bluuebunny/crossref_metadata_2025_split huggingface dataset:
Long-Term Evaluation of Electrospray Ionization Mass Spectrometric Analysis of Glycated Hemoglobin
Authors: Roberts Norman B , Amara Alieu B , Morris Michael , Green Brian N 
Published: January 2020

Abstract
Background: Electrospray ionization mass spectrometry (ESIMS) has been successfully applied to the identification of hemoglobin (Hb) variants and the presence of glucose adducts (mass difference of 162 Da) on the separate Hb α and β chains. To establish the potential of ESIMS as a routine and/or a reference method for the quantification of glycohemoglobin (HbA1c), we carried out a detailed evaluation over a 4-month period in a routine laboratory environment.
Methods: We optimized a procedure using ESIMS suitable for the routine quantitative analysis of HbA1c. We determined reliability and reproducibility over 4 months and assessed the potential for automated sample injection. We then compared values of 1022 blood samples from diabetic patients with a routine HPLC-based ion-exchange procedure (HA-8140; Menarini).
Results: Results of HbA1c measurement by ESIMS were available within 3 min. The analytical imprecision (CV) was 1.6–5.0% for both manual and automated injections. Data collection over the m/z 980-1400 range confirmed lower glycation of the α chain relative to the β chain (0.66:1). Only one glycation was observed per globin chain. The overall glycohemoglobin (i.e., the average of α- and β-chain glycations) measured by ESIMS (x) on 1022 blood samples was lower than by HPLC (y): y = 1.0432x + 0.4815. However, the β-chain glycation measured by ESIMS was up to 20% higher than the value measured by ion-exchange HPLC and showed a close conformity, particularly at 5–10% HbA1c, with the ion-exchange Diabetes Control and Complications Trial (DCCT)-corrected and the United Kingdom National External Quality Assessment Scheme DCCT mean return values.
Conclusions: ESIMS provides a precise measurement of HbA1c and, in particular, glycation of the β chain. The method is robust and could be proposed as a procedure to substantiate HbA1c measurement and/or calibration.
Evaluation of Blood Lead Proficiency Testing: Comparison of Open and Blind Paradigms
Authors: Parsons Patrick J , Reilly Andrew A , Esernio-Jenssen Debra , Werk Lloyd N , Mofenson Howard C , Stanton Noel V , Matte Thomas D 
Published: January 2020

Abstract
Background: Most proficiency testing (PT) programs operate with an open design in which clearly identified performance samples are distributed directly to participating laboratories on a shipping schedule announced in advance. In this study, we examine the effectiveness of assessing clinical laboratory performance for blood lead with an open PT by comparing its results with a double-blinded testing protocol.
Methods: Aliquots from up to 72 blood lead performance pools from the New York State Department of Health and the Wisconsin State Laboratory of Hygiene were disguised as routine patient specimens and submitted in two phases to up to 42 certified clinical laboratories for blood lead analysis. These 42 laboratories also received aliquots of the same performance samples for blood lead analysis directly from the “open” PT program provider.
Results: Data reported under blind and open strategies were scored against acceptable target ranges using the Clinical Laboratory Improvement Amendments of 1988 (CLIA ’88) criteria established for blood lead, i.e., ± 0.19 μmol/L (± 4 μg/dL) or ± 10%, whichever is greater. Performance differences between the strategies were also assessed. We found that 17.7% of all blind PT results were classified as unacceptable compared with only 4.5% of open PT results (P &lt;0.001). In phase 1, 13 of 22 laboratories (60%) exhibited a statistically significant difference (P &lt;0.05) between their blind and open PT performances, although for 6 laboratories the poorer blind performance may not necessarily have led to unsuccessful PT participation under CLIA ’88 criteria. Seven (32%) laboratories had unsuccessful aggregate performance (&lt;80%) under blind testing while maintaining successful performance in open testing. Of these seven, two had gross discrepancies motivating further investigation.
Conclusions: The data suggest that although ∼60% of clinical laboratories make special efforts to improve analytical performance on open PT samples relative to performance achieved for routine patient specimens, in most cases the differences are clinically insignificant and would not likely affect cumulative PT performance. Occasional use of blind PT may deter the inclination to treat performance samples more carefully.
High-Sensitivity C-Reactive Protein: A Novel and Promising Marker of Coronary Heart Disease
Authors: Rifai Nader , Ridker Paul M 
Published: January 2020

