bluuebunny/crossref_metadata_2025_split huggingface dataset:
K-ras Point Mutation Detection in Lung Cancer: Comparison of Two Approaches to Somatic Mutation Detection Using ARMS Allele-specific Amplification
Authors: Clayton Simon J , Scott Frank M , Walker Jill , Callaghan Kay , Haque Kemal , Liloglou Triantafillos , Xinarianos George , Shawcross Sue , Ceuppens Pete , Field John K , Fox Jayne C 
Published: January 2020

Abstract
Background: The use of sensitive molecular techniques to detect rare cells in a population is of increasing interest to the molecular pathologist, but detection limits often are poorly defined in any given molecular assay. We combined the approaches of real-time quantitative PCR with ARMSTM allele-specific amplification in a novel assay for detecting mutant K-ras sequences in clinical samples.
Methods: ARMS reactions were used to detect seven commonly occurring mutations in the K-ras oncogene. These mutations produce amino acid changes in codon 12 (Gly to Ala, Arg, Asp, Cys, Ser, or Val) and codon 13 (Gly to Asp). A control reaction was used to measure the total amount of amplifiable K-ras sequence in a sample so that the ratio of mutant to wild-type sequence could be measured. Quantitative data were confirmed for a selection of samples by an independent cloning and sequencing method. The assay was used to analyze 82 lung tumor DNA samples.
Results: The assay detected K-ras mutations in 44% of adenocarcinomas, which is equivalent to frequencies reported in the literature using ultrasensitive techniques. Forty-six percent of squamous carcinomas were also positive. The ratio of mutant sequence in the tumor DNA samples was 0.04–100%.
Conclusions: The assay is homogeneous, with addition of tumor DNA sample being the only step before results are generated. The quantitative nature of the assay can potentially be used to define the analytical sensitivity necessary for any specified diagnostic application of K-ras (or other) point mutation detection.
Use of Two Reporter Dyes without Interference in a Single-Tube Rapid-Cycle PCR: α1-Antitrypsin Genotyping by Multiplex Real-Time Fluorescence PCR with the LightCycler
Authors: von Ahsen Nicolas , Oellerich Michael , Schütz Ekkehard 
Published: January 2020

AbstractBackground: α1-Antitrypsin is the major plasma serine protease inhibitor. Its deficiency is mainly associated with the alleles PI*S and PI*Z and can lead to obstructive lung disease in adults and to liver cirrhosis during childhood.Methods: A multiplex PCR method has been established that uses two sets of primers to amplify the gene regions covering the PI*S or PI*Z mutations sites. Mutation detection was performed on the LightCycler by melting curve analysis of detection probes labeled with two different fluorescent dyes, LC-Red640 and LC-Red705.Results: Unequivocal genotyping results were obtained for all investigated samples in an assay time of ∼30 min. The color compensation procedure greatly improved the readability of the resulting diagnostic melting curves.Conclusions: To our knowledge, this is the first report of simultaneous detection of two mutations in a single tube by PCR of genomic DNA and the use of two different reporter dyes with the LightCycler color compensation feature. This approach is a rapid, convenient, and economic alternative to other methods described to date for the detection of α1-antitrypsin deficiency alleles.
Automated Assay for HER-2/neu in Serum
Authors: Payne Robert C , Allard Jeffrey W , Anderson-Mauser Linda , Humphreys John D , Tenney Donald Y , Morris David L 
Published: January 2020

AbstractBackground: The extracellular domain of the HER-2/neu oncogene product is increased in sera of some patients with epithelial cancers. Our aim was to develop an automated serum assay for the extracellular domain of the HER-2/neu protein.Methods: We used a monoclonal antibody labeled with fluorescein for capture and a monoclonal Fab′ fragment labeled with alkaline phosphatase for detection. Separation of bound and free detection conjugate was performed with magnetizable particles coated with monoclonal antibody to fluorescein. Alkaline phosphatase activity was measured kinetically at 405 or 450 nm.Results: The assay was linear from 0.1 to 250 μg/L. No hook effect was evident up to 10 000 μg/L. Within-run imprecision (CV) was 0.8–1.2%, and total imprecision was 1.1–1.7%. Cross-reactivity with human epidermal growth factor receptor, which has extensive homology with HER-2/neu extracellular domain, was &lt;0.6%. Human anti-mouse antibodies, heterophilic antibodies, and rheumatoid factor did not interfere, nor did the therapeutic monoclonal antibody Herceptin®. In 51 healthy females, the mean value was 9.3 μg/L with a range of 6.4–14.0 μg/L. No reagent lot-to-lot variability was detected over four lots of reagents tested.Conclusion: The Bayer Immuno 1TM assay for HER-2/neu was precise and resistant to interferences, characteristics that are essential for longitudinal monitoring of cancer patients.
Improved Prediction of Decreased Creatinine Clearance by Serum Cystatin C: Use in Cancer Patients before and during Chemotherapy
Authors: Štabuc Borut , Vrhovec Levin , Štabuc-Šilih Mirna , Cizej Tomaž Edvard 
Published: January 2020

