bluuebunny/crossref_metadata_2025 huggingface dataset:
Interlaboratory Study of the IFCC Method for Alanine Aminotransferase Performed with Use of a Partly Purified Reference Material
Authors: Schiele F , Muller J , Colinet E , Siest G , Arzoglou P , Brettschneider H , Calam D H , Ceriotti F , Férard G , Frei J
Published: January 2020

We present the results of a study on performance of a reference material for alanine aminotransferase (ALT, EC 2.6.1.2) and the corresponding IFCC-approved method in an interlaboratory trial involving 13 laboratories. The ALT material was partly purified from pig heart (specific activity, 150 kU/g) and was essentially free of six potentially contaminating enzyme activities, including aspartate aminotransferase (EC 2.6.1.1). The partly purified ALT was lyophilized in a triethanolamine-buffered matrix, pH 6.4, containing bovine serum albumin and saccharose. Under these conditions, the predicted yearly loss of activity was 0.02% at 4 degrees C and < 0.01% at -20 degrees C. The final blank-corrected results of the accepted set of data gave a mean (SD) of 128.5 (5.1) U/L. The among-laboratory SD was 4.6 U/L and the within-laboratory SD was 2.0 U/L. The certified ALT catalytic concentration in the reconstituted material was 129 U/L with a 0.95 confidence interval of +/- 4 U/L.
Apolipoprotein B Quantified by Particle-Concentration Fluorescence Immunoassay
Authors: Hallaway B J , Rastogi A , Kottke B A
Published: January 2020

We have developed a particle-concentration fluorescence immunoassay (PCFIA) for estimating apolipoprotein (apo) B concentrations in plasma. A two-step antigen-detection system with a polyclonal antibody to apo B bound to carboxyl-polystyrene particles binds the antigen, and a fluorescein-labeled monoclonal antibody detects the bound apo B. Narrow-cut low-density lipoproteins (d = 1.03-1.05 kg/L) were used as the primary standard. The assay compares well with the enzyme-linked immunosorbent assay. The PCFIA gives parallel responses with low-density lipoprotein, very-low-density lipoproteins, and plasma samples, and can be fully automated and completed in 3 h. In a pilot study of patients with diabetes, coronary artery disease (CAD), or both, we found statistically significant differences in apo B concentrations for patients with both CAD and diabetes compared with those for patients with diabetes alone or for control subjects (P < 0.01).
Standardization of Glycohemoglobin Determinations in the Clinical Laboratory: Three Years of Experience
Authors: Bodor G S , Little R R , Garrett N , Brown W , Goldstein D E , Nahm M H
Published: January 2020

Measurement of glycohemoglobin has been recommended for the long-term assessment of glycemic control in diabetic patients. Because different analytical methods measure different glycohemoglobin species, it has been difficult to compare results between laboratories. Here we report 3 years of experience with calibration of an affinity chromatography method for measuring total glycohemoglobin (GHb). Calibration was achieved by including in each assay three hemolysate calibrators for which values for HbA1c and GHb had been determined by repeated analyses by high-performance liquid chromatography (HPLC) and affinity chromatography, respectively. Calibration improved interassay precision (CV = 3.20-7.90% and < 5.0% before and after the introduction of calibration, respectively) and eliminated lot-to-lot variability. In 91 samples, HbA1c was estimated by the calibrated affinity chromatography assay and measured by an ion-exchange HPLC method. Estimated and HPLC-measured HbA1c showed no clinically significant differences during 36 months. The high degree of long-term precision, the disappearance of lot-to-lot variability, and the excellent comparability between analytical methods measuring different species of glycated hemoglobins demonstrate the advantages of calibration.
Relation Between Magnesium and Potassium Concentrations in Myocardium, Skeletal Muscle, and Mononuclear Blood Cells
Authors: Reinhart R A , Broste S K , Spencer S , Marx J J , Haas R G , Rae P
Published: January 2020

Potassium and magnesium were measured in 26 cardiac surgery patients (right atrial appendage), 23 autopsy subjects (right atrial appendage, left ventricular free wall, and skeletal muscle), and 9 healthy volunteers (mononuclear blood cells) to determine whether there was a relation between these two ions in the tissues measured. In the cardiac surgery patients, the potassium and magnesium concentrations were 46.35 +/- 3.89 and 4.40 +/- 0.58 (mean +/- SD, mumol/g wet weight tissue), respectively, and were significantly correlated (r = 0.54, P = 0.005). In the autopsy group, the respective concentrations were: for right atrial appendage, 30.54 +/- 10.18 and 3.66 +/- 0.70 mumol/g (r = 0.38, P = 0.14); left ventricular free wall, 60.69 +/- 17.93 and 7.74 +/- 1.73 mumol/g (r = 0.92, P = 0.0001); and skeletal muscle, 93.05 +/- 20.49 and 8.64 +/- 2.06 mumol/g (r = 0.91, P = 0.0001). In the healthy volunteer group, the results for potassium and magnesium in mononuclear blood cells were 42 +/- 9.9 and 3.99 +/- 0.70 fmol/cell, respectively (r = 0.94, P = 0.0001). Thus, potassium and magnesium concentrations were significantly correlated in all the tissues measured.
Spuriously Low Concentrations of Serum Iron Measured with Generation 14 Kodak Ektachem Slides: Prevalence, Possible Causes, and Partial Improvement with Generation 16 Slides
Authors: Kempinaire A , Laureys M , Goedhuys W , Demeulemeester L , Sevens C , Smeyers-Verbeke J , Cooreman W , Neels H , Gorus F K
Published: January 2020