Abstract
Background: Coronary heart disease remains the leading cause of morbidity and mortality in the industrialized world. Clinical and laboratory studies have shown that inflammation plays a major role in the initiation, progression, and destabilization of atheromas. C-Reactive protein (CRP), an acute phase reactant that reflects low-grade systemic inflammation, has been studied in a variety of cardiovascular diseases.
Approach: Findings from prospective clinical trials were examined to determine the prognostic utility of CRP in acute coronary syndromes, and observations from epidemiological studies were reviewed to determine the ability of CRP to predict future first coronary events. The analytical considerations of CRP measurement in these clinical applications were also examined.
Content: In patients with established coronary disease, CRP has been shown to predict adverse clinical events. In addition, prospective studies have consistently shown that CRP is a strong predictor of future coronary events in apparently healthy men and women. The relative risk associated with CRP is independent of other cardiovascular disease risk factors. High-sensitivity CRP (hs-CRP) assays are needed for risk assessment of cardiovascular disease. Such assays are currently available but may require further standardization because patients’ results will be interpreted using population-based cutpoints. Preventive therapies to attenuate coronary risk in individuals with increased hs-CRP concentrations include aspirin and statin-type drugs.
Summary: hs-CRP has prognostic utility in patients with acute coronary syndromes and is a strong independent predictor of future coronary events in apparently healthy subjects.
Absence of Diurnal Variation of C-Reactive Protein Concentrations in Healthy Human Subjects
Authors: Meier-Ewert Hans K , Ridker Paul M , Rifai Nader , Price Nick , Dinges David F , Mullington Janet M 
Published: January 2020

AbstractBackground: The concentration of C-reactive protein (CRP) in otherwise healthy subjects has been shown to predict future risk of myocardial infarction and stroke. CRP is synthesized by the liver in response to interleukin-6, the serum concentration of which is subject to diurnal variation.Methods: To examine the existence of a time-of-day effect for baseline CRP values, we determined CRP concentrations in hourly blood samples drawn from healthy subjects (10 males, 3 females; age range, 21–35 years) during a baseline day in a controlled environment (8 h of nighttime sleep).Results: Overall CRP concentrations were low, with only three subjects having CRP concentrations &gt;2 mg/L. Comparison of raw data showed stability of CRP concentrations throughout the 24 h studied. When compared with cutoff values of CRP quintile derived from population-based studies, misclassification of greater than one quintile did not occur as a result of diurnal variation in any of the subjects studied. Nonparametric ANOVA comparing different time points showed no significant differences for both raw and z-transformed data. Analysis for rhythmic diurnal variation using a method fitting a cosine curve to the group data was negative.Conclusions: Our data show that baseline CRP concentrations are not subject to time-of-day variation and thus help to explain why CRP concentrations are a better predictor of vascular risk than interleukin-6. Determination of CRP for cardiovascular risk prediction may be performed without concern for diurnal variation.
Standardization of Cardiac Troponin I Assays: Round Robin of Ten Candidate Reference Materials
Authors: Christenson Robert H , Duh Show Hong , Apple Fred S , Bodor Geza S , Bunk David M , Dalluge Joseph , Panteghini Mauro , Potter James D , Welch Michael J , Wu Alan H B , Kahn Stephen E 
Published: January 2020

AbstractBackground: Cardiac troponin I (cTnI) results vary 100-fold among assays. As a step toward standardization, we examined the performance of 10 candidate reference materials (cRMs) in dilution studies with 13 cTnI measurement systems.Methods: Solutions of 10 cTnI cRMs, each characterized by NIST, were shipped to the manufacturers of 13 cTnI measurement systems. Manufacturers used their respective diluents to prepare each cRM in cTnI concentrations of 1, 10, 25, and 50 μg/L. For the purpose of ranking the cRMs, the deviation of each cTnI measurement from the expected response was assessed after normalization with the 10 μg/L cTnI solution. Normalized deviations were examined in five formats. Parameters from linear regression analysis of the measured cTnI vs expected values were also used to rank performance of the cRMs.Results: The three cRMs demonstrating the best overall rankings were complexes of troponins C, I, and T. The matrices for these three cRMs values differed; one was reconstituted directly from the lyophilized form submitted by the supplier; one was submitted in liquid form, lyophilized at NIST, and subsequently reconstituted; and the third was evaluated in the liquid form received from the supplier. The cRM demonstrating the fourth best performance was a binary complex of troponins C and I supplied in lyophilized form and reconstituted before distribution.Conclusions: The cRMs demonstrating the best performance characteristics in 13 cTnI analytical systems will be included in subsequent activities of the cTnI Standardization Committee of the AACC.
Production of Recombinant Human Creatine Kinase (r-hCK) Isozymes by Tandem Repeat Expression of M and B Genes and Characterization of r-hCK-MB
Authors: Sunahara Yoshiko , Uchida Kohji , Tanaka Toshio , Matsukawa Hirokazu , Inagaki Manabu , Matuo Yuhsi 
Published: January 2020

