bluuebunny/crossref_metadata_2025_split huggingface dataset:
Immunochemical Assay of Hemoglobin with Nε-(Carboxymethyl)lysine at Lysine 66 of the β Chain
Authors: Iwamoto Hisahiko , Motomiya Yoshihiro , Miura Keisuke , Morisawa Masayo , Yoshimura Yoshimichi , Maruyama Ikuro 
Published: January 2020

AbstractBackground: N ε-(Carboxymethyl)lysine (CML), a well-characterized and major advanced glycation end product structure, is produced via a Maillard reaction by nonenzymatic glycation and/or oxidation. Although few of the carboxymethylation sites of lysine residues on proteins have been identified, it is known that the possible lysine glycation site in hemoglobin (Hb) is Lys-66 on the β chain. We aimed to develop an assay for the Hb with a CML (CML-Hb) site specific to Lys-66 on the Hb β chain and to determine whether the lysine residue at that site is carboxymethylated.Methods: Ala-His-Gly-Lys-Lys(CM)-Val-Leu-Gly-Ala-Phe-Ser-Cys, the peptide derived from the β chain of human Hb, was synthesized as an immunogen, and a monoclonal antibody against the peptide was prepared. A latex immunoassay method was established using the antibody on an automatic analyzer. In this study, 20 samples from healthy subjects and 80 samples from nondiabetic patients undergoing hemodialysis (HD) were analyzed.Results: The latex immunoassay method using the antibody correlated significantly with the ELISA method using the antibody (r = 0.95; P &lt;0.001). Between healthy subjects (n = 20) and nondiabetic HD patients (n = 80), a significant difference was seen in circulating CML-Hb (525 ± 76 vs 778 ± 137 pmol CML/mg of Hb; P &lt;0.0001).Conclusion: The latex method for the CML-Hb site specific to Lys-66 on the β chain can measure large numbers of samples on an automatic analyzer.
Near-Infrared Spectroscopy for Measuring Urea in Hemodialysis Fluids
Authors: Eddy Christopher V , Arnold Mark A 
Published: January 2020

AbstractBackground: Near-infrared spectroscopy is proposed as a method for providing real-time urea concentrations during hemodialysis treatments. The feasibility of such noninvasive urea measurements is evaluated in undiluted dialysate fluid.Methods: Near-infrared spectra were collected from calibration solutions of urea prepared in dialysate fluid. Spectra were collected over three distinct spectral regions, and partial least-squares calibration models were optimized and compared for each. Selectivity for urea was demonstrated with two-component samples composed of urea and glucose in the dialysate matrix. The clinical significance of this approach was assessed by measuring urea in real hemodialysate samples.Results: Urea absorptions within the combination and short-wavelength, near-infrared spectral regions provided sufficient spectral information for sound calibration models in the dialysate matrix. The combination spectral region had SEs of calibration (SEC) and prediction (SEP) of 0.38 mmol/L and 0.26 mmol/L, respectively, over the 4720–4600 cm−1 spectral range with 5 partial least-square factors. A second calibration model was established over the combination region from a series of solutions prepared with independently variable concentrations of urea and glucose. The best calibration model for urea in the presence of variable glucose concentrations had a SEC of 0.6 mmol/L and a SEP of 0.4 mmol/L for a 5-factor model over the 4600–4350 cm−1 spectral range. There was no significant decrease in SEP when the 4720–4600 cm−1 calibration model was used to measure urea in real samples collected during actual hemodialysis.Conclusions: Urea can be determined with sufficient sensitivity and selectivity for clinical measurements within the matrix of the hemodialysis fluid.
Determination of Acid α-Glucosidase Activity in Blood Spots as a Diagnostic Test for Pompe Disease
Authors: Umapathysivam Kandiah , Hopwood John J , Meikle Peter J 
Published: January 2020

