bluuebunny/crossref_metadata_2025 huggingface dataset:
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

Background: 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

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 >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

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

Background: 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

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

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, <4, 5, 25, and 3 μg/L, respectively, for the STC ELISAs. Corresponding detection limits for Immunalysis ELISAs were <1, <2, <4, 5, <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

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 <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 <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

Background: 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 >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.
Pitfalls in the Measurement of Circulating Vascular Endothelial Growth Factor
Authors: Jelkmann Wolfgang
Published: January 2020

Background: Vascular endothelial growth factor (VEGF) is a protein with antiapoptotic, mitogenic, and permeability-increasing activities specific for vascular endothelium. VEGF mRNA, which has five isoforms, is produced by nonmalignant cells in response to hypoxia and inflammation and by tumor cells in constitutively high concentrations. Because VEGF plays a crucial role in physiological and pathophysiological angiogenesis, measurements of circulating VEGF are of diagnostic and prognostic value, e.g., in cardiovascular failures, inflammatory diseases, and malignancies. However, there are major quantitative differences in the published results. This review attempts to identify reasons for these disparities.

Approach: The literature was reviewed through a Medline search covering 1995 to 2000. A selection of exemplary references had to be made for this perspective overview.

Content: Data are included from studies on healthy humans, gynecological patients, and persons suffering from inflammatory or malignant diseases. The results indicate that competitive immunoassays detect the total amount of circulating VEGF, which enables observations regarding the increase in VEGF in pregnancy and preeclampsia to be made. In these cases, capture immunoassays utilizing neutralizing antibodies are insufficient because of an accompanying increase in VEGF-binding soluble receptors (sFlt-1). Measurements of circulating free VEGF are useful for study of malignant diseases, which are associated with both genetically and hypoxia-induced overproduction of VEGF. The VEGF isoform specificity of the antibodies is also critical because both VEGF121 and VEGF165 are secreted. It is important to consider that platelets and leukocytes release VEGF during blood clotting.

Conclusions: Future efforts should concentrate on the balance between free VEGF, total VEGF, and sFlt-1. Plasma, rather than serum, should be used for analysis.
Carcinoembryonic Antigen as a Marker for Colorectal Cancer: Is It Clinically Useful?
Authors: Duffy Michael J
Published: January 2020

Background: Carcinoembryonic antigen (CEA) is one of the most widely used tumor markers worldwide. Its main application is mostly in gastrointestinal cancers, especially in colorectal malignancy. Although in use for almost 30 years, the clinical value of CEA in colorectal cancer is still not clear.

Methods: The literature relevant to the clinical value of CEA in colorectal cancer was reviewed. Particular attention was paid to studies involving metaanalyses and guidelines issued by Expert Panels.

Results: Although of little use in detecting early colorectal cancer, high preoperative concentrations of CEA correlate with adverse prognosis. Serial CEA measurements can detect recurrent colorectal cancer with a sensitivity of ∼80%, a specificity of ∼70%, and can provide a lead time of ∼5 months. CEA is the most frequent indicator of recurrence in asymptomatic patients and currently is the most cost-effective test for the preclinical detection of resectable disease. CEA is most useful for the early detection of liver metastasis in patients with diagnosed colorectal cancer. Overall, however, little evidence is available that monitoring of all patients with diagnosed colorectal cancer leads to enhanced patient outcome or quality of life.

Conclusions: Currently, the most useful application of CEA is in the detection of liver metastasis from colorectal cancers. Because of the relative success of surgery in resecting hepatic metastases, serial determinations of the marker are recommended for detecting cancer spread to the liver. In the future, preoperative concentrations of CEA may be included with the standard staging procedures for assessing prognosis.
DNA Melting Analysis for Detection of Single Nucleotide Polymorphisms
Authors: Lipsky Robert H , Mazzanti Chiara M , Rudolph Joseph G , Xu Ke , Vyas Gopal , Bozak David , Radel Marta Q , Goldman David
Published: January 2020

Background: Several methods for detection of single nucleotide polymorphisms (SNPs; e.g., denaturing gradient gel electrophoresis and denaturing HPLC) are indirectly based on the principle of differential melting of heteroduplex DNA. We present a method for detecting SNPs that is directly based on this principle.

