Need For Reference Materials Research Articles

Analysis of carbon nanotube levels in organic matter: an inter-laboratory comparison to determine best practice

Carbon nanotubes (CNTs) are increasingly being used in industrial applications, but their toxicological data in animals and humans are still sparse. To assess the toxicological dose-response of CNTs and to evaluate their pulmonary biopersistence, their quantification in tissues, especially lungs, is crucial. There are currently no reference methods or reference materials for low levels of CNTs in organic matter. Among existing analytical methods, few have been fully and properly validated. To remedy this, we undertook an inter-laboratory comparison on samples of freeze-dried pig lung, ground and doped with CNTs. Eight laboratories were enrolled to analyze 3 types of CNTs at 2 concentration levels each in this organic matrix. Associated with the different analysis techniques used (specific to each laboratory), sample preparation may or may not have involved prior digestion of the matrix, depending on the analysis technique and the material being analyzed. Overall, even challenging, laboratories’ ability to quantify CNT levels in organic matter is demonstrated. However, CNT quantification is often overestimated. Trueness analysis identified effective methods, but systematic errors persisted for some. Choosing the assigned value proved complex. Indirect analysis methods, despite added steps, outperform direct methods. The study emphasizes the need for reference materials, enhanced precision, and organized comparisons.

Developing a Global Library of Underwater Biological Sounds and a World Oceans passive acoustic monitoring day

The volumes of “Ocean Sound” recordings now being collected in aquatic systems around the world have reached a level that has necessitated a new wave of data sharing, processing, and analysis techniques to help detect, identify, and assess an increasing number of known and unknown sound sources and sound types. These new tools are matched with a need for reference material to assist the rapidly growing bioacoustics research community, and a process to standardize how they are characterized and reported. A working group of the International Quiet Ocean Experiment proposed the development of a Global Library of Underwater Biological Sound (GLUBS) to integrate new and existing applications to help address these needs. Two initial objectives of GLUBS have been to increase awareness of unknown sounds and to develop a community-wide project that can broaden our understanding of aquatic soundscapes around the world. To that end, we discuss recent developments in designing GLUBS, incorporating underwater sonifery as a searchable trait on the World Register of Marine Species, initiating a recent global community effort to simultaneously record aquatic soundscapes on World Oceans Day, and a drive to agree on a standard process for characterizing newly described sounds.

Abstract 6109: Multi-site evaluation of novel BRCA1/2 reference materials including large genomic rearrangements

Abstract Mutations in the tumor suppressor BRCA1 and BRCA2 genes can cause cell damage that significantly increases the risk of developing breast, ovarian, prostate, and pancreatic cancers. Large genomic rearrangements (LGRs) are defined as deletions, duplications or insertions; often involving a significant portion of an exon. Usually pathogenic, they have been reported to account for up to 27% of the BRCA1 and 5% of BRCA2 disease-causing mutations with a strong founder effect accounting for about 1/3 of all cancer diagnoses in some populations. Accurate detection of a BRCA1/2 pathogenic variants has immense impact on clinical management of disease including eligibility for PARP inhibitor therapy. However, these LGRs are frequently missed by PCR-based methods and NGS assays that do not detect partial or complete exon losses or gains. Given the difficulty in detecting LGRs, there is a need for improvement of BRCA1/2 testing algorithms including reference materials that incorporate challenging LGRs to support NGS assays that analyze for these mutations. Biosynthetic DNA constructs bearing clinically relevant BRCA1/2 variants including deletions up to 500 bp spanning 2 exons and duplications up to 170 bp, were mixed with purified genomic DNA from the GM24385 human reference cell line at 10% and 50% variant allele frequency (VAF) to represent somatic and inherited BRCA disease states, respectively. The same variants were engineered into GM24385 cells at the desired VAF (>5%) and the cells were formalin fixed and paraffin embedded using a proprietary method to mimic preserved tumor biopsies. In both materials, VAF was measured using digital PCR. The prepared purified gDNA and FFPE materials were sent to multiple clinical laboratories and analyzed using various NGS assays to assess variant detection performance and ensure that the product was compatible with clinical testing workflows. Digital PCR confirmed the presence of all 20 BRCA1 and BRCA2 variants within a 20% range of the target VAF in the purified gDNA material and between 11-23% in FFPE. NGS results were variable but confirmed the presence of most variants. A few variants were detected at very low levels but reported as not detected by the bioinformatic software due to limit of detection. We have successfully developed a reference material to support both BRCA1 and BRCA2 inherited and somatic genetic testing, providing laboratories and assay developers with a tool to challenge and optimize their assay’s detection and quantitation of pathogenic variants by NGS Provided as full process in FFPE format or in purified gDNA, these reference materials include 11 challenging LGRs, which are useful in evaluating the ability of different NGS platforms to detect such variants. This study has highlighted the need for reference materials that contain these challenging genomic alterations to improve genetic testing and ultimately, support PARP inhibitor treatment selection. Citation Format: Dana Ruminski Lowe, Benedicta Forson, Maria Cowen, Matthew G. Butler, Yves Konigshofer, Melissa Berenger, Krystyna Nahlik, Dianren Xia, Catherine Huang, Russell Garlick, Bharathi Anekella. Multi-site evaluation of novel BRCA1/2 reference materials including large genomic rearrangements. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6109.

