bluuebunny/crossref_metadata_2025 huggingface dataset:
ELISA-type titertray assay of IgM anti-GM1 autoantibodies
Authors: Bech E , Jakobsen J , Orntoft T F
Published: January 2020

We report an ELISA-type titertray assay for autoantibodies against the ganglioside GM1. Trays were coated with ganglioside GM1 and reacted with patients' sera; bound IgM was detected with rabbit antibody to human IgM. High-titer serum from a patient was used as calibrator, another patient's serum as the positive control, and the GM1-specific cholera toxin as the control for GM1 coating. Regression curves of serum titers obtained from different patients were linear and parallel. Intra- and interassay CVs were 4.0-7.8% and 5.5-16%, respectively. We detected antibodies at a titer of 1:250 in normal subjects. Analytical specificity of the calibrator serum against GM1 was demonstrated by immune thin-layer chromatography. Anti-GM1 antibodies were increased in patients with chronic inflammatory demyelinating polyradiculoneuropathy (P < 0.002) or multiple sclerosis (P < 0.01). In Guillain-Barré syndrome, preliminary longitudinal studies showed a decrease in anti-GM1 titer that was related to clinical recovery.
Technical and scientific developments in exposure marker methodology
Authors: Sampson E J , Needham L L , Pirkle J L , Hannon W H , Miller D T , Patterson D G , Bernert J T , Ashley D L , Hill R H , Gunter E W
Published: January 2020

Recent advances in techniques to measure markers of exposure to environmental toxicants in humans are changing the ways in which environmental scientists, epidemiologists, and policymakers characterize and interpret such exposure. In this article we review some major technical and scientific developments in exposure marker methodology for estimating internal dose, with special reference to studies conducted at the US Centers for Disease Control and Prevention. We consider important characteristics of laboratory methods, advances in laboratory technology, analytical standards, and quality assurance of laboratory measurements; comparisons with indirect methods for estimating exposures, such as exposure indices and questionnaires; human pharmacokinetic data; sampling problems; surveillance of human exposures to toxicants; and interpretation of measurements. With a view to increasing the reliability of exposure assessment, we make recommendations for obtaining more data on human exposure to toxicants.
Carcinogen adducts: use in diagnosis and risk assessment
Authors: Farmer P B
Published: January 2020

Exposure to genotoxic carcinogens results in the formation of covalently bound adducts of the carcinogens with cellular nucleophilic molecules, including DNA and protein. Quantitative measurements of these adducts may be used to monitor exposure to these carcinogens. The analytical methods required to detect the adducts need to be of exceptional sensitivity and include 32P-postlabeling, immunoassay, and physicochemical techniques (e.g., mass spectrometry or fluorescence measurements). Owing to its accessibility and long lifetime, hemoglobin is also suitable for carcinogen adduct measurement, and techniques based on gas chromatography-mass spectrometry or immunoassay have been developed for this purpose. Although the measurement of adducts is now accepted as a valid means of monitoring exposure to carcinogens, the value of such measurements in indicating carcinogenic risk in humans is less certain. However, adduct concentrations, particularly at low doses of carcinogen, have in several instances been shown to correlate with tumorigenicity in animal experiments.
Complex exposures: potentials for assessing integrated exposures
Authors: Ikeda M
Published: January 2020

To determine empirically the most probable mode of interaction of chemicals given in combination, major occupational health, environmental health, and toxicology journals published in the past 10 years were sorted by eye for studies on noncarcinogenic and nonteratogenic effects of industrial and environmental chemicals in combination. Of the 159 relevant studies, 49 reported "more than additive" effects; however, most of these had methodological weakness in extrapolating the observation to effects in humans; e.g., the dose used was much larger than expected exposure levels in real life. After excluding the studies that were based on unrealistic conditions, the "more than additive" effect was observed in < 5% of the studies reviewed. In the absence of information to suggest otherwise, therefore, the risk of underestimating toxicity by assuming the additiveness is probably < 5%.
Direct stopped-flow fluorescence polarization immunoassay of abused drugs and their metabolites in urine
Authors: Pérez-Bendito D , Gómez-Hens A , Gaikwad A
Published: January 2020

Kinetic methodology was applied to the direct determination of abused drugs (amphetamines, cocaine, and cannabinoids) in urine by stopped-flow fluorescence polarization immunoassay (SF-FPIA). This technique provides analytical data within a few seconds by measuring the variation of polarized fluorescence with time during development of immunochemical reactions. Methods based on this principle are particularly suitable for routine screening of these drugs in urine, being more expeditious than conventional FPIA methods. The dynamic ranges of the calibration curves were 20-300 micrograms/L for d,l-amphetamine, 15-300 micrograms/L for benzoylecgonine (a cocaine metabolite), and 10-400 micrograms/L for 11-nor-delta 8-tetrahydrocannabinol-9-carboxylic acid (a cannabinoid metabolite). The detection limits and within- and between-assay precision were better than those provided by conventional FPIA. Analytical recoveries ranged between 97.5% for d,l-amphetamine and 102.4% for the cannabinoid metabolite. The results for the three analytes were consistent with those obtained by conventional FPIA.
Candidate reference method for determining serum theophylline applied to target-setting in external quality assessment and routine method evaluation
Authors: Thienpont L M , Van Nieuwenhove B , Stöckl D , De Leenheer A P
Published: January 2020

