bluuebunny/crossref_metadata_2025 huggingface dataset:
HPLC Determination of Thiopurine Nucleosides and Nucleotides in Vivo in Lymphoblasts following Mercaptopurine Therapy
Authors: Dervieux Thierry , Chu Yaqin , Su Yi , Pui Ching-Hon , Evans William E , Relling Mary V
Published: January 2020

Background: Mercaptopurine is a prodrug requiring intracellular activation to thiopurine nucleotides to exert antileukemic effect. We developed a reversed-phase liquid chromatographic assay for the quantification of mercaptopurine, thioguanine, and methylmercaptopurine nucleoside and nucleotide concentrations in the target tissue, the leukemic lymphoblast.

Methods: Leukemic blasts were isolated from peripheral blood and bone marrow by a standard Ficoll-hypaque procedure. Proteins were removed by ultrafiltration in the presence of dithiothreitol. Thiopurine ribonucleotides were converted into their respective ribonucleosides by treatment of ultrafiltrate with acid phosphatase. Thiopurine nucleosides and bases were measured by direct injection of ultrafiltrate into the chromatographic system. Thiopurine nucleotide concentrations were calculated by subtracting the thiopurine nucleoside concentrations measured after treatment with acid phosphatase from those measured after direct injection of ultrafiltrate in the chromatographic system. Analytes were separated on a C18 Supelco column with ammonium phosphate-methanol eluent coupled with ultraviolet detection.

Results: CVs for intra- and interday precision were 1.1–14% (median, 4.9%), and recovery of added analyte was 89–126% (median, 105%) at low and high concentrations of analytes, except for mercaptopurine riboside. The median signal for each of the five metabolites in lymphoblast samples was 98% (range, 80–106%) of that in water. Detection limits for thiopurine bases and nucleosides ranged from 0.5 to 4.5 pmol/5 × 106 cells.

Conclusions: This method is suitable for measurement of thiopurine metabolite concentrations in lymphoblasts in children with acute lymphoblastic leukemia following a single dose of intravenous mercaptopurine.
Rapid Liquid Chromatography-Tandem Mass Spectrometry Method for Routine Analysis of Cyclosporin A Over an Extended Concentration Range
Authors: Keevil Brian G , Tierney David P , Cooper Donald P , Morris Michael R
Published: January 2020

Background: Cyclosporin A (CsA) is commonly measured by immunoassay techniques that have a limited analytical range. The consequence of this is that low CsA concentrations that may be clinically significant are difficult to measure and that high concentrations require sample dilution, which introduces error and increases cost. More specific assays, such as HPLC, do not have the required turnaround times for busy transplant clinics.

Methods: CsA was measured in whole blood from 180 cardiac and lung transplant recipients by a liquid chromatography-tandem mass spectrometry (MS) assay, and the results were compared with the Dade Behring Emit assay. Proteins were precipitated with acetonitrile containing ascomycin as internal standard. We used isocratic elution on a Supelco CN column (33 × 3.0 mm; 3- μm bead size) with a mobile phase of 65% aqueous acetonitrile containing ammonium acetate (2 mmol/L) and formic acid (1 g/L), at a flow rate of 0.5 mL/min, with a sample injection volume of 6 μL. We used positive-ion electrospray MS to monitor the ammonium adducts of the compounds of interest decomposing under controlled conditions to the most dominant fragments of the individual molecules. Calibration curves used linear least-squares regression with 1/x weighting.

Results: Maximum sensitivity was obtained by monitoring fragmentation of the ammonium adducts m/z 1220→m/z 1203 for CsA and m/z 809→m/z 765 for ascomycin. Sample throughput, including preparation time, was 30 samples in 1.5 h with an injection-to-injection cycle time of 1.5 min. The calibration curve was linear to 5000 μg/L, with a detection limit of 0.03 μg/L and a limit of quantification of 1 μg/L. Regression analysis [tandem MS method (y) and Emit assay (x)] yielded a slope of 1.09 (± 0.03), an intercept of 6.2 (± 4.5) μg/L, and Sy|x = 27 μg/L.

Conclusions: Tandem MS assay is a realistic alternative to immunoassay for the routine monitoring of CsA in transplant recipients. Its wide dynamic range has utility for pharmacokinetic studies of CsA.
Microtiter Format for Simultaneous Multianalyte Detection and Development of a PCR-Chemiluminescent Enzyme Immunoassay for Typing Human Papillomavirus DNAs
Authors: Roda Aldo , Mirasoli Mara , Venturoli Simona , Cricca Monica , Bonvicini Francesca , Baraldini Mario , Pasini Patrizia , Zerbini Marialuisa , Musiani Monica
Published: January 2020

Background: To allow multianalyte binding assays, we have developed a novel polystyrene microtiter plate containing 24 main wells, each divided into 7 subwells. We explored its clinical potential by developing a PCR-chemiluminescent immunoassay (PCR-CLEIA) for simultaneous detection and typing of seven high oncogenic risk human papillomavirus (HPV) DNAs in one well.

Methods: Seven different oligonucleotide probes, each specific for a high-risk HPV genotype, were separately immobilized in the subwells. Subsequently, a digoxigenin-labeled consensus PCR amplification product was added to the main well. The PCR product hybridized to the immobilized probe corresponding to its genotype and was subsequently detected by use of a peroxidase-labeled anti-digoxigenin antibody and chemiluminescence imaging with an ultrasensitive charge-coupled device camera. Results obtained for 50 cytologic samples were compared with those obtained with a conventional colorimetric PCR-ELISA.

