bluuebunny/crossref_metadata_2025 huggingface dataset:
Analytical evaluation of a sensitive enzyme immunoassay for determinations of creatine kinase isoenzyme MB
Authors: Jørgensen P J , Hørder M , Selmer J , Bøtker H E
Published: January 2020

We have evaluated a new sensitive immunometric assay for the determination of creatine kinase (CK; EC 2.7.3.2) MB isoenzyme (NovoClone CK-MB), involving an enzyme label and two monoclonal antibodies directed against the B subunit and the M subunit, respectively. The anti-CK-B antibodies are bound to the solid phase. The assay was modified to be extremely sensitive and thus to measure the concentration range below and close to the cutoff value used for the diagnosis of myocardial infarction. A reference interval of 0-6 micrograms/L was found for 315 outpatients without myocardial diseases (132 men and 183 women); the overall median of the log-gaussian distribution was 1.91 micrograms/L (2.03 and 1.79 micrograms/L for men and women, respectively). Total and within-assay imprecision (CV) was less than 6% at the upper reference limit. The detection limit was 0.1 microgram/L. The assay provides a favorable signal-to-noise ratio: the calibrators 0.0, 2.0, and 30.0 micrograms/L give absorbances at 492 nm of 0.040, 0.140, and 1.600 A. respectively. We conclude that the assay provides biochemical identification of individuals with myocardial damage but without myocardial infarction.
Continuous ex vivo and in vivo monitoring with chemical sensors
Authors: Fogt E J
Published: January 2020

The chemical species of principal clinical interest for continuous monitoring include oxygen, carbon dioxide, pH, potassium, and glucose. Although sensors exist for all of these analytes, relatively few continuous monitors are used for blood chemistries. Reviewing the available electrochemical and optical approaches along with the expected system performance requirements provides some insight into the reasons for this. In particular, the performance of sensor systems is significantly dependent on the environment in which they are used: transcutaneous, intravascular, intramuscular, etc. The future success and acceptance of "real-time" continuous monitoring of the critically ill patient probably does not require the development of new sensors for additional species. Rather, improvements in the stability of existing devices and protection of the sensor's chemistry from the environment are needed. Eventually, the development of new sensing technologies, e.g., near infrared spectroscopy, may lead to a completely noninvasive system capable of monitoring a broad spectrum of analytes.
Other aspects of metabolic monitoring in critically ill patients
Authors: Konstantinides F N , Kaproth P L , Cerra F B
Published: January 2020

Cardiopulmonary monitoring remains the mainstay of intensive-care unit utilization of clinical chemistry resources. Its focus has been on the restoration and maintenance of oxygen transport. Metabolic monitoring, a relatively new area of application for clinical chemistry technology, focuses on cell metabolism and on cell-cell interaction as a mechanism of metabolic regulation. This latter application of monitoring is developing as a result of a better understanding of the pathogenesis of organ dysfunction and disease processes in intensive-care unit patients. Some of the clinical chemistry technologies used include analyses for amino acids and polyunsaturated fatty acids, measurement of cytokine concentration and activity, nutritional assessment and monitoring, more sensitive monitors of liver function, and assessment of altered immunity in critically ill patients. Use of these technologies, along with specific support measures, offers new avenues for decreasing infectious complications and reducing mortality and morbidity of patients in intensive-care units.
How can new technology be introduced, evaluated, and financed in critical care?
Authors: Sibbald W J , Escaf M , Calvin J E
Published: January 2020

We briefly review issues impacting the introduction, evaluation, and cost of technology in critical care, providing a clinician's perspective. Where appropriate, we note important distinctions between health-care systems in Canada and the United States--primarily the result of significant differences in the methods for funding health care in the two countries. Finally, we discuss what processes might be reasonably considered for evaluating technology in critical care and discuss the probability of various consequences that will significantly affect the care we provide our patients if critical-care practitioners, industry, and health planners fail to jointly undertake this responsibility.
A systematic approach to materials handling in clinical laboratories
Authors: Nabb D P
Published: January 2020

I describe the automation/mechanization of several areas of sample processing at SmithKline Beecham Clinical Laboratories. By analyzing and implementing new systems for conveyance and sorting of incoming specimens, specimen processing, within-laboratory specimen delivery, specimen storage and retrieval, and client resupply, we greatly improved the productivity of the nontechnical aspects of laboratory operations.
Application of existing technology to meet increasing demands for automated sample handling
Authors: Chow A T , Kegelman J E , Kohli C , McCabe D D , Moore J F
Published: January 2020

As the clinical laboratory advances toward total automation, the marketplace is now demanding more-efficient sample-handling systems. These demands have arisen over a relatively short period of time, in part because of heightened concern over laboratory safety and the resulting manpower shortages. Adding sample-handling capabilities to existing instrumentation is often a challenge, because usually mechanical or system constraints are present that interfere. This challenge has been overcome in the DuPont Sample Management System (SMS), a second-generation general chemistry analyzer that incorporates the latest barcode and computer-interfacing technology. The development of the SMS system relies heavily on recent advances in technology, e.g., software modeling and computer-aided design. The SMS system includes a barcode scanner based on "charge-coupled device" technology, a random-access sample wheel, and new software that oversees the various functions.
The Ciba Corning ACS:180 benchtop immunoassay analyzer
Authors: Boland J , Carey G , Krodel E , Kwiatkowski M
Published: January 2020

