Real-World Clinical Performance Evaluation of a Fourth-Generation HIV Antigen/Antibody Differentiation Test.

BACKGROUND: HIV testing is still an important component of routine sexual health screening, assessment of at-risk individuals and as part of the care of pregnant women. To prevent further transmission of infection, it is important that HIV tests are highly sensitive and that positive cases are not missed. HIV serologic antigen/antibody tests are commonly used as they are capable of detecting recent and established infection. METHODS: In this study we assessed the performance of the Elecsys HIV Duo assay (Elecsys assay) against the Abbott Architect assay in 10 121 samples from US and non-US adult, pediatric, and pregnant populations including low-risk, high-risk, and known positive cohorts. Congruent repeatedly reactive and/or discrepant samples followed a confirmatory algorithm consisting of an antigen/antibody differentiation assay and a nucleic acid test, as per the study protocol. RESULTS: The overall sensitivity of the Elecsys assay was 100.00% (95% CI 99.81-100.00 [1977/1977]), and the specificity was 99.84% (95% CI 99.73-99.91 [8129/8142]). The Elecsys assay detected all positive samples within the study, including all 50 antigen-only positive samples and samples from different HIV subtypes, including group O, group M subtypes, HIV-2 positives, and HIV-1 and HIV-2 dual positives. CONCLUSIONS: The Elecsys HIV Duo assay was highly sensitive for diagnosis of HIV in a range of clinical samples from the United States and outside the United States and is suitable for routine use.

Comparative evaluation of technetium-99m-diethylenetriaminepentaacetic acid renal dynamic imaging versus the Modification of Diet in Renal Disease equation and the Chronic Kidney Disease Epidemiology Collaboration equation for the estimation of GFR.

PURPOSE: We compared the performance of technetium-99m-diethylenetriaminepentaacetic acid (99mTc-DTPA) renal dynamic imaging (RDI), the MDRD equation, and the CKD EPI equation to estimate glomerular filtration rate (GFR). METHODS: A total of 551 subjects, including CKD patients and healthy individuals, were enrolled in this study. Dual plasma sample clearance method of 99mTc-DTPA was used as the true value for GFR (tGFR). RDI and the MDRD and CKD EPI equations for estimating GFR were compared and evaluated. RESULTS: Data indicate that RDI and the MDRD equation underestimated GFR and CKD EPI overestimated GFR. RDI was associated with significantly higher bias than the MDRD and CKD EPI equations. The regression coefficient, diagnostic precision, and consistency of RDI were significantly lower than either equation. RDI and the MDRD equation underestimated GFR to a greater degree in subjects with tGFR ≥ 90 ml/min/1.73 m2 compared with the results obtained from all subjects. In the tGFR60-89 ml/min/1.73 m2 group, the precision of RDI was significantly lower than that of both equations. In the tGFR30-59 ml/min/1.73 m2 group, RDI had the least bias, the most precision, and significantly higher accuracy compared with either equation. In tGFR < 30 ml/min/1.73 m2, the three methods had similar performance and were not significantly different. CONCLUSIONS: RDI significantly underestimates GFR and performs no better than MDRD and CKD EPI equations for GFR estimation; thus, it should not be recommended as a reference standard against which other GFR measurement methods are assessed. However, RDI better estimates GFR than either equation for individuals in the tGFR30-59 ml/min/1.73 m2 group and thus may be helpful to distinguish stage 3a and 3b CKD.

Variability of Late-Night Salivary Cortisol in Cushing Disease: A Prospective Study.

Background: The frequency of variable hormonogenesis in patients with Cushing disease (CD) but without cyclical symptoms is unclear. Aim: To assess the frequency of variable hormonogenesis in patients presenting with CD. Methods: Over a 6-month period, patients with confirmed or suspected CD provided late-night salivary samples for up to 42 consecutive nights. Results: Of 19 patients confirmed to have CD, 16 provided at least 7 consecutive salivary samples, and 13 provided at least 21; these 16 patients are the subjects of this report. Twelve patients had at least three peak and two trough levels of late-night salivary cortisol (LNSC) but in only two patients were strict criteria for cyclical hormonogenesis fulfilled; variation was assessed as random in the others. Eight patients had de novo CD, and eight had recurrent/persistent disease. All patients with recurrent/persistent CD had two or more normal results, and in four of these patients, >50% of LNSC were normal. In six patients with de novo disease with at least one normal LNSC level, the maximum levels ranged from 1.55 to 15.5 times the upper limit of normal. Conclusions: Extreme fluctuations of cortisol production, measured by sequential LNSC, are common in CD. In newly diagnosed disease, this may only occasionally impair diagnostic ability, whereas in most patients with recurrent/persistent disease after pituitary surgery, LNSC is frequently within the reference range, with potential to cause diagnostic problems.

