bluuebunny/crossref_metadata_2025_split huggingface dataset:
Simultaneous determination of 6-thioguanine and methyl 6-mercaptopurine nucleotides of azathioprine in red blood cells by HPLC
Authors: Dervieux Thierry , Boulieu Roselyne 
Published: January 2020

Abstract6-thioguanine (6-TGN) and methyl 6-mercaptopurine nucleotides (Me6-MPNs) are the two major metabolites found in erythrocytes after administration of azathioprine. In an attempt to understand the role of these metabolites in the pharmacologic and toxic activity of thiopurines, we have developed a HPLC method for the simultaneous determination of 6-TGNs and Me6-MPNs in erythrocytes. A simple and rapid treatment procedure based on deproteinization by perchloric acid with dithiothreitol is described. The nucleotides were hydrolyzed to their own bases by heating the sample for 45 min at 100 °C. During acid hydrolysis Me6-MP was converted into a compound analyzed on a Purospher RP18-e column with 0.02 mol/L dihydrogenophosphate buffer–methanol as eluents. With this procedure, mean recoveries of 73.1% and 84.0% for 6-TGN and Me6-MPN derivatives, respectively, were found
A robust approach to reference interval estimation and evaluation
Authors: Horn Paul S , Pesce Amadeo J , Copeland Bradley E 
Published: January 2020

Abstract
We propose a new methodology for the estimation of reference intervals for data sets with small numbers of observations or for those with substantial numbers of outliers. We propose a prediction interval that uses robust estimates of location and scale. The SAS software can be readily modified to do these calculations. We compared four reference interval procedures (nonparametric, transformed, robust with a nonparametric lower limit, and transformed robust) for sample sizes of 20, 40, 60, 80, 100, and 120 from χ2 distributions of 1, 4, 7, and 10 df. χ2 distributions were chosen because they simulate the skewness of distributions often found in clinical chemistry populations. We used the root mean square error as the measure of performance and used computer simulation to calculate this measure. The robust estimator showed the best performance for small sample sizes. As the sample size increased, the performance values converged. The robust method for calculating upper reference interval values yields reasonable results. In two examples using real data for haptoglobin and glucose, the robust estimator provides slightly smaller upper reference limits than the other procedures. Lastly, the robust estimator was compared with the other procedures in a population where 5% of the values were multiplied by a factor of 5. The reference intervals were calculated with and without outlier detection. In this case, the robust approach consistently yielded upper reference interval values that were closer to those of the true underlying distributions. We propose that robust statistical analysis can be of great use for determinations of reference intervals from limited or possibly unreliable data.
Automated fluorescent analysis procedure for enzymatic mutation detection
Authors: Del Tito Benjamin J , Poff Herbert E , Novotny Mark A , Cartledge Donna M , Walker Ralph I , Earl Christopher D , Bailey Anne L 
Published: January 2020

AbstractThe Enzymatic Mutation Detection™ (EMD) assay detects mutations or polymorphisms in DNA. The assay procedure takes &lt;1 h and is followed by electrophoretic detection. We report an automated procedure, using fluorescently labeled probe and quantitative analysis on the ABI Prism™ 377 DNA Sequencer, that improves on earlier methods (1,2) by eliminating the need for sample purification, shortening the hybridization time, and increasing the signal-to-noise ratio. The EMD assay uses the bacteriophage resolvase T4 endonuclease VII, which cleaves the heteroduplex molecules at the mismatch site, forming two shorter fragments that are resolved by gel electrophoresis. Unlike existing mutation techniques, the EMD method uses a single protocol to identify point mutations, deletions, and insertions for all DNA fragments. Test DNA samples are assayed directly from PCR reactions, and fragments up to 4 kb in size have been assayed successfully. A independent analysis on the p53 tumor suppressor gene from clinical samples has shown 100% sensitivity and 94% specificity. Because the fluorescent EMD assay has been optimized for high signal-to-noise ratios, mutations can be identified in mixed samples containing up to a 20-fold excess of normal DNA.
Reference standardization and triglyceride interference of a new homogeneous HDL-cholesterol assay compared with a former chemical precipitation assay
Authors: Cobbaert Christa , Zwang Louwerens , Ceriotti Ferruccio , Modenese Annalisa , Cremer Peter , Herrmann Wolfgang , Hoss Gerhard , Jarausch Jochen , Türk Regina , März Winfried , Nauck Matthias 
Published: January 2020

