bluuebunny/crossref_metadata_2025 huggingface dataset:
Strand Displacement Amplification and Homogeneous Real-Time Detection Incorporated in a Second-Generation DNA Probe System, BDProbeTecET
Authors: Little Michael C , Andrews Jeffrey , Moore Richard , Bustos Silvia , Jones Lynda , Embres Chris , Durmowicz Gerard , Harris James , Berger Dolores , Yanson Karen , Rostkowski Christine , Yursis Daretta , Price James , Fort Thomas , Walters Adriann , Collis Matthew , Llorin Oscar , Wood Janet , Failing Frank , O’Keefe Christian , Scrivens Brian , Pope Bill , Hansen Tim , Marino Ken , Williams Keith , Boenisch Michael
Published: January 2020

Background: Amplified DNA probes provide powerful tools for the detection of infectious diseases, cancer, and genetic diseases. Commercially available amplification systems suffer from low throughput and require decontamination schemes, significant hands-on time, and specially trained laboratory staff. Our objective was to develop a DNA probe system to overcome these limitations.

Methods: We developed a DNA probe system, the BDProbeTecTMET, based on simultaneous strand displacement amplification and real-time fluorescence detection. The system uses sealed microwells to minimize the release of amplicons to the environment. To avoid the need for specially trained labor, the system uses a simple workflow with predispensed reagent devices; a programmable, expandable-spacing pipettor; and the 96-microwell format. Amplification and detection time was 1 h, with potential throughput up to 564 patient results per shift. We tested 122 total patient specimens obtained from a family practice clinic with the BD ProbeTecET and the Abbott LCx® amplified system for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae.

Results: Based on reportable results, the BDProbeTecET results for both organisms were 100% sensitive and 100% specific relative to the LCx.

Conclusions: The BDProbeTecET is an easy-to-use, high-throughput, closed amplification system for the detection of nucleic acid from C.trachomatis and N.gonorrhoeae and other organisms.
Assay of Procarboxypeptidase U, a Novel Determinant of the Fibrinolytic Cascade, in Human Plasma
Authors: Schatteman Katinka A , Goossens Filip J , Scharpé Simon S , Neels Hugo M , Hendriks Dirk F
Published: January 2020

Background: Procarboxypeptidase U (proCPU) is a novel proenzyme found in human plasma. The active form, carboxypeptidase U (CPU; EC 3.4.17.20), retards the rate of fibrinolysis through its ability to cleave C-terminal lysine residues on fibrin partially degraded by plasmin. This reduces the number of high-affinity plasminogen-binding sites on fibrin.

Methods: We developed an assay to determine the proCPU concentration in human plasma. The assay involved quantitative conversion of proCPU to active CPU by thrombin-thrombomodulin, a very efficient activator of proCPU, followed by determination of the enzymatic activity of CPU with the substrate hippuryl-l-arginine, using an HPLC-assisted determination of the released hippuric acid. Using this method, we established a reference interval based on 490 healthy individuals.

Results: The mean proCPU concentration, determined after activation of the zymogen in diluted plasma and expressed as CPU activity, was 964 U/L, with a SD of 155 U/L. The population showed a gaussian distribution. However, we noticed important differences related to age and the use of hormone preparations.

Conclusions: The sensitivity and precision of the method make it suitable for routine clinical determinations and as a reference procedure.
Hyperinsulinemia and Related Atherosclerotic Risk Factors in the Population at Cardiovascular Risk: A Community-based Study
Authors: Chien Kuo-Liong , Lee Yuan-Teh , Sung Fung-Chang , Hsu Hsiu-Ching , Su Ta-Chen , Lin Ruey S
Published: January 2020

Background: A population-based study was conducted in Taiwan to investigate the prevalence of insulin resistance and high serum insulin concentrations and their relationships with potential atherosclerotic risk factors.

Methods: We studied 2165 subjects, ages >35, from a community cohort.

Results: The distributions of fasting insulin were skewed to the right, with higher concentrations in women than in men. As age increased, insulin increased in women, but decreased in men. As fasting insulin concentrations increased, postloading insulin, glucose, blood pressure, body mass index, waist-to-hip ratio, total cholesterol, triglycerides, LDL-cholesterol, apoprotein B, plasminogen activator inhibitor 1, tissue plasminogen activator, and fibrinogen increased, but lipoprotein(a), HDL-cholesterol, and apoprotein A1 decreased. Multiple logistic regression showed that obesity, high LDL-cholesterol, and low HDL-cholesterol were significant predictors of hyperinsulinemic status.

Conclusion: The study subjects with insulin resistance syndrome and related risk factors may be at risk for atherosclerosis, thrombosis, and other coronary heart diseases.
Use of Bone Alkaline Phosphatase to Monitor Alendronate Therapy in Individual Postmenopausal Osteoporotic Women
Authors: Kress Barry C , Mizrahi Isaac A , Armour Karen W , Marcus Robert , Emkey Ronald D , Santora Arthur C
Published: January 2020

Background: Biochemical bone markers are sensitive to the changes in bone turnover that result from treatment of postmenopausal osteoporotic women with antiresorptive therapies. Although information is available on the use of bone markers in monitoring therapy in groups of subjects, less is known regarding how these markers perform in individual patients.

Methods: Serum bone alkaline phosphatase (bone ALP) concentrations, measured with the Tandem® Ostase® assay, were used to monitor the biochemical response of bone in postmenopausal women with osteoporosis receiving either 10 mg/day alendronate therapy (n = 74) or calcium supplementation (n = 148) for 24 months.

