bluuebunny/crossref_metadata_2025 huggingface dataset:
Seventy years of progestagen treatments for management of the sheep oestrous cycle: where we are and where we should go
Authors: Gonzalez-Bulnes Antonio , Menchaca Alejo , Martin Graeme B. , Martinez-Ros Paula
Published: January 2020

Management of the ovine oestrous cycle is mainly based on the use of exogenous hormones to mimic or enhance (progesterone and its analogues) or manipulate (prostaglandin F2α and its analogues) the activity of the corpus luteum, combined with the application of other hormones mimicking the pituitary secretion of gonadotrophins (e.g. equine chorionic gonadotrophin). These protocols have been applied without major change for decades but, now, there are two reasons to reconsider them: (1) our greatly improved knowledge of the dynamics of ovarian physiology, following the application of transrectal ultrasonography, indicates that modification of the protocols may improve fertility yields and (2) increasing concerns about animal health and welfare, food safety and the environmental impact of the treatments, as evidenced by public opinion and therefore market forces. Here, we offer an overview of these issues, introduce an updated protocol and suggest ways for future improvements to the protocols.
The relative pronoun strategy
Authors: Siegel Jeff
Published: January 2020

The Relative Pronoun strategy is commonly used for relativization in European languages such as English – for
example: The woman [
who won the lottery ] is my neighbour. In
this strategy the head nominal (here the woman) is indicated inside the relative clause by a clause-initial
pronominal element (the relative pronoun, here who). The Relative Pronoun strategy has been characterized as an
exclusively European areal feature (e.g. Comrie 1998). This article describes this
strategy in more detail, as well as previous accounts of its distribution, and goes on to demonstrate that the same strategy is
also found in Nama, a Papuan language of southern New Guinea.
Identifying and removing haplotypic duplication in primary genome assemblies
Authors: Guan Dengfeng , McCarthy Shane A , Wood Jonathan , Howe Kerstin , Wang Yadong , Durbin Richard
Published: January 2020

Motivation

Rapid development in long-read sequencing and scaffolding technologies is accelerating the production of reference-quality assemblies for large eukaryotic genomes. However, haplotype divergence in regions of high heterozygosity often results in assemblers creating two copies rather than one copy of a region, leading to breaks in contiguity and compromising downstream steps such as gene annotation. Several tools have been developed to resolve this problem. However, they either focus only on removing contained duplicate regions, also known as haplotigs, or fail to use all the relevant information and hence make errors.

Results

Here we present a novel tool, purge_dups, that uses sequence similarity and read depth to automatically identify and remove both haplotigs and heterozygous overlaps. In comparison with current tools, we demonstrate that purge_dups can reduce heterozygous duplication and increase assembly continuity while maintaining completeness of the primary assembly. Moreover, purge_dups is fully automatic and can easily be integrated into assembly pipelines.

Availability and implementation

The source code is written in C and is available at https://github.com/dfguan/purge_dups.

Supplementary information

Supplementary data are available at Bioinformatics online.
Ascorbic acid determination with an automated enzymatic procedure
Authors: Lee Winnie , Roberts Susan M , Labbe Robert F
Published: January 2020

Given the widespread interest in antioxidant nutrients, we have developed a new procedure that will permit the automated determination of plasma ascorbic acid (AA) concentration with a Roche Fara centrifugal analyzer. After the deproteinization of plasma with metaphosphoric acid, AA is oxidized to dehydroascorbic acid by AA oxidase. The product is coupled to o-phenylenediamine to produce a chromophore for which the absorbance is measured at 340 nm. The procedure allows much faster throughput than conventional HPLC methods while yielding results that correlate well and provide improved precision. Linearity extends beyond the reference range of 26.1–84.6 μmol/L, and severe hemolysis is the only interference identified.
Direct detection of mutations in the breast and ovarian cancer susceptibility gene BRCA1 by PCR-mediated site-directed mutagenesis
Authors: Rohlfs Elizabeth M , Learning William G , Friedman Kenneth J , Couch Fergus J , Weber Barbara L , Silverman Lawrence M
Published: January 2020

The tumor suppressor genes BRCA1 and BRCA2, which confer increased susceptibility to breast and (or) ovarian cancer, were recently identified. Mutation analysis of BRCA1 has demonstrated significant allelic heterogeneity; however, some distinct mutations have been detected in unrelated individuals. The most notable is the 185delAG mutation, which occurs at an estimated frequency of ∼1% in individuals of Ashkenazi Jewish descent [1]. Although consensus has not been reached regarding clinical testing for mutations in BRCA1, a tiered strategy may be appropriate, in which direct testing for the more common mutations is one component. Specific alleles can be detected by using PCR-mediated site-directed mutagenesis (PSM), which alters the PCR products derived from either the wild-type or mutant allele to create or destroy a restriction endonuclease recognition site. Recognition sites are introduced by a base substitution in one of the primers. The alleles are then resolved by electrophoresis of the digested PCR products. We have applied this technique to the detection of four BRCA1 mutations: 185delAG, 5382insC, E1250X, and R1443X. Another mutation, 1294del40, can be resolved from the wild-type allele by high-resolution gel electrophoresis alone. The PSM technique is sensitive, does not require radioactivity, and is specific for individual mutations.
Cation-exchange HPLC evaluated for presumptive identification of hemoglobin variants
Authors: Riou Jean , Godart Christian , Hurtrel Didier , Mathis Mireille , Bimet Catherine , Bardakdjian-Michau Josiane , Préhu Claude , Wajcman Henri , Galactéros Frédéric
Published: January 2020

