bluuebunny/crossref_metadata_2025 huggingface dataset:
The role of pinhole structures in Mo thin films on multi-layer graphene synthesis
Authors: Kizir Seda , van den Beld Wesley , Schurink Bart , van de Kruijs Robbert , Benschop Jos , Bijkerk Fred
Published: January 2020

In this study, the density and oxygen content of Mo thin films were varied by changing the deposition conditions, in order to understand their influence on the catalytic activity of Mo2C for the synthesis of multi-layer graphene (MLG). Structural and morphological analysis of Mo2C in relation to its catalytic activity indicate that the density of Mo plays a more critical role on MLG synthesis than the oxygen content. Results show that the pinholes present in relatively low density Mo layers act as catalytically active defect sites, promoting MLG synthesis.
‘Qué barbaridad, son latinos y deberían saber español primero’: Language Ideology, Agency, and Heritage Language Insecurity across Immigrant Generations
Authors: Tseng Amelia
Published: January 2020

This case study examines the consequences of community language attitudes and ideologies on later-generation heritage speakers through qualitative sociolinguistic and discourse analysis of 22 interviews with first- and second-generation Latinos of diverse backgrounds in a major US metropolitan area. The findings show that imposed deficit identities derived from ideologies of language purity, proficiency, and individual agency were misunderstood and stigmatized later-generation heritage speakers, leading to language insecurity and avoidance despite overtly positive attitudes toward Spanish maintenance. Results demonstrate the resilience of prescriptive/purist language attitudes and the tension inherent between these beliefs (albeit couched within positive heritage language attitudes) and speakers’ actual bilingualism. Further, they show that the ideologies of individual agency can paradoxically contribute to the imposition of deficit sociolinguistic identities on later-generation speakers and curtail their language use. The study renders visible connections between ideologies of language, identity, and agency and demonstrates how their reproduction within families and communities circumscribes later-generation heritage speakers’ linguistic identities and behavior.
GiniQC: a measure for quantifying noise in single-cell Hi-C data
Authors: Horton Connor A , Alver Burak H , Park Peter J
Published: January 2020

Single-cell Hi-C (scHi-C) allows the study of cell-to-cell variability in chromatin structure and dynamics. However, the high level of noise inherent in current scHi-C protocols necessitates careful assessment of data quality before biological conclusions can be drawn. Here, we present GiniQC, which quantifies unevenness in the distribution of inter-chromosomal reads in the scHi-C contact matrix to measure the level of noise. Our examples show the utility of GiniQC in assessing the quality of scHi-C data as a complement to existing quality control measures. We also demonstrate how GiniQC can help inform the impact of various data processing steps on data quality.

Availability and implementation

Source code and documentation are freely available at https://github.com/4dn-dcic/GiniQC.

Supplementary information

Supplementary data are available at Bioinformatics online.
Importance of the Detection Method for Thyroglobulin Antibodies for the Validity of Thyroglobulin Measurements in Sera from Patients with Graves Disease
Authors: Massart Catherine , Maugendre Didier
Published: January 2020

Background: The use of recovery tests has been proposed to disclose interferences from anti-thyroglobulin antibodies (TgAbs) in thyroglobulin (Tg) assays. We studied the value of a recovery test in Tg measurement by a new commercial IRMA.

Methods: Blood samples were collected from 153 patients with untreated Graves disease. Tg and TgAbs were measured by IRMA and RIA, respectively (Dynotest Tg-plus and Dynotest anti-Tgn; Brahms Diagnostica). The recoveries of added amounts of Tg were calculated for each serum.

Results: TgAbs were detected in 72 of the 153 patients (47%). The recovery test results for the 81 TgAb-negative sera (median, 101%; range, 80–115%) were identical to the results for the 91 controls (median, 102%; range, 80–124%). By contrast, significantly lower recovery test results were observed for the 72 TgAb-positive sera (median, 79%; range, 60–103%; Z = −8.363; P <0.0001). In the 34 of the 72 TgAb-positive sera with a normal recovery test, Tg concentrations were significantly lower (median Tg, 13.6 μg/L; range, 1.1–360 μg/L) than those measured in the TgAb-negative sera (median, 107 μg/L; range, 1.2–700 μg/L; Z = −3.797; P <0.0001).

Conclusions: Tg values were decreased in TgAb-positive sera even when the results of the recovery tests were normal. This test should not be used alone to determine the validity of a serum Tg measurement in Graves disease.
Influence of Adsorption and Deproteination on Potential Free Thyroxine Reference Methods
Authors: Holm Steen S , Andreasen Lisbeth , Hansen Steen H , Faber Jens , Staun-Olsen Poul
Published: January 2020

Background: There is a need for consensus concerning reference methods to be used for calibration of commercial free-thyroxine (FT4) assays.

Methods: Three different potential reference techniques were investigated for adsorption of T4 to membrane materials, including any in vitro solid surfaces to which T4 might adsorb, and for efficient separation of the T4-binding proteins from the free hormone fraction. We compared ultrafiltration with different commercial ultrafiltration units, ultrafiltration by dialysis tubing, equilibrium dialysis, and ultracentrifugation. We measured the adsorption to membranes and materials with l-[125I]T4. Separation efficiency was determined by measuring the T4-binding protein albumin in the ultrafiltrate and the dialysate as well as in the supernatant from the ultracentrifugation with a double-antibody sandwich ELISA technique.

Results: We found a constant relationship between the amount of T4 adsorbed to the dialysis or ultrafiltration membranes/materials and the initial T4 concentration in HEPES buffer (protein-free medium). T4 was considerably less adsorbed from serum than from HEPES buffer (P <0.001). Serum T4 was less adsorbed upon ultracentrifugation than during dialysis and ultrafiltration (P <0.001). It was difficult to completely separate FT4 from the binding proteins by ultrafiltration and ultracentrifugation. Separation by ultrafiltration depended on the material used.

