bluuebunny/crossref_metadata_2025 huggingface dataset:
Advanced multiplexed analysis with the FlowMetrixTM system
Authors: Fulton R Jerrold , McDade Ralph L , Smith Perry L , Kienker Laura J , Kettman John R
Published: January 2020

The FlowMetrixTM System is a multiplexed data acquisition and analysis platform for flow cytometric analysis of microsphere-based assays that performs simultaneous measurement of up to 64 different analytes. The system consists of 64 distinct sets of fluorescent microspheres and a standard benchtop flow cytometer interfaced with a personal computer containing a digital signal processing board and Windows95®-based software. Individual sets of microspheres can be modified with reactive components such as antigens, antibodies, or oligonucleotides, and then mixed to form a multiplexed assay set. The digital signal-processing hardware and Windows95-based software provide complete control of the flow cytometer and perform real-time data processing, allowing multiple independent reactions to be analyzed simultaneously. The system has been used to perform qualitative and quantitative immunoassays for multiple serum proteins in both capture and competitive inhibition assay formats. The system has also been used to perform DNA sequence analysis by multiplexed competitive hybridization with 16 different sequence-specific oligonucleotide probes.
Quantifying the bias associated with use of discrepant analysis
Authors: Lipman Harvey B , Astles J Rex
Published: January 2020

Discrepant analysis is a widely used technique for estimating the performance parameters of a laboratory test. In discrepant analysis, each specimen is initially tested with the candidate test and a comparison method, and when the results of the two tests disagree, a confirmatory test is used to resolve the discrepancy. Discrepant analysis usually produces biased estimates. This report quantifies this bias and shows that it is usually positive, leading to overestimation of the performance parameters of a laboratory test. The direction and magnitude of the bias are predictably influenced by the analytical sensitivity and specificity of the candidate test, comparison method, and confirmatory test. The proportion of abnormal specimens tested also affects the magnitude of the bias, particularly the estimates of analytical sensitivity and positive predictive value when this proportion is low. Alternative approaches are suggested.
Mechanisms of dopamine and dobutamine interference in biochemical tests that use peroxide and peroxidase to generate chromophore
Authors: Karon Brad S , Daly Thomas M , Scott Mitchell G
Published: January 2020

Dopamine and dobutamine have recently been shown to produce a negative interference in several biochemical tests that use peroxide and peroxidase to generate a chromophore. To define the chemical mechanism of this interference, we examined the effects of dopamine and dobutamine in various peroxidase-based biochemical tests. Dopamine interfered stoichiometrically with peroxidase-based tests that use 4-aminophenazone to form chromophore but interfered little in those that use other compounds to generate chromophore. Dopamine reacts with 4-aminophenazone in the presence of peroxide and peroxidase to form a novel quinone-imine dye, with a smaller absorptivity than the chromophore formed in the absence of dopamine. The smaller absorptivity of this novel chromophore results in negative interference by reducing the total absorbance at the wavelength used to measure analyte. In contrast, dobutamine interfered stoichiometrically with all peroxidase-based tests studied, regardless of whether 4-aminophenazone was used to form the chromophore. Dobutamine was rapidly oxidized by peroxide in the presence of peroxidase, thus depleting the peroxide necessary to generate chromophore. Dopamine and dobutamine demonstrate two distinct general mechanisms of interference in peroxidase-based biochemical tests.
Cardiac troponin I measurement with the ACCESS® immunoassay system: analytical and clinical performance characteristics
Authors: Christenson Robert H , Apple Fred S , Morgan David L , Alonsozana Gladys L , Mascotti Kristin , Olson Marilyn , McCormack Robert T , Wians Frank H , Keffer Joseph H , Duh Show-Hong
Published: January 2020

We evaluated the ACCESS® cardiac troponin I (cTnI) immunoassay as a marker for myocardial infarction (MI). Total imprecision was 6.0% to 13.5%, the minimum detectable concentration was 0.007 μg/L, and the limit of quantitation was 0.046 μg/L. Comparison of cTnI measurement between the ACCESS and Stratus systems (n = 114) showed a proportional difference: ACCESS cTnI = 0.0996 Stratus cTnI + 0.049 μg/L (r = 0.811). Fifty-nine of 61 ambulatory patients without cardiac symptoms had no detectable cTnI (95% range, 0.00 to 0.025 μg/L). The optimum cutoff for discriminating MI (n = 289, 45 with MI) was 0.15 μg/L by receiver operator characteristic curve analysis; at this cutoff, the ACCESS cTnI assay showed a sensitivity of 88.9% (95% CI, 79.7–98.1%) and specificity of 91.8% (95% CI, 88.4–95.2%). The ACCESS cTnI assay results showed 89.4% and 93.0% concordance with the MB isoenzyme of creatine kinase (CK-MB) mass and Stratus cTnI results, respectively, for classification of patients with suspected MI. The ACCESS cTnI assay appears to show sensitivity and specificity comparable with those of both CK-MB mass and Stratus cTnI assays for the diagnosis of MI in patients presenting within 12 h of onset of symptoms.
Molecular diagnosis of medium-chain acyl-CoA dehydrogenase deficiency by oligonucleotide ligation assay
Authors: Romppanen Eeva-Liisa , Mononen Tarja , Mononen Ilkka
Published: January 2020

