeptide agreement showed Pearson Rs of 0.987 and 0.984 for peptides GISSTVTGR and TPENYPNAGLTR, and method comparison indicated good correspondence (slopes 0.977, 1.033, and 1.085 for LFLEPTQADIALLK, GISSTVTGR, and TPENYPNAGLTR). Average within-laboratory imprecision of the cRMP was 8.9%, 11.9%, and 12.8% for the 3 peptides. CONCLUSIONS: A robust, antibody-independent, MS-based cRMP was developed as higher order RMP and an essential part of the apo(a) traceability chain and future RMS. The cRMP fulfils predefined analytical performance specifications, making it a promising RMP candidate in an SI-traceable MS-based RMS for apo(a).

Commutability Assessment of Candidate Reference Materials for Lipoprotein(a) by Comparison of a MS-based Candidate Reference Measurement Procedure with Immunoassays.

BACKGROUND: Elevated concentrations of lipoprotein(a) [Lp(a)] are directly related to an increased risk of cardiovascular diseases, making it a relevant biomarker for clinical risk assessment. However, the lack of global standardization of current Lp(a) measurement procedures (MPs) leads to inconsistent patient care. The International Federation for Clinical Chemistry and Laboratory Medicine working group on quantitating apolipoproteins by mass spectrometry (MS) aims to develop a next-generation SI (International system of units)-traceable reference measurement system consisting of a MS-based, peptide-calibrated reference measurement procedure (RMP) and secondary serum-based reference materials (RMs) certified for their apolipoprotein(a) [apo(a)] content. To reach measurement standardization through this new measurement system, 2 essential requirements need to be fulfilled: a sufficient correlation among the MPs and appropriate commutability of future serum-based RMs. METHODS: The correlation among the candidate RMP (cRMP) and immunoassay-based MPs was assessed by measuring a panel of 39 clinical samples (CS). In addition, the commutability of 14 different candidate RMs was investigated. RESULTS: Results of the immunoassay-based MPs and the cRMPs demonstrated good linear correlations for the CS but some significant sample-specific differences were also observed. The results of the commutability study show that RMs based on unspiked human serum pools can be commutable with CS, whereas human pools spiked with recombinant apo(a) show different behavior compared to CS. CONCLUSIONS: The results of this study show that unspiked human serum pools are the preferred candidate secondary RMs in the future SI-traceable Lp(a) Reference Measurement System.

Overcoming challenges regarding reference materials and regulations that influence global standardization of medical laboratory testing results.

BACKGROUND: Standardized results for laboratory tests are particularly important when their interpretation depends on fixed medical practice guidelines or common reference intervals. The medical laboratory community has developed a roadmap for an infrastructure to achieve standardized test results described in the International Organization for Standardization standard 17511:2020 In vitro diagnostic medical devices - Requirements for establishing metrological traceability of values assigned to calibrators, trueness control materials and human samples. Among the challenges to implementing metrological traceability are the availability of fit-for-purpose matrix-based certified reference materials (CRMs) and requirements for regulatory review that differ among countries. A workshop in December 2021 focused on these two challenges and developed recommendations for improved practices. DISCUSSION: The participants agreed that prioritization of measurands for standardization should be based on their impact on medical decisions in a clinical pathway. Ensuring that matrix-based CRMs are globally available for more measurands will enable fit-for-purpose calibration hierarchies for more laboratory tests. Regulation of laboratory tests is important to ensure safety and effectiveness for the populations served. Because regulations are country or region specific, manufacturers must submit recalibration changes intended to standardize results for regulatory review to all areas in which a measuring system is marketed. RECOMMENDATIONS: A standardization initiative requires collaboration and planning among all interested stakeholders. Global collaboration should be further developed for prioritization of measurands for standardization, and for coordinating the production and supply of CRMs worldwide. More uniform regulatory submission requirements are desirable when recalibration is implemented to achieve internationally standardized results.

Quantitative protein mass-spectrometry requires a standardized pre-analytical phase.