Conclusions: No investigated separation technique provides technically and theoretically correct separation of the free fraction of T4 from the protein-bound fraction. Equilibrium dialysis seems to be the least compromised.
Determination of Oligosaccharides in Pompe Disease by Electrospray Ionization Tandem Mass Spectrometry
Authors: Rozaklis Tina , Ramsay Steven L , Whitfield Phillip D , Ranieri Enzo , Hopwood John J , Meikle Peter J
Published: January 2020

Background: The development of therapies for lysosomal storage disorders has created a need for biochemical markers to monitor the efficacy of therapy and methods to quantify these markers in biologic samples. In Pompe disease, the concentration of a tetrasaccharide, consisting of four glucose residues, is reputedly increased in urine and plasma, but faster and more sensitive methods are required for the analysis of this, and other oligosaccharides, from biologic fluids.

Methods: We optimized the derivatization of storage oligosaccharides with 1-phenyl-3-methyl-5-pyrazolone for the measurement, by electrospray ionization tandem mass spectrometry, of oligosaccharide concentrations in urine (n = 6), plasma (n = 11), and dried-blood spots (n = 17) from Pompe-affected individuals. Age-matched control samples of urine (n = 10), plasma (n = 28), and blood spots (n = 369) were also analyzed.

Results: The mean tetrasaccharide concentration was increased in urine from infantile-onset (0.69–12 mmol/mol of creatinine) and adult-onset (0.22–3.0 mmol/mol of creatinine) Pompe individuals compared with age-matched controls. In plasma samples, an increased tetrasaccharide concentration was observed in some infantile patients (up to 22 μmol/L) compared with age-matched controls (mean, 2.2 μmol/L). The method developed was sensitive enough to determine oligosaccharide concentrations in a single 3-mm blood spot, but no differences were observed between blood spots from control and Pompe-affected individuals.

Conclusions: Measurements of oligosaccharide concentrations in urine by this new method have potential application for the diagnosis and monitoring of patients with Pompe disease. Plasma analysis may have limited application for infantile patients, but analysis of blood spots does not discriminate between controls and affected individuals.
Yield and Cost of Individual Common Diagnostic Tests in New Primary Care Outpatients in Japan
Authors: Takemura Yuzuru , Ishida Haku , Inoue Yuji , Beck J Robert
Published: January 2020

Background:Appropriate diagnostic testing involves considerations of cost-effectiveness. We examined the cost-effectiveness of individual tests in a panel of tests defined by the Japan Society of Clinical Pathology.

Methods: We studied 540 new, symptomatic primary care outpatients with a set of 30 common diagnostic tests [the Essential Laboratory Tests (2); ELT(2) panel] for clinical evaluation and identification of occult disease. A useful result (UR) of testing was defined as a finding that contributed to a change in a physician’s diagnosis or decision-making relating to a “tentative initial diagnosis” obtained from history and physical examination alone.

Results: The ELT(2) panel testing yielded 398 URs and uncovered 261 occult diseases among 540 patients. In total, 1592 tests contributed to either UR-generation or discovery of occult disease. The cost per effective test (cost required per test that contributed to either definition of effectiveness) ranged from ¥108 (∼US$0.92) for total cholesterol to ¥6200 (∼$52.50) for chest x-ray. Contribution rates and the cost per effective test varied among disease categories. We restructured panel components considering the effectiveness of each test. Subsets of the ELT(2) would have improved cost-effectiveness and achieved cost savings in five of eight disease categories.

Conclusions: Assembly of tests based on cost-effectiveness can improve clinical efficiency and decrease total cost of panel testing for selected patient groups.
Use of On-Site Testing for Drugs of Abuse
Authors: George Stephen , Braithwaite Robin A
Published: January 2020

Background: There is currently a profusion of near-patient testing devices that have been specifically targeted at drug dependency units and clinics. Some of these devices have been shown to produce accurate results. However, some devices suffer from inappropriate labeling, which together with the subjective interpretation of poorly defined reaction end-point markers, leads to misinterpretation of the results generated.

Methods: A literature search was conducted regarding the use and evaluation of near-patient testing devices for drugs-of-abuse screening. The results of this research, together our own practical evaluations of such devices, have been collated into this review.

Results: It is proposed that although near-patient testing devices may be useful in remote areas or where rapid action needs to be taken, it should be remembered that they provide only initial screening data and may yield false-positive or -negative results. Such devices need to be used with caution because a rapid but unconfirmed result may lead to misdiagnosis and inappropriate treatment for those who have a drug problem. It should be noted that a single result, which may be inaccurate, could lead to the cessation of treatment and a failure to provide care for those in greatest need. In addition, false-positive results may also have medico-legal implications, especially with the initiation of the drug testing and treatment orders.

Conclusions: Near-patient testing devices for drugs of abuse could be an expensive and potentially inaccurate means to monitor patient treatment and drug abuse status.
Duration of Detectable Methamphetamine and Amphetamine Excretion in Urine after Controlled Oral Administration of Methamphetamine to Humans
Authors: Oyler Jonathan M , Cone Edward J , Joseph Robert E , Moolchan Eric T , Huestis Marilyn A
Published: January 2020

Background: Confirmation of a workplace drug test requires urinary methamphetamine (MAMP) and amphetamine (AMP) concentrations ≥500 and 200 μg/L, respectively, but cutoffs at half those values (250/100 μg/L) have been proposed. We determined the urinary excretion of MAMP after oral ingestion and examined the effect of using lower cutoffs on detection of exposure.