Results: The method was specific and allowed detection of 50 genome copies of HPV 16, 18, 33, and 58, and 100 genome copies of HPV 31, 35, and 45. Intra- and interassay CVs for the method were 5.6% and 7.9%, respectively. All results obtained for clinical samples were confirmed by the conventional PCR-ELISA.

Conclusions: PCR-CLEIA allows rapid, single-tube simultaneous detection and typing of seven high-risk HPV DNAs with small reagent volumes. The principle appears applicable to the development of other single-tube panels of tests.
CYP3A5 Variant Allele Frequencies in Dutch Caucasians
Authors: van Schaik Ron HN , van der Heiden Ilse P , van den Anker John N , Lindemans Jan
Published: January 2020

Background: Enzymes of the cytochrome P450 3A (CYP3A) family are responsible for the metabolism of >50% of currently prescribed drugs. CYP3A5 is expressed in a limited number of individuals. The absence of CYP3A5 expression in ∼70% of Caucasians was recently correlated to a genetic polymorphism (CYP3A5*3). Because CYP3A5 may represent up to 50% of total CYP3A protein in individuals polymorphically expressing CYP3A5, it may have a major role in variation of CYP3A-mediated drug metabolism. Using sequencing, have been identified (Hustert et al. Pharmacogenetics 2001;11:773–9; Kuehl et al. Nat Genet 2001;27:383–91) variant alleles *2 through *7 for CYP3A5. Detection of CYP3A5 variant alleles, and knowledge about their allelic frequency in specific ethnic groups, is important to establish the clinical relevance of screening for these polymorphisms to optimize pharmacotherapy.

Methods: In a group of 500 healthy Dutch Caucasian blood donors, we determined the allelic frequency of the CYP3A5*2, *3, *4, *5, *6, and *7 alleles by use of newly developed PCR-restriction fragment length polymorphism assays.

Results: The frequency of the defective CYP3A5*3 allele in the Dutch Caucasian population was 91%, followed by the CYP3A5*2 (1%) and CYP3A5*6 (0.1%) alleles. The CYP3A5*4, *5, and *7 alleles were not detected.

Conclusions: On the basis of its allelic frequency, screening for the CYP3A5*3 allele in the Caucasian population is extremely relevant. In addition, screening for the CYP3A5*2 allele may be taken into consideration in individuals heterozygous for the CYP3A5*3 allele. The CYP3A5*4, *5, *6, and *7 alleles have low allelic frequencies that do not support initial screening.
Excretion and Detection of Cathinone, Cathine, and Phenylpropanolamine in Urine after Kath Chewing
Authors: Toennes Stefan W , Kauert Gerold F
Published: January 2020

Introduction: The stimulating herbal drug kath is uncommon in most countries, and information on its detection and interpretation of analytical results is limited. Therefore, a study with kath was carried out to compare the efficiencies of different analytical techniques used to detect drug use.

Methods: Four volunteers chewed kath leaves for 1 h; urine samples were collected up to 80 h afterward and analyzed by the Abbott fluorescence polarization immunoassay (FPIA), the Mahsan-AMP300 on-site immunoassay, the Bio-Rad Remedi HS HPLC system with photodiode array detection (DAD), and gas chromatography–mass spectrometry (GC-MS).

Results: FPIA gave negative results, whereas positive results were obtained with the Mahsan test during the first day. With HPLC, one peak could be observed up to 50 h, but its DAD spectrum could not be identified by the system. Further investigations indicated that the kath alkaloids coeluted and produced a mixed DAD spectrum. With GC-MS, the specific kath ingredient cathinone was detected up to 26 h, whereas cathine and norephedrine were still detectable in the last samples. Maximum concentrations of cathinone, cathine, and norephedrine in urine samples from the study were 2.5, 20, and 30 mg/L, respectively, whereas in authentic cases the concentrations were much higher.

Conclusion: GC-MS is superior to the screening techniques Mahsan-AMP300 and Remedi with respect to specificity and sensitivity for the detection of kath use in urine.
Trimolecular Complexes of λ Light Chain Dimers in Serum of a Patient with Multiple Myeloma
Authors: Abraham Roshini S , Charlesworth M Cristine , Owen Barbara AL , Benson Linda M , Katzmann Jerry A , Reeder Craig B , Kyle Robert A
Published: January 2020

Background: Patients with multiple myeloma often have Bence Jones proteins composed of free monoclonal light chains of the κ or λ type in their urine. Usually, these light chains exist as monomeric or dimeric forms, but rarely, larger molecules, such as tetramers, have been reported in the serum.

Methods and Results: We report the presence of trimeric complexes of λ light chain dimers in a patient who was diagnosed with a free λ light chain multiple myeloma 2 years earlier and subsequently underwent a stem cell transplant. Recently, the patient presented with a large serum M-spike (23 g/L) by protein electrophoresis. The spike consisted of monoclonal λ light chains without a heavy chain. The urine contained only 8 mg of λ light chain in a 24-h specimen. Quantitative analysis of the serum and urinary free light chains (FLCs) indicated the probability of larger aggregates of FLCs. Size-exclusion chromatography, electrophoresis, analytical ultracentrifugation, and mass spectrometric studies of the serum revealed almost exclusively the presence of trimolecular aggregates of λ light chain dimers without other multimeric species.