Abstract
Background: Pompe disease is an autosomal recessive disorder of glycogen metabolism that is characterized by a deficiency of the lysosomal acid α-glucosidase. Enzyme replacement therapy for the infantile and juvenile forms of Pompe disease currently is undergoing clinical trials. Early diagnosis before the onset of irreversible pathology is thought to be critical for maximum efficacy of current and proposed therapies. In the absence of a family history, the presymptomatic detection of these disorders ideally can be achieved through a newborn-screening program. Currently, the clinical diagnosis of Pompe disease is confirmed by the virtual absence, in infantile onset, or a marked reduction, in juvenile and adult onset, of acid α-glucosidase activity in muscle biopsies and cultured fibroblasts. These assays are invasive and not suited to large-scale screening.
Methods: A sensitive immune-capture enzyme activity assay for the measurement of acid α-glucosidase protein was developed and used to determine the activity of this enzyme in dried-blood spots from newborn and adult controls, Pompe-affected individuals, and obligate heterozygotes.
Results: Pompe-affected individuals showed an almost total absence of acid α-glucosidase activity in blood spots. The assay showed a sensitivity and specificity of 100% for the identification of Pompe-affected individuals.
Conclusions: The determination of acid α-glucosidase activity in dried-blood spots is a useful, noninvasive diagnostic assay for the identification of Pompe disease. With further validation, this procedure could be adapted for use with blood spots collected in newborn-screening programs.
Solid-Phase Amplification for Detection of C282Y and H63D Hemochromatosis (HFE) Gene Mutations
Authors: Turner Mark S , Penning Sarah , Sharp Angela , Hyland Valentine J , Harris Ray , Morris C Phillip , van Daal Angela 
Published: January 2020

Abstract
Background: There is a need for simple, rapid, and inexpensive methods for the detection of single-nucleotide polymorphisms. Our aim was to develop a single-tube ELISA-like PCR assay and evaluate it by detecting the common C282Y and H63D mutations found in the hemochromatosis gene (HFE) by use of clinical samples.
Methods: The method, termed solid-phase amplification (SPA), involves dual liquid- and solid-phase amplification of a target sequence by the use of two PCR primers, one of which is in two forms: the first is covalently immobilized to the wall of a microwell, and the second is free in solution. During allele-specific amplification, both the free and solid-phase amplicons are labeled by incorporation of digoxigenin (DIG)-dUTP. The amount of surface-bound amplicon is determined colorimetrically by the use of an alkaline phosphatase-anti-DIG-Fab conjugate and p-nitrophenyl phosphate.
Results: Two different amplicon-labeling methods were evaluated. Analysis of 173 clinical samples for the C282Y and H63D HFE point mutations with SPA revealed that only one sample was incorrectly diagnosed, apparently because of operator error, when compared with conventional restriction fragment length polymorphism assay results.
Conclusions: The SPA assay has potential for medium-scale mutation detection, having the advantage of being manipulatively simple and immediately adaptable for use in clinical laboratories with existing ELISA instrumentation.
Discrimination of Prostate Cancer from Benign Disease by Plasma Measurement of Intact, Free Prostate-specific Antigen Lacking an Internal Cleavage Site at Lys145-Lys146
Authors: Nurmikko Pauliina , Pettersson Kim , Piironen Timo , Hugosson Jonas , Lilja Hans 
Published: January 2020

AbstractBackground: The proportion of free prostate-specific antigen (PSA) is higher in the sera of patients with benign prostatic hyperplasia compared with patients with prostate cancer (PCa). We developed an immunoassay that measures intact, free PSA forms (fPSA-I), but does not detect free PSA that has been internally cleaved at Lys145-Lys146 (fPSA-N), and investigated whether this form could discriminate patients with PCa from those without PCa.Methods: The assay for fPSA-I uses a novel monoclonal antibody (MAb) that does not detect PSA that has been internally cleaved at Lys145-Lys146. A MAb specific for free PSA was used as a capture antibody, and purified recombinant proPSA was used as a calibrator. The concentrations of fPSA-I, free PSA (PSA-F), and total PSA (PSA-T) were analyzed in EDTA-plasma samples (n = 276) from patients who participated in a screening program for PCa (PSA-T, 0.83–76.3 μg/L).Results: The detection limit of the fPSA-I assay was 0.035 μg/L. Both the measured concentrations of fPSA-I and the concentrations of fPSA-N (calculated as PSA-F − fPSA-I) provided statistically significant discrimination of the two clinical groups. By contrast, PSA-F did not discriminate between these groups. Each of the ratios fPSA-I/PSA-F, fPSA-N/PSA-T, and PSA-F/PSA-T separated cancer samples from noncancer samples in a statistically significant manner (P &lt;0.0001). The ratio fPSA-I/PSA-F was significantly higher in cancer (median, 59%) compared with noncancer samples (47%).Conclusions: The ratio fPSA-I/PSA-F is significantly higher in cancer compared with noncancer. The percentages of both fPSA-N/PSA-T and fPSA-I/PSA-F may provide interesting diagnostic enhancements alone or in combination with other markers and require further studies.
Spectrophotometric Microassay for δ-Aminolevulinate Dehydratase in Dried-Blood Spots as Confirmation for Hereditary Tyrosinemia Type I
Authors: Schulze Andreas , Frommhold David , Hoffmann Georg F , Mayatepek Ertan 
Published: January 2020

