NIH, National Cancer Institute, Division of Cancer Treatment and Diagnosis (DCTD) NIH - National Institutes of Health National Cancer Institute DCTD - Division of Cancer Treatment and Diagnosis

Evaluation of pre-analytical factors affecting plasma DNA analysis.

Author(s): Markus H, Contente-Cuomo T, Farooq M, Liang WS, Borad MJ, Sivakumar S, Gollins S, Tran NL, Dhruv HD, Berens ME, Bryce A, Sekulic A, Ribas A, Trent JM, LoRusso PM, Murtaza M

Publication: Sci Rep, 2018, Vol. 8, Page 7375

PubMed ID: 29743667 PubMed Review Paper? No

Purpose of Paper

This paper compared cell-free DNA (cfDNA) extraction methods, blood collection protocols (tube type and delays), and use of prospectively collected versus retrospectively collected specimens on cfDNA yield and the percentage of low molecular weight (LMW) DNA. The effect of collection protocol on global nucleotide mismatch rate (GMR) was also investigated.

Conclusion of Paper

Significant differences between extraction kits on cfDNA yield and fragment size were observed. The highest median yields and fraction of LMW DNA were obtained using the spin column-based QIAamp Circulating Nucleic Acid kit. Plasma from specimens collected in K2EDTA tubes and processed after 1 h and those collected in Streck cell-free DNA tubes and processed after 24 or 72 had similar cfDNA yield, mean genome equivalents LMW DNA, fraction of LMW DNA, and global nucleotide mismatch rate (GMR). LMW fractions were significantly lower in retrospectively-analyzed archival specimens than in plasma prospectively collected for cfDNA analysis.

Studies

  1. Study Purpose

    This study compared results of using different cfDNA extraction kits on the cfDNA yield and fragment size using a commercially-available K2EDTA plasma pool purchased from Bioreclamation IVT. Plasma was thawed, aliquoted, and stored at -80˚C upon receipt.  cfDNA aliquots were centrifuged at 14,000 x g for 10 min and supernatants were used for DNA extraction with one of the seven kits. For each kit, 10 replicate extractions were performed following the manufacturer’s instructions. DNA was quantified by a droplet digital PCR assay that amplified five short amplicons with a mean size of 71 bp and four long amplicons with a mean size of 471 bp and determined genome equivalents by dividing by five and four, respectively. LMW DNA was quantified as the difference between the number of genome equivalents as determined by the short amplicons and the long amplicons.

    Summary of Findings:

    Significant differences between extraction kits were observed for cfDNA yield (P=5.01 x 10-11) and fragment size (P=.1.16 x 10-11). The highest median yields and fraction of LMW DNA were obtained using the spin column-based QIAamp Circulating Nucleic Acid kit. Although the yield using the QIAamp CNA kit was not significantly different from that using the second best performing kit (the spin column-based Norgen Plasma/Serum Cell-Free Circulating DNA Purification Midi Kit), the yields with the Norgen kit were much more variable among replicates.  Use of the best performing magnetic bead-based kit (Life Technologies MagMAX Cell-free DNA Isolation kit) resulted in a significantly lower yield than the QIAamp kit (P=0.946 x 10-5) and the remaining spin column-based (Analytik Jena PME free-circulating DNA Extraction Kit) and three magnetic bead-based kits (Promega Maxwell RSC ccfDNA Plasma kit, NeoGeneStar Circulating Cell Free DNA kit, and Omega Bio-Tek Mag-Bind Circulating DNA) resulted in even lower yields. However, the LMW fraction was comparable between the QIAamp Circulating Nucleic Acid kit and the Life Technologies MagMAX Cell-free DNA Isolation kit (89% and 90%, respectively), but was higher (Norgen Plasma/Serum Cell-Free Circulating DNA Purification Midi Kit and Promega Maxwell RSC ccfDNA Plasma kit) or lower (Analytik Jena PME free-circulating DNA Extraction Kit, NeoGeneStar Circulating Cell Free DNA kit and Omega Bio-Tek Mag-Bind Circulating DNA) using the remaining kits.