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is a recessively inherited defect in the mitochondrial β-oxidation of fatty acids. A single nucleotide change, the A985→G transition, in the MCAD gene accounts for ∼90% of all the disease-causing mutations in the patients. We have used PCR to amplify a segment of the human MCAD gene and typed the allelic sequence variation at base 985 by a colorimetric oligonucleotide ligation assay (OLA). PCR/OLA provides a technique that permits differentiation of the homozygotes, heterozygotes, and normals for the A985→G allele in the MCAD gene. Genotyping of 1908 random Finnish DNA samples by OLA identified 10 carriers of the mutant allele, but no homozygotes were found. The calculated carrier frequency for the A985→G mutation was 1:191 (95% confidence limits, 1:118–1:501), and the calculated frequency for the A985→G homozygotes was 1:147 000 (95% confidence limits, 1:56 000–1:1 004 000).
Plasma concentration, kinetic constants, and gene polymorphism of angiotensin I-converting enzyme in centenarians
Authors: Faure-Delanef Laurence , Baudin Bruno , Bénéteau-Burnat Bénédicte , Beaudoin Jean-Christophe , Giboudeau Jacqueline , Cohen Daniel
Published: January 2020

We have determined serum activity and kinetic constants of angiotensin I-converting enzyme (ACE), parallel to an insertion/deletion (I/D) polymorphism in its gene, in French centenarians and controls 20–70 years of age because this enzyme could have an impact on cardiovascular risk, and thus on longevity. Both the ACE D allele and ACE D/D genotype were more frequent in centenarians in comparison with controls, without sex-related differences nor significant correlation with a cardiovascular pathology. In centenarians, I/D polymorphism was correlated with circulating ACE activity (D/D genotype, 89.0 ± 36.8 U/L; I/D genotype, 63.5 ± 26.0 U/L; and I/I genotype, 55.1 ± 39.4 U/L). The Michaelis constants for two substrates were identical whatever the genotype and were not different between centenarians and controls, i.e., 0.30 ± 0.03 mmol/L for furylacryloyl-phenylalanyl-glycyl-glycine and 1.35 ± 0.05 mmol/L for hippuryl-histidyl-leucine; for the latter, the optimal pH and activating concentration of chloride did not depend on I/D polymorphism. The maximal velocities with both substrates reflected the distribution of serum ACE activity as a function of the genotypes, in centenarians and in controls. In conclusion, plasma ACE activity is subject to a similar genotypic influence in centenarians as in adults 20–70 years of age; however, ACE itself appears to be functionally similar for each genotype. Furthermore, the D allele as well as the higher serum ACE activities associated with the D/D genotype cannot discriminate individuals at high risk for cardiovascular diseases, major causes of mortality before the age of 100 years.
New sensitive method for the detection of the A3243G mutation of human mitochondrial deoxyribonucleic acid in diabetes mellitus patients by ligation-mediated polymerase chain reaction
Authors: Urata Michiyo , Wakiyama Machiko , Iwase Masanori , Yoneda Makoto , Kinoshita Sachiko , Hamasaki Naotaka , Kang Dongchon
Published: January 2020

An adenine-to-guanine mutation at nucleotide position (np) 3243 in the mitochondrial tRNALeu(UUR) gene is closely associated with various clinical phenotypes of diabetes mellitus. Because the mutation creates a new restriction site for the restriction enzyme ApaI, the mutation is usually detected and quantified by ApaI cleavage of the PCR products including np 3243. The sensitivity of the conventional method is, however, 5–10% heteroplasmy. The percentage of heteroplasmy of the mutation is usually highest in the affected tissues and is much lower in peripheral blood cells, which are used most frequently for the analysis. The sensitivity of the conventional method, however, is not sufficient to detect the mutation from peripheral blood cells. Utilizing ligation-mediated polymerase chain reaction, we have developed a feasible and sensitive method to detect 0.01% heteroplasmy of the 3243 mutation in peripheral leukocytes.
K-ras mutations in stools and tissue samples from patients with malignant and nonmalignant pancreatic diseases
Authors: Berndt Christoph , Haubold Katrin , Wenger Frank , Brux Brigitte , Müller Joachim , Bendzko Peter , Hillebrand Timo , Köttgen Eckhard , Zanow Jürgen
Published: January 2020

Mutant-enriched PCR and reverse dot blot hybridization in microplates were applied for examining K-ras status in stools and tissue samples from patients with pancreatic tumors and chronic pancreatitis. In tissue samples, K-ras mutations were found in 32 of 35 cases of ductal adenocarcinoma, in 5 of 7 periampullary cancers, in 1 cystadenocarcinoma, and in 3 of 5 patients with chronic pancreatitis. In stools, mutated K-ras was seen in 10 of 25 cases of ductal adenocarcinoma, in 1 case of cystadenocarcinoma, and in 2 of 6 cases of chronic pancreatitis. These data indicate that the K-ras status of stool samples may help identify pancreatic carcinoma and persons at risk for cancer development; however, it does not allow discrimination of malignant from nonmalignant diseases.
Measurement of LDL particle size in whole plasma and serum by high performance gel-filtration chromatography using a fluorescent lipid probe
Authors: Scheffer Peter G , Bakker Stephan J L , Heine Robert J , Teerlink Tom
Published: January 2020

We have recently described a technique for measuring LDL size by high performance gel-filtration chromatography (HPGC) with UV detection (Scheffer et al., Clin Chem 1997;43:1904–12). A drawback of this method is the necessity of LDL isolation before chromatography. We now describe a modification of this method based on selective detection of lipoproteins by postcolumn labeling with parinaric acid, a fluorescent lipid probe. Measuring the size of isolated LDL by HPGC in 56 subjects, we obtained diameters of 25.72 ± 0.60 nm with UV detection and of 25.74 ± 0.58 nm with fluorescence detection. The modified method is suitable for LDL size measurement in whole plasma or serum. LDL sizes measured in whole plasma correlated strongly with the respective values in isolated LDL (r = 0.976) but were on average 0.18 nm larger (P < 0.001). CVs for within- and between-series imprecision were <0.25%. The present method requires only 5 μL plasma or serum without sample preparation and is suitable for the unattended analysis of large series of samples.
Enzyme immunoassay of urinary mevalonic acid and its clinical application
Authors: Hiramatsu Makoto , Hayashi Akio , Hidaka Hideki , Ueshima Hirotsugu , Kanno Takashi
Published: January 2020

