bluuebunny/crossref_metadata_2025 huggingface dataset:
Cross-Reaction with Luteinizing Hormone β-Core Is Responsible for the Age-dependent Increase of Immunoreactive β-Core Fragment of Human Chorionic Gonadotropin in Women with Nonmalignant Conditions
Authors: Iles Ray K , Javid Mohammed K , Gunn Lionel K , Chard Tim
Published: January 2020

Background: The β-core fragment of human chorionic gonadotropin (hCGβcf), also termed “β-core” and urinary gonadotropin peptide (UGP), has been reported to be present in the urine of healthy women and to increase in concentration after menopause. This could reflect cross-reaction with the equivalent metabolite of luteinizing hormone (LH), the β-LH-core.

Methods: We measured immunoreactive LH, hCG, free α-subunit, and free β-subunit hCG (hCGβ), as well as β-core, using the S504 RIA and Triton UGP enzyme immunoassay in 274 urine samples from women with nonmalignant gynecological conditions. The molar cross-reaction of each assay with purified β-LH-core was determined.

Results: Cross-reaction with β-LH-core was 100% in the LH and the S504 β-core assay, 5% in the Triton UGP assay, and <0.1% in the hCG, free α-subunit, and free hCGβ assays. Median urine concentrations of all analytes showed an age-dependent increase. LH and free α-subunit concentrations were ∼103 pmol/mol creatinine; hCG and S504 β-core were ∼102 pmol/mol creatinine; free hCGβ and Triton UGP β-core were in the tens of pmol/mol creatinine. The S504 β-core concentrations were 10% of those of LH. S504 β-core was strongly correlated with LH, but not with hCG or with free hCGβ (LH, r2 = 0.45; hCG, r2 = 0.26; free hCGβ, r2 = 0.03). The concentrations of β-core detected by the Triton UGP assay, which has a 5% cross-reaction with β-LH-core, were 2% of LH and 5% of the S504 β-core concentrations. Triton UGP values correlated strongly with LH concentrations, but less well with S504 β-core, intact hCG, and free hCGβ (LH, r2 = 0.44; S504 β-core, r2 = 0.33; hCG, r2 = 0.32; free hCGβ, r2 = 0.19).

Conclusions: Immunoreactive β-core in women free of malignancies reflects cross-reaction with concentrations of the metabolite of LH, β-LH-core, within the health-related reference interval.
1H-NMR Spectroscopy of Body Fluids: Inborn Errors of Purine and Pyrimidine Metabolism
Authors: Wevers Ron A , Engelke Udo FH , Moolenaar Sytske H , Bräutigam Christa , de Jong Jan GN , Duran Ries , de Abreu Ronney A , van Gennip Albert H
Published: January 2020

Background: The diagnosis of inborn errors of purine and pyrimidine metabolism is often difficult. We examined the potential of 1H-NMR as a tool in evaluation of patients with these disorders.

Methods: We performed 1H-NMR spectroscopy on 500 and 600 MHz instruments with a standardized sample volume of 500 μL. We studied body fluids from 25 patients with nine inborn errors of purine and pyrimidine metabolism.

Results: Characteristic abnormalities could be demonstrated in the 1H-NMR spectra of urine samples of all patients with diseases in the pyrimidine metabolism. In most urine samples from patients with defects in the purine metabolism, the 1H-NMR spectrum pointed to the specific diagnosis in a straightforward manner. The only exception was a urine from a case of adenine phosphoribosyl transferase deficiency in which the accumulating metabolite, 2,8-dihydroxyadenine, was not seen under the operating conditions used. Similarly, uric acid was not measured. We provide the 1H-NMR spectral characteristics of many intermediates in purine and pyrimidine metabolism that may be relevant for future studies in this field.

Conclusion: The overview of metabolism that is provided by 1H-NMR spectroscopy makes the technique a valuable screening tool in the detection of inborn errors of purine and pyrimidine metabolism.
Determination of the Complex between Urokinase and Its Type-1 Inhibitor in Plasma from Healthy Donors and Breast Cancer Patients
Authors: Pedersen Anders N , Brünner Nils , Høyer-Hansen Gunilla , Hamer Peter , Jarosz David , Larsen Birthe , Nielsen Hans Jørgen , Stephens Ross W
Published: January 2020

Background: The complex between urokinase (uPA) and its type-1 inhibitor (PAI-1) is formed exclusively from the active forms of these components; thus, the complex concentration in a biological sample may reflect the ongoing degree of plasminogen activation. Our aim was to establish an ELISA for specific quantification of the uPA:PAI-1 complex in plasma of healthy donors and breast cancer patients.

Methods: A kinetic sandwich format immunoassay was developed, validated, and applied to plasma from 19 advanced-stage breast cancer patients, 39 age-matched healthy women, and 31 men.

Results: The assay detection limit was <2 ng/L, and the detection of complex in plasma was validated using immunoabsorption, competition, and recovery tests. Eighteen cancer patients had a measurable complex concentration (median, 68 ng/L; range, <16 to 8700 ng/L), whereas for healthy females and males the median signal values were below the detection limit (median, <16 ng/L; range, <16 to 200 ng/L; P <0.0001). For patient plasma, a comparison with total uPA and PAI-1 showed that the complex represented a variable, minor fraction of the uPA and PAI-1 concentrations of each sample.