Abstract
Background: Serum creatine kinase-MB isoenzyme (CK-MB) is widely used as a marker of myocardial injury. We prepared recombinant human CK (r-hCK) MB isoenzyme and examined its potential for use as a control material for assay of CK-MB in serum.
Methods: cDNAs encoding CK-M and CK-B subunits were inserted into the same plasmid vector, followed by transformation of Escherichia coli. The resulting three types of CK isoenzymes were purified by conventional chromatography.
Results: The ratio of MB to MM to BB was 50:40:10 on the basis of CK activity. Highly purified CK-MB with a specific activity of 533 U/mg was produced in a yield of 5.7 mg/g of packed cells. Purified r-hCK-MB had the isoelectric point (pI 5.3) and molecular size (46 kDa for the subunit) of native CK-MB. Its immunoreactivity in an ELISA using antibody against native heart enzyme was similar to that of cardiac CK-MB. The r-hCK-MB retained &gt;90% activity for at least 4 months at 11 °C in a delipidated serum matrix in a liquid form at a concentration of 118 U/L.
Conclusions: r-hCK-MB shows key properties of the native cardiac isoenzyme and may be useful as a control and calibrator for serum assays of CK-MB.
Simultaneous Absolute Quantification of Target and Control Templates by Real-Time Fluorescence Reverse Transcription-PCR Using 4-(4′-Dimethylaminophenylazo)benzoic Acid as a Dark Quencher Dye
Authors: Kreuzer Karl-Anton , Bohn Alexander , Lupberger Joachim , Solassol Jerome , le Coutre Philipp , Schmidt Christian Andreas 
Published: January 2020

Abstract
Background: Despite the many advantages of real-time fluorescence reverse transcription-PCR (RT-PCR) as a quantitative analytical tool, simultaneous quantification of target and reference templates within one reaction has not been reported. We developed such an assay with an internal reference template.
Methods: For quantification of target and reference sequences, we used two fluorescent probes in one reaction vessel on an ABI PRISM 7700 SDS instrument. Fluorescent probes were labeled with either 6-carboxy-fluorescein or hexachloro-6-carboxy-fluorescein as reporter dye and 4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL) as a dark quencher fluorophore. To test the sensitivity and specificity of this assay, serial dilutions of reference and target templates were analyzed in one PCR reaction. In the presence of 10 β-actin molecules as control templates, 105 bcr/abl molecules were amplified, and 105 β-actin molecules were amplified in the presence of 10 bcr/abl copies. We also performed single and duplex measurements on samples from five patients with documented Philadelphia chromosome-positive chronic myelogenous leukemia disease courses (72 samples) and three with minor bcr/abl+ acute myelogenous leukemias (26 samples).
Results: For M-bcr/abl duplex RT-PCR, the correlation coefficient (r) for starting template amounts and threshold cycle values was 0.99; for m-bcr/abl, r = 0.96, indicating a precise log-linear relation for 10–105 copies/100 ng of cDNA. In the same PCR reactions, r = 0.99 for β-actin (coamplified with M-bcr/abl or m-bcr/abl) for 103–107 copies/100 ng cDNA. The linear correlation coefficient for single and duplex measurements was 0.98 for M- and m-bcr/abl in patient samples.
Conclusions: DABCYL can be used as dark quencher fluorophore in real-time fluorescence PCR. The duplex fluorescence RT-PCR assay for bcr/abl and β-actin transcripts allows monitoring of bcr/abl+ leukemias.
Molecular Strategy for Detecting Metastatic Cancers with Use of Multiple Tumor-specific MAGE-A Genes
Authors: Miyashiro Isao , Kuo Christine , Huynh Kelly , Iida Aritoshi , Morton Donald , Bilchik Anton , Giuliano Armando , Hoon Dave S B 
Published: January 2020

