bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased oxidative stress in dilated cardiomyopathic heart failure
Authors: Yücel Doğan , Aydoğdu Sinan , Çehreli Sengül , Saydam Gülsevim , Canatan Hayrettin , Şeneş Mehmet , Topkaya Birsen Çiğdem , Nebioğlu Serpil
Published: January 2020

In the present study, we assessed oxidative stress in patients with dilated cardiomyopathy of ischemic or idiopathic etiology. For this reason we measured whole blood reduced glutathione, erythrocyte superoxide dismutase, susceptibility of erythrocyte membranes and erythrocytes to peroxidation, and SH content of erythrocyte membranes in 12 patients (8 men and 4 women, ages 31 to 66 years) with idiopathic dilated cardiomyopathy, in 11 patients (8 men and 3 women, ages 32 to 65 years) with ischemic dilated cardiomyopathy, and in 21 healthy volunteers (12 men and 9 women, ages 25 to 67 years). There was no statistically significant difference between the two patient groups for the indicators studied (P >0.05). Blood glutathione, erythrocyte superoxide dismutase, and membrane SH content of both groups of patients was decreased compared with controls (P <0.05), whereas erythrocyte and membrane susceptibility to peroxidation were increased (P <0.05). We conclude that patients with idiopathic or ischemic dilated cardiomyopathy exhibit abnormalities of a range of markers of increased oxidative stress. These abnormalities may contribute to contractile dysfunction, increased incidence of fatal arrhythmias, and sudden death.
Neopterin concentrations in cerebrospinal fluid and serum as an aid in differentiating central nervous system and peripheral infections in children
Authors: Millner Michael M , Franthal Wolfgang , Thalhammer Gabriela H , Berghold Andrea , Aigner Reingard M , Füger Gerhard F , Reibnegger Gilbert
Published: January 2020

Neopterin is a sensitive indicator for cellular immune activation. Its concentrations were determined in cerebrospinal fluid (CSF) and serum specimens from 91 children with no evidence of central nervous system (CNS) or peripheral inflammations, 43 with definite neuroborreliosis, 51 with other CNS infections, and 33 with peripheral infections. The aim of our study was (a) to establish a range of normal CSF neopterin concentrations in control children, and (b) to inquire into the diagnostic potential of neopterin measurements in both body compartments for aiding in differential diagnosis of inflammatory vs noninflammatory diseases, and CNS vs peripheral inflammations. CSF neopterin concentrations in controls were invariably low (up to 9.3 nmol/L), but in children with neuroborreliosis and, even more so, with other CNS infections neopterin concentrations were significantly (P <0.0001) increased. Children with peripheral infections, however, rarely showed raised CSF neopterin concentrations. Serum concentrations of neopterin, on the other hand, were not significantly different between controls and children with neuroborreliosis. Although serum concentrations were significantly different between controls and children with other CNS infections, diagnostic efficiency was poor for this comparison. Peripheral infections, in contrast, were associated with significantly higher (P <0.0001) serum neopterin concentrations when compared with controls. A classification tree was constructed on the basis of CSF and serum neopterin concentrations, allowing with high accuracy the discrimination between controls, children with CNS infections, and children with peripheral infections. Thus, on the basis of a comparatively large control group, our data underline the diagnostic validity of neopterin as an aid in differential diagnosis of inflammatory vs noninflammatory diseases, and confirm that CSF neopterin concentrations are not correlated with serum neopterin concentrations, and, therefore, CSF neopterin appears to be produced intrathecally.
Immunoradiometric assay of succinylated corticotropin: an improved method for quantification of ACTH
Authors: Kertesz Gilles , Bourcier Béatrice , Cailla Hélène , Jean Frédéric
Published: January 2020

In this paper we describe the development and the evaluation of a new type of immunoassay for human corticotropin (ACTH). We succeeded, by using an original approach based upon immunization with ACTH derivatized with succinic anhydride, in raising monoclonal antibodies against this poorly immunogenic peptide. Three of the antibodies were selected to develop an immunoassay for ACTH. The assay requires the prior succinylation of the plasma samples for optimal sensitivity and specificity. This acylation treatment is fast, reproducible, and, in addition, improves the stability of the ACTH molecule in plasma, thus facilitating sample handling. The assay is performed in only 3 h with a detection limit of 0.7 ng/L. Analytical evaluation showed excellent specificity, reproducibility, and reliability. A comparison with two commonly used but time-consuming ACTH IRMAs was carried out by assaying several plasma samples in parallel and gave in both cases very good correlation.
Evaluation of the Sysmex UF-100 automated urinalysis analyzer
Authors: Ben-Ezra Jonathan , Bork Linda , McPherson Richard A
Published: January 2020

Urinalysis is a high-volume procedure that currently requires significant labor to examine microscopic sediment. We evaluated the Sysmex UF-100 automated urinalysis analyzer for performing this task. Instrument accuracy was assessed by comparing continuous counts of microscopic elements from the UF-100 with ranges of cells (per low-power field or high-power field) from manual microscopy performed on centrifuged urines. Counts showed good agreement between methods (gamma statistic: 0.880–0.970) for all microscopic elements in 252 urine samples. Within-run imprecision of cell counts expressed as CV (mean cell count/μL) was for erythrocytes (RBC) 31% (5), 18% (50), 2.4% (800); for leukocytes (WBC) 14% (10), 11% (100), 8.5% (400); for squamous epithelial cells (SEC) 18% (5), 12% (30), 7.0% (100); for casts 45% (1), 17% (4); for bacteria 2–12% (entire range of 40–2500). Between-run imprecision on quality-control cell suspensions expressed as CV (mean cell count/μL) was for RBC 6.1% (50), 2.7% (256); for WBC 26.9% (54), 4.9% (228). Cells counted on dilution were 99.1% of expected for RBC, 102.0% for WBC, and 121.8% for bacteria. Carryover was <0.04% for RBC, <0.03% for WBC, <0.14% for SEC, <0.29% for bacteria. We conclude that the UF-100 can automatically perform reliable quantitative microscopic urinalysis in batches without operator interaction.
Rapid, quantitative nonisotopic assay for telomerase activity in human tumors
Authors: Gelmini Stefania , Caldini Anna , Becherini Lucia , Capaccioli Sergio , Pazzagli Mario , Orlando Claudio
Published: January 2020