Conclusion: The reported ELISA enables detection of the uPA:PAI-1 complex in blood and, therefore, the evaluation of the complex as a prognostic marker in cancer.
Validation of Accuracy-based Amino Acid Reference Materials in Dried-Blood Spots by Tandem Mass Spectrometry for Newborn Screening Assays
Authors: Chace Donald H , Adam Barbara W , Smith S Jay , Alexander J Richard , Hillman Steven L , Hannon W Harry
Published: January 2020

Background: Advances in technology and the earlier release of newborns from hospitals have pressed the demand for accurate calibration and improved interlaboratory performance for newborn screening tests. As a first step toward standardization of newborn screening aminoacidopathy tests, we have produced six-pool sets of multianalyte dried-blood-spot amino acid reference materials (AARMs) containing predetermined quantities of five amino acids. We describe here the production of the AARMs, validation of their amino acid contents, and characterization of their homogeneity and their stability in storage.

Methods: To each of six portions of a pool of washed erythrocytes suspended in serum we added Phe (0–200 mg/L), Leu (0–200 mg/L), Met (0–125 mg/L), Tyr (0–125 mg/L), and Val (0–125 mg/L). Six-pool sets (1300) were prepared, dried, and packaged. We used isotope-dilution mass spectrometry to estimate the endogenous amino acid concentrations of the AARMs and validate their final amino acid concentrations. We used additional tandem mass spectrometry analyses to examine the homogeneity of amino acid distribution in each AARM, and HPLC analyses to evaluate the stability of the amino acid contents of the AARMs.

Results: The absolute mean biases across the analytic range for five amino acids were 2.8–9.4%. One-way ANOVAs of the homogeneity results predicted no statistically significant differences in amino acid concentrations within the blood spots or within the pools (P >0.05). Regression slopes (0 ± 0.01) for amino acid concentrations vs storage times and their P values (>0.05) showed no evidence of amino acid degradation at ambient temperatures, 4 °C, or −20 °C during the intervals tested.

Conclusion: The validation, homogeneity, and stability of these blood spots support their use as a candidate national reference material for calibration of assays that measure amino acids in dried-blood spots.
Long-Distance PCR-based Screening for Large Rearrangements of the LDL Receptor Gene in Korean Patients with Familial Hypercholesterolemia
Authors: Kim Sung Han , Bae Ji Hyun , Chae Jae Jin , Kim Un Kyung , Choe Seong-Joon , Namkoong Yong , Kim Hyo-Soo , Park Young-Bae , Lee Chung Choo
Published: January 2020

Background: The LDL receptor is a cell-surface protein that regulates plasma cholesterol by specific uptake of LDL particles from the blood circulation. Familial hypercholesterolemia (FH) results from defective catabolism of LDL, which is caused by mutations in the LDL-receptor gene.

Methods: For the rapid and reliable detection of large rearrangements in the LDL-receptor gene, we established a screening method based on long-distance PCR as an alternative to Southern-blot hybridization. Using long-distance PCR, 45 unrelated Korean subjects heterozygous for FH were screened to assess the frequency and nature of major structural rearrangements in the LDL-receptor gene.

Results: Two different deletion mutations, FH6 (same type as FH3 and FH311) and FH 32, were detected in four families by long-distance PCR. Detailed restriction mapping and sequence analysis showed that FH6 was a 5.71-kb deletion extending from intron 8 to intron 12 and that FH32 was a 2-kb deletion extending from intron 6 to intron 7. Sequence analysis for the breakpoints of all deletions detected in Korean FH patients showed that only the left arms of the Alu repetitive sequences were involved in the deletion event.

Conclusions: The screening method based on long-distance PCR provides a powerful strategy for the detection of large rearrangements in the LDL-receptor gene and is a rapid and reliable screening alternative to Southern-blot hybridization.
Characterization of Apolipoprotein E-containing Lipoproteins in Cerebrospinal Fluid: Effect of Phenotype on the Distribution of Apolipoprotein E
Authors: Yamauchi Kazuyoshi , Tozuka Minoru , Hidaka Hiroya , Hidaka Eiko , Kondo Yoshiyuki , Katsuyama Tsutomu
Published: January 2020

Background: Apolipoprotein (apo) E, one of the main apolipoproteins in the central nervous system, may play an important role in lipid metabolism; however, the details of its function are poorly understood. In this study, we characterized apoE-containing lipoproteins in cerebrospinal fluid (CSF) and examined the effect of apoE phenotype on the distribution of apoE among the lipoprotein fractions.

Methods: CSF lipoproteins were fractionated by gel filtration and ultracentrifugation, and then characterized by electrophoresis, immunoblot, electron microscopy, and analysis of apoE, total cholesterol, and phospholipid concentrations.

Results: The ratio of sialylated to nonsialylated apoE was higher in CSF than in serum. However, the fundamental forms containing apoE homodimers or heterodimers [such as apo(E-AII) and apo(AII-E2-AII) complexes] were similar in CSF and serum. apoE-containing lipoproteins were fractionated at densities of <1.006, 1.063–1.125, and 1.125–1.21 kg/L. Neither apoE nor apoAI was detected in the fraction with a density range of 1.006–1.063 kg/L. The diameters of the lipoprotein particles with densities of <1.006, 1.063–1.125, and 1.125–1.21 kg/L were 16.7 ± 3.1, 14.0 ± 3.2, and 11.6 ± 2.8 nm (mean ± SD, n = 200), respectively. All of these lipoproteins exhibited a spherical structure. The distribution profile of apoE-containing lipoproteins was affected by the apoE phenotype. A relatively large amount of apoE-containing lipoproteins was isolated from the fraction with a density >1.125 kg/L obtained from CSF associated with apoE2 or apoE3. This tendency was more obvious in CSF associated with apoE2 than in CSF without apoE2. apoE-containing lipoproteins were predominantly observed in the fraction with a density of <1.006 kg/L obtained from CSF associated with apoE4.

