Ethylenediaminetetraacetic Acid Plasma Samples Research Articles

A new biomarker panel for differential diagnosis of cholangiocarcinoma: Results from an exploratory analysis.

Diagnosis of cholangiocarcinoma (CCA) can be challenging due to unclear imaging criteria and difficulty obtaining adequate tissue biopsy. Although serum cancer antigen 19-9 and carcinoembryonic antigen have been proposed as potential diagnostic aids, their use remains limited by insufficient sensitivity and specificity. This exploratory analysis aimed to identify individual- and combinations of serum biomarkers to distinguish CCA from hepatocellular carcinoma (HCC) and chronic liver disease (CLD) controls using samples from a published study. This prospective, multicenter, case-control study included patients aged ≥18 years at high-risk of HCC. Serum and ethylene diamine tetraacetic acid-plasma samples were collected prior to any treatment and confirmed diagnosis of HCC or CCA. Fourteen biomarkers (measured by electrochemiluminescence immunoassays or enzyme-linked immunosorbent assays) were subjected to univariate analysis and 13 included in a multivariate analysis (per selected combinations and exhaustive search). Overall, 55 CCA, 306 HCC, and 733 CLD control samples were analyzed. For distinguishing CCA from HCC, alpha-fetoprotein and matrix metalloproteinase-2 (MMP-2) showed the best individual performance (area under the curve (AUC) 86.6% and 84.4%, respectively); tissue inhibitor of metalloproteinase-1 (TIMP-1) was most able to distinguish CCA from CLD (AUC 94.5%) and from HCC + CLD (AUC 88.6%). The combination of MMP-2 and TIMP-1 was the best-performing two-marker panel, with AUC >90% for all comparisons. MMP-2 and TIMP-1 are promising biomarkers that could support differential diagnosis of CCA. Incorporating these assays into the diagnostic algorithm could provide additional diagnostic information in a non-invasive, rapid manner, and could supplement existing diagnostic methods.

Biomarkers of Alzheimer's disease and neurodegeneration in dried blood spots-A new collection method for remote settings.

We aimed to evaluate the precision of Alzheimer's disease (AD) and neurodegenerationbiomarkermeasurements from venous dried plasma spots (DPSv enous) for the diagnosis and monitoring of neurodegenerative diseases in remote settings. In a discovery (n=154) and a validation cohort (n=115), glial fibrillary acidic protein (GFAP); neurofilament light (NfL); amyloid beta (Aβ) 40, Aβ42; and phosphorylated tau (p-tau181 and p-tau217) were measured in paired DPSvenous and ethylenediaminetetraacetic acid plasma samples with single-molecule array. In the validation cohort, a subset of participants (n=99) had cerebrospinal fluid (CSF) biomarkers. All DPSvenous and plasma analytes correlated significantly, except for Aβ42. In the validation cohort, DPSvenous GFAP, NfL, p-tau181, and p-tau217 differed between CSF Aβ-positive and -negative individuals and were associated with worsening cognition. Our data suggest that measuring blood biomarkers related to AD pathology and neurodegeneration from DPSvenous extends the utility of blood-based biomarkers to remote settings with simplified sampling conditions, storage, and logistics. A wide array of biomarkers related to Alzheimer's disease (AD) and neurodegenerationwere detectable in dried plasma spots (DPSvenous). DPSvenous biomarkers correlated with standard procedures and cognitive status. DPSvenous biomarkers had a good diagnostic accuracy discriminating amyloid status. Our findings show the potential interchangeability of DPSvenous and plasmasampling. DPSvenous may facilitate remote and temperature-independent sampling for AD biomarker measurement. Innovative tools for blood biomarker sampling may help recognizing the earliest changes of AD.

Evaluation of the performance of a novel HIV-1 viral load assay for HIV quantification in China.

