bluuebunny/crossref_metadata_2025 huggingface dataset:
heterogeneity of creatine kinase isoenzyme MM in serum in myocardial infarction: interconversion of the "normal" and "abnormal" sub-bands by glutathione
Authors: Williams J , Williams K M , Marshall T
Published: January 2020

We used isoelectric focusing (IEF) in polyacrylamide gels to investigate the effects of glutathione on the sub-bands of serum creatine kinase (CK; EC 2.7.3.2) isoenzyme MM in acute myocardial infarction. The intensity of the "abnormal" sub-bands c (pI 7.25), e (pI 6.85), and g (pI 6.50) increased, and that of the "normal" sub-bands 1 (pI 6.91), 2 (pI 6.65), and 3 (pI 6.35) decreased, following serum incubation with reduced glutathione (GSH, final concentration 1.25 mmol/L). Further incubation with oxidized glutathione (GSSG, final concentration 5 mmol/L) reversed this change and restored the original pattern, whereas GSSG at 7.5 mmol/L caused sub-bands c, e, and g to disappear and sub-bands 1, 2, and 3 to be enhanced. Sequential incubation of serum with 2.5 mmol of GSSG and 7.5 mmol of GSH per liter produced the opposite sequence of events; i.e., the "abnormal" sub-bands disappeared then reappeared (and GSH at 10 mmol/L enhanced their reappearance). At higher concentrations, glutathione (GSH or GSSG) impaired the detection of the CK-MM sub-bands after IEF, an effect that was "quenched" by heat-inactivated serum of low CK activity. Likewise, the intensity of tissue CK-MM (corresponding to myocardium extracted into 100 mmol/L Tris HCl buffer, pH 7.4) was greatly enhanced by adding heat-inactivated serum to the tissue extract before IEF. We discuss the significance of these findings for the diagnosis of myocardial infarction.
Decreased storage stability of creatine kinase in a cardiac reperfusion solution
Authors: Dunphy G , Ely D
Published: January 2020

Creatine kinase (CK; EC 2.7.3.2) has been used as an indicator of myocardial cellular damage. In this study we used a Krebs-Henseleit (KH) solution to reperfuse isolated rat hearts after 24 h of cold preservation and collected the KH reperfusate for assay of CK to assess cellular damage. We wanted to determine the stability of CK in the KH solution at different cold-storage temperatures and albumin concentrations. CK activity (mean +/- SEM) after one week of refrigeration (5 degrees C) was 93% +/- 1% of control values, whereas CK activity in nitrogen-frozen (-200 degrees C) samples was only 1.6% +/- 1% of control values, and that in samples frozen at moderately low temperatures (-10 degrees C) was 63% +/- 1% of control values. To enhance stability, we added albumin at several concentrations (49, 25, 12, and 6 g/L) to reperfusion collections in which CK had been previously determined. Specimens were frozen (-10 degrees C), then re-analyzed for CK weekly for three weeks. CK activity was maintained (100% +/- 5%) only in samples containing 25 g/L or more albumin. These data suggest that refrigeration (5 degrees C) for one week maintains normal CK activity in KH solution; however, if prolonged storage is necessary, a stabilizer such as albumin (greater than or equal to 25 g/L) will maintain analyte stability in frozen storage (-10 degrees C) for at least three weeks.
Sensitive and rapid colorimetric immunoenzymometric assay of ferritin in biological samples
Authors: Ramm G A , Duplock L R , Powell L W , Halliday J W
Published: January 2020

We describe a rapid and sensitive enzyme-linked immunosorbent assay (ELISA) for quantifying ferritin in human and rat biological fluids. We used chlorophenol red beta-D-galactopyranoside as the colorimetric substrate of beta-galactosidase (EC 3.2.1.23), which is coupled to specific antibodies to either human or rat liver ferritin. The assay is sensitive (detection limit for human assay = 0.58 micrograms/L and for rat assay = 0.37 micrograms/L), accurate (average recovery for human assay = 93% and for rat assay = 92%), and precise (total CVs for human assay = 2.3-12.2% and for rat assay = 5.6-11.3%). The results correlated well with those of an established immunoradiometric technique (r = 0.99691). This assay has a prolonged shelf-life, is inexpensive, and utilizes a stable colorimetric substrate that requires relatively short incubation.
Heterophilic antibodies in human sera causing falsely increased results in the CA 125 immunofluorometric assay
Authors: Boerman O C , Segers M F , Poels L G , Kenemans P , Thomas C M
Published: January 2020

An in-house OC 125 monoclonal antibody-based "sandwich" immunofluorometric assay (IFMA) described previously (Clin Chem 1987; 33:2191-4) gave higher results for CA 125 in 37 of 123 serum samples than did a commercially available immunoradiometric assay (IRMA). Discordant results between the two assays became concordant when measurements of the samples were repeated with normal mouse serum (100 mL/L) included in the IFMA reagent. The murine immunoglobulins are thought to block the ability of the heterophilic antibodies in the human serum samples to cross-link the labeled antibody with the solid-phase antibody. Using an enzyme immunoassay, we demonstrated human anti-mouse antibodies (HAMA) in most of the discordant samples examined. We tested the ability of nonimmune sera from other animal species to lower the apparent CA 125 concentrations of the spurious samples and observed that rat, goat, and sheep serum were less effective than mouse serum. One serum sample was discovered to give a falsely increased CA 125 result with the IRMA, but this increase could be prevented by adding murine serum to the IRMA reagent. We conclude that falsely increased CA 125 results are best prevented by adding murine serum (or murine antibodies) to the assay buffer.
Light has a greater effect on direct bilirubin measured by the bilirubin oxidase method than by the diazo method
Authors: Ihara H , Aoki Y , Aoki T , Yoshida M
Published: January 2020

