bluuebunny/crossref_metadata_2025 huggingface dataset:
GC-MS determination of organic acids with solvent extraction after cation-exchange chromatography
Authors: Suh Ja Won , Lee Seon Hwa , Chung Bong Chul
Published: January 2020

We combined column and partition chromatography to isolate, purify, and quantify biological organic acids in urine and cerebrospinal fluid (CSF). Urine and CSF samples were introduced onto a preconditioned cation-exchange column (Dowex 50W × 8 resin) to remove the biological interferences. The effluent with water was extracted with ethyl acetate two times (pH 1 and 3) and the organic acids were quantitatively converted into their trimethylsilyl derivatives for detection by gas chromatography–mass spectrometry. The good quality-control data were obtained through precision and accuracy tests. Inter- and intraassay CVs were 0.01–10.2% and 0.02–12.2%, respectively. Analytical recoveries compared favorably with results from the commonly used solvent extraction method. This method was used for the measurement of the 14 organic acids in the urine and CSF of healthy volunteers. The values obtained were in the range of the published data.
Secretory carbonic anhydrase isoenzyme (CA VI) in human serum
Authors: Kivelä Jyrki , Parkkila Seppo , Waheed Abdul , Parkkila Anna-Kaisa , Sly William S , Rajaniemi Hannu
Published: January 2020

Carbonic anhydrase VI (CA VI) is a secretory isoenzyme that, by analogy to α-amylase, is produced in the salivary glands and delivered into saliva. To determine whether CA VI is transferred into the circulation and is detectable in human serum, we collected blood samples from four healthy subjects at 3-h intervals throughout a 24-h period and measured concentrations of CA VI by a specific time-resolved immunofluorometric assay. All serum samples contained CA VI, the concentrations being ∼22 times lower in serum than in the corresponding saliva samples. The presence of CA VI in serum was confirmed by Western blotting, which under reducing conditions identified a 42-kDa polypeptide band corresponding to the monomeric CA VI. The described time-resolved immunofluorometric assay for CA VI might be useful to identify or exclude diseases of the salivary glands in the differential diagnosis of patients whose serum amylase concentrations are increased.
Polyclonal pancreatic elastase assay is superior to monoclonal assay for diagnosis of acute pancreatitis
Authors: Keim Volker , Teich Niels , Reich Andrea , Fiedler Fritz , Mössner Joachim
Published: January 2020

We compared the clinical values for diagnosis of acute pancreatitis of two commercial assays for pancreatic elastase: an ELISA procedure with monoclonal antibodies and a RIA technique with polyclonal antibodies. In 14 patients with acute pancreatitis, serum concentrations of elastase determined by ELISA (ELISA-elastase) decreased much faster (half-life 0.4 days) than those of elastase determined by RIA (RIA-elastase) (2.2 days), amylase (0.8 days), or lipase (0.9 days). Serum samples from 253 additional patients with abdominal pain (32 of these with acute pancreatitis) were analyzed. In sera collected up to 48 h after the onset of disease, the ROC curves showed a slightly higher diagnostic value of RIA-elastase. In samples taken later, at a sensitivity of 90% the specificity of RIA-elastase was 95% (ELISA-elastase 40%). We conclude that serum ELISA-elastase is of much lower clinical value than RIA-elastase for diagnosis of acute pancreatitis.
C677T mutation of methylenetetrahydrofolate reductase gene determined in blood or plasma by multiple-injection capillary electrophoresis and laser-induced fluorescence detection
Authors: Ulvik Arve , Refsum Helga , Kluijtmans Leo A J , Ueland Per Magne
Published: January 2020

We constructed an assay to detect the common C677T mutation in the methylenetetrahydrofolate reductase gene. The mutation creates a Hinfl recognition site detected by restriction cleavage of a 198-bp fragment amplified in the polymerase chain reaction (PCR). Digested samples were subjected to capillary electrophoresis with laser-induced fluorescence detection (CE-LIF), with hydroxypropylmethylcellulose as the sieving matrix and SYBR Green I as the fluorescent dye. After amplification but before digestion, we added to the PCR mixture a fragment with the HinfI recognition site and a 15-bp truncation at the 3′ end. Using this procedure, we could (a) verify completeness of digestion and monitor injection, (b) assign genotypes on the basis of pattern recognition, and (c) develop a multiple-injection mode with simultaneous separation of as many as eight samples. A seminested PCR protocol in combination with CE-LIF allowed genotyping of plasma/serum samples 20 years old.
Passage of silver ions through membrane-mimetic materials, and its relevance to treatment of burn wounds with silver sulfadiazine cream
Authors: Tsipouras Nicholas , Rix Colin J , Brady Peter H
Published: January 2020

Treatment of acute burn wounds with silver sulfadiazine has raised concern of potential silver toxicity. As the wound heals, a barrier forms between the silver sulfadiazine and the blood, but this membrane is not impenetrable, and so silver absorption is still possible. In this work, we have modeled chemical systems to investigate the transport of silver sulfadiazine and silver chloride through cellulose, chitosan, collagen, and polyethylene membranes into the following media: synthetic serum electrolyte solution (SSES), SSES plus glutathione, and human serum, to simulate some of the chemical processes occurring at a burn wound during healing. Our results clearly indicate that membranes can retard the movement of silver ions, especially those that have silver-binding properties. This suggests that silver absorption at a healing wound will be minimized by entrapment of silver in the growing membrane network, and thus the likelihood of silver toxicity will be reduced.
Optical tweezers-based immunosensor detects femtomolar concentrations of antigens
Authors: Helmerson Kristian , Kishore Rani , Phillips William D , Weetall Howard H
Published: January 2020

