bluuebunny/crossref_metadata_2025_split huggingface dataset:
Capillary Electrophoresis-based Heteroduplex Analysis with a Universal Heteroduplex Generator for Detection of Point Mutations Associated with Rifampin Resistance in Tuberculosis
Authors: Thomas Gloria A , Williams Diana L , Soper Steven A 
Published: January 2020

Abstract
Background: Slab gel heteroduplex analysis (HDA), a popular scanning method for genetic mutations, uses DNA fragments typically generated by PCR to create homo- and heteroduplex molecules with conformational differences and sequence-dependent electrophoretic profiles. Use of a universal heteroduplex generator (UHG) enhances the subtle variations caused by single-base substitutions.
Methods: The HDA-UHG slab gel format was modified for an efficient capillary-based method. The effect of staining dyes TOPRO5 and YOPRO1 on the analysis of heteroduplexes was studied, as well as ultraviolet absorbance and laser-induced fluorescence (LIF) detection methods. In addition, the entangled polymers hydroxyethyl cellulose, methyl cellulose, and linear polyacrylamide were evaluated as separation matrices.
Results: This assay was able to detect the presence of Mycobacterium tuberculosis and its rifampin susceptibility directly from clinical specimens in dramatically reduced analysis time (30 min vs 2.5 h). Optimized conditions included 0.3% methyl cellulose as the separation matrix, on-line staining using 1 μmol/L YOPRO1, and LIF detection for quantitative and reproducible analysis of single-base substitutions in the rifampin resistance-determining region of rpoB that give rise to the rifampin-resistant phenotype of M. tuberculosis. We generated 95% confidence limits using the wild-type sequence and used these limits to determine rifampin susceptibility in samples.
Conclusions: Capillary electrophoresis, combined with the HDA-UHG technique, may be of value for rapid and efficient clinical diagnosis of rifampin-resistant tuberculosis strains.
Interpretation of Hemoglobin A1c (HbA1c) Values among Diabetic Patients
Authors: Skeie Svein , Thue Geir , Sandberg Sverre 
Published: January 2020

Abstract
Background: Few studies have examined patients’ views, knowledge, and understanding of glycohemoglobin A1c (HbA1c) testing. We explored such issues in patients with type 1 diabetes and used their statements to estimate analytical quality specifications for HbA1c testing.
Methods: We recruited 201 patients from a hospital outpatient clinic. A questionnaire was used to collect information on diabetes characteristics, perceived knowledge of HbA1c, last HbA1c value, HbA1c target value, and thresholds for action. Patients were asked to indicate the magnitude of change in HbA1c from 9.4% that they would consider to be a true (real) change; from their responses, we calculated patient-derived quality specifications for HbA1c.
Results: Fifty-eight percent of the patients felt they had “high” knowledge about HbA1c, and &gt;80% of responders knew their last HbA1c value, their target HbA1c, and the threshold value of HbA1c for treatment intensification. The mean acceptable HbA1c value was 7.5%. Patients with lower values on their most recent tests reported lower target values for HbA1c and lower values for the upper HbA1c threshold for treatment intensification. An analytical CV (CVa) of 3.1% would be satisfactory for 75% of patients when HbA1c is increasing (80% confidence), and a CVa of 3.2% would be satisfactory for 75% when HbA1c is decreasing (95% confidence).
Conclusions: Type 1 patients’ perceived knowledge about HbA1c testing is high. They are well informed about their own personal results and about target values and the upper HbA1c threshold for action. The patient-derived analytical quality specification for imprecision (CV) is 3.1%.
Quantitative Determination of Estradiol Fatty Acid Esters in Human Pregnancy Serum and Ovarian Follicular Fluid
Authors: Vihma Veera , Adlercreutz Herman , Tiitinen Aila , Kiuru Paula , Wähälä Kristiina , Tikkanen Matti J 
Published: January 2020

