Calibration Materials Research Articles

Application of ultrasonic waves to evaluate the rheological properties of non-Newtonian slurries: a preliminary study

An ultrasonic-wave system was developed to measure the rheological properties of non-Newtonian materials based on their responses to shear and pressure waves. This system was composed of a commercial shear wave transducer and various homemade devices, including a signal generator, a signal conditioner, and a transmission/receiving switch. The shear wave reflections at the interface between the delay line and the unknown test materials can be interpreted by the signals measured with calibration materials (e.g. air, water, olive oil and honey) that have well-known rheological properties. If the Voigt viscoelastic model is used to describe the dynamic behaviour of these test materials, the interpreted data can be used to determine the acoustic impedances, and thus solve for their shear moduli (G), kinematic viscosities (ν) and bulk densities (ρ). After the calibration procedures, the proposed system was used to test a consolidating kaolinite slurry with zero salinity and an initial suspended sediment concentration of 420 g l–1. The measurements showed that the shear modulus G is developed until consolidation for approximately 100 h. Afterward, the G gradually approached a value on the order of 500 N m–2 after approximately 200 h. This corresponded to an initial viscosity of approximately 7 × 10−5 m2 s–1, which decreased slowly over time to approximately 4 × 10−6 m2 s–1 after 300 h. With minor modification to capture pressure waves from the shear wave transducer, the bulk density can also be measured. The integrated system offers a non-invasive and efficient method for characterizing non-Newtonian fluids, providing valuable insights into their rheological properties, which are crucial for a wide range of industrial and scientific applications.

Mica Rb-Sr dating by laser ablation ICP-MS/MS using an isochronous calibration material and application to West African kimberlites

In-situ Rb-Sr dating of mica by laser ablation ICP-MS/MS has recently emerged as a new tool to date a range of geological processes including magmatic, metamorphic and hydrothermal events. The majority of age results presented to date are based on Rb/Sr calibration using the MicaMG pressed nano-powder pellet. However, some studies have reported low accuracy associated with mica Rb-Sr ages using this method, which are attributed to the different ablation properties of MicaMG and natural mica. In this work, we document the results of a systematic comparison between isotope-dilution and laser-ablation Rb/Sr ages of micas in Cretaceous diamondiferous kimberlites from Bultfontein (South Africa), Koidu and Tonguma (Sierra Leone), with additional in-situ data for the Mt. Dromedary mica (Australia). We adopt two different analytical strategies. While NIST610 SRM is employed to calibrate 87Sr/86Sr in both cases, the first approach follows the convention to utilise MicaMG as calibration material for Rb/Sr. In the second approach, Rb/Sr quantification is initially undertaken with NIST610, with a subsequent correction for Rb/Sr offset based on the age of isochronous micas (WBLK) from the Cretaceous Wimbledon lamproite (South Africa). We show that, using our instrumental set-up, employment of MicaMG as Rb/Sr calibration material commonly provides inaccurate results (generally 5–10% older than the solution-based ages). Conversely, our new approach returns accurate ages (within 3%) and represents a promising avenue to improve the accuracy of mica Rb-Sr age by laser ablation ICP-MS/MS especially once isochronous mica reference materials will be developed. Application of this methodology to multiple samples from Koidu and Tonguma shows that, contrarily to previous inferences based on age-dating of fewer samples, magmatism occurred at both localities in the 145–151 Ma interval. After a temporal hiatus, magmatism restarted exclusively in the Tonguma cluster between 135 and 139 Ma. The relative volumetric importance of these two magmatic stages in the Tonguma diamond mine awaits further evaluation.

Analytical performance specifications for combined uncertainty budget in the implementation of metrological traceability.

In addition to the correct implementation of calibration traceability, the definition and fulfillment of maximum allowable measurement uncertainty (MAU) are essential in assuring that laboratory measurements are clinically usable. Across the entire calibration hierarchy, three major contributors to the measurement uncertainty (MU) budget are identified, starting with the higher-order reference providers, extending through the in vitro diagnostic (IVD) manufacturers and their processes for assigning calibrator values, and ending with medical laboratories generating the random variability of results reported to clinicians. To understand if it is possible to achieve MAU and, consequently, to fix the possible drawbacks, the definition of combined MU budget limits across the entire calibration hierarchy has a central role. In particular, quality specifications for MU of reference and commercial calibrator materials should be defined according to the MAU on clinical samples. All involved stakeholders (i.e., higher-order reference providers, IVD manufacturers, medical laboratories) should be prepared to improve their performance whenever the clinical application of the test is made questionable by the failure to achieve MAU.