Abstract
A homogeneous HDL-c assay (HDL-H), which uses polyethylene glycol-modified enzymes and sulfated α-cyclodextrin, was assessed for precision, accuracy, and cholesterol and triglyceride interference. In addition, its analytical performance was compared with that of a phosphotungstic acid (PTA)/MgCl2 precipitation method (HDL-P). Within-run CVs were ≤1.87%; total CVs were ≤3.08%. Accuracy was evaluated in fresh normotriglyceridemic sera using the Designated Comparison Method (HDL-H = 1.037 Designated Comparison Method + 4 mg/L; n = 63) and in moderately hypertriglyceridemic sera by using the Reference Method (HDL-H = 1.068 Reference Method − 17 mg/L; n = 41). Mean biases were 4.5% and 2.2%, respectively. In hypertriglyceridemic sera (n = 85), HDL-H concentrations were increasingly positively biased with increasing triglyceride concentrations. The method comparison between HDL-H and HDL-P yielded the following equation: HDL-H = 1.037 HDL-P + 15 mg/L; n = 478. We conclude that HDL-H amply meets the 1998 NCEP recommendations for total error; its precision is superior compared with that of HDL-P, and its average bias remains below ±5% as long as triglyceride concentrations are ≤10 g/L and in case of moderate hypercholesterolemia.
Analytical approaches of European Union laboratories to drugs of abuse analysis
Authors: Badia Roser , de la Torre Rafael , Corcione Sergio , Segura Jordi 
Published: January 2020

AbstractWe report a survey on urine drug testing within a total of 269 laboratories of the European Union. Clinical laboratories predominated over forensic laboratories (59.5% vs 28.5%). Screening without identification/quantification was the common approach used by clinical laboratories, whereas screening with identification/quantification was the approach used by almost all forensic laboratories. Screening was primarily performed by immunoassay in both types of laboratories. Gas chromatography coupled to mass spectrometry was the main analytical method used for specific identification/quantification of drugs, but other methods (including immunoassays) were also used. Cutoff values applied varied by laboratory type, country, and method used. A high percentage of laboratories did not use or report cutoff values. Overall, countries of the European Union vary significantly in regards to drugs tested, analytical approach, and screening and identification cutoff values. It is recommended to clearly state the analytical method and the cutoff values used when reporting results for drugs of abuse testing.
Fully automated assay for total homocysteine, cysteine, cysteinylglycine, glutathione, cysteamine, and 2-mercaptopropionylglycine in plasma and urine
Authors: Pastore Anna , Massoud Renato , Motti Corradino , Russo Anna Lo , Fucci Giorgio , Cortese Claudio , Federici Giorgio 
Published: January 2020

AbstractWe describe a 6-min HPLC method to measure the total concentrations of the most important thiols in plasma and urine–cysteine, homocysteine, cysteinylglycine, and glutathione–as well as the concentrations in plasma and urine, respectively, of cysteamine and 2-mercaptopropionylglycine, two compounds used to treat disorders of cysteine metabolism. Precolumn derivatization with bromobimane and reversed-phase HPLC were performed automatically by a sample processor. Throughput was up to 100 samples in 24 h. The within-run CV ranged from 0.9% to 3.4% and the between-run CV ranged from 1.5% to 6.1%. Analytical recovery was 97–107%, with little difference between plasma and urine samples. The detection limit was ∼50 nmol/L for all the analytes studied. Thiol concentrations were determined in the plasma of 206 healthy donors and in the urine of 318 healthy donors distributed for age and sex. Mean values of plasma cysteine and homocysteine were significantly lower in infants (ages, &lt;1 y) compared with other age groups (P &lt;0.005). In adults, mean plasma homocysteine values were higher in males than in females (9.2 vs 6.7 μmol/L, P &lt;0.0001) and in the 6- to 10-year-old group (P &lt;0.05). Mean values for glutathione and cysteinylglycine were not sex- and age-dependent. In urine, both cysteine and homocysteine showed a wide range of variation.
A stochastic approximation method for assigning values to calibrators
Authors: Schlain Brian 
Published: January 2020