We have developed an enzyme immunoassay for mevalonic acid (MVA), using a specific monoclonal antibody. The intra- and interassay coefficients of variation calculated on two urine samples were 3.3% and 3.4%, respectively, in the intraassay precision test and 3.5% and 6.9% in the interassay evaluation. Pravastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor, was administered to nine healthy men, and in all cases, their MVA excretion rates then decreased. The more MVA that was excreted in the urine before the pravastatin administration, the greater a reduction of MVA excretion was observed. The daily MVA excretions in healthy men (n = 120) and women (n = 105) were 2.32 μmol/day (SD, 0.82 μmol/day) and 1.85 μmol/day (SD, 0.47 μmol/day), respectively. In streptozotocin-induced diabetic rats (n = 14), the plasma cholesterol concentrations and MVA excretion rates were increased, and a positive correlation was observed between the plasma cholesterol and the urinary MVA concentrations.
K-ras gene mutations in the diagnosis of fine-needle aspirates of pancreatic masses: prospective study using two techniques with different detection limits
Authors: Mora Josefina , Puig Pere , Boadas Jaume , Urgell Eulàlia , Montserrat Enric , Lerma Enrique , González-Sastre Francesc , Lluís Fèlix , Farré Antoni , Capellá Gabriel
Published: January 2020

Detection of K-ras mutations may be useful in the evaluation of pancreatic cancer. The aim of this study was to assess, in a prospective design, the diagnostic utility of K-ras mutation analysis in 62 consecutive fine-needle aspirates of pancreatic masses, using two PCR-based techniques—standard and enriched—with detection limits of a mutant allele in the presence of 102 or 103 wild-type alleles, respectively. Cytology alone offered a diagnostic sensitivity of 75%. The enriched higher sensitivity detection technique, in combination with cytology, offered a diagnostic sensitivity of 91% without false positives. The molecular analysis would have contributed to diagnosis in an additional 14 cases of pancreatic cancer. The standard technique contributed to diagnosis in an additional 9 cases. These results strongly support the use of the enriched method of detecting K-ras mutations as a complement to cytology in the evaluation of pancreatic masses.
Cardiac troponin I and minor cardiac damage: biochemical markers in a clinical model of myocardial lesions
Authors: Manuel del Rey José , Madrid Antonio Hernández , Valiño José Manuel , Rubí José , Mercader José , Moro Concepción , Ripoll Eduardo
Published: January 2020

Radiofrequency (RF) catheter ablation is the curative treatment of choice for many cardiac arrhythmias. After RF ablation there is always a small localized endomyocardial necrosis, necessary to abolish the arrhythmia. We designed this study to determine the serum concentrations of several cardiac markers in patients who underwent RF catheter ablation. The study shows a higher frequency of increase of serum cardiac troponin I (cTnI) than of creatine kinase (CK), the CK MB isoenzyme (CK-MB), or myoglobin. A pathological value of cTnI was found in 47 of 51 patients (92%) in the ablation group. The area under the ROC curve for cTnI was 0.9375, significantly higher than for the other biochemical markers (0.86, 0.76, and 0.75 for CK-MB, myoglobin, and CK, respectively), with P <0.05. We conclude that the serum concentration of cTnI is the best biochemical marker for detecting the minor myocardial damage produced by RF ablation.
Correlation between total homocysteine and cyclosporine concentrations in cardiac transplant recipients
Authors: Cole David EC , Ross Heather J , Evrovski Jovan , Langman Loralie J , Miner Steven ES , Daly Paul A , Wong Pui-Yuen
Published: January 2020

Increased circulating total homocysteine (tHcy) has been implicated as an independent risk factor for atherosclerotic disease. In cardiac transplant patients, accelerated coronary atherosclerosis is an important cause of late allograft failure; however, studies of tHcy in this at-risk group are limited. We sampled a cohort of 72 subjects 3.95 ± 3.14 (mean ± SD) years after transplantation and found that all had tHcy concentrations above our upper reference limit (15.0 μmol/L). The mean tHcy in the transplant group (25.4 ± 7.1 μmol/L) was significantly greater than in our reference group (9.0 ± 4.3 μmol/L; n = 457; P <0.001). We also examined the effect of age, gender, time since transplant, serum folate and cobalamin, total protein, urate, creatinine, albumin, and trough whole blood cyclosporine concentrations. In a multiple linear regression model, only creatinine (mean 144 ± 52 μmol/L; P = 0.021) and trough cyclosporine concentrations (191 ± 163 μg/L; P = 0.015) were independent positive predictors of tHcy, whereas serum folate (8.35 ± 7.43 nmol/L; P = 0.018) and time since transplant (P = 0.049) were significant negative predictors. We conclude that hyperhomocysteinemia is a common characteristic of cardiac transplant recipients. Our analysis suggests that folate and renal glomerular dysfunction are important contributory factors; however, whole blood cyclosporine concentrations may also predict the degree of hyperhomocysteinemia in this population and therefore influence interpretation of any apparent response to treatment.
Automated measurement of serum ferroxidase activity
Authors: Erel Ozcan
Published: January 2020

