Search

Aim: Lipoprotein (a) [Lp(a)] is a well-established risk factor for cardiovascular disease independent of low-density lipoprotein-cholesterol (LDL-C). The Lp(a) concentrations were inconsistent between the immunoassays. This study aimed to investigate whether harmonization of Lp(a) measurements can be achieved using a serum panel value assigned with the IFCC-endorsed mass spectrometry-based reference measurement procedure (IFCC-MS-RMP)., Methods: We measured the Lp(a) concentrations using five Lp(a) immunoassays in 40 panel sera provided by the Centers for Disease Control and Prevention (CDC), and 500 Japanese subjects enrolled in the Bunkyo Health Study. Of the five immunoassays, only the Roche Lp(a) assay was traceable to the WHO-IFCC reference material SRM2B. Lp(a) concentrations in CDC samples were also determined by IFCC-MS-RMP, provisionally calibrated to SRM2B. Lp(a) concentrations were expressed in mass units (mg/dL) for most reagents, but in SI units (nmol/L) for Roche's reagent and IFCC-MS-RMP., Results: In the CDC panel sera, all immunoassays, including Roche's reagent, showed good correlations with IFCC-MS-RMP. In the Bunkyo Health Study samples, all immunoassays showed good correlations with Roche's reagent (r s , 0.986-0.998) although the slopes of the regression lines ranged from 0.292 to 0.579. After recalibration with the CDC's panel sera, Lp(a) results of Bunkyo Health Study samples were converted to the equivalent values determined by the IFCC-MS-RMP, thus resulting in a marked reduction in the intermethod CV among the assays., Conclusion: We achieved harmonization of Lp(a) measurements with five immunoassays using a serum panel value assigned with the IFCC-MS-RMP.

Background: To evaluate the clinical performance and effectiveness of a multiplex apolipoprotein panel in the context of cardiovascular precision diagnostics, clinical samples of patients with recent acute coronary syndrome in the ODYSSEY OUTCOMES trial were measured by quantitative clinical chemistry proteomics (qCCP). The ISO15189-accredited laboratory setting, including the total testing process (TTP), served as a foundation for this study. Consequently, tailored quality assurance measures needed to be designed and implemented to suit the demands of a multiplex LC-MS/MS test., Methods: Nine serum apolipoproteins were measured in 23 376 samples with a laboratory-developed multiplex apolipoprotein test on 4 Agilent 6495 LC-MS/MS systems. A fit-for-purpose process was designed with tailored additions enhancing the accredited laboratory infrastructure and the TTP. Quality assurance was organized in 3 steps: system suitability testing (SST), internal quality control (IQC) evaluation with adjusted Westgard rules to fit a multiplex test, and interpeptide agreement analysis. Data was semi-automatically evaluated with a custom R script., Results: LC-MS/MS analyses were performed with the following between-run CVs: for apolipoprotein (Apo) (a) 6.2%, Apo A-I 2.3%, Apo A-II 2.1%, Apo A-IV 2.9%, Apo B 1.9%, Apo C-I 3.3%, Apo C-II 3.3%, Apo C-III 2.7%, and for Apo E 3.3% and an average interpeptide agreement Pearson r of 0.981., Conclusions: This is the first study of its kind in which qCCP was performed at this scale. This research successfully demonstrates the feasibility of high-throughput LC-MS/MS applications in large clinical trials. ClinicalTrials.gov Registration Number: NCT01663402., (© Association for Diagnostics & Laboratory Medicine 2024.)

Objectives: As high-sensitivity cardiac troponin T (hs-cTnT) is making the transition from diagnostic to prognostic use, a long-term stability study of 5th generation hs-cTnT according to EFLM CRESS recommendations was set up for investigation of frozen clinical specimens (two matrices)., Methods: Study samples collected in serum tubes and lithium heparin tubes with gel from patients admitted for suspected minor myocardial damage were measured directly after completion of the study (0 years), and after 3-year and 6-year storage at -80 °C, and recovery of hs-cTnT concentrations after long-term storage (%hs-cTnT concentration compared to 0-year) was calculated. Hs-cTnT changes were also compared to decisive delta changes, such as the ones proposed in the ESC NSTEMI 0 h/1 h algorithm (<3 or >5 ng/L for ruling out and ruling in suspected NSTEMI patients)., Results: Eighty-six patients were included in the study, whereof 28 both lithium heparin plasma and serum samples were collected simultaneously, in others only serum (n=30) or plasma (n=28). Multiple aliquots per patient were made, so that 479 serum and 473 plasma samples were available for analysis. Across the overall hs-cTnT measuring range, median recovery after 6 years was 105.4 % and 106.2 % for serum and plasma, respectively. Based on these decisive delta changes, serum showed consistent results upon long term storage (max 0.8 % of samples above delta threshold of >5 ng/L) as compared to heparin plasma (up to 19.2 % of samples above threshold)., Conclusions: Over 6 years of storage at -80 °C, recovery of hs-cTnT in serum and heparin plasma was similar and within common lot-to-lot variation. Yet, when evaluating absolute delta increments around hs-cTnT clinical decision points, long-term stored sera displayed better clinical performance compared to heparin plasma samples., (© 2024 the author(s), published by De Gruyter, Berlin/Boston.)

Objectives: Quantitative protein mass spectrometry (MS) is ideally suited for precision diagnostics and for reference standardization of protein analytes. At the Leiden Apolipoprotein Reference Laboratory we apply MS strategies to obtain detailed insight into the protein-to-peptide conversion in order to verify that quantifier peptides are not partly concealed in miscleaved protein backbone., Methods: Apolipoprotein(a) (apo(a)) was digested in a non-optimal manner to enhance the number of miscleaved peptides that were identified by high resolution liquid chromatography tandem-MS measurements. The protein-to-peptide conversion was carefully mapped with specific attention for miscleaved peptides that contain an apo(a) quantifier peptide. Four different isotopologues of each apo(a)-quantifier peptide were applied to evaluate linearity of internal peptide standards during measurement of specific real-life samples., Results: Two apo(a) quantifier peptides that were concealed in two different miscleaved peptides were included into a multiple reaction monitoring list in our targeted MS-based apo(a) quantifications to alert for potential protein digestion discrepancies. The presence of miscleaved peptides could be ruled out when applying our candidate reference measurement procedure (RMP) for apo(a) quantification., Conclusions: These data further corroborate the validity of our apo(a) candidate RMP as higher order method for certification of commercial Lp(a) tests that is endorsed by the International Federation of Clinical Chemistry and Laboratory Medicine. MS-based molecular detection and quantification of heterogeneous apo(a) proteoforms will allow manufacturers' transitioning from confounded lipoprotein(a) [Lp(a)] mass levels into accurate molar apo(a) levels., (© 2024 the author(s), published by De Gruyter, Berlin/Boston.)