We present a candidate reference method for the determination of serum theophylline by isotope dilution gas chromatography-mass spectrometry, using extractive alkylation in sample preparation. The maximum method bias was < 1.0%. The mean method imprecision (CV) was 0.63% (range 0.37-0.96%), calculated from the results of six measurements independently performed on 3 days. The maximum total error of the method, including the method bias and 95% confidence interval, was < 2.1%. Investigation of various substances and metabolites for interference proved the specificity of the proposed method. The method was used to evaluate three routine assays by linear regression analysis. Performance of the TDx II (Abbott) was excellent and of the Emit 2000 (Syva) assay was good. The Kodak Ektachem assay showed a high dispersion of results and a pronounced positive bias in the subtherapeutic range.
Findings of an external quality assessment scheme for determining aluminum in dialysis fluids and water
Authors: Taylor A , Briggs R J , Cevik C
Published: January 2020

The analytical performance of laboratories participating in the dialysis fluids and water aluminum program of the Guildford External Quality Assessment Scheme over the period 1986-1993 has been reviewed. For aluminum concentrations > 3.33 mumol/L in dialysis fluids, the between-laboratory CV has fallen from approximately 36% to 23%, whereas for specimens of water the reduction was from 36% to approximately 18%. Improvements at lower concentrations were less impressive. Performance of individual participants varied; only a few consistently provided accurate results. Many of the participants are able to measure serum aluminum well, so lack of expertise is not responsible for poor results. We suggest that matrix effects associated with different specimen types have a significant influence on performance and that due account is not always taken of these factors. Resolution of these problems would be aided by appropriate reference materials.
Carboxyhemoglobin determined in neonatal blood with a CO-oximeter unaffected by fetal oxyhemoglobin
Authors: Vreman H J , Stevenson D K
Published: January 2020

Measurements of carboxyhemoglobin (COHb) for clinical purposes are routinely made with CO-oximeters. However, fetal hemoglobin (HbF) interferes with this spectrophotometric method. The manufacturer (Ciba Corning Diagnostics) of a new CO-oximeter (CCD 270) claims that COHb measurements with this instrument are insignificantly affected by HbF. We examined this claim through CO-oximeter analysis of cord blood/adult blood mixtures and compared the results with those obtained by gas chromatography (GC). We also studied the influence of oxygen and bilirubin concentrations. Measurements of COHb with CCD 270 were significantly lower than the GC measurements, but the differences were not clinically important. Linear regression analysis of HbF and COHb measurements (n = 68) showed significant correlation (P < 0.0001) between the two factors for the older CCD 2500 CO-oximeter (R2 = 0.56), but not for the CCD 270 (R2 = 0.06) or GC (R2 = 0.02). Bilirubin concentrations, which affected COHb measurements with CCD 2500, did not significantly affect CCD 270 measurements. We conclude that COHb measurements with CCD 270 CO-oximeter are not affected by HbF or bilirubin concentrations.
Circadian rhythmic variations in serum concentrations of clinically important lipids
Authors: Rivera-Coll A , Fuentes-Arderiu X , Díez-Noguera A
Published: January 2020

Twenty-five apparently healthy adults (13 men and 12 women), mean age 29.5 years (SD 3.6 years), served as subjects in a 24-h study conducted in Barcelona (Spain) in spring of 1990. Six blood samples were collected from each subject at 4-h intervals over a 24-h period, beginning at 1000. The oral temperature was measured at 2-h intervals to facilitate an independent biological time reference. The serum concentrations of apolipoprotein (apo) A-I, apo B, cholesterol, high- and low-density lipoprotein cholesterol, and triglyceride were measured. The circadian rhythms of these quantities were studied with use of a periodic function resulting from the sum of two cosine functions of 24 and 12 h. For serum concentrations of apo A-I and apo B, only the 24- or 12-h components, respectively, were significant. However, the other quantities studied were significantly affected by both rhythmic components. The maximum daily rhythmic variation ranged from 5% (with respect to the daily mean) for apo A-I to 63% for triglyceride concentration. In all cases except triglyceride, the daily maximum values were measured in the afternoon samples, near the time of maximum oral temperature.
Structural and functional assessment of high-density lipoprotein heterogeneity
Authors: Dobiásová M , Frohlich J J
Published: January 2020

We studied the heterogeneity of high-density lipoproteins (HDL) in plasma of 110 subjects, using three different methods: (a) gradient gel electrophoresis (GGE); (b) electroimmunoassay, to measure the concentration of lipoprotein particles containing apoprotein (apo) AI but no apo-AII (LP AI); and (c) cholesterol esterification rate (FERHDL) in very-low- and low-density lipoprotein-depleted plasma. There were two study groups: patients with hypertension, whose plasma lipid profile was similar to their respective controls, and patients with hypoalphalipoproteinemia (hypoalpha), whose family members served as controls. Values for FERHDL were significantly higher in both risk groups than in their respective controls. LP AI was significantly decreased only in the hypoalpha subjects. Generally, LP AI and FERHDL were inversely related. LP AI correlated strongly with plasma HDL-cholesterol, apo AI, and LP AI/AII; FERHDL correlated with those values inversely. LP AI, but not FERHDL, correlated with HDL free cholesterol. On the other hand, FERHDL correlated strongly with plasma concentrations of triglycerides and with the plasma ratio of total/HDL-cholesterol while LP AI did not. GGE determination of the composition of HDL subspecies showed that both FERHDL and LP AI were significantly related to the content of HDL2b particles: FERHDL inversely, LP AI directly; the relative amount of HDL3b,c particles correlated only with FERHDL. We conclude that GGE and FERHDL can be used to quantify both the apparently protective (HDL2b) and risk-associated (HDL3b,c) particles, whereas the concentration of LP AI in plasma mainly reflects the concentration of the HDL2 subpopulation.
Plasma lipoprotein profiles of normocholesterolemic and hypercholesterolemic homozygotes for apolipoprotein E2(Arg158-->Cys) compared
Authors: Zhao S P , Smelt A H , Van den Maagdenberg A M , Van Tol A , Vroom T F , Gevers Leuven J A , Van der Laarse A , Van 't Hooft F M
Published: January 2020