Our objective was to assess the HIV-1 quantification performance of the Livzon HIV-1 viral load (VL) assay and the Roche Cobas HIV-1 assay to evaluate an HIV-1 VL testing reagent for application in China. We compared the Livzon and Roche Cobas HIV-1 VL assays using ethylenediaminetetraacetic acid plasma samples collected between May 2021 and November 2021 from patients with HIV-1 and healthy controls. We used Cohen's κ coefficient to measure agreement of qualitative values and Pearson's correlation coefficient (r) values and the coefficient of determination (R2 ) to determine the linear relationship between the two assays. We performed a Bland-Altman analysis to assess VL quantification agreement. In total, 11 plasma samples from patients with hepatitis B virus (HBV) or hepatitis C virus (HCV) and nine samples from healthy controls were undetectable on both assays. Overall agreement was seen in 419 of 500 specimens (91.40%), with a κ value of 0.59. Pearson's correlation coefficient between the two assays was 0.970. Using the Bland-Altman method, 95.14% (352/370) of paired VLs fell within the 95% confidence limits of agreement (-0.51 to 0.95 log10 copies/mL). Higher VLs had a better correlation and a smaller mean difference between the two assays. Pearson's correlation coefficient for the samples of subtype CRF01_AE, CRF07_BC, and CRF55_01B was 0.950, 0.935, and 0.952, respectively. The Livzon HIV-1 VL assay exhibits good precision and linearity and a high correlation with the Roche Cobas HIV-1 assay. The Livzon HIV-1 VL assay has salient advantages in terms of the lyophilized powder reagent, which gives the assay greater stability and sensitivity and can be readily used in low-resource areas.

Complement activation in severely ill patients with sepsis: no relationship with inflammation and disease severity

BackgroundSepsis is characterized by a dysregulated immune response to infection. The complement system plays an important role in the host defence to pathogens. However, exaggerated complement activation might contribute to a hyperinflammatory state. The interplay between complement activation and inflammation in relationship with adverse outcomes in sepsis patients is unclear.MethodsSecondary analysis of complement factors in a prospective study in 209 hospitalized sepsis patients, of whom the majority presented with shock. Concentrations of complement factors C3, C3a, C3c, C5, C5a, and soluble terminal complement complex were assessed in ethylenediaminetetraacetic acid plasma samples collected within 24 h after sepsis diagnosis using enzyme-linked immunosorbent assays.ResultsThe concentration of complement factors in plasma of severely ill sepsis patients indicated profound activation of the complement system (all P < 0.01 compared to healthy controls). Spearman rank correlation tests indicated consistent relationships between the different complement factors measured, but no significant correlations were observed between the complement factors and other inflammatory biomarkers such as leukocyte numbers, C-reactive protein and ferritin concentrations, or HLA-DR expression on monocytes. The concentration of complement factors was not associated with Sequential Organ Failure Assessment score, the incidence of septic shock, and mortality rates (all P > 0.05) in this cohort of patients with high disease severity.ConclusionsOnce an infection progresses to severe sepsis or septic shock, the complement pathway is already profoundly activated and is no longer related to a dysregulated inflammatory response, nor to clinical outcome. This implies that in this patient category with severe disease, the complement system is activated to such an extent that it no longer has predictive value for clinical outcome.

Determination of temporal reproducibility and variability of cancer biomarkers in serum and EDTA plasma samples using a proximity extension assay

BackgroundProximity extension assay (PEA) is a novel antibody-based proteomic technology. Sparse data have been published concerning the matrix effect of serum vs. ethylenediamine tetraacetic acid (EDTA) plasma and the reproducibility of results obtained using PEA technology.MethodsWe analyzed samples with the PEA-based 92-plex Olink® immuno-oncology (I-O) assay. To estimate the matrix effect, we analyzed paired serum and EDTA plasma samples from 12 patients with biliary tract cancer. To evaluate the reproducibility, we used data from 7 studies, where 6–8 serum samples from patients with pancreatic cancer were used as bridging samples on 3 versions of the panel over a 2.5-years period.ResultsFor the study of serum vs. plasma, 80 proteins were evaluable. The mean serum to EDTA plasma ratio ranged from 0.41–3.01. For 36 proteins, the serum and plasma values were not comparable due to high variability of the ratio, poor correlation, or possible concentration effect. For the bridging samples, the mean intra-study inter-assay coefficient of variation (CV) ranged from 11.3% to 26.1%. The mean inter-study CV was 42.0% before normalization and 26.2% after normalization. Inter-study results were well correlated (r ≥ 0.93), especially for studies using the same version of the panel (r ≥ 0.99).ConclusionFor 44 of 92 proteins included in the Olink® I-O panel, the variation between results obtained using serum and EDTA plasma was constant and results were well correlated. Furthermore, samples could be stored for several years and used on different versions of the same PEA panel without it effecting results.