The absolute δ18 O value for SLAP with respect to VSMOW reveals a much lower value than previously established.

SLAP is one of the two calibration materials for the isotopic water scale. By consensus the established δ18 O value is -55.5‰, although several expert laboratories measure significantly more negative δ18 OSLAP values. The real δ18 OSLAP value as such does not influence the isotopic water scale; however, knowledge of the size of isotopic scale contraction in stable isotope measurements is vital for second-order isotopes. This study describes the quantification of δ18 OSLAP with respect to δ18 OVSMOW . SLAP-like water was quantitatively mixed with highly 18 O-enriched water to mimic VSMOW. The 18 O concentration was determined using an electron ionization quadrupole mass spectrometer. The isotopic composition of the SLAP-like and VSMOW-like waters was measured using an optical spectrometer, alongside original VSMOW and SLAP. This study resulted in a much more negative δ18 O value for SLAP than expected. The averaged outcome of seven independent experiments is δ18 OSLAP = -56.33 ±0.03‰. There is a large discrepancy between the actual isotopic measurements of even the most carefully operating isotope laboratories and the true δ18 O value. Although this finding as such does not influence the use of the VSMOW-SLAP scale, it raises the intriguing question of what we actually measure with our instruments and why even a fully corrected measurement can be so far off. Our result has consequences for issues like the transfer of δ18 O from and to the VPDB scale, various fractionation factors, and Δ17 O. The absolute 18 O abundance for SLAP was calculated as (1887.98 ± 0.43) × 10-6 based on the absolute 18 O abundance of VSMOW and the presented δ18 OSLAP in this paper.

Calibration methods for laser ablation Rb–Sr geochronology: comparisons and recommendation based on NIST glass and natural reference materials

Abstract. In situ rubidium–strontium (Rb–Sr) geochronology, using laser ablation–inductively coupled plasma–tandem mass spectrometry (LA-ICP-MS/MS) technology, allows rapid dating of K-rich minerals such as micas (e.g. biotite, muscovite, and phlogopite) and K-feldspar (potassium-containing feldspar). While many studies have demonstrated the ability of the method, analytical protocols vary significantly, and to date, no studies have provided an in-depth comparison and synthesis in terms of precision and accuracy. Here we compare four calibration protocols based on commonly used reference materials (RMs) for Rb–Sr dating. We demonstrate that downhole fractionation trends (DHFs) for natural biotite, K-feldspar, and phlogopite contrast with that for the commonly used Mica-Mg nano powder reference material. Consequently, Rb–Sr dates calibrated to Mica-Mg can be up to 5 % inaccurate, and the degree of inaccuracy appears to be unsystematic between analytical sessions. Calibrating to Mica-Mg also introduces excess uncertainty that can be avoided with a more consistent primary calibration material. We propose a calibration approach involving (1) NIST-610 glass as the primary reference material (PRM) for normalisation and drift correction and (2) a natural mineral with similar DHF characteristics to the analysed samples as matrix correction RM (MCRM) to correct the Rb/Sr ratio for matrix-induced offsets. In this work, MDC phlogopite (the source mineral for Mica-Mg nano powder) was used as the MCRM, consistently producing accurate Rb–Sr dates for a series of natural biotites and K-feldspars with well-characterised expected ages. Biotite from the Banalasta Adamellite, Taratap Granodiorite, and Entire Creek pegmatite are also suitable RMs for Rb/Sr ratio calibration purposes, with consistently <1.5 % fully propagated uncertainties in our methodological approach. Until calibration using isochronous natural standards as the primary RM becomes possible in data reduction software, the two-step calibration approach described here is recommended.