Methods: We used a double-stranded DNA-specific fluorescent dye, SYBR Green I (SYBR) in an efficient system (PE 7700 Sequence Detector) in which DNA melting was controlled and monitored in a 96-well plate format. We measured the decrease in fluorescence intensity that accompanied DNA duplex denaturation, evaluating the effects of fragment length, dye concentration, DNA concentration, and sequence context using four naturally occurring polymorphisms (three SNPs and a single-base deletion/insertion).

Results: DNA melting analysis (DM) was used successfully for variant detection, and we also discovered two previously unknown SNPs by this approach. Concentrations of DNA amplicons were readily monitored by SYBR fluorescence, and DNA amplicon concentrations were highly reproducible, with a CV of 2.6%. We readily detected differences in the melting temperature between homoduplex and heteroduplex fragments 15–167 bp in length and differing by only a single nucleotide substitution.

Conclusions: The efficiency and sensitivity of DMA make it highly suitable for the large-scale detection of sequence variants.
Specific Reverse Transcription-PCR Quantification of Vascular Endothelial Growth Factor (VEGF) Splice Variants by LightCycler Technology
Authors: Wellmann Sven , Taube Tillmann , Paal Krisztina , Graf v. Einsiedel Hagen , Geilen Wilhelm , Seifert Georg , Eckert Cornelia , Henze Günter , Seeger Karlheinz
Published: January 2020

Background: Overexpression of vascular endothelial growth factor (VEGF) is associated with increased angiogenesis, growth and invasion in solid tumors, and hematologic malignancies. The expression of isoforms of VEGF, which mediate different effects, can be discriminated by splice-variant-specific quantitative reverse transcription-PCR (RT-PCR), but current methods have only modest sensitivity and precision and suffer from heteroduplex formation.

Methods: We used a real-time RT-PCR assay on the LightCycler system. Applicability for detection of different VEGF mRNAs and total VEGF message was tested on seven healthy tissues (each pooled from healthy donors) and seven correlated malignant tissues. Results were normalized to β2-microglobulin mRNA. Amplification of VEGF splice variants was performed exclusively with variant-specific reverse primers, whereas forward primer and fluorescent probe were common to obtain similar RT-PCR kinetics.

Results: Highly specific detection of VEGF splice variants was achieved with minor intra- and interassay variation (<0.22 threshold cycle). Total VEGF expression was higher in malignant tissues. In healthy tissues, the mRNA encoding diffusible variants VEGF121 and VEGF165 constituted on average 78% (SD = 9.3%) of the total VEGF message, and the cell-adherent variant VEGF189 constituted on average 22% (SD = 5.4%). In contrast, in malignant tissues VEGF121 and VEGF165 accounted for 94% (SD = 7.6%) and VEGF189 only 6% (SD = 3.7%).

Conclusions: Because of the ability for quantification of VEGF splice variants with high specificity, sensitivity, and reproducibility, this new LightCycler assay is superior to conventional semiquantitative competitive RT-PCR and immunological assays and may contribute to better understanding of VEGF-mediated angiogenesis.
Highly Sensitive, Automated Immunoassay for Immunoglobulin Free Light Chains in Serum and Urine
Authors: Bradwell Arthur R , Carr-Smith Hugh D , Mead Graham P , Tang Lian X , Showell Paul J , Drayson Mark T , Drew Roger
Published: January 2020

Background: Bence Jones proteins or monoclonal immunoglobulin κ and λ free light chains (FLCs) are important markers for identifying and monitoring many patients with B-cell tumors. Automated immunoassays that measure FLCs in urine and serum have considerable clinical potential.

Methods: Sheep antibodies, specific for FLCs, were prepared by immunization with pure κ and λ molecules and then adsorbed extensively against whole immunoglobulins. The antibodies were conjugated onto latex particles and used to assay κ and λ FLCs on the Beckman IMMAGETM protein analyzer.

Results: The unconjugated antibodies showed minimal cross-reactivity with intact immunoglobulins or other proteins. With latex-conjugated antibodies, κ and λ FLCs could be measured in normal sera and most normal urine samples. Patients with multiple myeloma had increased concentrations of the relevant serum FLC, whereas both FLCs were increased in the sera of patients with systemic lupus erythematosus.