A method is described for automated measurement of serum ceruloplasmin ferroxidase activity. In this method, Fe2+ ions are used as the substrate. In addition, a new calibration system without ceruloplasmin is also presented. Optimum assay reaction conditions were determined. Maximal catalytic activity was obtained at 0.45 mol/L acetate buffer, pH 5.8. The reagents and calibrator are stable for at least 6 months. Significant correlations between serum ferroxidase and p-phenylenediamine oxidase activities (r = 0.96; P <0.0001) and copper concentration (r = 0.93; P <0.0001) were found. The range for serum ceruloplasmin ferroxidase activity in healthy persons was 198-1107 U/L, and in patients with bronchial asthma it was 601-1912 U/L.
Lyophilized bovine hemoglobin as a possible reference material for the determination of hemoglobin derivatives in human blood
Authors: Maas Bart H A , Buursma Anneke , Ernst Rob A J , Maas Anton H J , Zijlstra Willem G
Published: January 2020

We investigated the suitability of a lyophilized bovine hemoglobin (LBH) preparation containing various fractions of oxyhemoglobin (O2Hb), carboxyhemoglobin (COHb), and methemoglobin (MetHb) for quality assessment in multicomponent analysis (MCA) of hemoglobin derivatives. It was demonstrated that a stable preparation of these components after reconstitution yields a hemoglobin solution that is spectrophotometrically equivalent with a fresh bovine hemoglobin solution. The preparation was found to be stable for at least 1 year when it is kept at 2–8 °C and for 1 h after reconstitution. We determined the fractions of O2Hb, COHb, and MetHb of several LBH preparations, using the complete spectra of 480–650 nm with 2-nm intervals and absorptivities as determined for pure LBH solutions. A field trial involving various types of multiwavelength hemoglobin photometers showed the suitability of LBH as a quality-control material. Computer models of the various common multiwavelength hemoglobin photometers may be useful for establishing more accurate target values of LBH preparations for each type of photometer and for studying the importance of the influence of specific factors such as wavelength selection, absorptivity values, and interfering dyes.
Degradation of cardiac troponin I: implication for reliable immunodetection
Authors: Katrukha Aleksei G , Bereznikova Anastasia V , Filatov Vladimir L , Esakova Tatiana V , Kolosova Olga V , Pettersson Kim , Lövgren Timo , Bulargina Tamara V , Trifonov Igor R , Gratsiansky Nikolai A , Pulkki Kari , Voipio-Pulkki Liisa-Maria , Gusev Nikolai B
Published: January 2020

We have analyzed by different immunological methods the proteolytic degradation of cardiac troponin I (cTnI) in human necrotic tissue and in serum. cTnI is susceptible to proteolysis, and its degradation leads to the appearance of a wide diversity of proteolytic peptides with different stabilities. N- and C-terminal regions were rapidly cleaved by proteases, whereas the fragment located between residues 30 and 110 demonstrated substantially higher stability, possibly because of its protection by TnC. We conclude that antibodies selected for cTnI sandwich immunoassays should preferentially recognize epitopes located in the region resistant to proteolysis. Such an approach can be helpful for a much needed standardization of cTnI immunoassays and can improve the sensitivity and reproducibility of cTnI assays.
Analytical and clinical evaluation of an electrochemiluminescence immunoassay for the determination of CA 125
Authors: van Ingen Huub E , Chan Daniel W , Hubl Walter , Miyachi Hayato , Molina Rafael , Pitzel Lutz , Ruibal Alvaro , Rymer Jean C , Domke Ingrid
Published: January 2020

The CA 125 II assay on the Elecsys® 2010 analyzer was evaluated in an international multicenter trial. Imprecision studies yielded within-run CVs of 0.8–3.3% and between-day CVs of 2.4–10.9%; CVs for total imprecision in the manufacturer’s laboratory were 2.4–7.8%. The linear range of the assay extended to at least 4500 kilounits/L (three decades). Interference from triglycerides (10.3 mmol/L), bilirubin (850 μmol/L), hemoglobin (1.1 mmol/L), anticoagulants (plasma), and several widely used drugs was undetectable. Method comparisons with five other CA 125 II assays showed good correlation but differences in standardization. A 95th percentile cutoff value of 35 kilounits/L was calculated from values measured in 593 apparently healthy (pre- and postmenopausal) women. In 95% of patients with benign gynecological diseases CA 125 was ≤190 kilounits/L; 63% of patients with newly diagnosed ovarian carcinoma had values >190 kilounits/L. A comparison of CA 125 values obtained with the Elecsys test and with other common CA 125 tests in monitored patients being treated for ovarian cancer showed identical patterns. In conclusion, the Elecsys CA 125 II assay is linear over a broad range, yields precise and accurate results, is free from interferences, and compares well with other assays.
Combinatorial search for diagnostic agents: Lyme antibody H9724 as an example
Authors: Tu Jian , Yu Zhiguang , Chu Yen-Ho
Published: January 2020