Features of the Choice of Knurl Material for Cold Calibration of Cylindrical Mid-Module Gear Wheels

Features of the Choice of Knurl Material for Cold Calibration of Cylindrical Mid-Module Gear Wheels

EFFECT OF VACANCIES AND VOID DEFECTS ON THE STRUCTURAL AND MECHANICAL BEHAVIOR OF TUNGSTEN UNDER HARSH TEMPERATURE AND PRESSURE CONDITIONS

The body-centered cubic transition metal tungsten is frequently used as a pressure calibration material at high temperatures and pressures due to its outstanding mechanical and thermal properties. In this study, molecular dynamics simulations were performed to investigate the behavior of tungsten under harsh temperature and pressure conditions and the impact of fundamental defects, particularly vacancies, and voids, on its physical, structural, and mechanical properties through their correlation with elastic constants. The study also covers mechanical stability, elastic properties, brittleness and ductility, and hardness. The simulations utilized two different embedded atom methods and one modified embedded atom method interatomic potentials. The results show that the fundamental structural characteristics and properties of pure tungsten crystal, including lattice constant, density, cohesive and vacancy formation energies, elastic constants, and moduli in the ground state for all three potentials, are in good agreement with previous experimental and theoretical calculations and results. The calculated results demonstrate that the elastic constants-related properties for defective structures also have the same trend as the perfect crystal. The presence of defects in the crystal causes a decrease in properties at all temperatures and pressures, directly correlated to the fraction of crystal defects. As the percentage of vacancies increases, a further reduction in the elastic constants is observed. Likewise, these findings reveal that the presence of scattered vacancies in the crystal structure causes a more significant decrease in the substance's properties than a void in the center of the crystal (with the same percentage). The presence of any vacancy weakens the interatomic bonds of the atoms around the vacancy, while the existence of a void in the center has less effect on the interatomic bonds of atoms further away from the center of the crystal.

50 Years of uranium isotopic reference materials at JRC-Geel

The history of uranium isotopic reference materials from JRC-Geel during the last 50 years is reviewed by presenting certification methods and relevant applications. The certified isotope ratios are traceable to the SI via gravimetrical preparation, either directly through gravimetrical mixing of highly enriched materials, or indirectly using existing gravimetrically prepared reference materials for calibration of mass spectrometers used for certification measurements. Due to developments of mass spectrometers and analytical methods, certification measurements have improved regarding precision, uncertainties and accuracy. This has led to a comprehensive set of uranium isotopic reference materials available for nuclear safety, security and the scientific community.

Experimental thermochemical study of aryl nitriles: A structural energetic approach

The present work comprised a study of the thermochemical properties of three aryl nitriles: Methyl p-cyanobenzoate, p-Acetylbenzonitrile, and p-Methoxybenzonitrile. This study was carried out using calorimetric and thermal analysis techniques. Through differential scanning calorimetry, we obtained the temperatures and enthalpies of fusion, as well as the heat capacities in the crystal phase. Specific combustion energies were determined through static bomb combustion calorimetry, from which, the enthalpies of formation in the condensed phase were derived. Additionally, isothermal thermogravimetry was used to measure the mass loss rate as a function of temperature. Using the Langmuir equation in conjunction with benzoic acid as a calibration material, vapor pressures were determined as a function of temperature. With the obtained vapor pressures and the Clausius-Clapeyron equation, enthalpies of sublimation were calculated. Moreover, entropies and Gibbs sublimation energies were determined at T = 298.15 K. Finally, using these properties, enthalpies of formation in the gaseous phase at T = 298.15 K were calculated for the three mentioned compounds. With these data, an analysis of the energetic effect of different substituents in benzonitrile is presented.

Identifying the effect of vitamin D on the HbA1c in patients of type 2 diabetes mellitus in Mosul city