Background: Although P5 (preventive, personalized, predictive, participatory, psychocognitive) medicine and patient-focused healthcare are gaining ground in various healthcare areas, the diagnosis of antithrombin deficiency (ATD) is still based on crude diagnostic tests, clustering patients into clinically heterogeneous subgroups whereby relevant thrombophilia phenotypes may go unnoticed. Clinical pathways and the majority of evidence are based on these tests; therefore, generic treatment is still the norm., Objectives: To unravel the heterogeneity of ATD, a mass spectrometry (liquid chromatography coupled to multiple-reaction-monitoring mass spectrometry [LC-MRM-MS])-based test for antithrombin was developed allowing molecular characterization of the antithrombin proteoforms in patient plasma. This study provides the first insight into the tests' clinical performance., Methods: Plasma from 91 unrelated ATD patients and 41 patients with a congenital disorder of glycosylation affecting antithrombin glycosylation were characterized functionally, genetically, and analyzed by LC-MRM-MS. An established data analysis strategy was applied for quantitation and molecular characterization of antithrombin proteoforms., Results: The test recognized patients with a quantitative defect, discriminated between type I and type II ATD, and identified variant proteoforms. Overall, the diagnostic sensitivity for ATD was 100% for LC-MRM-MS compared with 81.1% by the functional test. Type II ATD, a subtype prone to misdiagnosis, revealed an even larger difference of 100% identification by LC-MRM-MS vs 56.8% by functional test., Conclusion: The qualitative and quantitative mass spectrometry-based AT-test can serve as a platform for investigating the molecular basis of the clinical heterogeneity of ATD. This "precision diagnostics" approach for ATD can lower diagnostic uncertainty and modernize the ATD diagnostic and clinical pathways., Competing Interests: Declaration of competing interests There are no competing interests to disclose., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

European Union (EU) regulations on in vitro diagnostics (IVD) and on serious cross-border threats to health provide for the establishment of European Reference Laboratories (EURLs) and their harmonization and cooperation with National Reference Laboratories (NRLs). While the EURLs under the IVD Regulation will be operational by 1 October 2024, the EURLs under the Regulation on serious cross-border threats to health will be operational by January 2025. Although NRLs may have been operating for a long time on the basis of national legislation, they should now cooperate with each other and with EURLs in a network of centers of excellence for the authorization and post-market surveillance of IVDs and for the epidemiological surveillance and control of communicable diseases. The term "reference laboratory" has long been used colloquially to refer to many kinds of laboratories, regardless of their tasks, competencies, responsibilities and designation. A literature search and analysis confirmed this by showing that a considerable proportion of scientific publications in 2024 use the term "reference laboratory" inappropriately. In order to clarify the roles and functioning of EURLs and NRLs, we have evaluated the relevant current EU provisions and compared the findings with those of reference laboratories designated by other organizations, calibration (reference) laboratories and referral laboratories, which are simply referred to as "reference laboratories". With the forthcoming implementation of the EU regulations, at least the goals of providing safe and high-quality IVDs and adequate public health surveillance for communicable diseases appear to be achievable., (© 2024 Walter de Gruyter GmbH, Berlin/Boston.)

The ultimate goal of value-based laboratory medicine is maximizing the effectiveness of laboratory tests in improving patient outcomes, optimizing resources and minimizing unnecessary costs. This approach abandons the oversimplified notion of test volume and cost, in favor of emphasizing the clinical utility and quality of diagnostic tests in the clinical decision-making. Several key elements characterize value-based laboratory medicine, which can be summarized in some basic concepts, such as organization of in vitro diagnostics (including appropriateness, integrated diagnostics, networking, remote patient monitoring, disruptive innovations), translation of laboratory data into clinical information and measurable outcomes, sustainability, reimbursement, ethics (e.g., patient empowerment and safety, data protection, analysis of big data, scientific publishing). Education and training are also crucial, along with considerations for the future of the profession, which will be largely influenced by advances in automation, information technology, artificial intelligence, and regulations concerning in vitro diagnostics. This collective opinion paper, composed of summaries from presentations given at the two-day European Federation of Laboratory Medicine (EFLM) Strategic Conference "A vision to the future: value-based laboratory medicine" (Padova, Italy; September 23-24, 2024), aims to provide a comprehensive overview of value-based laboratory medicine, projecting the profession into a more clinically effective and sustainable future., (© 2024 Walter de Gruyter GmbH, Berlin/Boston.)

Plasma proteins involved in coagulation and fibrinolysis are essential to hemostasis. Consequently, their circulating levels and functionality are critical in bleeding and thrombosis development. Well-established laboratory tests to assess these are available; however, said tests do not allow high multiplicity, require large volumes of plasma and are often costly. A novel technology to quantify plasma proteins is quantitative protein mass spectrometry (QPMS). Aided by stable isotope-labeled internal standards a large number of proteins can be quantified in one single analytical run requiring <30 μL of plasma. This provides an opportunity to improve insight in the etiology and prognosis of bleeding and thrombotic disorders, in which the balance between different proteins plays a crucial role. This manuscript aims to give an overview of the QPMS potential applications in thrombosis and hemostasis research (quantifying the 38 proteins assigned to coagulation and fibrinolysis by the KEGG database), but also to explore the potential and hurdles if designed for clinical practice. Advantages and limitations of QPMS are described and strategies for improved analysis are proposed, using as an example the test requirements for antithrombin. Application of this technology in the future could represent a step towards individualized patient care., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Competing Interests: Conflict of interest: M.S. reports serving as a consultant or research support (or both) from CiVi, Resverlogix, Lexicon, Baxter, Esperion, Amarin, New Amsterdam, Tourmaline, Sanofi, and Regeneron Pharmaceuticals, Inc. E.R.: No disclosures. P.G.S. reports grants, personal fees, and non-financial support from Sanofi; grants and personal fees from Amarin, Servier, and Bayer; personal fees from Amgen, AstraZeneca, BMS, Boehringer Ingelheim, Idorsia, Pfizer, and Novartis. J.W.J. receives research grants from The Netherlands Heart Foundation, the Interuniversity Cardiology Institute of The Netherlands, and the European Commission Seventh Framework Programme; and research support from Amgen, Astellas, AstraZeneca, Daiichi Sankyo, Lilly, Merck-Schering-Plough, Novartis, Pfizer, Roche, and Sanofi. M.Sc. is an employee of Roche and may hold shares in the company. D.L.B. discloses the following relationships: Advisory Board: Angiowave, Bayer, Boehringer Ingelheim, Cardax, CellProthera, Cereno Scientific, Elsevier Practice Update Cardiology, High Enroll, Janssen, Level Ex, McKinsey, Medscape Cardiology, Merck, MyoKardia, NirvaMed, Novo Nordisk, PhaseBio, PLx Pharma, Regado Biosciences, and Stasys; Board of Directors: Angiowave (stock options), Boston VA Research Institute, Bristol Myers Squibb (stock), DRS.LINQ (stock options), High Enroll (stock), Society of Cardiovascular Patient Care, TobeSoft; Chair: Inaugural Chair, American Heart Association Quality Oversight Committee; Consultant: Broadview Ventures, Hims; Data Monitoring Committees: Acesion Pharma, Assistance Publique-Hôpitaux de Paris, Baim Institute for Clinical Research (formerly Harvard Clinical Research Institute, for the PORTICO trial, funded by St Jude Medical, now Abbott), Boston Scientific (Chair, PEITHO trial), Cleveland Clinic (including for the ExCEED trial, funded by Edwards), Contego Medical (Chair, PERFORMANCE 2), Duke Clinical Research Institute, Mayo Clinic, Mount Sinai School of Medicine (for the ENVISAGE trial, funded by Daiichi Sankyo; for the ABILITY-DM trial, funded by Concept Medical), Novartis, Population Health Research Institute; Rutgers University (for the NIH-funded MINT Trial); Honoraria: American College of Cardiology (Senior Associate Editor, Clinical Trials and News, ACC.org; Chair, ACC Accreditation Oversight Committee), Arnold and Porter law firm (work related to Sanofi/Bristol Myers Squibb clopidogrel litigation), Baim Institute for Clinical Research (formerly Harvard Clinical Research Institute; RE-DUAL PCI clinical trial steering committee funded by Boehringer Ingelheim; AEGIS-II executive committee funded by CSL Behring), Belvoir Publications (Editor in Chief, Harvard Heart Letter), Canadian Medical and Surgical Knowledge Translation Research Group (clinical trial steering committees), CSL Behring (AHA lecture), Cowen and Company, Duke Clinical Research Institute (clinical trial steering committees, including for the PRONOUNCE trial, funded by Ferring Pharmaceuticals), HMP Global (Editor in Chief, Journal of Invasive Cardiology), Journal of the American College of Cardiology (Guest Editor; Associate Editor), K2P (Co-Chair, interdisciplinary curriculum), Level Ex, Medtelligence/ReachMD (CME steering committees), MJH Life Sciences, Oakstone CME (Course Director, Comprehensive Review of Interventional Cardiology), Piper Sandler, Population Health Research Institute (for the COMPASS operations committee, publications committee, steering committee, and USA national co-leader, funded by Bayer), Slack Publications (Chief Medical Editor, Cardiology Today's Intervention), Society of Cardiovascular Patient Care (Secretary/Treasurer), WebMD (CME steering committees), Wiley (steering committee); Other: Clinical Cardiology (Deputy Editor), NCDR-ACTION Registry Steering Committee (Chair), VA CART Research and Publications Committee (Chair); Patent: Sotagliflozin (named on a patent for sotagliflozin assigned to Brigham and Women's Hospital who assigned to Lexicon; neither I nor Brigham and Women's Hospital receive any income from this patent); Research Funding: Abbott, Acesion Pharma, Afimmune, Aker Biomarine, Alnylam, Amarin, Amgen, AstraZeneca, Bayer, Beren, Boehringer Ingelheim, Boston Scientific, Bristol Myers Squibb, Cardax, CellProthera, Cereno Scientific, Chiesi, CinCor, Cleerly, CSL Behring, Eisai, Ethicon, Faraday Pharmaceuticals, Ferring Pharmaceuticals, Forest Laboratories, Fractyl, Garmin, HLS Therapeutics, Idorsia, Ironwood, Ischemix, Janssen, Javelin, Lexicon, Lilly, Medtronic, Merck, Moderna, MyoKardia, NirvaMed, Novartis, Novo Nordisk, Otsuka, Owkin, Pfizer, PhaseBio, PLx Pharma, Recardio, Regeneron, Reid Hoffman Foundation, Roche, Sanofi, Stasys, Synaptic, The Medicines Company, Youngene, 89Bio; Royalties: Elsevier (Editor, Braunwald's Heart Disease); Site Co-Investigator: Author contributions Abbott, Biotronik, Boston Scientific, CSI, Endotronix, St Jude Medical (now Abbott), Philips, SpectraWAVE, Svelte, Vascular Solut.

