Detection Of Human Blood Research Articles

Carotenoids and apocarotenoids determination in intact human blood samples by online supercritical fluid extraction-supercritical fluid chromatography-tandem mass spectrometry

A direct on-line method based on the coupling of supercritical fluid extraction and supercritical fluid chromatography with triple quadrupole mass spectrometry detection (SFE-SFC-QqQ/MS) for selected carotenoids determination and apocarotenoids detection in intact human blood was developed for the first time. Carotenoids and apocarotenoids were identified by using the available standard together with full scan, selected ion monitoring (SIM), and multiple reaction monitoring (MRM) experiments. Moreover, β-Cryptoxanthin, Zeaxanthin, β-Carotene and Capsanthin were directly quantified by the developed methodology, using a multiple reaction monitoring (MRM) approach; the determined average content of β-carotene was 123.8 nmol L−1 (range 18.7–485.1 nmol L−1), of β-cryptoxanthin was 385.3 nmol L−1 (range 72.5–1920.3 nmol L−1), of zeaxanthin was 396.9 nmol L−1 (range < LoD – 1795.8 nmol L−1) and of capsanthin was 38.9 nmol L−1 (range < LoD – 188.4 nmol L−1). Analyses were carried out on 10 μL aliquots of intact blood samples without any preliminary treatment; the online extraction and chromatographic separation time was just over 20 min. The method was validated in terms of linearity, precision, limits of detection and quantification, and accuracy. Interestingly, β-apo-12′-carotenal, apo-10′-zeaxanthinal, apo-12′-zeaxanthinal, apo-14′-zeaxanthinal, ε-apo-8-luteinal, ε-apo-12-luteinal and ε-apo-14-luteinal were detected in human blood, together with two zeaxanthin fatty acid esters, for the first time.

Stand-Off Detection of Alcohol Vapors Exhaled by Humans.

Early detection of humans under the influence of alcohol in public places (workplace, public gathering) is particularly important for safety reasons. In this article, the theoretical analysis of stand-off detection of alcohol in the air exhaled by humans as well as experimental results of the developed experimental setup is presented. The concept of differential absorption of two laser beams at different wavelengths was used. The idea of using standard deviation of the relative difference of the amplitudes of two signals to detect the alcohol was applied for the first time. The idea was verified by the experiments and it was shown that a reliable device can be developed that can efficiently detect alcohol concentration in the exhaled air at the level of 0.3 mg/L (0.63‰). Moreover, the concept of such device examining humans entering a specific area was proposed. The results of this article may be useful to scientists or engineers working on alcohol detection in human blood.

Xenon detection in human blood: Analytical validation by accuracy profile and identification of critical storage parameters

Xenon is a rare, mostly inert, noble gas that has applications in a wide range of fields, including medicine. Xenon acts on the human body as a useful organ-protective and anesthetic agent and has also been previously studied for potential applications in fields such as optics, aerospace and medical imaging. Recently, it was discovered that xenon can boost erythropoietin production, and it has been used as a performance-enhancing agent in international sports competitions such as the Sochi Olympic Games. Therefore, screening methods to detect the misuse of xenon by analysis of biological samples and to monitor anesthesia kinetics and efficiency are being investigated. The aim of this study was to develop and validate an analytical method to detect xenon in blood samples using gas chromatography coupled to tandem mass spectrometry (GC-MS/MS).Preliminary studies were conducted to determine the best parameters for chromatography and mass spectrometry for xenon. The analysis was performed using the multiple reaction monitoring (MRM) mode using the transitions m/z 129 → 129, 131 → 131 for xenon and 84 → 84, 86 → 86 for krypton, which was chosen as the internal standard. The LOD of GC-MS/MS was found to be 52 pmol on-column. Calibration lines and controls were made to obtain an accuracy profile at a range of 2.08–104 nmol with a β-expectation tolerance interval set at 80% and the acceptability limit set at ±30%. From the accuracy profile, the LOQ of 15 nmol on-column for the range of 2.08–104 nmol was obtained. The method was validated according to the guidelines of the French Society of Pharmaceutical Sciences and Techniques.The detection method was finally validated using blood from test persons subjected to a 15% or 30% xenon mixture with pure oxygen and air for 45 min. Even though the probes were already used for other projects, it was still possible to detect xenon.

