bluuebunny/crossref_metadata_2025_split huggingface dataset:
Improved Immunoradiometric Assay for Plasma Renin
Authors: Deinum Jaap , Derkx Frans HM , Schalekamp Maarten ADH 
Published: January 2020

AbstractBackground: Our renin IRMA overestimated renin in plasmas with high prorenin-to-renin ratios. We suspected that the overestimation of renin was caused less by cross-reactivity of the renin-specific antibody with prorenin than by a conformational change of prorenin into an enzymatically active form during the assay.Methods: Because the inactive form of prorenin converts slowly into an active form at low temperature, we raised the assay temperature from 22 °C to 37 °C, simultaneously shortening the incubation time from 24 to 6 h. The former IRMA was performed in &lt;1 working day with these modifications.Results: The comeasurement of prorenin as renin was eliminated. Reagents were stable at 37 °C, and the new and old IRMAs were comparable in terms of precision and accuracy. The functional lower limit of the assay (4 mU/L) was below the lower reference limit (9 mU/L). The modified IRMA agreed closely with the activities measured with an enzyme-kinetic assay. Results were not influenced by the plasma concentration of angiotensinogen. At normal angiotensinogen concentrations, the IRMA closely correlated with the classical enzyme-kinetic assay of plasma renin activity.Conclusion: The modified IRMA, performed at 37 °C, avoids interference by prorenin while retaining the desirable analytical characteristics of the older IRMA and requiring less time.
Quantification of Riboflavin, Flavin Mononucleotide, and Flavin Adenine Dinucleotide in Human Plasma by Capillary Electrophoresis and Laser-induced Fluorescence Detection
Authors: Hustad Steinar , Ueland Per Magne , Schneede Jørn 
Published: January 2020

AbstractBackground: Riboflavin is the precursor of flavin mononucleotide (FMN) and FAD, which serve as cofactors for several redox enzymes. We have developed a capillary electrophoresis method for the determination of riboflavin and its two coenzyme forms in human plasma.Methods: Trichloroacetic acid-treated plasma was subjected to solid-phase extraction on reversed-phase columns. The analytes were separated by micellar electrokinetic capillary chromatography in uncoated fused- silica capillaries filled with borate buffer containing 50 mmol/L sodium dodecyl sulfate, methanol, and N-methylformamide. Native fluorescence was monitored at 530 nm, using an argon laser operating at 488 nm as excitation source.Results: The assay was linear over a concentration range of two orders of magnitude, and the limit of detection was far below physiological concentrations for all vitamers. The within-day and between-day coefficients of variation were 4–9% and 6–12%, respectively. The reference values (median, 5–95 percentiles) obtained by analyzing plasma from 63 healthy subjects were 8.6 nmol/L (2.7–42.5 nmol/L) for riboflavin, 7.0 nmol/L (3.5–13.3 nmol/L) for FMN, and 57.9 nmol/L (44.5–78.1 nmol/L) for FAD.Conclusions: Capillary electrophoresis with laser-induced fluorescence detection allows determination of all riboflavin vitamers far below physiological concentrations. The method may become a useful tool for the assessment of riboflavin status in humans.
Degradation of Cardiac Troponin I in Serum Complicates Comparisons of Cardiac Troponin I Assays
Authors: Shi Qinwei , Ling Mingfu , Zhang Xiaochen , Zhang Minyuan , Kadijevic Lilly , Liu Shigui , Laurino Joseph P 
Published: January 2020