OBJECTIVES: Quantitative protein mass-spectrometry (QPMS) in blood depends on tryptic digestion of proteins and subsequent measurement of representing peptides. Whether serum and plasma can be used interchangeably and whether in-vitro anticoagulants affect the recovery is unknown. In our laboratory serum samples are the preferred matrix for QPMS measurement of multiple apolipoproteins. In this study, we investigated the effect of different matrices on apolipoprotein quantification by mass spectrometry. METHODS: Blood samples were collected from 44 healthy donors in Beckton Dickinson blood tubes simultaneously for serum (with/without gel) and plasma (heparin, citrate or EDTA). Nine apolipoproteins were quantified according to standard operating procedure using value-assigned native serum calibrators for quantitation. Tryptic digestion kinetics were investigated in the different matrices by following formation of peptides for each apolipoprotein in time, up to 22 h. RESULTS: In citrate plasma recovery of apolipoproteins showed an overall reduction with a bias of -14.6%. For heparin plasma only -0.3% bias was found compared to serum, whereas for EDTA-plasma reduction was more pronounced (-5.3% bias) and variable with >14% reduction for peptides of apoA-I, A-II and C-III. Digestion kinetics revealed that especially slow forming peptides showed reduced formation in EDTA-plasma. CONCLUSIONS: Plasma anticoagulants affect QPMS test results. Heparin plasma showed comparable results to serum. Reduced concentrations in citrate plasma can be explained by dilution, whereas reduced recovery in EDTA-plasma is dependent on altered proteolytic digestion efficiency. The results highlight the importance of a standardized pre-analytical phase for accurate QPMS applications in clinical chemistry.

Harmonization of indirect reference intervals calculation by the Bhattacharya method.

OBJECTIVES: The aim of this study was to harmonize the criteria for the Bhattacharya indirect method Microsoft Excel Spreadsheet for reference intervals calculation to reduce between-user variability and use these criteria to calculate and evaluate reference intervals for eight analytes in two different years. METHODS: Anonymized laboratory test results from outpatients were extracted from January 1st 2018 to December 31st 2019. To assure data quality, we examined the monthly results from an external quality control program. Reference intervals were determined by the Bhattacharya method with the St Vincent's hospital Spreadsheet firstly using original criteria and then using additional harmonized criteria defined in this study. Consensus reference intervals using the additional harmonized criteria were calculated as the mean of four users' lower and upper reference interval results. To further test the operation criteria and robustness of the obtained reference intervals, an external user validated the Spreadsheet procedure. RESULTS: The extracted test results for all selected laboratory tests fulfilled the quality criteria and were included in the present study. Differences between users in calculated reference intervals were frequent when using the Spreadsheet. Therefore, additional criteria for the Spreadsheet were proposed and applied by independent users, such as: to set central bin as the mean of all the data, bin size as small as possible, at least three consecutive bins and a high proportion of bins within the curve. CONCLUSIONS: The proposed criteria contributed to the harmonization of reference interval calculation between users of the Bhattacharya indirect method Spreadsheet.

ISO 15189 is a sufficient instrument to guarantee high-quality manufacture of laboratory developed tests for in-house-use conform requirements of the European In-Vitro-Diagnostics Regulation.

The EU In-Vitro Diagnostic Device Regulation (IVDR) aims for transparent risk-and purpose-based validation of diagnostic devices, traceability of results to uniquely identified devices, and post-market surveillance. The IVDR regulates design, manufacture and putting into use of devices, but not medical services using these devices. In the absence of suitable commercial devices, the laboratory can resort to laboratory-developed tests (LDT) for in-house use. Documentary obligations (IVDR Art 5.5), the performance and safety specifications of ANNEX I, and development and manufacture under an ISO 15189-equivalent quality system apply. LDTs serve specific clinical needs, often for low volume niche applications, or correspond to the translational phase of new tests and treatments, often extremely relevant for patient care. As some commercial tests may disappear with the IVDR roll-out, many will require urgent LDT replacement. The workload will also depend on which modifications to commercial tests turns them into an LDT, and on how national legislators and competent authorities (CA) will handle new competences and responsibilities. We discuss appropriate interpretation of ISO 15189 to cover IVDR requirements. Selected cases illustrate LDT implementation covering medical needs with commensurate management of risk emanating from intended use and/or design of devices. Unintended collateral damage of the IVDR comprises loss of non-profitable niche applications, increases of costs and wasted resources, and migration of innovative research to more cost-efficient environments. Taking into account local specifics, the legislative framework should reduce the burden on and associated opportunity costs for the health care system, by making diligent use of existing frameworks.