Design of a Fiber Optic Biosensor for Cholesterol Detection in Human Blood

A fiber optic sensor is to detect the cholesterol content in blood serum-based biosensor using plastic optical fiber that has been designed. The fiber optic sensor designed with sensing area was a fiber optic grated by 5 scratches then given bending treatment by 5 cm. The first test was by UV-Vis spectrometer with linearity of 0.96. The second test was by light spectrometer with linearity in white LED of 0.94. Optical fiber sensors were made to work well on a range of blood serum concentration of 140 mg/dL to 250 mg/dL.

Robust Ki67 detection in human blood by flow cytometry for clinical studies.

Ki67 is a prognostic and/or predictive biomarker in patients with malignancies. Flow cytometry is a powerful technology for single-cell multiparameter analysis. We developed and validated a multicolor quantitative flow cytometry assay for detection of intracellular Ki67 expression in various immune cell subsets from human blood. The assay was optimized and showed excellent precisions. Assessment of the sample stability indicated that percentage changes from the fresh sample for the reportable results of interest were within 20%, up to 72 h after blood collection in the Cyto-Chex® BCT tube. The validated assay is sufficiently robust to analyze clinical samples. Easy access to peripheral blood enables continuous monitoring of Ki67 expression in blood as a biomarker, for example, for immunotherapy studies.

MODIFIKASI PRINSIP PEMERIKSAAN β-D-GLUCAN UNTUK MENDETEKSI CANDIDA ALBICANS DALAM SERUM

Candida albicans β-D-glucans examination has been used for invasive fungal detection in human blood and was approved by the Food and Drug Administration. However, the method is rarely used in Indonesia and also because the cost is hardly affordable. A modification of the method using enzymatic reaction is hoped will provide a simple and affordable measurement in human blood serum. β-D-glucans as heterogeneous molecules constitute the major carbohydrates fractions of cell wall and readily detected in supernatants of Candida albicans cultures are hydrolyzed by β glucanase to form D-glucose. This additional glucose is measured using Megazyme GOPOD-Format Procedure® at 510 nm. Candida albicans were identified and cultured was derived from a patient of Dr. Moewardi General Hospital, Surakarta in July, 2012. The results show that β-D-glucans from Candida albicans is measureable to the amount of μg/100 μL serum using this modification principle

Recombinant single chain variable fragment antibodies (scFv) against Pro(144)-Leu(155) fragment of human protein C

The aim of this work was to obtain the recombinant single chain variable fragments of antibodies (scFv) against human protein C, the key component of blood anticoagulation system. For this purpose a peptide that mimics a Pro144-Leu155 sequence of protein C was synthesized and the murine immune scFv library against this peptide was constructed. The protein C specific scFv 9E were selected from the constructed library by the phage-display method. The scFv 9E dissociation constant was found to be 2∙10(-9) M. It was shown that scFv 9E were suitable for protein C detection by ELISA and Western blotting. Selected scFv could be further used for protein C investigation and for the development of quantitative methods for protein C detection in human blood.

Highly precise plasmonic and colorimetric sensor based on enzymatic etching of nanospheres for the detection of blood and urinary glucose

A highly precise glucose sensor was developed based on plasmon peak shift induced by the glucose oxidase mediated etching of Au–Ag nanoparticles. The platform enabled quantitative glucose detection in human blood and urine samples.

Analytical approaches for the detection of emerging therapeutics and non-approved drugs in human doping controls

The number and diversity of potentially performance-enhancing substances is continuously growing, fueled by new pharmaceutical developments but also by the inventiveness and, at the same time, unscrupulousness of black-market (designer) drug producers and providers. In terms of sports drug testing, this situation necessitates reactive as well as proactive research and expansion of the analytical armamentarium to ensure timely, adequate, and comprehensive doping controls. This review summarizes literature published over the past 5 years on new drug entities, discontinued therapeutics, and ‘tailored’ compounds classified as doping agents according to the regulations of the World Anti-Doping Agency, with particular attention to analytical strategies enabling their detection in human blood or urine. Among these compounds, low- and high-molecular mass substances of peptidic (e.g. modified insulin-like growth factor-1, TB-500, hematide/peginesatide, growth hormone releasing peptides, AOD-9604, etc.) and non-peptidic (selective androgen receptor modulators, hypoxia-inducible factor stabilizers, siRNA, S-107 and ARM036/aladorian, etc.) as well as inorganic (cobalt) nature are considered and discussed in terms of specific requirements originating from physicochemical properties, concentration levels, metabolism, and their amenability for chromatographic-mass spectrometric or alternative detection methods.