AbstractBackground: Serum cystatin C, a cysteine protease inhibitor, has been suggested as a new marker of glomerular filtration rate (GFR). This study explored the possibility of replacing the creatinine clearance (CrCl) estimation of GFR with cystatin C in early detection of renal impairment in cancer patients on chemotherapy.Methods: Serum creatinine and cystatin C concentrations as well as 24-h CrCl were determined simultaneously in 72 cancer patients. Among them, 60 were treated with combined chemotherapy with cisplatin (CDDP). Creatinine was determined enzymatically with a spectrophotometric method. Serum cystatin C was determined by a particle-enhanced turbidimetric immunoassay.Results: Cystatin C and creatinine correlated significantly (P = 0.001) with CrCl. The correlation was significantly better for cystatin C than creatinine (r = 0.84 vs 0.74; P = 0.01). Stepwise regression analysis identified no differences for the correlations between cystatin C and CrCl in patients with or without metastases (r = 0.82 and 0.84, respectively) as well as before treatment and before the fourth cycle of chemotherapy (r = 0.70 and 0.75, respectively). A cystatin C cutoff concentration of 1.33 mg/L had 87% sensitivity and 100% specificity for detecting CrCl &lt;78 mL/min. ROC analysis indicated that cystatin C was superior to serum creatinine for predicting CrCl &lt;78 mL/min (P &lt;0.04).Conclusions: Serum cystatin C is superior to serum creatinine for detection of decreased CrCl and potentially for the estimation of GFR in cancer patients independent of the presence of metastases or chemotherapy.
Strontium as a Marker for Intestinal Calcium Absorption: The Stimulatory Effect of Calcitriol
Authors: Dijkgraaf-ten Bolscher Marieke , Netelenbos J Coen , Barto Rob , van der Vijgh Wim J F 
Published: January 2020

AbstractBackground: Intestinal strontium absorption is becoming accepted as a clinical and diagnostic tool for assessing intestinal calcium absorption in humans. However, little is known about whether intestinal strontium absorption, like that of calcium, is stimulated by calcitriol in healthy humans.Methods: The effect of calcitriol on intestinal strontium absorption was measured in eight healthy men, ages 20–60 years. Before administration of calcitriol, two tests were performed with an interval of 10 days for calculating the within-subject variation (SER). Before the third test, 0.5 μg of calcitriol was given twice daily for 3 days. In each test, the fractional strontium absorption (Fc240) and the area under the concentration-time curve (AUC0–240) 4 h after an oral strontium load of 2.5 mmol were calculated.Results: The within-subject SER of Fc240 and AUC0–240 was 1.7 ± 0.7 and 0.83 ± 0.1, respectively. The stimulatory effect of calcitriol on Fc240 and AUC0–240 was 35% (21.8 ± 2.0 to 28.8 ± 2.4; P = 0.003) and 61% (8.97 ± 0.97 to 14.4 ± 1.3 mmol · L−1 · min; P = 0.001), respectively.Conclusions: Although the reproducibility of AUC0–240 and its sensitivity to calcitriol were better than those of Fc240, the Fc240 of strontium is preferred for a clinical test because of its simplicity, requiring only two instead of five blood samples.
Development and Evaluation of Three Immunofluorometric Assays That Measure Different Forms of Osteocalcin in Serum
Authors: Käkönen Sanna-Maria , Hellman Jukka , Karp Matti , Laaksonen Pirjo , Obrant Karl J , Väänänen H Kalervo , Lövgren Timo , Pettersson Kim 
Published: January 2020