Results: Bone ALP decreased significantly from baseline at 3 months (P ≤0.0001), reaching a nadir between 3 and 6 months of alendronate therapy. The magnitude of the bone ALP decrease in the treated osteoporotic population was consistent with normalization to premenopausal concentrations. Of the 74 alendronate-treated subjects, 63 (85.1%) demonstrated a decrease from baseline in bone ALP by 6 months that exceeded the least significant change of 25%. The bone ALP decrease from baseline exceeded 25% in 72 (97%) by the end of the study.

Conclusion: The bone ALP assay is a sensitive and reliable tool that may be used to monitor the reduction in bone turnover after alendronate therapy in individual postmenopausal osteoporotic women.
Stereospecific Analysis and Enantiomeric Disposition of 3,4-Methylenedioxymethamphetamine (Ecstasy) in Humans
Authors: Fallon John K , Kicman Andrew T , Henry John A , Milligan Peter J , Cowan David A , Hutt Andrew J
Published: January 2020

Background: Little is known concerning the enantioselective disposition of 3,4-methylenedioxymethamphetamine (MDMA; ecstasy) in humans. In addition, the potential of utilizing the stereochemical composition of an analyte in biological media for forensic purposes requires investigation.

Methods: The enantiomers of MDMA and its demethylated metabolite, 3,4-methylenedioxyamphetamine (MDA), present in plasma and urine extracts were derivatized with (−)-(R)-α-methoxy-α-trifluoromethylphenylacetyl chloride and analyzed by gas chromatography–mass spectrometry and gas chromatography, respectively. The enantioselective disposition of MDMA and MDA was determined following oral administration of racemic MDMA (40 mg) to eight male volunteers.

Results: The plasma concentrations of (R)-MDMA exceeded those of the S-enantiomer [ratio R:S of the area under the curve (AUC), 2.4 ± 0.3], and the plasma half-life of (R)-MDMA (5.8 ± 2.2 h) was significantly longer than that of the S-enantiomer (3.6 ± 0.9 h). The majority of the recovered material in urine was excreted within 24 h after dosing, with the recovery of (R)-MDMA (21.4% ± 11.6%) being significantly greater than that of (S)-MDMA (9.3% ± 4.9%), and with (S)- and (R)-MDA accounting for 1.4% ± 0.5% and 1.0% ± 0.3% of the dose, respectively. Mathematical modeling of plasma enantiomeric composition vs sampling time demonstrated the applicability of using stereochemical data for the prediction of time elapsed after drug administration.

Conclusions: Analytical methods for determining the enantiomeric composition of MDMA and MDA in plasma and urine were developed. The disposition of MDMA in humans is stereoselective, with the more active S-enantiomer having a reduced AUC and shorter half-life than (R)-MDMA. The determination of stereochemical composition may be applicable for forensic purposes.
The Use of Infrared Spectrophotometry for Measuring Body Water Spaces
Authors: Jennings Graham , Bluck Leslie , Wright Antony , Elia Marinos
Published: January 2020

Background: The conventional method of measuring total body water by the deuterium isotope dilution method uses gas isotope ratio mass spectrometry (IRMS), which is both expensive and time-consuming. We investigated an alternative method, using Fourier transform infrared spectrophotometry (FTIR), which uses less expensive instrumentation and requires little sample preparation.

Method: Total body water measurements in human subjects were made by obtaining plasma, saliva, and urine samples before and after oral dosing with 1.5 mol of deuterium oxide. The enrichments of the body fluids were determined from the FTIR spectra in the range 1800–2800 cm−1, using a novel algorithm for estimation of instrumental response, and by IRMS for comparison.

Results: The CV (n = 5) for repeat determinations of deuterium oxide in biological fluids and calibrator solutions (400–1000 μmol/mol) was found to be in the range 0.1–0.9%. The use of the novel algorithm instead of the integration routines supplied with the instrument gave at least a threefold increase in precision, and there was no significant difference between the results obtained with FTIR and those obtained with IRMS.

Conclusion: This improved infrared method for measuring deuterium enrichment in plasma and saliva requires no sample preparation, is rapid, and has potential value to the clinician.
The Lipoprotein Lipase HindIII Polymorphism: Association with Total Cholesterol and LDL-Cholesterol, but not with HDL and Triglycerides in 342 Females
Authors: Larson Ilona , Hoffmann Michael M , Ordovas Jose M , Schaefer Ernst J , März Winfried , Kreuzer Jörg
Published: January 2020

Background: Lipoprotein lipase (LPL) is the rate-limiting enzyme in the hydrolysis of core triglycerides in chylomicrons and VLDL.

Methods: We investigated the association between the HindIII polymorphism of the LPL gene and fasting glucose, lipid, and lipoprotein concentrations in 683 Caucasians. We first stabilized the study subjects, using an 8-day diet and exercise intervention program before obtaining blood samples. The use of this standardization period reduced the variance of all glucose and lipid concentrations.

Results: In our study, the HindIII allele frequencies for females and males were 0.29 and 0.34 for H− and 0.71 and 0.66 for H+, respectively. We found in females, but not in males, a significant association between the HindIII genotype and total cholesterol (P = 0.007) and LDL-cholesterol (P = 0.018), with females homozygous for the rare H− allele having the lowest, heterozygotes (H−/+) having intermediate, and women homozygous for the common H+ allele having the highest of each of these lipid traits. With regard to triglycerides, HDL-cholesterol, and glucose, no significant effect of the HindIII genotype was noted in either gender.

Conclusions: These results suggest that in a gender-specific manner, the rare LPLHindIII H− allele has a cholesterol-lowering and, therefore, potentially cardioprotective effect compared with the common H+ allele.
Estimation of Prostate Cancer Probability by Logistic Regression: Free and Total Prostate-specific Antigen, Digital Rectal Examination, and Heredity Are Significant Variables
Authors: Virtanen Arja , Gomari Mehran , Kranse Ries , Stenman Ulf-Håkan
Published: January 2020

Background: Despite low specificity, serum prostate-specific antigen (PSA) is widely used in screening for prostate cancer. Specificity can be improved by measuring free and total PSA and by combining these results with clinical findings. Methods such as neural networks and logistic regression are alternatives to multistep algorithms for clinical use of the combined findings.

