David Gancberg

Considerations for the development of a reference method for sequencing of haploid DNA--an opinion paper on behalf of the IFCC Committee on Molecular Diagnostics, International Federation of Clinical Chemistry and Laboratory Medicine.

Abstract Following the completion of sequencing of the human genome, there has been a very rapid increase in the development of new molecular diagnostic tests. However, the numerous genetic tests and genetic testing technologies offered do not always satisfy essential quality criteria required to ensure confidence in the results that are produced. This is of particular importance for genetic tests since many patients may be tested for a particular genetic defect only once in their lifetime. Thus, there is a pressing need for comprehensive guidelines for the validation of molecular diagnostic tests and procedures, including DNA sequencing, the latter being a fundamental aspect of the development and validation of most genetic tests. To that end, the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) Committee for Molecular Diagnostics has prepared the following paper that describes a possible approach to the development of a reference method for sequencing of haploid DNA. We discuss various aspects which should be considered before, during and after applying the sequencing procedure, in order to achieve results with a known level of confidence, including robustness and assessments of quality. Clin Chem Lab Med 2009;47:1343–50.

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

Abstract Background: 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. Methods: 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. Results: 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. Conclusions: 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. Clin Chem Lab Med 2008;46:463–9.

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

Abstract 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 worldwide.

Nucleic acid reference materials (NARMs): definitions and issues

Abstract Molecular diagnostics is one of the most rapidly growing areas of laboratory medicine. This rapid growth of clinical molecular tests has outpaced the availability and development of reference methods and reference materials. Such methods and materials are important for the development, validation, and interpretation of diagnostic methods and tests. Yet, there is a lack of harmonization between the numerous international organizations currently either certifying or defining reference materials. The objective of this position paper is to review and clarify the definition, attributes and applications for the use of reference materials in the context of molecular diagnostics. Clin Chem Lab Med 2010;48:1531–5.