Conclusion: Monoclonal λ light chains may present as hexameric aggregates that cannot be cleared by renal excretion.
Electrophoretic Variant of a Lactate Dehydrogenase Isoenzyme and Selective Promoter Methylation of the LDHA Gene in a Human Retinoblastoma Cell Line
Authors: Maekawa Masato , Inomata Motoko , Sasaki Masao S , Kaneko Akihiro , Ushiama Mineko , Sugano Kokichi , Takayama Jun , Kanno Takashi
Published: January 2020

Background: Lactate dehydrogenase (LD), a tetrameric product of the genes LDHA and LDHB, may be increased in sera of cancer patients. A variant isoenzyme with electrophoretic mobility between LD2 and LD3 (LD2ex) has been described in patients, but its molecular nature is largely unknown.

Methods: A newly established retinoblastoma cell line, NCC-RbC-51 (R51), showed an isoenzyme pattern with only two bands, LD1 and LD2ex. We investigated the isoenzymes by Northern blot, Western blot, and methylation analysis and PCR.

Results: Northern blot analysis revealed that R51 cells expressed no wild-type/somatic LDHA mRNA, but did express a small amount of LDHA-related mRNA with a slightly higher molecular mass. Western blot analysis confirmed the anti-LDHA-reactive protein with a 3-kDa higher molecular mass. Treatment of R51 cells with the demethylating agent 5-aza-2′-deoxycytidine restored the expression of the LD2, -3, -4, and -5 isoenzymes. PCR analysis of sodium bisulfite-treated genomic DNA revealed that the CpG island in the promoter region around exon a of the LDHA gene was completely methylated. Reverse transcription-PCR analysis and direct sequencing revealed that R51 cells expressed a RNA with the sequence of the human homolog of a murine testis-specific variant that has exon 0 as the 5′ noncoding sequence. LDHB was expressed normally in R51 cells.

Conclusions: The somatic LDHA in R51 cells is transcriptionally silenced by promoter hypermethylation around exon a, leaving only LDHB to be expressed normally and a testis-specific variant transcript of LDHA containing exon 0. LD2ex possibly results from tetramerization of three wild-type LDHB molecules and one variant LDHA product.
Analysis of Clinically Relevant Single-Nucleotide Polymorphisms by Use of Microelectronic Array Technology
Authors: Santacroce Rosa , Ratti Antonia , Caroli Francesco , Foglieni Barbara , Ferraris Alessandro , Cremonesi Laura , Margaglione Maurizio , Seri Marco , Ravazzolo Roberto , Restagno Gabriella , Dallapiccola Bruno , Rappaport Eric , Pollak Eleanor S , Surrey Saul , Ferrari Maurizio , Fortina Paolo
Published: January 2020

Background: Microelectronic DNA chip devices represent an emerging technology for genotyping. We developed methods for detection of single-nucleotide polymorphisms (SNPs) in clinically relevant genes.

Methods: Primer pairs, with one containing a 5′-biotin group, were used to PCR-amplify the region encompassing the SNP to be interrogated. After denaturation, the biotinylated strand was electronically targeted to discrete sites on streptavidin-coated gel pads surfaces by use of a Nanogen Molecular Workstation. Allele-specific dye-labeled oligonucleotide reporters were used for detection of wild-type and variant sequences. Methods were developed for SNPs in genes, including factor VII, β-globin, and the RET protooncogene. We genotyped 331 samples for five DNA variations in the factor VII gene, >600 samples from patients with β-thalassemia, and 15 samples for mutations within the RET protooncogene. All samples were previously typed by various methods, including DNA sequence analysis, allele-specific PCR, and/or restriction enzyme digestion of PCR products.

Results: Analysis of amplified DNA required 4–6 h. After mismatched DNA was removed, signal-to-noise ratios were >5. More than 940 samples were typed with the microelectronic array platform, and results were totally concordant with results obtained previously by other genotyping methods.

Conclusions: The described protocols detect SNPs of clinical interest with results comparable to those of other genotyping methods.
Diagnostic Accuracy of α1-Acid Glycoprotein Fucosylation for Liver Cirrhosis in Patients Undergoing Hepatic Biopsy
Authors: Rydén Ingvar , Påhlsson Peter , Lindgren Stefan
Published: January 2020

Background: Increased fucosylation of serum glycoproteins has previously been reported in patients with liver disease. We analyzed α1-acid glycoprotein (AGP) fucosylation in serum samples from patients investigated for suspected liver disease to evaluate its value as a biochemical marker for liver cirrhosis.

Methods: We used a novel lectin immunoassay adapted to the AutoDELFIA system to analyze AGP fucosylation in 261 consecutive patients admitted for liver biopsy at Malmö University Hospital in Southern Sweden. The results were compared with histopathologic findings. In addition, AGP fucosylation was compared with other biochemical markers described as useful in the diagnosis of liver cirrhosis. The biochemical markers were compared by ROC curve analysis.

Results: AGP fucosylation was significantly (P <0.05) higher in patients with liver cirrhosis (n = 65) than in healthy controls (n = 72), patients with normal histology (n = 29), patients with steatosis only (n = 38), patients with viral or chronic hepatitis without cirrhosis (n = 71), and patients with other liver diseases without histologic signs of cirrhosis (n = 58). By calculating the AGP fucosylation index (AGP-FI = AGP fucosylation/AGP serum concentration), we obtained a high diagnostic accuracy. The areas under the ROC curves for AGP-FI were 0.83 and 0.74 for men and women, respectively, compared with 0.82 for hyaluronic acid and 0.77 for the aspartate aminotransferase/alanine aminotransferase ratio in both men and women.

