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Preanalytical robustness of blood collection tubes with RNA stabilizers.

Author(s): Stellino C, Hamot G, Bellora C, Trouet J, Betsou F

Publication: Clin Chem Lab Med, 2019, Vol. , Page

PubMed ID: 31112504 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of stabilization tube type (PAXgene versus Tempus), partial-filling, tube inversion, and room temperature versus refrigerated storage on the yield and integrity of RNA, peripheral blood mononuclear cell (PBMC) stabilization, and microRNA (miRNA, miR) levels in blood. Blood was collected from seven healthy donors into four 11 mL Vacutainers without additive. The blood from each donor was immediately pooled and then transferred into 8 PAXgene Blood RNA and 8 Tempus Blood RNA tubes. For each tube type, four tubes were filled (2.5 mL for PAXgene and 3 mL for Tempus) and the other four tubes were half-filled (1.25 mL for PAXgene and 1.5 mL for Tempus). After filling, two tubes of each type and volume were inverted 8-10 times while the remaining were not inverted. Similarly, half of the tubes were then stored at room temperature (18-24˚C) for 24 h while the remainder were stored for 24 h at 2-8˚C. All tubes were then stored at -20˚C for 24 h and then stored at -80˚C until processing. RNA was extracted from PAXgene blood using a QIAcube instrument and from Tempus blood using a Tempus Spin RNA Isolation Kit. DNase steps were included in both extraction methods and extracted RNA was aliquoted to avoid freeze-thaw cycling. Due to a run failure with the QIAcube extraction, only specimens from 5-6 individuals were available for further analysis. RNA yield was determined spectrophotometrically. Integrity was determined using a bioanalyzer. The real-time PCR ratio of the cycle threshold values of 5’ and 3’ amplicons of GAPDH was used to determine the mRNA quality index with higher numbers indicating degradation. Levels of RNU24 and miR-16 were quantified by TaqMan assays. The ratio in the change in copies of IL8 to EDEM3 as quantified by TaqMan real-time PCR was used to determine the PBMC preanalytical score with higher scores being undesirable.

   Summary of Findings:

   RNA from blood stored in PAXgene tubes had a slightly higher mRNA quality index (indicating more degradation) and lower PBMC preanalytical score (indicating less PBMC stabilization) than that from blood in Tempus tubes (P=0.02 and P=0.007, respectively), but the authors state the differences were clinically irrelevant. There were no significant differences between the tube types with regard to RIN, miR-16 levels, or RNU23 levels.

   In PAXgene tubes, specimens that were half-filled had lower RNA yield (P=0.02) and higher RNU24 levels (P<0.001) and PBMC score (P<0.001), specimens that were not-inverted had lower RIN (7.1 versus 8.6 when inverted, P<0.001), and specimens that were stored at room temperature rather than refrigerated had lower RIN (8.4 versus 7.3, P<0.001), less miR-16 (P<0.001), and a lower mRNA quality index (0.7 versus 0.5, P<0.001). The authors state the effects on mRNA quality and PBMC scores were technically insignificant. Closer examination revealed a significant interaction in PAXgene specimens between inversion and filling status with more of an effect of inversion on full tubes (P=0.02) and inversion and storage temperature with more of an effect on RIN of room temperature storage when tubes were not-inverted (P=0.02).

   For Tempus specimens, those that were half-filled had higher RIN (8.7 versus 7.7, P<0.001), specimens that were not-inverted had lower RNA yield (P=0.05) and RIN (7.6 versus 8.8, P<0.001), and specimens that were stored at room temperature rather than refrigerated had lower spectrophotometric purity as determined by the ratio of the absorbance at 260 to 280 (1.91 versus 2.01, P=0.03). Further analysis of the Tempus specimens revealed significant interaction between all three factors on RIN and showed that inversion had a larger impact on RIN when the tubes were stored at room temperature than refrigerated (P<0.001 and P=0.02, respectively).

   Overall, the authors conclude that the investigated stabilization tubes are generally robust in regards to the preanalytical factors examined.

   Biospecimens

   • Bodily Fluid - Blood

   Preservative Types

   • Frozen
   • Other Preservative
   • PAXgene

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   RNA	Spectrophotometry
   RNA	Real-time qRT-PCR
   RNA	Automated electrophoresis/Bioanalyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Aliquot size/volume	Filled tube Half-filled tube
   Biospecimen Acquisition	Type of collection container/solution	PAXgene Blood RNA tube Tempus Blood RNA tube
   Real-time qRT-PCR Specific	Targeted nucleic acid	GAPDH 5' GAPDH 3' miR-16 RNU24 IL8 EDEM3
   Storage	Storage temperature	2-8˚C Room temperature
   Biospecimen Aliquots and Components	Biospecimen mixing	Inverted 8-10 times Not inverted

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