Purpose: Measurement of endogenous uracil (U) is increasingly being used as a dose-individualization method in the treatment of cancer patients with fluoropyrimidines. However, instability at room... Show morePurpose: Measurement of endogenous uracil (U) is increasingly being used as a dose-individualization method in the treatment of cancer patients with fluoropyrimidines. However, instability at room temperature (RT) and improper sample handling may cause falsely increased U levels. Therefore we aimed to study the stability of U and dihydrouracil (DHU) to ensure proper handling conditions.Methods: Stability of U and DHU in whole blood, serum, and plasma at RT (up to 24 h) and long-term stability (>= 7 days) at - 20 degrees C were studied in samples from 6 healthy individuals. U and DHU levels of patients were compared using standard serum tubes (SSTs) and rapid serum tubes (RSTs). The performance of our validated UPLC-MS/MS assay was assessed over a period of 7 months.Results: U and DHU levels significantly increased at RT in whole blood and serum after blood sampling with increases of 12.7 and 47.6% after 2 h, respectively. A significant difference (p = 0.0036) in U and DHU levels in serum was found between SSTs and RSTs. U and DHU were stable at - 20 degrees C at least 2 months in serum and 3 weeks in plasma. Assay performance assessment fulfilled the acceptance criteria for system suitability, calibration standards, and quality controls.Conclusion: A maximum of 1 h at RT between sampling and processing is recommended to ensure reliable U and DHU results. Assay performance tests showed that our UPLC-MS/MS method was robust and reliable. Additionally, we provided a guideline for proper sample handling, processing and reliable quantification of U and DHU. Show less
Muscle-specific kinase (MuSK) myasthenia gravis (MG) is a neuromuscular autoimmune disease belonging to a growing group of IgG4 autoimmune diseases (IgG4-AIDs), in which the majority of pathogenic... Show moreMuscle-specific kinase (MuSK) myasthenia gravis (MG) is a neuromuscular autoimmune disease belonging to a growing group of IgG4 autoimmune diseases (IgG4-AIDs), in which the majority of pathogenic autoantibodies are of the IgG4 subclass. The more prevalent form of MG with acetylcholine receptor (AChR) antibodies is caused by IgG1-3 autoantibodies. A dominant role for IgG4 in autoimmune disease is intriguing due to its antiinflammatory characteristics. It is unclear why MuSK autoantibodies are predominantly IgG4. We hypothesized that MuSK MG patients have a general predisposition to generate IgG4 responses, therefore resulting in high levels of circulating IgG4. To investigate this, we quantified serum Ig isotypes and IgG subclasses using nephelometric and turbidimetric assays in MuSK MG and AChR MG patients not under influence of immunosuppressive treatment. Absolute serum IgG1 was increased in both MuSK and AChR MG patients compared to healthy donors. In addition, only MuSK MG patients on average had significantly increased and enriched serum IgG4. Although more MuSK MG patients had elevated serum IgG4, for most the IgG4 serum levels fell within the normal range. Correlation analyses suggest MuSK-specific antibodies do not solely explain the variation in IgG4 levels. In conclusion, although serum IgG4 levels are slightly increased, the levels do not support ubiquitous IgG4 responses in MuSK MG patients as the underlying cause of dominant IgG4 MuSK antibodies. Show less
Aim Serum levels of Calcitonin Gene-Related Peptide (CGRP)-like immunoreactivity (CGRP-LI) in migraine patients before and after starting treatment with erenumab were measured to evaluate the... Show moreAim Serum levels of Calcitonin Gene-Related Peptide (CGRP)-like immunoreactivity (CGRP-LI) in migraine patients before and after starting treatment with erenumab were measured to evaluate the association with clinical treatment response. Methods Blood samples were collected from the cubital fossa before (T0) and 2-4 weeks after (T1) starting treatment with erenumab. Clinical response was monitored using a daily headache e-diary. Serum levels of CGRP-LI, assessed using radioimmunoassay, were compared between T0 and T1, correcting for migraine reduction. In addition, for both T0 and T1, linear regression models were constructed using migraine reduction as outcome and serum CGRP-LI as independent variable, corrected for age, gender and monthly migraine days (MMD) at baseline. Results Serum CGRP-LI did not differ between T0 and T1 (p = 0.30). However, there was an interaction between time and reduction in MMD (p = 0.01). Absolute reduction in MMD in the third month after treatment with erenumab was associated with serum CGRP-LI at T1, 2-4 weeks after starting treatment with erenumab (p = 0.003), but not with serum CGRP-LI at T0 (p = 0.24). Conclusion Lower serum CGRP-LI 2-4 weeks after starting treatment with erenumab was associated with a higher reduction in migraine days after three months of treatment. Although the underlying mechanisms remain to be determined, this suggests that changes in CGRP levels, shortly after starting erenumab, are important for its clinical effect. Show less