AbstractBackground: Lipophilic estradiol derivatives carried by lipoprotein particles in blood may mediate antioxidant or endocrine effects. We developed a new quantitative method to determine the concentration of circulating lipophilic estradiol fatty acid esters in human early- and late-pregnancy serum and in ovarian follicular fluid.Methods: After extraction from serum or follicular fluid, estradiol fatty acid esters were separated from nonesterified estradiol by Sephadex LH-20 column chromatography. The estradiol ester fraction was hydrolyzed by saponification and further purified by several chromatographic steps. The hydrolyzed estradiol esters were measured by time-resolved fluoroimmunoassay.Results: The average estradiol fatty acid ester concentration in serum increased 10-fold during pregnancy, from 40.4 pmol/L (expressed as pmol/L estradiol; range, 25.0–64.2 pmol/L) in early pregnancy (n = 8) to 404 pmol/L (196–731 pmol/L) in late pregnancy (n = 10). The ratio of estradiol ester to nonesterified estradiol remained relatively constant during pregnancy, at 0.4–0.6%. In 10 follicular fluid samples, the mean estradiol ester concentration was 106 nmol/L (56.9–262 nmol/L). Compared with serum, a greater proportion of estradiol in follicular fluid (3.0–10%) was in the esterified form.Conclusion: The new method provides a means to measure circulating estradiol fatty acid ester concentrations in human pregnancy serum.
Sensitive and Rapid Quantification of Busulfan in Small Plasma Volumes by Liquid Chromatography–Electrospray Mass Spectrometry
Authors: Mürdter Thomas E , Coller Janet , Claviez Alexander , Schönberger Frank , Hofmann Ute , Dreger Peter , Schwab Matthias 
Published: January 2020

Abstract
Background: High-dose busulfan is widely used in conditioning regimens before hematopoietic stem cell transplantation in both adults and children. Large interindividual variability in pharmacokinetics after oral administration has been reported; therefore, therapeutic drug monitoring of busulfan may decrease the incidence of drug-related toxicity (for example, hepatic venoocclusive disease) and may also improve therapeutic efficacy.
Methods: Busulfan concentrations were quantified using 200 μL of plasma and liquid–liquid extraction with diethyl ether after the addition of [2H8]busulfan as the internal standard. Separation and detection of busulfan and [2H8]busulfan were achieved with a LUNA C8 column (5 μm; 150 × 2 mm i.d.) at 30 °C, a HP 1100 liquid chromatography system, and a HP 1100 single-quadrupole mass spectrometer. Busulfan and [2H8]busulfan were detected as ammonium adducts in selected-ion monitoring mode at m/z 264.2 and 272.2, respectively.
Results: The calibration curve was linear at 5–2000 μg/L busulfan. Intra- and interassay imprecision (CV) and bias were both &lt;11%. The limits of detection and quantification were 2 and 5 μg/L, respectively. Extraction recovery of busulfan was &gt;87%. Analysis of pharmacokinetics in four patients receiving high-dose busulfan indicated that minimum busulfan concentrations before the next dose were 405–603 μg/L, with no interference observed.
Conclusions: The new rapid and sensitive liquid chromatographic–mass spectrometric assay is an appropriate method for quantification of busulfan in human plasma, making therapeutic drug monitoring of busulfan faster and easier in clinical practice.
Laboratory-related Outcomes in Healthcare
Authors: Bruns David E 
Published: January 2020

Abstract
Outcomes studies, long common on the therapeutic side of medicine, are appearing in the diagnostic arena. Outcomes can be defined as results of medical interventions (therapies or tests) in terms of health or cost. The studies of outcomes are important because funding for medical interventions increasingly depends on them; a major accrediting agency even defines “quality” entirely in terms of outcomes.
The study of laboratory-related outcomes is complex. Multiple steps occur between testing and outcomes, physicians act unpredictably on test results, and outcomes studies have high costs relative to potential profit from the test. Study design often must specify the action that is to follow a test result.
The model outcomes study is a randomized controlled trial (RCT). The CONSORT statement, which is used as a guideline for RCTs of therapies, is largely applicable to studies of diagnostic interventions. Recent laboratory-related RCTs have addressed questions such as: “Does routine testing before cataract surgery decrease morbidity or mortality?” and “Does fecal occult bleed testing decrease the incidence of colorectal cancer?”
RCTs of tests are sometimes impractical. Other approaches include simulation modeling and the use of intervention and control periods of testing. As for RCTs, these approaches require careful attention to study design, data analysis, and interpretation and reporting of results.
Evolution of Methods for Measurement of HDL-Cholesterol: From Ultracentrifugation to Homogeneous Assays
Authors: Warnick G Russell , Nauck Matthias , Rifai Nader 
Published: January 2020

