bluuebunny/crossref_metadata_2025 huggingface dataset:
Development and validation of sensitive method for determination of serum cotinine in smokers and nonsmokers by liquid chromatography/atmospheric pressure ionization tandem mass spectrometry
Authors: Bernert John T , Turner Wayman E , Pirkle James L , Sosnoff Connie S , Akins James R , Waldrep Mary K , Ann Qinghong , Covey Thomas R , Whitfield Wanda E , Gunter Elaine W , Miller Barbara B , Patterson Donald G , Needham Larry L , Hannon W Harry , Sampson Eric J
Published: January 2020

We describe a sensitive and specific method for measuring cotinine in serum by HPLC coupled to an atmospheric pressure chemical ionization tandem mass spectrometer. This method can analyze 100 samples/day on a routine basis, and its limit of detection of 50 ng/L makes it applicable to the analysis of samples from nonsmokers potentially exposed to environmental tobacco smoke. Analytical accuracy has been demonstrated from the analysis of NIST cotinine standards and from comparative analyses by both the current method and gas chromatography/high-resolution mass spectrometry. Precision has been examined through the repetitive analysis of a series of bench and blind QC materials. This method has been applied to the analysis of cotinine in serum samples collected as part of the Third National Health and Nutrition Examination Survey (NHANES III).
Subnanogram-concentration measurement of buprenorphine in human plasma by electron-capture capillary gas chromatography: application to pharmacokinetics of sublingual buprenorphine
Authors: Everhart E Thomas , Cheung Polly , Shwonek Peter , Zabel Karen , Tisdale Eileen C , Jacob Peyton , Mendelson John , Jones Reese T
Published: January 2020

We describe a sensitive and specific method for the measurement of buprenorphine in human plasma. The method involves a structural analog as an internal calibrator, careful control of pH during sample extraction to maximize drug recovery, and back-extraction into acid followed by reextraction to eliminate endogenous interferences. After evaporation, sample residues are derivatized with heptafluorobutyric anhydride and analyzed by separation on a fused-silica polymethylsiloxane capillary column and electron-capture detection. Calibration curves were linear in the ranges 0.1–2.0 μg/L and 2.0–20 μg/L, with within-run CVs of 9.7% at 0.1 μg/L to 5.0% at 20 μg/L, and total CVs of 15.9% at 0.1 μg/L to 6.5% at 10 μg/L. The limit of quantification was 0.1 μg/L. The method was utilized in studies to determine the absolute bioavailability of sublingual doses of 2 mg of buprenorphine in 1 mL of 300 mL/L ethanol and the bioequivalence of sublingual 8-mg tablet and 300 mL/L ethanol solution formulations.
IMP-dehydrogenase inhibition in human lymphocytes and lymphoblasts by mycophenolic acid and mycophenolic acid glucuronide
Authors: Griesmacher Andrea , Weigel Günter , Seebacher Gernot , Müller Mathias M
Published: January 2020

Inosine 5′-monophosphate dehydrogenase (IMP-DH) activities were measured in human lymphocytes (exhibiting type I IMP-DH activity) and human lymphoblasts (exhibiting type II IMP-DH activity) in the presence of various amounts of mycophenolic acid (MPA) (0–20 μmol/L) and MPA glucuronide (MPAG) (0–200 μmol/L). Moreover, the influences of human serum albumin (HSA) and human plasma on the MPA- and MPAG-mediated effects were investigated. In the presence of water, 2.5 μmol/L MPA decreased the IMP-DH activity measured in lymphocytes by 60%, whereas in lymphoblasts a 80% inhibition was detectable. In the presence of ≥10 μmol/L MPA, lymphocytic as well as lymphoblastic IMP-DH activities were reduced in a similar manner. The concentration of MPAG required for 50% inhibition was for both cell types >25 μmol/L and <50 μmol/L, respectively. MPAG (200 μmol/L) reduced lymphocytic as well as lymphoblastic IMP-DH activity by ∼80%. With 100 g/L HSA or human plasma as diluent, the inhibitory effects of MPA and MPAG were significantly (P <0.05) diminished, whereas HSA concentrations ≤25 g/L only slightly influenced the inhibition of IMP-DH activity by MPA and MPAG. In summary, it can be clearly demonstrated that not only MPA but also MPAG contributes to the inhibition of both IMP-DH isoenzymes, which might be relevant for the immunosuppressive properties of mycophenolate mofetil in transplant patients.
Calibration of ion-exchange HPLC measurements of glycohemoglobin: effect on interassay precision
Authors: Gerlo Erik , Gorus Frans
Published: January 2020

To investigate the effect of calibration with lyophilized calibrators on the interassay precision of glycohemoglobin (glyHb) measurements, we used an ion-exchange HPLC system equipped with a Pharmacia Mono S® HR 5/5 column. Calibration of analytical runs substantially increased interassay variation (CV), from 1.7% to 4.4% and from 0.9% to 3.2% for control samples with low (6.5%) and high (14%) glyHb percentages, respectively. Standardization of glyHb results, though essential for interlaboratory comparisons, should not be done at the expense of assay precision, as may occur with thoughtless use of lyophilized calibrators. We therefore recommend the use of carefully determined conversion factors for standardization of glyHb results obtained with ion-exchange HPLC systems that are capable of excellent long-term interassay precision.
Glycohemoglobin filter assay for doctors’ offices based on boronic acid affinity principle
Authors: Frantzen Frank , Grimsrud Kjersti , Heggli Dag-Erik , Faaren Arne Ludvig , Løvli Trond , Sundrehagen Erling
Published: January 2020