We compared the effect of light on direct-reacting bilirubin (DBIL) measurement by the bilirubin oxidase (EC 1.3.3.5; BOX) method and by the Jendrassik-Gróf diazo method. DBIL concentrations determined by the BOX method in the sera of hyperbilirubinemic infants treated with phototherapy yielded falsely higher values than those by the direct diazo method. A similar tendency was noted when DBIL concentrations in infants' sera irradiated with light in vitro were determined by both methods, although by HPLC none of these sera had detectable DBIL (i.e., conjugated plus delta bilirubin). In general, DBIL concentrations after photoirradiation remained unchanged when measured by the diazo method, but significantly increased when the BOX method was used. Indeed, photoirradiation gave rise to material that acted like a photobilirubin product, which was oxidized at pH 3.7 and therefore was measured as DBIL. Such false increases in DBIL values generated by the BOX method may have clinical diagnostic implications in monitoring jaundiced neonates and in differentiating between physiological jaundice and incipient pathological jaundice.
Differential assay of zidovudine and its glucuronide metabolite in serum and urine with a radioimmunoassay kit
Authors: Tadepalli S M , Puckett L , Jeal S , Kanics L , Quinn R P
Published: January 2020

We developed an ancillary procedure for the ZDV-Trac RIA (Incstar) to allow simultaneous determination of both zidovudine (3'-azido-3'-deoxythymidine, ZDV, AZT, Retrovir) and its metabolite, the glucuronide of ZDV (3'-azido-3'-deoxy-5'-O-beta-D-glucopyranuronosylthymidine, ZDVG, GAZT), in human serum and urine. Using the ZDV-Trac RIA, we measured ZDV concentrations before and after ZDVG in samples was hydrolyzed to ZDV by beta-glucuronidase (EC 3.2.1.31); ZDVG concentration was calculated as the difference between the two results. This method enables rapid evaluation of a large number of samples with a total turn-around time of 6 h. The lower detection limit of the RIA was 0.27 micrograms/L; the measurements varied linearly with ZDV concentrations from 0.27 to 217 micrograms/L, with the 50% inhibitory concentration being approximately 10 micrograms/L. Analytical recoveries of inhouse serum and urine controls for both ZDV and ZDVG exceeded 90%. Coefficients of variation (CVs) of serum controls were less than 6% for ZDV and less than 11% for ZDVG; for urine controls, CVs for both ZDV and ZDVG were less than 6%. Results for ZDVG concentrations obtained by HPLC and by the ZDV-Trac RIA system compared well: r = 0.978, slope 1.0, for serum samples, and r = 0.993, slope 1.09, for urine samples.
Simultaneous determination of disopyramide and mono-N-dealkyldisopyramide enantiomers in plasma and urine by use of a chiral cellulose-derivative column
Authors: Echizen H , Ochiai K , Kato Y , Chiba K , Ishizaki T
Published: January 2020

This assay allows simultaneous determination of the enantiomers of both disopyramide and its active metabolite, mono-N-dealkyldisopyramide, in 1 mL of plasma or 0.1 mL of urine within approximately 35 min by HPLC with a chiral cellulose-derivative column and ultraviolet detection. Recoveries for the analytes and the internal standard (racemic verapamil) with an extraction from alkalinized plasma or urine into diethyl ether were greater than 90%. Intra- and interassay CVs for disopyramide enantiomers were less than 5.5% at 2.5 mg/L in plasma and less than 6.5% at 25 mg/L in urine; for mono-N-dealkyldisopyramide enantiomers they were less than 6.3% and less than 8.9%, respectively. Intra- and interassay relative errors for determining these analytes in plasma and urine at 2.5 and 25 mg/L, respectively, ranged from -5.9% to +2.5%. The calibration curves for the respective analytes were linear (r = 0.995 or greater, P less than 0.01) from 0.025 to 5.0 mg/L in plasma and from 0.5 to 10 mg/L in urine. The lower detection limits (signal-to-noise ratio of 3) for S(+)-disopyramide and the other analytes were 0.010 and 0.025 mg/L, respectively. We evaluated clinical applicability of this method by determining steady-state plasma concentrations and urinary excretions of the respective analytes in a pediatric patient being treated with racemic disopyramide.
Ultrasensitive Time-Resolved Fluorescence Method for α-Fetoprotein
Authors: Christopoulos Theodore K , Lianidou Evrikiia S , Diamandis Eleftherios P
Published: January 2020

We have examined the maximum sensitivity of a newly developed and optimized time-resolved fluorescence immunoassay system. The system, originally described elsewhere Clin Biochem 1988;21:139-50), has undergone significant improvements in sensitivity through improvements of the labeled reagent used. We have chosen an α-fetoprotein [Illegible Text] assay as a model and used monoclonal "capture" antibodies and monoclonal or polyclonal biotinylated antibodies in "sandwich-type" assay configurations. Streptavidin labeled with the europium chelator 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid was used for detection. We can measure as few as 3 x 105 molecules of [Illegible Text] with the optimized system. We have applied this assay to measure AFP in the serum of normal individuals after a 10-fold sample dilution. We conclude that this system is extremely sensitive and can be used in immunoassay or other applications where biotinylated reagents can be applied.
Strategies of organization and service for the critical-care laboratory
Authors: Fleisher M , Schwartz M K
Published: January 2020

