bluuebunny/crossref_metadata_2025_split huggingface dataset:
TaqMan PCR-based Gene Dosage Assay for Predictive Testing in Individuals from a Cancer Family with INK4 Locus Haploinsufficiency
Authors: Laurendeau Ingrid , Bahuau Michel , Vodovar Nicolas , Larramendy Claire , Olivi Martine , Bieche Ivan , Vidaud Michel , Vidaud Dominique 
Published: January 2020

Abstract
Background: A genetic syndrome of cutaneous malignant melanoma and nervous system tumors recently has been characterized and shown to be linked to the INK4 locus in the 9p21 region. Hemizygosity at adjacent physically mapped microsatellite markers indicated deletion of p16, p19, and p15 clustered tumor suppressors. Because individuals from this family could benefit from predictive testing in terms of cancer prevention, we developed a direct test without need to analyze parental DNAs to comply with the rules of individual consent and secrecy.
Methods: We developed an assay using TaqManTM real-time quantitative PCR, with p15 as the test sequence and albumin (ALB) as the reference gene. The normalized ratio of p15/ALB is expected to yield a value of ∼1 in individuals without the deletion, whereas a ratio of ∼0.5, indicating p15 haploinsufficiency, is expected in predisposed individuals.
Results: All patients harboring the previously defined at-risk haplotype were correctly identified using this approach. In six individuals with deletions, the p15/ALB ratios were 0.472–0.556 (SD, 0.013–0.078). In the five individuals without deletions, the ratios were 0.919–1.019 (SD, 0.006–0.075).
Conclusions: This is the first report of a high-throughput, automatable gene dosage assay successfully applied to the identification of a germ-line deletion. This approach, not limited by marker informativeness or the need for harvesting live cells, can be applied to any condition with haploinsufficiency and extended to the characterization of most abnormalities of the ploidy.
Optimization of Allopurinol Challenge: Sample Purification, Protein Intake Control, and the Use of Orotidine Response as a Discriminative Variable Improve Performance of the Test for Diagnosing Ornithine Carbamoyltransferase Deficiency
Authors: Arranz José A , Riudor Encarnació , Rodés Margarita , Roig Manuel , Climent Consuelo , Rubio Vicente , Sentís Margarita , Burlina Alberto 
Published: January 2020

Abstract
Background: The diagnosis of heterozygosity for X-linked ornithine carbamoyltransferase (OCT) deficiency has usually been based on measurement of the increase of orotate and orotidine excretion after an allopurinol load. We examined the choices of analyte, cutoff, and test conditions to obtain maximal test accuracy.
Methods: Urine orotate/orotidine responses to allopurinol load in 37 children (13 OCT-deficient and 24 non-OCT-deficient) and 24 women (7 at risk for carrier status and 17 not related to OCT-deficient children) were analyzed by liquid chromatography after sample purification by anion-exchange chromatography. Diagnostic accuracy was evaluated by nonparametric ROC curves.
Results: Sample purification was necessary to prevent interferences. Orotate and orotidine excretion increased with increased protein intake during the test. At a cutoff of 8 mmol orotidine/mol creatinine, sensitivity was 1.0 and specificity was 0.92 in mild forms of OCT deficiency. Results in monoplex carrier women may differ greatly from those expected because of the genetics of this deficiency.
Conclusions: Standardization of protein intake is required in the allopurinol loading test. A negative response in the face of clinical suspicion should be followed with a repeat test during a protein intake not &lt;2.5 g · kg−1 · day−1. Measurements of orotidine provide better clinical sensitivity than measurements of orotate.
Rapid, Homogeneous Genotyping of the 4G/5G Polymorphism in the Promoter Region of the PAI1 Gene by Fluorescence Resonance Energy Transfer and Probe Melting Curves
Authors: Nauck Markus , Wieland Heinrich , März Winfried 
Published: January 2020

