Standard Method Procedure Research Articles

A novel method to efficiently isolate medullary thymic epithelial cells from murine thymi based on UEA-1 MicroBeads

ObjectiveThe central mechanism for establishing a self-tolerant and functional T cell repertoire includes the promiscuous expression of otherwise tissue-restricted proteins by medullary thymic epithelial cells (TEC). We here demonstrate a novel and highly efficient method for isolating this rare key cell type. MethodsWe combined the enrichment of medullary TEC via UEA-1 MicroBeads with the subsequent depletion of residual CD45+ hematopoietic cells via specific size exclusion and compared our results to the standard Percoll enrichment method and isolation procedure via flow cytometric cell sorting. ResultsThe addition of 2 μl UEA-1 MicroBeads per 108 thymus cells turned out best for optimal enrichment (an average of 22% purity compared to 1.2% for Percoll) and yield (an average of 1.73 × 105 medullary TEC per thymus compared to 5.16 × 104 for Percoll). After depletion of residual CD45+ cells, our method not only reached a purity of 75.5% but also turned out less stressful for the cells as compared to flow cytometric cell sorting. ConclusionWe here provide a fast and versatile procedure for enriching medullary TEC that yields higher purity and recovery rates than the standard Percoll enrichment method Our enrichment procedure in combination with CD45+ depletion via specific size exclusion is comparable to the current gold standard flow cytometric cell sorting method. Significance statementWe developed a fast and versatile procedure to isolate a high number medullary TEC to investigate the biochemical processes of medullary TEC in more depths.

Ash melting behaviour of wheat straw blends with wood and reed

Abstract Combustion of fuel blends is becoming more important. During combustion, interactions between ash-forming elements and compounds of different fuel blends can occur. Blending without sufficient knowledge and analysis can easily lead to operational problems, such as slagging, fouling and corrosion. Focus in our study is on the ash behaviour of wheat straw pellets blended with reed (Phragmites australis), wood pellets, and Douglas fir wood chips. The standard method (procedure) of CEN/TS 15370 and STA or simultaneous thermal analysis (thermogravimetric analysis (TGA) coupled with differential thermal analysis (DTA)) were applied to characterize the ash behaviour of the fuel blends studied. The ash melting behaviour of the blend of fuels depends not only on the composition, but also on the content of mineral matter of a single fuel. Low melting eutectics are possible when the K and/or Si-containing fuels are blended.

Environmental Condition and Trophic Status of Lake Rawa Pening in Central Java

Due to continuous problems of eutrophication, Lake Rawa Pening has been included into the 15 priority lakes in Indonesia to be saved from damage. This study aimed to clarify the current environmental conditions and trophic status of Lake Rawa Pening as a basis to control the eutrophication. Sediment loads, water quality, and nutrient concentrations were measured in the tributaries of lake inflow, within the lake, and at the point of lake outflow. The study was conducted in May, June, July, and August 2013. Water transparency, temperature, pH, turbidity, conductivity, and dissolved oxygen were measured in situ. Nitrogen, nitrate, total phosphorus, orthophosphate, TSS, and chlorophyll-a parameters were analyzed using standard method procedures. The Trophic State Index was used to determine the trophic state level. Hydroclimatological conditions showed that seasonally, fluctuation of water volume and discharge of lake followed the pattern of rainfall fluctuation. The sediment loads and nutrient concentration in the tributary inflow were more abundant than those in the lake and lake outflow. The results indicated that Lake Rawa Pening acts as sediment and nutrient sinks. Spatially and temporally, Lake Rawa Pening showed high variation of water quality. High concentration of nutrients observed during the wet and dry seasons indicated that the nutrients in the lake originated not only from external but also from internal sources. The overall results show that Lake Rawa Pening is a eutrophic lake, in which phosphorus seems to play a major role in causing eutrophication and massive growth of water hyacinth. <br /><br /><br />

The Impact of Rapid Left Ventricular Pacing during Pediatric Aortic Valvuloplasty on Postprocedural Aortic Insufficiency.

