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Evaluating transport conditions of conventional, widely used EDTA blood tubes for gene expression analysis in comparison to expensive, specialized PAXgene tubes in preparedness for radiological and nuclear events.

Author(s): Schüle S, Ostheim P, Muhtadi R, Stewart S, Kaletka G, Hermann C, Port M, Abend M

Publication: Int J Radiat Biol, 2023, Vol. , Page 1-9

PubMed ID: 37676284 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared yield, integrity, and expression of radio-sensitive genes in RNA isolated from blood stored for up to 4 h at room temperature; stored for up to 4 h at room temperature followed by storage for 24 h at room temperature, 4°C, or -20°C; and blood that was irradiated before storage. Blood was collected from 11 healthy volunteers into EDTA tubes.  As a control for all experiments, an aliquot of blood was immediately transferred to PAXgene tubes. To investigate the effect of storage, blood from 3 volunteers was stored for 0, 1, 2, 3, or 4 h at room temperature before transfer to PAXgene tubes. To investigate the effects of storage and shipment, blood from 3 volunteers was stored for 0 or 4 h at room temperature before storage at room temperature, 4°C, or -20°C for 24 h (simulated shipping). To investigate the impact of ionizing radiation and shipment, blood was exposed to X-rays filtered with 3 mm beryllium and 3 mm aluminum to give a mean photon energy of 100 keV (approximately 1.0 gy/h at 13 mA) at 37°C and then stored at 37°C for 8 h followed by 0 or 4 h of storage at room temperature, and then 24 h at room temperature or 4°C. After experimental storage, specimens were transferred to PAXgene tubes and stored at room temperature for 2 h before transfer to -20°C. Before RNA extraction, blood was thawed and cells were pelleted by centrifugation (conditions not specified). RNA was extracted from the pellet using the QIAsymphony PAXgene Blood RNA Kit and a QIAsymphony SP instrument and stored at -20°C. RNA was quantified by spectrophotometer and fragment size was profiled using a 4200 TapeStation instrument. Levels of DXR, DDB2, POU2AF1, and WNT3 were quantified by real-time PCR and normalized to 18S rRNA. Differential expression was defined as a fold change of ≤0.5 or ≥2 and a p-value <0.05.

   Summary of Findings:

   The yield, RIN, and CT values of the 4 RNAs investigated were unaffected by storage of blood at room temperature for ≤ 4 h before being transferred to PAXgene tubes. The RIN was lower, 18S rRNA Cq values higher, and POU2AF1 levels >2-fold higher when blood was stored at room temperature for 4 h and then at -20°C for 24 h before being transferred to PAXgene tubes compared to values from blood that was immediately transferred to PAXgene tubes (P=0.049, P=0.001, and P<0.05 respectively). A non-significant reduction in RIN and an increase in 18S rRNA Cq values was observed when blood was stored at -20°C for 24 h before being transferred to PAXgene tubes.  RNA yield, RIN, and 18S rRNA Cq values were comparable among blood specimens that were immediately transferred and those that were stored for 0 or 4 h at room temperature followed by 24 h at room temperature or 4°C before transfer. Importantly, storage-associated changes in WNT3, DDB2, FDXR, and POU2AF1 were less than 2-fold with the exceptions of WNT3 in blood stored for 24 h at -20°C (not significant) and the aforementioned significant increase in POU2AF1 in blood stored at room temperature for 4 h followed by storage at -20°C for 24 h. RNA yield, RIN, and 18S rRNA values were comparable in RNA extracted from irradiated specimens (stored at 37°C for 8 h, followed by 0 or 4 h at room temperature, and 24 h at room temperature or 4°C) and non-irradiated specimens (stored at 37°C for 8 h). However, normalized expression of FDXR and DDB were much higher (33- to 61-fold and 9- to 17-fold higher, respectively) in irradiated specimens regardless of storage relative to non-irradiated specimens that were immediately transferred to PAXGene tubes, but statistical significance was limited to FDXR in irradiated specimens stored for 4 h at room temperature followed by 24 h at room temperature. Normalized expression of POU2AF1 and WNT3 were generally lower in irradiated specimens relative to non-irradiated specimens that were immediately transferred to PAXgene tubes; but while fold changes in POU2AF1 were >2-fold, they were not statistically significant, and statistically significant changes in WNT3 were mostly < than 2-fold.  

   Biospecimens

   • Cell - Blood
   • Bodily Fluid - Blood

   Preservative Types

   • PAXgene
   • Frozen

   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)
   Storage	Time at room temperature	0 h 1 h 2 h 3 h 4 h
   Real-time qRT-PCR Specific	Targeted nucleic acid	WNT3 DDB2 FDXR POU2AF1 18S rRNA
   Storage	Storage conditions	Irradiated Not irradiated
   Storage	Storage duration	0 h at room temperature followed by 24 h at room temperature 4 h at room temperature followed by 24 h at room temperature 0 h at room temperature followed by 24 h at 4°C 4 h at room temperature followed by 24 h at 4°C 0 h at room temperature followed by 24 h at -20°C 4 h at room temperature followed by 24 h at -20°C

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