Harder, A.V.E.; Vijfhuizen, L.S.; Henneman, P.; Dijk, K.W. van; Duijn, C.M. van; Terwindt, G.M.; Maagdenberg, A.M.J.M. van den 2021
Background Migraine is a common brain disorder but reliable diagnostic biomarkers in blood are still lacking. Our aim was to identify, using proton nuclear magnetic resonance (H-1-NMR) spectroscopy... Show moreBackground Migraine is a common brain disorder but reliable diagnostic biomarkers in blood are still lacking. Our aim was to identify, using proton nuclear magnetic resonance (H-1-NMR) spectroscopy, metabolites in serum that are associated with lifetime and active migraine by comparing metabolic profiles of patients and controls. Methods Fasting serum samples from 313 migraine patients and 1512 controls from the Erasmus Rucphen Family (ERF) study were available for H-1-NMR spectroscopy. Data was analysed using elastic net regression analysis. Results A total of 100 signals representing 49 different metabolites were detected in 289 cases (of which 150 active migraine patients) and 1360 controls. We were able to identify profiles consisting of 6 metabolites predictive for lifetime migraine status and 22 metabolites predictive for active migraine status. We estimated with subsequent regression models that after correction for age, sex, BMI and smoking, the association with the metabolite profile in active migraine remained. Several of the metabolites in this profile are involved in lipid, glucose and amino acid metabolism. Conclusion This study indicates that metabolic profiles, based on serum concentrations of several metabolites, including lipids, amino acids and metabolites of glucose metabolism, can distinguish active migraine patients from controls. Show less
Background: Diagnosis of infections in returning international travellers can be challenging because of the broad spectrum of potential infectious etiologies potentially involved. Viral metagenomic... Show moreBackground: Diagnosis of infections in returning international travellers can be challenging because of the broad spectrum of potential infectious etiologies potentially involved. Viral metagenomic next-generation sequencing (mNGS) has the potential to detect any virus present in a patient sample and is increasingly being used for difficult to diagnose cases. The aim of this study was to analyze the performance of mNGS for viral pathogen detection in the clinical setting of international travellers returning with febrile illness. Methods: Thirty-eight serum samples from international travellers returning with febrile illness and presenting at the outpatient clinic of the Leiden University Medical Center in the Netherlands in the time period 2015-2016 were selected retrospectively. Samples were processed for viral metagenomic sequencing using a probe panel capturing all known vertebrate viruses. Bioinformatic analysis was performed using Genome Detective software for metagenomic virus detection. Metagenomic virus findings were compared with viral pathogen detection using conventional methods. Results: In 8 out of the 38 patients (21%), a pathogenic virus was detected by mNGS. All viral pathogens detected by conventional assays were also detected by mNGS: dengue virus (n=4 patients), Epstein-Barr virus (n=2), hepatitis B virus (n=1). In addition, mNGS resulted in additional pathogenic findings in 2 patients (5%): dengue virus (n=1), and hepatitis C virus (n=1). Non-pathogenic viruses detected were: GB virus C (n=1) and torque teno viruses (n=3). High genome coverage and depth using capture probes enabled typing of the dengue viruses detected. Conclusions: Viral metagenomics has the potential to assist the detection of viral pathogens and co-infections in one step in international travellers with a febrile syndrome. Furthermore, viral enrichment by probes resulted in high genome coverage and depth which enabled dengue virus typing. Show less
Background: Serum biomarkers may inform and improve care in traumatic brain injury (TBI). We aimed to correlate serum biomarkers with clinical severity, care path and imaging abnormalities in TBI,... Show moreBackground: Serum biomarkers may inform and improve care in traumatic brain injury (TBI). We aimed to correlate serum biomarkers with clinical severity, care path and imaging abnormalities in TBI, and explore their incremental value over clinical characteristics in predicting computed tomographic (CT) abnormalities.Methods: We analyzed six serum biomarkers (S100B, NSE, GFAP, UCH-L1, NFL and t-tau) obtained <24 h post-injury from 2867 patients with any severity of TBI in the Collaborative European NeuroTrauma Effectiveness Research (CENTER-TBI) Core Study, a prospective, multicenter, cohort study. Univariable and multivariable logistic regression analyses were performed. Discrimination was assessed by the area under the receiver operating characteristic curve (AUC) with 95% confidence intervals.Findings: All biomarkers scaled with clinical severity and care path (ER only, ward admission, or ICU), and with presence of CT abnormalities. GFAP achieved the