bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiplexed, Targeted Gene Expression Profiling and Genetic Analysis on Electronic Microarrays
Authors: Weidenhammer Elaine M , Kahl Brenda F , Wang Ling , Wang Larry , Duhon Melanie , Jackson Jo Ann , Slater Matthew , Xu Xiao
Published: January 2020

Background: Electronic microarrays comprise independent microelectrode test sites that can be electronically biased positive or negative, or left neutral, to move and concentrate charged molecules such as DNA and RNA to one or more test sites. We developed a protocol for multiplexed gene expression profiling of mRNA targets that uses electronic field-facilitated hybridization on electronic microarrays.

Methods: A multiplexed, T7 RNA polymerase-mediated amplification method was used for expression profiling of target mRNAs from total cellular RNA; targets were detected by hybridization to sequence-specific capture oligonucleotides on electronic microarrays. Activation of individual test sites on the electronic microarray was used to target hybridization to designated subsets of sites and allow comparisons of target concentrations in different samples. We used multiplexed amplification and electronic field-facilitated hybridization to analyze expression of a model set of 10 target genes in the U937 cell line during lipopolysaccharide-mediated differentiation. Performance of multiple genetic analyses (single-nucleotide polymorphism detection, gene expression profiling, and splicing isoform detection) on a single electronic microarray was demonstrated using the ApoE and ApoER2 genes as a model system.

Results: Targets were detected after a 2-min hybridization reaction. With noncomplementary capture probes, no signal was detectable. Twofold changes in target concentration were detectable throughout the (∼64-fold) range of concentrations tested. Levels of 10 targets were analyzed side by side across seven time points. By confining electronic activation to subsets of test sites, polymorphism detection, expression profiling, and splicing isoform analysis were performed on a single electronic microarray.

Conclusions: Microelectronic array technology provides specific target detection and quantification with advantages over currently available methodologies for targeted gene expression profiling and combinatorial genomics testing.
Stabilization of mRNA Expression in Whole Blood Samples
Authors: Rainen Lynne , Oelmueller Uwe , Jurgensen Stewart , Wyrich Ralf , Ballas Cynthia , Schram Jim , Herdman Chris , Bankaitis-Davis Danute , Nicholls Nancy , Trollinger David , Tryon Victor
Published: January 2020

Background: Accurate quantification of mRNA in whole blood is made difficult by the simultaneous degradation of gene transcripts and unintended gene induction caused by sample handling or uncontrolled activation of coagulation. This study was designed to compare a new blood collection tube (PAXgeneTM Blood RNA System) and a companion sample preparation reagent set with a traditional sample collection and preparation method for the purpose of gene expression analysis.

Methods: We collected parallel blood samples from healthy donors into the new sample collection tubes and control EDTA tubes and performed serial RNA extractions on samples stored for 5 days at room temperature and for up to 90 days at 4 and 20 °C. Samples were analyzed by Northern blot analysis or reverse transcription-PCR (RT-PCR).

Results: Specific mRNA concentrations in blood stored in EDTA tubes at any temperature changed substantially, as determined by high-precision RT-PCR. These changes were eliminated or markedly reduced when whole blood was stored in PAXgene tubes. Loss of specific mRNAs, as measured by RT-PCR, reflected total RNA depletion as well as specific mRNA destruction demonstrated by Northern blot analysis. The salutary effects of PAXgene on mRNA stabilization extended to blood samples from eight unrelated donors.

Conclusions: Compared with whole blood collected in EDTA tubes and extracted by an organic method, the PAXgene Blood RNA System reduced RNA degradation and inhibited or eliminated gene induction in phlebotomy whole blood samples. Storage of whole blood samples in PAXgene tubes can be recommended for clinically related blood samples that will be analyzed for total or specific RNA content.
Comprehensive Screening of Urine Samples for Inborn Errors of Metabolism by Electrospray Tandem Mass Spectrometry
Authors: Pitt James J , Eggington Mary , Kahler Stephen G
Published: January 2020

Background: Detection of abnormal metabolites in urine is important for the diagnosis of many inborn errors of metabolism (IEM). Rapid, comprehensive screening methods are needed.

Methods: We used electrospray ionization tandem mass spectrometry in positive- and negative-ion modes to detect selected metabolites in urine. For positive-ion analysis, samples were dried and butylated, whereas for negative-ion analysis, samples were merely diluted with the mobile phase. Analysis was by direct injection with multiple reaction monitoring for 32 metabolites in positive mode (amino acids and acylcarnitines) and 30 metabolites in negative mode (organic acids). Run time was 2.1 min in each mode.

Results: Interbatch CVs ranged from 4.8% to 32%, enabling quantification of many metabolites. The procedure was applied to controls (278 and 120 in positive- and negative-ion mode, respectively) and 108 IEM individuals representing 37 different IEM. In 105 IEM individuals, representing 36 different IEM, concentrations of one or more diagnostic metabolites were above the 99th percentiles of the control values.

Conclusions: The procedure is faster and less labor-intensive than conventional methods of testing for IEM by amino and organic acid profiling and has similar diagnostic sensitivity. The ability to include a greater range of metabolites offers the potential of a more comprehensive screening procedure.
Automated Time-resolved Immunofluorometric Assay for Toxoplasma gondii-specific IgM and IgA Antibodies: Study of More Than 130 000 Filter-Paper Blood-Spot Samples from Newborns
Authors: Sørensen Tina , Spenter Johanne , Jaliashvili Irakli , Christiansen Michael , Nørgaard-Pedersen Bent , Petersen Eskild
Published: January 2020

Background: To screen for congenital toxoplasmosis, we developed a time-resolved immunofluorometric assay for the simultaneous detection of Toxoplasma gondii-specific IgM and IgA in filter-paper samples collected from newborns 4–7 days after birth.