Conclusions: AGP fucosylation appears to be useful in identifying patients with liver cirrhosis among patients investigated for liver disease. The lectin immunoassay showed satisfactory reproducibility and is suitable for routine use in a clinical laboratory.
Delayed Cytokine mRNA Expression Kinetics after T-Lymphocyte Costimulation: A Quantitative Measure of the Efficacy of Cyclosporin A-based Immunosuppression
Authors: Härtel Christoph , Fricke Lutz , Schumacher Nina , Kirchner Holger , Müller-Steinhardt Michael
Published: January 2020

Background: Because cyclosporin A (CsA) and glucocorticoids inhibit the production of interleukin-2 (IL-2) and other cytokines, quantitative analysis of cytokine mRNA might constitute a pharmacodynamic measure for immunosuppressive drug effects. We investigated whether immunosuppressive drugs influence cytokine mRNA expression kinetics during T-cell costimulation.

Methods: We used a human whole blood assay to determine basal (unstimulated) IL-2, IL-4, and tumor necrosis factor-α (TNF-α) mRNA concentrations and expression kinetics after anti-CD3/anti-CD28 monoclonal antibody costimulation in kidney transplant recipients undergoing CsA-based immunosuppressive triple therapy and in healthy controls (ex vivo study I). The effect of CsA on IL-2 mRNA expression kinetics was also determined ex vivo in patients undergoing CsA monotherapy (ex vivo study II) and after in vitro addition of CsA.

Results: In ex vivo study I, basal TNF-α mRNA but not IL-2 and IL-4 mRNA was decreased in kidney transplant patients. We observed shifts in peak IL-2 and IL-4 (from 8 to 24 h) and TNF-α (from 4 to 8 h of costimulation) mRNA expression in kidney transplant patients after T-cell costimulation. In patients undergoing CsA monotherapy (ex vivo study II), the inhibitory effect of CsA was detectable as an individually delayed increase in IL-2 mRNA during costimulation. In vitro addition of CsA also induced a dose-independent displacement of IL-2 mRNA expression kinetics (i.e., a delay).

Conclusions: A delayed increase in cytokine mRNA expression during T-cell costimulation may represent a sensitive effect of immunosuppression. The single analysis of one absolute or peak mRNA value could be misleading. For prospective studies involving measurement of cytokine mRNA, we therefore suggest the parameter “area of cytokine mRNA expression over time”, which should include absolute cytokine mRNA values at two different time points of mRNA kinetics.
Assessment and Recommendations on Factors Contributing to Preanalytical Variability of Urinary Pyridinoline and Deoxypyridinoline
Authors: Vesper Hubert W , Demers Laurence M , Eastell Richard , Garnero Patrick , Kleerekoper Michael , Robins Simon P , Srivastava Apurva K , Warnick G Russell , Watts Nelson B , Myers Gary L
Published: January 2020

Background: Pyridinoline (PYD) and deoxypyridinoline (DPD) are two of the most extensively characterized biochemical bone markers, but the interpretation of results is hampered by biologic and other preanalytical variability. We reviewed factors contributing to preanalytical variation of pyridinium cross-links in urine.

Methods: We searched four databases for English-language reports on PYD and/or DPD in urine. Searches were restricted to humans, except for studies of stability, when the search was expanded to other species. The 599 identified articles were supplemented with references from those articles and with articles known to the authors.

Results: The mean reported within-day variability was 71% for PYD (range, 57–78%) and 67% for DPD (range, 53–75%). The mean interday variability was 16% for both DPD and PYD (range for PYD, 12–21%; range for DPD, 5–24%). The mean intersubject variabilities across studies were 26% for PYD (range, 12–63%) and 34% for DPD (range, 8–98%) for healthy premenopausal women and 36% (range, 22–61%) and 40%, (range, 27–54%) for postmenopausal women, respectively. Specimen instability and errors in creatinine measurements were additional sources of variability.

Conclusions: Intra- and intersubject variability can be reduced by collecting specimens at a specific time of the day and by maintaining similar patient status at each specimen collection regarding factors such as medications and dietary supplements.
Collagen Markers Deoxypyridinoline and Hydroxylysine Glycosides: Pediatric Reference Data and Use for Growth Prediction in Growth Hormone-deficient Children
Authors: Rauch Frank , Georg Mareile , Stabrey Angelika , Neu Christina , Blum Werner F , Remer Thomas , Manz Friedrich , Schoenau Eckhard
Published: January 2020

Background: In children and adolescents, markers of bone and collagen metabolism reflect the dynamics of skeletal growth and development. The aim of this study was to assess the relationship of the urinary collagen markers deoxypyridinoline (DPD) and hydroxylysine (Hyl) and its glycosides [galactosyl-Hyl (Gal-Hyl) and glucosyl-Gal-Hyl] with growth.

Methods: Urine samples from 240 apparently healthy children and adolescents (6–19 years; 124 girls) and from 51 prepubertal children with growth hormone (GH) deficiency (3–14 years; 14 girls) were analyzed. Urinary Hyl and its glycosides were quantified by HPLC, and DPD was assessed by chemiluminescence assay. Urinary concentrations of all markers were related to urinary creatinine.

Results: Multiple regression analysis revealed that only age and height velocity were independently associated with these markers in healthy children. In GH-deficient patients, the urinary excretion of both analytes after 4 weeks of GH therapy correlated significantly with the height increase during the first treatment year (r = 0.79 for Gal-Hyl; r = 0.70 for DPD; P <0.001 each). In a multivariate linear regression model using Gal-Hyl concentrations at 4 weeks, baseline concentrations of insulin-like growth factor 1 and height velocity after 3 months accounted for 80% of the variability in height gain during the first treatment year. A model using DPD concentrations at 4 weeks, in place of Gal-Hyl concentrations, as well as baseline concentrations of insulin-like growth factor 1 and height velocity after 3 months accounted for 83% of the variability.

