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... Show moreBACKGROUND: 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. Show less
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... Show moreBackground 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 & GE;3 (n = 132) in comparison to the general population (n = 1438). Relationships between the biomarkers and total protein, & beta;2-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 & GE;3, B2M, NGAL, and KIM-1 were increased up to 1.3-fold. IGFBP7, TIMP2, NGAL, and CXCL9 were strongly correlated (all r & GE; 0.8); correlations with B2M and TP were smaller (all r & LE; 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. Show less
Lentjes, E.G.W.M.; Bui, H.N.; Ruhaak, L.R.; Kema, I.P.; Coene, K.L.M.; Ouweland, J.M.W. van den 2023
Background: Over the past decade the use of LC-MS/MS has increased significantly in the hospital laboratories. Clinical laboratories have switched from immunoassays to LC-MS/MS methods due to the... Show moreBackground: Over the past decade the use of LC-MS/MS has increased significantly in the hospital laboratories. Clinical laboratories have switched from immunoassays to LC-MS/MS methods due to the promise of improve-ments in sensitivity and specificity, better standardization with often non-commutable international standards, and better between-laboratory comparison. However, it remains unclear whether routine performance of the LC-MS/MS methods have met these expectations. Method: This study examined the EQAS results, from the Dutch SKML, of serum cortisol, testosterone, 25OH-vita-minD and cortisol in urine and saliva over 9 surveys (2020 to first half of 2021). Results: The study found a significant increase in the number of compounds and in the number of results measured in the different matrices, with LC-MS/MS over a period of eleven years. In 2021, approximately 4000 LC-MS/MS results were submitted (serum: urine: saliva = 58:31:11%) compared to only 34 in 2010. When compared to the individual immunoassays, the LC-MS/MS based methods for serum cortisol, testosterone and 25OH-vitaminD showed comparable but also higher between-laboratory CVs in different samples of the surveys. For cortisol, testosterone and 25OH-vitaminD the median CV was 6.8%, 6.1% and 4.7% respectively for the LC-MS/MS compared to 3.9-8.0%,4.5-6.7%, and 7.5-18.3% for immunoassays. However, the bias and imprecision of the LC-MS/MS was better than that of the immunoassays. Conclusion: Despite the expectation that LC-MS/MS methods would result in smaller between-laboratory dif-ferences, as they are relatively matrix independent and better to standardize, the results of the SKML round robins do not reflect this for some analytes and may be in part explained by the fact that in most cases laboratory developed tests were used. Show less
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... Show moreIn 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. Show less
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... Show moreBackground: 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). Show less
Dikaios, I.; Althaus, H.; Angles-Cano, E.; Ceglarek, U.; Coassin, S.; Cobbaert, C.M.; ... ; IFCC Working Grp Apolipoprot Mass 2023
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.... Show moreBackground: 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. Show less
Diederiks, N.; Ravensbergen, C.J.; Treep, M.; Wezel, M. van; Kuruc, M.; Ruhaak, L.R.; ... ; Mesker, W.E. 2023
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... Show moreIn 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 deter-mining 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. Show less
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... Show moreBackground: 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 labdeveloped 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. Show less
Duijl, T.T. van; Ruhaak, L.R.; Kooten, C. van; Fijter, J.W. de; Cobbaert, C.M. 2022
BackgroundLC-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... Show moreBackgroundLC-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.MethodsNGAL, 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.ResultsThe 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.ConclusionsLC-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. Show less
Kruijt, M.; Treep, M.M.; Cobbaert, C.M.; Ruhaak, L.R. 2022
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... Show moreBackground: 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 & mu;mol/L, and a provisional reference interval of 1.07 to 1.49 & mu;mol/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. Show less
Kruijt, M.; Pol, L.M. van der; Eikenboom, J.; Verburg, H.J.; Cobbaert, C.M.; Ruhaak, L.R. 2021