Telomerase is a ribonucleoprotein enzyme that adds TTAGGG repeats onto human telomeres, preventing their shortening. The activation of this enzyme is an important step in cell immortalization and carcinogenesis and seems to represent a new and promising marker in cancer diagnosis and management. Telomerase activity is usually detected in cellular protein extract by the telomeric repeat amplification protocol (TRAP) assay, which can provide only a qualitative (presence/absence) evaluation. Here we present a modification of this method that can provide quantitative information without requiring time-consuming post-PCR procedures such as gel electrophoresis with radioactive materials and autoradiography. The detection and measurement of telomerase activity is performed by evaluating the amount of double-stranded DNA generated in the telomerase reaction and PCR amplification, with the use of the sensitive DNA fluorescent dye PicoGreen®. In a subset of tumors, the presence of telomerase activity was confirmed by the conventional TRAP assay. By this method we evaluated telomerase activity in unselected groups of breast (n = 15), ovarian (n = 12), endometrial (n = 12), gastric (n = 20), and renal (n = 12) carcinomas, in meningiomas (n = 8), and in pheochromocitomas (n = 10). The results indicate substantial differences of telomerase activity among cancer groups; however, a large variability among patients of the same group is observed. Kidney, ovarian, and breast carcinomas showed the highest mean values (31.8 ± 28.9, 29.2 ± 26.7, and 35.3 ± 15.9 ng DNA/μg protein, respectively, mean ± SD), whereas gastric and endometrial cancers had a lower activity (17.2 ± 8.8 and 13.5 ± 7.9 ng DNA/μg protein, respectively). Very low or no detectable telomerase activity was found in meningiomas (with the exception of one malignant atypical variant) and pheochromocitomas (9.7 ± 12.9 and 2.8 ± 2.1 ng DNA/μg protein, respectively). In conclusion, our method seems to be an accurate and reasonable procedure for measuring telomerase activity in human cancers.
Rapid pathogen detection using a microchip PCR array instrument
Authors: Belgrader Phillip , Benett William , Hadley Dean , Long Gary , Mariella Raymond , Milanovich Fred , Nasarabadi Shanavaz , Nelson William , Richards James , Stratton Paul
Published: January 2020

An array of PCR microchips for rapid, parallel testing of samples for pathogenic microbes is described. The instrument, called the Advanced Nucleic Acid Analyzer (ANAA), utilizes 10 silicon reaction chambers with thin-film resistive heaters and solid-state optics. Features of the system include efficient heating and real-time monitoring, low power requirements for battery operation, and no moving parts for reliability and ruggedness. We analyzed cultures of Erwinia herbicola vegetative cells, Bacillus subtilis spores, and MS2 virions, which simulated pathogenic microbes such as Yersinia pestis, Bacillus anthracis spores, and Venezuelan equine encephalitis, respectively. Detection of microbes was achieved in as little as 16 min with detection limits of 105–107 organisms/L (102–104 organisms/mL).
HPLC detection of fetal blood in meconium: improved sensitivity compared with qualitative methods
Authors: Chen Dan , Wilhite Timothy R , Smith Carl H , Blinder Morey A , Landt Michael
Published: January 2020

We describe an HPLC-based method for the detection and quantification of fetal hemoglobin in stools of newborns. The new procedure is an alternative to the classic qualitative test for adult hemoglobin in meconium based on the differential stability of hemoglobin species in dilute base (Apt test). The HPLC method, based on a commercial device for hemoglobin characterization (Bio-Rad Variant), readily separates fetal and adult hemoglobin from non-hemoglobin components of meconium. To validate the method, blood and meconium were mixed in various proportions and then prepared for analysis with extraction in saline. The HPLC method accurately identified hemoglobin species even when the blood constituted only 5 mL per 100 g of the meconium specimen, and nearly quantitative recovery of hemoglobin was obtained at a blood content of 20 mL per 100 g of the meconium. Analysis time was 6.5 min, and preparation of sample was simple. HPLC detection of fetal blood in stools or other specimens markedly improves detection/characterization of blood in meconium.
Validity of linear regression in method comparison studies: is it limited by the statistical model or the quality of the analytical input data?
Authors: Stöckl Dietmar , Dewitte Katy , Thienpont Linda M
Published: January 2020

