bluuebunny/crossref_metadata_2025 huggingface dataset:
Changes in oxygen measurements when whole blood is stored in iced plastic or glass syringes
Authors: Mahoney John J , Harvey James A , Wong Ronald J , Van Kessel Antonius L
Published: January 2020

Tonometered whole-blood and plasma specimens were tested in plastic and glass syringes to determine whether clinically significant changes in gas tensions occur during sample storage. When whole blood was tonometered with 60 and 100 mL/L (6% and 10%) oxygen and then stored for 30 min in iced plastic syringes, the pO2 of the samples remained stable (mean change = +0.4 and +0.8 mmHg, respectively). However, for 140 mL/L (14%) oxygen tonometry, the pO2 increased significantly (mean change = +8.4 mmHg; P less than 0.0001). When tonometered plasma was stored in iced plastic syringes, the pO2 increased progressively at all three concentrations, with the smallest change occurring at 140 mL/L (mean change = +12.6 mmHg) and the greatest at 60 mL/L oxygen (mean change = +20.9 mmHg). In contrast, when iced glass syringes were used for storing plasma or whole blood, no clinically significant changes in pO2 were found at any of the tonometered oxygen values for 60 min. When whole blood was stored in plastic syringes at ambient temperature for 30 min, again no clinically significant changes in pO2 were found at these tonometry conditions. Apparently, some blood gas samples stored in iced plastic syringes may yield clinically significant errors in oxygen tension.
Effects of serum-isolated vs synthetic bilirubin-albumin complexes on dye-binding methods for estimating serum albumin
Authors: Ihara H , Nakamura H , Aoki Y , Aoki T , Yoshida M
Published: January 2020

Bush and Reed reported (Clin Chem 1987;33:821-3) that the reaction of albumin with bromcresol purple but not with bromcresol green underestimated the concentration of albumin in synthetically obtained bilirubin-albumin (Bd) by 29%. Their unproven assumption was that chemically synthesized Bd behaved in a manner indistinguishable from the natural Bd in icteric serum. Here we verify that Bd, whether synthetically obtained or isolated from serum, causes an underestimation of albumin in the bromcresol purple but not in the bromcresol green method. The molar ratio of Bd from either source to underestimated albumin approximates 1.0, suggesting that one molecule of Bd would react equivalently to a molecule of albumin in the bromcresol purple method. This underestimation might falsely suggest hypoalbuminemia in patients with increased serum Bd.
Isopropylidine maltoheptosyl fructofuranoside, doubly blocked substrate for determination of endoamylase activity
Authors: Ogawa Z , Matsubayashi Y , Satoh S , Orita N , Itoh H
Published: January 2020

We synthesized o-(4,6-o-isopropylidene-alpha-D-glucopyranosyl)-(1----4)- [o-alpha-D-glucopyranosyl-(1----4])5-o-alpha-D-glucopyranosyl-(1----2)- alpha-D-fructofuranoside (IPG7F) and developed an assay for determining the activity of amylase in human serum and urine by using this substrate. Glucoamylase, alpha-glucosidase, and mannitol dehydrogenase are used as coupling enzymes. The coupled reactions are monitored by continuously measuring the oxidation rate of NADH. In this procedure, various substances in the test specimens do not interfere with the detection of amylase activity. Exactly one molecule of NADH is oxidized by one attack of amylase on the substrate, although four products can be produced in the reaction. The within-assay coefficient of variation (CV) ranged from 1.0% to 4.1% and the between-assay CV ranged from 2.6% to 5.3%. The results of our new assay correlate well with those of the amylase assay involving p-nitrophenol maltoheptaoside as substrate (r = 0.978) and with those of the amylase assay involving maltopentaose (r = 0.987).
Development and application of monoclonal antibodies to human cardiac myoglobin in a rapid fluorescence immunoassay
Authors: Silva D P , Landt Y , Porter S E , Ladenson J H
Published: January 2020

Myoglobin (Mb) is considered a useful marker for early detection of myocardial infarction and for monitoring cardiac reperfusion after thrombolytic therapy. We developed eight monoclonal antibodies to human cardiac Mb, characterized their epitopic reactivity, and determined which combinations of the antibodies are useful in two-site immunoassays. We configured two of the monoclonal antibodies in a one-step, two-site particle concentration fluorescence immunoassay (PCFIA) for measurement of Mb. The PCFIA has rapid kinetics of reaction, being complete in 15 min, and has a linear analytical range of 20-675 micrograms/L for human Mb. Although the PCFIA has a high-dose "hook" effect, this is of no analytical importance at concentrations of Mb less than or equal to 148,000 micrograms/L. The assay is not subject to interference from icterus (bilirubin less than or equal to 360 mg/L), has no cross-reaction with hemoglobin (less than or equal to 42 g/L), and may be performed with either plasma or serum in approximately 1 h. The intra- and interassay imprecisions (CV) of the method are less than 10% for concentrations of Mb within the normal range and less than 4% at higher concentrations. A comparison of the PCFIA with a commercial radioimmunoassay showed that results of the two assays correlate well (PCFIA = 0.88 x RIA + 18, r = 0.990, n = 171).
Influence of intermediate-density lipoproteins on the accuracy of the Friedewald formula
Authors: Sentí M , Pedro-Botet J , Nogués X , Rubiés-Prat J
Published: January 2020

