Unveiling a new era with liquid chromatography coupled with mass spectrometry to enhance parathyroid hormone measurement in patients with chronic kidney disease.

Precise determination of circulating parathyroid hormone (PTH) concentration is crucial to diagnose and manage various disease conditions, including the chronic kidney disease-mineral and bone disorder. However, the lack of standardization in PTH assays is challenging for clinicians, potentially leading to medical errors because the different assays do not provide equivalent results and use different reference ranges. Here, we aimed to evaluate the impact of recalibrating PTH immunoassays by means of a recently developed LC-MS/MS method as the reference. Utilizing a large panel of pooled plasma samples with PTH concentrations determined by the LC-MS/MS method calibrated with the World Health Organization (WHO) 95/646 International Standard, five PTH immunoassays were recalibrated. The robustness of this standardization was evaluated over time using different sets of samples. The recalibration successfully reduced inter-assay variability with harmonization of PTH measurements across different assays. By recalibrating the assays based on the WHO 95/646 International Standard, we demonstrated the feasibility for standardizing PTH measurement results and adopting common reference ranges for PTH assays, facilitating a more consistent interpretation of PTH values. The recalibration process aligns PTH results obtained from various immunoassays with the LC-MS/MS method, providing more consistent and reliable measurements. Thus, establishing true standardization across all PTH assays is crucial to ensure consistent interpretation and clinical decision-making.

[Clinical metabolomics : an innovative approach towards preventive and personalized medicine]. / La métabolomique clinique : une approche novatrice vers une médecine préventive et personnalisée.

In order to improve our healthcare system, it is undeniable that the future of modern medicine must focus on a more preventive and personalized approach, notably based on the individual characteristics specific to each patient. In this perspective, clinical metabolomics, which focuses on metabolites, emerges as a particularly interesting and promising approach. Indeed, this science reflects the internal and external stimuli received by an individual, thus capturing their physiological and/or pathological state. Close to the phenotype, it represents the interface between the patient, their genes, and their environment in the broadest sense. Its translational nature requires the conjunction of several expertise areas, both in analytical, biostatistical, and clinical levels. Combined with other data, it allows the generation of predictive or diagnostic models useful for early detection and monitoring of pathologies, taking into account notably the individual characteristics of patients. There are, of course, many obstacles and challenges to overcome for metabolomics to transition into clinical practice, but it is evident that this innovative approach will, in the years to come, find its place among the tools available to clinicians in a more personalized vision of patient care.

Dans le but d'améliorer notre système de santé, il est indéniable que l'avenir de la médecine moderne doit se porter sur une approche plus préventive et personnalisée, basée, notamment, sur les caractéristiques individuelles propres au patient. Dans cette optique, la métabolomique clinique, qui s'intéresse aux métabolites, apparaît comme une approche particulièrement intéressante et prometteuse. En effet, cette science est le reflet des stimuli internes et externes que reçoit un individu et permet donc de capturer son état physiologique et/ou pathologique. Proche du phénotype, elle représente l'interface entre le patient, ses gènes et son environnement au sens large. Sa nature translationnelle nécessite la conjonction de plusieurs expertises, tant au niveau analytique que bio-statistique et clinique. Combinée à d'autres données, elle permet de générer des modèles prédictifs ou diagnostiques utiles pour détecter précocement et suivre des pathologies, en tenant compte, notamment, des caractéristiques individuelles des patients. Il reste, bien entendu, de nombreux obstacles et défis à relever pour que la métabolomique passe dans la pratique clinique. Cependant, il apparaît évident que cette approche novatrice trouvera, dans les années à venir, sa place parmi les outils à disposition des cliniciens dans une vision préventive et plus personnalisée de la prise en charge du patient.

Concise review on the combined use of immunocapture, mass spectrometry and liquid chromatography for clinical applications.

