bluuebunny/crossref_metadata_2025_split huggingface dataset:
Rapid Single-Tube Screening of the C282Y Hemochromatosis Mutation by Real-Time Multiplex Allele-specific PCR without Fluorescent Probes
Authors: Donohoe Gerard G , Laaksonen Maija , Pulkki Kari , Rönnemaa Tapani , Kairisto Veli 
Published: January 2020

AbstractBackground: An accurate determination of the major HFE mutation (C282Y), which is associated with hereditary hemochromatosis, is important in diagnosis and risk assessment for this disease. We report a single-tube high-throughput PCR method for the detection of C282Y.Methods: We combined three previously described principles: allele-specific PCR, mutagenically separated PCR, and amplicon identification by specific dissociation curves. PCR amplification was performed with fluorescence detection or conventional thermocycler using the same primers, reactant constituents, and cycling protocol. Primer cross-reactions were prevented by deliberate primer:primer and primer:template mismatches.Results: PCR products were identified by their characteristic melting temperatures based on SYBR Green I fluorescence. For each of the 256 random and 17 known HFE C282Y samples, mutant homozygous, wild-type, and heterozygous samples were unequivocally distinguished.Conclusions: This homogeneous assay is rapid, reproducible, does not require fluorescent oligonucleotide probes, and correctly identifies HFE genotypes.
Multicenter Evaluation of Five Assays for Myoglobin Determination
Authors: Zaninotto Martina , Pagani Franca , Altinier Sara , Amboni Paolo , Bonora Roberto , Dolci Alberto , Pergolini Patrizia , Vernocchi Arialdo , Plebani Mario , Panteghini Mauro ,   for the Italian Society of Clinical Biochemistry and Clinical Molecular Biology–Italian Society of Laboratory Medicine Working Group on Markers of Myocardial Damage
Published: January 2020

AbstractBackground: Lacking assay standardization, different myoglobin methods may produce results that differ significantly.Methods: A multicenter study was carried out to compare the analytical performance of five commercially available assays for myoglobin measurement. Linearity, imprecision, interferences, and method comparison were studied according to NCCLS guidelines, whereas reference values were determined following IFCC recommendations.Results: The BNA and Opus showed relatively high imprecision (all but one total CV &gt;7.4%). Other assays showed lower CVs, but they varied among laboratories, particularly at a normal myoglobin concentration (Access, 6.0–11%; Hitachi, 3.8–5.8%; Stratus, 3.4–6.5%). Results were lower in anticoagulated samples on the Access, in heparin and citrate samples on the Stratus, and in citrate samples on the BNA and Opus, and increased in heparin and EDTA samples on the Hitachi. Use of separator gel produced results significantly lower (P &lt;0.001) on the Hitachi and higher (P = 0.016) on the Opus. Bilirubin, turbidity, and hemoglobin had no effect on evaluated methods, but rheumatoid factor affected the Access. In method comparisons, high correlation coefficients (≥0.98) were obtained. The Stratus gave higher results; however, the Access and BNA gave the lowest. The following upper reference limits (μg/L) for men and women, respectively, were obtained: Access, 70 and 52; BNA, 51 and 49; Hitachi, 67 and 58; Opus, 80 and 50; and Stratus, 86 and 63.Conclusion: The possibility of high imprecision and marked disagreement among commercial myoglobin assays should be carefully considered in clinical practice.
Determination of S-Adenosylmethionine and S-Adenosylhomocysteine in Plasma and Cerebrospinal Fluid by Stable-Isotope Dilution Tandem Mass Spectrometry
Authors: Struys Eduard A , Jansen Erwin E W , de Meer Kees , Jakobs Cornelis 
Published: January 2020