We used optical tweezers (optical trapping technology) to measure the force required to separate antigen–antibody bonds. Under competitive-binding conditions, we used the force determination to detect and measure protein antigen concentrations as small as 1 fmol/L (10−15 mol/L).
Single-strand conformation polymorphism analysis with high throughput modifications, and its use in mutation detection in familial hypercholesterolemia
Authors: Humphries Steve E , Gudnason Vilmundur , Whittall Ros , Day Ian N M ,   for the International Federation of Clinical Chemistry Scientific Divison: Committee on Molecular Biology Techniques
Published: January 2020

The identification of the specific mutation causing an inherited disease in a patient is the framework for the development of a rationale for therapy and of DNA-based tests for screening relatives. We present here a review of the single-strand conformational polymorphism (SSCP) method, which allows DNA fragments that have been amplified with specific primers and PCR to be scanned rapidly for any sequence variation. The general principles of the method are described, as are the major factors that must be considered in developing an optimal SSCP strategy, namely the length of the PCR fragment and the temperature of the gel run. Options for sample denaturing gel characteristics and detection of DNA fragments are discussed. In addition, several modifications are presented that have been developed for high-throughput mutational analysis. The application of these techniques to screen for mutations in the LDL receptor gene in patients with familial hypercholesterolemia are described.
Reversible myocardial ischemic injury is not associated with increased creatine kinase activity in plasma
Authors: Ishikawa Yuichi , Saffitz Jeffrey E , Mealman Terrence L , Grace Ann M , Roberts Robert
Published: January 2020

Creatine kinase (CK) isoenzymes MM, MB, and BB are located primarily in the cell cytosol, and increased CKMB in plasma is the hallmark of myocardial infarction. However, whether CK is released with reversible ischemic injury remains controversial. Here, we assessed plasma CK activity—cytosolic and mitochondrial CK—in serial samples (every 10 min for 60 min, then hourly or every 4 h for 48 h) from 46 conscious dogs after transient or sustained coronary occlusion. Four dogs were sham-operated (controls); four underwent sustained coronary occlusion (96 h); and 38 underwent transient coronary occlusion (10–40 min) followed by 48 h of reperfusion. In postmortem histological examination of the dogs’ hearts by light and electron microscopy, we looked for ischemia or necrosis. The presence of cell swelling and glycogen depletion was indicative of ischemia, whereas the added presence of cell disruption indicated necrosis. Coronary occlusion for ≥20 min consistently increased plasma mitochondrial and total CK activity and produced histologically evident myocardial necrosis. In contrast, after 10 to 15 min of coronary occlusion, 12 of 14 animals, despite extensive severe reversible ischemia, showed no increase in plasma CK; the remaining 2, which had increased plasma CK, had subendocardial necrosis. Thus, cytosolic or mitochondrial CK is released from the heart only when there has been irreversible myocardial injury—a finding with significant diagnostic and therapeutic implications.
Clinical evaluation of the Byk LIA-mat CA125 II assay: discussion of a reference value
Authors: Bonfrer Johannes M G , Korse Catharina M , Verstraeten Rob A , van Kamp Gerard J , Hart Guus A M , Kenemans Peter
Published: January 2020

The Byk LIA-mat CA125 II assay was compared with the Centocor IRMA CA125 II. Serum samples studied (n = 1012) were obtained from 652 apparently healthy females, 61 pregnant women, and 299 patients with benign and malignant gynecological tumors. The CA125 II assay value at the 95th percentile of the total healthy group was 29 kU/L for the LIA-mat and 32 kU/L for the Centocor assay. For the LIA-mat assay the 95th percentile was 31 kU/L (Centocor 36 kU/L) for the group <45 years and 21 kU/L (Centocor 25 kU/L) for women >55 years of age. By using ROC curves we found the optimal pretreatment Byk LIA-mat CA125 II value differentiating between benign and malignant ovarian tumors to be 95 kU/L. Pretreatment CA125 values >1000 kU/L were detected in serum samples of patients with advanced epithelial ovarian cancer.
Application of isotope-selective nondispersive infrared spectrometry (IRIS) for evaluation of [13C]octanoic acid gastric-emptying breath tests: comparison with isotope ratio–mass spectrometry (IRMS)
Authors: Schadewaldt Peter , Schommartz Bernd , Wienrich Gregor , Brösicke Herbert , Piolot Ralf , Ziegler Dan
Published: January 2020

Suitability of isotope-selective nondispersive infrared spectrometry (IRIS) for evaluation of [13C]octanoic acid gastric-emptying breath test was assessed and compared with standard isotope ratio–mass spectrometry (IRMS). The estimated bias of IRMS and IRIS measurements of baseline-corrected 13CO2exhalation amounted to ± 0.1 and ± 0.6 Δδ values (n = 360), respectively. In breath tests performed on 60 diabetic patients, the gastric emptying parameters were calculated by nonlinear regression analysis of the time course of 13CO2exhalation: half-emptying time (t1/2,breath, 90 ± 39 min), lag phase (tlag,breath, 34 ± 27 min), and gastric emptying coefficient (GEC, 2.9 ± 0.5). A reasonable linear correlation was found between the two methods (y = IRIS, x = IRMS) with respect to Δδ values (y = 0.35 + 0.92x, r = 0.985, Sy|x = ±0.6, n = 1116) and a rather good agreement of the computed gastric emptying parameters was obtained (t1/2,breath: y = 0.99x + 4.06, Sy|x = ±6.3; tlag,breath: y = 0.97x + 0.96, Sy|x = ±3.4; GEC: y = 0.97x − 0.01, Sy|x = ±0.09).
Enzymatic assay of d-mannose in serum
Authors: Etchison James R , Freeze Hudson H
Published: January 2020

