bluuebunny/crossref_metadata_2025_split huggingface dataset:
Rapid Detection of Point Mutations by Fluorescence Resonance Energy Transfer and Probe Melting Curves in Candida Species
Authors: Loeffler Juergen , Hagmeyer Lars , Hebart Holger , Henke Norbert , Schumacher Ulrike , Einsele Hermann 
Published: January 2020

Abstract
Background: The LightCyclerTM combines rapid amplification of nucleic acids in glass capillaries with melting curve analysis based on fluorescence resonance energy transfer for the sensitive detection of point mutations in various settings, such as drug resistance and hereditary diseases. Point mutations leading to an altered structure of lanosteroldemethylase, the target enzyme of the fungistatic azoles, are an important mechanism of acquired resistance in Candida albicans.
Methods: We screened 13 fluconazole-resistant C. albicans and 21 fluconazole-resistant C. tropicalis strains (minimum inhibitory concentration &gt;128 mg/L), isolated from patients with AIDS, for the presence of defined point mutations by comparing conventional cycle sequencing with a newly designed LightCycler-based assay.
Results: In C. tropicalis, 5 of 21 isolates showed the wild-type sequence, and 8 of 21 showed the homozygous nucleotide exchange thymine to cytosine at position 1554 (T1554C). A heterozygous genotype was detected in 8 of 21 isolates by the LightCycler, but in only 3 of 21 isolates by conventional cycle sequencing. In 2 of 13 C. albicans isolates, a homozygous point mutation leading to an amino acid exchange at position 464 (glycine to serine) was detected in both assays.
Conclusion: The LightCycler technique offers standardized, fast, sensitive, and reproducible detection of point mutations in different Candida spp.
ELISA for Urinary Trehalase with Monoclonal Antibodies: A Technique for Assessment of Renal Tubular Damage
Authors: Ishihara Reiko , Taketani Shigeru , Sasai-Takedatsu Misa , Adachi Yasushi , Kino Minoru , Furuya Akiko , Hanai Nobuo , Tokunaga Rikio , Kobayashi Yohnosuke 
Published: January 2020

AbstractBackground: α,α-Trehalase, located on renal proximal tubules, is a glycoprotein that hydrolyses α,α-trehalose to two glucose molecules. Urinary trehalase reflects damage to renal proximal tubules, but its activity has not been measured routinely because measurement of catalytic activity is rather complicated and because conventional assays for enzyme activity might not reflect all of the trehalase protein because of enzyme inactivation in urinary samples.Methods: We established novel monoclonal antibodies for human trehalase and a sandwich ELISA for quantification of urinary trehalase. We determined the urinary trehalase protein concentration with this ELISA and trehalase catalytic activity, and the results of these two methods were compared.Results: The ELISA system was more sensitive than the detection of enzyme activity and could detect a subtle difference in the amount of trehalase present in renal diseases. The within- and between-assay CVs in the ELISA were 6.7–7.6% and 6.2–8.2%, respectively. Highly significant increases in both the quantity and activity were seen in patients with nephrotic syndrome (acute phase), Lowe syndrome, and Dent disease. The quantities were 70- to 200-fold greater, whereas enzyme activities were, at most, 10-fold higher than those of control subjects. In the detection of small amounts of trehalase in patients with chronic glomerulonephritis and renal anomalies, quantities were better than enzyme activities.Conclusions: We have established an ELISA system for quantification of urinary trehalase that uses novel monoclonal antibodies. Our ELISA system is simpler and more sensitive than a conventional activity assay and reflects trehalase protein. This ELISA can be a useful as a common tool for clinical assessment of renal proximal tubular damage.
Laboratory Automation: Trajectory, Technology, and Tactics
Authors: Markin Rodney S , Whalen Scott A 
Published: January 2020