AbstractBackground: Many studies have convincingly shown that survivors of myocardial infarction have impaired fibrinolytic activity because of increased concentrations of plasma plasminogen activator inhibitor-1 (PAI-1). A single guanosine insertion/deletion polymorphism in the promoter region of the PAI1 gene, commonly called 4G/5G, has been shown to be associated with plasma PAI-1 activity. Our aim was to develop and validate a homogeneous assay for rapid genotyping of the 4G/5G polymorphism.Methods: In this report we present a single-tube method for genotyping of the 4G/5G polymorphism that combines both rapid-cycle PCR with real-time monitoring of the amplification process and generation of allele-specific fluorescent probe melting profiles on the LightCyclerTM. Two fluorescently labeled hybridization probes recognizing adjacent sequences in the amplicon were present in the reaction mixture. The shorter detection probe spanned the polymorphic site, perfectly matching the 5G allele. After annealing, the fluorophores were in resonance energy transfer, providing real-time monitoring of the amplification process. At the completion of the PCR, fluorescence was monitored as the temperature increased through the Tm of the probe/product duplex, and a characteristic melting profile for each genotype was obtained.Results: With this method, 32 samples were genotyped within 30 min without the need of any post-PCR sample manipulation. The genotypes of 100 DNA samples determined with the LightCycler were identical to those obtained with conventional PCR and restriction fragment length analysis.Conclusion: The genotyping of the 4G/5G polymorphism with the LightCycler is a rapid, reliable method that is suitable for typing both small and large numbers of samples.
Isocitrate as Calcium Ion Activity Buffer in Coagulation Assays
Authors: RÅnby Mats , Gojceta Tony , Gustafsson Kerstin , Hansson Kenny M , Lindahl Tomas L 
Published: January 2020

AbstractBackground: Ca2+ activity close to the physiological concentration of 1.3 mmol/L is essential in blood coagulation. Is this also true for the performance of global diagnostic coagulation assays? We searched for compounds that would buffer Ca2+ activity at ∼1.3 mmol/L without disturbing coagulation reactions and investigated whether such Ca2+ buffering improves diagnostic efficacy in global diagnostic coagulation tests.Methods: Buffering was investigated by mixing CaCl2 and 11 candidate compounds and determining Ca2+ activity. The best candidates were added to mixtures of plasma and thromboplastin to detect interference with coagulation reactions. The best of these candidates, isocitrate, was used to modify an activated partial thromboplastin time (APTT), buffering final Ca2+ activity to ∼1.3 mmol/L. Plasma samples from 22 healthy individuals and 120 patients were analyzed with original and modified APTT to determine whether diagnostic efficacy was improved.Results: Two suitable Ca2+ buffers, citrate and isocitrate, were found. Isocitrate was preferred as being less coagulation inhibitory, a better Ca2+ buffer, and possibly a better anticoagulant. The isocitrate-modified APTT showed a final Ca2+ activity of 1.60 ± 0.07 mmol/L, compared with 2.73 ± 0.20 mmol/L for the original APTT. The means and SDs for the healthy individuals were determined for both procedures, and the values were used to express patient deviation from normality (difference from mean divided by SD). The deviation was greater for the modified APTT; 4.3 ± 5.7, compared with 3.6 ± 5.0 (P &lt;0.005) for the original APTT.Conclusions: Isocitrate can be used to buffer Ca2+ activity at physiological concentrations and can serve as an anticoagulant. APTT with isocitrate-buffered Ca2+ activity shows signs of improved diagnostic efficacy.
Raging Hormones: Closing the Loop from Bench to Bedside. The Twenty-Second Arnold O. Beckman Conference in Clinical Chemistry
Authors: Ashwood Edward R 
Published: January 2020

AbstractThe Twenty-second Annual 1999 Arnold O. Beckman Conference was held on February 21 and 22, 1999, in San Antonio, Texas. In keeping with the tradition of the conference, the goal of this year’s meeting was to strengthen attendees’ knowledge of clinical endocrinology and the use of the clinical laboratory. The conference was divided into four sessions: basic science, diabetes, bone, and diagnostic endocrinology.
Quantitative Reverse Transcription-PCR Assay with an Internal Standard for the Detection of Prostate-specific Antigen mRNA
Authors: Ylikoski Alice , Sjöroos Minna , Lundwall Åke , Karp Matti , Lövgren Timo , Lilja Hans , Iitiä Antti 
Published: January 2020

