Reference materials (RMs) for analysis of the human factor II (prothrombin) gene G20210A mutation

The Scientific Committee of Molecular Biology Techniques (C-MBT) in Clinical Chemistry of the IFCC has initiated a joint project in co-operation with the European Commission, Joint Research Centre, Institute of Reference Materials and Measurements to develop and produce plasmid-type reference materials (RMs) for the analysis of the human prothrombin gene G20210A mutation. Although DNA tests have a high impact on clinical decision-making and the number of tests performed in diagnostic laboratories is high, issues of quality and quality assurance exist, and currently only a few RMs for clinical genetic testing are available. A gene fragment chosen was produced that spans all primer annealing sites published to date. Both the wild-type and mutant alleles of this gene fragment were cloned into a pUC18 plasmid and two plasmid RMs were produced. In addition, a mixture of both plasmids was produced to mimic the heterozygous genotype. The present study describes the performance of these reference materials in a commutability study, in which they were tested by nine different methods in 13 expert laboratories. This series of plasmid RMs are, to the best of our knowledge, the first plasmid-type clinical genetic RMs introduced worldwid

Reference Measurement Systems in Genetic Testing: Development of DNA-based Reference Materials

Recent progress in molecular pathology and biotechnology has made DNA-based assays important tools in patient care. DNA tests allow the detection of genetic alterations responsible for inheritable disease conditions, higher risk of developing diseases or altered drug effects. These tests have a high impact on clinical decision-making and thousands of different tests are already in routine use. Thus, quality issues and standardisation of DNA tests are particularly important. Reference materials (RMs) and certified reference materials (CRMs) are recognised as an excellent tool for checking analytical accuracy and are valuable in creating the necessary reference points in the development of comprehensive measurement systems. (C)RMs are used for method development and validation, calibration, statistical quality control within a laboratory and to assess the performance of laboratories in proficiency testing schemes. At present, the lack of (C)RMs for molecular genetic tests means that there are limitations in benchmarking against which a laboratory can judge the performance of its assays. Thus, the need for appropriate (C)RMs became increasingly urgent. In the frame of these projects, prototypes for different types of genetic RMs were processed for inherited diseases and genetic risk factors, such as hereditary hemochromatosis, fragile X syndrome, cystic fibrosis, the factor V Leiden (G1691A) mutation and Factor II (prothrombin) G20210A variant. These prototype RMs were evaluated for their suitability and different possibilities for the preservation and packaging of purified DNA were investigated in order to improve the stability and recovery rate of DNA-based RMs. Utilizing these results and experiences, a set of plasmid-type RMs for the analysis of the human prothrombin gene G20210A mutation were developed and produced. These candidate CRMs were characterised using quantitative real-time PCR techniques for homogeneity, stability, sequence identity and fitness for purpose. The material is homogeneous and stable at -20 °C. The sequence of the insert was confirmed by DNA sequence analysis. Each vial contains an indicative volume of 50 μL corresponding to approximately 1 ng of plasmid DNA. This series of plasmid CRMs was certified according to the ISO Guides 30−35 and is now available from the IRMM to support the validation and the harmonisation of polymerase chain reaction (PCR)-based methods used for detection of the G20210A mutation in the human prothrombin gene. The CRMs IRMM/IFCC-490 (G20210/G20210 wild-type), IRMM/IFCC-491 (20210A/20210A homozygous mutant) and IRMM/IFCC-492 (G20210/20210A heterozygous mutant) have been the first clinical genetic CRMs introduced worldwide. In addition, these reference plasmids were used also for the production of quality control materials for rare sequence variants to carry out a European proficiency study for the assessment of the competence of clinical laboratories to recognize and correctly interpret unusual genotyping results caused by rare SNPs. Our experiences and knowledge gained from these projects can be used in the development and production of further DNA-based CRMs for any molecular genetic test

Characterization of two different toxins of Wickerhamomyces anomalus (pichia anomala) VKM Y-159

Wickerhamomyces anomalus VKM Y-159 strain produces two types of toxin designated as WAKT a and WAKT b, encoded by chromosomal genes. The WAKT a toxin is heat-labile, pronase sensitive acting in pH range 3-4 affecting on several yeasts including pathogenic Candida species while the WAKT b toxin is protease- and thermo- resistant, acting in pH range 3-7 on two species, Candida alai and Candida norvegica. The rapid decrease of the number of viable cells after toxin treatment demonstrates that both toxins have cytocidic effect. © 2012 Akadémiai Kiadó, Budapest

Differential sensitivity of the species of Candida parapsilosis sensu lato complex against statins

Candida parapsilosis sensu stricto, Candida orthopsilosis and Candida metapsilosis are human fungal pathogens with clinical importance. The recently reclassified three closely related species have significant variation in virulence, clinical prevalence and susceptibility characteristics to different antifungal compounds. The aim of this study was to investigate the in vitro activity of atorvastatin and fluvastatin against C. metapsilosis, C. orthopsilosis and C. parapsilosis. Susceptibility tests showed that C. parapsilosis was the most sensitive while C. orthopsilosis was the least susceptible species to both drugs. On the basis of the differential sensitivity, we developed a simple, reliable and highly cost-effective plate assay to distinguish these closely related species. Applying this method, 54 isolates belonging to the C. parapsilosis sensu lato complex deposited in Szeged Microbial Collection could be sorted into the three species with 100 % probability

Determination of reference values of MDR-ABC transporter activities in CD3+ lymphocytes of healthy volunteers using a flow cytometry based method

BACKGROUND: MDR transporters are important biomarkers of drug resistance in cancer and in autoimmune conditions. We determined the MDR1, MRP1 and BCRP activity in CD3+ lymphocytes using a flow cytometry based method from 120 healthy volunteers in order to describe normal reference values of the activity of these transporters. The effects of gender and age were also determined. METHODS: The Solvo MDQ Kit™ was used for measurements. In this assay, fluorescent reporter substrates (Calcein-AM for MDR1 and MRP1 and mitoxantrone for BCRP, respectively) are trapped in the cytoplasm and pumped out by MDR proteins depending on the presence or absence of specific inhibitors (verapamil for MDR1 and MRP1, indomethacin for MRP1 and KO134 for BCRP, respectively), allowing for quantitative, standardized assessment. Cell surface staining was applied to select CD3+ cells. RESULTS: MAF values of MRP1 and BCRP are independent from age. MAFC and MAF of MDR1 show negative correlation with the age of the studied subjects (P = 0.003, r = -0.27 and P = 0.0001, r = -0.34, respectively). No difference was detected in any of the four MAF values between men and women. Gender does not affect the presence or lack of correlation between MAF values and age. CONCLUSIONS: The determination of the functional activity of MDR-ABC transporters is achievable using a flow cytometry based standardized method. Having established the normal range of MAF values on CD3+ lymphocytes of a healthy population, our results allow for the development of novel flow cytometry based diagnostic tools