Abstract
Laboratory automation is in its infancy, following a path parallel to the development of laboratory information systems in the late 1970s and early 1980s. Changes on the horizon in healthcare and clinical laboratory service that affect the delivery of laboratory results include the increasing age of the population in North America, the implementation of the Balanced Budget Act (1997), and the creation of disease management companies. Major technology drivers include outcomes optimization and phenotypically targeted drugs. Constant cost pressures in the clinical laboratory have forced diagnostic manufacturers into less than optimal profitability states. Laboratory automation can be a tool for the improvement of laboratory services and may decrease costs. The key to improvement of laboratory services is implementation of the correct automation technology. The design of this technology should be driven by required functionality. Automation design issues should be centered on the understanding of the laboratory and its relationship to healthcare delivery and the business and operational processes in the clinical laboratory. Automation design philosophy has evolved from a hardware-based approach to a software-based approach. Process control software to support repeat testing, reflex testing, and transportation management, and overall computer-integrated manufacturing approaches to laboratory automation implementation are rapidly expanding areas. It is clear that hardware and software are functionally interdependent and that the interface between the laboratory automation system and the laboratory information system is a key component. The cost-effectiveness of automation solutions suggested by vendors, however, has been difficult to evaluate because the number of automation installations are few and the precision with which operational data have been collected to determine payback is suboptimal. The trend in automation has moved from total laboratory automation to a modular approach, from a hardware-driven system to process control, from a one-of-a-kind novelty toward a standardized product, and from an in vitro diagnostics novelty to a marketing tool. Multiple vendors are present in the marketplace, many of whom are in vitro diagnostics manufacturers providing an automation solution coupled with their instruments, whereas others are focused automation companies. Automation technology continues to advance, acceptance continues to climb, and payback and cost justification methods are developing.
Branched-Chain Keto-Acids and Pyruvate in Blood: Measurement by HPLC with Fluorimetric Detection and Changes in Older Subjects
Authors: Pailla Karine , Blonde-Cynober Françoise , Aussel Christian , De Bandt Jean-Pascal , Cynober Luc 
Published: January 2020

AbstractBackground: Measurement of keto-acids is important in various clinical situations. The aim of the present work was to develop a rapid HPLC method for the determination of keto-acids in human serum and to assess the concentrations of these acids in young adults and institutionalized elderly adults. This method was applied to the determination of blood keto-acid concentrations of young adults and institutionalized elderly people, divided into age groupsMethods: Four keto-acids (α-ketoisocaproate, α-ketoisovalerate, α-keto-β-methylvalerate, and pyruvate) were derivatized with o-phenylenediamine to give fluorescent derivatives. After the sample preparation step (75 min to prepare 20 samples), the derivatives were separated chromatographically on a reversed-phase column using a binary gradient.Results: The fluorometric detection of the four keto-acids was rapid, &lt;12 min. The method is repeatable and reproducible: the CVs were &lt;6% and &lt;11%, respectively, for each of the keto-acids. We found no significant difference between males and females. Concentrations of the branched-chain keto-acids decreased after age 60 years, especially α-ketoisocaproate, which decreased ∼40%.Conclusions: The proposed method allows rapid and reliable measurement of keto-acids. The data demonstrate that changes in branched-chain keto-acids concentrations in serum occur with age.
Serum and Urinary Prostate-specific Antigen and Urinary Human Glandular Kallikrein Concentrations Are Significantly Increased after Testosterone Administration in Female-to-Male Transsexuals
Authors: Obiezu Chrisitna V , Giltay Erik J , Magklara Angeliki , Scorilas Andreas , Gooren Louis J G , Yu He , Howarth David J C , Diamandis Eleftherios P 
Published: January 2020

Abstract
Background: The genes that encode prostate-specific antigen (PSA) and human glandular kallikrein (hK2) are up-regulated by androgens and progestins in cultured cells, but no published studies have described the effect of androgen administration in women on serum and urinary PSA or hK2.
Methods: We measured serum and urinary PSA and hK2 before, and 4 and 12 months post testosterone treatment by immunofluorometric methods in 32 female-to-male transsexuals.
Results: Mean serum PSA increased from 1.1 ng/L to 11.1 ng/L and then to 22 ng/L by 4 and 12 months post treatment, respectively; the corresponding mean values in urine were 17, 1420, and 18 130 ng/L, respectively. Serum hK2, another kallikrein closely related to PSA, remained undetectable at the three time points. However, urinary hK2 concentration rose from below the detection limit (&lt;6 ng/L) before treatment to 18 and 179 ng/L by the 4th and the 12th month of treatment, respectively. All changes were statistically significant (P &lt;0.001) at 4 months.
Conclusions: Testosterone administration increases serum and urinary PSA and urinary hK2 in women. These measurements may be useful as indicators of androgenic stimulation in women.
Manifold-assisted Reverse Transcription-PCR with Real-Time Detection for Measurement of the BCR-ABL Fusion Transcript in Chronic Myeloid Leukemia Patients
Authors: Barbany Gisela , Hagberg Anette , Olsson-Strömberg Ulla , Simonsson Bengt , Syvänen Ann-Christine , Landegren Ulf 
Published: January 2020

