bluuebunny/crossref_metadata_2025_split huggingface dataset:
Dynamic Reaction in a Homogeneous HDL-Cholesterol Assay Visualized by Electron Microscopy
Authors: Kondo Akira , Muranaka Yoshinori , Ohta Isao , Kanno Takashi 
Published: January 2020

AbstractBackground: Measurement of HDL-cholesterol (HDL-C) by homogeneous assays with automated analyzers is replacing precipitation methods. However, in this reaction-type assay, interactions between the reagents and lipoproteins remain unknown.Methods: Electron microscopy was used to investigate the reactions in a homogeneous HDL-C assay. Negative staining with 10 g/L uranyl acetate was performed for lipoprotein visualization by electron microscopy. Observations of the interactions between lipoproteins and the reagents of a polyanion-polymer/detergent assay were achieved by cooling the reaction mixture in ice water. This treatment also allowed observation of the time course of the reaction.Results: In the first-reagent reaction (polyanion-polymer), every lipoprotein aggregated almost completely. In the second-reagent reaction (enzymes and detergent), only HDL in the lipoprotein aggregates was selectively resolved and reacted enzymatically. Reagent 1 contains two important substances: polyanion and synthetic polymer. Using x-ray microanalysis, we confirmed that aggregation of lipoproteins in the first reaction occurred through interaction with the phosphotungstate of the polyanion.Conclusion: Electron microscopy morphologically revealed the dynamic reaction in a homogeneous HDL-C assay.
Freedom from Drug Interference in New Immunoassays for Urinary Catecholamines and Metanephrines
Authors: Wassell Julie , Reed Paul , Kane John , Weinkove Cyril 
Published: January 2020

Abstract
Background: Determination of urinary free catecholamine and total (i.e., free plus conjugated) metanephrine excretion is considered the most clinically sensitive biochemical test for pheochromocytoma. In this study, we evaluated new immunoassay methods for the measurement of these analytes for potential drug-based interference.
Methods: Urine samples collected from patients on a variety of medications were grouped by specific drug type. The significance of any difference in the free catecholamine or total metanephrine concentrations in the different groups was assessed by one-way ANOVA. A group of patients receiving no medication was included as a control (no analytical interference). Additionally, analytical accuracy, detection limit, and precision were determined.
Results: No significant differences were found in the concentrations of free catecholamines or total metanephrines in urine from patients taking the medications investigated and the control group: P = 0.649 (fE), 0.221 (fNE), 0.149 (tM), and 0.170 (tNM). For free catecholamines, intraassay CVs were 4.6–18%; interassay CVs were 10–25%. For total metanephrines, intraassay CVs were 9.6–27%; interassay CVs were 5.8–22%. Detection limits were 0.009 and 0.027 μmol/L for fE and fNE and 0.119 and 0.346 μmol/L for tM and tNM, respectively.
Conclusions: None of the drugs examined in this study interfered in the measurement of free catecholamines or total metanephrines by these immunoassays. The technique is easier to use, requires less equipment, and is more accessible than HPLC. In combination, these assays are suitable as initial screening tests for pheochromocytoma.
Cross-Reaction with Luteinizing Hormone β-Core Is Responsible for the Age-dependent Increase of Immunoreactive β-Core Fragment of Human Chorionic Gonadotropin in Women with Nonmalignant Conditions
Authors: Iles Ray K , Javid Mohammed K , Gunn Lionel K , Chard Tim 
Published: January 2020