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., (© 2024 the author(s), published by De Gruyter, Berlin/Boston.)

Early kidney injury may be detected by urinary markers, such as beta-2 microglobulin (B2M), tissue inhibitor of metalloproteinases-2 (TIMP-2), insulin-like growth factor-binding protein 7 (IGFBP7), kidney injury molecule-1 (KIM-1) and/or neutrophil gelatinase-associated lipocalin (NGAL). Of these biomarkers information on pathophysiology and reference ranges in both healthy and diseased populations are scarce. Differences in urinary levels of B2M, TIMP-2, IGFBP7, KIM-1 and NGAL were compared 24 h before and after nephrectomy in 38 living kidney donors from the REnal Protection Against Ischaemia-Reperfusion in transplantation study. Linear regression was used to assess the relation between baseline biomarker concentration and kidney function 1 year after nephrectomy. Median levels of urinary creatinine and creatinine standardized B2M, TIMP-2, IGFBP7, KIM-1, NGAL, and albumin 24 h before nephrectomy in donors were 9.4 mmol/L, 14 μg/mmol, 16 pmol/mmol, 99 pmol/mmol, 63 ng/mmol, 1390 ng/mmol and 0.7 mg/mmol, with median differences 24 h after nephrectomy of - 0.9, + 1906, - 7.1, - 38.3, - 6.9, + 2378 and + 1.2, respectively. The change of donor eGFR after 12 months per SD increment at baseline of B2M, TIMP-2, IGFBP7, KIM-1 and NGAL was: - 1.1, - 2.3, - 0.7, - 1.6 and - 2.8, respectively. Urinary TIMP-2 and IGFBP7 excretion halved after nephrectomy, similar to urinary creatinine, suggesting these markers predominantly reflect glomerular filtration. B2M and NGAL excretion increased significantly, similar to albumin, indicating decreased proximal tubular reabsorption following nephrectomy. KIM-1 did not change considerably after nephrectomy. Even though none of these biomarkers showed a strong relation with long-term donor eGFR, these results provide valuable insight into the pathophysiology of these urinary biomarkers., (© 2024. The Author(s).)

Background: This was a cross-sectional study on the correlation between abdominal aortic calcification (AAC) and Modic changes (MC). Little is known regarding the etiology of MC in the lumbar spine. Currently, insufficient vascularization of the endplate has been proposed to contribute to the appearance of MC. Our objective was to investigate whether AAC, a marker for a poor vascular status, is associated with MC in patients suffering from degenerative disc disease., Methods: Radiologic images of patients (n = 130) suffering from degenerative lumbar disc disease were reviewed. Type and severity of MC were assessed using magnetic resonance images, and severity of AAC was evaluated using computed tomography images or fluoroscopy. Both items were dichotomized into minimal and relevant grades. The correlation between them was studied using Spearman's correlation test, with age as a covariate., Results: Of the patients, 113 (87%) demonstrated MC (31% type I, 63% type II, and 6% type III) (55% relevant grade), and 68% had AAC (44% relevant grade). Spearman statistical analysis revealed that AAC was correlated with age (P < 0.001), whereas MC were not (P = 0.142). AAC severity was significantly correlated with MC, remaining so after age adjustment (P < 0.05). While MC type I lacked correlation with AAC, MC type II were significantly correlated with AAC (0.288, P = 0.015); however, this association lost significance after adjusting for age (P = 0.057)., Conclusions: AAC and MC (mainly MC type II) are associated, indicating that reduced blood supply or even a poor systemic vascularization status due to atherosclerotic disease may play a role in the formation of MC. Future studies focusing on the etiology of MC should pay more attention to patients' vascular status and determinants of abdominal aorta calcification., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., (© 2024. The Author(s).)

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., (© 2024 Walter de Gruyter GmbH, Berlin/Boston.)