Two peptide libraries, Ac-MXXXXXBBRM and Ac-VXXXXXBBRM, were constructed on TentaGel solid support to search for ligands that bind tightly with the H9724 Lyme antibody. By using an on-bead ELISA, approximately 120 ligands were selected as candidates for further study. Matrix-assisted laser desorption ionization mass spectrometry analysis of the candidate ligands indicated a high rate of occurrence of certain amino acids at the randomized positions. On the basis of the initial screening results, a small library was designed and iteratively synthesized. Subsequent library screenings led to the identification of four peptides, Ac-PQEEGX-NH2 (X = R, K, A, D), that showed specific affinity to the antibody. This combination of solid-phase screening and iterative synthesis is an effective strategy for rapid identification of ligands that bind tightly with disease-specific antibodies and should be applicable, at least in principle, to other ligand-receptor systems. This combinatorial library approach can also be a useful tool for the discovery of novel diagnostic agents.
Radioimmunoassay for monitoring zidovudine in dried blood spot specimens
Authors: Mei Joanne V , Hannon W Harry , Dobbs Trudy L , Bell Carol J , Spruill Cathy , Gwinn Marta
Published: January 2020

We modified and evaluated a RIA for serum and used the modified RIA to measure zidovudine in dried blood spot specimens (DBSs) routinely collected for newborn screening and tested anonymously for maternally acquired HIV antibodies in the national HIV Seroprevalence Survey Among Childbearing Women. DBS calibration and quality-control materials were used to adapt the serum assay to the DBS matrix. The assay had a limit of detection of 24 μg/L serum and was used to measure zidovudine from both whole DBSs and the eluate remaining after HIV antibody screening. We initiated a pilot study to investigate the assay’s performance and assess its potential to determine the implementation of the US Public Health Service recommendations that HIV-infected pregnant women and newborns receive zidovudine treatment to reduce the risk of perinatal HIV transmission.
Total testing process applied to therapeutic drug monitoring: impact on patients’ outcomes and economics
Authors: Schumacher Gerald E , Barr Judith T
Published: January 2020

The Total Testing Process (TTP) refers to the sequence of 11 steps of laboratory testing, beginning with a clinical question prompted by the patient–clinician encounter and concluding with the impact of the test result on patient care. TTP when applied to therapeutic drug monitoring (TDM) emphasizes that TDM must be considered a process involving a series of steps and interrelated activities and not viewed simply as a numerical value for a serum drug concentration. TTP is also an ideal format for organizing and identifying the system-related and patient-centered variables used in outcomes assessment of TDM, as well as providing a template for collecting the cost data needed for economic analyses. Examples are provided for improving application of TDM by practitioners, clinical laboratories, and educators.
Analytical goals in therapeutic drug monitoring
Authors: Bowers Larry D
Published: January 2020

The practice of therapeutic drug monitoring is based on several disciplines, including pharmacokinetics, pharmacodynamics, and chemical analysis. The impact of the analysis on the determination of pharmacokinetic parameters is not well appreciated. Analytical goals in therapeutic drug monitoring should be established by determining the nature of the problem to be solved, selecting the appropriate matrix and methodology to solve the problem, and developing valid analytical schemes that are performed competently with appropriate quality and interpreted within the framework of the problem.
Strategies for physician education in therapeutic drug monitoring
Authors: Bates David W , Soldin Steven J , Rainey Petrie M , Micelli Joseph N
Published: January 2020

Although therapeutic drug monitoring (TDM) is probably very useful overall, studies suggest that it could be used better. Many drug concentrations appear to have inappropriate indications or suboptimal timing, particularly in the inpatient setting. Undermonitoring is also a concern. Thus, it may be possible to both improve the quality of TDM and reduce the overall costs of care. Here we review approaches for improving the use of TDM and present some illustrative experiences. Specific approaches discussed include use of traditional approaches such as lectures and newsletters, multidisciplinary quality improvement efforts, formal TDM services, and use of the computer as a tool for education and behavior change. Computerized methods appear to hold substantial potential, particularly as more organizations develop better information systems, but other approaches are also effective and are complementary. To be most successful, interventions should consider all stages of the process.
Pharmacodynamic monitoring of immunosuppressive drugs
Authors: Yatscoff Randall W , Aspeslet Launa J , Gallant Heather L
Published: January 2020

Pharmacodynamic (PD) monitoring measures the biological response to a drug, which alone—or coupled with pharmacokinetics— provides a novel method for optimization of drug dosing. PD monitoring has been investigated by us and other investigators primarily for four immunosuppressive drugs: cyclosporine (CsA), azathioprine (AZA), mycophenolate mofetil (MMF), and rapamycin (RAPA). PD monitoring of CsA and MMF involves measuring the activity of the enzymes calcineurin and inosine monophosphate dehydrogenase, respectively. The PD of AZA is assessed by measuring the activity of thiopurine methyltransferase, which is induced by a metabolite of AZA, 6-mercaptopurine. The PD for RAPA involves measuring the activity of a P70 S6 kinase in lymphocytes. To date, the most detailed studies have been performed with PD monitoring of CsA and MMF. Similarities exist in the PD responses to CsA and MMF in renal-transplant patients. At trough concentrations in blood, both drugs reduce the activity of their target enzymes by only 50%; however, considerable interpatient variability is evident. Throughout the dosing interval, the enzyme activities parallel the respective drug concentrations. AZA treatment of renal-transplant patients who exhibited an increase in thiopurine methyltransferase activity from time of transplantation resulted in fewer episodes of active rejection. Additional clinical trials are currently underway to relate various pharmacokinetics and PD parameters to clinical response, to ascertain which provides the best guide for dosing. PD monitoring may provide an alternative approach to additional measurements of drug concentrations.
HPLC determination of ketamine, norketamine, and dehydronorketamine in plasma with a high-purity reversed-phase sorbent
Authors: Bolze Sébastien , Boulieu Roselyne
Published: January 2020