Abstract
Background: Circulating prostate cells can be detected with a reverse transcription-PCR (RT-PCR) assay for prostate-specific antigen (PSA) mRNA. We have developed a new quantitative RT-PCR method for measuring PSA mRNA.
Methods: The method uses a PSA-like internal standard (IS) mRNA that is added into the sample at the beginning of the RNA extraction and coamplified by RT-PCR with the PSA in the sample. After PCR amplification, the IS and PSA products are selectively detected by hybridization in a microtitration plate using probes labeled with fluorescent europium chelates.
Results: The method was validated with PSA and IS mRNAs and PSA-expressing cells to obtain a detection limit of 50 PSA mRNA copies (i.e., signal 2 times the mean of zero signal), linearity up to 106 copies, and detection of a single PSA-expressing cell. In preliminary evaluations, 60% (n = 10) of the prostate cancer patients with skeletal metastases gave results above the detection limit (500 PSA mRNA copies in 5 mL of blood). The total number of PSA copies ranged from 900 ± 200 to 44 100 ± 4900 (mean ± SD) in the samples, corresponding to ∼1–100 PSA-expressing cells in 5 mL of blood. In the controls (n = 34), none of the healthy females and 2 of 19 healthy males had detectable PSA mRNA [700 ± 100 and 2000 ± 900 (mean ± SD) PSA mRNA copies in 5 mL of blood for the 2 males].
Conclusions: The assay provides sensitive and quantitative detection of PSA mRNA expression from blood samples and can be used to establish the clinically significant number of PSA mRNA copies in prostate cancer.
Analytical Validation of the PRO-Trac II ELISA for the Determination of Tacrolimus (FK506) in Whole Blood
Authors: MacFarlane Gordon D , Scheller Daniel G , Ersfeld Diana L , Shaw Leslie M , Venkatarmanan Raman , Sarkozi Laszlo , Mullins Richard , Fox Bonnie R 
Published: January 2020

AbstractBackground: The analytical validation of multiple lots of the PRO-TracTM II ELISA (DiaSorin) for the determination of tacrolimus in whole blood is described.Methods: The analytical parameters assessed included analytical sensitivity, dilution linearity, functional sensitivity, values in samples containing no tacrolimus, intra- and interassay precision, supplementation and recovery, metabolite cross-reactivity, interference studies, and method comparisons HPLC-tandem mass spectrometry (HPLC/MS/MS) and the IMx® Tacrolimus II multiparticle enzyme immunoassay. Where appropriate, assessments were performed according to NCCLS guidelines.Results: The mean analytical detection limit was &lt;0.25 μg/L for all lots, whereas the functional sensitivity was 1.0 μg/L. Excellent linear correlation (r = 0.985) was observed for dilution linearity. The intraassay imprecision was &lt;7%, and the total imprecision by ANOVA was &lt;10%. Recovery was 109% ± 11%. Metabolite cross-reactivity was consistent with previous reports for this antibody. No interference was observed for 35 tested drugs. Method comparison with HPLC/MS/MS showed no statistically significant differences. Samples exhibited stability through four freeze/thaw cycles and for 1 week at room temperature.Conclusion: These data demonstrate that the PRO-Trac II ELISA is a robust, accurate, and precise tool for the assessment and management of tacrolimus blood concentrations in transplant patients.
Simple Method for Determination of Thiocyanate in Urine
Authors: Haque M Rezaul , Bradbury J Howard 
Published: January 2020