AbstractBackground: Up to a 20-fold variation in serum cardiac troponin I (cTnI) concentration may be observed for a given patient sample with different analytical methods. Because more limited variation is seen for control materials and for purified cTnI, we explored the possibility that cTnI was present in altered forms in serum.Methods: We used four recombinantly engineered cTnI fragments to study the regions of cTnI recognized by the Stratus®, Opus®, and ACCESS® immunoassays. The stability of these regions in serum was analyzed with Western blot.Results: The measurement of several control materials and different forms of purified cTnI using selected commercial assays demonstrated five- to ninefold variation. Both the Stratus and Opus assays recognized the N-terminal portion (NTP) of cTnI, whereas the ACCESS assay recognized the C-terminal portion (CTP) of cTnI. Incubation of recombinant cTnI in normal human serum produced a marked decrease in cTnI concentration as determined with the ACCESS, but not the Stratus, immunoassay. Western blot analysis of the same samples using cTnI NTP- and CTP-specific antibodies demonstrated preferential degradation of the CTP of cTnI.Conclusions: The availability of serum cTnI epitopes is markedly affected by the extent of ligand degradation. The N-terminal half of the cTnI molecule was found to be the most stable region in human serum. Differential degradation of cTnI is a key factor in assay-to-assay variation.
Adulteration of Urine by “Urine Luck”
Authors: Wu Alan HB , Bristol Ben , Sexton Karen , Cassella-McLane Gina , Holtman Verena , Hill Dennis W 
Published: January 2020

Abstract
Background: In vitro adulterants are used to invalidate assays for urine drugs of abuse. The present study examined the effect of pyridinium chlorochromate (PCC) found in the product “Urine Luck”.
Methods: PCC was prepared and added to positive urine controls at concentrations of 0, 10, 50, and 100 g/L. The controls were assayed for methamphetamine, benzoylecgonine (BE), codeine and morphine, tetrahydrocannabinol (THC), and phencyclidine (PCP) with the Emit II (Syva) and Abuscreen Online (Roche) immunoassays, and by gas chromatography/mass spectrometry (GC/MS). Two tests were also developed to detect PCC in urine: a spot test to detect chromate ions using 10 g/L 1,5-diphenylcarbazide as the indicator, and a GC/MS assay for pyridine. We tested 150 samples submitted for routine urinalysis, compliance, and workplace drug testing for PCC, using these assays.
Results: Response rates decreased at 100 g/L PCC for all Emit II drug assays and for the Abuscreen morphine and THC assays. In contrast, the Abuscreen amphetamine assay produced apparently higher results, and no effect was seen on the results for BE or PCP. The PCC did not affect the GC/MS recovery of methamphetamine, BE, PCP, or their deuterated internal standards, but decreased GC/MS recovery of the opiates at both intermediate (50 g/L) and high (100 g/L) PCC concentrations and apparent concentrations of THC and THC-d3 at all PCC concentrations. Two of 50 samples submitted for workplace drug testing under chain-of-custody conditions were positive for PCC, whereas none of the remaining 100 specimens submitted for routine urinalysis or compliance drug testing were positive.
Conclusions: PCC is an effective adulterant for urine drug testing of THC and opiates. Identification of PCC use can be accomplished with use of a spot test for the oxidant.
National Academy of Clinical Biochemistry Standards of Laboratory Practice: Recommendations for the Use of Cardiac Markers in Coronary Artery Diseases
Authors: Wu Alan HB , Apple Fred S , Gibler W Brian , Jesse Robert L , Warshaw Myron M , Valdes Roland 
Published: January 2020

AbstractThe Sixth Conference on the “Standards of Laboratory Practice Series”, sponsored by the National Academy of Clinical Biochemistry (NACB), was held on August 4–5, 1998, at the Annual Meeting of the American Association for Clinical Chemistry, in Chicago, IL. An expert committee was assembled to write recommendations on the use of cardiac markers in coronary artery diseases. The NACB Committee prepared a preliminary draft of the guidelines, made them available on the World Wide Web (www.nacb.org), and distributed them before the presentations. The recommendations were divided into four areas: the use of markers in the triage of patients with chest pain, acute coronary syndromes, clinical applications other than acute myocardial infarction and research, and assay platforms and markers of acute myocardial infarction. The recommendations were revised and subsequently re-presented in part at the “Biomarkers in Acute Cardiac Syndromes Conference”, sponsored by the Jewish Hospital Heart and Lung Institute, Louisville KY, on October 16–17, 1998. This report lists each recommendation, its scientific justification, and a summary of discussions from conference participants and reviewers.Approximately 100 individuals responded to various versions of these recommendations via direct correspondences, telephone calls to Committee members, electronic mail correspondence to the Committee Chairman, or oral questions and comments raised during one of the two conference presentations. Some of the recommendations were changed to reflect the consensus opinion. In cases in which there was no consensus, the Committee included pertinent discussion without necessarily changing the original recommendations. At times, the Committee members felt that although a particular recommendation might not be the current standard of care today, they anticipate that it likely will be adopted in the near future.
Detection of Five Rare Cystic Fibrosis Mutations Peculiar to Southern Italy: Implications in Screening for the Disease and Phenotype Characterization for Patients with Homozygote Mutations
Authors: Castaldo Giuseppe , Fuccio Antonella , Cazeneuve Cécile , Picci Luigi , Salvatore Donatello , Raia Valeria , Scarpa Maurizio , Goossens Michel , Salvatore Francesco 
Published: January 2020