Methods: The assay was performed on the AutoDELFIATM, and results were calculated based on the ToxoM WHO Third International Reference Serum. Comparison with an in-house μ-capture immunoassay was carried out retrospectively on filter-paper samples from children with confirmed congenital toxoplasmosis. Prospectively the assay was compared with a μ-capture immunoassay on 68 394 samples and a commercially available assay on another 69 467 samples. Before serum was requested from the newborn, positive samples were tested for IgA and IgM separately and in an IgM-immunosorbent agglutination assay developed for filter-paper samples.

Results: Intra- and interassay variations (CVs) were 8% and 16%, respectively. The cutoff of 5 units/mL produced a 0.5% retest rate. The assay detected 13 of 18 (72%) samples from newborns diagnosed with congenital toxoplasmosis in the retrospective study. Prospectively, the assay identified 24 newborns who were later diagnosed with congenital toxoplasmosis. Results for all 24 cases were positive by the respective comparison method. No cases were detected solely by the IgA antibodies in the sample.

Conclusion: Neonatal screening for congenital toxoplasmosis can be automated by use of purified europium-labeled antigen for detection of T. gondii-specific IgM and IgA eluted from filter-paper samples.
Genotyping of Essential Hypertension Single-Nucleotide Polymorphisms by a Homogeneous PCR Method with Universal Energy Transfer Primers
Authors: Bengra Chikh , Mifflin Theodore E , Khripin Yuri , Manunta Paolo , Williams Scott M , Jose Pedro A , Felder Robin A
Published: January 2020

Background: Human hypertension is a complex, multifactorial disease with a heritability of more than 30–50%. A genetic screening test based on analysis of multiple single-nucleotide polymorphisms (SNPs) to assess the likelihood of developing hypertension would be helpful for disease management.

Methods: Tailed allele-specific primers were designed to amplify by PCR six biallelic SNP loci [three in G protein-coupled receptor kinase type 4 (GRK4): R65L, A142V, and A486V; two in angiotensinogen: −6G→A and M235T; and one in aldosterone synthase: −344C→T] associated with essential hypertension. PCRs of SNP loci were coupled (via a common sequence of 21 nucleotide tails) to incorporate Universal Amplifluor™ primers labeled with fluorescein or sulforhodamine in a homogeneous format. Use of Amplifluors in SNP PCRs produced labeled amplicons, the fluorescence of which was quantified by a microplate reader and then analyzed via an Excel macro to provide genotypes for all six SNP loci. Unique restriction endonucleases were identified for five SNP loci that could independently confirm homogeneous PCR results when needed.

Results: We developed six homogeneous PCR assays that were set up, performed, and fluorometrically analyzed in 96-well microplates. Allele frequencies were determined for six SNPs in 60 Italian hypertensive patients and a control group of 60 normotensive persons. A significant correlation (P = 0.034) between one SNP [GRK4 (A486V)] and the hypertensive patients was observed. Genotyping results for five of six SNPs were confirmed by digesting corresponding amplicons with locus-specific restriction endonucleases.

Conclusions: We developed a simple and homogeneous fluorescent protocol that has been used to determine the SNP genotype for six loci in a population of hypertensive and normotensive persons. We also observed a significant association (P = 0.034) between one SNP (A486V) and an Italian population of mildly hypertensive patients.
Serum Paraoxonase Activity: A New Additional Test for the Improved Evaluation of Chronic Liver Damage
Authors: Ferré Natàlia , Camps Jordi , Prats Eduard , Vilella Elisabet , Paul Antoni , Figuera Lídia , Joven Jorge
Published: January 2020

Background: Paraoxonase 1 (PON1) is an ester hydrolase present in serum and in the liver. The aims of the present study were to investigate the following: (a) the relationship between serum PON1 activity alterations and the degree of liver damage in patients with chronic liver disease; (b) the influence of genetic variability on serum PON1 activity; and (c) the efficacy of serum PON1 activity measurement, alone and in combination with standard liver function tests, in the assessment of liver damage.

Methods: We studied 68 patients with liver cirrhosis, 107 patients with chronic hepatitis, and 368 apparently healthy volunteers. Baseline and salt-stimulated PON1 activities were measured by the hydrolysis of paraoxon. PON1 genotyping at positions 55 and 192 was analyzed by PCR and restriction isotyping.

Results: Baseline and stimulated PON1 activities were decreased (P <0.001) in chronic hepatitis and in liver cirrhosis. PON1 activity was significantly correlated with serum total proteins, albumin, and bilirubin in patients but not in controls. There were no significant differences with respect to allele and genotype frequencies between patients and controls. The combination of baseline serum PON1 with five standard biochemical tests had a higher classification accuracy (94% of patients; 96% of controls) than the five standard tests alone (75% of patients; 96% of controls). ROC plots demonstrated a high diagnostic accuracy for baseline serum PON1 [area under the curve, 0.89 (95% confidence interval, 0.86–0.93) in chronic hepatitis and 0.96 (95% confidence interval, 0.94–0.99) in cirrhosis]. Baseline PON1 provided the highest ROC area for cirrhosis vs controls.

Conclusions: The significant decrease of PON1 activity in chronic liver diseases is related to the degree of hepatic dysfunction and not to allelic or genotypic differences. Addition of serum PON1 activity measurement to the current battery of tests may improve the evaluation of chronic liver diseases.
Bleomycin-detectable Iron Assay for Non-Transferrin-bound Iron in Hematologic Malignancies
Authors: von Bonsdorff Leni , Lindeberg Enni , Sahlstedt Leila , Lehto Jari , Parkkinen Jaakko
Published: January 2020

Background: A microwell modification of the bleomycin assay for determining non-transferrin-bound iron (NTBI) was evaluated and compared with a chelation method.