We developed an isocratic, selective, and very sensitive HPLC method for the determination of ketamine and its two main metabolites in plasma. The compounds were extracted from plasma by a liquid–liquid extraction with a dichloromethane:ethyl acetate mixture followed by an acidic back-extraction. Separation was achieved on a new stationary phase, Purospher RP-18 end-capped, with a mobile phase containing acetonitrile:0.03 mol/L phosphate buffer (23:77 by vol) adjusted to pH 7.2. Because of the high column efficiency and the significant improvement of peak symmetry, the quantification limit could be down to 5 μg/L for ketamine and norketamine (NK). The intraday and interday CVs ranged from 1.7% to 5.8% and 3.1% to 10.2% for all compounds respectively. The method is sensitive enough for monitoring ketamine, NK, and dehydroketamine in plasma during pharmacokinetic studies after an intravenous bolus of a low dose of ketamine.
Adrenomedullin(1–52) measured in human plasma by radioimmunoassay: plasma concentration, adsorption, and storage
Authors: Lewis Lynley K , Smith Mary W , Yandle Timothy G , Richards A Mark , Nicholls M Gary
Published: January 2020

We describe a specific and sensitive RIA for human adrenomedullin (AM)(1–52). The detection limit and the concentration required for 50% inhibition of binding were 0.1 and 1.2 fmol/tube, respectively. Cross-reactivities with AM(1–12), AM(13–52), calcitonin gene-related peptide, amylin, and other vasoactive hormones were negligible. AM immunoreactivity in normal subjects ranged from 2.7 to 10.1 pmol/L (n = 44). We investigated factors influencing the recovery and measurement of AM in the assay. Recovery of labeled AM (>80%) was markedly higher than that of unlabeled AM (56%). Immunoreactivity of exogenous AM added to plasma decreased up to 70% over four freeze–thaw cycles, whereas endogenous AM was stable. Alkali-treated casein (1 g/L) reduced adsorption of AM to surfaces and significantly increased assay precision compared with bovine serum albumin (P <0.0001). HPLC separation of extracted plasma verified the presence of AM(1–52). We suggest that considerable care is needed to ensure that accurate and reproducible results are obtained from studies quantifying this peptide.
Microchip systems for immunoassay: an integrated immunoreactor with electrophoretic separation for serum theophylline determination
Authors: Chiem Nghia H , Harrison D Jed
Published: January 2020

A glass microchip is described in which reagents and serum samples for competitive immunoassay of serum theophylline can be mixed, reacted, separated, and analyzed. The device functions as an automated microfluidic immunoassay system, creating a lab-on-a-chip. Electroosmotic pumping was used to control first the mixing of 50-fold-diluted serum sample with labeled theophylline tracer in a 1:1 ratio, followed by 1:1 mixing and reaction with anti-theophylline antibody. The 51-nL on-chip mixer gave the same concentration as dilution performed off-chip, within 3%. A 100-pL plug of the reacted solution was then injected into an electrophoresis separation channel integrated within the same chip. Measurements of free and bound tracer by fluorescence detection gave linear calibration curves of signal vs log[theophylline] between 0 and 40 mg/L, with a slope of 0.52 ± 0.03 and an intercept of −0.04 ± 0.04 after a 90-s reaction time. A detection limit of 0.26 mg/L in serum (expressed before the dilution step, actual concentration of 1.3 μg/L at the detector) was obtained. Recovery values were 107% ± 8% for 15 mg/L serum samples.
A competitive reverse transcription–PCR to study apolipoprotein ε gene expression
Authors: Rexin Martin , Feussner Giso
Published: January 2020

We developed a rapid and simple competitive reverse transcription-polymerase chain reaction for the quantification of apoε mRNA in human monocyte-derived macrophages. The method was applied, and its reliability was shown in patients with the familial lipoprotein disorder, type III hyperlipoproteinemia. Type III hyperlipoproteinemic patients express markedly higher concentrations of apoε mRNA when compared with healthy controls. Patients with this disease are usually (>90%) homozygous for a receptor binding-defective isoform of apolipoprotein apo E (apo E2). The higher expression of apoε mRNA in the patients could, therefore, be a physiological mechanism to compensate for functionally defective apo E. The developed procedure might be valuable in assessment of apoε gene expression in human disease.
Direct determination of urinary iodine by inductively coupled plasma mass spectrometry using isotope dilution with iodine-129
Authors: Haldimann Max , Zimmerli Bernhard , Als Claudine , Gerber Hans
Published: January 2020

An inductively coupled mass spectrometric method was developed for the direct determination of iodine in urine. The application of isotope dilution analysis with added 129I offers new possibilities for automatic and accurate determinations. The sample preparation consists of dilution with an ammonia solution containing 129I. The validation was made by comparison with the results obtained in another laboratory by a spectrophotometric method based on the Sandell–Kolthoff reaction. Different regression models, including maximum likelihood estimation, were used to compare the methods. None of the models revealed analytical bias between the two methods. The urine samples analyzed for validation were from three persons previously exposed to an iodine bath and covered a concentration range of 0.2 to 2.8 μmol/L. A detection limit of 0.02 μmol/L, a within-run CV of 2.5%, and a between-run CV of 11.9% were estimated for the proposed method.
Serum kynurenine-to-tryptophan ratio increases with progressive disease in HIV-infected patients
Authors: Huengsberg Mia , Winer John B , Gompels Mark , Round Rachel , Ross Jonathan , Shahmanesh Mohsen
Published: January 2020