We describe a new clinical laboratory instrument, the ACS:180, used to automate heterogeneous immunoassay testing. The ACS:180 automates immunoassays in which paramagnetic particles are the solid phase and changes in chemiluminescence are measured. The system can accommodate both competitive and sandwich-type assay configurations. The microprocessor-based instrument fully automates each step of the assay, including sample and reagent addition, separation and wash of paramagnetic particles, and generation and acquisition of the chemiluminescent signal. The instrument has the flexibility to operate in random-access or batch mode. The time from application of sample to first result is less than 15 min; throughput is as much as 180 tests per hour.
Precision requirements for cost-effective operation of analytical processes
Authors: Westgard J O , Burnett R W
Published: January 2020

Current performance criteria for analytical methods are often based on recommendations developed many years ago. A common criterion for imprecision requires that two times the standard deviation (s) of the method be less than the allowable total error (TEa). Unfortunately, when this criterion is minimally satisfied, commonly used control procedures cannot achieve reliable detection of medically important errors. Studies of the power functions for statistical quality-control (QC) procedures show that the magnitude of medically important errors must be at least 3s to fall near the plateau of the power curves and be readily detected by current QC procedures. For methods that just meet the precision criterion 2s less than TEa, however, medically important errors will fall on the rising portion of the power curves and typically be detected less than half of the time. From a "reverse engineering" perspective, the 2s less than TEa criterion is inadequate because it does not allow for the known performance limitations (lack of sensitivity) of commonly used control procedures. A strong case can be made for using a criterion of at least '4s less than TEa, which calls for a twofold improvement in imprecision over, the current minimum requirements. This recommendation is consistent with current industrial guidelines for process capability and would lead to more reliable detection of medically important errors.
The ontogeny of human hepatic microsomal glucose-6-phosphatase proteins
Authors: Burchell A , Gibb L , Waddell I D , Giles M , Hume R
Published: January 2020

We have studied 250 human liver biopsy samples to determine the ontogeny of the microsomal glucose-6-phosphatase (EC 3.1.3.9) system. Human hepatic glucose-6-phosphatase enzyme activity develops at 11 weeks' gestation and slowly increases to approximately 10% of adult activity at term. In the first week after birth, activity rises to adult values. Increases in enzyme activity coincide with increasing concentrations of the glucose-6-phosphatase enzyme protein. The phosphate/pyrophosphate transport protein (T2) of the human hepatic glucose-6-phosphatase complex develops at a different rate from that of the enzyme. Our study shows that the development of rat and human glucose-6-phosphatase activities are completely different. We conclude that deficiencies of the proteins in the microsomal glucose-6-phosphatase complex can be diagnosed with much more certainty perinatally than prenatally.
Use of serum cholesterol/triglyceride ratio to discern for which individuals the Friedewald formula can be used confidently
Authors: González Estrada M , Rodríguez Ferrer C R , Astarloa I R , Lahera E M
Published: January 2020

The values of low-density lipoprotein cholesterol obtained according to the Friedewald formula (Clin Chem 1972; 18:499-502), or by the De Long transformation (J Am Med Assoc 1986;256:2372-7), were compared with the values obtained when the individual cholesterol/triglyceride ratio of very-low-density lipoprotein was used for estimating the contribution of this lipoprotein to the total cholesterol. We found that these formulas gave the greatest errors for individuals with a low serum cholesterol/triglyceride ratio. We propose criteria for deciding when the numerically calculated value of low-density cholesterol is appropriate, and when it is not.
Three highly sensitive "bedside" serum and urine tests for pregnancy compared
Authors: Christensen H , Thyssen H H , Schebye O , Berget A
Published: January 2020

We examined three enzyme-linked immunosorbent assay (ELISA) kits for human choriogonadotropin (hCG) (pregnancy tests) for use with urine and serum samples: the Tandem Icon II hCG Urine and Tandem Icon II hCG Serum, the NovoClone Target hCG Test, and the Abbott TestPacks hCG-urine and hCG-serum. Paired comparison of the results from each kit indicated that the NovoClone Target assay showed significantly lower diagnostic sensitivity (P less than 0.05) than did the Tandem Icon II or Abbott TestPack, both for urine and for serum samples. None of the products demonstrated any significant difference (P greater than 0.05) in diagnostic specificity, but the NovoClone Target kit showed several serious false-negative results with both urine and serum. Paired testing of urine kits vs serum kits also showed no significant differences (P greater than 0.05) in diagnostic sensitivity or specificity. We found the Abbott kits to be the most convenient to use and to read.
Detection of simultaneous self-administration of testosterone and epitestosterone in healthy men
Authors: Dehennin L
Published: January 2020

Combined self-administration of testosterone (T) and epitestosterone (ET) by athletes counteracts the efficiency of the corresponding urinary glucuronides (G) ratio, (T/ET)G, as an indicator of T abuse. I therefore propose 5-androstene-3 beta, 17 alpha-diol (5A3 beta 17 alpha), the immediate metabolic precursor of ET, as a new reference compound for the expression of relative excretions of T and ET. Thus (T/5A3 beta 17 alpha)G and (ET/5A3 beta 17 alpha)G become potential criteria to indicate joint administration of T and ET, since their respective threshold values (2.5 and 1.5) are exceeded even when (T/ET)G remains below the critical value of 6.
Simplified isoelectric focusing/immunoblotting determination of apoprotein E phenotype
Authors: Kataoka S , Paidi M , Howard B V
Published: January 2020