Abstract
Background: The search for the eight most frequent mutations (i.e., ΔF508, G542X, W1282X, N1303K, 1717-1G→A, R553X, 2183AA→G, and I148T) by allele-specific oligonucleotide dot-blot analysis revealed 78% of 396 cystic fibrosis alleles in Southern Italy. The observation of frequent haplotypes on the unidentified cystic fibrosis alleles suggested that a few mutations could account for a large number of unidentified alleles.
Methods: We screened most of the coding sequence of the cystic fibrosis transmembrane regulator gene by denaturing gradient gel electrophoresis to determine the spectrum of these mutations in 68 unrelated cystic fibrosis patients bearing one or both unidentified mutations.
Results: The screening revealed five mutations, R1158X, 711+1G→T, 4016insT, L1065P, and G1244E, each of which had a frequency of 1.3–1.8% (7% collectively). The 7% increase in the detection rate (85% vs 78%) reduces by &gt;50% the residual risk of being cystic fibrosis carriers for couples who had tested negative by molecular analysis. We therefore designed a second allele-specific oligonucleotide set to analyze the five mutations. Among the patients analyzed, one patient homozygous for the L1065P mutation expressed a mild pulmonary and intestinal form of the disease with pancreatic insufficiency. Two other patients, homozygous for mutations R1158X and 4016insT, both expressed a severe cystic fibrosis phenotype.
Conclusions: Five cystic fibrosis mutations are peculiar to patients from Southern Italy. The method described for their analysis is efficient, inexpensive, and can be semi-automated by use of a robotic workstation. The results obtained in patients from Southern Italy may have an impact on laboratories in other countries, given the large migrations of populations from Southern Italy to other countries in the last two centuries.
BRCA1 Gene Mutations in Sporadic Ovarian Carcinomas: Detection by PCR and Reverse Allele-specific Oligonucleotide Hybridization
Authors: Tong Dan , Stimpfl Margit , Reinthaller Alexander , Vavra Norbert , Müllauer-Ertl Silvia , Leodolter Sepp , Zeillinger Robert 
Published: January 2020

Abstract
Background: Although germline mutations in BRCA1 play a central role in familial breast and ovarian cancers, to date, no somatic mutations in BRCA1 have been reported in sporadic breast cancer, and only five somatic mutations have been identified in the sporadic ovarian carcinomas. Because loss of heterozygosity appears frequently at the BRCA1 locus in nonfamilial breast and ovarian carcinomas, we searched for mutations in the BRCA1 gene in sporadic ovarian tumors.
Methods: We developed a detection system based on PCR and reverse allele-specific oligonucleotide hybridization on membrane strips for the simultaneous detection of 17 frequently occurring mutations in the BRCA1 gene.
Results: As little as 2% mutant DNA in a sample could be detected. Two of 122 DNA samples isolated from sporadic ovarian tumor biopsies contained the Cys61Gly mutation. Both mutations were germline mutations. One of these was an ovarian metastasis of a primary fallopian tube carcinoma. The tubal carcinoma was also confirmed to contain the Cys61Gly mutation.
Conclusions: This is the first report that a germline BRCA1 mutation is associated with primary tubal carcinoma. The 17 specific mutations in the BRCA1 gene do not play a major role in the tumorigenesis and progression of sporadic ovarian cancer.
Optimal Temperature Selection for Mutation Detection by Denaturing HPLC and Comparison to Single-Stranded Conformation Polymorphism and Heteroduplex Analysis
Authors: Jones Alistair C , Austin Jehannine , Hansen Nancy , Hoogendoorn Bastiaan , Oefner Peter J , Cheadle Jeremy P , O’Donovan Michael C 
Published: January 2020