    Biospecimens
    Preservative Types
    • Frozen
    Diagnoses:
    • Not specified
    Platform:
    AnalyteTechnology Platform
    DNA Digital PCR
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Analyte Extraction and Purification Analyte isolation method QIAamp Circulating Nucleic Acid kit
    Norgen Plasma/Serum Cell-Free Circulating DNA Purification Midi Kit
    Analytik Jena PME free-circulating DNA Extraction Kit
    Life Technologies MagMAX Cell-free DNA Isolation kit
    Promega Maxwell RSC ccfDNA Plasma kit
    NeoGeneStar Circulating Cell Free DNA kit
    Omega Bio-Tek Mag-Bind Circulating DNA
  2. Study Purpose

    This study compared cfDNA yield, percentage low molecular weight DNA, and global nucleotide mismatch rate (GMR) in blood collected in EDTA tubes and processed within 1 h and in blood collected in BCT tubes and processed 24 and 72 h later. Blood was collected from 23 healthy volunteers into one K2EDTA Vacutainer tube and two Streck Cell-free DNA tubes. Plasma was obtained by centrifugation at 820 x g for 10 min at room temperature 1 h (K2EDTA tube), 24 h (BCT), and 72 h (BCT) after collection. Plasma was aliquoted and recentrifuged at 16000 x g for 10 min and supernatants were stored frozen at -80˚C until cfDNA extraction using the QIAamp Circulating Nucleic Acid kit. cfDNA yield and fragment size were determined by ddPCR. Sequencing libraries were constructed from all three specimens from 12 individuals and from an additional seven of the specimens using ThruPLEX DNA-Seq with enrichment using NimbleGen SeqCap EZ Human Exome v3 kit. Libraries were sequenced using an Illumina miSeq.

    Summary of Findings:

    The cfDNA yield and mean genome equivalents LMW DNA were comparable in plasma collected in K2EDTA tubes subjected to a 1 h processing delay and specimens collected in cell-free DNA tubes and subjected to 24 or 72 h processing delays. Similarly, the fraction of LMW DNA was comparable for EDTA plasma, BCT plasma subjected to a 24 h delay, and BCT plasma subjected to a 72 h delay (87%, 88%, and 90%, respectively). Similarly, the GMR was modestly to strongly correlated among the tube types (r=0.647-0.758, P<0.05) and there was no significant difference between tube types in GMR when adjusted based on depth of coverage.

    Biospecimens
    Preservative Types
    • Frozen
    • Streck/BCT
    Diagnoses:
    • Normal
    Platform:
    AnalyteTechnology Platform
    DNA Next generation sequencing
    DNA Digital PCR
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Biospecimen Acquisition Type of collection container/solution K2 EDTA Vacutainer
    Streck Cell-free DNA Blood Collection Tube
    Storage Time at room temperature 1 h
    24 h
    72 h
    Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
  3. Study Purpose

    This study compared the cfDNA fragment size in archival plasma specimens and plasma collected prospectively for cfDNA analysis. Archival specimens consisted of EDTA plasma from 43 patients with melanoma and 27 patients with rectal cancer that were processed and stored at -80˚C within 24 h of collection. Prospectively collected specimens consisted of EDTA plasma collected from 37 patients with melanoma, 11 with glioblastoma, and 32 with cholangiocarcinoma that were processed and stored at -80˚C within 1-3 h of collection. Additional prospective specimens were collected from 69 healthy individuals into EDTA tubes and processed after 1 h and into Streck tubes that were processed after 24 or 72 h. DNA was extracted using the QIAamp Circulating Nucleic Acid kit. cfDNA yield and fragment size were determined by ddPCR.

    Summary of Findings:

    LMW fractions were significantly lower in retrospectively-analyzed archival specimens than in plasma prospectively collected for cfDNA analysis (82.5% versus 91.1%, P2.302 x 10-7), which the authors attribute to increased contamination with peripheral cell DNA.

    Biospecimens
    Preservative Types
    • Frozen
    • