bluuebunny/crossref_metadata_2025 huggingface dataset:
Material Softening and Structural Instability
Authors: Chen G. , Baker G.
Published: February 2004

This note discusses the relationship between two kinds of instability problems: material failure and structural instability. Material failure is governed by the second-order work at the material point concerned, whereas structural instability is governed by the second-order work of the whole structure. Structural instability is not only related to material instability but also to the structural topology, boundary conditions, and the mathematical model used. Material failure only indicates that the structure cannot support some forms of loading further. If the mathematical modelling does not reflect these forms of the loading, the structure may be stable but with material failure. The important conclusion is that at a structural level, we should examine global not local stability. As an example, the stability of localized and non-localized solutions is evaluated with the aid of the second-order work expressions. A theoretical explanation is presented to the interesting phenomenon in softening solids that increasing the finite element space will reveal more unstable solutions and will “turn” those that were previously found “stable” into unstable solutions.
Surface Integral Methods in Computational Aeroacoustics—From the (CFD) Near-Field to the (Acoustic) Far-Field
Authors: Lyrintzis Anastasios S.
Published: February 2004

A review of recent advances in the use of surface integral methods in Computational AeroAcoustics (CAA) for the extension of near-field CFD results to the acoustic far-field is given. These integral formulations (i.e. Kirchhoff's method, permeable (porous) surface Ffowcs-Williams Hawkings (FW-H) equation) allow the radiating sound to be evaluated based on quantities on an arbitrary control surface if the wave equation is assumed outside. Thus only surface integrals are needed for the calculation of the far-field sound, instead of the volume integrals required by the traditional acoustic analogy method (i.e. Lighthill, rigid body FW-H equation). A numerical CFD method is used for the evaluation of the flow-field solution in the near field and thus on the control surface. Diffusion and dispersion errors associated with wave propagation in the far-field are avoided. The surface integrals and the first derivatives needed can be easily evaluated from the near-field CFD data. Both methods can be extended in order to include refraction effects outside the control surface. The methods have been applied to helicopter noise, jet noise, propeller noise, ducted fan noise, etc. A simple set of portable Kirchhoff/FW-H subroutines can be developed to calculate the far-field noise from inputs supplied by any aerodynamic near/mid-field CFD code.
A Boundary Element Method for Aerodynamics and Aeroacoustics of Bodies in Arbitrary Motions
Authors: Morino L. , Bernardini G. , Gennaretti M.
Published: February 2004

The validation for a boundary element formulation for the combined potential–flow aerodynamic and aeroacoustic analysis of bodies in arbitrary motion (applicable for instance to a tiltrotor during the conversion phase) is presented. The formulation, introduced in the past by the authors, is based on the velocity potential for compressible flows. The distinguishing feature being validated is the fact that the boundary integral representation is written for a surface that moves in arbitrary motion with respect to a frame of reference that in turn moves in arbitrary motion with respect to the undisturbed air. Thus, the integrals are evaluated on the emission surface, which is the locus of the emitting points at the moving–frame locations that they had when the signal influencing a given point at a given time was emitted. Numerical validation results are presented for helicopter rotors in hover and forward flight.
A Three-Dimensional Parallel Discontinuous Galerkin Solver for Acoustic Propagation Studies
Authors: Crivellini A. , Bassi F.
Published: February 2004

The paper presents recent developments of a computational code for the numerical investigation of acoustic propagation. The code solves the three-dimensional linear Euler equations using a Discontinuous Galerkin (DG) method for the spatial discretization and an explicit high-order low-storage Runge-Kutta method for advancing the solution in time. Thanks to DG discretization, high-order accurate numerical solutions on arbitrary unstructured hybrid grids have been easily computed. The code has been parallelized using MPI and preliminary results on a small 10-processor Linux cluster seem very promising.
Aeroacoustic Predictions Using High-Order Shock-Capturing Schemes
Authors: Ekaterinaris John A.
Published: February 2004

High-order accurate, finite-difference methods, such as the compact centered schemes with spectral-type or characteristic-based filters and the weighted essentially non-oscillatory (WENO) schemes, which are used in high resolution CFD solutions and for DNS or LES of compressible turbulence, are applied to aeroacoustics. Implicit and explicit schemes are used for time marching. The accuracy of the numerical solutions is evaluated for test problems. It is found that these methods are appropriate for sound propagation in complex flows that require use of curvilinear coordinates. Therefore they are applicable for the prediction of sound generation from both smooth subsonic flows, and transonic or supersonic flows with discontinuities.
Numerical Evidence of Mode Switching in the Flow-Induced Oscillations by a Cavity
Authors: Gloerfelt Xavier , Bogey Christophe , Bailly Christophe
Published: February 2004

A Direct Noise Computation (DNC) has been performed for a turbulent boundary layer past a rectangular cavity, matching one configuration of Karamcheti1 experiments. An LES approach with periodic boundary conditions in the spanwise direction is used to evaluate the solution at a reasonable computational cost. The two components in the pressure spectra found experimentally are well reproduced. The acoustic field appears to be dominated by the low-frequency component whereas the experimental visualization indicates a radiation at the higher frequency. The mechanism giving rise to the lower frequency is investigated providing evidence on the possibility of switching between two cavity modes and that the strong coupling of the separated shear layer with the recirculation flow within the cavity is likely to participate to the low-frequency modulation. Moreover, an extrapolation method is proposed and applied to obtain the far-field from the near acoustic field.
Internal loop mutations in the ribosomal protein L30 binding site of the yeast L30 RNA transcript
Authors: WHITE SUSAN A. , HOEGER MARGARET , SCHWEPPE JAMES J. , SHILLINGFORD AMANDA , SHIPILOV VALERIE , ZARUTSKIE JENNIFER
Published: February 2004