Conclusions: We developed sensitive, automated immunoassays for κ and λ FLC measurements in serum and urine that should facilitate the assessment of patients with light chain abnormalities.
Measurement of Immunoreactive Angiotensin-(1–7) Heptapeptide in Human Blood
Authors: Nussberger Juerg , Brunner Dorette B , Nyfeler Juerg A , Linder Lilly , Brunner Hans R
Published: January 2020

Background: The renal enzyme renin cleaves from the hepatic α2-globulin angiotensinogen angiotensin-(1–10) decapeptide [Ang-(1–10)], which is further metabolized to smaller peptides that help maintain cardiovascular homeostasis. The Ang-(1–7) heptapeptide has been reported to have several physiological effects, including natriuresis, diuresis, vasodilation, and release of vasopressin and prostaglandins.

Methods: To investigate Ang-(1–7) in clinical settings, we developed a method to measure immunoreactive (ir-) Ang-(1–7) in 2 mL of human blood and to estimate plasma concentrations by correcting for the hematocrit. A sensitive and specific antiserum against Ang-(1–7) was raised in a rabbit. Human blood was collected in the presence of an inhibitor mixture including a renin inhibitor to prevent peptide generation in vitro. Ang-(1–7) was extracted into ethanol and purified on phenylsilylsilica. The peptide was quantified by radioimmunoassay. Increasing doses of Ang-(1–7) were infused into volunteers, and plasma concentrations of the peptide were measured.

Results: The detection limit for plasma ir-Ang-(1–7) was 1 pmol/L. CVs for high and low blood concentrations were 4% and 20%, respectively, and between-assay CVs were 8% and 13%, respectively. Reference values for human plasma concentrations of ir-Ang-(1–7) were 1.0–9.5 pmol/L (median, 4.7 pmol/L) and increased linearly during infusion of increasing doses of Ang-(1–7).

Conclusions: Reliable measurement of plasma ir-Ang-(1–7) is achieved with efficient inhibition of enzymes that generate or metabolize Ang-(1–7) after blood sampling, extraction in ethanol, and purification on phenylsilylsilica, and by use of a specific antiserum.
Fluorescence-based Detection of the CETPTaqIB Polymorphism: False Positives with the TaqMan-based Exonuclease Assay Attributable to a Previously Unknown Gene Variant
Authors: Teupser Daniel , Rupprecht Wolfgang , Lohse Peter , Thiery Joachim
Published: January 2020

Background: Previous studies have shown an association between the TaqIB polymorphism of the cholesteryl ester transfer protein (CETP) gene with plasma CETP and HDL concentrations and the progression of coronary artery disease (CAD). The aim of the present study was to determine the performance of two new fluorescence-based detection systems in the analysis of the TaqIB genotype.

Methods: CAD patients (n = 150) with known TaqIB genotype, as determined by restriction fragment length polymorphism (RFLP) analysis, were selected, including three groups of 50 patients, carrying the B1/1, B1/2, and B2/2 genotypes, respectively. The genotypes were also analyzed by fluorescence-based allele-specific TaqMan PCR and melting curve analysis (LightCycler). In addition, DNA sequencing was applied.

Results: The TaqIB genotypes obtained by fluorescence analysis corresponded to those determined by RFLP analysis with the exception of three heterozygous patients (B1/2), who were misclassified as homozygous B2 carriers with the TaqMan system. Melting curve analysis of these samples demonstrated an additional melting point at 59.1 °C, which was also found in four patients homozygous for the B1 allele. DNA sequencing revealed a previously unknown C270T nucleotide exchange in intron 1 of the CETP gene, only nine base pairs from the TaqIB site.

Conclusions: Determination of the TaqIB polymorphism with the TaqMan system led to misclassifications because of a previously unknown C270T polymorphism of the CETP gene. The base substitution was detected with the LightCycler because of the occurrence of an additional melting point. Our data indicate the importance of thorough evaluation of new gene analysis systems before using them on a routine basis.
Quantification of Cellular Acid Sphingomyelinase and Galactocerebroside β-Galactosidase Activities by Electrospray Ionization Mass Spectrometry
Authors: Zhou Xinfeng , Tureček František , Scott C Ronald , Gelb Michael H
Published: January 2020

Background: Diagnosis of Niemann-Pick (A and B) and Krabbe diseases is achieved by measurement of the lysosomal enzymes acid sphingomyelinase (ASM) and galactocerebroside β-galactosidase (GCG), respectively. Conventional assays use radiolabeled or fluorescent substrates and do not allow simultaneous determination of two or more enzymes in the sample.

Methods: We developed a sensitive and specific method to assay ASM and GCG in skin fibroblast homogenates using biotinylated substrate conjugates. The products were purified by bioaffinity capture on streptavidin-agarose beads and, following release, were analyzed by electrospray ionization mass spectrometry. Quantification was achieved using stable-isotope-labeled internal standards that were chemically identical to the products of the enzymatic reactions.