We compared plasma lipoprotein profiles of 15 individuals with normocholesterolemic (plasma cholesterol 4.81 +/- 0.90 mmol/L) familial dysbetalipoproteinemia (NFD) and 15 patients with hypercholesterolemic (plasma cholesterol 10.61 +/- 2.32 mmol/L) familial dysbetalipoproteinemia (HFD), matched for age and sex. All subjects were homozygous for apoE2(Arg158-->Cys). Compared with 15 normolipidemic controls (plasma cholesterol 5.47 +/- 0.92 mmol/L), subjects with NFD and HFD had greater cholesterol concentrations of large very-low-density lipoprotein (VLDL1), small VLDL (VLDL2), and intermediate-density lipoprotein, each of which was correlated to their plasma total cholesterol concentration. VLDL1 and VLDL2 subfractions were enriched in cholesteryl ester, and plasma cholesteryl ester transfer protein activities were increased in both NFD and HFD; however, absolute changes were larger in HFD than in NFD. Concentrations of low-density lipoprotein cholesterol were lower in HFD (1.89 +/- 0.48 mmol/L) and NFD (1.56 +/- 0.36 mmol/L) than in normolipidemic controls (3.35 +/- 0.73 mmol/L). We conclude that all subjects homozygous for apoE2(Arg158-->Cys) show features of dysbetalipoproteinemia.
Premature termination mutations in two patients with deficiency of lactate dehydrogenase H(B) subunit
Authors: Sudo K , Maekawa M , Kanno T , Li S S , Akizuki S , Magara T
Published: January 2020

Two patients with low lactate dehydrogenase (LD) activity were discovered during healthcare examinations and were found to be homozygous for LD-H (heart) subunit deficiency by electrophoretic isoenzyme analysis of serum and erythrocyte hemolysate. The molecular nature of the genetic mutations was characterized by amplification by the polymerase chain reaction and DNA sequencing. In one case, a single-base substitution (T-->G transversion) at codon 147 of the LD-H(B) gene resulted in a nonsense mutation; in the other case, a deletion of 2 base pairs had occurred at codon 139, resulting in a frameshift translation and premature termination.
Endotoxin binding to platelets in blood from patients with a sepsis syndrome
Authors: Salden H J , Bas B M
Published: January 2020

Endotoxin, the lipopolysaccharide cell wall constituent of Gram-negative bacteria, produces symptoms of the Gram-negative sepsis syndrome. By measuring endotoxin in blood from septic patients it may be possible to select a subpopulation of patients in which mortality can be prevented by treatment with anti-endotoxin antibodies. We evaluated the performance of an endotoxin-free blood-collection tube. Within-run and between-run CVs of our endotoxin assay were 4-18% and 8-20%, respectively. In endotoxin-positive samples (LPS > or = 6 ng/L), the concentration of endotoxin in platelet-rich plasma was significantly higher (P < 0.001) than in platelet-poor plasma. Apparent binding of endotoxin to platelets ranged from 0% to 92%. The correlation between the apparent percentage binding of LPS to platelets and the platelet count in platelet-rich plasma is linear and positive, but LPS is not bound solely to platelets. We conclude that endotoxin must be measured in platelet-rich plasma.
Data utilization, not data acquisition, is the main problem
Authors: Altshuler C H
Published: January 2020

Previous studies focusing on the value of laboratory testing have sometimes been flawed because they had not always determined whether the data generated had been appropriately used by clinicians. It has been well documented that neither clinicians nor administrators always use objective data effectively. When data are not used appropriately by clinicians, not only are morbidity and mortality increased, but also care costs are higher. Failures by administrators result in lost opportunities to identify and correct system deficiencies. To provide information support to assure good data utilization, a prototype desktop workstation has been developed and partially implemented. Experience with such a system suggests that not only may it help provide better care more economically, but it can also aid in implementation of Practice Guidelines (Agency for Health Care Policy and Research) and Clinical Indicator (Joint Commission on Accreditation of Healthcare Organizations) programs. Laboratorians can be influential in this effort and promote the use of new knowledge in patient care in a timely and responsible manner.
Cost-effectiveness analysis for evaluation of screening programs: hereditary hemochromatosis
Authors: Buffone G J , Beck J R
Published: January 2020

A significant body of research over the last 10-20 years supports the hypothesis that screening for hereditary hemochromatosis (HH) may be cost-effective, given the low-cost, low-risk therapeutic options available for most homozygous individuals. The factors that confound a straightforward test of this hypothesis include the fact that the disease is not fully penetrant and that, to achieve the anticipated life-year gains, therapy must be instituted before disease complications become irreversible. Recent articles and editorials, as well as practice guidelines prepared by the College of American Pathologists, recommend screening for HH with transferrin saturation and ferritin testing, and with percutaneous liver biopsy for those with positive laboratory test results. Patients at risk would be treated with phlebotomy for life and monitored with ferritin testing. We present a cost-effectiveness analysis that evaluates the efficacy of using a screening strategy to accomplish the desired healthcare goals.
Laboratory diagnostics in light of massive changes in official health policies
Authors: Müller B
Published: January 2020