Methods: Volunteers (n = 8) ingested four 10-mg doses of MAMP · HCl daily over 7 days, and five of them ingested four 20-mg doses 4 weeks later. After ingestion, the volunteers collected all urine specimens for 2 weeks. After solid-phase extraction, MAMP and AMP were measured by gas chromatography–positive chemical ionization mass spectrometry with dual silyl derivatization.

Results: MAMP and AMP were generally detected in the first or second void (0.7–11.3 h) collected after drug administration, with concentrations of 82–1827 and 12–180 μg/L, respectively. Peak MAMP concentrations (1871–6004 μg/L) after single doses occurred within 1.5–60 h. MAMP ≥500 μg/L was first detected in the first or second void (1–11 h) at 524-1871 μg/L. Lowering the MAMP cutoff to 250 μg/L changed the initial detection time little. AMP ≥200 μg/L was first detected in the 2nd–13th (7–20 h) post-administration voids. At a cutoff of 100 μg/L, AMP was first confirmed in the second to eighth void (4–13 h). Reducing the cutoff to 250/100 μg/L extended terminal MAMP detection by up to 24 h, increased total detection time by up to 34 h, and increased the total number of positive specimens by 48%.

Conclusions: At the lower cutoff, initial detection times are earlier, detection windows are longer, and confirmation rates are increased. Elimination of the AMP requirement would increase detection rates and allow earlier detection.
Human Adrenal Cells in Culture Produce Both Ouabain-like and Dihydroouabain-like Factors
Authors: El-Masri M Adnan , Clark Barbara J , Qazzaz Hassan M , Valdes Roland
Published: January 2020

Background: Ouabain-like factor (OLF) and its newly discovered reduced species, dihydroouabain-like factor (Dh-OLF), are mammalian cardenolides whose structural and functional characteristics are similar to the plant-derived compounds ouabain and dihydroouabain. These endogenous compounds are believed to be produced by the adrenals and to constitute part of an hormonal axis that may regulate the catalytic activity of the α-subunit of Na+,K+-ATPase. We developed antibodies sufficiently specific to distinguish between OLF and Dh-OLF, and in this study demonstrate the selective secretion of OLF and Dh-OLF from human H295R-1 adrenocortical cells in culture.

Methods: We used reversed-phase HPLC, inhibition of Na+,K+-ATPase catalytic activity, and two enzyme immunoassays developed with antibodies specific to ouabain and dihydroouabain to purify and characterize the secretion of these two compounds by human adrenal cells in culture. Purified antisera had high titers (1 × 106 for ouabain and 8 ×105 for dihydroouabain) and were specific to their corresponding antigens.

Results: Human H295R-1 cells grown in serum-free medium secreted 0.18 ± 0.03 pmol of OLF and 0.39 ± 0.04 pmol of Dh-OLF per 106 cells in 24 h. Both OLF and Dh-OLF inhibited the ouabain-sensitive catalytic activity of the sodium pump (0.03 μmol/L OLF inhibited 29% of the catalytic activity; 0.07 μmol/L Dh-OLF inhibited 17%). Stimulation of the cell culture by dibutryl cAMP increased the secretion of Dh-OLF 50% over control (unstimulated), whereas the secretion of OLF did not increase significantly.

Conclusions: OLF and Dh-OLF are secreted by human adrenal cells, and antibodies specific to these two compounds can be developed, using the plant-derived counterparts as antigens. The secretion of Dh-OLF is responsive to a cAMP-dependent stimulation mechanism, whereas OLF is not. Our data suggest that either the secretory or biosynthetic pathways for production of these two compounds by human adrenal cells may have different control mechanisms or that they may be linked via a precursor–product relationship.
Molecular and Biochemical Studies of Acute Intermittent Porphyria in 196 Patients and Their Families
Authors: Kauppinen Raili , von und zu Fraunberg Mikael
Published: January 2020

Background: Acute intermittent porphyria (AIP) is a metabolic disease with clinical manifestations that mimic other abdominal, neurologic, or mental crises. We studied the diagnostic accuracy of current laboratory tests during an acute attack and in remission.

Methods: Since 1966, we have studied all known Finnish AIP patients (n = 196) and their families (n = 45) and identified the porphobilinogen deaminase (PBGD) mutation in each family. Diagnoses or exclusions of AIP were based on clinical data (including family history), biochemical tests, and in 239 cases, mutation testing. We retrospectively evaluated the diagnostic accuracy of erythrocyte PBGD activity, urinary excretion of porphobilinogen (PBG) and δ-aminolevulinic acid, and urinary and fecal excretion of porphyrins in these patients.

Results: Measurement of urinary PBG identified all 35 AIP patients studied during an acute attack. The mean excretion of PBG was 50-fold above the reference interval, although the intraindividual increases were modest (1.6- to 4.0-fold). In the mutation-screened population, urinary PBG analysis identified only 85% of 81 AIP patients studied during remission, but by ROC curve analysis it was nonetheless the best of the biochemical tests. It was increased ≤2-fold in 29% of healthy relatives. Erythrocyte PBGD activity was decreased in only 84% of AIP patients, with results within the reference interval mainly in the variant form of AIP; it was decreased in 23% of healthy relatives.