AbstractA new procedure is provided for transferring analyte concentration values from a reference material to production calibrators. This method is robust to calibration curve-fitting errors and can be accomplished using only one instrument and one set of reagents. An easily implemented stochastic approximation algorithm iteratively finds the appropriate analyte level of a standard prepared from a reference material that will yield the same average signal response as the new production calibrator. Alternatively, a production bulk calibrator material can be iteratively adjusted to give the same average signal response as some prespecified, fixed reference standard. In either case, the outputted value assignment of the production calibrator is the analyte concentration of the reference standard in the final iteration of the algorithm. Sample sizes are statistically determined as functions of known within-run signal response precisions and user-specified accuracy tolerances.
Optimized serum pancreolauryl test for differentiating patients with and without chronic pancreatitis
Authors: Domínguez-Muñoz J Enrique , Malfertheiner Peter 
Published: January 2020

Abstract
The serum pancreolauryl test has limited sensitivity for detecting mild pancreatic insufficiency. The aim of this study was to optimize the serum pancreolauryl test so as to increase the probability of positive results in patients with chronic pancreatitis. The study had three parts. First, the sampling time was optimized by analyzing retrospectively the frequency of fluorescein peaks at different times from 0 to 240 min in 560 consecutive patients. Second, the calculation of serum fluorescein concentrations by means of a standard calibration factor was prospectively compared in 271 consecutive patients with a modification involving a specimen-specific calibration factor for each patient. Third, the clinical utility of the intravenous injection of secretin before ingestion of the test meal was prospectively evaluated in a further 32 patients. As a result, the optimized serum pancreolauryl test developed differs from the former version of the test in utilizing intravenous administration of secretin before the test meal, calculation of serum fluorescein based on specimen-specific calibration factors, and blood samples taken only at 0 (basal), 120, 150, 180, and 240 min. This optimized pancreolauryl test was abnormal more frequently in patients with chronic pancreatitis than was the formerly used test, especially for cases of mild and moderate disease.
Misleading serum free thyroxine results during low molecular weight heparin treatment
Authors: Stevenson Heather P , Pooler G , Archbold R , Johnston Paul , Young Ian S , Sheridan Brian 
Published: January 2020

Abstract
Measured free thyroxine concentrations in serum increase markedly after intravenous heparin administration, but the effect of heparin administered subcutaneously has not been adequately documented. We found in vitro increases of up to 63% in measured FT4 after a single dose of subcutaneous heparin (enoxaparin, 2000 units) in nine healthy volunteers, and the magnitude of these increases was correlated with initial serum triglyceride concentrations (r = 0.93, P &lt;0.005) and in vitro free fatty acid release (r = 0.88, P &lt;0.005). In 10 cardiac inpatients receiving repeated doses of enoxaparin (2000 units twice daily), measured FT4 increased by up to 171% in specimens taken 2–6 h after injection. When specimens were obtained 10 h after injection, the effect appeared to be minimized, with in vitro increases of &lt;40%, but such increases may still be sufficient to cause interpretative errors. If FT4 estimation is absolutely necessary in patients receiving enoxaparin, specimens should be taken ≥10 h postdose and analyzed within 24 h.
Standards of laboratory practice: analgesic drug monitoring
Authors: White Suzanne , Wong Steven H Y 
Published: January 2020

AbstractAnalgesics are the most commonly consumed over-the-counter preparations in the United States. They are used in the treatment of various pain syndromes and other medical conditions. Although analgesics are generally perceived to be safe agents, serious toxicity may occur in the setting of acute overdose, chronic abuse, or overuse. The indications for therapeutic drug monitoring in patients using these medications appropriately is as yet not well defined. The emphasis of this discussion, therefore, is on recommendations for monitoring in situations where toxicity is suspected. Preanalytical, analytical, and practice issues including drug interactions, frequency of monitoring, pertinent ancillary tests, reporting, and special patient groups at risk for toxicity are reviewed. Recent information from a major manufacturer of evacuated tubes arguing against the use of gel tubes for blood collection for drug monitoring is included. Colorimetric/enzymatic/immunoassays for the routine/stat monitoring of acetaminophen and salicylate and diflunisal cross-reactivity with most of the currently used salicylate assays are presented. Achiral and chiral chromatographic assays and newly introduced columns such as restricted access media and/or automated chromatographic systems are reviewed for the analysis of ibuprofen, naproxen, and the recently introduced tramadol. Finally, concepts regarding future directions including drug chirality and chiral analysis are presented.
A homogeneous fluorescence assay for PCR amplicons: its application to real-time, single-tube genotyping
Authors: Whitcombe David , Brownie Jannine , Gillard Helen L , McKechnie Doug , Theaker Jane , Newton Clive R , Little Stephen 
Published: January 2020