AbstractBackground: BCR-ABL fusion mRNA expression in bone marrow or peripheral blood can be used as a measure of minimal residual disease in patients with chronic myeloid leukemia (CML).Methods: We used an oligo(dT)-coated manifold support to capture the mRNA directly from the cell lysate. After reverse transcription, the cDNA was eluted from the manifold support, and BCR-ABL and GAPDH mRNAs were quantified in real time using the TaqMan fluorogenic detection system.Results: The detection limit of the method was one positive K562 cell among 105 negative cells. GAPDH was chosen as a reference gene based on the low variation between samples from different stages of the disease and the low signal in the absence of reverse transcription. The day-to-day variation of the method (CV) was 32%. In 43 blood samples from 13 CML patients, mRNA quantification agreed well with cytogenetic data.Conclusions: The proposed procedure constitutes a reproducible and sensitive BCR-ABL mRNA quantification method and is suitable to monitor minimal residual disease in CML patients.
Identification of Lignans as Major Components in the Phenolic Fraction of Olive Oil
Authors: Owen Robert W , Mier Walter , Giacosa Attilio , Hull William E , Spiegelhalder Bertold , Bartsch Helmut 
Published: January 2020

Abstract
Background: Because olive oil is an important component of the Mediterranean diet, it is necessary to establish unequivocal identification of the major potential antioxidant phenolic compounds it contains.
Methods: The major phenolic antioxidants in extra virgin olive oil were isolated and purified. Structural analysis was conducted using several spectroscopic techniques, including mass spectrometry and nuclear magnetic resonance (NMR). In particular, detailed 1H and 13C NMR data are presented, and several assignment errors in the literature are corrected.
Results: The data show for the first time that the lignans (+)-1-acetoxypinoresinol and (+)-pinoresinol are major components of the phenolic fraction of olive oils. These lignans, which are potent antioxidants, are absent in seed oils and virtually absent in refined virgin oils but are present at concentrations of up to 100 mg/kg (mean ± SE, 41.53 ± 3.93 mg/kg; range, 0.65–99.97 mg/kg) in extra virgin oils. As with the simple phenols and secoiridoids, there is considerable interoil variation in lignan concentrations. Foods containing high amounts of lignan precursors have been found to be protective against breast, colon, and prostate cancer.
Conclusion: Lignans, as natural components of the diet, may be important modulators of cancer chemopreventive activity.
Rapid Detection of the CYP2D6*3, CYP2D6*4, and CYP2D6*6 Alleles by Tetra-Primer PCR and of the CYP2D6*5 Allele by Multiplex Long PCR
Authors: Hersberger Martin , Marti-Jaun Jacqueline , Rentsch Katharina , Hänseler Edgar 
Published: January 2020

Abstract
Background: Interindividual differences in CYP2D6 activity range from total absence of metabolism of certain drugs to ultrafast metabolism and can produce adverse effects or lack of therapeutic effect under standard therapy. Several mutations have been described in the CYP2D6 gene that abolish CYP2D6 activity. However, four mutations explain the majority of the poor metabolizers. We describe four single-tube assays to detect these mutations.
Methods: Three tetra-primer PCR assays were developed to detect the mutations in the CYP2D6*3, *4, and *6 alleles. In these single-tube assays, the CYP2D6 locus is amplified directly, followed by the allele-specific amplification on this new template. In addition, a multiplex long PCR was developed to genotype the CYP2D6*5 allele. Two long PCR amplifications for detection of the deletion of CYP2D6 (*5) and for detection of the CYP2D6 gene region were combined in one tube.
Results: Analysis of 114 alleles showed no CYP2D6*3 allele, and allele frequencies of 28.1% for CYP2D6*4, 2.6% for CYP2D6*5, and 0.9% for CYP2D6*6. Re-analysis of the DNA samples by restriction fragment length polymorphism and sequencing analysis confirmed these results. Furthermore, re-analysis of sequenced genomic DNA by tetra-primer PCR analysis (7–11 times) always showed identical results.
Conclusions: Our set of single-tube assays allows rapid and reproducible genotyping of the majority of CYP2D6 poor metabolizers.
Analytical Quality of Near-Patient Blood Cholesterol and Glucose Determinations
Authors: du Plessis Marita , Ubbink Johan B , Vermaak W J Hayward 
Published: January 2020