For sera with iron (Fe) concentrations < 4 mumol/L, Kodak Ektachem slides Generation (GEN) 14 (without ascorbic acid) yielded systematically lower results for Fe than did liquid Ferrozine-based reagents from Baker containing ascorbic acid (10 g/L, final concentration) and adapted to Cobas-Bio. During an 8-month comparison period, outliers (defined as [Fe]Cobas - [Fe]Kodak > 4 mumol/L) were seen in 21 of the 8731 sera (0.24%) tested, corresponding to < 5% of the sera with [Fe]Kodak < 4 mumol/L. In vitro addition of ascorbic acid and (or) Fe identified at least two types of outliers: type 1 (approximately 70%), characterized by [Fe]Kodak > 0.4 mumol/L, by (supra)normal Fe recovery in Kodak slides in the presence or absence of ascorbic acid (10 g/L), and by between-method differences in serum Fe (Cobas - Kodak) that were significantly correlated with serum Zn content (P < 0.0004); and type 2 (approximately 30%), tentatively ascribed to contamination by EDTA, with serum Fe by Kodak < 0.4 mumol/L and Fe recovery near 0%, both of which could be significantly and dose-dependently increased by addition of ascorbic acid (5-20 g/L). For both types of outliers, flameless atomic absorption spectrometry (AAS) yielded results that were significantly higher than concentrations by Kodak with GEN 14. Use of GEN 16 slides (containing ascorbic acid) improved concordance of Kodak results with Cobas, and hence with flameless AAS, for both types of outliers; abolished Zn dependency of results; and increased Fe results in sera with type 2 outliers, although these remained substantially lower than by Cobas. However, like other ascorbic acid-containing reagents, GEN 16 slides were more sensitive to interference by dextran-bound Fe, as assessed during in vitro addition experiments and comparisons involving samples from Fe-dextran-treated patients. GEN 16 slides are hence expected to more frequently overestimate the physiologically available protein-bound Fe in hemodialysis patients. In hospital laboratories, this new interference will probably arise more frequently than the spuriously low results with GEN 14, hence warranting further efforts in optimizing Fe slides.
The Two mRNAS Encoding Rat Intestinal Alkaline Phosphatase Represent Two Distinct Nucleotide Sequences
Authors: Engle M J , Alpers D H
Published: January 2020

Rat duodenal mucosa contains two mRNAs (2.7 and 3.0 kb) encoding intestinal alkaline phosphatase (IAP), but the mechanism for their production has not been clear. By means of the polymerase chain reaction (PCR), we isolated a fragment that identifies a second rat IAP (rIAP-II), differing from the rIAP-I sequence in the coding and 3' untranslated regions and encoding a COOH-terminal sequence predicted to be hydrophilic. By means of probes unique to each sequence, rIAP-I identified the 2.7-kb mRNA, and rIAP-II the 3.0-kb mRNA. The different structures and differential regulation of the mRNAs after fat feeding demonstrate the presence of two rat IAP transcripts.
Differential Release of Human Intestinal Alkaline Phosphatase in Duodenal Fluid and Serum
Authors: Deng J T , Hoylaerts M F , Van Hoof V O , De Broe M E
Published: January 2020

Human intestinal alkaline phosphatase (IAP) can be released by the enterocyte into duodenal fluid as a mixture of three isoforms. A proportion of the enzyme is associated with triple-layered membrane vesicles (vesicular IAP). Although, occasionally, free hydrophilic IAP dimers are present, the remaining enzyme usually consists of a mixture of hydrophobic IAP dimers and more complex hydrophobic IAP structures of larger size, both entities being identified as "intestinal variant" alkaline phosphatase (VAR IAP). The hydrophobicity of VAR IAP stems exclusively from its attached glycosyl-phosphatidylinositol (GPI) anchor. Both vesicular IAP and VAR IAP are converted to hydrophilic enzyme upon removal of the GPI tail by phospholipase D (PLD) present in duodenal fluid. The IAP released into the vascular bed consists mainly of VAR IAP; vesicular IAP is absent. The enzyme characteristics of VAR IAP partially purified from duodenal fluid and from serum are identical. In plasma, VAR IAP appears to associate with (lipo)protein complexes and is thus protected from further degradation by plasma PLD. Such complex formation may explain why, in the serum of a healthy reference population, VAR IAP was more abundant than hydrophilic dimeric IAP.
Amplified Enzyme-Linked Immunoassay of Human Proinsulin in Serum (Detection Limit: 0.1 pmol/L)
Authors: Dhahir F J , Cook D B , Self C H
Published: January 2020

We describe an amplified enzyme-linked immunoassay of human proinsulin in serum that detects intact proinsulin and both the 32/33 and 65/66 split forms. The method uses the IgG fraction of a polyclonal antibody raised in a guinea pig against intact proinsulin, which we used to coat plastic microtiter plates. A sandwich was formed with proinsulin by using a monoclonal antibody against C-peptide labeled with alkaline phosphatase. We quantified the reaction by using the enzyme amplification procedure, which detected as little intact proinsulin as 0.1 pmol/L. We found no cross-reactivity with C-peptide in the assay, and decreased recovery attributable to the presence of insulin could be demonstrated only with a 30-fold excess of this hormone over proinsulin.
Simultaneous Determinations of Liver- and Bone-Type Alkaline Phosphatase by Curve-Fitting of Inhibition Kinetic Data. I. Development and Evaluation of an Absorbance-Based Method
Authors: Fitzpatrick Carol P , Pardue Harry L
Published: January 2020