Values of low-density lipoprotein (LDL) cholesterol (C) according to the Friedewald formula (Clin Chem 1972;18:499-502) were compared with those obtained by lipoprotein fractionation in 98 healthy subjects (control group), 135 specimens from patients with peripheral vascular and cerebrovascular disease (atherosclerotic group), and 45 with chronic renal failure on hemodialysis (CRF group). All had concentrations of total cholesterol between 3.23 and 7.76 mmol/L (1.25-3.00 g/L) and triglycerides less than 3.39 mmol/L (less than 3.00 g/L). The percentage error of calculated LDL-C was 4% in controls with a cholesterol/triglycerides (C/TG) ratio for very-low-density lipoprotein (VLDL) of 0.20, but greater than 60% in those with a (C/TG)VLDL ratio of 0.40. The percentage of error in sera of patients with atherosclerosis and chronic renal failure was higher than in controls with a similar mean (C/TG)VLDL ratio. The percentage of error of calculated LDL-C increases progressively with the increase in the C/TG intermediate-density lipoprotein (IDL) ratio, both in controls and in the atherosclerotic and CRF groups. Similar findings are observed when the mean percentage of error of measured LDL-C is evaluated. The percentage of error from calculated LDL-C in the atherosclerotic and CRF groups is significantly lower than that obtained by comparison of LDL-C separated by ultracentrifugation when the "broad cut" LDL (IDL plus LDL, both by ultracentrifugation) was used. The high percentage of errors found in the groups of patients studied underlines the need for caution when assessing the reliability of the Friedewald formula, particularly in cases in which disturbances in IDL composition are suspected.
Simple, sensitive measurement of carbon monoxide in plasma
Authors: Chalmers A H
Published: January 2020

A simple, sensitive method for estimating carbon monoxide in plasma is described. In this method, the carbon monoxide in plasma is trapped with hemoglobin and subsequently estimated by dithionite reduction. The method has an intra- and interassay precision (CV) of 10.7% and 12.8%, respectively, at a concentration of 1.12 mg of carbon monoxide per liter and has a detection limit of 0.1 mg/L. The reference interval for carbon monoxide in plasma from 17 men and eight women ranged from 0.14 to 0.60 mg/L (mean 0.36 mg/L).
2,3-Butanediol in plasma from an alcoholic mistakenly identified as ethylene glycol by gas-chromatographic analysis
Authors: Jones A W , Nilsson L , Gladh S A , Karlsson K , Beck-Friis J
Published: January 2020

2,3-Butanediol was mistakenly identified as ethylene glycol in plasma specimens from two alcoholic patients. The cyclic phenylboronate ester derivatives of 2,3-butanediol and ethylene glycol had the same retention time when OV-17 was used as the stationary phase for gas chromatography. This led to incorrect diagnosis of ethylene glycol poisoning and unnecessary invasive therapy. Plasma from two chronic alcoholics contained 2,3-butanediol at 3.5 and 3.4 mmol/L. The elimination half-life of 2,3-butanediol was 3.9 days when ethanol was administered during therapy for suspected ethylene glycol poisoning. Low concentrations of 2,3-butanediol might be present in blood of chronic alcoholics as a result of a novel pathway of intermediary metabolism associated with some forms of alcoholism. However, a more likely explanation for fairly high concentrations of 2,3-butanediol is enzymatic production from 2-butanone. This ketone occurs in denatured alcohol preparations often consumed by alcoholics in Sweden.
Time-resolved fluorescence detection of enzyme-amplified lanthanide luminescence for nucleic acid hybridization assays
Authors: Templeton E F , Wong H E , Evangelista R A , Granger T , Pollak A
Published: January 2020

A new nonisotopic detection method based on time-resolved fluorescence for nucleic acid hybridization assays with alkaline phosphatase labels has been developed: enzyme-amplified lanthanide luminescence (EALL). EALL combines the amplification of an enzyme label with the sensitivity and background elimination of time-resolved fluorescence detection of lanthanide ion luminescence. The detection system for alkaline phosphatase makes use of a phosphorylated salicylic acid derivative that, upon dephosphorylation, gives a product capable of forming a luminescent terbium chelate. We demonstrate DNA hybridization assays by using two substrates, one for membrane and one for solution-based formats. Using the substrate that produces a more adhesive product allows performance of dot-blot and Southern blot assays on nylon membranes; results can be recorded with a time-resolved photographic camera system, or with an ultraviolet transilluminator-based system. Less than 4 pg of target sequence can be detected in a dot-blot assay after incubation with substrate for 2-4 h. DNA microwell-plate hybridization assays with the more soluble substrate/product pair can be quantified with time-resolved fluorescence plate readers, giving a similar detection sensitivity. EALL is thus a practical time-resolved fluorescence-based alternative to other detection systems for DNA hybridization assays.
The integrated blood-collection system as a vehicle into complete clinical laboratory automation
Authors: Columbus R L , Palmer H J
Published: January 2020

A rationale is offered and methodology illustrated for integrating the fundamental steps involved in the collection and processing of blood for laboratory evaluation. The approach taken in the development of these concepts and components greatly extends the possibilities of laboratory systems integration without upsetting established modalities. A prototype design of the integrated blood-collection system integrates blood collection, cellular separations, sample transfer to stable storage without chemical mediators, and sample presentation for chemical analysis (e.g., precision metering) while preserving patient identification. A sophisticated, multi-chambered blood-collection container is the site of all blood sample processing and transfer steps. This device is supported by a compact, robotic centrifuge of unique design and a transfer mechanism to facilitate sample delivery for analysis within a diagnostic instrument. The confluence of these individual components into a single integrated system provides the means to completely automate the processing of blood samples, after sample collection, eliminating all manual transfer steps and any external exposure of blood interfaces outside the diagnostic instrument. Configurational derivatives of the Integrated Blood-Collection System offer choice of skin or venipuncture procedure, rapid plasma extraction for micro- or macro-collected volumes, and sample delivery by either aspiration or direct metering of discrete 10-microL samples from the collection container. The skin-puncture configuration provides the opportunity within a single device to collect and process up to 500 microL of sample by capillarity from a skin prick.
Time course of changes in pancreatic enzymes, isoenzymes and, isoforms in serum after endoscopic retrograde cholangiopancreatography
Authors: Panteghini M , Pagani F , Alebardi O , Lancini G , Cestari R
Published: January 2020