Conclusions: These urinary bone and collagen markers give some early indication of growth response, but the prediction of an individual marker is too imprecise to serve as a basis for clinical decisions. Markers of bone and collagen metabolism might be more useful as components of multivariate growth prediction models.
Biochemical and Genetic Markers of Iron Status and the Risk of Coronary Artery Disease: An Angiography-based Study
Authors: Bozzini Claudia , Girelli Domenico , Tinazzi Elisa , Olivieri Oliviero , Stranieri Chiara , Bassi Antonella , Trabetti Elisabetta , Faccini Giovanni , Pignatti Pier Franco , Corrocher Roberto
Published: January 2020

Background: Iron may promote coronary atherosclerotic disease (CAD) by increasing lipid peroxidation. Studies on biochemical or genetic markers of body iron stores as risk factors for CAD have yielded conflicting results.

Methods: We studied 849 individuals with a clear-cut definition of the CAD phenotype, i.e., with (CAD; n = 546) or without (CAD-free; n = 303) angiographically documented disease. We determined serum ferritin, as a biochemical estimate of iron stores, and the C282Y mutation in the HFE gene, i.e., the main cause of hemochromatosis in Caucasians. The relationships of ferritin with serum markers of either inflammation [C-reactive protein (CRP)] or lipid peroxidation (malondialdehyde) were also investigated.

Results: Mean ferritin concentrations were slightly higher in CAD vs CAD-free individuals, but this difference disappeared after adjusting for sex and CRP. Ferritin was significantly correlated with CRP (Spearman’s test, ρ = 0.129; P <0.001). Heterozygotes for Cys282Tyr were 4.8% among the CAD group and 6.6% among the CAD-free group (P = 0.26). The prevalence of high concentrations of stored iron, defined as ferritin concentrations above the sex-specific upper quintiles of the control distribution, was also similar in the two groups. There was a higher prevalence of “iron depletion” in CAD-free vs CAD females (20% vs 8.8%, respectively), but this difference disappeared after adjustment for age and other cardiovascular risk factors (odds ratio, 0.66; 95% confidence interval, 0.21–2.08). No differences in iron markers were found in CAD patients with or without myocardial infarction.

Conclusions: Our results do not support a role for biochemical or genetic markers of iron stores as predictors of the risk of CAD or its thrombotic complications.
Molecular Diagnosis of Analbuminemia: A Novel Mutation Identified in Two Amerindian and Two Turkish Families
Authors: Galliano Monica , Campagnoli Monica , Rossi Antonio , Wirsing von König Carl Heinz , Lyon Andrew W , Cefle Kivanc , Yildiz Alaattin , Palanduz Sukru , Ozturk Sukru , Minchiotti Lorenzo
Published: January 2020

Background: Analbuminemia is a rare autosomal recessive disorder in which individuals have little or no circulating albumin, usually the most abundant plasma protein. We describe a new mutation associated with analbuminemia.

Methods: We studied four apparently unrelated patients who had congenital analbuminemia: two of Amerindian and two of Turkish origin. The 14 exons and the flanking intron sequences of the albumin gene were amplified by PCR and screened for mutations by single-strand conformational polymorphism and heteroduplex analysis. The mutated DNA fragments were sequenced directly.

Results: In all four cases, analbuminemia was caused by the same mutation, an AT deletion at nucleotides 2430–2431, the 91st and 92nd bases of exon 3. This novel defect, named Kayseri, produces a frameshift leading to a premature stop two codons downstream. The predicted translation product would consist of 54 amino acid residues.

Conclusions: The AT deletion at nucleotides 2430–2431 is a novel mutation associated with analbuminemia.
Cardiac Troponin T and Creatine Kinase MB Content in Skeletal Muscle of the Uremic Rat
Authors: Fredericks Salim , Murray Joanne F , Carter Nicholas D , Chesser Alistair MS , Papachristou Stavroula , Yaqoob Magdi M , Collinson Paul O , Gaze David , Holt David W
Published: January 2020

Background: The assertion that creatine kinase MB (CK-MB) and the developmental isoforms of cardiac troponin T (cTnT) are expressed by skeletal muscle in some clinical settings is an extrapolation from nonuremic rodent studies. We studied the content of CK-MB and cTnT in skeletal muscle of the renal-insufficient rat.

Methods: Skeletal muscles (gastrocnemius) were collected from both five-sixths nephrectomized rats (n = 11) and sham-operated controls (n = 11). cTnT content was analyzed by Elecsys (Roche), immunoblotting, and immunohistochemistry with antibodies M7 and M11-7 (Roche). CK isoenzymes were analyzed electrophoretically.

Results: Trace concentrations of cTnT were detected in some of the skeletal muscle samples [controls (3 of 11) and uremic rats (1 of 11)] at concentrations <0.01% of that detected in heart. By contrast, positive staining appeared in both groups with M11-7 by immunoblotting and immunohistochemistry. No immunoreactivity was detected in skeletal muscle using M7 in the immunoblot format, although immunoreactivity was detected by immunohistochemistry in all samples. The median percentages of CK-MB were 6.0% and 4.1% for the skeletal muscle from control and uremic rats, respectively.

Conclusion: The detection of cTnT and CK-MB in skeletal muscle does not differ for uremic rats compared with sham-operated controls. cTnT isoforms detected by qualitative methods are not detected with the cTnT immunoassay. Observations with rodents should not necessarily be extrapolated to humans.
Quantitative Measurement of Lipoprotein Particles Containing Both Apolipoprotein AIV and Apolipoprotein B in Human Plasma by a Noncompetitive ELISA
Authors: Ferrer Fanny , Bigot-Corbel Edith , N′Guyen Patrick , Krempf Michel , Bard Jean-Marie
Published: January 2020

Background: A reliable method for plasma would be useful to investigate the role of apolipoprotein (apo) AIV when associated with apo B-containing or triglyceride-rich lipoproteins.