AbstractBackground: Available methods for the determination of nanomolar concentrations of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) in plasma and cerebrospinal fluid (CSF) are time-consuming. We wished to develop a method for their rapid and simultaneous measurement.Methods: We used tandem mass spectrometry (MS/MS) for the simultaneous determination of SAM and SAH, with stable-isotope-labeled internal standards. The 13C5-SAH internal standard was enzymatically prepared using SAH-hydrolase and [13C5]adenosine. The method comprises a weak anion-exchange solid-phase extraction procedure serving as clean-up step for the deproteinized plasma and CSF samples. After clean-up, samples were injected on a C18 HPLC column, which was connected directly to the tandem mass spectrometer, operating in MS/MS mode.Results: In plasma samples, the intraassay CVs for SAM and SAH were 4.2% and 4.0%, respectively, and the interassay CVs were 7.6% and 5.9%, respectively. In CSF, the intraassay CVs for SAM and SAH were 6.8% and 6.9%, respectively, and the interassay CVs were 4.2% and 5.5%, respectively. Mean recovery of SAM and SAH for both matrices at two concentrations was 93%. Detection limits for SAM and SAH in samples were 7.5 and 2.5 nmol/L, respectively. Concentrations of SAM and SAH in plasma from healthy subjects were within the previously reported ranges. In 10 CSF samples, the mean concentrations (range) were 248 (137–385) nmol/L for SAM and 11.3 (8.9–14.1) nmol/L for SAH.Conclusions: SAM and SAH can be analyzed by MS/MS, taking optimal advantage of the speed and high sensitivity and specificity of this relatively new analytical technique.
Association of Serum Bilirubin Concentration with Risk of Coronary Artery Disease
Authors: Mayer Michael 
Published: January 2020

AbstractBackground: Lipid oxidation and formation of oxygen radicals are important elements of arterial plaque formation and atherosclerosis, and are involved in the pathophysiology of coronary artery disease (CAD). Because bilirubin has antioxidant properties, it has been suggested that it may have a protective role in the atherosclerotic process.Approach: This review examines in vitro and in vivo studies indicating that bilirubin inhibits lipid oxidation and oxygen radical formation. Experimental and epidemiological evidence is presented that suggests that bilirubin may serve as a physiological antioxidant providing protection against atherosclerosis and CAD. Special attention is focused on studies that noted an inverse relationship between plasma bilirubin concentration and cardiovascular morbidity.Content: Serum bilirubin concentrations in the upper portion of the reference interval reportedly reduce atherogenic risk and provide protection against CAD. In contrast, serum bilirubin concentrations in the lower portion of the reference interval may be associated with increased risk of ischemic heart disease.Summary: Taken together, the evidence presented in this review supports the concept that bilirubin, via its antioxidant potential, has antiatherogenic properties and that an inverse relationship exists between circulating bilirubin concentrations and risk of CAD.
Influence of DNA Target Melting Behavior on Real-Time PCR Quantification
Authors: Wilhelm Jochen , Hahn Meinhard , Pingoud Alfred 
Published: January 2020

Abstract
Background: Quantitative real-time PCR is increasingly used to quantify copy numbers of nucleic acids for clinical applications. We observed that the measurements of allele imbalances of the tumor suppressor gene p16 and the oncogene ErbB-2 yielded results with variable precision under certain experimental conditions.
Methods: We used the LightCyclerTM real-time PCR system to quantify different genomic target sequences using hybridization probes or SYBR Green for detection.
Results: With two primer/template systems (p16 and ErbB-2), we observed sinusoidal scattering of the threshold cycle values depending on the capillary position in the thermostated reaction chamber. This scattering depended on the denaturation temperature only when complete genomic DNA was used as template and did not occur when PCR product or restricted or boiled genomic DNA was used or the denaturation temperature in the first cycles was increased (and other targets, such as p53, HBB, IGF-1, GAPDH, and PBGD, did not show this behavior).
Conclusions: Before a primer system is used for precise quantitative real-time PCR, the dependence of the quantification results on the positions of reaction tubes in the thermocycler should be tested. Our data indicate that amplification efficiencies, especially in the first cycles, depend not only on the priming efficiencies of the primers and the melting temperature of the amplicon, but also on the melting behavior of the amplicon’s genomic vicinity. Complete denaturation of genomic DNA is necessary to maximize precision of quantitative PCR. Higher denaturation temperatures in the initial cycles or boiling of DNA before the PCR can improve the accuracy of quantification in some cases.
Evaluation of Indicators of Cobalamin Deficiency Defined as Cobalamin-induced Reduction in Increased Serum Methylmalonic Acid
Authors: Bolann Bjørn J , Solli Jan Dag , Schneede Jörn , Grøttum Kjell A , Loraas Arne , Stokkeland Morgan , Stallemo Asbjørn , Schjøth Arne , Bie Rolf Bruun , Refsum Helga , Ueland Per Magne 
Published: January 2020