Antithrombin deficiency diagnostics by first-line activity tests suffer from a lack of sensitivity sometimes resulting in diagnostic uncertainty. We here present a case of a woman with recurrent... Show moreAntithrombin deficiency diagnostics by first-line activity tests suffer from a lack of sensitivity sometimes resulting in diagnostic uncertainty. We here present a case of a woman with recurrent pregnancy loss who was screened for inherited thrombophilia. Antithrombin activity was borderline low, resulting in uncertainty about the correct diagnosis. Using a mass spectrometry-based test, the antithrombin protein of the patient was characterized at the molecular level and a heterozygous p.Pro73Leu mutation was identified. The mutation, also known as antithrombin "Basel," increases the risk of venous thromboembolism and obstetric complications. This case is illustrative of current antithrombin deficiency screening, in which diagnoses may be missed by traditional diagnostics. Next-generation protein diagnostics by mass spectrometry provides molecular insight into the proteoforms present in vivo. This information is essential for laboratory specialists and clinicians to unambiguously diagnose patients and will aid in evolving healthcare from traditional to precision diagnostics. Show less
We have recently introduced multiple reaction monitoring (MRM) mass spectrometry as a novel tool for glycan biomarker research and discovery. Herein, we employ this technique to characterize the... Show moreWe have recently introduced multiple reaction monitoring (MRM) mass spectrometry as a novel tool for glycan biomarker research and discovery. Herein, we employ this technique to characterize the site-specific glycan alterations associated with primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC). Glycopeptides associated with disease severity were also identified. Multinomial regression modelling was employed to construct and validate multi-analyte diagnostic models capable of accurately distinguishing PBC, PSC, and healthy controls from one another (AUC = 0.93 +/- 0.03). Finally, to investigate how disease-relevant environmental factors can influence glycosylation, we characterized the ability of bile acids known to be differentially expressed in PBC to alter glycosylation. We hypothesize that this could be a mechanism by which altered selfantigens are generated and become targets for immune attack. This work demonstrates the utility of the MRM method to identify diagnostic site-specific glycan classifiers capable of distinguishing even related autoimmune diseases from one another. Show less
Cobbaert, C.M.; Althaus, H.; Brkovic, I.B.; Ceglarek, U.; Coassin, S.; Delatour, V.; ... ; IFCC Working Grp Standardization A 2021
Current dyslipidemia management in patients with atherosclerotic cardiovascular disease (ASCVD) is based on traditional serum lipids. Yet, there is some indication from basic research that serum... Show moreCurrent dyslipidemia management in patients with atherosclerotic cardiovascular disease (ASCVD) is based on traditional serum lipids. Yet, there is some indication from basic research that serum apolipoproteins A-I, (a), B, C-I, C-II, C-III, and E may give better pathophysiological insight into the root causes of dyslipidemia. To facilitate the future adoption of clinical serum apolipoprotein (apo) profiling for precision medicine, strategies for accurate testing should be developed in advance.Recent discoveries in basic science and translational medicine set the stage for the IFCC Working Group on Apolipoproteins by Mass Spectrometry. Main drivers were the convergence of unmet clinical needs in cardiovascular disease (CVD) patients with enabling technology and metrology. First, the residual cardiovascular risk after accounting for established risk factors demonstrates that the current lipid panel is too limited to capture the full complexity of lipid metabolism in patients. Second, there is a need for accurate test results in highly polymorphic and atherogenic apolipoproteins such as apo(a). Third, sufficient robustness of mass spectrometry technology allows reproducible protein quantification at the molecular level. Fourth, several calibration hierarchies in the revised ISO 17511:2020 guideline facilitate metrological traceability of test results, the highest achievable standard being traceability to SI.This article outlines the conceptual approach aimed at achieving a novel, multiplexed Reference Measurement System (RMS) for seven apolipoproteins based on isotope dilution mass spectrometry and peptide-based calibration. This RMS should enable standardization of existing and emerging apolipoprotein assays to SI, within allowable limits of measurement uncertainty, through a sustainable network of Reference Laboratories. Show less
