bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mechanism of Interference of a Polymerized Hemoglobin Blood Substitute in an Alkaline Phosphatase Method
Authors: Chance Jeffrey J , Norris Edward J , Kroll Martin H 
Published: January 2020

Abstract
Background: Hemoglobin-based oxygen carriers can cause profound interferences in many analytical procedures. We determined the mechanism of interference in the assay of alkaline phosphatase activity and identified approaches that might be used to correct for this interference.
Methods: Interference of a polymerized hemoglobin blood substitute with the assay of alkaline phosphatase was examined with a Hitachi 917 analyzer and ultraviolet-visible spectrophotometry.
Results: Hemoglobin-based oxygen carrier solutions had substantial absorbance at 415 nm, the wavelength of analysis used to measure the formation of 4-nitrophenol. In addition to offsetting the initial absorbance at the analytical wavelength, polymerized hemoglobin gave rise to a strong negative interference plot because of alkali denaturation of the substitute. The same interference mechanism was also observed for native hemoglobin (hemolysate), indicating that the interference was not derived from the polymerization process. The interference can be corrected by implementing a rate-correction procedure, or the interference can be avoided by measurement at 450 nm.
Conclusions: The interference of polymerized hemoglobin in the alkaline phosphatase assay is a result of an absorbance offset caused by alkali denaturation of hemoglobin. The interference can be corrected or avoided by modifying the calculation or the analytical wavelength. The correction strategy may also be applicable to improving the hemolysis index for this method.
Measurement of Human Plasma Phospholipid Transfer Protein by Sandwich ELISA
Authors: Oka Tomoichiro , Kujiraoka Takeshi , Ito Mayumi , Nagano Makoto , Ishihara Mitsuaki , Iwasaki Tadao , Egashira Tohru , Miller Norman E , Hattori Hiroaki 
Published: January 2020

AbstractBackground: Plasma phospholipid transfer protein (PLTP) plays a central role in the remodeling of HDLs. Reliable and accurate methods for assaying PLTP concentration are required.Methods: A sandwich ELISA for PLTP has been developed, using two monoclonal antibodies against recombinant human PLTP (rhPLTP) expressed in Chinese hamster ovary cells. The ELISA allows for the quantification of PLTP in the range 0.625–15.0 ng/assay (1.2–30.0 mg/L). Intra- and interassay CVs were &lt;3.0% and &lt;4.2% respectively. The assay was used to quantify plasma PLTP concentrations in 132 Japanese subjects (75 males and 57 females).Results: PLTP concentrations were 12.0 ± 3.0 mg/L (mean ± SD; range, 4.9–20.5 mg/L). No sex difference was observed. Plasma PLTP concentration was positively correlated with HDL-cholesterol (r = 0.72; P &lt;0.001), apolipoprotein (apo) A-I (r = 0.62; P &lt;0.001) and HDL2-cholesterol (r = 0.72; P &lt;0.001), and was negatively correlated with triacylglycerol (r = −0.45; P &lt;0.001). There was no correlation with plasma apo A-II. These results agree with other evidence that plasma PLTP is associated with large apo A-I-containing lipoproteins. There was no correlation (r = −0.01) between plasma PLTP and plasma phosphatidylcholine transfer activity (range, 3.5–10.5 μmol · mL−1 · h−1), suggesting that PLTP may exist in active and inactive forms.Conclusion: This new ELISA will be of value for further studies of PLTP in health and disease.
Bioconjugates of Intelligent Polymers and Recognition Proteins for Use in Diagnostics and Affinity Separations
Authors: Hoffman Allan S 
Published: January 2020

Abstract
Polymers that respond to small changes in environmental stimuli with large, sometimes discontinuous changes in their physical state or properties are often called “intelligent” or “smart” polymers. We have conjugated these polymers to different recognition proteins, including antibodies, protein A, streptavidin, and enzymes. These bioconjugates have been prepared by random polymer conjugation to lysine amino groups on the protein surface, and also by site-specific conjugation of the polymer to specific amino acid sites, such as cysteine sulfhydryl groups, that are genetically engineered into the known amino acid sequence of the protein. We have conjugated several different smart polymers to streptavidin, including temperature-, pH-, and light-sensitive polymers. The preparation of these conjugates and their many fascinating applications are reviewed here.
Interlaboratory Study on Thermal Cycler Performance in Controlled PCR and Random Amplified Polymorphic DNA Analyses
Authors: Saunders Ginny C , Dukes Juliet , Parkes Helen C , Cornett Johanne H 
Published: January 2020