Methods: The bleomycin assay reagent and sample volumes were halved, and measurements were done in microwell plates. Samples from patients treated for hematologic malignancies were studied. The chelation method was based on mobilization of NTBI with a chelator and measurement of the ultrafiltered iron-chelator complex. NTBI results were also compared with transferrin saturation and the distribution of transferrin iron forms by urea-polyacrylamide gel electrophoresis.

Results: The bleomycin assay intraassay imprecision (CV) was 7.7% and 8.2% and the interassay imprecision was 18% and 9.8% for a low (0.2 μmol/L) and a high (1.5 μmol/L) control, respectively. Hemolysis increased measured NTBI. A detection limit of 0.1 μmol/L was established based on the interference of nonvisible hemolysis and on accuracy studies. In patient samples, NTBI exceeded the detection limits only when transferrin saturation was >80%. Compared with the chelation method, the bleomycin assay gave clearly lower NTBI concentrations. The chelation method also gave positive results at <80% transferrin saturation. The recovery of iron added as ferric nitrilotriacetate to serum was 33% by the bleomycin assay and 64% by the chelation assay.

Conclusions: The microwell version of the bleomycin assay is reproducible. When hemolyzed samples were excluded, bleomycin-detectable iron was found only when the transferrin saturation was >80%, suggesting high specificity. Bleomycin-detectable iron constitutes only a portion of the NTBI measured by the chelation method.
Predominant Hematopoietic Origin of Cell-free DNA in Plasma and Serum after Sex-mismatched Bone Marrow Transplantation
Authors: Lui Yanni YN , Chik Ki-Wai , Chiu Rossa WK , Ho Cheong-Yip , Lam Christopher WK , Lo YM Dennis
Published: January 2020

Background: Despite current interest in the biology and diagnostic applications of cell-free DNA in plasma and serum, the cellular origin of this DNA is poorly understood. We used a sex-mismatched bone marrow transplantation model to study the relative contribution of hematopoietic and nonhematopoietic cells to circulating DNA.

Methods: We studied 22 sex-mismatched bone marrow transplantation patients. Paired buffy coat and plasma samples were obtained from all 22 patients. Matching serum samples were also obtained from seven of them. Plasma DNA, serum DNA, and buffy coat were quantified by real-time PCR of the SRY and β-globin gene DNA. To investigate the effects of blood drawing and other preanalytical variables on plasma DNA concentrations, blood samples were also collected from 14 individuals who had not received transplants. The effects of blood sampling by syringe and needle, centrifugation, and time delay in blood processing were studied.

Results: The median percentage of Y-chromosome DNA in the plasma in female patients receiving bone marrow from male donors (59.5%) differed significantly (P <0.001) from that in the male patients receiving bone marrow from female donors (6.9%). This indicated that plasma DNA in the bone marrow transplantation recipients was predominantly of donor origin. Compared with paired plasma samples, serum samples had a median 14-fold higher DNA concentration, with the additional DNA being of donor origin. Control experiments indicated that none of the three tested preanalytical variables contributed to a significant change in cell-free DNA concentration.

Conclusions: After bone marrow transplantation, the DNA in plasma and serum is predominantly hematopoietic in origin. Apart from the biological implications of this observation, this finding suggests that plasma and serum can be used as alternative materials for the study of postbone marrow transplantation chimerism.
Comparison of the Emit Immunoassay with HPLC for Therapeutic Drug Monitoring of Mycophenolic Acid in Pediatric Renal-Transplant Recipients on Mycophenolate Mofetil Therapy
Authors: Weber Lutz T , Shipkova Maria , Armstrong Victor W , Wagner Natalie , Schütz Ekkehard , Mehls Otto , Zimmerhackl Lothar B , Oellerich Michael , Tönshoff Burkhard
Published: January 2020

Background: HPLC is currently the preferred method for accurate measurement of mycophenolic acid (MPA). This study was designed to validate the Emit compared with HPLC in relation to clinical outcome measurements.

Methods: Pediatric renal-transplant recipients (n = 50) on an immunosuppressive triple regimen consisting of cyclosporin A, prednisone, and mycophenolate mofetil (600 mg/m2 twice per day) were investigated in an open-label prospective study. Pharmacokinetic profiles over 12 h were obtained at 1 week, 3 weeks, 3 months, and 6 months posttransplant. Plasma MPA was measured by both reversed-phase HPLC and the Emit immunoassay.

Results: There was an association between the risk of acute rejection episodes and low area under the curve values from t0 to t12h (AUC0–12) for MPA (MPA-AUC0–12) or predose concentrations of MPA derived from both HPLC and Emit measurements. According to ROC analysis, an AUC value of 33.8 mg · h/L for MPA from t0 to t12h (MPA-AUC0–12) determined by HPLC had a diagnostic sensitivity of 80% and a diagnostic specificity of 57%. The corresponding value of the Emit was 36.1 mg · h/L. For the predose concentration (MPA-c12), a concentration of 1.2 mg/L determined by HPLC and 1.4 mg/L determined by Emit gave a sensitivity of 80% and a specificity of 60%, respectively. There was no association of any pharmacokinetic variables derived from total MPA measurements with an increased risk of side effects related to mycophenolate mofetil.

