Effect Of Sample Dilution Research Articles

Effects of Sample Dilution on Nuclear Magnetic Resonance-Derived Metabolic Profiles of Human Urine.

Traditionally, a relatively big urine volume (e.g., 500 μL) is used in nuclear magnetic resonance (NMR)-based human metabolomics, which is not feasible for studies with limited/precious samples. Although urine may be diluted before conventional high-throughput metabolomics analysis, the comprehensive effect of urine dilution on metabolic profiles is unknown. Here, for the first time, we systematically investigated the effect of urine dilution on 1H NMR metabolic profiles, by evaluating signal detectability, integration, signal-to-noise ratio (SNR), chemical shift (δ) and its variation, and signal overlapping of 47 metabolites in 10 volunteers. We observed significant linear changes along with increased dilution, including decreased integration and SNR, altered δ, decreased intersample variation of δ, and increased separation between overlapped signals, e.g., lactate and threonine, β-d-glucose and an unassigned signal, and histidine and 3-methylhistidine. We further tested the 40% dilution level (i.e., employing 300 μL urine) in an epidemiological study containing 1018 pregnant women from the Tongji-Shuangliu Birth Cohort, showing acceptable detectability and chemical shift variability for most of the 47 metabolites profiled. It indicated that mild (e.g., 40%) dilution of human urine can largely preserve the high-abundance metabolites profiled, reduce intersample chemical shift variations, and increase separations of overlapped signals, which is an improvement of routine sample preparation methods in NMR-based metabolomics and is applicable for studies with limited urine volumes, including large-scale epidemiological studies.

Voltammetric Determination of Lead and Copper in Wine by Modified Glassy Carbon Electrode

This paper describes the determination of Pb and Cu with a Nafion-modified glassy carbon electrode and MnCo2O4 nanoparticles as working electrode for anodic stripping voltammetry. Pb and Cu were accumulated in HCl/KCl (0.1 mol dm-3) at a potential of -1.4 V (vs. Ag/AgCl electrode) for 480 s, followed by a linear sweep anodic stripping voltammetry (ASV) scan from -1.0 to +0.5 V. Under optimum conditions, the calibration curves were linear in the range of 0.01 - 8 and 0.01 - 5 mg dm-3 for Pb and Cu, respectively. Effect of sample dilution, accumulation time and potential were optimized. A study of interfering substances was performed. A significant increase in current was obtained at the modified electrode in comparison with the bare glassy carbon electrode. The modified electrode was successfully applied for determination of Pb and Cu in wine samples after a simple preparation procedure. Pb and Cu content in wine was used for estimation of the target hazard quotient (THQ) values for minimal and maximal levels of the metals.

Effect of Different Heparin Volumes on Blood Gas Analysis

AbstractBackground and Aim: Blood gas analysis is an important laboratory test for diagnosis and treatment of a variety of medical conditions in emergency rooms. Inaccurate sample collection is one of the reasons for errors in blood gas analysis. This study was conducted to evaluate different volumes of heparin in syringes and the effect of sample dilution on blood gas analysis as one of the main factors affecting the results of blood gas analysis.Methods: One hundred children (4 months to 12 years) who presented to Loghman Hakim Hospital, Tehran, Iran, were enrolled in this study. Two samples were taken from each patient. For the first sample, the syringes were filled with heparin sodium and then emptied completely to achieve a very thin layer anticoagulant coating. For the second sample, the syringes were filled with 0.1 mL of liquid heparin sodium 5000 U/mL (5%). The blood gas parameters including pH, PO2, PCO2, HCO3 and BE (base excess) were measured. Data were analyzed using SPSS version 18.Results: All parameter had lower levels in second samples (0.1 mL heparin 5%) except for PO2 compared to the first samples (P < 0.001).Conclusion: This study found that a small amount of heparin in the syringe changed the result of blood gas analysis. Keywords: Blood Gas Analysis; Heparin; pH; PCO2; HCO3.

An LC-MS/MS approach to assess total and free protein target in the serum of cynomolgus monkey.