AbstractBackground: Hereditary tyrosinemia type I (HT) fulfills the criteria for inclusion in neonatal screening programs, but measurement of tyrosine lacks clinical specificity and quantitative assay of succinylacetone is laborious. We developed a semiquantitative assay based on inhibition of δ-aminolevulinate dehydratase (ALA-D) by succinylacetone.Methods: Preincubation of 3-mm discs from dried-blood spots and reaction of the enzyme with δ-aminolevulinic acid as substrate were performed in microtiter plates. After separation of the supernatant and 10 min of color reaction with modified Ehrlich reagent, the formation of porphobilinogen was measured at 550 nm in a plate reader.Results: The detection limit for succinylacetone was 0.3 μmol/L; imprecision (CV) was &lt;5.5% within-run and 10–16% between-run. Storage of blood spots at ambient temperature for several days led to a significant decrease of ALA-D activity. Enzyme activity was lost in filter cards at 45 °C, but remained stable at 2–37 °C. Enzyme activity was decreased in EDTA blood. The absorbance at 550 nm was 0.221 (± 0.073) in healthy neonates and 0.043–0.100 in 11 patients with HT. All neonates with increased tyrosine (above the 99.5th centile) in neonatal screening (97 of 47 000) had normal results by the new assay.Conclusions: The spectrophotometric microassay for ALA-D is a simple and sensitive test for HT. This represents a basis for further examination of its general reliability as a confirmatory test if tyrosine is found to be increased.
Interlaboratory Variation of Biochemical Markers of Bone Turnover
Authors: Seibel Markus J , Lang Matthias , Geilenkeuser Wolf-Jochen 
Published: January 2020

Abstract
Background: Biochemical markers of bone metabolism are used to assess skeletal turnover, but the variability of marker assays is still an issue of practical concern. We describe the results of an international proficiency testing program for biochemical bone markers among clinical laboratories.
Methods: Two serum and two urine pools (normal and increased marker concentrations) were sent on dry ice to 79 laboratories for analysis within 2 weeks of receipt.
Results: Data were submitted by 73 laboratories. The within-method interlaboratory CVs (CVILs) were as follows: serum bone-specific alkaline phosphatase (n = 47 laboratories), 16–48%; serum osteocalcin (n = 31), 16–42%; urinary free deoxypyridinoline (n = 30), 6.4–12%; urinary total deoxypyridinoline and pyridinoline (n = 29), 27–28%; urinary N-terminal cross-linked telopeptide of type I collagen (n = 10), 39%; serum C-terminal cross-linked telopeptide of type I collagen (ICTP; n = 8), 22–27%; urinary hydroxyproline (n = 13), 12%. Analytical results showed both systematic and nonsystematic deviations. In identical samples, results obtained for the same marker by the same method differed up to 7.3-fold. In urine-based assays, correction for urinary creatinine slightly increased CVs.
Conclusion: Even with identical assays and methods, results for most biochemical markers of bone turnover differ markedly among laboratories.
Successful Testing Protocols in Virology
Authors: Kelley Violet A , Caliendo Angela M 
Published: January 2020

AbstractMolecular methods have had a significant impact on the diagnosis of viral infections because of their superior sensitivity and rapid turnaround time compared with conventional diagnostic methods. These characteristics have allowed molecular tests to play a central role in the use of testing protocols for managing viral infections. Several examples of such protocols are reviewed in this report, including the use of molecular testing for early disease detection to improve overall disease management and to direct antiviral therapy.
Laboratory Investigation of Thrombophilia
Authors: Tripodi Armando , Mannucci Pier Mannuccio 
Published: January 2020