An alternative pathway of Trp metabolism involves the conversion of Trp to kynurenine by indoleamine-2,3-dioxygenase, which leads to synthesis of the neurotoxin, quinolinic acid. This study explores the relationship of indoleamine-2,3-dioxygenase activity with stages of HIV infection. Sera from 206 HIV-positive and 72 seronegative subjects were analyzed for Trp and kynurenine. The kynurenine-to-Trp (KT) ratio was calculated. The mean KT ratio of seronegative controls was 36.6 ± 10.9, and the median ratio was 34.9. The upper limit of the seronegative KT ratio, defined as mean + 2 SD, was 58.4. Patients with HIV infection showed a reciprocal relationship between the KT ratio, the CD4 count, and the stage of the disease. The median KT ratios for asymptomatic and AIDS patients were 50.5 and 117.0, respectively. This study shows that the serum Trp concentration is markedly decreased and that the kynurenine concentration is increased with immune stimulation in HIV infection. This may lead to changes in quinolinic acid and explain some of the pathogenesis of AIDS dementia.
Performance of Deming regression analysis in case of misspecified analytical error ratio in method comparison studies
Authors: Linnet Kristian
Published: January 2020

Application of Deming regression analysis to interpret method comparison data presupposes specification of the squared analytical error ratio (λ), but in cases involving only single measurements by each method, this ratio may be unknown and is often assigned a default value of one. On the basis of simulations, this practice was evaluated in situations with real error ratios deviating from one. Comparisons of two electrolyte methods and two glucose methods were simulated. In the first case, misspecification of λ produced a bias that amounted to two-thirds of the maximum bias of the ordinary least-squares regression method. Standard errors and the results of hypothesis-testing also became misleading. In the second situation, a misspecified error ratio resulted only in a negligible bias. Thus, given a short range of values in relation to the measurement errors, it is important that λ is correctly estimated either from duplicate sets of measurements or, in the case of single measurement sets, specified from quality-control data. However, even with a misspecified error ratio, Deming regression analysis is likely to perform better than least-squares regression analysis.
Antioxidant enzymes and fatty acid status in erythrocytes of Down syndrome patients
Authors: Pastor M-Cruz , Sierra Cristina , Doladé María , Navarro Elisabet , Brandi Nuria , Cabré Eduard , Mira Aurea , Serés Agustí
Published: January 2020

The excess of genetic information in patients with Down syndrome (DS) produces an increase in the catalytic activity of superoxide dismutase (SOD1), an antioxidant enzyme coded on chromosome 21. It has been suggested that an increase in oxidative stress in DS patients may cause adverse effects in the cell membranes through the oxidation of polyunsaturated fatty acids (PUFAs). The aim of this study was to evaluate the cellular antioxidant system by determining the catalytic activity of the SOD1, glutathione peroxidase (GPx), catalase (CAT), and glutathione reductase (GR) enzymes and the concentrations of α-tocopherol in red blood cells (RBCs) in a group of 72 DS patients. The profile of fatty acids in the phospholipids of RBC membranes was also evaluated. The activity of the erythrocyte antioxidant enzymes is significantly higher in the DS group than in the control group (SOD1, 635 ± 70 U/g Hb vs 476 ± 67 U/g Hb; CAT, 1843 ± 250 U/g Hb vs 1482 ± 250 U/g Hb; GPx, 23.2 ± 5.3 U/g Hb vs 21.5 ± 3.6 U/g Hb; and GR, 9.32 ± 1.4 U/g Hb vs 6.9 ± 1.3 U/g Hb, respectively). No differences were observed in RBC α-tocopherol concentrations between the two groups studied. Long-chain n6 PUFA (C20:3n6, C20:4n6) concentrations were increased in DS patients, suggesting enhanced Δ-6-desaturase activity. The long-chain n3 PUFA (docosahexenoic acid) does not appear to be affected by increased oxidative stress, probably because of the existence of compensatory antioxidant mechanisms.
Urinary measurement of transforming growth factor-β and type IV collagen as new markers of renal injury: application in diabetic nephropathy
Authors: Ellis Demetrius , Forrest Kimberly Y-Z , Erbey John , Orchard Trevor J
Published: January 2020

Urinary samples were concentrated rapidly and efficiently and were used to develop several protein assays that may be of value in monitoring individuals with progressive renal disorders. Transforming growth factor-β1 (TGF-β1) and retinol binding protein (RBP) were measured with modification of commercially available methods used to assay serum specimens; type 3 collagen (T3C) was measured with a new immunonephelometric assay. The precision characteristics of these assays are comparable with those reported for microalbuminuria. The clinical utility of measuring a panel of these markers was demonstrated in urine samples from 16 control subjects and from 46 individuals with insulin-dependent diabetes mellitus (IDDM) with various albumin excretion rates (AERs). TGF-β1 and T3C were used as markers of cytokine expression and of the renal fibrogenic process, whereas RBP excretion served as a marker of tubular injury or dysfunction. Compared with controls, T3C excretion was significantly increased in 18 normoalbuminuric and further increased in 13 microalbuminuric (AER 20 ≤ 200 μg/min) IDDM subjects. RBP excretion was increased in macroalbuminuric IDDM subjects (AER >200 μg/min, overt nephropathy). Significant correlations were also found between AER and RBP in all but macroalbuminuric individuals, whereas TGF-β1 correlated with T3C excretion in controls and in normoalbuminuric diabetic subjects. Urinary RBP but not AER was an excellent predictor of diabetic nephropathy as defined by serum creatinine (P = 0.0001). This underscores the importance of an early tubulopathy in the subsequent development of glomerulopathy and overt nephropathy. The data suggest that longitudinal monitoring of a panel of urinary markers such as that used in the current study may better define their relevance in progressive glomerulosclerosis and may also provide greater insight into the mechanisms underlying such process.
4-Chlorotestosterone acetate metabolites in cattle after intramuscular and oral administrations
Authors: Le Bizec Bruno , Montrade Marie-Pierre , Monteau Fabrice , Gaudin Isabelle , Andre François
Published: January 2020