Develop LC-MS/MS-based assays to measure total and free complement C5 in cynomolgus monkey serum as a target engagement biomarker for pharmacokinetic/pharmacodynamic correlation study. Materials & methods/results: The C5-specific signature peptide derived from pellet digestion of serum proteins with and without prior immunodepletion of the drug-bound C5 by protein A beads was quantified to assess free and total C5 levels, respectively. Conditions for immunodepletion by protein A were optimized to ensure complete depletion of IgGs (and drug-bound C5). The effect of sample dilution on drug-target dissociation and thus free C5 measurement was evaluated by applying a mathematical simulation. The procedure described here allows for the assessment of protein target engagement, aiding in pharmacokinetic/pharmacodynamic correlation analysis and human dose projection.

A Gyrolab Assay for the Quantitation of Free Complement Protein C5a in Human Plasma.

Complement protein C5a is recognized as an important component of the alternative complement pathway. Its role is prominent enough to garner interest not only as a biomarker, but also as a potential therapeutic target. Bioanalytical challenges have been posed in proper quantitation of free C5a due to interference from its precursor, C5. Additionally, free therapeutic target quantitation can be difficult due to effects of sample dilution and prolonged sample incubation when therapeutic is used as capture reagent. Gyrolab technology enables quantitation of free target analyte with minimal sample dilution and rapid sample incubations, thus enabling in vitro results that are more representative of in vivo pharmacodynamics. When coupled with strategic sample pretreatment, Gyrolab offers an opportunity to quantitate free C5a in human plasma with an assay that vastly diminishes C5 interference. A Gyrolab assay for the quantitation of free C5a in human plasma was developed and validated. Validation results confirmed that proper sample pretreatment and use of the Gyrolab platform yield accurate and reliable results. Due to the advantages that it provides, Gyrolab has become our default technology of choice for quantitation of free target.

Study of the peak splitting in an alternative method for the radiochemical purity measurement of 99mTechnetium-sestamibi

One of the alternative methods for the measurement of radiochemical purity (RCP) of Technetium-sestamibi uses Whatman 3MM paper as the stationary phase and ethyl acetate as the mobile phase. However, alternative method shows peak splitting and shouldering of the radiopharmaceutical. This could be because of higher levels of analyte. We aimed to assess the effect of sample dilution (1 : 1) on this method and to compare these RCP values with the reference method. The diluted samples showed a unique peak in the radiopharmaceutical zone, but the RCP values were significantly different (P<0.05) and correlated poorly with the reference method.

Impact of DNA extraction, sample dilution, and reagent contamination on 16S rRNA gene sequencing of human feces.

Culture-independent methods have granted the possibility to study microbial diversity in great detail, but technical issues pose a threat to the accuracy of new findings. Biases introduced during DNA extraction can result in erroneous representations of the microbial community, particularly in samples with low microbial biomass. We evaluated the DNA extraction method, initial sample biomass, and reagent contamination on the assessment of the human gut microbiota. Fecal samples of 200mg were subjected to 1:10 serial dilutions; total DNA was obtained using two commercial kits and the microbiota assessed by 16S ribosomal RNA (rRNA) gene sequencing. In addition, we sequenced multiple technical controls. The two kits were efficient in extracting DNA from samples with as low as 2mg of feces. However, in instances of lower biomass, only one kit performed well. The number of reads from negative controls was negligible. Both DNA extraction kits allowed inferring microbial consortia with similar membership but different abundances. Furthermore, we found differences in the taxonomic profile of the microbial community. Unexpectedly, the effect of sample dilution was moderate and did not introduce severe bias into the microbial inference. Indeed, the microbiota inferred from fecal samples was distinguishable from that of negative controls. In most cases, samples as low as 2mg did not result in a dissimilar representation of the microbial community compared with the undiluted sample. Our results indicate that the gut microbiota inference is not much affected by contamination with laboratory reagents but largely impacted by the protocol to extract DNA.

Alterations due to dilution and anticoagulant effects in hematologic analysis of rodent blood samples on the Sysmex XT-2000iV.

