Implementation of a proficiency testing for the assessment of the preanalytical phase of blood samples used for RNA based analysis

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• 作者：Kalle Gü ; nther^a ; ^g ; ¹ ; ; Francesca Malentacchi^b ; ^g ; ¹ ; ; Paolo Verderio^c ; ^g ; ; Sara Pizzamiglio^c ; ^g ; ; Chiara Maura Ciniselli^c ; ^g ; ; Ales Tichopad^d ; ^e ; ^g ; ; Mikael Kubista^d ; ^f ; ^g ; ; Ralf Wyrich^a ; ^g ; ; Mario Pazzagli^b ; ^g ; ^{m.pazzagli@dfc.unifi.it} ; ; Stefania Gelmini^b ; ^g ;
• 关键词：n ; Total number ; IQR ; Interquartile range ; RT-qPCR ; Reverse Transcriptase Quantitative Polymerase Chain Reaction ; qPCR ; Quantitative Polymerase Chain Reaction ; IL1B ; Interleukin-1¦Â gene ; IL8 ; Interleukin-8 gene ; FOS ; c-fos gene ; GAPDH ; Glyceraldehyde-3-P
• 刊名：Clinica Chimica Acta
• 出版年：2012
• 出版时间：11 April, 2012
• 年：2012
• 卷：413
• 期：7-8
• 页码：779-786
• 全文大小：841 K

文摘

Background

Although important improvements of downstream molecular in vitro diagnostics assays based on RNA from blood were made, the pre-analytical workflow is still poorly defined.

Methods

We performed a multicenter study within the EU-granted SPIDIA project to investigate blood collection and shipping influence on the following RNA quality parameters: yield, purity, integrity, RT-qPCR interference and IL1B, IL8, FOS and GAPDH gene expression. Two models were designed: Exp A. Ten laboratories collected blood from an own donor into two different tubes (with or without stabilizer) and extracted RNA at two different times; Exp B. Blood was drawn from a single donor and shipped to ten laboratories in two different tubes (with or without stabilizer) for RNA extraction.

Results

In both models and collection tubes, reliable results were obtained for purity, yield, GAPDH expression, and interferences. A substantial variation in RIN (Exp A) and in transcription levels of IL1B, IL8 and FOS (Exp B) was observed for blood collected in tube without stabilizer tubes. Overall the variability was higher among data obtained from unstabilized blood samples.

Conclusions

We defined the experimental setup for a larger ring trial throughout Europe. The chosen downstream analyses verified their potential, serving as adequate markers to test the quality of blood RNA.