Abstract
We have developed a method whereby a single TaqMan™ probe can be used for many PCR reactions. We demonstrate its application as an integrated system for the direct measurement of allele-specific amplicon generation coupled to the suppression of primer-dimer accumulation in PCR. The system uses a 5′-exonuclease assay of amplicon annealed fluorogenic probes that operates in conjunction with the Amplification Refractory Mutation System, whereby relative changes in reporter fluorescent emission are monitored in real-time using an analytical thermal cycler. We have called this system Three-STAR, and it is universal in that it can either use a single probe for the detection of any one target DNA sequence or a single pair of probes for genotyping any bi-allelic polymorphism. Three-STAR is, therefore, particularly useful for the single-tube genotype analysis of a variety of human DNA polymorphisms and mutations.
Evaluation of a homogeneous assay for high-density lipoprotein cholesterol: limitations in patients with cardiovascular, renal, and hepatic disorders
Authors: Simó Josep M a , Castellano Isabel , Ferré Natàlia , Joven Jorge , Camps Jordi 
Published: January 2020

Abstract
We evaluated the performance of a homogeneous assay for the automated measurement of high-density lipoprotein cholesterol (HDL-C) and compared it with a conventional precipitation technique in the following groups of people: control subjects (group A), clinically-healthy elderly (group B), myocardial infarction patients (group C), nephrotic syndrome patients (group D), and liver cirrhosis patients (group E). The performance of the technique was acceptable with respect to precision, accuracy, linearity, and detection limit. Triglycerides up to 40 mmol/L and bilirubin up to 150 μmol/L did not cause interferences. Hemoglobin decreased HDL-C measurements. Samples were stable at −20 °C for up to four months. Bland–Altman plots showed a good agreement between both techniques in the control group but with a progressive divergence in the patient groups B to E. Results indicate limitations of the technique in certain clinical conditions and, coincidentally, the need for reliable calibration materials.
Analytical and clinical performance of a homogeneous enzymatic LDL-cholesterol assay compared with the ultracentrifugation-dextran sulfate-Mg2+ method
Authors: Rifai Nader , Iannotti Elizabeth , DeAngelis Kristen , Law Terence 
Published: January 2020

Abstract
LDL-cholesterol (LDL-C) concentration is currently determined in most clinical laboratories by the Friedewald calculation. This approach has several limitations and may not meet the current total error requirement in LDL-C measurement of ≤ 12%. We evaluated the analytical and clinical performance of the direct N-geneous LDL-C assay (Equal Diagnostics). The N-geneous method correlated highly with the modified beta-quantification assay (r = 0.95; y = 0.91x + 70.6 mg/L; n = 199), showed no significant effect of increased triglyceride or other common interferants, and performed adequately in serum samples from nonfasting individuals. This assay demonstrated a mean total error of 6.75% over a wide range of LDL-C concentrations. In addition, at the medical decision cutoff points, this LDL-C assay showed positive predictive values of 78–95% and negative predictive values of 84–99%. We conclude that the N-geneous LDL-C meets the currently established analytical performance goals and appears to have a role in the diagnosis and management of hypercholesterolemic patients.
Determination of ciprofloxacin in plasma and urine by HPLC with ultraviolet detection
Authors: Kamberi Marika , Tsutsumi Kimiko , Kotegawa Tsutomu , Nakamura Koichi , Nakano Shigeyuki 
Published: January 2020

Abstract
A simple, sensitive isocratic method for the detection and quantification of ciprofloxacin in plasma and urine has been developed. The assay consisted of reversed-phase HPLC with ultraviolet detection. Plasma proteins were removed by a fast and efficient procedure. For the urine samples, the only required sample preparation was dilution. Separation was achieved on a C18 reversed-phase column. The quantification limit was 0.01 mg/L in plasma and 0.5 mg/L in urine. This method was sufficiently sensitive for pharmacokinetic studies.
Non-separation assay for glycohemoglobin
Authors: Blincko Stuart , Edwards Raymond 
Published: January 2020

