Circulating tumoral DNA: Preanalytical validation and quality control in a diagnostic laboratory.
Author(s): Nikolaev S, Lemmens L, Koessler T, Blouin JL, Nouspikel T
Publication: Anal Biochem, 2017, Vol. , Page
PubMed ID: 29137972 PubMed Review Paper? No
Purpose of Paper
This paper investigated the stability of cell-free DNA in EDTA, Streck BCT, PAXgene, and Roche cell-free DNA (cfDNA) tubes stored under different conditions. The authors also investigated if the Kappa hgDNA assay could be used for cell-free DNA quality control as well as quantification.
Conclusion of Paper
Storage of blood in EDTA tubes at room temperature for more than 8 h, at 39˚C for 5 h, at 22˚C for 19 h, or at 4˚C for 48 h or in cfDNA tubes for 5 days with cycling between 39˚C for 5 h and 22˚C for 19 h resulted in an increased high molecular weight DNA contamination, but genomic DNA contamination was found when blood was stored for 7 days at room temperature or 39˚C and 22˚C in BCT or PAXgene tubes.
The Kapa hgDNA assay was found to be useful for quantification of cell-free DNA as quantity was strongly correlated with amplification of the 41 bp product and the ratio of the 305 to 41 bp products obtained by the assay was found to be an indicator of genomic contamination and DNA damage.
Studies
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Study Purpose
This study investigated the stability of cell-free DNA in EDTA, Streck BCT, PAXgene, and Roche cfDNA tubes stored under different conditions. The authors also investigated if the Kappa hgDNA assay could be used for cell-free DNA quality control as well as quantification. Blood from an unspecified number of healthy volunteers was collected into EDTA, PAXgene, BCT, and cfDNA tubes. Specimens were stored at 4˚C, room temperature, 36˚C, or 39˚C for 0 h, 3 h, 8 h 16 h, 24 h, 32 h, 40 h, 48 h, 3 days, 4 days, 5 days, 6 days, and 7 days before centrifugation at 1600 x g for 10 min at 4˚C. The resultant plasma was centrifuged at 16000 x g for 10 min at 4˚C. DNA was extracted using a modification of the Qiagen circulating DNA extraction kit and quantified by Qubit fluorimetry. DNA integrity was evaluated by capillary electrophoresis and by the Kapa hgDNA real-time PCR assay.
Summary of Findings:
Storage of blood in EDTA tubes for more than 24 h resulted in an increased high molecular weight DNA contamination with significant increases in the ratio of 305 to 41 bp real-time PCR products noted with processing delays as short as 8 h. Although no change in the ratio of 305 to 41 bp real-time PCR products was observed with up to 7 days of room temperature storage in BCT or PAXgene tubes even when shipped during the storage period, it increased significantly compared to immediately processed specimens with storage in cfDNA (P<0.01) or EDTA tubes (P<0.005). Similarly, there was no genomic DNA contamination apparent when specimens in BCT or PAXgene tubes were exposed to expected ambient temperatures (30˚C for 12 h followed by 22˚C for 12 h) for 7 days, but the genomic DNA contamination was observed by day 5 when stored in cfDNA tubes. Although genomic contamination was evident after storage in EDTA tubes at 39˚C for 5 h followed by 22˚C for 19 h, no genomic contamination occurred in specimens stored in PAXgene, BCT, or cfDNA tubes. Genomic contamination in EDTA tubes was only detected by capillary electrophoresis in specimens stored at 4˚C for 5 days or more, but a significant increase in contamination was noted after 2 days at 4˚C using the Kappa assay (P<0.05).
Amplification of the 41 bp real-time PCR product was strongly correlated with the quantity of DNA as determined by flourimetry (R2=0.9). The real-time PCR assay was capable of detecting genomic DNA contamination as the ratio of the 305 to 41 bp products increased from 0.2 in cell-free DNA to 0.85 in genomic DNA. The ratio of 305 to 41 bp products in cell-free DNA decreased when DNA was damaged by depurination (incubation at pH 3.0 for >45 min) or UV light exposure.
Biospecimens
Preservative Types
- PAXgene
- Streck/BCT
- Other Preservative
- None (Fresh)
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA Electrophoresis DNA Real-time qPCR DNA Automated electrophoresis/Bioanalyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Type of collection container/solution EDTA tube
Streck BCT
PAXgene tube
Roche Cell-free DNA tube
Biospecimen Preservation Type of fixation/preservation Blood collection tube additive
EDTA
PAXgene
Storage Time at room temperature 0 h
3 h
8 h
16 h
24 h
32 h
40 h
48 h
7 days
Real-time qPCR Specific Length of gene fragment 41 bp
129 bp
305 bp
Storage Storage temperature 4˚C
30˚C
39˚C for 5 h and 22˚C for 19 h
Storage Between site transportation method Mailed
Not transported
Storage Storage duration 1 day
2 days
3 days
4 days
5 days
6 days
7 days