The use of 4-chlorotestosterone acetate by farmers for cattle fattening was recently demonstrated although the use of this anabolic steroid is strictly forbidden in the European Union. We investigated the metabolism of 4-chlorotestosterone acetate in the bovine species after intramuscular and oral administration. Nineteen metabolites were detected in urine after intramuscular injection, and eight metabolites were identified. For this purpose, preparative HPLC, mass spectrometry with different ionization modes (electronic impact and chemical ionization), and different acquisition techniques were used (high resolution, selected ion monitoring, and scan measurement). Metabolite stereoisomerism was determined on the basis of retention time and organic synthesis. 4-Chloroepitestosterone (M2), 4-chloroandrost-4-en-3α-ol-17-one (M3), and 4-chloroandrost-4-ene-3,17-dione (M4) were identified as the main urinary markers of intramuscular administration. On the other hand, 4-chloroandrost-4-ene-3α,17β-diol (M7), 4-chloroandrostan-3β-ol-17-one (M5), and M2 were the primary indicators of an oral administration. In addition, we have shown that 95% of the metabolites were sulfo-conjugated, except for M3, which was partially conjugated to glucuronic acid. Finally, the main metabolites (M2, M3, and M4) were easily identified for 1.5 months after intramuscular administration.
Indirect enzyme-linked immunosorbent assay for the quantitative estimation of lysergic acid diethylamide in urine
Authors: Kerrigan Sarah , Brooks Donald E
Published: January 2020

A new antibody to lysergic acid diethylamide (LSD) was used to develop a novel indirect ELISA for the quantification of drug in urine. Evaluation of the new assay with the commercially available LSD ELISA (STC Diagnostics) shows improved performance. The test requires 50 μL of urine, which is used to measure concentrations of drug in the μg/L to ng/L range. The limit of detection was 8 ng/L compared with 85 ng/L in the commercial assay, and analytical recoveries were 98–106%. Our test detected 0.1 μg/L of LSD in urine with an intraassay CV of 2.4% (n = 8) compared with 6.0% for a 0.5 μg/L sample in the commercial assay (n = 20). The upper and lower limits of quantification were estimated to be 7 μg/L and 50 ng/L, respectively. Specificity was evaluated by measuring the extent of cross-reactivity with 24 related substances. Drug determination using the new assay offers both improved sensitivity and precision compared with existing methods, thus facilitating the preliminary quantitative estimation of LSD in urine at lower concentrations with a greater degree of certainty.
Paraquat-induced renal injury studied by 1H nuclear magnetic resonance spectroscopy of urine
Authors: Bairaktari Eleni , Katopodis Kostas , Siamopoulos Kostas C , Tsolas Orestes
Published: January 2020

The herbicide paraquat (1,1′-dimethyl-4,4′-bipyridylium dichloride; PQ), is a poison known to cause delayed mortality due to lung and kidney injuries. High-resolution proton nuclear magnetic resonance (1H NMR) spectroscopy has been extensively applied in evaluating nephrotoxicity by the characteristic perturbations in the excretion pattern of low molecular weight endogenous metabolites. The application of the method allows the rapid localization of the renal injury noninvasively. In this study, we report 1H NMR and conventional clinical chemistry urinalysis in two patients suffering from paraquat intoxication after overdose with suicidal intent. The alterations in the urine NMR spectrum suggest necrosis of the pars recta of the proximal renal tubules. The molecule of paraquat is also clearly detected in the same spectrum. In conclusion, the rapid screening of urine by NMR spectroscopy provides information about both the identity of the poison and the abnormal pattern of endogenous metabolites that characterize the location of the injury in renal tubules and reveals alterations in unusual metabolites that are not commonly measured.
Optimized PCR labeling in mutational and microsatellite analysis
Authors: Esposito Diana Liberata , Palmirotta Raffaele , Verı̀ Maria Concetta , Mammarella Sandra , D’Amico Franca , Curia Maria Cristina , Aceto Gitana , Crognale Stefania , Creati Beatrice , Mariani-Costantini Renato , Battista Pasquale , Cama Alessandro
Published: January 2020

To optimize the labeling and visualization of PCR products we tested different variables, including deoxynucleotide concentration and ratio, dilution of labeled product, number of PCR cycles, and use of one-step or nested labeling protocols. Labeling was achieved using a fixed amount of labeled dATP, whose relative specific activity was varied by adding increasing amounts of cold dATP. Optimal PCR-labeling intensity was reached at dATP concentrations between 0.9 and 7.0 μmol/L, with a peak at 1.8 μmol/L. This concentration corresponded to an optimal ratio between the increase in specific activity and the decrease in DNA yield. Nucleotide imbalances >1:2 were not advantageous. Mutational analysis by single-strand conformational polymorphism (SSCP) was used to validate PCR-labeling protocols. The limiting nucleotide concentrations did not affect SSCP. Clear SSCP patterns were obtained using DNA templates of different sizes derived from several genes. SSCP patterns obtained using one-step or nested PCR-labeling protocols were equivalent and were visualized after overnight exposure, using [α35S]dATP as the label. Dilutions of labeled products ranging between 1:10 and 1:2.5 influenced SSCP patterns, and the lowest dilution tested produced better-defined and more-intense signals. Optimized SSCP conditions allowed the detection of novel and previously characterized nucleotide variants. Clear microsatellite typing was also obtained using optimized protocols and [α35S]dATP as the label.