highest discrimination for predicting CT abnormalities (AUC 0.89 [95%CI: 0.87-0.90]), with a 99% likelihood of better discriminating CT-positive patients than clinical characteristics used in contemporary decision rules. In patients with mild TBI, GFAP also showed incremental diagnostic value: discrimination increased from 0.84 [95%CI: 0.83-0.86] to 0.89 [95%CI: 0.87-0.90] when GFAP was included. Results were consistent across strata, and injury severity. Combinations of biomarkers did not improve discrimination compared to GFAP alone.Interpretation: Currently available biomarkers reflect injury severity, and serum GFAP, measured within 24 h after injury, outperforms clinical characteristics in predicting CT abnormalities. Our results support the further development of serum GFAP assays towards implementation in clinical practice, for which robust clinical assay platforms are required.(C) 2020 The Authors. Published by Elsevier B.V. Show less
Grootveld, R. van; Dam, G.J. van; Dood, C. de; Vries, J.J.C. de; Visser, L.G.; Corstjens, P.L.A.M.; Lieshout, L. van 2018
Schistosomiasis is a parasitic disease affecting over 250 million people in the tropics. In non-endemic regions, imported Schistosoma infections are commonly diagnosed by serology, but based on... Show moreSchistosomiasis is a parasitic disease affecting over 250 million people in the tropics. In non-endemic regions, imported Schistosoma infections are commonly diagnosed by serology, but based on antibody detection an active infection cannot be distinguished from a cured infection and it may take more than 8 weeks after exposure before seroconversion occurs. In endemic populations, excellent results have been described in diagnosing low-grade active Schistosoma infections by the detection of the adult worm-derived circulating anodic antigen (CAA) utilising robust lateral flow (LF) assays combined with up-converting phosphor (UCP) reporter technology. The purpose of this study is to explore the diagnostic value of the UCP-LF CAA assay in a non-endemic setting. CAA concentrations were determined in 111 serum samples originating from 81 serology-positive individuals. In nine individuals, serum could be collected before travel and an additional five provided samples before and after seroconversion occurred. Based on detectable CAA levels, an active infection was seen in 56/81 (69%) of the exposed individuals, while the 10 controls and the 9 sera collected before travel were tested negative for CAA. Positive CAA levels were observed starting 4 weeks after exposure and in four cases CAA was detected even before Schistosoma-specific antibodies became positive. Higher serum CAA levels were seen in migrants than in travellers and CAA concentrations dropped sharply when testing follow-up samples after treatment. This explorative study indicates the UCP-LF CAA serum assay to be a highly accurate test for detecting active low-grade Schistosoma infections in a non-endemic routine diagnostic setting. Show less
IntroductionThe Alzheimer's Disease Research Summits of 2012 and 2015 incorporated experts from academia, industry, and nonprofit organizations to develop new research directions to transform our... Show moreIntroductionThe Alzheimer's Disease Research Summits of 2012 and 2015 incorporated experts from academia, industry, and nonprofit organizations to develop new research directions to transform our understanding of Alzheimer's disease (AD) and propel the development of critically needed therapies. In response to their recommendations, big data at multiple levels are being generated and integrated to study network failures in disease. We used metabolomics as a global biochemical approach to identify peripheral metabolic changes in AD patients and correlate them to cerebrospinal fluid pathology markers, imaging features, and cognitive performance.MethodsFasting serum samples from the Alzheimer's Disease Neuroimaging Initiative (199 control, 356 mild cognitive impairment, and 175 AD participants) were analyzed using the AbsoluteIDQ-p180 kit. Performance was validated in blinded replicates, and values were medication adjusted.Results Multivariable-adjusted analyses showed that sphingomyelins and ether-containing phosphatidylcholines were altered in preclinical biomarker-defined AD stages, whereas acylcarnitines and several amines, including the branched-chain amino acid valine and α-aminoadipic acid, changed in symptomatic stages. Several of the analytes showed consistent associations in the Rotterdam, Erasmus Rucphen Family, and Indiana Memory and Aging Studies. Partial correlation networks constructed for Aβ1–42, tau, imaging, and cognitive changes provided initial biochemical insights for disease-related processes. Coexpression networks interconnected key metabolic effectors of disease.DiscussionMetabolomics identified key disease-related metabolic changes and disease-progression-related changes. Defining metabolic changes during AD disease trajectory and its relationship to clinical phenotypes provides a powerful roadmap for drug and biomarker discovery. Show less