We compared the application of ordinary linear regression, Deming regression, standardized principal component analysis, and Passing–Bablok regression to real-life method comparison studies to investigate whether the statistical model of regression or the analytical input data have more influence on the validity of the regression estimates. We took measurements of serum potassium as an example for comparisons that cover a narrow data range and measurements of serum estradiol-17β as an example for comparisons that cover a wide data range. We demonstrate that, in practice, it is not the statistical model but the quality of the analytical input data that is crucial for interpretation of method comparison studies. We show the usefulness of ordinary linear regression, in particular, because it gives a better estimate of the standard deviation of the residuals than the other procedures. The latter is important for distinguishing whether the observed spread across the regression line is caused by the analytical imprecision alone or whether sample-related effects also contribute. We further demonstrate the usefulness of linear correlation analysis as a first screening test for the validity of linear regression data. When ordinary linear regression (in combination with correlation analysis) gives poor estimates, we recommend investigating the analytical reason for the poor performance instead of assuming that other linear regression procedures add substantial value to the interpretation of the study. This investigation should address whether (a) the x and y data are linearly related; (b) the total analytical imprecision (sa,tot) is responsible for the poor correlation; (c) sample-related effects are present (standard deviation of the residuals ≫ sa,tot); (d) the samples are adequately distributed over the investigated range; and (e) the number of samples used for the comparison is adequate.
Simplified interpretative format for assessing test interference: studies with hemoglobin-based oxygen carrier solutions
Authors: Kazmierczak Steven C , Catrou Paul G , Boudreau Donald
Published: January 2020

Substances such as hemoglobin that interfere with analytical processes are recognized as a frequent source of error in laboratory medicine. Standard guidelines for assessment of test interferences assume that interference effects are not related to the concentration of the analyte being measured. However, previous investigations have demonstrated that interference effects can be markedly different, depending on the concentrations of interferent and analyte within the specimen. An experimental protocol for investigating these different types of interference effects has been developed. This protocol utilizes an orthogonally arranged matrix with progressively increasing concentrations of analyte and interferent. Evaluation of the measured analyte concentrations in specimens within the matrix using multiple regression analysis allows the magnitude, direction, and significance of each type of interference to be determined. Unfortunately, implementation of the interference data derived from the multiple regression analysis for judging the clinical acceptability of test results when an interferent is present is difficult. We describe a two-dimensional graphical format for evaluating the clinical acceptability of test results, based on criteria established under the Clinical Laboratory Improvement Amendments of 1988, in specimens containing hemoglobin-based oxygen carrier solutions.
Regression-based reference limits: determination of sufficient sample size
Authors: Virtanen Arja , Kairisto Veli , Uusipaikka Esa
Published: January 2020

Regression analysis is the method of choice for the production of covariate-dependent reference limits. There are currently no recommendations on what sample size should be used when regression-based reference limits and confidence intervals are calculated. In this study we used Monte Carlo simulation to study a reference sample group of 374 age-dependent hemoglobin values. From this sample, 5000 random subsamples, with replacement, were constructed with 10–220 observations per sample. Regression analysis was used to estimate age-dependent 95% reference intervals for hemoglobin concentrations and erythrocyte counts. The maximum difference between mean values of the root mean square error and original values for hemoglobin was 0.05 g/L when the sample size was ≥60. The parameter estimators and width of reference intervals changed negligibly from the values calculated from the original sample regardless of what sample size was used. SDs and CVs for these factors changed rapidly up to a sample size of 30; after that changes were smaller. The largest and smallest absolute differences in root mean square error and width of reference interval between sample values and values calculated from the original sample were also evaluated. As expected, differences were largest in small sample sizes, and as sample size increased differences decreased. To obtain appropriate reference limits and confidence intervals, we propose the following scheme: (a) check whether the assumptions of regression analysis can be fulfilled with/without transformation of data; (b) check that the value of v, which describes how the covariate value is situated in relation to both the mean value and the spread of the covariate values, does not exceed 0.1 at minimum and maximum covariate positions; and (c) if steps 1 and 2 can be accepted, the reference limits with confidence intervals can be produced by regression analysis, and the minimum acceptable sample size will be ∼70.
Cytochrome P450 1B1 mRNA measured in blood mononuclear cells by quantitative reverse transcription-PCR
Authors: Dassi Cristina , Signorini Stefano , Gerthoux Piermario , Cazzaniga Mariangela , Brambilla Paolo
Published: January 2020

Cytochrome P450 (CYP) 1B1 activates polycyclic aromatic hydrocarbons and aryl aromatic hydrocarbons to carcinogens. We describe a competitive reverse transcription-PCR (RT-PCR) assay for the quantification of CYP1B1 mRNA in blood mononuclear cells (BMCs) by simultaneous RT and PCR amplification of cellular RNA with decreasing amounts of an internal standard. The concentration of CYP1B1 mRNA is derived from the ratio between the intensities of the bands corresponding to the amplified products. To reduce the variability of mRNA extraction efficiency, the measured amount of CYP1B1 has been calculated in relation to the β-actin gene products. We measured CYP1B1 expression in the BMCs of 75 human subjects; no significant differences in the CYP1B1:β-actin ratio were detected between women (range, 0.47–4.35; median, 2.0) and men (range, 0.72–3.85; median, 2.09). The analytical imprecision (CV) of duplicates was 14% (n = 25 pairs), and the intraindividual CV for two samples, 1 month apart, was 22% (n = 20). No significant differences were detected in smokers (n = 25; range, 0.77–3.55; median, 2.14) compared with nonsmokers (n = 50; range, 0.47–4.35; median, 2.0). The method has a wide range of linearity, good sensitivity and precision, and is suitable for studies of individual susceptibility as indicated by CYP1B1 expression in BMCs.
Identification of urinary oligosaccharides by matrix-assisted laser desorption ionization time-of-flight mass spectrometry
Authors: Klein André , Lebreton Alexandra , Lemoine Jérome , Périni Jean-Marc , Roussel Philippe , Michalski Jean-Claude
Published: January 2020