Abstract 1978: Reference materials for measurable residual disease (MRD) monitoring

Abstract Here we describe reference materials for measurable residual disease (MRD) assay design and validation as well as initial data from testing. The analytical validation of liquid biopsy-based assays that attempt to monitor for the disappearance and reemergence of cancer can be challenging due to the need for reference materials that allow for the assessment of sensitivity and specificity at variant allele frequencies (VAFs) that can be over an order of magnitude below those that can be detected reliably by typical circulating tumor DNA (ctDNA) assays. At such low VAFs, a given plasma sample may only contain limited somatic variants - and only at a few copies - which is why some MRD assays focus on monitoring only patient-specific somatic variants and do not analyze other parts of the genome. However, this also means that validation should include the steps needed to monitor patient-specific somatic variants. Therefore, we designed our reference materials for a tumor/normal workflow as a set of three components. First, one cell line is a source of normal DNA that can be used to assess specificity. Second, a germline SNP-matched cell line provides hundreds to thousands of additional somatic variants. Third, blends of fragmented and sized DNA are used to mimic circulating cell-free DNA (ccfDNA) and serve as the input for MRD assays at VAFs from 0.1% to 0.01% (one mutant copy per 3.5 to 35 ng of ccfDNA) and at 0%. As a proof of concept, we analyzed three cell lines for somatic variants by Whole Exome Sequencing (WES). Using the resulting data, digital PCR assays were designed and two different error corrected NGS assays were customized to measure some of the identified variants. Based on past experiments, A>G (T>C) and C>T (G>A) changes had the highest background in NGS data, so these were generally avoided. The tumor and normal samples were used to assess the performance of a given customized assay to measure a variant. Digital PCR was used to verify VAFs in the ccfDNA format, which proved challenging for 0.01% given the expectation of approximately one positive droplet per assay well. NGS was then used to look for the variants using around 50 ng of input per reaction. While the detection of 0.1% VAFs appeared to be feasible with customized off-the-shelf assays, 0.01% was difficult given the need to measure a variant-containing region over 10,000 times to observe a somatic variant - if it was included in the library - and to measure each starting molecule around 10 times for error correction, which required a depth of greater than 100,000 for 20 of several hundred identified candidate variants. While we did not evaluate the reference materials with emerging clinical MRD assays and focused our initial testing on customized off-the-shelf assays, these reference materials should enable the design, validation, and evaluation of MRD assays. Citation Format: Yves Konigshofer, Matthew G. Butler, Jessica Dickens, Omoshile Clement, Andrew Anfora, Dan Brudzewsky, Bharathi Anekella, Russell K. Garlick. Reference materials for measurable residual disease (MRD) monitoring [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1978.

ELEMENTAL CHARACTERIZATION OF COAL FLY ASH BY NEUTRON ACTIVATION ANALYSIS TOWARDS REFERENCE MATERIAL CERTIFICATION

<p>Coal combustion by-products, such as coal fly ash, has known as a significant source of potentially toxic elements that may affect the environment since the increase of coal utilization as power fuel in Indonesia. Issuing the policies to protect the environment and public health is often based on analytical results. The quality of data analysis is very important since many crucial policies related to public health and environmental protections are based on the analytical results. Considering the need for reference material in ensuring the validity and reliability of the analytical results, reference material candidate of coal fly ash is being developed. Elemental characterization as one of the important parts in certifying the reference material is carried out using Neutron Activation Analysis (NAA) since it considered as high accuracy analytical method and commonly used to certified many reference materials throughout the world. Our NAA facility performance was assessed through optimizing analytical conditions by varying samples weight, method validation, and comparison test with other techniques as well. As much as 25, 100, and 250 mg of sample weight were assessed for its homogeneity effect. Method validation using SRM NIST 1633b had shown good results with bias not more than 10% compared to its certificate values. A comparison test with other results also showed a great agreement.</p><p> </p>