AbstractBackground: The β-core fragment of human chorionic gonadotropin (hCGβcf), also termed “β-core” and urinary gonadotropin peptide (UGP), has been reported to be present in the urine of healthy women and to increase in concentration after menopause. This could reflect cross-reaction with the equivalent metabolite of luteinizing hormone (LH), the β-LH-core.Methods: We measured immunoreactive LH, hCG, free α-subunit, and free β-subunit hCG (hCGβ), as well as β-core, using the S504 RIA and Triton UGP enzyme immunoassay in 274 urine samples from women with nonmalignant gynecological conditions. The molar cross-reaction of each assay with purified β-LH-core was determined.Results: Cross-reaction with β-LH-core was 100% in the LH and the S504 β-core assay, 5% in the Triton UGP assay, and &lt;0.1% in the hCG, free α-subunit, and free hCGβ assays. Median urine concentrations of all analytes showed an age-dependent increase. LH and free α-subunit concentrations were ∼103 pmol/mol creatinine; hCG and S504 β-core were ∼102 pmol/mol creatinine; free hCGβ and Triton UGP β-core were in the tens of pmol/mol creatinine. The S504 β-core concentrations were 10% of those of LH. S504 β-core was strongly correlated with LH, but not with hCG or with free hCGβ (LH, r2 = 0.45; hCG, r2 = 0.26; free hCGβ, r2 = 0.03). The concentrations of β-core detected by the Triton UGP assay, which has a 5% cross-reaction with β-LH-core, were 2% of LH and 5% of the S504 β-core concentrations. Triton UGP values correlated strongly with LH concentrations, but less well with S504 β-core, intact hCG, and free hCGβ (LH, r2 = 0.44; S504 β-core, r2 = 0.33; hCG, r2 = 0.32; free hCGβ, r2 = 0.19).Conclusions: Immunoreactive β-core in women free of malignancies reflects cross-reaction with concentrations of the metabolite of LH, β-LH-core, within the health-related reference interval.
1H-NMR Spectroscopy of Body Fluids: Inborn Errors of Purine and Pyrimidine Metabolism
Authors: Wevers Ron A , Engelke Udo FH , Moolenaar Sytske H , Bräutigam Christa , de Jong Jan GN , Duran Ries , de Abreu Ronney A , van Gennip Albert H 
Published: January 2020

Abstract
Background: The diagnosis of inborn errors of purine and pyrimidine metabolism is often difficult. We examined the potential of 1H-NMR as a tool in evaluation of patients with these disorders.
Methods: We performed 1H-NMR spectroscopy on 500 and 600 MHz instruments with a standardized sample volume of 500 μL. We studied body fluids from 25 patients with nine inborn errors of purine and pyrimidine metabolism.
Results: Characteristic abnormalities could be demonstrated in the 1H-NMR spectra of urine samples of all patients with diseases in the pyrimidine metabolism. In most urine samples from patients with defects in the purine metabolism, the 1H-NMR spectrum pointed to the specific diagnosis in a straightforward manner. The only exception was a urine from a case of adenine phosphoribosyl transferase deficiency in which the accumulating metabolite, 2,8-dihydroxyadenine, was not seen under the operating conditions used. Similarly, uric acid was not measured. We provide the 1H-NMR spectral characteristics of many intermediates in purine and pyrimidine metabolism that may be relevant for future studies in this field.
Conclusion: The overview of metabolism that is provided by 1H-NMR spectroscopy makes the technique a valuable screening tool in the detection of inborn errors of purine and pyrimidine metabolism.
Determination of the Complex between Urokinase and Its Type-1 Inhibitor in Plasma from Healthy Donors and Breast Cancer Patients
Authors: Pedersen Anders N , Brünner Nils , Høyer-Hansen Gunilla , Hamer Peter , Jarosz David , Larsen Birthe , Nielsen Hans Jørgen , Stephens Ross W 
Published: January 2020

AbstractBackground: The complex between urokinase (uPA) and its type-1 inhibitor (PAI-1) is formed exclusively from the active forms of these components; thus, the complex concentration in a biological sample may reflect the ongoing degree of plasminogen activation. Our aim was to establish an ELISA for specific quantification of the uPA:PAI-1 complex in plasma of healthy donors and breast cancer patients.Methods: A kinetic sandwich format immunoassay was developed, validated, and applied to plasma from 19 advanced-stage breast cancer patients, 39 age-matched healthy women, and 31 men.Results: The assay detection limit was &lt;2 ng/L, and the detection of complex in plasma was validated using immunoabsorption, competition, and recovery tests. Eighteen cancer patients had a measurable complex concentration (median, 68 ng/L; range, &lt;16 to 8700 ng/L), whereas for healthy females and males the median signal values were below the detection limit (median, &lt;16 ng/L; range, &lt;16 to 200 ng/L; P &lt;0.0001). For patient plasma, a comparison with total uPA and PAI-1 showed that the complex represented a variable, minor fraction of the uPA and PAI-1 concentrations of each sample.Conclusion: The reported ELISA enables detection of the uPA:PAI-1 complex in blood and, therefore, the evaluation of the complex as a prognostic marker in cancer.
Validation of Accuracy-based Amino Acid Reference Materials in Dried-Blood Spots by Tandem Mass Spectrometry for Newborn Screening Assays
Authors: Chace Donald H , Adam Barbara W , Smith S Jay , Alexander J Richard , Hillman Steven L , Hannon W Harry 
Published: January 2020

