bluuebunny/crossref_metadata_2025_split huggingface dataset:
Sample Preparation and Storage Can Change Arsenic Speciation in Human Urine
Authors: Feldmann Jörg , Lai Vivian W-M , Cullen William R , Ma Mingsheng , Lu Xiufen , Le X Chris 
Published: January 2020

AbstractBackground: Stability of chemical speciation during sample handling and storage is a prerequisite to obtaining reliable results of trace element speciation analysis. There is no comprehensive information on the stability of common arsenic species, such as inorganic arsenite [As(III)], arsenate [As(V)], monomethylarsonic acid, dimethylarsinic acid, and arsenobetaine, in human urine.Methods: We compared the effects of the following storage conditions on the stability of these arsenic species: temperature (25, 4, and −20 °C), storage time (1, 2, 4, and 8 months), and the use of additives (HCl, sodium azide, benzoic acid, benzyltrimethylammonium chloride, and cetylpyridinium chloride). HPLC with both inductively coupled plasma mass spectrometry and hydride generation atomic fluorescence detection techniques were used for the speciation of arsenic.Results: We found that all five of the arsenic species were stable for up to 2 months when urine samples were stored at 4 and −20 °C without any additives. For longer period of storage (4 and 8 months), the stability of arsenic species was dependent on urine matrices. Whereas the arsenic speciation in some urine samples was stable for the entire 8 months at both 4 and −20 °C, other urine samples stored under identical conditions showed substantial changes in the concentration of As(III), As(V), monomethylarsonic acid, and dimethylarsinic acid. The use of additives did not improve the stability of arsenic speciation in urine. The addition of 0.1 mol/L HCl (final concentration) to urine samples produced relative changes in inorganic As(III) and As(V) concentrations.Conclusions: Low temperature (4 and −20 °C) conditions are suitable for the storage of urine samples for up to 2 months. Untreated samples maintain their concentration of arsenic species, and additives have no particular benefit. Strong acidification is not appropriate for speciation analysis.
Zinc Protoporphyrin: A Metabolite with a Mission
Authors: Labbé Robert F , Vreman Hendrik J , Stevenson David K 
Published: January 2020

AbstractZinc protoporphyrin (ZnPP) is a normal metabolite that is formed in trace amounts during heme biosynthesis. The final reaction in the biosynthetic pathway of heme is the chelation of iron with protoporphyrin. During periods of iron insufficiency or impaired iron utilization, zinc becomes an alternative metal substrate for ferrochelatase, leading to increased ZnPP formation. Evidence suggests that this metal substitution is one of the first biochemical responses to iron depletion, causing increased ZnPP to appear in circulating erythrocytes. Because this zinc-for-iron substitution occurs predominantly within the bone marrow, the ZnPP/heme ratio in erythrocytes reflects iron status in the bone marrow. In addition, ZnPP may regulate heme catabolism through competitive inhibition of heme oxygenase, the rate-limiting enzyme in the heme degradation pathway that produces bilirubin and carbon monoxide. Physiological roles, especially relating to carbon monoxide and possibly nitric oxide production, have been suggested for ZnPP. Clinically, ZnPP quantification is valuable as a sensitive and specific tool for evaluating iron nutrition and metabolism. Diagnostic determinations are applicable in a variety of clinical settings, including pediatrics, obstetrics, and blood banking. ZnPP analytical methodologies for clinical studies are discussed. In addition to diagnostic tests and metabolic studies, ZnPP has a potential therapeutic application in controlling bilirubin formation in neonates as a preventive measure for hyperbilirubinemia. Biochemical research techniques, both in vivo and in vitro, are described for further studies into the role of ZnPP in metabolism and physiology.
Ribonuclease-resistant RNA Controls (Armored RNA) for Reverse Transcription-PCR, Branched DNA, and Genotyping Assays for Hepatitis C Virus
Authors: WalkerPeach Cindy R , Winkler Matthew , DuBois Dwight B , Pasloske Brittan L 
Published: January 2020