Methods: We compared multilayer perceptron (MLP) and logistic regression (LR) analysis for predicting prostate cancer in a screening population of 974 men, ages 55–66 years. The study sample comprised men with PSA values >3 μg/L. Explanatory variables considered were age, free and total PSA and their ratio, digital rectal examination (DRE), transrectal ultrasonography, and a family history of prostate cancer.

Results: When at least 90% sensitivity in the training sets was required, the mean sensitivity and specificity obtained were 87% and 41% with LR and 85% and 26% with MLP, respectively. The cancer specificity of an LR model comprising the proportion of free to total PSA, DRE, and heredity as explanatory variables was significantly better than that of total PSA and the proportion of free to total PSA (P <0.01, McNemar test). The proportion of free to total PSA, DRE, and heredity were used to prepare cancer probability curves.

Conclusion: The probability calculated by logistic regression provides better diagnostic accuracy for prostate cancer than the presently used multistep algorithms for estimation of the need to perform biopsy.
Possible Mechanism for in Vitro Complement Activation in Blood and Plasma Samples: Futhan/EDTA Controls in Vitro Complement Activation
Authors: Pfeifer Philippe H , Kawahara Marleen S , Hugli Tony E
Published: January 2020

Background: Ongoing in vitro complement (C) activation in citrate or EDTA plasma has prevented an accurate analysis of C-activation products generated in vivo. The aim of this study was to characterize handling and storage conditions required to prevent in vitro C activation in blood and plasma samples collected with Futhan/EDTA.

Methods: BiotrakTM RIAs were used to quantitatively measure C3a and C4a in blood and/or plasma samples from healthy individuals (controls) and from liver transplant patients. Blood samples were routinely drawn into either EDTA (1 g/L) tubes or into tubes containing both EDTA (1 g/L) and Futhan (0.1 g/L) and immediately centrifuged at 2000g for 15 min at 4 °C.

Results: In controls, C4a, but not C3a, in fresh samples (time 0) was higher in EDTA plasma than in Futhan/EDTA plasma (n = 20; P = 0.002). Futhan/EDTA prevented C3a and C4a generation in blood and plasma samples held at room temperature (22–23 °C) for 1 h and in plasma held for 24 h at 4 °C or −70 °C. The mean C3a concentration (1.76 mg/L; n = 19) at time 0 in EDTA plasma samples from liver transplant patients was significantly higher than for controls (0.34 mg/L; n = 11). In these patients, the mean C3a in EDTA samples increased to 13.8 mg/L after 60 min at room temperature, but there was no change in the C3a concentration of an EDTA plasma from a control. In the patients, C3a concentrations were lower in Futhan/EDTA plasma than in EDTA at time 0 and after 60 min at room temperature (1.40 and 2.02 mg/L, respectively). The mean patient C4a was 4.02 mg/L in EDTA plasma at time 0 vs 0.24 mg/L for controls; it increased to 16.9 mg/L after 60 min at room temperature compared with 0.76 mg/L for controls. The mean patient C4a was 0.83 mg/L in Futhan/EDTA plasma at time 0 vs 0.1 mg/L for controls. Neither patient nor control C4a concentrations increased vs time in Futhan/EDTA.

Conclusion: The combination of Futhan (0.1 g/L) and EDTA (1 g/L) eliminates in vitro C activation.
Type 2 Diabetes: An Overview
Authors: Lebovitz Harold E
Published: January 2020

Type 2 diabetes is a heterogeneous disorder. Clinical expression of the disorder requires both genetic and environmental factors. One theory concerning its etiology is that it is the result of the evolution of a thrifty genotype that had survival benefits in the past but is detrimental in the current environment. An opposing theory is that it represents an adult metabolic response to fetal malnutrition. Hyperglycemia in type 2 diabetes results from absolute or relative insulin deficiency. Most often relative insulin deficiency is attributable to an inability to adequately compensate for insulin resistance. Insulin resistance may be caused by a variety of genetic or metabolic factors. The most common etiological factor in insulin resistance is central obesity. Insulin resistance is associated with a cluster of metabolic abnormalities that include glucose intolerance, hypertension, a unique dyslipidemia, a procoagulant state, and an increase in macrovascular disease. Clinical intervention studies have demonstrated that reduction in the chronic microvascular and macrovascular complications of type 2 diabetes requires treatment of hyperglycemia to achieve hemoglobin A1c <7.0%, blood pressure ≤130/80 mmHg, and plasma LDL-cholesterol ≤2.6 mmol/L (≤100 mg/dL). Oral antihyperglycemic agents increase endogenous insulin secretion, decrease insulin resistance, or lower postprandial plasma glucose rise by delaying absorption of complex carbohydrates. Long-term glycemic control in type 2 diabetes requires progressive, stepwise, combination treatment with oral agents and eventually combination treatment with oral agents and insulin.
Hormonal Control of Calcium Homeostasis
Authors: Mundy Gregory R , Guise Theresa A
Published: January 2020

Calcium homeostasis in the extracellular fluid is tightly controlled and defended physiologically. Hypercalcemia always represents considerable underlying pathology and occurs when the hormonal control of calcium homeostasis is overwhelmed. The major hormones that are responsible for normal calcium homeostasis are parathyroid hormone and 1,25-dihydroxyvitamin D; these hormones control extracellular fluid calcium on a chronic basis. Over- or underproduction of these hormones or the tumor peptide, parathyroid hormone-related peptide, are the major causes of aberrant extracellular fluid calcium concentrations. These hormonal defense mechanisms are reviewed here.
The Endocrinology of Aging
Authors: Perry Horace M
Published: January 2020