We describe an approach for the simultaneous determination of isoenzymes of alkaline phosphatase (EC 3.1.3.1) based on the kinetic behavior of inhibition reactions. Data for absorbance vs time, collected while enzymes are being inhibited, are fitted with suitable models to obtain results related to activities of the individual isoenzymes. The primary focus is on two-component mixtures of the bone and liver isoenzymes of alkaline phosphatase, but some results are reported for three- and four-component mixtures. Factors studied include choices of inhibitors, buffers, pH, ionic strength, substrate concentration, kinetic models, data ranges, data densities, and data-processing approaches and programs. Criteria used to select optimal conditions include measurement times, detection limits, useful range, and agreement between expected and computed results for mixtures of isoenzymes. For two-component mixtures, a linear least-squares fit of isoenzyme content computed with the curve-fitting method (y) v a comparison method (x) gave y = 0.96 (+/- 0.05)x + 3.8 (+/- 3)% with r = 0.97 and standard error of the estimate of 9.4% for a range from 15 to 300 U/L. The pooled relative standard deviation (CV) for results was about 5%. Results were degraded for three- and four-component samples.
Simultaneous Determinations of Liver- and Bone-Type Alkaline Phosphatase by Curve-Fitting of Inhibition Kinetic Data. II. Development and Evaluation of a Fluorescence-Based Method
Authors: Fitzpatrick Carol P , Pardue Harry L
Published: January 2020

We describe a kinetic method for the determination of alkaline phosphatase (ALP) isoenzymes, based on fluorescence detection of 4-methylumbelliferone. Several different buffer-inhibitor combinations and substrate concentrations were evaluated. Best results were obtained for inhibition with 2.9 mol/L urea in amino-2-methyl-1-propanol buffer. With this combination, normal concentrations of bone- and liver-type ALP could be determined from kinetic data during an 8-min measurement period. We computed initial velocities from parameters for first-order fits during 1.2 half-lives of the response for liver-type ALP. A linear least-squares fit of initial velocities (y) determined in this way vs results obtained with a comparison procedure (x) gave good correlations. We also estimated total signal changes, delta S, from first-order fits during four half-lives. Isoenzyme content correlated well with parameters computed from the first-order fits. Values for standard errors of the estimates represent 5% and 3% of median responses for activities and isoenzyme content, respectively. When compared with an absorbance-based method described previously, this method had threefold shorter measurement times, but imprecisions were 1.6- to 1.8-fold larger.
Improved Detection of Ischemia-Induced Increases in Coronary Sinus Adenosine in Patients with Coronary Artery Disease
Authors: Feldman Marc D , Ayers Carlos R , Lehman Marcia R , Taylor Heidi E , Gordon Vicki L , Sabia Peter J , Ras Don , Skalak Thomas C , Linden Joel
Published: January 2020

Attempts to monitor coronary sinus adenosine as a clinical marker of myocardial ischemia in humans have been disappointing. Accordingly, procedures have been developed for detecting adenosine in blood collected from the human coronary sinus. Collection involves using a double-lumen metabolic catheter, which allows blood to be mixed with a stop solution at the catheter tip, thereby minimizing adenosine formation and degradation. A five-component stop solution almost completely arrests adenosine formation and degradation. Adenosine analysis is improved by using both boronate and C18 Sep-Pak columns to purify and concentrate adenosine in human plasma before HPLC. Plasma adenosine in the coronary sinus of patients with and without coronary artery disease, measured before and during peak atrial pacing, showed a twofold atrial pacing-induced increase in adenosine in the patients with coronary artery disease (n = 9, P less than 0.001) but no change in the patients with normal epicardial coronary arteries (n = 6). These preliminary results indicate that coronary sinus adenosine may provide an index of myocardial ischemia in patients with coronary artery disease.
Detection of Benzodiazepine Intake in Therapeutic Doses by Immunoanalysis of Urine: Two Techniques Evaluated and Modified for Improved Performance
Authors: Beck Olof , Lafolie Pierre , Hjemdahl Paul , Borg Stefan , Odelius Gudrun , Wirbing Peter
Published: January 2020

We evaluated the EMIT (enzyme-multiplied immuno technique) and FPIA (fluorescence polarization immunoassay) urine screening systems for detection of benzodiazepine intake. Healthy male volunteers were given single oral therapeutic doses of alprazolam (2 mg), chlordiazepoxide (25 mg), flunitrazepam (1 mg), lorazepam (3.75 mg), nitrazepam (5 mg), and triazolam (0.25 mg), after which urine was collected for the next 32 h. The EMIT method failed to detect the intake of flunitrazepam, lorazepam, and nitrazepam. FPIA did not detect the intake of chlordiazepoxide, flunitrazepam, lorazepam, nitrazepam, and triazolam. Modification of the EMIT method to include enzymatic hydrolysis did not significantly alter the results obtained with this method. A modification of the FPIA method to include enzymatic hydrolysis and a lower cutoff value improved the results considerably, so that we reliably detected all studied substances but flunitrazepam. We conclude that (a) both EMIT and FPIA techniques, when used as intended by the manufacturers, are unreliable for the detection of intake of therapeutic doses of these benzodiazepines, and (b) the described modification of the FPIA should provide a much improved tool for detection of benzodiazepine intake.
Two Automated Fully Enzymatic Assays for Lipase Activity in Serum Compared: Positive Interference from Post-Heparin Lipase Activity
Authors: Demanet C , Goedhuys W , Haentjens M , Huyghens L , Blaton V , Gorus F
Published: January 2020

