Serum Aliquots Research Articles

A rare missense variant in the milk fat globule-EGF factor 8 (MFGE8) increases T2DM susceptibility and cardiovascular disease risk with population-specific effects.

The milk fat globule-epidermal growth factor 8 (MFGE8), also called lactadherin, is an integrin ligand and a known mediator of inflammation and atherosclerosis in T2DM in studies using animal models. However, its role in the pathophysiology of human T2DM, obesity, and cardiovascular disease has been poorly explored. Aim of thisstudy was toinvestigate the role ofa missense variant (rs371227978 C/T: Arg148His) in the MFGE8 gene identified through exome sequencing for its association with T2DM and cardiometabolic traits. Exome-wide sequencing was performed using DNA samples from 68 Sikh individuals from multi-generation pedigrees affected with diabetes onIllumina's GAIIx using "SureSelect Human All Exon" panels. We further replicated this variant by de novo genotyping in a total of 4242 individuals of the Asian Indian Diabetic Heart Study/Sikh Diabetes Study using custom TaqMan genotyping assay. We also measured circulating concentrations of Mfge8 using frozen serum aliquots by enzyme-linked immunosorbent assay. Overall, only 1.78% of 4242 individuals were carriers of this variant with MAF being 0.009. Except for the significant correlation of this variant with T2DM and triglycerides, no other quantitative risk phenotype was significant. The minor per allele-associated increased risk for T2DM showed odds ratio of 1.95 (95% CI 1.18-3.23; p = 0.008) in unadjusted model and was 1.73 (95% CI 1.02-2.93; p = 0.043) after adjusting for the age, gender, and BMI. However, there was a strong correlation between serum Mfge8 concentrations with T2DM, (r2 = 0.38; p = 0.001), fasting glucose (r2= 0.36; p = 0.002), and triglycerides (r2 = 0.33; p = 0.005). Our data revealed a significant dose-related increase in MFGE8 genotypes for affecting serum Mfge8 (p = 2.1 × 10-3) and triglyceride concentrations (p = 3.2 × 10-3). For a per riskallele-associated increase of 4.74ng/ml ± SD of 1.62ng/ml of the Mfge8 concentration was found to increase T2DM risk to 1.7 fold (95% CI from 1 to 3 fold). Here, we report for the first time a novel population-specific rare variant in the MFGE8 gene linked with the increased Mfge8 concentrations and the risk for developing T2DM and cardiovascular risk factors in a population of Punjabi Sikhs from India. In view of a strong evidence from animal studies supporting the role of Mfge8 in obesity, insulin resistance, and the development of atherosclerosis in T2DM, our findings are important and timely. If validated in a large independent dataset, early screening of Mfge8 in blood levels may especially benefit those patients with genetically elevated Mfge8 levels to preventing or reducing the risk of T2DM and cardiovascular disease.

Is the Osmolal Concentration of Ethanol Greater Than Its Molar Concentration?

Background: Recent data suggested that the osmolal gap attributed to ethanol as determined by the difference between measured serum osmolality and calculated serum osmolarity is greater than its molar concentration. The increased osmotic activity of ethanol is thought to be due to its binding to water molecules. This study is conducted to determine the true osmotic contribution of ethanol to serum osmolality.Methods: Baseline serum osmolality and ethanol concentration were measured on each serum sample. Varying amounts of ethanol were added to aliquots of serum in which the baseline serum ethanol concentration was undetectable. Repeat serum osmolality and serum ethanol concentration were measured after addition of ethanol.Results: The range of serum ethanol concentration was 27.3–429.8 mg/dL. The serum osmolal gap attributed solely to ethanol is calculated based on the difference between measured serum osmolality before and measured serum osmolality after addition of ethanol. Our results demonstrated that the contribution of ethanol to serum osmolality can be calculated by dividing the serum ethanol level in mg/dl by 4.6. In addition, the relationship between serum ethanol concentration and osmolal gap due to ethanol was assessed by linear regression analysis. Linear regression analysis relating the osmolal gap due to ethanol and ethanol concentration yielded the following equation: Osmolal Gap (mOsm/kg H2O) = 0.23 (Ethanol [mg/dL]) – 1.43.Conclusion: The osmolal concentration of ethanol can be calculated based on its molar concentration. We found no evidence for ethanol binding to water molecules over the range of ethanol concentration in this study.