Determining the optimal screening interval for type 2 diabetes mellitus using a risk prediction model.

BACKGROUND: Progression to diabetes mellitus (DM) is variable and the screening time interval not well defined. The American Diabetes Association and US Preventive Services Task Force suggest screening every 3 years, but evidence is limited. The objective of the study was to develop a model to predict the probability of developing DM and suggest a risk-based screening interval. METHODS: We included non-diabetic adult patients screened for DM in the Cleveland Clinic Health System if they had at least two measurements of glycated hemoglobin (HbA1c), an initial one less than 6.5% (48 mmol/mol) in 2008, and another between January, 2009 and December, 2013. Cox proportional hazards models were created. The primary outcome was DM defined as HbA1C greater than 6.4% (46 mmol/mol). The optimal rescreening interval was chosen based on the predicted probability of developing DM. RESULTS: Of 5084 participants, 100 (4.4%) of the 2281 patients with normal HbA1c and 772 (27.5%) of the 2803 patients with prediabetes developed DM within 5 years. Factors associated with developing DM included HbA1c (HR per 0.1 units increase 1.20; 95%CI, 1.13-1.27), family history (HR 1.31; 95%CI, 1.13-1.51), smoking (HR 1.18; 95%CI, 1.03-1.35), triglycerides (HR 1.01; 95%CI, 1.00-1.03), alanine aminotransferase (HR 1.07; 95%CI, 1.03-1.11), body mass index (HR 1.06; 95%CI, 1.01-1.11), age (HR 0.95; 95%CI, 0.91-0.99) and high-density lipoproteins (HR 0.93; 95% CI, 0.90-0.95). Five percent of patients in the highest risk tertile developed DM within 8 months, while it took 35 months for 5% of the middle tertile to develop DM. Only 2.4% percent of the patients in the lowest tertile developed DM within 5 years. CONCLUSION: A risk prediction model employing commonly available data can be used to guide screening intervals. Based on equal intervals for equal risk, patients in the highest risk category could be rescreened after 8 months, while those in the intermediate and lowest risk categories could be rescreened after 3 and 5 years respectively.

Identification of Filamin-A and -B as potential biomarkers for prostate cancer.

AIM: A novel strategy for prostate cancer (PrCa) biomarker discovery is described. MATERIALS & METHODS: In vitro perturbation biology, proteomics and Bayesian causal analysis identified biomarkers that were validated in in vitro models and clinical specimens. RESULTS: Filamin-B (FLNB) and Keratin-19 were identified as biomarkers. Filamin-A (FLNA) was found to be causally linked to FLNB. Characterization of the biomarkers in a panel of cells revealed differential mRNA expression and regulation. Moreover, FLNA and FLNB were detected in the conditioned media of cells. Last, in patients without PrCa, FLNA and FLNB blood levels were positively correlated, while in patients with adenocarcinoma the relationship is dysregulated. CONCLUSION: These data support the strategy and the potential use of the biomarkers for PrCa.

Comparison of symmetric dimethylarginine with creatinine, cystatin C and their eGFR equations as markers of kidney function.