Abstract
Background: Intercomparisons of PCR-based data between laboratories require an assurance of assay reproducibility. We performed an interlaboratory study to investigate the contribution made by a variety of thermal cyclers to PCR performance as measured by interblock reproducibility and intrablock repeatability.
Methods: Two standardized assays designed to minimize the introduction of non-thermal-cycler-dependent variations were evaluated by 18 laboratories in the United Kingdom, using 33 thermal cyclers of various makes and models. We used a single-product (590 bp) PCR, established in our laboratory as a robust and specific reaction. The second reaction, a multiproduct random amplified polymorphic DNA (RAPD) PCR, was known to be more susceptible to small changes in block temperature and was therefore considered a way of assessing block uniformity with respect to temperature. Assay repeatability data were analyzed with respect to temperature calibration status, the type of temperature control mechanism, thermal cycler age, and the presence of oil overlay or heated lid systems.
Results: All (100%) of the laboratories produced the correct target for the single-product PCR assay, although substantial variation in yield in replicate reactions was observed in 9.4% of these. The RAPD reaction generated results that varied extensively both within the same block and between different thermal cyclers. For eight replicates of a positive sample, 88% intrablock repeatability was demonstrated in calibrated thermal cyclers, which decreased to 63% in noncalibrated instruments.
Conclusions: Irrespective of the make and model of thermal cycler, temperature-calibrated instruments consistently generated more repeatable RAPD data than noncalibrated instruments. Guidelines are offered on optimizing and monitoring thermal cycler performance.
Effect of Urgent Clinician Notification of Low Hemoglobin Values
Authors: Froom Paul , Mahameed Tayser , Havis Rosa , Barak Mira 
Published: January 2020

Abstract
Background: Urgent clinician notification of low hemoglobin values is a common practice. The effect of such notification for samples obtained during an office visit is uncertain.
Methods: We notified the attending physicians in 100 consecutive cases of outpatients with hemoglobin &lt;80 g/L. We reviewed the medical charts of these patients with their personal physicians 1–2 months later. We considered transfusions unnecessary only when given to a clinically stable patient with probable iron deficiency anemia in the absence of chronic diseases that produce the anemia.
Results: Overall, 47 (47%) of the patients were referred to the emergency room (ER), and 31 of the 47 (66%) were transfused. Increasing age and decreasing hemoglobin were associated with increased referral of patients to the ER. Six of 31 transfusions were unnecessary. An emergency transfusion was clearly indicated in only 3 of the 31 patients; the other patients were clinically stable. A modified notification strategy excluding microcytic samples &gt;50 g/L would have detected these three patients. Over the follow-up period, no patient not referred to the ER died from complications of anemia, nor were any patients hospitalized with acute symptoms of low hemoglobin.
Conclusion: In some settings, urgent notification of medical outpatient clinics of patients with hemoglobin &lt;80 g/L may lead to unnecessary ER visits.
Novel Technique for Scanning of Codon 634 of the RET Protooncogene with Fluorescence Resonance Energy Transfer and Real-Time PCR in Patients with Medullary Thyroid Carcinoma
Authors: Ruiz Agustín , Antiñolo Guillermo , Marcos Irene , Borrego Salud 
Published: January 2020

Abstract
Background: The multiple endocrine neoplasia 2 (MEN 2) syndromes [MEN 2A, MEN 2B, and familial medullary thyroid carcinoma (FMTC)] are caused by germline mutations of the RET protooncogene. Because 85% of MEN 2A patients and 30% of FMTC patients have mutations at codon 634, the recommended molecular analyses begin at exon 11, where codon 634 is located.
Methods: We scanned codon 634 of the RET protooncogene with real-time PCR and fluorescence resonance energy transfer (FRET), using a unique pair of internal probes to detect mutations localized at codon 634. We compared results with sequencing results in 66 patients.
Results: The method detected all codon 634 mutations available in our laboratory (Cys634Tyr, Cys634Arg, Cys634Phe, Cys634Trp). Comparing this method with the direct sequencing of exon 11 in a cohort of 66 patients with MTC, the system identified all 14 MTC patients carrying germline mutations at codon 634. One apparent false-positive result occurred among 52 patients.
Conclusions: The simultaneous scanning of multiple mutations is possible with the FRET system. The method allows rapid characterization of germline mutations at codon 634 in MTC patients.
Commutability of Calibration and Control Materials for Serum Lipase
Authors: Cattozzo Giampaolo , Franzini Carlo , Melzi d’Eril Gianvico 
Published: January 2020