Circulating Sex Steroid Measurements of Men by Mass Spectrometry Are Highly Reproducible after Prolonged Frozen Storage

Long-term studies investigating hormone-dependent cancers and reproductive health often require prolonged frozen storage of serum which assumes that the steroid molecules and measurements are stable over that time. Previous studies of reproducibility of circulating steroids have relied upon flawed historical rather than contemporaneous controls. We measured serum testosterone (T), dihydrotestosterone (DHT), estradiol (E2) and estrone (E1) in 150 randomly selected serum samples by liquid chromatography-mass spectrometry (LC-MS) from men 70 years or older (mean age 77 years) in the CHAMP study. The original measurements in 2009 were repeated 10 years later using the identical serum aliquot (having undergone 2-4 freeze-thaw cycles in the interim) in 2019 together with another never-thawed aliquot of the same serum sample. The results of all three sets of measurements were evaluated by Passing-Bablok regression and Bland-Altman difference analysis. Serum androgens (T, DHT) and estrogens (E2, E1) measured by LC-MS display excellent reproducibility when stored for 10 years at -80 C without thawing. Serum T and DHT displayed high level of reproducibility across all three sets of measurements. Multiple freeze-thaw cycles over those storage conditions do not significantly affect serum T, DHT and E1 concentrations but produce a modest increase (21%) in serum E2 measurements.

Reference Values for 30 Common Biochemistry Analytes Across 5 Different Analyzers in Neonates and Children 30 Days to 18 Years of Age.

Age-specific reference intervals (RIs) have been developed for biochemistry analytes in children. However, the ability to interpret results from multiple laboratories for 1 individual is limited. This study reports a head-to-head comparison of reference values and age-specific RIs for 30 biochemistry analytes for children across 5 analyzer types. Blood was collected from healthy newborns and children 30 days to <18 years of age. Serum aliquots from the same individual were analyzed on 5 analyzer types. Differences in the mean reference values of the analytes by the analyzer types were investigated using mixed-effect regression analysis and by comparing maximum variation between analyzers with analyte-specific allowable total error reported in the Westgard QC database. Quantile regression was used to estimate age-specific RIs using power variables in age selected by fractional polynomial regression for the mean, with modification by sex when appropriate. The variations of age-specific mean reference values between analyzer types were within allowable total error (Westgard QC) for most analytes, and common age-specific reference limits were reported as functions of age and/or sex. Analyzer-specific reference limits for all analytes on 5 analyzer types are also reported as functions of age and/or sex. This study provides quantitative and qualitative measures of the extent to which results for individual children can or cannot be compared across analyzer types, and the feasibility of RI harmonization. The reported equations enable incorporation of age-specific RIs into laboratory information systems for improving evidence-based clinical decisions in children.

Fabrication of Microarrays for the Analysis of Serological Antibody Isotypes against Food Antigens.

Food intolerance is delayed adverse food reactions which follow consumption of specific foods. The underlying mechanisms are not well understood, but food intolerance is often considered as a type 2 hypersensitivity reaction mediated by immunoglobulin G (IgG) antibody. To understand the causes of food intolerance, it is important to investigate sensitization patterns of food-specific IgGs (sIgG) in relation to dietary patterns and physical conditions. Conventional approaches to measure serological IgGs often require large volumes of serum, thus are not suitable for highly multiplexed assays. To overcome this impracticality, we developed a highly sensitive method to screen the sIgGs and other antibody isotypes against 66 antigens with minimal amount of serums. We prepared a microarray by immobilizing food antigens on activated glass slides. Human sera and their dietary information were obtained from 30 subjects. Aliquots (200 nl) of sera were analyzed against 66 food antigens in parallel. sIgG levels were determined and analyzed in relation to subjects’ dietary patterns. The levels of antibody isotypes were also examined to understand the relationship between allergy and food intolerance. The developed microarray showed exceptional performances in antibody screening and demonstrated the potential to be used as an automated assay system.

Comparison of sample preparation approaches and validation of an extraction method for nitrosatable pesticides and metabolites in human serum and urine analyzed by liquid chromatography – Orbital ion trap mass spectrometry