A battery of relatively simple tests allows the presumptive identification of hemoglobin (Hb) variants, making unnecessary structural analysis by protein chemistry methods or DNA sequencing. The primary step in this strategy involves the use of a matrix of electrophoretic mobilities obtained under various experimental conditions. This leads to an unambiguous result in ∼90% of the cases. Additional tests are required to characterize with more confidence the remaining 10%. We describe here the use of cation-exchange HPLC on the Bio-Rad Variant automated analyzer with the “β Thalassemia Short” program. By comparing the elution time of 125 human Hb mutants, we found that some variants with almost identical pI values or produced by the same type of amino acid substitution displayed different elution times. We present several examples in which use of the HPLC profile helped establish the diagnosis.
Sensitive bioaffinity assays with individual microparticles and time-resolved fluorometry
Authors: Lövgren Timo , Heinonen Pia , Lehtinen Päivi , Hakala Harri , Heinola Johanna , Harju Raimo , Takalo Harri , Mukkala Veli-Matti , Schmid Ruth , Lönnberg Harri , Pettersson Kim , Iitiä Antti
Published: January 2020

Future immunoassays and nucleic acid hybridization assays will be performed in miniaturized formats that utilize microchips or microparticles. This will require a sensitive detection technology that allows spatial resolution. By using fluorescent europium chelates and time-resolved microfluorometry, one can detect 11 000 europium molecules on individual microparticles. In a miniaturized noncompetitive immunoassay of prostate-specific antigen (PSA), we quantitatively detected 5 ng/L (0.05 amol per particle) of the analyte on an individual microparticle with excellent precision over the whole measurement range (CV <10%). Using a hybridization assay, we also could detect the ΔF508 mutation for cystic fibrosis on individual microparticles. Consequently, fluorescent lanthanide chelate labels and time-resolved microfluorometry qualify as the next generation of technology in this field.
Immunoassay of human chorionic gonadotropin, its free subunits, and metabolites
Authors: Cole Laurence A
Published: January 2020

Multiple hCG-related molecules are present in pregnancy serum and urine samples. These include nonnicked hCG (the hormone), nicked hCG, hyper- and hypoglycosylated hCG, hCG missing the C-terminal extension, free α-subunit, large free α-subunit, free β-subunit, nicked free β-subunit, and β-core fragment. Over 100 immunoassays are sold for quantifying hCG-related molecules in serum or urine. Each measures nonnicked hCG and one of seven combinations of the other hCG-related molecules. This is the source of interassay discordance in hCG determinations. Whereas minor variations are noted in different kit results in normal pregnancy samples (more than twofold variation), much larger variations may be found in two immunoassay results in irregular gestations (spontaneous abortion, aneuploidy, preeclampsia, cancers, and trophoblast disease). Care is needed in choosing an immunoassay. What the assay measures may be more important than its cost or speed. This article reviews the structure of hCG and related molecules. It examines the stability and degradation of hCG, and recognition of hCG-related molecules by different types of immunoassay. Also reviewed are new assays for specifically detecting these other hCG-related molecules.
Basal concentrations of free and esterified monohydroxylated fatty acids in human blood platelets
Authors: Guichardant Michel , Thevenon Chantal , Pageaux Jean Francois , Lagarde Michel
Published: January 2020

Monohydroxylated fatty acids (HO-FA), namely 12-hydroxyeicosatetraenoic and 12-hydroxyheptadecatrienoic acids, are enzymatically formed in response to platelet activation. Different techniques, including gas chromatography (GC) and liquid chromatography–mass spectrometry (LC-MS), have been described to measure HO-FA in activated cells, but they are not well-adapted to resting cells. Measurements of free and esterified HO-FA at basal concentration require the prevention of platelet activation. For this purpose, such an activation was minimized by adding various inhibitors to the anticoagulant. Platelet recovery was greater in the protected group than in controls (473 × 109 ± 4.0 × 109 platelets/L vs 410 × 109 ± 4.53 × 109 platelets/L, respectively) (mean ± SEM, n = 9, P <0.05). Lipids were extracted and immediately hydrogenated to avoid fatty acid autoxidation occurring during the workup. Unesterified and esterified HO-FA were analyzed by GC-MS, and the former were lower in the protected group (1.52 ± 0.84 pmol/109 platelets) than in the unprotected one (12.63 ± 10.52 pmol/109 platelets) (mean ± SEM, n = 9, P <0.05). Interestingly, only traces of HO-FA were detected in both the triglyceride and sterol ester fractions, and they were also weakly esterified in phospholipids.
Competitive enzyme immunoassay with monoclonal antibody for homovanillic acid measurement in human urine samples
Authors: Taran Frédéric , Frobert Yveline , Créminon Christophe , Grassi Jacques , Olichon Didier , Mioskowski Charles , Pradelles Philippe
Published: January 2020