A new method of urinary oligosaccharides identification by matrix-assisted laser desorption time-of-flight mass spectrometry is presented. The method involves three steps: coupling of the urinary oligosaccharides with 8-aminonaphthalene-1,3,6-trisulfonic acid; fast purification over a porous graphite carbon extraction column; and mass spectrometric analysis. Identification of urinary oligosaccharides is based on the patterns and values of the pseudomolecular ions observed. We report here the patterns in urines from patients with Pompe disease, alpha and beta mannosidoses, galacto-sialidosis, and GM1 gangliosidosis. The protocols described here allowed facile and sensitive identification of the pathognomonic oligosacchariduria present in lysosomal diseases and can be extended to any pathological oligosacchariduria.
Heterozygosity for the C282Y mutation in the hemochromatosis gene is associated with increased serum iron, transferrin saturation, and hemoglobin in young women: a protective role against iron deficiency?
Authors: Datz Christian , Haas Thomas , Rinner Heinrich , Sandhofer Friedrich , Patsch Wolfgang , Paulweber Bernhard
Published: January 2020

Genetic hemochromatosis (GH) is the most common autosomal-recessive disorder (1 in 300 in populations of Celtic origin). Homozygosity for a C282Y mutation in the hemochromatosis (HFE) gene is the underlying defect in ∼80% of patients with GH, and 3.2–13% of Caucasians are heterozygous for this gene alteration. Because the high frequency of this mutation may result from a selection advantage, the hypothesis was tested that the C282Y mutation confers protection against iron deficiency in young women. To address this question the genotype of codon 282 was determined in a cohort of 468 unrelated female healthcare workers, ages 18–40 years. In all study participants, a complete blood count was obtained, and erythrocyte distribution width, serum iron, transferrin, transferrin saturation, and ferritin were measured. Two individuals were homozygous for the C282Y mutation, 44 were heterozygous, and 416 were homozygous for the wild-type allele. Heterozygous women had significantly higher values for hemoglobin (P = 0.006), serum iron (P = 0.013), and transferrin saturation (P = 0.006) than women homozygous for the wild-type allele. Our data provide evidence for a protective role of the C282Y mutation in the HFE gene against iron deficiency in young women and suggest that a more efficient utilization of nutritional iron may have contributed to the high prevalence of the mutation in Caucasian populations.
Percent free prostate-specific antigen in assessing the probability of prostate cancer under optimal analytical conditions
Authors: Gion Massimo , Mione Riccardo , Barioli Paola , Barichello Mario , Zattoni Filiberto , Prayer-Galetti Tommaso , Plebani Mario , Aimo Giuseppe , Terrone Carlo , Manferrari Fabio , Madeddu Giuseppe , Caberlotto Livio , Fandella Andrea , Pianon Carlo , Vianello Luisa
Published: January 2020

Although general consensus exists that percent free prostate-specific antigen (PSA) is superior to total immunoreactive PSA for prostate cancer (CaP) detection, its diagnostic performance is not yet well established. Analytical problems may account for difficulties in evaluating percent free PSA because the free PSA concentration is substantially lower than that of total PSA. The aim of the present study was to establish the diagnostic performances of the IMMULITE percent free PSA assay from Diagnostics Products Corp. under experimental conditions optimized to minimize analytical variability. Eighty-five patients with untreated primary CaP and 261 with untreated benign prostate hypertrophy (BPH) were prospectively enrolled. The Diagnostics Products IMMULITE total (Third Generation) and free PSA were measured by the same technician, using the same instrument and the same reagent batch. We calculated the post-test probability to express how the likelihood of the diagnosis of CaP changed after the percent free PSA was determined. Areas under the ROC curves of percent free PSA were better than those of total PSA in every evaluated range of total PSA. The percent free PSA could have reduced the rate of unnecessary biopsies by 47% in patients with total PSA ≥4 μg/L with only 3.8% false-negative results. The post-test probability of percent free PSA was, however, <50% in men 50–70 years of age, using cutoff points providing sensitivity from 99% to 80%. Percent free PSA is superior to total PSA in distinguishing primary CaP from BPH in patients with total PSA between 2 and 30 μg/L. In men with low total PSA, the diagnostic performance of the percent free PSA assay may be optimized by controlling methodological variability. The percent free PSA assay is effective in reducing the rate of unnecessary biopsies in men with total PSA >4 μg/L. However, the post-test probability provided by percent free PSA is relatively low in asymptomatic patients 50–70 years of age.
Characterization and immunological determination of the complex between prostate-specific antigen and α2-macroglobulin
Authors: Zhang Wan-Ming , Finne Patrik , Leinonen Jari , Vesalainen Satu , Nordling Stig , Rannikko Sakari , Stenman Ulf-Håkan
Published: January 2020