Abstract
Background: It would be useful to develop a simple kit method for determination of thiocyanate in urine, which could be used to monitor cyanide overload in cassava-consuming populations.
Methods: The method was based on the quantitative oxidation of thiocyanate in acid permanganate at room temperature in a closed vial with liberation of HCN, which reacted with a picrate paper. For semiquantitative analysis in the field, the colored picrate paper was matched with a color chart prepared using known amounts of KSCN. In the laboratory, a more accurate result was obtained by elution of the colored complex in water and measurement of the absorbance at 510 nm. Over the range 0–100 mg/L, there was a linear relationship given by the equation: thiocyanate content (mg/L) = 78 × absorbance.
Results: The picrate thiocyanate method gave no interference with urine samples containing protein at less than 7 g/L, 21 amino acids, histamine, glucose, NaCl, urea, blood, and linamarin. For 53 urine samples analyzed by an accurate column method and the thiocyanate picrate method, a regression line gave very good agreement (r2 = 1.000). Quantitative recoveries of thiocyanate added to urine samples were obtained with the picrate method.
Conclusions: A simple picrate kit for determination of thiocyanate in urine was developed and is available free of charge for workers in developing countries.
Codeine Concentration in Hair after Oral Administration Is Dependent on Melanin Content
Authors: Kronstrand Robert , Förstberg-Peterson Sophie , KÅgedal Bertil , Ahlner Johan , Larson Göran 
Published: January 2020

AbstractBackground: Analysis of drugs in hair has been used on a qualitative basis to estimate earlier exposure to drugs. Clinical applications are rare because of the lack of dose–response relationships in the studies performed to date, and questions remain regarding the mechanisms of drug incorporation into hair. Several human studies have shown differences in drug accumulation between pigmented and nonpigmented hair. However, the melanin concentration in hair was not determined and correlated to the amount of drug incorporated.Methods: Nine human subjects were given codeine as a single oral dose, and plasma codeine concentrations were determined for 24 h, using gas chromatography–mass spectrometry. Hair samples were obtained weekly for a month. Total melanin, eumelanin, and codeine were measured quantitatively in hair samples by spectrophotometry, HPLC, and gas chromatography–mass spectrometry, respectively.Results: There was an exponential relationship between codeine and melanin concentrations in hair, (r2 = 0.95 with total melanin and r2 = 0.83 with eumelanin). After normalizing the results by the area under the curve for codeine in plasma, we obtained r2 = 0.86 for codeine vs total melanin and r2 = 0.90 vs eumelanin.Conclusions: Our results stress the importance of melanin determination when measuring drugs in hair. We postulate that analysis of drug concentration in hair may be worthwhile in the monitoring of drug compliance if the results are normalized for melanin content.
A Collection Method and High-Sensitivity Enzyme Immunoassay for Sweat Pyridinoline and Deoxypyridinoline Cross-Links
Authors: Sarno Mark , Powell Helen , Tjersland Gayle , Schoendorfer Donald , Harris Holden , Adams Kimberly , Ogata Peggy , Warnick G Russell 
Published: January 2020

AbstractBackground: Collagen cross-link molecules such as pyridinoline (PYD), deoxypyridinoline (DPD), and N-terminal cross-linked peptides (NTX) have been measured in urine as indices of bone resorption. However, very little is known regarding the excretion of pyridinolines into other biological fluids. We report a collection device, normalizing analyte, and high-sensitivity immunoassay for quantitative analysis of free pyridinoline cross-links in sweat.Methods: Flame atomic emission and ion-selective electrode techniques were used to measure potassium as a sweat volume marker. The Pyrilinks immunoassay for urine free pyridinolines was optimized to increase sensitivity for measurements in sweat. The precision, accuracy, and detection limit of this assay were characterized. To assess values and variability of sweat pyridinolines in human subjects, a nonocclusive skin patch was used to collect sweat samples from a reference group and from a mixed group experiencing accelerated bone resorption, postmenopausal women and men receiving gonadotropin-releasing hormone for prostate cancer.Results: The immunoassay intra- and interassay variations were ≤10% and &lt;16%, respectively, with a detection limit of 309 pmol/L. Linearity upon dilution and analytical recovery ranged from 93% to 109% and 85% to 122%, respectively. Sweat PYD values normalized to potassium output yielded a weekly intraindividual biological variability of 14.7%. The mean increase in the population experiencing increased bone resorption vs the reference group was 36% (P &lt;0.05) for sweat PYD/K vs 23–40% (P &lt;0.05) for urinary PYD/Cr, DPD/Cr, and NTX/Cr.Conclusion: We conclude that this new platform sweat collection technology and PYD immunoassay show potential as an indicator of bone resorption.
Determination of Glucose in Dried Serum Samples by Fourier-Transform Infrared Spectroscopy
Authors: Petibois Cyril , Rigalleau Vincent , Melin Anne-Marie , Perromat Annie , Cazorla Georges , Gin Henri , Déléris Gérard 
Published: January 2020