Immunocapture is now a well-established method for sample preparation prior to quantitation of peptides and proteins in complex matrices. This short review will give an overview of some clinical applications of immunocapture methods, as well as protocols with and without enzymatic digestion in a clinical context. The advantages and limitations of both approaches are discussed in detail. Challenges related to the choice of mass spectrometer are also discussed. Top-down, middle-down, and bottom-up approaches are discussed. Even though immunocapture has its limitations, its main advantage is that it provides an additional dimension of separation and/or isolation when working with peptides and proteins. Overall, this short review demonstrates the potential of such techniques in the field of proteomics-based clinical medicine and paves the way for better personalized medicine.

Analytical performance specifications for the measurement uncertainty of 24,25-dihydroxyvitamin D examinations.

OBJECTIVES: The exploration of the metabolites in the degradation pathways of vitamin D (VTD) has gained importance in recent years and simultaneous quantitation of twenty-five-hydroxy vitamin D (25(OH)D) mass concentration together with 24,25-dihydroxyvitamin D (24,25(OH)2D) has been proposed as a newer approach to define VTD deficiency. Yet, no data are available on 24,25(OH)2D biological variation (BV). In this study, we evaluated 24,25(OH)2D's BV on the European Biological Variation Study (EuBIVAS) cohort samples to determine if analytical performance specifications (APS) for 24,25(OH)2D could be generated. METHODS: Six European laboratories recruited 91 healthy participants. 25(OH)D and 24,25(OH)2D concentrations in K3-EDTA plasma were examined weekly for up to 10 weeks in duplicate with a validated LC-MS/MS method. The Vitamin D Metabolite Ratio (24,25(OH)2D divided by 25(OH)D × 100) was also calculated at each time point. RESULTS: Linear regression of the mean 24,25(OH)2D concentrations at each blood collection showed participants were not in steady state. Variations of 24,25(OH)2D over time were significantly positively associated with the slopes of 25(OH)D concentrations over time and the concentration of 25(OH)D of the participant at inclusion, and negatively associated with body mass index (BMI), but not with age, gender, or location of the participant. The variation of the 24,25(OH)2D concentration in participants over a 10 weeks period was 34.6%. Methods that would detect a significant change linked to the natural production of 24,25(OH)2D over this period at p<0.05 would need a relative measurement uncertainty (u%)<14.9% while at p<0.01, relative measurement uncertainty should be <10.5%. CONCLUSIONS: We have defined for the first time APS for 24,25(OH)2D examinations. According to the growing interest in this metabolite, several laboratories and manufacturers might aim to develop specific methods for its determination. The results presented in this paper are thus necessary prerequisites for the validation of such methods.

Validation of an LC-MS/MS Method Using Solid-Phase Extraction for the Quantification of 1-84 Parathyroid Hormone: Toward a Candidate Reference Measurement Procedure.

BACKGROUND: Parathyroid hormone (PTH) measurement is important for patients with disorders of calcium metabolism, including those needing bone-turnover monitoring due to chronic kidney disease-mineral bone disorder. There are currently 2 generations of PTH immunoassays on the market, both having cross-reactivity issues and lacking standardization. Therefore, we developed an LC-MS/MS higher-order method for PTH analysis. METHODS: The method was calibrated against the international standard for 1-84 PTH (WHO 95/646). Antibody-free sample preparation with the addition of an isotope-labeled internal standard was performed by solid-phase extraction. Extracts were analyzed by LC-MS/MS. EDTA-K2 plasma was used throughout the development and validation. Bias and uncertainty sources were tested according to ISO 15193. Clinical Laboratory Standards Institute guidelines and reference measurement procedures were consulted for the design of the validation. Patient samples and external quality controls were compared between LC-MS/MS and 2 third-generation immunoassays. RESULTS: The method was validated for 1-84 PTH from 5.7 to 872.6 pg/mL. The interassay imprecision was between 1.2% and 3.9%, and the accuracy ranged from 96.2% to 103.2%. The measurement uncertainty was <5.6%. The comparison between LC-MS/MS and the immunoassays showed a proportional bias but moderate to substantial correlation between methods. CONCLUSIONS: This LC-MS/MS method, which is independent of antibodies, is suitable for a wide range of PTH concentrations. The obtained analytical performance specifications demonstrate that development of a reference measurement procedure will be possible once a higher order reference standard is available.