A fast competitive enzyme immunoassay (EIA) for measuring homovanillic acid in human urine samples was developed with a monoclonal antibody and acetylcholinesterase as enzyme label. Enzyme detection was performed by an easy colorimetric assay. Monoclonal antibodies were screened on the basis of sensitivity, specificity, and correlation studies. EIA has a detection limit of 0.5 μmol/L, a CV <10% in the 1.25–10 μmol/L range, and intra- and interassay CVs of <10%. Cross-reactivity with vanillylmandelic acid was 0.5% and <8% for other structurally related catecholamine metabolites. Parallelism of the EIA was shown in dilution studies and the correlation with routine HPLC assay in 62 normal and pathologic samples was EIA = 1.492 (HPLC) − 3.46, Sy|x = 47.52, range = 4–1800 μmol/L, r2 = 0.977. Additional data concerning the validity of this assay were provided by HPLC analysis of urinary immunoreactive material.
Plasma mitomycin C concentrations determined by HPLC coupled to solid-phase extraction
Authors: Paroni Rita , Arcelloni Cinzia , De Vecchi Elena , Fermo Isabella , Mauri Davide , Colombo Renzo
Published: January 2020

The aim of this study was to set up a method for quantification of plasma mitomycin C (MMC) concentrations during intravesical chemotherapy delivered in the presence of local bladder hyperthermia (HT). In comparison with existing methods, this assay, characterized by relative simplicity and efficiency, resulted in the facilitation of performance with nondedicated instrumentation or nonspecialized staff. Purification from plasma matrix was carried out by solid-phase extraction under vacuum. The purified drug was then collected directly into the vials of the HPLC autosampler. Chromatographic analysis was performed on a reversed-phase C18 column with water:acetonitrile (85:15 by vol) as the mobile phase and the UV detector set at 365 nm. The use of porfiromycin as internal standard provided a method with good within-day precision (CV 6.0% at 5 μg/L, n = 6), linearity (0.5–50 μg/L), and specificity. The lower limit of detection (≤0.5 μg/L) proved to be suitable for plasma pharmacokinetics monitoring in two tested patients treated with MMC+HT for superficial bladder cancer.
Capillary electrophoresis system for hemoglobin A1c determinations evaluated
Authors: Doelman Cees J A , Siebelder Carla W M , Nijhof Wim A , Weykamp Cas W , Janssens Jacques , Penders Theo J
Published: January 2020

Hb A1c is the analyte of choice for monitoring metabolic control in patients with diabetes mellitus. Here we present a new analytical technique for measuring Hb A1c, capillary electrophoresis. The Hb A1c determination is not influenced by the labile Hb A1c fraction or by carbamylated or acetylated hemoglobin derivatives. Also, hemoglobin variants (Hb F, Hb S, and Hb C) do not interfere. This new application of capillary electrophoresis seems to be a valuable analytical tool for measuring Hb A1c in the clinical laboratory.
Automated measurement of mouse apolipoprotein B: convenient screening tool for mouse models of atherosclerosis
Authors: Levine Daniel M , Williams Kevin Jon
Published: January 2020

Although mice are commonly used for studies of atherosclerosis, investigators have had no convenient way to quantify apolipoprotein (apo) B, the major protein of atherogenic lipoproteins, in this model. We now report an automated immunoturbidimetric assay for mouse apo B with an NCCLS imprecision study CV <5%. Added hemoglobin up to 50 g/L did not interfere with the assay, nor did one freeze–thaw cycle of serum samples. Assay linearity extends to apo B concentrations of 325 mg/L. We have used the assay to determine serum apo B concentrations under several atherogenic conditions, including the apo E “knock-out” genotype and treatment with a high-cholesterol diet. Our assay can be used to survey inbred mouse strains for variants in apo B concentration or regulation. Moreover, the mouse can now be used as a convenient small-animal model to screen compounds that may lower apo B concentrations.
Plasma malondialdehyde as biomarker for oxidative stress: reference interval and effects of life-style factors
Authors: Nielsen Flemming , Mikkelsen Bo Borg , Nielsen Jesper Bo , Andersen Helle Raun , Grandjean Philippe
Published: January 2020

Malondialdehyde (MDA) is one of the most frequently used indicators of lipid peroxidation. To generate reliable reference intervals for plasma malondialdehyde (P-MDA), a reference sample group was established in Funen, Denmark. The group consisted of 213 individuals (107 men, 106 women), ages 20–79 years. P-MDA was measured in EDTA-treated plasma after derivatization by thiobarbituric acid (TBA) and separation on HPLC. UV detection was performed at 532 nm. A reference interval was calculated as recommended by IFCC with REFVAL 3.42. The estimated reference limits (0.025 and 0.975 fractals) for the group were 0.36 and 1.24 μmol/L. The data were analyzed for gender- and age-related differences. Analysis of variance showed no interaction between gender and age, but separate analyses showed an independent effect of gender (P = 0.03), but not of age (P = 0.11). Daily smokers had a slightly higher average concentration of P-MDA than nonsmokers (P = 0.05), and P-MDA correlated with daily exposure to cigarette smoke (r = 0.162; P = 0.03). A positive correlation was also demonstrated between P-MDA and weekly alcohol consumption (r = 0.153; P = 0.03). Within-subject and day-to-day variations of P-MDA indicated that the potential of P-MDA as a biomarker for individuals is questionable. However, on a group basis, the present data support that P-MDA may be a potential biomarker for oxidative stress.
Mistakes in a stat laboratory: types and frequency
Authors: Plebani Mario , Carraro Paolo
Published: January 2020

