bluuebunny/crossref_metadata_2025_split huggingface dataset:
Impact of the Third Cholesterol Report from the Adult Treatment Panel of the National Cholesterol Education Program on the Clinical Laboratory
Authors: Warnick G Russell , Myers Gary L , Cooper Gerald R , Rifai Nader 
Published: January 2020

Abstract
Background: The US National Cholesterol Education Program has recently released the third report of the Adult Treatment Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults. Incorporating new evidence and more consistent with other international intervention programs, these more complex guidelines will considerably expand indications for treatment. The implications for clinical laboratories are summarized in this report.
Content: LDL-cholesterol (LDL-C) remains the major focus for classification and treatment, whereas diabetes, the presence of multiple risk factors, including the metabolic syndrome, and increased triglycerides (TGs), will now require more intensive management. For screening, a fasting lipoprotein profile is recommended, adding LDL-C and TGs to the previous measurements of total cholesterol and HDL-cholesterol (HDL-C). Lowering the cutpoints defining optimal LDL-C [100 mg/dL (2.58 mmol/L)] and normal TGs [150 mg/dL (1.70 mmol/L)] and raising the cutpoint for low HDL-C to 40 mg/dL (1.03 mmol/L) will select more patients for treatment. A new marker, non-HDL-C, becomes a secondary target in treating high TGs.
Conclusions: Laboratories will need to adjust reporting formats and interpretations and can expect more requests for tests to characterize secondary causes of dyslipidemia, e.g., diabetes, and for the so-called “emerging risk factors”, e.g., lipoprotein(a), homocysteine, and C-reactive protein.
Effect of Disease Complications on Hospital Costs
Authors: Young Donald S , Sachais Bruce S , Jefferies Leigh C 
Published: January 2020

Abstract
Background: To test the hypothesis that complications increase the use of resources in managing patients in hospitals, we examined the costs of managing patients with the same disease with and without complications.
Methods: We used a database developed by the University HealthSystems Consortium that contains the costs of managing more than 1 million patients in 60 University hospitals. We created a simplified database of the costs of 457 445 patients in 111-paired diagnosis-related groups (DRGs) that were classified as either having or not having complications and/or comorbidities. Costs were calculated from the ratio of costs to charges within the individual hospitals.
Results: The median costs of managing patients with complications were higher than those for managing patients without complications, confirming the appropriateness of the dual classification. Notably, these extra costs were largely incurred through increased length of stay. Of note, the cost per day for DRGs with complications and/or comorbidities was most often less than that for the corresponding uncomplicated conditions. Although accommodation costs generally were the largest single component of total costs for both complicated and uncomplicated conditions, in only 31 DRGs (15 with complications, 16 without) did they account for more than one-half the total costs. Laboratory and drug costs were higher for complicated conditions, but as a proportion of total costs were comparable for complicated and uncomplicated conditions.
Conclusions: Complications in patients are associated with increased hospital costs, although the costs per day of hospitalization are often less than in patients without such complications.
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

AbstractBackground: 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

Abstract
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

AbstractBackground: 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

AbstractBackground: Enzymes of the cytochrome P450 3A (CYP3A) family are responsible for the metabolism of &gt;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

Abstract
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

AbstractBackground: 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

AbstractBackground: 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

Abstract
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, &gt;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 &gt;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

AbstractBackground: 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 &lt;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

AbstractBackground: 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.