Objectives: Urinary tract infection (UTI) is common among older women. However, diagnosis is challenging because of frequent chronic lower urinary tract symptoms, cognitive impairment, and a high prevalence of asymptomatic bacteriuria (ASB). Current urine diagnostics lack specificity, leading to unnecessary treatment and antimicrobial resistance. This study aimed to evaluate the diagnostic accuracy of 12 urine biomarkers for diagnosing UTI in older women., Methods: In this case-control study, cases were women ≥65 years with ≥2 new-onset lower urinary tract symptoms, pyuria, and one uropathogen ≥10 4  CFU/mL. Controls were asymptomatic and classified as ASB (one uropathogen ≥10 5  CFU/mL), negative culture, or mixed flora. Urine biomarker concentrations were measured through liquid chromatography-mass spectrometry and ELISA. Diagnostic accuracy parameters of individual biomarkers and a biomarker model were derived from receiver operating characteristic curves., Results: We included 162 community-dwelling and institutionalized older women. Five urine inflammatory biomarkers demonstrated high discriminative ability (area under the curve ≥0.80): interleukin 6, azurocidin, neutrophil gelatinase-associated lipocalin, tissue inhibitor of metalloproteinases 2, and C-X-C motif chemokine 9. Azurocidin exhibited the highest diagnostic accuracy (sensitivity 86% [95% CI 75%-93%] and specificity 89% [95% CI 82%-94%] at 16.7 ng/mmol creatinine). A combined biomarker and pyuria model showed improved diagnostic accuracy in patients with UTI and ASB, compared with pyuria alone., Discussion: We identified several urine biomarkers that accurately differentiated older women with UTI from asymptomatic women, including ASB. These findings represent a potential advancement towards improved diagnostics for UTI in older women and warrant validation in a diverse population., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., (© 2023. The Author(s).)

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., Competing Interests: Disclosures Dr Szarek reports serving as a consultant or receiving research support, or both, from CiVi, Resverlogix, Lexicon, Baxter, Esperion, Amarin, NewAmsterdam, Sanofi, and Regeneron Pharmaceuticals, Inc. E. Reijnders reports no disclosures. Dr Jukema reports research grants from the Netherlands Heart Foundation, the Interuniversity Cardiology Institute of the Netherlands, and the European Commission Seventh Framework Programme, and research support from Amgen, Astellas, AstraZeneca, Daiichi-Sankyo, Lilly, Merck-Schering-Plough, Novartis, Pfizer, Roche, and Sanofi. Dr Bhatt was on advisory boards of Angiowave, Bayer, Boehringer Ingelheim, Cardax, CellProthera, Cereno Scientific, Elsevier Practice Update Cardiology, High Enroll, Janssen, Level Ex, McKinsey, Medscape Cardiology, Merck, MyoKardia, NirvaMed, Novo Nordisk, PhaseBio, PLx Pharma, Regado Biosciences, and Stasys; was on boards of directors of Angiowave (stock options), Boston VA Research Institute, Bristol Myers Squibb (stock), DRS.LINQ (stock options), High Enroll (stock), Society of Cardiovascular Patient Care, and TobeSoft; was inaugural chair of the American Heart Association quality oversight committee; was a consultant for Broadview Ventures and Hims; was on data monitoring committees of Acesion Pharma, Assistance Publique-Hôpitaux de Paris, Baim Institute for Clinical Research (formerly Harvard Clinical Research Institute, for the PORTICO trial, funded by St Jude Medical, now Abbott), Boston Scientific (chair, PEITHO trial), Cleveland Clinic (including for the ExCEED trial, funded by Edwards), Contego Medical (chair, PERFORMANCE 2), Duke Clinical Research Institute, Mayo Clinic, Mount Sinai School of Medicine (for the ENVISAGE trial, funded by Daiichi Sankyo; and for the ABILITY-DM trial, funded by Concept Medical), Novartis, Population Health Research Institute, and Rutgers University (for the National Institutes of Health–funded MINT Trial); received honoraria from American College of Cardiology (senior associate editor, “Clinical Trials and News,” ACC.org; chair, ACC accreditation oversight committee), Arnold and Porter law firm (work related to Sanofi/Bristol Myers Squibb clopidogrel litigation), Baim Institute for Clinical Research (formerly Harvard Clinical Research Institute; RE-DUAL PCI clinical trial steering committee, funded by Boehringer Ingelheim; AEGIS-II executive committee, funded by CSL Behring), Belvoir Publications (editor-in-chief, Harvard Heart Letter), Canadian Medical and Surgical Knowledge Translation Research Group (clinical trial steering committees), CSL Behring (American Heart Association lecture), Cowen and Company, Duke Clinical Research Institute (clinical trial steering committees, including for the PRONOUNCE trial, funded by Ferring Pharmaceuticals), HMP Global (editor-in-chief, Journal of Invasive Cardiology), Journal of the American College of Cardiology (guest editor and associate editor), K2P (co-chair, interdisciplinary curriculum), Level Ex, Medtelligence/ReachMD (CME steering committees), MJH Life Sciences, Oakstone CME (course director, Comprehensive Review of Interventional Cardiology), Piper Sandler, Population Health Research Institute (for the COMPASS operations committee, publications committee, steering committee, and USA national co-leader, funded by Bayer), Slack Publications (chief medical editor, Cardiology Today’s Intervention), Society of Cardiovascular Patient Care (secretary/treasurer), WebMD (CME steering committees), and Wiley (steering committee); additional disclosures: Clinical Cardiology (deputy editor), NCDR-ACTION registry steering committee (chair), and VA CART research and publications committee (chair); patent: sotagliflozin (named on a patent for sotagliflozin assigned to Brigham and Women’s Hospital, assigned to Lexicon; Dr Bhatt nor Brigham and Women’s Hospital receives any income from this patent); research funding: Abbott, Acesion Pharma, Afimmune, Aker Biomarine, Alnylam, Amarin, Amgen, AstraZeneca, Bayer, Beren, Boehringer Ingelheim, Boston Scientific, Bristol Myers Squibb, Cardax, CellProthera, Cereno Scientific, Chiesi, CinCor, Cleerly, CSL Behring, Eisai, Ethicon, Faraday Pharmaceuticals, Ferring Pharmaceuticals, Forest Laboratories, Fractyl, Garmin, HLS Therapeutics, Idorsia, Ironwood, Ischemix, Janssen, Javelin, Lexicon, Lilly, Medtronic, Merck, Moderna, MyoKardia, NirvaMed, Novartis, Novo Nordisk, Otsuka, Owkin, Pfizer, PhaseBio, PLx Pharma, Recardio, Regeneron, Reid Hoffman Foundation, Roche, Sanofi, Stasys, Synaptic, The Medicines Company, Youngene, and 89Bio; royalties: Elsevier (editor, Braunwald’s Heart Disease); site coinvestigator: Abbott, Biotronik, Boston Scientific, CSI, Endotronix, St Jude Medical (now Abbott), Philips, SpectraWAVE, Svelte, and Vascular Solutions; trustee: American College of Cardiology; unfunded research: FlowCo and Takeda. Dr Bittner reports grant support from Sanofi, Regeneron Pharmaceuticals, Amgen, Astra Zeneca, DalCor, Esperion, and Novartis; consulting fees from Pfizer; honoraria from Medscape; and fees for participating on data safety monitoring boards for the National Institutes of Health and for Verve Therapeutics. Dr Diaz reports research grants from Sanofi, DalCor Pharmaceuticals, Population Health Research Institute, Duke Clinical Research Institute, the TIMI group, Amgen, Cirius, Montreal Health Innovations Coordinating Center, and Lepetit; and personal fees, as a member of the Executive Steering Committee, from Amgen and Cirius. Dr Fazio is an employee of Regeneron Pharmaceuticals Inc and may hold shares in the company. G. Garon is an employee of Sanofi and may hold shares in the company. Dr Goodman reports research grant support (eg, steering committee or data and safety monitoring committee) or speaker/consulting honoraria (eg, advisory boards) from Amgen, Anthos Therapeutics, AstraZeneca, Bayer, Boehringer Ingelheim, Bristol Myers Squibb, CSL Behring, Daiichi-Sankyo/American Regent, Eli Lilly, Esperion, Ferring Pharmaceuticals, HLS Therapeutics, JAMP Pharma, Merck, Novartis, Novo Nordisk A/C, Pendopharm/Pharmascience, Pfizer, Regeneron, Sanofi, Servier, and Valeo Pharma; and salary support/honoraria from the Heart and Stroke Foundation of Ontario/University of Toronto (Polo) chair, Canadian Heart Research Centre and MD Primer, Canadian VIGOUR Centre, Cleveland Clinic Coordinating Centre for Clinical Research, Duke Clinical Research Institute, New York University Clinical Coordinating Centre, PERFUSE Research Institute, and the TIMI Study Group (Brigham Health). Dr Harrington reports research grants from the Patient-Centered Outcomes Research Institute, National Institutes of Health, CSL, and Janssen; consulting for Atropos Health, Bitterroot Bio, Bridge Bio, Bristol Myers Squibb, Foresight, and Element Science; and served on the boards of directors for the American Heart Association (unpaid) and Cytokinetics. Dr Ruhaak reports no disclosures. Dr Schwertfeger is an employee of Roche and may hold shares in the company. Dr Tsimikas is a coinventor and receives royalties from patents owned by University of California San Diego and is a cofounder and has an equity interest in Oxitope, LLC, and its affiliates, Kleanthi Diagnostics, LLC, and has a dual appointment at the University of California San Diego and Ionis Pharmaceuticals. Dr White reports grant support paid to the institution for serving on a steering committee for the ODYSSEY OUTCOMES trial (Evaluation of Cardiovascular Outcomes After an Acute Coronary Syndrome During Treatment With Alirocumab) from Sanofi-Aventis and Regeneron Pharmaceuticals, for ACCELERATE (A Study of Evacetrapib in High-Risk Vascular Disease) from Eli Lilly and Company, for STRENGTH (Outcomes Study to Assess Statin Residual Risk Reduction With EpaNova in High CV Risk Patients With Hypertriglyceridemia) from Omthera Pharmaceuticals, for CAMELLIA-TIMI (A Study to Evaluate the Effect of Long-term Treatment With BELVIQ [Lorcaserin HC] on the Incidence of Major Adverse Cardiovascular Events and Conversion to Type 2 Diabetes Mellitus in Obese and Overweight Subjects With Cardiovascular Disease or Multiple Cardiovascular Risk Factors) from Eisai Inc, for HEART-FID (Randomized Placebo-Controlled Trial of FCM as Treatment for Heart Failure With Iron Deficiency) from American Regent, and for ISCHEMIA (International Study of Comparative Health Effectiveness With Medical and Invasive Approaches) and the MINT Trial (Myocardial Ischemia and Transfusion) from the National Institutes of Health; received grants to the institution and personal fees as a steering committee member for the Dal-Gene study (Effect of Dalcetrapib versus Placebo on CV Risk in a Genetically Defined Population With a Recent ACS) from DalCor Pharma UK Inc, for the AEGIS-II study (The Safety and Tolerability of CSL112, a Reconstituted, Infusible, Plasma-Derived Human ApoA-I, After Acute Myocardial Infarction: The ApoA-I Event Reducing in Ischemic Syndromes I) from CSL Behring, for the SCORED trial (Effect of Sotagliflozin on Cardiovascular and Renal Events in Patients With Type 2 Diabetes and Moderate Renal Impairment Who Are at Cardiovascular Risk) and the SOLOIST-WHF trial (Effect of Sotagliflozin on Cardiovascular Events in Patients With Type 2 Diabetes Post Worsening Heart Failure) from Sanofi-Aventis Australia Pty Ltd, and for the CLEAR Outcomes Study (Evaluation of Major Cardiovascular Events in Patients With, or at High Risk for, Cardiovascular Disease Who Are Statin Intolerant Treated With Bempedoic Acid [ETC-1002] or Placebo) from Esperion Therapeutics Inc; and was on advisory boards for CSL Behring and Genentech, Inc (an affiliate of F. Hoffmann–La Roche Ltd, “Roche”; Lytics Post-PCI advisory board at European Society of Cardiology). Dr Steg reports grants, personal fees, and nonfinancial support from Sanofi; grants and personal fees from Amarin, Servier, and Bayer; and personal fees from Amgen, AstraZeneca, BMS, Boehringer Ingelheim, Idorsia, Pfizer, and Novartis. Dr Cobbaert reports research collaboration with Roche Diagnostics. Dr Schwartz reports research grants to the University of Colorado from AstraZeneca, Resverlogix, Sanofi, Silence Therapeutics, and The Medicines Company; and is coinventor of pending US patent 62/806,313 (“Methods for Reducing Cardiovascular Risk”) assigned in full to the University of Colorado.