Application of Total Quality Management concepts to laboratory testing requires that the total process, including preanalytical and postanalytical phases, be managed so as to reduce or, ideally, eliminate all defects within the process itself. Indeed a “mistake” can be defined as any defect during the entire testing process, from ordering tests to reporting results. We evaluated the frequency and types of mistakes found in the “stat” section of the Department of Laboratory Medicine of the University-Hospital of Padova by monitoring four different departments (internal medicine, nephrology, surgery, and intensive care unit) for 3 months. Among a total of 40 490 analyses, we identified 189 laboratory mistakes, a relative frequency of 0.47%. The distribution of mistakes was: preanalytical 68.2%, analytical 13.3%, and postanalytical 18.5%. Most of the laboratory mistakes (74%) did not affect patients’ outcome. However, in 37 patients (19%), laboratory mistakes were associated with further inappropriate investigations, thus resulting in an unjustifiable increase in costs. Moreover, in 12 patients (6.4%) laboratory mistakes were associated with inappropriate care or inappropriate modification of therapy. The promotion of quality control and continuous improvement of the total testing process, including pre- and postanalytical phases, seems to be a prerequisite for an effective laboratory service.
Determination of urinary free cortisol by HPLC
Authors: Turpeinen Ursula , Markkanen Helene , Välimäki Matti , Stenman Ulf-Håkan
Published: January 2020

We here report a reversed-phase HPLC method for the determination of free cortisol in human urine, using methylprednisolone as the internal standard. Before chromatography, samples were extracted with a C18 solid-phase extraction column and the steroids were separated on a LiChrospher 100 C18 column with a mobile phase of methanol/acetonitrile/water (43/3/54 by vol). Linearity, precision, and accuracy of the method were established. The detection limit was 10 pmol of cortisol, and total CVs were <8%. With various solid-phase extraction columns the recovery of cortisol was 36–97%; recovery of the internal standard was 43–85%. Study of interference by 6 other steroids and metabolites and 24 drugs showed that carbamazepine and digoxin partly overlapped with cortisol, but this interference could be reduced by modification of the mobile phase. The HPLC method was compared with an RIA and an automated immunoassay method. The results obtained by HPLC averaged 40% of the RIA values.
Application of the Department of Health and Human Services proposed waived status requirements for in vitro diagnostic testing devices: case study
Authors: Ehrmeyer Sharon S , Laessig Ronald H
Published: January 2020

The CLIA’88 classified all clinical laboratory testing as waived, moderate, or high complexity. The eight original waived tests were characterized as simple, accurate, error-free, risk-free, and suitable for home use by nonlaboratory professionals. The subjective nature of the classification process was challenged immediately. The Clinical Laboratory Improvement Advisory Committee asked the CDC and the Health Care Financing Administration to develop objective criteria that included assessment of performance by field-test and in-house data. We examined the efficacy of the CDC protocol with empirical data from the HemoCue B-Hemoglobin Test System® submission, to assess operator competency, intra-/interoperator and between-site imprecision, and accuracy. Non-laboratory-trained operators demonstrated 2–3% imprecision (40–200 g/L). Accuracy studies yielded a slope of 1.01, an intercept of 3.53 g/L, and r of 1.00 (52–230 g/L). Results met the protocol’s Tonks’ criterion for imprecision (less than one-fourth of the reference range).
Proficiency testing of creatine kinase and creatine kinase-2: the experience of the Ontario Laboratory Proficiency Testing Program
Authors: Henderson A Ralph , Krishnan Suba , Webb Sharon , Cheung C Mark , Nazir Darius J , Richardson Harold
Published: January 2020

The Ontario Laboratory Proficiency Testing Program has regularly monitored the analytical performance of total creatine kinase (CK) (≈230 participants) and CK isoenzyme-2 (CK-MB) (≈160 participants) throughout the entire province. Consistently, a wide dispersion of results has been observed not only between different analyzer systems but also among identical analyzers. Accordingly, the results of the last three proficiency surveys for these analytes were examined statistically to establish both the extent of these variations and the range of values reported for the male upper reference ranges. The results of many of the analyzer systems were significantly different from each other, as were many of the reference ranges. This unsatisfactory situation may only be remedied by the use of reference materials as shown by others. The consequences of these findings also effect the reliability of epidemiological surveys such as the WHO MONICA Project (Circulation 1994;90:583–612), which monitors deaths due to heart disease and includes cardiac enzyme results in its criteria.
Rebound increase of plasminogen activator inhibitor type 1 after cessation of thrombolytic treatment for acute myocardial infarction is independent of type of plasminogen activator used
Authors: Genser Norbert , Lechleitner Peter , Maier Josef , Dienstl Franz , Artner-Dworzak Erika , Puschendorf Bernd , Mair Johannes
Published: January 2020

