One of the benefits of Digital PCR (dPCR) is the potential for unparalleled precision enabling smaller fold change measurements. An example of an assessment that could benefit from such improved precision is the measurement of tumour-associated copy number variation (CNV) in the cell free DNA (cfDNA) fraction of patient blood plasma. To investigate the potential precision of dPCR and compare it with the established technique of quantitative PCR (qPCR), we used breast cancer cell lines to investigate HER2 gene amplification and modelled a range of different CNVs. We showed that, with equal experimental replication, dPCR could measure a smaller CNV than qPCR. As dPCR precision is directly dependent upon both the number of replicate measurements and the template concentration, we also developed a method to assist the design of dPCR experiments for measuring CNV. Using an existing model (based on Poisson and binomial distributions) to derive an expression for the variance inherent in dPCR, we produced a power calculation to define the experimental size required to reliably detect a given fold change at a given template concentration. This work will facilitate any future translation of dPCR to key diagnostic applications, such as cancer diagnostics and analysis of cfDNA.
The international standardizing organizations - International, ISO, and IUPAC - cooperated to produce the International Harmonized Protocol for the Proficiency Testing of (Chemical) Analytical Laboratories. The Working Group that produced the protocol agreed to revise that Protocol in the light of recent developments and the experience gained since it was first published. This revision has been prepared and agreed upon in the light of comments received following open consultation.
Standard additions is a calibration technique devised to eliminate rotational matrix effects in analytical measurement. Although the technique is presented in almost every textbook of analytical chemistry, its behaviour in practice is not well documented and is prone to attract misleading accounts. The most important limitation is that the method cannot deal with translational matrix effects, which need to be handled separately. In addition, because the method involves extrapolation from known data, the method is often regarded as less precise than external calibration (interpolation) techniques. Here, using a generalised model of an analytical system, we look at the behaviour of the method of standard additions under a range of conditions, and find that, if executed optimally, there is no noteworthy loss of precision.
Synopsis: ISO, IUPAC and AOAC INTERNATIONAL have co-operated to produce agreed protocols or guidelines on the "Design, Conduct and Interpretation of Method Performance Studies" [1] on the "Proficiency Testing of (Chemical) Analytical Laboratories" [2] and on "Internal Quality Control in Analytical Chemistry Laboratories" [3]. The Working Group that produced these protocols/guidelines was asked to prepare guidelines on the use of recovery information in analytical measurement. Such guidelines would have to outline minimum recommendations to laboratories producing analytical data on the internal quality control procedures to be employed.A draft of the guidelines was discussed at the Seventh International Symposium on the Harmonization of Quality Assurance Systems in Chemical Laboratory, sponsored by IUPAC/ISO/AOAC INTERNATIONAL, held in Orlando, USA, 4-5 September 1996. Proceedings from that Symposium are available [4].The purpose of these guidelines is to outline the conceptual framework needed for considering those types of analysis where loss of analyte during the analytical procedure is inevitable. Certain questions cannot be satisfactorily addressed, and hence remain irreducibly complex, unless such a conceptual framework is established. The questions at issue involve (a) the validity of methods for estimating the recovery of the analyte from the matrix of the test material, and (b) whether the recovery estimate should be used to correct the raw data to produce the test result. The types of chemical analysis most affected by these considerations are those where an organic analyte is present at very low concentrations in a complex matrix."Protocol for the Design, Conduct and Interpretation of Method Performance Studies", W. Horwitz, Pure Appl. Chem. 60, 855- 864 (1988), revised, 67, 331-343 (1995)."The International Harmonized Protocol for the Proficiency Testing of (Chemical) Analytical Laboratories", M. Thompson and R. Wood, Pure Appl. Chem. 65, 2123-2144 (1993). (Also published in J. AOAC International 76, 926-940 (1993). "Harmonized Guidelines for Internal Quality Control in Analytical Chemistry Laboratories", M. Thompson and R. Wood, Pure Appl. Chem. 67, 49-56 (1995)."Quality Assurance for Analytical Laboratories", edited M. Parkany, Royal Society of Chemistry, London, UK, 1996.
Background: Accurate quantification of DNA using quantitative real-time PCR at low levels is increasingly important for clinical, environmental and forensic applications. At low concentration levels (here referring to under 100 target copies) DNA quantification is sensitive to losses during preparation, and suffers from appreciable valid non-detection rates for sampling reasons. This paper reports studies on a real-time quantitative PCR assay targeting a region of the human SRY gene over a concentration range of 0.5 to 1000 target copies. The effects of different sample preparation and calibration methods on quantitative accuracy were investigated.
A number of approaches for evaluating recovery and its contribution to uncertainty budgets for analytical methods are considered in detail. The recovery, R, for a particular sample is considered as comprising three elements, R m , R s and R rep . These relate to the recovery for the method; the effect of sample matrix and/or analyte concentration on recovery; and how well the behaviour of spiked samples represents that of test samples. The uncertainty associated with R, u(R), will have contributions from u(R m ), u(R s ) and u(R rep ). The evaluation of these components depends on the method scope and the availability, or otherwise, of representative certified reference materials. Procedures for evaluating these parameters are considered and illustrated with worked examples. Techniques discussed include the use of certified reference materials and spiking studies, and the use of extraction profiling to predict recoveries. All the approaches discussed evaluate the recovery and its uncertainty for the analytical method as a whole. It is concluded that this is a useful approach as it reduces the amount of experimental work required. In addition, most of the required data are frequently available from method validation studies.
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