A Microfluidics-Based Screening Tool to Assess the Impact of Blood Plasma Factors on Microvascular Integrity.

This study provides a method to assess the impact of circulating plasma factors on microvascular integrity by using a recently developed microvessel-on-a-chip platform featuring the human endothelium that is partly surrounded by the extracellular matrix. The system is high-throughput, which allows parallel analysis of organ-level microvessel pathophysiology, including vascular leakage. Ethylenediaminetetraacetic acid plasma samples are mixed with inhibitors for recalcification of the plasma samples to avoid activation of the coagulation- or complement system. Moreover, the assay is validated by spiking vascular endothelial growth factor, histamine, or tumor necrosis factor alpha to recalcified plasma and confirms their modulation of microvessel barrier function at physiologically relevant concentrations. Finally, this study shows that perfusing the microvessels with recalcified plasma samples of coronavirus disease-2019 patients, with a confirmed proinflammatory profile, results in markedly increased leakage of the microvessels. The assay provides opportunities for diagnostic screening of inflammatory or endothelial disrupting plasma factors associated with endothelial dysfunction.

Liquid chromatography-tandem mass spectrometry measurement of 26 steroid hormones in human serum and plasma samples.

The measurement of steroid hormones provided critical information in the clinical evaluation of endocrine disorders. In this study, we developed a high-throughput solid-phase extraction method for the analysis of 26 steroids in human serum and plasma samples by liquid chromatography-tandem mass spectrometry. Chromatographic conditions and sample preparation were optimized to achieve good separation and maximum sensitivity for these analytes. Under the optimum conditions, good linearities were achieved in the quantitative range for each steroid hormone with the correlation coefficients (r) larger than 0.99. The limits of quantitation of the method were in the range from 0.0005 to 0.7901ng/mL. The recoveries were in the range of 87.2-114.2% with intra- and interday precision lower than 9.94%. This method has already been applied to series of samples from clinical trials, and there was no significant difference between serum and ethylenediaminetetraacetic acid plasma samples.

Collection and Storage of Human Plasma for Measurement of Oxylipins

Oxylipins derived from omega-3 and -6 fatty acids are actively involved in inflammatory and immune processes and play important roles in human disease. However, as the interest in oxylipins increases, questions remain regarding which molecules are detectable in plasma, the best methods of collecting samples, and if molecules are stable during collection and storage. We thereby built upon existing studies by examining the stability of an expanded panel of 90 oxylipins, including specialized pro-resolving lipid mediators (SPMs), in human plasma (n = 5 subjects) during sample collection, processing, and storage at −80 °C. Oxylipins were quantified using liquid chromatography-tandem mass spectrometry (LC/MS/MS). Blood samples collected in ethylenediaminetetraacetic acid (EDTA) or heparin followed by up to 2 h at room temperature prior to processing showed no significant differences in oxylipin concentrations compared to immediately processed samples, including the SPMs lipoxin A4 and resolvin D1. The majority of molecules, including SPMs, remained stable following storage for up to 1 year. However, in support of previous findings, changes were seen in a small subset of oxylipins including 12-HETE, TXB2, 14-HDHA, and 18-HEPE. Overall, this study showed that accurate measurements of most oxylipins can be obtained from stored EDTA or heparin plasma samples using LC/MS/MS.

Plasma neutrophil gelatinase-associated lipocalin and Interleukin-18 as predictors of acute kidney injury in renal transplant recipients: A pilot study.