Comparison of two LC-MS/MS methods for the quantification of 24,25-dihydroxyvitamin D3 in patients and external quality assurance samples.

OBJECTIVES: In-house developed liquid-chromatography mass spectrometry (LC-MS/MS) methods are used more and more frequently for the simultaneous quantification of vitamin D metabolites. Among these, 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) is of clinical interest. This study assessed the agreement of this metabolite in two validated in-house LC-MS/MS methods. METHODS: 24,25(OH)2D3 was measured in 20 samples from the vitamin D external quality assurance (DEQAS) program and in a mixed cohort of hospital patients samples (n=195) with the LC-MS/MS method at the Medical University of Graz (LC-MS/MS 1) and at the University of Liège (LC-MS/MS 2). RESULTS: In DEQAS samples, 24,25(OH)2D3 results with LC-MS/MS 1 had a proportional bias of 1.0% and a negative systemic difference of -0.05%. LC-MS/MS 2 also showed a proportional bias of 1.0% and the negative systemic bias was -0.22%. Comparing the EQA samples with both methods, no systemic bias was found (0.0%) and the slope was 1%. The mean difference of 195 serum sample measurements between the two LC-MS/MS methods was minimal (-0.2%). Both LC-MS/MS methods showed a constant bias of 0.31 nmol/L and a positive proportional bias of 0.90%, respectively. CONCLUSIONS: This study is the first to assess the comparability of 24,25(OH)2D3 concentrations in a mixed cohort of hospitalized patients with two fully validated in-house LC-MS/MS methods. Despite different sample preparation, chromatographic separation and ionization, both methods showed high precision measurements of 24,25(OH)2D3. Furthermore, we demonstrate the improvement of accuracy and precision measurements of 24,25(OH)2D3 in serum samples and in the DEQAS program.

Evolution of the slopes of ST2 and galectin-3 during marathon and ultratrail running compared to a control group.

Background Previous studies have suggested that exercising may induce cardiac damage. Galectin-3 (Gal-3) and soluble suppression of tumorigenicity 2 (ST2) are very interesting biomarkers for heart failure and myocardial fibrosis. We aimed to compare the kinetics of emerging fibrosis cardiac biomarkers as Gal-3 and ST-2 in endurance runners, and recreational runners before and after a running event represented by a marathon and an ultratrail event. Methods Blood samples were taken from 19 healthy non-elite marathon runners (42 km), 27 ultratour runners (67 km), and 14 recreational runners who represented the control group (10 km) just before the run (T0), just after (T1) and 3 h after (T2), in order to analyze Gal-3, ST2, hsTnT, NT-proBNP, CKMB and hsCRP. We compared the percentage of evolution and the slopes obtained from T0 to T1 (pT0T1) and from T1 to T2 (pT1T2), between the different groups of runners participating in three different races. Results Plasma cardiac biomarker concentrations increased significantly from baseline to immediately post-exercise and most of the time decreased over the subsequent 3-h period. For pT0T1 and pT1T2, the markers Gal-3 and ST2 showed a significant difference between types of run (p < 0.05 and p < 0.0001, respectively). During the recovery time, Gal-3 returned to the baseline values but not ST2 which continued to increase. Conclusions Gal-3 and ST2 are considered as a reflection of cardiac fibrosis and remodeling. The evolution of both was different, particularly after the recovery time. ST2 values exceeding cutoff values at any time.

Simultaneous measurement of 25(OH)-vitamin D and 24,25(OH)2-vitamin D to define cut-offs for CYP24A1 mutation and vitamin D deficiency in a population of 1200 young subjects.