Conclusions: Measurement of urinary PBG is the best biochemical test for AIP, although it is unspecific and does not distinguish AIP from other acute porphyrias. Because the acute increase in PBG is often modest, the medical history, signs, and symptoms must be evaluated carefully during an acute attack. In addition, because biochemical analyses often remain indeterminate in remission, mutation analysis is needed to exclude or confirm the diagnosis of AIP.
Evaluation of Coagulation Markers for Early Diagnosis of Acute Coronary Syndromes in the Emergency Room
Authors: Derhaschnig Ulla , Laggner Anton N , Röggla Martin , Hirschl Michael M , Kapiotis Stylianos , Marsik Claudia , Jilma Bernd
Published: January 2020

Background: Diagnosis of acute coronary syndromes (ACS) is a major challenge for emergency physicians. Because soluble fibrin (sF) has been suggested as a potential early marker of impending myocardial ischemia, we were interested whether a sF bedside test could help in early identification of patients with ACS in the emergency department.

Methods: We evaluated plasma coagulation markers, including a newly developed sF bedside test, prothrombin fragment (F1+2), sF, and D-dimer, in a cross-sectional trial with 184 patients suggestive of ACS.

Results: Whereas 76% (13 of 17) of patients with unstable angina pectoris (UAP) had a positive sF bedside test, only 10 of 33 patients (30%) with non-ST-segment-elevation myocardial infarction and 10 of 44 patients (23%) with ST-elevation myocardial infarction tested positive. Three percent of controls (1 of 33) and 11% of patients (6 of 57) with preexisting stable angina had a positive sF bedside test (P <0.001 for noncardiac chest pain vs ACS), yielding an overall specificity of 92% and a sensitivity of 35%. The sensitivity of the established coagulation markers was significantly less to detect ACS (11% for F1+2, 20% for thrombus precursor protein, and 18% for D-dimer; P <0.02 vs sF bedside test). The sF bedside test presented the earliest objective indicator of impending myocardial damage in the majority (10 of 13) of ACS patients with a normal or nondiagnostic electrocardiogram (ECG).

Conclusions: A sF bedside test offers a specific tool for early identification of patients with ACS in an emergency department setting, although its sensitivity seems sufficient only for the early identification of patients with UAP. A sF bedside test could be useful, particularly in UAP patients with a nondiagnostic ECG.
False-Positive Immunoassay Results: A Multicenter Survey of Erroneous Immunoassay Results from Assays of 74 Analytes in 10 Donors from 66 Laboratories in Seven Countries
Authors: Marks Vincent
Published: January 2020

Background: Analytical interference in immunoassays can produce serious errors, but it is generally considered rare with modern analytical systems.

Method: Blood was collected from 10 donors with illnesses known to be associated with rheumatoid factor. Immunoassays for 74 analytes were performed in 66 clinical laboratories. Each sample was measured in duplicate, and again in duplicate after the addition of a proprietary heterophil blocking reagent, with the laboratory’s routinely used reagents and equipment. Reagents were typically supplied by the manufacturers of the closed analytical systems. Both competitive and sandwich immunoassays were investigated.

Results: Overall ∼8.7% of the 3445 results were considered to be “false positive”, many of them seriously so. Twenty-one percent of the erroneously high results (1.8% of all results) were potentially misleading and were corrected by blocking reagent, although 49% of such seriously high results (4.2% of all results) were not. A further 39% of the false-positive results (2.6% of all results) would not necessarily have appeared likely to produce adverse clinical consequences but were substantially lowered by the addition of the blocking reagent.

Conclusions: Random errors can occur with all types of immunoassays tested and can be difficult to identify even when repeated in another laboratory. Clinicians need to be aware of these limitations.
Wrong Biochemistry Results: Two Case Reports and Observational Study in 5310 Patients on Potentially Misleading Thyroid-stimulating Hormone and Gonadotropin Immunoassay Results
Authors: Ismail Adel AA , Walker Paul L , Barth Julian H , Lewandowski Kryzsztof C , Jones Rick , Burr William A
Published: January 2020

Background: Immunoassays are used in almost all medical and surgical specialties, but they suffer from interference from proteins such as antibodies in some patients’ sera. Such interferences are usually reported in the literature only as case reports after the introduction of a new assay.

Methods: We undertook a prospective observational study on 5310 patients for whom the common immunoassay tests for thyroid-stimulating hormone (TSH) and/or gonadotropins were requested. All TSH and gonadotropin results were critically assessed for a mismatch between the clinical details and analytical results to identify samples suspected of analytical unreliability. These were tested further by three approaches to screen for interference.

Results: From the 5310 sets of results, 59 patients’ samples were identified as suspect and were tested further. Analytically incorrect results were found in 28 (0.53% of the total studied). The magnitude of interference varied, but in 23 of 28 patients (82%), it was considered large enough to have a potentially adverse effect on cost and/or the clinical care of these patients. Two cases, described in detail, illustrate the adverse effect of error on patient care and cost, and the second highlights the difficulties and limitations of current approaches for identifying interference and inaccuracy in immunoassays.

Conclusions: Because millions of TSH/gonadotropin tests are carried out in UK hospital laboratories alone, our data suggest that thousands of patients could be adversely affected by errors from interferences. Early identification of interference in cases with unusual results could be valuable.
Pyrosequencing as an Alternative to Single-Strand Conformation Polymorphism Analysis for Detection of N-ras Mutations in Human Melanoma Metastases
Authors: Sivertsson Åsa , Platz Anton , Hansson Johan , Lundeberg Joakim
Published: January 2020

Background: Mutations in codons 12, 13, and 61 of the N-ras gene are common alterations in cutaneous malignant melanoma. We evaluated pyrosequencing, a simple and rapid method used mainly for single-nucleotide polymorphism analysis, as a possible alternative to single-strand conformation polymorphism (SSCP) analysis and sequencing of N-ras.