We developed a rapid, accurate method for phenotyping apoprotein E that can be used for large-scale population studies. In this method, adapted from the method of Kamboh et al. (J Lipid Res 1988;29:1535-43), 10-microL plasma samples are incubated with dithiothreitol and Tween-20 for 15 min and then applied to 5% polyacrylamide gels containing ampholyte (pH 4.5-8) and urea (3 mol/L). After 2 h of isoelectric focusing, the apoprotein E bands are made visible by immunoblotting. Utilizing whole plasma, this method does not require time-consuming ultracentrifugation, delipidation of samples, or dialysis. Small amounts of plasma are required, electrofocusing time is short, and as many as 160 samples can be processed per day. Identification of phenotype is easily accomplished by noting the location and number of protein bands instead of their intensity. Because identification of phenotype is not affected by sialylation, neuraminidase treatment is not necessary. Agreement in identification of 301 individuals from blinded duplicates was 96%, and there was 98% concordance of results for 431 samples that had undergone genetic typing. This method is thus well suited for large-scale population studies.
Total interleukin-6 in plasma measured by immunoassay
Authors: Brailly H , Montero-Julian F A , Zuber C E , Flavetta S , Grassi J , Houssiau F , vanSnick J
Published: January 2020

Determinations of total cytokine concentration in biological fluids by immunoassays face two major problems: the biochemical heterogeneity of the analyte and the interference of cytokine-binding proteins. We developed an ultrasensitive enzyme immunoassay for interleukin-6 (IL-6), using monoclonal antibodies and acetylcholinesterase as the tracer enzyme. The antibodies recognized recombinant and glycosylated forms of IL-6 equally. The antibodies measured dimeric recombinant IL-6, yet we could not detect IL-6 oligomers in plasma samples. We investigated the potential interference of soluble IL-6 receptor (sIL-6R), which is present at high concentrations in plasma samples (1 to 2 nmol/L). Heat treatment of the sample obviated the sIL-6R interference. Using calibrators in a plasma matrix, we demonstrated by fractionation, dilution, and recovery experiments that the immunoassay accurately measured total IL-6 in both normal and pathological serum and plasma samples.
StatPal II pH and Blood Gas Analysis System evaluated
Authors: Wong R J , Mahoney J J , Harvey J A , Van Kessel A L
Published: January 2020

We evaluated a new portable instrument, the PPG StatPal II pH and Blood Gas Analysis System, designed for "point-of-care" measurements of blood gases and pH. Inaccuracy (% of target value) and imprecision (CV%) were assessed by blood tonometry and comparison with a Corning 178. Within-day results for PCO2 inaccuracy and imprecision ranged from 98.2% to 102.9% and 3.3% to 3.9%, respectively; for PO2, these were 95.5% to 102.3% and 2.3% to 3.0%, respectively. Between-day results for PCO2 inaccuracy and imprecision ranged from 99.2% to 99.3% and from 2.9% to 3.2%, respectively; for PO2, the ranges were 96.2% to 98.2% and 2.6% to 3.0%, respectively. Two PCO2 outliers (in 645 samples = 0.3%) were observed. In general, tonometry recovery, measurement stability, and pH bias results for the StatPal II and Corning 178 were comparable. We conclude that the StatPal II performs within acceptable ranges of inaccuracy and imprecision.
Association of low serum concentration of bilirubin with increased risk of coronary artery disease
Authors: Schwertner H A , Jackson W G , Tolan G
Published: January 2020

We examined serum bilirubin and various liver-function enzymes as possible risk factors for angiographically documented coronary artery disease (CAD). The studies involved a "training" set of 619 men for whom complete data on all risk factors considered were available, and a "test" set of 258 men for whom some risk factor data were not available. In both study groups, the liver enzymes were not related to CAD; however, In[total bilirubin] was inversely and statistically significantly related to the presence of CAD, both univariately and multivariately after adjustment for the established risk factors of age, total cholesterol, high-density lipoprotein cholesterol, smoking history, and systolic blood pressure. A 50% decrease in total bilirubin was associated with a 47% increase in the odds of being in a more severe CAD category. Our data suggest that serum bilirubin is an inverse and independent risk factor for CAD, with an association equivalent in degree to that of systolic blood pressure.
Quantitative determination of CD4/CD8 molecules by a cell marker ELISA
Authors: Franke L , Nugel E , Döcke W D , Porstmann T
Published: January 2020

Determination of percentages of CD4+ and CD8+ T cells from patients with human immunodeficiency virus infection is usually done by flow cytometric analysis. We compared a cell marker ELISA with flow cytometry for quantitation of CD4 and CD8 molecules on T lymphocytes, and correlated the values both with the number of CD4+ and CD8+ T lymphocytes and with clinical data. Results by cell marker ELISA (y) correlated well with those by flow cytometric analysis (x); r = 0.69, P < 0.001 (y = 0.01x + 3.9) for CD4; r = 0.81, P < 0.001 (y = 0.03x + 5.4) for CD8; n = 343. The ELISA detected changes in numbers of CD8 molecules on the cells earlier than flow cytometry recognized changes in CD8+ T-cell counts. The advantages of the ELISA are the small sample volume required (0.5 mL of blood), its internal standardization by a CD4+/CD8+ cell line, and its simple and fast performance. The cell marker ELISA appears to be an efficient alternative to flow cytometry.
Manipulation and flow of biological fluids in straight channels micromachined in silicon
Authors: Wilding P , Pfahler J , Bau H H , Zemel J N , Kricka L J
Published: January 2020