Yeast ribosomal protein L30 binds to an asymmetric, purine-rich internal loop in its transcript to repress its own splicing and translation. The protein-bound form of the stem-internal loop–stem RNA is an example of a kink-turn RNA structural motif. Analysis of kink-turn motifs reveals that in (2 + 5) internal loops, the identities of five nucleotides are very important, while the remaining two may be varied. Previous SELEX experiments on the L30 binding site showed an identical pattern of sequence variation with five nucleotides highly conserved and two positions variable. In this work, internal loop residues were mutated and tested for protein binding in vitro and in vivo. The two sheared G-A pairs, which cannot be mutated without severely weakening L30 binding, make sequence specific contacts with other portions of the RNA and L30 protein. In contrast, the lone nucleotide that protrudes into the protein and an unpaired adenosine make no sequence-specific contacts, and may be mutated without compromising L30 binding. The internal loop allows the formation of a very tight bend that brings the two stems together with cross-strand stacking of two adenines and an interhelical ribose contact. Replacement of a ribonucleotide with a deoxynucleotide adjacent to the internal loop weakens protein binding significantly. In the absence of L30, some of the internal loop residues involved in the formation of the kink-turn motif are protected from chemical modification, indicating that some elements of kink-turn structure may form in the free L30 RNA.
Translation of cellular inhibitor of apoptosis protein 1 (c-IAP1) mRNA is IRES mediated and regulated during cell stress
Authors: VAN EDEN MARC E. , BYRD MARSHALL P. , SHERRILL KYLE W. , LLOYD RICHARD E.
Published: February 2004

Cellular inhibitor of apoptosis protein 1 (c-IAP1) can regulate apoptosis through its interaction with downstream TNF receptor effectors (TRAF1 and TRAF2), by binding to and inhibiting certain caspases, and by controlling the levels of specific proapoptotic stimuli (e.g., Smac/DIABLO) within the cell. Studies involving the expression of c-IAP1 mRNA and protein in cells and tissues have provided evidence suggesting c-IAP1 expression may be posttranscriptionally controlled. Because the 5′-UTR of c-IAP1 mRNA is unusually long, contains multiple upstream AUG codons, and has the potential to form thermodynamically stable secondary structures, we investigated the possibility it contained an internal ribosome entry site (IRES) that may regulate its expression. In the present study, the c-IAP1 5′-UTR exhibited IRES activity when dicistronic RNA constructs were translated in rabbit reticulocyte lysate (RRL) and in transiently transfected cells. IRES-mediated translation was similar to that exhibited by the hepatitis C virus IRES but varied significantly in RRL and in HeLa, HepG2, and 293T cells, indicating the c-IAP1 IRES was system and cell type specific. IRES-mediated translation was maintained in mono- and dicistronic constructs in which the UTR was inserted downstream from a stable hairpin that prevented cap-dependent ribosome scanning. In cells, the presence or absence of a methylated cap did not significantly affect the translation of polyadenylated, monocistronic RNAs containing the c-IAP1 5′-UTR. IRES-mediated translation was stimulated in transfected cells treated with low doses of pro-apoptotic stimuli (i.e., etoposide and sodium arsenite) that inhibited endogenous cellular translation.
Evidence for a base triple in the free HIV-1 TAR RNA
Authors: HUTHOFF HENDRIK , GIRARD FREDERIC , WIJMENGA SYBREN S. , BERKHOUT BEN
Published: February 2004

We propose the existence of a novel base triple in the HIV-1 TAR hairpin. This triple is supported by covariation of loop residue 31 with residue 22, which is part of an unusual base pair with U40 below the 3-nucleotide bulge. A set of mutants was constructed to test the involvement of bases A22, U31, and U40 in a triple interaction. RNA structure probing, trans-activation assays, and structure modeling are consistent with the existence of this base triple in a bent conformation of the free TAR element. However, disruption of the base triple does not affect binding of a Tat-derived peptide. We therefore compared the structure of free and Tat-bound TAR RNA by footprinting and site-specific cross-linking analyses. These studies indicate that the Tat arginine-rich motif, in addition to its known binding site at the bulge, is in close contact with U31 in the TAR loop. Because binding of Tat to TAR is known to coincide with the formation of a base triple with residues U23, A27, and U38, we hypothesize that Tat binding and the associated straightening of TAR triggers the disruption of the (A22-U40)U31 triple.
Interaction of the Bacillus subtilis RNase P with the 30S ribosomal subunit
Authors: BARRERA ALESSANDRA , PAN TAO
Published: February 2004

Ribonuclease P (RNase P) is a ribozyme required for the 5′ maturation of all tRNA. RNase P and the ribosome are the only known ribozymes conserved in all organisms. We set out to determine whether this ribonucleoprotein enzyme interacts with other cellular components, which may imply other functions for this conserved ribozyme. Incubation of the Bacillus subtilis RNase P holoenzyme with fractionated B. subtilis cellular extracts and purified ribosomal subunits results in the formation of a gel-shifted complex with the 30S ribosomal subunit at a binding affinity of ~40 nM in 0.1 M NH4Cl and 10 mM MgCl2. The complex does not form with the RNase P RNA alone and is disrupted by a mRNA mimic polyuridine, but is stable in the presence of high concentrations of mature tRNA. Endogenous RNase P can also be detected in the 30S ribosomal fraction. Cleavage of a pre-tRNA substrate by the RNase P holoenzyme remains the same in the presence of the 30S ribosome, but the cleavage of an artificial non-tRNA substrate is inhibited eightfold. Hydroxyl radical protection and chemical modification identify several protected residues located in a highly conserved region in the RNase P RNA. A single mutation within this region significantly reduces binding, providing strong support on the specificity of the RNase P-30S ribosome complex. Our results also suggest that the dimeric form of the RNase P is primarily involved in 30S ribosome binding. We discuss several models on a potential function of the RNase P-30S ribosome complex.
Characterization of deadenylation in trypanosome extracts and its inhibition by poly(A)-binding protein Pab1p
Authors: MILONE JOSEPH , WILUSZ JEFFREY , BELLOFATTO VIVIAN
Published: February 2004