Abstract Background: Diabetes mellitus causes metabolic disturbances and defectiveness in most tissues and cells of the body, especially in the nerves, blood vessels, eyes, kidneys, and other body tissues. It is known that type 2 diabetes mellitus is one of the most prevalent diseases in the city of Mosul, due to several reasons, such as excessive carbohydrate intake, psychological disorders, obesity, and lack of exercise. Because of these and other reasons, there was a need to conduct scientific research to clarify the main reasons for diabetes, modify the method of treatment and change the lifestyle to avoid falling into these chronic diseases. Objective: Studying the effect of vitamin D deficiency on patients with type 2 diabetes mellitus for the population of Mosul city for the purpose of modifying the metabolism in the body. Materials and Methods: Fifty eight study subjects were collected from patients with type 2 diabetes mellitus (diagnosed by a specialist and HbA1c test results) with 20 control samples of approximately the same ages and body mass index. Thereafter, We have investigate the durations of improving vitamin D for more than one year and record all medical history of the patients. Eleven study subjects were excluded due to drug interactions. Forty nine subjects of a study comprised 33 females and 16 males who were included in the study after making sure that they did not take medication. None of them were pregnant or breastfeeding. Their mean age was (51.8 ± 0.2 years) and their mean BMI was (26.8 ± 0.5). The examination was carried out using immunofluorescence and dry chemistry using the I CHROMA TM II device with the materials of the device, which were provided by the manufacturer with all the materials for calibration and quality control. Results: Forty nine study subjects were examined by HbA1c and vitamin D level by immunofluorescence technique, and the mean results were (9.3 ± 0.1%) for HbA1c and (14.3 ± 1 ng/mL) for vitamin D. After treatment with vitamin D for 1 month. The results showed a good improvement in the results of the HbA1c examination, as their average results were (7.1 ± 0.1%), with an improvement in the results of the vitamin D examination by (48.8 ± 1 ng/mL). Conclusions: There was a clear improvement in the level of sugar in the blood of patients with type 2 diabetes mellitus after treatment with vitamin D and an improvement in its level in the blood.

Improvement of heat release methodologies using mass loss calorimeter and oxygen consumption hood to assess flammability of wood plastic composites

The mass loss calorimeter provides a robust method for evaluating flammability of very sooty materials such as wood plastic composites as a lower cost and rugged alternative to the reliable and effective cone calorimeter. The high amounts of smoke and carbon monoxide associated with these materials resulted in a much-reduced heat release rate, resulting in incomplete combustion of the diffusion flame, which required modifications to the standards in measuring heat release rate via oxygen consumption or carbon dioxide production or thermopiles. In addition to liquid fuels of ethylene glycol and methanol, the solids polystyrene and polymethyl methacrylate were used as calibration materials for the mass loss calorimeter and the instrumented heat release rate hood. Because of concerns about the flue gas thermopile methodology for heat release rate measurements, a second thermopile was attached to the exterior of the metal pipe chimney typically used for heat release rate calculations in the mass loss calorimeter. A heat balance analysis indicated that this is an effective design compared with alternative designs. Estimates of flaming heat release rate are reported using both signals from the thermopiles for calculation of heat release rates and confirmed with the updated oxygen consumption analysis under an instrumented hood. This special fire test arrangement was used to assess the flammability of four different wood plastic composites, some with fire-retardant treatments, such as ammonium polyphosphate and brominated, all of which tended to have high smoke production leading to high-radiant energy losses. The reduction of heat release rate with 10% by content of fire-retardant treatment was confirmed by both heat release rate measures. The average heat release rate decreased 19% to 39% when fire retardants were added, relative to this wood flour–polyethylene composite.

Intrinsic variability of fluorescence calibrators impacts the assignment of MESF or ERF values to nanoparticles and extracellular vesicles by flow cytometry

Flow cytometry allows to characterize nanoparticles (NPs) and extracellular vesicles (EVs) but results are often expressed in arbitrary units of fluorescence. We evaluated the precision and accuracy of molecules of equivalent soluble fluorophores (MESF) beads for calibration of NPs and EVs. Firstly, two FITC-MESF bead sets, 2 and 6 um in size, were measured on three flow cytometers. We showed that arbitrary units could not be compared between instruments but after calibration, comparable FITC MESF units were achieved. However, the two calibration bead sets displayed varying slopes that were consistent across platforms.Further investigation revealed that the intrinsic uncertainty related to the MESF beads impacts the robust assignment of values to NPs and EVs based on extrapolation into the dim fluorescence range. Similar variations were found with PE MESF calibration.Therefore, the same calibration materials and numbers of calibration points should be used for reliable comparison of submicron sized particles.