Results: The method demonstrated excellent linearity of ASM and GCG enzymatic product formation with the amount of cellular protein and incubation time. The range of ASM activities in fibroblast lysates from six healthy patients was 39–70 nmol · mg−1 · h−1 compared with 3.7–5.1 nmol · mg−1 · h−1 in cell lysates from two patients affected with Niemann-Pick A disease. The GCG activities toward the corresponding substrate conjugate were 4.0–6.8 nmol · mg−1 · h−1 in cell lysates from healthy patients compared with 0.1–0.2 nmol · mg−1 · h−1 in cell lysates from two patients affected with Krabbe disease. The amounts of substrate conjugates needed per analysis were 15 nmol (14 μg) for both ASM and GCG.

Conclusions: Electrospray mass spectrometry combined with the use of biotinylated substrate conjugates and bioaffinity purification represents a new approach for the diagnosis of lysosomal storage diseases as demonstrated for Niemann-Pick A and Krabbe diseases. No radioactive substrates are used, and the method uses a single instrumental platform to determine both ASM and GCG in one cell sample.
Oxidative Stress and Homocysteine in Coronary Artery Disease
Authors: Cavalca Viviana , Cighetti Giuliana , Bamonti Fabrizia , Loaldi Alessandro , Bortone Luana , Novembrino Cristina , De Franceschi Michela , Belardinelli Romualdo , Guazzi Maurizio D
Published: January 2020

Background: Oxidative stress is present in cardiovascular diseases (CVDs), and hyperhomocysteinemia, an independent risk factor for these diseases, may play a role by inducing production of oxygen free radicals.

Methods: To evaluate the possible role of homocysteine (Hcy) in inducing oxidative stress in coronary artery disease (CAD), plasma Hcy was measured in 68 consecutive cardiovascular patients, and plasma malondialdehyde (MDA), both free and total (free + bound), was measured in 40 patients with CAD (18 with chronic stable angina and 22 with unstable angina). As controls, we tested 70 healthy volunteers. Hcy was measured by an immunoenzymatic method and MDA, an index of lipid peroxidation, by gas chromatography–mass spectrometry.

Results: Plasma Hcy concentrations were significantly higher in cardiovascular patients than in controls (10.2 vs 8.9 μmol/L; P <0.0002), with no significant difference between values in the stable and unstable angina subgroups. Similarly, total MDA was significantly higher in the CAD group than in the controls (2.6 vs 1.3 μmol/L; P <0.00001), again with no significant difference between stable and unstable angina patients. By contrast, free MDA, which was significantly higher in the CAD patients than the controls (0.4 vs 0.2 μmol/L; P <0.00001), was also significantly higher in the unstable than in the stable angina group (0.5 vs 0.3 μmol/L; P <0.03). However, no correlation was observed among Hcy and free and total MDA.

Conclusions: Our findings show that a moderate increase of Hcy is associated with CVD but that Hcy at the detected values cannot be considered completely responsible for oxidative damage. That lipid peroxidation is involved in CAD is shown by our observation of significantly increased plasma free and total MDA concentrations compared with controls. Moreover, free MDA values discriminated between unstable and chronic stable angina, and could thus represent a new diagnostic tool.
Heparin-induced Release of Protein-bound Solutes during Hemodialysis Is an in Vitro Artifact
Authors: De Smet Rita , Van Kaer Jacqueline , Liebich Hartmut , Lesaffer Gerrit , Verstraete Alain , Dhondt Annemieke , Duym Phillippe , Lameire Norbert , Vanholder Raymond
Published: January 2020

Background: Several studies have pointed to a release of drugs or protein-bound solutes from their binding sites during heparinization. The effect is attributed to the metabolism of triglycerides to free fatty acids (FFAs), which compete with drugs for protein binding sites. This study evaluated the impact of intradialytic heparin on the free concentration of the uremic toxin p-cresol and on FFAs.

Methods: Blood samples from hemodialysis (HD) patients, before and during HD, were collected with selected anticoagulation strategies. We assessed the effects of standing time, temperature, pH, and the addition of a lipase inhibitor, tetrahydrolipstatin (THL) to blood samples on the free p-cresol concentration. p-Cresol was analyzed by HPLC with fluorescence detection. We measured FFAs by gas chromatography, and the free fractions of added valproic acid and phenytoin were evaluated by fluorescence polarization immunoassay.