Conclusions: The Emit assay appears to have a comparable diagnostic efficacy to HPLC for assessing the risk of acute rejection in pediatric renal-transplant recipients. However, because of the cross-reactivity of the antibody used in the Emit assay with the active MPA acyl glucuronide metabolite, the decision thresholds for the Emit were higher than those calculated from HPLC measurements.
The Tortuous Road to the Adoption of katal for the Expression of Catalytic Activity by the General Conference on Weights and Measures
Authors: Dybkær René
Published: January 2020

Background: The “unit” for “enzymic activity” (U = 1 μmol/min) was recommended by the International Union of Biochemistry and Molecular Biology (IUB) in 1961 and is widely used in medical laboratory reports. The general trend in metrology, however, is toward global standardization through defining units coherent with the International System of Units (SI).

Approach: Several proposals were advanced from the IFCC, International Union of Pure and Applied Chemistry, and IUB regarding the definition for enzymic activity as well as the terms for kind-of-quantity, units, symbol, and dimension. In 1977, international agreement was reached between these bodies and WHO that “catalytic activity” (z), of a catalyst in a given system is defined by the rate of conversion in a measuring system (in mol/s) and expressed in “katal” (symbol, kat; equal to 1 mol/s). The katal is invariant of the measurement procedure, but the numerical quantity value is not. Gaining support for the katal from the final arbiter, the General Conference on Weights and Measures, was slow, but Resolution 12 of 1999 adopted the katal (symbol, kat) as a special name and symbol for the SI-derived unit, mol/s, used in measuring catalytic activity.

Conclusions: Laboratory results for amounts of catalysts, including enzymes, measured by their catalytic activity can now officially be expressed in katals and are traceable to the SI provided that the specified indicator reaction reflects first-order kinetics. The conversion from “unit” is: 1 U = 16.667 × 10−9 kat. Further derived quantities have coherent units such as kat/L, kat/kg, and kat/kat = 1.
Detection of Epitestosterone Doping by Isotope Ratio Mass Spectrometry
Authors: Aguilera Rodrigo , Hatton Caroline K , Catlin Don H
Published: January 2020

Background: Epitestosterone is prohibited by sport authorities because its administration will lower the urinary testosterone/epitestosterone ratio, a marker of testosterone administration. A definitive method for detecting epitestosterone administration is needed.

Methods: We developed a gas chromatography-combustion-isotope ratio mass spectrometry method for measuring the δ13C values for urinary epitestosterone. Sample preparation included deconjugation with β-glucuronidase, solid-phase extraction, and semipreparative HPLC. Epitestosterone concentrations were determined by gas chromatography-mass spectrometry for urines obtained from a control group of 456 healthy males. Epitestosterone δ13C values were determined for 43 control urines with epitestosterone concentrations ≥40 μg/L (139 nmol/L) and 10 athletes’ urines with epitestosterone concentrations ≥180 μg/L (624 nmol/L), respectively.

Results: The log epitestosterone concentration distribution was gaussian [mean, 3.30; SD, 0.706; geometric mean, 27.0 μg/L (93.6 nmol/L)]. The δ13C values for four synthetic epitestosterones were low (less than or equal to −30.3‰) and differed significantly (P <0.0001). The SDs of between-assay precision studies were low (≤0.73‰). The mean δ13C values for urine samples obtained from 43 healthy males was −23.8‰ (SD, 0.93‰). Nine of 10 athletes’ urine samples with epitestosterone concentrations >180 μg/L (624 nmol/L) had δ13C values within ± 3 SD of the control group. The δ13C value of epitestosterone in one sample was −32.6‰ (z-score, 9.4), suggesting that epitestosterone was administered. In addition, the likelihood of simultaneous testosterone administration was supported by low δ13C values for androsterone and etiocholanolone.

Conclusions: Determining δ13C values for urinary epitestosterone is useful for detecting cases of epitestosterone administration because the mean δ13C values for a control group is high (−23.8‰) compared with the δ13C values for synthetic epitestosterones.
Multicenter Evaluation of an Automated Assay for Troponin I
Authors: Uettwiller-Geiger Denise , Wu Alan HB , Apple Fred S , Jevans Anthony W , Venge Per , Olson Marilyn D , Darte Claude , Woodrum David L , Roberts Sean , Chan Stephen
Published: January 2020

Background: Cardiac troponin I (cTnI) is a powerful tool to aid in the diagnosis of myocardial infarction and cardiac muscle damage. We describe an assay that overcomes problems of early assays that were often affected by cTnI degradation, assay interference, poor sensitivity, and imprecision.

Methods: The analytical performance of the Access® AccuTnITM assay (Beckman Coulter) was evaluated at five institutions. Controls, zero calibrator, and diluted patient samples were used to determine precision, detection limit, functional sensitivity, and linearity. The 97.5 and 99 percentiles of a reference population were determined. Common interferents and heterophilic patient samples were tested. Equimolarity was determined by assaying samples with various ratios of free and complexed cTnI. Matched samples drawn into serum, EDTA, lithium heparin, and sodium heparin sample tubes were compared.

Results: Total imprecision (CVs) was 4.0–8.8% between 0.40 and 31 μg/L cTnI. The detection limit was <0.01 μg/L. The 97.5 percentile upper reference limit (URL) was 0.03 μg/L (CV = 20%), and the 99 percentile URL was 0.04 μg/L (CV = 14%). Total CVs of 10% and 20% were seen at and above 0.06 and 0.03 μg/L, respectively. The assay was linear to >60 μg/L and not affected by common assay interferents. An equimolar response was observed with free, complexed, phosphorylated, and dephosphorylated forms of cTnI. Results were 4% lower in serum and 14% lower in EDTA plasma than in lithium heparin plasma (P <0.01), independent of cTnI concentration.