We present a new filter assay for the determination of glycohemoglobin as a unique application of the boronic acid affinity principle. With the use of a water-soluble blue-colored boronic acid derivative and a specific precipitation method for hemoglobin, total hemoglobin including bound boronic acid is precipitated and collected on a filter strip before quantification. Hemoglobin and boronic acid are quantified by a dual-wavelength reflectometric measurement, and the result is reported directly as percent glycohemoglobin. The test is simple, quick, and designed as a doctors’ office test for the monitoring and management of diabetes. The imprecision of the assay is <4% over the range 3–18% Hb A1c, and the method is linear up to at least 20% Hb A1c. Comparisons with four well-established glycohemoglobin methods yielded correlation coefficients ranging from 0.94 to 0.99, with slopes from 0.94 to 1.01.
Stability of long-chain and short-chain 3-hydroxyacyl-CoA dehydrogenase activity in postmortem liver
Authors: Hale Daniel E , Cornell John E , Bennett Michael J
Published: January 2020

Inherited enzyme defects in mitochondrial fatty acid oxidation (FAO) are associated with acute metabolic crisis and sudden death. Necropsy findings may be subtle, yielding no diagnosis and precluding genetic counseling. Preliminary identification of an FAO disorder requires the use of sophisticated tools (e.g., GC/MS) and specific body fluids, and the diagnosis rests on molecular analysis or enzyme assay. At present, confirmation of long-chain or short-chain 3-hydroxyacyl-CoA dehydrogenase deficiency relies on measurement of enzyme activity. Here, we report our examination of the effect of storage temperature (25, 4, −20, and −70 °C) and the postmortem interval on enzyme activities in rat and human liver. Enzyme activity decreases 50% in 30 h in samples stored at 25 °C, whereas 55 h at 4 °C is required to reach this value; freezing minimizes this loss. Regardless of rate of degradation, however, the short-chain to long-chain activity ratio remains constant—which should make it possible to differentiate postmortem degradation from enzyme deficiency.
Rapid screening method for detecting mutations in the 21-hydroxylase gene
Authors: Oriola Josep , Plensa Isabel , Machuca Isabel , Pavía Carles , Rivera-Fillat Francisca
Published: January 2020

Impaired synthesis of adrenal steroid hormones because of steroid 21-hydroxylase deficiency is one of the most common inborn errors of metabolism. To expedite molecular diagnosis in families with 21-hydroxylase deficiency, we have designed a rapid strategy to determine nine of the most common mutations in the 21-hydroxylase gene. According to the mutation to be detected, we apply either of two simple strategies: digestion with adequate restriction enzyme or use of the amplification-created restriction site (ACRS) approach and subsequent restriction analysis. Both procedures are rapid and, being nonradioactive, are safer to perform; moreover determination of zygosity in the analyzed mutations requires only one tube per mutation.
Antioxidative enzyme activities in human erythrocytes
Authors: Andersen Helle Raun , Nielsen Jesper B , Nielsen Flemming , Grandjean Philippe
Published: January 2020

Reliable and standardized methods are necessary to determine the expression of antioxidative enzymes and their role in maintaining health. In addition, the variability of the enzyme activities within the general population caused by age, gender, and life-style factors must be described. This study describes methodological conditions that are suitable for analyzing copper–zinc superoxide dismutase (CuZn-SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and glutathione reductase (GR) in human erythrocytes with a high degree of reproducibility. Intervals for the enzyme activities have been established in a randomly selected population of 220 individuals between 20 and 89 years of age. An age-related decrease was observed in CuZn-SOD and GR activities, whereas no age-related changes were demonstrated for GSH-Px and CAT. The GSH-Px activity was positively associated with the intake of dietary supplements and negatively correlated with tobacco consumption. These factors probably account for the fact that women tended to have higher GSH-Px activity.
Clinical and technical evaluation of ACS™BR serum assay of MUC1 gene-derived glycoprotein in breast cancer, and comparison with CA 15-3 assays
Authors: Bon Gijsbert G , von Mensdorff-Pouilly Silvia , Kenemans Peter , van Kamp Gerard J , Verstraeten Rob A , Hilgers Jo , Meijer Sybren , Vermorken Jan B
Published: January 2020

The mucin glycoprotein-detecting assay CA 15-3 is a valuable tool for monitoring the course of disease in breast cancer patients. Assays of CA 15-3 are based on the use of two MAbs to polymorphic epithelial mucin (PEM). We evaluated the technical and clinical performance of the Chiron ACSTM BR, an automated competitive chemiluminescence assay using a single MAb, B27.29, and compared the assay’s results with those of the Centocor CA 15-3 RIA, the Abbott IMx CA 15-3, and the Boehringer Mannheim Enzymun-Test CA 15-3. The study population consisted of 253 healthy women, 66 patients with benign breast disease, 168 breast cancer patients, and 76 patients with other carcinomas. In the technical evaluation, we assessed the precision and linearity on dilution of the ACS BR assay. Cutoff values (upper limits of values seen in healthy subjects) were determined for all four assays. Agreement between the assays was studied by linear regression analysis. The ACS BR assay gave within- and between-assay CVs of 2.2% and 3.9%, respectively. Three samples from healthy women gave discordant values by ACS BR and were not included in the calculations. All four assays exhibit a highly similar pattern when monitoring breast cancer disease; the closest agreement of values was obtained between ACS BR and Centocor CA 15-3. We conclude that the ACS BR assay is a fast and reliable immunoassay for measuring PEM in serum. Although it detects a slightly different epitope on the PEM molecule than is targeted in other assays, for cancer serum samples it agreed better with the original Centocor CA 15-3 assay than did the other two CA 15-3 assays tested.
Precipitation methods for high-density lipoprotein cholesterol measurement compared, and final evaluation under routine operating conditions of a method with a low sample-to-reagent ratio
Authors: Demacker Pierre N M , Hessels Marja , Toenhake-Dijkstra Helga , Baadenhuijsen Henk
Published: January 2020