Results: In blood samples (n = 22) not submitted to a specific treatment, free p-cresol increased from 9.9 ± 5.1 to 31.9 ± 22.3 μmol/L after 30 min of heparin HD (P <0.001) and correlated significantly with FFAs (r = 0.80; P = 0.002; n = 12). There was no increase in free p-cresol during heparin-free HD (n = 6) and trisodium citrate HD (n = 9). In addition, p-cresol in ultrafiltrates (n = 3) did not correspond to the free p-cresol in heparinized blood, suggesting that the increase in free p-cresol was artifactual. The release of p-cresol in the test tube was enhanced by standing time (n = 6), sample temperature (n = 6), and alkaline pH (n = 6). Inhibition of lipase activity with THL prevented the increase of FFAs (n = 6) and the release of free p-cresol during HD (n = 22). These results were corroborated by the study of the free fraction of valproic acid (n = 6) and phenytoin (n = 6).

Conclusions: The free concentrations of protein-bound solutes in plasma of heparinized patients are influenced by external factors that alter the lipase activity in the test tube. The free fraction does not increase during HD when lipase activity is neutralized at the time of blood sampling, so that previously reported increases are probably artifacts.
Biochemical Markers of Bone Turnover and Response of Bone Mineral Density to Intervention in Early Postmenopausal Women: An Experience in a Clinical Laboratory
Authors: Chailurkit La-or , Ongphiphadhanakul Boonsong , Piaseu Noppawan , Saetung Sunee , Rajatanavin Rajata
Published: January 2020

Background: Markers of bone formation and resorption may be useful as early indicators of response to therapy. Our aim in this study was to investigate the use of bone markers for monitoring of intervention for bone loss in early postmenopausal women and to assess the relationships between these markers and changes in bone mineral density (BMD).

Methods: Subjects were randomly assigned to the following groups: a control group; a group receiving calcium alone; groups receiving calcium plus low or conventional doses of conjugated equine estrogen; and groups receiving calcium plus low or conventional doses of calcitriol. At baseline and at 1 and 3 months after intervention, we measured serum intact osteocalcin, serum N-terminal midfragment osteocalcin, serum C-terminal telopeptide of type I collagen (CTx), urinary deoxypyridinoline cross-links, and urinary CTx. The BMD of the lumbar spine and the femoral neck was measured at baseline and after 1 and 2 years of intervention.

Results: No marker changed significantly in the control group except urinary CTx, which increased at 3 months. Serum CTx decreased in all regimens at 1 or 3 months of intervention. In addition, the changes of all markers at 3 months were inversely associated with the change in the BMD of the lumbar spine at 1 or 2 years (r = −0.144 to −0.314), whereas only the changes of bone resorption markers at 3 months were inversely correlated with the changes in femoral BMD at 1 or 2 years (r = −0.143 to −0.366).

Conclusions: Biochemical markers of bone turnover appear to be of use in assessing early response to therapy. Bone resorption markers, especially serum CTx, are better indicators than bone formation markers for estimating the response to intervention in early postmenopausal women. However, the early changes in bone markers were weakly related to the later changes in BMD.
Diagnostic Accuracy of the Anti-Citrulline Antibody Assay for Rheumatoid Arthritis
Authors: Bizzaro Nicola , Mazzanti Giovanni , Tonutti Elio , Villalta Danilo , Tozzoli Renato
Published: January 2020

Background: Rheumatoid arthritis (RA) is the most common autoimmune rheumatic disease, but specific and practicable tests for its diagnosis are lacking. We evaluated the diagnostic accuracy of a new commercial ELISA in detecting anti-cyclic citrullinated peptide (CCP) antibodies for the diagnosis of RA.

Methods: Anti-CCP antibodies were determined in 330 serum samples: 98 from RA patients and 232 from controls, including patients with connective tissue diseases, other rheumatic diseases, viral infections, Lyme disease, autoimmune thyroiditis, cancer, and monoclonal gammopathy, and sex- and age-matched healthy subjects. Intra- and interassay CVs were 5–13% and 9–17%, respectively. Rheumatoid factor (RF) was also assayed in every sample, and results were compared to anti-CCP for sensitivity and specificity.