Physiologic systems have substantial reserves in younger individuals. The process of aging and intercurrent pathologic processes gradually eliminate these reserves. Changes in endocrine systems, including menopause in women, androgen deficiency in men, loss of skeletal mass, decrease in growth hormone serum concentrations, and increased incidence of type 2 diabetes are all more common or certain in older individuals. This review summarizes the progression of each of these processes with age, the potential outcomes of the untreated process, and the treatment outcomes for these age-related losses. Maintenance of a premenopausal lipid profile presumably protects against cardiovascular events. Maintenance of skeletal mass reduces fracture risk and risk for loss of mobility and independence. Testosterone replacement in hypogonadal older men improves strength and presumably function and independence. Growth hormone therapy is reported to have similar effects. Improvement of long-term outcomes in older type 2 diabetics, however, is more difficult to demonstrate.
Drug Monitoring of Antiretroviral Therapy for HIV-1 Infection: Method Validation and Results of a Pilot Study
Authors: Moyer Thomas P , Temesgen Zelalem , Enger Robert , Estes Lynn , Charlson Joel , Oliver Lawrence , Wright Alan
Published: January 2020

Background: Antiretroviral therapy for HIV-1 infection has become increasingly complex. The availability of new and potent drugs and progress in understanding the pathogenesis of HIV-1 infection have led to the establishment of new treatment paradigms. The varying dosing regimens, associated toxicities, and the potential for drug-drug and food-drug interactions further complicate treatment. This complexity contributes to patient nonadherence. Because clinicians have no tools to monitor adherence or drug-drug interactions and because response requires that therapy exceed the known inhibiting concentration, serum monitoring of antiretroviral therapy may play a role in improving treatment of HIV-1 infection. We report methods to quantify serum concentrations of antiretroviral drugs used to treat HIV-1 infection, precision and interference studies of these methods, and results observed in a pilot evaluation of blood serum concentrations from 12 human subjects.

Methods: HPLC offers adequate sensitivity to measure peak or trough serum concentrations of delavirdine, lamivudine, nevirapine, indinavir, nelfinavir, ritonavir, and saquinavir and peak serum concentrations of stavudine, zidovudine, and didanosine with reasonable precision.

Results: Peak indinavir serum concentrations in most patients were in the range of 1–10 mg/L, and trough concentrations were in the range of 0.1–0.5 mg/L. Peak stavudine concentrations were in the range of 0.3–1.3 mg/L, and trough concentrations were in the range of 0.1–0.5 mg/L. Peak zidovudine concentrations were in the range of 0.1–1.1 mg/L.

Conclusions: Because this was a blood serum concentration-seeking pilot study to evaluate analytic performance, we do not report on the correlation of drug response to blood concentration. However, the concentrations observed in patients are generally consistent with blood concentrations reported from studies of monotherapy.
Inhibition of Stimulated Interleukin-2 Production in Whole Blood: A Practical Measure of Cyclosporine Effect
Authors: Stein C Michael , Murray John J , Wood Alastair JJ
Published: January 2020

Background: Prediction of cyclosporine (CSA) efficacy and toxicity in individual patients is difficult. There is no practical, biologically relevant, pharmacodynamic measure of CSA effect. A major effect of CSA is to decrease interleukin-2 (IL-2) production; however, measurement of this effect in isolated lymphocytes as a marker of response to CSA has been problematic.

Methods: CSA inhibition of phytohemagglutinin-P (PHA)-stimulated IL-2 production, measured by ELISA, was studied ex vivo in whole blood drawn before, and after subjects received 4 mg/kg oral CSA.

Results: Four hours after CSA was administered, the mean (± SD) CSA concentration was 702 ± 196 μg/L and PHA-stimulated IL-2 production decreased by 68.7% ± 17.2% (P <0.0001; n = 17). Twenty-four hours after CSA was administered, concentrations were low (64 ± 24 μg/L), with no inhibition of IL-2 production. A rapid, concentration-dependent response occurred. Maximum CSA concentrations (944 ± 187 μg/L) and maximum inhibition of IL-2 production (86.9% ± 13.7%) occurred 90 min after subjects received CSA. In vitro, 32.5–1200 μg/L CSA also inhibited PHA-stimulated IL-2 production in whole blood in a dose-dependent fashion with a similar IC50 (∼300–400 μg/L) ex vivo and in vitro.

Conclusion: In the search for a pharmacodynamic marker to better guide immunosuppressive therapy, the relationship between this simple, biologically relevant measure of CSA effect and clinical outcome should be determined.
Predictive Value of Cord Blood Hematological Indices and Hemoglobin Barts for the Detection of Heterozygous α-Thalassemia-2 in an African-Caribbean Population
Authors: van der Dijs Fey PL , Volmer Marcel , van Gijssel-Wiersma Dieuwke G , Smit Jan W , van Veen Reind , Muskiet Frits AJ
Published: January 2020

Background: Cord blood hemoglobin Barts (HbBarts) and hemocytometric indices may be used for classification of newborns into those without α-thalassemia-2 (αα/αα) and with heterozygous α-thalassemia-2 (-α3.7/αα). We investigated by logistic regression analysis whether the combination of HbBarts and hemocytometric indices improves classification compared with classification based on a single analyte.

Methods: HbBarts percentages and hemocytometric indices were determined in cord blood of 208 consecutive newborns in Curaçao (Netherlands Antilles). Of these, 157 had αα/αα and 51 had -α3.7/αα, as established by DNA analysis.

