Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Circulating Cell-Free DNA and RNA Analysis as Liquid Biopsy: Optimal Centrifugation Protocol.

Author(s): Sorber L, Zwaenepoel K, Jacobs J, De Winne K, Goethals S, Reclusa P, Van Casteren K, Augustus E, Lardon F, Roeyen G, Peeters M, Van Meerbeeck J, Rolfo C, Pauwels P

Publication: Cancers (Basel), 2019, Vol. 11, Page

PubMed ID: 30935089 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of tube type and centrifugation protocol on the yield of cfDNA and cfRNA, genomic DNA contamination, and detection of KRAS-mutated ctDNA and ctRNA. Blood from 15 patients with KRAS-mutated advanced cancer and 18 healthy patients were collected into EDTA and Streck BCT tubes. Plasma was isolated from blood in EDTA tubes within 2 h of collection and from blood in Streck tubes after 24 h at room temperature. Plasma was obtained by: 1) centrifugation for 10 min at 400 x g; 2) centrifugation for 10 min at 400 x g, storage of the supernatant at -80˚C and recentrifugation for 1 min at maximum speed; 3) centrifugation for 20 min at 120 x g, centrifugation for 20 min at 360 x g (to separate plasma from platelets), platelet pellet washed in PBS and centrifuged at 360 g for 5 min; 4) centrifugation for 10 min at 1600 x g followed by centrifugation of the supernatant at 6000 x g for 10 min (Streck tubes only); or 5) centrifugation for 10 min at 1900 x g followed by centrifugation at 16000 x g. For centrifugation protocol 5, the effect of storing blood on ice and centrifugation at 4˚C was compared with room temperature storage and centrifugation of EDTA tubes. The effects of centrifugation speed on ctDNA fraction was investigated using blood from 8 cancer patients collected in EDTA (4 patients) and Streck tubes (4 patients). All plasma specimens were stored at -80˚C until cfDNA and cfRNA extraction. cfDNA was isolated using the Maxwell RSC ccfDNA large volume plasma kit. cfDNA concentration and KRAS mutational status determined using the ddPCR KRAS Screening Multiplex Kit and cfDNA concentration was also determined by real-time PCR amplification of an 82 bp fragment of Long Interspersed Nuclear Elements (LINE-1). The percentage of long DNA was determined by amplification of unspecified 425 and 82 bp amplicons. cfRNA was isolated using the miRNeasy Serum/Plasma Kit and reverse transcribed using the miScript II RT Kit. cfRNA yield and KRAS status were determined using the ddPCR KRAS Screening Multiplex Kit.

   Summary of Findings:

   Significantly more total cfDNA was obtained in plasma isolated by centrifugation for 10 min at 400 x g than in plasma obtained by the other protocols (P≤0.001, all for EDTA and P≤0.002, all for Streck) as determined by ddPCR, but there were no significant differences between the other plasma specimens.  Similarly, the quantification cycle (Cq) value for real-time PCR amplification of LINE-1 was lower in EDTA plasma obtained by centrifugation for 10 min at 400 x g than that obtained using any other method (P≤0.004) or Streck plasma obtained by centrifugation at 400 x g followed by at max speed for 1 min (P=0.043) or by centrifugation at 120 x g for 20 min, followed by 360 x g for 20 min (P=0.013). However, plasma obtained by centrifugation at 400 x g had a higher percentage of long (>400 bp) fragments, indicating more genomic contamination than other plasma specimens (P<0.0001, all for EDTA and P<0.024, all for Streck). When plasma centrifuged at 400 x g for 10 min was subsequently centrifuged for 1 min at maximum speed, the percentage of long fragments was still higher than in plasma obtained by centrifugation for 20 min at 120 x g followed by 20 min at 360 x g (P=0.003 for EDTA, and NS for Streck), centrifugation at 1600 x g for 10 min followed by 6000 x g for 10 min (not performed for EDTA, P=0.035 for Streck) and centrifugation at 1900 x g for 10 min followed by 16000 x g for 10 min (P=0.00011 for EDTA, regardless of refrigeration and P=0.031 for Streck). For all centrifugation protocols, the percentage of long fragments was higher in EDTA plasma than Streck plasma (P=0.001-0.011). KRAS ctDNA concentration and allele frequency was unaffected by centrifugation protocol with the exception of one patient in which plasma obtained by centrifugation at 400 x g was considered to be KRAS-wildtype and plasma generated by the other protocols was KRAS-mutated. Importantly, the KRAS-mutated ctDNA was not detected in the pellet except in the case with a very high mutated KRAS allele frequency (20.6-51.9%).

   For EDTA tubes, plasma obtained by centrifugation at 400 x g (platelet-rich plasma) had significantly higher levels of cfRNA than plasma generated by other methods; however, this was due to the higher concentration of platelets which had the highest cfRNA levels. As expected, centrifugation at 1900 x g for 10 min followed by 16000 x g for 10 min resulted in the lowest levels of cfRNA. cfRNA levels were very low in plasma from Streck tubes, regardless of centrifugation protocol, precluding analysis of centrifugation protocol effects. KRAS-mutated ctRNA was only detected in platelets from EDTA blood and was not detected in specimens from Streck tubes.

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Blood

   Preservative Types

   • None (Fresh)
   • Streck/BCT
   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma
   • Neoplastic - Not specified
   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Digital PCR
   DNA	Real-time qPCR
   RNA	Digital PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Type of collection container/solution	EDTA tube Streck tube
   Biospecimen Aliquots and Components	Centrifugation	Multiple durations compared Multiple speeds compared Multiple temperatures compared Different number of centrifugation steps compared
   Digital PCR Specific	Targeted nucleic acid	KRAS

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