Abstract
Until recently, laboratory diagnosis of thrombophilia was based on investigation of the plasmatic anticoagulant pathways to detect antithrombin, protein C, and protein S deficiencies and on the search for dysfibrinogenemia and anti-phospholipid antibodies/lupus anticoagulants. More recently, laboratory investigations have been expanded to include activated protein C (APC) resistance, attributable or not to the presence of the factor V Leiden mutation; hyperprothrombinemia attributable to the presence of the prothrombin gene mutation G20210A; and hyperhomocysteinemia attributable to impairment of the relevant metabolic pathway because of enzymatic and/or vitamin deficiencies. All of the above are established congenital or acquired conditions associated with an increased risk of venous and, more rarely, arterial thrombosis.
Testing is recommended for patients who have a history of venous thrombosis and should be extended to their first-degree family members. Because most of the tests are not reliable during anticoagulation, it is preferable to postpone laboratory testing until after discontinuation of treatment.
Whenever possible, testing should be performed by means of functional assays. DNA analysis is required for the prothrombin gene mutation G20210A. Laboratory diagnosis for anti-phospholipid antibodies/lupus anticoagulant should be performed by a combination of tests, including phospholipid-dependent clotting assays and solid-phase anti-cardiolipin antibodies. Hyperhomocysteinemia can be diagnosed by HPLC methods or by fluorescence polarization immunoassays.
Instability of Lipoprotein(a) in Plasma Stored at −70 °C
Authors: Simó Josep M , Camps Jordi , Vilella Elisabet , Gómez Federico , Paul Antonio , Joven Jorge 
Published: January 2020

AbstractBackground: There is considerable evidence to suggest that plasma lipoprotein(a) [Lp(a)] concentration is a cardiovascular risk factor. Confusing results in epidemiologic studies, however, suggest that the effects of storage should be further investigated. The influence of the assay method, the initial plasma Lp(a) concentration, and the apolipoprotein(a) [apo(a)] genotype are all factors that should be considered.Methods: Blood was obtained from 65 survivors of premature myocardial infarction and 95 age-matched controls. The plasma samples were stored in sterile conditions at −70 °C for 5 years in the presence of antioxidant and antiproteolytic substances. Plasma Lp(a) was measured by immunoturbidimetry, and apo(a) alleles were determined by pulsed-field gel electrophoresis and Southern blotting.Results: Plasma Lp(a) was significantly higher in patients. The mean kringle number for the smallest isoform was also lower in patients than in controls, but no differences were found in the distribution of the largest isoform. All patients and controls were heterozygotes. During storage, mean Lp(a) decreased significantly in samples from patients (−23%; P &lt;0.001) but not in samples from controls (−9%; P, not significant). This was not related to the kringle number and was limited to samples with initial plasma Lp(a) concentrations between 41 and 345 mg/L.Conclusions: Plasma Lp(a) from patients is less stable than Lp(a) from controls, and the difference is not related to distribution of apo(a) genotypes but may be concentration-dependent. Differential sample stability may complicate the interpretation of several studies.
Whole-Blood Calcineurin Activity Is Not Predicted by Cyclosporine Blood Concentration in Renal Transplant Recipients
Authors: Caruso Raffaele , Perico Norberto , Cattaneo Dario , Piccinini Giampiero , Bonazzola Samantha , Remuzzi Giuseppe , Gaspari Flavio 
Published: January 2020

AbstractBackground: In transplant patients, current cyclosporine (CsA) dose monitoring with classic pharmacokinetics has demonstrated limitations. Evaluation of the activity of calcineurin (CN), the serine-threonine phosphatase enzyme target of CsA, has been proposed as a reliable way to optimize CsA dosing.Methods: CN activity was measured in whole blood in an attempt to overcome the high variability of results obtained previously with peripheral blood mononuclear cells (PBMCs). We also explored, in vitro, a possible relationship between the CsA concentration and CN inhibition in whole blood. Finally, we assessed whether the CsA blood trough concentration correlates with whole-blood CN activity in kidney transplant recipients (n = 15) on maintenance immunosuppression with CsA.Results: In 14 healthy individuals, less scattered CN activity values were documented in whole blood than in the PBMC fraction. Whole-blood CN activity was higher than the sum of the enzyme activity in each cell blood fraction. After ex vivo incubation of whole blood from healthy subjects (n = 5) with increasing concentrations of CsA (50–1000 μg/L for 1 h), a concentration-dependent inhibition of CN activity was found comparable to that in the PBMC fraction. Moreover, in 15 kidney transplant recipients, no relationship was found between CsA pharmacokinetic parameters and CN activity at time 0. However, a highly significant correlation was found between CN area under the CN activity-time curve, which represents the extent of the CN daily inhibition, and CN activity at time 0 (r = 0.79; P &lt;0.01) and at 12 h postdosing (r = 0.96; P &lt;0.01).Conclusions: Measuring CN activity in whole-blood samples is a reproducible method. In kidney transplant recipients, CsA trough concentrations do not predict baseline CN activity. Moreover, a single CN activity monitoring at baseline or at time 12 h post-CsA dosing may be a useful surrogate for the inhibition of this enzyme by CsA during 12 h.
Direct Serum Total Iron-binding Capacity Assay Suitable for Automated Analyzers
Authors: Siek Gordon , Lawlor Joseph , Pelczar Donna , Sane Meena , Musto Joseph 
Published: January 2020