The stability of mRNAs is an important point in the regulation of gene expression in eukaryotes. The mRNA turnover pathways have been identified in yeast and mammals. However, mRNA turnover pathways in trypanosomes have not been widely studied. Deadenylation is the first step in the major mRNA turnover pathways of yeast and mammals. To better understand mRNA degradation processes in these organisms, we have developed an in vitro mRNA turnover system that is functional for deadenylation. In this system, addition of poly(A) homopolymer activates the deadenylation of poly(A) tails. The trypanosomal deadenylase activity is a 3′→5′ exonuclease specific for adenylate residues, generates 5′-AMP as a product, is magnesium dependent, and is inhibited by neomycin B sulfate. These characteristics suggest similarity with other eukaryotic deadenylases. Furthermore, this activity is cap independent, indicating a potential difference between the trypanosomal activity and PARN, but suggesting similarity to Ccr4p/Pop2p activities. Extracts immunodepleted of Pab1p required the addition of poly(A) competition to activate deadenylation. Trypanosomal Pab1p functions as an inhibitor of the activity under in vitro conditions. Pab1p appears to be one of several mRNA stability proteins in trypanosomal extracts.
Prediction of RNA-binding proteins from primary sequence by a support vector machine approach
Authors: HAN LIAN YI , CAI CONG ZHONG , LO SIEW LIN , CHUNG MAXEY C.M. , CHEN YU ZONG
Published: February 2004

Elucidation of the interaction of proteins with different molecules is of significance in the understanding of cellular processes. Computational methods have been developed for the prediction of protein–protein interactions. But insufficient attention has been paid to the prediction of protein–RNA interactions, which play central roles in regulating gene expression and certain RNA-mediated enzymatic processes. This work explored the use of a machine learning method, support vector machines (SVM), for the prediction of RNA-binding proteins directly from their primary sequence. Based on the knowledge of known RNA-binding and non-RNA-binding proteins, an SVM system was trained to recognize RNA-binding proteins. A total of 4011 RNA-binding and 9781 non-RNA-binding proteins was used to train and test the SVM classification system, and an independent set of 447 RNA-binding and 4881 non-RNA-binding proteins was used to evaluate the classification accuracy. Testing results using this independent evaluation set show a prediction accuracy of 94.1%, 79.3%, and 94.1% for rRNA-, mRNA-, and tRNA-binding proteins, and 98.7%, 96.5%, and 99.9% for non-rRNA-, non-mRNA-, and non-tRNA-binding proteins, respectively. The SVM classification system was further tested on a small class of snRNA-binding proteins with only 60 available sequences. The prediction accuracy is 40.0% and 99.9% for snRNA-binding and non-snRNA-binding proteins, indicating a need for a sufficient number of proteins to train SVM. The SVM classification systems trained in this work were added to our Web-based protein functional classification software SVMProt, at http://jing.cz3.nus.edu.sg/cgi-bin/svmprot.cgi. Our study suggests the potential of SVM as a useful tool for facilitating the prediction of protein–RNA interactions.
Transient Ischemic Attack Reported with Paroxetine Use
Authors: Manos Gail H , Wechsler Steven M
Published: February 2004

OBJECTIVE

To report the occurrence of a transient ischemic attack (TIA) temporally related to the initiation of paroxetine.

CASE SUMMARY

A 57-year-old white man with a history of intermittent atrial fibrillation and hypercholesterolemia developed slurred speech and a facial droop 3 days after starting paroxetine. He was diagnosed with a TIA, hospitalized, and given anticoagulation treatment. The presenting symptoms resolved, but recurred when paroxetine was restarted 2 days later.

DISCUSSION

Platelets secrete serotonin, which mediates vasoconstriction through stimulation of 5-HT2a receptors. This is counterbalanced by the release of the vasodilator nitric oxide upon serotonin stimulation of endothelial 5-HT1 receptors. In conditions such as atherosclerosis, the damage to the endothelium leads to a greater vasoconstrictive response. Paroxetine has been reported to weakly inhibit norepinephrine reuptake and nitric oxide production in addition to increasing serotonergic activity, potentially compounding the vasoconstrictive response. An objective causality assessment revealed that the TIA was probably an adverse event resulting from use of paroxetine.

CONCLUSIONS

Use of paroxetine and other selective serotonin-reuptake inhibitors may result in changes of the vasculature and subsequent ischemic events in predisposed patients.
Use of Epoetin Alfa in Critically III Patients
Authors: Pajoumand Mehrnaz , Erstad Brian L , Camamo James M
Published: February 2004

OBJECTIVE

To discuss the controversies regarding the use of epoetin alfa (EPO) for reducing red blood cell (RBC) transfusions in critically ill patients with anemia.

DATA SOURCES

A MEDLINE search (1966–July 2003) was conducted using the search terms anemia; critical illness; erythropoietin; epoetin alfa; and erythropoietin, recombinant. References of selected articles were reviewed for studies that may have been missed by the computerized search.