Background Simultaneous measurement of 25(OH)D and 24,25(OH)2D is a new tool for predicting vitamin D deficiency and allows evaluating CYP24A1 lack of function. Interpretation of 24,25(OH)2D should be performed according to 25(OH)D levels and a ratio, called the vitamin D metabolite ratio (VMR) has been proposed for such a purpose. Unfortunately, the VMR can be expressed in different ways and cannot be used if 24,25(OH)2D concentrations are undetectable. Here, we propose evaluating the enzyme activity taking into consideration the probability that a normal population presents undetectable 24,25(OH)2D concentrations according to 25(OH)D levels. We thus retrospectively measured 25(OH)D and 24,25(OH)2D in a population of 1200 young subjects to evaluate the 25(OH)D threshold above which the enzyme was induced. Methods Serum samples from 1200 infants, children, adolescent and young adults were used to simultaneously quantify 25(OH)D and 24,25(OH)2D by LCMS/MS. Results Median (interquartile range [IQR]) levels were 20.6 (14.4-27.2) ng/mL for 25(OH)D. 172 subjects (14.3%) presented 24,25(OH)2D values below the LOQ. When 25(OH)D values were <11 ng/mL, 63.1% of subjects presented undetectable 24,25(OH)2D concentrations. Percentage decreased with increasing 25(OH)D values to become 19.7% for 25(OH)D comprised between 12 and 15 ng/mL, 5.1% for 25(OH)D between 16 and 20 and 0.7% for 25(OH)D >21 ng/mL. Conclusions We suggest using a statistical approach to evaluate CYP24A1 function according to 25(OH)D concentrations. Our results also show that vitamin D deficiency, as defined biochemically, could be around 20 ng/mL in infants, children, adolescent and young adults and that vitamin D deficiency could be evaluated on a more individual basis.

The pathway through LC-MS method development: in-house or ready-to-use kit-based methods?

Historically, the determination of low concentration analytes was initially made possible by the development of rapid and easy-to-perform immunoassays (IAs). Unfortunately, typical problems inherent to IA technologies rapidly appeared (e.g. elevated cost, cross-reactivity, lot-to-lot variability, etc.). In turn, liquid chromatography tandem mass spectrometry (LC-MS/MS) methods are sensitive and specific enough for such analyses. Therefore, they would seem to be the most promising candidates to replace IAs. There are two main choices when implementing a new LC-MS/MS method in a clinical laboratory: (1) Developing an in-house method or (2) purchasing ready-to-use kits. In this paper, we discuss some of the respective advantages, disadvantages and mandatory requirements of each choice. Additionally, we also share our experiences when developing an in-house method for cortisol determination and the implementation of an "ready-to-use" (RTU) kit for steroids analysis.

Analytical and clinical validation of the new Abbot Architect 25(OH)D assay: fit for purpose?

BACKGROUND: We provide a clinical and analytical evaluation of the reformulated version of the Abbott Architect 25-hydroxyvitamin D assay. We compared this assay with three commercial automated immunoassays and against a VDSP-traceable liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) in six different populations. We also supplemented 40 healthy volunteers with either 600,000 IU of vitamin D2 or 100,000 of vitamin D3 to evaluate the performance of the immunoassays vs. the LC-MS/MS. METHODS: Precision and limit of quantification were assessed, 25(OH)D2 and C3-epimer recovery were calculated. Two hundred and forty samples obtained in healthy Caucasians and Africans, osteoporotic, hemodialyzed and intensive care patients and 3rd trimester pregnant women were analyzed by all methods. Correlation was studied using Passing-Bablok and Bland-Altman analysis. Concordance correlation coefficient (CCC) was calculated to evaluate agreement between immunoassays and LC-MS/MS. We verified if patients were homogeneously classified with the immunoassays when they took vitamin D2 or vitamin D3 after 1, 7 and 28 days. RESULTS: We observed excellent analytical features and showed a very good correlation to the LC-MS/MS results in the overall population. Compared to the other immunoassays, concordance of the new Abbott assay with the LC-MS/MS was at least similar, and often better in diseased populations. Althought the cross-reactivity with 25(OH)D2 was not of 100%, there was no significant difference in the classifications of the patients, either supplemented with D2 or D3 or after 7 or 28 days. CONCLUSIONS: This modified version of the Abbott Architect assay is clearly improved compared to the previous one and presents a better agreement with the LC-MS/MS.