Kinetics of the catalytic activities of total amylase (AMY; EC 3.2.1.1), pancreatic (P)-AMY isoenzyme, P2 and P3 isoforms, and pancreatic lipase (LPS; EC 3.1.1.3), and of the mass concentration of LPS in serum were studied in 10 patients who underwent endoscopic retrograde cholangiopancreatography (ERCP) and showed a distinct pancreatic injury. The temporal characteristics of enzyme changes described were (a) the maximal rate (Ka) at which enzymes are released into blood, (b) the time lag from ERCP until maximum concentration value, (c) the peak value of each serum enzyme, and (d) the rate (Kd) at which each enzyme is cleared from serum. LPS activity and mass concentrations increased and decreased faster than AMY and isoamylases, and the time of the LPS peak tended to be earlier than that of the other enzymes, but not significantly. The average peak increase of LPS values was higher than that of total AMY, P-AMY, and P2 isoform (P less than 0.001). The P-AMY time-activity curve was a composite of curves attributable to its isoforms; the isoforms increased and peaked sequentially, with P3 returning to normal more slowly than did P2. LPS mass and activity concentrations showed excellent parallelism, with no important differences. At 50 h after ERCP, only LPS values still exceeded the upper reference limit, returning to normal 70 h after the examination.
One-Step, Labeled-Antibody Assay for Measuring Free Thyroxin. I. Assay Development and Validation
Authors: Christofides N D , Sheehan C P , Midgley J E M
Published: January 2020

We describe a one-step, labeled-antibody radioassay for measuring free thyroxin (FT4) in serum or plasma, based on a novel principle. FT4 in the sample competes with a gross molar excess (over antibody) of a cross-reactant (L-triiodothyronine, T3), chemically coupled to magnetizable polymer particles, for binding to avid 125l-labeled monoclonal anti-thyroxin antibodies. As in conventional immunoassays, 125I counts bound to the solid phase (T3-magnetizable particles) are inversely proportional to sample FT4 concentration. We demonstrate here the development and technical validity of this new method.
Methods for Measuring Plasma Hemoglobin in Micromolar Concentration Compared
Authors: Fairbanks Virgil F , Ziesmer Steven C , O'Brien Peter C
Published: January 2020

Of eight methods examined for measuring plasma hemoglobin in micromolar concentration, all exhibited acceptable linearity, reproducibility, and concurrence except when specimens were icteric or lipemic or contained methemoglobin or methemalbumin. Measurement of absorbance at 578 nm with an Allen correction permits precise assay of plasma oxyhemoglobin concentration as low as 0.01 g/L (1 mg/dL, 0.16 mumol/L), unaffected by hyperlipidemia or hyperbilirubinemia. Discrepancies between methods occurred in 11.6% of a consecutive series of 50 nonicteric patients' plasma specimens. Examination of absorption spectra is helpful when discrepancies are observed between methods. The presence of methemalbumin or methemoglobin in plasma is not recognized by methods that measure only oxyhemoglobin. Increased ceruloplasmin or beta-carotene does not significantly affect results.
Measurement of low-density-lipoprotein cholesterol in serum: a status report
Authors: Rifai Nader , Warnick G Russell , McNamara Judith R , Belcher John D , Grinstead Gregory F , Frantz Ivan D
Published: January 2020

Current recommendations of the Adult Treatment Panel and the Children and Adolescents Treatment Panel of the National Cholesterol Education Program make the concentration of low-density lipoproteins cholesterol (LDL-C) in serum the basis for the classification and treatment of hypercholesterolemia. Numerous methodologies for the determination of serum LDL-C concentrations, in research and clinical laboratories, have been described. Here, we review the principles, performance, and limitations of major current methodologies for determining LDL-C concentrations. These methods include sequential and density-gradient ultracentrifugation, chromatographic and electrophoretic techniques, and precipitation methods. In addition, the advantages and disadvantages of estimating LDL-C concentration by the Friedewald equation, the most commonly used approach in clinical laboratories, are addressed.
One-Step, Labeled-Antibody Assay for Measuring Free Thyroxin. II. Performance in a Multicenter Trial
Authors: Sheehan C P , Christofides N D
Published: January 2020

The technical and diagnostic performance of a labeled antibody radioassay for free thyroxin (Amerlex-MAB* FT4) was examined in a multicenter trial. The overall mean within-assay precision (CV) in eight centers was 6.2% at 30 pmol/L and 3.6% at 13 pmol/L concentrations of FT4. Between-assay precision was 5.4% at 18 pmol/L and 6.8% at 50 pmol/L. The euthyroid central 95% reference range for FT4 was 11.3 to 24.3 pmol/L. Results of the method correlated well with those of an analog radioimmunoassay: [Amerlex-MAB* FT4] = 1.09 [Amerlex-M FT4] + 1.50 pmol/L (r = 0.96, n = 732). Clinical performance of the assay was better than that of first-generation analog tracer assays for sera from patients with nonthyroidal illness or binding-protein abnormalities.
New Enzymatic Method with Tryptophanase for Determining Potassium in Serum
Authors: Kimura Shigeki , Asari Seishi , Hayashi Sadao , Yamaguchi Yoshihisa , Fushimi Ryo , Amino Nobuyuki , Miyai Klyoshi
Published: January 2020

