bluuebunny/crossref_metadata_2025_split huggingface dataset:
Clinical and Analytical Evaluation of an Enzyme Immunoassay for Myelin Basic Protein in Cerebrospinal Fluid
Authors: Ohta Mitsuhiro , Ohta Kiyoe , Ma Jie , Takeuchi Juji , Saida Takahiko , Nishimura Masataka , Itoh Nobuyuki 
Published: January 2020

AbstractBackground: RIA of myelin basic protein (MBP) in cerebrospinal fluid (CSF) is commonly used as a biochemical marker of demyelination in patients with multiple sclerosis (MS). Our aim was to develop a sufficiently sensitive ELISA for MBP and evaluate it clinically in patients with MS.Methods: The ELISA used anti-bovine MBP antibody coated on plates and biotinylated anti-MBP antibody. The bound antibody complex was quantified with streptavidin-horseradish peroxidase. MBP was determined in CSF from 84 MS patients and 55 patients with other neurological diseases.Results: The respective within- and between-assay CVs were 4.7% and 7.2% at 200 ng/L, and 6.3% and 8.8% at 2000 ng/L. The detection limit was 30 ng/L. Most of the MS patients with acute exacerbations had markedly increased MBP in the CSF. Longitudinal studies of six MS patients with recurrent exacerbation confirmed this observation. MBP concentrations from 78 MS patients, as tested with our ELISA, correlated well with those obtained by RIA (r = 0.9; P &lt;0.01), but the detection limit of the ELISA was much lower than that of the RIA.Conclusions: This convenient ELISA with higher sensitivity than the existing assays is a suitable routine assay that provides a diagnostic indicator of myelin breakdown in the central nervous system; moreover, it is an excellent indicator of MS disease activity.
Lower Cardiac Troponin T and I Results in Heparin-Plasma Than in Serum
Authors: Stiegler Hugo , Fischer Yuriko , Vazquez-Jimenez Jaime F , Graf Jürgen , Filzmaier Karsten , Fausten Bernd , Janssens Uwe , Gressner Axel M , Kunz Dagmar 
Published: January 2020

AbstractBackground: The use of plasma rather than serum for determination of cardiac troponins can improve turnaround time and potentially avoid incomplete serum separation that may produce falsely increased results. We investigated the influence of incomplete serum separation and the effect of heparin-plasma on cardiac troponin concentrations.Methods: Serum and heparin-plasma samples were drawn simultaneously from 100 patients (50 patients with acute coronary syndrome and 50 patients after open heart surgery) and measured on three different analytical systems, two for determination of cardiac troponin I (cTnI; Abbott AxSYM and Bayer ACS:Centaur) and one for cardiac troponin T (cTnT; Roche Elecsys cTnT STAT). Serum samples were reanalyzed after a second centrifugation to assess the influence of incomplete serum separation.Results: Mean results (± 95% confidence interval) in heparin-plasma compared with serum were 101% ± 2% (AxSYM cTnI), 94% ± 3% (ACS:Centaur cTnI), and 99% ± 3% (Elecsys cTnT). Differences &gt;20% were seen in 11% of results on the ACS:Centaur, 9% of results on Elecsys cTnT, and 2% of results on the AxSYM. For the Elecsys cTnT assay, the magnitude of the difference between serum and plasma was independent of the absolute concentration and confined to individual samples, and was reversed by treatment with heparinase. A second centrifugation had no effect on serum results by any of the assays.Conclusion: The concentrations of troponins measured in heparin-plasma are markedly lower than in serum in some cases.
Evaluation of Two Different Homogeneous Assays for LDL-Cholesterol in Lipoprotein-X-positive Serum
Authors: Fei Hua , Maeda Satoshi , Kirii Hirokazu , Fujigaki Suwako , Maekawa Naoya , Fujii Hidehiko , Wada Hisayasu , Saito Kuniaki , Seishima Mitsuru 
Published: January 2020