Performance characteristics of the VIDAS® 25-OH Vitamin D Total assay - comparison with four immunoassays and two liquid chromatography-tandem mass spectrometry methods in a multicentric study.

BACKGROUND: The study was conducted to evaluate the analytical and clinical performance of the VIDAS® 25-OH Vitamin D Total assay. The clinical performance of the assay was compared with four other immunoassays against the results of two different liquid chromatography/mass spectrometry methods (LC-MS/MS) standardized to NIST reference materials. METHODS: VIDAS® 25-OH Vitamin D Total assay precision, linearity, detection limits and sample matrix comparison were assessed following CLSI guidelines. For method comparison, a total of 150 serum samples ranging from 7 to 92 ng/mL were analyzed by all the methods. Correlation was studied using Passing-Bablok regression and Bland-Altman analysis. The concordance correlation coefficient (CCC) was calculated to evaluate agreement between immunoassays and the reference LC-MS/MS method. In addition, samples containing endogenous 25(OH)D2 were used to assess each immunoassay's ability to detect this analyte. Pregnancy and hemodialysis samples were used to the study the effect of vitamin D binding protein (DBP) concentration over VIDAS® assay performance. RESULTS: The VIDAS® 25-OH Vitamin D Total assay showed excellent correlation to the LC-MS/MS results (y=1.01x+0.22 ng/mL, r=0.93), as obtained from two different sites and distinct LC-MS/MS methods. The limit of quantification was determined at 8.1 ng/mL. Cross-reactivity for 25(OH)D2 was over 80%. At concentrations of 10.5, 26 and 65.1 ng/mL, within-run CVs were 7.9%, 3.6% and 1.7%, while total CVs (between runs, calibrations, lots and instruments) were 16.0%, 4.5% and 2.8%. The VIDAS® performance was not influenced by altered DBP levels, though under-recovery of 25(OH)D as compared to LC-MS/MS was observed for hemodialysis samples. CONCLUSIONS: The VIDAS® 25-OH Vitamin D Total assay is therefore considered suitable for assessment of vitamin D status in clinical routine.

Analytical and clinical evaluation of the new Fujirebio Lumipulse®G non-competitive assay for 25(OH)-vitamin D and three immunoassays for 25(OH)D in healthy subjects, osteoporotic patients, third trimester pregnant women, healthy African subjects, hemodialyzed and intensive care patients.

BACKGROUND: In this study, we provide a short analytical evaluation of the new Fujirebio Lumipulse®G non-competitive immunoassay for 25(OH)D. Clinical performance was compared with three commercial competitive automated immunoassays against a Vitamin D Standardization Program (VDSP)-traceable liquid chromatography-tandem mass spectrometry (LC-MS/MS) in six different clinically relevant populations. METHODS: Lumipulse®G 25(OH)D precision, measurement uncertainty, recovery, limit of quantification were assessed, as well as 25(OH)D2 and C3-epimer recovery. For method comparison, 250 serum samples obtained in healthy Caucasians and Africans, osteoporotic, hemodialyzed and intensive care patients and 3rd trimester pregnant women were analyzed by all methods. Correlation was studied using Passing-Bablok and Bland-Altman analysis. Concordance correlation coefficient (CCC) was calculated to evaluate agreement between immunoassays and the LC-MS/MS. RESULTS: The Lumipulse®G 25(OH)D assay presented interesting analytical features and showed excellent correlation to the LC-MS/MS results (y=1.00×-1.35 ng/mL), as obtained in healthy Caucasian individuals. In the other special populations, Lumipulse®G presented a concordance with LC-MS/MS which was generally higher than competitors, even if all methods significantly under-recovered 25(OH)D in hemodialyzed patients. Intra-assay CV ranged from 12.1% at 9.6 ng/mL to 2.1% at 103.7 ng/mL and inter-assay CV ranged from 16.2 to 3.7% at the same concentrations, respectively. Measurement uncertainty, with a probability of 95%, were respectively 33.1 and 7.6% at these concentrations. LOQ was found to be at 4.6 ng/mL. Mean (95% CI) 25(OH)D2 revovery was 77% (74-81) and no cross-reactivity was observed with C3-epimer. CONCLUSIONS: Fujirebio Lumipulse®G 25-OH Vitamin D Total assay is therefore considered suitable for assessment of vitamin D status in clinical routine.