Conclusions: The lipoproteins in CSF have a unique composition that is different from that of the lipoproteins in plasma. However, the differences in diameter between the CSF fractions were not as large as for the serum fractions. Our data suggest that the apoE phenotype may affect the distribution profile of apoE-containing lipoproteins in the CSF. This would mean that the metabolism of apoE-containing lipoproteins depends on the apoE isoform present.
5′ Nuclease Assays for the Loci CCR5-+/Δ32, CCR2-V64I, and SDF1-G801A Related to Pathogenesis of AIDS
Authors: Yuan Chiu-Chin , Peterson Raymond J , Wang Cheng-Dian , Goodsaid Frederico , Waters David J
Published: January 2020

Background: Variations within the human genome play important roles in human disease. To study variations related to susceptibility to AIDS, we have developed 5′ nuclease assays that eliminate post-PCR molecular biology steps.

Methods: TaqMan assays based on the 5′ nuclease activity of Taq polymerase and fluorescent resonance energy transfer were developed to score alleles at the biallelic loci CCR5-+/Δ32, CCR2-V64I and SDF1-G801A. For each assay, 72 samples were analyzed. Data collection and analysis were performed on the Prism 7700 Sequence Detection System. For comparison with gel electrophoresis methods, each locus was also scored on a subset of 24 samples, using restriction enzymes or single-strand conformational polymorphism (SSCP).

Results: Clear allelic discrimination was obtained on each of the 72 samples for all three TaqMan assays. The TaqMan scores for the subset of 24 samples were concordant with the restriction enzyme and SSCP scores.

Conclusions: Because of its simplicity, speed, and potential for automation and miniaturization, TaqMan is an excellent candidate for investigation of genetic variation in clinical, research, and forensic settings.
Serum Procalcitonin Concentrations in Term Delivering Mothers and Their Healthy Offspring: A Longitudinal Study
Authors: Assumma Marcello , Signore Fabrizio , Pacifico Lucia , Rossi Naila , Osborn John F , Chiesa Claudio
Published: January 2020

Background: The reported sensitivities and specificities of procalcitonin (PCT) concentrations for the diagnosis of neonatal infection vary widely. A postnatal increase of PCT has been observed in healthy term newborns with a peak at ≈24 h of age, and many questions remain regarding maternal and perinatal factors that may influence the normal PCT kinetics during the immediate postnatal period.

Methods: We prospectively investigated the association between the serum PCT values obtained from 121 mothers at delivery and serum PCT in their healthy, term offspring at birth as well as at 24 and 48 h of age. We also analyzed whether obstetric and perinatal factors would alter maternal and neonatal PCT response.

Results: PCT concentrations in the babies at birth were significantly higher than in the mothers (P <0.0001), with even larger differences at 24 and 48 h of age. None of the variables identified from maternal and perinatal histories had a significant effect on maternal PCT response. In the healthy neonate, the variables that significantly affected the concentration of PCT at birth were the mothers’ PCT (P <0.01), maternal group B streptococcus colonization (P <0.05), and rupture of membranes ≥18 h (P <0.01). The coefficient of linear correlation between the mother’s PCT concentration and that of the baby at birth was 0.32 (P <0.01). The only variable that significantly altered the PCT concentration at both 24 (P <0.01) and 48 (P <0.01) h of age was rupture of membranes ≥18 h. Nonetheless, the PCT response observed during the 48-h period after birth among healthy babies born to mothers with risk factors for infection was well below that reported previously among age-matched neonates with sepsis.

Conclusions: The postnatal increase of PCT observed in the healthy neonate with peak values at 24 h of age most likely represents endogenous synthesis. In estimating the sensitivities and specificities of PCT for diagnosis of sepsis throughout the initial 48 h of life, it is important to consider the normal PCT kinetics and the pattern(s) of PCT response in the healthy neonate.
Determination of Apolipoprotein B-48 in Plasma by a Competitive ELISA
Authors: Lorec Anne-Marie , Juhel Christine , Pafumi Yan , Portugal Henri , Pauli Anne-Marie , Lairon Denis , Defoort Catherine
Published: January 2020

Background: Apolipoprotein B-48 (apoB-48) is produced by the small intestine, as part of chylomicrons, and appears to be a suitable marker for clinical studies of postprandial lipoproteins and related cardiovascular risk. Our aim was to develop, for routine analysis, an assay to quantify apoB-48 in plasma samples.

Methods: A microtiter plate was coated with a C-terminal apoB-48-specific heptapeptide. Plasma samples were incubated with appropriate detergent to allow competition between immobilized antigen and plasma apoB-48. Appropriate calibration curves were obtained in the ELISA, using calibrated lymph and chylomicrons.

Results: Treatment of plasma samples with the mild detergent Triton X-100 allowed an efficient competition between immobilized antigen and plasma apoB-48. No cross-reactivity was found with apoB-100, as checked by ELISA and Western blot analysis. Intra- and interassay CVs were 5.4% and 5.5%, respectively. In healthy subjects, apoB-48 concentrations markedly increased in the postprandial state, in parallel with triglycerides.

Conclusions: This new ELISA allows determination of the concentration of apoB-48 in normolipidemic plasma.
Sensitivity and Specificity of Immunological Methods for the Detection of Anti-Topoisomerase I (Scl70) Autoantibodies: Results of a Multicenter Study
Authors: Bizzaro Nicola , Tonutti Elio , Villalta Danilo , Bassetti Danila , Tozzoli Renato , Manoni Fabio , Pirrone Stefano , Piazza Anna , Rizzotti Paolo , Pradella Marco ,   for The Italian Society of Laboratory Medicine Study Group on the Diagnosis of Autoimmune Diseases
Published: January 2020

Background: The ability of immunometric methods to identify anti-topoisomerase I (Scl70) antibodies is controversial. We wished to quantify the performance of the currently available commercial systems for the assay of anti-topoisomerase I antibodies in a large multicenter study.