Clinical pathology of rodents is hindered by sample volume limitations. A single whole heparinized blood sample is often submitted for hematologic and clinical chemistry analysis in exploratory research settings, and sample dilution may be required. Published information on the potential impact of sample dilution and heparin use on hematology variables in rodents is sparse. The purpose of the study was to evaluate the effects of sample dilution and of anticoagulant on hematologic analysis of mouse and rat blood samples on the Sysmex XT-2000iV. Mice and rats were obtained from various ends of study research projects, and whole blood was collected via terminal cardiocentesis in lithium heparin, and additionally in EDTA when paired samples were obtained from rats. Hematology analytes were measured on the Sysmex XT-2000iV straight and diluted from ×2 to ×5. Significant differences between heparinized samples analyzed straight vs diluted were found for MCV and MCHC, with a bias for several additional variables. Significant differences between paired heparinized and EDTA-anticoagulated samples at each dilution point were found for most variables, with the largest differences found in platelet count. Evidence of platelet clumping presumably due to heparin exposure was noted in numerous samples. Dilution-induced changes occur in some hematologic variables and may render dilution unacceptable in the exploratory research environment. Many variables, most notably platelet count, differ based on the anticoagulant used, and values from heparinized vs EDTA-anticoagulated samples should not be directly compared.

Effect of sample dilution on matrix effects in pesticide analysis of several matrices by liquid chromatography-high-resolution mass spectrometry.

This study used two LC columns of different adsorbents and liquid chromatography-electrospray ionization-high-resolution mass spectrometry to study the relationship between matrix effects (ME), the LC separations, and elution patterns of pesticides and those of matrix components. Using calibration standards of 381 pesticides at three dilution levels of 1×, 1/10×, and 1/100×, 108 samples were prepared in solvent and five different sample matrices for the study. Results obtained from principal component analysis and slope ratios of calibration curves provided measurements of the ME and showed the 1/100× sample dilution could minimize suppression ME for most pesticides analyzed. Should a pesticide coeluting with matrix components have a peak intensity of 25 times or higher, the suppression for that pesticide would persist even at 1/100× dilution. The number of pesticides had enhancement ME increased with increasing dilution from 1× to 1/100×, with those early eluting, hydrophilic pesticides affected the most.

Evaluation of the Effects of Sample Dilution and Volume in Hydrophilic Interaction Liquid Chromatography

The effects of sample dilution and volume on the peak shapes in hydrophilic interaction liquid chromatography (HILIC) were evaluated using fluorescence-labeled thiols as model compounds and ZIC-HILIC as the HILIC column. The content of acetonitrile, which was selected as the diluent of the aqueous samples in this study, was varied 0−95 % in the injection samples, and the numbers of theoretical plates (NTPs) and retention times were compared using a mobile phase composed of ammonium formate buffer/acetonitrile (25:75, v/v). Although the NTPs and retention times decreased with decreasing acetonitrile content, the peak shapes were acceptable for samples with acetonitrile contents down to 50 % based on asymmetry factor. Furthermore, the sample volume had a serious effect for samples with low acetonitrile contents. Although a high content of acetonitrile in samples is still recommended, the capacity for the aqueous solution in the injection samples under HILIC conditions should vary with the composition of the mobile phase and may be larger than previously thought. These findings should be helpful in deciding the sample composition under HILIC conditions, particularly in bioanalysis, where aqueous solution is often contained in the injection samples and the sample dilution with an organic solvent may decrease the detection sensitivity.

Sample Dilution Influences the Determination of Antioxidant Capacity in Food: How to Minimize It?