We established a simple and rapid enzymatic method for measuring potassium ion in serum by using tryptophanase (EC 4.1.99.1) purified from Escherichia coli K12 strain (E. coli K12 IFO 3301). The presence of pyridoxal 5-phosphate promotes this enzymatic reaction, and potassium and (or) ammonium ions further accelerate it, with ammonium and potassium ions providing equivalent acceleration. We eliminated endogenous ammonium ion by using glutamate dehydrogenase (GLDH; EC 1.4.1.4), then produced ammonium ion in the presence of tryptophanase, tryptophan, and pyridoxal 5-phosphate. The concentration of formed ammonium ion, which was proportional to that of potassium ion in sample, was determined by adding GLDH to produce NADP+ in the presence of 2-oxoglutarate and NADPH; we then read the change of absorbance at 340 nm. The standard curve was linear for potassium ion concentrations up to 7.00 mmol/L. The within-assay variation (CV) was 0.89% at 5.51 mmol/L and 1.32% at 3.37 mmol/L. The day-to-day CVs were 0.99% at 6.85 mmol/L and 1.71% at 3.52 mmol/L. Analytical recoveries ranged from 98.7% to 108.9%. The correlation coefficient between values obtained with this enzymatic assay (y) and by flame photometry (x) was 0.995: y = 0.984x + 0.091 mmol/L (Sy.x = 0.105, n = 100). The presence of hemoglobin, bilirubin, or other cations little affects this system.
Biological Variation Data Applied to the Selection of Serum Lipid Ratios Used as Risk Markers of Coronary Heart Disease
Authors: Ortolá J , Castiñeiras M J , Fuentes-Arderiu X
Published: January 2020

The biological variation of several relative lipid quantities, calculated as the ratios between the concentrations of various serum lipids and apolipoproteins, has been estimated over a one-year period. The medians of the within-subject biological coefficient of variation, separated by sex when significant differences exist, were 15.4% for [apolipoprotein A-I]/[apolipoprotein B], 6.8% for [high-density lipoprotein (HDL)-cholesterol]/[cholesterol], 10.5% and 17.6% (women and men, respectively) for [HDL2-cholesterol]/[HDL-cholesterol], 13.6% for [HDL2-cholesterol]/[HDL3-cholesterol], 10.6% for [low-density lipoprotein (LDL)-cholesterol]/[apolipoprotein B], 10.6% and 8.7% (women and men, respectively) for [LDL-cholesterol]/[cholesterol], and 6.3% for [LDL-cholesterol]/[HDL-cholesterol]. From these data, we have calculated the critical difference for significant change detection, the index of individuality, and the goal for the between-day imprecision. Concerning within-subject biological variation, the best ratios for the detection of risk of coronary heart disease and the monitoring of intervention are [LDL-cholesterol]/[HDL-cholesterol] and [HDL-cholesterol]/[cholesterol]. The index of individuality obtained in this study indicates that the use of population-based reference values is inadequate for interpreting the ratios studied.
Serum Steroid Hormonal Profiles by Reversed-Phase Liquid Chromatography in Patients with 17-Hydroxylase Deficiency and in an Affected Family
Authors: Wei Jl-Qing , Wei Ji-Lu , Lucarelli Claudio , Zhou Xian-Teng , Wang De-Quan , Dai Wen-Ju , Li Shan , Li Si-Min , Liu Rong-Tang
Published: January 2020

We used an optimized isocratic reversed-phase high-performance liquid-chromatographic procedure to separate and measure 12 steroid hormones, and studied the steroid hormone profiles in sera from three patients with 17-hydroxylase deficiency (17-OHD). Two of the patients were sisters, one of whom (II-3), expressing normotension and primary amenorrhea, was diagnosed on the basis of chromatographic data and followed up for seven years. The untreated patients had obvious abnormalities on chromatograms of serum extracts, characterized by markedly increased corticosterone (B) and decreased or undetectable cortisol (F) and cortisone (E). The concentration of 11-deoxycorticosterone was much greater in the patient with classical symptoms than in the normotensive patient. In all three patients, concentrations of aldosterone were within the normal range, but concentrations of progesterone were much lower than in the patients with 21-hydroxylase deficiency. We evaluated the responses to corticotropin and dexamethasone. HPLC evaluation of the serum steroid profiles before and after corticotropin stimulation in the affected family showed that in the parents and one other sibling, concentrations of F before and after stimulation were within the normal ranges. The sums of the ratio of B to F before and the ratio of B to F after corticotropin stimulation (sigma B/F) in the parents and the other sibling were 0.292, 0.496, and 0.614, respectively, all much higher than the normal value (mean +/- SD: 0.164 +/- 0.038). Thus the sigma B/F value may be a hormonal marker of heterozygotes carrying this defect.
VALAB: Expert System for Validation of Biochemical Data
Authors: Valdiguié P M , Rogari E , Philippe H
Published: January 2020

In large laboratories that use "high-throughput" equipment, it is now possible to use artificial intelligence techniques to aid decision making and validation of data. This paper describes an artificial intelligence project, VALAB, that has been carried out in our laboratory. VALAB, an expert system that permits real-time validation of data, is designed to be equivalent to validation by the laboratory director. The decision produced by the expert system is based on several factors, including correlation between repeated laboratory results, physiological association between different variables, the hospital department from which the test was ordered, and the patient's age and sex. In 200 abnormal chemistry profiles randomly selected, VALAB's ability to detect abnormal cases (i.e., sensitivity = 0.75) was exceeded by only one of seven laboratory experts. However, all seven experts outperformed VALAB's measured specificity of 0.63. The VALAB system incorporates greater than 4000 rules. Operational since November 1988, it has validated greater than 50,000 medical patients' reports in real time.
Prevalence of Anti-Thyroid Peroxidase Antibodies in Serum in the Elderly: Comparison with Other Tests for Anti-Thyroid Antibodies
Authors: Roti Elio , Gardini Eliana , Minelli Roberta , Bianconi Lina , Braverman Lewis E
Published: January 2020

