bluuebunny/crossref_metadata_2025_split huggingface dataset:
Miniature Single-Particle Immunoassay for Prostate-specific Antigen in Serum Using Recombinant Fab Fragments
Authors: Härmä Harri , Tarkkinen Piia , Soukka Tero , Lövgren Timo 
Published: January 2020

AbstractBackground: Quantitative, miniaturized nucleic acid assays and immunoassays can be developed with single microparticles, microfluorometric detection, and intrinsically fluorescent lanthanide chelates in a multiple assay format to decrease reagent consumption, cost, and assay time. We used recombinant Fab fragments to capture and detect free and total prostate-specific antigen (PSA) from serum in a submicroliter volume single-particle immunoassay.Methods: Genetically engineered thiol-Fab or thiolated monoclonal antibodies (mAbs) were covalently attached onto uniformly sized 60-μm maleimide-activated microparticles. Free and total PSA were detected with europium- or terbium-labeled Fab fragments on a single microparticle using a microfluorometer in a time-resolved mode.Results: The detection limit of the free- and total-PSA assays (mean + 3 SD of zero calibrator) was 0.35 μg/L, with a total volume of 330 nL per particle. An excellent correlation was found in microparticle and microtiter-well assays for 21 serum samples: slopes for free and total PSA were 1.06 ± 0.03 and 1.03 ± 0.02, respectively (Sy|x = 0.084 and 0.057 μg/L), with intercepts of 0.013 ± 0.018 and 0.013 ± 0.017 μg/L (R &gt;0.99). Furthermore, the particle-immobilized Fab fragment had a PSA binding capacity 1.5-fold higher than the intact mAb capacity on a single microparticle. Capacity, kinetics, and sensitivity of the Fab fragment and intact mAb assays in the microparticle and microtiter well formats are discussed.Conclusions: With site-specific (cysteine tail) covalent attachment of Fab fragments on a microparticle, subattomole amounts of PSA can be detected quantitatively.
Quantification of Melanoma Cell-specific MART-1 mRNA in Peripheral Blood by a Calibrated Competitive Reverse Transcription-PCR
Authors: Sørensen Boe Sandahl , Schmidt Henrik , von der Maase Hans , Straten Per Thor , Nexø Ebba 
Published: January 2020

AbstractBackground: Reverse transcription-PCR (RT-PCR) amplification of melanoma cell-specific mRNA can detect melanoma cells in the peripheral blood of patients with malignant melanoma. We present a method to quantify mRNA coding for the melanoma-specific melanoma antigen recognized by T cells #1 (MART-1) in RNA isolated from peripheral blood.Methods: To establish a calibration curve, we measured the concentration of MART-1 mRNA in SK-MEL-28 melanoma cells grown in vitro by competitive RT-PCR. Serial dilutions of these cells were used as calibrators in the assay. The assay was conducted by adding a fixed amount of a RNA internal standard to RNA isolated from either peripheral blood or the calibrators before RT-PCR amplification with MART-1 primers in a nested PCR design. The amount of MART-1 mRNA in blood samples was calculated from the calibration curve.Results: Addition of melanoma cells grown in vitro to blood from healthy donors demonstrated that the method can detect a single SK-MEL-28 melanoma cell in 1 mL of blood (1.5 × 10−21 mol MART-1 mRNA/mL). MART-1 mRNA was observed in 4 of 12 blood samples from patients with malignant melanoma, at concentrations of 3–18 × 10−21 mol MART-1 mRNA/mL of blood. No MART-1 mRNA was detected in blood samples from 25 controls without malignant melanoma. Intra- and interassay CVs were 15% (n = 12; mean = 44 × 10−21 mol MART-1 mRNA/mL) and 33% (15 samples analyzed in two different analytical runs; mean = 30 × 10−21 mol MART-1 mRNA/mL), respectively.Conclusions: Our method is the first competitive RT-PCR assay for quantification of melanoma cells in blood samples that compensates for the variation of both the reverse transcription and PCR reactions. The method allows the inclusion of control samples for continuous quality assessment.
Saposins A, B, C, and D in Plasma of Patients with Lysosomal Storage Disorders
Authors: Chang Melissa H Y , Bindloss Colleen A , Grabowski Gregory A , Qi Xiaoyang , Winchester Bryan , Hopwood John J , Meikle Peter J 
Published: January 2020