In the acidic environment of the stomach, nitrosatable pesticide residues may react with nitrite to form potentially carcinogenic pesticide-associated N-nitroso compounds (PANOCs). The objective of this study was to develop a method for the analysis of 10 nitrosatable pesticides and breakdown products in human serum and urine. Three sample preparation methods were evaluated for extraction of target analytes from the biomatrices. Deproteinization by methanol for 300-μL aliquots of serum with a final extract volume of 225 μL resulted in excessive ion enhancement of some analytes and suppression of others. Three types of solid-phase extraction cartridges were tested for optimal analyte retention from 200-μL aliquots of serum with a final extract volume of 400 μL; this approach resulted in significant analyte loss for some compounds. The Quick, Easy, Cheap, Effective, Rugged, and Safe approach resulted in a suitable method for extraction of the analytes from each biomatrix. Biofluid samples (500 μL) were spiked to 100 μg L−1 with analytical standards and extracted using 500 μL of acetonitrile (ACN) with 4% acetic acid (AcOH) for serum and 0.1% AcOH in ACN for urine. For extraction, 200 mg magnesium sulfate (MgSO4) and 50 mg sodium acetate were added for serum and 200 mg MgSO4 and 50 mg sodium chloride were added for urine. Final extract volumes for both biomatrices using the QuEChERS method was 400 μL after dilution. Samples were analyzed via ultra-high pressure liquid chromatography/high-resolution accurate mass orbital ion trap mass spectrometry. Mean recoveries for target analytes in serum and urine ranged between 74 and 120% (%RSD < 12) and 96 to 116% (%RSD ≤ 10), respectively. These methods may be used in large-scale biomonitoring studies to analyze PANNs and their parent compounds in human serum and urine.

Cost-Effective Automated Preparation of Serum Samples for Reproducible Quantitative Clinical Proteomics.

Reproducible sample preparation remains a significant challenge in large-scale clinical research using selected reaction monitoring-mass spectrometry (SRM-MS), which enables a highly sensitive multiplexed assay. Although automated liquid-handling platforms have tremendous potential for addressing this issue, the high cost of their consumables is a drawback that renders routine operation expensive. Here we evaluated the performance of a liquid-handling platform in preparing serum samples compared with a standard experiment while reducing the outlay for consumables, such as tips, wasted reagents, and reagent stock plates. A total of 26 multiplex assays were quantified by SRM-MS using four sets of 24 pooled human serum aliquots; the four sets used a fixed number (1, 4, 8, or 24) of tips to dispense digestion reagents. This study demonstrated that the use of 4 or 8 tips is comparable to 24 tips (standard experiment), as evidenced by their coefficients of variation: 13.5% (for 4 and 8 tips) versus 12.0% (24 tips). Thus we can save 37% of the total experimental cost compared with the standard experiment, maintaining nearly equivalent reproducibility. The routine operation of cost-effective liquid-handling platforms can enable researchers to process large-scale samples with high throughput, adding credibility to their findings by minimizing human error.

Anti-Müllerian hormone as a diagnostic tool for PCOS under different diagnostic criteria in an unselected population.

Is anti-Müllerian hormone (AMH) a valid tool to diagnose polycystic ovary syndrome (PCOS) according to different subsets of criteria among an unselected group of women? In this cross-sectional study, AMH concentrations were measured in an unselected group of women. The ability of AMH to diagnose PCOS according to National Institutes of Health (NIH), Rotterdam-2003 and Androgen Excess and PCOS Society (AE-PCOS) criteria was tested by using frozen serum aliquots (n=392) that had been collected from a previous prevalence study of PCOS. The respective age and body mass index adjusted area under the curve (aAUC, 95% confidence interval) values were 0.80 (0.71-0.89), 0.74 (0.67-0.81) and 0.71 (0.64-0.79). When the definition of polycystic ovary morphology (PCOM) was set to an antral follicle count (AFC) of 20 instead of 12, the prevalence of syndrome dropped from 19.9% to 10.2% and from 15.3% to 8.9% according to Rotterdam-2003 and AE-PCOS criteria, respectively. In patients with Phenotype A, who had hyperandrogenism, ovulatory dysfunction and PCOM, AMH had an aAUC of 0.85 (0.77-0.92) to diagnose the syndrome. In Phenotypes B (hyperandrogenism + ovulatory dysfunction), C (hyperandrogenism + PCOM) or D (ovulatory dysfunction + PCOM), AMH had poor to fair ability to diagnose the syndrome. AMH has poor to fair validity to diagnose PCOS among an unselected group of women, except for patients bearing all features of the syndrome (Phenotype A). This finding is valid using the NIH, Rotterdam-2003 and AE-PCOS criteria and even after revising the definition of PCOM as AFC ≥20.