Prostate-specific antigen (PSA) rapidly forms a complex with α2-macroglobulin (A2M) in vitro; however, PSA complexed with A2M (PSA-A2M) is not detected by conventional immunoassays for PSA because it is encapsulated by the A2M. In this study, we show that denaturation of PSA-A2M at high pH renders PSA immunoreactive. Part of the complexed PSA is released in free form and part remains bound to denatured A2M. These forms can be measured by a conventional immunoassay for PSA. This finding enabled us to design a dissociation assay for the detection of PSA-A2M, which was based on the removal of immunoreactive PSA in serum by immunoadsorption, denaturation of PSA-A2M at high pH, and measurement of the released PSA immunoreactivity by a conventional PSA immunoassay. This PSA-A2M assay was calibrated with PSA-A2M formed in vitro. The detection limit of the assay was 0.14 μg/L. Inter- and intraassay coefficients variation were 4–9% and 8–14%, respectively. When purified PSA was incubated with A2M, the loss of PSA immunoreactivity was highly correlated with the PSA-A2M formed, as measured by the dissociation assay for PSA-A2M (r = 0.99; P <0.0001). The concentration of PSA-A2M in serum correlated with that of total PSA both in prostate cancer (PCa) and benign prostatic hyperplasia (BPH); however, the ratio of PSA-A2M in relation to total PSA was significantly higher in BPH than in PCa (P <0.0003). ROC curve analysis suggested that measurement of the ratio of PSA-A2M to total PSA in serum improves the diagnostic accuracy for PCa compared with assays for total PSA only.
Comparison of two homogeneous assays with a precipitation method and an ultracentrifugation method for the measurement of HDL-cholesterol
Authors: Arranz-Peña Maria-Luisa , Tasende-Mata Juan , Martin-Gil Francisco J
Published: January 2020

We report on the performance of four HDL-cholesterol assays: a homogeneous method based on polyethylene glycol-modified enzymes/α-cyclodextrin sulfate (PEGME; Kyowa); a homogeneous method based on polyanion–polymer/detergent (PPD; Daiichi); the usual precipitation method with phosphotungstic acid/MgCl2 (PTA); and an ultracentrifugation (UC) procedure. The homogeneous HDL-cholesterol assays (performed with automated analyzers) were precise and correlated well with the PTA and UC assays. The specificity and accuracy of the PEGME method were better than those of the PPD method.
Assay of β-N-acetylhexosaminidase isoenzymes in different biological specimens by means of determination of their activation energies
Authors: Pérez Luis F , Tutor J Carlos
Published: January 2020

The activation energy (Ea) of β-N-acetylhexosaminidase (Hex, EC 3.2.1.52) was determined with 3,3′-dichlorophenylsulfonphthaleinyl-N-acetyl-β-d-glucosaminide as substrate, with a much higher value being found for the Hex B isoenzyme (Ea = 75.1 kJ/mol) than for the Hex A isoenzyme (Ea = 41.8 kJ/mol). This fact allowed for the development of a fast and reliable thermodynamic method to determine the isoenzyme composition of Hex in different biological specimens (serum/plasma, saliva, cerebrospinal fluid, seminal plasma, urine, and leukocyte lysates). The results in serum given by the proposed method may be superimposed upon those obtained by the heat inactivation assay of O’Brien et al. (N Engl J Med 1970;273:15–20), and the catalytic activity calculated for Hex A offers a good correlation with that obtained by using the specific substrate 4-methylumbelliferyl-N-acetyl-β-d-glucosaminide-6 sulfate (n = 25, r = 0.953).
Direct enzyme immunometric measurement of plasma big endothelin-1 concentrations and correlation with indicators of left ventricular function
Authors: Haug Cornelia , Koenig Wolfgang , Hoeher Martin , Kochs Matthias , Hombach Vinzenz , Gruenert Adolf , Osterhues Hans
Published: January 2020

Recent studies have suggested that the plasma concentrations of endothelin-1, a potent vasoconstrictive peptide, are increased in patients with congestive heart failure. This study aimed to evaluate a new direct ELISA for big endothelin-1 (the precursor of endothelin-1), in comparison with a big endothelin-1 ELISA using plasma sample extraction, and to investigate whether plasma big endothelin-1 concentrations correlate with indicators of left ventricular function. The direct ELISA yielded significantly (P <0.001) lower results than the assay with extracted samples (0.9 ± 0.5 pmol/L vs 2.7 ± 1.9 pmol/L; n = 90); however, the results of the two assays were closely correlated (r = 0.86, P <0.001). Plasma big endothelin-1 concentrations exhibited a significant (P <0.001) negative correlation (r = −0.46, r = −0.40) with the left ventricular ejection fraction and a significant positive correlation (r = 0.40, P <0.001; r = 0.36, P <0.01) with the left ventricular end-diastolic pressure and the left ventricular end-diastolic (r = 0.42, r = 0.38, P <0.001) and end-systolic (r = 0.52, r = 0.47, P <0.001) volume indices. Plasma big endothelin-1 concentrations were notably greater in patients with New York Heart Association (NYHA) class II–IV symptoms than in patients without cardiac disease or in patients categorized to NYHA class I. These data suggest that plasma big endothelin-1 concentrations, measured by a direct ELISA, correlate with hemodynamic indicators and symptoms of left ventricular dysfunction.
Rapid, convenient radioimmunoassay of estrone sulfate
Authors: Ranadive Girish N , Mistry Jehangir S , Damodaran Kalyani , Khosravi M Javad , Diamandi Anastasia , Gimpel Terry , Castracane V Daniel , Patel S , Stanczyk Frank Z
Published: January 2020