Abstract
Background: Practical improvements are needed to allow measurement of glucose concentrations by Fourier- transform infrared (FT-IR) spectroscopy. We developed a new method that allows determination of the glucose concentration in dried sera.
Methods: We studied 32 serum samples after fourfold dilution and desiccation before FT-IR analyses on a spectrometer operated at a resolution of 2.0 cm−1. We integrated all spectral windows at the surface of the spectrum in the C—O region. For comparison, glucose was measured in the sera by a glucose oxidase method.
Results: One peak within the spectrum was most specific for glucose (997–1062 cm−1). Its surface integration showed a strong relationship with reference data (r = 0.998; P &lt;0.001). FT-IR analyses of five glucose solutions were performed to determine its specific absorption at the same peak. In this way, glucose concentrations in serum spectra could be measured. For the first time while using FT-IR spectroscopy, no manipulation of spectra nor use of internal standard was necessary to obtain results in high accordance with glucose concentration measured by a conventional (glucose-oxidase) method (Sy|x = 0.25 mmol/L; r = 0.998).
Conclusions: FT-IR spectroscopy appears to be an easy and accurate method to determine glucose concentration and could be widely used to simultaneously identify and quantify several metabolites in biological fluids or tissues.
External Quality Assessment of Methylmalonic Acid and Total Homocysteine
Authors: Møller Jan , Rasmussen Karsten , Christensen Lene 
Published: January 2020

Abstract
Background: The use of analyses for methylmalonic acid (MMA) and total homocysteine (HCY) in plasma has become widespread. Realizing the need for external quality assessment for these measurements, we started a program in 1997. The results for 1998 are reviewed in this report.
Methods: Fourteen laboratories participated with 15 sets of results for MMA, and 28 laboratories participated with 34 sets of results for HCY. Results for four identical samples, made up from the same unmodified serum (MMA) or EDTA-plasma (HCY) pool, sent out under different identifications, were used for assessing the imprecision. Samples made up from the same pools supplemented with MMA or l-HCY to three concentrations were used for assessing the recovery. By using literature data for the biological variation, quality goals for both analytes were calculated.
Results: The overall within-laboratory CV was 12% for MMA and 7.5% for HCY. Gas chromatography–mass spectrometric HCY results had lower imprecision than the HPLC or immunoassay results. For MMA, no significant between-laboratory component of variance was found. Only results for HCY obtained with HPLC methods showed significant between-laboratory variance.
Conclusions: Eight of the 15 participants achieved the minimum imprecision goal for MMA vs 9 of the 34 participants for HCY. The minimum quality goals for bias as approximated by the recovery were achieved by 13 participants for MMA and 26 for HCY.
Preanalytical Factors (Biological Variation) and the Measurement of Serum Soluble Intercellular Adhesion Molecule-1 in Humans: Influence of the Time of Day, Food Intake, and Physical and Psychological Stress
Authors: Dugué Benoit , Leppänen Esa , Gräsbeck Ralph 
Published: January 2020

AbstractBackground: Adhesion proteins such as soluble intercellular adhesion molecule-1 (sICAM-1) may be important markers for early atherosclerosis and some other diseases. To devise optimum specimen collection procedures, we investigated the effects on serum sICAM-1 of several preanalytical factors (factors that occur before a specimen is analyzed) such as the time of the day, food ingestion, and physical and psychological stress.Methods: Three sets of experiments were conducted: (a) 30 subjects were investigated during the morning, after an overnight fast, and then after the usual breakfast and at 1200; (b) 20 subjects were studied before and after exposure to thermal stress (sauna + swimming in ice-cold water); and (c) 15 volunteers were investigated after their driving license examination and during a (stress-free) control session. Conventional methods and kits were used to determine the blood picture and serum sICAM-1.Results: All of these preanalytical factors induced a significant increase (∼10%) in the concentration of sICAM-1.Conclusion: It is advisable to consider timing, food intake, and stress when collecting specimens and analyzing data on the concentration of sICAM-1 in serum.