Results: Between-group differences were significant for erythrocytes, mean cell volume, mean cell hemoglobin (MCH), mean cell hemoglobin concentration, platelets, hemoglobin F0 (HbF0), and HbBarts. The Logit equation of the logistic regression model, using MCH (pg) and HbBarts (%), was: 42.7164 + 5.7916(HbBarts) − 1.3110(MCH). A sensitivity of 100% was reached at a Logit value of −3.70. The corresponding specificity was 62.2%, and the predictive value of a positive test (PV+) was 46.3% (95% confidence interval, 37.0–55.7%). The relative information gains were as follows: 88% for the HbBarts-MCH combination, 26% for MCH (not significant), and 0% for HbBarts compared with the 24.6% -α3.7/αα prevalence.

Conclusion: Combined use of cord blood HbBarts and MCH improves classification compared with classification based on single hemocytometric indices.
Development, Characterization, and Use of Monoclonal Antibodies Made to Antigens Expressed on the Surface of Fetal Nucleated Red Blood Cells
Authors: Alvarez Francisco V , Olander Jitka , Crimmins Dan , Prieto Belén , Paz Ana , Alonso Rebeca , Porter Sharon , Hess Jay , Crist Robert D , Landt Yvonne , Ladenson Jack H
Published: January 2020

Background: Current methods for obtaining fetal cells for prenatal diagnosis are invasive and carry a small (0.5–1.0%) but definite risk of miscarriage. An attractive alternative would be isolation of fetal cells from peripheral maternal blood using antibodies with high specificity and avidity.

Methods: To generate antibodies, we purified nucleated red blood cells (NRBCs) from fetal livers and used them as the immunogen to generate monoclonal antibodies (mAbs) directed against surface antigens.

Results: The four antibodies recognized at least two conformationally sensitive epitopes of the transferrin receptor. Isolation of NRBCs from 252 maternal blood samples using these antibodies in magnetic activated cell sorting after an initial density gradient centrifugation yielded 0–419 NRBCs per 25 mL of maternal blood. One antibody, 2B7.4, not only isolated the highest number of NRBCs (>10 in 90% of the samples) but also isolated these NRBCs in 78 consecutive maternal samples.

Conclusion: Antibody 2B7.4 shows promise for the isolation of NRBCs from maternal blood and should allow studies concerning the source of these cells, fetal vs maternal, and the factors controlling their prevalence.
Aptamers: An Emerging Class of Molecules That Rival Antibodies in Diagnostics
Authors: Jayasena Sumedha D
Published: January 2020

Antibodies, the most popular class of molecules providing molecular recognition needs for a wide range of applications, have been around for more than three decades. As a result, antibodies have made substantial contributions toward the advancement of diagnostic assays and have become indispensable in most diagnostic tests that are used routinely in clinics today. The development of the systematic evolution of ligands by exponential enrichment (SELEX) process, however, made possible the isolation of oligonucleotide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. These oligonucleotide sequences, referred to as “aptamers”, are beginning to emerge as a class of molecules that rival antibodies in both therapeutic and diagnostic applications. Aptamers are different from antibodies, yet they mimic properties of antibodies in a variety of diagnostic formats. The demand for diagnostic assays to assist in the management of existing and emerging diseases is increasing, and aptamers could potentially fulfill molecular recognition needs in those assays. Compared with the bellwether antibody technology, aptamer research is still in its infancy, but it is progressing at a fast pace. The potential of aptamers may be realized in the near future in the form of aptamer-based diagnostic products in the market. In such products, aptamers may play a key role either in conjunction with, or in place of, antibodies. It is also likely that existing diagnostic formats may change according to the need to better harness the unique properties of aptamers.
Analytical Characterization of Electrochemical Biosensor Test Strips for Measurement of Glucose in Low-Volume Interstitial Fluid Samples
Authors: Collison Michael E , Stout Philip J , Glushko Tatyana S , Pokela Kristen N , Mullins-Hirte Debra J , Racchini Joel R , Walter Melissa A , Mecca Steve P , Rundquist Joanna , Allen John J , Hilgers Michael E , Hoegh Thomas B
Published: January 2020

Background: Minimally invasive interstitial fluid (ISF) sampling and glucose measurement technologies were integrated into a hand-held device for diabetic glucose monitoring investigations.

Methods: Conventional electrochemical test strip technology (Bayer Glucometer Elite®) was adapted to measure glucose in small (0.5–2.0 μL) samples of ISF. Test strip glucose measurements were performed on a commercial potentiostat and were compared to various reference glucose methodologies (YSI 2300 analyzer, microhexokinase procedure, Bayer Glucometer Elite). Characterizations of the integrated ISF sampling-glucose test strip design included accuracy and precision in various sample media (saline, ISF surrogates, diabetic ISF samples), sample volume dependence, test strip sterilization studies (electron beam, γ irradiation), and diabetic ISF sampling and glucose measurements.

Results: Glucose measurements were free from significant media effects. Sample volume variations (0.6–3.2 μL) revealed only modest dependence of glucose measurement bias on sample volume (−1.5% per microliter). Sterilization treatments had only a minor impact on glucose response and test strip aging and no significant impact on interferent responses of the glucose test strips. Diabetic subject testing under minimum fasting conditions of at least 2 h with integrated ISF sampling and glucose measurement gave low ISF glucose measurement imprecision (CV, 4%) and mean glucose results that were indistinguishable from reference (microhexokinase) ISF glucose measurements and from capillary blood glucose measurements (Glucometer Elite).

