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Clinical Chemistry 52: 1584-1591, 2006. First published June 1, 2006; 10.1373/clinchem.2005.066019
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(Clinical Chemistry. 2006;52:1584-1591.)
© 2006 American Association for Clinical Chemistry, Inc.

Laboratory Management

EQUAL-quant: An International External Quality Assessment Scheme for Real-Time PCR

Simon C. Ramsden1,a, Sarah Daly2, Wolf-Jochen Geilenkeuser3, Graeme Duncan4, Fabienne Hermitte4, Ettore Marubini5, Michael Neumaier6, Claudio Orlando7, Vladimir Palicka8, Angelo Paradiso9, Mario Pazzagli7, Sara Pizzamiglio10 and Paolo Verderio10

1 National Genetics Reference Laboratory (Manchester), St. Mary’s Hospital, Manchester, UK.
2 Molecular Diagnostic Centre, Manchester Royal Infirmary, Manchester, UK.
3 DGKL-Reference Institute for Bioanalytics, Bonn, Germany.
4 Ipsogen, Luminy Biotech Enterprises, Marseilles, France.
5 Institute of Medical Statistics and Biometry, University of Milan, Milan, Italy.
6 Institute for Clinical Chemistry, University Hospital Mannheim of the University of Heidelberg, Mannheim, Germany.
7 Clinical Biochemistry Unit, Department of Clinical Physiopathology, University of Florence, Florence, Italy.
8 Institute for Clinical Biochemistry and Diagnostics, School of Medicine, University Hospital Sokolska, Hradec Králové, Czech Republic.
9 National Cancer Institute, Bari, Italy.
10 Operative Unit of Medical Statistics and Biometry, Instituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy.

aAddress correspondence to this author at: National Genetics Reference Laboratory (Manchester), St. Mary’s Hospital, Hathersage Road, Manchester, M13 0JH, UK. Fax 44-161-276-6606; e-mail simon.ramsden{at}cmmc.nhs.uk.

Background: Quantitative gene expression analysis by real-time PCR is important in several diagnostic areas, such as the detection of minimum residual disease in leukemia and the prognostic assessment of cancer patients. To address quality assurance in this technically challenging area, the European Union (EU) has funded the EQUAL project to develop methodologic external quality assessment (EQA) relevant to diagnostic and research laboratories among the EU member states. We report here the results of the EQUAL-quant program, which assesses standards in the use of TaqManTM probes, one of the most widely used assays in the implementation of real-time PCR.

Methods: The EQUAL-quant reagent set was developed to assess the technical execution of a standard TaqMan assay, including RNA extraction, reverse transcription, and real-time PCR quantification of target DNA copy number.

Results: The multidisciplinary EQA scheme included 137 participating laboratories from 29 countries. We demonstrated significant differences in performance among laboratories, with 20% of laboratories reporting at least one result lacking in precision and/or accuracy according to the statistical procedures described. No differences in performance were observed for the >10 different testing platforms used by the study participants.

Conclusions: This EQA scheme demonstrated both the requirement and demand for external assessment of technical standards in real-time PCR. The reagent design and the statistical tools developed within this project will provide a benchmark for defining acceptable working standards in this emerging technology.




The following articles in journals at HighWire Press have cited this article:


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C. Orlando, P. Verderio, R. Maatman, J. Danneberg, S. Ramsden, M. Neumaier, D. Taruscio, V. Falbo, R. Jansen, C. Casini-Raggi, et al.
EQUAL-qual: A European Program for External Quality Assessment of Genomic DNA Extraction and PCR Amplification
Clin. Chem., July 1, 2007; 53(7): 1349 - 1357.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Chem.Home page
A. Dorn-Beineke, P. Ahmad-Nejad, U. Pfeiffer, S. Ramsden, M. Pazzagli, and M. Neumaier
Improvement of Technical and Analytical Performance in DNA Sequencing by External Quality Assessment-Based Molecular Training
Clin. Chem., November 1, 2006; 52(11): 2072 - 2078.
[Abstract] [Full Text] [PDF]


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