AbstractBackground: Adoption of automated homogeneous assays for HDL-cholesterol (HDL-C) is increasing, driven by the need of clinical laboratories to cope with increasing workloads while containing costs. However, performance characteristics of homogeneous assays often differ in important aspects from those of the earlier precipitation methods. This review provides an overview of the new generation of homogeneous assays for HDL-C within the historical context of the evolution of methods and the efforts to standardize measurements of the lipoproteins.Approach: This is a narrative review based on method evaluations conducted in the laboratories of the authors as well as on relevant publications, especially comparative evaluation studies, from the literature. Publications considered here have been collected by the authors over the past 30 years of involvement as methods for HDL-C made the transition from their early use in lipid research laboratories to clinical laboratories and the recent emergence of homogeneous assays.Content: The presentation includes descriptions of methodologies, including homogeneous, precipitation, electrophoresis, and ultracentrifugation assays. Reference methods and recommended approaches for assessing accuracy are described. Accuracy and imprecision are summarized in the context of the National Cholesterol Education Program (NCEP) standards for analytical performance. The effects of interfering substances and preanalytical sources of variation are presented.Summary: Homogeneous assays have been shown to be reasonably well suited for use in routine clinical laboratories, generally meeting the NCEP criteria for precision, accuracy, and total error. However, discrepant results compared with the reference methods have been observed with some of the assays, and the sources of discrepancies are not well characterized. Some homogeneous reagents have not been thoroughly evaluated. At least three of the reagents have experienced successive adjustments in formulation; hence, the reagents may not yet be fully optimized. For these reasons, the homogeneous assays cannot be confidently recommended for use in long-term clinical trials and other research applications without thorough validation.
Effects of Blood-Processing Protocols on Fetal and Total DNA Quantification in Maternal Plasma
Authors: Chiu Rossa W K , Poon Leo L M , Lau Tze K , Leung Tse N , Wong Eric M C , Lo Y M Dennis 
Published: January 2020

Abstract
Background: Recently, apoptotic cells have been found in plasma obtained by centrifugation of blood from pregnant women, raising the question of what constitutes plasma and whether plasma is truly cell free. We compared the effects of different blood-processing protocols on the quantification, DNA composition, and day-to-day fluctuation of fetal and total DNA in maternal plasma.
Methods: Blood samples were collected from healthy pregnant women. The blood sample from each individual was simultaneously processed by different means, including the following: Percoll separation, centrifugation, microcentrifugation, and filtration. The resulting plasma aliquots were subjected to real-time quantitative amplification of the β-globin (for total DNA) and SRY (for fetal DNA) genes. The differences in the β-globin and SRY DNA concentrations and the degree of variation between the various plasma aliquots were assessed statistically.
Results: Different protocols of blood processing significantly affected the quantification and the day-to-day fluctuation of total (P &lt;0.001), but not fetal (quantification, P = 0.336; fluctuation, P = 0.206), DNA in maternal plasma. The quantitative difference could be attributed to the fact that efficacies of different protocols for generating cell-free plasma vary. Processing blood samples by centrifugation followed by filtration or microcentrifugation is effective in producing cell-free plasma.
Conclusions: Standardization in plasma-processing protocols is needed for maternal plasma DNA analysis, especially for quantification of total DNA in maternal plasma. Such preanalytic factors may also affect other applications of plasma DNA analysis.
Association of Increased Ferritin with Premature Coronary Stenosis in Men
Authors: Haidari Mehran , Javadi Ebrahim , Sanati Arashmidos , Hajilooi Mehrdad , Ghanbili Jafar 
Published: January 2020

Abstract
Background: Body iron status has been implicated in atherosclerotic cardiovascular disease. The main hypothesis is that high iron status is associated with increased oxidation of LDL. We investigated the potential role of ferritin as an additional risk factor promoting atherosclerosis among a young population with coronary artery disease (CAD).
Methods: Four hundred consecutive patients (218 males, 182 females) referred for diagnostic coronary angiography were examined, and risk factors for CAD, lipids, C-reactive protein (CRP), and ferritin concentrations were recorded for all participants.
Results: Ferritin was higher in the male patients with CAD (121 μg/L; range, 56–258 μg/L) than in the men without significant CAD (73 μg/L; range, 32–138 μg/L; P &lt;0.002). Multiple logistic regression analysis, after adjustment for the established coronary risk factors, showed ferritin as an independent discriminating risk factor for CAD (P &lt;0.01). Men in the highest quartile of ferritin had an odds ratio (OR) of 1.62 [95% confidence interval (95% CI), 1.12–2.42; P &lt;0.01] compared with men in the lowest quartile of ferritin. The association between ferritin and CAD was more pronounced in male patients ≤50 years (OR = 2.65; 95% CI, 1.35–5.51; P &lt;0.003). Ferritin was significantly higher in diabetic male patients in comparison with nondiabetic male patients [168 μg/L (range, 74–406 μg/L) vs 106 μg/L (range, 44–221 μg/L), respectively; P &lt;0.002]. No association was observed between ferritin and CAD among the female patients.
Conclusion: Our data suggest that increased ferritin might be an independent predictor of premature CAD in male Iranian patients.
Nitrogen Metabolism and Bone Metabolism Markers in Healthy Adults during 16 Weeks of Bed Rest
Authors: Scheld Kerstin , Zittermann Armin , Heer Martina , Herzog Birgit , Mika Claudia , Drummer Christian , Stehle Peter 
Published: January 2020