Correction of EELS dispersion non-uniformities for improved chemical shift analysis

We outline a simple routine to correct for non-uniformities in the energy dispersion of a post-column electron energy-loss spectrometer for use in scanning transmission electron microscopy. We directly measure the dispersion and its variations by sweeping a spectral feature across the full camera to produce a calibration that can be used to linearize datasets post-acquisition, without the need for reference materials. The improvements are illustrated using core excitation electron energy-loss spectroscopy (EELS) spectra collected from NiO and diamond samples. The calibration is rapid and will be of use in all EELS analysis, particularly in assessments of the chemical states of materials via the chemical shift of core-loss excitations.

Evaluation of Cystic Endometrial Hyperplasia and the Normal Estrous Cycle in Longitudinal Sections of Uterus from Female Harlan Sprague-Dawley Rats.

The National Toxicology Program (NTP) has recently introduced the practice of examining longitudinal histological sections of the female rodent uterus to improve the identification of non-neoplastic lesions, preneoplastic lesions, and uterine tumors. This practice has created a need for reference material that includes normal histology, spontaneous lesions, and inducible lesions in longitudinal as well as transverse sections of the body of the uterus, uterine horns, cervix and vagina. Using 3 archived NTP reproductive and developmental toxicity studies, the authors reviewed longitudinal and transverse sections of uteri from female Hsd:Sprague Dawley SD® (Hsd:SD) rats for cystic endometrial hyperplasia (CEH). The purposes of this review were to (1) evaluate if existing criteria for CEH in transverse uterine sections could be applied to longitudinal sections to develop diagnostic features of CEH in longitudinal uterine sections of rat uterus and (2) create an atlas of the normal estrous cycle phases in longitudinal sections of young and mature adult Hsd:SD rat uteri. The information provided in this original article should help facilitate the examination of longitudinal sections of the uterus in future commercial and governmental rodent studies.

Digital PCR method for detection and quantification of specific antimicrobial drug-resistance mutations in human cytomegalovirus

Antimicrobial drug resistance is one of the biggest threats to human health worldwide. Timely detection and quantification of infectious agents and their susceptibility to antimicrobial drugs are crucial for efficient management of resistance to antiviral drugs. In clinical settings, viral drug resistance is most often associated with prolonged treatment of chronic infections, and assessed by genotyping methods; e.g., sequencing and PCR. These approaches have limitations: sequencing can be expensive and does not provide quantification; and qPCR quantification is hampered by a lack of reference materials for standard curves. In recent years, digital PCR has been introduced, which provides absolute quantification without the need for reference materials for standard curves. Using digital PCR, we have developed a rapid, sensitive and accurate method for genotyping and quantification of the most prevalent mutations that cause human cytomegalovirus resistance to ganciclovir.

Human Biomonitoring of Mycotoxins in Blood, Plasma and Serum in Recent Years: A Review.

This manuscript reviews the state-of-the-art regarding human biological monitoring (HBM) of mycotoxins in plasma, serum and blood samples. After a comprehensive and systematic literature review, with a focus on the last five years, several aspects were analyzed and summarized: (a) the biomarkers analyzed and their encountered levels, (b) the analytical methodologies developed and (c) the relationship between biomarker levels and some illnesses. In the literature reviewed, aflatoxin B1-lysine (AFB1-lys) and ochratoxin A (OTA) in plasma and serum were the most widely studied mycotoxin biomarkers for HBM. Regarding analytical methodologies, a clear increase in the development of methods for the simultaneous determination of multiple mycotoxins has been observed. For this purpose, the use of liquid chromatography (LC) methodologies, especially when coupled with tandem mass spectrometry (MS/MS) or high resolution mass spectrometry (HRMS) has grown. A high percentage of the samples analyzed for OTA or aflatoxin B1 (mostly as AFB1-lys) in the reviewed papers were positive, demonstrating human exposure to mycotoxins. This review confirms the importance of mycotoxin human biomonitoring and highlights the important challenges that should be faced, such as the inclusion of other mycotoxins in HBM programs, the need to increase knowledge of mycotoxin metabolism and toxicokinetics, and the need for reference materials and new methodologies for treating samples. In addition, guidelines are required for analytical method validation, as well as equations to establish the relationship between human fluid levels and mycotoxin intake.