Methods: Fifty Italian clinical laboratories analyzed 36 serum samples: 27 from individuals with scleroderma/systemic sclerosis, and 9 from a control group. The scleroderma/systemic sclerosis samples were positive in our laboratories by both ELISA and immunoblot (IB), and the control samples were negative. The laboratories used 42 immunoenzymatic (ELISA), 21 IB, 3 counterimmunoelectrophoresis, and 2 dot-blot methods, produced by 23 different manufacturers.

Results: We obtained 2389 results. The ELISA methods showed 99.2% specificity and 97.2% sensitivity for detection of anti-Scl70 antibodies. For IB methods, specificity was 97.6% and sensitivity was 96.1%. The Western-blot method had poor analytical specificity (27% false positives for anti-extractable nuclear antigen antibodies other than anti-Scl70).

Conclusions: Excluding Western blots, commercial ELISA and IB reagents as used in clinical laboratories have a sensitivity and a specificity >95% for determination of anti-Scl70 antibodies.
A Golden Age of Clinical Chemistry: 1948–1960
Authors: Rosenfeld Louis
Published: January 2020

This segment of history aims to inform the new, and remind the not-so-new, members of the profession about the relatively recent period that initiated the dominant role played by technological innovation in the modern investigation of disease. The 12 years from 1948 to 1960 were notable for introduction of the Vacutainer tube, electrophoresis, radioimmunoassay, and the AutoAnalyzer. Also appearing during this interval were new organizations, publications, programs, and services that established a firm foundation for the professional status of clinical chemists. It was a golden age.
Miniature Single-Particle Immunoassay for Prostate-specific Antigen in Serum Using Recombinant Fab Fragments
Authors: Härmä Harri , Tarkkinen Piia , Soukka Tero , Lövgren Timo
Published: January 2020

Background: Quantitative, miniaturized nucleic acid assays and immunoassays can be developed with single microparticles, microfluorometric detection, and intrinsically fluorescent lanthanide chelates in a multiple assay format to decrease reagent consumption, cost, and assay time. We used recombinant Fab fragments to capture and detect free and total prostate-specific antigen (PSA) from serum in a submicroliter volume single-particle immunoassay.

Methods: Genetically engineered thiol-Fab or thiolated monoclonal antibodies (mAbs) were covalently attached onto uniformly sized 60-μm maleimide-activated microparticles. Free and total PSA were detected with europium- or terbium-labeled Fab fragments on a single microparticle using a microfluorometer in a time-resolved mode.

Results: The detection limit of the free- and total-PSA assays (mean + 3 SD of zero calibrator) was 0.35 μg/L, with a total volume of 330 nL per particle. An excellent correlation was found in microparticle and microtiter-well assays for 21 serum samples: slopes for free and total PSA were 1.06 ± 0.03 and 1.03 ± 0.02, respectively (Sy|x = 0.084 and 0.057 μg/L), with intercepts of 0.013 ± 0.018 and 0.013 ± 0.017 μg/L (R >0.99). Furthermore, the particle-immobilized Fab fragment had a PSA binding capacity 1.5-fold higher than the intact mAb capacity on a single microparticle. Capacity, kinetics, and sensitivity of the Fab fragment and intact mAb assays in the microparticle and microtiter well formats are discussed.

Conclusions: With site-specific (cysteine tail) covalent attachment of Fab fragments on a microparticle, subattomole amounts of PSA can be detected quantitatively.
Quantification of Melanoma Cell-specific MART-1 mRNA in Peripheral Blood by a Calibrated Competitive Reverse Transcription-PCR
Authors: Sørensen Boe Sandahl , Schmidt Henrik , von der Maase Hans , Straten Per Thor , Nexø Ebba
Published: January 2020

Background: Reverse transcription-PCR (RT-PCR) amplification of melanoma cell-specific mRNA can detect melanoma cells in the peripheral blood of patients with malignant melanoma. We present a method to quantify mRNA coding for the melanoma-specific melanoma antigen recognized by T cells #1 (MART-1) in RNA isolated from peripheral blood.

Methods: To establish a calibration curve, we measured the concentration of MART-1 mRNA in SK-MEL-28 melanoma cells grown in vitro by competitive RT-PCR. Serial dilutions of these cells were used as calibrators in the assay. The assay was conducted by adding a fixed amount of a RNA internal standard to RNA isolated from either peripheral blood or the calibrators before RT-PCR amplification with MART-1 primers in a nested PCR design. The amount of MART-1 mRNA in blood samples was calculated from the calibration curve.

Results: Addition of melanoma cells grown in vitro to blood from healthy donors demonstrated that the method can detect a single SK-MEL-28 melanoma cell in 1 mL of blood (1.5 × 10−21 mol MART-1 mRNA/mL). MART-1 mRNA was observed in 4 of 12 blood samples from patients with malignant melanoma, at concentrations of 3–18 × 10−21 mol MART-1 mRNA/mL of blood. No MART-1 mRNA was detected in blood samples from 25 controls without malignant melanoma. Intra- and interassay CVs were 15% (n = 12; mean = 44 × 10−21 mol MART-1 mRNA/mL) and 33% (15 samples analyzed in two different analytical runs; mean = 30 × 10−21 mol MART-1 mRNA/mL), respectively.