This study aims to determine efficacy and safety of rapid left ventricular pacing (RLVP) during balloon aortic valvuloplasty and effect on development of postprocedural aortic insufficiency (AI) in children. This is a retrospective comparison of 56 children (mean age 18.3 month; 1 day-15 years of age) who underwent valvuloplasty by using RLVP with standard method (without pacing) during same time period (74 children; mean age 12.6 month; 1 day-18 years of age). The systolic valvular gradient decreased from a mean 67 ± 20.4 mm Hg (26-120 mm Hg) before the procedure to a mean 27.6 ± 17.8 mm Hg (0-120 mm Hg) after the procedure. Procedure failed in two of the patients (3.6%) who received the RLVP; only two patients developed severe AI. Among those patients who underwent the standard method procedure failed in 6 patients (8.3%), whereas severe AI was seen in 15 (20.2%). RLVP decreased the incidence of postprocedure severe AI significantly (P < .01) but it had no effect on the gradient reduction of the procedure (P > .05). RLVP did not change the procedure duration, but decreased the duration of fluoroscopy significantly (P < .01). RLVP can be used effectively and safely for pacing during balloon aortic valvuloplasty procedures in all age groups; it decreases procedural failure rate, eases the procedure and prevents the development of AI through the stabilization of the balloon.

Performance evaluation of membrane bioreactor for treating industrial wastewater: A case study in Isfahan Mourchekhurt industrial estate

Aims: The aim of this study was to evaluate a membrane bioreactor (MBR) system for optimization effluent quality by feeding of the influent (raw wastewater and anaerobic reactor effluent) in Isfahan - Mourchekurt Industrial Estate Centralized Wastewater Treatment Plant. Materials and Methods: The MBR was equipped with two flat sheets membrane with 0.2 μm pore size, were operated in parallel style and feed simultaneously with raw industrial wastewater (MBR1) and anaerobic reactor effluent (MBR2). The average organic loading rates in two reactors were 1.37 and 0.52 (kg chemical oxygen demand [COD]/m 3 .day), respectively. All analyses were implemented according to the standard methods procedure. Results: The average concentration of COD was lower than 100 mg/L and 50 mg/L in both reactor effluent, respectively, and it was 1000 MPN/100 mL for MBR1 and these were Conclusion: The MBR technology was used to treat the combined industrial wastewater was efficient, and its effluent can be perfectly used for water reuse. The MBR performance was improved by applying an anaerobic pretreatment unit.

An Efficient Spectral Element Model with Electric DOFs for the Static and Dynamic Analysis of a Piezoelectric Bimorph

An efficient spectral element (SE) model for static and dynamic analysis of a piezoelectric bimorph is proposed. It combines an equivalent single layer (ESL) model for the mechanical displacement field with a sublayer approximation for the electric potential. The 2D Gauss-Lobatto-Legendre (GLL) shape functions are used to discretize the displacements and then the governing equation of motion is derived following the standard SE method procedure. It is shown numerically that the present SE model can well predict both the global and local responses such as mechanical displacements, natural frequencies, and the electric potentials across the bimorph thickness. In the case of bimorph sensor application, it is revealed that the distribution of the induced electric potential across the thickness does not affect the global natural frequencies much. Furthermore, the effects of the order of Legendre polynomial and the mesh size on the convergence rate are investigated. Comparison of the present results for a bimorph sensor with those from 3D finite element (FE) simulations establishes that the present SE model is accurate, robust, and computationally efficient.

Validation of analytical methods in GMP: the disposable Fast Read 102® device, an alternative practical approach for cell counting