Critical-care medicine requires rapidity of treatment decisions and clinical management. To meet the objectives of critical-care medicine, the critical-care laboratory must consider four major aspects of laboratory organization in addition to analytical responsibilities: specimen collection and delivery, training of technologists, selection of reliable instrumentation, and efficient data dissemination. One must also consider the advantages and disadvantages of centralization vs decentralization, the influence of such a laboratory on patient care and personnel needs, and the space required for optimal operation. Centralization may lead to workflow interruption and increased turnaround time (TAT); decentralization requires redundancy of instrumentation and staff but may shorten TAT. Minimal TAT is the hallmark of efficient laboratory service. We surveyed 55 laboratories in 33 hospitals and found that virtually all hospitals with 200 or more beds had a critical-care laboratory operating as a satellite of the main laboratory. We present data on actual TAT, although these were available in only eight of the 15 routine laboratories that provided emergency service and in eight of the 40 critical-care laboratories. In meeting the challenges of an increasing workload, a reduced clinical laboratory work force, and the need to reduce TAT, changes in traditional laboratory practice are mandatory. An increased reliance on whole-blood analysis, for example, should eliminate delays associated with sample preparation, reduce the potential hazards associated with centrifugation, and eliminate excess specimen handling.
New in vitro analytical approaches for clinical chemistry measurements in critical care
Authors: Meyerhoff M E
Published: January 2020

The effective management of patients in intensive care units, operating rooms, and emergency rooms requires frequent measurement of a select group of analytes, preferably at or near the patient's bedside. Tests recognized as being essential for such management include blood gases and related variables (pH, pO2, pCO2, HCO3-, hematocrit/hemoglobin, O2 saturation), electrolytes (Na+, K+, Ca2+, Cl-), and in some cases, certain metabolites (glucose, lactate, urea, creatinine). This report describes the measurement principles, practical instrumental designs, analytical performance, and limitations of several newer electrochemical sensor-based approaches useful for in vitro determination of these species in undiluted whole-blood samples. Considerable attention is given to the most recent advances in ion-selective electrode technology as they relate to blood gas and electrolyte determinations. Similar attention is given to modern enzyme-electrode techniques, which are useful for direct measurements of metabolites in whole blood. The challenges of integrating these new analytical methods into convenient, multi-analyte, user-friendly, bedside or stat-lab instruments are also discussed.
Robotics in the medical laboratory
Authors: Felder R A , Boyd J C , Margrey K , Holman W , Savory J
Published: January 2020

Robotic systems specifically designed for the automation of laboratory tasks are now available commercially. Equipped with computer, analytical hardware, and supporting software, these devices may soon revolutionize the concept of the clinical laboratory and usher in a new era in laboratory testing. We review the types of robots and motion-control software currently available and discuss examples of their applications that extend across many analytical areas. Several ongoing projects are concerned with the systematic integration of robotic devices with other laboratory automation. The integrated robotic laboratories emerging from this work portend a bright future for robotic automation. Many challenges remain, however, in training the individuals needed to develop and manage robotic laboratories, and in making this new technology cost-efficient.
A fully robotic assay for human hormone analysis
Authors: Sasaki M , Ogura K
Published: January 2020

To create a more convenient analytical system for the determination of hormones in human serum, we attempted to make a robotic system by combining robotic equipment and the DELFIA system (time-resolved fluoroimmunoassay; Pharmacia Co.). To combine the two systems, we constructed various peripheral instruments, created software for the computer, and introduced these to the robot at our laboratory. We developed this system to operate more exactly and smoothly than manual procedures during analytical movement steps such as taking samples, adding reagents, mixing samples in kit strips, and measuring the results of the fluoroimmunoassay. With this system the robot can measure in random sequence eight hormones--thyrotropin, triiodothyronine, thyroxin, prolactin, lutropin, follitropin, choriogonadotropin, and cortisol--according to physicians' orders placed through the computer. Consequently, this robotic system can operate fully automatically, from delivering the samples into the robotic system to printing out graph reports for physicians. The technologist merely puts the sample rack on the conveyor belt.
Clearance of different multiple forms of human gamma-glutamyltransferase
Authors: Grøstad M , Huseby N E
Published: January 2020

Several multiple forms of gamma-glutamyltransferase (EC 2.3.2.2) have been described in serum. Most of these are large complexes between the enzyme and circulating lipoproteins. One dominating form is complexed with high-density lipoprotein; a small, hydrophilic form is present in minor amounts. We purified the two forms by immunoaffinity chromatography, injected the purified forms into rabbits, and studied the clearance of the two forms by measuring the change in enzyme activity and in enzyme protein concentration with an enzyme-linked immunosorbent assay. The half-life of the hydrophilic enzyme was 9 h; that of the lipoprotein-enzyme complex was 20 h. This indicates that the lipoprotein-enzyme complex accumulates in serum relative to the hydrophilic enzyme, suggesting in part an explanation of the dominance of the larger form in disease.
Direct solid-phase time-resolved fluoroimmunoassay of 17 alpha-hydroxyprogesterone in serum and dried blood spots on filter paper
Authors: Gonzalez R R , Mäentausta O , Solyom J , Vihko R
Published: January 2020