Conclusions: Our method is the first competitive RT-PCR assay for quantification of melanoma cells in blood samples that compensates for the variation of both the reverse transcription and PCR reactions. The method allows the inclusion of control samples for continuous quality assessment.
Saposins A, B, C, and D in Plasma of Patients with Lysosomal Storage Disorders
Authors: Chang Melissa H Y , Bindloss Colleen A , Grabowski Gregory A , Qi Xiaoyang , Winchester Bryan , Hopwood John J , Meikle Peter J
Published: January 2020

Background: Early diagnosis of lysosomal storage disorders (LSDs), before the onset of irreversible pathology, will be critical for maximum efficacy of many current and proposed therapies. To search for potential markers of LSDs, we measured saposins A, B, C, and D in patients with these disorders.

Methods: Four time-delayed fluorescence immunoquantification assays were used to measure each of the saposins in plasma from 111 unaffected individuals and 334 LSD-affected individuals, representing 28 different disorders.

Results: Saposin A was increased above the 95th centile of the control population in 59% of LSD patients; saposins B, C, and D were increased in 25%, 61%, and 57%, respectively. Saposins were increased in patients from several LSD groups that in previous studies did not show an increase of lysosome-associated membrane protein-1 (LAMP-1).

Conclusion: Saposins may be useful markers for LSDs when used in conjunction with LAMP-1.
Flow Cytometric Assessment of LDL Ligand Function for Detection of Heterozygous Familial Defective Apolipoprotein B-100
Authors: Raungaard Bent , Heath Finn , Hansen Peter Steen , Brorholt-Petersen Jens Uffe , Jensen Henrik Kjærulf , Færgeman Ole
Published: January 2020

Background: Familial defective apolipoprotein (apo) B-100 (FDB) is caused by a mutation in the apoB gene and characterized by decreased binding of LDL to LDL receptors because of reduced function of the apoB-100 ligand. FDB may be associated with severe hypercholesterolemia and cannot always be distinguished from familial hypercholesterolemia phenotypically.

Methods: We used a fluorescence flow cytometry assay with Epstein-Barr virus-transformed lymphocytes to detect reduced LDL ligand function by competitive binding with fluorescently conjugated LDL (DiI-LDL). The assay was tested and validated using LDL from patients heterozygous for the Arg3500-Gln mutation and their first-degree relatives. Knowing the actual apoB genotype of patients and relatives allowed us to assess the ability of the assay to predict the results of DNA analysis. The results were compared to measurements of LDL ligand function in unrelated healthy control subjects to characterize functionally the Arg3500-Gln mutation.

Results: Fluorescence was significantly increased in cells incubated with DiI-LDL in competition with unlabeled LDL from FDBR3500Q heterozygotes compared with cells incubated with DiI-LDL in competition with unlabeled LDL from relatives or unrelated healthy control subjects. Thus, patients heterozygous for the Arg3500-Gln mutation had significantly reduced LDL ligand function. The binding affinity of LDL from FDBR3500Q heterozygotes was 32% of that in non-FDB relatives and healthy controls. The assay had a diagnostic sensitivity of 0.95 and diagnostic specificity of 0.89.

Conclusions: The diagnostic accuracy of the assay was too low to allow reliable diagnosis of individual cases of heterozygous FDBR3500Q. However, fluorescence flow cytometry may supplement genetic identification of FDB and functionally characterize gene mutations associated with major reductions in LDL ligand function.
Comparison of a New Method for the Direct and Simultaneous Assessment of LDL- and HDL-Cholesterol with Ultracentrifugation and Established Methods
Authors: Benlian Pascale , Cansier Christophe , Hennache Geneviève , Khallouf Oumayma , Bayer Pascale , Duron Françoise , Carrat Fabrice , Couderc Rémi , Chazouillères Olivier , Bardet Jean , Bouchard Philippe , Poupon Raoul , Masliah Joëlle , Béréziat Gilbert
Published: January 2020

Background: Automated electrophoresis combined with enzymatic cholesterol staining might improve routine assessment of LDL- and HDL-cholesterol (LDLC and HDLC), as an alternative to the Friedewald equation and precipitation. A new method (Hydrasys; SEBIA) that adapts the cholesterol esterase/cholesterol oxidase reaction within urea-free gels was evaluated.

Methods: Fresh sera from 725 subjects (512 dyslipidemics) were analyzed by electrophoresis, in parallel with sequential ultracentrifugation, β-quantification, calculation, and precipitation.

Results: Electrophoresis was linear up to 4 g/L cholesterol, with a detection limit of 0.042 g/L cholesterol/band. Within-run, between-run, between-batch, and between-operator imprecision (CVs) were 1.6%, 2.0%, 1.5%, and 2.7% for LDLC, and 3.9%, 4.3%, 5.5%, and 4.9% for HDLC, and remained unchanged up to 6.3 g/L plasma triglycerides (TGs). Precision decreased with very low HDLC (<0.25 g/L). Serum storage for 3–7 days at +4 or −80 °C did not interfere significantly with the assay. Agreement with β-quantification was stable for LDLC up to 5.07 g/L (r = 0.94), even at TG concentrations >4 g/L (r = 0.91). Bias (2.88% ± 12%) and total error (7.84%) were unchanged at TG concentrations up to 18.5 g/L. Electrophoresis predicted National Cholesterol Education Program cut-points with <0.04 g/L error, exactly and appropriately classified 79% and 96% of the subjects, and divided by 2.4 (all subjects) and 5.8 (TGs >1.5 g/L) the percentage of subjects underestimated by calculation. One-half of the patients with TGs >4 g/L had LDLC >1.30 g/L. For HDLC, correlation was better with precipitation (r = 0.87) than ultracentrifugation (r = 0.76). Error (−0.10% ± 26%) increased when HDLC decreased (<0.35 g/L). Direct assessment of the LDLC/HDLC ratio detected 45% more high-risk subjects than the calculation/precipitation combination.