Conclusions: Conventional single-use, electrochemical glucose test strip and ISF collection technologies can be readily integrated to provide real-time ISF sampling and glucose measurements for diabetic monitoring applications.
Genetic Analysis of the CYP2D6 Locus in a Hong Kong Chinese Population
Authors: Garcia-Barceló Mercè , Chow Lok Yee , Chiu Helen Fung Kum , Wing Yun Kowk , Lee Dominic Tak Shing , Lam Kwok Lim , Waye Mary Miu Yee
Published: January 2020

Background: The cytochrome P450 CYP2D6 enzyme debrisoquine 4-hydroxylase metabolizes many different classes of commonly used drugs, such as tricyclic antidepressants and neuroleptics. Genetic polymorphism of the CYP2D6 gene is responsible for pronounced interindividual and interracial differences in the metabolism of these drugs. The CYP2D6*10 allele and its variants are the most frequent alleles found in Orientals, and they are responsible for diminished debrisoquine 4-hydroxylase activity because of the presence of a C188→T mutation in exon 1.

Methods: One hundred nineteen Hong Kong Chinese subjects were genotyped by means of allele-specific PCR, PCR, and restriction enzyme analysis for 10 CYP2D6 alleles (CYP2D6*1, *2, *4D, *5, *8/*14, *10A, *10B, *15, *16, and J9).

Results: CYP2D6*10B was the most prevalent allele, and CYP2D6*10/CYP2D6*10 was the most frequent genotype, representing 46.22% of the population.

Conclusions: There was no significant difference in the prevalence of the alleles analyzed between our study and the Chinese populations genotyped previously. This is the largest study in terms of the number of CYP2D6 alleles analyzed in an Oriental population and the first one conducted in a Hong Kong Chinese population.
Increased Glutathionyl Hemoglobin in Diabetes Mellitus and Hyperlipidemia Demonstrated by Liquid Chromatography/Electrospray Ionization-Mass Spectrometry
Authors: Niwa Toshimitsu , Naito Chika , Mawjood Abdul Hassan Mohammed , Imai Kiyohiro
Published: January 2020

Background: Erythrocytes contain a large amount of glutathione (GSH), which protects cells from oxidative injury. The purpose of this study was to examine whether hemoglobin (Hb) is modified with glutathione by oxidation of the thiol groups in diabetes mellitus and hyperlipidemia, and to determine the oxygen affinity of glutathionyl Hb.

Methods: Hb samples obtained from patients with type 2 diabetes, patients with hyperlipidemia, and healthy subjects were analyzed by liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS). Glutathionyl Hb was synthesized in vitro by incubating Hb with GSH. The oxygen affinity of glutathionyl Hb was determined by measuring its oxygen dissociation curve.

Results: We first demonstrated that the concentration of glutathionyl Hbβ chains is markedly increased in the diabetic patients and hyperlipidemic patients compared with healthy subjects. The in vitro synthesis of glutathionyl Hb by incubation of Hb with GSH was enhanced by adding H2O2, a reactive oxygen species, into the incubation solution. The glutathionyl Hb prepared in vitro by incubating Hb with GSH showed a marked increase in oxygen affinity and a marked decrease in the Hill coefficient compared with Hb incubated without GSH.

Conclusions: Glutathionyl Hb may be useful as a clinical marker of oxidative stress. The increased concentrations of glutathionyl Hb with high oxygen affinity and low cooperativity in diabetes and hyperlipidemia may lead to reduced tissue oxygen delivery.
Discriminating Capacity of Indole Markers in the Diagnosis of Carcinoid Tumors
Authors: Meijer Wim G , Kema Ido P , Volmer Marcel , Willemse Pax H B , de Vries Elisabeth G E
Published: January 2020

Background: We evaluated the discriminating capacity of the indole markers urinary 5-hydroxyindoleacetic acid (5-HIAA), urinary serotonin, and platelet serotonin in the diagnosis of carcinoid tumors.

Methods: Indole markers were measured in 688 patients with suspected carcinoid disease. The initial values of indole markers from patients in whom a carcinoid tumor was confirmed during follow-up (n = 98) were used for ROC analysis. Two groups served as reference populations. The first consisted of 45 healthy individuals (“healthy controls”). The second was a random sample of 40 patients, drawn from the 590 (688 minus 98) patients with carcinoid-like symptoms but without a carcinoid tumor (“clinically suspected patients”).

Results: ROC curve analysis showed platelet serotonin to have the highest discriminating capacity, especially in foregut carcinoids. Cutoff values for platelet serotonin obtained from ROC analysis with healthy controls as reference group (5.4 nmol/109 platelets) gave a sensitivity of 74%, specificity of 91%, positive predictive value of 63%, and negative predictive value of 95% when applied to the initial 688 patients. Using the cutoff value with the clinically suspected patients as the reference group (9.3 nmol/109 platelets) gave a sensitivity of 63%, specificity of 99%, positive predictive value of 89%, and negative predictive value of 93%. Indole markers were increased in 169 (25%) of 688 patients. In 76 (45%) of these 169 patients, a carcinoid tumor was present. Slight increases of markers were associated with non-carcinoid neuroendocrine tumors, non-neuroendocrine tumors, and disturbed bowel motility.

Conclusions: ROC curve analysis shows that platelet serotonin is the most discriminating indole marker for the diagnosis of carcinoid tumors. Platelet serotonin especially improves the diagnosis of carcinoids producing small amounts of serotonin.
C-Reactive Protein and Cardiac Troponin T in Risk Stratification: Differences in Optimal Timing of Tests Early after the Onset of Chest Pain
Authors: de Winter Robbert J , Fischer Johan , Bholasingh Radha , van Straalen Jan P , de Jong Thyra , Tijssen Jan G P , Sanders Gerard T
Published: January 2020

Background: Increased C-reactive protein (CRP) is an important prognostic indicator for early risk stratification in patients with an acute coronary syndrome (ACS), independent of, and in combination with, increased cardiac troponin T (cTnT). However, increases in both cTnT and CRP also occur secondary to myocardial damage.