Plasma concentrations of tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor type 1 (PAI-1), and D-dimer were investigated in 50 patients treated intravenously for acute myocardial infarction with either streptokinase (n = 23), urokinase (n = 17), or recombinant t-PA (rt-PA, n = 10). The fibrinolytic variables were measured by enzyme immunoassay on admission; 1, 2, 4, 6, 8, 12, and 24 h later; and then daily until day 7 after admission. In each subgroup of patients treated with different thrombolytic agents, PAI-1 increased significantly (P <0.01) ∼3 h after cessation of thrombolytic therapy. PAI-1 peak concentrations did not differ significantly (P = 0.82) among these three subgroups. t-PA and D-dimer did not differ significantly (P >0.14) among subgroups except for higher t-PA in the rt-PA group attributable to detection of the therapeutically administered exogenous rt-PA by the t-PA assay. Our findings demonstrate a marked PAI-1 increase after thrombolytic therapy for acute myocardial infarction, which seems to be a common, drug-independent antifibrinolytic rebound phenomenon in response to thrombolytic treatment.
High-pressure-mediated dissociation of immune complexes demonstrated in model systems
Authors: Cheung Charles Y , Green David J , Litt Gerald J , Laugharn James A
Published: January 2020

The use of pressure to disrupt immune complexes was demonstrated in two model systems: prostate-specific antigen (PSA) and anti-PSA antibody; and epiglycanin, a mucin glycoprotein, and an antibody specific to that protein. Dissociation of the anti-PSA antibody from the immobilized PSA antigen was observed when pressures of 415 MPa and 550 MPa (1 MPa ∼144 psi) were applied at room temperature (∼21 °C). Application of pressures ranging from 140 MPa to 550 MPa resulted in dissociation of antibody from epiglycanin. In both cases, the rebinding of dissociated antibody to immobilized antigen indicated that the effect of high pressure on the binding of the immune complexes was reversible. These findings suggest that application of high hydrostatic pressure has the potential to be used to significantly improve the sensitivity and specificity of clinical assays.
Antibody interference in thyroid assays: a potential for clinical misinformation
Authors: Després Normand , Grant Andrew M
Published: January 2020

Measurements of thyrotropin and of total and free thyroxine and triiodothyronine are widely used diagnostic methods for thyroid function evaluation. However, some serum samples will demonstrate a nonspecific binding with assay reagents that can interfere with the measurement of these hormones. Several recent case reports have described the presence of such interferences resulting in reported abnormal concentrations of thyroid hormones inconsistent with the patient’s thyroid state. Circulating thyroid hormone autoantibodies, described in thyroid and nonthyroid disorders, are an important class of interference factor and can bind to hormone tracers used in various immunoassays. Two additional categories of interfering antibodies may particularly interfere within two-site immunoassays for thyrotropin. These include heterophile antibodies, especially human anti-mouse antibodies, and rheumatoid factors, which can cause interferences by immunoglobulin aggregation and (or) cross-linking of both capture and signal antibodies. Here we review the nature of these disturbances; their occurrence, prevalence, and detection; and the clinical consequences of the failure to recognize such interference.
Comparison of different analytical methods for assessing total antioxidant capacity of human serum
Authors: Cao Guohua , Prior Ronald L
Published: January 2020

Three assays were compared for the determination of total antioxidant capacity in human serum: the oxygen radical absorbance capacity (ORAC) assay, the Randox Trolox-equivalent antioxidant capacity (Randox-TEAC) assay, and the ferric reducing ability (FRAP) assay. There was a weak but significant linear correlation between serum ORAC and serum FRAP. There was no correlation either between serum ORAC and serum TEAC or between serum FRAP and serum TEAC. The effect of dilution on the serum TEAC value and the use of inhibition percentage at a fixed time, without considering the length of inhibition time in the quantitation of results, adversely affected the Randox-TEAC assay. The FRAP assay is simple and inexpensive but does not measure the SH-group-containing antioxidants. The ORAC assay has high specificity and responds to numerous antioxidants. By utilizing different extraction techniques in the ORAC assay, one can remove serum proteins and also make some gross differentiation between aqueous and lipid-soluble antioxidants. However, the ORAC assay requires ∼60 min more than the FRAP or Randox-TEAC assay to quantitate results.
Teaching the microscopic examination of urine sediment to second year medical students using the Urinalysis-Tutor computer program
Authors: Phillips Carla , Henderson Paul J , Mandel Lynn , Kim Sara , Schaad Doug , Cooper Mindy , Bien Claudia , Orkand Adam , Wener Mark H , Fine James S , Astion Michael L
Published: January 2020

The microscopic examination of urine sediment is a common diagnostic tool taught to medical students, medical technologists, and others. The urine microscopic exam is difficult to teach because supervised instruction and textbook-based teaching suffer from numerous drawbacks. Here, we describe Urinalysis-Tutor, a computer program that uses digitized microscope images and computer-based teaching techniques to systematically teach the urine microscopic exam. In addition, we report the results of a 2-year study that evaluated the effectiveness of the program in 314 second year medical students who were required to use the program. The program contained two, 20-question exams. In the first year of the study (1996), one of the exams was chosen as the pretest and the other as the posttest; the pretest had to be completed before the students viewed the contents of the program, and the posttest was taken after finishing the tutorial. In 1997, the order of the two exams was reversed. In 1996, 159 students completed the study. The mean pretest score was 34% (SD, 14%), the mean posttest score was 71% (SD, 13%), and the improvement was significant (P <0.001, paired t-test). In 1997, 155 students participated. The mean pretest score was 41% (SD, 11%), the mean posttest score was 71% (SD, 13%), and the improvement was significant (P <0.001, paired t-test). The study shows that Urinalysis-Tutor helps medical students learn to interpret the microscopic appearance of urine sediment and that it is feasible to implement this tutorial in a medical school class.
Useful laboratory tests for studying thrombogenesis in acute cardiac syndromes
Authors: Fareed Jawed , Hoppensteadt Debra A , Leya Fred , Iqbal Omer , Wolf Helmut , Bick Roger
Published: January 2020