Conclusions: Electrophoresis provides reliable quantification of LDLC, improving precision, accuracy, and concordance over calculation, particularly with increasing plasma TGs. Implementation of methods to detect low cholesterol concentrations could extend the applications for HDLC assessment.
Simple and Sensitive Binding Assay for Measurement of Adenosine Using Reduced S-Adenosylhomocysteine Hydrolase
Authors: Kloor Doris , Yao Kozo , Delabar Ursula , Osswald Hartmut
Published: January 2020

Background: Adenosine has been suggested to play an important role in the regulation of renal function. We developed a simple and sensitive binding assay for the detection of adenosine based on the displacement of [3H]adenosine from S-adenosylhomocysteine (SAH) hydrolase in its reduced form.

Methods: SAH hydrolase was purified to apparent homogeneity from bovine kidney by standard chromatographic methods. SAH hydrolase was converted in its reduced form, which had the advantage that the SAH hydrolase is enzymatically inactive. This reduced enzyme retains its ability to bind adenosine with high affinity. To determine adenosine in urine or tissues, samples must be deproteinized (e.g., with 10 g/L sulfosalicylic acid or 0.6 mol/L perchloric acid).

Results: The reduced SAH hydrolase bound adenosine with a dissociation constant of 33.0 ± 2 nmol/L. Displacement of adenosine binding by the adenine 5′-nucleotides, adenine and hypoxanthine, required >1000-fold higher concentrations than adenosine itself. The intra- and interassay imprecision (CV) was <3.9% and 7.8%, respectively, and the values obtained showed acceptable correlation with those by HPLC.

Conclusions: The highly sensitive adenosine-binding protein assay is a simple test that allows detection of adenosine in samples with small volumes without purification, and is in this respect superior to HPLC.
Clinical Outcomes of Point-of-Care Testing in the Interventional Radiology and Invasive Cardiology Setting
Authors: Nichols James H , Kickler Thomas S , Dyer Karen L , Humbertson Sandra K , Cooper Peg C , Maughan William L , Oechsle Denise G
Published: January 2020

Background: Point-of-care testing (POCT) can provide rapid test results, but its impact on patient care is not well documented. We investigated the ability of POCT to decrease inpatient and outpatient waiting times for cardiovascular procedures.

Methods: We prospectively studied, over a 7-month period, 216 patients requiring diagnostic laboratory testing for coagulation (prothrombin time/activated partial thromboplastin time) and/or renal function (urea nitrogen, creatinine, sodium, and potassium) before elective invasive cardiac and radiologic procedures. Overall patient management and workflow were examined in the initial phase. In phase 2, we implemented POCT but utilized central laboratory results for patient management. In phase 3, therapeutic decisions were based on POCT results. The final phase, phase 4, sought to optimize workflow around the availability of POCT. Patient wait and timing of phlebotomy, availability of laboratory results, and therapeutic action were monitored. Split sampling allowed comparability of POCT and central laboratory results throughout the study.

Results: In phase 1, 44% of central laboratory results were not available before the scheduled time for procedure (n = 135). Mean waiting times (arrival to procedure) were 188 ± 54 min for patients who needed renal testing (phase 2; n = 14) and 171 ± 76 min for those needing coagulation testing (n = 24). For patients needing renal testing, POCT decreased patient wait times (phases 3 and 4 combined, 141 ± 52 min; n = 18; P = 0.02). For patients needing coagulation testing, wait times improved only when systematic changes were made in workflow (phase 4, 109 ± 41 min; n = 12; P = 0.01).

Conclusions: Although POCT has the potential to provide beneficial patient outcomes, merely moving testing from a central laboratory to the medical unit does not guarantee improved outcomes. Systematic changes in patient management may be required.
The Role of Total Laboratory Automation in a Consolidated Laboratory Network
Authors: Seaberg Richard S , Stallone Robert O , Statland Bernard E
Published: January 2020

Background: In an effort to reduce overall laboratory costs and improve overall laboratory efficiencies at all of its network hospitals, the North Shore–Long Island Health System recently established a Consolidated Laboratory Network with a Core Laboratory at its center.

Methods: We established and implemented a centralized Core Laboratory designed around the Roche/Hitachi CLAS Total Laboratory Automation system to perform the general and esoteric laboratory testing throughout the system in a timely and cost-effective fashion. All remaining STAT testing will be performed within the Rapid Response Laboratories (RRLs) at each of the system’s hospitals.

Results: Results for this laboratory consolidation and implementation effort demonstrated a decrease in labor costs and improved turnaround time (TAT) at the core laboratory. Anticipated system savings are ∼$2.7 million. TATs averaged 1.3 h within the Core Laboratory and less than 30 min in the RRLs.

Conclusions: When properly implemented, automation systems can reduce overall laboratory expenses, enhance patient services, and address the overall concerns facing the laboratory today: job satisfaction, decreased length of stay, and safety. The financial savings realized are primarily a result of labor reductions.
Stand-Alone Automated Solutions Can Enhance Laboratory Operations
Authors: Orsulak Paul J
Published: January 2020

Clinical laboratory automation has developed over the past decade as one means of consolidating testing, reducing costs, and improving the effectiveness of laboratory testing. Most of the developments have been aimed at core clinical laboratory operations, and have primarily addressed preanalytical and analytical processing of traditional specimens arriving in blood collection or similar aliquot tubes. Much less attention has been given to specialized applications such as processing specimens for urine toxicology, and only recently have vendors attacked the problems associated with sorting and maintaining the laboratory’s inventory of specimens. This report highlights selected developments in these areas, describes one approach to cost-effective custom platform development, and discusses the advantages and pitfalls to solving problems with laboratory automation.
Laboratory Automation and Optimization: The Role of Architecture
Authors: Wing Alexander K
Published: January 2020