Analysis of minute sample volumes is a major analytical challenge that requires an understanding of fluid flow in microstructures. Accordingly, flow dynamics of biological fluids and cell suspensions in straight glass-capped silicon microchannels (40 to 150 microns wide, 20 and 40 microns deep) were studied. We demonstrated that these microstructures are appropriate components for microfluidic analytical devices. Different fluids were easily manipulated in the microchannels, and measurements of flow rate as a function of pressure for whole human blood, serum, plasma, and cell suspensions revealed non-Newtonian behavior. By means of micromachined filters (5 microns) located in channels, blood cells and microparticles were effectively separated from nanoliter-sized samples, clearly indicating the future role of microstructures for a variety of analytical purposes.
Simultaneous determination of endogenous retinoic acid isomers and retinol in human plasma by isocratic normal-phase HPLC with ultraviolet detection
Authors: Meyer E , Lambert W E , De Leenheer A P
Published: January 2020

We describe a rapid and simple procedure for the simultaneous quantitation of endogenous 13-cis-retinoic acid, all-trans-retinoic acid, and retinol by isocratic normal-phase HPLC with ultraviolet detection, in 0.5 mL of human plasma. A silica adsorption column was eluted with n-hexane:2-propanol:acetic acid (200:0.7:0.135 by vol) at 0.9 mL/min, and the effluent monitored at 350 nm. The arotinoid ethylsulfonic acid Ro 15-1570 was used as the internal standard. High sensitivity, allowing quantitation of physiological concentrations, was achieved, particularly for the retinoic acid isomers. The detection limits were 0.5 microgram/L in plasma for both 13-cis- and trans-retinoic acid, and 10 micrograms/L for retinol. The CVs for between-day determinations of the lowest quality-control concentration (n = 12) were 4.8% for 13-cis-retinoic acid, 3.4% for trans-retinoic acid, and 3.0% for retinol. The mean (+/- SD) concentrations of 13-cis-retinoic acid (1.79 +/- 0.56 microgram/L), trans-retinoic acid (1.35 +/- 0.42 microgram/L), and retinol (533 +/- 58 micrograms/L) measured in plasma from 22 healthy volunteers agreed well with those previously reported.
Measurement of blood volume with an enriched stable isotope of chromium (53Cr) and isotope dilution by combined gas chromatography-mass spectrometry
Authors: Veillon C , Patterson K Y , Nagey D A , Tehan A M
Published: January 2020

We used an established isotope dilution gas chromatography-mass spectrometry method to measure blood volumes with an enriched stable isotope of chromium. The results were compared with those of the conventional method, which involves radioactive 51Cr. The two methods were compared simultaneously in two male subjects of different sizes, and essentially identical volumes were obtained. The isotope dilution method is insensitive to contamination with natural (unenriched) chromium and can be used to measure all of the stable isotopes of chromium. The method has potential applications in simultaneous erythrocyte survival studies. The absence of radioactivity makes possible an accurate means of studying blood volume changes in children and during pregnancy.
Serotonin, catecholamines, histamine, and their metabolites in urine, platelets, and tumor tissue of patients with carcinoid tumors
Authors: Kema I P , de Vries E G , Slooff M J , Biesma B , Muskiet F A
Published: January 2020

We monitored long-term (median 11 months) concentrations of platelet serotonin and urinary serotonin, 5-hydroxyindoleacetic acid, and seven catecholamine metabolites in 44 patients with carcinoid tumors. Tumor serotonin and catecholamine contents (11 patients) and urinary histamine and N-methylhistamine (15 patients) were determined. Consistently increased concentrations of indoles, notably platelet serotonin, were observed in 96%, 43%, and 0% of patients with mid-, fore-, and hindgut carcinoids, respectively. Urinary dopamine metabolites, notably 3-methoxytyramine, were consistently increased in 38%, 20%, and 7% of patients with mid-, hind-, and foregut carcinoids, respectively. For urinary norepinephrine/epinephrine metabolites, notably normetanephrine and metanephrine, these data were 33%, 20%, and 14%, respectively. Midgut carcinoid tumors had the highest serotonin contents, whereas concentrations of catecholamines were independent of primary localization. There was no consistent relation between biogenic amine contents in tumors and urinary excretion of the amine metabolites. Occurrence of carcinoid syndrome was related to increased serotonin production rate. Increased histamine production is not an important feature in patients with lung carcinoids or liver-metastasized ileum carcinoids.
Free light chains of immunoglobulins: clinical laboratory analysis
Authors: Levinson S S , Keren D F
Published: January 2020

The increased sensitivity of immunofixation electrophoresis (IFE) over prior electrophoretic methods has led to renewed interest in the study of free light chains. Here, we discuss problems associated with the identification of monoclonal free light chains (Bence Jones proteins) in urine. Besides reviewing the nature of the sample specimens and the assays themselves, we discuss the physiology, biochemistry, genetics, and immunological properties of these molecules. Direct measurement of kappa/lambda ratios may ultimately be useful, but all commercial methods available now lack sufficient sensitivity. IFE is the preferred method because of its sensitivity and ease of interpretation. There are, however, difficulties associated with the interpretation of urinary IFE patterns, because the technique does not include an intrinsic mechanism for antibody-antigen titration and because of its great sensitivity in the absence of quantification. Problems of interpretation are discussed.
Tiglylglycine excreted in urine in disorders of isoleucine metabolism and the respiratory chain measured by stable isotope dilution GC-MS
Authors: Bennett M J , Powell S , Swartling D J , Gibson K M
Published: January 2020