AbstractBackground: To effectively assess and correct for intermethod variability, calibration and control materials (CCMs) must show the same intermethod behavior as patient sera, i.e., they must be commutable. We describe the commutability of selected CCMs for lipase assays, the impact of noncommutability of CCMs in normalizing patient results, and characteristics of reagents that affect assay specificity and commutability.Methods: Lipase was measured in 98 patient sera and in 29 commercial CCMs, with 2 commercial methods using different substrates and with 4 experimental methods using 1,2-o-dilauryl-rac-glycero-3-glutaric acid-(6′-methylresorufin) ester as substrate and colipase as cofactor, but differing in the stabilizing proteins used and in the size of the substrate micelles.Results: The noncommutability rate, i.e., the frequency of aberrant intermethod behavior of CCMs in comparison with patient sera, was 27% for liquid CCMs and 47% for lyophilized CCMs. The normalized residuals, measuring the degree of noncommutability, were −2.3 to 2.4 for CCMs with “normal” lipase activity, and −3.5 to 21.7 for CCMs with higher lipase activity. Recalculation of patient results with CCMs as calibrators decreased or increased the original bias according to whether the CCMs were commutable.Conclusions: For the lipase methods in this study, the frequency of noncommutability of CCMs is affected by assay-specific characteristics, including size of substrate micelles and the presence or absence of added proteins.
Enabling Large-Scale Pharmacogenetic Studies by High-Throughput Mutation Detection and Genotyping Technologies
Authors: Shi Michael M 
Published: January 2020

AbstractBackground: Pharmacogenetics is a scientific discipline that examines the genetic basis for individual variations in response to therapeutics. Pharmacogenetics promises to develop individualized medicines tailored to patients’ genotypes. However, identifying and genotyping a vast number of genetic polymorphisms in large populations also pose a great challenge.Approach: This article reviews the recent technology development in mutation detection and genotyping with a focus on genotyping of single nucleotide polymorphisms (SNPs).Content: Novel mutations/polymorphisms are commonly identified by conformation-based mutation screening and direct high-throughput heterozygote sequencing. With a large amount of public sequence information available, in silico SNP mapping has also emerged as a cost-efficient way for new polymorphism identification. Gel electrophoresis-based genotyping methods for known polymorphisms include PCR coupled with restriction fragment length polymorphism analysis, multiplex PCR, oligonucleotide ligation assay, and minisequencing. Fluorescent dye-based genotyping technologies are emerging as high-throughput genotyping platforms, including oligonucleotide ligation assay, pyrosequencing, single-base extension with fluorescence detection, homogeneous solution hybridization such as TaqMan®, and molecular beacon genotyping. Rolling circle amplification and InvaderTM assays are able to genotype directly from genomic DNA without PCR amplification. DNA chip-based microarray and mass spectrometry genotyping technologies are the latest development in the genotyping arena.Summary: Large-scale genotyping is crucial to the identification of the genetic make-ups that underlie the onset of diseases and individual variations in drug responses. Enabling technologies to identify genetic polymorphisms rapidly, accurately, and cost effectively will dramatically impact future drug and development processes.
Capillary and Microchip Electrophoresis for Rapid Detection of Known Mutations by Combining Allele-specific DNA Amplification with Heteroduplex Analysis
Authors: Tian Huijun , Brody Lawrence C , Fan Saijun , Huang Zhili , Landers James P 
Published: January 2020