Virtually 100% of the German population has health insurance. Of this 100%, approximately 90% are members of the Statutory Health Insurance plan. This insurance plan assumes responsibility for practically all of the costs of treatment; self-participation by the patient in the costs is minimal. To counteract the financial deficits of the Statutory Health Insurance funds, the Health Care Reform Act was introduced in 1993, bringing with it massive economy measures for everyone involved in the health sector. For the practitioner sector, this new legislation provides for, among other things, revision of the structure and the reimbursement of laboratories. In this context, the originally agreed-upon introduction of lump-sum payments as reimbursement for laboratory tests was abandoned, in the face of vigorous resistance by the medical profession. Instead, the system of reimbursement for each individual test continues to apply. However, the number of tests is to be limited for each specific group of doctors. In addition, the laboratory fee in the practitioner sector is being reduced by 20%.
Artificial neural network predictions of urinary calculus compositions analyzed with infrared spectroscopy
Authors: Volmer M , Wolthers B G , Metting H J , de Haan T H , Coenegracht P M , van der Slik W
Published: January 2020

Infrared (IR) spectroscopy is used to analyze urinary calculus (renal stone) constituents. However, interpretation of IR spectra for quantifying urinary calculus constituents in mixtures is difficult, requiring expert knowledge by trained technicians. In our laboratory IR spectra of unknown calculi are compared with references spectra in a computerized library search of 235 reference spectra from various mixtures of constituents in different proportions, followed by visual interpretation of band intensities for more precise semiquantitative determination of the composition. To minimize the need for this last step, we tested artificial neural network models for detecting the most frequently occurring compositions of urinary calculi. Using constrained mixture designs, we prepared various samples containing ammonium hydrogen urate, brushite, carbonate apatite, cystine, struvite, uric acid, weddellite, and whewellite for use as a training set. We assayed known artificial mixtures as well as selected patients' samples from which the semiquantitative compositions were determined by computerized library search followed by visual interpretation. Neural network analysis was more accurate than the library search and required less expert knowledge because careful visual inspection of the band intensities could be omitted. We conclude that neural networks are promising tools for routine quantification of urinary calculus compositions and for other related types of analyses in the clinical laboratory.
Quantification of apolipoprotein A-IV in human plasma by immunonephelometry
Authors: Schwarz S , Haas B , Luley C , Schäfer J R , Steinmetz A
Published: January 2020

We developed a nephelometric procedure for determining concentrations of human plasma apolipoprotein (apo) A-IV. Results obtained correlate well with those measured by an established electroimmunodiffusion assay (r = 0.98; n = 56). Intra- and interassay CVs were 2.4% and 2.0%, respectively, indicating excellent precision and reproducibility. Various conditions of sample treatment, such as adequate storage, freezing, and thawing, did not affect results significantly. However, keeping samples at room temperature for 4 days led to a slight increase in measured values. Preincubation with a cholesterinesterase-detergent mixture abolished interference from triglyceride-rich lipoproteins, allowing assay of samples containing triglycerides as great as 10 g/L. The assay is easily applicable to clinical laboratories for routine diagnostic use, as shown with hypertriglyceridemic plasmas and samples with a broad range of apo A-IV concentrations.
Multicenter evaluation of automated immunoturbidimetric assays for measurement of apolipoproteins A-I and B in serum and plasma
Authors: Kahn S E , Labbé R F , Pfadenhauer E H , Kanonchoff A D , Joseph R T
Published: January 2020

Results of a multicenter evaluation of automated assays for measurement of apolipoproteins (apo) A-I and B with the Paramax analytical system are reported. Apo A-I and apo B response surface models were used to optimize concentrations of critical assay variables. Overall imprecision for apo A-I controls at concentrations of 1.01-1.61 g/L was 3.7-6.6%; overall imprecision for apo B controls at 1.00-1.61 g/L was 2.8-6.9%. There was no interference in apo A-I measurements. Albumin concentrations > 59 g/L resulted in a negative interference, and collection in sodium heparin caused a positive interference in apo B results. Apo A-I and apo B assays demonstrated acceptable agreement with comparative methods, although the Paramax apo B assays had a negative bias with respect to comparison methods. In 116 healthy individuals, serum apo A-I ranged from 0.97 to 2.05 g/L and serum apo B ranged from 0.51 to 1.32 g/L.
Improved determination of small amounts of free hydroxyproline in biological fluids
Authors: Lange M , Mályusz M
Published: January 2020

A fast, reliable, and sensitive (< 20 pmol) method for the quantification of 4-hydroxyproline is described. It ensures good separation of imino acid peaks, eliminates interference by primary amino acids, and guarantees full (96-105%) recovery of hydroxyproline (HYP). Interfering primary amino acids are derivatized by o-phthaldialdehyde and removed from the sample by use of a discardable C18 column. HYP is measured photometrically at 254 nm after a second derivatization with phenylisothiocyanate and isocratic separation on a reversed-phase HPLC column.
Quantification of skeletal alkaline phosphatase in osteoporotic serum by wheat germ agglutinin precipitation, heat inactivation, and a two-site immunoradiometric assay
Authors: Farley J R , Hall S L , Ilacas D , Orcutt C , Miller B E , Hill C S , Baylink D J
Published: January 2020