BackgroundThe quality and safety of advanced therapy products must be maintained throughout their production and quality control cycle to ensure their final use in patients. We validated the cell count method according to the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use and European Pharmacopoeia, considering the tests’ accuracy, precision, repeatability, linearity and range.MethodsAs the cell count is a potency test, we checked accuracy, precision, and linearity, according to ICH Q2. Briefly our experimental approach was first to evaluate the accuracy of Fast Read 102® compared to the Bürker chamber. Once the accuracy of the alternative method was demonstrated, we checked the precision and linearity test only using Fast Read 102®. The data were statistically analyzed by average, standard deviation and coefficient of variation percentages inter and intra operator.ResultsAll the tests performed met the established acceptance criteria of a coefficient of variation of less than ten percent. For the cell count, the precision reached by each operator had a coefficient of variation of less than ten percent (total cells) and under five percent (viable cells). The best range of dilution, to obtain a slope line value very similar to 1, was between 1:8 and 1:128.ConclusionsOur data demonstrated that the Fast Read 102® count method is accurate, precise and ensures the linearity of the results obtained in a range of cell dilution. Under our standard method procedures, this assay may thus be considered a good quality control method for the cell count as a batch release quality control test. Moreover, the Fast Read 102® chamber is a plastic, disposable device that allows a number of samples to be counted in the same chamber. Last but not least, it overcomes the problem of chamber washing after use and so allows a cell count in a clean environment such as that in a Cell Factory. In a good manufacturing practice setting the disposable cell counting devices will allow a single use of the count chamber they can then be thrown away, thus avoiding the waste disposal of vital dye (e.g. Trypan Blue) or lysing solution (e.g. Tuerk solution).

Concentration of selected heavy metals in water of the Juru River, Penang, Malaysia

This study was conducted to determine heavy metal lead (Pb), cadmium (Cd), Zinc (Zn) and copper (Cu) concentrations in water body of the Juru River, Penang, Malaysia. A total of 20 sampling stations were chosen and water at 1 m below the surface was sampled using Van Dorn water sampler in December 2009 and April 2010. Collected water samples were kept in ice and acidified to pH 2 using nitrite acid (analytical grade HNO 3 ). Heavy metal concentrations were determined using inductively couple plasma (ICP model Perkin elmer/elan 9000) following standard method procedures. For comparison purposes, the river was divided into three different zones in the first and second samplings that is, upstream zone, middle reach zone and estuary zone and each zone consists of 7, 7, 6 sampling sites, respectively. Generally, results indicate that Pb and Cd and Cu in the first sampling were higher than the second sampling while the concentrations of Zn in the second sampling were higher than the first sampling. Also the results indicate that the Cd and Pb and Zn in the estuary zone were the lowest as compared to other zones. Meanwhile the highest concentration of Cu was in the middle zone. In conclusion, the lowest concentrations of some heavy metals were in estuary zone, it seems to be naturally due to dilution to heavy metals from sea water. Average metal concentrations from 20 water samples indicate that studied metal concentrations were still lower than Malaysian’s National Water Quality Standards (INWQS) guideline. Keywords: Heavy metals, pollution, river, inductively couple plasma

Laboratory study of feasibility of compaction grouting of soil

Compaction grouting has been utilized for various ground improvement applications in geotechnical engineering. However, there is no standard laboratory method or experimental procedure to determine the feasibility of compaction grouting of soil. Therefore, decision on the adoption of the technology and choices of operation parameters are based on past experience and empirical rules of thumb. A new laboratory apparatus and new experimental procedures were developed to establish the feasibility of compaction grouting of soil. The concept is analogous to the Proctor test for compaction. The relationship between the input grouting energy and the resulting compaction as a function of initial water content and relative compaction of the soil is quantified experimentally. The quantification can provide the necessary data for the geotechnical engineer to evaluate the viability and applicability of compaction grouting for their specific project needs. The design, fabrication, and assembly of the new apparatus are presented with results to demonstrate the workability of the apparatus.

Determination of Aluminum Traces in Hemodialysis and Tap Water Using Standard Method's Procedure Modified and Flow Injection Ionic Exchange Preconcentration

A procedure for the spectrophotometric determination of aluminum traces in water using a flow injection (FI) preconcentration system has been proposed. The flow system was made up of a peristaltic pump, an injector‐commutator, and a minicolumn filled with 300.0 mg of Amberlite IR‐120 cationic exchange resin. After the preconcentration step, aluminum was eluted by a 4.0 mol l−1 HCl solution. In a second stage, out of the flow system, the eluate was neutralized with a 4.0 mol l−1 NaOH solution. The aluminum was submitted to the reaction with eriochrome cyanine to form a chelate in solution buffered to pH 5.85, according to the Standard Method's procedure with some modifications, and this was followed by spectrophotometric detection. Chemical and flow variables were studied in the preconcentration system. The precision of the proposed method was calculated for a solution containing 46.8 µg l−1 of Al(III), when 40.0 ml of solution were preconcentrated (n=7), and their respective relative standard deviation (RSD) was 1.8%. The detection limit obtained was 1.7 µg l−1 of Al(III) (sample volume=40.0 ml). The proposed method was successful in determining aluminum in water certified reference material.