The pipeline of biomarker translation from bench to bedside is challenging and limited biomarkers have been adopted to routine clinical care. Ideally, biomarker research and development should be... Show moreThe pipeline of biomarker translation from bench to bedside is challenging and limited biomarkers have been adopted to routine clinical care. Ideally, biomarker research and development should be driven by unmet clinical needs in health care. To guide researchers, clinical chemists and clinicians in their biomarker research, the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) has developed a structured questionnaire in which the clinical gaps in current clinical pathways are identified and desirable performance specifications are predefined. In kidney injury, the high prevalence of the syndrome acute kidney injury (AKI) in the hospital setting has a significant impact on morbidity, patient survival and health care costs, but the use of biomarkers indicating early kidney injury in daily patient care remains limited. Routinely, medical labs measure serum creatinine, which is a functional biomarker, insensitive for detecting early kidney damage and cannot distinguish between renal and prerenal AKI. The perceived unmet clinical needs in kidney injury were identified through the EFLM questionnaire. Nephrologists within our tertiary care hospital emphasized that biomarkers are needed for (1) early diagnosis of in-hospital AKI after a medical insult and in critically ill patients, (2) risk stratification for kidney injury prior to a scheduled (elective) intervention, (3) kidney injury monitoring in patients scheduled to receive nephrotoxic medication and after kidney transplantation and (4) differentiation between prerenal AKI and structural kidney damage. The biomarker search and selection strategy resulted in a rational selection of an eleven-protein urinary panel for kidney injury that target these clinical needs. To assess the clinical utility of the proposed biomarker panel in kidney injury, a multiplexed LC-MS test is now in development for the intended translational research. Show less
Acute kidney injury (AKI) is an important risk factor for chronic kidney disease, renal replacement therapy (RRT), and mortality. However, predicting AKI with currently available markers remains... Show moreAcute kidney injury (AKI) is an important risk factor for chronic kidney disease, renal replacement therapy (RRT), and mortality. However, predicting AKI with currently available markers remains problematic. We assessed the predictive value of urinary tissue inhibitor of metalloprotease-2 (TIMP-2) and insulin-like growth factor-binding protein 7 (IGFBP7) regarding the need for RRT, and 30-day mortality, in elective cardiac surgery patients. In 344 elective cardiac surgery patients, we measured urinary TIMP-2 and IGFBP7 and serum creatinine at baseline and directly after surgery. Discrimination of both urinary biomarkers was assessed by the C-statistic. Model improvement for each biomarker when added to a basic model containing serum creatinine and duration of surgery was tested by the net-reclassification index (cf-NRI) and integrated discrimination index (IDI). At baseline, mean age was 66 years and 67% were men. Of all patients, 22 required RRT following surgery. IGFBP7 pre- and post-surgery and change in TIMP-2 during surgery predicted RRT with a C-statistic of about 0.80. However, a simple model including baseline serum creatinine and duration of surgery had a C-statistic of 0.92, which was improved to 0.93 upon addition of post-surgery TIMP-2 or IGFBP7, with statistically significant cf-NRIs but non-significant IDIs. Post-surgery TIMP-2 and IGFBP predicted 30-day mortality, with C-statistics of 0.74 and 0.80. In conclusion, in elective cardiac surgery patients, pre- and peri-operative clinical variables were highly discriminating about which patients required RRT after surgery. Nonetheless, in elective cardiac surgery patients, urinary TIMP-2 and IGFBP7 improved prediction of RRT and 30-day mortality post-surgery. Show less
Lipoprotein(a) (Lp(a)) is an independent risk factor in the development of atherosclerotic cardiovascular diseases (ASCVD) and calcific aortic valve disease (CAVD). Lp(a) is an LDL-like particle to... Show moreLipoprotein(a) (Lp(a)) is an independent risk factor in the development of atherosclerotic cardiovascular diseases (ASCVD) and calcific aortic valve disease (CAVD). Lp(a) is an LDL-like particle to which apolipoprotein (a) (apo(a)) is covalently bound. Apo(a) contains a variable number of kringle IV repeats, a kringle V and a protease domain. Serum/plasma Lp(a) concentrations are traditionally expressed as total particle mass in mg/L. Concern has arisen lately as flawed Lp(a) mass tests have masked its clinical utility.The determinants of variability in Lp(a) composition were investigated, including the apo(a) size polymorphism, post-translational modifications -N- and O-glycosylation- and the lipid:protein ratio. Depending on the number of kringle IV-2 repeats, the theoretical protein content of the Lp(a) particle varies between 30 and 46 (w/w) %, which inescapably confounds Lp(a) mass measurements.The authors advocate that reporting of Lp(a) particle concentrations in mass units is metrologically inappropriate and should be abandoned, as it results in systematically biased Lp(a) results. Enabling technology, such as mass spectrometry, allows unequivocal molecular characterization of the apo(a) measurand(s) and accurate quantitation of apo(a) in molar units, unaffected by apo(a) size polymorphism. To guarantee that Lp(a)/apo(a) tests are fit-for-clinical-purpose, basic metrology principles should be implemented upfront during test development. Show less