Tiglyglycine (TG), an intermediate product of the catabolism of isoleucine, is increased in the urine of patients with beta-ketothiolase deficiency or with disorders of propionate metabolism. It is also implicated as a useful diagnostic marker in disorders of the respiratory chain. We present a method for the synthesis of TG and tiglyl[13C, 15N]glycine and the development of a stable isotope dilution mass spectrometric assay for TG. We compare data from controls with that from subjects with beta-ketothiolase deficiency and propionyl-CoA carboxylase deficiency, and with six patients with enzyme-confirmed disorders of the respiratory chain. TG was increased in the urine from all of the patient groups. The increased TG excretion did not persist in one patient with a respiratory chain defect, which suggests that, in some patients, multiple sample analysis may be necessary to identify a respiratory chain defect. This is the first urinary compound to be implicated as a potential marker of disorders of the respiratory chain.
Immunoassay with recombinant Trypanosoma cruzi antigens potentially useful for screening donated blood and diagnosing Chagas disease
Authors: Pastini A C , Iglesias S R , Carricarte V C , Guerin M E , Sánchez D O , Frasch A C
Published: January 2020

A new enzyme immunoassay (EIA), the Dia Kit Bio-Chagas assay (Gador S.A.), is potentially useful for detecting antibodies to Trypanosoma cruzi in the diagnosis of infected individuals and the screening of blood in blood banks. The EIA is carried out on test strips of plastic backing covered with a nitrocellulose membrane to which a mixture of recombinant T. cruzi antigens 1, 2, shed acute-phase antigen, 13, and 30 has been applied as a horizontal line. A horizontal line of human IgG is included to monitor the test procedure. The test strips exhibited homogeneity in the adsorption of the mixture of recombinant antigens (CV = 6.0%) and the human IgG (CV = 8.6%). The EIA results obtained with sera positive by xenodiagnosis showed 100% agreement between both types of tests; tested against sera with positive and negative matched results of indirect hemagglutination, immunofluorescence, and ELISA, the EIA results agreed for 99.1% (347 of 350) and 99.6% (299 of 300) of the samples, respectively.
Digoxin immunoassay with cross-reactivity of digoxin metabolites proportional to their biological activity
Authors: Miller J J , Straub R W , Valdes R
Published: January 2020

Our objective was to identify commercially available digoxin immunoassays whose cross-reactivity with digoxin metabolites paralleled the pharmacological activity of the metabolites. We measured the immunoreactivity of digoxigenin bis- and monodigitoxosides, digoxigenin, and dihydrodigoxin in four immunoassays and compared the immunoactivities with pharmacological activities from studies involving whole-animal and receptor (Na,K-ATPase)-based assays. Correlation coefficients for comparisons of immunoassay reactivity and human heart receptor reactivities were: ACS, 0.96; TDx, 0.60; Stratus, 0.57; and Magic, 0.42. Comparison with other biological assays showed a similar trend. The major difference in metabolite cross-reactivities among the immunoassays was that of digoxigenin (ACS, 0.7%; TDx, 103%; Stratus, 108%; Magic, 153%), which has approximately 10% bioactivity relative to digoxin. Measured recovery of mixtures of digoxin and metabolites confirmed these findings. We conclude that the monoclonal antibody in the ACS digoxin assay closely mimics Na,K-ATPase in detecting digoxin and its metabolites. This finding provides a basis for developing therapeutic drug monitoring immunoassays capable of approximating the true pharmacological activity of a mixture of drug metabolites.
Serum cystatin C, determined by a rapid, automated particle-enhanced turbidimetric method, is a better marker than serum creatinine for glomerular filtration rate
Authors: Kyhse-Andersen J , Schmidt C , Nordin G , Andersson B , Nilsson-Ehle P , Lindström V , Grubb A
Published: January 2020

We describe a fully automated particle-enhanced turbidimetric assay for cystatin C in undiluted serum and EDTA-plasma. The throughput is 90 samples per hour and urgent samples can be analyzed in 7 min. The assay range (0.4-14.1 mg/L) covers the concentration range in health and disease. The within- and between-run imprecision is 0.9% and 2.2%, respectively. Analytical recovery of additions of recombinant cystatin C averaged 98%. Rheumatoid factors (< or = 323,000 IU/L), bilirubin (< or = 150 mumol/L), hemoglobin (< or = 1.2 g/L), and triglycerides (< or = 8.5 mmol/L) do not interfere in the assay. In view of the superior (by ROC analysis) diagnostic accuracy of serum concentrations of cystatin C for reduced glomerular filtration rate (GFR) in comparison with creatinine, cystatin C seems an attractive alternative to creatinine for estimation of GFR.
Chemistry of the fructosamine assay: D-glucosone is the product of oxidation of Amadori compounds
Authors: Baker J R , Zyzak D V , Thorpe S R , Baynes J W
Published: January 2020