Homogeneity and stability of a secondary microbiological reference material candidate for Salmonella in fish matrix

Reference material (RM) is defined as a material, sufficiently homogeneous and stable with respect to one or more specified properties which has been established to be fit for its intended use in a measurement process. RM is one of the tools used to assess laboratory performance on an ongoing basis, as quality control in conducting testing and can be used to calibrate an equipment and to validate or verify the testing method. Currently microbiological reference materials with fish matrices are not much available so that there is a need for RM with fisheries matrices specification. Comprehensive studies of homogeneity and stability are need to be able to develop of any reference material. Initially, a feasibility study revealed a suitable processing procedure for fish matrix, the fish protein hydrolysate (FPH) was prepared by enzymatic hydrolysis, freeze-drying, and milling. Microbiological reference material with fish matrix were shown to be sufficiently homogeneous, and storage at temperatures of -20°C for 4 weeks statistically showed the effect of storage time (P value that was significant at 5% significance level). These results provide the basis for the development of a RM for Salmonella in fish matrix.

Improved light scattering measurement with microscopy data for nanoparticle number-based size distribution

Size is the key feature of nanoparticle properties. Nanoparticle size distribution can be measured by microscopy technique and light scattering posterior to a separation technique. Discrepancies can be observed, especially in the case of polydisperse samples. Microscopy is more precise for size and shape measurement, while light scattering provides better information about concentration. An experiment is set up to investigate the feasibility of bringing comparability of Asymmetrical Flow Field-Flow Fractionation followed by Multi-Angle Light Scattering and AFM measurements. A sample made from a mixture of stock samples is built, after detailed characterisation of the stock samples. By obtaining comparability of MALS and AFM measurements, a new way to calibrate MALS instrument without the need for reference material is opened: using aliquots of the sample itself as a reference.

Detection of transgenic rice line TT51-1 in processed foods using conventional PCR, real-time PCR, and droplet digital PCR

To assess the effects of food processing on the detection and quantification of transgenic rice TT51-1 in processed food by polymerase chain reaction (PCR) technology, we monitored the presence of TT51-1 components in rice crackers at different processing stages using conventional PCR, quantitative real-time PCR (qPCR), and droplet digital PCR (ddPCR) with standard or validated primers and probes. In conventional PCR, relatively longer amplification targets, such as the Bacillus thuringiensis (Bt) gene (301 bp) and the event-specific target (274 bp), were barely detected in baked, fried or microwaved samples. In qPCR, the amplification fluorescence signal was detected in boiled, dried, baked, and microwaved samples, but barely observed in fried samples. Conventional PCR with the same primers used in qPCR detected the corresponding shorter targets in all samples. The conventional PCR results were mainly consistent with the results of qPCR. The results indicate that food processing directly affects the detection of transgenic components, and suggest that relatively shorter fragments should be selected as the amplification targets for this type of analysis. We established qPCR and duplex ddPCR methods for quantifying TT51-1. The results of an orthogonal experiment indicated that the optimal conditions for TT51-1/PLD duplex ddPCR were 500/250 nM of primers/probe combined with 58 °C annealing temperature. Both methods were feasible for quantitative detection of TT51-1 in processed samples, with duplex ddPCR being a more attractive method for detecting transgenic components in processed food due to its stability, accuracy, PCR inhibitor resistance, and the lack of a need for reference materials.

Quantitative Analysis with Droplet Digital PCR.

Digital PCR-based methods, such as droplet digital PCR, are one of the best tools for determination of absolute nucleic-acid copy numbers. These techniques avoid the need for reference materials with known target concentrations. Compared to real-time PCR, they provide higher accuracy of quantification at low target concentrations, and have higher resilience to inhibitors. In this Chapter, we describe the droplet digital PCR workflow for the detection and quantification of flavescence dorée phytoplasma.