Abstract
Background: The associations between nitrogen metabolism and bone turnover during bed rest are still not completely understood.
Methods: We measured nitrogen balance (nitrogen intake minus urinary nitrogen excretion) and biochemical metabolic markers of calcium and bone turnover in six males before head-down tilt bed rest (baseline), during 2, 10, and 14 weeks of immobilization, and after reambulation.
Results: The changes in nitrogen balance were highest between baseline and week 2 (net change, −5.05 ± 1.30 g/day; 3.6 ± 0.6 g/day at baseline vs −1.45 ± 1.3 g/day at week 2; P&lt;0.05). In parallel, serum intact osteocalcin (a marker of bone formation) was already reduced and renal calcium and phosphorus excretions were increased at week 2 (P &lt;0.05). Fasting serum calcium and phosphorus values and renal excretion of N-telopeptide (a bone resorption marker) were enhanced at weeks 10 and 14 (P &lt;0.05–0.001), whereas serum concentrations of parathyroid hormone, calcitriol, and type I collagen propeptide (a marker of bone collagen formation) were decreased at week 14 (P &lt;0.05–0.01). Significant associations were present between changes of serum intact osteocalcin and 24-h calcium excretion (P &lt;0.001), nitrogen balance and 24-h phosphorus excretion (P &lt;0.001), nitrogen balance and renal N-telopeptide excretion (P &lt;0.05), and between serum osteocalcin and nitrogen balance (P &lt;0.025).
Conclusions: Bone formation decreases rapidly during immobilization in parallel with a higher renal excretion of intestinally absorbed calcium. These changes appear in association with the onset of a negative nitrogen balance, but decreased bone collagen synthesis and enhanced collagen breakdown occur after a time lag of several weeks.
Telomerase Detection in Body Fluids
Authors: Hess Jennifer L , Highsmith W Edward 
Published: January 2020

Abstract
Background: Telomerase is a ribonucleoprotein that maintains chromosomal telomere length. Telomerase is not active in nonmalignant somatic cells, but is activated in most human cancers. Telomerase activity in easily obtainable body fluids that bathe tumors may be a useful cancer marker, especially when used in conjunction with conventional cytology.
Approach: Results from studies that assayed telomerase activity in easily obtainable body fluids are reviewed.
Content: The telomerase repeat amplification protocol (TRAP) assay has been used to measure telomerase activity in body fluids, including ascites, pleural effusions, pelvic washes, bronchial washings, bronchial lavage, urine, bladder washings, oral rinses, and plasma. Telomerase activity has sensitivities of 60–90% as a tumor marker with clinical specificities for cancer of ∼90%. Telomerase activity is more sensitive than conventional cytology, the sensitivity of which was 40–65% in various studies.
Summary: Telomerase activity in body fluids, as measured by the TRAP assay, is a sensitive potential tumor marker that might help increase the cancer detection rate and the cancer treatment success rate when combined with conventional cytology.
Stability of Endogenous and Added RNA in Blood Specimens, Serum, and Plasma
Authors: Tsui Nancy BY , Ng Enders KO , Lo YM Dennis 
Published: January 2020