The fully enzymatic Wako and Ektachem assays for pancreatic lipase in serum were found to yield precise (especially Ektachem) and consistent results that were significantly correlated (r = 0.995). Less good concordance was found with the turbidimetric aca method, results being on several occasions above normal by Wako or Ektachem but within the reference limit by the aca. Supplementation of aca packs with colipase generally increased lipase activities measured by the aca in these samples, thereby improving the correlation with Wako and Ektachem. In vitro addition experiments documented that both Wako and Ektachem were relatively insensitive to common chemical interferences. However, post-heparin lipase activities produced a positive interference in both assays, resulting in seemingly increased lipase activities that were suggestive of pancreatic disease. Substituting sodium glycocholate (6 mmol/L) for deoxycholic acid largely eliminated this positive interference in the Wako assay. Because Ektachem reagents cannot be modified in this way, we added sodium glycocholate (2-12 mmol/L) to sera; however, this failed to dissipate the in vivo effect of heparin on the Ektachem results.
Multianalyte Immunoassay Based on Spatially Distinct Fluorescent Areas Quantified by Laser-Excited Solid-Phase Time-Resolved Fluorometry
Authors: Kakabakos S E , Christopoulos T K , Diamandis E P
Published: January 2020

We describe a new multianalyte immunoassay principle and apply it to the simultaneous immunoassay of lutropin, follitropin, choriogonadotropin, and prolactin in serum. The method is based on the coating of distinct areas of polystyrene with analyte-specific antibodies. These antibodies react with the analyte and immobilize it in a specific area while another biotinylated antibody also reacts with the analyte to form a sandwich. After addition of streptavidin labeled with the fluorescent europium chelate of 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid, fluorescent areas are formed, the intensity of which is related to the amount of each analyte present in the sample. The fluorescent areas are quantified on the dry solid phase with laser-excited time-resolved fluorometric measurements. The assays developed are highly sensitive, precise, and accurate. We believe that this system shows potential for multianalyte immunoassay of diverse groups of compounds in disciplines such as endocrinology, infectious disease, hematology, and oncology.
Simultaneous Determination of Lactulose and Mannitol in Urine of Burn Patients by Gas-Liquid Chromatography
Authors: Shippee R L , Johnson A A , Cioffi W G , Lasko J , LeVoyer T E , Jordan B S
Published: January 2020

Ratios of lactulose/mannitol excretion in urine have been used to assess the extent of intestinal permeability in various disease and trauma conditions. Reported studies have used this technique to correlate altered gastrointestinal mucosal permeability to translocation of bacteria and endotoxin, leading to occult sepsis in burn patients. Enzymatic methods of analysis for urine concentrations of mannitol and lactulose were used in these studies. We have found that urine from patients with severe burns frequently contains compounds that interfere with the enzymatic methods. We describe using gas-liquid chromatography to determine mannitol and lactulose simultaneously in the urine of burn patients. To avoid the multiple peaks for the anomeric forms of the reducing sugars during precolumn trimethylsilyl derivatization, we converted the sugars to oximes before the silylation step. The method gave good recoveries of mannitol and lactulose added to burn patients' urine samples. Unlike the enzymatic methods, gas-liquid chromatography eliminates the effect of interfering compounds and allows for the simultaneous determination of both sugars in urine samples.
Specific Detection and Properties of Enzyme Hydrolyzing Phosphonate Ester in Serum
Authors: Han G Y , Fan X H , Jin X B , Wang D P
Published: January 2020

An enzyme capable of hydrolyzing 4-methylumbelliferyl phenylphosphonate to 4-methylumbelliferone and phenylphosphonic acid has been detected in human serum. It has a Km value of 1.72 x 10(-4) mol/L, has an optimum pH of 8.8-9.1 in Tris buffer, and shows maximum activity at 60 degrees C (30 min). The enzymic activity can be inhibited by Na3PO4, EDTA, and cysteine. We saw no effect of CuSO4, adenosine, thymidine, NaN3, diethyl p-nitrophenyl phosphate, p-chloromercuribenzoate, isopropyl fluorophosphate, or eserine on the enzymic activity. The enzyme cannot hydrolyze substrates of phosphodiesterase I or alkaline phosphatase. The enzyme is considered a phosphonate esterase.
Reference Intervals for 24-h Urinary Normetanephrine, Metanephrine, and 3-Methoxy-4-Hydroxymandelic Acid in Hypertensive Patients
Authors: Kairisto V , Koskinen P , Mattila K , Puikkonen J , Virtanen A , Kantola I , Irjala K
Published: January 2020

The urinary excretion of normetanephrine, metanephrine, and 3-methoxy-4-hydroxymandelic acid was quantified by HPLC in hypertensive patients who were being routinely investigated for the exclusion of pheochromocytoma. The data were used to calculate reference intervals for these analytes. Age- and sex-specific 95% reference intervals for 24-h urinary excretion were as follows (mumol/24 h): normetanephrine in men ages 16-35 years (n = 17) 0.7-3.4, in men older than 35 years (n = 121) 0.8-5.1, in women ages 16-35 years (n = 41) 0.5-2.1, and in women older than 35 years (n = 144) 0.6-3.3 mumol/24 h; metanephrine in men (n = 138) 0.3-2.0, and in women (n = 185) 0.2-1.3; and 3-methoxy-4-hydroxymandelic acid in men (n = 142) 10-54, and in women (n = 184) 9-38. Normetanephrine or metanephrine excretion rates or both exceeded the proposed reference intervals in seven of the eight patients with pheochromocytoma. Determination of 3-methoxy-4-hydroxymandelic acid excretion did not yield any additional diagnostic information.
Acyclic Adenine Nucleoside Phosphonates in Plasma Determined by High-Performance Liquid Chromatography with Fluorescence Detection
Authors: Naesens L , Balzarini J , De Clercq E
Published: January 2020