Three methods for quantifying skeletal alkaline phosphatase (ALP; EC 3.1.3.1) activity/immunoactivity in serum--heat inactivation, wheat germ agglutinin (WGA) precipitation, and an immunoradiometric assay--were tested for recovery and specificity and applied to 81 sera collected from 14 postmenopausal osteoporotic subjects. The heat-inactivation and WGA precipitation assays showed relative recoveries of 91-100% and 16-32%, respectively, for skeletal ALP with complete specificity (no cross-reactivity with hepatic or intestinal ALP); the IRMA showed a relative recovery of 86-100% and 11-14% cross-reactivity with hepatic ALP. There was a closer correlation between the heat-inactivation assay and IRMA (r = 0.833) than between the WGA precipitation assay and IRMA (r = 0.673) or between the heat-inactivation and WGA-precipitation assays (r = 0.568). The WGA precipitation assay failed to detect skeletal ALP in three serum samples that contained significant amounts as determined by the heat-inactivation assay and the IRMA.
Microfabrication in silicon microphysiometry
Authors: Baxter G T , Bousse L J , Dawes T D , Libby J M , Modlin D N , Owicki J C , Parce J W
Published: January 2020

Over the past 5 years, microphysiometry has proved an effective means for detecting physiological changes in cultured cells, particularly as a functional assay for the activation of many cellular receptors. To demonstrate the clinical relevance of this method, we have used it to detect bacterial antibiotic sensitivity and to discriminate between bacteriostatic and bacteriocidal concentrations. The light-addressable potentiometric sensor, upon which microphysiometry is based, is well suited for structural manipulations based on photolithography and micromachining, and we have begun to take advantage of this capability. We present results from a research instrument with eight separate assay channels on a 5-cm2 chip. We discuss the planned evolution of the technology toward high-through-put instruments and instruments capable of performing single-cell measurements.
PCR in a silicon microstructure
Authors: Wilding P , Shoffner M A , Kricka L J
Published: January 2020

Devices for performing polymerase chain reactions (PCR) have been developed for use with photolithographed silicon. Microchambers capable of holding between 5.0 and 10 microL of PCR reagents were constructed by etching specific areas of rectangular silicon chips (17 x 15 mm), which were then capped with Pyrex glass. These silicon devices (PCRChips), which were etched to depths of 40-80 microns, permitted free flow of fluids in the microchannels and microchambers. Access to the microchambers was through holes in the silicon. Thermal cycling of the PCR reagents was achieved by placing the disposable PCRChip in a small holder containing a computer-controlled Peltier heater-cooler. Successful amplification was demonstrated by electrophoresis of products in agarose gel containing ethidium bromide, and the migration of the product was compared with that obtained in a commercially available thermal cycler. The thermal characteristics of the silicon, coupled with the high surface area to volume ratio in the new devices, are particularly advantageous features for amplification by PCR.
Feasibility studies for simultaneous immunochemical multianalyte drug assay by capillary electrophoresis with laser-induced fluorescence
Authors: Chen F T , Evangelista R A
Published: January 2020

We present a method for the simultaneous quantification of multiple drug analytes in urine, based on combining immunochemical binding with capillary electrophoretic separation. Two fluorescent drug-cyanine (Cy) dye conjugates were prepared as competing species for the immunoassay. Morphine was derivatized with Cy5 (lambda max = 652 nm, epsilon = 215,000 mol-1cm-1 L), phencyclidine (PCP) with Cy5.5 (lambda max = 675 nm, epsilon = 200,000 mol-1cm-1L). The high-efficiency resolving power of the capillary electrophoresis system (20 microns x 27 cm column) separated the individual labeled drugs, and the antigen-antibody complexes were detected by laser-induced fluorescence (laser: 10 mW He-Ne at 632.8 nm) with Cy5 diacid as internal standard. Simultaneous competitive immunoassay of morphine and PCP in urine showed that the free labeled-drug peak areas were proportional to the concentrations of the drug species present in the urine sample. This immunoassay can be performed routinely and reproducibly in < 5 min with analytical detection limits of 4 nmol/L for PCP and 40 nmol/L for morphine.
Role of low-density lipoproteins in atherogenesis and development of coronary heart disease
Authors: Grundy S M
Published: January 2020

There is a strong association between increased blood concentrations of low-density lipoprotein (LDL) and severity of coronary atherosclerosis. Multiple mechanisms affect hypercholesterolemia, e.g., diet, aging, hormones, and genetics. LDL receptors apparently are also important--through down-regulation, defects in structure, or decreased numbers--as are changes in LDL binding characteristics caused by alterations in apolipoprotein B content or structure. Current concepts of LDL metabolism are extensively reviewed, including the role of modified or oxidized LDL in atherogenesis.
Serum apolipoproteins A-I and B as markers of coronary artery disease risk in early life: the Bogalusa Heart Study
Authors: Srinivasan S R , Berenson G S
Published: January 2020