We review laboratory tests that evaluate thrombogenesis during acute coronary syndromes. These tests have been found to be valuable research tools in more clearly understanding the pathophysiology of acute coronary syndromes. In particular, we describe tissue factor, tissue factor pathway inhibitor, prothrombin fragment 1.2, thrombin-antithrombin complex, fibrinopeptide A, tissue plasminogen activator (t-PA), plasminogen activator inhibitor-1 (PAI-1), t-PA-PAI complex, Bβ 15–42-related peptides, fibrinogen degradation products, fibrin degradation products, D-dimer, platelet factor 4, β-thromboglobulin, 5-hydroxytryptamine, thromboxane B2, prostacyclin, endothelin, angiotensin-converting enzyme, soluble thrombomodulin, C1-esterase inhibitor, anaphylotoxins C3a, C4a, and C5a, bradykinin, tumor necrosis factor, leukotriene C4, platelet activating factor, anti-phospholipid antibody, and von Willebrand factor. Some of these tests may prove to be useful in clinical diagnosis and management of acute coronary syndromes. Clinical outcome studies are needed to determine which tests may be cost effective and medically useful.
Simultaneous Serum Assays of Lipase and Interleukin-6 for Early Diagnosis and Prognosis of Acute Pancreatitis
Authors: Pezzilli Raffaele , Morselli-Labate Antonio Maria , Miniero Rita , Barakat Bahjat , Fiocchi Manuela , Cappelletti Onda
Published: January 2020

Background: There are no systems for the rapid diagnosis and prognosis of acute pancreatitis in the Emergency Department. Our aim was to evaluate whether the combined use of serum lipase and interleukin-6 or serum lipase and C-reactive protein is able to simultaneously establish both the diagnosis and the prognosis of acute pancreatitis.

Methods: Eighty patients with acute abdomen were studied on admission to the Emergency Room. Forty patients had nonpancreatic acute abdomen, and 40 had acute pancreatitis (25 had mild acute pancreatitis and 15 had severe pancreatitis). Forty healthy subjects comparable for sex and age were also studied as controls. Lipase, interleukin-6, and C-reactive protein were determined on serum in all subjects.

Results: Using lipase to discriminate between patients with nonpancreatic acute abdomen and patients with acute pancreatitis (cutoff values ranging from 419 to 520 U/L), one patient with acute pancreatitis was not identified correctly. To discriminate between patients with severe acute pancreatitis and those with mild pancreatitis in the remaining 39 patients, interleukin-6 (cutoff value, <3.7 μg/L) had a sensitivity of 100% (15 of 15) and a specificity of 83% (20 of 24); 75 of 80 (94%) patients were classified correctly. C-reactive protein (cutoff values ranging from 6 to 7 mg/L) showed a lower prognostic efficiency than interleukin-6: sensitivity of 87% (13 of 15) and specificity of 46% (11 of 24). Sixty-four of 80 patients (80%) were classified correctly. The area under the ROC curve for interleukin-6 (0.911 ± 0.049) was significantly (P = 0.013) greater than that for C-reactive protein (0.685 ± 0.090).

Conclusion: The combined use of serum lipase and interleukin-6 is useful in simultaneously establishing both the diagnosis and the prognosis of acute pancreatitis.
Plastic Microchip Electrophoresis for Analysis of PCR Products of Hepatitis C Virus
Authors: Chen Yu-Hung , Wang Wei-Chang , Young Kung-Chia , Chang Ting-Tsung , Chen Shu-Hui
Published: January 2020

Background: Electrophoresis on polymeric rather than glass microstructures is a promising separation method for analytical chemistry. Assays on such devices need to be explored to allow assessment of their utility for the clinical laboratory.

Methods: We compared capillary and plastic microchip electrophoresis for clinical post-PCR analysis of hepatitis C virus (HCV). For capillary electrophoresis (CE), we used a separation medium composed of 10 g/L hydroxypropyl methyl cellulose in Tris-borate-EDTA buffer and 10 μmol/L intercalating dye. For microchip electrophoresis, the HCV assay established on the fused silica tubing was transferred to the untreated polymethylmethacrylate microchip with minimum modifications.

Results: CE resolved the 145-bp amplicon of HCV in 15 min. The confidence interval of the migration time was <3.2%. The same HCV amplicon was resolved by microchip electrophoresis in <1.5 min with the confidence interval of the migration time <1.3%.

Conclusion: The polymer microchip, with advantages that include fast processing time, simple operation, and disposable use, holds great potential for clinical analysis.
Single-Step Mutation Scanning of the 6-Pyruvoyltetrahydropterin Synthase Gene in Patients with Hyperphenylalaninemia
Authors: Romstad Anne , Guldberg Per , Blau Nenad , Güttler Flemming
Published: January 2020

Background: Deficiency of 6-pyruvoyltetrahydropterin synthase (PTPS) is a recessively inherited disorder that leads to depletion of 5,6,7,8-tetrahydrobiopterin, the obligatory cofactor for hydroxylation of phenylalanine, tyrosine, and tryptophan. A marker for neonatal detection of PTPS deficiency is hyperphenylalaninemia (HPA). Molecular analysis would provide a simple and reliable means for distinguishing PTPS deficiency from other potential causes of HPA.