We developed a specific, simple, and rapid RIA for the direct quantification of estrone sulfate (E1S) and established its performance characteristics. The assay has a dynamic range of 0.05–90 μg/L with a detection limit of 0.009 μg/L. Intraassay CVs were 9.2%, 4.5%, and 4.6% at 0.35, 9.0, and 60 μg/L, respectively. Interassay CVs were 8.8%, 5.1%, and 5.5% at 0.076, 0.5, and 12 μg/L, respectively. Linearity of dilution studies showed values of 80–105% of expected, and recovery of E1S added to serum samples ranged from 82% to 102%. Cross-reactivities with structurally related estrogens were <5%. When compared with a conventional assay (involving hydrolysis of E1S and indirect measurement of estrone), the present RIA showed excellent correlation (r = 0.99, slope = 1.54, Sy‖x = 2.14, n = 71). Mean E1S concentrations measured with this RIA for normal men (n = 20) and women in follicular (n = 20) and luteal (n = 25) phases of their menstrual cycle were 0.96, 0.96, and 1.74 μg/L, respectively. Mean E1S concentrations for oral contraceptive users (n = 20) and postmenopausal women without hormone replacement therapy (n = 21) or on hormone replacement therapy (n = 22) were 0.74, 0.13, and 2.56 μg/L, respectively. Serum concentrations of E1S in pregnant women in their first (n = 14), second (n = 17), and third (n = 15) trimesters were 20, 66, and 105 μg/L, respectively. Availability of this simple RIA should provide a useful tool for the assessment of estrogen status in women.
Increased concentrations of serum pentosidine in rheumatoid arthritis
Authors: Rodríguez-García Javier , Requena Jesús R , Rodríguez-Segade Santiago
Published: January 2020

Advanced glycosylation end products (AGEs) are thought to play an important role in the development of diabetic complications. Oxidative reactions are essential for the formation of some AGEs, termed glycoxidation products. Increased concentrations of pentosidine, one of such products, are found in tissue and serum in diabetes mellitus and in end-stage renal disease, suggesting that hyperglycemia and impaired renal function are important factors in AGE accumulation. We hypothesized that increased concentrations of pentosidine would also be found in pathological conditions associated with increased oxidative stress. We measured pentosidine in sera of patients with rheumatoid arthritis (RA), systemic lupus erythematosus, and diabetes. Increased serum pentosidine was found in RA (108.4 ± 146.5 nmol/L, P <0.002) and in diabetes (69.6 ± 42.4 nmol/L, P <0.001) as compared with healthy subjects (48.3 ± 12.0 nmol/L). These results prove that AGEs may accumulate in the absence of hyperglycemia or impaired kidney function.
Determination of glycated albumin by enzyme-linked boronate immunoassay (ELBIA)
Authors: Ikeda Kazuyoshi , Sakamoto Yuichiro , Kawasaki Yukie , Miyake Takehiro , Tanaka Kimikazu , Urata Takeyoshi , Katayama Yoshiaki , Ueda Shoichi , Horiuchi Seikoh
Published: January 2020

A new affinity method for quantification of glycated albumin by an enzyme-linked boronate-immunoassay (ELBIA) has been established, based on the interaction between boronic acids and the cis-diols of glycated human serum albumin (HSA) trapped by anti-HSA antibody. To evaluate the ELBIA, we first examined the accuracy of the conventional boronate affinity chromatographic (BAC) method. In the BAC method, 8.1–18.9% of nonglycated albumin calibrator nonspecifically bound to the boronate affinity column, values that were regarded as the column blank. In the modified BAC method, therefore, we subtracted the column blank value from the measured glycated albumin value to obtain the true value. Because glycated albumin values measured by ELBIA were exactly the same as reported by the modified BAC method, we suggest that the ELBIA results reflect the real status of albumin glycation. We have also developed a fully automated ELBIA system, allowing multiple, rapid, and precise measurements of glycated albumin.
Rapid detection of a recombinant hotspot associated with Charcot–Marie–Tooth disease type 1A duplication by a PCR-based DNA test
Authors: Chang Jan-Gowth , Jong Yuh-Jyh , Wang Wen-Pin , Wang Jyh-Chwan , Hu Chaur-Jong , Lo Man-Chi , Chang Chih-Peng
Published: January 2020

A 1.5-Mb duplication on chromosome 17p11.2-p12 (CMT1A duplication) caused by a misalignment of the CMT1A repeat sequences (CMT1A-REPs) is associated with Charcot–Marie–Tooth disease type 1A (CMT1A). A hotspot of crossover breakpoints located in a 3.2-kb region of the CMT1A-REPs accounts for three-quarters of the rearrangements in CMT1A patients. We developed a PCR-based diagnostic method to detect a recombination hotspot associated with the CMT1A duplication. Thirty-one CMT1A Chinese patients from different families and 50 healthy people over 65 years of age were studied. Twenty-seven of the 31 cases demonstrated the 3.2-kb hotspot crossover, of which there were two subgroups. The type 1 crossover breakpoint was located at the distal CMT1A-REP around the PmeI site, and accounted for 24 of the 27 cases with a 3.2-kb hotspot crossover in CMT1A duplication patients. The type 2 crossover breakpoint was located at the distal CMT1A-REP around the base 3625 region, accounting for 3 of the 27 cases. The results correlated very well with the results of Southern transfer analysis. This study has a potentially important role in the diagnosis of CMT1A disease.
Assessment of simple colorimetric procedures to determine smoking status of diabetic subjects
Authors: Smith Robert F , Mather Hugh M , Ellard Gordon A
Published: January 2020

The performance of a simple colorimetric assay for urinary nicotine metabolites to assess smoking status in diabetic subjects (n = 251) was investigated. Several variations of the colorimetric assay and a qualitative extraction procedure were evaluated in comparison with a cotinine immunoassay as the “gold standard.” Among these, the best overall performance was achieved with the qualitative test (sensitivity 95%; specificity 100%). The quantitative measurement of total nicotine metabolites performed less well (sensitivity 92%; specificity 97%) but could be improved by incorporating a blank extraction (sensitivity 98%; specificity 98%). Allowance for diuresis appeared to offer no advantage over the other methods. These results support previous findings regarding the use of these colorimetric procedures in nondiabetic subjects and, contrary to other recent observations, their performance was not impaired in diabetic patients.
Radioimmunoassay of bound and free melatonin in plasma
Authors: Di Wei-Li , Kadva Aban , Djahanbakhch Ovrang , Silman Robert
Published: January 2020