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.

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, β2-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., (© American Association for Clinical Chemistry 2023.)

Concern continues about whether the measurement of apolipoprotein B (apoB) is adequately standardized, and therefore, whether apoB should be applied widely in clinical care. This concern is misplaced. Our objective is to explain why and what the term "standardization" means. To produce clinically valid results, a test must accurately, precisely, and selectively measure the marker of interest. That is, it must be standardized. Accuracy refers to how closely the result obtained with 1 method corresponds to the result obtained with the standard method, precision to how reproducible the result is on repeated testing, and selectivity to how susceptible the method is to error by inclusion of other classes of lipoprotein particles. Multiple expert groups have determined that the measurement of apoB is adequately standardized for clinical care, and that apoB can be measured inexpensively, using widely available automated methods, more accurately, precisely, and selectively than low-density lipoprotein cholesterol or non-high-density lipoprotein cholesterol. ApoB is a standard superior to low-density lipoprotein cholesterol and high-density lipoprotein cholesterol because it is a defined molecule, whereas the cholesterol markers are the mass of cholesterol within lipoprotein particles defined by their density, not by their molecular structure. Nevertheless, the standardization of apoB is being further improved by the application of mass spectrophotometric methods, whereas the limitations in the standardization and, therefore, the accurate, precise, and selective measurement of low-density lipoprotein cholesterol and high-density lipoprotein cholesterol are unlikely to be overcome. We submit that greater accuracy, precision, and selectivity in measurement is a decisive advantage for apoB in the modern era of intensive lipid-lowering therapies.