STUDY SELECTION AND DATA EXTRACTION

Studies pertaining to the use of EPO for anemia of critical illness with an emphasis on data obtained from controlled trials.

DATA SYNTHESIS

Anemia is a common complication in patients admitted to the intensive care unit (ICU). Two prospective, randomized studies have demonstrated decreased transfusion requirements associated with EPO administration in medical/surgical patients who were in the ICU for at least 3 days and had hematocrit concentrations <38%. No differences were found in length of stay or mortality. A multicenter trial found that a restrictive strategy of RBC transfusion (hemoglobin goal 7–9 g/dL) was associated with in-hospital mortality lower than that with a more liberal approach, which calls into question the 38% hematocrit goal in the EPO trials. Furthermore, preliminary results from an economic analysis of EPO use in the ICU setting have demonstrated that EPO is not cost-effective.

CONCLUSIONS

Given the controversies surrounding EPO administration in critically ill patients, institutions are encouraged to develop EPO guidelines to help ensure the most appropriate use of this expensive product. Additional studies regarding patients most likely to benefit from EPO therapy, the most effective dosing regimen, and use of adjunctive therapies are needed.
Methamphetamine and Amphetamine Pharmacokinetics in Oral Fluid and Plasma after Controlled Oral Methamphetamine Administration to Human Volunteers
Authors: Schepers Raf J F , Oyler Jonathan M , Joseph Robert E , Cone Edward J , Moolchan Eric T , Huestis Marilyn A
Published: February 2004

Background: Methamphetamine (METH) and amphetamine (AMP) concentrations in 200 plasma and 590 oral fluid specimens were used to evaluate METH pharmacokinetics and pharmacodynamics after oral administration of sustained-release METH.

Methods: Eight participants received four oral 10-mg S-(+)-METH hydrochloride sustained-release tablets within 7 days. Three weeks later, five participants received four oral 20-mg doses. Blood samples were collected for up to 24 h and oral fluid for up to 72 h after drug administration.

Results: After the first oral dose, initial plasma METH detection was within 0.25–2 h; cmax was 14.5–33.8 μg/L (10 mg) and 26.2–44.3 μg/L (20 mg) within 2–12 h. In oral fluid, METH was detected as early as 0.08–2 h; cmax was 24.7–312.2 μg/L (10 mg) and 75.3–321.7 μg/L (20 mg) and occurred at 2–12 h. The median oral fluid-plasma METH concentration ratio was 2.0 across 24 h and was highly variable. Neutral cotton swab collection yielded significantly higher METH and AMP concentrations than citric acid candy-stimulated expectoration. Mean (SD) areas under the curve for AMP were 21% ± 25% and 24% ± 11% of those observed for METH in plasma and oral fluid, respectively. After a single low or high dose, plasma METH was >2.5 μg/L for up to 24 h in 9 of 12 individuals (mean, 7.3 ± 5.5 μg/L at 24 h); in oral fluid the detection window was at least 24 h (mean, 18.8 ± 18.0 μg/L at 24 h). The plasma and oral fluid 24-h METH detection rates were 54% and 60%, respectively. After four administrations, METH was measurable for 36–72 h (mean, 58.3 ± 14.5 h).

Conclusions: Perceived advantages of oral fluid for verifying METH exposure compared with urine include simpler specimen collection and reduced potential for adulteration, but urine offers higher analyte concentrations and a greater window of detection.
Homogeneous Real-Time Detection of Single-Nucleotide Polymorphisms by Strand Displacement Amplification on the BD ProbeTec ET System
Authors: Wang Sha-Sha , Thornton Keith , Kuhn Andrew M , Nadeau James G , Hellyer Tobin J
Published: February 2004

Background: The BD ProbeTec™ ET System is based on isothermal strand displacement amplification (SDA) of target nucleic acid coupled with homogeneous real-time detection using fluorescent probes. We have developed a novel, rapid method using this platform that incorporates a universal detection format for identification of single-nucleotide polymorphisms (SNPs) and other genotypic variations.

Method: The system uses a common pair of fluorescent Detector Probes in conjunction with unlabeled allele-specific Adapter Primers and a universal buffer chemistry to permit analysis of multiple SNP loci under generic assay conditions. We used Detector Probes labeled with different dyes to facilitate differentiation of two alternative alleles in a single reaction with no postamplification manipulation. We analyzed six SNPs within the human β2-adrenergic receptor (β2AR) gene, using whole blood, buccal swabs, and urine samples, and compared results with those obtained by DNA sequencing.

Results: Unprocessed whole blood was successfully genotyped with as little as 0.1–1 μL of sample per reaction. All six β2AR assays were able to accommodate ≥20 μL of unprocessed whole blood. For the 14 individuals tested, genotypes determined with the six β2AR assays agreed with DNA sequencing results.

Conclusion: SDA-based allelic differentiation on the BD ProbeTec ET System can detect SNPs rapidly, using whole blood, buccal swabs, or urine.
HPLC Determination of Erythrocyte Methotrexate Polyglutamates after Low-Dose Methotrexate Therapy in Patients with Rheumatoid Arthritis
Authors: Dervieux Thierry , Orentas Lein Diana , Marcelletti John , Pischel Ken , Smith Katie , Walsh Michael , Richerson Russell
Published: February 2004

Background: Methotrexate (MTX) may produce antiarthritic effects through polyglutamation to methotrexate polyglutamates (MTXPGs), a process that covalently attaches sequential γ-linked glutamic residues to MTX. We sought to develop an innovative HPLC method for the quantification of these metabolites in erythrocytes.