Abstract
The determination of glycohemoglobin [HbA1c, HbA1, or total glycohemoglobin (GHb)] has become an established procedure in the management of diabetes mellitus. Here, we describe the development of a simple, fluorescence, non-separation assay for the percentage of GHb (%GHb). The fluorescence of an eosin-boronic acid derivative when it was mixed with hemolysates of unwashed erythrocytes was quenched in proportion to the percentage of glycohemoglobin. Measurement of the fluorescence intensity gave an estimate of GHb in the sample, and measurement of light absorbance gave an estimate of total hemoglobin. A combination of the two measurements gave the assay response. Comparison with HPLC (Menarini-Arkray HA-8140 fully automated analyzer) for the percentage of HbA1 (%HbA1) gave %GHb(NETRIA) = 1.1(SD ±0.03)%HbA1 +0.6(SD ±0.3), Sy‖x = 0.821, r = 0.972, n = 80; comparison for HbA1c gave %GHb(NETRIA) = 1.3(SD ±0.04)%HbA1c + 1.8(SD ±0.3), Sy‖x = 0.813, r = 0.973, n = 80. Precision, estimated as the percentage of the CV of the %GHb assay results, was &lt;2% (intraassay, range 5–22% GHb) and &lt;4.2% (interassay, range 4–16% GHb). Dilution of a high-percentage GHb sample lysate showed that the assay was linear, and addition of glucose (60 mmol/L), bilirubin (250 μmol/L), and triglycerides (14 mmol/L) to low, medium, and high %GHb samples showed no clinical interference in assay results.
Rapid sequencing of the p53 gene with a new automated DNA sequencer
Authors: Bharaj Bhupinder S , Angelopoulou Katerina , Diamandis Eleftherios P 
Published: January 2020

Abstract
p53 is the most commonly mutated gene in human cancers. Approximately 90% of the p53 gene mutations are localized between domains encoding exons 5 to 8. Sequencing methods currently available are tedious and time-consuming and are not suitable for routine laboratory testing. In an effort to identify a simple and rapid sequencing method, we analyzed 16 preselected breast tumors and 18 preselected ovarian tumors, using a newly developed automated DNA sequencer. p53 gene mutations had been previously identified in these tumors, using a conventional automated sequencing procedure. Exons 5 to 8 were amplified by PCR, and the PCR products were subsequently subjected to cycle sequencing with the Sanger chain termination method, using Cy5.5-labeled primers. The sequencing mixture was then resolved on a newly developed automated DNA sequencer that can sequence ∼300 bases of DNA in 30 min. Of these 16 breast tumors, two had mutations in exon 5, four in exon 6, three in exon 7, and three in exon 8. Of the 18 ovarian tumors, two had mutations in exon 5, five in exon 6, two in exon 7, and three in exon 8. In all cases, we identified the same mutations by both the new and the conventional sequencing procedures. Most mutations affected an arginine codon. These data demonstrate that the new method has the capability to provide accurate sequencing information in a fraction of the time and labor in comparison with current automated sequencing techniques. When such procedures are used, DNA sequencing may become a routine tool for identifying clinically important mutations for diagnosis and prognosis of patients with genetic, malignant, infectious, and other diseases.
Highly specific radioimmunoassay for human insulin based on immune exclusion of all insulin precursors
Authors: Deberg Michelle , Houssa Paule , Frank Bruce H , Sodoyez-Goffaux Françoise , Sodoyez Jean-Claude 
Published: January 2020

AbstractWe describe a rapid and simple insulin RIA in which proinsulin and conversion intermediates do not interfere. Three monoclonal antibodies (S1, S2, and S53) were selected for their specificity (directed, respectively, against the B10 region, the junction between A chain and C-peptide, and the junction between B chain and C-peptide), their affinity constant (∼1010 L/mol), and their interactive properties in mixture. S2 and S53 were able to bind simultaneously to the same proinsulin molecule, whereas neither could bind simultaneously with S1. Preincubation of serum samples with an excess of S2 resulted in capture of proinsulin and conversion intermediates modified at the junction between B chain and C-peptide into immune complexes that no longer reacted with S1. Similarly, preincubation with S53 prevented proinsulin and conversion intermediates modified at the junction between A chain and C-peptide from reacting with S1. Preincubation with an excess of both S2 and S53 left insulin as the sole reactant with S1. Thus, separation of insulin precursors from insulin by mutually exclusive antibodies is feasible, and on the basis of this new principle, a highly specific RIA for insulin was designed. The detection limit was 11 pmol/L, and the inter- and intraassay coefficients of variation were 11% and 5%, respectively. The potential of the assay for use in clinical studies was verified by application to serum samples from control subjects and patients with diabetes or insulinoma.