The increasing automation of laboratory equipment has had far-reaching impacts on the organizational structure and spatial requirements of clinical laboratories. This report explores the changing role of the laboratory in the healthcare environment and shows the architectural impact of these changes, both inside and outside of the laboratory space.
HPLC Analysis of Lipid-derived Polyunsaturated Fatty Acid Peroxidation Products in Oxidatively Modified Human Plasma
Authors: Browne Richard W , Armstrong Donald
Published: January 2020

Background: Lipid peroxidation is a prominent manifestation of free radical activity and oxidative stress in biological systems. Diverse methodologies have been developed that measure a variety of lipid peroxidation products used as markers of lipid peroxidation processes.

Methods: Hydroxy and hydroperoxy polyunsaturated fatty acid (PUFA) peroxidation products were analyzed in human blood plasma by reversed-phase HPLC after liquid-liquid extraction of total lipids and alkaline hydrolysis of lipid esters to liberate free PUFAs. An isocratic mobile phase containing 1 g/L acetic acid-acetonitrile-tetrahydrofuran (52:30:18, by volume) over 60 min duration, with ultraviolet absorbance detection at 236 nm by photodiode array, enabled the resolution and quantification of 13 regioisomeric hydroxy and hydroperoxy PUFAs.

Results: As little as 250 μL of human plasma was utilized with an analytical range of 0.033–1.6 μmol/L for each compound. Intra- and interassay CVs for all compounds detected in normal or oxidatively modified human plasma were 3.2–11% and 4.7–12%, respectively. Analytical recoveries were 87–103%. Analysis of human plasma exposed to artificial oxidation with Cu2+ ion and hydrogen peroxide, a free radical-generating reaction, showed marked increases in hydroxy and hydroperoxy PUFA concentrations.

Conclusion: Lipid-derived hydroxy and hydroperoxy PUFAs may be useful as clinical markers of lipid peroxidation and oxidative stress in the peripheral circulation.
Progression Criteria for Cancer Antigen 15.3 and Carcinoembryonic Antigen in Metastatic Breast Cancer Compared by Computer Simulation of Marker Data
Authors: Sölétormos György , Hyltoft Petersen Per , Dombernowsky Per
Published: January 2020

Background: We investigated the utility of computer simulation models for performance comparisons of different tumor marker assessment criteria to define progression or nonprogression of metastatic breast cancer.

Methods: Clinically relevant values for progressive cancer antigen 15.3 and carcinoembryonic antigen concentrations were combined with representative values for background variations in a computer simulation model. Fifteen criteria for assessment of longitudinal tumor marker data were obtained from the literature and computerized. Altogether, 7200 different patients, each based on 50 measurements, were simulated. With a sampling interval of 4 weeks, the monitoring period for each event was ∼3.8 years.

Results: Modulation of the background variation, the starting concentrations, and the cutoffs enabled identification of criteria that were robust against false-positive signals of progression.

Conclusions: The computer simulation model is a fast, effective, and inexpensive approach for comparing the diagnostic potential of assessment criteria during clinically relevant conditions of steady-state and progressive disease. The model systems can be used to generate tumor marker assessment criteria for a variety of malignancies and to compare and optimize their diagnostic performance.
Reminders of Drug Effects on Laboratory Test Results
Authors: Kailajärvi Marita , Takala Timo , Grönroos Paula , Tryding Nils , Viikari Jorma , Irjala Kerttu , Forsström Jari
Published: January 2020

Drug effects on laboratory test results are difficult to take into account without an online decision support system. In this study, drug effects on hormone test results were coded using a drug-laboratory effect (DLE) code. The criteria that trigger the reminders were defined. To issue reminders, it was necessary to write a computer program linking the DLE knowledge base with databases containing individual patient medication and laboratory test results. During the first 10 months, 11% of the results from hormone samples were accompanied by one or more DLE reminders. The most common drugs to trigger reminders were glucocorticoids, furosemide, and metoclopramide. Physicians facing the reminders completed a questionnaire on the usefulness of the reminders. All respondents considered them useful. In addition, DLE reminders had caused 74% of respondents to refrain from additional, usually performed examinations. In conclusion, drug effects on laboratory tests should always be considered when interpreting laboratory results. An online reminder system is useful in displaying potential drug effects alongside test results.
Quantification of DNA Using the Luminescent Oxygen Channeling Assay
Authors: Patel Rajesh , Pollner Reinhold , de Keczer Steve , Pease John , Pirio Marcel , DeChene Neal , Dafforn Alan , Rose Sam
Published: January 2020

Background: Simplified and cost-effective methods for the detection and quantification of nucleic acid targets are still a challenge in molecular diagnostics.

Methods: Luminescent oxygen channeling assay (LOCITM) latex particles can be conjugated to synthetic oligodeoxynucleotides and hybridized, via linking probes, to different DNA targets. These oligomer-conjugated LOCI particles survive thermocycling in a PCR reaction and allow quantified detection of DNA targets in both real-time and endpoint formats. The endpoint DNA quantification format utilized two sensitizer bead types that are sensitive to separate illumination wavelengths. These two bead types were uniquely annealed to target or control amplicons, and separate illuminations generated time-resolved chemiluminescence, which distinguished the two amplicon types.

Results: In the endpoint method, ratios of the two signals allowed determination of the target DNA concentration over a three-log range. The real-time format allowed quantification of the DNA target over a six-log range with a linear relationship between threshold cycle and log of the number of DNA targets.