Method: We used a sandwich ELISA to quantify lipoprotein B:AIV particles (Lp B:AIVf; lipoproteins containing at least apo B and apo AIV) in plasma. The method used microtiter plates coated with purified anti-apo B immunoglobulins that selectively retained apo B-containing particles. Lipoproteins containing both apo B and apo AIV were distinguished from those containing only apo B by use of a peroxidase-labeled anti-apo AIV antibody. These subspecies were revealed by ABTS® reagent and further quantified by spectrophotometry. Results were expressed in mg/L apo AIV associated with apo B. This method was applied to samples with different cholesterol and triglyceride concentrations.

Results: The developed sandwich ELISA method identified and quantified Lp B:AIVf in plasma samples. Within- and between-run CVs were ∼10%, and analytical recoveries were 95–107%. Results were not significantly influenced by addition of triglycerides or by storage at −20 °C (up to 9 months). Under these conditions, plasma Lp B:AIVf concentrations were statistically higher in hypercholesterolemic and mixed hyperlipidemic individuals (53 ± 13 mg/L; P <0.001 and 70 ± 18 mg/L; P <0.001, respectively) than in normolipidemic individuals (43 ± 12 mg/L). Lp B:AIVf concentration appeared to be well correlated with total cholesterol, triglycerides, LDL-cholesterol, and apo B. These results were in contrast to total apo AIV, which was not different between dyslipidemic and normolipidemic individuals.

Conclusions: The developed ELISA method for Lp B:AIVf in plasma combines specificity, reliability, and speed. The increase in Lp B:AIVf concentrations in various dyslipidemic states, together with a lack of change in total apo AIV concentrations, suggests a redistribution of apo AIV toward apo B-containing lipoproteins when these lipoproteins accumulate.
Long-Term Analytical Performance of Hemostasis Field Methods as Assessed by Evaluation of the Results of an External Quality Assessment Program for Antithrombin
Authors: Meijer Piet , de Maat Moniek PM , Kluft Cornelis , Haverkate Frits , van Houwelingen Hans C
Published: January 2020

Background: It is important for a laboratory to know the stability of performance of laboratory tests over time. The aim of this study was to adapt from the field of clinical chemistry a method to assess the long-term analytical performance of hemostasis field methods.

Methods: The linear regression model was used to compare the laboratory results with the consensus mean value of a survey. This model was applied to plasma antithrombin activity using the data for 82 laboratories, collected between 1996 and 1999 in the European Concerted Action on Thrombosis (ECAT) external quality assessment program. The long-term total, random, and systematic error were calculated. The variables introduced to define the long-term performance in this model were the long-term analytical CV (LCVa) and the analytical critical difference (ACD), which indicates the minimum difference necessary between two samples measured on a long-term time-scale to consider them statistically significantly different.

Results: The systematic error (bias) ranged from 4.5 to 103 units/L. The random error ranged from 24.4 to 242 units/L. For the majority of the laboratories, random error was the main component (>75%) of the total error. The LCVa, after adjustment for the contribution of the bias, ranged from 2.8% to 48%. The ACD ranged from 78 to 1290 units/L with a median value of 190 units/L. No statistically significant differences were observed for either LCVa or ACD between the two different measurement principles for antithrombin activity based on the inhibition of either thrombin or factor Xa.

Conclusions: This linear regression model is useful for assessing the total error, random error, and bias for hemostasis field methods. The LCVa and ACD for measurement on a long-term time-scale appear to be useful for assessing the long-term analytical performance.
Computer-supported Interpretation of Protein Profiles after Capillary Electrophoresis
Authors: Jonsson Magnus , Carlson Joyce
Published: January 2020

Background: Electrophoretic patterns of proteins in serum/plasma are useful in the diagnosis and evaluation of many diseases. Capillary zone electrophoresis (CZE) allows rapid and automated protein separation and produces digital absorbance data, appropriate for mathematical analysis. We previously demonstrated success in detection of monoclonal immunoglobulins in such a system. This study tests new algorithms to produce rapid standardized computer-supported interpretation of the entire electropherogram.

Methods: Data from Beckman Paragon CZE 2000 electropherograms were compared with quantitative protein data from >800 routine clinical samples. Algorithms were designed to produce semiquantitative analyses of major proteins and to define different patterns of inflammation based on the electropherogram.

Results: The algorithms produced reliable semiquantitative evaluations of prealbumin, albumin, α1-antitrypsin, haptoglobin, and transferrin, but were less accurate for α1-acid glycoprotein. Some genetic variants of albumin and deficiency variants of α1-antitrypsin were easily recognized. Complex clinical traits such as degree and type of inflammation could be evaluated. When used together with previously developed algorithms addressing immunoglobulins, the new algorithms provide relevant clinical interpretation. Selected outputs indicate the need for reflex testing or evaluation by specialists.