The effect of time, temperature and P-chloro-mercuriphenylsulfonic acid during serum storage on HDLᵼ-C and HDL₃-C concentration

Aims: Accurate measurement of clinical laboratory parameters plays an essential role in the correct interpretation of clinical biochemistry abnormalities. The purpose of this study was evaluation the time and temperature effect on HDLᵼ-C, HDL₂-C and HDL₃-C stability during storage. Methods: 50 adult healthy subjects were participated. For the isolation of HDLᵼ-C, we used precipitation method and HDLᵼ-C data was analyzed by the Abell-Kendal cholesterol reference method. The remaining serum were dispensed into 12 sample tubes and divided into two groups. One of each group was stored upright at room temperature (approximately 25 oC) while another at 4 oC and the half of each group tubes were received p-chloro - mercuriphenylsulfonic acid (PCMPS). The stored serum aliquots from all temperature and time points were analyzed on 1, 2, 3 days post collection. Results: HDLᵼ-C concentration at the temperature of room in 24 hours is not changed significantly but over the time decreased (7.2% in 3 days). In addition of PCMPS inhibitor, the concentration is increased by 17.3% in 3 days. But in 4oC, with or without PCMPS, there is no a significant change in the HDLᵼ-C concentration. HDL₃-C was found to be the most stable lipoprotein studied because of non-significant effect of storage time and temperature on it. Conclusion: The results suggest 4oC as the ideal storage condition for the preservation of human serum samples for HDLᵼ-C assay. Also it is suggested that HDL concentration estimation should be performed in the first 24 hours of samples collection. PCMPS addition didn’t affect HDL subtypes concentration in 4oC.

Gentamicin and Vancomycin Interference on Results of Clinical Chemistry Parameters on Abbott Architect c8000.

Gentamicin and vancomycin are nephrotoxic antibiotics. Little is known about the influence of drug concentrations on results of clinical chemistry tests. To investigate gentamicin and vancomycin interference on results of 33 commonly measured biochemistry tests. The study was carried out in the University Department of Chemistry, Medical School University Hospital Sestre Milosrdnice (Zagreb, Croatia). For each drug, 10 aliquots of pooled serum were prepared. In order to cover toxic concentrations, pool serum samples were spiked with drugs to obtain 0 to 50 μg/mL of gentamicin and 0 to 200 μg/mL of vancomycin. Biochemistry tests were measured in duplicate on the Architect c8000 analyzer, and drug concentrations were measured on Architect i2000 SR (both Abbott Laboratories, Abbott Park, Illinois). For each tested concentration, bias was calculated against the initial measurement. Acceptance criteria were defined as measurement uncertainty of the commercial control with the value close to the measured range of the pool sample. For gentamicin, all bias values were below established criteria. For vancomycin, significant changes were observed for potassium, direct bilirubin, and immunoglobulin A. Significant bias was already detected at low vancomycin concentration (2.98 μg/mL) for direct bilirubin (bias = 9.7%; acceptable = 8%). Potassium bias at the highest vancomycin concentration (204.4 μg/mL) exceeded acceptance criteria (bias = 4.5%; acceptable = 4%). For immunoglobulin A, no apparent trend was observed, and bias is attributed to increased method imprecision. Gentamicin did not interfere with the results of clinical chemistry tests. Direct bilirubin concentration is falsely increased in the presence of vancomycin, and potassium is affected at high concentrations.

GC-MS measurement of biological NG-hydroxy-L-arginine, a stepmotherly investigated endogenous nitric oxide synthase substrate and arginase inhibitor.

L-Arginine is converted by nitric oxide synthase (NOS) to L-citrulline and nitric oxide (NO). NG-Hydroxy-L-arginine (NOHA) is the isolable intermediate of this reaction. NOHA has been identified in biological samples by gas chromatography-mass spectrometry (GC-MS) and quantified by high-performance liquid chromatography (HPLC). Reportedly, NOHA concentrations in human plasma and serum range over four orders of magnitude (e.g., 2nM-34µM). The natural occurrence of NOHA in urine has not been reported thus far. Here, we report a validated stable-isotope dilution GC-MS method for the quantitative determination of NOHA in 10-µL aliquots of human serum and urine samples. The method is based on a two-step derivatization of NOHA to the methyl ester pentafluoropropionyl (PFP) derivatives using newly synthesized trideuteromethyl ester NOHA (d3Me-NOHA) as the internal standard and GC-MS quantification. NOHA was found to form a methyl ester-NG,Nδ,Nα-pentafluoropropionyl derivative, i.e., Me-(PFP)3 (M, 642) with the NG-hydroxy group remaining non-derivatized. Selected-ion monitoring of mass-to-charge (m/z) ratio of 458 for endogenous NOHA and m/z 461 for d3Me-NOHA in the negative-ion chemical ionization mode revealed NOHA concentrations of the order of 0.2µM in human serum and 3µM in urine samples. Accuracy (recovery,%) was 91.6 ± 1.6% in serum and 39.9 ± 4.5% in urine. Inorganic nitrate was found to decrease NOHA recovery from urine presumably through the reaction of the OH group of NOHA with nitric acid. Imprecision (RSD, %) ranged between 1.4 and 14.8% in serum, and between 5.3 and 18.4% in urine in the investigated concentration range (0-15µM NOHA). Ten healthy kidney donors excreted in the urine (mean ± SEM) 13.9 ± 1.81µmol NOHA per day before and 10.9 ± 1.4µmol NOHA per day after kidney donation (P = 0.24). Similar results were observed for dimethylamine (DMA), the major urinary metabolite of asymmetric dimethylarginine (ADMA). Changes in NOHA and DMA correlated positively (r = 0.718, P = 0.019). This is the first report on the occurrence and measurement of NOHA in human urine and on the effect of human unilateral nephrectomy on urinary NOHA and DMA. Healthy kidney donation may be useful as a model for kidney disease.