Conclusions: This is the first report of the use of an oligomer-labeled latex particle assay capable of producing DNA quantification and sequence-specific chemiluminescent signals in a homogeneous format. It is also the first report of the generation of two signals from a LOCI assay. The methods described here have been shown to be easily adaptable to new DNA targets because of the generic nature of the oligomer-labeled LOCI particles.
Linked Linear Amplification: A New Method for the Amplification of DNA
Authors: Reyes Antonio A , Ugozzoli Luis A , Lowery Jimmie D , Breneman John W , Hixson Craig S , Press Richard D , Wallace R Bruce
Published: January 2020

Background: Linked Linear Amplification (LLA) is a new nucleic acid amplification method that uses multiple cycles of primer extension reactions. The presence of nonreplicable elements in LLA primers renders primer extension products unusable as templates for further amplification, leading to linear accumulation of products. Through the use of nested primers, linear reactions can be “linked”, providing total amplification yields comparable to those obtained by PCR.

Methods: The LLA model predicts (a) that amplification yield will approach that of PCR as the number of primers increases and (b) that the unique composition of LLA products will give lower carryover amplification efficiency compared with PCR. To test these hypotheses, the human β-globin gene was amplified by 10-, 14-, or 18-primer LLA and the yield was compared with PCR. Carryover contamination was simulated by reamplifying a dilution series of LLA or PCR products. To demonstrate the clinical utility of the method, LLA coupled with allele-specific oligonucleotide (ASO) capture was used to detect the factor V Leiden mutation in a panel of 111 DNA samples.

Results: Fourteen- and 18-primer LLA gave amplification yields comparable to PCR. However, LLA carryover amplification efficiency was four orders of magnitude lower than that of PCR. The LLA-ASO assay detected the correct factor V Leiden genotype in all 111 samples.

Conclusions: LLA is a robust target amplification method that is comparable to PCR in yield. However, LLA is more resistant to false results caused by carryover amplicon contamination.
Atomic Emission Method for Total Boron in Blood during Neutron-Capture Therapy
Authors: Laakso Juha , Kulvik Martti , Ruokonen Inkeri , Vähätalo Jyrki , Zilliacus Riitta , Färkkilä Markus , Kallio Merja
Published: January 2020

Background: Boron neutron-capture therapy (BNCT) is a drug-targeted binary radiotherapy for cancer. The 10B capture of thermal neutrons induces secondary radiation within cells during irradiation. The most widely used boron carrier is 4-dihydroxyborylphenylalanine (BPA). The duration and timing of the irradiation is adjusted by monitoring 10B concentrations in whole blood.

Methods: We developed a new method for boron determination that uses inductively coupled plasma atomic emission spectrometry (ICP-AES) and protein removal with trichloroacetic acid before analysis. This method was compared with the established but tedious inductively coupled plasma mass spectrometry (ICP-MS), which uses wet ashing as sample pretreatment. Erythrocyte boron concentrations were determined indirectly on the basis of plasma and whole blood boron concentrations and the hematocrit. The hematocrit was determined indirectly by measuring calcium concentrations in plasma and whole blood.

Results: Within- and between-day CVs were <5%. The recoveries for boron in whole blood were 95.6–96.2%. A strong correlation was found between results of the ICP-AES and ICP-MS (r = 0.994). Marked differences in plasma and erythrocyte boron concentrations were observed during and after infusion of BPA fructose complex.

Conclusions: The present method is feasible, accurate, and one of the fastest for boron determination during BNCT. Our results indicate that it is preferable to determine boron in plasma and in whole blood. Indirect erythrocyte-boron determination thus becomes possible and avoids the impact of preanalytical confounding factors, such as the influence of the hematocrit of the patient. Such an approach enables a more reliable estimation of the irradiation dose.
Use of Up-Converting Phosphor Reporters in Lateral-Flow Assays to Detect Specific Nucleic Acid Sequences: A Rapid, Sensitive DNA Test to Identify Human Papillomavirus Type 16 Infection
Authors: Corstjens Paul , Zuiderwijk Michel , Brink Antoinette , Li Shang , Feindt Hans , Niedbala R Sam , Tanke Hans
Published: January 2020

Background: A lateral-flow (LF) device using the new reporter up-converting phosphor technology (UPTTM) was applied to DNA (hybridization) assays for the detection of specific nucleic acid sequences, thereby aiming to perform the test outside well-equipped laboratories. The methodology reported here is sensitive and provides a rapid alternative for more elaborate gel electrophoresis and Southern blotting. In a preliminary study, it was applied to screen for the presence of human papillomavirus type 16 (HPV16) in a defined series of cervical carcinomas.

Methods: A LF assay was used to capture haptenized DNA molecules and hybrids, which were immunolabeled (before LF) with 400-nm UPT particles. These particles emit visible light after excitation with infrared in a process called up-conversion. Because up-conversion occurs in only the phosphor lattice, autofluorescence of other assay components is virtually nonexistent.

Results: The use of the UPT reporter in LF-DNA tests, as compared with colloidal gold, improved the detection limit at least 100-fold. UPT LF-DNA tests were successfully applied to detect (in a blind test) the presence of HPV16 in DNA extracts obtained from cervical carcinomas. Test results matched 100% with previous characterization of these carcinomas.

Conclusions: The use of UPT in LF assays to detect specific nucleic acids provides low attamole-range sensitivity. Hybridization and consecutive detection of PCR-amplified HPV16 sequences were successful in a background of 10 μg of fish-sperm DNA. The sensitivity of UPT detection in these complex mixtures indicates that detection of viral infections without PCR or other amplification technique is achievable.