We describe a new and improved enzymatic assay for determining the concentration of d-mannose in sera. Serum d-glucose is selectively converted to glucose-6 phosphate with the highly specific thermostable glucokinase (EC 2.7.1.2) from Bacillus stearothermophilus. The anionic reaction products and excess substrates are removed by a rapid and simple anion-exchange chromatography step in microcentrifuge spin columns. d-Mannose in the glucose-depleted sample is then assayed spectrophotometrically by using coupled enzymatic reactions. The quantitative elimination of glucose from the serum samples allowed the accurate and reproducible assay of serum mannose in the 0–200 μmol/L range. Recovery of mannose added to serum (5–200 μmol/L) was 94% ± 4.4%. The intraassay CV was 6.7% at 40 μmol/L mannose (n = 5; 39.6 ± 1.6 μmol/L) and 4.4% at 80 μmol/L (n = 11; 75.0 ± 1.8 μmol/L); the interassay CV at these concentrations was 12.2% (n = 7; 36.9 ± 2.1 μmol/L) and 9.8% (n = 7; 74.2 ± 2.7 μmol/L), respectively. Sera from 11 healthy human volunteers contained an average of 54.1 ± 11.9 μmol/L mannose (range 36–81 μmol/L).
New sandwich ELISA for human urinary N-acetyl-β-d-glucosaminidase isoenzyme B as a useful clinical test
Authors: Numata Yoshito , Morita Atsushi , Kosugi Yoko , Shibata Kazunori , Takeuchi Nozomu , Uchida Kiyohisa
Published: January 2020

We have developed a new ELISA for quantifying N-acetyl-β-d-glucosaminidase (NAG) isoenzyme B in human urine after raising monoclonal antibodies against the isoenzyme from human placenta. Though the obtained antibodies reacted not only to isoenzyme B but also to A, we could detect isoenzyme B selectively by a two-step sandwich ELISA with a pair of selected antibodies at low pH in the first reaction. The detected limit was 0.5 μg/L for a sample volume of 25 μL. Within-run CVs ranged from 2.5% to 5.4% and between-run CVs ranged from 6.2% to 9.1%. Recoveries of NAG isoenzyme B added to each of three urine samples ranged from 91% to 114%. The dilution curves of urine samples showed good linearity. The cross-reactivity of NAG isoenzyme A was practically negligible (2–3%). The mean value for NAG isoenzyme B in spot urines from healthy adults was 2.9 μg/g creatinine. This ELISA method is rapid and precise enough for routine determination of NAG isoenzyme B in human urine.
Quality-control (QC) performance measures and the QC planning process
Authors: Parvin Curtis A
Published: January 2020

Numerous outcome measures can be used to characterize and compare the performance of alternative quality-control (QC) strategies. The performance measure traditionally used in the QC planning process is the probability of rejecting an analytical run when a critical out-of-control error condition exists. Another performance measure that naturally fits within the total allowable error paradigm is the probability that a reported test result contains an analytical error that exceeds the total allowable error specification. In general, the out-of-control error conditions associated with the greatest chance of reporting an unacceptable test result are unrelated to the traditionally defined “critical” error conditions. If the probability of reporting an unacceptable test result is used as the primary performance measure, worst-case QC performance can be determined irrespective of the magnitude of any out-of-control error condition that may exist, thus eliminating the need for the concept of a “critical” out-of-control error.
Quantitative colorimetric determination of urinary p-aminophenol with an automated analyzer
Authors: Van Bocxlaer Jan F , Clauwaert Karine M , Lambert Willy E , De Leenheer André P
Published: January 2020

We developed an automated colorimetric method for the quantitative determination of p-aminophenol with a Cobas Mira analyzer. The procedure can be used for the biological monitoring of human exposure to aniline. An absorbed aniline dose is extensively oxidized to p-aminophenol, which is excreted in urine mainly as glucurono- and sulfo- conjugates. After enzymatic hydrolysis, we reacted the free compound with resorcinol in the presence of manganese ions to form an indophenol dye, which is measured at 550 nm. Excellent accuracy (102.8%, 103.9%, and 96.8% at 2.5, 50, and 90 mg/L, respectively) and precision (7.7%, 2.1%, and 0.8% CV for within-run and 11.1%, 4.7%, and 4.6% for total reproducibility at 2.5, 50, and 90 mg/L, respectively) were achieved over a linear concentration range of 2.0 to 100 mg/L. The detection limit was 0.9 mg/L and no significant interference (except for o-aminophenol) was found for several investigated drugs and related compounds. The proposed method was used for a stability study and to analyze several samples from an occupational health screen.
Interference with testing for lysergic acid diethylamide
Authors: Ritter Detlef , Cortese Cherise M , Edwards Linda C , Barr Judith L , Chung Hyung D , Long Christopher
Published: January 2020

We found a high rate (4.2%) of positive results for lysergic acid diethylamide (LSD) by Emit in 1898 urine samples that were submitted primarily from psychiatric patients for drugs-of-abuse (DOA) testing. Specimens that tested positive for LSD by Emit subsequently tested negative for LSD with two RIAs. Furthermore, LSD was not detected in randomly selected Emit-positive urine samples by gas chromatography–mass spectrometry. Normal urine samples tested positive for LSD by Emit when they were supplemented with therapeutic medications that were prescribed for patients with positive urine LSD results by Emit. These therapeutic drugs interfered specifically with the Emit assay for LSD, since other Emit DOA tests were not affected by these medications at the tested concentrations.
Detection of anabolic steroid administration: ratio of urinary testosterone to epitestosterone vs the ratio of urinary testosterone to luteinizing hormone
Authors: Perry Paul J , MacIndoe John H , Yates William R , Scott Shane D , Holman Timothy L
Published: January 2020