Abstract
Background: Detection of mutations by gel electrophoresis and allele-specific amplification by PCR (AS-PCR) is not easily scaled to accommodate a large number of samples. Alternative electrophoretic formats, such as capillary electrophoresis (CE) and microchip electrophoresis, may provide powerful platforms for simple, fast, automated, and high-throughput mutation detection after allele-specific amplification.
Methods: DNA samples heterozygous for four mutations (185delAG, 5382insC, 3867G→T, and 6174delT) in BRCA1 and BRCA2, and homozygous for one mutation (5382insC) in BRCA1 and two mutations (16delAA and 822delG) in PTEN were chosen as the model system to evaluate the capillary and microchip electrophoresis methods. To detect each mutation, three primers, of which one was labeled with the fluorescent dye 6-carboxyfluorescein and one was the allele-specific primer (mutation-specific primer), were used to amplify the DNA fragments in the range of 130–320 bp. AS-PCR was combined with heteroduplex (HD) analysis, where the DNA fragments obtained by AS-PCR were analyzed with the conditions developed for CE-based HD analysis (using a fluorocarbon-coated capillary and hydroxyethylcellulose). The CE conditions were transferred into the microchip electrophoresis format.
Results: Three genotypes, homozygous wild type, homozygous mutant, and heterozygous mutant, could be identified by CE-based AS-PCR-HD analysis after 10–25 min of analysis time. Using the conditions optimized with CE, we translated the AS-PCR-HD analysis mutation detection method to the microchip electrophoresis format. The detection of three heterozygous mutations (insertion, deletion, and substitution) in BRCA1 could be accomplished in 180 s or less.
Conclusions: It is possible to develop a CE-based method that exploits both AS-PCR and HD analysis for detecting specific mutations. Fast separation and the capacity for automated operation create the potential for developing a powerful electrophoresis-based mutation detection system. Fabrication of multichannel microchip platforms may enable mutation detection with high throughput.
Development of a Rapid, Quantitative Method for LDL Subfractionation with Use of the Quantimetrix Lipoprint LDL System
Authors: Hoefner Daniel M , Hodel Shannon D , O’Brien John F , Branum Earl L , Sun Deborah , Meissner Irene , McConnell Joseph P 
Published: January 2020

AbstractBackground: Recent evidence suggests that the presence of small, dense LDL is independently associated with increased risk of developing coronary artery disease. Current methods to subfractionate LDL are time-consuming and/or technically demanding. Therefore, we have sought the development of a less complex LDL subfractionation procedure.Methods: LDL subfractions were separated using the Quantimetrix LipoprintTM LDL System. High-resolution 3% polyacrylamide gel tubes were scanned densitometrically (610 nm) with a Helena EDC system. A computerized method to identify and quantitatively score the resolved LDL subfractions was developed. Results from the Quantimetrix method were compared using 51 plasma samples with values obtained by nondenaturing gradient gel electrophoresis (NDGGE) and nuclear magnetic resonance (NMR) spectroscopy.Results: LDL subfractionation scores correlated significantly (P &lt;0.05) with triglyceride, HDL-cholesterol, apolipoprotein B100, and LDL-cholesterol/apolipoprotein B100 (r = 0.591, −0.392, 0.454, and −0.411, respectively). For 51 samples, the Quantimetrix method classified 21 with small, 14 with intermediate, and 16 with large LDL. Of the 21 samples classified as small by Quantimetrix, 20 (95%) were classified as small (n = 18) or intermediate (n = 2) by NDGGE. All of the 16 specimens classified as large by Quantimetrix were either large (n = 14) or intermediate (n = 2) by NDGGE. LDL score was inversely correlated (r = −0.674; P &lt;0.0001) with LDL particle size determined by NMR spectroscopy.Conclusions: A quantitative method for the assessment of LDL particle size phenotype was developed using the Quantimetrix Lipoprint LDL System. The method can be performed in less than 3 h in batch mode and is suitable for routine use in clinical laboratories.
Development of a Routine Method for the Determination of Trace Metals in Whole Blood by Magnetic Sector Inductively Coupled Plasma Mass Spectrometry with Particular Relevance to Patients with Total Hip and Knee Arthroplasty
Authors: Case C Patrick , Ellis Lyndon , Turner Justine C , Fairman Ben 
Published: January 2020