We evaluated six precipitation methods for high-density lipoprotein cholesterol (HDL-chol) determination: the heparin/Mn2+precipitation reagent method (Hep), two variants of the phosphotungstic acid/Mg2+ method (Tung-L and Tung-B), the dextran sulfate 50 000/Mg2+ method (Dex), the PEG 6000 method (PEG), and the PEG 6000/dextran sulfate 15 000 (PEG/Dex) method. The Tung-B and PEG/Dex precipitation methods have a low sample/precipitation reagent volume ratio (<0.4). The Tung-B, Dex, PEG, and PEG/Dex methods gave similar values, averaging within 0.1 mmol/L of each other, showing that the precipitation selectivity of these methods is comparable. The precipitation efficiency of Tung-B and Peg/Dex, however, was superior. Ultrafiltration of the supernatants was needed only at triglyceride concentrations >16.4 mmol/L (undiluted sample) or >28.0 mmol/L (sample diluted twofold); however, ultrafiltration without dilution was the most accurate method. Results of Tung-B under routine conditions (33 technicians) agreed well with those of the PEG method for 406 normo- and hyperlipidemic plasma samples. By comparison with the HDL-chol method from the Centers for Disease Control and Prevention, the Tung-B method showed a total error of 10.6%, which fulfills the criteria of the National Cholesterol Education Program for HDL-chol analysis. In conclusion, with motivated personnel, Tung-B is a reliable, cost-effective method for routine HDL-chol analysis.
Positive interference in lithium determinations from clot activator in collection container
Authors: Sampson Maureen , Ruddel Mark , Albright Suzanne , Elin Ronald J
Published: January 2020

We describe positive interference with the ion-selective electrode determination of lithium (Lytening 2Z analyzer; Dade) when blood is collected in a 10-mL plain red-top plastic Vacutainer Plus Tube® (Becton Dickinson) containing a silica clot activator and silicone surfactant (prod. no. 36–7820). We evaluated both the original tube (blue-labeled) and a new tube formulated to contain less silicone surfactant (striped-labeled). We determined that the interference is from either the silica clot activator or the silicone surfactant used to fix the silica to the tube and is inversely related to the volume of blood in the tube. Long-term intermittent exposure of the Li ion-selective electrode to the silica clot activator or surfactant results in decreased Li values—in terms of both the positive interference by the silica clot activator or surfactant and the actual Li determinations. Moreover, this long-term interference with the Li ion-selective electrode for patients’ specimens is undetected by the Dade control material (QCLytes).
Exhaled human breath measurement method for assessing exposure to halogenated volatile organic compounds
Authors: Pleil Joachim D , Lindstrom Andrew B
Published: January 2020

The organic constituents of exhaled human breath are representative of blood-borne concentrations through gas exchange in the blood/breath interface in the lungs. The presence of specific compounds can be an indicator of recent exposure or represent a biological response of the subject. For volatile organic compounds (VOCs), sampling and analysis of breath is preferred to direct measurement from blood samples because breath collection is noninvasive, potentially infectious waste is avoided, and the measurement of gas-phase analytes is much simpler in a gas matrix rather than in a complex biological tissue such as blood. To exploit these advantages, we have developed the “single breath canister” (SBC) technique, a simple direct collection method for individual alveolar breath samples, and adapted conventional gas chromatography–mass spectrometry analytical methods for trace-concentration VOC analysis. The focus of this paper is to describe briefly the techniques for making VOC measurements in breath, to present some specific applications for which these methods are relevant, and to demonstrate how to estimate exposure to example VOCs on the basis of breath elimination. We present data from three different exposure scenarios: (a) vinyl chloride and cis-1,2-dichloroethene from showering with contaminated water from a private well, (b) chloroform and bromodichloromethane from high-intensity swimming in chlorinated pool water, and (c) trichloroethene from a controlled exposure chamber experiment. In all cases, for all subjects, the experiment is the same: preexposure breath measurement, exposure to halogenated VOC, and a postexposure time-dependent series of breath measurements. Data are presented only to demonstrate the use of the method and how to interpret the analytical results.
Diagnosis of Duchenne/Becker muscular dystrophy and quantitative identification of carrier status by use of entangled solution capillary electrophoresis
Authors: Fortina Paolo , Cheng Jing , Shoffner Mann A , Surrey Saul , Hitchcock Wendy M , Kricka Larry J , Wilding Peter
Published: January 2020

