One purpose of the EC funded project, SPIDIA, is to develop evidence-based quality guidelines for the pre-analytical handling of blood samples for RNA molecular testing. To this end, two pan-European External Quality Assessments (EQAs) were implemented. Here we report the results of the second SPIDIA-RNA EQA. This second study included modifications in the protocol related to the blood collection process, the shipping conditions and pre-analytical specimen handling for participants. Participating laboratories received two identical proficiency blood specimens collected in tubes with or without an RNA stabilizer. For pre-defined specimen storage times and temperatures, laboratories were asked to perform RNA extraction from whole blood according to their usual procedure and to return extracted RNA to the SPIDIA facility for further analysis. These RNA samples were evaluated for purity, yield, integrity, stability, presence of interfering substances, and gene expression levels for the validated markers of RNA stability: FOS, IL1B, IL8, GAPDH, FOSB and TNFRSF10c. Analysis of the gene expression results of FOS, IL8, FOSB, and TNFRSF10c, however, indicated that the levels of these transcripts were significantly affected by blood collection tube type and storage temperature. These results demonstrated that only blood collection tubes containing a cellular RNA stabilizer allowed reliable gene expression analysis within 48 h from blood collection for all the genes investigated. The results of these two EQAs have been proposed for use in the development of a Technical Specification by the European Committee for Standardization.

Becton Dickinson Franklin Lakes New Jersey United States of America

Department of Biomedical Experimental and Clinical Sciences University of Florence Florence Italy

DiaGenic ASA Oslo Norway

Institute of Biotechnology Academy of Sciences of the Czech Republic Prague Czech Republic

Institute of Biotechnology Academy of Sciences of the Czech Republic Prague Czech Republic; TATAA Biocenter AB Gothenburg Sweden

Labonnet Ltd St Ramat Hasharon Israel

Qiagen GmbH Hilden Germany

Unit of Medical Statistics Biometry and Bioinformatics Fondazione Istituto di Ricovero e Cura a Carattere Scientifico Istituto Nazionale dei Tumori Milan Italy

Zobrazit více v PubMed

Pazzagli M, Malentacchi F, Simi L, Orlando C, Wyrich R, et al. (2013) SPIDIA-RNA: first external quality assessment for the pre-analytical phase of blood samples used for RNA based analyses. Methods. 59: 20–31. PubMed

Malentacchi F, Pazzagli M, Simi L, Orlando C, Wyrich R, et al. (2013) SPIDIA-DNA: An External Quality Assessment for the pre-analytical phase of blood samples used for DNA-based analyses. Clin Chim Acta. 25: 274–286. PubMed

Bustin SA (2010) Why the need for qPCR publication guidelines? – The case for MIQE. Methods 50: 217–26. PubMed

Rainen L, Oelmueller U, Jurgensen S, Wyrich R, Ballas C, et al. (2002) Stabilization of mRNA expression in whole blood samples. Clin Chem 48: 1883–90. PubMed

Tichopad A, Bar T, Pecen T, Kitchen RR, Kubista M, et al. (2010) Quality control for quantitative PCR based on amplification compatibility test. Methods 50: 308–12. PubMed

Kim SJ, Dix DJ, Thompson KE, Murrell RN, Schmid JE, et al. (2007) Effects of storage, RNA extraction, genechip type, and donor sex on gene expression profiling of human whole blood. Clin Chem 53: 1038–45. PubMed

Pahl A, Brune K (2002) Gene expression changes in blood after phlebotomy: implications for gene expression profiling. Blood 100: 1094–95. PubMed

Härtel C, Bein G, Müller-Steinhardt M, Klüter H (2001) Ex vivo induction of cytokine mRNA expression in human blood samples. J Immunol Methods 249: 63–71. PubMed

McCulloch CE, Searle SR (2001) Generalized, Linear, and mixed models. New York: John Wiley & Sons. 281–284.

Günther K, Malentacchi F, Verderio P, Pizzamiglio S, Ciniselli CM, et al. (2012) Implementation of a proficiency testing for the assessment of the preanalytical phase of blood samples used for RNA based analysis. Clinica Chimica Acta 413: 779–86. PubMed

Efron B, Tibshirana RJ (1993) An Introduction to the Bootstrap. London: Chapman and Hall 436pp.

Iglewicz B, Hoaglin DC (1993) How to Detect and Handle Outliers. In: ASCQ Quality Press, Milwaukee, 87pp.

Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using Real Time Quantitative PCR and the 2−ΔΔCt methods. Methods 25: 402–8. PubMed

Laurell H, Iacovoni JS, Abot A, Svec D, Maoret JJ, et al. (2012) Correction of RT-qPCR data for genomic DNA-derived signals with ValidPrime.Nucleic Acid Res. 40: e51. PubMed PMC

Fleige S, Pfaffl M (2006) RNA integrity and the effect on the real-time qRT-PCR performance. Molecular Aspects of Medicine. 27: 126–39. PubMed

Fleige S, Walf V, Huch S, Prgomet C, Sehm J, et al. (2006) Comparison of relative mRNA quantification models and the impact of RNA integrity in quantitative real-time RT-PCR. Biotechnol. Let 28: 1601–13. PubMed

Nussbaumer C, Gharehbaghi-Schnell E, Korschineck I (2006) Messanger RNA profiling: a novel method for body fluid identification by real-time PCR. Forensic science international 157: 181–6. PubMed

Ovstebo R, Lande K, Kierulf P, Haug KBF (2007) Quantification of relative change in specific mRNA from frozen whole blood-methodological considerations and clinical implications. Clin Chim Med 45: 171–6. PubMed

Madej RM, Cao Z, Hall L, Neuwald PD, Williams LO (2005) Proficiency testing (external quality assessment) for molecular methods, approved guideline. CLSI document MM14-A, Vol. 25, No. 24. Clinical and Laboratory Standards Institute; 1–51.

Effects of post-mortem and physical degradation on RNA integrity and quality