Urine neutrophil gelatinase-associated lipocalin (NGAL) and interleukin-18 (IL- 18) have shown promise for predicting renal graft recovery. However, urinary flow rate variations may cause variable biomarker dilution. Plasma NGAL and IL-18 may form a biomarker panel that may help predict delayed graft function and slow graft function (SGF) in renal transplant recipients within the first two postoperative days earlier than serum creatinine. There are only a few studies in the literature using plasma NGAL for predicting renal graft recovery. Hence, we planned this study. This observational single-center, prospective cohort study was conducted in renal transplant recipients above 18 years of age. In 22 consecutive renal transplant recipients, we collected ethylenediaminetetraacetic acid-plasma samples 1 h before surgery and subsequently at 6 h, 24 h, and 48 h after surgery for NGAL and IL-18 by sandwich enzyme-linked immuno-sorbent assay technique. Serum creatinine was measured as a part of routine transplant protocol. In renal transplant recipients, neither serum levels of NGAL and IL-18 nor their trends could reliably predict SGF. The only significant correlation existed between serum creatinine at day 2 and IL-18 at day 2 with P = 0.023. Serum NGAL did not correlate with serum creatinine in this setting of renal transplantation. Patients with immediate graft function had a greater percentage decrease of creatinine at day 1 and day 2 (P = 0.002 and 0.001) The percentage change in IL-18 at 24 h and 48 h after transplant from baseline could predict the occurrence of early graft loss (EGL) (P = 0.05, 0.04). The cutoffs were -4.12% at day 1 and +3.39% at day 2 with area under receiver operator characteristics of 0.82 and 0.83, respectively. The percentage change in IL-18 may be a useful marker of EGL in renal transplant recipients. Serum NGAL and creatinine were not able to predict EGL.

Plasma steroid profiles before and after ACTH stimulation test in healthy horses

This study describes steroid profiles in equine plasma before and after ACTH stimulation. In human medicine, other steroids have been shown to have a more pronounced reaction to an ACTH stimulation test than cortisol. This study aimed to determine if the same was true for the horse. A total of 11 clinically healthy horses were selected for this study. Ethylenediaminetetraacetic acid plasma samples were taken before and 60 min after stimulation with 1 μg/kg BW of synthetic ACTH administered intravenously. The samples were analyzed for cortisol, 11-deoxycortisol, 21-deoxycortisol, cortisone, corticosterone, 11-deoxycorticosterone, androstenedione, 17-OH-progesterone, progesterone, and testosterone with a liquid chromatography–tandem mass spectrometry (LC-MS/MS). Cortisol, 11-deoxycortisol, cortisone, corticosterone, and 11-deoxycorticosterone showed a significant increase after ACTH stimulation. In conclusion, the LC-MS/MS represents a viable method to measure glucocorticoids and related precursors or metabolites in equine plasma samples. In addition, we were able to show a more pronounced increase of 11-deoxycorticosterone, 11-deoxycortisol, and corticosterone compared with cortisol. These 3 metabolites could potentially serve as more sensitive biomarkers for stress in horses than cortisol.

Effects of Long-Term Storage at -80 °C on the Human Plasma Metabolome.

High-quality biological samples are required for the favorable outcome of research studies, and valid data sets are crucial for successful biomarker identification. Prolonged storage of biospecimens may have an artificial effect on compound levels. In order to investigate the potential effects of long-term storage on the metabolome, human ethylenediaminetetraacetic acid (EDTA) plasma samples stored for up to 16 years were analyzed by gas and liquid chromatography-tandem mass spectrometry-based metabolomics. Only 2% of 231 tested plasma metabolites were altered in the first seven years of storage. However, upon longer storage periods of up to 16 years and more time differences of few years significantly affected up to 26% of the investigated metabolites when analyzed within subject age groups. Ontology classes that were most affected included complex lipids, fatty acids, energy metabolism molecules, and amino acids. In conclusion, the human plasma metabolome is adequately stable to long-term storage at −80 °C for up to seven years but significant changes occur upon longer storage. However, other biospecimens may display different sensitivities to long-term storage. Therefore, in retrospective studies on EDTA plasma samples, analysis is best performed within the first seven years of storage.

Proteomic Profiles in Advanced Age-Related Macular Degeneration Using an Aptamer-Based Proteomic Technology.