Biological reference materials for extracellular vesicle studies

Extracellular vesicles (EVs) mediate normal physiological homeostasis and pathological processes by facilitating intercellular communication. Research of EVs in basic science and clinical settings requires both methodological standardization and development of reference materials (RM). Here, we show insights and results of biological RM development for EV studies. We used a three-step approach to find and develop a biological RM. First, a literature search was done to find candidates for biological RMs. Second, a questionnaire was sent to EV researchers querying the preferences for RM and their use. Third, a biological RM was selected, developed, characterized, and evaluated.The responses to the survey demonstrated a clear and recognized need for RM optimized for the calibration of EV measurements. Based on the literature, naturally occurring and produced biological RM, such as virus particles and liposomes, were proposed as RM. However, none of these candidate RMs have properties completely matching those of EVs, such as size and refractive index distribution. Therefore, we evaluated the use of nanoerythrosomes (NanoE), vesicles produced from erythrocytes, as a potential biological RM. The strength of NanoE is their resemblance to EVs. Compared to the erythrocyte-derived EVs (eryEVs), NanoE have similar morphology, a similar refractive index (1.37), larger diameter (70% of the NanoE are over 200nm), and increased positive staining for CD235a and lipids (Di-8-ANEPPS) (58% and 67% in NanoE vs. 21% and 45% in eryEVs, respectively).Altogether, our results highlight the general need to develop and validate new RM with similar physical and biochemical properties as EVs to standardize EV measurements between instruments and laboratories.

Highly monodisperse, lanthanide‐containing polystyrene nanoparticles as potential standard reference materials for environmental “nano” fate analysis

ABSTRACTFor the safe commercialization of nanoparticle technology, there is a need for reference materials that can be used in studies related to the environmental fate of nanoparticles. This work produced metal‐containing polystyrene nanoparticles with target parameters such as high monodispersity, tailorable size range (33–193 nm), and variable surface charge. In addition, the combination of organic and inorganic components made them detectable by all of the main analytical techniques routinely used in nanoparticle characterization, e.g., TEM, DCS, DLS, and ICP‐MS (the latter when interfaced to chromatographic instrumentation). The lanthanides Gd, Dy, and Nd were investigated as the inorganic component because of their high response when analyzed by ICP‐MS, and because of their low environmental abundance. Particles were prepared by emulsion polymerization using one of two stabilizers: the nonionic surfactant Pluronic F68 or the anionic surfactant sodium dodecyl sulfate © 2015 The Authors Journal of Applied Polymer Science Published by Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015, 132, 42061.

Enabling consistency in pluripotent stem cell-derived products for research and development and clinical applications through material standards.

There is a need for physical standards (reference materials) to ensure both reproducibility and consistency in the production of somatic cell types from human pluripotent stem cell (hPSC) sources. We have outlined the need for reference materials (RMs) in relation to the unique properties and concerns surrounding hPSC-derived products and suggest in-house approaches to RM generation relevant to basic research, drug screening, and therapeutic applications. hPSCs have an unparalleled potential as a source of somatic cells for drug screening, disease modeling, and therapeutic application. Undefined variation and product variability after differentiation to the lineage or cell type of interest impede efficient translation and can obscure the evaluation of clinical safety and efficacy. Moreover, in the absence of a consistent population, data generated from in vitro studies could be unreliable and irreproducible. Efforts to devise approaches and tools that facilitate improved consistency of hPSC-derived products, both as development tools and therapeutic products, will aid translation. Standards exist in both written and physical form; however, because many unknown factors persist in the field, premature written standards could inhibit rather than promote innovation and translation. We focused on the derivation of physical standard RMs. We outline the need for RMs and assess the approaches to in-house RM generation for hPSC-derived products, a critical tool for the analysis and control of product variation that can be applied by researchers and developers. We then explore potential routes for the generation of RMs, including both cellular and noncellular materials and novel methods that might provide valuable tools to measure and account for variation. Multiparametric techniques to identify "signatures" for therapeutically relevant cell types, such as neurons and cardiomyocytes that can be derived from hPSCs, would be of significant utility, although physical RMs will be required for clinical purposes.

Uranium mining and milling: the need for reference materials in environmental radioactivity monitoring programmes

Environmental radioactivity monitoring programmes are now implemented in many countries around legacy uranium mining and milling sites and current uranium mining sites and include, for example, the analyses of soil, plant, meat and water samples. Often, there are legal requirements on periodic reporting of this monitoring. The results are used to assess compliance with the standards for radiation exposure of the public. To provide quality assurance, there is a need for reference materials (RMs) for use in monitoring programmes related to assessment of environmental contamination, uranium waste management and radiation protection in uranium areas. Reliable uranium supply to satisfy the increasing demand for low-carbon electricity production needs to meet the increasing environmental and health safety standards and public concerns for responsible information.