Results: At a cutoff value of 50 units, sensitivity was 41% (confidence interval, 31–50%) and specificity was 97.8% (95–100%). Anti-CCP-positive RA patients had a mean antibody concentration of 1100 units (range, 57–3419 units), and anti-CCP-negative RA patients and controls had mean values of 7.6 and 6.8 units, respectively (range, 1–39 units). The area under the ROC curve was 0.71 (95% confidence interval, 0.63–0.78). RF had a higher sensitivity (62%) and a lower specificity (84%) than anti-CCP. When the two antibodies were used together, specificity was 99.6%.

Conclusion: Anti-CCP antibody testing may be useful if performed concomitantly with RF assay to diagnose patients with suspected early RA.
Capillary Electrophoresis-based Heteroduplex Analysis with a Universal Heteroduplex Generator for Detection of Point Mutations Associated with Rifampin Resistance in Tuberculosis
Authors: Thomas Gloria A , Williams Diana L , Soper Steven A
Published: January 2020

Background: Slab gel heteroduplex analysis (HDA), a popular scanning method for genetic mutations, uses DNA fragments typically generated by PCR to create homo- and heteroduplex molecules with conformational differences and sequence-dependent electrophoretic profiles. Use of a universal heteroduplex generator (UHG) enhances the subtle variations caused by single-base substitutions.

Methods: The HDA-UHG slab gel format was modified for an efficient capillary-based method. The effect of staining dyes TOPRO5 and YOPRO1 on the analysis of heteroduplexes was studied, as well as ultraviolet absorbance and laser-induced fluorescence (LIF) detection methods. In addition, the entangled polymers hydroxyethyl cellulose, methyl cellulose, and linear polyacrylamide were evaluated as separation matrices.

Results: This assay was able to detect the presence of Mycobacterium tuberculosis and its rifampin susceptibility directly from clinical specimens in dramatically reduced analysis time (30 min vs 2.5 h). Optimized conditions included 0.3% methyl cellulose as the separation matrix, on-line staining using 1 μmol/L YOPRO1, and LIF detection for quantitative and reproducible analysis of single-base substitutions in the rifampin resistance-determining region of rpoB that give rise to the rifampin-resistant phenotype of M. tuberculosis. We generated 95% confidence limits using the wild-type sequence and used these limits to determine rifampin susceptibility in samples.

Conclusions: Capillary electrophoresis, combined with the HDA-UHG technique, may be of value for rapid and efficient clinical diagnosis of rifampin-resistant tuberculosis strains.
Interpretation of Hemoglobin A1c (HbA1c) Values among Diabetic Patients
Authors: Skeie Svein , Thue Geir , Sandberg Sverre
Published: January 2020

Background: Few studies have examined patients’ views, knowledge, and understanding of glycohemoglobin A1c (HbA1c) testing. We explored such issues in patients with type 1 diabetes and used their statements to estimate analytical quality specifications for HbA1c testing.

Methods: We recruited 201 patients from a hospital outpatient clinic. A questionnaire was used to collect information on diabetes characteristics, perceived knowledge of HbA1c, last HbA1c value, HbA1c target value, and thresholds for action. Patients were asked to indicate the magnitude of change in HbA1c from 9.4% that they would consider to be a true (real) change; from their responses, we calculated patient-derived quality specifications for HbA1c.

Results: Fifty-eight percent of the patients felt they had “high” knowledge about HbA1c, and >80% of responders knew their last HbA1c value, their target HbA1c, and the threshold value of HbA1c for treatment intensification. The mean acceptable HbA1c value was 7.5%. Patients with lower values on their most recent tests reported lower target values for HbA1c and lower values for the upper HbA1c threshold for treatment intensification. An analytical CV (CVa) of 3.1% would be satisfactory for 75% of patients when HbA1c is increasing (80% confidence), and a CVa of 3.2% would be satisfactory for 75% when HbA1c is decreasing (95% confidence).

Conclusions: Type 1 patients’ perceived knowledge about HbA1c testing is high. They are well informed about their own personal results and about target values and the upper HbA1c threshold for action. The patient-derived analytical quality specification for imprecision (CV) is 3.1%.
Quantitative Determination of Estradiol Fatty Acid Esters in Human Pregnancy Serum and Ovarian Follicular Fluid
Authors: Vihma Veera , Adlercreutz Herman , Tiitinen Aila , Kiuru Paula , Wähälä Kristiina , Tikkanen Matti J
Published: January 2020

Background: Lipophilic estradiol derivatives carried by lipoprotein particles in blood may mediate antioxidant or endocrine effects. We developed a new quantitative method to determine the concentration of circulating lipophilic estradiol fatty acid esters in human early- and late-pregnancy serum and in ovarian follicular fluid.