Our goal in this study was to determine whether the urinary ratio of testosterone to luteinizing hormone (T/LH) as an indicator of exogenous anabolic steroid (AS) use is superior to the urinary ratio of testosterone to epitestosterone (T/E). After 2 weekly placebo injections, 19 subjects were given testosterone cypionate (TC) injections of 250 or 500 mg/week for 14 weeks followed by 14 weekly placebo injections. Patients were considered to have ceased taking TC if they tested negative 9 weeks after their last injection. For detection of illicit or supraphysiological TC (AS) use, the urinary T/E ratio of ≥6 yielded a false-negative rate of 46% and a false-positive rate of 4%. However, a urinary T/LH ratio of ≥30 produced a false-negative rate of only 24% and a false-positive rate of 13%. We conclude that the urinary T/LH ratio of ≥30 is a more sensitive marker of AS use than the urinary T/E ratio of ≥6 and remains sensitive for twice as long as urinary T/E.
Microdialysis-HPLC for plasma levodopa and metabolites monitoring in parkinsonian patients
Authors: Dethy Sophie , Laute Marie Aline , Van Blercom Nadège , Damhaut Philippe , Goldman Serge , Hildebrand Jerzy
Published: January 2020

We used in vitro microdialysis-HPLC to determine l-3,4-dihydroxyphenylalanine (l-DOPA) and its metabolites in plasma of patients with advanced Parkinson disease. Blood samples and clinical evaluations were obtained 0, 30, 60, 90, 120, and 150 min after oral administration of carbidopa/l-DOPA (25/100 mg, 12.5/125 mg, and 50/200 mg). In vitro recoveries for l-DOPA and metabolites ranged from 22% to 36%. Linear correlation was found between metabolite concentrations in the dialysate and in the surrounding medium. There was a significant positive correlation between l-DOPA dose and plasma concentration of l-DOPA and homovanillic acid (P <0.04). Clinical response was maximum 60 min after l-DOPA administration. Threshold l-DOPA plasma concentration averaged 7.74 ± 3.3 μmol/L. Motor effect is longer with the highest l-DOPA peak concentration (P <0.01). Microdialysis-HPLC is readily applicable, reproducible, and allows monitoring of plasma l-DOPA and metabolites in parkinsonian patients.
Potential of electrospray mass spectrometry for quantifying glycohemoglobin
Authors: Roberts Norman B , Green Brian N , Morris Michael
Published: January 2020

An electrospray ionization–mass spectrometric procedure has been developed for determining glycohemoglobin. Whole-blood samples from 78 diabetic and 50 nondiabetic subjects (glycation range 3–15%, as determined by electrospray mass spectrometry) were diluted 500-fold in an acidic denaturing solvent and introduced directly into a mass spectrometer. The resulting mass spectra were then processed to estimate the percentage of glycohemoglobin present in the sample. Total analysis time, including plotting the spectra and computing the percentage of glycation, was ∼3 min. The imprecision (CV) of the method was <5.1% for inter- and intrabatch analyses for total glycohemoglobin in the range 3.6–14%. Comparison of the mass spectrometric results with those from established affinity chromatographic procedures showed good overall agreement. The relative glycation of the α- and β-chains was determined directly and was shown to be constant (0.64:1) over the glycation range measured. Only single glucose attachment to both the α- and β-chains was observed.
Accurate and precise isotope dilution mass spectrometry method for determining glucose in whole blood
Authors: Hannestad Ulf , Lundblad Arne
Published: January 2020

An accurate and precise method to determine glucose concentration in whole blood is presented. The method, based on isotope dilution gas chromatography–mass spectrometry (ID GC-MS), was developed to be used as a Reference Method for determining glucose concentration in capillary or venous whole blood. Blood samples and standards are pipetted manually with “microcap” micropipettes, which makes it possible to collect samples even at the patient’s bedside. Glucose is quantified as its aldononitrile pentaacetate. [13C6]Glucose is used as an internal standard. Assay of Seronorm and Pathonorm L and H controls by ID GC-MS gave within-run CVs of 0.66%, 0.96%, and 0.92%, respectively. For whole blood with glucose concentrations in the low, normal, and high ranges, the within-run CVs were 1.27%, 0.91%, and 0.78%, respectively. The between-run CV for glucose calculated from 36 separate single analyses of Seronorm was 1.44%. In an accuracy assessment test of the HemoCue blood glucose analyzer, 140 capillary blood samples were measured in parallel after split-sampling. For all samples the HemoCue analyzer results had a mean bias of +2.0% compared with the ID GC-MS results.
Modified and improved anti-acetylcholine receptor (AChR) antibody assay: comparison of analytical and clinical performance with conventional anti-AChR antibody assay
Authors: Ferrero Bruno , Aimo Giuseppe , Pagni Roberto , Bergamasco Bruno , Bongioanni Maria R , Bergamini Lodovico , Durelli Luca
Published: January 2020