The influence of sample dilution on the measurement of antioxidant capacity was analyzed. To ensure the reproducibility of results, it is necessary to realize such scarce investigations. This study focuses on different antioxidant capacity assays commonly used for the analysis of pure substances and food extracts. For all compounds and foods tested in most of the four assays (Trolox equivalent antioxidant capacity, 2,2-diphenyl-1-picrylhydrazyl, and oxygen radical absorbance capacity), effects of sample dilution on the measured (and recalculated) antioxidant capacity were observed, with differences up to 28 % between dilutions. An extrapolation method was proposed to obtain a “real value” thus to minimize the effects of the sample dilution. This extrapolation method is relatively simple, based on a linear regression of 4 or 5 appropriate dilutions of the sample and applicable to the various assays. The use of such a method will improve the consistency of interlaboratory antioxidant capacity data and thus permit better comparisons. In contrast, there was no dilution problem with ferric reducing antioxidant power assays.

Effect of sample dilution on estimated values of antioxidant capacity by photochemiluminiscence method

This paper describes principles of a proper dilution of samples used for the determination of antioxidant capacity by means of a photochemiluminiscence method using the instrument Photochem (Analytik Jena AG). The authors used the method ACL (Lipid-soluble Antioxidant Capacity), which is one of two methods enabling to measure in this instrument. It was demonstrated that values measured by the photochemiluminiscence method ACL were influenced by the degree of sample dilution. When studying effects of dilution of samples of wine and rutin, it was demonstrated that there is a non-linear correlation between the degree of dilution and measured values of antioxidant capacity. At low molar amount of substance (i.e. at 0.5 and 1.0 nmol), the measured value of rutin inhibition was higher than the same that molar amount of trolox. At higher molar amounts (i.e. 3; 4 and 5 nmol), the inhibition value of trolox was higher. This dependent change can be explained by means of a different effectiveness of antioxidants and a different stability of their products with radicals.

Linear and Nonlinear Regimes of Electrospray Signal Response in Analysis of Urine by Electrospray Ionization-High Field Asymmetric Waveform Ion Mobility Spectrometry-MS and Implications for Nontarget Quantification

Quantitative nontarget analysis is intended to provide a measurement of concentration of newly identified components in complex biological or environmental samples for which authentic or labeled standard do not exist. Electrospray ionization-high field asymmetric waveform ion mobility spectrometry-mass spectrometry (ESI-FAIMS-MS) has unique advantages that allowed us to develop a novel approach for quantification of nontarget analytes. In the nontarget analysis of urinary metabolites by ESI-FAIMS-MS, we find it practical and beneficial to analyze highly diluted urine samples. We show that urine extracts can be analyzed directly at very high dilutions (up to 20,000 times) by extending MS analysis times during slow FAIMS scanning. We explore the effects of sample dilution on ionization efficiency and ionization suppression in direct electrospray of complex sample matrixes. We consistently observe two distinct regimes in ESI operation related to the limited ionization capacity of this method. In the linear dynamic concentration range below the limiting ionization capacity, the analytical sensitivity of an analyte is constant and does not depend on matrix composition and concentration. Once the capacity of ESI is exceeded, all species exhibit log-log linearity in signal response. We show how quantification can be carried out using two different approaches, one for analytes which can be detected in the linear regime and another for those only detected in the suppression regime that overcomes the effects of ionization suppression. Our new insight into ionization suppression effects in ESI is of broad interest to anyone using ESI as an ionization technique for the MS analysis of complex samples.

Measurement and validation of the nature of salivary adiponectin

Adiponectin (Ad) is an adipocyte-derived hormone that plays an essential role in regulating insulin sensitivity, inflammation, and atherogenesis. Levels of some hormones in saliva change in a fashion similar to that in plasma in response to a disease or physiological condition. Since saliva is an easy to obtain biological fluid, measurements of salivary hormonal changes are preferred in diagnoses and treatments. Therefore, it was of interest to examine the nature of salivary Ad. While there have been two publications in the literature reporting presence of Ad in human saliva, the nature of salivary Ad has not been characterized. To this end, we investigated the effect of sample dilution on the measurement of Ad in saliva. To our surprise, we observed an increase in measurable level of Ad in saliva on sample dilution. One explanation for this paradoxical observation may be the presence of inhibitor(s) of Ad/anti-Ad binding in saliva that following dilution relieves the inhibitory effect. Working with this hypothesis, we were able to demonstrate the presence of an inhibitor in saliva that co-eluted with the dimeric form of Ad and was capable of inhibiting Ad assay. The presence of such inhibitor(s) may lead to underestimation of Ad in saliva.