Conclusion: AccuTnI is a sensitive and precise assay for the measurement of cTnI.
Setting Performance Goals and Evaluating Total Analytical Error for Diagnostic Assays
Authors: Krouwer Jan S
Published: January 2020

Background: Total analytical error has been a useful metric both to assess laboratory assay quality and to set goals. It is often estimated by combining imprecision (SD) and average bias in the equation: total analytical error = bias + 1.65 × imprecision. This indirect estimation model (referred to as the simple combination model) leads to different estimates of total analytical error than that of a direct estimation method (referred to as the distribution-of-differences method) or of simulation.

Methods: A review of the literature was undertaken to reconcile the different estimation approaches.

Results: The simple combination model can underestimate total analytical error by neglecting random interference bias and by not properly treating other error sources such as linear drift and outliers. A simulation method to estimate total analytical error is outlined, based on the estimation and combination of total analytical error source distributions. Goals for each total analytical error source can be established by allocation of the total analytical error goal. Typically, the allocation is cost-based and uses the probability of combinations of error sources. The distribution-of-differences method, simple combination model, and simulation method to evaluate total analytical error are compared. Outlier results can profoundly influence quality, but their rates are seldom reported.

Conclusions: Total analytical error should be estimated either directly by the distribution-of-differences method or by simulation. A systems engineering approach that uses allocation of the total analytical error goal into error source goals provides a cost-effective approach to meeting total analytical error. Because outliers can cause serious laboratory error, the inclusion of outlier rate estimates from large studies (e.g., those conducted by manufacturers) would be helpful in assessing assay quality.
Longitudinal Concentrations of Vitamin B12 and Vitamin B12-binding Proteins during Uncomplicated Pregnancy
Authors: Koebnick Corinna , Heins Ulrike A , Dagnelie Pieter C , Wickramasinghe Sunitha N , Ratnayaka Indrika D , Hothorn Torsten , Pfahlberg Annette B , Hoffmann Ingrid , Lindemans Jan , Leitzmann Claus
Published: January 2020

Background: Because reference values for vitamin B12 concentrations and vitamin B12-binding capacities for pregnant women have not been established, the reference values for nonpregnant women are often applied to assess vitamin B12 status. The aim of the present study was to describe ranges of biochemical indices of vitamin B12 status, including red blood cell (RBC) vitamin B12, saturated and unsaturated cobalamin-binding proteins, and binding capacities in all trimesters of uncomplicated pregnancy.

Methods: A total of 39 healthy pregnant women with long-term daily intake of vitamin B12 >2.6 μg/day and uncomplicated pregnancies participated in the study throughout their pregnancies. RBCs and serum vitamin B12, holo-haptocorrin, unsaturated cobalamin-binding proteins, unsaturated and total vitamin B12-binding capacities, total homocysteine (tHcy), and RBC count were assessed in weeks 9–12, 20–22, and 36–38 of gestation.

Results: Significant changes in vitamin B12 status occurred in the course of pregnancy. Serum vitamin B12 concentrations and percentage of saturation of vitamin B12-binding proteins decreased steadily throughout pregnancy. In the third trimester, 35% of the participants had serum vitamin B12 concentrations <150 pmol/L and 68.6% had <15% saturation of total vitamin B12-binding capacities, but no women had RBC vitamin B12 concentrations <148 pmol/L. However, the decrease in these indices was not associated with reduced hemoglobin concentrations or RBC count or with increased tHcy concentrations.

Conclusions: Our findings suggest that the reference values for vitamin B12 status in nonpregnant women may not be applicable to pregnant women.
Phospholipid Autoantibodies and the Antiphospholipid Antibody Syndrome: Diagnostic Accuracy of 23 Methods Studied by Variation in ROC Curves with Number of Clinical Manifestations
Authors: Wasmuth Jan-Christian , Oliver y Miñarro Desamparados , Homrighausen Angela , Leifeld Ludger , Rockstroh Jürgen K , Sauerbruch Tilman , Spengler Ulrich
Published: January 2020

Background: We analyzed the diagnostic accuracies for the diagnosis of antiphospholipid syndrome (APS) of 23 antiphospholipid antibody (APL-Ab) assays targeted at different antigen preparations and immunoglobulin isotypes.

Methods: In 144 patients with suspected APS, anti-cardiolipin (aCL) and anti-β2-glycoprotein I (aβ2GPI) antibodies were measured with 23 different ELISAs from three manufacturers. Data were analyzed by ROC curves. In the absence of an accepted criterion standard, the endpoint “diagnosis of APS” was varied according to the number (two through five) of signs and symptoms of APS.

Results: Although the presence of lupus anticoagulant was associated significantly with APL-Ab in 10 of 23 assays (P = 0.01–10−4) and recurrent arterial or venous occlusions were significantly associated with APL-Ab of IgM isotype in 5 of 6 assays (P = 0.02–10−4), sensitivity for detection of APS did not exceed 67%. With the exception of IgA APL-Ab, the diagnostic accuracy of the assays improved when the diagnosis of APS was based on an increasing number of simultaneous features of APS. For most methods, areas under the ROC curves were >0.8 irrespective of the method’s subclass specificity and antigen preparation (aCL or aβ2GPI), if the clinical diagnosis of APS was based on four or more signs and symptoms of APS.