Autoimmune thyroid disease, especially chronic thyroiditis, is prevalent in elderly women and is probably the major cause of hypothyroidism in this population. The reported prevalence of chronic thyroiditis is variable, depending on the area of residence and the method(s) used to detect the presence of anti-thyroid antibodies. The recent finding that thyroid peroxidase (TPO) is the antigen for the thyroid anti-microsomal antibody (AbM) has resulted in the development of sensitive radioimmunoassays (RIA) to detect the presence of AbTPO. We have determined the prevalence of AbTPO (by RIA) in sera from 342 elderly subjects, 248 women and 94 men (mean age 80 years) residing in Reggio Emilia, Italy, and compared the results with other methods for detecting anti-thyroid antibodies, including anti-thyroglobulin (AbTg) and AbM measured by passive hemagglutination (PH) of tanned erythrocytes, and AbM measured by RIA. The prevalence of positive AbTPO was 2.3% in the men and 10.2% in the women, only slightly higher than the prevalence of AbM. However, in the antibody-positive sera, the mean value for AbTPO was approximately 20-fold greater than the upper limit of the normal range, whereas the mean value for AbM was only threefold greater. The prevalence of positive titers for AbM and AbTg measured by PH was far lower, 1.2% and 3.2%, respectively, and those sera weakly positive for AbM and AbTg by PH were strongly positive for AbTPO by RIA. AbTPO RIA may be more useful than AbM and AbTg hemagglutination and AbM RIA for detecting the presence of autoimmune thyroid disease.
Denaturing Gradient Gel Electrophoresis for Rapid Detection of Latent Carriers of a Subtype of Acute Intermittent Porphyria with Normal Erythrocyte Porphobilinogen Deaminase Activity
Authors: Bourgeois F , Gu Xue-Fan , Deybach J C , Te Velde M P , Rooij F de , Nordmann Y , Grandchamp B
Published: January 2020

Acute intermittent porphyria is an autosomal dominant disorder defined by a partial deficiency of porphobilinogen deaminase (EC 4.3.1.8). Clinical manifestations of the disease are characterized by acute attacks of neurological dysfunction often linked to environmental factors. Early diagnosis of gene carriers is important in the prevention of attacks and is usually achieved by determining the porphobilinogen deaminase activity in erythrocytes. However, in a subtype of acute intermittent porphyria, the enzymatic defect is restricted to nonerythropoietic cells. Different mutations have already been described that account for this phenotype in two unrelated families. We previously detected asymptomatic carriers by using mutation-specific probes after in vitro amplification of the target DNA sequence. In this study, we investigated the DNA of eight unrelated subjects with the same subtype of acute intermittent porphyria by using the polymerase chain reaction, with subsequent analysis of the amplified products by denaturing gradient gel electrophoresis. Five of these patients shared the same single-base change. This technique was quite simple and efficient for detecting asymptomatic carriers. Importantly, it is potentially useful for studying families with the same phenotypic subtype of the disease and possibly different mutations in the same DNA region.
Serum Creatinine as an Index of Renal Function: New Insights into Old Concepts
Authors: Perrone R D , Madias N E , Levey A S
Published: January 2020

The serum creatinine concentration is widely interpreted as a measure of the glomerular filtration rate (GFR) and is used as an index of renal function in clinical practice. Glomerular filtration of creatinine, however, is only one of the variables that determines its concentration in serum. Alterations in renal handling and metabolism of creatinine and methodological interferences in its measurement may have a profound impact on the serum concentration of creatinine. We review the fundamental principles of physiology, metabolism, and analytical chemistry that are necessary to correctly interpret the serum creatinine concentration. These principles are then applied to important clinical circumstances, including aging, pregnancy, diabetes mellitus, drug administration, and acute and chronic renal failure. Despite numerous limitations, serum creatinine remains a useful clinical tool, but more accurate measures of renal function are frequently necessary.
Highly Sensitive, Specific Enzyme-Linked Immunosorbent Assay of Neopterin and Biopterin in Biological Samples
Authors: Ogiwara S , Kiuchi K , Nagatsu T , Teradaira R , Nagatsu I , Fujita K , Sugimoto T
Published: January 2020

An enzyme immunosorbent assay of neopterin and biopterin on a polystyrene microtiter plate has been developed. A conjugate of neopterin or biopterin to bovine serum albumin was used to raise a specific antiserum against neopterin or biopterin in rabbits. An incubation mixture of the antiserum and samples prepared from human serum underwent another antigen-antibody reaction with the hapten fixed on the microtiter plate. The amount of antibody bound to the fixed hapten, which is inverse to the amount of hapten in the sample, was determined by using anti-rabbit IgG-horseradish peroxidase conjugate in a usual manner by measuring absorbance at 490 nm after reaction with o-phenylenediamine and hydrogen peroxide. The minimal detectable amounts of neopterin and biopterin were approximately 0.1 pmol. The specificity of the assay was so high that the assay system for neopterin completely distinguished it from biopterin, as judged from the cross-reaction of 0.002%, and vice versa. The amounts of neopterin and biopterin in human serum determined by the present method agreed well with those determined by high-performance liquid chromatography. We used the present method to determine the concentrations of neopterin in serum from healthy control subjects and patients with cancers and systemic lupus erythematosus; the results were consistent with literature data.
Two-Site Direct Immunoassay Specific for Active Renin
Authors: Simon D , Hartmann D J , Badouaille G , Caillot G , Guyenne T T , Corvol P , Pau B , Marchand J
Published: January 2020

