Evaluation of Streck BCT and PAXgene Stabilised Blood Collection Tubes for Cell-Free Circulating DNA Studies in Plasma.
Author(s): Warton K, Yuwono NL, Cowley MJ, McCabe MJ, So A, Ford CE
Publication: Mol Diagn Ther, 2017, Vol. , Page
PubMed ID: 28631163 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of storing blood in EDTA, Streck, and PAXgene tubes and proteinase K digestion duration on the yield and fragment size of cell-free DNA (cfDNA).
Conclusion of Paper
cfDNA yields were higher from specimens collected in EDTA tubes than Streck or PAXgene tubes when plasma was obtained within 1 h of blood collection and the proteinase K digestion step during extraction was limited to 30 min, but yields were comparable between tube types when the digestion was extended to 1 h. Storage of blood at room temperature for 4 days in EDTA tubes resulted in higher DNA yields, electrophoretic patterns consistent with internucleosomal cleavage, and decreases in the ratio of short to long Alu amplicons compared to blood from specimens centrifuged after 1 h at 4˚C. In contrast, storage of blood in PAXgene or Streck tubes for 4 days at room temperature had no effect on the yield of DNA and did not result in an electrophoretic ladder. While a very slight increase in genomic DNA contamination was detected for specimens stored in Streck tubes, a similar increase was not observed for specimens stored in PAXgene tubes and the ratio of the short to long Alu PCR products appeared higher for two of three specimens when blood was stored for 4 days in Streck tubes rather than processed immediately.
Studies
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Study Purpose
This study investigated the effects storing blood in EDTA, Streck, and PAXgene tubes and proteinase K digestion duration on the yield and fragment size of cell-free DNA (cfDNA). Nine sets of blood specimens were collected from eight healthy blood donors into two each of Streck BCT tubes, PAXgene Blood ccfDNA tubes, and EDTA tubes. One tube of each type was stored for 1 h at 4˚C and the other at room temperature for 4 days. After storage, blood was centrifuged at 2500 x g for 10 min at 4˚C and plasma was transferred to a new tube and centrifuged at 3500 x g for 10 min at 4˚C. Plasma was then transferred to another tube, mixed well, aliquoted and stored for less than a month at -70˚C. cfDNA was obtained from plasma using the QIAamp Circulating Nucleic Acids (CNA) extraction kit using a 30 min or 1 h proteinase K digestion. cfDNA was stored at -20˚C until quantitation by real-time PCR amplification of TP-53. cfDNA integrity was assessed in three specimens by amplifying two different length amplicons of Alu (115 bp and 247 bp) and by electrophoresis. DNA concentrations were adjusted to account for differences in the preservative volume.
Summary of Findings:
The cfDNA yield was higher from specimens collected in EDTA tubes than Streck (-28%, P=0.004) or PAXgene (-26%, P=0.01) tubes when plasma was obtained within 1 h of blood collection and the proteinase K digestion step during extraction was limited to 30 min; however, the Streck and PAXgene specimens yielded 19% and 15% more cfDNA, respectively, than EDTA specimens when the digestion was extended to 60 min, but the difference was not significant and the variability in yields increased. DNA yields increased 9-fold (30 min digest) to 22-fold (60 min digest) when blood was stored at room temperature in EDTA tubes for 4 days before processing, but cfDNA levels were unaffected by storage of blood for 4 days at room temperature in Streck or PAXgene tubes before plasma isolation.
The characteristic 180 bp and 360 bp cfDNA bands were observed when blood was processed within 1 h, regardless of collection tube type. The 180 bp and 360 bp cfDNA remained the predominant DNA in plasma from blood stored for 4 days at room temperature in PAXgene or Streck tubes, but a small increase in genomic DNA was observed for specimens stored in Streck tubes. In contrast, a more intense 180 bp band as well as an internucleosomal cleavage ladder pattern were observed when blood was stored for 4 days at room temperature in EDTA tubes before processing. Consistent with the electrophoretic patterns, specimens stored in EDTA tubes had the lowest ratio of the short to long Alu PCR products and specimens collected in PAX gene tubes and processed within 1 h had the highest ratio. Interestingly, the decrease in the ratio of the short to long Alu PCR products with blood storage was similar in PAXgene and EDTA specimens and two of three Streck specimens appeared to have a slight increase in the ratio of the short to long Alu PCR products after storage.
Biospecimens
Preservative Types
- None (Fresh)
- Streck/BCT
- PAXgene
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA Electrophoresis DNA Real-time qPCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Type of collection container/solution Streck BCT tube
PAXgene tube
EDTA tube
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
Biospecimen Preservation Type of fixation/preservation Blood collection tube additive
PAXgene
EDTA
Real-time qPCR Specific Targeted nucleic acid Alu
TP-53
Analyte Extraction and Purification Protein digestion 30 min
1 h
Real-time qPCR Specific Length of gene fragment 115 bp
247 bp
Storage Storage duration 1 h at 4˚C
4 days at room temperature