OBJECTIVES: Symmetric dimethylarginine (SDMA) is a catabolic product of arginine-methylated proteins and is an emerging biomarker for kidney function. A limited number of studies in selected populations have shown good correlation between SDMA and a few known markers of glomerular filtration rate (GFR). However, a comprehensive comparison of SDMA with all existing serum endogenous markers in a population with varied kidney function and against measured GFR is lacking. The objective of this study was to compare the correlations of SDMA, creatinine, cystatin C and their eGFR equations against GFR measured by iothalamate clearance in an adult population with varied kidney function. DESIGN & METHODS: Left-over serum and plasma specimens were collected from 40 adults with normal and reduced kidney function. GFR was measured using a radioactive iothalamate procedure. Creatinine and cystatin C were measured on Roche Cobas 8000. SDMA was measured by a published liquid chromatography-tandem mass spectrometry method. RESULTS: SDMA correlated highly with measured GFR (r=-0.84), which was better than creatinine (r=-0.70) but equivalent to cystatin C (r=-0.86) and the eGFR equations [MDRD and CKD-EPI (separate and combined)]. CONCLUSIONS: SDMA is a strong marker of kidney function and further studies are needed to establish an eGFR formula that includes it for widespread clinical use.

Sensitive, Simple, and Robust Nano-Liquid Chromatography-Mass Spectrometry Method for Amyloid Protein Subtyping.

Amyloidosis is a rare condition characterized by deposits of insoluble proteins in the form of ß-pleated sheets. These deposits interfere with the normal structure and function of varying tissues. Thirty-one amyloid proteins have been identified, and the correct identification is critical due to the varying treatments. Immunohistochemistry, the most routine method for identification of amyloid proteins, suffers from limitations. Mass spectrometry (MS)-based methods offer better sensitivity and specificity. We describe here a sensitive, simple, and robust MS-based method for the identification of amyloid proteins. Amyloid deposits are excised from formalin-fixed tissue by laser microdissection and is put through protein extraction followed by trypsin digestion. The resulting peptides are separated by nano-liquid chromatography and analyzed by high-resolution Orbitrap mass spectrometry. The mass spectrometry data are then searched against a human protein database for identification and semi-quantification.

Quantitation of Free Metanephrines in Plasma by Liquid Chromatography-Tandem Mass Spectrometry.

Plasma metanephrines are measured to aid in the diagnosis of pheochromocytomas. In patients with pheochromocytomas there is excessive production of catecholamines and metanephrines. Measurement of plasma free metanephrines is one of the first-line clinical tests that are used for the diagnosis and follow-up of pheochromocytoma. We describe here a liquid chromatography-tandem mass spectrometry method to measure free metanephrines in plasma. Free metanephrine and normetanephrine are extracted via solid-phase extraction. After extraction and evaporation, the reconstituted supernatant is analyzed by high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). The MS/MS is set to selective reaction monitoring mode (180.1 → 148.1 m/z for metanephrine, 183.1 → 168.1 for d3-metanephrine, 166.1 → 134.1 m/z for normetanephrine, and 169.1 → 137.2 m/z for d3-normetanephrine) with positive electrospray ionization. Quantitation is based on peak area ratio of the analyte to its respective deuterated internal standard. The assay is linear from 5.9 to 4090.0 pg/mL for metanephrine and 22.0 to 4386.7 pg/mL for normetanephrine with precision of <6 % over the ranges.

Quantification of Metanephrine and Normetanephrine in Urine Using Liquid Chromatography-Tandem Mass Spectrometry.

Measuring urinary metanephrines aides in the diagnosis of pheochromocytomas-catecholamine producing tumors. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) allows for greater sensitivity and simpler sample preparation as compared with other techniques. Here we describe a simple LC-MS/MS method for measuring metanephrines in urine. Each urine sample was treated with diphenylboronic acid to create boronate complexes, and then applied to a Bond-Elut Plexa cartridge. After solid phase extraction, samples were concentrated and analyzed on an Atlantis T3 column with chromatographic run time totaling 8.5 min. MS/MS was set in positive electrospray ionization mode with multiple reaction monitoring for data collection. The assay was linear from 0.2 to 27.4 µmol/L and 0.3 to 14.6 µmol/L for metanephrine and normetanephrine, respectively. Intra-assay and total precision at three concentration levels over 10 days were <5 % for metanephrine and <10 % for normetanephrine.

Recent advances in MS methods for nicotine and metabolite analysis in human matrices: clinical perspectives.

Tobacco smoking is a major global health issue and represents the leading cause of preventable death in the developed countries. Nicotine is a major alkaloid found in tobacco products and its detection with its metabolites in human matrices is generally used for assessing tobacco consumption and second hand exposure. Several analytical techniques have been developed for the detection of nicotine and its metabolites, and MS coupled with chromatography is considered the standard reference method because of its superior sensitivity and specificity. In this work, we reviewed nicotine metabolism, clinical MS and the latest (2009-2014) development of MS-based techniques for measurement of nicotine and metabolites in human matrices. Appropriate biomarker and matrix selection are also critically discussed.