We developed a modified anti-acetylcholine receptor (AChR) antibody (Ab) assay based on a radioreceptor assay and a calibration curve. We compared the analytical and clinical performances of this modified assay with those of the conventional anti-AChR Ab radioreceptor assay. Serum specimens were from patients with myasthenia gravis (MG) (n = 156) and from control subjects (n = 106). The modified assay demonstrated lower within-assay (4.0–6.6%) and between-assay (5.3–7.8%) CVs, greater linearity, lower cost, and shorter assay time than the conventional method. ROC curve analysis indicated almost identical specificity and sensitivity (>0.92) for these two anti-AChR Ab assays. The modified and conventional assays were also equivalent for blocking anti-AChR Ab assay. Moreover, the modified anti-AChR Ab assay, differently from the conventional assay, allowed us to reveal anti-AChR Ab concentration differences among different clinical grades of MG.
Determination of d-lactate by enzymatic methods in biological fluids: study of interferences
Authors: Martí Ramon , Varela Encarna , Segura Rosa M , Alegre José , Suriñach José M , Pascual Carles
Published: January 2020

Analysis of nondeproteinized samples with an enzymatic method to determine d-lactate indicated interferences. The presence of l-lactate dehydrogenase (LD) and l-lactate in the sample led to underestimation of d-lactate content when a sample blank was processed and overestimation when it was omitted. We proved that this interference is not due to lack of d-LD stereospecificity. Moreover, assessment of d-LD and l-LD KM for NAD+ allowed us to rule out the different affinities for this coenzyme as a cause of the interference. Our results underline the importance of deproteinizing samples for d-lactate analysis when enzymatic methods are used. The ultrafiltration procedure we propose is convenient and shows acceptable mean recovery (108%) and good imprecision (within-run CV = 4.2% and 3.0% for d-lactate at 31 and 107 μmol/L, respectively; between-run CVs were 7.3% at 49 μmol/L d-lactate and 3.1% at 115 μmol/L d-lactate).
Free triiodothyronine concentration in serum of 1050 euthyroid children is inversely related to their age
Authors: Verheecke Paul
Published: January 2020

Free triiodothyronine was assayed in serum of 1050 euthyroid children with the Amerlex-MAB method. We studied 50 samples for each year of age, from <1 to 20, and no child had more than one sample taken. A gradient of values was observed, increasing by 31% from 5.04 pmol/L as the mean of 20-year-old patients to 6.59 pmol/L in the serum of children younger than 1 year. For practical reasons the lower normal limit was proposed to remain at 3.4 pmol/L for all patients; the higher limit can be set at 8.3 (0 to 3 years), 7.8 (4 to 7 years), 7.1 (8 to 11 years), 6.8 (12 to 15 years), and 6.7 (16 to 19 years). Preferably, the regression line of the mean + 2 SD can be used, resulting in a high normal limit of 8.3 pmol/L at birth to 6.3 pmol/L at 20 years, decreasing by 0.1 pmol/L per year.
Evaluation of the Menarini–Arkray HA 8140 hemoglobin A1c analyzer
Authors: John W Garry , Braconnier Francois , Miedema Kor , Aulesa Carlos , Piras Giampiero
Published: January 2020

We describe a multinational evaluation of the Menarini– Arkray HA 8140 hemoglobin (Hb) A1canalyzer, which utilizes a high degree of automation, including bar code reading, cap piercing, and whole-blood sampling. Within- and between-batch CVs were <2%. Linearity was confirmed throughout the working range of the analyzer. Common Hb variants, including Hb S, Hb C, and Hb F, did not interfere with the Hb A1c separation, and the potentially interfering labile Schiff base was effectively removed during the chromatographic procedure. The HA 8140 analyzer displayed good correlation to the Bio-Rad Variant analyzer, Tinaquant immunoassay, affinity chromatography, and an optimized “in-house” HPLC Hb A1c method. The methods when compared by Altman and Bland plots showed bias (upper, lower 95% confidence limits) of: Variant minus HA 8140 = 0.99 (0.23, 1.74), Tinaquant minus HA 8140 = 0.14 (−0.71, 0.98); affinity minus HA 8140 (after log transformation) = 1.13 (0.90, 1.41), and “in house” HPLC minus HA 8140 (after log transformation) = 0.91 (0.82, 1.01).
Direct colorimetric monoclonal antibody enzyme immunoassay for estradiol-17β in saliva
Authors: Tamate Kenichi , Charleton Margaret , Gosling James P , Egan Declan , Ishikawa Mutsuo , Fottrell Patrick F , Kane Marian M
Published: January 2020

We developed a direct microtiter plate enzyme immunoassay to measure estradiol-17β in saliva. The assay has a commercially available monoclonal antibody, raised against estradiol-17β–6-carboxymethyloxime–bovine serum albumin, and a homologous horseradish peroxidase conjugate measured colorimetrically. The detection limit (equivalent to B0 − 3 SD) is 365 amol/well or 7.3 pmol/L when 50-μL samples are assayed. Cross-reactivity with estrone and estriol, testosterone, or progesterone is <0.2%. Estradiol-17β was measured in daily samples over five natural menstrual cycles and eight cycles stimulated as a preliminary to in vitro fertilization, and the concentrations and fluctuations found agreed with previously published data. This method gives results in ∼3 h and may be useful for fertility monitoring and management.
Serum enzymes in heat stroke: prognostic implication
Authors: Alzeer Abdulaziz H , El-Hazmi Mohsen A F , Warsy Arjumand S , Ansari Ziauddin A , Yrkendi Mohammed S
Published: January 2020