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)., Competing Interests: Potential conflicts of interest. M. M. C. L. reports grants or contracts as the principal investigator on the Embrace Study. L. G. V. reports grants or contracts as the co-investigator on the Embrace Study. J. E. S. reports consulting fees from Viiv Expert Board HIV, unrelated to this manuscript; payment or honoraria from Nederlandse Internisten Vereniging: Centraal Onderwijs Interne Geneeskunde Infection and Immunity (a course for internists in training); and participation as the Chair of Dutch Infection Prevention Guideline Committee “Urinary Catheterization.” M. E. N. reports payment for expert testimony for the development of guidelines for primary and secondary care, focusing on GERD and Dyspepsia, Ondansetron, and Non-Alcoholic Fatty Liver Disease, and is a committee member for these guidelines (paid to author); unpaid membership to Network Academic Primary Care, the Netherlands; the Nederlands Huisartsen Genootschap Primary Care Practice Accreditation Board; and the Advisory Board of SIR Institute for Pharmacy Practice and Policy. S. P. C. reports royalties for textbook editing on geriatric emergency medicine, including urinary tract infections; consulting fees as clinical lead of the UK Frailty Improvement Network; and travel support for teaching or speaking on geriatric care across Europe. C. M. C. serves as Chair of the International Federation of Clinical Chemistry and Laboratory Medicine Scientific Division (independent) and reports receipt of equipment, materials, or services from Sysmex Corporation for reagents for flowcytometric measurements of samples on the Sysmex urine flowcytometry analyzer at the Clinical Chemistry and Laboratory Medicine Department, as reported under “funding.” All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

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., (© American Association for Clinical Chemistry 2023.)

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)., (©American Association for Clinical Chemistry 2023.)

Background: Antithrombin deficiency is a rare but severe disorder leading to high risk of thrombosis. The current clinical care pathway relies on activity tests, which only provide overall functional information on the in vitro activity of antithrombin. However, antithrombin exists in many different forms, also known as proteoforms, with varying clinical phenotypes. Precision diagnostics, facilitated by mass spectrometry, provides a strategy to improve patient diagnostics by molecular characterization., Objectives: To develop and analytically validate a mass spectrometry-based test for molecular characterization of antithrombin., Methods: The test was analytically validated based on predefined analytical performance specifications. The validation covered imprecision, carryover, linearity, stability, analytical specificity, a provisional reference interval, and an explorative method comparison., Results: The test passed the predefined analytical performance specifications with a mean within-laboratory imprecision of 5.9%, linearity between 0.08 and 2.58 μmol/L, and a provisional reference interval of 1.07 to 1.49 μmol/L. When measuring samples with a suspected quantitative deficiency, the test showed a good correlation with a commercial activity test (Pearson r  = 0.88)., Conclusion: The test passed the validation, and we now envision the use of the test for exploration of the clinical relevance of specific antithrombin proteoforms. Puzzling cases of antithrombin deficiency, for instance, due to ambiguous activity results or an atypical clinical presentation, can be investigated by the LC-MRM mass spectrometry test serving as an add-on to the activity test and providing a molecular diagnosis. Clinical studies are planned to investigate the potential of the test to improve antithrombin diagnostics. Furthermore, the molecular information gained using the test may aid in establishing better risk stratification and a basis for personalized medicine., (© 2023 The Authors.)

Background: The STAtins Reduce Thrombophilia trial showed that, in patients with prior venous thrombosis, rosuvastatin decreased various coagulation factor levels., Objectives: Here, we investigated the hypothesis that statins decrease coagulation factor levels through shared mechanisms of synthesis or regulatory pathways with apolipoproteins., Methods: We measured the levels of apolipoprotein (Apo)A-I, A-II, A-IV, (a), B-100, B-total, C-I, C-II, C-III, and E in patients (n = 126) randomized to 28 days of rosuvastatin use. We assessed the association between apolipoproteins and coagulation factors at baseline using linear regression. The mean difference in apolipoprotein levels between baseline and after 28 days of rosuvastatin use was determined through linear regression, adjusting for age, sex, and body mass index. Coagulation factors were added to this model to determine if the lowering of apolipoproteins by rosuvastatin was linked with coagulation factor levels., Results: At baseline, levels of all apolipoproteins, except Apo(a), were positively associated with FVII, FIX, and FXI. Apolipoproteins levels, except for ApoA-I, A-IV, and Apo(a), were decreased after 28 days of rosuvastatin. ApoB-100 showed the largest mean decrease of -0.43 g/L (95% CI = -0.46 to -0.40). The decrease in ApoC-I and C-III levels was associated with a decrease in FVII, whereas the decrease in apoA-II, B-100, and B-total was associated with a decrease in FXI. The decrease in apolipoproteins was neither associated with FVIII or vWF decrease nor with endogenous thrombin potential changes., Conclusions: Rosuvastatin decreases the level of several apolipoproteins, but this decrease was associated only with a decrease in FVII and XI and not with FVIII/vWF., (© 2023 The Author(s).)

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., (© 2023 Walter de Gruyter GmbH, Berlin/Boston.)

In the pursuit of personalized diagnostics and tailored treatments, quantitative protein tests contribute to a more precise definition of health and disease. The development of new quantitative protein tests should be driven by an unmet clinical need and performed in a collaborative effort that involves all stakeholders. With regard to the analytical part, mass spectrometry (MS)-based platforms are an excellent tool for quantification of specific proteins in body fluids, for example focused on cancer. The obtained readouts have great potential in determining tumor aggressiveness to facilitate treatment decisions, and can furthermore be used to monitor patient response. Internationally standardized TNM classifications of malignant tumors are beneficial for diagnosis, however treatment outcome and survival of cancer patients is poorly predicted. To this end, the importance of the tumor microenvironment has endorsed the introduction of the tumor-stroma ratio as a prognostic parameter in solid primary tumor types. Currently, the stromal content of tumor tissues is determined via routine diagnostic pathology slides. With the development of liquid chromatography (LC)-MS methods we aim at quantification of tumor-stroma specific proteins in body fluids. In this mini-review the analytical aspect of this developmental trajectory is further detailed., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: M.K. is the vice president of the issuing organization of the patent-pending Stroma Liquid BiopsyTM product, the Biotech Support Group (BSG). All remaining authors have declared no conflict of interest., (© 2022 THE AUTHORS.)

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., (© 2022 Walter de Gruyter GmbH, Berlin/Boston.)

Background and Aims: There is an ongoing need to recognize early kidney injury and its progression in structural chronic pathologies. The proteins 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), C-X-C motif chemokine 9 (CXCL9), transforming growth factor-beta 1 (TGF-β1), solute carrier family 22 member 2 (SLC22A2), nephrin, cubilin, and uromodulin (UMOD) have been proposed as early kidney injury biomarkers. To guide clinical interpretation, their urinary concentrations should be accompanied by reference intervals, which we here establish in a representative Dutch middle-aged population., Materials and Methods: The 24 h urine samples from 1443 Caucasian middle-aged men and women were analyzed for the biomarkers by quantitative LC-MS/MS. Biomarker excretion per 24 h were calculated, and urine creatinine and osmolality were measured for dilution normalization. This population was characterized by demographic and anthropometric parameters, comorbid conditions, and conventional kidney function measures., Results: NGAL, IGFBP7, TIMP2, KIM-1, and UMOD could be quantified in this population, whereas nephrin, SLC22A2, and CXCL9 were below their detection limits. Urine creatinine and osmolality were correlated to urine volume (r = -0.71; -0.74) and to IGFBP7 (r = 0.73; 0.71) and TIMP2 (r = 0.71; 0.69). Crude and normalized biomarker concentrations were affected by sex, but not by age, body mass index, smoking, kidney function, or common comorbid conditions. The reference intervals (men; women) were 18-108; 21-131 pmol IGFBP7/mmol creatinine, 1-63; 4-224 pmol NGAL/mmol creatinine, 7-48; 7-59 pmol TIMP2/mmol creatinine, &lt;1-9; &lt;1-12 pmol KIM-1/mmol creatinine, and 0.1-1.2; 0.1-1.7 mg UMOD/mmol creatinine., Conclusion: We present dilution-normalized and sex-stratified urinary reference intervals of kidney injury biomarkers in a middle-aged Caucasian population.