New biomarkers for primary mitral regurgitation.

BACKGROUND: Mitral regurgitation is a frequent valvular heart disease affecting around 2.5 % of the population with prevalence directly related to aging. Degeneration of mitral valve is broadly considered as a passive ongoing pathophysiological process and little is known about its physiological deregulation. The purpose of this study was to highlight new biomarkers of mitral regurgitation in order to decipher the underlying pathological mechanism as well as to allow the diagnosis and the monitoring of the disease. RESULTS: Modulation of various blood proteins expression was examined in patients suffering from different grades of mitral regurgitation (mild, moderate and severe) compared to healthy controls. To this end, several routine clinical assays and the multi analyte profile technology targeting 184 proteins were used. High-density lipoprotein, apolipoprotein-A1, haptoglobin and haptoglobin-α2 chain levels significantly decreased proportionally to the degree of mitral regurgitation when compared to controls. High-density lipoprotein and apolipoprotein-A1 levels were associated with effective regurgitant orifice area and regurgitant volume. Apolipoprotein-A1 was an independent predictor of severe mitral regurgitation. Moreover, with ordinal logistic regression, apolipoprotein-A1 remained the only independent factor associated with mitral regurgitation. In addition, myxomatous mitral valves were studied by immunocytochemistry. We observed an increase of LC3, the marker of autophagy, in myxomatous mitral valves compared with healthy mitral valves. CONCLUSION: These potential biomarkers of mitral regurgitation highlighted different cellular processes that could be modified in myxomatous degenerescence: reverse cholesterol transport, antioxidant properties and autophagy.

One-year follow-up of platelet-rich plasma infiltration to treat chronic proximal patellar tendinopathies.

Infiltration of Platelet-Rich Plasma (PRP) may be considered as a recent therapeutic option for chronic tendinopathies. The aim of this study is to evaluate the clinical status and the return to sports activities in patients with chronic proximal patellar tendinopathies. Twenty subjects with chronic proximal patellar tendinopathy benefited from 1 infiltration of PRP coupled with a standardized eccentric rehabilitation. The follow-up (up to 1 year) was assessed by means of a Visual Anologue Scale (VAS), the International Knee Documentation Committee (IKDC) form and the Victorian Institute of Sport Assessment (VISA-P) score. Moreover, subjects had to answer an information questionnaire concerning their life and sports activities. Seventy percents of the patients reported a favourable evolution with decrease of pain, and returned to sports activities. With time, VAS dropped significantly and both IKDC and VISA-P scores improved also significantly. This study confirms that a local injection of PRP coupled with a program of eccentric rehabilitation for treating a chronic jumper's knee, improves pain symptoms and the functionalities of the subjects' knee up to 1 year after injection.

Exuberant inflammatory reaction as a side effect of platelet-rich plasma injection in treating one case of tendinopathy.