Abstract
Background: Comparison and evaluation of molecular diagnostic assays for the detection and quantification of hepatitis C virus (HCV) RNA have been limited by the lack of RNA controls and calibrators. Armored RNA® technology is a means for producing RNA that is completely protected from plasma ribonucleases. This method produces recombinant pseudoviral particles that are noninfectious and contain predefined RNA sequences.
Methods: A consensus 412-base sequence from the 5′NCR/Core region of HCV subtype 2b was derived from 34 individually sequenced HCV genotype 2b variants. A DNA fragment encoding the consensus HCV-2b sequence was synthesized de novo, cloned, and expressed as an Armored RNA control. The resulting HCV-2b Armored RNA (AR-HCV-2b) contained the complete HCV-2b consensus RNA sequence encapsidated within a protective protein coat.
Results: AR-HCV-2b was fully recoverable from human plasma incubated at 4 °C for &gt;300 days. The particles were tested in three clinical assay formats: AmplicorTM HCV Monitor 1.0, QuantiplexTM HCV RNA 2.0, and INNO-LiPATM HCV II. When added into seronegative, nonviremic plasma, AR-HCV-2b showed reproducible signals and linear dilutions in both the Amplicor and Quantiplex assays. AR-HCV-2b was correctly identified as subtype 2b in the INNO-LiPA line probe assay.
Conclusion: The HCV-2b Armored RNA control is a versatile, durable, ribonuclease-resistant viral RNA control that is compatible in three different clinical assay formats.
Application of a Thermodynamic Nearest-Neighbor Model to Estimate Nucleic Acid Stability and Optimize Probe Design: Prediction of Melting Points of Multiple Mutations of Apolipoprotein B-3500 and Factor V with a Hybridization Probe Genotyping Assay on the LightCycler
Authors: von Ahsen Nicolas , Oellerich Michael , Armstrong Victor William , Schütz Ekkehard 
Published: January 2020

Abstract
Background: PCR-based mutation detection is prone to methodological errors, e.g., in restriction length fragment polymorphism (RFLP) and allele-specific amplification (ASA), false PCR results may occur because of technical faults or atypical new mutations.
Methods: We investigated the ability of a genotyping assay based on hybridization of labeled oligonucleotides to detect and discriminate known and as yet unknown mutations in the factor V and apolipoprotein B-100 genes. Expected melting points were calculated using a nearest-neighbor model for nucleic acid duplex stability and compared with experimental findings derived from LightCycler melting curves. A method for genotyping the apolipoprotein B-100 G10699A and C10698T mutations is presented.
Results: All mismatches tested for in the probed sequence could be detected with a single probe. The measured melting points were in good agreement with their values predicted using the nearest-neighbor model (r = 0.96; y = 0.98x + 1.18; Sy|x= 0.96; n = 24).
Conclusions: This procedure not only allows the identification of the mutation of interest but also enables the discrimination from other potential mutations in the vicinity of the former. The nearest-neighbor model is valid for hybridization probe assays on the LightCycler and should be of general value in setting up such assays. We have shown for two clinically relevant genotyping examples that the LightCycler mutation detection system has superior discriminatory performance compared with conventional RFLP or ASA PCR-based methods for molecular diagnostic purposes. With this method, in every hybridization probe assay, all mutations under a properly designed probe should be detectable, but they will not necessarily be discriminated from each other in all cases.
Hyperglycosylated Human Chorionic Gonadotropin (Invasive Trophoblast Antigen) Immunoassay: A New Basis for Gestational Down Syndrome Screening
Authors: Cole Laurence A , Shahabi Shohreh , Oz Utku A , Bahado-Singh Ray O , Mahoney Maurice J 
Published: January 2020

Abstract
Background: Serum human chorionic gonadotropin (hCG) and hCG free β-subunit tests are used in combination with unconjugated estriol and α-fetoprotein in the triple screen test, and with the addition of inhibin-A in the quadruple marker test for detecting Down syndrome in the second trimester of pregnancy. These tests have a limited detection rate for Down syndrome: ∼40% for hCG or free β-subunit alone, ∼60% for the triple screen test, and ∼70% for the quadruple marker test, all at 5%, or a relatively high, false-positive rate. New tests are needed with higher detection and lower false rates. Hyperglycosylated hCG (also known as invasive trophoblast antigen or ITA) is a new test. It specifically detects a unique oligosaccharide variant of hCG associated with Down syndrome pregnancies. We evaluated this new Down syndrome-directed test in prenatal diagnosis.
Methods: Hyperglycosylated hCG was measured in urine samples from women undergoing amniocentesis for advanced maternal age concerns at 14–22 weeks of gestation, 1448 with normal karyotype and 39 with Down syndrome fetuses.
Results: The median hyperglycosylated hCG value was 9.5-fold higher in Down syndrome cases (9.5 multiples of the normal karyotype median). The single test detected 80% of Down syndrome cases at a 5% false-positive rate. Urine hyperglycosylated hCG was combined with urine β-core fragment (urine breakdown product of serum hCG free β-subunit), serum α-fetoprotein, and maternal age-related risk. This urine-serum combination detected 96% of Down syndrome cases at a 5% false-positive rate, 94% of cases at a 3% false-positive rate, and 71% of cases at a 1% false-positive rate. These detection rates exceed those of any previously reported combination of biochemical markers.
Conclusions: Hyperglycosylated hCG is a new base marker for Down syndrome screening in the second trimester of pregnancy. The measurement of hyperglycosylated hCG can fundamentally improve the performance of Down syndrome screening protocols.
Analysis of Nicotine, 3-Hydroxycotinine, Cotinine, and Caffeine in Urine of Passive Smokers by HPLC-Tandem Mass Spectrometry
Authors: Tuomi Tapani , Johnsson Tom , Reijula Kari 
Published: January 2020