Methods: Two alternative approaches were developed. In the first approach, MTXPGs from 50 μL of packed erythrocytes were converted to MTX in the presence of plasma γ-glutamyl hydrolase and mercaptoethanol at 37 °C. In the second approach, MTXPG species (up to the hepta order of glutamation) from 100 μL packed erythrocytes were directly quantified in a single run. In both methods, the MTXPGs were extracted from the biological matrix by a simple perchloric acid deproteinization step with direct injection of the extract into the HPLC. The chromatography used a C18 reversed-phase column, an ammonium acetate/acetonitrile buffer, and postcolumn photo-oxidation of MTXPGs to fluorescent analytes.

Results: Intra- and interday imprecision (CVs) were <10% at low and high concentrations of analytes for both methods. The limit of quantification was 5 nmol/L. In 70 patients with rheumatoid arthritis receiving weekly low-dose MTX, the mean (SD) total MTXPG concentration measured after conversion of MTXPGs to MTX was similar to the total MTXPG concentration calculated from the sum of individual MTXPG species [117 (56) vs 120 (59) nmol/L; r = 0.97; slope = 1.0]. The triglutamate predominated over all other MTXPG species (36% of total), the pentaglutamate was the highest order of glutamation detected, and a stability study revealed no change in the polyglutamation pattern in erythrocytes 48 h after phlebotomy when the specimen was stored at 2–8 °C.

Conclusion: The proposed method for quantification of erythrocyte MTXPGs is rapid, sensitive, and accurate and can be applied to the routine monitoring of MTX therapy.
Evaluation of Cell-free Fetal DNA as a Second-Trimester Maternal Serum Marker of Down Syndrome Pregnancy
Authors: Farina Antonio , LeShane Erik S , Lambert-Messerlian Geralyn M , Canick Jacob A , Lee Thomas , Neveux Louis M , Palomaki Glenn E , Bianchi Diana W
Published: February 2004

Background: Second-trimester cell-free fetal DNA (studied only in pregnancies with male fetuses) is higher in maternal serum samples from women carrying Down syndrome fetuses than in unaffected pregnancies. In this study we evaluated the potential performance of fetal DNA as a screening marker for Down syndrome.

Methods: Data on maternal serum fetal DNA concentrations and the corresponding concentrations of the quadruple serum markers were available from 15 Down syndrome cases, each matched for gestational age and length of freezer storage, with 5 control samples. Analyte values were expressed as multiple(s) of the control or population median. Screening performance of fetal DNA, both alone and when added to estimates of quadruple marker performance, was determined after modeling using univariate and multivariate gaussian distribution analysis.

Results: The median fetal DNA concentration in Down syndrome cases was 1.7 times higher than in controls. In univariate analysis, fetal DNA gave a 21% detection rate at a 5% false-positive rate. When added to quadruple marker screening, fetal DNA increased the estimated detection rate from 81% to 86% at a 5% false-positive rate.

Conclusions: Cell-free fetal DNA, measured in maternal serum, can modestly increase screening performance above what is currently available in the second trimester. If and when maternal serum fetal DNA can be measured in pregnancies with both male and female fetuses, the utility and cost-effectiveness of adding it as a Down syndrome screening marker should be assessed.
Comparison of Two Assays for Human Kallikrein 2
Authors: Blijenberg Bert G , Wildhagen Mark F , Bangma Chris H , Finlay Judith A , Väisänen Ville , Schröder Fritz H
Published: February 2004

Background: We compared two recently developed research assays for the measurement of human kallikrein 2 (hK2) in serum: one fully automated assay (Beckman Coulter Access® immunoanalyzer) and one manual assay based on the DELFIA® technology.

Methods: We used two subsets of clinical specimens consisting of 48 samples from prostate cancer patients and 210 samples from participants in an ongoing screening study (ERSPC). Both subsets were measured in the Rotterdam laboratory, and the prostate cancer samples were used for analytical comparison with the originating sites for the assays: Beckman Coulter Research Department (San Diego, CA) and Turku University (Turku, Finland).

Results: Both the Beckman Coulter and the Turku assays performed very similarly between the Rotterdam laboratory and the originating sites: the R2 value for both comparisons was 0.99, and the slope difference between sites was <20%. Deming regression analysis of the DELFIA (y) and Access (x) assays yielded the following: for the prostate cancer group, y = 1.17x − 0.01 (R2 = 0.88; n = 48); and for the ERSPC group, y = 0.62x − 0.01 (R2 = 0.77). Breakdown of the latter group into subgroups (nondiseased, benign prostatic hyperplasia, and prostate cancer samples) gave only minor differences. The Access calibrators were underrecovered by 13% in the DELFIA assay, whereas the DELFIA calibrators were overrecovered by 45% in the Access assay.

Conclusion: The DELFIA and Access assays for hK2, which have similar analytical features, show differences that cannot be explained by calibration.
Benign Prostate-specific Antigen (BPSA) in Serum Is Increased in Benign Prostate Disease
Authors: Linton Harry J , Marks Leonard S , Millar Lisa S , Knott Christine L , Rittenhouse Harry G , Mikolajczyk Stephen D
Published: February 2004

Background: BPSA is a “benign” form of free prostate-specific antigen (PSA) that is increased in prostate transition zone tissues of men with pathologic benign prostatic hyperplasia (BPH). We developed an immunoassay to determine the concentration of BPSA in the serum of men with BPH.