Abstract
Background: Circulating RNA in plasma/serum is an emerging field for noninvasive molecular diagnosis. Because RNA is widely thought to be labile in the circulation, we investigated the stability and various preanalytical factors that may affect RNA concentrations in blood specimens.
Methods: Blood samples were collected from 65 healthy volunteers. The effects of two preanalytical variables were studied: (a) time delay in processing of EDTA blood and clotted blood after venesection, and (b) freezing and thawing of plasma and serum. The lability of free added RNA in plasma was also investigated. Plasma/serum RNA was measured by a real-time quantitative reverse transcription-PCR assay for glyceraldehyde 3-phosphate dehydrogenase mRNA, whereas DNA was measured by a real-time quantitative PCR assay for the β-globin gene.
Results: No significant difference was found for plasma RNA concentrations obtained from uncentrifuged EDTA blood that had been left at 4 °C for 0, 6, and 24 h (P =0.182). On the other hand, the serum RNA concentrations increased significantly over 24 h when uncentrifuged clotted blood was stored at 4 °C (P &lt;0.05). In comparison, &gt;99% of the free added RNA could no longer be amplified after incubation in plasma for 15 s. Never-frozen plasma, freeze-thawed plasma, and thawed plasma left at room temperature for 1 h showed no significant differences in RNA concentration (P =0.465). No significant difference was observed for freeze-thawed serum (P = 0.430).
Conclusions: Plasma RNA is stable in uncentrifuged EDTA blood stored at 4 °C, but to obtain a stable serum RNA concentration, uncentrifuged clotted blood should be stored at 4 °C and processed within 6 h. A single freeze/thaw cycle produces no significant effect on the RNA concentration of plasma or serum.
Comprehensive Scanning of the Entire Mitochondrial Genome for Mutations
Authors: Wong Lee-Jun C , Liang Min-Hui , Kwon Haeyoung , Park John , Bai Ren-Kui , Tan Duan-Jun 
Published: January 2020

Abstract
Background: Definitive molecular diagnosis of mitochondrial disorders has been greatly hindered by the tremendous clinical and genetic heterogeneity, the heteroplasmic condition of pathogenic mutations, and the presence of numerous homoplasmic mitochondrial DNA (mtDNA) variations with unknown significance. We used temporal temperature gradient gel electrophoresis (TTGE) to detect heteroplasmic mutations from homoplasmic variations in the whole mitochondrial genome.
Methods: We screened 179 unrelated patients by TTGE with use of 32 overlapping primer pairs. Mutations were identified by direct sequencing of the PCR products and confirmed by PCR with allele-specific oligonucleotide or restriction fragment length polymorphism analysis.
Results: We detected 71 heteroplasmic and 647 homoplasmic banding patterns. Sequencing of the heteroplasmic fragments identified 68 distinct novel mutations and 132 reported sequence variations and mutations; most of them occurred only once. The deleterious nature of some of the novel mutations was established by analyzing the asymptomatic family members and the biochemical and molecular characteristics of the mutation. When the number of mutations was normalized to the size of the region, the occurrence of mutations was 2.4 times more frequent in the tRNA genes than in the mRNA (protein coding) regions.
Conclusions: Screening by TTGE detects low proportions of mutant mtDNA and distinguishes heteroplasmic from homoplasmic variations. Results from comprehensive molecular analysis should be followed up with clinical correlation to establish a guideline for complete mutational analysis of the entire mitochondrial genome and to facilitate the diagnosis of mitochondrial disorders.
Simple Method for Quantification of Bence Jones Proteins
Authors: Salomo Morten , Gimsing Peter , Nielsen Lars B 
Published: January 2020

AbstractBackground: Quantification of free monoclonal light chains in urine [Bence Jones proteins (BJPs)] is used to diagnose multiple myeloma and to evaluate response to treatment. We have developed and evaluated an optimized approach for quantification of BJPs.Methods: High-resolution gel electrophoresis of unconcentrated urine and albumin calibrators was carried out on Sebia’s Hydrasys instrument with Hydragel HR agarose gels. After staining with acid violet, the gels were scanned densitometrically. The staining intensities of BJP bands relative to the staining intensities of albumin solutions were used to determine the BJP concentrations. Results for patient samples were compared with conventional agarose gel electrophoresis on concentrated samples.Results: The relationships between staining intensity and the protein concentrations of albumin and BJPs were linear up to protein concentrations of ∼2000 mg/L. The detection limit was ∼20 mg/L. The interassay imprecision (CV) was ∼8% (n = 23, duplicate analysis), and the results (y) showed a close positive relationship to the comparison method: slope = 0.82 (confidence interval, 0.75-0.88); y-intercept = 34 (−14 to 81) mg/L; n = 29; r2 = 0.96.Conclusions: Agarose gel electrophoresis of unconcentrated urine samples together with a series of albumin calibrators followed by acid violet staining and densitometric scanning is sufficiently reproducible and sensitive to quantify clinically relevant BJPs.