bluuebunny/crossref_metadata_2025_split huggingface dataset:
Immunoradiometric assay of succinylated corticotropin: an improved method for quantification of ACTH
Authors: Kertesz Gilles , Bourcier Béatrice , Cailla Hélène , Jean Frédéric 
Published: January 2020

Abstract
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

Abstract
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 &lt;0.04% for RBC, &lt;0.03% for WBC, &lt;0.14% for SEC, &lt;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

Abstract
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

AbstractAn 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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

AbstractCytochrome 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

AbstractA 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

Abstract
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

Abstract
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, &lt;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 &gt;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

AbstractProstate-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 &lt;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 &lt;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

AbstractWe 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

AbstractThe 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).