Abstract
Background: Present methods for measuring serum total iron-binding capacity (TIBC) involve manipulation of samples or performance of two assays on each sample. We developed a direct automated assay (DTIBC) for TIBC.
Methods: We added to serum a saturating amount of iron bound to an excess of chelating dye at a low pH, recorded a blank reading that represented the sum of the saturating amount of iron plus the serum iron, and then added a strong neutral pH buffer. The decrease in absorbance (as transferrin extracts iron from the iron-dye complex) is directly proportional to the TIBC. TIBC values for 125 patients were determined by DTIBC, alumina column TIBC (AC), magnetic particle TIBC (MTIBC), and the UIBC method (UIBC) on Roche COBAS FARA and Mira chemistry analyzers. In a separate study, TIBC values for 128 patients were determined on an Olympus AU400 by the DTIBC and the MTIBC methods.
Results: Methods comparisons on the COBAS analyzers yielded the following results: DTIBC = 1.05(MTIBC) − 1.0 μmol/L (r = 0.987; Sy|x = 2.6 μmol/L); DTIBC = 1.07(AC) − 1.0 μmol/L (r = 0.982; Sy|x = 3.0 μmol/L); and DTIBC = 1.14(UIBC) + 3.4 μmol/L (r = 0.982; Sy|x = 3.0 μmol/L). A similar correlation study using the Olympus AU400 yielded DTIBC = 1.00(MTIBC) − 0.1 μmol/L (r = 0.983; Sy|x = 2.7 μmol/L). The assay was linear from 12.5 to 125 μmol/L (70–700 μg/dL) TIBC on the COBAS FARA. Within- and between-run imprecision (CV) was ≤4.8% at two concentrations. Plasma samples were unsuitable for the method. No interference was seen with common interferants other than ascorbate, deferoxamine, and ferrous sulfate, and only at concentrations well above normal.
Conclusion: The new DTIBC assay is suitable for routine use in clinical laboratories and may improve the quality of iron metabolism studies.
Compliance of General Practitioners with a Guideline-based Decision Support System for Ordering Blood Tests
Authors: van Wijk Marc AM , van der Lei Johan , Mosseveld Mees , Bohnen Arthur M , van Bemmel Jan H 
Published: January 2020

Abstract
Background: Guidelines are viewed as a mechanism for disseminating a rapidly increasing body of knowledge. We determined the compliance of Dutch general practitioners with the recommendations for blood test ordering as defined in the guidelines of the Dutch College of General Practitioners.
Methods: We performed an audit of guideline compliance over a 12-month period (March 1996 through February 1997). In an observational study, a guideline-based decision support system for blood test ordering, BloodLink, was integrated with the electronic patient records of 31 general practitioners practicing in 23 practices (16 solo). BloodLink followed the guidelines of the Dutch College of General Practitioners. We determined compliance by comparing the recommendations for test ordering with the test(s) actually ordered. Compliance was expressed as the percentage of order forms that followed the recommendations for test ordering.
Results: Of 12 668 orders generated, 9091 (71%) used the decision-support software rather than the paper order forms. Twelve indications accounted for &gt;80% of the 7346 order forms that selected a testing indication in BloodLink. The most frequently used indication for test ordering was “vague complaints” (2209 order forms; 30.1%). Of the 7346 order forms, 39% were compliant. The most frequent type of noncompliance was the addition of tests. Six of the 12 tests most frequently added to the order forms were supported by revisions of guidelines that occurred within 3 years after the intervention period.
Conclusions: In general practice, noncompliance with guidelines is predominantly caused by adding tests. We conclude that noncompliance with a guideline seems to be partly caused by practitioners applying new medical insight before it is incorporated in a revision of that guideline.