Residual Ozone Measurement: Indigo Sensitivity Coefficient Adjustment

The determination of residual ozone using indigotrisulfonate (Standard Methods 4500–O3B) assumes a molar absorptivity of–20,000 M -1cm -1 and a constant sensitivity coefficient of 0.42 L mg-1cm-1. Data are presented showing large molar absorptivity differences and subsequent differences in the calculated sensitivity coefficients for various sources of indigo. The variation in molar absorptivity (up to 15%) emphasizes the need for measuring the sensitivity coefficient on a regular basis. A revised simple calculation that compensates for decomposing indigo reagents is proposed. A modified equation that can be used with the current Standard Method procedure is given. However, for the most accurate results, it is recommended that the dry indigo reagent be periodically calibrated.

Peristaltic pumps and Fourier transforms in flame atomic absorption spectrometry: use of standard additions method and on-line dilution procedures

The use of periodically pulsed peristaltic pumps to introduce samples in flame atomic absorption spectrometry allows the signal obtained during the sampling time to be treated with Fourier transform. In this work two applications of this methodology are presented: the standard additions method and the on-line dilution of the sample. In both cases two peristaltic pumps are used. For the standard additions method, the process is carried out in two experiments using three standards. The developed procedure has been applied to copper, calcium and chromium determination in the presence of several interferents. The RSD and accuracy were lower than ±2% and 5%, respectively. The second application permits high on-line dilutions of sample. In this case, two periodically pulsed peristaltic pumps rotate in opposite ways. This method and the use of 0.5 mm id tubes and a sampling time of 13 s, permits copper concentrations as high as 2000 µg ml−1 to be determined.

Determination of oil and grease by solid phase extraction and infrared spectroscopy

An alternative procedure using solid phase extraction for the analysis of oil and grease in waters was developed. The use of a PTFE filter as a solid phase allows retention of oil and grease and further analysis of carbon–hydrogen bonds can be run over the filter. This method avoids the use of any kind of solvent. Experiments used n-hexadecane as a standard oil in different water matrices. The detection limit was 0.034 mg/l. Recoveries of diesel oil, pump vacuum oil, olive oil, sunflower oil and mixture of oils, depend strongly on the oil used for the calibration line, but in general satisfactory results were obtained. Recoveries of isooctane and benzene, used in the standard methods procedure, gave poor results. The method is fast, clean and sensitive, and represents a way of measuring oil and grease in water with high reproducibility. The method was compared with standard methodologies in tests involving real samples.

Copper circulation in two tidally influenced marinas studied with the use of a Nafion polymer probe

Being able to measure the levels, and predict the movement of an anthropogenic trace metal released into a marine or estuarine environment is a major focus in coastal pollution studies. One major source of trace metal contamination in these environments is from ionic-copper-containing antifouling paints on ship hulls. Because ionic copper [Cu (I) + Cu (II)] complexes with organic ligands in the aquatic environment, there is a need for a quick and reliable measurement method to determine its levels. The organic dye, 2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline (bathocuproine = BCP), embedded in the polymer Nafion 117, is such a reliable method. This procedure provides for the development of a probe capable of directly measuring Cu (I) in seawater. Measurement numbers are within 17% of the values obtained with the aqueous BCS standard method procedure. With this Nafion probe, it is possible to test for Cu (I), and, with a method modification, for Cu (II). The probe also allows the study of the movement of the ionic copper relative to the water movements in a basin. Results from this study indicate that ionic copper circulates, mixes, and releases from the basins or harbors with the tide. The circulation and mixing are functions of the basin flushing rate and exchange ratio. Nafion impregnated with bathocuproine (BCP) is a rapid method useful for detecting Cu (I) releases into the marine environment. ©1999 John Wiley & Sons, Inc. Field Analyt Chem Technol 3:3–18, 1999