New Potential Sulfide Reference Materials for Microbeam S‐Fe‐Cu Isotope Measurements

S‐Fe‐Cu isotope systems are powerful tracers for revealing geochemical processes. However, the microanalysis of S‐Fe‐Cu isotopes is critically limited by the lack of suitable reference materials. Herein, we present three potential reference materials Ll‐Cpy (chalcopyrite), Ll‐Po (pyrrhotite) and Ll‐Sp (sphalerite) for in situ S‐Fe‐Cu isotope measurements. Numerous in situ S‐Fe‐Cu isotope measurements were performed over two years to assess isotopic homogeneity. The bulk S isotopic compositions were determined independently in seven laboratories by isotope ratio mass spectrometry (IRMS); the preferred δ34SV‐CDT for Ll‐Cpy, Ll‐Po, Ll‐Sp are 6.13 ± 0.37‰ (2s), 6.42 ± 0.37‰ (2s) and 6.28 ± 0.38‰ (2s), respectively. The bulk Fe isotope ratios in Fe‐bearing Ll‐Cpy and Ll‐Po were determined using solution nebulisation multi‐collector inductively coupled plasma‐mass spectrometry, and the obtained δ56FeIRMM‐014 values are 0.57 ± 0.07‰ (2s) and ‐0.62 ± 0.07‰ (2s), respectively. The mean bulk δ65CuNIST SRM 976 value of Ll‐Cpy is 0.57 ± 0.06‰ (2s). All the bulk values are in good agreement with the long‐term statistical results of laser ablation‐MC‐ICP‐MS and proposed as the recommended values. These sulfides are well characterised and isotopically homogeneous (at 30–40 μm spatial resolution), and can be used as potential calibration materials for in situ S‐Fe‐Cu isotope measurements.

Genome length determination in adeno-associated virus vectors with mass photometry

Recombinant adeno-associated viruses (rAAVs) are attractive therapeutic viral vectors for gene delivery. To ensure the efficacy and safety of rAAV-based therapies, comprehensive characterization of the AAV capsids is essential. Mass photometry (MP) provides the advantage of short analysis times, low sample consumption and high accuracy of molecular mass determination. Despite having just recently emerged, MP has already been used to characterize AAV genome content and quantify filled/empty capsid ratios. In this study, we explored three approaches for the application of MP to assess genome length in AAVs. In approach 1, genome length in intact AAVs was approximated with good precision (%CV <2.6%) and accuracy (±5%) by using a straightforward protein-based calibration. In approach 2, genome length was determined even more accurately (±1%, %CV <2.9%) considering a calibration with a set of additional AAVs of different genome length. In approach 3, genome length was assessed after genome release from the capsid by heating in 1% sodium dodecyl sulfate followed by surfactant removal with a precision of %CV <0.7% and an accuracy of ±5%. In conclusion, the three developed MP-based approaches are fast, precise, and accurate methods for genome length determination in AAVs, differing in their used calibration materials and efforts.

Calibration Method and Material Constants of an Anisotropic, Linearly Elastic and Perfectly Plastic Mohr–Coulomb Constitutive Model for Opalinus Clay

AbstractNagra, the cooperative for developing and implementing a long-term radioactive waste depository in Switzerland, identified Opalinus Clay as the most suitable host rock for deep geological containment. This paper deals with those features of Opalinus Clay that are important for the design and construction of the underground structures. Consolidated drained (CD) and consolidated undrained (CU) triaxial compression tests on specimens from deep boreholes revealed that Opalinus Clay exhibits pronounced stiffness and strength anisotropy, dependency of stiffness on the initial confining pressure, slightly non-linear pre-failure stress–strain behaviour, and a drop in axial resistance after a certain amount of shearing. Within the scope of establishing a rigorous—yet practical—design approach for the repository tunnels and caverns, the simplest possible constitutive model capable of reproducing the main aspects of the Opalinus Clay behaviour is adopted. The non-associated linear elastic and perfectly plastic MC model is chosen as a starting point, on account of its wide use in tunnel engineering practice, its simplicity, and the clear physical meaning of its parameters. This paper presents a systematic and robust calibration method for an extended version of this model, which considers the pronounced strength and stiffness anisotropy of Opalinus Clay. The paper additionally provides the full suite of the equations that describe the model behaviour under triaxial CU or CD testing conditions and for any bedding orientation relative to the specimen axis. The equations are employed to determine ranges of material constants for two varieties of Opalinus Clay, based upon the results of 73 CU and CD tests. A thorough comparison between the model predictions and the experimental response is conducted, to demonstrate the versatility and limitations of the constitutive model and of the proposed calibration approach.