Methods and Results: In 156 consecutive patients, early release kinetics of CRP and cTnT were analyzed. The cutoff values were 3.0 mg/L for CRP and 0.1 μg/L for cTnT. In the 75 patients with a CRP below the cutoff on admission, there was little change in CRP until 8 h after the onset of symptoms. At 12 h after the onset of symptoms, the cumulative proportions of abnormal CRP and cTnT in non-ST elevation ACS patients were 27% and 89%, respectively (P <0.01). During the first 24 h after the onset of symptoms, the median time above the cutoff was 20 h for CRP and 5 h for cTnT (P <0.0001). CRP was below the cutoff on admission significantly more often among patients receiving thrombolytic therapy than in patients without an indication for reperfusion therapy (51% vs 28%; P = 0.004).

Conclusions: Increased CRP as an early independent risk indicator should be measured as soon as possible after the onset of symptoms, whereas increased cTnT is most reliable at 12 or more hours after the onset of symptoms.
Clinical Evaluation of the First Medical Whole Blood, Point-of-Care Testing Device for Detection of Myocardial Infarction
Authors: Apple Fred S , Anderson F Philip , Collinson Paul , Jesse Robert L , Kontos Michael C , Levitt M Andrew , Miller Elizabeth A , Murakami MaryAnn M
Published: January 2020

Background: Validation of whole blood, point-of-care testing devices for monitoring cardiac markers to aid clinicians in ruling in and ruling out myocardial infarction (MI) is necessary for both laboratory and clinical acceptance.

Methods: This study evaluated the clinical diagnostic sensitivity and specificity of the First Medical Cardiac Test device operated by nursing and laboratory personnel that simultaneously measures cardiac troponin I (cTnI), creatine kinase (CK) MB, myoglobin, and total CK on the Alpha Dx analyzer in whole blood for detection of MI. Over a 6-month period, 369 patients initially presenting to the emergency department with chest pain were evaluated for MI using modified WHO criteria. Eighty-nine patients (24%) were diagnosed with MI.

Results: In whole blood samples collected at admission and at 3- to 6-h intervals over 24 h, ROC curve-determined MI decision limits were as follows: cTnI, 0.4 μg/L; CKMB, 7.0 μg/L; myoglobin, 180 μg/L; total CK, 190 μg/L. Based on peak concentrations within 24 h after presentation, the following sensitivities (± 95% confidence intervals) were found: cTnI, 93% ± 5.5%; myoglobin, 81% ± 9.7%; CKMB, 90% ± 6.3%; total CK, 86% ± 7.5%. Sensitivities were maximal at >90% for both cTnI and CKMB at >12 h in MI patients, without differences between ST-segment elevation and non-ST-segment elevation MI patients.

Conclusions: The First Medical point-of-care device provides cardiac marker assays that can be used by laboratories and clinicians in a variety of hospital settings for ruling in and ruling out MI.
The Haptoglobin 2-2 Phenotype Affects Serum Markers of Iron Status in Healthy Males
Authors: Langlois Michel R , Martin Marie-Elise , Boelaert Johan R , Beaumont Carole , Taes Youri E , De Buyzere Marc L , Bernard Dirk R , Neels Hugo M , Delanghe Joris R
Published: January 2020

Background: Human iron status is influenced by environmental and genetic factors. We hypothesized that the genetic polymorphism of haptoglobin (Hp), a hemoglobin-binding plasma protein, could affect iron status.

Methods: Reference values of serum iron status markers were compared according to Hp phenotypes (Hp 1-1, Hp 2-1, Hp 2-2; determined by starch gel electrophoresis) in 717 healthy adults. Iron storage was investigated in peripheral blood monocyte-macrophages by measuring cytosolic L- and H-ferritins and by in vitro uptake of radiolabeled (125I) hemoglobin-haptoglobin complexes.

Results: In males but not in females, the Hp 2-2 phenotype was associated with higher serum iron (P <0.05), transferrin saturation (P <0.05), and ferritin (P <0.01) concentrations than Hp 1-1 and 2-1, whereas soluble transferrin receptor concentrations were lower (P <0.05). Moreover, serum ferritin correlated with monocyte L-ferritin content (r = 0.699), which was also highest in the male Hp 2-2 subgroup (P <0.01). In vitro, monocyte-macrophages took up a small fraction of 125I-labeled hemoglobin complexed to Hp 2-2 but not to Hp 1-1 or 2-1.

Conclusions: The Hp 2-2 phenotype affects serum iron status markers in healthy males and is associated with higher L-ferritin concentrations in monocyte-macrophages because of a yet undescribed iron delocalization pathway, selectively occurring in Hp 2-2 subjects.
Intraoperative Parathyroid Hormone Analysis: A Study of 200 Consecutive Cases
Authors: Sokoll Lori J , Drew Helen , Udelsman Robert
Published: January 2020

Background: Immunoassays for parathyroid hormone (PTH), with short incubation times and results available in <15 min, have allowed intraoperative monitoring of the success of parathyroid surgery. The purpose of this study was to evaluate the analytical performance of a rapid PTH assay and its clinical performance in a series of 200 patients.

Methods: PTH was measured with a modified immunochemiluminometric assay with a 7-min incubation time (QuiCk-IntraOperative™ Intact PTH assay). The rapid assay was compared with results in a central laboratory (immunoradiometric assay) in 44 EDTA-plasma specimens. The rapid assay was used intraoperatively in 200 consecutive cases with specimens analyzed before and 5–10 min after resection of the hypersecreting parathyroid gland(s).