9-(2-Phosphonylmethoxyethyl)adenine (PMEA), (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine (HPMPA), and (R,S)-9-(3-fluoro-2-phosphonylmethoxypropyl)adenine (FPMPA) are selective antiviral agents with activity against a broad spectrum of DNA viruses and retroviruses. A highly sensitive method has been developed to determine concentrations of these compounds in human plasma. Plasma samples containing PMEA, HPMPA, or FPMPA were treated with chloroacetaldehyde to yield the corresponding highly fluorescent 1,N6-ethenoadenine derivatives. Chromatographic separation combined with excitation at 254 nm and fluorescence detection at 425 nm allowed quantification of PMEA, HPMPA, and FPMPA within a concentration range of 0.25-4000 mumol/L. This method proved useful in accurately determining low PMEA concentrations in sera from PMEA-treated monkeys and cats. The assay should be applicable to the quantification of PMEA, HPMPA, and FPMPA in plasma and urine of humans treated with any of these drugs.
Methylmalonic Acid Concentration in Serum Not Affected in Hepatic Disease
Authors: Hagelskjaer L , Rasmussen K
Published: January 2020

Accumulation of methylmalonic acid may provide an early clue to deficiency of cobalamin (vitamin B12) in tissue. Metabolic abnormalities involving precursors of methylmalonic acid are frequently observed in patients with hepatic diseases. To establish whether methylmalonic acid accumulates and thereby gives false-positive test results for cobalamin deficiency, we measured the concentration of methylmalonic acid in serum of patients with various hepatic diseases. Many of the patients had increased concentrations of cobalamin in serum. In serum from 70 patients, the mean concentration of methylmalonic acid (252, SE 25 nmol/L) did not differ significantly from that found in healthy subjects (211, SE 12 nmol/L). We conclude that the assay of methylmalonic acid in serum may be useful for evaluating cobalamin status in hepatic disease with functional cobalamin deficiency despite an artificially increased normal or high concentration of cobalamin in serum.
Advanced Glycosylation End Products in the Mesenteric Artery
Authors: MacDonald E , Lee W K , Hepburn S , Bell J , Scott P J , Dominiczak M H
Published: January 2020

We measured advanced glycosylation end products in the mesenteric artery of 37 patients (ages 29-82 years), 34 of whom were nondiabetic. Samples of arterial tissue were obtained during bowel resectioning. Advanced glycosylation end products were measured as collagen-linked fluorescence (excitation wavelength 370 nm, emission wavelength 440 nm) after collagenase digestion of tissue samples. Mean fluorescence of the arterial samples was 15 U/mg (range 5.3-27). Collagen fluorescence correlated with patients' age (r = 0.57; P less than 0.001). No difference in the collagen-linked fluorescence was observed between men and women (P = 0.63), hypertensive and normotensive patients (P = 0.44), smokers and nonsmokers (P = -0.52), and patients with and without symptomatic coronary heart disease (P = 0.7). This study demonstrates, for the first time, the relationship between collagen-linked fluorescence and patients' age in human arterial tissue ex vivo.
Elimination of Fibrinogen Interference in a Dye-Binding Method for Iron
Authors: Ooi D S , Perkins S L , Tokessy N E
Published: January 2020

Serum iron concentrations greater than 90 mumol/L were measured in samples from hemodialysis patients by a Ferrozine dye-binding method. Reanalysis by coulometry showed these results to be spuriously high. Turbidity resulting from precipitation of fibrinogen was identified as the source of the interference. High concentrations of heparin enhanced the effect. We conclude that the persistence of fibrinogen in serum samples from patients treated with anticoagulants is a potential interference in Ferrozine dye-binding methods performed without prior deproteinization. Adding guanidine.HCl to the acid buffer reagent is a simple way to eliminate this interference.
Method-Dependent Differences in Apparent Sodium Concentrations in Plasma after Exchange Transfusions with Citrated Blood
Authors: Schots J , De Raeve H , Boeyckens A , Umbrain V , Verbeek F , Gorus F K
Published: January 2020

Shortly after exchange transfusions (n = 7) with citrate-dextrose-phosphate (CDP)-treated blood, but not after regular transfusions (n = 9), sodium concentrations in plasma were significantly and paradoxically lower (3-10 mmol/L) by Ektachem 700 XR determination [with use of electrolyte reference fluid (ERF) generation 00] than by flame photometry. Similar between-method differences could also be generated by in vitro supplementation of plasma pools with CDP solution, with trisodium citrate, or to a lesser extent with Na2HPO4. For four potentiometric methods the analytical recovery of sodium added as CDP was significantly lower than by flame photometry. Within each potentiometric method the recovery was also less than the value for sodium added as NaCl. A significant association of sodium ions with citrate3- ions and, to a lesser extent, HPO4(2-) ions could theoretically account for at least part of the observations, conferring greater medical relevance to potentiometric measurements in this particular clinical situation.
Genetic Variants of Alpha 1-Antitrypsin and Hemoglobin Typed by Isoelectric Focusing in Preselected Narrow pH Gradients with Phastsystem
Authors: Jeppsson J O , Einarsson R
Published: January 2020