AbstractBackground: The purpose of this study was to evaluate the performance of two homogeneous assays for LDL-cholesterol (LDL-C), a polyethylene/cyclodextrin (PC) assay and a detergent (D) assay, which are based on different principles, in cholestatic serum.Methods: We compared serum LDL-C concentrations determined by the two assays for healthy normolipidemic subjects (n = 42) and cholestatic patients (n = 51). LDL-C concentrations obtained with the homogeneous assays were also compared with those obtained by HPLC for patients’ sera. In the interference study, conjugated bile acids were added to normal serum, and their effects on the two assays were examined. The effects of lipoprotein-X (LP-X), intermediate-density lipoprotein (IDL), and apolipoprotein (apo) E-rich HDL on the LDL-C assays were also investigated by adding these lipoproteins to normal serum.Results: The LDL-C concentrations obtained with the D assay were higher than those obtained with the PC assay in the serum with high LP-X. The bias for LDL-C vs LP-X in cholestatic serum correlated with LP-X concentration (r = 0.582; P &lt;0.0001; n = 51). In the interference study, no effect of bile acids on the LDL-C assays was observed. However, the D assay measured 51.0% of the cholesterol in LP-X, whereas no reactivity was observed for LP-X in the PC assay. In addition, the D assay and the PC assay measured IDL-cholesterol at 31.2% and 52.4%, respectively, and measured apo E-rich HDL-C at 7.6% and 17.8%, respectively.Conclusions: Although both homogeneous LDL-C assays are suitable for most cases, the present study showed that each homogeneous assay has a different limitation for cholestatic serum with gross alterations in lipoproteins.
Discrimination of Prohibited Oral Use of Salbutamol from Authorized Inhaled Asthma Treatment
Authors: Bergés Rosa , Segura Jordi , Ventura Rosa , Fitch Kenneth D , Morton Alan R , Farré Magı́ , Mas Marta , de la Torre Xavier 
Published: January 2020

AbstractBackground: The administration of salbutamol is permitted only by inhalation by the International Olympic Committee (IOC) for the management of asthma and exercise-induced asthma in athletes. The establishment of criteria to distinguish between the IOC authorized use (inhaled) and the IOC prohibited use (oral) of salbutamol appeared possible using simultaneous evaluation of variables based on the concentration of nonconjugated enantiomers of salbutamol excreted in urine.Methods: Urine was collected from asthmatic and nonasthmatic swimmers who had received various preexercise doses of oral (five doses of 4 mg) or inhaled (two doses of 100 μg) salbutamol. Urine was also obtained from subjects who had received the maximum dosage of inhaled salbutamol advisable for competing athletes to provide protection from exercise-induced asthma and treatment of asthma (1600 μg in 24 h, 800 μg being in the last 4 h). All samples were analyzed to determine the total amount of unchanged salbutamol excreted in urine and the ratio between the S and R enantiomers.Results: The discriminant function D = −3.776 + 1.46 × 10−3 {[S(+)] + [R(−)]} + 1.012 {[S(+)]/[R(−)]} can be used to classify data into two groups, inhaled and oral. The confirmatory criterion suggested (cutoff at D = 1.06, 4 SD from the mean D value of the inhaled distribution) has been verified in different sets of samples showing suspicious concentrations by conventional screening procedures in doping control. An 11.8% false-negative (oral classified as inhaled) rate is assumed with the confirmatory criterion proposed, but virtually no false positives (inhaled classified as oral) are obtained (&lt;1 in 33 000).Conclusions: The overall procedure recommended is to screen all samples and to apply the confirmation criterion proposed to samples showing free racemic salbutamol concentrations &gt;500 μg/L by gas chromatography-mass spectrometry or free + conjugated racemic salbutamol concentrations &gt;1400 μg/L by ELISA.
Streptavidin-Polyvinylamine Conjugates Labeled with a Europium Chelate: Applications in Immunoassay, Immunohistochemistry, and Microarrays
Authors: Scorilas Andreas , Bjartell Anders , Lilja Hans , Moller Christina , P. Diamandis Eleftherios 
Published: January 2020