We describe a direct, solid-phase time-resolved fluoroimmunoassay (TRFIA) for measuring 17 alpha-hydroxyprogesterone (17OHP) in serum and blood spots on filter paper. We used 17OHP-3-carboxymethyloxime (17OHP3CMO) coupled to polylysine as the label, which enabled incorporation of up to 34 atoms of europium per molecule of 17OHP, for a very high specific activity. The assay is based on competition between labeled 17OHP3CMO and 17OHP in blood specimens for polyclonal rabbit anti-17OHP antibodies. The antibody-label complex is separated by binding to anti-rabbit antibodies coated onto microtiter strips. The assay buffer contains danazol to displace 17OHP from steroid-binding proteins in serum. For serum samples, the assay is accomplished in 1 h of incubation at room temperature. The blood spot assay with filter paper discs involves incubation overnight at 4 degrees C. Results for both types of specimens from the same subjects correlated well. The lowest measurable concentrations of 17OHP (nmol/L) were 0.10 (3 SD) and 0.75 (3 SD) for serum and dried blood on filter paper, respectively. Intra- and interassay CVs were about 5-15% for both types of samples.
Quantitative nephelometric assay for determining myoglobin evaluated
Authors: Delanghe J R , Chapelle J P , Vanderschueren S C
Published: January 2020

A recently introduced automated nephelometric immunoassay involving shell/core particles for determination of myoglobin (Behringwerke) was evaluated with the BNA Nephelometer. Method precision was good: the intra-assay CV varied between 1.5% and 6.1%; with daily calibration, the interassay CV ranged between 1.5% and 7.5%. For usual sample dilutions, the assay response varied linearly with myoglobin concentrations up to 23.1 nmol/L. After automatic dilution by the instrument, concentrations up to 2310 nmol/L could be measured without high-dose "hook" effect. Further manual dilution allowed measurement of myoglobin concentrations up to 26,000 nmol/L. Calibration was stable for at least seven days. We detected no significant interferences from hemoglobin, haptoglobin, bilirubin, iodine-containing contrast media, and rheumatoid factors. Treating lipemic samples with Lipoclean (Behringwerke) decreased test results. Simultaneously drawn serum and plasma samples from the same subject showed no consistent differences in myoglobin concentrations. The mean reference myoglobin concentration was 1.380 (SD 0.82) nmol/L for men and 0.878 (SD 0.45) nmol/L for women. In patients with renal insufficiency, serum creatinine values were moderately related to serum myoglobin values (r = 0.465). Although a commercial radioimmunoassay (Byk-Sangtec) and the nephelometric assay intercorrelated well (r = 0.929), values obtained by nephelometry were significantly lower (P less than 0.05). By both assays, results for heart and skeletal muscle tissue extracts showed no correlation, a finding that suggests the existence of multiple forms of myoglobin in human tissues. We conclude that immunonephelometry is a rapid, practical, and reliable method for measuring myoglobin in serum.
A step forward in enzymatic measurement of creatinine
Authors: Fossati P , Ponti M , Passoni G , Tarenghi G , Melzi d'Eril G V , Prencipe L
Published: January 2020

We describe an improved enzymatic ultraviolet absorbance method for assaying creatinine in serum, plasma, and urine. Creatinine is hydrolyzed by creatinine iminohydrolase (EC 3.5.4.21) to ammonia and N-methylhydantoin. The ammonia produced combines with 2-oxoglutarate and NADPH in the presence of glutamate dehydrogenase to yield glutamate and NADP+. The consumption of NADPH, measured by a two-point fixed-time assay, is proportional to the amount of creatinine in the sample. The assay is carried out in two steps: The first step eliminates background absorbance in hyperlipemic samples and endogenous ammonia through a "clearing system" and an isocitrate dehydrogenase-based "ammonia scavenger system"; the second step starts creatinine measurement. The method affords a simple, rapid, and sensitive assay with good precision and extended linearity; it employs working solutions stable at least 4 months. Test results compare closely with those of the isotope dilution-mass spectrometry Definitive Method, the HPLC procedure, and the fuller's earth method. The proposed method is not subject to interference from several metabolites or from the 72 drugs tested. Because it is easily automated, the method is suitable for routine work in clinical laboratories.
Rapid, sensitive fluorometric determination of serum triglyceride by measuring lipase-liberated fatty acids
Authors: Voysey J E , Wilton D C
Published: January 2020

A continuous fluorescence displacement assay was developed for the measurement of long-chain fatty acids and utilized in the study of triglyceride lipase-catalyzed reactions (Wilton, DC. Biochem J 1991;276:129-33). We now describe a method for the rapid measurement of triglyceride in serum with the fluorescence displacement assay. The method involves the hydrolysis of a diluted sample equivalent to 0.1 microL of original serum with excess lipase (EC 3.1.1.3) from Rhizopus arrhizus and measuring the fatty acid released after a set time interval, normally 1 min. Under the conditions of the timed assay, about 2 mol of fatty acid are released per mole of triglyceride. The released fatty acid is monitored by fluorescence change and is proportional to the concentration of triglyceride in the original serum sample. The effective range of serum triglyceride concentration that could be measured was 0.5-10 mmol/L (0.44-8.8 g/L), based on triolein standard. The assay is unique in that it measures lipase-liberated fatty acids rather than liberated glycerol and as such is not subject to many of the criticisms of the glycerol-based methods. Comparison of fasted serum samples established a high correlation between the fatty acid and glycerol methods.
Metabolic profiling of amniotic fluid by proton nuclear magnetic resonance spectroscopy: correlation with fetal maturation and other clinical variables
Authors: Bock J L
Published: January 2020

Proton NMR spectroscopy at 600 MHz was performed on 70 specimens of human amniotic fluid, representing pregnancies at different stages of maturation and with different fetomaternal complications. Spectra with good signal-to-noise characteristics were obtained in 3.5 min of acquisition time, directly on untreated fluid, with presaturation to suppress the intense water resonance. Metabolites that generally gave rise to well-resolved resonances included lactate, glucose, creatinine, choline derivatives, citrate, acetate, alanine, glycine, glutamate, succinate, and others. For three of these metabolites--creatinine, glucose, and lactate--the peak intensities correlated well with results of conventional chemical analysis. The NMR spectra of third-trimester specimens were readily distinguishable from those of second-trimester specimens, and several peak intensities correlated with fetal maturation during the third trimester. Significant correlations with maternal preeclampsia and fetal open spina bifida were also observed.
Clinical significance of urinary cyclic guanosine monophosphate in diagnosis of heart failure
Authors: Jakob G , Mair J , Vorderwinkler K P , Judmaier G , König P , Zwierzina H , Pichler M , Puschendorf B
Published: January 2020