Acute Immunotoxic Effects of Perfluorononanoic Acid (PFNA) in C57BL/6 Mice.

otrganic perfluorochemicals (PFCs) have become an environmental concern due to widespread detection in human blood and experimental evidence for immune, developmental, and liver toxicity. Whereas the blood concentrations of many PFCs are declining, blood levels of Perfluorononanoic Acid (PFNA) are rising in the United States. The purpose of the present studies was to determine the effects of PFNA on lymphoid organs and immune cells of C57BL/6 mice. The present study demonstrates that PFNA produces immunotoxic effects in both male and female C57BL/6 mice as evidenced by splenic atrophy, decreased splenocyte numbers, and a marked reduction in thymocyte viability. The current study also demonstrates that the effects of PFNA on different leukocyte populations are not uniform. The CD4+CD8+ double-positive thymocytes were particularly sensitive to PFNA in which the proportion of this population was >95% decreased relative to the entire CD4+ thymocyte population in PFNA-treated mice. Interestingly, PFNA also markedly increased serum levels of TNFα in response to LPS in mice. Collectively, the present studies demonstrate that PFNA decreases lymphocyte viability and alters the immune response to LPS in C57BL/6 mice.

Development of Nanoprobe for the Determination of Blood Cholesterol

Measurements of serum cholesterol levels are important in the diagnosis and classification of hyper lipoproteinemias. Elevated cholesterol levels may occur with hypothyroidism, nephrotic syndrome, diabetes, and various liver diseases. The present investigation was carried out for the development of rapid, highly sensitive and economic cholesterol biosensor for the determination of blood cholesterol. The PVP encapsulated ZnS nanoparticals have been synthesized and characterized by Fourier Transform InfraRed (FTIR) spectroscopy, UV-visible spectroscopy, and Atomic force microscopy techniques in the present investigation. The size of the nanoparticles is found to be ranging between 21-22 nm. The surface morphology of the sensing area of functionalized MWCNT was examined using Scanning Electron Microscopy (SEM). The length of the MWCNTs is found to be approximately 2 microns. In the IR spectrum of functionalized MWCNTs, the presence of bands at 1636 cm-1 and 3434 cm-1 confirmed the presence of carbonyl and hydroxyl moieties of carboxylic acid group. Cholesterol Oxidase was immobilized on to the ZnS nanoparticles and Multiwall Carbon Nanotubes (MWCNTs) which they were placed on a glassy carbon electrode surface using Nafion by LBL assembly technique. Cyclic voltammetric study reveals that the fast electron transfer between electrodes was achieved through the incorporation of ChOx into the Nafion-ZnS-MWCNTs film. ZnS and MWCNTs played an important role in facilitating the electron transfer between the ChOx and the electrode surface. The cholesterol bioelectrode shows detection range of 10-450 mg/dl and good linearity is obtained in 50-450 mg/dl (1.3 -11.6 mM) range with linear regression coefficient 'R2' as 0.986. The Michaelis-Menten constant Km value obtained as 0.84 mM using Lineweaver-Burke plot by plotting 1/V vs 1/Conc. The low Km value 0.84 mM indicates high affinity of immobilized ChOx to cholesterol. It exhibits optimum pH 7.0 and optimum temperature 35°C. Therefore, the bioelectrode fabricated in this study is promising for cholesterol detection in human blood.

In Vivo Expression of Salmonella enterica Serotype Typhi Genes in the Blood of Patients with Typhoid Fever in Bangladesh

Background Salmonella enterica serotype Typhi is the cause of typhoid fever. It is a human-restricted pathogen, and few data exist on S. Typhi gene expression in humans.Methodology/Principal FindingsWe applied an RNA capture and amplification technique, Selective Capture of Transcribed Sequences (SCOTS), and microarray hybridization to identify S. Typhi transcripts expressed in the blood of five humans infected with S. Typhi in Bangladesh. In total, we detected the expression of mRNAs for 2,046 S. Typhi genes (44% of the S. Typhi genome) in human blood; expression of 912 genes was detected in all 5 patients, and expression of 1,100 genes was detected in 4 or more patients. Identified transcripts were associated with the virulence-associated PhoP regulon, Salmonella pathogenicity islands, the use of alternative carbon and energy sources, synthesis and transport of iron, thiamine, and biotin, and resistance to antimicrobial peptides and oxidative stress. The most highly represented group were genes currently annotated as encoding proteins designated as hypothetical, unknown, or unclassified. Of the 2,046 detected transcripts, 1,320 (29% of the S. Typhi genome) had significantly different levels of detection in human blood compared to in vitro cultures; detection of 141 transcripts was significantly different in all 5 patients, and detection of 331 transcripts varied in at least 4 patients. These mRNAs encode proteins of unknown function, those involved in energy metabolism, transport and binding, cell envelope, cellular processes, and pathogenesis. We confirmed increased expression of a subset of identified mRNAs by quantitative-PCR.Conclusions/SignificanceWe report the first characterization of bacterial transcriptional profiles in the blood of patients with typhoid fever. S. Typhi is an important global pathogen whose restricted host range has greatly inhibited laboratory studies. Our results suggest that S. Typhi uses a largely uncharacterized genetic repertoire to survive within cells and utilize alternate energy sources during infection.