Abstract
Background: The favorable properties of lanthanide chelates compared with conventional fluorescent probes have attracted considerable interest. A Eu3+ chelator, 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA), has been synthesized previously.
Methods: We here describe immunoassay, immunohistochemistry, and microarray applications of a new streptavidin-based universal polyvinylamine (PVA) detection reagent that is multiply labeled with the europium chelate of BCPDA. Solid-phase time-resolved immunofluorometric assays for biotinylated mouse IgG and prostate-specific antigen (PSA) were developed using the new conjugate as a detection reagent. The new conjugate was also used for the immunohistochemical localization of PSA expression in paraffin-embedded prostatic tissues. A model microarray with spotted biotinylated antibody as target was also performed.
Results: Approximately 50–100 BCPDA moieties were covalently bound to PVA, which was then linked to streptavidin via biotin interaction. The macromolecular complex successfully recognized and bound biotinylated detection reagents, e.g., antibodies. The new reagent enabled measurement of solid phase-immobilized biotinylated mouse IgG with a detection limit of ∼1 pg/assay and demonstrated excellent linearity. In an ELISA-type sandwich PSA assay that included two PSA monoclonal antibodies using the new conjugate as detection reagent, we detected 0.001 μg/L PSA (∼100 fg or ∼3 amol/assay). Serum samples analyzed for PSA by this method and a commercial assay gave highly correlated results. The new reagent enabled excellent immunohistochemical localization of PSA expression in prostate tissues. Using the new reagent in a model microarray experiment with biotinylated mouse IgG as target, we demonstrated excellent spatial resolution of 5- to 10-nL microspots.
Conclusions: The new detection reagent may find important applications in biotechnology.
Patient-derived Quality Specifications for Instruments Used in Self-Monitoring of Blood Glucose
Authors: Skeie Svein , Thue Geir , Sandberg Sverre 
Published: January 2020

Abstract
Background: Instruments for self-monitoring of glucose (SMBG) are increasingly used by diabetic patients. Information is limited on how patients use and interpret SMBG results, and no quality specifications for such instruments are based on the opinions of patients.
Methods: Type 1 diabetic patients (n = 201) filled in a questionnaire eliciting daily limits for blood glucose (BG) and changes of BG considered significant at different glucose concentrations. From these responses, patient-derived quality specifications were calculated in different clinical situations with low, intermediate, and high BG concentrations.
Results: Mean age of the patients was 31.8 years, mean diabetes duration was 14.7 years, and mean SMBG duration was 10.0 years with a mean frequency of 11.2 measurements/week. The threshold for hypoglycemic symptoms was 3.0 mmol/L (54 mg/dL), and the mean daily BG target window was 4.3–10.4 mmol/L (77–187 mg/dL). The mean absolute BG changes producing actions from the patients ranged from 1.1 mmol/L (20 mg/dL) to 3.6 mmol/L (65 mg/dL). The analytical quality specifications for imprecision depended on the clinical situation. Excluding the hypoglycemic situation, the analytical CV needed to fulfill the expectations of 75% of the patients was 6.4–9.7%. The analytical quality specification for CV at hypoglycemic concentrations was 3.1%.
Conclusions: Instruments for self-measurements of glucose with an imprecision (CV) of ≤5% and bias ≤5% meet the expectations of &gt;75% of patients in clinical situations other than hypoglycemia.
Fluorescence-based, Nonradioactive Method for Efficient Detection of the Pentanucleotide Repeat (TTTTA)n Polymorphism in the Apolipoprotein(a) Gene
Authors: Rubin Jill , Pearson Thomas A , Reed Roberta G , Berglund Lars 
Published: January 2020