Abstract
Background: A method is described for the simultaneous analysis of nicotine and two of its major metabolites, cotinine and 3-hydroxycotinine, as well as for caffeine from urine samples. The method was developed to assess exposure of restaurant and hotel workers to environmental tobacco smoke.
Methods: The method includes sample pretreatment and reversed-phase HPLC separation with tandem mass spectrometric identification and quantification using electrospray ionization on a quadrupole ion trap mass analyzer. Sample pretreatment followed standard protocols, including addition of base before liquid-liquid partitioning against dichloromethane on a solid matrix, evaporation of the organic solvent using gaseous nitrogen, and transferring to HPLC vials using HPLC buffer. HPLC separation was run on-line with the electrospray ionization-tandem mass spectrometric detection.
Results: The detection limits of the procedure were in the 1 μg/L range, except for nicotine (10 μg/L of urine). Still lower detection limits can be achieved with larger sample volumes. Recoveries of the sample treatment varied from 99% (cotinine) to 78% (3-hydroxycotinine).
Conclusions: The method described is straightforward and not labor-intensive and, therefore, permits a high throughput of samples with excellent prospects for automation. The applicability of the method was demonstrated in a small-scale study on restaurant employees.
Increased Serum Transferrin Saturation Is Associated with Lower Serum Transferrin Receptor Concentration
Authors: Looker Anne C , Loyevsky Mark , Gordeuk Victor R 
Published: January 2020

AbstractBackground: Serum transferrin receptor (sTfR) concentrations are increased in iron deficiency. We wished to examine whether they are decreased in the presence of potential iron-loading conditions, as reflected by increased transferrin saturation (TS) on a single occasion.Methods: We compared sTfR concentrations between 570 controls with normal iron status and 189 cases with increased serum TS on a single occasion; these latter individuals may be potential cases of iron overload. Cases and controls were selected from adults who had been examined in the third National Health and Nutrition Examination Survey (1988–1994) and for whom excess sera were available to perform sTfR measurements after the survey’s completion. Increased TS was defined as &gt;60% for men and &gt;55% for women; normal iron status was defined as having no evidence of iron deficiency, iron overload, or inflammation indicated by serum ferritin, TS, erythrocyte protoporphyrin, and C-reactive protein.Results: Mean sTfR and mean log sTfR:ferritin were ∼10% and 24% lower, respectively, in cases than in controls (P &lt;0.002). Cases were significantly more likely to have an sTfR value &lt;2.9 mg/L, the lower limit of the reference interval, than were controls (odds ratio = 1.8; 95% confidence interval, 1.04–2.37).Conclusion: Our results support previous studies that suggested that sTfR may be useful for assessing high iron status in populations.
Comparison of Pituitary and Recombinant Human Thyroid-stimulating Hormone (rhTSH) in a Multicenter Collaborative Study: Establishment of the First World Health Organization Reference Reagent for rhTSH
Authors: Rafferty Brian , Gaines Das Rose 
Published: January 2020