AbstractBackground: Circulating human osteocalcin (hOC) has been used as a marker of bone formation. Our aim was to validate three immunofluorometric assays (IFMAs), measuring different forms of hOC.Methods: The two-site IFMAs were based on previously characterized monoclonal antibodies. Assay 2 recognized intact hOC, assays 4 and 9 measured the NH2-terminal mid-fragment and the intact hOC. In addition, assay 9 required hOC to be γ-carboxylated.Results: A 76–79% increase of serum immunoreactive hOC was found in the postmenopausal group compared with the premenopausal group with all IFMAs. With EDTA-plasma samples, the observed increases were lower (49–65%). The hOC concentration in the postmenopausal group receiving hormone replacement therapy was 42–44% lower than that in the postmenopausal control group in both serum and EDTA-plasma samples. The depressed carboxylation in warfarin-treated patients was accompanied by lower results in assay 9. The ratio of assay 9 to assay 4 totally discriminated the warfarin-treated patients from the controls. Assay 9 showed the smallest decreases in measured hOC after storage of serum or plasma for 4 weeks at 4 °C, followed by assay 4 and assay 2. Results from the last assay were &lt;17% of their initial values after 4 weeks of storage. No diurnal variation was observed with assay 9 as opposed to the two other IFMAs.Conclusion: The three assays with their distinct specificity profiles (intact vs fragmented and carboxylated vs decarboxylated hOC) may provide valuable tools for investigating the significance of different hOC forms in various bone-related diseases.
Serum Dehydroepiandrosterone, Dehydroepiandrosterone Sulfate, and Pregnenolone Sulfate Concentrations in Patients with Hyperthyroidism and Hypothyroidism
Authors: Tagawa Noriko , Tamanaka Junko , Fujinami Aya , Kobayashi Yoshiharu , Takano Toru , Fukata Shuji , Kuma Kanji , Tada Hisato , Amino Nobuyuki 
Published: January 2020

AbstractBackground: Dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEA-S) have been suggested to have protective effects against cardiovascular disease, cancer, immune-modulated diseases, and aging. We examined serum concentrations of DHEA, DHEA-S, and pregnenolone sulfate (PREG-S) in patients with thyroid dysfunction.Methods: Steroids extracted with methanol from serum sample were separated into an unconjugated fraction (DHEA) and a monosulfate fraction (DHEA-S and PREG-S), using a solid-phase extraction and an ion-exchange column. After separation of unconjugated steroids by HPLC, the DHEA concentration was measured by enzyme immunoassay. The monosulfate fraction was treated with arylsulfatase, and the freed steroids were separated by HPLC. The DHEA and PREG fractions were determined by gas chromatography–mass spectrometry, and the concentrations were converted into those of DHEA-S and PREG-S.Results: Serum concentrations of DHEA, DHEA-S, and PREG-S were all significantly lower in patients with hypothyroidism (n = 24) than in age- and sex-matched healthy controls (n = 43). By contrast, in patients with hyperthyroidism (n = 22), serum DHEA-S and PREG-S concentrations were significantly higher, but the serum DHEA concentration was within the reference interval. Serum concentrations of these three steroids correlated with serum concentrations of thyroid hormones in these patients. Serum albumin and sex hormone-binding globulin concentrations were not related to these changes in the concentration of steroids.Conclusions: Serum concentrations of DHEA, DHEA-S, and PREG-S were decreased in hypothyroidism, whereas serum DHEA-S and PREG-S concentrations were increased but DHEA was normal in hyperthyroidism. Thyroid hormone may stimulate the synthesis of these steroids, and DHEA sulfotransferase might be increased in hyperthyroidism.
Analysis of the Chimeric CYP21P/CYP21 Gene in Steroid 21-Hydroxylase Deficiency
Authors: Lee Hsien-Hsiung , Chang Jan-Gowth , Tsai Chang-Hai , Tsai Fuu-Jen , Chao Hsiang-Tai , Chung Bon-chu 
Published: January 2020

Abstract
Background: A single nonfunctional chimeric gene with its 5′ and 3′ ends corresponding to CYP21P and CYP21, respectively, is caused by unequal gene crossover in the CYP21 genes during meiosis. The presence of the chimeric CYP21P/CYP21 molecule can not be detected by conventional PCR methods and therefore may be lost in PCR amplification. This leads to a false result and diagnostic discordance.
Methods: We developed a rapid and direct method to detect a chimeric CYP21P/CYP21 gene that uses a 3′-specific primer for the CYP21 gene and two different 5′ primers for both CYP21 and CYP21P to amplify the wild-type CYP21 and the chimeric CYP21P/CYP21 genes. A secondary PCR that can differentiate the chimeric from the wild-type gene was also performed. The PCR product was directly analyzed on agarose gel.
Results: After careful titration, we found that earlier failure to detect the chimeric CYP21P/CYP21 gene could be caused by unequal concentrations of two independent alleles as the PCR template or by the lack of primers to amplify chimeric molecules. We successfully amplified the chimeric gene using our improved method.
Conclusions: The chimeric CYP21P/CYP21 is present in a large portion of congenital adrenal hyperplasia patients. By adding a CYP21P/CYP21-specific primer, we were able to amplify and detect both homozygous and heterozygous chimeric genes. Therefore, our new PCR-based assay is a more effective way to analyze congenital adrenal hyperplasia mutations.
Routine α-Amylase Assay Using Protected 4-Nitrophenyl-1,4-α-d-maltoheptaoside and a Novel α-Glucosidase
Authors: Lorentz Klaus 
Published: January 2020