Effect of sample dilution on serum free light chain concentration by immunonephelometric assay

BackgroundFree light chains (FLC) are useful biomarkers in the assessment of plasma cell disorders. Concerns have been raised about some technical aspects of the assay. This report examined the occurrence of dilution anomalies and/or antigen excess. MethodsFLC were determined on a BNII instrument at at-least two dilutions (100- and 2000-fold) in 2088 consecutive samples from 865 different patients. In part of them, the 400-fold dilution was also available. ResultsHigher than 2-fold differences and inconsistencies [“<” or “>” result at one dilution not consistent with the result obtained at another dilution] between the 100- and 2000-fold dilutions were found in 12.7% of patients for κ FLC and in 3.1% of patients for λ FLC. More than 4-fold differences between results obtained at the 2000-fold and the 100-fold dilutions were observed in 5.4% of patients for κ FLC and in 1.2% of patients for λ FLC. ConclusionsThe FLC assay on BNII suffers from sample dilution anomalies and/or failure of antigen excess detection in a substantial fraction of patients. Laboratory professionals and clinicians should be alert to such analytical difficulties.

Airborne Endotoxin from Indoor and Outdoor Environments: Effect of Sample Dilution on the Kinetic Limulus Amebocyte Lysate (LAL) Assay

Airborne endotoxins in occupational environments are a potential respiratory hazard to individuals. In this study, airborne endotoxins were collected using open-face and button aerosol samplers from inside animal housing units and downwind from agricultural production sites and a wastewater treatment plant. Filter extracts were then diluted to examine the effect of interfering substances on the kinetic Limulus amebocyte lysate (LAL) assay. In most cases, the overall endotoxin concentration was shown to decrease with increasing dilution up to 1000-fold, suggesting the presence of enhancing substances in the filter extracts. This dilution-dependent effect was most prominent in the open-face endotoxin samples, while button samples displayed little effect. Using a joinpoint regression model, it was determined that a dilution factor of 50 to 100 was generally sufficient to eliminate the presence of enhancing substances. After screening the data for dilution dependent effects, the airborne endotoxin concentrations were determined. The highest endotoxin concentrations, ranging from 2841 to 49,066 endotoxin units m−3 of air, were found inside swine farrowing and finishing barns. Airborne endotoxin concentrations were 10- to 100-fold lower inside a dairy barn and downwind of other agricultural production sites and the wastewater treatment plant. Examination of dilution-dependent effects should be considered essential when utilizing the LAL assay, especially if values are to be used for regulatory purposes.

Multi-elemental characterization of organic liquid samples by use of a 13 MeV 6Li 3+ beam

Evaporated amniotic fluid (AF) targets have been bombarded with a 13 MeV 6Li 3+ beam. Forward angle data have served to identify the 1H( 6Li, 1H) 6Li reaction as an alternative for hydrogen characterization of such organic samples. Detected 6Li ions elastically scattered from C, N, O, Na and Cl can also be used to determine the concentrations of these elements in AF. The analyzed AF samples have been diluted with distilled water. The effect of sample dilution on the improvement of spectrum energy resolution has been observed, confirming previous reports. The hydrogen concentration determined in the studied AF targets is shown to vary linearly with sample dilution. Two detector arrangements have been used to find out which detection scheme is the most convenient. The combination of a counter telescope and a single detector, set up at the same polar angle on the opposite side of the beam, seems to be the best choice to have a reliable particle identification and an adequate energy resolution simultaneously.