We measured concentrations of guanosine 3',5'-monophosphate (cGMP) in plasma and urine of healthy subjects and patients with congestive heart failure, renal impairment, neoplastic disease, and hepatic cirrhosis. There was no correlation between cGMP concentrations in urine and in plasma. In all patients except those with renal impairment, urinary cGMP concentrations were significantly higher than in healthy persons. Only patients with heart failure or renal impairment showed significantly increased plasma cGMP concentrations. In contrast, cGMP in urine does not relate to the clinically assessed severity of heart failure (New York Heart Association functional classes). Determination of cGMP in plasma results in higher sensitivity and specificity for diagnosing heart failure than measurement of cGMP in urine.
Measurement of apolipoproteins A-I and B during the National Health and Nutrition Examination Survey (NHANES) III
Authors: Bachorik P S , Lovejoy K L , Carroll M D , Johnson C L , Albers J J , Marcovina S M
Published: January 2020

We measured apolipoproteins (apo) A-I and B by rate immunonephelometry (rate INA) during Phase 1 of the National Health and Nutrition Examination Survey (NHANES) III. We also made the measurements by radial immunodiffusion (RID) in a 20% subset of the samples. Aliquots of this subset were also analyzed in the Northwest Lipid Research Laboratories by fixed-time INA calibrated to the World Health Organization (WHO)-International Federation of Clinical Chemistry (IFCC) First International Reference Materials for Apolipoproteins A-I and B. The CVs for the rate INA and RID measurements were: apoA-I, 4.5-7.7% and 2.5-7.6%, respectively; apoB, 2.3-5.3% and 2.3-6.4%, respectively. In NHANES III, rate INA values (x) can be transformed to WHO-IFCC Reference Material-based values (y) as follows: for apoA-I, y = 0.87x + 251.8 mg/L (r = 0.93, SEslope = 0.13, SEintercept = 17, n = 708); for apoB (mg/L), y = 1.068x + 112.8 mg/L (r = 0.98, SEslope = 0.08, SEintercept = 7, n = 646).
Semiportable electrochemical instrument for determining carbon monoxide in breath
Authors: Vreman H J , Stevenson D K , Oh W , Fanaroff A A , Wright L L , Lemons J A , Wright E , Shankaran S , Tyson J E , Korones S B
Published: January 2020

Measurements of carbon monoxide (CO) in breath can be used for the diagnosis of hemolytic disease. A small, semiportable, easy-to-operate CO instrument was developed at Stanford University and tested at 12 Neonatal Research Network Centers of the National Institute of Child Health and Human Development. A syringe pump delivers 7.7 mL of sample per minute through an activate carbon filter to an electrochemical (EC) sensor having a sensitivity of 0.10 +/- 0.01 V per 1 microL/L CO in air. The electronically processed sensor signal is displayed on a digital multimeter. For a typical end-tidal CO measurement, corrected for inhaled CO, three 10- to 12-mL breath and room air samples are manually or mechanically collected and analyzed. CO determination in breath samples from 108 healthy, 1-day-old infants of nonsmoking mothers compared favorably with determinations by gas chromatography (GC), 1.3 +/- 0.8 vs 1.2 +/- 0.8 (mean +/- SD), respectively, with a regression equation of EC = 0.95 GC+0.13 (r2 = 0.98). The results demonstrate that the EC-CO instrument yields results that are comparable with those obtained by the more difficult to perform GC assay.
Endogenous serum antibodies that interfere with a common thyroid hormone uptake assay: characterization and prevalence
Authors: Ritter D , Brown W , Nahm M H , Ladenson J H , Scott M G
Published: January 2020

We identified individuals whose serum contained a substance that produced falsely decreased thyroid hormone (T)-uptake values determined by the Emit (Syva) procedure. Investigation of this interference was prompted by identification of a patient with T-uptake values inconsistent with clinical assessment. IgG depletion and supplementation studies with this patient's serum suggested that the interference was due to endogenous antibodies with specificity for the thyroxine-glucose-6-phosphate dehydrogenase conjugate in the Emit T-uptake assay. The prevalence of the interference was examined by prospectively comparing routine Emit T-uptake values of 1710 patients' samples to T-uptake values obtained by another method. Discrepant samples were also assayed by a radioactive binding triiodothyronine-uptake assay. We identified eight samples that had falsely decreased T-uptake values by Emit, for an overall prevalence of 0.46%. Among 45 consecutive patients with a T-uptake value < 20%, five patients, or 11%, were falsely decreased by Emit and three of these were clearly due to an interfering IgG. We suggest that samples with abnormally low T-uptake values determined by the Emit method be confirmed by an alternative method.
High-performance thin-layer chromatography of free porphyrins for diagnosis of porphyria
Authors: Lai C K , Lam C W , Chan Y W
Published: January 2020