In this method for automatically running and staining isoelectric focusing (IEF) gels, pre-made dehydrated polyacrylamide gels were rehydrated before assays run with the PhastSystem (Pharmacia LKB Biotechnology). The typing of genetic variants of hemoglobin and alpha 1-antitrypsin in narrow pH gradients (pH 6.7-7.7 and 4.2-4.9, respectively) was simple, convenient, and reproducible. The clinically important variants of alpha 1-antitrypsin (ZZ and SZ) were identified from serum or dried blood on filter paper. The fast screening of abnormal hemoglobin samples (HbS) for cases in clinical medicine was easily performed. The total analysis time for the phenotyping with conventional protein staining was approximately 60 min.
Multisite Immunochemiluminometric Assay for Simultaneously Measuring Whole-Molecule and Amino-Terminal Fragments of Human Parathyrin
Authors: Klee G G , Preissner C M , Schryver P G , Taylor R L , Kao P C
Published: January 2020

The immunochemiluminometric assay described uses immobilized anti-human parathyrin (parathyroid hormone, hPTH)(1-44) and anti-hPTH(44-68) antisera and acridinium ester-labeled anti-hPTH(1-34) to simultaneously measure both intact hPTH and its amino-terminal fragments. Results by the assay correlate well with those by a cAMP-based bioassay and the Nichols Allegro immunoradiometric assay. The minimal detection limit is 0.08 pmol/L. The normal range is 1.0-5.0 pmol/L, and values are higher in older women. About 90% of study patients with surgically proven parathyroid adenomas had above-normal preoperative PTH concentrations, whereas patients with hypercalcemia of malignancy had normal or suppressed values. This assay was designed to detect both intact PTH and amino-terminal PTH fragments; however, chromatographic fractionation of pools of primary and secondary hyperparathyroid plasma showed virtually no amino-terminal fragment activity. Nonetheless, the design is important because the absence of carboxyl-terminal binding sites prevents interference by carboxyl-terminal fragments and because bioactive amino-terminal fragments will react in the assay if they are present in the patients' sera or if they are produced by in vitro proteolysis of intact PTH.
Immunoassay in Urine of a Specific Marker for Proximal Tubular S3 Segment
Authors: Verpooten G F , Nuyts G D , Hoylaerts M F , Nouwen E J , Vassanyiova Z , Dlhopolcek P , De Broe M E
Published: January 2020

Urinary intestinal alkaline phosphatase (EC 3.1.3.1; IAP) is a marker of the S3 segment of the human kidney proximal tubule. An accurate enzyme-antigen immunoassay (EAIA) with a high-affinity specific monoclonal antibody (IAP250) developed for this marker has a detection limit below the lowest IAP activity found in urine samples of normal subjects. The intra- and interassay CVs were less than 5%. Mean analytical recovery of pure IAP added to urine was 102% (SD 6%), and the EAIA results correlated well with immunoreactivity (measured by a sandwich ELISA), suggesting that the EAIA detected all of the IAP in urine. In healthy individuals (ages 20-80 years) the IAP concentrations, expressed as urinary creatinine ratios, ranged from 0.1 to 2.0 U/g (5-95 percentiles) without major differences related to sex and age. Workers exposed to mercury, which affects the S3 segment, showed an increased IAP elimination; abusers of analgesics, which affect more distal parts of the nephron, did not. As opposed to currently measured markers, the EAIA offers easy, accurate, and precise measurement of early alterations in the S3 segment.
International Federation of Clinical Chemistry Standardization Project for Measurements of Apolipoproteins A-I and B. II. Evaluation and Selection of Candidate Reference Materials
Authors: Albers J J , Marcovina S M , Kennedy H
Published: January 2020

The first phase of an international collaborative study for standardization of test systems for measuring apolipoprotein (apo) A-I and apo B demonstrated that uniformity of apo A-I and apo B measurements can be achieved if suitable common reference materials are used to calibrate the different systems. The objective of the second phase was to evaluate the linearity and parallelism or proportionality of the candidate reference materials selected in phase one and to determine whether any of them could be proposed as international reference materials. We evaluated the proposed reference materials with 37 test systems for apo A-I and 38 for apo B, involving 23 manufacturers and five research laboratories. Two lyophilized preparations were proposed for apo A-I, SP1 from Behringwerke AG and SP2 from Daiichi Pure Chemicals Co., and two liquid preparations were proposed for apo B, SP3 from Behringwerke AG and SP4 from Reagents Applications. The linearity of the candidate reference materials was compared with the linearity of a frozen serum pool or interim serum reference material distributed to all the participants and with that of a fresh serum pool prepared by each participant. SP1 and SP3 exhibited linearity and parallelism similar to that of the fresh frozen serum pool and had among-laboratory CVs less than or similar to those obtained on normolipidemic serum samples (approximately 6% for apo A-I and approximately 7% for apo B).
Marijuana Testing in Urine: Use of a Hexadeuterated Internal Standard for Extended Linearity, and Ion Trap vs Mass Selective Detector Gas Chromatograph/Mass Spectrometer Systems
Authors: Joern W A
Published: January 2020

The use of a hexadeuterated internal standard for the assay of the marijuana carboxy metabolite in urine resulted in two significant improvements. First, the linearity of the procedure was increased considerably because of the minimal chromatographic peak overlap of the internal standard and carboxy metabolite derivatives. Second, again because of minimal peak overlap, the same extract could be analyzed with similar results by both the ion trap detector and mass selective detector gas chromatograph/mass spectrometer systems.
Performance of an Immunoradiometric Assay of Erythropoietin and Results for Specimens from Anemic and Polycythemic Patients
Authors: Andre M , Ferster A , Toppet M , Fondu P , Dratwa M , Bergmann P
Published: January 2020