Abstract
Background: Denaturing HPLC (DHPLC) is a semi-automated method for detecting unknown DNA sequence variants. The sensitivity of the method is dependent on the temperature at which the analysis is undertaken, the selection of which is dependent on operator experience. To circumvent this, software has been developed for predicting the optimal temperature for DHPLC analysis. We examined the utility of this software.
Methods: To maximize the relevance of our data for other investigators, we have screened 42 different amplimers from CFTR, TSC1, and TSC2. The samples consisted of 103 unique sequence heterozygotes and 126 wild-type homozygous controls.
Results: At the temperature recommended by the software, 96% (99 of 103) of heterozygotes and all of the wild-type controls were correctly classified. This compares favorably with sensitivities of 85% for single-stranded conformation polymorphism and 82% for gel-based heteroduplex analyses of the same fragments.
Conclusions: Software-optimized DHPLC is a highly sensitive method for mutation detection. However, where sensitivity &gt;96% is required, our data suggest that in addition to the recommended temperature, fragments should also be run at the recommended temperature plus 2 °C.
Real-Time Reverse Transcription-PCR Assay for Future Management of ERBB2-based Clinical Applications
Authors: Bièche Ivan , Onody Peter , Laurendeau Ingrid , Olivi Martine , Vidaud Dominique , Lidereau Rosette , Vidaud Michel 
Published: January 2020

Abstract
Background: Gene amplification/overexpression of ERBB2 (HER2, neu) is a major event in human breast tumorigenesis. ERBB2-based therapeutic agents and ERBB2-specific gene therapy are under development. These new perspectives call for a sensitive and accurate method to screen breast cancer patients for ERBB2 alterations.
Methods: We have developed and validated a real-time quantitative reverse transcription (RT)-PCR assay, based on fluorescent TaqMan methodology, to quantify ERBB2 gene expression at the mRNA level in breast tumors. This recently developed method of nucleic acid quantification in homogeneous solutions has the potential for a wide dynamic range, interlaboratory agreement, and high-throughput capacity without tedious post-PCR processing. The ERBB2 mRNA signal was normalized to the signal for TATA box-binding protein mRNA.
Results: The dynamic range was &gt;1000-fold. The relationship between Ct and log starting concentration was linear (r2 ≥0.99). The mean (SD) normalized expression of ERBB2 in healthy breast tissue was 0.95 (0.37). Overexpression (&gt;5 SD above mean for healthy breast) of the ERBB2 gene was observed (at 3.2- to 135-fold) in 23 (17%) of 134 breast tumor RNA samples. As expected, ERBB2 overexpression was present in all tumors with ERBB2 gene amplification but was uncommon and at a low ratio (&lt;5) in breast cancers without gene amplification.
Conclusions: This new simple, rapid, semi-automated assay is a major alternative to fluorescence in situ hybridization and immunochemistry for gene alteration analysis in human tumors and may be a powerful tool for large randomized, prospective cooperative group trials and to support future ERBB2-based biological and gene therapy approaches.
Detection of Mitochondrial DNA Mutations by Temporal Temperature Gradient Gel Electrophoresis
Authors: Chen Tian-Jian , Boles Richard G , Wong Lee-Jun C 
Published: January 2020

Abstract
Background: A unique requirement for the molecular diagnosis of mitochondrial DNA (mtDNA) disorders is the ability to detect heteroplasmic mtDNA mutations and to distinguish them from homoplasmic sequence variations before further testing (e.g., sequencing) is performed. We evaluated the potential utility of temporal temperature gradient gel electrophoresis (TTGE) for these purposes in patients with suspected mtDNA mutations.
Methods: DNA samples were selected from patients with known mtDNA mutations and patients suspected of mtDNA disorders without detectable mutations by routine analysis. Six regions of mtDNA were PCR amplified and analyzed by TTGE. Electrophoresis was carried out at 145 V with a constant temperature increment of 1.2 °C/h. Mutations were identified by direct sequencing of the PCR products and confirmed by PCR/allele-specific oligonucleotide or PCR/restriction fragment length polymorphism analysis.
Results: In the experiments using patient samples containing various amounts of mutant mtDNA, TTGE detected as little as 4% mutant heteroplasmy and identified heteroplasmy in the presence of a homoplasmic polymorphism. In 109 specimens with 15 different known mutations, TTGE detected the presence of all mutations and distinguished heteroplasmic mutations from homoplasmic polymorphisms. When 11% of the mtDNA genome was analyzed by TTGE in 104 patients with clinically suspected mitochondrial disorders, 7 cases of heteroplasmy (≈7%) were detected.
Conclusions: TTGE distinguishes heteroplasmic mutation from homoplasmic polymorphisms and appears to be a sensitive tool for detection of sequence variations and heteroplasmy in patients suspected of having mtDNA disorders.
The Influence of an Expert System for Test Ordering and Interpretation on Laboratory Investigations
Authors: Smith Beverly J , McNeely Michael DD 
Published: January 2020