A Multi-Center Assessment of Thyroid Function Test Precision in Chemiluminescence Immunoassay (CLIA) Systems

Background: Chemiluminiscence immunoassay (CLIA) is exclusively pragmatic technology for the analysis of biomarker for diagnosis of thyroid disorders. However, performance characteristics of different chemiluminescence immunoassay (CLIA) systems supplied by different manufacturers in diverse set up for thyroid function test (TFT) has not yet been studied well.&#x0D; Objective: Our aim is to evaluate laboratory results by assessment of the reproducibility and repeatability of TFTs in three different diagnostic set up to assure the quality of thyroid hormone assay using chemiluminescence immunoassay (CLIA) instruments: Advia Centaur CP (Siemens), Access 2 (Beckman Coulter) and Liaison (Diasorin).&#x0D; Materials and Methods: Among the adult male and female individuals visited for thyroid hormone assay, 51 normal individuals were selected for the study. Three aliquots of serum samples were distributed to assess the reproducibility of three different CLIA equipments operated in three diagnostic centers. Additional three aliquots of serum were analyzed weekly for TFT (fFT3, fFT4 and TSH) to check the repeatability of assay in ADVIA Centaur CP set up. Assay precision was determined by reproducibility and repeatability of test results.&#x0D; Results: Results of TFTs of serum samples obtained from three different interlaboratory assays using different CLIA systems have achieved good precision showing minimal variance (P&gt;0.05) and acceptable reproducibility. Results are also precise with adequate repeatability showing minimal variance (P &gt;0.05) obtained from the three different intra-laboratory assays in a single CLIA system using ADVIA Centaur CP by same team.&#x0D; Conclusion: Our study elucidates the thyroid hormone assay performance of CLIA systems in three centers, which has shown assay precision with good reproducibility and repeatability of thyroid hormone assay. Thus, the analysis of precision as an essential component of quality control is necessary to deliver precise diagnostic services.

Early detection of metabolic dysregulation using water T2 analysis of biobanked samples.

BackgroundThe ability to use frozen biobanked samples from cohort studies and clinical trials is critically important for biomarker discovery and validation. Here we investigated whether plasma and serum water transverse relaxation times (T2) from frozen biobanked samples could be used as biomarkers for metabolic syndrome (MetS) and its underlying conditions, specifically insulin resistance, dyslipidemia, and subclinical inflammation.MethodsPlasma and serum aliquots from 44 asymptomatic, non-diabetic human subjects were biobanked at –80°C for 7–9 months. Water T2 measurements were recorded at 37°C on 50 µL of unmodified plasma or serum using benchtop nuclear magnetic resonance relaxometry. The T2 values for freshly drawn and once-frozen-thawed (“frozen”) samples were compared using Huber M-values (M), Lin concordance correlation coefficients (ρc), and Bland–Altman plots. Water T2 values from frozen plasma and serum samples were compared with >130 metabolic biomarkers and analyzed using multi-variable linear/logistic regression and ROC curves.ResultsFrozen plasma water T2 values were highly correlated with fresh (M=0.94, 95% CI 0.89, 0.97) but showed a lower level of agreement (ρc=0.74, 95% CI 0.62, 0.82) because of an average offset of −5.6% (−7.1% for serum). Despite the offset, frozen plasma water T2 was strongly correlated with markers of hyperinsulinemia, dyslipidemia, and inflammation and detected these conditions with 89% sensitivity and 91% specificity (100%/63% for serum). Using optimized cut points, frozen plasma and serum water T2 detected hyperinsulinemia, dyslipidemia, and inflammation in 23 of 44 subjects, including nine with an early stage of metabolic dysregulation that did not meet the clinical thresholds for prediabetes or MetS.ConclusionPlasma and serum water T2 values from once-frozen-thawed biobanked samples detect metabolic dysregulation with high sensitivity and specificity. However, the cut points for frozen biobanked samples must be calibrated independent of those for freshly drawn plasma and serum.