Methods: We evaluated the sensitivity and accuracy of the pyrosequencing method for identification of mutations in N-ras codons 12, 13, and 61. Nucleotide dispensation orders were created to produce distinct pyrogram peak profiles for the most frequent mutations in codon 61 and codons 12 and 13, respectively.

Results: The detection limits for the two most common codon 61 mutations found in malignant melanoma, which code for Arg and Lys, were 30% and 15%, respectively. To evaluate the pyrosequencing method on clinical samples, we performed a parallel analysis of 82 melanoma metastases using SSCP analysis and pyrosequencing. All mutations detected by SSCP analysis and confirmed by sequencing were also correctly identified by pyrosequencing. Codon 61 mutations were identified in 26 of the 82 samples (32%), whereas no mutations were found in codons 12 and 13. Four types of codon 61 mutations, Arg (17%), Lys (10%), Leu (4%), and His (1%), were identified.

Conclusion: Pyrosequencing is an attractive alternative to SSCP analysis for N-ras mutation detection in malignant melanoma tumor samples because it displays the same sensitivity and accuracy as SSCP analysis and is simple and rapid.
Detection of Specific Antinuclear Reactivities in Patients with Negative Anti-nuclear Antibody Immunofluorescence Screening Tests
Authors: Hoffman Ilse EA , Peene Isabelle , Veys Eric M , De Keyser Filip
Published: January 2020

Background: For detection of anti-nuclear antibodies (ANAs) and antibodies to extractable nuclear antigens (ENAs), samples frequently are screened with indirect immunofluorescence (IIF); further determination of anti-ENA antibodies is performed only when the result is positive. However, because anti-ENA reactivities are found in samples with low fluorescence intensities, we determined anti-ENA antibodies in samples with negative IIF and thus calculated the sensitivity of IIF for specific ANAs.

Methods: We collected 494 samples consecutively referred by rheumatologists for routine ANA testing. IIF on HEp-2 and HEp-2000 (HEp-2 cells transfected with Ro60 cDNA) and line immunoassay (LIA) for the detection of specific ANAs were performed on all samples.

Results: Fluorescence intensities and patterns on HEp-2 were strongly correlated with those on HEp-2000 [Spearman ρ = 0.852 (P <0.001) and 0.838 (P <0.001), respectively]. Sixty-eight of 494 samples were positive on LIA, of which only 72% (confidence interval, 68–76%) were detected with HEp-2 and 75% (confidence interval, 70–78%) with HEp-2000. Of 291 samples negative on both substrates, 12 were positive on LIA. Connective tissue diseases were diagnosed in four of these patients and suspected in at least three others.

Conclusion: The HEp-2 and HEp-2000 substrates perform comparably for fluorescence intensities and patterns and for detecting specific ANAs, but some patients with negative IIF show reactivity on LIA. We recommend testing for fine reactivities, regardless of the IIF result, when the clinical suspicion for rheumatic connective tissue disease is high.
Carbohydrate-deficient Transferrin Isoforms Measured by Capillary Zone Electrophoresis for Detection of Alcohol Abuse
Authors: Legros Franz J , Nuyens Vincent , Minet Eddy , Emonts Philippe , Boudjeltia Karim Zouaoui , Courbe Anne , Ruelle Jean-Luc , Colicis Jacques , de L’Escaille Francois , Henry Jean-Pol
Published: January 2020

Background: Measurements of carbohydrate-deficient transferrin (CDT) are used as markers of alcohol abuse. We developed a capillary zone electrophoresis (CZE) method aimed at improving accuracy of CDT testing.

Methods: We studied 111 alcohol abusers with Alcohol Use Disorders Identification Test scores >11 and 50 teetotalers. CZE was performed with a P/ACE 5500, fused-silica capillaries, and a CEofix CDT reagent set. After iron saturation, sera were loaded by low-pressure injection, separated at 28 kV, and monitored at 214 nm. We identified the transferrin isoforms by migration times, treatment with 100 U/L neuraminidase, and immunosubtraction with anti-human transferrin and anti-C-reactive protein antibodies. We compared CZE results with current biological markers of alcohol abuse, including the %CDT turbidimetric immunoassay.

Results: Migration times of the isoforms were identical in both populations. Asialotransferrin was missing in teetotalers but present in 92% of alcohol abusers. Disialotransferrin was higher in those who consumed excessive amounts of alcohol, whereas mean trisialotransferrin concentration was not affected by alcohol abuse. At cutoffs to maximize sensitivity and specificity, these values were 0.92 and 1 [mean ROC area (MRa), 0.96; 95% confidence interval (CI), 0.93–0.99] for asialotransferrin; 0.84 and 0.94 for the sum of asialo- + disialotransferrin (MRa, 0.94; 95% CI, 0.91–0.98); 0.79 and 0.94 for disialotransferrin (MRa, 0.89; 95% CI, 0.84–0.94); 0.62 and 0.53 for trisialotransferrin (MRa, 0.58; 95% CI, 0.49–0.68); 0.79 and 0.82 for a 3% %CDT; and 0.83 and 0.69 for a 2.6% cutoff (MRa, 0.87; 95% CI, 0.81–0.92). Current markers lack sensitivity (<0.65). Transferrins were not significantly correlated with serum enzymes and mean erythrocyte volume.

Conclusions: CZE-isolated desialylated transferrin isoforms allowed differentiation between chronic alcohol abusers and teetotalers.
Quantitative Evaluation of Urinalysis Test Strips
Authors: Penders Joris , Fiers Tom , Delanghe Joris R
Published: January 2020

Background: Urine test strip results are generally reported in categories (i.e., ordinal scaled), but automated strip readers are now available that can report quantitative data. We investigated the possible use of these meters to complement flow cytometry of urine and compared reflectance readings with quantitative determinations of urinary glucose and microalbumin.

Methods: We compared URISYS 2400 (Roche) quantitative reflectance data with data from the UF-100 (Sysmex) and biochemical data for 436 nonpathologic and pathologic urine samples.

Results: Reproducibility of the reflectance signal was good for high- and low-concentration urine pools for protein (0.8% and 0.9% and 1.5% and 2.2% within and between runs, respectively), leukocyte esterase (1.1% and 1.0%; 5.1% and 1.2%), hemoglobin (1.7% and 1.1%; 8.9% and 1.1%) and glucose (2.1% and 0.5%; 6.5% and 2.3%). Fair agreement was obtained between UF-100 and test strip reflectance data for erythrocytes and hemoglobin (r = −0.680) and leukocytes and leukocyte esterase (r = −0.688). Higher correlations were observed for biochemical and test strip data comparing protein and albumin (r = −0.825) and glucose data (r = −0.851). The lower limits of detection for erythrocytes and leukocytes were 8 × 106/L and 19 × 106/L, respectively. The protein test (n = 220) detected 86% (95% confidence interval, 78–92%) of samples with <30 mg/L albumin with a specificity of 84% (95% confidence interval, 76–91%).

Conclusions: In urine test strip analysis, quantitative hemoglobin and leukocyte esterase reflectance data are complementary with flow cytometric results and glucose and albumin results.
Insulin: Discovery and Controversy
Authors: Rosenfeld Louis
Published: January 2020

During the first two decades of the 20th century, several investigators prepared extracts of pancreas that were often successful in lowering blood sugar and reducing glycosuria in test animals. However, they were unable to remove impurities, and toxic reactions prevented its use in humans with diabetes. In the spring of 1921, Frederick G. Banting, a young Ontario orthopedic surgeon, was given laboratory space by J.J.R. Macleod, the head of physiology at the University of Toronto, to investigate the function of the pancreatic islets. A student assistant, Charles Best, and an allotment of dogs were provided to test Banting’s hypothesis that ligation of the pancreatic ducts before extraction of the pancreas, destroys the enzyme-secreting parts, whereas the islets of Langerhans, which were believed to produce an internal secretion regulating sugar metabolism, remained intact. He believed that earlier failures were attributable to the destructive action of trypsin. The name “insuline” had been introduced in 1909 for this hypothetic substance. Their experiments produced an extract of pancreas that reduced the hyperglycemia and glycosuria in dogs made diabetic by the removal of their pancreases. They next developed a procedure for extraction from the entire pancreas without the need for duct ligation. This extract, now made from whole beef pancreas, was successful for treating humans with diabetes. Facilitating their success was a development in clinical chemistry that allowed blood sugar to be frequently and accurately determined in small volumes of blood. Success with purification was largely the work of J.B. Collip. Yield and standardization were improved by cooperation with Eli Lilly and Company. When the Nobel Prize was awarded to Banting and Macleod for the discovery of insulin, it aggravated the contentious relationship that had developed between them during the course of the investigation. Banting was outraged that Macleod and not Best had been selected, and he briefly threatened to refuse the award. He immediately announced that he was giving one-half of his share of the prize money to Best and publicly acknowledged Best’s contribution to the discovery of insulin. Macleod followed suit and gave one-half of his money award to Collip. Years later, the official history of the Nobel Committee admitted that Best should have been awarded a share of the prize.
Analysis of Catecholamines in Urine by Positive-Ion Electrospray Tandem Mass Spectrometry
Authors: Kushnir Mark M , Urry Francis M , Frank Elizabeth L , Roberts William L , Shushan Bori
Published: January 2020

Background: Determination of urinary catecholamines (CATs) is considered important for clinical diagnosis of pheochromocytoma, paraganglioma, and neuroblastoma. The major disadvantages of existing tests include relatively long instrumental analysis time and potential interference from drugs and drug metabolites that are structurally similar to CATs.

Methods: CATs were extracted from a 300-μL aliquot of urine by a two-step liquid-liquid extraction method specific for compounds containing a catechol group. Chromatographic separation did not require the use of ion-pairing reagents, which typically hinder MS detection but are frequently used in HPLC analysis of CATs. Instrumental analysis was performed by electrospray ionization tandem mass spectrometry (ESI-MS/MS) in the multiple-reaction monitoring mode. Stable-isotope-labeled CATs were used as internal standards.

Results: Epinephrine (E), norepinephrine (NE), and dopamine (D) were measured within 3.5 min instrumental run time. Quantification limits were 2.5 μg/L for E and D and 10 μg/L for NE. The total imprecision (CV) was ≤9.6%; extraction recoveries were 71% ± 12%.

Conclusions: HPLC with ESI-MS/MS in combination with sample preparation specific to catechol group-containing compounds allows rapid testing for disorders associated with increased CAT concentrations. The method is free of interferences from drugs and drug metabolites, which commonly interfere with HPLC methods.
Point-of-Care Time-resolved Immunofluorometric Assay for Human Pregnancy-associated Plasma Protein A: Use in First-Trimester Screening for Down Syndrome
Authors: Qin Qiu-Ping , Christiansen Michael , Pettersson Kim
Published: January 2020

Background: Screening for Down syndrome in the first trimester by a combination of fetal nuchal translucency thickness and maternal serum pregnancy-associated plasma protein A (PAPP-A) and free β-human chorionic gonadotropin has been shown to be effective and efficient. We aimed to develop a fast point-of-care assay that could be placed in one-stop clinics for the measurement of PAPP-A.