Automated Liver Cell Processing Facilitates Large Scale Isolation and Purification of Porcine Hepatocytes

An automated method for large scale isolation and purification of porcine hepatocytes is described. Liver cells were harvested by a two-step portal vein perfusion with ethylenediaminetetraacetate and collagenase. Hepatocyte purification was carried out using either a standard manual processing method (Procedure A) or an automated processing method using a filtration chamber and a programmable cell washer (Procedure B). Both methods produced high cell yields (Procedure A: 1.30 +/- 0.55 x 10(10) viable hepatocytes/liver; Procedure B: 1.38 +/- 0.32 x 10(10) viable hepatocytes/liver) and viability (Procedure A: 89 +/- 6.5%; Procedure B: 92 +/- 3.9%). Hepatocyte purity was significantly greater after Procedure B than after Procedure A (93.1 +/- 3.1% versus 83.1 +/- 3%, p < 0.01). Isolated hepatocytes by either method were morphologically intact, as demonstrated by transmission electron microscopy showing integrity of plasma membranes and intracellular organelles. Cultured hepatocytes isolated by either method were functionally intact, although those isolated by Procedure A showed significantly lower activity of microsomal 7-ethoxycoumarin-O-deethylase activity (p < 0.05) and mitochondrial succinate dehydrogenase activity (p < 0.01). In conclusion, use of the automated hepatocyte processing method resulted in efficient large scale preparation of porcine hepatocytes, with higher purity and greater retention of differentiated liver metabolic functions, and was found to be less time consuming and less labor intensive.

Epstein-Barr virus (EBV) replicative gene expression in tumour cells of AIDS-related non-Hodgkin's lymphoma in relation to CD4 cell number and antibody titres to EBV.

To determine whether activation of Epstein-Barr virus (EBV) replication in tumour cells of AIDS-related non-Hodgkin's lymphoma (ARNHL) is correlated with CD4+ cell counts and influences antibody response to EBV [anti-Z Epstein-Barr replicative activator (ZEBRA), anti-early antigen (EA), anti-viral capsid antigen (VCA)]. Retrospective study based on immunohistochemistry and in situ hybridization to detect EBV replicative gene products in tissue samples from patients affected by ARNHL and correlation with CD4+ cell counts and results of EBV serology (including anti-ZEBRA activity) in sera from the same patients. Seventeen out of 22 cases of ARNHL were selected for the presence of EBV [Epstein-Barr early region (EBER) RNA-positive]. Immunohistochemistry was performed with anti-ZEBRA, anti-EA-restricted, anti-VCA antibodies and in situ hybridization with BHLF1/NotI oligoprobes on tumour samples. Results were statistically correlated with those of CD4+ cell counts (17 out of 17) and with anti-EBV antibody titres (13 out of 17) assessed using standard immunofluorescence method and enzyme-linked immunosorbent assay procedure using recombinant ZEBRA protein and synthetic peptides as antigens. BZLF1 (ZEBRA) or early gene products (EA-R and EA-D/BHLF1/NotI) were detected in a small proportion (< 0.01-5%) of tumour cells in eight of these 17 cases by immunohistochemistry and in situ hybridization. Demonstration of replicative gene expression did not correlate with either low CD4+ cell counts (P > 0.05) or anti-EBV antibody titres (P > 0.05). Anti-ZEBRA activity was not significantly increased in patients affected with ARNHL, the cells of which expressed replicative gene products (P > 0.05). The degree of immunodeficiency does not clearly enhance replicative gene expression in tumour cells of ARNHL. EBV serology, including anti-ZEBRA activity, is not a reliable tool for predicting the occurrence of such proliferations.

Electromagnetic scattering from 3-D arbitrary coated cavities via a connection scheme using triangular patches

The electromagnetic scattering from arbitrarily shaped 3-D cavities with coated walls is presented. In this formulation, the cavity is divided into smaller sections. Each section is individually analyzed with a boundary integral equation, followed by the standard moment method procedure to arrive at an admittance matrix for each section. These admittance matrices for different sections are then cascaded using a connection scheme based on microwave network theory. Once the admittance matrix of the whole cavity is obtained, the electric field on the aperture and thus the backscattered field can be obtained by the generalized network formulation. Triangular patch basis functions are used to provide easy mesh generation using standard CAD tools. To model wall treatment, the impedance boundary condition is applied. The numerical results generated by the present approach show good agreement with the modal solutions for regular geometries. An example for a cavity with coated walls is also presented.