The technique of measuring thrombin generation with fluorogenic substrates: 3. The effects of sample dilution

Assessing the clotting function inevitably brings about dilution of plasma. With the existing techniques of thrombin generation (TG) measurement, dilution ranges from 2:3 to 1:8. However, the possibility that dilution alters procoagulant and anticoagulant pathways differently has not been examined. We investigated the effects of dilution on the thrombin generation process and found that the anticoagulant pathways are far more affected by dilution than the procoagulant pathways. That is, when prothrombin and antithrombin concentrations are kept constant, dilution of plasma does not significantly affect tissue factor (TF)-driven thrombin generation. We demonstrate that dilution of plasma slows down the inhibitory activity of tissue factor pathway inhibitor (TFPI) to a greater extent when compared with the down regulation by diluting procoagulant factors. Dilution of plasma has also a negative effect on the participation of the antihaemophiliac factors VIII and IX in TG driven by contact activation or low TF concentration. We also investigated the effect of dilution on the participation of the anticoagulant system that consists of thrombomodulin, protein C and protein S (APC system). We found that plasma dilution causes a loss of sensitivity towards TM and APC. Furthermore, at high dilutions (> 1:12) a second wave of prothrombinase-activity was observed that could be attributed to the suppression of protein S-dependent inhibition. In conclusion, the mechanism of TG is profoundly disturbed by plasma dilution. As a consequence, the less a plasma sample is diluted, the better a TG experiment represents the physiological process.

Effects of dilution on dissolved oxygen depletion and microbial populations in the biochemical oxygen demand determination

The biochemical oxygen demand (BOD) value is still a key parameter that can determine the level of organics, particularly the content of biodegradable organics in water. In this work, the effects of sample dilution, which should be done inevitably to get appropriate dissolved oxygen (DO) depletion, on the measurement of 5-day BOD (BOD(5)), was investigated with and without seeding using natural and synthetic water. The dilution effects were also evaluated for water samples taken in different seasons such as summer and winter because water temperature can cause a change in the types of microbial species, thus leading to different oxygen depletion profiles during BOD testing. The predation phenomenon between microbial cells was found to be dependent on the inorganic nutrients and carbon sources, showing a change in cell populations according to cell size after 5-day incubation. The dilution of water samples for BOD determination was linked to changes in the environment for microbial growth such as nutrition. The predation phenomenon between microbial cells was more important with less dilution. BOD(5) increased with the specific amount of inorganic nutrient per microbial mass when the natural water was diluted. When seeding was done for synthetic water samples, the seed volume also affected BOD due to the rate of organic uptake by microbes. BOD(5) increased with the specific bacterial population per organic source supplied at the beginning of BOD measurement. For more accurate BOD measurements, specific guidelines on dilution should be established.

Effects of sample dilution, peroxidase concentration, and chloride ion on the measurement of unbound bilirubin in premature newborns

Objectives To assess the effects of sample dilution, peroxidase concentration, and chloride ion (Cl −) on plasma unbound bilirubin ( B f) measurements made using a commercial peroxidase methodology (UB Analyzer) in a study population of ill, premature newborns. Design and methods B f was measured with a UB Analyzer in 74 samples at the standard 42-fold sample dilution and compared with B f measured at a 2-fold sample dilution using a FloPro Analyzer. B f was measured at two peroxidase concentrations to determine whether the peroxidase steady state B f ( B fss) measurements were significantly less than the equilibrium B f ( B feq), in which case it was necessary to calculate B feq from the two B fss measurements. B f was also measured before and after adding 100 mmol/L Cl − to the UB Analyzer assay buffer. Results B feq at the 42-fold dilution was nearly 10-fold less than but it correlated significantly with B feq at the 2-fold dilution (mean 8.2 ± 5.2 nmol/L versus 73.5 ± 70 nmol/L, respectively, p < 0.0001; correlation r = 0.6). The two UB Analyzer B fss measurements were significantly less than B feq in 42 of 74 (57%) samples, and Cl − increased B feq in 66 of 74 (89%) samples by a mean of 82 ± 67%. Conclusions B fss measured by the UB Analyzer at the standard 42-fold sample dilution using assay buffer without Cl − and a single peroxidase concentration is significantly less than the B feq in undiluted plasma. Accurate B f measurements can be made only in minimally diluted serum or plasma.