We measured serum creatine kinase (CK), lactate dehydrogenase (LD), aspartate aminotransferase (AST), and serum alanine aminotransferase (ALT) in 26 heat stroke (HS) victims and 10 control (non-heat-exhausted) subjects during annual Hajj in Makkah, Saudi Arabia. On admission to the HS treatment unit, serum CK, AST, ALT, and LD were higher in HS victims than controls (P <0.05), and at 6, 12, and 24 h were higher than baseline concentration. The patient group was divided into three groups, (a) those who had a quick recovery, (b) those who were critically ill until the end of the Hajj period (7 days), and (c) those who died. Serum enzymes at the time of admission were significantly higher (P <0.05) in the nonsurviving group (n = 6) and the severely ill (n = 9) than in those who had a quick recovery (n = 11). ROC curves were plotted for each enzyme. The most useful indicator was LD, as it could distinguish significantly between the groups who died and those who had a quick recovery (area under the curve = 0.991 ± 0.0286). It was followed by CK and AST as useful prognostic factors. When compared with ROC curves for body temperature, anion gap, and serum potassium, the enzyme results were superior prognostic indicators.
Plasma protein abnormalities in nephrotic syndrome: effect on plasma colloid osmotic pressure and viscosity
Authors: Joven Jorge , Clivillé Xavier , Camps Jordi , Espinel Eugenia , Simó Jose , Vilella Elisabet , Oliver Angel
Published: January 2020

The concentrations of 25 plasma proteins were measured in 22 patients with membranous nephropathy. For some large proteins, the plasma concentrations were increased; there were also large proteins with low plasma concentrations, but small or medium-sized proteins showed uniformly lower plasma concentration than the controls. Plasma colloid osmotic pressure (π) and viscosity (η) were not interrelated but showed positive and significant correlations with plasma concentrations of small and medium-sized proteins (π) and plasma concentrations of large proteins (η), respectively. Nephrotic plasma is not efficient in maintaining plasma π but highly efficient in maintaining plasma η. High plasma fibrinogen concentrations and low antithrombin III concentrations may predispose to thrombosis, and low IgG concentrations may account for the higher predisposition to bacterial infection. The relative composition of nephrotic plasma is heavily dependent on the size of the different proteins. Plasma π and η are also maintained by the relative preponderance of different plasma proteins.
Hormonal doping and androgenization of athletes: a secret program of the German Democratic Republic government
Authors: Franke Werner W , Berendonk Brigitte
Published: January 2020

Several classified documents saved after the collapse of the German Democratic Republic (GDR) in 1990 describe the promotion by the government of the use of drugs, notably androgenic steroids, in high-performance sports (doping). Top-secret doctoral theses, scientific reports, progress reports of grants, proceedings from symposia of experts, and reports of physicians and scientists who served as unofficial collaborators for the Ministry for State Security (“Stasi”) reveal that from 1966 on, hundreds of physicians and scientists, including top-ranking professors, performed doping research and administered prescription drugs as well as unapproved experimental drug preparations. Several thousand athletes were treated with androgens every year, including minors of each sex. Special emphasis was placed on administering androgens to women and adolescent girls because this practice proved to be particularly effective for sports performance. Damaging side effects were recorded, some of which required surgical or medical intervention. In addition, several prominent scientists and sports physicians of the GDR contributed to the development of methods of drug administration that would evade detection by international doping controls.
Analytical advances in detection of performance-enhancing compounds
Authors: Bowers Larry D
Published: January 2020

The use and abuse of performance-enhancing substances has been an issue in sports since the ancient Greeks. The availability of numerous synthetic steroids and recombinant peptide hormones has made testing an analytical challenge. Recent advances in mass spectrometry have provided an opportunity to decrease detection limits. The Atlanta Olympic Games in 1996 marked the first time every specimen was screened by gas chromatography (GC) coupled to high-resolution mass spectrometry (MS). A further improvement may be seen with GC/MS/MS and quadrupole ion traps. Electrospray HPLC/MS has also been applied to the detection and confirmation of peptide hormones in urine. The ability to detect subtle differences in oligosaccharide structure may provide a way to detect abuse of recombinant glycoproteins. Simply decreasing detection limits is not enough; new technology also allows development of a foundation on which to base interpretation. Application of HPLC/MS/MS has allowed direct measurement of steroid conjugates in urine. The relative importance of sulfate, glucuronide, and other conjugates and metabolites of testosterone and epitestosterone can now be assessed. In the international sports arena, the impact of genetic metabolic disposition must also be considered if we are to provide an equitable system. Further research will establish more-refined criteria for the detection threshold of abused substances.
A homogeneous method for genotyping with fluorescence polarization
Authors: Gibson Neil J , Gillard Helen L , Whitcombe David , Ferrie Richard M , Newton Clive R , Little Stephen
Published: January 2020

We combined the amplification refractory mutation system (ARMS™) and fluorescence polarization (FP) to give a homogeneous genomic DNA genotype analysis method. Oligonucleotide probes labeled with the fluorescein dyes fluorescein isothiocyanate and 5-([4,6-dichlorotriazin-2-yl]amino)fluorescein and the rhodamine dye 6-carboxyrhodamine were included in amplification mixes and were annealed to PCR products after amplification. Hybridization was accompanied by an increase in the FP of the probe. We demonstrated homogeneous genotyping by analyzing human DNA samples for ΔF508 mutation status of the cystic fibrosis transmembrane conductance regulator gene. The genotypes determined with the method described herein were in full agreement with those obtained by the conventional application of ARMS. We also demonstrated the simultaneous detection of two PCR products in a single reaction. The assay method described is homogeneous and so obviates the necessity to open reaction vessels after amplification. This therefore eliminates PCR carryover contamination.
Increased alkaline phosphatase isoforms in autoimmune diseases
Authors: Hanna Atef N , Waldman W James , Lott John A , Koesters Stephen C , Hughes Anna M , Thornton David J
Published: January 2020