Conclusion: Despite considerable heterogeneity in the individual test results, a single test of IgG or IgM isotype targeted at either aCL or aβ2GPI antibodies has excellent diagnostic accuracy when the criterion for diagnosis requires four or more typical manifestations of APS.
Technical Evaluation of a New Immunoradiometric and a New Immunoluminometric Assay for Thyroglobulin
Authors: Morgenthaler Nils G , Froehlich Juergen , Rendl Johann , Willnich Marita , Alonso Christine , Bergmann Andreas , Reiners Christoph
Published: January 2020

Background: After removal of differentiated thyroid carcinoma (DTC), serum thyroglobulin (Tg) can indicate persistent or recurrent disease. We describe two novel two-step assays designed to measure low Tg concentrations.

Methods: We evaluated prototypes of the new IRMA, DYNOtest® Tg-pluS, and the new immunoluminometric assay (ILMA), LUMItest® Tg-pluS. In the first step, a high-salt incubation buffer leads to dissociation of Tg-Tg antibody complexes in serum and is intended to reduce nonspecific interference and interference of potential Tg autoantibodies in the system. We studied recovery of human Tg (from thyroid glands) added to horse serum. We also studied 58 patients with DTC in whom Tg values under thyroid-stimulating hormone (TSH) suppression and TSH stimulation (without thyroxine) were available.

Results: The detection limits were 0.04 μg/L Tg for the IRMA and 0.02 μg/L for the ILMA. Intraassay imprecision (CV) was <10% over the range of the calibration curve in both assays. The day-to-day CV was <20% at 0.2 μg/L for the IRMA and at 0.06 μg/L for the ILMA. No high-dose hook effect was seen with up to 200 000 μg/L added Tg or in dilutions of 12 patient sera with Tg values of 307–38 880 μg/L. Mean recovery of 50 μg Tg/L was 96% in those patients. Among 77 samples with Tg antibody values of 65.2–8150 kilounits/L, recovery by the IRMA was disturbed in 7 cases (9%) and by the ILMA in 9 cases (12%). Tg increased as measured in both assays in 50 of 58 patients after thyroxine withdrawal.

Conclusions: The new assays have improved precision for Tg <1 μg/L, and even low measured Tg concentrations respond physiologically to thyroxine withdrawal. The assays are free of a high-dose hook effect up to a Tg concentration of at least 38 000 μg/L and may further reduce Tg antibody interference.
Independent Risk Factor for Moderate to Severe Internal Carotid Artery Stenosis: T786C Mutation of the Endothelial Nitric Oxide Synthase Gene
Authors: Ghilardi Giorgio , Biondi Maria Luisa , DeMonti Marco , Bernini Mara , Turri Olivia , Massaro Federico , Guagnellini Emma , Scorza Roberto
Published: January 2020

Background: NO synthesized from l-arginine by the constitutive endothelial NO synthase (eNOS) plays a key role in the atherosclerotic process. We investigated whether common variants in the NOS3 gene (a T786C mutation in the 5′ flanking region and the polymorphism on exon 7 that produced the Glu298Arg polymorphism in the protein) are associated with an increased risk of moderate to severe internal carotid artery (ICA) stenosis.

Methods: We studied 88 patients consecutively operated for ICA stenosis and 133 healthy controls. A T786C mutation in the 5′ flanking region and the polymorphism in exon 7 that produces the Glu298Asp polymorphism in the protein were explored by PCR and fluorescent probe analysis.

Results: Genotype distribution was significantly different between patients and controls only for T786C, the CC genotype frequency being 26% and 13%, respectively [odds ratio (OR), 2.26; 95% confidence interval (CI), 1.14–4.46; P = 0.018]. Moreover, the CC genotype was significantly more frequent in a subgroup of patients with ulcerative plaques compared with patients with nonulcerative lesions (44% vs 17%; OR, 3.82; 95% CI, 1.79–8.14; P = 0.003). Multiple logistic regression analysis using the most frequent risk factors and the eNOS gene variant showed that the CC genotype is an independent risk factor for ICA stenosis (P = 0.023).

Conclusion: C allele homozygosity in position 786 of the eNOS promoter seems to be an independent risk factor for the development of moderate to severe ICA stenosis, especially ulcerative lesions.
The Proportion of Prostate-specific Antigen (PSA) Complexed to α1-Antichymotrypsin Improves the Discrimination between Prostate Cancer and Benign Prostatic Hyperplasia in Men with a Total PSA of 10 to 30 μg/L
Authors: Martínez Manuel , España Francisco , Royo Montserrat , Alapont José M , Navarro Silvia , Estellés Amparo , Aznar Justo , Vera César D , Jiménez-Cruz Juan F
Published: January 2020

Background: The aim of this study was to assess the diagnostic accuracy of the proportion of prostate-specific antigen (PSA) complexed to α1-antichymotrypsin (PSA-α1ACT:PSA ratio) in the differential diagnosis of prostate cancer (CaP) and benign prostatic hyperplasia (BPH) in men with total PSA of 10–30 μg/L.

Methods: We used our immunoassays (ELISAs) for total PSA and PSA-α1ACT complex to study 146 men. In 123, total PSA was between 10 and 20 μg/L; 66 of these had CaP and 57 BPH. In 23 men, total PSA was between 20 and 30 μg/L; 14 of these had CaP and 9 BPH. We calculated the area under the ROC curves (AUC) for total PSA, PSA-α1ACT complex, and PSA-α1ACT:PSA ratio, and determined the cutoff points that gave sensitivities approaching 100%.

Results: In the total PSA range between 10 and 20 μg/L, the AUC was significantly higher for the PSA-α1ACT:PSA ratio (0.850) than for total PSA (0.507) and PSA-α1ACT complex (0.710; P <0.0001). A cutoff ratio of 0.62 would have permitted diagnosis of all 66 patients with CaP (100% sensitivity) and avoided 19% of unnecessary biopsies (11 of 57 patients). In the total PSA range between 20 and 30 μg/L, the AUC for the PSA-α1ACT:PSA ratio (0.980; 95% confidence interval, 0.82–0.99) was greater than the AUC for total PSA (0.750; 95% confidence interval, 0.51–0.89; P = 0.042). In this range, a cutoff point of 0.64 would have permitted the correct diagnosis of all 14 patients with CaP and 6 of the 9 with BPH.