AbstractBackground: In contrast to numerous methods for measuring α-amylase activity, the approved IFCC reference method offers an invariable time-independent constant product pattern, thus avoiding possibly changing stoichiometric calculations. However, reference methods do not lend themselves to routine use, so that such methods need to be modified.Methods: Ethylidene-blocked 4-nitrophenylmaltoheptaoside (EPS-G7) is degraded to glucose and 4-nitrophenol in a coupled assay with a bacterial α-glucosidase under the following measurement conditions: 3.5 mmol/L EPS-G7, 7.1 kU/L α-glucosidase, 70 mmol/L sodium chloride, 1 mmol/L calcium chloride, 50 mmol/L HEPES, pH 7.15, at 37 °C. The increase of absorbance is continuously monitored for 3 min at 405 nm after a lag phase of 2 min.Results: Catalytic concentrations up to 15-fold higher than the upper reference limit can be determined without dilution. Precision studies in manual performance show CVs of 1.4–2.6% (within-run) and 1.9–2.8% (day-to-day). There was no interference from 100 mmol/L glucose, 30 mmol/L triacylglycerols, 610 μmol/L bilirubin, and 2.95 g/L hemoglobin. The method closely correlates with other chromogenic assays. The preliminary 0.95 reference interval for adults, not dependent on age and sex, is 33.6–96.2 U/L.Conclusion: The procedure is a robust adaptation of the reference method to routine use at 37 °C with increased sensitivity, fewer interferences, and reduced cost.
Proteasome Inhibition Measurements: Clinical Application
Authors: Lightcap Eric S , McCormack Teresa A , Pien Christine S , Chau Vincent , Adams Julian , Elliott Peter J 
Published: January 2020

AbstractBackground: PS-341, a selective inhibitor of the proteasome, currently is under evaluation as an anticancer agent in multiple phase I clinical trials. In animal-model studies, PS-341 was rapidly removed from the vascular compartment and distributed widely, quickly approaching the limits of detection. An accurate pharmacodynamic assay has been developed as an alternative or complement to pharmacokinetic measurements.Methods: Fluorogenic kinetic assays for both the chymotryptic and tryptic activities of the proteasome have been optimized for both whole blood and blood cells. Using the ratio of these activities and the catalytic mechanism of the proteasome, we developed a novel method of calculating percentage of inhibition, using two structurally unrelated inhibitors (PS-341 and lactacystin).Results: This ratio method was demonstrated to be sensitive (detection limit of 13% inhibition with 10 μg of cell lysate), specific to the proteasome (PS-341 provides &gt;98% inhibition), accurate (112% analyte recovery), and precise (0% ± 5% inhibition at 0 nmol/L PS-341 and 74.5% ± 1.7% inhibition at 200 nmol/L PS-341). Using these assays, we found that both erythrocytes and leukocytes contain proteasome at 3 μmol/L. Pharmacodynamic results for PS-341 obtained from the whole-blood ratio method were comparable to those using leukocytes determined by another method.Conclusions: The described assay provides a reliable method for studying the pharmacodynamics of proteasome inhibitors and is now in use in concurrent phase I clinical trials with PS-341.
Quantitative Beutler Test for Newborn Mass Screening of Galactosemia Using a Fluorometric Microplate Reader
Authors: Fujimoto Akie , Okano Yoshiyuki , Miyagi Tomiko , Isshiki Gen , Oura Toshiaki 
Published: January 2020