Features of the choice of burnisher material for cold calibration of cylindrical mid-module gear wheels

The experience is analyzed and the possibilities of replacing high-speed steel in the manufacture of rolling tools with high-chromium steels are shown. The modes of thermal and forging treatments in the manufacture of billets for cylindrical burnishers are given. Keywords: finishing calibration, burnisher, steel, heat treatment. tkm1410@yandex.ru, valpal-1938@mail.ru

A-010 Systemwide Implementation of High Sensitivity Troponin T in a Multi-center Hospital Set Up with Instrument Variability: The Baystate Health Experience

Abstract Background High-sensitivity cardiac troponin (hs-cTn) testing helps in rapid risk stratification in patients with acute coronary syndromes resulting in early identification of low-risk patients eligible for early discharge. A high sensitivity troponin test can measure the 99th percentile upper reference limit with an analytical imprecision ≤10% and it can measure cTn above the limit of detection in ≥50% of healthy subjects. We carried out method validation of the Elecsys Gen 5 hsTnT assay with variable instrument capacities like Roche cobas e801, e601, and e411 in our health system. The analytical variability of these instruments was studied and standardized to implement the Gen 5 hsTnT assay systemwide for clinical use in four hospitals. Methods The cTnT control samples with low, medium, and high concentrations were used for precision studies (N = 20). The calibration materials at five levels ranging from 8 ng/L to 10 000 ng/L were used for evaluating linearity, accuracy, and analytical measurement range. The observed and assigned values were compared using linear regression analysis. Heparinized plasma samples were tested using the Gen 5 hsTnT assay. Method comparisons were carried out on different Roche cobas analyzers in each hospital splitting remnant specimens. Analyzer e801 performance was compared with a reference laboratory. Simultaneously, the e601 and e411 analyzers were compared with the e801. Statistical analysis was carried out using EP Evaluator and GraphPad Prism. Results The Roche e801 demonstrated imprecision of 0.5%–2.2% (N = 20), the e601 had imprecision of 0.4%–1.4% (N = 20) and the e411 had 0.8%–4.8% (N = 20) across the three levels of control samples tested. These were well below the expected imprecision criteria of ≤10% for troponins. Deming regression analysis of the 5th GEN assays showed linear relationship across platforms: y (e801 5th GEN) = 1.007x (Reference Lab 5th GEN) + 50.65, R = 0.9899 with positive bias of 8% (N = 45); y (e601 5th GEN) = 0.929x(e801 5th GEN) + (−1.426), R = 0.9995 with negative bias of 7% (N = 48); y (e411 5th GEN) = 0.942x(e801 5th GEN) + (−7.044), R = 1.000 with negative bias of 7% (N = 49). The accuracy, reportable range, and linearity of Gen 5 hsTnT were tested on e801, e611 and e411 analyzers over a measured range of 8–10 000 ng/L. Allowable systematic error (SEa) was 1.5 ng/L (conc) or 5.0% (e801); 1.5 ng/L (conc) or 7.5% (e611 and e411). The maximum deviation for a mean recovery from 100% was 2.4% (e801), 2.7% (e601) and 6.2% (e411). The 5 of 5 mean recoveries were accurate within the SEa for all the three analyzers. The 15 out of 15 results were accurate within the allowable total error (TEa) of 3 ng/L (conc) or 10.0% (e801), 3 ng/L (conc) or 15.0% (e601 and e411). The three analyzers passed reportable range and accuracy tests, and the results were linear. Conclusion The TEa was determined from instrument specific imprecision, systemwide medical decision limits and parameters of rule out criteria. The standardization of analytical performance characteristics is important in a large health system with instrument variability and provision of transfer of care across health systems.