The functional properties of the apolipoproteins (apoB and apoA-I) governing lipoprotein metabolism make these variables conceptually important as markers for coronary artery disease risk. This aspect was examined in a biracial (black-white) population of children as part of the Bogalusa Heart Study. White children, especially boys, showed lower concentrations of apoA-I and ratios of cholesterol to apoB within low-density lipoprotein (LDL) than did black children. Persistence of apoB concentrations over time, coupled with its strong linkage with apoB gene locus, underscores the value of detecting apoB excess early in life. Further, the impact of apoE genotypes on apoB and apoA-I levels is already evident in childhood. We found that, as a screening test for detecting increased LDL cholesterol, apoB is superior to total cholesterol. Low values for apoA-I concentration, the apoA-I to apoB ratio, and LDL cholesterol to apoB ratio in children are strongly related to parental incidence of myocardial infarction; no such relationship is seen with respect to lipoprotein cholesterols. Thus, expanding screening strategies might be useful for identifying individuals with adverse apolipoprotein profiles early in life.
Relation of high-density lipoprotein subfractions and apolipoprotein E isoforms to coronary disease
Authors: Wilson P W
Published: January 2020

Although a variety of methods have become available for the determination of high-density lipoprotein (HDL) subfractions in plasma, a review of published data from nine studies of coronary disease outcomes and 10 investigations of coronary artery disease severity do no suggest that measurement of HDL2-cholesterol (C) and HDL3-C offer any advantage in the prediction of coronary disease over the determination of total HDL-C alone. Apolipoprotein E is typically present in plasma as one of six isoforms, the six being encoded by three common alleles, epsilon 2, epsilon 3, and epsilon 4. The epsilon 3 allele is the most common, the epsilon 4 allele has been reported to be associated with higher cholesterol concentrations, and the epsilon 2 and epsilon 4 alleles are both associated with higher triglyceride concentrations. Clinical and arteriographic studies of coronary disease suggest that vascular disease risk is increased among persons with the epsilon 4 allele.
Multicenter evaluation of enhanced chemiluminescence labeled-antibody immunoassay (Amerlite-MAB) for free thyroxine
Authors: Christofides N D , Sheehan C P
Published: January 2020

The technical and diagnostic performance of the Amerlite-MAB enhanced luminescence assay for free thyroxine (FT4) was assessed in a multicenter evaluation trial. The euthyroid central 95% reference range for FT4 (1393 subjects) was 11.5-27.7 pmol/L (by cumulative frequency plot with two-tailed 2.5% cutoffs). Results (y) agreed with those of similar radioactive method (Amerlex-MAB FT4) (x): y = 1.06 x + 0.54, Sylx = 335.5, n = 235). Mean within-assay precision (CV) in six centers was 5.7% at 6 pmol/L and 2.6% at 51 pmol/L FT4; between-assay precision (CV) was 7.2% and 3.6%, respectively. Diagnostic performance was assessed in all patient groups usually encountered, including those with nonthyroidal illness and extreme binding-protein anomalies. In elderly euthyroid subjects, the proportion of above-normal FT4 values exceeded that in younger age-groups. These increases often accompanied normal estimates of thyrotropin and, in some cases, might have arisen by interference from medication that is taken more often by the elderly.
Indirect estimation of thyroid hormone-binding proteins to calculate free thyroxine index: comparison of nonisotopic methods that use labeled thyroxine ("T-uptake")
Authors: Faix J D , Rosen H N , Velazquez F R
Published: January 2020

There are many alternative ways of estimating free thyroxine (T4) when thyrotropin screening results are abnormal. In addition to free T4 immunoassays, the menu of most automated immunoassay instruments includes a nonisotopic version of the original triiodothyronine (T3)-uptake assay called "T-uptake." We evaluated the ability of five such assays (Access, ES-300, IMx, Magnum Opus, and Stratus) to accurately estimate the free thyroxine index (FTI) in euthyroid, hyperthyroid, and hypothyroid patients with abnormal concentrations of thyroid hormone-binding proteins, and in patients with nonthyroidal illness. For comparison, we calculated a similar FTI, using either T3-uptake or direct measurement of thyroxine-binding globulin (TBG). Euthyroid reference ranges were comparable. Of euthyroid patients with increased TBG, 12-32% and 5-20% had increased or suppressed FTI, respectively, depending on the T-uptake method used. Except for IMx, 6-35% of hypothyroid patients with increased TBG had inappropriately increased FTI. Patients with nonthyroidal illness had comparable results regardless of the method used, and T-uptake methods were variably affected by known inhibitors of thyroid hormone binding. The most reliable T-uptake method appeared to be the IMx, which, despite claims that it measures all thyroid hormone-binding proteins, correlated best with TBG concentrations.
Apolipoprotein E2/E3/E4 genotyping with agarose gels
Authors: Dallinga-Thie G M , van Linde-Sibenius Trip M , Kock L A , De Bruin T W
Published: January 2020

Here we report an improved, simple method to assign the human apolipoprotein (apo) E genotype and its isoforms, apo E2, apo E3, and apo E4. Genomic DNA was amplified with specific primers that included the polymorphic region of amino acids 112 and 158. Digestion of the product with Hhal resulted in unique fragments that were separated on Meta-Phor agarose instead of polyacrylamide. The pattern of unique DNA fragments obtained unequivocally characterizes the different apo E alleles.
Direct solid-phase sequence analysis of the human p53 gene by use of multiplex polymerase chain reaction and alpha-thiotriphosphate nucleotides
Authors: Berg C , Hedrum A , Holmberg A , Pontén F , Uhlén M , Lundeberg J
Published: January 2020