Generation of an equine biobank to be used for Functional Annotation of Animal Genomes project.

The Functional Annotation of Animal Genomes (FAANG) project aims to identify genomic regulatory elements in both sexes across multiple stages of development in domesticated animals. This study represents the first stage of the FAANG project for the horse, Equus caballus. A biobank of 80 tissue samples, two cell lines and six body fluids was created from two adult Thoroughbred mares. Ante-mortem assessments included full physical examinations, lameness, ophthalmologic and neurologic evaluations. Complete blood counts and serum biochemistries were also performed. At necropsy, in addition to tissue samples, aliquots of serum, ethylenediaminetetraacetic acid (EDTA) plasma, heparinized plasma, cerebrospinal fluid, synovial fluid, urine and microbiome samples from all regions of the gastrointestinal and urogenital tracts were collected. Epidermal keratinocytes and dermal fibroblasts were cultured from skin samples. All tissues were grossly and histologically evaluated by a board-certified veterinary pathologist. The results of the clinical and pathological evaluations identified subclinical eosinophilic and lymphocytic infiltration throughout the length of the gastrointestinal tract as well as a mild clinical lameness in both animals. Each sample was cryo-preserved in multiple ways, and nuclei were extracted from selected tissues. These samples represent the first published systemically healthy equine-specific biobank with extensive clinical phenotyping ante- and post-mortem. The tissues in the biobank are intended for community-wide use in the functional annotation of the equine genome. The use of the biobank will improve the quality of the reference annotation and allow all equine researchers to elucidate unknown genomic and epigenomic causes of disease.

Evaluation of the Siemens ADVIA Centaur high-sensitivity cardiac troponin I assay in serum

BackgroundInternational recommendations on high-sensitivity cardiac troponin (hs-cTn) testing recommend that laboratories select only one sample type for testing. We evaluated the Siemens ADVIA Centaur hs-cTnI assay in serum and thereby provide information on imprecision, long-term storage stability, freeze-thaw stability, method comparison to other hs-cTnI assays, and clinical performance. MethodsPatients with chest pain onset <6 h who already had Roche hs-cTnT, Beckman hs-cTnI and Abbott hs-cTnI results recorded and had non-thawed and frozen serum aliquots formed the study population (n = 134 patients with 305 serum aliquots obtained at either 0, 3 or 6 h stored below -70 °C since 2003) for measurement with the Siemens hs-cTnI assay in 2018. Receiver-operating characteristic curve analyses for myocardial infarction (MI) using the highest obtained hs-cTn concentration was performed. Additional comparison testing on serum samples stored frozen (at -70 °C for <1 month in 2018) for the Siemens and Abbott hs-cTnI assays were performed, as well as precision testing in serum pools and freeze-thaw stability testing. ResultsThe Siemens hs-cTnI assay had an area under the curve (AUC) of 0.978 (95%CI: 0.937–0.996) for MI in the study cohort (Roche hs-cTnT AUC = 0.965 and Abbott AUC = 0.973). The Siemens hs-cTnI assay yielded higher cTnI concentrations than the other hs-cTn assays, with the same proportional bias (slope = 1.4) between the Siemens and Abbott hs-cTnI assays obtained from serum samples collected in 2003 and 2018. Over 3 months, a low serum pool of 3.5 ng/l achieved a CV of 20% (SD = 0.7, n = 42) and a high serum pool of 820 ng/l achieved a CV of 2.3% (SD = 20, n = 42). Three different serum pools recovered within 10% from baseline concentration after 5 freeze-thaw cycles for the Siemens hs-cTnI assay. ConclusionsIn serum, the Siemens ADVIA Centaur hs-cTnI assay had excellent clinical performance for MI in an early chest pain onset population, acceptable precision at normal and highly elevated cTnI concentrations, long-term storage stability (15 y storage below -70 °C) and acceptable freeze-thaw stability, all of which supports serum as an acceptable sample type to use in clinical studies and in clinical practice.

Highly Sensitive Glycan Profiling of Hepatitis B Viral Particles and a Simple Method for Dane Particle Enrichment.