Platelet-rich plasma (PRP) contains a large number of growth factors, which may enhance tendon healing processes. Local injection of PRP represents a relatively new treatment for tendinopathies. To date, no side effects have been reported after injection of PRP to treat tendinopathy. We report a case of exuberant inflammatory reaction after 1 injection of PRP to treat jumper's knee in a 35-year-old male type 1 diabetic patient. Injections of PRP should be proposed only after careful consideration in cases of patients with morbidity risks linked to insulin-dependent diabetes.

Development and validation of an LC-MS/MS method for the simultaneous quantitation of angiotensin (1-7), (1-8), (1-9) and (1-10) in human plasma.

Cardiovascular diseases have cast a significant negative impact on the lives of millions worldwide. Over the years, extensive efforts have been dedicated to enhancing diagnostic and prognostic tools for these diseases. A growing body of evidence indicates that the angiotensin convertase enzyme (ACE) and the angiotensin convertase enzyme 2 (ACE2), and angiotensin peptide levels could hold a pivotal role in assisting clinicians with the management of cardiovascular conditions, notably hypertension and heart failure. However, despite the considerable body of knowledge in this domain, a void remains in the field of analytical methodologies for these molecules. In this study, we present a fully validated LC-MS/MS method for the precise quantitation of plasma angiotensin (1-7), (1-8), (1-9), and (1-10), following the guidelines set by the Clinical and Laboratory Standards Institute (CLSI). Our method not only enables the accurate quantification of angiotensin peptides but also provides a means to assess ACE and ACE2 activity. Remarkably, our method achieved a Lower Limit of Measurement Interval (LLMI) as low as 5 pg/mL. This has enabled the detection of angiotensin (1-7), (1-8), (1-9) and (1-10) and the accurate quantitation of angiotensin (1-7), (1-8) and (1-10) in all analyzed groups, including healthy controls, patients with high blood pressure, and patients with chronic kidney disease. To our knowledge, our method represents the most sensitive approach allowing for simultaneous quantitation of these four angiotensin peptides. A distinct advantage of our method, when compared to immunoassays, is its high sensitivity combined with comprehensive chromatographic separation of all currently known angiotensin peptides. This combination translates to an exceptional level of selectivity, underscoring the value and potential of our methodology in advancing cardiovascular disease research.

Technical and clinical evaluation of the VITROS® Immunodiagnostic Products 25-OH Vitamin D Total Assay--comparison with marketed automated immunoassays and a liquid chromatography-tandem mass spectrometry method.

BACKGROUND: The study was conducted to evaluate the technical and clinical performance of the VITROS® Immunodiagnostic Products 25-OH Vitamin D Total Assay, and compare it with the performance of five marketed automated assays and a liquid chromatography/mass spectrometry reference method (LC-MS/MS). METHODS: Three hundred patient serum samples were used to compare the correlation of the VITROS® 25-OH Vitamin D Total Assay with both the other immunoassays and the LC-MS/MS method, using Passing-Bablok regression and Bland-Altman analyses. Concordance of the diagnosis of vitamin D status was calculated to test the agreement between the different assays. In addition, samples containing vitamin D2 were used to test the assay's ability to detect the D2 form of the vitamin. RESULTS AND CONCLUSIONS: These results from the VITROS® 25-OH Vitamin D Total Assay generally correlated well with those from most of the marketed immunoassays. Cross-reactivity of the D2 form was calculated as being close to 100%. Additionally, we found substantial variability in performance amongst the various assays, which suggests the need for optimisation and recalibration of commercial methods.

Inconsistencies in laboratory investigations of hypertension in a young woman taking ethinylestradiol/drospirenone association.