Verhoeven, A.; Slagboom, E.; Wuhrer, M.; Giera, M.; Mayboroda, O.A. 2017
Glycosylation is the most abundant and complex protein modification, and can have a profound structural and functional effect on the conjugate. The oligosaccharide fraction is recognized to be... Show moreGlycosylation is the most abundant and complex protein modification, and can have a profound structural and functional effect on the conjugate. The oligosaccharide fraction is recognized to be involved in multiple biological processes, and to affect proteins physical properties, and has consequentially been labeled a critical quality attribute of biopharmaceuticals. Additionally, due to recent advances in analytical methods and analysis software, glycosylation is targeted in the search for disease biomarkers for early diagnosis and patient stratification. Biofluids such as saliva, serum or plasma are of great use in this regard, as they are easily accessible and can provide relevant glycosylation information. Thus, as the assessment of protein glycosylation is becoming a major element in clinical and biopharmaceutical research, this review aims to convey the current state of knowledge on the N-glycosylation of the major plasma glycoproteins alpha-1-acid glycoprotein, alpha-1-antitrypsin, alpha-1B-glycoprotein, alpha-2-HS-glycoprotein, alpha-2-macroglobulin, antithrombin-III, apolipoprotein B-100, apolipoprotein D, apolipoprotein F, beta-2-glycoprotein 1, ceruloplasmin, fibrinogen, immunoglobulin (Ig) A, IgG, IgM, haptoglobin, hemopexin, histidine-rich glycoprotein, kininogen-1, serotransferrin, vitronectin, and zinc-alpha-2-glycoprotein. In addition, the less abundant immunoglobulins D and E are included because of their major relevance in immunology and biopharmaceutical research. Where available, the glycosylation is described in a site-specific manner. In the discussion, we put the glycosylation of individual proteins into perspective and speculate how the individual proteins may contribute to a total plasma N-glycosylation profile determined at the released glycan level. Show less
Rojas, L.B.; Weigelt, K.; Wit, H. de; Ozcan, B.; Oudenaren, A. van; Sempertegui, F.; ... ; Leenen, P.J.M. 2016
Given the natural history of colorectal and breast cancer, early diagnosis appears to be the most appropriate tool to reduce disease-related mortality.[6;7] Currently, there is no early diagnostic... Show moreGiven the natural history of colorectal and breast cancer, early diagnosis appears to be the most appropriate tool to reduce disease-related mortality.[6;7] Currently, there is no early diagnostic test with high sensitivity, specificity and positive predictive value, which can be used as a routine screening tool. Therefore, there is a need for new biomarkers for both types of cancer that can improve early diagnosis, monitoring of disease progression and therapeutic response and detect disease recurrence. Proteomic expression profiles generated with mass spectrometry have been suggested as potential tools for the early diagnosis of cancer and other diseases. Because it is still in its infancy, many problems have to be overcome before clinical proteomics can be transferred form bench to bedside. Chapter 2 gives an insight in the different fields of translational research in colorectal cancer by our group. In chapter 3 reliability of human serum protein profiling using MALDI-TOF mass spectrometry is analysed. We present a pipeline for pre-processing, statistical data analysis and presentation of MALDI-TOF spectra. This novel analysis method was used to assess the effect of variable pre-analytical conditions on human serum protein profiles, and their effect on reproducibility. In line with the logistic conditions in a routine clinical setting, the effects of sample handling and storage, and also circadian rhythm factors on the serum protein profiles were analysed. In chapter 4 and 5 the feasibility of mass spectrometry based protein profiling for the discrimination of colorectal cancer patients from healthy individuals was assessed. In addition to standardizing technical factors and biological variations, we performed blinded tests and employed a randomised block design experimentation to minimize impact of potential confounding factors and to avoid bias. Especially, validation of our classifier, as a possible pitfall, was given much attention. Therefore, we performed a linear discriminant analysis with double cross-validation to separate cancer patients from healthy subjects. Chapter 6 reports on results from an identical designed protein profiling study for the detection of breast cancer. In chapter 7 a first validated study on the detection of breast cancer based on mass spectrometry generated protein profiles is described. In this study the same randomised blocked design and double cross validation is used, however the classifier was validated in an independent set of new patients and controls. Finally, the results and conclusions of all above mentioned studies and especially the current status of clinical proteomics in cancer are discussed in chapter 8. A Dutch summary of this thesis is written in chapter 9. Show less