In this simple high-performance thin-layer chromatographic technique for evaluating porphyrin excretion, porphyrins rapidly extracted from urine or feces are separated on reversed-phase octadecylsilyl (C18)-bonded silica thin-layer chromatography plates. We observed > 12 distinct bands of different porphyrins by viewing the plates under long-wave ultraviolet light. Positive screening tests can readily be characterized by the relative fluorescence of various free porphyrins. The two patients presented in this paper are possibly the first two cases of porphyria cutanea tarda reported in Hong Kong Chinese.
Random and continuous-access immunoassays with chemiluminescent detection by Access automated analyzer
Authors: Patterson W , Werness P , Payne W J , Matsson P , Leflar C , Melander T , Quast S , Stejskal J , Carlson A , Macera M
Published: January 2020

The Access Immunoassay System is an automated random and continuous-access analyzer for use with heterogeneous enzyme immunoassays. The instrument stores refrigerated reagent packs for as many as 24 different immunoassays. Throughput is 50-100 tests per hour. One- and two-step, and sandwich and competitive formats, each with various incubation times, can be accommodated, and sample sizes can vary from 10 to 200 microL. A paramagnetic microparticle solid phase combines with a chemiluminescent substrate for signal generation. Within-run CVs for noninfectious disease assays were 2.0% to 9.2%; total CVs were 3.4% to 11.1%. Regression analysis of method comparison studies with established procedures yielded slopes of 0.84 to 1.12 and correlation coefficients > or = 0.94 for 12 of 14 assays (range 0.83-0.99). Compared with culture methods, the Access assay for Chlamydia in urogenital specimens demonstrated sensitivity, specificity, and positive and negative predictive values of 90%, 99.7%, 95%, and 99%, respectively.
Congenital adrenal hypoplasia, Duchenne muscular dystrophy, and glycerol kinase deficiency: importance of laboratory investigations in delineating a contiguous gene deletion syndrome
Authors: Cole D E , Clarke L A , Riddell D C , Samson K A , Seltzer W K , Salisbury S
Published: January 2020

We describe an infant with adrenal insufficiency who was subsequently diagnosed with Duchenne muscular dystrophy (DMD) and hyperglycerolemia due to glycerol kinase deficiency. Karyotyping showed a deletion on the short arm of the X chromosome (p21.1 to p22.1). Molecular mapping revealed that the deletion extended from the 3' end of the DMD gene to a site telomeric to the loci for X-linked congenital adrenal hypoplasia and glycerol kinase deficiency. These results are diagnostic for an Xp21 contiguous gene deletion syndrome--so named because the deletion manifests as a distinctive cluster of otherwise unrelated single-gene disorders in the same individual. The Xp21 syndrome should be considered in any infant with adrenal insufficiency. Measurement of serum triglycerides (without glycerol blanking) and creatine kinase activity are simple screening tests that may facilitate early diagnosis and appropriate genetic counseling about risks of recurrence in subsequent offspring.
Reference intervals developed from data for hospitalized patients: computerized method based on combination of laboratory and diagnostic data
Authors: Kouri T , Kairisto V , Virtanen A , Uusipaikka E , Rajamäki A , Finneman H , Juva K , Koivula T , Näntö V
Published: January 2020

We utilized the databases of a hospital information system to select for determination of reference values various individual hospitalized patients on the basis of their diagnoses at discharge. The nonparametric 2.5-97.5% "health-related" reference intervals were calculated for hemoglobin concentration, mean corpuscular volume (MCV), and erythrocyte count for both sexes. After excluding patients with diseases possibly affecting erythrocyte variables, we obtained a final group of 1786 women and 1450 men, ages 20-65 years, who were studied in age groups of 20-30, 30-45, 45-55, and 55-65 years. The upper reference limits of the MCV results obtained from hospitalized patients were higher than those produced conventionally from healthy individuals, as would be intuitively suggested by clinical experience. This method, based on selection by diagnosis, could be applicable to various analytes measured in hospital laboratories, provided sufficient data are available as databases.
Simple and rapid detection of cholesteryl ester transfer protein deficiency by using nonradioisotopic single-strand conformation polymorphism analysis
Authors: Arai T , Tsukada T , Nakayama T
Published: January 2020

We developed a polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) analysis technique by using silver staining to detect the G-->A point mutation of intron 14 of the cholesteryl ester transfer protein (CETP) gene, which is a common mutation in CETP deficiency. After conventional PCR, amplified DNA was heated and subjected to electrophoresis in polyacrylamide gel containing glycerol. The gel was then stained with silver to identify DNA bands. With this technique, we examined 40 subjects with high concentrations of high-density lipoprotein cholesterol (> or = 1 g/L) and found one homozygote and eight heterozygotes. The procedure is suitable for screening because it is quick and does not require radioactive materials or restriction enzymes.
Detergent extraction and enzymatic analysis for fecal long-chain fatty acids, triglycerides, and cholesterol
Authors: Lee M J , Crook T , Noel C , Levinson U M
Published: January 2020

We report a procedure for determining fecal long-chain fatty acids (LCFA), triglycerides, and cholesterol after detergent extraction with 10 mL/L Triton X-100, 6 mL/L Brij 30, and 0.1 mol/L HCl in isotonic saline by enzymatic analysis of the extraction supernates. Mean recoveries of calibrators ranged from 105% to 117%. Assays of fecal extracts were linear with concentration from 0 to 1000 mumol/L for LCFA, from 10 to 90 mumol/L for triglycerides, and from 0 to 300 mumol/L for cholesterol. Within-run CVs were < 3% for medium and high concentrations, and 3%, 14%, and 28% for low concentrations of LCFA, cholesterol, and triglycerides, respectively. Correlation with gravimetric determination resulted in coefficients (r2) of 0.72 for total fat, 0.63 for neutral fat, and 0.66 for nonesterified fat. Assays of extracts and residues prepared for gravimetric determination indicated that approximately 90% of triglycerides, cholesterol, and LCFA were extracted by organic solvents and that the extracts contain a significant amount of other fats. The proposed method appears accurate, precise, specific, and suitable for routine analysis.
Immunological recognition and clinical significance of nicked human chorionic gonadotropin in testicular cancer
Authors: Hoermann R , Berger P , Spoettl G , Gillesberger F , Kardana A , Cole L A , Mann K
Published: January 2020