The chemistry of the fructosamine assay was studied by using the Amadori compound, N alpha-formyl-N epsilon-fructose-lysine (fFL), an analog of glycated lysine residues in protein. Previously (Clin Chem 1993;39:2460-5), we reported that free lysine was formed from fFL at 70% yield during incubation with alkaline nitroblue tetrazolium (NBT) under the conditions routinely used for the fructosamine assay (sodium carbonate buffer, pH 10.35 at 37 degrees C). Here, we show that D-glucosone is the primary carbohydrate oxidation product formed from Amadori compounds in the fructosamine assay. Glucosone, which decomposes under alkaline assay conditions with a half-life of < 30 min, reaches a maximum concentration of approximately 50% of the initial fFL concentration after 10 min of incubation. Like fFL, glucosone reduces NBT to the purple monoformazan dye, but its decomposition is not accelerated by the presence of NBT. The dicarbonyl-trapping reagent, aminoguanidine, inhibits the fructosamine assay by approximately 25% when fFL is the substrate, but by nearly 100% with glucosone as substrate. Studies with serum samples from diabetics and nondiabetics indicate that glucosone formation does not have a significant effect on the clinical usefulness of the fructosamine assay; however, corrections for glucosone formation may be required when the assay is used for estimating the extent of glycation of proteins.
Evaluation of preanalytical variables associated with measurement of prothrombin fragment 1.2
Authors: Greenberg C S , Hursting M J , Macik B G , Ortel T L , Kane W H , Moore B M
Published: January 2020

We have used a monoclonal antibody-based ELISA for plasma prothrombin fragment 1.2 (F1.2) to establish appropriate sample collection and storage conditions for this biomarker of thrombin generation. F1.2 concentrations were not altered by exogenous factor Xa, thrombin, or thromboplastin if blood was collected by routine venipuncture into tubes containing heparin as anticoagulant (but not citrate, acid-citrate-dextrose, EDTA, or oxalate) and if plasma antithrombin III concentration was > or = 30% of normal. Heparinized plasma F1.2 was stable for > or = 8 h at 20-25 degrees C, and if premixed with a stabilizing reagent, for > or = 4 years at -70 degrees C. Mean values for heparinized plasma F1.2 collected and stored by recommended procedures were increased in patients with thrombosis and conditions of increased thrombotic risk, and were sensitive to heparin and oral anticoagulant therapies. We conclude that plasma obtained by routine venipuncture into tubes with heparin as anticoagulant is an appropriate specimen for F1.2 measurements for most patients.
Immunoassay for quantifying type I collagen degradation products in urine evaluated
Authors: Bonde M , Qvist P , Fledelius C , Riis B J , Christiansen C
Published: January 2020

An enzyme-linked immunosorbent assay (ELISA) for measuring type I collagen degradation products in urine < 3 h was evaluated. The measuring range was 0.5-10.5 mg/L with a detection limit of 0.2 mg/L. Within-run and total CVs were 5.3% and 6.6%, respectively. Analytical recovery averaged 100%. The mean (+/- SD) concentrations in urine samples from a healthy premenopausal population (n = 102) were 250 +/- 110 mg/mol creatinine (Cr). A group of healthy postmenopausal women (n = 410) gave a mean value of 416 +/- 189 mg/mol Cr. Values obtained in the ELISA correlated well (r = 0.83) to HPLC values for the established bone resorption marker deoxypyridinoline (n = 214), slightly better than the correlation to hydroxyproline measurements (r = 0.78, n = 421). We conclude that the assay described here presents a useful tool for further elucidating the importance of type I collagen degradation products in urine.
Assay of plasma oxalate with soluble oxalate oxidase
Authors: Petrarulo M , Cerelli E , Marangella M , Cosseddu D , Vitale C , Linari F
Published: January 2020

We use oxalate oxidase from barley seedlings for the colorimetric determination of oxalate in plasma. The oxalate is converted to hydrogen peroxide, which, in the presence of peroxidase, is detected by a Trinder-like chromogenic system. Optimization of the assay, including deproteinization and elimination of interferences from reducing substrates, is described. Ascorbate additions (200 mumol/L) did not affect oxalate concentration in plasma, even after long frozen storage. Mean analytical recovery of oxalate averaged 102% +/- 6.9%, imprecision (CV) at 2.0 mumol/L was 7.2%, and the lower limit of quantification (CV = 20%) was 0.6 mumol/L. Results correlated well with those by chromatography (r = 0.999, Sy/x = 0.29 mumol/L, n = 32, range for x, y = 0-140 mumol/L). Plasma oxalate concentrations measured in 32 healthy subjects ranged from 0.6 to 2.9 mumol/L (mean 1.28, SD 0.71 mumol/L), which agrees with those measurable by using indirect radioisotopic dilution methods. Patients with primary hyperoxaluria and chronic renal failure exhibited markedly greater plasma concentrations of oxalate.
Effect on sports drug tests of ingesting meat from steroid (methenolone)-treated livestock
Authors: Kicman A T , Cowan D A , Myhre L , Nilsson S , Tomten S , Oftebro H
Published: January 2020

Anabolic-androgenic steroids are widely misused in human sports and are also used as growth promoters in livestock. Athletes who consume meat containing such hormone residues may risk failing a sports drug test. Prompted by an athlete's defense case, we questioned whether the consumption of small livestock given doses of anabolic steroid, orally or intramuscularly, could generate positive results in samples tested by our analytical procedures. We analyzed urine from eight men who consumed chickens that had been either fed with methenolone acetate (1 mg/day) from day 0 to 21 or injected with methenolone heptanoate depot (1 mg/intramuscular injection) on days 0, 7, and 14 and slaughtered on day 22. No methenolone or characteristic major metabolite was detected in samples from subjects who ate meat from the orally dosed chickens. However, 50% of the samples collected 24 h after consumption of the intramuscularly dosed chickens were confirmed positive. Hence, eating meat containing small amounts of injected hormone may constitute a serious liability to the athlete.
EDTA-plasma vs serum differences in cholesterol, high-density-lipoprotein cholesterol, and triglyceride as measured by several methods
Authors: Beheshti I , Wessels L M , Eckfeldt J H
Published: January 2020