Abstract
Background: Screening for diabetes and hypercholesterolemia is widely advocated, and extra-laboratory testing could play a major role in cost-effective population screening. We wished to assess the analytical quality and interchangeability of capillary blood cholesterol and glucose assays as performed on near-patient devices in pharmacies in Pretoria, South Africa.
Methods: Accuracy of near-patient and laboratory analyzers was assessed by analyses of human-serum-based reference material. To assess interchangeability in routine use, six volunteers visited each of 12 randomly selected pharmacies consecutively during a 3-week period to have their fasting blood glucose and cholesterol concentrations determined. For comparison purposes, a similar procedure was followed to evaluate the eight clinical chemistry laboratories servicing Pretoria and surroundings.
Results: The analytical performances in our laboratory of a single point-of-care instrument and of a laboratory analyzer compared well. Nevertheless, between-pharmacy analytical variation was larger than between-laboratory variation (11% vs 6.1% for cholesterol; 10% vs 7.6% for glucose). For glucose measurements, near-patient testing in pharmacies demonstrated a bias of −48.1% to 16.2%, whereas bias for laboratory measurements was −1.0% to 7.4%. Cholesterol assays showed a bias of −5.6% to 16.6% in pharmacies compared with −10.6% to 3.7% in laboratories. The percentage of closeness to the homeostatic set point for a single glucose and cholesterol determination done in any pharmacy was 24.6% and 23.6%, respectively. The corresponding values for laboratories were 16.9% and 15.6%, respectively.
Conclusions: Although modern point-of-care instruments allow high-quality blood analyses under ideal conditions, performance goals often are not achieved in practice as indicated by a higher uncertainty of cholesterol and glucose blood results when determined in pharmacies. Nonuniformity of calibration procedures, deficiencies in training, a lack of internal quality control, and the absence of an external quality assessment program may all contribute to the current state of affairs.
Analytical and Clinical Evaluation of Two Homogeneous Assays for LDL-Cholesterol in Hyperlipidemic Patients
Authors: Esteban-Salán Margarita , Guimón-Bardesi Amada , de la Viuda-Unzueta Jesús María , Azcarate-Ania María Nerea , Pascual-Usandizaga Pilar , Amoroto-Del-Río Eduardo 
Published: January 2020

Abstract
Background: LDL-cholesterol (LDL-C) concentrations are the primary basis for treatment guidelines established for hyperlipidemic patients. LDL-C concentrations are commonly monitored by means of the Friedewald formula, which provides a relative estimation of LDL-C concentration when the triglyceride concentration is &lt;2000 mg/L and there are no abnormal lipids. The Friedewald formula has several limitations and may not meet the current total error requirement of &lt;12% in LDL-C measurements.
Methods: We evaluated the analytical and clinical performance of two direct methods (Roche and Wako) by analyzing 313 fresh serum samples obtained from dyslipidemic patients in a lipid clinic and comparing them with modified β-quantification.
Results: Both homogeneous assays displayed excellent precision (CV &lt;2%). The Roche method showed a mean total error of 7.72%, and the Wako method showed a mean total error of 4.46% over a wide range of LDL-C concentrations. The Roche method correlated highly with the modified β-quantification assay (r = 0.929; y = 1.052x − 168 mg/L; n = 166) and showed a bias of −4.5% as a result of the assigned standard value. The Wako method also correlated highly with β-quantification (r = 0.966; y = 0.9125x + 104.8 mg/L; n = 145) without significant bias. The Roche method correctly classified 97% of patients with triglycerides &lt;2000 mg/L, 75% of patients with type IIb hyperlipemia (HPL), and 84% of patients with type IV HPL based on the cutpoints of 1300 and 1600 mg/L, compared with 98%, 78.4%, and 89%, respectively, for the Wako method. In dysbetalipoproteinemic patients, both methods have a 30% mean positive bias compared with β-quantification.
Conclusions: Both direct methods can be a useful alternative when ultracentrifugation is not available for the diagnosis and control of lipid-lowering medication for patients with mixed HPL, but not for patients with type III hyperlipidemia.
Flow Cytometry: Principles and Clinical Applications in Hematology
Authors: Brown Michael , Wittwer Carl 
Published: January 2020