AbstractBackground: The human melanoma-associated antigen family A (MAGE-A) has high specificity and expression in various malignancies, but individual family members are expressed at low frequency in any one particular type of cancer. We therefore developed a method to detect mRNAs from multiple MAGE-A genes in a single reaction.Methods: Universal MAGE-A (uMAGE-A) primers and probe were designed to reverse-transcribe, amplify, and detect by electrochemiluminescence (ECL) MAGE-A mRNAs on the Origen Analyzer. The assay was performed on total RNA of melanoma (n = 9 cell lines and 24 tumors), breast cancer (n = 7 and 26), and colorectal cancer (CRC; n = 5 and 12). We also evaluated blood from melanoma (n = 50), breast cancer (n = 16), and CRC (n = 21) patients.Results: The uMAGE-A mRNA was detectable in 0.01–1 ng of cell line RNA. The identity of the uMAGE-A cDNA products was confirmed by sequencing and polyacrylamide gel electrophoresis. The uMAGE-A assay increased detection of melanoma, breast cancer, and CRC tumor by 13%, 31%, and 25%, respectively, compared with a MAGE-A1 assay, and by 17%, 19%, and 25%, respectively, compared with a MAGE-A3 assay. The uMAGE-A assay detected circulating tumor cells in the blood of melanoma (24%), breast cancer (25%), and CRC (29%) patients.Conclusions: The uMAGE-A reverse transcription-PCR/ECL assay provides a practical and sensitive approach for detection of various metastatic cancers in tissues and blood.
Development of a Stable-Isotope Dilution Assay for γ-Aminobutyric Acid (GABA) Transaminase in Isolated Leukocytes and Evidence That GABA and β-Alanine Transaminases Are Identical
Authors: Schor Danielle S M , Struys Eduard A , Hogema Boris M , Gibson K Michael , Jakobs Cornelis 
Published: January 2020

Abstract
Background: Several methods have been published for measuring γ-aminobutyric acid transaminase (GABA-T) activity, but these methods are either impracticable because of the use of radioisotopes or insufficiently sensitive to determine small enzyme activities in leukocyte extracts. We developed a direct and sensitive enzyme method.
Methods: We developed a stable-isotope dilution method for the measurement of [15N]glutamic acid derived from [15N]GABA and α-ketoglutaric acid, catalyzed by GABA-T. The method for analysis of [15N]glutamic acid comprised a solid-phase extraction procedure to isolate this analyte from incubation samples. After derivatization, [15N]glutamic acid was quantified by gas chromatography–mass spectrometry relative to its 2H5-labeled internal standard. In addition to [15N]GABA, [15N]β-alanine was a cosubstrate.
Results: GABA-T-deficient lymphoblasts showed diminished enzyme activity, with both [15N]GABA and [15N]β-alanine as substrate. Vigabatrin inhibited the enzyme activity for both substrates.
Conclusions: The activity of GABA-T can be accurately determined by our procedure using 15N-labeled substrate, measuring the formation of [15N]glutamic acid. Our results with [15N]β-alanine indicate that GABA and β-alanine transaminases are identical.
Comparison of ELISAs for Opiates, Methamphetamine, Cocaine Metabolite, Benzodiazepines, Phencyclidine, and Cannabinoids in Whole Blood and Urine
Authors: Kerrigan Sarah , Phillips Jr William H 
Published: January 2020