AbstractBackground: Advances in technology and the earlier release of newborns from hospitals have pressed the demand for accurate calibration and improved interlaboratory performance for newborn screening tests. As a first step toward standardization of newborn screening aminoacidopathy tests, we have produced six-pool sets of multianalyte dried-blood-spot amino acid reference materials (AARMs) containing predetermined quantities of five amino acids. We describe here the production of the AARMs, validation of their amino acid contents, and characterization of their homogeneity and their stability in storage.Methods: To each of six portions of a pool of washed erythrocytes suspended in serum we added Phe (0–200 mg/L), Leu (0–200 mg/L), Met (0–125 mg/L), Tyr (0–125 mg/L), and Val (0–125 mg/L). Six-pool sets (1300) were prepared, dried, and packaged. We used isotope-dilution mass spectrometry to estimate the endogenous amino acid concentrations of the AARMs and validate their final amino acid concentrations. We used additional tandem mass spectrometry analyses to examine the homogeneity of amino acid distribution in each AARM, and HPLC analyses to evaluate the stability of the amino acid contents of the AARMs.Results: The absolute mean biases across the analytic range for five amino acids were 2.8–9.4%. One-way ANOVAs of the homogeneity results predicted no statistically significant differences in amino acid concentrations within the blood spots or within the pools (P &gt;0.05). Regression slopes (0 ± 0.01) for amino acid concentrations vs storage times and their P values (&gt;0.05) showed no evidence of amino acid degradation at ambient temperatures, 4 °C, or −20 °C during the intervals tested.Conclusion: The validation, homogeneity, and stability of these blood spots support their use as a candidate national reference material for calibration of assays that measure amino acids in dried-blood spots.
Long-Distance PCR-based Screening for Large Rearrangements of the LDL Receptor Gene in Korean Patients with Familial Hypercholesterolemia
Authors: Kim Sung Han , Bae Ji Hyun , Chae Jae Jin , Kim Un Kyung , Choe Seong-Joon , Namkoong Yong , Kim Hyo-Soo , Park Young-Bae , Lee Chung Choo 
Published: January 2020

AbstractBackground: The LDL receptor is a cell-surface protein that regulates plasma cholesterol by specific uptake of LDL particles from the blood circulation. Familial hypercholesterolemia (FH) results from defective catabolism of LDL, which is caused by mutations in the LDL-receptor gene.Methods: For the rapid and reliable detection of large rearrangements in the LDL-receptor gene, we established a screening method based on long-distance PCR as an alternative to Southern-blot hybridization. Using long-distance PCR, 45 unrelated Korean subjects heterozygous for FH were screened to assess the frequency and nature of major structural rearrangements in the LDL-receptor gene.Results: Two different deletion mutations, FH6 (same type as FH3 and FH311) and FH 32, were detected in four families by long-distance PCR. Detailed restriction mapping and sequence analysis showed that FH6 was a 5.71-kb deletion extending from intron 8 to intron 12 and that FH32 was a 2-kb deletion extending from intron 6 to intron 7. Sequence analysis for the breakpoints of all deletions detected in Korean FH patients showed that only the left arms of the Alu repetitive sequences were involved in the deletion event.Conclusions: The screening method based on long-distance PCR provides a powerful strategy for the detection of large rearrangements in the LDL-receptor gene and is a rapid and reliable screening alternative to Southern-blot hybridization.
Characterization of Apolipoprotein E-containing Lipoproteins in Cerebrospinal Fluid: Effect of Phenotype on the Distribution of Apolipoprotein E
Authors: Yamauchi Kazuyoshi , Tozuka Minoru , Hidaka Hiroya , Hidaka Eiko , Kondo Yoshiyuki , Katsuyama Tsutomu 
Published: January 2020