Conclusions: The diagnostic accuracy of the PSA-α1ACT:PSA ratio persists at high total PSA concentrations, increasing the specificity of total PSA. Prospective studies with large numbers of patients are needed to assess whether the ratio of PSA-α1ACT to total PSA is a useful tool to avoid unnecessary prostatic biopsy in patients with a total PSA >10 μg/L.
Simultaneous Quantification of Prostate-specific Antigen and Human Glandular Kallikrein 2 mRNA in Blood Samples from Patients with Prostate Cancer and Benign Disease
Authors: Ylikoski Alice , Pettersson Kim , Nurmi Jussi , Irjala Kerttu , Karp Matti , Lilja Hans , Lövgren Timo , Nurmi Martti
Published: January 2020

Background: Detection or quantification of circulating cancer cells has been proposed as an aid in detection and monitoring of several solid tumors. We investigated the classification accuracy of prostate-specific antigen (PSA) and human glandular kallikrein 2 (hK2) mRNA copy numbers in blood for the differentiation of patients with prostate cancer (PC) and benign disease.

Methods: PSA and hK2 mRNA expression was studied in blood samples from 51 men with PC and 19 men with benign disease. Among the PC patients, 10 had organ-confined disease (pT1–pT2). We used a multiplexed reverse transcription-PCR assay with two highly target-like mRNA internal standards for the simultaneous quantification of PSA and hK2 mRNA. An external calibration curve covered the range of 102–106 mRNA copies.

Results: PSA and hK2 mRNA were detected in 41 of 51 (median, 1200 copies/0.5 mL of blood) and 43 of 51 (median, 3800 copies/0.5 mL of blood) patients with PC, respectively, whereas only 1 of 19 men with benign disease was positive for both mRNAs (1500 PSA and 3100 hK2 mRNA copies/0.5 mL of blood; P <0.0001, Mann–Whitney U-test). Of the 10 patients with organ-confined PC, only 3 with low Gleason scores (≤5) were negative for both PSA and hK2 (P = 0.02, Mann–Whitney U-test).

Conclusions: The presence of PC cells in the blood circulation is an early event in PC progression, and quantitative assays for PSA and hK2 mRNA discriminate benign from PC cases. Further studies are needed to determine the diagnostic accuracy and prognostic value of the assays.
Proteomics and Bioinformatics Approaches for Identification of Serum Biomarkers to Detect Breast Cancer
Authors: Li Jinong , Zhang Zhen , Rosenzweig Jason , Wang Young Y , Chan Daniel W
Published: January 2020

Background: Surface-enhanced laser desorption/ionization (SELDI) is an affinity-based mass spectrometric method in which proteins of interest are selectively adsorbed to a chemically modified surface on a biochip, whereas impurities are removed by washing with buffer. This technology allows sensitive and high-throughput protein profiling of complex biological specimens.

Methods: We screened for potential tumor biomarkers in 169 serum samples, including samples from a cancer group of 103 breast cancer patients at different clinical stages [stage 0 (n = 4), stage I (n = 38), stage II (n = 37), and stage III (n = 24)], from a control group of 41 healthy women, and from 25 patients with benign breast diseases. Diluted serum samples were applied to immobilized metal affinity capture Ciphergen ProteinChip® Arrays previously activated with Ni2+. Proteins bound to the chelated metal were analyzed on a ProteinChip Reader Model PBS II. Complex protein profiles of different diagnostic groups were compared and analyzed using the ProPeak software package.

Results: A panel of three biomarkers was selected based on their collective contribution to the optimal separation between stage 0–I breast cancer patients and noncancer controls. The same separation was observed using independent test data from stage II–III breast cancer patients. Bootstrap cross-validation demonstrated that a sensitivity of 93% for all cancer patients and a specificity of 91% for all controls were achieved by a composite index derived by multivariate logistic regression using the three selected biomarkers.

Conclusions: Proteomics approaches such as SELDI mass spectrometry, in conjunction with bioinformatics tools, could greatly facilitate the discovery of new and better biomarkers. The high sensitivity and specificity achieved by the combined use of the selected biomarkers show great potential for the early detection of breast cancer.
Predictive Value of Routine Circulating Soluble Endothelial Cell Adhesion Molecule Measurements during Pregnancy
Authors: Krauss Thomas , Emons Günter , Kuhn Walther , Augustin Hellmut G
Published: January 2020

Background: The present study was aimed at determining whether routine prenatal measurements of circulating soluble intercellular adhesion molecule (sICAM-1; CD54) and soluble vascular cell adhesion molecule (sVCAM-1; CD106) in midgestation have predictive value for the identification of pregnant women destined to develop preeclampsia or other complications of pregnancy during late gestation.

Methods: Plasma sICAM-1 and sVCAM-1 were analyzed between weeks 22 and 29 of gestation in 1543 pregnant women and related to the outcome of pregnancy in a prospective longitudinal study.