AbstractBackground: Early diagnosis of lysosomal storage disorders (LSDs), before the onset of irreversible pathology, will be critical for maximum efficacy of many current and proposed therapies. To search for potential markers of LSDs, we measured saposins A, B, C, and D in patients with these disorders.Methods: Four time-delayed fluorescence immunoquantification assays were used to measure each of the saposins in plasma from 111 unaffected individuals and 334 LSD-affected individuals, representing 28 different disorders.Results: Saposin A was increased above the 95th centile of the control population in 59% of LSD patients; saposins B, C, and D were increased in 25%, 61%, and 57%, respectively. Saposins were increased in patients from several LSD groups that in previous studies did not show an increase of lysosome-associated membrane protein-1 (LAMP-1).Conclusion: Saposins may be useful markers for LSDs when used in conjunction with LAMP-1.
Flow Cytometric Assessment of LDL Ligand Function for Detection of Heterozygous Familial Defective Apolipoprotein B-100
Authors: Raungaard Bent , Heath Finn , Hansen Peter Steen , Brorholt-Petersen Jens Uffe , Jensen Henrik Kjærulf , Færgeman Ole 
Published: January 2020

AbstractBackground: Familial defective apolipoprotein (apo) B-100 (FDB) is caused by a mutation in the apoB gene and characterized by decreased binding of LDL to LDL receptors because of reduced function of the apoB-100 ligand. FDB may be associated with severe hypercholesterolemia and cannot always be distinguished from familial hypercholesterolemia phenotypically.Methods: We used a fluorescence flow cytometry assay with Epstein-Barr virus-transformed lymphocytes to detect reduced LDL ligand function by competitive binding with fluorescently conjugated LDL (DiI-LDL). The assay was tested and validated using LDL from patients heterozygous for the Arg3500-Gln mutation and their first-degree relatives. Knowing the actual apoB genotype of patients and relatives allowed us to assess the ability of the assay to predict the results of DNA analysis. The results were compared to measurements of LDL ligand function in unrelated healthy control subjects to characterize functionally the Arg3500-Gln mutation.Results: Fluorescence was significantly increased in cells incubated with DiI-LDL in competition with unlabeled LDL from FDBR3500Q heterozygotes compared with cells incubated with DiI-LDL in competition with unlabeled LDL from relatives or unrelated healthy control subjects. Thus, patients heterozygous for the Arg3500-Gln mutation had significantly reduced LDL ligand function. The binding affinity of LDL from FDBR3500Q heterozygotes was 32% of that in non-FDB relatives and healthy controls. The assay had a diagnostic sensitivity of 0.95 and diagnostic specificity of 0.89.Conclusions: The diagnostic accuracy of the assay was too low to allow reliable diagnosis of individual cases of heterozygous FDBR3500Q. However, fluorescence flow cytometry may supplement genetic identification of FDB and functionally characterize gene mutations associated with major reductions in LDL ligand function.
Comparison of a New Method for the Direct and Simultaneous Assessment of LDL- and HDL-Cholesterol with Ultracentrifugation and Established Methods
Authors: Benlian Pascale , Cansier Christophe , Hennache Geneviève , Khallouf Oumayma , Bayer Pascale , Duron Françoise , Carrat Fabrice , Couderc Rémi , Chazouillères Olivier , Bardet Jean , Bouchard Philippe , Poupon Raoul , Masliah Joëlle , Béréziat Gilbert 
Published: January 2020

AbstractBackground: Automated electrophoresis combined with enzymatic cholesterol staining might improve routine assessment of LDL- and HDL-cholesterol (LDLC and HDLC), as an alternative to the Friedewald equation and precipitation. A new method (Hydrasys; SEBIA) that adapts the cholesterol esterase/cholesterol oxidase reaction within urea-free gels was evaluated.Methods: Fresh sera from 725 subjects (512 dyslipidemics) were analyzed by electrophoresis, in parallel with sequential ultracentrifugation, β-quantification, calculation, and precipitation.Results: Electrophoresis was linear up to 4 g/L cholesterol, with a detection limit of 0.042 g/L cholesterol/band. Within-run, between-run, between-batch, and between-operator imprecision (CVs) were 1.6%, 2.0%, 1.5%, and 2.7% for LDLC, and 3.9%, 4.3%, 5.5%, and 4.9% for HDLC, and remained unchanged up to 6.3 g/L plasma triglycerides (TGs). Precision decreased with very low HDLC (&lt;0.25 g/L). Serum storage for 3–7 days at +4 or −80 °C did not interfere significantly with the assay. Agreement with β-quantification was stable for LDLC up to 5.07 g/L (r = 0.94), even at TG concentrations &gt;4 g/L (r = 0.91). Bias (2.88% ± 12%) and total error (7.84%) were unchanged at TG concentrations up to 18.5 g/L. Electrophoresis predicted National Cholesterol Education Program cut-points with &lt;0.04 g/L error, exactly and appropriately classified 79% and 96% of the subjects, and divided by 2.4 (all subjects) and 5.8 (TGs &gt;1.5 g/L) the percentage of subjects underestimated by calculation. One-half of the patients with TGs &gt;4 g/L had LDLC &gt;1.30 g/L. For HDLC, correlation was better with precipitation (r = 0.87) than ultracentrifugation (r = 0.76). Error (−0.10% ± 26%) increased when HDLC decreased (&lt;0.35 g/L). Direct assessment of the LDLC/HDLC ratio detected 45% more high-risk subjects than the calculation/precipitation combination.Conclusions: Electrophoresis provides reliable quantification of LDLC, improving precision, accuracy, and concordance over calculation, particularly with increasing plasma TGs. Implementation of methods to detect low cholesterol concentrations could extend the applications for HDLC assessment.
Simple and Sensitive Binding Assay for Measurement of Adenosine Using Reduced S-Adenosylhomocysteine Hydrolase
Authors: Kloor Doris , Yao Kozo , Delabar Ursula , Osswald Hartmut 
Published: January 2020