We evaluated a new commercially available two-site immunoradiometric assay (IRMA; BioMérieux 125I-EPO CoatRIA) for erythropoietin (EPO) in human serum. The precision (CV) was 4.1% intra-assay and 8% interassay for a serum pool with an EPO concentration of 17 int. units/L; the detection limit was 0.5 int. unit/L, one order of magnitude lower than by classical radioimmunoassay (RIA), although standardization of IRMA and RIA were similar. Results by both IRMA and RIA are compared for normal subjects, patients with nonrenal noninflammatory anemias, patients with beta-thalassemia major, hemodialysis patients, and patients with primary or secondary polycythemia. Values by IRMA compared well with those by RIA in the upper area of the range; IRMA and RIA values for EPO show parallel expected variations with the degree of anemia. However, because of its greater sensitivity and specificity, we consider the IRMA more appropriate than RIA for investigating patients with sub-normal EPO concentrations.
Design and Evaluation of an Anti-Evaporative Cover for Use with Liquid Containers
Authors: Burtis C A , Watson J S
Published: January 2020

We have designed an anti-evaporative cover for use with the sample and reagent cups and other liquid containers that are required in automated analytical systems. This cover, which is simple in design, consists of a baseplate and a cylindrical chimney. By increasing the height and decreasing the inner diameter of the chimney, evaporative losses can be reduced to less than 0.1%/h; thus, aliquots of sample and reagents can be allowed to remain in their cups and containers for several hours before an analytical error due to evaporation will be measurable. We have also modified a model that we previously developed and validated to estimate evaporative losses from open sample cups, to allow us to predict evaporative losses from cups and containers fitted with the new type of cover. This model confirms that the magnitude of the evaporative loss is inversely proportional to the resistance provided by the headspace above the sample in the cup and by the space defined by the chimney of the cover. With chimney heights ranging from 12 to 36 mm and their inner diameters from 1 to 4 mm, greater than 70% of the resistance to evaporation is provided by the cover.
Urinary Glycosaminoglycan Excretion in Rheumatic Diseases
Authors: Kéry V , Orlovská M , Stancíková M , Risko M , Zlnay D
Published: January 2020

We used Alcian Blue (AB) and dimethylmethylene blue (DMB) methods to measure glycosaminoglycan (GAG) excretion in the first morning urine specimens of patients with osteoarthritis (OA), ankylosing spondylitis (AS), and rheumatoid arthritis (RA) in different stages of disease. By the AB method, urinary GAG excretion in patients with RA was not different from healthy control subjects. However, the DMB method showed significant differences (in milligrams of GAG per gram of creatinine) for OA (median 25.4, range 14.3-44.0, P less than 0.01, n = 23) and RA patients (median 33.0; range 10.0-147.6; P less than 0.001, n = 63) in comparison with unaffected individuals (median 20.2; range 8.9-41.4, n = 38). We noted a significant difference in urinary GAG excretion between RA and OA patients (P less than 0.01) and between RA and AS (P less than 0.01) patients. The DMB method was further investigated by clinical decision analysis. The DMB method is simple and rapid and may be useful in diagnosing RA by distinguishing between RA and OA or AS.
Is Free Thyroxine Accurately Measurable at Room Temperature?
Authors: Ross H A , Benraad T J
Published: January 2020

Two recently developed two-step, or "back-titration" assay kits for free thyroxine (FT4)--one based on a europium-labeled derivative of T4, the other on conventional radiolabeled T4--were compared with both symmetrical dialysis and an indirect FT4 radioimmunoassay. Discrepancies between the two-step and the other two methods were observed, particularly in samples that had very low to zero thyroxine-binding globulin (TBG) content. In those instances the two-step methods gave values almost twice as high as the other methods. This effect could be largely reversed in one of the two-step assays and completely reversed in the other by performing the first incubation step at 37 degrees C rather than at room temperature, as prescribed by the manufacturers. When symmetrical dialysis is performed at both temperatures, FT4 at room temperature is about 40% of the amount determined at 37 degrees C, except in zero-TBG samples, where it averages almost 80% of the value at 37 degrees C. Moreover, we demonstrated that the affinity of TBG for T4 is much more temperature-dependent than the affinity of transthyretin and albumin for T4, so that the net temperature effect on the FT4 in a sample depends on the relative contribution of TBG to total binding. We conclude that performing FT4 assays at room temperature is principally incorrect and leads to falsely increased values when samples with very low TBG concentrations are analyzed.
A Study of Experimental Lancets for Blood Collection to Avoid Bone Infection of Infants
Authors: Meites S , Hamlin C R , Hayes J R
Published: January 2020

We describe a study made at two pediatric centers to test experimental lancets for blood collection by skin puncture of infants' heels or fingers. Our primary goal is to decrease the hazard of osseous injury while collecting adequate blood, by using three lancet widths at a constant length of 1.0 mm. The three widths used were 1.0, 1.25, and 1.5 mm. When success at skin puncture was defined rigidly on the basis of the blood volume obtained, the data show that success was related neither to the lancets' dimensions as tested nor to the age of the child, but rather to the phlebotomist's skill and experience, which improved with time.
The History of Proficiency Testing/Quality Control
Authors: Sunderman F W
Published: January 2020

The history and origins of proficiency testing of clinical laboratories are reviewed. Since the introduction of proficiency testing in the late 1940s, dramatic improvement in laboratory performance has been demonstrated. Nonetheless, the poor performance found two decades earlier was summoned to support the passage of the Clinical Laboratory Improvement Act of 1967 (CLIA '67).
How Good are Clinical Laboratories? An Assessment of Current Performance
Authors: Rej R , Jenny R W
Published: January 2020