Abstract
Background: ELISAs are widely utilized in forensic drug analysis. A comparative assessment of microtiter plate assays for the detection of six common classes of drug in blood and urine is described.
Methods: ELISAs for opiates, methamphetamine, benzodiazepines, cocaine metabolite, phencyclidine (PCP), and tetrahydrocannabinol (THC) metabolite were evaluated in a side-by-side study. The analytical performance of 12 commercially available ELISAs was determined in terms of binding characteristics, dose–response curves, limits of detection, sensitivity, intra- and interassay imprecision, and lot-to-lot reproducibility. Assay performance was also compared using 855 forensic casework samples.
Results: Detection limits in whole blood for morphine, d-methamphetamine, nordiazepam, benzoylecgonine, nordiazepam, PCP, and l-11-nor-9-carboxy-Δ9-THC were 3, 2, &lt;4, 5, 25, and 3 μg/L, respectively, for the STC ELISAs. Corresponding detection limits for Immunalysis ELISAs were &lt;1, &lt;2, &lt;4, 5, &lt;1, and 1 μg/L, respectively. Intraassay CVs (n = 8) at the immunoassay cutoff concentrations were 4.1–5.6% and 3.5–11% for STC and Immunalysis ELISAs, respectively. Corresponding interassay CVs were 3.1–10% and 6.5–20%. Of the 855 casework samples, there were a total of 92 discordant results (44 cannabinoid, 15 opiate, 15 methamphetamine, 11 benzodiazepine, and 7 cocaine metabolite). Gas chromatography–mass spectrometry analysis indicated a total of three unconfirmed positive results for Immunalysis assays and one unconfirmed positive for STC assays.
Conclusions: A comparative assessment of drugs-of-abuse assays from two manufacturers indicated some key differences in analytical performance. Overall, Immunalysis assays offered superior binding characteristics and detection limits, whereas STC assays offered improved overall precision and lot-to-lot reproducibility.
Automated Flow Cytometric Analysis of Cerebrospinal Fluid
Authors: Van Acker Jos T , Delanghe Joris R , Langlois Michel R , Taes Youri E , De Buyzere Marc L , Verstraete Alain G 
Published: January 2020

Abstract
Background: Recently, the UF-100 (Sysmex Corporation) flow cytometer was developed to automate urinalysis. We evaluated the use of flow cytometry in the analysis of cerebrospinal fluid (CSF).
Methods: UF-100 data were correlated with microscopy and biochemical data for 256 CSF samples. Microbiological analysis was performed in 144 suspected cases of meningitis.
Results: Good agreement was obtained between UF-100 and microscopy data for erythrocytes (r = 0.919) and leukocytes (r = 0.886). In some cases, however, incorrect classification of lymphocytes by the UF-100 led to underestimation of the leukocyte count. UF-100 bacterial count positively correlated (P &lt;0.001) with UF-100 leukocyte count (r = 0.666), CSF total protein (r = 0.754), and CSF lactate concentrations (r = 0.641), and negatively correlated with CSF glucose concentration (r = −0.405; P &lt;0.001). UF-100 bacterial counts were unreliable in hemorrhagic samples and in samples collected by ventricular drainage where interference by blood platelets and cell debris was observed. Another major problem was the UF-100 “bacterial” background signal in sterile CSF samples. Cryptococcus neoformans yeast cells and cholesterol crystals in craniopharyngioma were detected by the flow cytometer.
Conclusions: Flow cytometry of CSF with the UF-100 offers a rapid and reliable leukocytes and erythrocyte count. Additional settings offered by the instrument may be useful in the diagnosis of neurological disorders.
Europium Nanoparticles and Time-resolved Fluorescence for Ultrasensitive Detection of Prostate-specific Antigen
Authors: Härmä Harri , Soukka Tero , Lövgren Timo 
Published: January 2020

AbstractBackground: Nanoparticle-based detection technologies have the potential to improve detection sensitivity in miniature as well as in conventional biochemical assays. We introduce a detection technology that relies on the use of europium(III) nanoparticles and time-resolved fluorometry to improve the detection limit of biochemical assays and to visualize individual molecules in a microtiter plate format.Methods: Streptavidin was covalently coated on 107-nm nanoparticles containing &gt;30 000 europium molecules entrapped with β-diketones. In a model assay system, these nanoparticles were used to trace biotinylated prostate-specific antigen (PSA) in a microtiter plate format.Results: The detection limit (mean + 3 SD of the zero calibrator) of biotinylated PSA was 0.38 ng/L, corresponding to 10 fmol/L or 60 zeptomoles (60 × 10−21 moles) of PSA. Moreover, single nanoparticles, representing individual PSA molecules, were visualized in the same microtiter wells with a time-resolved fluorescence microscope using a ×10 objective. Single nanoparticles, possessing high specific activity, were also detected in solution by a standard time-resolved plate fluorometer.Conclusions: The universal streptavidin-coated europium(III) nanoparticle label is suitable for detection of any biotinylated molecule either in solution or on a solid phase. The europium(III) nanoparticle labeling technology is applicable to many areas of modern biochemical analysis, such as immunochemical and multianalyte DNA-chip assays as well as histo- and cytochemistry to improve detection sensitivities.