Use of capillary electrophoresis, a new and useful analytical tool, offers a variety of advantages for nucleic acid analyses, including rapid analysis, automation, high resolution, qualitative and quantitative results, and low consumption of both sample and reagents. We report the first example of the use of entangled solution capillary electrophoresis (ESCE) and laser-induced fluorescence detection (LIF) for separation-based diagnostics in the quantitative analysis of multiplex PCR products for determination of carrier status of Duchenne/Becker muscular dystrophy (DMD/BMD). This ap-proach greatly improved the speed, resolution, and sensitivity of information needed for the diagnosis of DMD/BMD compared with that from conventional diagnostic methods, and is of general utility for diagnosis of genetic diseases.
Measurement of antibodies against glutamic acid decarboxylase 65 (GADA): two new 125I assays compared with [35S]GAD 65-ligand binding assay
Authors: Borg Henrik , Fernlund Per , Sundkvist Göran
Published: January 2020

Recently, 65-kDa glutamic acid decarboxylase (GAD 65) antibodies (GADA) have been introduced as autoimmune markers in blood to confirm the diagnosis of insulin-dependent diabetes mellitus (IDDM). In this study, to evaluate two new assays that use 125I-labeled GAD 65, we assayed samples from 100 children with recent onset of diabetes and 100 control children; the results were compared with those of a [35S]GADA assay and with results for islet cell antibodies (ICA), the conventional autoimmune marker. Receiver operating characteristic (ROC) curve analysis showed one of the new assays (from RSR) to be more sensitive (P = 0.01) than the comparison ([35S]GADA) assay, whereas the second new assay (from Elias) was less sensitive (P <0.001). The GADA frequency at 97.5% specificity was greatest in the comparison assay: 63 of 100 vs 41 of 100 (P < 0.01) and 53 of 100 (P = 0.16) in the RSR and Elias assays, respectively. Almost all GADA-positive patients had ICA, but one-third of the ICA-positive patients was GADA-negative. Accordingly, adding GADA analysis results to ICA testing increased the frequency of detection of autoimmune markers only slightly (from 81% to 85%). In conclusion, at 97.5% specificity the [35S]GADA assay seemed to be more efficient than the 125I assays, although the difference was significant only for the Elias 125I assay. Antigen-specific antibodies other than GADA may explain the difference in GADA and ICA frequencies.
Calculation of LDL-cholesterol by using apolipoprotein B for classification of nonchylomicronemic dyslipemia
Authors: Planella Teresa , Cortés Mariano , Martínez-Brú Cecília , González-Sastre Francesc , Ordóñez-Llanos Jordi
Published: January 2020

In this paper we propose a calculation of LDL-cholesterol (LDL-C) not affected by hypertriglyceridemia by using lipid quantities directly measured in total serum. We also propose an algorithm for the classification of nonchylomicronemic dyslipemias. Plasma apolipoproteins (apo) A-I, B, total cholesterol (TC), triglycerides (TG), and cholesterol of lipoproteins were measured in a group of 38 normolipemic and 120 dyslipemic patients (42 phenotype IIa, 38 IIb, and 40 IV) classified according to TG and LDL-C values. Discriminant analysis was applied to obtain the best classification with the lowest number of quantities directly measured from total serum (TC, TG, and apo B), and multiple regression analysis was performed to find an equation to calculate LDL-C from these quantities. Apo B seems to be a useful discriminator between normolipemic and phenotype IIa patients, by using a cutoff value of 1.35 g/L obtained by ROC curve analysis. The proposed algorithm, based on lipid quantities measured by easily automated methods, is shown to be a good alternative for the classification of nonhyperchylomicronemic dyslipemia. LDL-C calculated from TC, TG, and apo B proved a better estimate of true LDL-C than the estimate obtained with Friedewald’s formula.
Evaluation of a rapid, new method for detecting serum IgG antibodies to Helicobacter pylori
Authors: Sharma Tarun K , Young Eugene L , Miller Sherry , Cutler Alan F
Published: January 2020

There is an increased need for rapid, inexpensive tests to diagnose Helicobacter pylori infection. Our objective was to determine the performance characteristics of an immunochromatographic test (ICT) for detection of anti-H. pylori IgG antibodies. A commercially available ICT kit (FlexSure® HP) was tested with a well-characterized cohort of banked sera as well as with fresh serum from randomly selected symptomatic patients. The ICT was evaluated with 107 stored sera and 96 prospective patients. The test correctly identified 65 of 68 H. pylori-infected and 37 of 39 noninfected stored sera and 54 of 57 infected and 30 of 39 noninfected patients. Sensitivity, specificity, and positive and negative predictive values were 96%, 95%, 97%, and 93% in stored serum and 95%, 77%, 86%, and 91% in fresh serum, respectively. We concluded that the ICT, reported at 4 min, is highly sensitive for detecting anti-H. pylori IgG antibodies in human serum. With a high negative predictive value, the test may be used to exclude H. pylori infection in symptomatic patients.
Designing in quality through design control: a manufacturer’s perspective
Authors: Lasky Fred D , Boser Robert B
Published: January 2020

Quality by design is a comprehensive program that begins with understanding user needs and continues through (but does not end with) monitoring customer acceptance. Management tools and processes such as ISO 9000 standards and the Food and Drug Administration Quality System Regulations exist to guide medical device manufacturers in quality practices. The goal is to deliver products acceptable for their intended use. Quality control begins with defining attributes ranging from color to accuracy and precision. Failure mode and effects analysis and risk analysis consider both probability and severity of potential malfunctions and their effects on patients or operators. Tools used to implement design and production practices include Program Evaluation and Review Technique (PERT) charts and industry-conceived concepts, such as Six Sigma techniques. Their use varies with manufacturer, depending on product and customer needs and the manufacturer’s specific quality practices. Verification confirms that input goals are met. Then, validation assures that intended clinical needs are continually satisfied by establishing adequate production specifications. Conformance is monitored to verify that stable, consistent processes are in place, and precise user instructions enable the device to satisfy its intended use. Finally, complaint tracking can help assess whether needs have been met. Modifications in service, hardware, or instructions (including quality control) might be required. Therefore, both manufacturers and users work in partnership for continual improvement. The manufacturer’s knowledge of design, production, and service needs of its devices enable it to recommend appropriate quality-control protocols for clinical testing.
Quality systems for unit-use testing devices
Authors: Phillips David L
Published: January 2020