AbstractBackground: The increasing use of recombinant-DNA-derived materials in therapy and diagnosis poses a new challenge for biological standardization, that of developing reference preparations appropriate for both the native and recombinant products. Here we report the results of an international collaborative study that was carried out under the auspices of WHO to assess the suitability of a preparation of recombinant thyroid-stimulating hormone (rTSH; 94/674) to serve as a potential standard for the calibration of diagnostic immunoassays compared with the International Reference Preparation (IRP) for human TSH (80/558).Methods: Coded samples were provided to the 33 laboratories in the study, and participants were asked to perform TSH assays currently in use in their laboratories. Twenty-eight laboratories contributed 93 immunoassays in 41 different method-laboratory combinations, and an additional 5 laboratories contributed bioassay data. All data were analyzed centrally at the National Institute for Biological Standards and Control.Results: The results obtained in different laboratories and with different assay systems revealed significant variability between estimates of rTSH relative to the IRP. These ranged from 5.51 mIU (95% limits, 3.95–7.67 mIU) per ampoule by RIA to 7.15 mIU (95% limits, 6.7–7.63 mIU) per ampoule by immunofluorometric assay. However, the results showed that the assignment of a value of 6.70 mIU per ampoule of 94/674 would give reasonable continuity with the IRP in many assay systems.Conclusions: The preparation was established as the First WHO Reference Reagent for TSH, human, recombinant, to provide a means of validating assay performance and to maintain continuity with the IRP without compromising clinical data.
Quantitative Abnormalities of Fetal DNA in Maternal Serum in Preeclampsia
Authors: Lo YM Dennis , Leung Tse N , Tein Mark SC , Sargent Ian L , Zhang Jun , Lau Tze K , Haines Christopher J , Redman Christopher WG 
Published: January 2020

Abstract
Background: There is much recent interest in the biologic and diagnostic implication of cell-free non-host DNA in the plasma and serum of human subjects. To determine if quantitative abnormalities of circulating non-host DNA may be associated with certain pathologic processes, we used circulating fetal DNA in preeclampsia as a model system. Methods: We studied 20 preeclamptic women and 20 control subjects of comparable gestational age (means, 32 and 33 weeks, respectively). Male fetal DNA in maternal serum was measured using real-time quantitative PCR for the SRY gene on the Y chromosome. Results: The imprecision (CV) of the assay was 2.7%. The median circulating fetal DNA was increased fivefold in 20 preeclamptic women compared with 20 control pregnant women (381 vs 76 genome-equivalents/mL, P &lt;0.001). Conclusions: These observations suggest that preeclampsia is associated with disturbances in the liberation and/or clearance mechanisms of circulating DNA. These results also raise the possibility that measurement of circulating DNA may prove useful as a marker for the diagnosis and/or monitoring of preeclampsia.
Variability of Glutathione S-Transferase α in Human Liver and Plasma
Authors: Mulder Theo PJ , Court Daniel A , Peters Wilbert HM 
Published: January 2020

AbstractBackground: Glutathione S-transferases are a family of enzymes involved in the binding, transport, and detoxification of a wide variety of endogenous and exogenous compounds. Little information is available about the variability of class α glutathione S-transferases in human liver, where they are highly expressed, or in serum.Methods: Both total class α glutathione S-transferase (GST-α, composed of GSTA1-1, GSTA1-2, and GSTA2-2) as well as GSTA1-1 concentrations were measured by specific and sensitive ELISA in liver cytosols of 35 organ donors and in plasma samples of 350 healthy controls.Results: The mean total GST-α and GSTA1-1 in liver cytosols were 25.1 ± 9.4 and 10.7 ± 5.3 μg/mg protein, respectively, and did not correlate with activities of aspartate aminotransferase or alanine aminotransferase. The mean total GST-α in liver was significantly higher in females compared with males (28.8 ± 10.0 vs 22.0 ± 7.8 μg/mg protein; P &lt;0.05). In contrast, the median total GST-α in plasma was lower in females compared with males (2.0 and 2.8 μg/L, respectively; P &lt;0.0001). The median ratios for GSTA1-1/total GST-α in liver and plasma were 0.42 and 0.58, respectively.Conclusions: GSTA1-1 constitutes approximately one-half of the total amount of α class GSTs in human plasma and liver. Total GST-α values are higher in female liver but lower in plasma compared with the respective values in males.
Performance and Specificity of Monoclonal Immunoassays for Cyclosporine Monitoring: How Specific Is Specific?
Authors: Steimer Werner 
Published: January 2020