Among the candidate cancer-prognostic genes is the p53 tumor suppressor gene, which, when mutated, plays an important role in the development of many types of cancers. To facilitate robust large-scale DNA analysis of microdissected tumor biopsies, we describe a multiplex/nested PCR approach for a simultaneous outer amplification of exons 4-9 of the human p53 gene with parallel amplification of the HLA-DQB1 locus, involving a total of 14 primers. This approach reduces the required number of cells for analysis and avoids any variation in the amplifications of the individual p53 exons during the common outer amplification step. The HLA sequencing allows sample identification because the DQB1 locus is highly polymorphic and is thereby patient-specific. The p53 and HLA amplicons are analyzed by solid-phase sequencing in a semiautomated format. To improve the DNA sequence quality, we used 2'-deoxyribonucleoside 5'-O-1-thiotriphosphates in the sequencing reactions.
Cholesterol content of circulating immune complexes in patients with coronary stenosis and subjects without evidence of atherosclerosis
Authors: Lecomte E , Herbeth B , Clerc G , Khalife K , Siest G , Artur Y
Published: January 2020

The biological variation factors for cholesterol in circulating immune complexes (CIC-cholesterol) were studied in 941 unselected supposedly healthy volunteers, ages 4 to 78 years. We found a complex effect of age, including the existence of two peaks of CIC-cholesterol, one in males between 11 and 14 years and in females between 11 and 30 years, and in both sexes another peak between 41 and 60 years, and in both sexes a decrease between 31 and 40 years. By use of multiple regression analysis and after adjustment for age, CIC-cholesterol was positively related to plasma cholesterol concentration and leukocyte count, values being lower in females than in males and among subjects taking anti-inflammatory drugs. In addition, CIC-cholesterol was measured in 76 coronary angiography patients and in 100 supposedly healthy controls, ages 30 to 77 years. We noticed a significant increase (P < or = 0.05) of CIC-cholesterol when patients were affected by coronary stenosis between 20% and 50% (71.8 +/- 52.5 mumol/L vs 46.2 +/- 45.9 mumol/L in controls), but this was less pronounced in those with > 50% of obstruction (58.9 +/- 54.3 mumol/L); however, serum total cholesterol was not modified or even surprisingly slightly decreased in the coronary angiography individuals. Nevertheless, an important overlap of values in controls and patients makes questionable the usefulness of this variable in clinical practice.
Improved RNase protection assay for quantifying LDL-receptor mRNA; estimation of analytical imprecision and biological variance in peripheral blood mononuclear cells
Authors: Petersen N E , Larsen L K , Nissen H , Jensen L G , Jensen A , Hyltoft Petersen P , Hørder  , Gregersen N , Kristiansen K
Published: January 2020

We have improved the protocol for RNA quantification by using RNase protection. Instead of precipitation and extraction with phenol and chloroform, we use a faster and more reliable precipitation based on guanidinium thiocyanate (GdSCN). The internal standard is produced by in vitro transcription of a DNA template constructed so as to allow simultaneous detection of the in vitro transcript and the low-density lipoprotein receptor (LDLR) mRNA by use of the same probe and hybridization. Addition of this internal standard at the step for RNA isolation reduced the analytical imprecision from 40.8% to 19.3%. Estimates of the within- and between-subject biological variations of the LDLR mRNA content in peripheral blood mononuclear cells (PBMCs) isolated from healthy volunteers were 21.5% and 13.6%, respectively, and the analytical imprecision was 22.6%. The mean content of LDLR mRNA in PBMCs from healthy individuals was 0.78 copies per cell.
Proposed confirmatory procedure for detecting 5 alpha-dihydrotestosterone doping in male athletes
Authors: Kicman A T , Coutts S B , Walker C J , Cowan D A
Published: January 2020

Currently, there is no recommended confirmatory procedure for detecting doping with the anabolic steroid 5 alpha-dihydrotestosterone (DHT) in sportsmen. To develop a method, we determined ratios of hormone concentrations in urine from 120 healthy men and used these to set discrimination limits. These limits were then applied to results for urine specimens from 10 volunteers given DHT percutaneously (125 mg twice daily for 4 days). The ratio of DHT to epitestosterone (EpiT) was chosen as the primary marker of DHT administration, and ratios of 5 alpha-androstane-3 alpha, 17 beta-diol (5 alpha-ADIOL) to EpiT, 5 alpha-ADIOL to luteinizing hormone, and 5 alpha-ADIOL to 5 beta-androstane-3 alpha, 17 beta-diol were proposed as secondary markers. To evaluate method suitability, we analyzed 194 samples from sports competitors; results for 193 samples were negative, but the ratios in 1 sample greatly exceeded all the chosen limits. In conclusion, we propose that a test scheme based on our recommendations be considered for implementation in all Olympic drug-testing laboratories.
Glycohemoglobin assays evaluated in a large-scale quality-control survey
Authors: Gillery P , Labbé D , Dumont G , Vassault A
Published: January 2020

We report the results of a national quality-control survey on glycohemoglobin (GHb), monitored in France by the Société Française de Biologie Clinique on behalf of the authority of the "Agence du Médicament." A sample of lyophilized hemolysate was sent to 3109 laboratories. Results were obtained from 2770 laboratories. HbA1C, HbA1, and total GHb were measured by 50%, 24%, and 26% of the participants, respectively. Of these measurements, 79% of the HbA1C results and 76% of the total GHb results, but only 48% of the HbA1 results, were within the +/- 20% limits of the indicated target values. Mean values for the hemolysate ranged from 8% to 11% for HbA1C, from 7% to 12% for HbA1, and from 11% to 13% for total GHb. The interlaboratory CVs ranged from 3% to 20%, according to method used. So, methods used for GHb assay, which are based on various principles, exhibit very different analytical performances. Nonetheless, this large-scale study indicates that some techniques can support transferability of results from laboratory to laboratory.
Cardiac troponin-I is not expressed in fetal and healthy or diseased adult human skeletal muscle tissue
Authors: Bodor G S , Porterfield D , Voss E M , Smith S , Apple F S
Published: January 2020