PurposeTo explore top-ranked plasma proteins related to neovascular age-related macular degeneration (AMD) and geographic atrophy (GA), and explore pathways related to neovascular AMD and GA.MethodsWe conducted a pilot study of patients with neovascular AMD (n = 10), GA (n = 10), and age-matched cataract controls (n = 10) who were recruited into an AMD registry. We measured 4001 proteins in ethylenediaminetetraacetic acid plasma samples using an aptamer-based proteomic technology. Relative concentrations of each of 4001 proteins were log (base 2) transformed and compared between cases of neovascular AMD and GA versus controls using linear regression. Pathway analysis was conducted using pathways downloaded from Reactome.ResultsIn this pilot study, higher levels of vinculin and lower levels of CD177 were found in patients with neovascular AMD compared with controls. Neuregulin-4 was higher and soluble intercellular adhesion molecule-1 was lower in patients with GA compared with controls. For neovascular AMD, cargo trafficking to the periciliary membrane, fibroblast growth factor receptor 3b ligand binding and activation, and vascular endothelial growth factor–related pathways were in the top ranked pathways. The top-ranked pathways for GA included several related to ErbB4 signaling.ConclusionsWe found different proteins and different pathways associated with neovascular AMD and GA. Vinculin and some of the top-ranked pathways have been previously associated with AMD, whereas others have not been described.Translational RelevanceBiomarkers identified in plasma likely reflect systemic alterations in protein expression and may improve our understanding of the mechanisms leading to AMD.

Influence of sample collection tube method, anticoagulant-containing plasma versus serum, on influenza virus hemagglutination inhibition titer and microneutralization titer serological assays

BackgroundThe hemagglutination-inhibition (HAI) assay is a critical component for measurement of immunogenicity in influenza vaccine development. It is unknown if the results can be influenced by sample type and anticoagulants. The purpose of this study was to evaluate the influence of different sample collection methods, in particular different anticoagulants, and choice of plasma or serum, on influenza virus serological assays.MethodsBlood samples from thirty donors previously immunized against influenza viruses were collected using six different types of blood collection tubes, two of which collect serum and four of which contain various anticoagulants for collecting plasma. Serum: (1) serum separator tubes (SST); and (2) Plus Plastic serum “red-top serum” tubes. Plasma: (3) spray-coated K2 ethylenediaminetetraacetic acid (EDTA) tubes: (4) Sodium Heparin tubes; (5) Citrate tubes with 3.2% sodium citrate solution; and (6) Glass Blood Collection tubes with acid citrate dextrose. Samples were tested against three different influenza viruses (A/California/07/2009 (H1N1pdm09), A/Texas/50/2012 (H3N2), and B/Massachusetts/2/2012) for hemagglutination inhibition titer and virus neutralization titer via a microneutralization (MN) assay, and data compared to that obtained for standard serum sample collected in SST.ResultsHAI and MN titers against type A viruses were within two dilutions compared to SST collection method over 96% of the time irrespective of sample type or anticoagulant. However, HAI titers for type B virus were more variable across different collection methods. EDTA plasma samples were greater than two dilutions higher than SST serum samples 70% (21 of 30 samples) of the time. In contrast, MN titers were within two dilutions over 96% of the time, with the highest deviation noted in acid citrate dextrose plasma samples (3 of 30 samples tested, 10%).ConclusionsThese data provide useful guidelines for sample collection and serology testing when screening: (i) influenza vaccine immunogenicity antibody response; (ii) antibody responses to newly emerging viral strains; and (iii) clinical samples for anti-influenza antibody activity.

Comparisons of metabolite profile from paired serum and ethylenediaminetetraacetic acid-plasma samples using dry chemistry technology: An emergency department perspective.