AbstractBackground: Apolipoprotein (apo) E, one of the main apolipoproteins in the central nervous system, may play an important role in lipid metabolism; however, the details of its function are poorly understood. In this study, we characterized apoE-containing lipoproteins in cerebrospinal fluid (CSF) and examined the effect of apoE phenotype on the distribution of apoE among the lipoprotein fractions.Methods: CSF lipoproteins were fractionated by gel filtration and ultracentrifugation, and then characterized by electrophoresis, immunoblot, electron microscopy, and analysis of apoE, total cholesterol, and phospholipid concentrations.Results: The ratio of sialylated to nonsialylated apoE was higher in CSF than in serum. However, the fundamental forms containing apoE homodimers or heterodimers [such as apo(E-AII) and apo(AII-E2-AII) complexes] were similar in CSF and serum. apoE-containing lipoproteins were fractionated at densities of &lt;1.006, 1.063–1.125, and 1.125–1.21 kg/L. Neither apoE nor apoAI was detected in the fraction with a density range of 1.006–1.063 kg/L. The diameters of the lipoprotein particles with densities of &lt;1.006, 1.063–1.125, and 1.125–1.21 kg/L were 16.7 ± 3.1, 14.0 ± 3.2, and 11.6 ± 2.8 nm (mean ± SD, n = 200), respectively. All of these lipoproteins exhibited a spherical structure. The distribution profile of apoE-containing lipoproteins was affected by the apoE phenotype. A relatively large amount of apoE-containing lipoproteins was isolated from the fraction with a density &gt;1.125 kg/L obtained from CSF associated with apoE2 or apoE3. This tendency was more obvious in CSF associated with apoE2 than in CSF without apoE2. apoE-containing lipoproteins were predominantly observed in the fraction with a density of &lt;1.006 kg/L obtained from CSF associated with apoE4.Conclusions: The lipoproteins in CSF have a unique composition that is different from that of the lipoproteins in plasma. However, the differences in diameter between the CSF fractions were not as large as for the serum fractions. Our data suggest that the apoE phenotype may affect the distribution profile of apoE-containing lipoproteins in the CSF. This would mean that the metabolism of apoE-containing lipoproteins depends on the apoE isoform present.
5′ Nuclease Assays for the Loci CCR5-+/Δ32, CCR2-V64I, and SDF1-G801A Related to Pathogenesis of AIDS
Authors: Yuan Chiu-Chin , Peterson Raymond J , Wang Cheng-Dian , Goodsaid Frederico , Waters David J 
Published: January 2020

Abstract
Background: Variations within the human genome play important roles in human disease. To study variations related to susceptibility to AIDS, we have developed 5′ nuclease assays that eliminate post-PCR molecular biology steps.
Methods: TaqMan assays based on the 5′ nuclease activity of Taq polymerase and fluorescent resonance energy transfer were developed to score alleles at the biallelic loci CCR5-+/Δ32, CCR2-V64I and SDF1-G801A. For each assay, 72 samples were analyzed. Data collection and analysis were performed on the Prism 7700 Sequence Detection System. For comparison with gel electrophoresis methods, each locus was also scored on a subset of 24 samples, using restriction enzymes or single-strand conformational polymorphism (SSCP).
Results: Clear allelic discrimination was obtained on each of the 72 samples for all three TaqMan assays. The TaqMan scores for the subset of 24 samples were concordant with the restriction enzyme and SSCP scores.
Conclusions: Because of its simplicity, speed, and potential for automation and miniaturization, TaqMan is an excellent candidate for investigation of genetic variation in clinical, research, and forensic settings.
Serum Procalcitonin Concentrations in Term Delivering Mothers and Their Healthy Offspring: A Longitudinal Study
Authors: Assumma Marcello , Signore Fabrizio , Pacifico Lucia , Rossi Naila , Osborn John F , Chiesa Claudio 
Published: January 2020

Abstract
Background: The reported sensitivities and specificities of procalcitonin (PCT) concentrations for the diagnosis of neonatal infection vary widely. A postnatal increase of PCT has been observed in healthy term newborns with a peak at ≈24 h of age, and many questions remain regarding maternal and perinatal factors that may influence the normal PCT kinetics during the immediate postnatal period.
Methods: We prospectively investigated the association between the serum PCT values obtained from 121 mothers at delivery and serum PCT in their healthy, term offspring at birth as well as at 24 and 48 h of age. We also analyzed whether obstetric and perinatal factors would alter maternal and neonatal PCT response.
Results: PCT concentrations in the babies at birth were significantly higher than in the mothers (P &lt;0.0001), with even larger differences at 24 and 48 h of age. None of the variables identified from maternal and perinatal histories had a significant effect on maternal PCT response. In the healthy neonate, the variables that significantly affected the concentration of PCT at birth were the mothers’ PCT (P &lt;0.01), maternal group B streptococcus colonization (P &lt;0.05), and rupture of membranes ≥18 h (P &lt;0.01). The coefficient of linear correlation between the mother’s PCT concentration and that of the baby at birth was 0.32 (P &lt;0.01). The only variable that significantly altered the PCT concentration at both 24 (P &lt;0.01) and 48 (P &lt;0.01) h of age was rupture of membranes ≥18 h. Nonetheless, the PCT response observed during the 48-h period after birth among healthy babies born to mothers with risk factors for infection was well below that reported previously among age-matched neonates with sepsis.
Conclusions: The postnatal increase of PCT observed in the healthy neonate with peak values at 24 h of age most likely represents endogenous synthesis. In estimating the sensitivities and specificities of PCT for diagnosis of sepsis throughout the initial 48 h of life, it is important to consider the normal PCT kinetics and the pattern(s) of PCT response in the healthy neonate.
Determination of Apolipoprotein B-48 in Plasma by a Competitive ELISA
Authors: Lorec Anne-Marie , Juhel Christine , Pafumi Yan , Portugal Henri , Pauli Anne-Marie , Lairon Denis , Defoort Catherine 
Published: January 2020