To investigate EDTA-plasma/serum (P/S) differences, we collected paired samples from 25 volunteers and measured total cholesterol (TC), triglyceride (TG) and high-density-lipoprotein cholesterol (HDLC), using the Cobas FARA, Ektachem 700, DuPont Dimension, and Baxter Paramax Analyzers. The mean (SD) P/S ratios for TC, HDLC, and TG concentrations were, respectively: 0.980 (0.0171), 1.063 (0.0704), and 0.961 (0.363) for Paramax; 0.976 (0.0189), 1.034 (0.1091), and 0.950 (0.557) for Dimension; 1.003 (0.0221), 1.059 (0.0304), and 0.988 (0.0179) for Ektachem; and 0.993 (0.0162), 1.063 (0.0830), and 1.013 (0.0410) for Cobas. We conclude that P/S ratios vary by analytical methods, and that HDLC ratios tend to be larger in magnitude and in the opposite direction from TC and TG. Both effects lead to significant biases in computed disease risk.
Insights into the ontogeny and activation of T cells
Authors: Mak T W
Published: January 2020

T lymphocytes recognize antigen peptides and major histocompatibility complex products through their T-cell antigen receptors (TcR), consisting of alpha and beta chains. The interaction between T cells and their target cells or antigen-presenting cells is also assisted by a series of other cell-surface polypeptides, most notably CD4 and CD8, which are selectively expressed on mature helper/inducer and killer/suppressor T cells, respectively. Upon engagement of their ligands, a series of signals is transduced intracytoplasmically via some of these molecules and their associated proteins. Perhaps the most important enzyme in this signal transduction process is the lymphocyte-specific tyrosine kinase lck. Another important component is the cell-surface tyrosine phosphatase CD45. This molecule is alternatively spliced and the different isoforms are expressed on the various hematopoietic and lymphopoietic cells. Signaling through the TcR-CD4 D8-lck-CD45 complex is thought to be insufficient to activate T lymphocytes. A costimulatory signal is believed to be essential, and many investigators have suggested that CD28, a ligand for B7/BB1, is such a signal. Immune responses are also controlled by a number of cytokines and soluble factors. Signaling through the tumor necrosis factor receptor p55 is required for clearance of intracellular pathogens. Transcriptional factors involved in controlling interferon production are also important in T-cell development and immune responses. In an attempt to gain a better understanding of the roles of these molecules in T-lymphocyte functions and ontogeny, we generated a series of mutant mice with disruptions in the genes coding for these molecules. We are analyzing the mutant mice to evaluate the importance of these genes in T-cell development.
The clinical immunology laboratory of the future
Authors: Hamilton R G
Published: January 2020

The clinical immunology laboratory assumes the responsibility of providing a complete array of analytical measurements for the diagnosis and management of patients with dysfunction of the humoral (antibody, complement) and cellular (T/B cells, phagocyte) immune system. To identify trends in the activities of the clinical immunology laboratory of the future, I first define the current status of the clinical/diagnostic immunology laboratory, examining six aspects: scope of work, analyte groups, assay methods, specimen types, instrumentation, and standardization. The scope of the clinical immunology laboratory's testing is expected to expand, with a trend toward consolidation of work into fewer, more well-equipped laboratories. An increase is expected in the total number of analytes examined as well as in the numbers of analyte groups and specificities. Assay methods will continue to improve in analytical and clinical sensitivity and specificity, with an emphasis toward less-complex procedures. The primary specimens evaluated will still be serum, urine, tissue, and cells, with a minor expansion into the use of other, less-accessible human body fluids. Instrumentation will move toward increased automation, with the concurrent development of "universal" automated immunoanalyzers for use in humoral and cellular immunology. Finally, standardization of immunological measurements with calibrated reference proteins and antibodies will promote interlaboratory agreement. I describe the diagnostic allergy laboratory to illustrate the immunology laboratory environment.
Plasma vs whole blood for therapeutic drug monitoring of patients receiving FK 506 for immunosuppression
Authors: Winkler M , Ringe B , Baumann J , Loss M , Wonigeit K , Pichlmayr R
Published: January 2020

By retrospective analysis of 13,000 blood samples obtained from 248 patients receiving FK 506 therapy, we compared the suitability of plasma with that of whole blood as the matrix for therapeutic drug monitoring of FK 506. The plasma concentrations did not correlate with the concentrations in whole blood (r = 0.56). In contrast to plasma samples (analyzed by enzyme immunoassay), FK 506 was detectable in all whole-blood samples (analyzed by enzyme immunoassay/microparticle enzyme immunoassay). The inter- and intraindividual variations of FK 506 measurements were greater in plasma than in whole blood. Moreover, plasma concentrations correlated only poorly with clinical events. There was a tendency to greater plasma concentrations being measured during episodes of toxicity, but no clear difference was evident between stable course and rejection. In whole-blood specimens, a correlation between reduced or increased FK 506 concentrations and rejection or toxicity, respectively, was observed. The discriminatory power of whole-blood values was greater for the differentiation between toxicity and stable course than between rejection and stable course. We therefore recommend whole blood rather than plasma as the matrix for therapeutic monitoring of FK 506 concentrations.
Detection of autoantibodies to amylase by ELISA: comparison of detection of macroamylase and free autoantibody
Authors: Hortin G L , Summerfield A L , Wilhite T R , Smith C H , Branum E L , O'Brien J F , Landt M
Published: January 2020