AbstractThe use of flow cytometry in the clinical laboratory has grown substantially in the past decade. This is attributable in part to the development of smaller, user-friendly, less-expensive instruments and a continuous increase in the number of clinical applications. Flow cytometry measures multiple characteristics of individual particles flowing in single file in a stream of fluid. Light scattering at different angles can distinguish differences in size and internal complexity, whereas light emitted from fluorescently labeled antibodies can identify a wide array of cell surface and cytoplasmic antigens. This approach makes flow cytometry a powerful tool for detailed analysis of complex populations in a short period of time. This report reviews the general principles in flow cytometry and selected applications of flow cytometry in the clinical hematology laboratory.
Reference Reagents for Prostate-specific Antigen (PSA): Establishment of the First International Standards for Free PSA and PSA (90:10)
Authors: Rafferty Brian , Rigsby Peter , Rose Matthew , Stamey Thomas , Gaines Das Rose 
Published: January 2020

Abstract
Background: Prostate-specific antigen (PSA) measurements in serum by immunoassay are widely used in the screening, diagnosis, and monitoring of patients with prostate cancer although the lack of common reference reagents has led in the past to wide differences in estimates. We report here the results of a WHO international collaborative study in which two preparations of PSA representative of the main immunoreactive components in serum, free PSA and PSA 90:10, and a preparation of recombinant DNA-derived PSA were assessed as potential standards for the calibration of diagnostic immunoassays for PSA.
Methods: Coded vials of the candidate materials and serum preparations containing PSA in the clinically important range were provided to the 10 laboratories in the study, and participants were asked to perform PSA assays currently in use in their laboratories. Data from 89 immunoassays by 26 different method-laboratory combinations were contributed to the study and analyzed centrally at the National Institute for Biological Standards and Control.
Results: Potency estimates of the preparations relative to the in-house calibrators were in good agreement with the target value of 1 μg of total PSA/vial, the preparation of free PSA giving 1.10 μg/vial (95% confidence interval, 0.99–1.21 μg/vial) and PSA 90:10, 1.11 μg/vial (95% confidence interval, 1.04–1.18 μg/vial). No immunoreactivity was detected in ampoules containing the recombinant material. Use of a common standard of PSA 90:10 significantly reduced the between-laboratory geometric coefficients of variation for serum samples included in the study and gave a much narrower range of potency estimates.
Conclusions: The preparation of free PSA was established by WHO as the First International Standard for PSA (free) with an assigned content of 1 μg of total PSA per vial. In addition, the preparation of bound PSA was established as the First International Standard for PSA (90:10) with an assigned content of 1 μg of total PSA per vial.
Determination of Acid α-Glucosidase Protein: Evaluation as a Screening Marker for Pompe Disease and Other Lysosomal Storage Disorders
Authors: Umapathysivam Kandiah , Whittle Alison M , Ranieri Enzo , Bindloss Colleen , Ravenscroft Elaine M , van Diggelen Otto P , Hopwood John J , Meikle Peter J 
Published: January 2020

AbstractBackground: In recent years, there have been significant advances in the development of enzyme replacement and other therapies for lysosomal storage disorders (LSDs). Early diagnosis, before the onset of irreversible pathology, has been demonstrated to be critical for maximum efficacy of current and proposed therapies. In the absence of a family history, the presymptomatic detection of these disorders ideally can be achieved through a newborn screening program. One approach to the development of such a program is the identification of suitable screening markers. In this study, the acid α-glucosidase protein was evaluated as a marker protein for Pompe disease and potentially for other LSDs.Methods: Two sensitive immunoquantification assays for the measurement of total (precursor and mature) and mature forms of acid α-glucosidase protein were used to determine the concentrations in plasma and dried blood spots from control and LSD-affected individuals.Results: In the majority of LSDs, no significant increases above control values were observed. However, individuals with Pompe disease showed a marked decrease in acid α-glucosidase protein in both plasma and whole blood compared with unaffected controls. For plasma samples, this assay gave a sensitivity of 95% with a specificity of 100%. For blood spot samples, the sensitivity was 82% with a specificity of 100%.Conclusions: This study demonstrates that it is possible to screen for Pompe disease by screening the concentration of total acid α-glucosidase in plasma or dried blood spots.