The Measurement of Platelet-Poor Plasma Serotonin: A Systematic Review of Prior Reports and Recommendations for Improved Analysis

Recent reports of new and important roles for serotonin (5-hydroxytryptamine, 5-HT) in the periphery have substantially increased interest in measuring peripheral serotonin. Nearly all circulating serotonin is found within platelets and this pool has been assessed by measuring serotonin in whole blood or in platelet-rich plasma. Measurement of the much smaller but potentially critically important pool of human free plasma serotonin in platelet-poor plasma (PPP) has proven much more difficult, with a wide range of reference values reported. To characterize the available data we carried out a systematic literature search of previous reports of PPP serotonin and attempted to determine the best estimate of true PPP serotonin concentration in humans. A total of 101 published reports that included PPP serotonin values in healthy controls were found and included in the summary statistical analyses. The distribution of PPP serotonin values demonstrated high skewness (+1.98), and the reported values ranged from 0.6 to 179 nmol/L, with a mean of 31.6 nmol/L, an SD of 38.9 nmol/L, and a median of 14.8 nmol/L. Reported concentrations for human PPP or free plasma serotonin were highly discrepant, with most reports giving erroneously high values that should be disregarded. Inherent difficulties in selectively measuring the extremely low concentrations of serotonin present in PPP and in preparing PPP without contamination from platelet-derived serotonin contributed to the problem, as did the failure of researchers to compare their results with those from prior studies. There is a clear and pressing need for reference materials for the measurement of plasma (PPP) serotonin.

The Association Between a CommonFCGR2APolymorphism and C-Reactive Protein and Coronary Artery Disease Revisited

the FcγRIIa receptor is responsible for the clearance of large immune complexes and recently has been proved to be a C-reactive protein (CRP) receptor as well. A polymorphism in the corresponding FCG2RA gene resulting in an amino acid change (R131H) has been implicated, with conflicting results in the pathogenesis of various autoimmune or inflammatory disorders (e.g., atherosclerosis and coronary artery disease [CAD]). we recently developed a real-time polymerase chain reaction and melting curve analysis method for the genotyping of the above polymorphism. We further looked at its validity with bioinformatics study and DNA sequencing. Then we genotyped 134 CAD patients and 45 angiographically normal controls and determined serum high-sensitivity CRP by nephelometry (Dade-Behring). Also, we used apparently healthy platelet donors (n = 206) as a larger control group. our method is accurate and devoid of problems with homologs and copy number variants. The need for reference materials is stressed. There were statistically significant differences (p < 0.05) between the CAD patients and each of the two other control groups, with the percentage of RR genotype rising from 6.5% and 11% in the control groups to an average of 19% in all CAD patients (17%, 24%, and 18.5% in stable angina, unstable angina, and myocardial infarction, respectively). In a logistic regression model that included known risk factors for CAD including CRP, the RR genotype remained a significant predictor for CAD (odds ratio: 6.3 [1.1-36.3]). Also after linear regression analysis, CRP levels were reduced in the RR carriers (vs. HH + HR), controlling for age, sex, and disease (marginal p = 0.07). with our accurate genotyping method, the RR genotype was correlated with atherothrombotic CAD events. The inverse correlation found between CRP levels and genotype supports the in vitro data of RR cells binding CRP stronger than HH.

Certification of reference materials for detection of the human prothrombin gene G20210A sequence variant

There is a need for reference materials (RMs) in the field of genetic testing for verification of test results obtained in patients and probands. For the frequent genetic variation G20210A in the prothrombin gene, it has been shown that purified plasmids containing the gene fragment harbouring the mutation constitute good candidate RMs. Plasmid-type RMs were characterised for homogeneity, stability, sequence identity and fitness for purpose. Their certification required the use of different real-time PCR methods for genotyping and quantification of the plasmid copy number. Homogeneity, stability and fitness for the purpose of the plasmids could be demonstrated. The long-term stability (up to 24 months) of the materials was confirmed by highly sensitive and specific quantitative real-time PCR methods. New types of certified RMs (CRMs) for genetic testing of the human prothrombin gene G20210A sequence variant are available. Their fitness for purpose was demonstrated and no evidence was found that they would not work with other methods as long as these are targeting the whole or parts of the prothrombin gene fragment inserted into the plasmids. The described CRMs support the efforts of the international community in development, validation and harmonisation of tests for molecular genetic testing.