Methods: We developed a two-site, one-step assay that uses two monoclonal antibodies (mAbs) to PAPP-A, based on a dry-reagent, all-in-one immunoassay concept with a stable fluorescent lanthanide chelate and time-resolved fluorometry. One antibody (mAb 10E1) was biotinylated, and the other (mAb 234-5) was europium-labeled, both via the ε-amino groups of surface lysine residues. The assay was performed on an AIO immunoanalyzer at 36 °C in single, streptavidin-coated microtitration wells that contained the dry reagents. PAPP-A, either in free or complexed form, was detected by the antibodies used.

Results: The assay procedure required 20 min and used 10 μL of sample. The calibration curve was linear from 5 to 10 000 mIU/L. The detection limit was 0.5 mIU/L. Intra- and interassay imprecision (CV) was ≤4.3% and 8.3%, respectively, for whole blood, plasma, or serum samples. Recovery was 93–96% for serum, 95–108% for heparin-derived whole blood, and 98–103% for heparin-derived plasma. Parallelism was observed in all three matrices. Results correlated [slope = 0.85 (confidence interval, 0.82–0.87); intercept = −33 (confidence interval, −58 to −9); Sy|x = 85 mIU/L; r = 0.991; n = 100] with those obtained by a Delfia assay. Heparin did not affect the assay, but EDTA markedly reduced PAPP-A values. PAPP-A was stable at 4 °C for at least 18 days in serum and for 8 days in heparin-derived whole blood or plasma.

Conclusions: The present assay appears suited for use in one-stop clinics for screening for Down syndrome in the first trimester, with results available within 1 h.
Performance of Four Homogeneous Direct Methods for LDL-Cholesterol
Authors: Miller W Greg , Waymack Parvin P , Anderson F Philip , Ethridge Steven F , Jayne Eduviges C
Published: January 2020

Background: Homogeneous LDL-cholesterol methods from Genzyme, Reference Diagnostics, Roche, and Sigma were evaluated for precision, accuracy, and specificity for LDL in the presence of abnormal lipoproteins.

Methods: Each homogeneous method was performed by a Roche/Hitachi 911 according to the vendors’ instructions, and the results were compared with the β-quantification reference method. We measured precision over 20 days using quality-control and frozen serum specimens. Sera from 100 study participants, including 60 with hyperlipidemias, were assayed by each method. Accuracy was evaluated from regression and total error analysis. Specificity was evaluated from the bias (as a percentage) vs concentration of triglycerides.

Results: The total CV was <2% for all methods. Regression slope and intercept (with 95% confidence intervals) were as follows: Genzyme, 0.955 (0.92 to 0.99) and 30.3 (−12 to 73) mg/L; Reference Diagnostics, 0.975 (0.93 to 1.02) and −8 (−63 to 47) mg/L; Roche, 1.067 (1.02 to 1.11) and −101 (−161 to −42) mg/L; and Sigma, 0.964 (0.91 to 1.02) and 164 (89 to 239) mg/L. The percentages of individual results with >12% bias were as follows: Genzyme, 8.0%; Reference Diagnostics, 11.0%; Roche, 10.0%; and Sigma, 30.0%. Total error calculated from mean systematic bias and all-sources random bias was as follows: Genzyme, 12.6%; Reference Diagnostics, 16.5%; Roche, 41.6%; and Sigma, 38.3%. Slopes of bias (as a percentage) vs triglycerides were P <0.001 for all methods except the Roche method, which was P = 0.094.

Conclusions: The evaluated methods show nonspecificity toward abnormal lipoproteins, thus compromising their ability to satisfy the National Cholesterol Education Program goal for a total error of <12%. These homogeneous LDL-cholesterol results do not improve on the performance of LDL-cholesterol calculated by the Friedewald equation at triglyceride concentrations <4000 mg/L.
Immunometric Assay Interference
Authors: Bjerner Johan , Nustad Kjell , Norum Lars F , Olsen Kari Hauge , Børmer Ole P
Published: January 2020

Background: The primary aim of the study was to reduce interference in an in-house two-site, two-step immunometric assay.

Methods: In the running laboratory routine, 11 261 samples were tested with a carcinoembryonic antigen (CEA) assay with bovine immunoglobulin but no murine immunoglobulins in the buffer, in parallel to our routine CEA assay, using 15 mg/L heat-treated nonspecific murine immunoglobulin (MAK33) in the buffer and with the Fc fragments removed from the capture antibody.

Results: The frequency of interference was estimated to be 4.0% (95% confidence interval, 3.3–4.7%). The addition of 15 mg/L native MAK33 had little effect (frequency, 3.9%; 95% confidence interval, 3.2–4.6%), whereas adding 15 mg/L heat-treated MAK33 reduced interference to 0.86% (0.61–1.12%), and adding 50 mg/L reduced it further to 0.06% (0–0.13%). Removing the Fc fragments by itself reduced interference to 0.10% (0.02–0.19%). There were no statistically significant differences for age (P <0.23) or gender (P <0.40) between patients with interference (n = 210) and a randomly selected interference-negative control group (n = 186). Interference was not constant in patients: 15 of 25 individuals positive for interference and with four or more samples screened for interference had an interference-negative sample either before or after the peak of interference.

Conclusions: In a two-site, two-step immunometric assay using mouse monoclonal antibodies, use of heat-treated nonspecific murine immunoglobulin in the buffer or removal of the Fc fragment from the capture antibody could improve performance.
Candidate Reference Method for Total Thyroxine in Human Serum
Authors: Tai Susan S-C , Sniegoski Lorna T , Welch Michael J
Published: January 2020

Background: There is a need for a critically evaluated reference method for thyroxine to provide an accuracy base to which routine methods can be traceable. We describe a candidate reference method involving isotope-dilution coupled with liquid chromatography/mass spectrometry.