B-001 Method Development of C-Reactive Protein in the Serum of Non-Human Primates

Abstract Objective The objective of this study is to validate C-Reactive Protein (CRP) measurement in Non-Human Primate (NHP) serum using the ADVIA 1800 analyzer. Methods CRP was validated in NHP serum using the ADVIA 1800 analyzer. Validation testing included intra-assay precision, inter-assay precision, accuracy, linearity of dilution, limit of quantitation (LOQ), limit of detection (LOD), reference interval, carry-over, correlation, and stability. Samples were collected into tubes containing no anti-coagulant. Intra-assay precision was determined by analyzing two biological samples and the two quality control levels of Bio-Rad Lyphochek Immunology Plus Control 10 consecutive times (in duplicate) within a single run. Inter-assay precision was determined by analyzing two biological samples and the two quality control levels of Bio-Rad Lyphochek Immunology Plus Control six times for six days over a 10-day period. Accuracy was calculated using the data obtained from the inter-assay precision testing. For linearity of dilution, calibrator material and diluted specimen samples were analyzed in duplicate. The LOQ was determined by diluting the low-level calibrator material to produce a value at the low end of the reportable range. Diluted material was tested six times within the same run. The LOD was determined by assaying the appropriate blank 10 times within the same run. The reference interval was determined by analyzing 21 serum samples. Correlation between the two ADVIA 1800 analyzers was determined by assaying 10 samples on both analyzers once. The carry-over of the assay was determined by analyzing the high-level quality control material followed by the low-level quality control material. Stability of serum samples was tested at room temperature, refrigerated, and frozen (at -20°C) for various durations of storage. Results Results show that measurement of CRP in NHP serum met the acceptance criteria for intra-assay precision, inter-assay precision, accuracy, linearity of dilution, limit of quantitation, carry-over, and stability. For intra-assay precision in specimen and control material, the %CV was within ±20%. The inter-assay precision for specimen and control material %CV was within ±20%. The total error observed (TEobs) was within ±20% for accuracy in control material and the obtained mean was within the acceptable range specified by the manufacturer. For linearity of dilution, the coefficient of determination was ≥0.9000 and TEobs for each dilution and calibrator level was lower than 20%. The LOQ was set at the lowest concentration for which the TEobs was within ±20%. The LOD was calculated by adding the mean concentration obtained and three times the standard deviation. The reference interval was set to the 2.5th to 97.5th percentile interval using Excel software. The carry-over was acceptable as it was ≤2%. Correlation between the two ADVIA 1800 analyzers was determined to describe any bias in result interpretation. Stability at all storage conditions was found to be acceptable with a mean %difference within ±20%. Conclusion CRP in NHP serum met the acceptance criteria for all required validation parameters. The ADVIA 1800 analyzer can be used for preclinical studies to test CRP in NHP serum.

A-080 Overcoming Challenges of Equilibrium Dialysis-Based Free Thyroxine Measurements

Abstract Background Thyroid function tests are widely used to detect diseases of the thyroid. Concerns about the accuracy and reliability of free thyroxine (FT4) measurements have been stated by the clinical laboratory community. To address these concerns, CDC established a reference measurement procedure (RMP) for FT4 based on equilibrium dialysis (ED). ED-based methods have advantages over traditional direct immunoassays. As with all analytical methods, factors affecting accuracy and reliability of ED-based methods need identification and understanding. To investigate potential sources of error, the CDC FT4 RMP was used to evaluate T4 stability at different of storage conditions, adsorption of FT4 to common labware surfaces, and the effect of dialysis membrane materials on the measured FT4 in serum. Methods FT4 was measured in serum using the IFCC RMP (1). FT4 was separated from the protein-bound form during the equilibrium dialysis step of the RMP. Dialysates were spiked with internal standard (13C6-T4) and the analyte was further isolated by C18 solid phase extraction (SPE) and liquid-liquid extraction (LLE) in ethyl acetate prior to injection on an UHPLC system coupled with a triple quadrupole mass spectrometer. Bracketed calibration and primary reference materials were used to determine FT4 concentration in serum. This procedure was used to evaluate stability of serum FT4 during storage at −70 to 5 °C. Short- and long-term T4 stability of calibrator solutions at −70 °C to ambient temperature and the extent of adsorption of T4 calibrator solutions to the commonly used labware surfaces were evaluated. Furthermore, the suitability of 4 different dialysis membranes was assessed. In addition, three alternatives to the currently used SPE and LLE extraction steps were tested: LLE using cyclohexane and ethyl acetate, LLE with ethyl acetate, and supported liquid extraction with cyclohexane and ethyl acetate. Results T4 calibrator solution was stable in 1.7% ammonium hydroxide in ethanol for at least 2.9 years at −70 °C and up to 7 days at ambient temperature. Exposure of various calibrator solutions such as T4 in ethanol, ethanol with 1.7% ammonium hydroxide, 10% acetonitrile with 0.1% formic acid to common labware resulted in up to 10.1% loss of the analyte. FT4 in serum and T4 in dialysates and extracts were stable at −70 to 5 °C. Use of alternative dialysis membranes resulted in up to −8.8% mean bias to the reference membranes. Recoveries of the analyte using four extraction methods were 85.4%–95.0%. None of the tested extraction conditions significantly changed the results obtained with the original FT4 RMPs. Conclusion Although FT4 RMP must strictly adhere to the ED conditions described for the RMP, other parts of the sample preparation procedures can deviate from the originally published procedure. Careful selection of procedure for calibrator solvents, dialysis membranes, labware, and storage conditions is required to ensure accurate and reproducible FT4 measurements.