Methods: The BPSA antigen was purified by HPLC, and murine monoclonal antibodies were prepared by standard methods. A fluorogenic ELISA was developed with high specificity for BPSA and no cross-reactivity with other forms of PSA.

Results: The BPSA immunoassay had a lower limit of detection of 6 ng/L and a cross-reactivity of <1% with all other clipped and nonclipped forms of PSA. The BPSA antibody was specific for the internal Lys182 cleavage site that characterizes BPSA. Biopsy-negative men with a median total PSA of 4.8 μg/L had a median of 0.22 μg/L BPSA, representing 25% of the free PSA in serum. BPSA ranged from 0% to 60% of the free PSA in serum. BPSA in a cohort of cancer serum also comprised 25% of the free PSA. Control serum from women or men without increased PSA had nondetectable BPSA.

Conclusions: BPSA is a significant percentage of the free PSA in BPH serum but not in control serum. The presence of prostate cancer does not alter the relative proportions of BPSA in sera with <10 μg/L PSA. BPSA has a wide distribution of concentrations in the serum and may provide clinical information for the study of men with BPH.
New Enzymatic Assay for Glycohemoglobin
Authors: Sakurabayashi Ikunosuke , Watano Tatsurou , Yonehara Satoshi , Ishimaru Kaori , Hirai Kaoru , Komori Tsuguki , Yagi Masayuki
Published: February 2004

Background: Previous methods to measure glycohemoglobin (GHb) have been time-consuming or imprecise; we therefore developed a new enzymatic assay for GHb.

Methods: Blood cells were first hemolyzed, and hemoglobin was digested with protease to yield fructosyl amino acid. Fructosyl amino acid oxidase acts on the fructosyl amino acid and generates hydrogen peroxide, which reacts with chromogens in the presence of peroxidase. Total hemoglobin was measured spectrometrically in the same reaction tube. The results were reported as the ratio of the concentrations of GHb and hemoglobin.

Results: The measured values were comparable to those determined with a HPLC method and with an immunoassay in blood samples from 2854 patients with diabetes. Regression analysis for the enzymatic assay (y) vs the HPLC method (x) produced the following: r = 0.979; slope, 0.994 [95% confidence interval (CI), 0.986–1.001]; y-intercept, 0.04% (95% CI, −0.09% to 0.01%); n = 2854. For the enzymatic assay (y) vs the immunoassay (x), the regression statistics were as follows: r = 0.982; slope, 1.002 (95% CI, 0.995–1.009); y-intercept, 0% (95% CI, −0.05% to 0.05%); n = 2854.

Conclusions: The values measured by the new enzymatic assay are sufficiently correlated with those of the conventional HPLC method and immunoassay, but the proposed assay for GHb is rapid and has high precision.
Detection of Anti-Human T-Lymphotropic Virus Type I Antibody in Whole Blood by a Novel Counting Immunoassay
Authors: Yamaguchi Kazunari , Yonemura Yuji , Okabe Hiroaki , Takahama Yoichi , Nagai Shinya , Yamaguchi Haruki , Hirai Kojiro
Published: February 2004

Background: Assays to screen for and confirm the presence of the antibody for human T-lymphotropic virus type I (HTLV-I) are currently performed with serum or plasma. We developed and evaluated a new counting immunoassay (CIA) for the detection of HTLV-I antibody in whole blood, using recombinant and synthetic peptide antigens.

Methods: We assessed the CIA for detection of HTLV-I antibody in whole blood and plasma. The CIA is an immunity-measuring method that combines latex agglutination with particle-counting technology. The numbers of agglutinated latex particles, single latex particles, and blood cells in a sample are measured based on differences in particle size between latex particles and blood cells.

Results: The CIA and ELISA methods were in agreement for all 24 plasma samples tested, including those from 6 patients with HTLV-I-associated diseases, 6 HTLV-I carriers, and 12 HTLV-I antibody-negative individuals. The concordance between the ELISA (plasma) and the CIA (whole blood) for samples from 24 patients was 100%. The concordance between a particle agglutination method (plasma) and the CIA (plasma or whole blood) for 1065 patients was 99.5%. The concordance between results obtained for 1065 pairs of plasma and whole blood samples with the CIA method was 100%. HTLV-I antibody in whole blood was stable for 3 days after blood collection. With this CIA method, results were available within 15 min.

Conclusions: The CIA method can be used in screening for HTLV-I. The use of whole blood rather than serum or plasma reduces the sample volume and number of blood collections required, as well as assay time.
Influence of Urinary Stones on the Composition of a 24-Hour Urine Sample
Authors: Laube Norbert , Pullmann Michael , Hergarten Stefan , Hesse Albrecht
Published: February 2004

Background: It can be assumed that stones in the urinary tract continuously increase in size by incorporating material from urine. Consequently, urine will exhibit depleted concentrations of lithogenic constituents when urinary stones are present in the patient’s urinary tract.

Methods: To calculate the influence of the depletion effect, we considered two different models of stone growth. In the first model, the increase in stone size depends only on the urinary concentration of a lithogenic substance; the second model also considers the surface area of the growing stone. The case of only one kidney being affected by stone formation is considered separately. We discuss example calculations involving the formation of calcium oxalate.

Results: The calculated depletion effects are of a nonnegligible order of magnitude. Assuming both a measured oxalate concentration of, e.g., 0.37 mmol/L and a reasonable in vivo stone growing rate of 10 mm3/day, a relative underestimation of the real “in situ” oxalate concentration between ∼21% (model 1) and ∼42% (model 2) occurs. The depletion effect increases markedly with increasing stone growth rate.