Over the past decades, the genome and proteome have been widely explored for biomarker discovery and personalized medicine. However, there is still a large need for improved diagnostics and... Show moreOver the past decades, the genome and proteome have been widely explored for biomarker discovery and personalized medicine. However, there is still a large need for improved diagnostics and stratification strategies for a wide range of diseases. Post-translational modification of proteins by glycosylation affects protein structure and function, and glycosylation has been implicated in many prevalent human diseases. Numerous proteins for which the plasma levels are nowadays evaluated in clinical practice are glycoproteins. While the glycosylation of these proteins often changes with disease, their glycosylation status is largely ignored in the clinical setting. Hence, the implementation of glycomic markers in the clinic is still in its infancy. This is for a large part caused by the high complexity of protein glycosylation itself and of the analytical techniques required for their robust quantification. Mass spectrometry-based workflows are particularly suitable for the quantification of glycans and glycoproteins, but still require advances for their transformation from a biomedical research setting to a clinical laboratory. In this review, we describe why and how glycomics is expected to find its role in clinical tests and the status of current mass spectrometry-based methods for clinical glycomics. (C) 2020 The Authors. Published by Elsevier B.V. on behalf of The Association for Mass Spectrometry: Applications to the Clinical Lab (MSACL). Show less
Merleev, A.A.; Park, D.; Xie, Y.X.; Kailemia, M.J.; Xu, G.G.; Ruhaak, L.R.; ... ; Maverakis, E. 2020
Alterations in the human glycome have been associated with cancer and autoimmunity. Thus, constructing a site-specific map of the human glycome for biomarker research and discovery has been a... Show moreAlterations in the human glycome have been associated with cancer and autoimmunity. Thus, constructing a site-specific map of the human glycome for biomarker research and discovery has been a highly sought-after objective. However, due to analytical barriers, comprehensive site-specific glycoprofiling is difficult to perform. To develop a platform to detect easily quantifiable, site-specific, disease-associated glycan alterations for clinical applications, we have adapted the multiple reaction monitoring mass spectrometry method for use in glycan biomarker research. The adaptations allow for highly precise site-specific glycan monitoring with minimum sample prep. Using this technique, we successfully mapped out the relative abundances of the most common 159 glycopeptides in the plasma of 97 healthy volunteers. This plasma glycome map revealed 796 significant (FDR<0.05) site-specific inter-protein and intra-protein glycan associations, of which the vast majority were previously unknown. Since age and gender are relevant covariants in biomarker research, these variables were also characterized. 13 glycopeptides were found to be associated with gender and 41 to be associated with age. Using just five age-associated glycopeptides, a highly accurate age prediction model was constructed and validated (r(2)=0.62 +/- 0.12). The human plasma site-specific glycan map described herein has utility in applications ranging from glycan biomarker research and discovery to the development of novel glycan-altering interventions. Show less
Elevated serum prostate-specific antigen (PSA) levels in body fluids may indicate prostate cancer (PCa), but it is noted that the clinical performance is rather poor. Specificity and sensitivity... Show moreElevated serum prostate-specific antigen (PSA) levels in body fluids may indicate prostate cancer (PCa), but it is noted that the clinical performance is rather poor. Specificity and sensitivity values of 20 and 94% at a cutoff value of 4.1 ng/mL, respectively, result in overdiagnosis and unnecessary interventions. Previous exploratory studies have indicated that the glycosylation of PSA potentially leads to improved PCa diagnosis based on qualitative analyses. However, the applied methods are not suited for a quantitative evaluation or implementation in a medical laboratory. Therefore, in this proof-of-principle study, we have evaluated the use of hydrophilic interaction liquid chromatography (HILIC) in combination with targeted quantitative mass spectrometry for the sialic acid linkage-specific analysis of PSA glyco-proteoforms based on either trypsin or ArgC peptides. The efficiency of PSA proteolysis was optimized as well as the glycopeptide separation conditions (buffer type, strength, and pH). The HILIC-based analysis of PSA glyco-proteoforms presented here has the potential for the clinical validation of patient cohorts. The method shows the feasibility of the use of a HILIC stationary phase for the separation of isomeric glycopeptides to detect specific glyco-proteoforms. This is the first step toward the development and evaluation of PSA glyco-proteoforms for use in a clinical chemistry setting aiming for improved PCa diagnosis or screening. Show less