Expressed Alu repeats as a novel, reliable tool for normalization of real-time quantitative RT-PCR data

Expressed Alu repeats are a reliable, accurate and universal reference for use in RT-qPCR normalization of human genes

Solubilization and purification of Escherichia coli expressed GST-fusion human vascular endothelial growth factors with N-Lauroylsarcosine

Vascular endothelial growth factor (VEGF) is a potent mitogen for tumor angiogenesis. Clinically, VEGF detection in human blood can be expected to be used in the very near future for cancer screening, prognosis, monitoring of therapy and diagnosis. VEGF has been identified as the target for the treatment of cancer. Though prokaryotic expression of VEGF has been done, the solubilization and purification is time consuming and empirical. In this study, VEGF165 and VEGF121 were cloned into pGEX-4T-1 vector, and GST-VEGF fusion proteins were expressed in Escherichia coli at 37°C. The inclusion bodies of GST-VEGF fusion proteins were solubilized with N-Lauroylsarcosine (sarkosyl). Briefly, the cell suspension with inclusion body was added with sarkosyl at a final concentration of 1.5%. After the disruption of cells, the clarified supernatant containing sarkosyl was added with Triton X-100 at a final concentration of 3%. The GST-VEGFs were purified by affinity chromatography on glutathione Sepharose 4B. The overall yield was approximately 10 – 12 mg/l cell culture. The binding assay showed that the GST-VEGF165 binds to VEGF receptor in a dose dependent manner. The current work provides a novel procedure for solubilization and purification of GST-VEGF fusion proteins, and no laborious procedures for separation of inclusion bodies and renaturation were needed.

Solubilization and purification of Escherichia coli expressed GST-fusion human vascular endothelial growth factors with N-Lauroylsarcosine

Vascular endothelial growth factor (VEGF) is a potent mitogen for tumor angiogenesis. Clinically, VEGF detection in human blood can be expected to be used in the very near future for cancer screening, prognosis, monitoring of therapy and diagnosis. VEGF has been identified as the target for the treatment of cancer. Though prokaryotic expression of VEGF has been done, the solubilization and purification is time consuming and empirical. In this study, VEGF165 and VEGF121 were cloned into pGEX-4T-1 vector, and GST-VEGF fusion proteins were expressed in Escherichia coli at 37°C. The inclusion bodies of GST-VEGF fusion proteins were solubilized with N-Lauroylsarcosine (sarkosyl). Briefly, the cell suspension with inclusion body was added with sarkosyl at a final concentration of 1.5%. After the disruption of cells, the clarified supernatant containing sarkosyl was added with Triton X-100 at a final concentration of 3%. The GST-VEGFs were purified by affinity chromatography on glutathione Sepharose 4B. The overall yield was approximately 10 – 12 mg/l cell culture. The binding assay showed that the GST-VEGF165 binds to VEGF receptor in a dose dependent manner. The current work provides a novel procedure for solubilization and purification of GST-VEGF fusion proteins, and no laborious procedures for separation of inclusion bodies and renaturation were needed.

Disease detection in human blood using microwaves

AbstractThis letter reports a novel in vitro test of blood analysis based on the measurement of the dielectric properties at microwave frequencies. The measurements were made using rectangular cavity perturbation technique at the S‐band of microwave frequency with the different samples of blood obtained from healthy donors as well as from patients. It is observed that an appreciably change in the dielectric properties of patient samples with the normal healthy samples and this measurements were in good agreement with clinical analysis. This measurement technique is nondestructive in nature. These results indicate an alternative in vitro method of diagnosing blood abnormalities using microwaves.© 2007 Wiley Periodicals, Inc. Microwave Opt Technol Lett 49: 1589–1592, 2007; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/mop.22512

Use of a nested polymerase chain reaction (N-PCR) to detect Trypanosoma cruzi in blood samples from chronic chagasic patients and patients with doubtful serologies