Conclusions: Metabolic status can be evaluated correctly only in patients who have been declared “stone-free”, e.g., after stone removal. Because the expected stone-related depletion effect in most cases is of high clinical relevance, we recommend estimating the effect of the order of magnitude of the depletion on actual urinary composition.
Comparison of Eight Computer Programs for Receiver-Operating Characteristic Analysis
Authors: Stephan Carsten , Wesseling Sebastian , Schink Tania , Jung Klaus
Published: February 2004

Background: ROC analysis is widely accepted to assess and compare diagnostic validity of laboratory tests. Within the last few years, many new ROC programs have become available but have not been systematically evaluated. The aim of this study was to assess different ROC programs regarding their ease of use, mathematical correctness, final output, and their compatibility with other graphics programs.

Methods: Eight available programs running under Windows (AccuROC, Analyse-It, CMDT, GraphROC, MedCalc, mROC, ROCKIT, and SPSS) were evaluated. ROC analyses of prostate-specific antigen and related values were performed from a dataset of 928 men with prostate cancer and benign prostatic hyperplasia and corresponding subsets. Criteria such as data input, data output, and correctness and completeness of results were used to evaluate the practicability of the programs.

Results: Although the programs produced equivalent results (areas under the curves and their characteristics), we observed deficiencies concerning input of data, processing of the output data, and completeness of the results. Analyse-It, AccuROC, and MedCalc exhibited good performance, but each program had different shortcomings. Only GraphROC could compare curves at a certain sensitivity or specificity cutoff.

Conclusions: Adequate ROC analysis and ROC plotting cannot be performed with a single program. Analyse-It, AccuROC, and MedCalc can be recommended with certain limitations. Further improvements of the programs are necessary.
RIA for Serum Holo-Transcobalamin: Method Evaluation in the Clinical Laboratory and Reference Interval
Authors: Loikas Saila , Löppönen Minna , Suominen Pauli , Møller Jan , Irjala Kerttu , Isoaho Raimo , Kivelä Sirkka-Liisa , Koskinen Pertti , Pelliniemi Tarja-Terttu
Published: February 2004

Background: Decreased serum holo-transcobalamin (holoTC) could be the earliest marker of cobalamin (Cbl) deficiency, but there has been no method suitable for routine use. We evaluated a new commercial holoTC RIA, determined reference values, and assessed holoTC concentrations in relation to other biochemical markers of Cbl deficiency.

Methods: The reference population consisted of 303 individuals 22–88 years of age, without disease or medication affecting Cbl or homocysteine metabolism. In elderly individuals (≥65 years), normal Cbl status was further confirmed by total homocysteine (tHcy; <19 μmol/L) and methylmalonic acid (MMA; <0.28 μmol/L) concentrations within established reference intervals. HoloTC in Cbl deficiency was studied in a population of 107 elderly individuals with normal renal function. The Cbl deficiency was graded as potential (total Cbl ≤150 pmol/L or tHcy ≥19 μmol/L), possible (total Cbl ≤150 pmol/L and either tHcy ≥19 μmol/L or MMA ≥0.45 μmol/L), and probable (tHcy ≥19 μmol/L and MMA ≥0.45 μmol/L).

Results: The intra- and between-assay imprecision (CV) for the holoTC RIA were 4–7% and 6–8%, respectively. A 95% central reference interval for serum holoTC was 37–171 pmol/L. All participants (n = 16) with probable Cbl deficiency, 86% of those with possible, and 30% of those with potential Cbl deficiency had holoTC below the reference limit (<37 pmol/L). The holoTC correlated with total Cbl (rs = 0.80; P <0.0001) and inversely with MMA (rs = −0.52; P <0.0001). HoloTC concentrations were significantly (P = 0.01) higher in women than in men.

Conclusions: The new holoTC RIA is precise and simple to perform. Low holoTC is found in individuals with biochemical signs of Cbl deficiency, but the sensitivity and specificity of low holoTC in diagnosis of Cbl deficiency need to be further evaluated.
Standardization of Immunoassays for Measurement of High-Sensitivity C-reactive Protein. Phase I: Evaluation of Secondary Reference Materials
Authors: Kimberly Mary M , Vesper Hubert W , Caudill Samuel P , Cooper Gerald R , Rifai Nader , Dati Francesco , Myers Gary L
Published: February 2004

Background: Inflammation contributes to the development and progression of atherosclerosis, and C-reactive protein (CRP) can be used as a marker to assess risk for cardiovascular diseases. As variability among existing high-sensitivity CRP (hsCRP) assays can lead to misclassification of patients and hamper implementation of population-based medical decision points, standardization of hsCRP assays is needed.

Methods: We evaluated five proposed secondary reference materials, including two diluted preparations of Certified Reference Material 470 (CRM470), two preparations of a serum-based material with recombinant CRP added, and one serum-based material with isolated CRP added. Twenty-one manufacturers participated in the comparison with 28 different assays. We examined imprecision, linearity, and parallelism with these materials and with fresh serum.

Results: All materials had similar imprecision; CVs for the undiluted materials were 2.1–3.7%. None of the materials was linear across all assays. Each had between one and three cases of nonlinearity, with one preparation of CRM470 having the fewest cases of nonlinearity. Although none of the materials was parallel across all assays, the differences in slope from fresh serum were similar across all assays.