Methods: We developed a method based on PCR in combination with denaturing gradient gel electrophoresis (DGGE) that rapidly scans the six coding sequences and all splice sites of the PTPS gene (PTS) for mutations. This method was used to examine the status of the PTS gene in control samples with known PTS mutations and in five patients with PTPS deficiency.

Results: Two features of the PTS gene posed particular problems in relation to DGGE analysis: the very high GC content of exon 1, and a 15-bp poly(dT) stretch in the acceptor splice site of intron 1. Both problems were solved by special design of amplification primers. PCR and DGGE conditions were adjusted to allow simultaneous analysis of all six regions of the PTS gene. Using this one-step approach, all control mutations were readily resolved. Among the five PTPS patients, four mutations were identified, including IVS1-3C→G, IVS2-7T→A, V57del, and V97M (289G→A). The IVS1-3C→G mutation was shown by reverse transcription-PCR analysis to produce multiple splice variants.

Conclusions: We have established a fast and reliable screening method for detection of mutations and small deletions/insertions in the PTS gene. This method should be useful for rapid diagnosis of PTPS deficiency in newborns with HPA.
Rapid Diagnosis of Herpes Simplex Encephalitis Using Microchip Electrophoresis of PCR Products
Authors: Hofgärtner Wolfgang T , Hühmer Andreas FR , Landers James P , Kant Jeffrey A
Published: January 2020

Background: Herpes simplex virus (HSV) is the most common cause of acute sporadic encephalitis in the United States. PCR of DNA extracted from cerebrospinal fluid (CSF) allows for reliable diagnosis of herpes simplex encephalitis (HSE). A faster turnaround time for HSE testing would improve patient management and lead to better outcomes. The aims of this study, therefore, were to develop a microchip-based electrophoretic method for rapid detection of HSV PCR products, and to compare the performance characteristics of liquid hybridization/gel retardation as an established clinical PCR product detection method with the new microchip-based method.

Methods: The study examined archival DNA from 33 selected CSF specimens submitted for HSV PCR testing to the clinical laboratory. One aliquot of the HSV PCR product was analyzed by liquid hybridization/gel retardation analysis, and a second PCR aliquot was analyzed directly with a microchip capillary electrophoresis system using an instrument built in-house. PCR samples were introduced directly into the microchip without a desalting step by use of a novel fluidic interface. Channel surfaces on the glass microchip were silanized, followed by derivatization with polyvinylpyrrolidone.

Results: Of the 33 CSF specimens tested by liquid hybridization analysis of HSV PCR products, 10 tested positive for HSV DNA, 2 gave a weakly positive result, and 21 tested negative. Total analysis time for detection of HSV DNA by gel retardation assay was 18 h. Microchip electrophoresis provided identical results in <110 s/sample, achieving 100% sensitivity and specificity compared with the established method.

Conclusions: Microchip-based electrophoresis can rapidly and accurately separate HSV PCR products, giving results identical to those obtained by liquid hybridization but with substantially decreased turnaround time. Clinical implementation of the new method will help to improve patient management and outcomes.
Coupled-Column Liquid Chromatographic Analysis of Catecholamines, Serotonin, and Metabolites in Human Urine
Authors: Panholzer Torsten J , Beyer Jürgen , Lichtwald Klaus
Published: January 2020

Acolumn-switching HPLC system was utilized for the simultaneous determination of epinephrine, norepinephrine, dopamine, serotonin, and their metabolites metanephrine, normetanephrine, 3,4-dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindoleacetic acid in human urine. The sample was injected directly onto a C18-alkyl-diol silica precolumn, which separated the analytes from matrix. The analytes were eluted from the precolumn onto the analytical column by the use of column-switching techniques and were then separated on the analytical column by means of ion-pair reversed-phase HPLC. The analytes were then oxidized to the corresponding quinones and converted into fluorescent derivatives by reaction with meso-1,2-diphenylethylenediamine.
Reverse Transcription-Competitive Multiplex PCR Improves Quantification of mRNA in Clinical Samples—Application to the Low Abundance CFTR mRNA
Authors: Loitsch Stefan M , Kippenberger Stefan , Dauletbaev Nurlan , Wagner Thomas OF , Bargon Joachim
Published: January 2020

Background: To monitor gene therapy, we wished to quantify cystic fibrosis transmembrane conductance regulator (CFTR) mRNA. We developed a PCR-based method to measure CFTR mRNA in clinical samples.

Methods: Expression was determined by reverse transcription-competitive multiplex PCR (RCMP) for CFTR and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts, and for serial dilutions of two internal cDNA standards consisting of CFTR and GAPDH mutants containing short deletions. The RCMP used simultaneous amplification of the gene of interest with a reporter gene in one reaction tube. The expression of CFTR was calculated with reference to the amount of GAPDH to correct for variations in initial RNA loading.

Results: Amplification of cDNAs derived from different amounts of RNA (1–4 μg) gave similar GAPDH/CFTR ratios, with a coefficient of variation (CV) below 7.5%. RCMP was applied on nasal and bronchial brushings and shows a high variability of CFTR expression in non-cystic fibrosis donors.