ALKALINITY MEASUREMENTS IN ANAEROBIC DIGESTION SYSTEMS AS INFLUENCED BY ORGANIC ACID LEVEL AND ENDPOINT PH

ABSTRACT The alkalinity measurement procedure proposed by Hill and Jenkins (1990) and the method recommended by Standard Methods (APHA, 1985) were compared using three operating anaerobic digesters treating liquid swine waste. The results of the comparison show great differences in the measured alkalinity. Based on correcting for the total volatile fatty acid (TVFA) level [i.e., calculating the true bicarbonate alkalinity (TBA)], the procedure of Standard Methods produced errors ranging from 30% for digester effluent to 566% for raw waste influent. The proposed method of Hill and Jenkins (1990) produced errors of from 1.6% to 54% for the same samples. It would therefore appear that volatile fatty acid (VFA) concentration, if assumed negligible, can introduce measurement errors of five to six times using the Standard Methods procedure. This error is greatest where TVFA level exceeds by five times the TBA level and would occur frequently in raw waste samples. In biological effluent samples, where TBA is high compared to TVFA, the error encountered using the Standard Methods procedure was in the 30 to 40% range. The method proposed by Hill and Jenkins (1990) is a simple means of correcting a majority of this error...

Measuring Alkalinity Accurately in Aqueous Systems Containing High Organic Acid Concentrations

ABSTRACT Alkalinity is one of the most important parameters in biological waste management systems. It is a measurement of the chemical buffering capacity and its level therefore is an indication of the biological health of aqueous waste treatment processes. Alkalinity is based primarily on the carbonate-bicarbonate-carbonic acid complex as the major contributor. The measurement of alkalinity for years has been based on the fact that titration to a pH endpoint of 4.3 or 3.7 {Standard Methods, 1985, 1980) would measure all the carbonic acid system. While this is true, in waste management processes where organic acids are significant, titration to this pH also measures much of the organic acid complex. This is a false alkalinity since the pKa of most organic acid systems is below 4.75. Jenkins et al. (1983) first expressed concern about this phenomenon. Described here is a simple titrametric procedure for determining alkalinity in wastewater samples that is approximately 10 times more accurate than the Standard Methods (1985) procedure. The study used synthetic controlled chemical solutions. The levels of organic acid, true bicarbonate alkalinity and endpoint titration pH were closely controlled and their influence on the measured alkalinity was determined. The results show that titration to pH 4.3, when organic acids are present, can result in great error (ca. 600%) in measured alkalinity. Using the procedure proposed here, the error is much less (<45%).

Evaluation of Methods for Determination of Moisture Content of Heavy Saltd Cod, Gadus morhua

Moisture contents of various grades of salted cod were determined directly by the standard oven method and Compu-Trac™ procedures. A predictive approach was also tested using data collected with a Hygrodynamics water activity meter. The coefficient of variation (cv) for moisture determination of 47 samples ranged from 0.2 to 2.3% and averaged 0.96% for the oven method and ranged from 0.1 to 1.2% moisture and averaged 0.63% for the CompuTrac™ method. The coefficient of determination (r2/for linear regression analysis of moisture determination by the two methods was 0.99 for the equation:(% H2O, oven method) = .96 (% H2O, Compu-Trac method) + 3.3%The desorption isotherm at 5°C for a single lot of intact heavy salt fish, 50.7% H2O which was stepwise dehydrated to 11.7% H2O, exhibited a typical hyperbolic plot: (% H2O)=77.24×100Aw)/(1.82+100Aw) with a coefficient of determination of 0.99. However, the relationship between Aw and % H2O for 26 commercials samples, ranging in water content from 27.2 to 52.0%, was ambiguous. The salt content of 26 commercial samples of heavy salt fish ranged from 37 to 49% of the total solids. The variable salt content of commercial fish was largely responsible for the lack of correlation between Aw and moisture content.