We found significant increases in ALP and ALP isoform band 10 in the serum of patients with early insulin-dependent diabetes, rheumatoid arthritis, and in those with multiple sclerosis during periods of disease exacerbation as compared with healthy controls. The ALP isoforms were assayed by isoelectric focusing. Our data suggest that the increase in ALP and ALP-10 closely reflects the abnormal activation of T lymphocytes that is common in autoimmune diseases, and that the source of the ALP-10 is activated T lymphocytes. ALP-10 is a sensitive but nonspecific marker of an active autoimmune process and appears to have the ability to detect abnormal T-cell activation. ALP-10 may be a useful test in the screening for autoimmune disorders.
Troponin I is released in bloodstream of patients with acute myocardial infarction not in free form but as complex
Authors: Katrukha Aleksei G , Bereznikova Anastasia V , Esakova Tatiana V , Pettersson Kim , Lövgren Timo , Severina Maria E , Pulkki Kari , Vuopio-Pulkki Liisa-Maria , Gusev Nikolai B
Published: January 2020

Fourteen monoclonal antibodies (mAbs) against human cardiac troponin I (cTnI) were generated by commonly used experimental techniques. All these antibodies, as well as antibody 414 (HyTest), were specific for human cTnI. Fifteen antibodies thus obtained were tested in a sandwich cTnI immunofluorescence assay (altogether 196 combinations). Ten pairs giving the highest sensitivity were selected for further investigation. The effect of TnI–TnC complex formation on antibody interaction with antigen was analyzed. The formation of TnI–TnC complex results in a significant decrease of the interaction of mAbs with TnI for seven of 10 analyzed pairs of antibodies. Using two pairs of cTnI-specific mAbs, one that recognized only free cTnI but not cTnI complexed with cTnC, and another that could be used for measurement of total cTnI (free cTnI and cTnI in complex with cTnC), we demonstrated that the main part of cTnI in serum collected from acute myocardial infarction patients is presented in the complex form. We concluded that effective and reliable immunological detection of TnI is possible only when antibodies used for assay development recognize both free TnI and TnI complexed with other troponin components.
Binding of nitric oxide to thiols and hemes in hemoglobin H: implications for α-thalassemia and hypertension
Authors: Reddy Poluru L , Bowie Lemuel J , Callistein Steven
Published: January 2020

Our earlier studies suggested an association between α-thalassemia and hypertension. We postulated that this association might involve trapping of the vasodilator nitric oxide (NO) by hemoglobin (Hb). Hb A has recently been shown to carry NO on its sulfhydryl groups in addition to its hemes. In this report we studied the interaction of purified Hb H as well as Hb A with NO. The number of reactive sulfhydryls were determined spectrophotometrically with bis-dithionitrobenzoate. Spectral studies and nitrosothiol measurements after treatment with NO or nitrosothiols indicated that all eight reactive sulfhydryls of Hb H were capable of binding NO. Hb A, however, was only able to bind and transfer two molecules of NO per tetramer. These findings support the biochemical basis for the association between α-thalassemia and hypertension.
Type A viral hepatitis: epidemiology, diagnosis, and prevention
Authors: Lemon Stanley M
Published: January 2020

Hepatitis A virus (HAV) infection occurs worldwide and is an important cause of acute viral hepatitis in the US. In this review, I cover the epidemiology, course of infection, clinical manifestations, serological responses, and prevention of this infection. Although most patients completely recover from this disease, elderly patients have a substantial mortality risk. Recently licensed vaccines are highly efficacious.
Mild liver enzyme abnormalities: eliminating hemochromatosis as cause
Authors: Witte David L
Published: January 2020

Chronic mild liver enzyme abnormalities are attributable to hereditary hemochromatosis in at least 3% of cases. Hemochromatosis formerly was diagnosed late with diabetes and hepatic and cardiac failure. Only recently have the autosomal recessive inheritance and subtle early presentations been understood. However, patients still wait many years and see many physicians before receiving a correct diagnosis. Increased serum transferrin saturation is currently the best test for detection of those likely to accumulate iron. Serum ferritin identifies those requiring treatment. When liver biopsy (controversial in asymptomatic individuals) is indicated, chemical measurement of liver iron content is helpful and therapeutic phlebotomy is the only effective treatment. Caucasian-type hemochromatosis (prevalence of 0.005) is associated with genetic abnormalities in HLA-H but also occurs in other ethnic groups. Those of African descent may have a different but also heritable iron-loading disease. Caucasian-type and to a lesser extent African iron loading are detectable early by laboratory testing. Early treatment restores normal expectations of length and quality of life in the Caucasian disease. Long-term treatment data are not yet available in African iron loading. Laboratory-initiated screening programs using unsaturated iron-binding capacity can eliminate symptomatic hemochromatosis.
Comparison of prostate-specific antigen (PSA) measured by four combinations of free PSA and total PSA assays
Authors: Junker Ralf , Brandt Burkhard , Zechel Christian , Assmann Gerd
Published: January 2020