AbstractBackground: Immunoassays designed for the selective measurement of cyclosporin A (CsA) inadvertently show cross-reactivity for CsA metabolites. The extent and clinical significance of the resulting overestimation is controversial. A comprehensive assessment of old and new methods in clinical specimens is needed.Methods: In a comprehensive evaluation, CsA was analyzed in 145 samples with the new CEDIA® assay and compared with the Emit® assay with the old and new pretreatments, the TDx® monoclonal and polyclonal assays, the AxSYM®, and HPLC. All samples were from patients with liver and/or kidney transplants.Results: The CEDIA offered the easiest handling, followed by the AxSYM, which showed the longest calibration stability. The TDx monoclonal assay provided the lowest detection limit and the lowest CVs. The mean differences compared with HPLC were as follows: Emit, 9–12%; CEDIA, 18%; AxSYM, 29%; and TDx monoclonal, 57%. The CycloTrac® RIA paralleled the Emit results. In contrast to the mean differences, substantial (&gt;200%) and variable overestimations of the CsA concentration were observed in individual patient samples. Metabolic ratios, estimates of the overall concentrations of several cross-reacting metabolites (nonspecific TDx polyclonal/specific reference method), correlated with the apparent biases of the various monoclonal assays. Metabolic ratios varied up to 10-fold, which translated into biases for individual samples between −7% and +174%. The higher the cross-reactivity of an assay was, the higher was the range of biases observed. The interindividual differences markedly exceeded other factors of influence (organ transplanted, hepatic function).Conclusion: Because assay bias cannot be predicted in individual samples, substantially erratic CsA dosing can result. The specificity of CsA assays for parent CsA remains a major concern.
TE671 Cell-based ELISA for Anti-Acetylcholine Receptor Antibody Determination in Myasthenia Gravis
Authors: Franciotta Diego , Martino Gianvito , Brambilla Elena , Zardini Elisabetta , Locatelli Vera , Bergami Alessandra , Tinelli Carmine , Desina Gaetano , Cosi Vittorio 
Published: January 2020

AbstractBackground: Acetylcholine receptor (AChR) from human muscles is the antigen used currently in radioimmunoprecipitation assays (RIPAs) for the determination of anti-AChR antibodies in the diagnosis of myasthenia gravis (MG). Our aim was to develop and validate an ELISA using TE671 cells as the source of AChR.Methods: After TE671 cell homogenization, the crude AChR extract was used for plate coating. Anti-AChR antibodies were determined in 207 MG patients and in 77 controls.Results: The mean intra- and interassay CVs (for two samples with different anti-AChR antibody concentrations) were 9.7% and 15.7%, respectively. Test sensitivity and specificity, for generalized MG, were 79.5% (95% confidence interval, 72.8–85.0%) and 96.1% (89.0–99.1%). The detection limit was 2 nmol/L. Anti-AChR antibody concentrations from 53 MG patients, as tested with our ELISA, showed good agreement with an RIPA with a mean difference (SD) of 1.0 (5.6) nmol/L.Conclusion: Our ELISA is a simple screening test for the diagnosis of MG and enables rapid and inexpensive patient follow-up.
Major Interference from Leukocytes in Reverse Transcription-PCR Identified as Neurotoxin Ribonuclease from Eosinophils: Detection of Residual Chronic Myelogenous Leukemia from Cell Lysates by Use of an Eosinophil-depleted Cell Preparation
Authors: Hämäläinen Mauri M , Eskola Jarkko U , Hellman Jukka , Pulkki Kari 
Published: January 2020

AbstractBackground: The extraction of RNA from leukocytes for reverse transcription-PCR (RT-PCR) is time-consuming and contributes to variation in analysis of the Philadelphia (Ph1) chromosome of chronic myelogenous leukemia (CML) by RT-PCR. To detect residual CML after bone marrow transplantation, mRNA from at least 105 leukocytes should be analyzed, but the RNase activity of the cells precludes simple leukocytes lysis as an alternative to RNA extraction. We sought to identify the main source of RNase activity of leukocytes.Methods: We used a three-step chromatographic process and amino acid sequence analysis. We selected eosinophil-free granulocytes by using a biotinylated CD16 antibody and selected mononuclear cells by fractionating the leukocytes with a Ficoll-Paque® density gradient.Results: Chromatography and amino acid sequencing identified eosinophil-derived neurotoxin (EDN) as the main source of leukocyte RNase. Depletion of eosinophils reduced the EDN content of cell lysates by ∼90%, allowing a signal from a lysate of 50 K562 Ph1-positive cells mixed with 105 CD16+ granulocytes that was equivalent to 77% of the signal in the absence of leukocytes. A similar lysate with mononuclear cells gave a signal equivalent to 53% of that without mononuclear cells. RNA extraction gave a signal equivalent to only 24% of the leukocyte-free control.Conclusion: The depletion of eosinophils during the preparation of leukocyte samples for RT-PCR efficiently reduces the risk of mRNA degradation by ribonucleases, enabling RT-PCR analysis directly from cell lysates with a better signal than can be obtained by RNA extraction.