Conclusions: Automation of both electrophoresis and interpretation can provide a rapid, inexpensive, standardized analysis that can hopefully improve the diagnostic information and clinical outcome for large groups of patients. It also provides objective criteria for clinical interpretations, to be validated or adjusted in future clinical studies.
Human Tissue Kallikreins: A Family of New Cancer Biomarkers
Authors: Diamandis Eleftherios P , Yousef George M
Published: January 2020

Kallikreins are a subgroup of the serine protease enzyme family. Until recently, it was thought that the human kallikrein gene family contained only three members. In the past 3 years, the entire human kallikrein gene locus was discovered and found to contain 15 kallikrein genes. Kallikreins are expressed in many tissues, including steroid hormone-producing or hormone-dependent tissues such as the prostate, breast, ovary, and testis. Most, if not all, kallikreins are regulated by steroid hormones in cancer cell lines. There is strong but circumstantial evidence linking kallikreins and cancer. Prostate-specific antigen (PSA; hK3) and, more recently, human glandular kallikrein (hK2) are widely used tumor markers for prostate cancer. Three other kallikreins, hK6, hK10, and hK11, are emerging new serum biomarkers for ovarian and prostate cancer diagnosis and prognosis. Several other kallikreins are differentially expressed at both the mRNA and protein levels in various endocrine-related malignancies, and they have prognostic value. The coexpression of many kallikreins in the same tissues (healthy and malignant) points to the possible involvement of kallikreins in cascade enzymatic pathways. In addition to their diagnostic/prognostic potential, kallikreins may also emerge as attractive targets for therapeutics.
Host Genetic Polymorphism Analysis in Cervical Cancer
Authors: Calhoun Eric S , McGovern Renee M , Janney Carol A , Cerhan James R , Iturria Stephen J , Smith David I , Gostout Bobbie S , Persing David H
Published: January 2020

Background: The natural history of cervical cancer comprises a latency period that probably involves long-term immunologic tolerance of human papillomavirus infection. Identifying host determinants of viral persistence may help to better understand the mechanisms of tolerance and may lead to the development of tests that can allow more focused follow-up of high-risk individuals.

Methods: Genotypic frequencies of 12 polymorphic loci in four candidate genes from 127 cervical cancer patients were compared with a control group of 108 female blood donors. Genotypes were determined by PCR amplification and direct sequencing of isolated genomic DNA.

Results: The tumor necrosis factor-α (TNFα) −238 polymorphism was significantly underrepresented in cervical cancer patients [heterozygotes (HETs), odds ratio (OR) = 0.33; 95% confidence interval (CI), 0.11–0.96], as was the TNFα −376 polymorphism (P = 0.02; 0% for any variant genotype in cases vs 4.7% in controls). The NRAMP1 3′ untranslated region STP+86 polymorphism also appeared to be inversely associated with cervical cancer, but this result did not reach statistical significance (HET, OR = 0.57; 95% CI, 0.32–1.02). The p53 codon 72 arginine allele showed a suggestive negative association with cervical cancer (HET, OR = 0.49; 95% CI, 0.14–1.63; homozygotes, OR = 0.35; 95% CI, 0.11–1.17). The remaining alleles tested showed no association with cervical cancer.

Conclusions: We identified host genetic polymorphisms that may be associated with cervical cancer risk, some of which have been linked to potential functional effects on cellular immune responses or antigen processing. We failed to confirm earlier reports of increased cervical cancer susceptibility in women who harbor the p53 P72R allele. Although our findings support the general hypothesis that host immunogenetic determinants other than class II MHC may be important in the development of cervical cancer, further analysis of the HLA gene cluster comprising the implicated TNFα single-nucleotide polymorphisms will be required to determine whether their association is linkage independent.
Application of a Multigene Reverse Transcription-PCR Assay for Detection of Mammaglobin and Complementary Transcribed Genes in Breast Cancer Lymph Nodes
Authors: Zehentner Barbara K , Dillon Davin C , Jiang Yuqiu , Xu Jiangchun , Bennington Angela , Molesh David A , Zhang XinQun , Reed Steven G , Persing David , Houghton Raymond L
Published: January 2020

Background: Mammaglobin mRNA expression is found in 70–80% of primary and metastatic breast tumor biopsies. The potential breast tumor markers B305D, B726P, and γ-aminobutyrate type A receptor π subunit (GABAπ) complement the expression of mammaglobin. Collectively the expression profile of these four genes could be used as a diagnostic and prognostic indicator for breast cancer.

Methods: A multigene reverse transcription-PCR (RT-PCR) assay was established to detect the expression of mammaglobin, GABAπ, B305D, and B726P simultaneously. Specific primers and TaqMan® probes were used to analyze combined mRNA expression profiles in primary breast tumors and metastatic lymph node specimens.

Results: The multigene RT-PCR assay detected substantial expression signals in 27 of 27 primary tumor and 50 of 50 metastatic breast lymph node samples. Specificity studies demonstrated no significant expression signal in 27 non-breast cancer lymph nodes, in 22 various healthy tissue samples, or in 14 colon tumor samples.

Conclusion: The novel RT-PCR-based assay described here provides a sensitive detection system for disseminated breast tumor cells in lymph nodes. In addition, this multigene assay could also be used to test peripheral blood and bone marrow samples.
Diagnosis and Molecular Monitoring of Acute Promyelocytic Leukemia Using DzyNA Reverse Transcription-PCR to Quantify PML/RARα Fusion Transcripts
Authors: Applegate Tanya L , Iland Harry J , Mokany Elisa , Todd Alison V
Published: January 2020

Background: PML/RARα fusion transcripts provide a readily accessible marker for diagnosis of acute promyelocytic leukemia (APL) and for monitoring response to therapy. Survival rates are improved by therapies guided by such monitoring. We assessed the potential of DzyNA reverse transcription-PCR (RT-PCR) for measurement of PML/RARα fusion transcripts.

Methods: Parallel single-tube DzyNA RT-PCR protocols were developed to allow real-time fluorescent quantification of PML/RARα fusion transcripts and a low abundance control transcript, normal BCR. Calibration curves, generated using cell line RNA, allowed estimation of these transcripts in RNA from patients with APL at various stages of the disease.