Chagas disease, caused by Trypanosoma cruzi, is an important endemic illness in Latin America. Serologic tests for T. cruzi detection in blood are sensitive, but their specificity is unsatisfactory. Direct detection of parasites in blood, either by xenodiagnosis or hemoculture, is highly specific but of low sensitivity. Molecular assays such as the Polymerase chain reaction (PCR), which amplifies certain repetitive sequences of nuclear DNA has been used as a good alternative tool for T. cruzi detection in human blood. The present study aimed to test PCR diagnosis in chagasic chronic patients and doubtful serologic patients attended in GEDOCH (Chagas Disease Study Group/UNICAMP, Brazil). A 149 bp fragment originated from nuclear DNA was specifically detected in chronic chagasic patients. The results of these tests were compared with serologic diagnosis performed using standard techniques and xenodiagnosis. We found that 43 out of 50 patients previously serodiagnosed as chagasic were positive using the N-PCR method. Thirteen of 30 patients with doubtful serologic results were confirmed as positive by N-PCR. Our results suggest that the N-PCR may be a complementary tool to serology in the diagnosis of Chagas disease, and that it is usefull for parasite detection in patients with chronic disease and patients with doubtful serologic results.

In vitro adduct formation of phosgene with albumin and hemoglobin in human blood.

The development of procedures for retrospective detection and quantitation of exposure to phosgene, based on adducts to hemoglobin and albumin, is described. Upon incubation of human blood with [(14)C]phosgene (0-750 microM), a significant part of radioactivity (0-13%) became associated with globin and albumin. Upon Pronase digestion of globin, one of the adducts was identified as the pentapeptide O=C-(V-L)-S-P-A, representing amino acid residues 1-5 of alpha-globin, with a hydantoin function between N-terminal valine and leucine. Micro-LC/tandem MS analyses of tryptic as well as V8 protease digests identified one of the adducts to albumin as a urea resulting from intramolecular bridging of lysine residues 195 and 199. The adducted tryptic fragment could be sensitively analyzed by means of micro-LC/tandem MS with multiple-reaction monitoring (MRM), enabling the detection in human blood of an in vitro exposure level of >/=1 microM phosgene.

A comparative study of different enzyme immunosorbent assays for human tumor necrosis factor-α

Enzyme immunosorbent assays (ELISA) for human tumor necrosis factor-α (TNF-α) are available from many companies. Although this cytokine is meanwhile well established in scientific work, its detection in human blood still leads often to conflicting results. We studied the analytical performance of three different commercial ELISA (Amersham, Immunotech and Medgenix Diagnostics) in plasma samples of 30 human immunodeficiency virus (HIV) infected individuals. All kits showed significantly different mean concentrations of TNF-α in the study population ( p < 0.05). Kit A: 30.9 pg/ml ± 46.6 vs. kit B: 46.9 pg/ml ± 24.8 vs. kit C: 11.8 pg/ml ± 11.6. Results of assays A and B ( r = 0.34) as well as A and C ( r = 0.31) were not significantly correlated. A good statistical relation between values was only found for assay B and assay C ( r = 0.80, p < 0.001). Three plasma samples were spiked with TNF-α standard of the corresponding kit to a final concentration of 200 pg/ml. The detection results of these samples were compared with the variation conceded by the manufacturer. Assay A detected two samples above the corresponding coefficient of intra-assay variation (recovery: 81.8% and 89.2%). All three values obtained with assay B were markedly out of its variation range (recovery: 88.7%, 84.6% and 89.0%) while assay C showed again two results above the intra-assay variation (recovery: 92.1% and 89.6%). In our observations we found relevant differences between commercial ELISA kits of different manufacturers, which made interpretation of TNF-α in human plasma samples difficult. Additionally, it should be taken into consideration that plasma specimen may contain cytokine-binding proteins or autoantibodies which are capable of blocking epitopes of TNF-α. This may lead to the loss of the necessary immunoreactivity which prevents detection antibodies from recognizing these molecules.

Detection of human blood by cloth-based enzyme immunoassay of immunoglobulin G

A specific and sensitive assay for the detection of human blood was developed using polyester cloth coated with goat anti-human IgG antibody to capture human IgG, an abundant and stable protein in blood. The captured IgG was detected by the reaction between goat anti-human IgG antibody-peroxidase conjugate and a chromogenic peroxidase substrate. Because the assay is simple and rapid, and permits simultaneous analysis of multiple samples, it has the potential to be used as a forensic test for human blood.