We describe a nonextraction procedure, and two extraction procedures, for RIA of melatonin in human plasma. All procedures showed a diurnal rhythm of melatonin in human subjects, with interindividual differences greater than interprocedure differences. However, further investigations demonstrated considerable variability of recovery in the nonextraction procedure, suggesting a variability of binding proteins between samples. Combining recovery and dialysis experiments in the extraction procedures, we demonstrated that chloroform was unable to extract albumin-bound melatonin from a human serum albumin solution but, paradoxically, was able to extract bound and free melatonin from a plasma sample. The methanol extraction procedure extracted free and bound melatonin from all sources. These results indicate that albumin binding can substantially affect the RIA procedures. We conclude that assays should be validated against free and bound melatonin and that the two forms should be independently investigated when assessing bioactivity.
Enzyme conversion immunoassay for determining total homocysteine in plasma or serum
Authors: Frantzen Frank , Faaren Arne Ludvig , Alfheim Ingrid , Nordhei Arne Kristian
Published: January 2020

A rapid and precise immunoassay for quantification of total homocysteine in blood samples is presented. The method avoids the use of radioisotopes and chromatographic separations and relies on enzymatic conversion of homocysteine to S-adenosyl-l-homocysteine, followed by quantification of S-adenosyl-l-homocysteine by an enzyme-linked immunoassay in microtiter format. The within- and between-assay imprecision is <6% and 8%, respectively, and results by the method show good correlation with those by HPLC. Including controls and calibrators in duplicates, 82 samples can be analyzed within 2.5 h. The procedure can be fully automated.
Assessment of sequence-based p53 gene analysis in human breast cancer: messenger RNA in comparison with genomic DNA targets
Authors: Williams Cecilia , Norberg Torbjörn , Ahmadian Afshin , Pontén Fredrik , Bergh Jonas , Inganäs Mats , Lundeberg Joakim , Uhlén Mathias
Published: January 2020

The high prevalence of p53 mutations in human cancers and the suggestion from several groups that the presence or absence of p53 mutations might have both prognostic and therapeutic consequences point to the importance of optimal methods for p53 determination. Several strategies exploring this have been described, based either on mRNA or genomic DNA as a template. However, no comparative study on the reliability of the two templates has been performed. The principal aim of this study was to study the concordance of RNA- and DNA-based direct sequencing methods in detecting p53 mutations in breast tumors. In 100 tumors, 22 mutations were detected by both methods. Furthermore, one stop mutation, two splice-site mutations, and one intron alteration were found only by genomic sequencing. In addition, the comparative study suggests that cells with missense mutations have increased steady-state concentrations of p53-specific mRNA, in contrast to cells with a gene encoding a truncated protein.
Semiautomated microtiter plate assay for monitoring peptidylprolyl cis/trans isomerase activity in normal and pathological human sera
Authors: Küllertz Gerhard , Lüthe Sabine , Fischer Gunter
Published: January 2020

An UV/VIS spectrophotometric assay technique was developed that was able to routinely monitor peptidylprolyl cis/trans isomerase (PPIase) activity of biological fluids in 96-well microtiter plates. The assay, based on monitoring the cis-to-trans isomerization of succinyl-Phe-cisPro-Phe-4-nitroanilide as substrate in a chymotrypsin-coupled reaction, yields a throughput of 96 samples per 30 min. The assay’s capacity was exemplified by dealing with the PPIase activity in several normal and pathological human sera. Reference values of 151 healthy subjects (83 females, 69 males, 17 to 60 years old) were found to possess significant sex-specific differences. PPIase activity factor K of the sera was significantly greater in males (5th, 50th, 95th percentiles: 17, 36, 55 K) than females (14, 30, 48 K). PPIase activities of sera from healthy donors (n = 151) were significantly higher (Mann–Whitney rank-sum test P <0.0001) than those of patients (n = 47). PPIase activity in serum samples stored at 4 °C was stable for at least 20 h.
Prenatal and postnatal diagnoses of thalassemias and hemoglobinopathies by HPLC
Authors: Fucharoen Suthat , Winichagoon Pranee , Wisedpanichkij Raewadee , Sae-Ngow Busara , Sriphanich Rungrat , Oncoung Warangkana , Muangsapaya Wanna , Chowthaworn Jew , Kanokpongsakdi Sujin , Bunyaratvej Ahnond , Piankijagum Anong , Dewaele Chris
Published: January 2020

The conventional approach to qualitative and quantitative analyses of hemoglobin (Hb) molecules for the diagnoses of hemoglobinopathies requires a combination of tests. We used an automated HPLC (VARIANT™) system to study α-thalassemia and β-thalassemia syndromes in Thailand. The beta-thalassemia short program is applicable to the diagnosis of α-thalassemia and β-thalassemia disorders, including Hb H, EA Bart’s disease, and EF Bart’s disease, in adults, newborns, and fetuses. The system cannot quantify accurately certain Hb molecules, such as Hb H and Hb Bart’s. The alpha-thalassemia short program was therefore developed and used to quantify Hb Bart’s to detect α-thalassemia genotypes in cord blood. This automated HPLC system is an alternative approach to the diagnosis of complicated thalassemia syndromes in Thailand and Southeast Asia.
A non-(1–84) circulating parathyroid hormone (PTH) fragment interferes significantly with intact PTH commercial assay measurements in uremic samples
Authors: Lepage Raymond , Roy Louise , Brossard Jean-Hugues , Rousseau Louise , Dorais Claude , Lazure Claude , D’Amour Pierre
Published: January 2020