The Clinical Laboratory Improvement Act of 1967 and Amendments of 1988 (CLIA '67 and CLIA '88) were enacted to ensure that clinical laboratories within the U.S. provide a quality of service that meets clinical needs for good patient care. Approved proficiency-testing programs are to judge the quality of laboratory testing by promulgated performance criteria. We examine the quality of analytical results reported in 1991 to the New York State Department of Health Proficiency Testing program in light of these criteria and analytical goals, based on medical usefulness. Analytical performance is examined for cholesterol, potassium, sodium, calcium, glucose, aspartate aminotransferase, digoxin, and theophylline. In general, proposed CLIA '88 performance standards are compatible with the current state of practice for the population of laboratories examined. Exceptions appear to be digoxin and sodium (failure rate exceeding average) and most therapeutic substances (low failure rate). Sources of analytical bias relative to an accuracy-based target value must be characterized as method-, laboratory-, or matrix-dependent if regulatory programs are to achieve the objective of improving analytical accuracy across all testing sites.
Proficiency Testing Linked to the National Reference System for the Clinical Laboratory: a Proposal for Achieving Accuracy
Authors: Lasky F D
Published: January 2020

I propose using proficiency testing (PT) to achieve one of the important goals of CLIA: accurate and reliable clinical testing. Routine methods for the clinical laboratory are traceable to Definitive (DM) or Reference Methods (RM) or to Methodological Principles (MP) through a modification of the National Reference System for the Clinical Laboratory. PT is the link used to monitor consistent field performance. Although PT has been effective as a relative measure of laboratory performance, the technical limitations of PT fluids and of routine methods currently in use make it unlikely that PT alone can be used as a reliable measure of laboratory accuracy. Instead, I recommend calibration of routine systems through correlation to DM, RM, or MP with use of patients' specimens. The manufacturer is in the best position to assume this responsibility because of also being responsible for consistent, reliable product. Analysis of different manufactured batches of reagent would be compared with predetermined goals for precision and accuracy, as illustrated with data from product testing of Kodak Ektachem clinical chemistry slides. Adoption of this proposal would give manufacturers of PT materials, manufacturers of analytical systems, PT providers, and government agencies time to understand and resolve sources of error that limit the utility of PT for the job required by law.
The Impact of Legislation on Future Technology Development
Authors: Wilding P
Published: January 2020

In this paper I examine some of the factors influencing technology development and attempt to explain whether CLIA '88 enhances the opportunities or exacerbates the problems for U.S.-based technology development. The viewpoint I express is concentrated on the clinical chemistry segment of the diagnostics arena.
Laboratory Monitoring of Androgenic Activity in Benign Prostate Hypertrophy Treated with a 5 Alpha-Reductase Inhibitor
Authors: Matzkin H , Chayen R , Goldfarb H , Gilad S , Braf Z
Published: January 2020

Testosterone and androstenedione are metabolized by 5 alpha- and 5 beta-reductases to androsterone (A) and etiocholanolone (E), respectively. These are excreted in the urine as conjugates, and the A/E ratio in normal men is usually greater than or equal to 1.5 (as opposed to 1 in women) because of the high 5 alpha-reductase activity in the prostate. The A/E ratio can be determined simply by gas chromatography after acid hydrolysis of a urine sample, extraction of steroids, and formation of trimethylsilyl derivatives. A timed collection of urine is unnecessary because the ratio of A/E is used rather than absolute values. In men suffering from benign prostate hypertrophy who are treated with Finasteride (a 5 alpha-reductase inhibitor), the A/E ratio decreases to less than 0.5. The A/E ratio decrease can be detected long before there is clinical improvement.
Detection of Hepatitis B Virus DNA in Serum by a Nonisotopic Hybridization Technique
Authors: Akar A , Bournique B , Scholler R
Published: January 2020

We developed a nonisotopic technique, Hepagene, for measuring hepatitis B virus (HBV) DNA in human serum by using a sulfonated probe that is detected by a sandwich immunoenzymatic reaction. The detection limit, determined by serum dilution tests, was 2.5 ng/L. The precision of the Hepagene test was demonstrated by the accurate reproducibility observed for low (3 ng/L) and medium (38 ng/L) concentrations of HBV DNA assayed in 24 different series. Specificity was established by assaying HBV DNA in sera from 98 patients by the Hepagene technique or by a solution hybridization assay with an 125I-labeled probe. Results by both techniques agreed for 94 sera (96%), with 68 being concordant for HBV DNA negativity and 26 for positivity. HBV DNA titers assayed by both methods also agreed. Hepagene represents the first nonisotopic HBV DNA assay involving a sulfonated probe and with performance characteristics equivalent to those of classical radioactive hybridization techniques.
Colorimetric Determination of Potassium in Plasma and Serum by Reflectance Photometry with a Dry-Chemistry Reagent
Authors: Ng R H , Sparks K M , Statland B E
Published: January 2020

We evaluated a colorimetric assay of potassium in plasma and serum with the Boehringer Mannheim Reflotron reflectance photometric analyzer, which is designed for near-patient testing in hospitals and physicians' offices. This potassium method does not require calibration or instrument maintenance by the operator. Analysis of 30 microL of plasma or serum takes approximately 140 s. Within-day imprecision (CV) was 1.0-1.2%. Total CVs over a 1-month period were 1.0-1.4%. Patients' results from the Reflotron correlated well with those from the IL 643 flame photometer and the Beckman Synchron CX3 ion-selective electrode methods. The accuracy of Reflotron values was also verified with Standard Reference Material 956 from the National Institute of Standards and Technology.