Applicability of chronic kidney disease epidemiology collaboration equations in a Chinese population.

BACKGROUND: Accurate estimated glomerular filtration rates (eGFR) is an important step in the diagnosis of chronic kidney disease (CKD). The Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation, based on creatinine alone (eGFRcr), was developed to improve on the Modification of Diet in Renal Disease equation, in particular by addressing the systematic underestimation of high GFR. Whether the CKD-EPI equation, based on cystatin C alone (eGFRcys), or the combined creatinine-cystatin C CKD-EPI equation (eGFRcr-cys C), actually perform better than the CKD-EPI equation based on creatinine (eGFRcr) remains unknown, especially in Asians including Chinese populations, where eGFR equations may overestimate true GFR. METHODS: A standard dual plasma sampling method (DPSM) of estimating (99m)Tc-diethylene triamine penta-acetic acid clearance was used to determine the reference or measured GFR (mGFR). Linear regression analysis, Bland-Altman analysis, bias, absolute bias and accuracy (P30) were used to compare the performance of the combined creatinine-cystatin C equation (eGFRcr-cys) and equations based on each marker alone (eGFRcr and eGFRcys) in Chinese subjects, including both patients with CKD and healthy individuals. RESULTS: We enrolled 617 Chinese participants (49.11% female, 47.11 ± 17.25 years old), with a mean mGFR of 73.80 ± 37.55 mL/min/1.73 m(2). The predictive abilities (r), the accuracy (P15, P30, P50), bias and absolute bias of the eGFRcr-cys equation were superior to eGFRcr equation and the eGFRcys equation in overall samples. Bland-Altman analysis also demonstrated a consistent result. When compared in subgroups, the accuracy (P30) of all three equations exceeded 90% at mGFR ≥90 mL/min/1.73m(2); the eGFRcr-cys equation had the highest accuracy (P30: 95.56%). At mGFR 60-89 mL/min/1.73 m(2), the accuracies (P30) of the eGFRcr-cys and eGFRcr equations exceeded the acceptable level (≥70%), and there was no significant difference between them (P = 0.58). At mGFR <60 mL/min/1.73 m(2), the accuracy (P30) of all three equations was below 70%, but the eGFRcr-cys equation had the greatest precision. CONCLUSIONS: The performances of the eGFRcr-cys and eGFRcr equations were similar to superior to that of the eGFRcys equation at higher GFR levels in an Asian population, especially in normal and mild to moderate kidney disease. Further improvement is needed for these equations at GFR <60 mL/min per 1.73 m(2).

A simple, fast, and sensitive method for the measurement of serum nicotine, cotinine, and nornicotine by LC-MS/MS.

The measurement of nicotine and its metabolites has been used to monitor tobacco use. A high-sensitivity method (<1 ng/mL) is necessary for the measurement in serum or plasma to differentiate nonsmokers from passive smokers. Here, we report a novel LC-MS/MS method to quantify nicotine, cotinine, and nornicotine in serum with high sensitivity. Sample preparation involved only protein precipitation, followed by online turbulent flow extraction and analysis on a porous graphitic carbon column in alkaline conditions. The chromatography time was 4 min. No significant matrix effects or interference were observed. The lower limit of quantification was 0.36, 0.32, and 0.38 ng/mL for nicotine, cotinine, and nornicotine, respectively, while accuracy was 91.6-117.1%. No carryover was observed up to a concentration of 48 , 550, and 48 ng/mL for nicotine, cotinine, and nornicotine, respectively. Total CV was <6.5%. The measurement of nicotine and cotinine was compared with an independent LC-MS/MS method and concordant results were obtained. In conclusion, this new method was simple, fast, sensitive, and accurate. It was validated to measure nicotine, cotinine, and nornicotine in serum for monitoring tobacco use.

High levels of zinc-protoporphyrin identify iron metabolic abnormalities in pulmonary arterial hypertension.