Unit-use testing or single-test-system analysis has existed for many years. Quality-control and quality-assurance procedures have generally used conventional methods and lyophilized or aqueous control materials. Because these materials were readily available and generally accepted, they became part of the quality-assurance program for many early unit-use test systems such as the DuPont aca®. Over the years, these control products became standard and are now required as part of good laboratory practice. Technically speaking, however, conventional quality-control methods and materials cannot completely control the test system when used in a unit-use or single-test-system device. When conventional control material is run on a unit-use single-test system, only that testing unit is checked. One cannot test every unit with control material because by definition these are single-test systems: Once the control has been run, the patient’s sample cannot be run. Conventional quality-assurance and quality-control methods do not, of themselves, assure quality. A one-size-fits-all, or “two levels per day of use” as outlined in the CLIA ’88 regulations, is not appropriate. The divergence between HCFA-approved practices and those of the deemed agencies, coupled with the financial aspects of this quality-control method, led to the formation of the Subcommittee on Unit Use Testing of the National Committee on Clinical Laboratory Standards to develop guidelines for manufacturers, users, and regulators to use in developing new quality systems.
Decision-making laboratory computer systems as essential tools for achievement of total quality
Authors: Blick Kenneth E
Published: January 2020

Areas other than the analytical process should be the focus of concern about quality issues in the laboratory because nearly 95% of errors occur at the nonanalytical front and back ends of the testing process. Until now, computer systems have been designed to handle the more predictable aspects of laboratory testing, necessitating that the infrequent and unpredictable data events be handled by manual systems. The manual systems are termed “workarounds” and indeed, because they occur sporadically, they are frequently not handled predictably. Here, I describe and give examples of an expert laboratory computer system that can be designed to handle both predictable and unpredictable data events without the use of manual workarounds. This expert system works in concert with a dynamic database allowing such data events to be detected in real time and handled predictably, thus providing a tool to address quality assurance issues throughout the testing process. The system performs up to 31 separate actions or tasks based on data events that in the past were handled by human workarounds.
Simultaneous identification and quantitation of codeine, morphine, hydrocodone, and hydromorphone in urine as trimethylsilyl and oxime derivatives by gas chromatography–mass spectrometry
Authors: Broussard Larry A , Presley Lance C , Pittman Thomas , Clouette Randy , Wimbish Gary H
Published: January 2020

Following enzymatic hydrolysis of urine, a gas chromatography–mass spectrometry method for the simultaneous determination of codeine, morphine, hydrocodone, and hydromorphone uses hydroxylamine to form oxime derivatives of the keto-opiates (i.e., hydrocodone, hydromorphone, oxycodone, and oxymorphone). These trimethylsilyl-derivatized forms no longer interfere with the detection and quantitation of codeine and morphine. Samples are extracted on solid-phase columns and quantitated by deuterated internal calibrations of each analyte with selected ion monitoring. Codeine, morphine, hydrocodone, and hydromorphone are completely separated, allowing simultaneous quantitation without interference and a chromatographic analysis time <9 min.
Evaluation of selected-ion storage ion-trap mass spectrometry for detecting urinary anabolic agents
Authors: Bowers Larry D , Borts David J
Published: January 2020

Limits of detection are important issues for GC/MS screening for anabolic agents and for confirmation of various drugs of abuse. We compared a quadrupole ion trap (QIT) operated in two different selected-ion storage modes and a quadrupole mass filter (QMF) operated in the selected-ion monitoring mode. Results with the model compound tetrachlorobenzene indicate that, for simultaneous monitoring of more than four ions, the QIT operated in a frequency-modulated selected-ion storage mode has better limits of detection than the QMF. Use of a single-ion storage technique gave results similar to those of the QMF. We also evaluated both QIT selected-ion storage approaches for the limits of detection of the trimethylsilyl derivatives of four anabolic steroid metabolites and the β-agonist clenbuterol. We found no improvement in detection limits over that of a similar method with selected-ion monitoring and a QMF when four anabolic steroid metabolites and clenbuterol were extracted from a urine matrix. The lack of improvement in the limit of detection resulted from matrix background signals at masses similar to those of the steroids.
Semiautomated procedures for evaluation of carbohydrate-deficient transferrin in the diagnosis of alcohol abuse
Authors: Bean Pamela , Liegmann Karsten , Løvli Trond , Westby Christina , Sundrehagen Erling
Published: January 2020

