Specialized Blood Collection Tubes for Liquid Biopsy: Improving the Pre-analytical Conditions.
Author(s): Sorber L, Zwaenepoel K, Jacobs J, De Winne K, Van Casteren K, Augustus E, Lardon F, Prenen H, Peeters M, Van Meerbeeck J, Roeyen G, Rolfo C, Pauwels P
Publication: Mol Diagn Ther, 2019, Vol. , Page
PubMed ID: 31838654 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to investigate the effects of blood collection tube (BCT) type and storage temperature on hemolysis, cell-free DNA (cfDNA) yield and integrity, and the KRAS mutational allele frequency in blood stored upright at room temperature for 24 h, followed by storage on a shaker at 6°C or room temperature for 24 h, and then upright for 24 h at room temperature. Results were compared to those in EDTA blood processed to plasma within 2 h of collection.
Conclusion of Paper
cfDNA yield and integrity, concentration of KRAS mutations, and the KRAS allele frequency were statistically comparable among the tube types but of the 8 specimens with >5% long fragments (>425 bp), half were collected in PAXgene genes. Importantly, the allele frequency of KRAS was much lower in a PAXgene specimen with 74% long DNA than in other specimens. While hemolysis of one or more specimens occurred in each of the tested tube types, hemolysis was most common in specimens stored in Roche tubes. Specimens stored in Streck tubes were less likely to be hemolyzed if stored at room temperature but those in PAXgene were less likely to be hemolyzed if stored at 6°C.
Studies
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Study Purpose
The purpose of this study was to investigate the effects of BCT type and storage temperature on hemolysis, cfDNA yield and integrity, and the KRAS mutational allele frequency in blood stored at room temperature or a combination of 6°C and room temperature. Results were compared to those in EDTA blood processed to plasma within 2 h of collection. Blood was collected from 10 patients with KRAS-mutated metastatic cancer into EDTA tubes and BCTs made by Streck, Roche, and PAXgene. EDTA blood was immediately processed to plasma by centrifugation at 1900 x g for 10 min followed by recentrifugation of the plasma at 16000 x g for 10 min. The BCTs were inverted ten times, stored upright at room temperature for 24 h, stored on a rocking platform (50 rpm) at room temperature or 6°C for 24 h, and then stored upright at room temperature for another 24 h. Plasma was obtained by dual centrifugation of Streck tubes at 1600 x g for 10 min followed by 6000 x g for 10 min, Roche tubes at 1600 x g for 10 min followed by 16000 x g for 10 min, and PAXgene tubes at 1900 x g for 15 min followed by 1900 x g for 10 min. All plasma was stored frozen at -80°C until analysis. Hemolysis was determined by visual inspection of photographs. cfDNA was isolated from plasma using the QIAamp Circulating Nucleic Acid Kit. Total cfDNA and KRAS-mutated cfDNA were quantified using the droplet digital PCR (ddPCR) KRAS Screening Multiplex Kit. cfDNA integrity was determined by qPCR of a 435 bp to 82 bp DNA fragment.
Summary of Findings:
Hemolysis was apparent in at least one specimen of each type when stored at room temperature or 6°C but depended on tube type when stored at 6°C (P=0.034) or room temperature (P=0.008) during shaking. After 6°C storage during shaking, a hemolysis score of 3 was found in all five specimens in Roche tubes, one PAXgene specimen, and one Streck specimen; a score of 2 was found in one PAXgene and one Streck tube; a score of 1 was found in one PAXgene and two Streck specimens; and a score of 0 was found in two PAXgene specimens and one Streck specimen but the matched EDTA specimens processed immediately had a score of 2 in one specimen and 0 in the remaining four specimens. After room temperature storage during shaking, a hemolysis score of 3 was found in four specimens in Roche tubes and two PAXgene specimens, a score of 2 was found in two PAXgene specimens and one Roche specimen, a score of 1 was found in one PAXgene and three Streck specimens, and a score of 0 was found in two Streck specimens. The matched EDTA specimens had a score of 0 in four specimens and a score of 1 in the remaining specimen. cfDNA yield and integrity were statistically comparable among the tube types but of the 8 specimens with >5% long fragments (>425 bp), four were collected in PAXgene, two in Streck, one in Roche, and one in EDTA tubes. Further, two of the PAXgene specimens had 54% and 74% long fragments while all other specimens had <15% long fragments. Importantly, the concentration of KRAS mutations and the allele frequency were comparable among the tube types but were much lower in the specimen with 74% long DNA (1.76% versus mean of 21.2%).
Biospecimens
Preservative Types
- Other Preservative
- None (Fresh)
- PAXgene
- Streck/BCT
Diagnoses:
- Neoplastic - Not specified
Platform:
Analyte Technology Platform DNA Digital PCR Cell count/volume Macroscopic observation DNA Real-time qPCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Type of collection container/solution EDTA tube
Streck BCT
Roche BCT
PAXgene BCT
Storage Storage temperature Upright at room temperature for 24 h, at 6°C on shaker for 24 h followed by at room temperature for 24 h
Upright at room temperature for 24h, at room temperature on a shaker for 24 h followed by at room temperature for 24 h