A sensitive immunoradiometric assay, without an enzymatic step and specific for active human renin, was developed with use of two monoclonal antibodies (MAbs). In this assay system, the first MAb was coupled to magnetic beads (Magnogel); the second one, directed against the active form of the enzyme, was radiolabeled with 125I. The specificity of this assay was demonstrated in experiments measuring the active plasma renin concentration in the presence or absence of inactive renin. The assay, performed in two steps, was sensitive enough to detect 0.9 pg of renin per tube (3.5 ng/L). Intra- or interassay CVs were < 10%. Concentrations of active plasma renin measured in normotensive subjects were between 7 and 40 ng/L.
Evaluation of the Abbott IMx® Automated Immunoassay of Prostate-Specific Antigen
Authors: Vessella R L , Noteboom J , Lange P H
Published: January 2020

We detail the performance characteristics of the new IMx PSA immunoassay developed by Abbott Laboratories, addressing PSA recovery, assay reproducibility, standard curve storage, lower limit of detection, dilution linearity, and correlation with the Hybritech Tandem-R PSA immunoassay. We analyzed 686 sera for PSA retrospectively, testing 555 of these concurrently with the IMx and the Tandem-R immunoassays. The IMx PSA standard curve was linear from 0 to 100 micrograms/L, and curve storage was maintained for 4 weeks. The lower limit of detection of the IMx PSA assay was < or = 0.03 microgram/L; allowing for the assay precision yielded a biological detection limit of 0.06 microgram/L. We conservatively set the clinical threshold at 0.1 microgram/L. Regression analysis of dilution linearity involving 10 samples (0.44-200 micrograms/L) resulted in coefficients of correlation ranging from 0.9972 to 1.000. Reproducibility studies with 18 specimens within the range of 0.39-413.67 micrograms/L gave intra- and interassay CVs < 6.5%. The interassay 95% confidence interval for a specimen containing 0.06 microgram of PSA per liter was 0.03-0.09 microgram/L. Correlation between IMx and Tandem-R PSA assay results was excellent: r = 0.9909 and slope = 0.95. Overall, the IMx PSA immunoassay, with the conveniencies of automation, curve storage, and nonisotopic handling, provided an improved lower limit of PSA detection, which allows for earlier indication of residual or recurrent disease after radical prostatectomy.
Improved Measurement of Low-Density-Lipoprotein Susceptibility to Copper-Induced Oxidation: Application of a Short Procedure for Isolating Low-Density Lipoprotein
Authors: Kleinveld H A , Hak-Lemmers H L , Stalenhoef A F , Demacker P N
Published: January 2020

Low-density-lipoprotein (LDL) oxidation may provide the crucial link between plasma LDL and atherosclerotic-lesion formation. Oxidation can be induced in vitro by incubating LDL with cells or metal ions and can be measured by continuously monitoring conjugated-diene absorbance at 234 nm. Measurement of LDL oxidizability was improved by performing the assay with 0.05 g of LDL-protein per liter of phosphate buffer containing 1 mumol of EDTA, by initiating oxidation by adding CuCl2 (5 mumol/L) at 30 degrees C, and by using a short-run ultracentrifugation method for isolating LDL, which reduced the time needed for obtaining purified LDL and thus reduced in vitro oxidation. LDL apolipoprotein analysis and oxidizability determination showed that this method is better than the longer sequential-isolation procedure. Adding butylated hydroxytoluene (BHT) to plasma as an antioxidant unpredictably increased the LDL oxidation lag time, making BHT unsuitable as an antioxidant. Adding EDTA appeared to be sufficient to prevent in vitro oxidation. Additionally, the diene production correlated highly with the concentration of thiobarbituric acid-reactive substances (r = 0.97). No relation between the vitamin E content of LDL and the oxidation lag time was found.
Laboratory Evaluation of the GlucocardTM Blood Glucose Test Meter
Authors: Lewis B D
Published: January 2020

The Glucocard (Kyoto Daiichi Kagaku) blood glucose meter is designed for self-monitoring of blood glucose concentrations in capillary blood through use of an electrochemical test strip. Evaluated in this laboratory, the Glucocard had CVs of 4.6%, 6.6%, and 3.5% at blood glucose concentrations of 2.4, 4.1, and 18.9 mmol/L, respectively. The meter's response varied linearly with blood glucose concentration between 2.2 and 27.8 mmol/L. Hemolysis, urate, ascorbate, and acetaminophen interfered by > 5%. Different hematocrits, in the range 0.20-0.70, did not affect the measured glucose concentration. Comparison with glucose results measured in whole blood with a NOVA Stat Profile 5 instrument yielded the following: Glucocard = 0.898 NOVA--0.184 (r = 0.995). The main advantages of the Glucocard are its small sample volume (5 microL), wide linear range, and fully automated sample-handling steps, which reduce user-related variability.
AxonTM Clinical Chemistry Analyzer Evaluated According to ECCLS Protocol
Authors: Brenna S , Prencipe L
Published: January 2020