AbstractBackground: Early detection of cobalamin deficiency is clinically important, and there is evidence that such deficiency occurs more frequently than previously anticipated. However, serum cobalamin and other commonly used tests have limited ability to diagnose a deficiency state.Methods: We investigated the ability of hematological variables, serum cobalamin, plasma total homocysteine (tHcy), serum and erythrocyte folate, gastroscopy, age, and gender to predict cobalamin deficiency. Patients (n = 196; age range, 17–87 years) who had been referred from general practice for determination of serum cobalamin were studied. Cobalamin deficiency was defined as serum methylmalonic acid (MMA) &gt;0.26 μmol/L with at least 50% reduction after cobalamin supplementation. ROC and logistic regression analyses were used.Results: Serum cobalamin and tHcy were the best predictors, with areas under the ROC curve (SE) of 0.810 (0.034) and 0.768 (0.037), respectively, but age, intrinsic factor antibodies, and gastroscopy gave additional information.Conclusions: When cobalamin deficiency is suspected in general practice, serum cobalamin should be the first diagnostic test, and the result should be interpreted in relation to the age of the patient. When a definite diagnosis cannot be reached, MMA and tHcy determination will provide additional discriminative information, but MMA, being more specific, is preferable for assessment of cobalamin status.
A Reference Method Laboratory Network for Cholesterol: A Model for Standardization and Improvement of Clinical Laboratory Measurements
Authors: Myers Gary L , Kimberly Mary M , Waymack Parvin P , Smith S Jay , Cooper Gerald R , Sampson Eric J 
Published: January 2020

AbstractBackground: Accurate and precise measurement of blood cholesterol plays a central role in the National Cholesterol Education Program’s strategy to reduce the morbidity and mortality attributable to coronary heart disease. Matrix effects hamper the ability of manufacturers to adequately calibrate and validate traceability to the National Reference System for Cholesterol (NRS/CHOL). CDC created the Cholesterol Reference Method Laboratory Network (CRMLN) to improve cholesterol measurement by assisting manufacturers of in vitro diagnostic products with validation of the traceability of their assays to the NRS/CHOL.Methods: CRMLN laboratories established the CDC cholesterol reference method (modification of the Abell-Levy-Brodie-Kendall chemical method) and are standardized using CDC frozen serum reference materials. CRMLN laboratories use common quality-control materials and participate in monthly external performance evaluations conducted by CDC. The CRMLN performance criteria require member laboratories to agree with CDC within ± 1.0% and maintain a CV ≤2.0%.Results: From 1995 to 2000, the CRMLN laboratories met the accuracy criterion 97% of the time and the precision criterion 99% of the time. During this time period, the CRMLN maintained an average bias to CDC of 0.01% and an average collective CV of 0.33%.Conclusions: CDC established the CRMLN as the first international reference method laboratory network. The CRMLN assists manufacturers in the validation of the calibration of their diagnostic products so that clinical laboratories can measure blood cholesterol more reliably. The CRMLN can serve as a model for other clinical analytes where traceability to a hierarchy of methods is needed and matrix effects of the field methods with processed calibrators or reference materials are present.
Influence of Increased Fruit and Vegetable Intake on Plasma and Lipoprotein Carotenoids and LDL Oxidation in Smokers and Nonsmokers
Authors: Chopra Mridula , O’Neill Maura E , Keogh Nora , Wortley Gary , Southon Susan , Thurnham David I 
Published: January 2020