Abstract
Background: The apolipoprotein(a) [apo(a)] gene is a major predictor of plasma lipoprotein(a) concentrations, an independent risk factor for cardiovascular disease. The apo(a) gene contains a pentanucleotide repeat (PNR) polymorphism, 1.4 kb upstream from the apo(a) gene reading frame. This polymorphism has been suggested to be important in control of apo(a) gene expression.
Methods: We developed a fluorescence-based, nonradioactive procedure to detect the PNR polymorphism. After amplification of the polymorphism by PCR, the respective PCR products were separated by denaturing polyacrylamide gel electrophoresis and detected using a 3′-end fluorescently labeled oligonucleotide as a probe. We used the method to characterize the PNR polymorphism pattern in 313 individuals, 195 Caucasians and 118 African Americans. The new method efficiently separated DNAs corresponding to the different PNR repeats.
Results: Among both ethnic groups, alleles containing eight PNRs were most common. Smaller PNRs were more common among African Americans, and larger PNRs were more common among Caucasians.
Conclusions: We developed a nonradioactive technique that separates the PNR polymorphism in the apo(a) gene and can be used in other studies involving closely sized polymorphisms.
Hepatic Carnitine Palmitoyltransferase I Deficiency: Acylcarnitine Profiles in Blood Spots Are Highly Specific
Authors: Fingerhut Ralph , Röschinger Wulf , Muntau Ania C , Dame Torsten , Kreischer Jens , Arnecke Ralf , Superti-Furga Andrea , Troxler Heinz , Liebl Bernhard , Olgemöller Bernhard , Roscher Adelbert A 
Published: January 2020

AbstractBackground: In carnitine palmitoyltransferase I (CPT-I) deficiency (MIM 255120), free carnitine can be increased with no pathologic acylcarnitine species detectable. As inclusion of CPT-I deficiency in high-risk and newborn screening could prevent potentially life-threatening complications, we tested whether CPT-I deficiency might be diagnosed by electrospray ionization-tandem mass spectrometry (ESI-MS/MS).Methods: A 3.2-mm spot of whole blood dried on filter paper was extracted with 150 μL of methanol. After derivatization of carnitine and acylcarnitines to their butyl esters, the samples were analyzed by ESI-MS/MS with 37.5 pmol of l-[2H3]carnitine and 7.5 pmol of l-[2H3]palmitoylcarnitine as internal standards.Results: In all dried-blood specimens from each of three patients with CPT-I deficiency, we found an invariably increased ratio of free carnitine to the sum of palmitoylcarnitine and stearoylcarnitine [C0/(C16 + C18)]. The ratio in patients was between 175 and 2000, or 5- to 60-fold higher than the ratio for the 99.9th centile of the normal newborn population in Bavaria (n = 177 842). No overlap with the values of children that were known to be supplemented with carnitine was detected [C0/(C16 + C18), 34 ± 30; mean ± SD; n = 27].Conclusions: ESI-MS/MS provides a highly specific acylcarnitine profile from dried-blood samples. The ratio of free carnitine to the sum of palmitoylcarnitine and stearoylcarnitine [C0/(C16 + C18)] is highly specific for CPT-I deficiency and may allow presymptomatic diagnosis.
High-Throughput Genomic and Proteomic Analysis Using Microarray Technology
Authors: Huang Joe Xi , Mehrens Dorothy , Wiese Rick , Lee Sandy , Tam Sun W , Daniel Steve , Gilmore James , Shi Michael , Lashkari Deval 
Published: January 2020