BACKGROUND:No data is available evaluating the difference in serum versus plasma sample assay of commonly tested parameters in the emergency department, where the sample processing time can be significantly reduced if plasma is used for analysis instead of conventionally used serum. Hence, this study aimed to evaluate the differences in serum versus plasma sample estimation of commonly evaluated biochemical parameters using dry chemistry technology.MATERIALS AND METHODS:Paired blood samples were collected from a single venipuncture of 405 patients admitted to the emergency department. Dry chemistry autoanalyzer (Vitros-350, Ortho Clinical Diagnostics) was used to process all the samples.RESULTS:Data from 401 patients were analyzed. Percentage differences between serum versus plasma samples for all analytes ranged from 0.0% to 57.44% and were <±4% for a majority of parameters, except uric acid (−6.25%), albumin (+11.90%), chloride (–5.05%), phosphorus (−6.06%), creatine phosphokinase (CPK) total (−57.44%), amylase (−37.53%), lipase (−42.74%), lactate dehydrogenase (LDH) (−8.53%), and C-reactive protein (−7.44%). For albumin, CPK total, amylase, and lipase, the difference between serum and plasma samples was more than the accepted upper range recommended by College of American Pathologists.CONCLUSION:Glucose, urea, creatinine, bilirubin, total protein, serum glutamate-pyruvate transaminase, total cholesterol, high-density lipoprotein cholesterol, triglycerides, sodium, and CPK-mb can be reliably assayed from either serum or plasma samples in emergency/routine practice. CPK total, amylase, and lipase should always be assayed from serum and not plasma due to significant variations. Uric acid, chloride, phosphorous, and LDH only in emergency situations should be assayed from plasma. For routine assays, serum should be preferred.

Standardization of Sampling for Isolation of Exosome-Like Small-Extracellular Vesicles from Peripheral Blood from Reproductive-Aged Women

Exosome-like small-extracellular vesicles (sEVs) are extracellular vesicles that act in intercellular communication and are involved in several biologic and pathologic processes. While sEVs increase the stability of their cargo molecules, there is still a need for standardization of sampling and isolation of these microvesicles. We aimed to determine the best sampling method for isolation of sEVs from peripheral blood from reproductive-aged women. Material and Methods: We included samples of plasma from our biobank collected in 2014 by venipuncture in heparin tubes and stored at -80°C. We also included blood samples collected in heparin tubes and Ethylenediamine tetraacetic acid (EDTA) tubes and stored at -80°C for one to two weeks prior processing. All blood samples were collected from the same nine reproductive-aged female volunteers. sEVs were isolated from plasma by ultracentrifugation and filtration and indirectly quantified using Pierce BCA Protein Assay kit. Transmission electron microscopy (TEM) and Nano Tracking Analysis (NTA) were performed to confirm the isolation of sEVs. Results and Discussion: TEM and NTA confirmed the isolation of sEVs. Protein concentration of short-time stored heparin samples was not statistically different from long-time stored heparin samples (1847.2 ± 651.4 vs. 2363.2 ± 1025.1, p = 0.14). There was no difference between heparin and EDTA plasma samples recently collected (2363.2 ± 1025.1 vs. 2044.8 ± 653.2, p = 0.44). In conclusion, blood samples may be collected using heparin or EDTA for isolation of sEVs. Long-time stored plasma samples maintain sEVs integrity and may be used, especially in comparative studies.

Plasma Exosomes From HLA-Sensitized Kidney Transplant Recipients Contain mRNA Transcripts Which Predict Development of Antibody-Mediated Rejection.

Sensitization to HLA remains a significant immunologic barrier to successful transplantation. Identifying immune mechanisms responsible for antibody-mediated rejection (AMR) is an important goal. Here, we explored the possibility of predicting the risk for AMR by measuring mRNA transcripts of AMR-associated genes in plasma exosomes from kidney transplant patients. Total RNA was extracted from exosomes purified from 152 ethylenediaminetetraacetic acid-plasma samples of 64 patients (18 AMR, 8 cell-mediated rejection [CMR], 38 no rejection in desensitized [DES] and non-DES control groups) for reverse transcription into cDNA, preamplification and then real time quantitative polymerase chain reaction (qPCR) for 21 candidate genes. The mRNA transcript levels of each gene were calculated. Comparisons were made among 4 patient groups for each gene and also for a gene combination score based on selected genes. Among 21 candidate genes, we identified multiple genes (gp130, CCL4, TNFα, SH2D1B, CAV1, atypical chemokine receptor 1 [duffy blood group]) whose mRNA transcript levels in plasma exosomes significantly increased among AMR compared with CMR and/or control patients. A gene combination score calculated from 4 genes of gp130, SH2D1B, TNFα, and CCL4 was significantly higher in the AMR than the CMR (P < 0.0001) and no rejection control groups (P < 0.01 vs DES control, P < 0.05 vs non-DES control). Our results suggest that plasma exosomes may contain information indicating clinical conditions of kidney transplant patients. mRNA transcript profiles based on gp130, SH2D1B, TNFα, and CCL4 in plasma exosomes may be used to predict on-going and/or imminent AMR.