Results: DzyNA RT-PCR calibration curves were linear for both transcripts over a broad range and demonstrated interassay variations of 12% (mean, 658 ng) and 10% (mean, 263 ng), respectively. The protocols detected low concentrations of transcripts and resolved twofold dilutions. PML/RARα mRNA was quantified in 10 patients at diagnosis and in 1 patient over a 7-year period. Monitoring of transcript concentrations effectively reflected the disease course in one patient and demonstrated that an increase in PML/RARα transcripts can be detected 4–6 months before hematologic relapse, with no false-positive results.

Conclusion: DzyNA RT-PCR has potential for use in clinical practice as a tool for diagnosis of APL and forsubsequent monitoring of minimal residual disease and detection of molecular relapse.
Determination of the Factor V Leiden Single-Nucleotide Polymorphism in a Commercial Clinical Laboratory by Use of NanoChip Microelectronic Array Technology
Authors: Evans Jess G , Lee-Tataseo Cindy
Published: January 2020

Background: Methods for analysis of the single-nucleotide polymorphism (SNP) known as factor V Leiden (FVL) are described. The technique provides rapid, highly accurate detection of the point mutation that encodes for replacement of arginine-506 with glutamine. After formal assay qualification, 758 clinical samples that had previously been analyzed by the InvaderTM Monoplex Assay were tested as research samples in a commercial clinical laboratory.

Methods: Primers specific for factor V (FV) were prepared, and PCR was performed. Samples were analyzed using the NanoChip® Molecular Biology Workstation with fluorescently labeled reporters for wild-type and SNP sequences.

Results: Of the 635 samples classified by the Third WaveTM assay as FV wild type, 10 were identified as heterozygous FVL by the NanoChip technique. Similarly, of the 114 putative heterozygous samples, 4 were wild type, and of the 9 reported homozygous samples, 6 were homozygous, 2 were heterozygous, and 1 was FV wild type by the NanoChip assay. All 17 results that were discordant with the Third Wave analysis were confirmed by DNA sequencing to be correctly classified by the NanoChip technology. The Nanochip system was 100% accurate in characterizing wild-type, heterozygous, and homozygous samples compared with accuracies of 99.2%, 90.2%, and 100% for the comparable Third Wave analysis.

Conclusions: The NanoChip microelectronic chip array technology is an accurate and convenient method for FVL screening of research samples in a clinical laboratory environment.
Transcriptional control of hyphal morphogenesis inCandida albicans
Authors: Villa Sonia , Hamideh Mohammad , Weinstock Anthony , Qasim Mohammad N , Hazbun Tony R , Sellam Adnane , Hernday Aaron D , Thangamani Shankar
Published: January 2020

Candida albicans is a multimorphic commensal organism and opportunistic fungal pathogen in humans. A morphological switch between unicellular budding yeast and multicellular filamentous hyphal growth forms plays a vital role in the virulence of C. albicans, and this transition is regulated in response to a range of environmental cues that are encountered in distinct host niches. Many unique transcription factors contribute to the transcriptional regulatory network that integrates these distinct environmental cues and determines which phenotypic state will be expressed. These hyphal morphogenesis regulators have been extensively investigated, and represent an increasingly important focus of study, due to their central role in controlling a key C. albicans virulence attribute. This review provides a succinct summary of the transcriptional regulatory factors and environmental signals that control hyphal morphogenesis in C. albicans.
Data Interfaces: Tensions between Copyright and Competition Law – A New Swiss Court Practice for an Old Problem
Authors: Weber Rolf H
Published: January 2020

The reconciliation of intellectual property rights laws with competition rules is a difficult task. For decades, courts and legal doctrine have been trying to draw fine lines in order to balance corresponding (yet partly differing) interests. Recently, the Swiss Federal Administrative Court ruled on the tensions between copyright and competition law, particularly assessing data interfaces in the credit/debit card payment transactions business. The Court applied a restrictive approach in respect of the exclusively protected sphere of the rightholder–its behaviour of building entry barriers for potential competitors was considered non-compliant with the Cartel Act. This contribution outlines the general principles applied in relation to the reconciliation of intellectual property rights with competition rules in the Swiss law environment and analyses the detailed, concrete reasoning of the recent decision in the light of the needs of the growing information society.
Let's talk about the interregnum: Gramsci and the crisis of the liberal world order
Authors: Babic Milan
Published: January 2020

The liberal international order (LIO) is in crisis. Numerous publications, debates and events have time and again made it clear that we are in the midst of a grand transformation of world order. While most contributions focus on either what is slowly dying (the LIO) or what might come next (China, multipolarity, chaos?), there is less analytical engagement with what lies in between those two phases of world order. Under the assumption that this period could last years or even decades, a set of analytical tools to understand this interregnum is urgently needed. This article proposes an analytical framework that builds on Gramscian concepts of crisis that will help us understand the current crisis of the LIO in a more systematic way. It addresses a gap in the literature on changing world order by elaborating three Gramsci-inspired crisis characteristics—processuality, organicity and morbidity—that sketch the current crisis landscape in a systematic way. Building on this framework, the article suggests different empirical entry points to the study of the crisis of the LIO and calls for a research agenda that takes this crisis seriously as a distinct period of changing world orders.
Implementation and Widespread of Inflammatory Bowel Disease Vaccination and Immunization Guidelines: Not an Easy Task
Authors: Martinelli Massimo , Miele Erasmo
Published: January 2020

Current IBD vaccination and immunization guidelines need to be widespread.