Methods: An isotopically labeled internal standard, thyroxine-d5, was added to serum, followed by equilibration, protein precipitation, and ethyl acetate and solid-phase extractions to prepare samples for liquid chromatography-mass spectrometry electrospray ionization (LC/MS-ESI) analysis. For separation, a Zorbax Eclipse XDB-C18 column was used with a mobile phase consisting of 1 mL/L acetic acid in acetonitrile-water (32:68 by volume) for positive ions and a Zorbax Extend-C18 column with a mobile phase consisting of 2 mL/L ammonium hydroxide in methanol-water (32:68 by volume) for negative ions. [M + H]+ ions at m/z 778 and 783 for thyroxine and its labeled internal standard were monitored for positive ions and [M − H]− ions at m/z 776 and 781 for negative ions. Samples of frozen serum pools were prepared and measured in three separate sets.

Results: Within-set CVs were 0.2–1.0%. The correlation coefficients of all linear regression lines (measured intensity ratios vs mass ratios) were 0.999–1.000. Positive- and negative-ion measurements agreed with a mean difference of 0.45% at three concentrations (50, 110, and 168 μg/L). The detection limits (at a signal-to-noise ratio of ∼3 to 5) were 30 and 20 pg for positive and negative ions, respectively. The results from the LC/MS-ESI method were within 1 SD of the composite means from many routine clinical methods, although it appears that the clinical method means may be biased high by 4–5 μg/L across the concentrations. Some routine clinical methods may be biased by up to 20% at low concentrations.

Conclusions: This well-characterized LC/MS-ESI method for total serum thyroxine with a theoretically sound approach, demonstrated good accuracy and precision, and low susceptibility to interferences qualifies as a candidate reference method. Use of this reference method as an accuracy base may reduce the apparent biases in routine methods along with the high interlaboratory imprecision.
Use of Constant Denaturant Capillary Electrophoresis of Pooled Blood Samples to Identify Single-Nucleotide Polymorphisms in the Genes (Scnn1a and Scnn1b) Encoding the α and β Subunits of the Epithelial Sodium Channel
Authors: Xue Ming-Zhan , Bonny Olivier , Morgenthaler Stephan , Bochud Murielle , Mooser Vincent , Thilly William G , Schild Laurent , Leong-Morgenthaler Phaik-Mooi
Published: January 2020

Background: The epithelial sodium channel (ENaC) is composed of three homologous subunits: α, β, and γ. Mutations in the Scnn1b and Scnn1g genes, which encode the β and the γ subunits of ENaC, cause a severe form of hypertension (Liddle syndrome). The contribution of genetic variants within the Scnn1a gene, which codes for the α subunit, has not been investigated.

Methods: We screened for mutations in the COOH termini of the α and β subunits of ENaC. Blood from 184 individuals from 31 families participating in a study on the genetics of hypertension were analyzed. Exons 13 of Scnn1a and Scnn1b, which encode the second transmembrane segment and the COOH termini of α- and β-ENaC, respectively, were amplified from pooled DNA samples of members of each family by PCR. Constant denaturant capillary electrophoresis (CDCE) was used to detect mutations in PCR products of the pooled DNA samples.

Results: The detection limit of CDCE for ENaC variants was 1%, indicating that all members of any family or up to 100 individuals can be analyzed in one CDCE run. CDCE profiles of the COOH terminus of α-ENaC in pooled family members showed that the 31 families belonged to four groups and identified families with genetic variants. Using this approach, we analyzed 31 rather than 184 samples. Individual CDCE analysis of members from families with different pooled CDCE profiles revealed five genotypes containing 1853G→T and 1987A→G polymorphisms. The presence of the mutations was confirmed by DNA sequencing. For the COOH terminus of β-ENaC, only one family showed a different CDCE profile. Two members of this family (n = 5) were heterozygous at 1781C→T (T594M).

Conclusion: CDCE rapidly detects point mutations in these candidate disease genes.
Blood Glutathione Disulfide: In Vivo Factor or in Vitro Artifact?
Authors: Rossi Ranieri , Milzani Aldo , Dalle-Donne Isabella , Giustarini Daniela , Lusini Lorenzo , Colombo Roberto , Di Simplicio Paolo
Published: January 2020

Background: The reported mean concentration of glutathione disulfide (GSSG) in human blood/erythrocytes varies widely (1 to >500 μmol/L), as does that of reduced glutathione (GSH) to a lesser extent. We have identified and investigated possible pitfalls in measurement of both GSH and GSSG.

Methods: We measured GSH and GSSG using a spectrophotometer with a modification of the GSH recycling method; the same samples were also measured by reversed-phase HPLC after derivatization of thiols (dithiothreitol was used to reduce disulfides) with monobromobimane. The thiol-bimane adduct was measured by a fluorescence detector.

Results: Measured GSH/GSSG concentrations were affected by the following: (a) oxidation of thiols in acidified samples; (b) oxidation after restoring neutral-alkaline pH; (c) oxidation during acid deproteinization; (d) shift in the GSH/GSSG equilibrium because of irreversible blocking of free thiols; and (e) reaction of electrophiles with amino groups. In particular, oxidation during sample deproteinization with acid influenced and produced artifacts (30–150 μmol/L GSSG was produced by this procedure); this phenomenon was directly correlated with the presence of oxygenated hemoglobin, being minimized by both oxygen deprivation and incubation in an atmosphere of 5% carbon monoxide.

Conclusions: GSSG is present in healthy human blood at low concentrations (2–6 μmol/L), and most published data on GSSG may be affected by artifacts.