Carbohydrate-deficient transferrin (CDT) may now be the most valuable biological marker for diagnosis of alcohol abuse. We compared the diagnostic performance of two new CDT tests, Axis %CDT turbidimetric immunoassay (TIA) and Axis %CDT HPLC, against Specialty Laboratories’ isoelectric focusing/immunoblotting/laser densitometry (IEF/IB/LD). Both Axis tests include one-half the concentration of trisialotransferrin isoforms in their CDT quantitation schemes. Considering an alcohol abuse prevalence of 7%, Axis %CDT TIA shows a sensitivity of 87% at 98% specificity and a positive predictive value (PPV) of 0.75; %CDT HPLC shows a sensitivity of 87% at 100% specificity for a PPV of 1, and the IEF/IB/LD shows 81% sensitivity at 94% specificity for a PPV of 0.5. All three CDT tests show the same negative predictive value (0.98). Both Axis procedures perform better than IEF/IB/LD in the diagnosis of alcohol abuse; %CDT TIA is available in several semiautomated, cost-effective formats.
Apolipoprotein E genotyping by capillary electrophoretic analysis of restriction fragments
Authors: De Bellis Gianluca , Salani Giuliana , Panigone Silvia , Betti Ferruccio , Invernizzi Luigia , Luzzana Massimo
Published: January 2020

We present the genotyping of apolipoprotein (apo) E by means of restriction fragment analysis of amplified genomic DNA by high-performance capillary electrophoresis and a replaceable non-gel-sieving matrix. This procedure streamlines the genotyping of apo E in large-scale population studies because of the automation and speed of capillary electrophoresis.
Measurement of serum iohexol by determination of iodine with inductively coupled plasma–atomic emission spectroscopy
Authors: Braselton W Emmett , Stuart Kirk J , Kruger John M
Published: January 2020

We used inductively coupled plasma–atomic emission spectroscopy to measure serum iodine to determine plasma clearance of iohexol, an iodinated radiographic contrast agent. We determined I at 178.276 nm on the phosphorus 178.287 nm channel of the polychromator by utilization of spectrum shifter offset software, while correcting for P with the sequential P 214.914 nm emission line. Determination of I on the polychromator provided excellent precision in the measurement of serum I, even though the interelement correction of P was done with a sequential P line. Total imprecision (CV) (n = 13) was 16% (at 13.7 mg/L I), 8.6% (28.7 mg/L), 3.6% (59.0 mg/L), 2.6% (120.5 mg/L), 1.7% (237.8 mg/L), 1.2% (478.7 mg/L), and 1.8% (597 mg/L). The linear range was 15 to 600 mg/L. Iohexol added to serum (mg/L I) and recoveries (%) were 15 (91.3%), 30 (95.7%), 60 (98.3%), 120 (100.4%), 240 (99.1%), 480 (99.7%), and 600 (99.5%). Studies on dogs and cats administered a single intravenous injection of iohexol indicated that a dose of 300 mg I/kg body weight was sufficient for measurement of glomerular filtration rate by using a single compartment model for plasma clearance with three samples drawn 3 to 7 h after treatment. With this protocol, correlation coefficients were >0.99 on the β phase of the plasma disappearance curve.
Prostate-specific antigen expression in a case of intracystic carcinoma of the breast: characterization of immunoreactive protein and literature surveys
Authors: Mannello Ferdinando , Sebastiani Maurizio , Amati Silvana , Gazzanelli Giancarlo
Published: January 2020

A case is presented of female breast intracystic carcinoma with prostate-specific antigen (PSA) expressed in high amounts in aspirated cystic fluid (55 μg/L). Tumor extract analysis revealed the presence of both estrogen and progesterone receptors (0.38 and 1.87 nmol/L, respectively) and high quantities of PSA too (19.52 μg/L). Chromatographic analysis of cystic fluid revealed two peaks of PSA, at the expected positions for free and bound serine protease. A major proportion of 33-kDa free form was also confirmed by Western blotting analysis. Free PSA was heat-stable at 56 °C and displayed no change after freezing–thawing. These findings are discussed in the context of a detailed literature survey. Our data support the contention that PSA immunoreactivity in intracystic fluid of breast carcinoma is partly the result of secretory activity by the neoplastic cells and that the steroid receptors can also modulate its expression.
Molecular biological xxxmethods in diagnosis and treatment of liver diseases
Authors: Worman Howard J
Published: January 2020

Molecular biology is making a tremendous impact on the diagnosis and treatment of liver diseases. Methods such as the polymerase chain reaction are changing the way physicians diagnose and monitor patients with viral hepatitis. Assays based on recombinant protein antigens allow for detection of specific autoantibodies in diseases such as primary biliary cirrhosis. The diagnosis of inherited metabolic diseases, such as hemochromatosis and Wilson disease, is being revolutionized by discovery of the defective genes involved and the development of methods to rapidly sequence DNA and identify mutations. Treatments and preventive measures are now possible with use of drugs and vaccines produced by recombinant DNA technology. Gene therapy and nucleic acid-based therapeutics are also realistic future treatment options for individuals with liver diseases.
History of posttransfusion hepatitis
Authors: Tobler Leslie H , Busch Michael P
Published: January 2020

The risk of hepatitis virus transmission from transfusions has declined dramatically from that of the 1940s when posttransfusion hepatitis (PTH) was first appreciated. Introduction of hepatitis B surface antigen screening and conversion to volunteer donors for whole-blood donations in the late 1960s and early 1970s led to substantial reduction in PTH cases. However, up to 10% of the recipients continued to develop PTH, most cases of which were attributed to an unknown non-A, non-B viral agent. Implementation of surrogate marker testing (i.e., alanine aminotransferase and anti-hepatitis B virus core antigen) for residual non-A, non-B hepatitis in the late 1980s reduced the per unit risk of PTH from 1 in 200 to about 1 in 400. Hepatitis C virus was discovered in 1989 and quickly was established as the causative agent of >90% of non-A, non-B PTH. Introduction of progressively improved antibody assays in the early 1990s reduced the risk of PTH due to hepatitis C virus to about 1 in 100 000. Although additional hepatitis viruses exist (e.g., hepatitis G virus), these appear to be minor contributors to clinical PTH, which has been virtually eradicated.
Hepatitis C—diagnosis and monitoring
Authors: Urdea Mickey S , Wuestehube Linda J , Laurenson Patricia M , Wilber Judith C
Published: January 2020