Abstract
Background: Adenosine has been suggested to play an important role in the regulation of renal function. We developed a simple and sensitive binding assay for the detection of adenosine based on the displacement of [3H]adenosine from S-adenosylhomocysteine (SAH) hydrolase in its reduced form.
Methods: SAH hydrolase was purified to apparent homogeneity from bovine kidney by standard chromatographic methods. SAH hydrolase was converted in its reduced form, which had the advantage that the SAH hydrolase is enzymatically inactive. This reduced enzyme retains its ability to bind adenosine with high affinity. To determine adenosine in urine or tissues, samples must be deproteinized (e.g., with 10 g/L sulfosalicylic acid or 0.6 mol/L perchloric acid).
Results: The reduced SAH hydrolase bound adenosine with a dissociation constant of 33.0 ± 2 nmol/L. Displacement of adenosine binding by the adenine 5′-nucleotides, adenine and hypoxanthine, required &gt;1000-fold higher concentrations than adenosine itself. The intra- and interassay imprecision (CV) was &lt;3.9% and 7.8%, respectively, and the values obtained showed acceptable correlation with those by HPLC.
Conclusions: The highly sensitive adenosine-binding protein assay is a simple test that allows detection of adenosine in samples with small volumes without purification, and is in this respect superior to HPLC.
Clinical Outcomes of Point-of-Care Testing in the Interventional Radiology and Invasive Cardiology Setting
Authors: Nichols James H , Kickler Thomas S , Dyer Karen L , Humbertson Sandra K , Cooper Peg C , Maughan William L , Oechsle Denise G 
Published: January 2020

Abstract
Background: Point-of-care testing (POCT) can provide rapid test results, but its impact on patient care is not well documented. We investigated the ability of POCT to decrease inpatient and outpatient waiting times for cardiovascular procedures.
Methods: We prospectively studied, over a 7-month period, 216 patients requiring diagnostic laboratory testing for coagulation (prothrombin time/activated partial thromboplastin time) and/or renal function (urea nitrogen, creatinine, sodium, and potassium) before elective invasive cardiac and radiologic procedures. Overall patient management and workflow were examined in the initial phase. In phase 2, we implemented POCT but utilized central laboratory results for patient management. In phase 3, therapeutic decisions were based on POCT results. The final phase, phase 4, sought to optimize workflow around the availability of POCT. Patient wait and timing of phlebotomy, availability of laboratory results, and therapeutic action were monitored. Split sampling allowed comparability of POCT and central laboratory results throughout the study.
Results: In phase 1, 44% of central laboratory results were not available before the scheduled time for procedure (n = 135). Mean waiting times (arrival to procedure) were 188 ± 54 min for patients who needed renal testing (phase 2; n = 14) and 171 ± 76 min for those needing coagulation testing (n = 24). For patients needing renal testing, POCT decreased patient wait times (phases 3 and 4 combined, 141 ± 52 min; n = 18; P = 0.02). For patients needing coagulation testing, wait times improved only when systematic changes were made in workflow (phase 4, 109 ± 41 min; n = 12; P = 0.01).
Conclusions: Although POCT has the potential to provide beneficial patient outcomes, merely moving testing from a central laboratory to the medical unit does not guarantee improved outcomes. Systematic changes in patient management may be required.
The Role of Total Laboratory Automation in a Consolidated Laboratory Network
Authors: Seaberg Richard S , Stallone Robert O , Statland Bernard E 
Published: January 2020