Abstract
Background: Epidemiological studies suggest a cardioprotective role for carotenoid-rich foods. Smokers have a high risk of cardiovascular disease and low dietary intake and plasma concentrations of carotenoids. The aim of this study was to determine the carotenoid response of smokers and nonsmokers to increased intake of 300–400 g of vegetables and its effect on LDL oxidation.
Methods: After a depletion period of 8 days, 34 healthy females (18 nonsmokers, 16 smokers) were supplemented with β-carotene- and lutein-rich (green) and lycopene-rich (red) vegetable foods, each for 7 days.
Results: Baseline concentrations (mean ± SD) of plasma β-carotene (0.203 ± 0.28 μmol/L vs 0.412 ± 0.34 μmol/L; P &lt;0.005) and lutein (0.180 ± 0.10 vs 0.242 ± 0.11 μmol/L; P &lt;0.05) but not lycopene (0.296 ± 0.10 vs 0.319 ± 0.33 μmol/L) were significantly lower in smokers compared with nonsmokers. After supplementation, the change (supplementation minus depletion) in plasma β-carotene (0.152 ± 0.43 vs 0.363 ± 0.29 μmol/L in smokers vs nonsmokers; P = 0.002) and LDL lutein (0.015 ± 0.03 vs 0.029 ± 0.03 μmol/mmol cholesterol; P = 0.01) was significantly lower in smokers than nonsmokers. Green-vegetable supplementation had no effect on the resistance of LDL to oxidation (lag-phase) in either group. After red-vegetable supplementation, plasma and LDL lycopene concentrations were increased in both groups, but only nonsmokers showed a significant increase in the lag-phase (44.9 ± 9.5 min at baseline, 41.4 ± 6.5 min after depletion, and 49.0 ± 8.9 min after supplementation; P &lt;0.01) compared with depletion.
Conclusions: In this short-term intervention study, a dietary intake of &gt;40 mg/day of lycopene by a group of nonsmoking individuals significantly reduced the susceptibility of LDL to oxidation, whereas an equivalent increase in lycopene by a group of smokers showed no such effect.
Use of a Reference Material Proposed by the International Federation of Clinical Chemistry and Laboratory Medicine to Evaluate Analytical Methods for the Determination of Plasma Lipoprotein(a)
Authors: Marcovina Santica M , Albers John J , Scanu Angelo M , Kennedy Hal , Giaculli Federico , Berg Kåre , Couderc Rémy , Dati Francesco , Rifai Nader , Sakurabayashi Ikunosuke , Tate Jillian R , Steinmetz Armin 
Published: January 2020

AbstractBackground: As part of the NIH/National Heart, Lung and Blood Institute Contract for the Standardization of Lipoprotein(a) [Lp(a)] Measurements, a study was performed in collaboration with the IFCC Working Group for the Standardization of Lp(a) Assays. The aims of the study, performed with the participation of 16 manufacturers and 6 research laboratories, were to evaluate the IFCC proposed reference material (PRM) for its ability to transfer an accuracy-based value to the immunoassay calibrators and to assess concordance in results among different methods.Methods: Two different purified Lp(a) preparations with protein mass concentrations determined by amino acid analysis were used to calibrate the reference method. A Lp(a) value of 107 nmol/L was assigned to PRM. After uniformity of calibration was demonstrated in the 22 evaluated systems, Lp(a) was measured on 30 fresh-frozen sera covering a wide range of Lp(a) values and apolipoprotein(a) [apo(a)] sizes.Results: The among-laboratory CVs for these samples (6–31%) were, in general, higher than those obtained for PRM (2.8%) and the quality-control samples (14%, 12%, and 9%, respectively), reflecting the broad range of apo(a) sizes in the 30 samples and the sensitivity of most methods to apo(a) size heterogeneity. Thus, although all of the assays were uniformly calibrated through the use of PRM, no uniformity in results was achieved for the isoform-sensitive methods.Conclusions: Linear regression analyses indicated that to various degrees, apo(a) size heterogeneity affects the outcome of the immunochemical methods used to measure Lp(a). We have also shown that the inaccuracy of Lp(a) values determined by methods sensitive to apo(a) size significantly affects the assessment of individual risk status for coronary artery disease.
Erythrocyte Folate Analysis: Saponin Added During Lysis of Whole Blood Can Increase Apparent Folate Concentrations, Depending on Hemolysate pH
Authors: Wright Anthony J A , Finglas Paul M , Southon Susan 
Published: January 2020