Abstract
Background: Joint-replacement surgery has revolutionized the treatment of osteoarthritis and is still the most effective therapy. A recent clinical trend reintroducing metal-on-metal bearing surfaces has in turn stimulated a requirement for accurate measurement of the concentrations of relevant metals in both pre- and postoperative patients. Thus, there is a need for cost-effective, multielement methods for trace metal analysis in whole blood to monitor possible increases in wear metal concentrations.
Methods: A method was developed to allow routine analysis of whole blood samples for molybdenum, cobalt, chromium, and nickel. Sample preparation consisted of a simple 1:10 dilution of whole blood with a solution of 10 mL/L Triton X-100, 0.0002 mol/L EDTA, and 0.01 mol/L ammonium hydroxide. Final determination was performed by a double-focusing magnetic sector inductively coupled plasma mass spectrometer operated in medium-resolution mode (resolution, 3400). Online addition of rhodium was used for internal standardization.
Results: Detection limits in whole blood were 0.06 μg/L for chromium, cobalt, and molybdenum and 0.30 μg/L for nickel. Base concentrations of 0.22, 0.17, 0.62, and 0.99 μg/L for chromium, cobalt, molybdenum, and nickel, respectively, in whole blood have been found. Polyatomic interferences on all four elements have been shown to be resolved from the analyte masses by use of a resolution of &gt;3000.
Conclusions: The simple, rapid method of sample preparation is effective in minimizing potential contamination and enables 60 samples (run time, 8 h) to be analyzed before cleaning the instrument is necessary. A resolution &gt;3000 was sufficient to separate polyatomic interferences from the masses of interest. The method was used to analyze a large number of blood samples taken from primary patients awaiting total hip arthroplasty. The method is sensitive enough to provide base concentrations for chromium, cobalt, and molybdenum in whole blood. The results for nickel were compromised by high signals for blank samples.
Miniaturization Technologies for Molecular Diagnostics
Authors: McGlennen Ronald C 
Published: January 2020

Abstract
Background: Molecular diagnostics devices are becoming smaller. With the advancement of miniaturization technologies, microchip-based systems will soon be available for genetic testing. The purpose of this review is to highlight the underlying principles in miniaturization, the strategies being developed for bioanalysis, and the potential impact on the practice of this rapidly growing medical discipline.
Approach: The author discusses DNA microchips and their practical importation into the clinical laboratory, based on his background in medical device and microchip design and development. His discussion is supported by a body of literature covering both biomedical and electrical engineering and more recent publications in the field of molecular genetics and pathology.
Content: This review is descriptive and intended to outline the technologic and methodologic approaches to the creation of an integrated genetic analysis instrument based on miniature components. The review draws on published scientific evaluations of these devices without regard to the companies involved in their development.
Summary: The intent of this review is that the reader will better understand the variety of technical approaches toward the miniaturization of molecular genetic testing for the clinical laboratory. With insight into the principles underlying the operation of these chips and the integrated systems, the end user can better evaluate the value to the field in terms of making molecular genetics testing simpler, faster, and less expensive.
Cardiac Troponin T Predicts Long-Term Outcomes in Hemodialysis Patients
Authors: Ooi Daylily S , Zimmerman Deborah , Graham Janet , Wells George A 
Published: January 2020

Abstract
Background: Increased plasma troponin T (cTnT), but not troponin I (cTnI), is frequently observed in end-stage renal failure patients. Although generally considered spurious, we previously reported an associated increased mortality at 12 months.
Methods: We studied long-term outcomes in 244 patients on chronic hemodialysis for up to 34 months, correlating the outcomes to plasma cTnT in routine predialysis samples. In addition, subsequent plasma samples at least 1 year later and within 6 months of data analysis were available in 97 patients and were used to identify patients with increasing plasma cTnT. The endpoints used were death and new or worsening coronary, cerebro-, and peripheral vascular disease and neuropathy.
Results: Transplantation occurred more frequently in patients with low initial cTnT: 31%, 13%, and 3% in the groups with cTnT &lt;0.010, 0.010–0.099, and ≥0.100 μg/L, respectively. In the same groups, total deaths occurred in 6%, 43%, and 59% and cardiac deaths in 0%, 14%, and 24% of patients. In patients with follow-up samples, the group with increasing cTnT had a significantly increased death (relative risk, 2.0; P = 0.028). The increase was mainly in cardiac and sudden deaths.
Conclusions: Higher plasma cTnT predicts long-term all-cause mortality in hemodialysis patients, even at concentrations &lt;0.100 μg/L, as does an increasing cTnT concentration over time.