Hepatitis B virus (HBV) is a double-stranded DNA virus composed of three types of viral particles. The virions are called Dane particles and the others are noninfectious subviral particles (SVPs). In blood, SVPs are detected in abundance, about 1000-10000 fold higher than Dane particles. Dane particles are hazardous because of their strong infectivity, unlike SVPs. Dane particles are covered with an envelope of glycoprotein called HBV surface antigen (HBsAg). HBsAg glycosylation is involved in viral particle formation and secretion. In this study, we established a novel and highly sensitive method for viral glycan profiling of HBsAg using small aliquots of patient serum. Our lectin microarray system could sensitively profile the glycans exposed on HBV while retaining the intact viral particle structure under nonreducing conditions. Several typical lectins were chosen from the lectin microarray results. Specifically, jacalin, which recognizes O-glycan, showed specific and strong reactivity to the M-HBsAg required for Dane particle secretion. Employing the lectin-fractionation method using jacalin, HBV particles were fractionated into jacalin-bound and unbound fractions from patient serum. We measured HBsAg titer and viral DNA load in each fraction using clinical tests. Interestingly, the jacalin-bound fraction contained a major fraction of the HBV viral DNA load. Thus, in this study we have presented a glycan profiling method for HBsAg on the intact HBV particle and an easy and simple method to enrich Dane particles from patient serum by jacalin fractionation.

Abstract 1556: Does surgical treatment affect serum levels of vascular endothelial growth factor (VEGF) in orofacial tumors

Abstract Introduction: Angiogenesis, the generation of new vessels, is regulated by several growth factors. The most important of them is vascular endothelial growth factor (VEGF), which is closely related to tumor progression and prognosis. To my knowledge no study in sub-Saharan Africa has evaluated the level of VEGF following surgery in African patients. This study therefore aims to evaluate the pattern and clinical implication of VEGF expression in benign and malignant neoplasms and determine the effect of surgical intervention on the levels of circulating VEGF. Methods: This case-control study was carried out at the University of Benin Teaching Hospital (UBTH), Benin City, Nigeria, between October 2015 and February 2017. Cases were drawn from among histologically confirmed orofacial tumors, while controls were from consecutive, ambulatory dental patients, matched for age and sex. Calculated sample size for minimum effect was 77 for both cases and controls. Blood samples were taken from cases and controls, pre- and 1-month post-surgery, allowed to clot and serum aliquots were stored at -600 C till analyses. Post-surgery tissue was processed and stained with anti-VEGF antibody for immunohistochemical analysis. Serum VEGF was analyzed using Sandwich ELISA, and mean levels as well as median were measured. Spearman correlation was estimated, t-test measured significance (at p ≤ 0.05), and one-way ANOVA measured between group differences. Results: Following 12% attrition, 81 subjects were retained in the study (55 cases [20 malignant, 35 benign] and 26 controls). The most common malignant neoplasms were head and neck squamous cell carcinoma (35%) and polymorphous low-grade adenocarcinoma (20%), while the benign tumors were mostly unicystic ameloblastoma (42.9%) and solid multicystic ameloblastoma (31.4%). Before surgical treatment, there were significantly different serum levels of VEGF in benign (48.11 ± 25.19 pg/ml) vs. malignant cases (1065.00 ± 412.14 pg/ml), and compared with controls (45.42 ± 29.83 pg/ml), p &amp;lt;0.001. Also, the level of circulating VEGF correlated strongly and positively with tumor tissue expression in malignant cases (r = 0.674, p &amp;lt;0.001). After definitive surgical intervention, there was a significant reduction in the level of serum VEGF in both benign (45.50 ± 24.71 pg/mL) and malignant cases (51.22 ± 16.84 pg/mL), p &amp;lt;0.001. One-way ANOVA revealed no significant differences between cases and controls post-surgery. Conclusion: There were elevated levels of serum VEGF in benign and malignant orofacial neoplasia, which correlate positively with the expression of VEGF in tumor tissue. Surgery caused a significant reduction of circulating VEGF to normal levels irrespective of age, gender, previous medical history, perceived duration of the lesion and lymph node involvement. Citation Format: Aimuamwosa Obarisiagbon, Peter Egbor, Ozoemene Ndubuisi Obuekwe, Osagie Akpata, Akin Ojo, Nosayaba Osazuwa-Peters. Does surgical treatment affect serum levels of vascular endothelial growth factor (VEGF) in orofacial tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1556.

Activity-Based Detection of Cannabinoids in Serum and Plasma Samples.