Results: Intraassay imprecision was 12% at 28 ng/L and 11% at 278 ng/L. Regression analysis of results of the rapid PTH assay and the IRMA PTH assay in 44 parathyroidectomy patients yielded y = 1.26x − 12 ng/L, Sy|x = 26.3 ng/L, r = 0.984, and in 40 of 44 patients with values <200 ng/L, y = 1.02x + 1.9, Sy|x = 13.9, r = 0.947. In the 195 cases using intraoperative PTH testing with complete results and defined clinical outcomes, the overall accuracy of the assay in predicting surgical success was 88% using the criterion of a 50% decrease at 5–10 min and 97% including the subset of patients with delayed decreases of PTH.

Conclusions: The rapid PTH assay had excellent analytical performance and excellent agreement with the PTH immunoradiometric assay and predicted the success of parathyroid surgery in this large series of consecutive patients.
Rapid Screening Method for Osteoclast Differentiation in Vitro That Measures Tartrate-resistant Acid Phosphatase 5b Activity Secreted into the Culture Medium
Authors: Alatalo Sari L , Halleen Jussi M , Hentunen Teuvo A , Mönkkönen Jukka , Väänänen H Kalervo
Published: January 2020

Background: Osteoclasts secrete tartrate-resistant acid phosphatase (TRAP; EC 3.1.3.2) 5b into the circulation. We studied the release of TRAP 5b from osteoclasts using a mouse in vitro osteoclast differentiation assay.

Methods: We developed and characterized a polyclonal antiserum in rabbits, using purified human osteoclastic TRAP 5b as antigen. The antiserum was specific for TRAP in Western analysis of mouse osteoclast culture medium and was used to develop an immunoassay. We cultured mouse bone marrow-derived osteoclast precursor cells for 3–7 days with or without clodronate in the presence of vitamin D and analyzed the number of osteoclasts formed and the amount of TRAP 5b activity released into the culture medium.

Results: TRAP 5b activity was not secreted from osteoclast precursor cells. Addition of clodronate-containing liposomes decreased in a dose-dependent manner the number of osteoclasts and TRAP 5b activity released in 6-day cultures. The amount of TRAP 5b activity in the medium detected by the immunoassay correlated significantly with the number of osteoclasts formed (r = 0.94; P <0.0001; n = 120).

Conclusions: The TRAP 5b immunoassay can be used to replace the laborious and time-consuming microscopic counting of osteoclasts in the osteoclast differentiation assay and to test the effects of potential therapeutic agents on osteoclast differentiation, enabling fast screening of large amounts of potential therapeutic agents.
Determination of the Glycoforms of Human Chorionic Gonadotropin β-Core Fragment by Matrix-assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry
Authors: Jacoby Eli S , Kicman Andrew T , Laidler Paul , Iles Ray K
Published: January 2020

Background: Metabolism of human chorionic gonadotropin (hCG) in the serum and kidney yields the terminal urinary product hCG β-core fragment (hCGβcf), comprising two disulfide-linked peptides (β6-β40 and β55-β92) of which one (β6-β40) retains truncated N-linked sugars. Hyperglycosylated hCGβcf may indicate choriocarcinoma or Down syndrome, but the glycosylation profile of hCGβcf has not been thoroughly evaluated.

Methods: hCGβcf, purified from pregnancy urine, was reduced by “on-target” dithiothreitol (DTT) reduction and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The mass ([M+H]+) of the primary sequence of the glycosylated peptide β6-β40 was subtracted from the m/z values of the discrete peaks observed to give the masses of the carbohydrate moieties. Carbohydrate structure was predicted by sequentially subtracting the masses of the monosaccharide residues corresponding to N-linked carbohydrates of the hCG β-subunit reported in the literature.

Results: Mass spectra of hCGβcf revealed a broad triple peak at m/z 8700–11300. After reduction, the triple peak was replaced by a discrete set of peaks between m/z 4156 and 6354. A peak at m/z 4156.8 corresponded to the nonglycosylated peptide (β55-β92). The remaining nine peaks indicated that urinary hCGβcf comprises a set of glycoforms smaller and larger than the trimannosyl core.

Conclusions: hCGβcf comprises a wider set of glycoforms than reported previously. Peaks of highest mass indicate evidence of hyperglycosylated carbohydrate moieties. The data support previous reports that hCGβcf oligosaccharides lack sialic acid and galactose residues. No indication was found of a β6-β40 peptide that was entirely devoid of carbohydrate.
Evaluation of LabRespond, a New Automated Validation System for Clinical Laboratory Test Results
Authors: Oosterhuis Wytze P , Ulenkate Herman J L M , Goldschmidt Henk M J
Published: January 2020

Background: Manual validation of laboratory test results is time-consuming, creating a demand for expert systems to automate this process. We have started to set up the program “LabRespond”, which covers five validation levels: administrative, technical, sample, patient, and clinical validation. We present the evaluation of a prototype of an automated patient validation system based on statistical methods, in contrast to the commercially available program “VALAB”, a rule-based automated validation system.

Methods: In the present study, 163 willfully altered, erroneous test results out of 5421 were submitted for validation to LabRespond, VALAB, and to a group of clinical chemists (n = 9) who validated these test results manually. The test results rejected by three or more clinical chemists (n = 281) served as a secondary reference standard.

Results: The error recovery rates of clinical chemists ranged from 23.9% to 71.2%. The recovery rates of LabRespond and VALAB were 77.9% and 71.8%, respectively (difference not significant). The false-positive rates were 82.7% for LabRespond, 83.6% for VALAB, and 27.8–86.7% for clinical chemists. Using the consensus of three or more clinical chemists as the secondary reference standard, we found error recovery rates of 64.8% for LabRespond and 72.2% for VALAB (P = 0.06). Compared with VALAB, LabRespond detected more (P = 0.003) erroneous test results of the type that were changed from abnormal to normal.

Conclusions: The statistical plausibility check used by LabRespond offers a promising automated validation method with a higher error recovery rate than the clinical chemists participating in this study, and a performance comparable to VALAB.