Methods: After extraction from serum or follicular fluid, estradiol fatty acid esters were separated from nonesterified estradiol by Sephadex LH-20 column chromatography. The estradiol ester fraction was hydrolyzed by saponification and further purified by several chromatographic steps. The hydrolyzed estradiol esters were measured by time-resolved fluoroimmunoassay.

Results: The average estradiol fatty acid ester concentration in serum increased 10-fold during pregnancy, from 40.4 pmol/L (expressed as pmol/L estradiol; range, 25.0–64.2 pmol/L) in early pregnancy (n = 8) to 404 pmol/L (196–731 pmol/L) in late pregnancy (n = 10). The ratio of estradiol ester to nonesterified estradiol remained relatively constant during pregnancy, at 0.4–0.6%. In 10 follicular fluid samples, the mean estradiol ester concentration was 106 nmol/L (56.9–262 nmol/L). Compared with serum, a greater proportion of estradiol in follicular fluid (3.0–10%) was in the esterified form.

Conclusion: The new method provides a means to measure circulating estradiol fatty acid ester concentrations in human pregnancy serum.
Sensitive and Rapid Quantification of Busulfan in Small Plasma Volumes by Liquid Chromatography–Electrospray Mass Spectrometry
Authors: Mürdter Thomas E , Coller Janet , Claviez Alexander , Schönberger Frank , Hofmann Ute , Dreger Peter , Schwab Matthias
Published: January 2020

Background: High-dose busulfan is widely used in conditioning regimens before hematopoietic stem cell transplantation in both adults and children. Large interindividual variability in pharmacokinetics after oral administration has been reported; therefore, therapeutic drug monitoring of busulfan may decrease the incidence of drug-related toxicity (for example, hepatic venoocclusive disease) and may also improve therapeutic efficacy.

Methods: Busulfan concentrations were quantified using 200 μL of plasma and liquid–liquid extraction with diethyl ether after the addition of [2H8]busulfan as the internal standard. Separation and detection of busulfan and [2H8]busulfan were achieved with a LUNA C8 column (5 μm; 150 × 2 mm i.d.) at 30 °C, a HP 1100 liquid chromatography system, and a HP 1100 single-quadrupole mass spectrometer. Busulfan and [2H8]busulfan were detected as ammonium adducts in selected-ion monitoring mode at m/z 264.2 and 272.2, respectively.

Results: The calibration curve was linear at 5–2000 μg/L busulfan. Intra- and interassay imprecision (CV) and bias were both <11%. The limits of detection and quantification were 2 and 5 μg/L, respectively. Extraction recovery of busulfan was >87%. Analysis of pharmacokinetics in four patients receiving high-dose busulfan indicated that minimum busulfan concentrations before the next dose were 405–603 μg/L, with no interference observed.

Conclusions: The new rapid and sensitive liquid chromatographic–mass spectrometric assay is an appropriate method for quantification of busulfan in human plasma, making therapeutic drug monitoring of busulfan faster and easier in clinical practice.
Laboratory-related Outcomes in Healthcare
Authors: Bruns David E
Published: January 2020

Outcomes studies, long common on the therapeutic side of medicine, are appearing in the diagnostic arena. Outcomes can be defined as results of medical interventions (therapies or tests) in terms of health or cost. The studies of outcomes are important because funding for medical interventions increasingly depends on them; a major accrediting agency even defines “quality” entirely in terms of outcomes.

The study of laboratory-related outcomes is complex. Multiple steps occur between testing and outcomes, physicians act unpredictably on test results, and outcomes studies have high costs relative to potential profit from the test. Study design often must specify the action that is to follow a test result.

The model outcomes study is a randomized controlled trial (RCT). The CONSORT statement, which is used as a guideline for RCTs of therapies, is largely applicable to studies of diagnostic interventions. Recent laboratory-related RCTs have addressed questions such as: “Does routine testing before cataract surgery decrease morbidity or mortality?” and “Does fecal occult bleed testing decrease the incidence of colorectal cancer?”

RCTs of tests are sometimes impractical. Other approaches include simulation modeling and the use of intervention and control periods of testing. As for RCTs, these approaches require careful attention to study design, data analysis, and interpretation and reporting of results.