SPIDIA-RNA: Second External Quality Assessment for the Pre-Analytical Phase of Blood Samples Used for RNA Based Analyses

<div><p>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.</p></div

Biomarkers for Monitoring Pre-Analytical Quality Variation of mRNA in Blood Samples

<div><p>There is an increasing need for proper quality control tools in the pre-analytical phase of the molecular diagnostic workflow. The aim of the present study was to identify biomarkers for monitoring pre-analytical mRNA quality variations in two different types of blood collection tubes, K₂EDTA (EDTA) tubes and PAXgene Blood RNA Tubes (PAXgene tubes). These tubes are extensively used both in the diagnostic setting as well as for research biobank samples. Blood specimens collected in the two different blood collection tubes were stored for varying times at different temperatures, and microarray analysis was performed on resultant extracted RNA. A large set of potential mRNA quality biomarkers for monitoring post-phlebotomy gene expression changes and mRNA degradation in blood was identified. qPCR assays for the potential biomarkers and a set of relevant reference genes were generated and used to pre-validate a sub-set of the selected biomarkers. The assay precision of the potential qPCR based biomarkers was determined, and a final validation of the selected quality biomarkers using the developed qPCR assays and blood samples from 60 healthy additional subjects was performed. In total, four mRNA quality biomarkers (USP32, LMNA, FOSB, TNRFSF10C) were successfully validated. We suggest here the use of these blood mRNA quality biomarkers for validating an experimental pre-analytical workflow. These biomarkers were further evaluated in the 2^nd ring trial of the SPIDIA-RNA Program which demonstrated that these biomarkers can be used as quality control tools for mRNA analyses from blood samples.</p></div

Blood collection tube and/or storage temperature and Gene Expression.

<p>Overall distribution of FOS and IL8 according to blood collection tube (3A and 3C, respectively) and to storage temperature/collection tube in RNA D (3B and 3D, respectively). The box horizontal sides identify the 25th and 75th centile, the horizontal line inside the box the median, the two whiskers correspond to minimum and maximum, and the dashed line indicates the T₀ value zero.</p

Classification of the proficiency of the laboratories.

a<p><b>all “in control” or “warning”</b>: labs with all parameters in control or warning, without missing;</p>b<p><b>one “out of control” and/or one or more “missing”</b>: labs with only one out of control (D1: n = 16, D2: n = 13); labs with only one missing (D1: n = 0, D2: n = 1) or only more than one missing (D1: n = 0, D2: n = 1); labs with one out of control and one missing (D1: n = 0, D2: n = 1); labs with one out of control and more than one missing (D1: n = 0, D2: n = 0);</p>c<p><b>two or more “out of control” with or without “missing</b>”: labs with two out of control with at least one missing (D1: n = 2, D2: n = 3) or without missing (D1: n = 5, D2: n = 6); labs with more than two out of control with at least one missing (D1: n = 3, D2: n = 3) or without missing (D1: n = 5, D2: n = 3).</p><p>Classification of the proficiency of the laboratories.</p

Time-course profile of EDTA down-regulation biomarkers in the validation study.

<p>1A: ATP2B4_S (mixed model contrasts: T₂₄ vs T₀, p-value <0.0001; T₄₈ vs T₀, p-value <0.0001); 1B: TNFRSF10C_S (mixed model contrasts: T₂₄ vs T₀, p-value <0.0001; T₄₈ vs T₀, p-value <0.0001). ΔCq = (Cq_biomarker – Cq_meanref) with Cq_meanref = mean of the Cq values of the 3 reference genes.</p

Relative Transcript levels of FOS and IL1B in individual and pools samples.

<p>Overall distribution of FOS (A) and IL1B (B) according to time storage. Each dot represents the expression levels of each individual (black) or pool (gray) samples for each time; the dashed lines indicate the time-trend for each sample. The continuous lines indicate the overall trend for individual (black) and pool (gray) samples. The horizontal dot-dashed line indicates the expected value of T₀.</p