Iron homeostasis influences the development of pulmonary arterial hypertension (PAH) associated with hypoxia or hematologic disorders. To investigate whether severity of idiopathic PAH (IPAH) is impacted by alterations in iron metabolism, we assessed iron metabolic markers, including levels of zinc-protoporphyrin (Zn-pp), transferrin receptor, and red blood cell numbers and morphology in IPAH, associated PAH and sleep apnea-induced pulmonary hypertension patients in comparison to healthy controls and asthmatics. Despite similarly normal measures of iron metabolism, Zn-pp levels in IPAH and sleep apnea patients were elevated approximately twofold, indicating deficient iron incorporation to form heme and levels were closely related to measures of disease severity. Consistent with high Zn-pp, PAH patients had increased red cell distribution width (RDW). In an expanded cohort including patients with IPAH and familial disease, the RDW was validated and related to clinical parameters of severity; including pulmonary artery pressures and 6-minute walk distances. These results reveal an increased prevalence of subclinical functional iron deficiency in primary forms of PAH that is quantitatively related to disease severity. This suggests that altered iron homeostasis influences disease progression and demonstrates the importance of closely monitoring iron status in PAH patients.

Quantitative measurement of plasma free metanephrines by ion-pairing solid phase extraction and liquid chromatography-tandem mass spectrometry with porous graphitic carbon column.

Plasma free metanephrine and normetanephrine are the best biomarkers for diagnosing pheochromocytoma. In the past few years, liquid chromatography-tandem mass spectrometry has become the preferred technology to measure plasma metanephrine and normetanephrine because of its high sensitivity and specificity, as well as fast and simple sample preparation. In this study, we report a liquid chromatography-tandem mass spectrometry method for measuring plasma metanephrine and normetanephrine. A solid phase extraction method using ion-pairing reagent and C18 stationary phase was used for sample preparation. We tested a porous graphitic carbon column and a HILIC column for chromatographic separation, and the former one showed better resolution with no interference from plasma matrix. This method was linear from 7.2-486.8 pg/mL for metanephrine and 18.0-989.1 pg/mL for normetanephrine with an accuracy of 92.2-111.8% and 92.1-115.0%, respectively. Inter-assay and intra-assay CV for metanephrine and normetanephrine at two different concentration levels ranged from 2.0% to 10.9%. In conclusion, this liquid chromatography-tandem mass spectrometry method using ion-pairing solid phase extraction and porous graphitic column was simple and efficient for measuring plasma metanephrines.

A simple liquid chromatography-tandem mass spectrometry method for measuring metanephrine and normetanephrine in urine.

BACKGROUND: Measuring urinary fractionated metanephrines is one of the initial tests in the diagnosis of pheochromocytoma. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) represents the most specific and accurate technology for this purpose. The goal of this work was to develop a simple LC-MS/MS method for measuring metanephrines in urine. METHODS: Each urine sample was complexed with diphenylboronic acid, and purified on a Bond-Elute Plexa cartridge. The extract was concentrated and analyzed on a short Atlantis T3 column in 8.5 min. Metanephrines and their deuterated internal standards were monitored in positive electrospray ionization mode by multiple reaction monitoring. RESULTS: Ion suppression was observed, but was compensated for by the respective internal standard. The analytical measurement range was 0.2-27.4 µmol/L and 0.3-14.6 µmol/L for metanephrine and normetanephrine, respectively. The intra-assay and total coefficient of variation throughout the linear ranges was 2.03%-2.11% and 2.20%-3.80% for metanephrine, and 4.50%-8.09% and 9.00%-10.00% for normetanephrine, respectively. Comparison with a commercial HPLC method using patient samples (n=65) by Passing-Bablok regression showed a slope of 1.000 and 1.014, y-intercept of -0.080 and -0.067, a correlation coefficient of 0.8830 and 0.9022, and a mean difference of 14.0% and -0.43% for metanephrine and normetanephrine, respectively. CONCLUSIONS: This simple method for urine metanephrines is suitable for clinical use.

The MMACHC proteome: hallmarks of functional cobalamin deficiency in humans.