Abstract
Background: High-density microarrays are ideally suited for analyzing thousands of genes against a small number of samples. The next step in the discovery process is to take the resulting genes of interest and rapidly screen them against thousands of patient samples, tissues, or cell lines to further investigate their involvement in disease risk or the response to medication.
Methods: We used a microarray technology platform for both single-nucleotide polymorphisms (SNPs) and protein expression. Each microarray contains up to 250 elements that can be customized for each application. Slides contained either a 16- or 96-microarray format (4000–24 000 elements per slide), allowing the corresponding number of samples to be rapidly processed in parallel.
Results: Results for SNP genotyping and protein profiling agreed with results of restriction fragment length polymorphism (RFLP) analysis or ELISA, respectively. Genotyping analyses, using the microarray technology, on large sample sets over multiple polymorphisms in the NAT2 gene were in full agreement with traditional methodologies, such as sequencing and RFLP analysis. The multiplexed protein microarray had correlation coefficients of 0.82–0.99 (depending on analyte) compared with ELISAs.
Conclusions: The integrated microarray technology platform is adaptable and versatile, while offering the high-throughput capabilities needed for drug development and discovery applications.
Denaturing HPLC for Identification of Clonal T-Cell Receptor γ Rearrangements in Newly Diagnosed Acute Lymphoblastic Leukemia
Authors: zur Stadt Udo , Rischewski Johannes , Schneppenheim Reinhard , Kabisch Hartmut 
Published: January 2020

Abstract
Background: Denaturing HPLC (DHPLC) can be used to screen DNA for known and unknown mutations. We describe a novel, HPLC-based method for discrimination among polyclonal, oligoclonal, and/or clonal T-cell receptor γ (TCR-γ) rearrangements in samples from children with newly diagnosed acute lymphoblastic leukemia.
Methods: TCR rearrangements were PCR amplified from initial leukemic samples and, after heteroduplex-induction, the clonality status of each product was evaluated. To attain this, we used DHPLC on a high-resolution micropellicular matrix. Running conditions were established by melting-curve analysis of known clonal and polyclonal products and melting-point prediction software. Elution profiles were studied at 50 °C (native) and, to achieve optimal separation, at different column temperatures between 56 and 64 °C.
Results: For VγI-Jγ1.3/2.3 rearrangements, an analysis temperature of 60 °C with a linear triethylammoniumacetate—acetonitrile gradient separated clonal bands from the polyclonal background amplification. Less than 15% clonal PCR product was detectable in mixtures of initial leukemic cell DNA and polyclonal DNA. Biallelic rearrangements produced two sharp peaks. Clonality of PCR products from 100 initial leukemic samples was completely identified in all investigated cases.
Conclusions: Heteroduplex analysis with standardized DHPLC conditions simplifies the detection of unknown clonal or polyclonal TCR rearrangements in newly diagnosed leukemias. Clonal targets for detection of minimal residual disease are available after a short, automated analysis of PCR amplified rearrangements.
Celiac Disease
Authors: Aleanzi Mabel , Demonte Ana María , Esper Cecilia , Garcilazo Silvia , Waggener Marta 
Published: January 2020

Abstract
Background: Selective deamidation of glutamine residues by tissue transglutaminase (tTG) turns gliadin peptides into stronger activators of T cells from celiac disease (CD) patients. We examined the possibility that these modified peptides could be more specific epitopes for circulating antibodies than are native peptides.
Methods: Two native synthetic peptides and their respective modified sequences were used as antigens for ELISA assays: peptide-1, with residues 56–75 of α-type gliadin; and peptide-2, with residues 134–153 of γ-type gliadin. We examined 40 CD patients [31 not being treated with a gluten-free diet (GFD) and 9 being treated with a GFD] and 30 non-CD patients.
Results: An enhanced response against deamidated peptides was observed in 4 (IgA) and 22 (IgG) of 31 untreated CD patients for peptide-1 and in 25 (IgA) and 29 (IgG) patients for peptide-2. Higher anti-gliadin antibody and anti-tTG IgA concentrations correlated with increased IgA reactivity to modified peptides. Among the nine treated CD patients, eight also displayed an improved IgG signal for the deamidated sequence. Deamidation of peptides did not increase the reactivity of non-CD sera.
Conclusions: Selective deamidation specifically increases circulating antibody recognition of gliadin peptides in CD patients. This suggests that deamidated gliadin peptides are more specific CD B-cell epitopes than native peptides; this finding may be relevant for designing improved diagnostic tests.
Determination of l-Pipecolic Acid in Plasma Using Chiral Liquid Chromatography-Electrospray Tandem Mass Spectrometry
Authors: Rashed Mohamed S , Al-Ahaidib Lujane Y , Aboul-Enein Hassan Y , Al-Amoudi Mohamed , Jacob Minnie 
Published: January 2020