We compared prostate-specific antigen (PSA) assay systems [i.e., free PSA (f-PSA) and the corresponding total PSA (t-PSA) assay] from four different manufacturers as well as the f-PSA/t-PSA ratios with regard to their ability to discriminate between benign prostate hyperplasia (BPH) and prostate cancer (PCA). ROC analysis showed similar areas under the curves (AUCs) with different assay systems. For the entire patient population the AUCs of the f-PSA/t-PSA ratio were not or slightly increased compared with the sole measurement of t-PSA (t-PSA, 0.792–0.820; f-PSA/t-PSA ratio, 0.685–0.859). In contrast, for only those patients who showed t-PSA concentrations within the diagnostic gray area of 4–25 μg/L t-PSA, the AUCs were greater for the f-PSA/t-PSA ratio than for measurement of t-PSA alone (t-PSA, 0.608–0.647; f-PSA/t-PSA ratio, 0.690–0.806). These results were confirmed by the predictive values of the negative results (NPVs) of the t-PSA assays and the f-PSA/t-PSA ratios (assay thresholds corresponding to a 95% detection limit). Compared with the sole t-PSA measurement there was no mentionable increase in the NPVs due to the f-PSA/t-PSA ratio for the entire patient population, but an increase up to 49% when limited to t-PSA concentrations within 4–25 μg/L. We therefore conclude that the f-PSA/t-PSA ratio may be helpful for differential diagnosis of BPH and PCA within the diagnostic gray area of 4–25 μg/L t-PSA.
Quality control for qualitative assays: quantitative QC procedure designed to assure analytical quality required for an ELISA of hepatitis B surface antigen
Authors: Green George A , Carey R Neill , Westgard James O , Carten Tami , Shablesky LeeAnn , Achord Daniel , Page Eileen , Van Le Anh
Published: January 2020

An assay for hepatitis B surface antigen (HBsAg) should reliably detect 0.2 μg/L, the lowest reported concentration in an asymptomatic blood donor. The difference between this concentration and the assay cutoff defines the analytical quality requirement in a total error format. The design of a statistical QC procedure is critically dependent on the precision of the assay. The precision of a developmental ELISA of HBsAg under study ranged from 17.5% to 9.6% for controls containing 0.07 to 1.50 μg/L, respectively. Use of one positive control with the 13s QC rule provided an 85% chance of detecting a critical loss of assay sensitivity; use of two positive controls increased the chance of detecting critical loss of assay sensitivity to nearly 100%. These rules are based on the precision of this developmental assay, and must be developed individually for other assays. The development of the proposed QC procedures illustrates how quantitative QC can be provided for qualitative assays.
Factor V Leiden and other coagulation factor mutations affecting thrombotic risk
Authors: Bertina Rogier M
Published: January 2020

Five genetic defects have been established as risk factors for venous thrombosis. Three are protein C, protein S, and antithrombin deficiencies, defects in the anticoagulant pathways of blood coagulation. Together they can be found in ∼15% of families with inherited thrombophilia. Their laboratory diagnosis is hampered by the large genetic heterogeneity of these defects. The other two genetic risk factors, resistance to activated protein C associated with the factor V Leiden mutation and increased prothrombin associated with the prothrombin 20210 A allele, are much more prevalent and together can be found in 63% of the thrombophilia families. Because both defects are caused by a single mutation, DNA analysis is the basis of their laboratory diagnosis.
Hartman interferometer: versatile integrated optic sensor for label-free, real-time quantification of nucleic acids, proteins, and pathogens
Authors: Schneider Bernard H , Edwards John G , Hartman Nile F
Published: January 2020

The Hartman interferometer, a proprietary integrated optic sensor, provides a basis for a broad range of biomedical diagnostics, including antibody-based and gene probe-based assays. As with other evanescent-wave optical sensors, the interferometer measures the refractive index change resulting from biomolecular binding on a waveguide surface. The exciting promise of evanescent-wave sensors lies, in general, in their potential to be used as label-free, real-time transducers that can operate in a true mix-and-read fashion and provide fast, quantitative results. One of the major issues facing their development, however, is creating a simple, low-cost configuration for multianalyte testing. The Hartman interferometer addresses this challenge by relying on linearly polarized light and a planar waveguide format, thereby avoiding the problems associatedwith circular polarization and channel waveguides. We report preliminary experiments that demonstrate the applicability of this sensor configuration to detection of a wide range of protein, nucleic acid, and pathogen analytes.
Fundamentals of quality assessment of molecular amplification methods in clinical diagnostics
Authors: Neumaier Michael , Braun Andreas , Wagener Christoph ,   for the International Federation of Clinical Chemistry Scientific Division Committee on Molecular, Techniques Biology
Published: January 2020

The increasing interest in molecular biology diagnostics is a result of the tremendous gain of scientific knowledge in genetics, made possible especially since the introduction of amplification techniques. High expectations have been placed on genetic testing, and the number of laboratories now using the relevant technology is rapidly increasing—resulting in an obvious need for standardization and definition of laboratory organization. This communication is an effort towards that end. We address aspects that should be considered when structuring a new molecular diagnostic laboratory, and we discuss individual preanalytical and analytical procedures, from sampling to evaluation of assay results. In addition, different means of controlling contamination are discussed. Because the methodology is in constant change, no general standards can be defined. Accordingly, this publication is intended to serve as a recommendation for good laboratory practice and internal quality control and as a guide to troubleshooting, primarily in amplification techniques.
Study of arsenic–protein binding in serum of patients on continuous ambulatory peritoneal dialysis
Authors: Zhang Xinrong , Cornelis Rita , De Kimpe Jurgen , Mees Louis , Lameire Norbert
Published: January 2020

Arsenic (As) bound to serum proteins in patients on continuous ambulatory peritoneal dialysis (CAPD) was studied. A prior experiment by ultrafiltration showed that 5.57% of total As was bound to serum proteins for 14 CAPD patients. Further identification of the As species and protein molecules in serum of three CAPD patients with high As concentrations was carried out by combining the separation methods of size-exclusion, anion-exchange, and affinity fast-protein liquid chromatography, detected by hydride generation atomic absorption spectrometry. The results indicated that only inorganic As species are bound to serum proteins. Transferrin is the main carrier. The concentrations of As bound to proteins in serum for the three patients were 0.44 ± 0.12, 0.19 ± 0.09, and 0.59 ± 0.09 μg/L (n = 3), respectively.