AbstractBackground: Apolipoprotein B-48 (apoB-48) is produced by the small intestine, as part of chylomicrons, and appears to be a suitable marker for clinical studies of postprandial lipoproteins and related cardiovascular risk. Our aim was to develop, for routine analysis, an assay to quantify apoB-48 in plasma samples.Methods: A microtiter plate was coated with a C-terminal apoB-48-specific heptapeptide. Plasma samples were incubated with appropriate detergent to allow competition between immobilized antigen and plasma apoB-48. Appropriate calibration curves were obtained in the ELISA, using calibrated lymph and chylomicrons.Results: Treatment of plasma samples with the mild detergent Triton X-100 allowed an efficient competition between immobilized antigen and plasma apoB-48. No cross-reactivity was found with apoB-100, as checked by ELISA and Western blot analysis. Intra- and interassay CVs were 5.4% and 5.5%, respectively. In healthy subjects, apoB-48 concentrations markedly increased in the postprandial state, in parallel with triglycerides.Conclusions: This new ELISA allows determination of the concentration of apoB-48 in normolipidemic plasma.
Sensitivity and Specificity of Immunological Methods for the Detection of Anti-Topoisomerase I (Scl70) Autoantibodies: Results of a Multicenter Study
Authors: Bizzaro Nicola , Tonutti Elio , Villalta Danilo , Bassetti Danila , Tozzoli Renato , Manoni Fabio , Pirrone Stefano , Piazza Anna , Rizzotti Paolo , Pradella Marco ,   for The Italian Society of Laboratory Medicine Study Group on the Diagnosis of Autoimmune Diseases
Published: January 2020

AbstractBackground: The ability of immunometric methods to identify anti-topoisomerase I (Scl70) antibodies is controversial. We wished to quantify the performance of the currently available commercial systems for the assay of anti-topoisomerase I antibodies in a large multicenter study.Methods: Fifty Italian clinical laboratories analyzed 36 serum samples: 27 from individuals with scleroderma/systemic sclerosis, and 9 from a control group. The scleroderma/systemic sclerosis samples were positive in our laboratories by both ELISA and immunoblot (IB), and the control samples were negative. The laboratories used 42 immunoenzymatic (ELISA), 21 IB, 3 counterimmunoelectrophoresis, and 2 dot-blot methods, produced by 23 different manufacturers.Results: We obtained 2389 results. The ELISA methods showed 99.2% specificity and 97.2% sensitivity for detection of anti-Scl70 antibodies. For IB methods, specificity was 97.6% and sensitivity was 96.1%. The Western-blot method had poor analytical specificity (27% false positives for anti-extractable nuclear antigen antibodies other than anti-Scl70).Conclusions: Excluding Western blots, commercial ELISA and IB reagents as used in clinical laboratories have a sensitivity and a specificity &gt;95% for determination of anti-Scl70 antibodies.
A Golden Age of Clinical Chemistry: 1948–1960
Authors: Rosenfeld Louis 
Published: January 2020

AbstractThis segment of history aims to inform the new, and remind the not-so-new, members of the profession about the relatively recent period that initiated the dominant role played by technological innovation in the modern investigation of disease. The 12 years from 1948 to 1960 were notable for introduction of the Vacutainer tube, electrophoresis, radioimmunoassay, and the AutoAnalyzer. Also appearing during this interval were new organizations, publications, programs, and services that established a firm foundation for the professional status of clinical chemists. It was a golden age.