Conclusion: This method is precise and reproducible and advantageous for use with limited amounts of tissue, such as from biopsies or from nasal or bronchial brushings.
A Two-Year Study of Microscopic Urinalysis Competency Using the Urinalysis-Review Computer Program
Authors: Astion Michael L , Kim Sara , Nelson Amanda , Henderson Paul J , Phillips Carla , Bien Claudia , Mandel Lynn , Orkand Adam R , Fine James S
Published: January 2020

Background: The microscopic examination of urine sediment is one of the most commonly performed microscope-based laboratory tests, but despite its widespread use, there has been no detailed study of the competency of medical technologists in performing this test. One reason for this is the lack of an effective competency assessment tool that can be applied uniformly across an institution.

Methods: This study describes the development and implementation of a computer program, Urinalysis-ReviewTM, which periodically tests competency in microscopic urinalysis and then summarizes individual and group test results. In this study, eight Urinalysis-Review exams were administered over 2 years to medical technologists (mean, 58 technologists per exam; range, 44–77) at our academic medical center. The eight exams contained 80 test questions, consisting of 72 structure identification questions and 8 quantification questions. The 72 structure questions required the identification of 134 urine sediment structures consisting of 63 examples of cells, 25 of casts, 18 of normal crystals, 8 of abnormal crystals, and 20 of organisms or artifacts.

Results: Overall, the medical technologists correctly identified 84% of cells, 72% of casts, 79% of normal crystals, 65% of abnormal crystals, and 81% of organisms and artifacts, and correctly answered 89% of the quantification questions. The results are probably a slight underestimate of competency because the images were analyzed without the knowledge of urine chemistry results.

Conclusions: The study shows the feasibility of using a computer program for competency assessment in the clinical laboratory. In addition, the study establishes baseline measurements of competency that other laboratories can use for comparison, and which we will use in future studies that measure the effect of continuing education efforts in microscopic urinalysis.
Four Common Mutations of the Cystathionine β-Synthase Gene Detected by Multiplex PCR and Matrix-assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry
Authors: Harksen Angela , Ueland Per Magne , Refsum Helga , Meyer Klaus
Published: January 2020

Background: A deficiency of cystathionine β-synthase (CBS) is the most frequent cause of homocystinuria. The effect of therapy is related to the underlying CBS genotype, which makes early diagnosis of this genetic defect important. Our aim was to develop a fast and reliable method based on matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry for the determination of common mutations of the CBS gene.

Methods: We used MALDI-TOF mass spectrometry to detect four common CBS mutations (G307S, T272M, I278T, and V320A). The method is based on multiplex PCR of exons 7, 8, and 9, followed by single nucleotide extension in the presence of dideoxy NTPs of four primers targeted at the separate mutation sites. The extension products, as well as the 3-hydroxypicolinic acid matrix, were incubated with cation-exchange beads to remove disturbing salt contaminants.

Results: The above-mentioned mutations were determined in samples from 12 homocystinuria patients. The MALDI-TOF spectra allowed unambiguous discrimination between primers and extension products (>9 Da) in the mass range between 4500 and 7500 Da. No labeled primers or ddNTPs were required. The genotyping was verified by reference technique.

Conclusion: Our results demonstrate fast, simple, and unambiguous multiplex genotyping of four common CBS mutations by MALDI-TOF mass spectrometry.
Effect of the Ratio of Free to Total Prostate-specific Antigen on Interassay Variability in Proficiency Test Samples
Authors: Fox M Pat , Reilly Andrew A , Schneider Erasmus
Published: January 2020

Background: Up to sevenfold differences were observed between total prostate-specific antigen (PSA) methods for New York State Proficiency Test samples prepared with seminal fluid PSA in human female serum. Because the PSA was mainly in its free form under these conditions, we wanted to determine whether a defined mixture of free and complexed PSA would reduce the interassay differences.

Methods: We prepared a series of five solutions of 60 g/L bovine serum albumin with 10 μg/L total PSA consisting of varied proportions of free, noncomplexible PSA, and α1-antichymotrypsin (ACT)-complexed PSA from 0% to 100%. Two hundred seventy laboratories measured the total PSA in these samples, and 16 laboratories also analyzed the samples for free PSA. The results were used to calculate free/total PSA ratios.

Results: Interassay CVs for total PSA measurements were ∼7% at 10–15% free PSA but became gradually larger as the free/total PSA ratio increased. Measured free-PSA concentrations were similar within each sample (mean CV, 12%), and the results were relatively independent of the proportion of free PSA in the samples. Twofold discrepancies between actual and expected ratios were observed with some methods at 100% free PSA and to a lesser degree at 30% free PSA. At 100% free PSA, the relatively higher total-PSA values measured by nonequimolar methods yielded low free/total PSA ratios of 50–60%. In contrast, the lower total PSA values obtained by equimolar methods yielded ratios close to the expected 100%.

Conclusions: Preparing proficiency test samples with a 10:90 mixture of free, noncomplexible PSA:PSA-ACT is a viable alternative to the use of seminal fluid PSA. Furthermore, the method used to measure total PSA may have a substantial impact on the calculated proportion of free PSA and hence may have clinical relevance.