Conclusions: All materials performed similarly with regard to imprecision, linearity, and parallelism. As one preparation of CRM470 had slightly better characteristics than the other materials and because CRM470 had been certified previously as a reference material for the acute-phase reactant range, it will be used in the next phase to standardize hsCRP assays.
Evaluation of Electronic Microarrays for Genotyping Factor V, Factor II, and MTHFR
Authors: Erali Maria , Schmidt Ben , Lyon Elaine , Wittwer Carl
Published: February 2004

Background: Genetic risk factors associated with venous thrombosis include mutations in the factor V (Leiden), factor II (prothrombin), and methylenetetrahydrofolate reductase (MTHFR) genes. We evaluated a method using electronically addressable microarrays for the detection of mutations in these genes that have been associated with vascular disease.

Methods: The NanoChip® Molecular Biology Workstation (Nanogen) uses electronic microarrays for mutation detection. Factor V, factor II, and MTHFR genotypes identified in the NanoChip system on 225 samples were compared with genotypes from LightCycler® assays (Roche). We determined within- and between-cartridge signal and ratio variation and analyzed the effect of additional mutations at or near the detection area used for the NanoChip assays.

Results: Genotypes determined for all three mutations on the NanoChip platform were in complete concordance with LightCycler results. Within-cartridge signal variation as measured by the CV of fluorescence signals was <10% for each allele when present. The within-cartridge CV for heterozygous mutant/wild-type ratios was <8.5%, and between-cartridge CV was <18%. A dilution study showed that results could be obtained in this assay with 6 ng of nucleic acid per PCR, the lowest input tested. The presence of additional sequence variations near the expected mutations can produce equivocal or discrepant results.

Conclusions: Mutation detection using the NanoChip Molecular Biology Workstation was accurate and reproducible for the three assays evaluated.
Quantitative Analysis of Pleural Fluid Cell-free DNA as a Tool for the Classification of Pleural Effusions
Authors: Chan Michael H M , Chow Kai Ming , Chan Anthony T C , Leung Chi Bon , Chan Lisa Y S , Chow Katherine C K , Lam Ching Wan , Lo Y M Dennis
Published: February 2004

Background: Recently, much interest has been focused on the quantification of DNA in miscellaneous body fluids. In this study, the application is extended to classifying pleural effusions by measuring cell-free DNA in pleural fluid.

Methods: We recruited 50 consecutive patients with pleural effusions with informed consent. Pleural fluids were centrifuged at 13 000g, with supernatants aliquoted for extraction and analysis of β-globin DNA sequence by quantitative real-time PCR. Serum and pleural fluid biochemistries were performed to classify pleural effusions using the modified criteria of Light et al. (Ann Intern Med 1972;77:507–13). The ROC curve was plotted to determine the cutoff DNA concentration for classifying pleural fluids as transudates or exudates. Indicators of diagnostic accuracy were calculated for both pleural fluid DNA and modified criteria of Light et al., using the discharge, microbiologic, and histologic diagnoses as the reference standard.

Results: The area under the ROC curve was 0.95 [95% confidence interval (CI), 0.84–0.99]. At 509 genome-equivalents/mL, pleural fluid DNA alone correctly classified 46 of 50 pleural effusions with 91% sensitivity (95% CI, 76–98%), 88% specificity (95% CI, 64–98%), and positive and negative likelihood ratios of 7.7 (95% CI, 3.1–19.5) and 0.10 (95% CI, 0.04–0.27), respectively. With the modified criteria of Light et al., 43 of 50 pleural effusions were correctly classified with 97% sensitivity (95% CI, 91–100%) and 67% specificity (95% CI, 45–89%). There were significant correlations between cell-free DNA and both lactate dehydrogenase and total protein in pleural fluid, suggesting their common origin.

Conclusions: Pleural fluid DNA concentrations are markedly increased in exudative effusions, making it a potential new tool to evaluate the etiologic causes of pleural effusions.
Detection of Autoantibodies to Protein Tyrosine Phosphatase-like Protein IA-2 with a Novel Time-resolved Fluorimetric Assay
Authors: Westerlund-Karlsson Annette , Suonpää Katriina , Ankelo Matti , Ilonen Jorma , Knip Mikael , Hinkkanen Ari E
Published: February 2004

Background: Circulating autoantibodies to pancreatic glutamic acid decarboxylase (GAD65; the 65-kDa isoform of glutamic acid decarboxylase), protein tyrosine phosphatase-like protein IA-2, and insulin can be used as predictive markers of type 1 diabetes. We developed a novel assay for the detection of IA-2 autoantibodies (IA-2As) in serum based on time-resolved fluorimetry, hypothesizing that this kind of assay could provide several advantages over methods described to date, including radiobinding assays (RBAs) and ELISAs.

Methods: The intracellular part of IA-2 (IA-2ic) was biotinylated and bound to streptavidin-coated 96-well plates by simultaneous incubation with serum samples and glutathione S-transferase (GST)-IA-2ic fusion protein. GST-IA-2ic captured by autoantibodies in the serum was detected with europium-labeled anti-GST antibody, and the signal was measured in a time-resolved fluorimeter. A serum sample panel from 100 patients with newly diagnosed type 1 diabetes and 100 unaffected controls was analyzed with the new assay and a conventional RBA.

Results: Among the 100 serum samples from patients with type 1 diabetes, the time-resolved fluorimetric assay identified 74 IA-2A-containing sera, whereas the RBA detected 80 IA-2A-positive samples. Five of the six samples positive in the RBA but not detected by the time-resolved fluorimetric assay were only weakly positive in the RBA. The performance time of the time-resolved fluorimetric assay was 2.5 h compared with 10–12 h required by the RBA.

Conclusions: The time-resolved fluorimetric assay provides a simple, nonradioactive analysis method for the detection of IA-2As with a specificity and a sensitivity comparable to the RBA method. This assay allows substantial reduction in performance time compared with the conventional RBA.