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.

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., (© 2022 the author(s), published by De Gruyter, Berlin/Boston.)

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., (© 2022 the author(s), published by De Gruyter, Berlin/Boston.)

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., (© 2022 the author(s), published by De Gruyter, Berlin/Boston.)

Virus-specific cellular and humoral responses are major determinants for protection from critical illness after SARS-CoV-2 infection. However, the magnitude of the contribution of each of the components to viral clearance remains unclear. Here, we studied the timing of viral clearance in relation to 122 immune parameters in 102 hospitalised patients with moderate and severe COVID-19 in a longitudinal design. Delayed viral clearance was associated with more severe disease and was associated with higher levels of SARS-CoV-2-specific (neutralising) antibodies over time, increased numbers of neutrophils, monocytes, basophils, and a range of pro-inflammatory cyto-/chemokines illustrating ongoing, partially Th2 dominating, immune activation. In contrast, early viral clearance and less critical illness correlated with the peak of neutralising antibodies, higher levels of CD4 T cells, and in particular naïve CD4+ T cells, suggesting their role in early control of SARS-CoV-2 possibly by proving appropriate B cell help. Higher counts of naïve CD4+ T cells also correlated with lower levels of MIF, IL-9, and TNF-beta, suggesting an indirect role in averting prolonged virus-induced tissue damage. Collectively, our data show that naïve CD4+ T cell play a critical role in rapid viral T cell control, obviating aberrant antibody and cytokine profiles and disease deterioration. These data may help in guiding risk stratification for severe COVID-19.

Background: Mutations in genes encoding lipoprotein lipase (LPL) or its regulators can cause severe hypertriglyceridemia (HTG). Thus far, the effect of genetic HTG on the lipid profile has been mainly determined via conventional techniques., Objective: To show detailed differences in the (apo)lipoprotein profile of patients with genetic HTG by combining LC-MS and NMR techniques., Methods: Fasted serum from 7 patients with genetic HTG and 10 normolipidemic controls was used to measure the concentration of a spectrum of apolipoproteins by LC-MS, and to estimate the concentration and size of lipoprotein subclasses and class-specific lipid composition using NMR spectroscopy., Results: Patients with genetic HTG compared to normolipidemic controls had higher levels of apoB48 (fold change [FC] 11.3, P<0.001), apoC-I (FC 1.5, P<0.001), apoC-II (FC 4.3, P=0.007), apoC-III (FC 3.4, P<0.001), and apoE (FC 4.3, P<0.001), without altered apoB100. In addition, patients with genetic HTG had higher concentrations of TG-rich lipoproteins (i.e., chylomicrons and very low-density lipoproteins [VLDL]; FC 3.0, P<0.001), but lower LDL (FC 0.4, P=0.001), of which medium and small-sized LDL particles appeared even absent. While the correlation coefficient between NMR and enzymatic analysis in normolipidemic controls was high, it was considerably reduced in patients with genetic HTG., Conclusion: The lipoprotein profile of patients with genetic HTG is predominated with large lipoproteins (i.e., chylomicrons, VLDL), explaining high levels of apoC-I, apoC-II, apoC-III and apoE, whereas small atherogenic LDL particles are absent. The presence of chylomicrons in patients with HTG weakens the accuracy of the NMR-based model as it was designed for normolipidemic fasted individuals., Competing Interests: Declarations of Competing Interest None, (Copyright © 2022. Published by Elsevier Inc.)

Background: LC-MS/MS has enabled the translation of many novel biomarkers to the clinical laboratory, but its potential for measurement of urinary proteins is still unexplored. In this study we examined the correlation and agreement between immunoassay and LC-MS/MS in the quantitation of kidney injury biomarkers and evaluated the application of technical LC-MS/MS meta-data assessment to ensure test result validity., Methods: NGAL, IGFBP7, TIMP2, and KIM-1 were quantified in 345 urine samples with one multiplex lab-developed test that combines immunocapture with mass spectrometry read-out and 4 singleplex sandwich-type immunoassays. Assay performance and imprecision were monitored by 2 urine-based quality controls. Ion ratios, signal intensity, and retention time were monitored over all study samples., Results: The LC-MS/MS retention time drift was ≤1.2%, ion ratios were within 20% of the target values at concentrations of >100 pmol/L, and peptides originating from the same protein were in agreement (slopes between 1.03 and 1.41). The interassay CV was between 9.3% and 19.1% for LC-MS/MS analysis and between 4.2% and 10.9% for immunoassay. Direct LC-MS/MS analysis was correlated with immunoassay in the quantitation of NGAL (r = 0.93; range: 0.01-37 nmol/L), IGFBP7 (r = 0.80; range: 0.01-2.6 nmol/L), TIMP2 (r = 0.85; range: 0.01-6.3 nmol/L), and KIM-1 (r = 0.70; range 0.01-0.4 nmol/L), but the analytical methodologies differed in measurands and calibration strategies., Conclusions: LC-MS/MS is explored as a next-generation technology for multiplex urinary protein measurement. It has great potential to overcome nonselectivity and lack of standardization because of its capability of directly measuring well-defined molecular proteins., (© American Association for Clinical Chemistry 2022.)

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., (© 2022 Walter de Gruyter GmbH, Berlin/Boston.)

The aim of this study was to determine reference intervals in an outpatient population from Vall d'Hebron laboratory using an indirect approach previously described in a Dutch population (NUMBER project). We used anonymized test results from individuals visiting general practitioners and analysed during 2018. Analytical quality was assured by EQA performance, daily average monitoring and by assessing longitudinal accuracy between 2018 and 2020 (using trueness verifiers from Dutch EQA). Per test, outliers by biochemically related tests were excluded, data were transformed to a normal distribution (if necessary) and means and standard deviations were calculated, stratified by age and sex. In addition, the reference limit estimator method was also used to calculate reference intervals using the same dataset. Finally, for standardized tests reference intervals obtained were compared with the published NUMBER results. Reference intervals were calculated using data from 509,408 clinical requests. For biochemical tests following a normal distribution, similar reference intervals were found between Vall d'Hebron and the Dutch study. For creatinine and urea, reference intervals increased with age in both populations. The upper limits of Gamma-glutamyl transferase were markedly higher in the Dutch study compared to Vall d'Hebron results. Creatine kinase and uric acid reference intervals were higher in both populations compared to conventional reference intervals. Medical test results following a normal distribution showed comparable and consistent reference intervals between studies. Therefore a simple indirect method is a feasible and cost-efficient approach for calculating reference intervals. Yet, for generating standardized calculated reference intervals that are traceable to higher order materials and methods, efforts should also focus on test standardization and bias assessment using commutable trueness verifiers., Competing Interests: The authors have declared that no competing interests exist.