Interference by macroprolactin in assays for prolactin: will the In Vitro Diagnostics Regulation lead to a solution at last?

Cross reactivity with high molecular weight complexes of prolactin known as macroprolactin is a common cause of positive interference in assays for serum prolactin. All prolactin assays currently available are affected with 5-25% of results indicating hyperprolactinaemia falsely elevated due to macroprolactinaemia - hyperprolactinaemia due to macroprolactin with normal concentrations of bioactive monomeric prolactin. Macroprolactinaemia has no pathological significance but, if it is not recognised as the cause, the apparent hyperprolactinaemia can lead to clinical confusion, unnecessary further investigations, inappropriate treatment and waste of healthcare resources. Macroprolactinaemia cannot be distinguished from true hyperprolactinaemia on clinical grounds alone but can be detected by a simple laboratory test based on the precipitation of macroprolactin with polyethylene glycol. Laboratory screening of all cases of hyperprolactinaemia to exclude macroprolactinaemia has been advised as best practice but has not been implemented universally and reports of clinical confusion caused by macroprolactinaemia continue to appear in the literature. Information provided by manufacturers to users of assays for prolactin regarding interference by macroprolactin is absent or inadequate and does not comply with the European Union Regulation covering in vitro diagnostic medical devices (IVDR). As the IVDR is implemented notified bodies should insist that manufacturers of assays for serum prolactin comply with the regulations by informing users that macroprolactin is a source of interference which may have untoward clinical consequences and by providing an estimate of the magnitude of the interference and a means of detecting macroprolactinaemia. Laboratories should institute a policy for excluding macroprolactinaemia in all cases of hyperprolactinaemia.

Moving average quality control of routine chemistry and hematology parameters - a toolbox for implementation.

OBJECTIVES: Moving average quality control (MA QC) is a patient-based real-time quality control system. Advantages compared to conventional periodic internal quality control (IQC) include absence of commutability problems and continuous monitoring of performance. We implemented MA QC for multiple routine hematology and chemistry parameters. We describe the evaluation process and provide practical tools to aid MA QC implementation. METHODS: Nine parameters (serum sodium, calcium, bicarbonate and free thyroxine, hemoglobin [Hb], mean corpuscular volume, mean corpuscular hemoglobin concentration [MCHC], reticulocyte count and erythrocyte sedimentation rate [ESR]) were chosen for initial consideration. Using data extractions from the laboratory information system (LIS; General Laboratory Information Management System), evaluation of usefulness and optimization of MA QC settings was performed using bias detection curves. After this, MA QC settings were incorporated in our LIS for further evaluation and implementation in routine care. RESULTS: Three out of nine parameters (Hb, ESR, and sodium) were excluded from MA QC implementation due to high variation and technical issues in the LIS. For the six remaining parameters, MA QC showed added value to IQC and was therefore implemented in the LIS. For three parameters a direct MA alarm work-up method was set up, including newly developed built-in features in the LIS. For the other parameters, we identified MA utilization beyond real-time monitoring. CONCLUSIONS: Implementation of MA QC has added value for our laboratory setting. Additional utilization beyond real-time QC monitoring was identified. We find MA QC especially useful for trend monitoring, detection of small shifts after maintenance and inter-analyzer comparisons.

Longitudinal Serum Protein Analysis of Women with a High Risk of Developing Breast Cancer Reveals Large Interpatient Versus Small Intrapatient Variations: First Results from the TESTBREAST Study.