AbstractBackground: l-Pipecolic acid (L-PA), an important biochemical marker for the diagnosis of peroxisomal disorders, is usually determined as the racemate. We developed a chiral liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the analysis of L-PA in plasma.Methods: We used a narrow bore chiral macrocyclic glycopeptide teicoplanin column for the enantioseparation of d-pipecolic acid (D-PA) and L-PA and interfaced the column directly to the electrospray source of a tandem mass spectrometer. We used phenylalanine-d5 as internal standard added to 50 μL of plasma followed by deproteinization, evaporation, and injection. The analysis was performed in the selected-reaction monitoring mode using two transitions: m/z 130→m/z 84 for PA, and m/z 171→m/z 125 for phenylalanine-d5. L-PA eluted at 7 min, and D-PA eluted at 11.7 min, whereas phenylalanine-d5 eluted at 6 min. The turnaround time for the assay was 20 min.Results: Linear calibration curves were obtained in the range of 0.5–80 μmol/L. At a plasma concentration of 1.0 μmol/L, the signal-to-noise ratio was 50:1. The intra- and interassay variations were 3.1–7.9% and 5.7–13%, respectively, at concentrations of 1–50 μmol/L. Mean recoveries of L-PA added to plasma were 95% (5 μmol/L) and 102% (50 μmol/L). The method clearly distinguished between healthy individuals and peroxisomal disease patients.Conclusions: The novel LC-MS/MS method is simple, rapid, and stereoselective, and uses only 50 μL of plasma, no derivatizing reagents, and a commercially available internal standard. Sample preparation is not complex and is faster than for all other methods.
The Ratio of Trypsin-2-α1-Antitrypsin to Trypsinogen-1 Discriminates Biliary and Alcohol-induced Acute Pancreatitis
Authors: Andersén Jan M , Hedström Johan , Kemppainen Esko , Finne Patrik , Puolakkainen Pauli , Stenman Ulf-Håkan 
Published: January 2020

Abstract
Background: Rapid determination of the etiology of acute pancreatitis (AP) enables institution of appropriate treatment. We evaluated the ability of trypsinogen-1, trypsinogen-2, trypsin-1-α1-antitrypsin (AAT), and trypsin-2-AAT in serum to identify the etiology of AP.
Methods: The study consisted of 67 consecutive patients with AP admitted to Helsinki University Central Hospital. Forty-two had alcohol-induced AP, 16 had biliary AP, and 9 had unexplained etiology. Serum samples were drawn within 12 h after admission. Trypsinogen-1, trypsinogen-2, trypsin-1-AAT, and trypsin-2-AAT were determined by time-resolved immunofluorometric assays. Logistic regression was used to estimate the ability of the serum analytes to discriminate between alcohol-induced and biliary AP. The validity of the tests was evaluated by ROC curve analysis.
Results: Patients with alcohol-induced AP had higher median values of trypsin-1-AAT (P = 0.065), trypsinogen-2 (P = 0.034), and trypsin-2-AAT (P &lt;0.001) than those with biliary AP, who had higher values of amylase (P = 0.002), lipase (P = 0.012), and alanine aminotransferase (P = 0.036). The ratios of trypsin-2-AAT to trypsinogen-1, lipase, or amylase efficiently discriminated between biliary and alcohol-induced AP (areas under ROC curves, 0.92–0.96).
Conclusions: Trypsinogen-2 and trypsin-2-AAT are markedly increased in AP of all etiologies, whereas trypsinogen-1 is increased preferentially in biliary AP. The trypsin-2-AAT/trypsinogen-1 ratio is a promising new marker for discrimination between biliary and alcohol-induced AP.