AbstractBackground: The Laboratory Advisory System (LAS) is an expert system interface that works interactively with clinicians to assist them with test selection and result interpretation throughout the laboratory investigation of a patient.Methods: To study the influence of the LAS on laboratory investigations, a repeated-measures experiment using clinical vignettes was conducted. To collect baseline data on how laboratory investigations are currently conducted, clinicians investigated one-half of the vignettes using a conventional (noncomputer) approach. To determine the influence of the LAS on clinicians’ behavior, the other half of the vignettes were investigated using the LAS.Results: Clinicians using the LAS (compared with conventional practice) ordered fewer laboratory tests during the diagnostic process (mean, 17.8 vs 32.7), completed the diagnostic workup with fewer sample collections (mean, 5.8 vs 7.5), generated lower laboratory costs (mean, $194 vs $232), shortened the time required to reach a diagnosis (mean, 1 day vs 3.2 days), showed closer adherence to established clinical practice guidelines, and exhibited a more uniform and diagnostically successful investigation.Conclusion: The LAS enhances the outcome of the investigation and improves laboratory utilization.
Estrogen Replacement Therapy, Serum Lipids, and Polymorphism of the Apolipoprotein E Gene
Authors: Garry Philip J , Baumgartner Richard N , Brodie Steven G , Montoya George D , Liang Hwa Chi , Lindeman Robert D , Williams Thomas M 
Published: January 2020

AbstractBackground: Pharmacogenomics, the study of genetic loci that modulate drug responsiveness, may help to explain why estrogen replacement therapy (ERT) has differential effects on serum lipid and lipoprotein concentrations in postmenopausal women who inherit distinct alleles of the apolipoprotein E gene (APOE).Methods: We compared total-cholesterol, triglyceride, and lipoprotein (LDL and HDL) concentrations in 66 postmenopausal women receiving ERT ([+]ERT) with 174 postmenopausal women not receiving ERT ([−]ERT), controlling for three APOE genotypes divided into three groups: E2 (ε2/ε3, n = 31), E3 (ε3/ε3, n = 160), and E4 (ε3/ε4 + ε4/ε4, n = 49).Results: Mean total-cholesterol concentrations were lower in all three [+]ERT groups compared with their [−]ERT counterparts but were statistically significant only for women in group E4 (P = 0.014). The mean LDL-cholesterol concentrations were significantly lower in all three [+]ERT groups compared with their [−]ERT counterparts (P ≤0.005). Although all three groups of [+]ERT women tended to have higher mean HDL-cholesterol concentrations compared with their [−]ERT counterparts, the differences were not statistically significant. [+]ERT women in groups E2 and E3 had significantly higher (P &lt;0.05) triglyceride concentrations than their [−]ERT counterparts. In [+]ERT women, the ratios of total and LDL-cholesterol to HDL-cholesterol were significantly higher in group E3 and E4 women compared with E2 women (P &lt;0.006). Group E4 [+]ERT women had ratios of total and LDL-cholesterol to HDL-cholesterol that were comparable to group E2 [−]ERT women.Conclusions: Triglyceride concentrations in group E2 [+]ERT women may need to be monitored more closely than those in E3 or E4 [+]ERT women. Group E4 women should probably be targeted for ERT. Results suggest that APOE genotypes have a differential effect on serum lipids and lipoproteins in [+]ERT postmenopausal women.