Introduction: The definition of the International Normalized Ratio (INR) depends on a reference measurement procedure for the prothrombin time (PT) determined with international standards for thromboplastins. The agreed water bath temperature for PT determination in the reference measurement procedure is 37°C. The aim of the study was to assess the influence of small deviations of the agreed reaction temperature on PT and INR determined with World Health Organization international standards for thromboplastins rTF/16 (recombinant human) and RBT/16 (rabbit brain)., Methods: Prothrombin time was determined, with a manual hook technique, in glass test tubes in a water bath at a controlled temperature. The PT reaction temperatures were varied between 28 and 40°C. Pooled normal plasma and pooled coumarin plasma (INR ≈ 2.8) were used as test plasmas. The data were fitted to a quadratic relationship between PT and temperature., Results: Prothrombin times with rTF/16 were shortened by increasing the reaction temperature up to approximately 39-40°C. PTs with RBT/16 were shortened by increasing the reaction temperature up to approximately 34-37°C, but were prolonged at higher temperatures. The apparent INR change of the coumarin plasma at 37.0°C was 0.06/°C and 0.11/°C for rTF/16 and RBT/16, respectively., Conclusions: Reaction temperature had a significant effect on PT and the apparent INR with the International Standards. At 37.0°C, the apparent INR of coumarin plasma determined with RBT/16 was more responsive to temperature change than the apparent INR determined with rTF/16. The required accuracy of the water bath temperature should be 37.0 ± 0.1°C., (© 2021 The Authors. International Journal of Laboratory Hematology published by John Wiley & Sons Ltd.)

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-β1, 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.

Introduction: Guidelines of management of dyslipidemias and prevention of cardiovascular disease (CVD) are based on firm scientific evidence obtained by randomized controlled trials (RCTs). However, the role of elevated low-density lipoprotein-cholesterol (LDL-C)as a risk factor of CVD and therapies to lower LDL-C are frequently disputed by colleagues who disagree with the conclusions of the RCTs published. This review focuses on this dispute, and evaluates the current approach of management of dyslipidemias and CVD prevention to find modern alternatives for more precise diagnosis and therapy of dyslipidemic patients., Areas Covered: Recent interest in lipoprotein(a) (Lp(a)) and remnants lipoproteins and in therapies that do not influence LDL-C levels primarily, such as anti-inflammatory drugs and icosapent ethyl, has revitalized our concern to optimize the care for patients with increased CVD risk without focusing simply on reduction of LDL-C by therapy with statins, ezitemibe, and proprotein convertase subtilisin-kexin type 9 (PCSK9) inhibitors., Expert Opinion: The limited characterization of study populations by measurement of total cholesterol (TC), high-density lipoprotein-cholesterol (HDL-C) and triglycerides (TG) followed by measurement or calculation of LDL-C should be extended by a more integral approach in order to realize precision diagnostics and precision medicine, for the sake of personalized patient care.

Laboratory medicine in the European Union is at the dawn of a regulatory revolution as it reaches the end of the transition from IVDD 98/79/EC (https://eur-lex.eur-opa.eu/legal-content/EN/TXT/?uri=CELEX%3A31998L0079&qid=1628781352814) to IVDR 2017/746 https://eur-lex.europa.eu/eli/reg/2017/746. Without amendments and contingency plans, implementation of the IVDR in May 2022 will lead the healthcare sector into uncharted waters due to unpreparedness of the EU regulatory infrastructure. Prospective risk analyses were not made by the European Commission, and if nothing happens it can be anticipated that the consequences will impact all stakeholders of the medical test pipeline, may seriously harm patients and may prevent caregivers from making appropriate clinical decisions due to non-availability of medical tests. Finally, it also may discourage manufacturers and academia from developing specialty tests, thereby hampering innovation in medical diagnostic care. We hereby inform laboratory professionals about the imminent diagnostic collapse using testimonies from representative stakeholders of the diagnostic supply chain and from academia developing innovative in-house tests in domains of unmet clinical needs. Steps taken by the EFLM Task Force on European Regulatory Affairs, under the umbrella of the Biomedical Alliance in Europe, will be highlighted, as well as the search for solutions through dialogue with the European Commission. Although we recognize that the IVDR promotes positive goals such as increased clinical evidence, surveillance, and transparency, we need to ensure that the capabilities of the diagnostic sector are not damaged by infrastructural unpreparedness, while at the same time being forced to submit to a growing bureaucratic and unsupportive structure that will not support its " droit d'exister "., (© 2021 Christa Cobbaert et al., published by De Gruyter, Berlin/Boston.)

Invasive bilirubin measurements remain the gold standard for the diagnosis and treatment of infants with severe neonatal hyperbilirubinemia. The present paper describes different methods currently available to assess hyperbilirubinemia in newborn infants. Novel point-of-care bilirubin measurement methods, such as the BiliSpec and the Bilistick, would benefit many newborn infants, especially in low-income and middle-income countries where the access to costly multi-analyzer in vitro diagnostic instruments is limited. Total serum bilirubin test results should be accurate within permissible limits of measurement uncertainty to be fit for clinical purposes. This implies correct implementation of internationally endorsed reference measurement systems as well as participation in external quality assessment programs. Novel analytic methods may, apart from bilirubin, include the determination of bilirubin photoisomers and bilirubin oxidation products in blood and even in other biological matrices. IMPACT: Key message: Bilirubin measurements in blood remain the gold standard for diagnosis and treatment of severe neonatal hyperbilirubinemia (SNH). External quality assessment (EQA) plays an important role in revealing inaccuracies in diagnostic bilirubin measurements. What does this article add to the existing literature? We provide analytic performance data on total serum bilirubin (TSB) as measured during recent EQA surveys. We review novel diagnostic point-of-care (POC) bilirubin measurement methods and analytic methods for determining bilirubin levels in biological matrices other than blood. Impact: Manufacturers should make TSB test results traceable to the internationally endorsed total bilirubin reference measurement system and should ensure permissible limits of measurement uncertainty., (© 2021. The Author(s), under exclusive licence to the International Pediatric Research Foundation, Inc.)

Severe neonatal hyperbilirubinemia (SNH) is a serious condition that occurs worldwide. Timely recognition with bilirubin determination is key in the management of SNH. Visual assessment of jaundice is unreliable. Fortunately, transcutaneous bilirubin measurement for screening newborn infants is routinely available in many hospitals and outpatient settings. Despite a few limitations, the use of transcutaneous devices facilitates early recognition and appropriate management of neonatal jaundice. Unfortunately, however, advanced and often costly screening modalities are not accessible to everyone, while there is an urgent need for inexpensive yet accurate instruments to assess total serum bilirubin (TSB). In the near future, novel icterometers, and in particular optical bilirubin estimates obtained with a smartphone camera and processed with a smartphone application (app), seem promising methods for screening for SNH. If proven reliable, these methods may empower outpatient health workers as well as parents at home to detect jaundice using a simple portable device. Successful implementation of ubiquitous bilirubin screening may contribute substantially to the reduction of the worldwide burden of SNH. The benefits of non-invasive bilirubin screening notwithstanding, any bilirubin determination obtained through non-invasive screening must be confirmed by a diagnostic method before treatment. IMPACT: Key message: Screening methods for neonatal hyperbilirubinemia facilitate early recognition and timely treatment of severe neonatal hyperbilirubinemia (SNH). Any bilirubin screening result obtained must be confirmed by a diagnostic method. What does this article add to the existing literature? Data on optical bilirubin estimation are summarized. Niche research strategies for prevention of SNH are presented. Impact: Transcutaneous screening for neonatal hyperbilirubinemia contributes to the prevention of SNH. A smartphone application with optical bilirubin estimation seems a promising low-cost screening method, especially in low-resource settings or at home., (© 2021. The Author(s), under exclusive licence to the International Pediatric Research Foundation, Inc.)

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., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)