AbstractBackground: The analysis of red cell folate (RCF) depends on complete hemolysis of erythrocytes, and it is assumed that complete hemolysis is achieved by 10-fold dilution of whole blood with hypotonic solutions of 10 g/L ascorbic acid/ascorbate. This report challenges this assumption.Methods: The conventional method of erythrocyte lysis was modified to include saponin, a known effective hemolyzing agent. The influence of saponin was determined at various lysate pHs, using the microbiological (Lactobacillus rhamnosus) folate assay. The effect of saponin during lysate preparation was subsequently compared with either the effect of 30 s of sonication or a single 1-h freeze-thaw cycle.Results: Saponin addition was found to increase assayable RCF up to ninefold, depending on lysate pH. Sonication of lysates had no effect, and freezing-thawing lysates once did not always guarantee complete hemolysis. Lysates created with 10 g/L ascorbic acid (a historically widely used diluent) without pH adjustment produced assayable folate concentrations significantly lower than optimal.Conclusions: A lysing agent should be incorporated into RCF assays to guarantee complete hemolysis. Ten-fold dilution of blood with 10 g/L ascorbic acid, without pH adjustment, produces lysates with pHs (pH 4.0) below the point (pH 4.7) at which hemoglobin can denature irreversibly. The optimum pH for hemolysates is ∼5.0.
Real-Time Quantification of Human Telomerase Reverse Transcriptase mRNA in Tumors and Healthy Tissues
Authors: de Kok Jacques B , Ruers Theo J M , van Muijen Goos N P , van Bokhoven Adrie , Willems Hans L , Swinkels Dorine W 
Published: January 2020

Abstract
Background: Expression of the hTERT gene, which codes for the catalytic subunit of telomerase, is associated with malignancy. We recently developed a real-time reverse transcription-PCR assay, based on TaqMan technology, for accurate and reproducible determination of hTERT mRNA expression (Lab Investig 1999;79:911–2). This method may be of interest for molecular tumor diagnostics in tissues and corresponding body fluids, washings, or brushes.
Methods: In this study, we measured hTERT expression in a subset of healthy tissues and tumors to select those tumor types with the best potential for quantification of hTERT in corresponding body fluids. To demonstrate the use of the method in body fluids, we quantified hTERT expression in voided urine of patients with bladder cancer and controls.
Results: Real-time measurement of hTERT expression could discriminate between all healthy and malignant tissue samples from pancreas, lung, esophagus, and bladder, but not for colon tissues. Moreover, in five of nine (55%) urine samples, hTERT could be quantified.
Conclusions: The present study demonstrates that accurate quantitative measurement of hTERT expression has high potential for discrimination between healthy and tumor cells in tissues and urine and supports future measurements in pancreatic fluid, bronchoalveolar lavage fluid, esophageal brushings, and urine or bladder washings.
Development of Conventional and Real-Time PCR Assays for the Rapid Detection of Group B Streptococci
Authors: Ke Danbing , Ménard Christian , Picard François J , Boissinot Maurice , Ouellette Marc , Roy Paul H , Bergeron Michel G 
Published: January 2020

AbstractBackground: Group B streptococci (GBS), or Streptococcus agalactiae, are the leading bacterial cause of meningitis and bacterial sepsis in newborns. Currently available rapid methods to detect GBS from clinical specimens are unsuitable for replacement of culture methods, mainly because of their lack of sensitivity.Methods: We have developed a PCR-based assay for the rapid detection of GBS. The cfb gene encoding the Christie-Atkins-Munch-Petersen (CAMP) factor was selected as the genetic target for the assay. The PCR primers were initially tested by a conventional PCR method followed by gel electrophoresis. The assay was then adapted for use with the LightCyclerTM. For this purpose, two fluorogenic adjacent hybridization probes complementary to the GBS-specific amplicon were designed and tested. In addition, a rapid sample-processing protocol was evaluated by colony-forming unit counting and PCR. A total of 15 vaginal samples were tested by both standard culture method and the two PCR assays.Results: The conventional PCR assay was specific because it amplified only GBS DNA among 125 bacterial and fungal species tested, and was able to detect all 162 GBS isolates from various geographical areas. This PCR assay allowed detection of as few as one genome copy of GBS. The real-time PCR assay was comparable to conventional PCR assay in terms of sensitivity and specificity, but it was more rapid, requiring only ∼30 min for amplification and computer-based data analysis. The presence of vaginal specimens had no detrimental effect on the sensitivity of the PCR with the sample preparation protocol used. All four GBS-positive samples identified by the standard culture method were detected by the two PCR assays.Conclusion: These assays provide promising tools for the rapid detection and identification of GBS.