The prospective, multicenter TESTBREAST study was initiated with the aim of identifying a novel panel of blood-based protein biomarkers to enable early breast cancer detection for moderate-to-high-risk women. Serum samples were collected every (half) year up until diagnosis. Protein levels were longitudinally measured to determine intrapatient and interpatient variabilities. To this end, protein cluster patterns were evaluated to form a conceptual basis for further clinical analyses. Using a mass spectrometry-based bottom-up proteomics strategy, the protein abundance of 30 samples was analyzed: five sequential serum samples from six high-risk women; three who developed a breast malignancy (cases) and three who did not (controls). Serum samples were chromatographically fractionated and an in-depth serum proteome was acquired. Cluster analyses were applied to indicate differences between and within protein levels in serum samples of individuals. Statistical analyses were performed using ANOVA to select proteins with a high level of clustering. Cluster analyses on 30 serum samples revealed unique patterns of protein clustering for each patient, indicating a greater interpatient than intrapatient variability in protein levels of the longitudinally acquired samples. Moreover, the most distinctive proteins in the cluster analysis were identified. Strong clustering patterns within longitudinal intrapatient samples have demonstrated the importance of identifying small changes in protein levels for individuals over time. This underlines the significance of longitudinal serum measurements, that patients can serve as their own controls, and the relevance of the current study set-up for early detection. The TESTBREAST study will continue its pursuit toward establishing a protein panel for early breast cancer detection.

Update on apolipoprotein B.

PURPOSE OF REVIEW: The 2019 European Society of Cardiology/European Atherosclerosis Society Guidelines concluded that apolipoprotein B (apoB) was a more accurate measure of cardiovascular risk and a better guide to the adequacy of lipid lowering than low-density lipoprotein cholesterol (LDL-C) or non-high-density lipoprotein cholesterol (HDL-C). Also, they stated that apoB can be measured more accurately than LDL-C or non-HDL-C. This strong endorsement of the central role of apoB contrasts with the limited endorsement of apoB by the 2018 American College of Cardiology/American Heart Association Multisociety Guidelines. Nevertheless, both retained LDL-C as the primary metric to guide statin/ezetimibe/Proprotein convertase subtilisin/kexin type 9 (PCSK9) therapy. RECENT FINDINGS: This essay will review the most important recent advances in knowledge about apoB with particular emphasis on the results of Mendelian randomization studies and a new discordance analysis in subjects on statin therapy. We will also lay out why using LDL-C to guide the adequacy of lipid lowering therapy represents an interpretive error of the results of the statin/ezetimibe/PCSK9 inhibitor randomized clinical trials and therefore why apoB should be the primary metric to guide statin/ezetimibe/PCSK9 therapy. SUMMARY: There is now a robust body of evidence demonstrating the superiority of apoB over LDL-C and non-HDL-C as a clinical marker of cardiovascular risk. LDL-C is not the appropriate marker to assess the benefits of statin/ezetimibe/PCSK9 therapy.

Development and Provisional Validation of a Multiplex LC-MRM-MS Test for Timely Kidney Injury Detection in Urine.

Kidney injury is a complication frequently encountered in hospitalized patients. Early detection of kidney injury prior to loss of renal function is an unmet clinical need that should be targeted by a protein-based biomarker panel. In this study, we aim to quantitate urinary kidney injury biomarkers at the picomolar to nanomolar level by liquid chromatography coupled to tandem mass spectrometry in multiple reaction monitoring mode (LC-MRM-MS). Proteins were immunocaptured from urinary samples, denatured, reduced, alkylated, and digested into peptides before LC-MRM-MS analysis. Stable-isotope-labeled peptides functioned as internal standards, and biomarker concentrations were attained by an external calibration strategy. The method was evaluated for selectivity, carryover, matrix effects, linearity, and imprecision. The LC-MRM-MS method enabled the quantitation of KIM-1, NGAL, TIMP2, IGFBP7, CXCL9, nephrin, and SLC22A2 and the detection of TGF-ß1, cubilin, and uromodulin. Two to three peptides were included per protein, and three transitions were monitored per peptide for analytical selectivity. The analytical carryover was <1%, and minimal urine matrix effects were observed by combining immunocapture and targeted LC-MRM-MS analysis. The average total CV of all quantifier peptides was 26%. The linear measurement range was determined per measurand and found to be 0.05-30 nmol/L. The targeted MS-based method enables the multiplex quantitation of low-abundance urinary kidney injury biomarkers for future clinical evaluation.

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.