CASE PRESENTATION: A 34-year-old woman presented to the emergency department for arterial hypertension. Blood analysis requested by the endocrinologist showed very high level of aldosterone (1805 ng/L, normal values: <264 ng/L) and high level of renin activity (2.8 ng/mL/h, normal values: 0.1-2.0 ng/mL/h). The patient reported the use of Yasmin® (ethinylestradiol 30 µg/drospirenone 3 mg) continuously (without hormone-free week between cycles) as oral contraception. Medical imaging examinations revealed no anomaly in the kidneys and the adrenal glands. On the endocrinologist advice, patient stopped the intake of Yasmin®. Aldosterone and renin levels were measured several times after the discontinuation of the oral contraception and a diminution of these levels was observed with a complete normalization of both levels 26 days after the synthetic hormones discontinuation. DISCUSSION: The literature shows that ethynilestradiol/drospirenone association can interfere with the renin-angiotensin-aldosterone system and increase the levels of aldosterone and/or renin. We reported here a clinical case illustrating the significant impact of this medication on the renin-angiotensin-aldosterone axis of a young woman. However, this association is not listed among the drugs interfering with the aldosterone and renin-level measurements. CONCLUSION: Considering the data in the literature and our clinical case, we suggest adding drospirenone and the ethinylestradiol/drospirenone association in the list of drugs interfering with aldosterone and renin level determination.

Evaluation of analytical and clinical performance of the AFIAS Tn-I plus assay - a new point-of-care.

BACKGROUND: The objective of this evaluation was to determine the analytical and clinical performance of the AFIAS point-of-care (POC) Tn-I Plus assay (Boditech Med Inc). DESIGN AND METHODS: Limit of detection (LOD), limit of quantification (LOQ), repeatability, reproducibility, inter- and intra-individual CV were evaluated using the CLSI guidelines. The study was also designed to estimate the 99th percentile upper reference limit (URL) and to assess the diagnostic sensitivity and specificity. RESULTS: The precision repeatability CVs were 6.7-8.5% and reproducibility was 7.5-7.6%. The LOD and LOQ were consistent with the manufacturer's specified values of 0.010 ng/mL and 0.030 ng/mL, respectively. The 99th percentile URLs for males (aged 18-75 years) and females (aged 17-65 years) in serum were 0.0300 ng/mL (7.8% CV) and 0.0239 ng/mL (9.4% CV) respectively. Overall 99th percentile URL was 0.0296 ng/mL (8.2% CV). For the overall apparently healthy population, the percentage of measurable cardiac troponin I (cTn-I) values below the 99th percentile (i.e. 0.0296 ng/mL) and above the assay's LOD (= 0.010 ng/mL) was 47,68% (391/820 samples). The diagnostic sensitivity and specificity were 100% with 95% CI (97% - 100%) and 95.2% with 95% CI (93.6% - 96.5%), respectively. No significant differences were observed for the diagnosis of acute myocardial infarction (AMI) between AFIAS Tn-I plus and Abbott ARCHITECT High Sensitive Troponin-I. CONCLUSION: The clinical performance of AFIAS Tn-I Plus assay for AMI is comparable to the established Abbott ALINITY STAT High Sensitive Troponin-I. This assay is suitable for routine use in clinical laboratories.

Innovative workflow for the identification of cathepsin K cleavage sites in type I collagen.

Since the late 1990s, cathepsin K cleavage sites in type I collagen have been extensively studied due to its ability to release bone resorption biomarkers such as CTX and NTX. However, gel-based methods and N-sequencing used in these studies lack sensitivity, especially for small to medium peptides. In this work, we propose a degradomics mass spectrometry-based workflow that combines protein digestion, Nano-LC-UDMSE, and several software tools to identify cathepsin K cleavage sites. This workflow not only identified previously known cleavage sites, but also discovered new ones. Multiple cleavage hotspots were found and described in type I α1 and type I α2 collagen, many of which coincided with pyridinoline crosslinks, known to stabilize the triple helix. Our results allowed us to establish a chronology of digestion and conclude that cathepsin K preferentially cleaves the extremities of type I collagen before the helical part. We also found that cathepsin K preferentially cleaves amino acid residues with long and hydrophobic lateral chains at the beginning of digestion, whereas no preferred amino acid residues were identified later in the digestion. In conclusion, our workflow successfully identified new cleavage sites and can be easily applied to other proteins or proteases.