Cloning of the hepatitis C virus (HCV) genome was a tremendous advance in the development of tests for diagnosis and monitoring of HCV-infected patients. Serological tests, including enzyme-linked immunoassays and RIBATM strip immunoblot assays, are primarily used to screen blood donations and to diagnose and confirm HCV infection. Tests for HCV RNA, including polymerase chain reaction (PCR)-based assays and the branched-DNA (bDNA) assay, are used for therapeutic monitoring and prognostics. Here, we present the development and future potential of these diagnostic tests. We also provide examples of how these tests are used to follow the progression of disease, select and adjust treatment protocols, and evaluate the efficacy of therapeutic regimens.
International collaborative study to evaluate a recombinant L ferritin preparation as an International Standard
Authors: Thorpe Susan J , Walker Dawn , Arosio Paolo , Heath Alan , Cook James D , Worwood Mark
Published: January 2020

A recombinant L ferritin preparation, lyophilized in ampoules and designated 94/572, was evaluated by 18 laboratories in 9 countries for its suitability as an International Standard (IS). The preparation was assayed in a wide range of in-house and commercial immunoassays against the 2nd IS for ferritin (of spleen origin; 80/578). The immunological reactivity of the recombinant material was similar to that of the 2nd IS for ferritin in the majority of assays and demonstrated adequate stability in accelerated degradation studies. On the basis of the results presented here, the WHO Expert Committee on Biological Standardization established 94/572 as the 3rd IS for ferritin, recombinant.
Multicenter evaluation of a homogeneous assay for HDL-cholesterol without sample pretreatment
Authors: Nauck Matthias , März Winfried , Jarausch Jochen , Cobbaert Christa , Sägers Anja , Bernard Dirk , Delanghe Joris , Honauer Gunter , Lehmann Paul , Oestrich Evelyn , von Eckardstein Arnold , Walch Stephan , Wieland Heinrich , Assmann Gerd
Published: January 2020

We evaluated a new homogeneous assay for the measurement of HDL-cholesterol (HDL-C) in six European laboratories. The assay includes two reagents and is applicable to most autoanalyzers, which allows full automation. The total CVs of the new method ranged between 1.3% and 6.7%. Thereby determined HDL-C values were in good agreement with those obtained by precipitation with phosphotungstic acid/MgCl2 or by a combination of ultracentrifugation and precipitation (0.956< r <0.994). The assay was linear up to at least 1500 mg/L HDL-C. Hemoglobin did not interfere, whereas icteric samples with bilirubin >100 mg/L showed discrepancies between the homogeneous and the precipitation assay. Lipemia up to total triglyceride concentrations of 8000 mg/L did not interfere with the homogeneous HDL-C assay. The homogeneous HDL-C assay was easy to handle and produced similar results in all laboratories participating in this study. This method will significantly facilitate the screening of individuals at increased risk for cardiovascular disease.
Blood substitutes: evolution and future applications
Authors: Scott Mitchell G , Kucik Dennis F , Goodnough Lawrence T , Monk Terri G
Published: January 2020

The development of oxygen-carrying blood substitutes has progressed significantly in the last decade with phase I and phase II clinical trials of both hemoglobin-based and perfluorocarbon-based oxygen carriers nearing completion. As these products approach clinical use it is important for the laboratory medicine community to be aware of their effects on routine laboratory testing and the settings in which they might be used. Here we review the forces driving the development of oxygen-carrying blood substitutes, the clinical settings in which they might be used, the major categories of oxygen carriers in clinical trials, and the challenges faced by these products as they approach clinical use.
Effect of hemoglobin- and Perflubron-based oxygen carriers on common clinical laboratory tests
Authors: Ma Zhongmin , Monk Terri G , Goodnough Lawrence T , McClellan Adrain , Gawryl Maria , Clark Terri , Moreira Paulo , Keipert Peter E , Scott Mitchell G
Published: January 2020

Polymerized hemoglobin solutions (Hb-based oxygen carriers; HBOCs) and a second-generation perfluorocarbon (PFC) emulsion (Perflubron) are in clinical trials as temporary oxygen carriers (“blood substitutes”). Plasma and serum samples from patients receiving HBOCs look markedly red, whereas those from patients receiving PFC appear to be lipemic. Because hemolysis and lipemia are well-known interferents in many assays, we examined the effects of these substances on clinical chemistry, immunoassay, therapeutic drug, and coagulation tests. HBOC concentrations up to 50 g/L caused essentially no interference for Na, K, Cl, urea, total CO2, P, uric acid, Mg, creatinine, and glucose values determined by the Hitachi 747 or Vitros 750 analyzers (or both) or for immunoassays of lidocaine, N-acetylprocainamide, procainamide, digoxin, phenytoin, quinidine, or theophylline performed on the Abbott AxSym or TDx. Gentamycin and vancomycin assays on the AxSym exhibited a significant positive and negative interference, respectively. Immunoassays for TSH on the Abbott IMx and for troponin I on the Dade Stratus were unaffected by HBOC at this concentration. Tests for total protein, albumin, LDH, AST, ALT, GGT, amylase, lipase, and cholesterol were significantly affected to various extents at different HBOC concentrations on the Hitachi 747 and Vitros 750. The CK-MB assay on the Stratus exhibited a negative interference at 5 g/L HBOC. HBOC interference in coagulation tests was method-dependent—fibrometer-based methods on the BBL Fibro System were free from interference, but optical-based methods on the MLA 1000C exhibited interferences at 20 g/L HBOC. A 1:20 dilution of the PFC-based oxygen carrier (600 g/L) caused no interference on any of these chemistry or immunoassay tests except for amylase and ammonia on the Vitros 750 and plasma iron on the Hitachi 747.
Interferences of o-raffinose cross-linked hemoglobin in three methods for serum creatinine
Authors: Ali Arlene C Y , Mihas Chrystina C , Campbell Janet A
Published: January 2020

