SPIDIA-RNA: First external quality assessment for the pre-analytical phase of blood samples used for RNA based analyses

Abstract

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. This report is the summary of the results of that trial. 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. Each participating laboratory described their respective laboratory policies and practices as well as blood collection tubes typically used in performing this type of testing. 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). Laboratories were requested to perform RNA extraction according to the laboratory's own procedure as soon as possible upon receipt of the tubes for one tube and 24h after the first extraction for the second tube. 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. All the results obtained by participants for each RNA quality parameter were classified as "in control", "warning", "out of control" and "missing" by consensus mean analysis. 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. Analyzing the results of quality testing of submitted RNA samples we observed a data distribution of purity, yield, and presence of assay interference in agreement with expected values. The RNA Integrity Number (RIN) values distribution was, on the other hand, much wider than the optimal expected value, which led to an "in control" classification, even for partly degraded RNA samples. On the other hand, RIN values below 5 significantly correlated with a reduction of GAPDH expression levels. Furthermore, the distribution of the values of the four transcripts investigated (c-fos, IL-1β, IL-8, and GAPDH) was wide and the RNA instability between samples separated by 24h were similar. Assuming the presence of at least two quality parameters "out of control" as an indication of a c...

Preanalytical Variables Affecting the Integrity of Human Biospecimens in Biobanking

“…Preanalytical collection factors affecting RNA quantity, quality, and gene expression analyses are tube type (51 ), sterility (7 ), tube additive (36 ), biospecimen type, volume of blood, and short-term storage temperature until extraction (48 ), and lag time until extraction (52,53 ). RNA yields when extracted from Tempus-stabilized tubes are higher than from PAXgenestabilized tubes.…”

“…Higher RNA yields have been obtained from cord blood (3-4 times higher) than from adult blood (51 ). Suboptimal blood volumes collected in the tubes may affect gene expression (51,53 ).…”

“…Preanalytical storage factors affecting analyte quantity, quality, or gene expression are temperature, storage time, concentration of RNA, and repeated thaws (53,119,120 ). As for RNA, new technologies for dry storage at room temperature have been developed (121)(122)(123).…”

Quality Markers Addressing Preanalytical Variations of Blood and Plasma Processing Identified by Broad and Targeted Metabolite Profiling

“…It is well known in the field of routine clinical chemistry that preanalytics are considered to account for approximately 68% of diagnostic misinterpretations (2 ). Defined preanalytical protocols are a prerequisite for reproducible and reliable results (3)(4). There is an increasing awareness of the essential need for securing high-quality biospecimens and implementing QC tools for assuring reproducible and credible results in biomarker studies (5 ).…”

SPIDIA-DNA: An External Quality Assessment for the pre-analytical phase of blood samples used for DNA-based analyses

“…In the absence of known reference values for each of the factors investigated, we measured the consistency of a given participant's results against all values excluding outliers (90%) of the results provided by the other participants as previously described [22]. Briefly, a two-step statistical procedure based on a distribution-free approach was adopted in order to process the data corresponding to each of the following variables: DNA purity (DNA1, 2, 3, 4), DNA concentration (DNA1, 2, 3), DNA yield (DNA4), DIRVs (i.e.…”