We assessed the analytical performance of the Axon system (Bayer Diagnostici), according to the European Committee for Clinical Laboratory Standards guidelines, for assay of 12 analytes: cholesterol, creatinine, glucose, total protein, urea, uric acid, alkaline phosphatase, alpha-amylase, aspartate aminotransferase, creatine kinase, sodium, and potassium. The field evaluation lasted approximately 5 months and involved the collection of approximately 10,000 data points with the Axon. The following results were obtained: The highest CVs for controls and human sera at different concentration/activity values were 2.2% for within-run imprecision (n = 60; 3 days, pooled estimate) and 3.5% for the between-day imprecision (n = 20 days). Close correlation was found with results for patients' specimens assayed with comparative instruments (Hitachi 717 for substrates and enzymes, Beckman Synchron EL/E4A for electrolytes). No drift was observed during 8 h of operation. The linearity range was broad, sometimes exceeding the manufacturer's claims. No sample-, reagent-, or cuvette-related carryover was found. Measurement of control sera gave results within +/- 5% of the assigned values. We conclude that good reliability and practicability make the Axon system suitable for laboratories with various needs.
Influence of Obesity on Plasma Fructosamine Concentration
Authors: Woo J , Cockram C , Lau E , Chan A , Swaminathan R
Published: January 2020

The influence of obesity on plasma fructosamine concentration was studied in 68 diabetic and 1335 nondiabetic subjects from a Chinese community. Obese nondiabetic men (body mass index > 25 kg/m2) had lower fructosamine concentrations than nonobese nondiabetic men (body mass index < or = 25 kg/m2); the pattern was similar for diabetic women. Stepwise multiple-regression analysis showed that, apart from known factors (total protein, albumin, and indices of glycemic control), fructosamine was also associated with body mass index and plasma fasting triglycerides. However, the contribution of these were small except in diabetic women. We conclude that the effect of obesity on fructosamine is small.
Using 16O35CL to Correct for Chloride Interference Improves Accuracy of Urine Arsenic Determinations by Inductively Coupled Plasma Mass Spectrometry
Authors: Kershisnik M M , Kalamegham R , Ash K O , Nixon D E , Ashwood E R
Published: January 2020

We have observed inaccurate urine arsenic values with the method of isobaric fractionation, which was designed to correct for the 40Ar35Cl interference with 75As quantitation by inductively coupled plasma mass spectrometry. Isobaric fractionation, which is based on ion intensities at m/z 77 and 82, consistently underestimates the 40Ar35Cl interference and overestimates urine arsenic. We present an improved method for identifying the argon-chloride interference. We observed that signal intensities for the species 16O35Cl and 40Ar35Cl are proportional (I75 = 0.0295 x I51 - 14.7, r2 = 0.998; where Ix is the normalized ion intensity at m/z X) in water and urine, over a broad range of chloride concentrations (0-800 mmol/L). The proportionality constant is remarkably stable within a run (mean and SD, 0.0295 +/- 0.0023, based on 10 replicates of five chloride calibrators, 0, 100, 200, 400, and 800 mmol/L). Increased sensitivity (50-fold) for detecting the 40Ar35Cl interference provides improved accuracy for urine arsenic quantitation as demonstrated by a split-sample comparison with graphite-furnace atomic absorption spectrophotometry.
Automated Analysis for Free and Short-Chain Acylcarnitine in Plasma with a Centrifugal Analyzer
Authors: Roe D S , Terada N , Millington D S
Published: January 2020

We describe a fully automated, spectrophotometric assay of free and total carnitine in plasma ultrafiltrates. The method, suitable for routine application in most hospital laboratories, incorporates the hydrolysis of acylcarnitines to free carnitine within the program of a Cobas Fara II centrifugal analyzer. The hydrolysis is monitored and calibrated with standard solutions containing octanoylcarnitine. Results correlated well with those from a reference isotope-dilution mass spectrometric assay. The ability to analyze a batch of samples for both free and total carnitine within 90 min enables analysis of > or = 100 samples per day. Used in conjunction with acylcarnitine species identification by mass spectrometry, the Cobas assay facilitates the diagnosis of carnitine-deficiency syndromes and specific metabolic disorders.
Analytical Goal-Setting for Monitoring Patients When Two Analytical Methods are Used
Authors: Petersen P H , Fraser C G , Westgard J O , Larsen M L
Published: January 2020

Serial results from an individual are often obtained using more than one method. Results should be transferable over time and locale. Every method has inherent analytical error, and goals are required to delineate the maximum allowable random (imprecision) and systematic (inaccuracy, bias) errors to facilitate optimal patient care. Based on Harris's proposal [Am J Clin Pathol 1979;72:374-82] that desirable imprecision should be less than or equal to one-half the within-subject biological variation, if the methods have negligible imprecision, then the maximum allowable bias between two methods used for monitoring is one-third of the within-subject biological variation. A more general model has been developed that relates the analytical imprecisions of two methods, and the bias between them, to biological variation. Applying the general formula derived in specific clinical monitoring situations in which a known change in serial results (occurring at a stated probability) stimulates clinical action allows goals for the imprecisions of the two methods and allows the difference in bias between them to be determined quantitatively.
Immunoluminometric Assay of Chromogranin A in Serum with Commercially Available Reagents
Authors: Bender H , Maier A , Wiedenmann B , O'Connor D T , Messner K , Schmidt-Gayk H
Published: January 2020