Oxidation of PTH: in vivo feature or effect of preanalytical conditions?

Posttranslational oxidation of parathyroid hormone (PTH) modifies its biological activity. Measurement of non-oxidized PTH (n-oxPTH) could be an improvement in assessing PTH status, as intact PTH may rather reflect oxidative stress. However, it is debated whether oxidation of PTH occurs in vivo, or whether it is mainly an in vitro artifact. The aim of this study was to investigate the influence of different preanalytical conditions on the oxidation of PTH within a wide range of plasma PTH concentrations and oxidation propensity. n-oxPTH was separated from its oxidized form using an affinity column capturing the oxidized PTH. n-oxPTH was measured in eluate using commercially available PTH assays. The study included ethylenediaminetetraacetic acid plasma samples from 17 patients undergoing hemodialysis and 32 healthy subjects. We determined effects of storage temperature, time until centrifugation and freeze-thaw cycles. PTH and n-oxPTH concentrations were measured in each sample using six different immunoassays. n-oxPTH concentrations remained unchanged up to 180 min until centrifugation, two freeze-thaw cycles or after storage at -20°C or -80°C up to 79 days. Various methods for n-oxPTH and PTH measurements yielded highly comparable results, apart from standardization differences between various PTH and n-oxPTH assays. n-oxPTH concentrations were stable under our study conditions, indicating negligible ex vivo oxidation of PTH. In addition, PTH immunoassays have a different sensitivity for n-oxPTH than for total PTH. For this reason, the n-oxPTH/total PTH ratio cannot be used in absence of a n-oxPTH standard. Clinical implications of determining n-oxPTH require additional study.

Determination of Aflatoxin B1-Lysine in Pig Serum and Plasma by Liquid Chromatography-Tandem Mass Spectrometry.

Aflatoxin B1 (AFB1) is a hepatocarcinogen produced by certain Aspergillus species growing on crops. After biotransformation in the liver, AFB1 generates several metabolites, one of which is AFB1 bound to lysine on serum albumin. AFB1-lysine (AFB1-lys) is a digest product of AFB1-albumin and is considered a biomarker of exposure to AFB1 in humans and animals. The objectives of this paper were to evaluate the performance characteristics of a new analytical method for determination of AFB1-lys levels in pig serum, heparinized and ethylenediaminetetraacetic acid (EDTA) plasma and to evaluate the interference of these anticoagulants in AFB1-lys quantification. Blank blood samples were obtained from eight crossbreed 91-day-old barrows fed AFB1-free diets. Pooled samples (n = 3) and individual samples of serum, EDTA and heparinized plasma collected from five pigs were enzymatically digested with pronase at 37°C for 4 h. AFB1-lys was isolated by solid-phase extraction and quantified by liquid chromatography coupled to tandem mass spectrometry. The analytical method was applied for determination of AFB1-lys in serum and EDTA plasma collected from five 49-day-old crossbreed barrows fed ad libitum diets containing 1.1 mg of AFB1 per kg of feed during 7 days (three animals) or 42 days (two animals). Samples of heparinized plasma were only available from animals intoxicated for 42 days. All animals had lower levels of AFB1-lys in EDTA plasma samples (24.78-37.40 ng/mL), when compared to serum (49.32-252.07 ng/mL-1) or heparinized plasma (176.81 and 264.24 ng/mL-1). EDTA did not interfere in AFB1-lys standard detection, but our findings suggest that EDTA should be avoided during blood collection since it affects the pronase activity in AFB1-albumin adduct digestion and, consequently, causes a reduction in the AFB1-lys levels. Hence, determination of AFB1-lys in serum and heparinized plasma is an approach to assess an individual's exposure of swine to AFB1.

Metabolomic Quality Assessment of EDTA Plasma and Serum Samples.