The interferences of o-raffinose cross-linked hemoglobin (HemolinkTM) were examined and compared in two serum creatinine methods on the Hitachi 717 [Boehringer Mannheim (BMC) and Synermed] and in an enzymatic creatinine method on the Vitros 750 (Johnson & Johnson). Interference was considered significant when the change in creatinine concentration from the control exceeded the 95% confidence limits of each method. Significant interference was observed for the BMC/Hitachi 717 method with Hemolink ≥5 g/L. No interference was observed for the Synermed/Hitachi 717 assay with Hemolink up to 30 g/L in normal samples and up to 50 g/L in samples with increased creatinine. No interference was observed for the Vitros 750 assay with Hemolink up to 50 g/L at both normal and increased concentrations of creatinine. Although the BMC/Hitachi 717 method was considered unacceptable, the Synermed/Hitachi 717 and the Vitros 750 methods allow accurate quantification of serum creatinine in the presence of Hemolink.
Statistically accurate estimation of hormone concentrations and associated uncertainties: methodology, validation, and applications
Authors: Straume Martin , Johnson Michael L , Veldhuis Johannes D
Published: January 2020

We describe a data reduction procedure to assign statistically accurate estimates of unknown hormone concentrations, with associated uncertainties, based on experimental uncertainties in sample replicates and the fitted calibration curve. Three mathematical calibration curve functions are considered. The one providing optimal statistical characterization of reference calibrators is chosen for unknown evaluation. Experimental error is addressed by assigning and propagating uncertainty estimates for each measured response (including zero-dose responses) by an empirically determined discrete uncertainty profile and by propagating calibration curve uncertainty. Discrete uncertainty profiles account for both response precision (replicability) and accuracy (deviation from predicted calibration curves) without relying on assumed theoretical response variance–assay response relations. The validity of assigning variable response weighting by this procedure was assessed by Monte Carlo simulations based on chemiluminescence growth hormone calibration curves. Much-improved accuracy and estimated precision are achieved for unknown hormone concentrations, particularly extremely low concentrations, by using this variable response weighting procedure.
Plasma apolipoproteins A-I and B in survivors of myocardial infarction and in a control group
Authors: Graziani Maria Stella , Zanolla Luisa , Righetti Gabriella , Marchetti Cristina , Mocarelli Paolo , Marcovina Santica M
Published: January 2020

The values of apolipoproteins (apo) A-I and B were determined in a population sample of hospital outpatients with a standardized method to verify if the cutpoints calculated in a cross-sectional study in the US are usable with other populations. We also tested the apolipoproteins’ ability to discriminate between healthy people and survivors of myocardial infarction. In the studied population the apo A-I value corresponding to the HDL-cholesterol decisional centile is 1.12 g/L for males and 1.17 g/L for females; the apo B value corresponding to the LDL-cholesterol decisional centile is 1.23 g/L for males and 1.14 g/L for females. These values are quite close to the cutpoints proposed for the American population (1.20 g/L for both apolipoproteins). In comparison with the LDL- and HDL-cholesterol decisional concentrations, the cutpoints for apolipoproteins allow a correct classification of a greater percentage of postmyocardial infarction patients (16% higher for apo B and 5% for apo A-I). Standardized assays coupled with a reference database allow a better clinical use of apolipoprotein measurements.
Exon 5 of the p53 gene is a target for deletions in ovarian cancer
Authors: Angelopoulou Katerina , Levesque Michael A , Katsaros Dionyssios , Shipman Rob , Diamandis Eleftherios P
Published: January 2020

Missense point mutations, leading to inactivation of the p53 tumor suppressor gene product, are currently the most frequent alterations in human cancer. Little, however, is known about small intragenic deletions or insertions occurring in this locus of chromosome 17. We have analyzed 56 primary ovarian tumors for the presence of such abnormalities. The analysis was based on multiplex PCR amplification of exons 1 through 11 of the p53 gene and fragment analysis of the generated PCR products. Mutations were detected in 14% (8 of 56) of the tumors. Deletions were much more prevalent than insertions (seven vs one). Six of the deletions and the insertion affected exon 5, and the other deletion was in exon 7. Two deletions and the insertion did not disrupt the reading frame; the protein product was expressed in the tumor at high concentrations in all three cases. The other five deletions generated a frameshift, which is predicted to result in the production of a truncated protein product. In the case of the deletions, a 2–5-bp repeat was present close to the detected deletion, whereas the insertion duplicated the sequence immediately upstream of the insertion site. Overall our findings indicate that small intragenic p53 deletions/insertions are not rare events in ovarian cancer, and that p53 exon 5 is the target in the vast majority (88%) of the cases.