AbstractBackground: In an effort to reduce overall laboratory costs and improve overall laboratory efficiencies at all of its network hospitals, the North Shore–Long Island Health System recently established a Consolidated Laboratory Network with a Core Laboratory at its center.Methods: We established and implemented a centralized Core Laboratory designed around the Roche/Hitachi CLAS Total Laboratory Automation system to perform the general and esoteric laboratory testing throughout the system in a timely and cost-effective fashion. All remaining STAT testing will be performed within the Rapid Response Laboratories (RRLs) at each of the system’s hospitals.Results: Results for this laboratory consolidation and implementation effort demonstrated a decrease in labor costs and improved turnaround time (TAT) at the core laboratory. Anticipated system savings are ∼$2.7 million. TATs averaged 1.3 h within the Core Laboratory and less than 30 min in the RRLs.Conclusions: When properly implemented, automation systems can reduce overall laboratory expenses, enhance patient services, and address the overall concerns facing the laboratory today: job satisfaction, decreased length of stay, and safety. The financial savings realized are primarily a result of labor reductions.
Stand-Alone Automated Solutions Can Enhance Laboratory Operations
Authors: Orsulak Paul J 
Published: January 2020

AbstractClinical laboratory automation has developed over the past decade as one means of consolidating testing, reducing costs, and improving the effectiveness of laboratory testing. Most of the developments have been aimed at core clinical laboratory operations, and have primarily addressed preanalytical and analytical processing of traditional specimens arriving in blood collection or similar aliquot tubes. Much less attention has been given to specialized applications such as processing specimens for urine toxicology, and only recently have vendors attacked the problems associated with sorting and maintaining the laboratory’s inventory of specimens. This report highlights selected developments in these areas, describes one approach to cost-effective custom platform development, and discusses the advantages and pitfalls to solving problems with laboratory automation.
HPLC Analysis of Lipid-derived Polyunsaturated Fatty Acid Peroxidation Products in Oxidatively Modified Human Plasma
Authors: Browne Richard W , Armstrong Donald 
Published: January 2020

Abstract
Background: Lipid peroxidation is a prominent manifestation of free radical activity and oxidative stress in biological systems. Diverse methodologies have been developed that measure a variety of lipid peroxidation products used as markers of lipid peroxidation processes.
Methods: Hydroxy and hydroperoxy polyunsaturated fatty acid (PUFA) peroxidation products were analyzed in human blood plasma by reversed-phase HPLC after liquid-liquid extraction of total lipids and alkaline hydrolysis of lipid esters to liberate free PUFAs. An isocratic mobile phase containing 1 g/L acetic acid-acetonitrile-tetrahydrofuran (52:30:18, by volume) over 60 min duration, with ultraviolet absorbance detection at 236 nm by photodiode array, enabled the resolution and quantification of 13 regioisomeric hydroxy and hydroperoxy PUFAs.
Results: As little as 250 μL of human plasma was utilized with an analytical range of 0.033–1.6 μmol/L for each compound. Intra- and interassay CVs for all compounds detected in normal or oxidatively modified human plasma were 3.2–11% and 4.7–12%, respectively. Analytical recoveries were 87–103%. Analysis of human plasma exposed to artificial oxidation with Cu2+ ion and hydrogen peroxide, a free radical-generating reaction, showed marked increases in hydroxy and hydroperoxy PUFA concentrations.
Conclusion: Lipid-derived hydroxy and hydroperoxy PUFAs may be useful as clinical markers of lipid peroxidation and oxidative stress in the peripheral circulation.
Progression Criteria for Cancer Antigen 15.3 and Carcinoembryonic Antigen in Metastatic Breast Cancer Compared by Computer Simulation of Marker Data
Authors: Sölétormos György , Hyltoft Petersen Per , Dombernowsky Per 
Published: January 2020

AbstractBackground: We investigated the utility of computer simulation models for performance comparisons of different tumor marker assessment criteria to define progression or nonprogression of metastatic breast cancer.Methods: Clinically relevant values for progressive cancer antigen 15.3 and carcinoembryonic antigen concentrations were combined with representative values for background variations in a computer simulation model. Fifteen criteria for assessment of longitudinal tumor marker data were obtained from the literature and computerized. Altogether, 7200 different patients, each based on 50 measurements, were simulated. With a sampling interval of 4 weeks, the monitoring period for each event was ∼3.8 years.Results: Modulation of the background variation, the starting concentrations, and the cutoffs enabled identification of criteria that were robust against false-positive signals of progression.Conclusions: The computer simulation model is a fast, effective, and inexpensive approach for comparing the diagnostic potential of assessment criteria during clinically relevant conditions of steady-state and progressive disease. The model systems can be used to generate tumor marker assessment criteria for a variety of malignancies and to compare and optimize their diagnostic performance.
Reminders of Drug Effects on Laboratory Test Results
Authors: Kailajärvi Marita , Takala Timo , Grönroos Paula , Tryding Nils , Viikari Jorma , Irjala Kerttu , Forsström Jari 
Published: January 2020