Handling and processing of blood can significantly alter the molecular composition and consistency of biobank samples and can have a major impact on the identification of biomarkers. It is thus crucial to identify tools to determine the quality of samples to be used in biomarker discovery studies. In this study, a non-targeted gas chromatography/time-of-flight mass spectrometry (GC-TOFMS) metabolomic strategy was used with the aim of identifying quality markers for serum and plasma biobank collections lacking proper documentation of preanalytical handling. The effect of postcentrifugation delay was examined in serum stored in tubes with gel separation plugs and ethylenediaminetetraacetic acid (EDTA) plasma in tubes with or without gel separation plugs. The change in metabolic pattern was negligible in all sample types processed within 3 hours after centrifugation regardless of whether the samples were kept at 4°C or 22°C. After 8 and 24 hours postcentrifugation delay before aliquoting, there was a pronounced increase in the number of affected metabolites, as well as in the magnitude of the observed changes. No protective effect on the metabolites was observed in gel-separated EDTA plasma samples. In a separate series of experiments, lactate and glucose levels were determined in plasma to estimate the effect of precentrifugation delay. This separate experiment indicates that the lactate to glucose ratio may serve as a marker to identify samples with delayed time to centrifugation. Although our data from the untargeted GC-TOFMS analysis did not identify any specific markers, we conclude that plasma and serum metabolic profiles remain quite stable when plasma and serum are centrifuged and separated from the blood cells within 3 hours.

Vascular endothelial growth factor pathway in endometriosis: genetic variants and plasma biomarkers

To study single nucleotide polymorphisms (SNPs) involved in angiogenesis (VEGF, PLGF, VEGFR1, VEGFR2, HIF-1α) and plasma levels of the corresponding proteins (VEGF, PLGF, sVEGFR1, sVEGFR2) in women with and without endometriosis. Allele frequencies of vascular endothelial growth factor (VEGF) pathway SNPs and plasma levels of the corresponding proteinswere investigated in patients with endometriosis and in controls. University hospital. Samples of DNA from 1,931 Caucasian patients were included (1,109 patients with endometriosis and 822 controls). An additional study group included 973 DNA samples from volunteers, self-reported to be healthy without laparoscopic evaluation. Women who underwent a laparoscopy for subfertility and/or pain and healthy volunteers without laparoscopic evaluation. Functional SNPs of the VEGF, VEGFR1, VEGFR2, HIF-1α genes and Hap Map tagging SNPs of thePLGF gene were genotyped by using iPLEX technology on a Sequenom MassArray and TaqMan SNP Genotyping Assay.The VEGF levels were determined in ethylenediaminetetraacetic acid plasma samples by using Bio-Plex Protein Array System. PLGF, sVEGFR1, and sVEGFR2 levels were measured in ethylenediaminetetraacetic acid plasma samples by using ELISA Quantikine kits. A significant association was found between the rs2268613 polymorphism in the PLGF gene and PLGF plasma levels. Inall study subjects, women with the AA variant of the rs2268613 PLGF gene had significantly lower PLGF plasma levels (median[interquartile range] 9.36 [8.19-10.43] pg/mL) than those with the AG variant (12.1 [11.81-20.84] pg/mL; P(a)=.0085, P(b)=.04), both before and after multiple testing. Plasma levels of VEGF were elevated in endometriosis patients (especially in minimal-mild endometriosis during the menstrual cycle phase) compared with laparoscopic controls but had a moderate diagnosticperformance (area under the curve, 0.73) in this discovery dataset. At a cut-off plasma level of VEGF >3.88 pg/mL, minimal-mild stages of endometriosis were diagnosed with a sensitivity of 74% and a specificity of 80% during the menstrual phase of cycle. The associations between the presence of endometriosis and SNPs in PLGF (rs2268614), HIF-1α (rs11549465), and VEGFR1 (rs9582036) genes lost statistical significance after multiple testing. Genetic variants in the PLGF rs2268613 gene may influence plasma levels of the corresponding protein. Plasma levels of VEGF were elevated in endometriosis patients compared with controls. The associations between the presence of endometriosis and SNPs in PLGF (rs2268614), HIF-1α (rs11549465), and VEGFR1 (rs9582036) genes lost statistical significance after multiple testing.