Chromogranin A (Cg A) is a useful marker of neuroendocrine neoplasia in humans. Here we describe an immunoluminometric assay (ILMA) for measuring Cg A in serum, with use of a new tube as a solid phase. The new tube has a large surface area and is coated with a polyclonal antibody. Optimized coating conditions provide a high IgG adsorption to the polystyrene wall. Serum is added to the coated tube and incubated for 2 h or overnight. After washing the tubes, acridinium ester-labeled monoclonal antibody against Cg A is added and incubation is continued for 2 h. The tubes are washed again and the bound luminescence is measured. The assay is very sensitive (detection limit 1 microgram/L, if 25 microL of serum is used), is specific for human Cg A, and offers a wide dynamic range (1-1000 micrograms/L). The range in healthy humans is 10-53 micrograms/L (median 30 micrograms/L). The correlation of serum Cg A to creatinine and parathyroid hormone is described. We also report the influence of intravenous calcium injection on Cg A concentrations in serum.
Pitfalls in the Measurement of Plasma Osmolality Pertinent to Research in Vasopressin and Water Metabolism
Authors: Bohnen N , Terwel D , Markerink M , Ten Haaf J A , Jolles J
Published: January 2020

The reliability of measurements of plasma osmolality is known to be biased by technical artifacts, such as the anticoagulant and the osmometric technique used; the resulting measurement errors therefore may cause errors in interpretation of data. In assessing the potential biasing influence of procedural variables, we found that the temperature at which fresh plasma samples were stored, the duration of storage, and the freezing and thawing of samples appeared to significantly (P < 0.01) affect osmolality values around the narrow physiological range. These factors should be considered in the interpretation of studies on the osmoregulation of vasopressin secretion. In particular, the results suggest that data obtained for any but fresh samples, whether frozen-thawed samples or samples stored at room temperature, are unreliable.
Two-Site Immunoradiometric Assay of Intact Salmon Calcitonin
Authors: Deftos L J
Published: January 2020

We developed a two-site immunoradiometric assay (IRMA) of salmon calcitonin (SCT) that detects intact SCT(1-32) and not peptide fragments of the hormone. This was accomplished by using monoclonal antibodies prepared against the peptide fragments SCT(1-11) and SCT(11-32). Two antibodies with specificity for each of the peptides were purified from their respective ascites and evaluated in a two-site format wherein one of the antibodies was adsorbed to polystyrene beads and the other was radioiodinated. In this assay format, the antibody pair detected intact SCT(1-32) but did not react with either SCT(1-11) or SCT(11-32). The sensitivity of the assay could be increased by exchanging the antibodies with respect to bead adsorption and radioiodination. Furthermore, by increasing the incubation time and volume of incubation of sample with the polystyrene-bead-adsorbed antibody, the effective detection limit of the assay could be improved. This assay system can be used to detect intact SCT when the presence of fragments of the hormone might otherwise complicate interpretation of assay data.
Decreases in Albumin/Creatinine and N-Acetylglucosaminidase/Creatinine Ratios in Urine Samples Stored At -20 Degrees C
Authors: Manley S E , Burton M E , Fisher K E , Cull C A , Turner R C
Published: January 2020

The effects of storage for 6 months or 2 years at -20 degrees C were studied in urine samples from Type II diabetic patients by assaying albumin by immunoturbidity, N-acetylglucosaminidase (EC 3.2.1.30) by methoxynitrovinylphenol release, and creatinine by the Jaffé method. There were significant decreases (P < 0.001) in albumin/creatinine ratios from 1.14 (0.63-2.98) to 0.83 (0.32-2.12) g/mol (median + interquartile ranges) after 6 months (n = 97), and from 1.64 (0.74-5.72) to 1.00 (0.37-4.54) g/mol after 2 years (n = 89). The percentage of samples with results below the detection limit of the albumin assay (2 mg/L) increased from 5% to 21% after 6 months and from 0% to 34% after 2 years. N-Acetylglucosaminidase/creatinine ratios decreased (P < 0.001) from 520 (358-832) to 380 (263-695) U/mol after 6 months and from 520 (330-865) to 258 (82-462) U/mol after 2 years. The effect of storage was greater in samples with concentrations in the normal range (< 2.5 g/mol for albumin/creatinine, < 500 U/mol for N-acetylglucosaminidase/creatinine). Samples with albumin concentrations more than twice the normal range were still detected as abnormal after storage at -20 degrees C; e.g., 18% were > 5 g/mol (albumin/creatinine) initially, with 17% > 5 g/mol after 6 months of storage. We therefore recommend storage of urine samples at 4 degrees C for no longer than 7 days before assay.
Measurement of Cyclosporine by Liquid Chromatography and Three Immunoassays in Blood from Liver, Cardiac, and Renal Transplant Recipients
Authors: McBride J H , Kim S S , Rodgerson D O , Reyes A F , Ota M K
Published: January 2020

In an effort to replace HPLC for whole-blood determination of cyclosporine (CsA), we compared HPLC with radioimmunoassay (RIA; INCSTAR, Cyclo-Trac SP assay), fluorescence polarization immunoassay (FPIA; Abbott TDx), and in-house modified enzyme-multiplied immunoassay technique (EMIT; Syva Co.). For blood samples from 200 various transplant recipients, RIA = 1.262 (HPLC) - 8.16, r = 0.983; FPIA = 1.200 (HPLC) + 19.90, r = 0.981; and EMIT = 1.038 (HPLC) + 11.28, r = 0.985. For segregation by transplant type, RIA, FPIA, and EMIT demonstrated positive biases of 27%, 12%, and 3%, respectively, for liver transplant recipients (n = 50) when compared with HPLC. Heart transplant recipients (n = 50) gave positive bias values of 23%, 14%, and 4% for RIA, FPIA, and EMIT, respectively. Adult renal transplant recipients (n = 50) demonstrated positive bias values of 30%, 31%, and 0% for RIA, FPIA, and EMIT, respectively. For pediatric renal transplant recipients (n = 50), positive biases of 40%, 31%, and 9% were obtained for RIA, FPIA, and EMIT, respectively. We conclude that the modified EMIT represents the best replacement for HPLC.