Results: Plasma sICAM-1 and sVCAM-1 in uncomplicated pregnancies were normally distributed and varied over a small range (sICAM-1, SD = 22.5%; sVCAM-1, SD = 25.5%). Of all analyzed uncomplicated pregnancies, 54 (3.95%) were identified with concentrations of sICAM-1 or sVCAM-1 above the mean + 2 SD. In contrast, of 177 pregnancies with complications (prevalence, 11.5%), 97 (55%) had sICAM-1 or sVCAM-1 concentrations above the same cutoffs weeks before the onset of disease. The sensitivities of sICAM-1 and sVCAM-1 measurements were 66% for preeclampsia and hemolysis, elevated liver enzymes, and low platelets (HELLP syndrome), 42% for gestational hypertension, 50% for fetal retardation, 46% for preterm labor, 50% for gestational diabetes mellitus, 67% for gestational proteinuria, and 70% for infections during pregnancy. Taken together, routine prenatal sICAM-1 and sVCAM-1 measurements had an overall predictive value of 64%.

Conclusions: Midgestation measurements of circulating sICAM-1 and sVCAM-1 have a high predictive value (area under the curve of combined sICAM-1 and sVCAM-1 measurements determined by ROC analysis, 0.85) and may identify up to 55% of pregnant women who will later develop a severe pregnancy-related complication.
Plasma and Oral Fluid Pharmacokinetics and Pharmacodynamics after Oral Codeine Administration
Authors: Kim Insook , Barnes Allan J , Oyler Jonathan M , Schepers Raf , Joseph Robert E , Cone Edward J , Lafko Diana , Moolchan Eric T , Huestis Marilyn A
Published: January 2020

Background: The ease, noninvasiveness, and safety of oral fluid collection have increased the use of this alternative matrix for drugs-of-abuse testing; however, few controlled drug administration data are available to aid in the interpretation of oral fluid results.

Methods: Single oral codeine doses (60 and 120 mg/70 kg) were administered to 19 volunteers. Oral fluid and plasma were analyzed for free codeine, norcodeine, morphine, and normorphine by solid-phase extraction combined with gas chromatography–mass spectrometry (SPE/GC-MS). Physiologic and subjective effects were examined.

Results: Mean (SE) peak codeine concentrations were 214.2 ± 27.6 and 474.3 ± 77.0 μg/L in plasma and 638.4 ± 64.4 and 1599.3 ± 241.0 μg/L in oral fluid. The oral fluid-to-plasma ratio for codeine was relatively constant (∼4) from 1 to 12 h. The mean half-life (t1/2) of codeine was 2.2 ± 0.10 h in plasma and 2.2 ± 0.16 h in oral fluid. Significant dose-related miosis and increases in sedation, psychotomimetic effect, and “high” occurred after the high dose. Mean codeine oral fluid detection time was 21 h with a 2.5 μg/L cutoff, longer than that of plasma (12–16 h). Detection times with the proposed Substance Abuse and Mental Health Services Administration cutoff (40 μg/L) were only 7 h. Norcodeine, but not morphine or normorphine, was quantified in both plasma and oral fluid.

Conclusions: The disposition of codeine over time was similar in plasma and oral fluid, but because of high variability, oral fluid codeine concentrations did not reliably predict concurrent plasma concentrations. Oral fluid testing is a useful alternative matrix for monitoring codeine exposure with a detection window of 7–21 h for single doses, depending on cutoff concentrations. These controlled drug administration data should aid in the interpretation of oral fluid codeine results.
Validation of a High-Throughput Liquid Chromatography–Tandem Mass Spectrometry Method for Urinary Cortisol and Cortisone
Authors: Taylor Robert L , Machacek Dwaine , Singh Ravinder J
Published: January 2020

Background: Urinary free cortisol and cortisone measurements are useful in evaluation of Cushing syndrome, apparent mineralocorticoid excess, congenital adrenal hyperplasia, and adrenal insufficiency. To reduce analytical interference, improve accuracy, and shorten the analysis time, we developed a liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for urinary cortisol and cortisone.

Methods: We added 190 pmol (70 ng) of stable isotope cortisol-9,11,12,12-d4 to 0.5 mL of urine as an internal standard before extraction. The urine was extracted with 4.5 mL of methylene chloride, washed, and dried, and 10 μL of the reconstituted extract was injected onto a reversed-phase C18 column and analyzed using a tandem mass spectrometer operating in the positive mode.

Results: Multiple calibration curves for urinary cortisol and cortisone exhibited consistent linearity and reproducibility in the range 7–828 nmol/L (0.25–30 μg/dL). Interassay CVs were 7.3–16% for mean concentrations of 6–726 nmol/L (0.2–26.3 μg/dL) for cortisol and cortisone. The detection limit was 6 nmol/L (0.2 μg/dL). Recovery of cortisol and cortisone added to urine was 97–123%. The regression equation for the LC-MS/MS (y) and HPLC (x) method for cortisol was: y = 1.11x + 0.03 μg cortisol/24 h (r2 = 0.992; n = 99). The regression equation for the LC-MS/MS (y) and immunoassay (x) methods for cortisol was: y = 0.66x − 12.1 μg cortisol/24 h (r2 = 0.67; n = 99).

Conclusion: The sensitivity and specificity of the LC-MS/MS method for urinary free cortisol and cortisone offer advantages over routine immunoassays or chromatographic methods because of elimination of drug interferences, high throughput, and short chromatographic run time.
Optimal Monetary Policy when Information is Market-Generated
Authors: Benhima Kenza , Blengini Isabella
Published: January 2020

The nature of the private sector’s information changes the optimal conduct of monetary policy. When firms observe their individual demand and use it as a signal of real shocks, the optimal policy consists in maximising the information content of that signal. When real shocks are deflationary (like labour supply shocks), the optimal policy is countercyclical and magnifies price movements, which contrasts with the exogenous information case, where optimal monetary policy is procyclical and stabilises prices. When the central bank communicates its information to the public, this policy is still optimal if firms pay limited attention to central bank announcements.