Abstract
Drug effects on laboratory test results are difficult to take into account without an online decision support system. In this study, drug effects on hormone test results were coded using a drug-laboratory effect (DLE) code. The criteria that trigger the reminders were defined. To issue reminders, it was necessary to write a computer program linking the DLE knowledge base with databases containing individual patient medication and laboratory test results. During the first 10 months, 11% of the results from hormone samples were accompanied by one or more DLE reminders. The most common drugs to trigger reminders were glucocorticoids, furosemide, and metoclopramide. Physicians facing the reminders completed a questionnaire on the usefulness of the reminders. All respondents considered them useful. In addition, DLE reminders had caused 74% of respondents to refrain from additional, usually performed examinations. In conclusion, drug effects on laboratory tests should always be considered when interpreting laboratory results. An online reminder system is useful in displaying potential drug effects alongside test results.
Quantification of DNA Using the Luminescent Oxygen Channeling Assay
Authors: Patel Rajesh , Pollner Reinhold , de Keczer Steve , Pease John , Pirio Marcel , DeChene Neal , Dafforn Alan , Rose Sam 
Published: January 2020

Abstract
Background: Simplified and cost-effective methods for the detection and quantification of nucleic acid targets are still a challenge in molecular diagnostics.
Methods: Luminescent oxygen channeling assay (LOCITM) latex particles can be conjugated to synthetic oligodeoxynucleotides and hybridized, via linking probes, to different DNA targets. These oligomer-conjugated LOCI particles survive thermocycling in a PCR reaction and allow quantified detection of DNA targets in both real-time and endpoint formats. The endpoint DNA quantification format utilized two sensitizer bead types that are sensitive to separate illumination wavelengths. These two bead types were uniquely annealed to target or control amplicons, and separate illuminations generated time-resolved chemiluminescence, which distinguished the two amplicon types.
Results: In the endpoint method, ratios of the two signals allowed determination of the target DNA concentration over a three-log range. The real-time format allowed quantification of the DNA target over a six-log range with a linear relationship between threshold cycle and log of the number of DNA targets.
Conclusions: This is the first report of the use of an oligomer-labeled latex particle assay capable of producing DNA quantification and sequence-specific chemiluminescent signals in a homogeneous format. It is also the first report of the generation of two signals from a LOCI assay. The methods described here have been shown to be easily adaptable to new DNA targets because of the generic nature of the oligomer-labeled LOCI particles.
Linked Linear Amplification: A New Method for the Amplification of DNA
Authors: Reyes Antonio A , Ugozzoli Luis A , Lowery Jimmie D , Breneman John W , Hixson Craig S , Press Richard D , Wallace R Bruce 
Published: January 2020

Abstract
Background: Linked Linear Amplification (LLA) is a new nucleic acid amplification method that uses multiple cycles of primer extension reactions. The presence of nonreplicable elements in LLA primers renders primer extension products unusable as templates for further amplification, leading to linear accumulation of products. Through the use of nested primers, linear reactions can be “linked”, providing total amplification yields comparable to those obtained by PCR.
Methods: The LLA model predicts (a) that amplification yield will approach that of PCR as the number of primers increases and (b) that the unique composition of LLA products will give lower carryover amplification efficiency compared with PCR. To test these hypotheses, the human β-globin gene was amplified by 10-, 14-, or 18-primer LLA and the yield was compared with PCR. Carryover contamination was simulated by reamplifying a dilution series of LLA or PCR products. To demonstrate the clinical utility of the method, LLA coupled with allele-specific oligonucleotide (ASO) capture was used to detect the factor V Leiden mutation in a panel of 111 DNA samples.
Results: Fourteen- and 18-primer LLA gave amplification yields comparable to PCR. However, LLA carryover amplification efficiency was four orders of magnitude lower than that of PCR. The LLA-ASO assay detected the correct factor V Leiden genotype in all 111 samples.
Conclusions: LLA is a robust target amplification method that is comparable to PCR in yield. However, LLA is more resistant to false results caused by carryover amplicon contamination.