New ELISAs for detecting macroamylase or free autoantibodies to amylase were tested with 48 samples that had been characterized by gel chromatography and electrophoresis. The macroamylase ELISA, with anti-IgG or anti-IgA for detection, detected macroamylase in 28 of 33 samples known to contain macroamylase (85% sensitivity), whereas the ELISA for free autoantibody to amylase was positive for only 11 samples. Specificities of both ELISAs were 93%. Among 28 true positives detected with the macroamylase ELISA, 22 contained IgA, 3 contained IgG, and 3 contained both immunoglobulins. Detection of IgM added no true positives. ELISA responses (y) were proportional to log [macroamylase concentration by chromatography (x)] from 0 to 1200 U/L: y = 5.15 x + 1.66; r = 0.72; Sy x = 1.65. As new tools for detecting macroenzymes consisting of enzyme-autoantibody complexes, the ELISAs show that some autoantibodies are detected more sensitively as antibody-antigen complexes than as free antibody.
Analysis of fatty acid oxidation intermediates in cultured fibroblasts to detect mitochondrial oxidation disorders
Authors: Pourfarzam M , Schaefer J , Turnbull D M , Bartlett K
Published: January 2020

We describe a method for the diagnosis of mitochondrial fatty acid oxidation disorders that is based on the analysis of acylcarnitine and acyl-coenzyme A (acyl-CoA) esters generated during fatty acid oxidation by permeabilized skin fibroblasts. This method requires only small amounts of cultured fibroblasts with minimal preparation, and no isolation of mitochondrial fractions is necessary. During oxidation of [U-14C]hexadecanoate, normal human fibroblasts from patients with fatty acid oxidation defects show a completely different pattern of intermediates, and in each case the observed profile reflects the site of the defect. The diagnosis and likely site of a mitochondrial fatty acid oxidation defect can be made readily from two 80-cm2 culture flasks of fibroblasts with this technique.
Establishment of creatinine clearance reference values for older women
Authors: Sokoll L J , Russell R M , Sadowski J A , Morrow F D
Published: January 2020

Age-adjusted reference ranges for creatinine clearance were determined in 279 women, ages 40-95 years, who were housed in a metabolic research unit and consumed a meat-free diet. Creatinine clearance, but not serum creatinine, declined with age by 0.63 mL/min per 1.73 m2 per year. Serum and urine creatinine concentrations, used to calculate clearances, were analyzed by a kinetic Jaffé procedure. In a subset of 100 subjects, fasting serum creatinine values averaged 8.3 +/- 5.2 (SD) mumol/L higher when measured by the kinetic Jaffé procedure than by an enzymatic method (creatinine PAP). The Cockcroft-Gault formula for estimating creatinine clearance from serum creatinine in women was validated, and the modification factor for the male equation was determined to be 0.84 (95% confidence interval 0.83-0.86) confirming the suggested 15% correction. A prediction formula derived from this population was similar in accuracy to the Cockcroft-Gault formula.
Urea and lactate determined in 1-microL whole-blood samples with a miniaturized thermal biosensor
Authors: Xie B , Harborn U , Mecklenburg M , Danielsson B
Published: January 2020

A miniaturized flow-injected thermal biosensor was developed for the determination of urea and L-lactate in undiluted blood in 1-microL samples. The sensor employed a small enzyme column constructed of stainless steel tubing and microbead thermistors. Urease and lactate oxidase/catalase were separately immobilized onto controlled-pore glass beads, which, in turn, were charged into the enzyme column. With a flow rate of 70 microL/min, linear analytical ranges from 0.2 to at least 50 mmol/L and 0.2 to 14 mmol/L were obtained for urea and lactate, respectively. The relative standard deviations (CVs) for measurements of analyte in buffer were 0.91% for urea and 1.84% for lactate. For urea in whole blood, the CV for 50 determinations was 4.1%. Contrived samples containing various concentrations of urea and L-lactate in whole blood were determined with this sensor and with a spectrophotometric method. Comparisons of the results gave correlation coefficients of 0.989 and 0.984 for 30 blood urea and 30 blood lactate assays in concentrations ranging from 4 to 20.9 mmol/L and from 1.7 to 12.7 mmol/L, respectively.
Hirsutism and acne in women: coordinated radioimmunoassays for eight relevant plasma steroids
Authors: Fiet J , Gosling J P , Soliman H , Galons H , Boudou P , Aubin P , Belanger A , Villette J M , Julien R , Brérault J L
Published: January 2020

We developed and validated a coordinated set of RIAs for the following eight steroids in single small aliquots (< or = 1 mL) of plasma: androstenedione, dehydroepiandrosterone, 11-deoxycortisol, 21-deoxycortisol (21-DF), 11 beta-hydroxyandrostenedione, 17 alpha-hydroxypregnenolone (17-Hpreg), 17 alpha-hydroxyprogesterone, and testosterone. Samples were extracted and then chromatographed on celite microcolumns. Radioiodinated tracers were used for two of the assays (17-Hpreg and 21-DF). Tritiated tracers and scintillation proximity assay counting were used to give separation-free procedures for the other six assays, which considerably improved their practicability and reproducibility. The basal and postadrenocorticotropic hormone plasma values for these steroids in normal women sampled in the follicular phase are presented. Finally, the measurement of the eight steroids as a diagnostic method is evaluated with reference to data from 203 patients with hirsutism and (or) acne.