Cardiac troponin-I (cTnI) is not found in sera of patients with skeletal muscle disease in the absence of myocardial injury. It is not known, however, whether trace amounts of cTnI are expressed in regenerating human skeletal muscle, as has been observed with creatine kinase MB. Using immunohistochemical and biochemical techniques, we investigated cTnI expression in various human muscle tissues: human heart tissue (n = 5), normal adult skeletal muscle (n = 3), and fetal heart (n = 3) and skeletal muscle (n = 3) obtained, respectively, during heart transplant, from autopsy, or from a tissue bank. Specimens from diagnostic tissue biopsies were used as diseased skeletal muscle: polymyositis (PM), n = 13; Duchenne muscular dystrophy (DMD), n = 6. Frozen sections 8 microns thick were stained immunohistochemically for either cTnI or TnI (cardiac or skeletal) by using monoclonal antibodies (MAb) 2B1.9 (cTnI specific) or 3C5.10 (reactive with all TnI isoforms), respectively. cTnI was measured in tissue homogenates by an immunofluorometric assay. Cardiac muscle was stained by both MAbs. Normal fetal and adult skeletal muscle, and samples from all of the PM and DMD patients, stained only with the nonspecific MAb (3C5.10), confirming the sole presence of skeletal TnI. No cTnI was detectable by immunoassay in any skeletal muscle sample. We conclude that cTnI is not expressed in human skeletal muscle during development or during regenerative muscle disease processes such as PM or DMD.
Plasma alpha-glutathione S-transferase assessed as a marker of liver damage in patients with chronic hepatitis C
Authors: Vaubourdolle M , Chazouillères O , Briaud I , Legendre C , Serfaty L , Poupon R , Giboudeau J
Published: January 2020

alpha-Glutathione S-transferase (alpha-GST; EC 2.5.1.18) has been advocated as a better marker of hepatocellular damage than the transaminases in toxic and autoimmune hepatitis. We have assessed the potential interest of plasma alpha-GST determination in 94 anti-hepatitis C virus-positive patients with histologically proven chronic hepatitis C (34 women, 60 men, ages 40.0 +/- 11.9 years). Blood samples were assayed for aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyltransferase, alkaline phosphatase, and alpha-GST on the same day a liver biopsy was performed. alpha-GST concentrations were significantly above reference values in 64% of patients (compared with 58% for AST, 68% for ALT), and this increase was seen in 52% of patients with normal values for transaminases and a Knodell score > 3. Furthermore, there was a significant correlation between alpha-GST and lobular necrosis score (r = 0.31; P < 0.01). Our findings suggest that association of plasma alpha-GST with ALT may improve the biochemical assessment of liver damage in patients with chronic hepatitis C.
Time-resolved immunofluorometric assay of p53 protein
Authors: Levesque M A , D'Costa M , Angelopoulou K , Diamandis E P
Published: January 2020

A common feature of human tumor tissue is mutant p53 protein accumulation. Here we evaluate a new "sandwich" immunoassay for p53 protein incorporating modifications to a previously reported method, including the use of microtiter plates coated directly with the anti-p53 monoclonal antibody DO-1, a detergent- and mouse serum-containing sample diluent, and a labeled secondary antibody diluent containing goat serum. The use of CM-1 antiserum to probe the immunocaptured p53 and the detection of bound complexes by a labeled secondary antibody allows coupling to a time-resolved fluorescence detection system. The new assay yielded p53 concentrations comparable with those by the previous assay for breast tumor cytosols (n = 198), nondiseased breast tissues (n = 70), and five transformed cell lines, but showed differences in p53 values measured in sera from patients without cancer (n = 78). These serum differences were found to reflect nonspecific interferences affecting the original method, which implies that the new immunoassay has improved specificity for serum p53 quantification.
Cobas Integra: clinical laboratory instrument with continuous and random-access capabilities
Authors: Palmer S M , Kaufman R A , Salamone S J , Blake-Courtney J , Bette W , Wahl H P , Furrer F
Published: January 2020

The Cobas Integra from Roche Diagnostic Systems is a new clinical laboratory analyzer with continuous and random-access features for routine chemistries, specific proteins, electrolytes, hormones, therapeutic drugs, and drugs of abuse. The system maintains 68 test-specific reagent cassettes on board, along with multiple ion-selective electrodes (ISEs) for electrolyte determinations. This gives the Cobas Integra the capability of analyzing as many as 72 analytes without having to load additional reagents. We describe the basic analyzer configuration and the subsystems for absorbance, fluorescence polarization, and ISE measurements. Performance characteristics for precision, methods comparison, and on-board stability are given for assays representative of the various test groups. The 29 Cobas Integra tests evaluated in the present study show good agreement (r > or = 0.98 and slopes generally 0.90 to 1.12) with the respective methods available on either the Olympus AU5000, Hitachi 911 or 717, Behring BNA, or Abbott TDx systems. Total assay precision (CV) ranged from 0.8% to 8.5%, and calibration curves were stable for as long as 20 weeks. Test throughput, which is dependent on pipetting sequence, was determined to be up to 600 tests per hour without ISE and up to 750 tests per hour with ISE; the time to first result was 2.0-10.0 min.