B-011 Analytical Validation of a New Vasoactive Intestinal Peptide Radioimmunoassay

Abstract Background Vasoactive intestinal peptide (VIP), a potent vasodilator, is produced by neuronal cells located in the central nervous system, the digestive, respiratory, and urogenital tracts, and the exocrine, thyroid, and adrenal glands. Increased circulating VIP is clinically useful to detect VIP-producing tumors in patients with chronic diarrheal diseases. Objective Develop a sensitive replacement radioimmunoassay (RIA) for VIP quantitation in human plasma due to the diminished supply of the antibody used in the previous in-house method. Method Patient VIP in EDTA plasma competes with labeled (125I) VIP for a limited number of primary rabbit polyclonal antibody (BMA Biomedicals, Augst, Switzerland) binding sites. Antibody-bound VIP is then complexed with a goat anti-rabbit secondary antibody and precipitated out of the solution with the use of polyethylene glycol and centrifugation. After the supernatant is discarded, the 125I signal from the pellet is counted on the Wizard2 gamma counter (Perkin Elmer, Waltham, MA). The measured signal is inversely proportional to the amount of VIP present in the patient’s sample. Method validation of the new assay included determination of imprecision (pooled human EDTA plasma), limits of detection and quantification, analytical measurement range (AMR), accuracy by spike recovery, interferences, cross-reactivity, stability, reference interval determination, and agreement with the current Mayo laboratory-developed assay. Results Stability studies on freshly collected EDTA plasma showed VIP is stable for 3 days ambient, 7 days refrigerated, and 14 days frozen. Intra-assay imprecision studies showed 8.3, 2.3, and 1.3%CV for mean VIP concentrations of 41, 144, and 304 pg/mL. Inter-assay imprecision was 12.9, 7.6, 5.2, and 7.5%CV for mean VIP concentrations of 30, 81, 180, and 308 pg/mL. The assay limit of detection was 29 pg/mL. The limit of quantitation was 30 pg/mL (%CV = 13%) and was established via precision profile. The AMR was 30–500 pg/mL (slope of 0.98, intercept of 3.0 pg/mL, and R2 of 0.97). The mean % spike recovery using a different preparation lot of calibrator material was 89% (range 83%–93%). The assay was not affected by up to 1000 mg/dL hemoglobin, 1000 mg/dL triglycerides, or 10 mg/dL bilirubin. Peptide histidine methionine 27, neurotensin, gastric inhibitory peptide, motilin, secretin, and glucagon do not cross-react in the assay. The 97.5th reference limit was &amp;lt;86 pg/mL (95% CI 81–90, n = 127). Positive percent agreement (PPA), negative percent agreement (NPA), and overall percent agreement (OPA) with the current Mayo VIP assay were 97, 82, and 90% respectively. Conclusion We have developed a replacement VIP RIA. This assay has a lower limit of quantitation and a broader AMR than the current assay. Concordance with the current assay was acceptable.