Evidence Based Guidelines for the Pre-analytical Phase of RNA Testing in Blood Samples

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SELECTBIO

The diagnostic use of in vitro molecular assays can be limited by the lack of guidelines for collection, handling, stabilization and storage of patient specimens. One of the major goals of the EC funded project SPIDIA (www.spidia.eu) is to develop evidence-based quality guidelines for the pre-analytical phase of blood samples used for molecular testing which requires intracellular RNA analytes. To this end, a survey and a pan-European external quality assessment (EQA) were implemented. With the European Federation of Laboratory Medicine (EFLM) support, 124 applications for participation in the trial were received from 27 different European countries, and 102 laboratories actually participated in the trial. The participating laboratories received two identical blood specimens: in an EDTA tubes (unstabilized blood; n = 67) or in tubes designed specifically for the stabilization of intracellular RNA in blood (PAXgene_ Blood RNA tubes; n = 35). Participants (n = 93) returned the two extracted RNAs to SPIDIA facility for analysis, and provided details about the reagents and protocols they used for the extraction. At the SPIDIA facility responsible for coordinating the study, the survey data were classified, and the extracted RNA samples were evaluated for purity, yield, integrity, stability, and the presence of interfering substances affecting RT-qPCR assays. All participants received a report comparing the performance of the RNA they submitted to that of the other participants. From the survey data, the most variable parameters were the volume of blood collected and the time and storage temperature between blood collection and RNA extraction. The RNA Integrity Number (RIN) values distribution was much wider than the optimal expected value. When RIN values were below 5, a significantly reduction of GAPDH expression levels was observed. A significant difference was observed in the expression levels of c-fos and IL8 according to the blood collection tubes (EDTA vs PAXgene tubes) after normalization versus the T0 value. The presence of a stabilizer maintained gene expression levels of IL8 and c-fos close to those measured in T0 samples in RNA C and D stored at RT (24h and 48h after blood collection, respectively). Our results demonstrated that the use of blood collection tubes containing cellular RNA stabilizers allows a reliable gene expression analysis at least within 48 h from blood collection. The results of the SPIDIA RNA EQAs studies have been used for the development of a Technical Specification by the European Committee for standardization (CEN).