Abstract
Background: The Beutler enzyme spot test is an effective assay for newborn mass screening of galactosemia, but it is qualitative and relies on visual interpretation. We describe a quantitative, instrumental modification of the assay.
Methods: We modified the macroscopic visual Beutler enzyme spot test by adding extraction of blood components from filter paper, deproteinization with acetone-methanol, and quantification and recording by a fluorescent microplate reader and personal computer. All handling was performed in microplates. The measurement time was 90 min.
Results: Fluorescence intensity (FI) of healthy controls correlated with hematocrit and galactose-1-phosphate uridyltransferase (GALT) activity. Patients with GALT deficiency were distinguished clearly from healthy subjects and heterozygous carriers by FI. FI decreased to 75% of the initial activity after storage at 25 °C for 3 days and to 40% after storage at 37 °C for 7 days. Screening of 46 742 newborns yielded 1 false-positive result (in a heterozygous carrier), 1 patient with glucose-6-phosphate dehydrogenase deficiency, and no apparent false negatives as judged by concurrent measurements of galactose and galactose-1-phosphate.
Conclusions: The quantitative Beutler test can provide precise GALT activity in newborn mass screening, and can take into consideration the influence of high temperature and humidity, duration between sampling and testing, and anemia. This method is clinically useful, simple, automated, and highly reliable for newborn mass screening of galactosemia.
Determination of Gentamicins C1, C1a, and C2 in Plasma and Urine by HPLC
Authors: Isoherranen Nina , Soback Stefan 
Published: January 2020

Abstract
Background: Gentamicin is an aminoglycoside antibiotic complex containing gentamicins C1, C1a, and C2. Few methods have been described for analysis of the three gentamicin components separately in biological fluids, and none has been used in pharmacokinetic studies. Determination of the three gentamicins separately may have pharmacokinetic and toxicological implications. The present study describes development of an HPLC method for the analysis of gentamicin C1, C1a, and C2 components in plasma and urine.
Methods: The three components were isolated by preparative chromatography and their identities verified by thin-layer chromatography, HPLC, mass spectrometry, nuclear magnetic resonance spectroscopy, and melting point determination. The gentamicins were extracted from the biological matrix by use of Tris buffer and polymer phase solid-phase extraction. Derivatization was carried out in the solid-phase extraction cartridge with 1-fluoro-2,4-dinitrobenzene. The 2,4-dinitrophenyl derivatives were separated with reversed-phase HPLC and quantified by the ultraviolet absorbance at 365 nm.
Results: The detector response was linear from the limit of quantification to 50 mg/L for the individual components. The limit of quantification was 0.07 mg/L for gentamicin C1 and 0.1 mg/L for gentamicins C2 and C1a. The recovery of the gentamicin components was 72% from plasma and 98% from urine. The method was validated for human and dog plasma and urine.
Conclusions: The method was repeatable and enabled the analysis of gentamicins C1, C1a, and C2 in plasma and urine in concentrations covering the therapeutic range of the drug, thus being suitable for therapeutic drug monitoring and pharmacokinetic studies.
Toward a Checklist for Reporting of Studies of Diagnostic Accuracy of Medical Tests
Authors: Bruns David E , Huth Edward J , Magid Erik , Young Donald S 
Published: January 2020

Abstract
Background: “Diagnostic accuracy” refers to the ability of medical tests to provide accurate information about diagnosis, prognosis, risk of disease, and other clinical issues. Published reports on diagnostic accuracy of medical tests frequently fail to adhere to minimal clinical epidemiological standards, and such failures lead to overly optimistic assessments of evaluated tests. Our aim was to enumerate key items for inclusion in published reports on diagnostic accuracy, with a related aim of making the reports more useful for systematic reviews.
Methods: We examined published reports on shortcomings of studies of diagnostic accuracy. We prepared an initial draft of a checklist to address common errors and presented it at a meeting of editors. After incorporation of comments from editors, we published a revised version in Clinical Chemistry in 1997 for comment from readers. One of us (E.M.) additionally circulated copies of the draft to methodologists and others interested in Evidence-Based Medicine. We updated the checklist with input from these sources.
Results: The updated document lists items for inclusion in the title, abstract, methods, results, and discussion sections of published papers. Depending on the nature of the study, the total number of items for a single paper is ∼40. We invite comments on this document, which is freely available at Clinical Chemistry Online, where it can accessed readily from the Table of Contents for the July 2000 issue at www.clinchem.org/content/vol46/issue7/. Comments (eLetters) can be posted there for general reading.
Conclusions: The suggested revisions incorporated in this report appear useful to ensure inclusion of additional information that can allow assessment of the validity of the conclusions and the applicability of the study in other settings. The list can be useful in formulating guidelines and a checklist, which will require testing by authors and study of their effect on published studies of diagnostic accuracy.