We have previously shown that the Nichols assay for intact parathyroid hormone (I-PTH) reacts with a non-(1–84) molecular form of PTH. This form behaves as a carboxy-terminal fragment and accumulates in renal failure, accounting for 40–60% of the measured immunoreactivity. We wanted to see whether this was a common event with other commercial two-site I-PTH assays. We thus compared the ability of three commercial kits [Nichols (NL), Incstar (IT), and Diagnostic System Laboratories (DSL)] to measure I-PTH in 112 renal failure patients and to detect hPTH(1–84) and non-(1–84)PTH on HPLC profiles of serum pools from uremic patients with I-PTH concentrations of 10–100 pmol/L. The behavior of synthetic hPTH(7–84), a fragment possibly related to non-(1–84)PTH was also compared with hPTH(1–84) in the three assays. The I-PTH concentrations measured with the three assays in the 112 uremic samples were highly related (r2 ≥ 0.89, P <0.0001), and the values measured with NL were, on average, 23% higher than IT. Values measured with DSL were 23% and 56% higher than IT for values less than and more than 40 pmol/L, respectively. The three assays detected two HPLC peaks on four different profiles corresponding to hPTH(1–84) and non-(1–84)PTH. This last peak represented 36 ± 8.4% of the immunoreactivity with NL, 24 ± 5.5% with IT, and 25 ± 2.8% with DSL (NL vs IT or DSL: P <0.05). These differences were confirmed by a 50% lower immunoreactivity to hPTH(7–84) compared with hPTH(1–84) for IT and DSL but not for NL. These results suggest that most of the two-site I-PTH assays would cross-react with non-(1–84)PTH material, thus explaining about one-half of the 2–2.5 × higher I-PTH concentrations reported in uremic patients without bone involvement than in subjects without uremia.
Intrinsic and routine quality of serum total potassium measurement as investigated by split-sample measurement with an ion chromatography candidate reference method
Authors: Thienpont Linda M , Van Nuwenborg Jean E , Stöckl Dietmar
Published: January 2020

We evaluated the intrinsic quality of eight routine test systems for the measurement of serum total potassium (K+), as well as the routine quality of four of these systems, using a group of 60 single-donation serum samples that had been certified with an ion chromatography reference method. The intrinsic quality of the tests was evaluated by analysis of the sera in the manufacturers’ application laboratories under strict internal quality control. The routine quality was evaluated by analysis of the same sera in five (per system) routine laboratories under daily working conditions. The results of the study were interpreted in light of the most stringent specifications derived from the biological variation of K+, which require limits of 6.3% for total error and 1.6% for systematic error. The study revealed that the intrinsic quality of all systems was excellent. None of the test systems yielded a substantial number of results outside the 6.3% total error limit, and only one test system exceeded the 1.6% systematic error limit. The majority of the routine laboratories reproduced the manufacturers’ intrinsic quality. In particular, most laboratories satisfied the 6.3% total error limit. However, several laboratories exceeded the 1.6% systematic error limit. Generally, there was a considerable difference in quality between the participating laboratories. This showed that the major problems for serum K+ analysis (for samples with no unusual matrices and with concentrations within the reference interval) are at the routine laboratory.
Standards of laboratory practice: antiepileptic drug monitoring
Authors: Warner Ann , Privitera Michael , Bates David
Published: January 2020

Discussion and development of standards for appropriate monitoring led to the following key recommendations for ordering, sampling, and analyzing antiepileptic drugs: Monitoring should usually be done on trough specimens after steady-state has been reached and always with an appropriate medical indication; non-steady-state concentrations may be indicated in selected situations. Monitoring of free phenytoin and free valproic acid is indicated in specific situations and should be done in serum. The metabolite of primidone, phenobarbital, should be measured concurrently with parent drug, but the active metabolite of carbamazepine does not need to be monitored unless the patient is exhibiting an unusual toxic response that cannot be otherwise explained. Assays used for antiepileptic drug monitoring should display a long-term CV of <10% and preferably <5%. Subtherapeutic and supratherapeutic drug concentrations should be investigated on a regular basis as part of a quality assurance process.
Rapid capillary zone electrophoresis in isoelectric histidine buffer: high resolution of the poly-T tract allelic variants in intron 8 of the CFTR gene
Authors: Gelfi Cecilia , Perego Marilena , Righetti Pier Giorgio , Cainarca Silvia , Firpo Stefania , Ferrari Maurizio , Cremonesi Laura
Published: January 2020

The poly-T tract in intron 8 of the cystic fibrosis conductance transmembrane regulator (CFTR) gene exists in three variants, 5T, 7T, and 9T. The 7T and 9T variants generate a predominantly normal transcript, whereas the 5T variant engenders an anomalous product. The analysis of the poly-T tract is assuming increasing relevance, both to assess the implication of the CFTR gene in congenital bilateral absence of the vas deferens and to evaluate genotype-phenotype correlation in cystic fibrosis. Mapping of the poly-T tract has been performed by cumbersome and time-consuming methodologies. Capillary zone electrophoresis, combined with laser-induced fluorescence detection, was introduced for a rapid separation of the poly-T tract amplified products. As separation buffer, we adopted 200 mmol/L histidine (pH = pI = 7.6), and the capillary was filled with 10% polyacrylamide, allowing separations in less than 10 min. Capillary zone electrophoresis results were in perfect agreement with dot-blot analysis.