Cobalamin (Cbl, B(12)) is an essential micronutrient required to fulfill the enzymatic reactions of cytosolic methylcobalamin-dependent methionine synthase and mitochondrial adenosylcobalamin-dependent methylmalonyl-CoA mutase. Mutations in the MMACHC gene (cblC complementation group) disrupt processing of the upper-axial ligand of newly internalized cobalamins, leading to functional deficiency of the vitamin. Patients with cblC disease present with both hyperhomocysteinemia and methylmalonic acidemia, cognitive dysfunction, and megaloblastic anemia. In the present study we show that cultured skin fibroblasts from cblC patients export increased levels of both homocysteine and methylmalonic acid compared to control skin fibroblasts, and that they also have decreased levels of total intracellular folates. This is consistent with the clinical phenotype of functional cobalamin deficiency in vivo. The protein changes that accompany human functional Cbl deficiency are unknown. The proteome of control and cblC fibroblasts was quantitatively examined by two dimensional difference in-gel electrophoresis (2D-DIGE) and liquid chromatography-electrospray ionization-mass spectrometry (LC/ESI/MS). Major changes were observed in the expression levels of proteins involved in cytoskeleton organization and assembly, the neurological system and cell signaling. Pathway analysis of the differentially expressed proteins demonstrated strong associations with neurological disorders, muscular and skeletal disorders, and cardiovascular diseases in the cblC mutant cell lines. Supplementation of the cell cultures with hydroxocobalamin did not restore the cblC proteome to the patterns of expression observed in control cells. These results concur with the observed phenotype of patients with the cblC disorder and their sometimes poor response to treatment with hydroxocobalamin. Our findings could be valuable for designing alternative therapies to alleviate the clinical manifestation of the cblC disorder, as some of the protein changes detected in our study are common hallmarks of known pathologies such as Alzheimer's and Parkinson's diseases as well as muscular dystrophies.

A fast and simple assay for busulfan in serum or plasma by liquid chromatography-tandem mass spectrometry using turbulent flow online extraction technology.

Busulfan is used in myeloablative preparation regimens for hematopoietic bone marrow transplantation. Due to its narrow therapeutic range therapeutic drug monitoring of busulfan is recommended. In this study a fast and simple method for measuring busulfan in serum or plasma by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed utilizing turbulent flow online extraction technology. Serum or plasma was mixed with acetonitrile containing d(8)-busulfan. After centrifugation the supernatant was injected onto a turbulent flow preparatory column then transferred to a C18 analytical column monitored by a tandem mass spectrometer set at positive electrospray ionization. The analytical cycle time was 4.0min. The method was linear from 0.15 to 41.90µmol/L with an accuracy of 87.9-103.0%. Inter- and intra-assay CVs across four concentration levels were 2.1-7.8%. No significant carryover or ion suppression was observed. No interference was observed from commercial control materials containing more than 100 compounds. Comparison with a well established LC-MS/MS method using patient specimens (n=45) showed a mean bias 1.3% with Deming regression of slope 1.02, intercept -0.02µmol/L, and a linear correlation coefficient 0.9883. The LC-MS/MS method coupled with turbulent flow online sample cleaning technology described here offers reliable busulfan quantitation in serum or plasma with minimum manual sample preparation and was fully validated for clinical use.

Epidemiology of vitamin D in health and disease.

Results from ecological, case-control and cohort studies have shown that vitamin D reduces the risk of bone fracture, falls, autoimmune diseases, type 2 diabetes, CVD and cancer. However, there is still epidemic vitamin D insufficiency especially among individuals living at high latitudes or with dark skin. Serum levels of 25-hydroxyvitamin D (25(OH)D) are considered the best biomarker of vitamin D nutritional status. Appropriate sunshine exposure or oral supplementation is necessary to maintain sufficient vitamin D status, which is generally accepted as serum 25(OH)D>75 nmol/l. Immunoassays, especially RIA, have been primarily used to measure serum 25(OH)D while liquid chromatography-MS (LC-MS) is considered the 'gold standard'. There is significant disparity among the immunoassays, and all immunoassays have considerable bias compared with LC-MS methods. Because of the variations among the results from these different assays, it is necessary that assay-specific reference ranges be established or standardisation of the assays take place. The present review focuses on ecological, case-control, and cohort studies that investigated the role of vitamin D in health and disease. In addition, analytical techniques used in laboratory evaluation of vitamin D nutritional status are also critically reviewed. The majority of the literature included in the present review is selected from that searchable in PubMed up to the end of September 2008.