We studied the physical properties, immunological recognition, and clinical significance of nicked human chorionic gonadotropin (hCGn) in testicular cancer. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, the beta-subunit of hCGn (hCG beta n) dissociated into two peptides, and, by reversed-phase chromatography, hCG beta n was found to be less hydrophobic than hCG beta. Immunologically, hCGn lacked two epitopes specific for holo-hCG (c1, c2), whereas at least five hCG beta epitopes (beta 1-beta 5) were preserved, and, as a result, recognition of hCGn by different hCG assays varied widely. In 309 sera and 88 urine samples from patients with seminomatous or nonseminomatous testicular cancer, hCG-only, hCG+hCGn, and hCG+hCGn+hCG beta+hCG beta n+hCG beta core-fragment assays gave parallel results. hCGn was more abundant in urine than in serum samples. In conclusion, hCGn lacks two conformationally dependent epitopes of hCG, causing a change in hydrophobicity and explaining its failure to react in certain holo-hCG assays. Recognition of hCGn, however, does not seem to be crucial in the routine use of serum hCG as a tumor marker in patients with testicular cancer.
Libyan family with hypercholesterolemia and increased high-density lipoprotein cholesterol in plasma
Authors: Sheriff D S , el Fakhri M , Ghwarsha K
Published: January 2020

Genetic deficiencies of cholesteryl ester transport protein (CETP) and hepatic lipase activities have been associated with hyperalpha-lipoproteinemias. Here we present a family of 11 members, of which 9, including the father, mother, 5 sons, and 2 daughters, show a marked increase in high-density lipoprotein (HDL) cholesterol alone with low plasma concentrations of triglycerides. Analyses of lecithin:cholesterol acyltransferase (LCAT) activity, cholesteryl ester transfer between HDL fractions, hepatic lipase (HL) activity, and lipoprotein lipase (LPL) activity in these cases showed that a decrease in the heparin-releasable HL activity was the possible cause of the marked increase of HDL2 fractions observed in nine of them. Such a defect in HL activity could significantly affect HDL metabolism in particular and lipoprotein metabolism in general. Evidently, a marked increase in serum total cholesterol due to abnormal metabolism of HDL cholesterol, separate from known causes of altered low-density lipoprotein cholesterol metabolism, e.g., a clearance or a receptor defect, is not uncommon. The coordinated action of HL, LCAT, LPL, and CETP may be essential for normal metabolism of plasma lipoproteins.
Ultrasensitive time-resolved immunofluorometric assay of glutathione transferase alpha in serum
Authors: Tiainen P , Karhi K K
Published: January 2020

Glutathione transferase Alpha (GSTA) is a very sensitive marker of acute centrilobular liver damage. We purified glutathione transferases from human liver and separated the isoenzymes. Polyclonal rabbit antibodies specific for Alpha-class isoenzymes were produced and labeled with Eu(3+)-chelate. We set up a sandwich-type time-resolved immunofluorometric assay (TR-IFMA) to measure GSTA in serum. The detection limit was 0.03 microgram/L, and measuring range was from 0.03 to 100 micrograms/L. The reference range for serum GSTA in this assay was 0.7-6.0 micrograms/L for women and 0.7-14 micrograms/L for men. This TR-IFMA is practical, and the modified rapid assay can be completed in 3 h. Therefore it is applicable for diagnostic purposes in acute liver damage, e.g., associated with drug toxicity or hypoperfusion of the liver.
Catecholamines in depression: an update
Authors: Potter W Z , Manji H K
Published: January 2020

Despite extensive research, the biochemical abnormalities underlying the predisposition to and the pathogenesis of affective disorders remain to be clearly established. Efforts to study norepinephrine (NE) output and function have utilized biochemical assays, neuroendocrine challenge strategies, and measures of peripheral blood cell receptors; the cumulative database points to a dysregulation of the noradrenergic system. Depressed patients (in particular, melancholic, unipolar subjects) excrete disproportionately greater amounts of NE and its major extraneuronal metabolite, normetanephrine, than do controls. Depressed patients also show subsensitive neuroendocrine (growth hormone) and biochemical (inhibition of adenylate cyclase) responses to alpha 2-adrenergic agonists, suggesting that subsensitivity of nerve terminal alpha 2 autoreceptors may underlie the exaggerated plasma NE observed in response to various challenges in affective disorders. Future advances in brain imaging techniques and in the molecular biology of adrenergic receptor-coupled signal transduction systems offer promise for meaningful advances in our understanding of the pathophysiology of affective disorders.
Screening for familial defective apolipoprotein B-100 with improved U937 monocyte proliferation assay
Authors: Van den Broek A J , Hollaar L , Schaefer H I , Van der Laarse A , Schuster H , Defesche J C , Kastelein J J , Van 't Hooft F M
Published: January 2020