Synthetic cannabinoids are the largest group of new psychoactive substances monitored by the European Monitoring Centre of Drugs and Drug Addiction. The rapid proliferation of novel analogs makes the detection of these new derivatives challenging and has initiated considerable interest in the development of so-called "untargeted" screening strategies to detect these compounds. We developed new, stable bioassays in which cannabinoid receptor activation by cannabinoids led to recruitment of truncated β-arrestin 2 (βarr2) to the cannabinoid receptors, resulting in functional complementation of a split luciferase, allowing readout via bioluminescence. Aliquots (500 μL) of authentic serum (n = 45) and plasma (n = 73) samples were used for simple liquid-liquid extraction with hexane:ethyl acetate (99:1 v/v). Following evaporation and reconstitution in 100 μL of Opti-MEM® I/methanol (50/50 v/v), 10 μL of these extracts was analyzed in the bioassays. Truncation of βarr2 significantly (for both cannabinoid receptors; P = 0.0034 and 0.0427) improved the analytical sensitivity over the previously published bioassays applied on urine samples. The new bioassays detected cannabinoid receptor activation by authentic serum or plasma extracts, in which synthetic cannabinoids were present at low- or sub-nanogram per milliliter concentration or in which Δ9-tetrahydrocannabinol was present at concentrations >12 ng/mL. For synthetic cannabinoid detection, analytical sensitivity was 82%, with an analytical specificity of 100%. The bioassays have the potential to serve as a first-line screening tool for (synthetic) cannabinoid activity in serum or plasma and may complement conventional analytical assays and/or precede analytical (mass spectrometry based) confirmation.

Convallatoxin, the active cardiac glycoside of lily of the valley, minimally affects the ADVIA Centaur digoxin assay

Lily of the valley is a poisonous plant due to the presence of the cardiac glycoside convallatoxin which is known to interfere with serum digoxin measurement using the LOCI digoxin assay and other digoxin assays. We evaluated potential interference of convallatoxin as well as extract of lily of the valley with the ADVIA Centaur digoxin assay by comparing results obtained using the LOCI digoxin assay. Aliquots of a drug-free serum pool and a digoxin serum pool were supplemented with nanograms to 1μg quantities of convallatoxin or 1.0 and 2.5μL of lily of the valley extract per milliliter of serum followed by measurement of digoxin concentrations using the LOCI and ADVIA Centaur digoxin assays. Apparent digoxin concentrations were minimal using the ADVIA Centaur digoxin assay when aliquots of drug-free serum were supplemented with convallatoxin or extract of lily of the valley but apparent digoxin levels were very high using the LOCI digoxin assay. Moreover, minimal interference in serum digoxin measurement using the ADVIA Centaur digoxin assay was observed when aliquots of serum digoxin pool were further supplemented with lily of the valley extract. As expected, the LOCI digoxin assay showed significant interference of convallatoxin in serum digoxin measurement. Significant interference of convallatoxin in serum digoxin measurement using the LOCI digoxin assay could be minimized using the ADVIA Centaur digoxin assay.

Quantitative Analysis of l-Arginine, Dimethylated Arginine Derivatives, l-Citrulline, and Dimethylamine in Human Serum Using Liquid Chromatography\u2013Mass Spectrometric Method

Nitric oxide (NO) is a small molecule involved in the regulation of many physiological processes. It plays a crucial role in the regulation of nervous system, immune and inflammatory responses, and blood flow. NO is synthesized by nitric oxide synthase (NOS) during two-step oxidation of l-arginine to l-citrulline. Intermediates and derivatives of NO metabolism, such as l-arginine, l-citrulline, asymmetrical dimethylarginine (ADMA), symmetrical dimethylarginine (SDMA), and dimethylamine (DMA), are investigated as potential biomarkers. In this article, we present a novel analytical method that allowed for simultaneous analysis of l-arginine, ADMA, SDMA, l-citrulline, and DMA, in a single-step extraction and derivatization using benzoyl chloride. In brief, aliquots of serum were mixed with internal standard solution mixture (50 µM D6-DMA, 20 µM D7-ADMA, and 100 µM D7-arginine) and 0.025 M borate buffer, pH 9.2 (10:1:5). The derivatization process was performed at 25 °C for 5 min using 10% benzoyl chloride. A reverse phase column was used for chromatographic separation. Quantitation was performed using following ions (m/z): 279.1457, 286.1749, 307.1717, 314.2076, 280.1297, 150.0919, and 156.1113 for l-arginine, D7-arginine, ADMA, SDMA, D7-ADMA, l-citrulline, DMA, and D6-DMA, respectively. The method was validated, and its assay linearity, accuracy and precision, recovery, and limits of detection (1.7 µM l-arginine, 0.03 µM ADMA, 0.02 µM SDMA, 0.36 µM l-citrulline, 0.06 µM DMA) and quantification (3.2 µM l-arginine, 0.08 µM ADMA, 0.05 µM SDMA, 1.08 µM l-citrulline, 0.19 µM DMA) were determined. The method is sensitive, reliable, repeatable, and reproducible. It can be applied in the routine clinical/diagnostic laboratory.Graphical abstract