Frostegård et al. (J Lipid Res 1990;31:37-44) demonstrated that the proliferation of the human monocyte cell line U937 is critically dependent on the uptake of low-density lipoprotein (LDL) via the apo B, E (LDL) receptor, a characteristic that was used to detect patients with familial defective apolipoprotein B-100 (FDB). Here we applied this principle to develop a simple and reproducible assay for the detection of patients with functionally defective LDL. We added serum to U937 cells in cholesterol-free incubation medium and determined the increase in cell number after a 72-h incubation at 37 degrees C by using an electronic cell counter. Sera from 10 normolipidemic individuals and from 34 patients with type IIa hyperlipoproteinemia stimulated the growth of U937 cells in proportion to the exogenous cholesterol concentration (r = 0.83, P < 0.001) and the LDL-cholesterol concentration (r = 0.81, P < 0.001). However, sera from 16 patients with FDB stimulated less cell proliferation than did sera from patients with type IIa hyperlipoproteinemia with equal LDL-cholesterol concentrations. With a 15% reduction in growth as the cutoff value, this test had a sensitivity and specificity for diagnosis of FDB of 87.5% and 100%, respectively. The improved U937 monocyte proliferation assay can be used for screening hypercholesterolemic patients to detect individuals with functionally defective LDL.
The fitting, acceptance, and processing of standard curve data in automated immunoassay systems, as exemplified by the Serono SR1 analyzer
Authors: Daniels P B
Published: January 2020

Methodologies for establishing standard curves on automated immunoassay systems are dependent on the computational capability of the instrument, its throughput, and, perhaps, the technical experience of the user. Here, factors constraining this approach are discussed in general and the procedures used for the Serono SR1 analyzer are considered in detail. The SR1 is a moderate-throughput, fully automated immunoassay system capable of performing tests for (currently) 18 analytes. The associated user-generated standard curves are utilized not only for interpolation of results but also for evaluation of system performance. The choices of curve-fitting method, weighting of data, acceptance criteria, and outlier rejection are taken out of the hands of the user and are made a vital and integral part of the system. I justify this approach and demonstrate how a sensible mathematical approach must be tailored to the configuration of the instrument.
Enzymatic measurement of phosphatidylglycerol in amniotic fluid
Authors: Jones G W , Ashwood E R
Published: January 2020

We describe an enzymatic method for measuring phosphatidylglycerol (PG) at concentrations as low as 0.2 mumol/L in amniotic fluid. Amniotic fluid (1.5 mL) is centrifuged at 10,000g for 20 min to obtain a lamellar body pellet, which is reconstituted with 0.5 mL of buffer. The PG is measured by a two-step enzymatic scheme. Recovery studies demonstrated that the pellet contains > 97% of the PG present in amniotic fluid. Between-run CVs were 28%, 5.7%, and 2.6% for amniotic fluid controls with means of 0.32, 3.9, and 10.7 mumol/L, respectively (n = 20). The enzymatic procedure was not significantly affected by blood, meconium, bilirubin, or other phospholipids. Lecithin/sphingomyelin ratio (n = 101) and fluorescence polarization (n = 127) compared with log(PG) showed correlation coefficients of 0.832 and -0.866, respectively. This test's ability to detect low concentrations of PG in amniotic fluid may make it a better predictor of fetal lung immaturity than previous methods.
Two-site enzyme immunoassay of cholesteryl ester transfer protein with monoclonal and oligoclonal antibodies
Authors: Mezdour H , Kora I , Parra H J , Tartar A , Marcel Y L , Fruchart J C
Published: January 2020

We developed a sandwich-type enzyme immunoassay to measure cholesteryl ester transfer protein (CETP) mass in human plasma. A specific monoclonal antibody (TP-4) that recognizes an epitope located in the C-terminal domain was used for antigen capture and an anti-CETP peptide antibody directed against the 290-306 residue was used for detection. Bound antibodies were revealed with an antibody-peroxidase conjugate specific for rabbit IgG. The presence of 10 mL/L Triton X-100 in the incubation buffer increased antigen exposure of CETP in plasma. The curves for CETP in standard plasma and partially purified CETP were parallel. This technique is rapid (results within 6 h), accurate, precise (mean intra- and interassay CVs 3.6% and 8.4%, respectively), and simple to perform. Assay sensitivity is at microgram concentrations, with a working range of 20-200 micrograms/L. In 40 normolipidemic healthy subjects, the mean CETP concentration in plasma was 1.1 +/- 0.4 mg/L. A strong correlation between CETP concentration and CETP activity (r = 0.91, n = 42) was observed. In plasma, the bulk of CETP was found in high-density lipoprotein fractions. Therefore, this assay may be a useful tool for investigations of CETP and its significance in relevant diseases.
Enzymes in diagnostics: achievements and possibilities of recombinant DNA technology
Authors: Kopetzki E , Lehnert K , Buckel P
Published: January 2020

We discuss, from an industrial point of view, the scope and possibilities of recombinant DNA technology for "diagnostic enzyme" production and application. We describe the construction of enzyme-overproducing strains and show how to simplify downstream processing, increase product quality and process profitability, improve diagnostic enzyme properties, and adjust enzymes to harsh assay conditions. We also consider some safety and environmental aspects of enzyme production. Other aspects of diagnostic enzymes that we cover are the facilitation of enzyme purification by attachment of short amino acid tails, the introduction of tails or tags for site-specific conjugation or oriented immobilization, the construction of bi- or multifunctional enzymes, and the production of enzyme-based diagnostic tests as demonstrated by the homogeneous immunoassay system of CEDIA tests. We use as examples of diagnostic enzymes glucose-6-phosphate dehydrogenase (EC 1.1.1.49), glucose oxidase (EC 1.1.3.4), alkaline phosphatase (EC 3.1.3.1), alpha-glucosidase (EC 3.2.1.20), pyruvate oxidase (EC 1.2.3.3), creatinase (EC 3.5.3.3), and beta-galactosidase (EC 3.2.1.23).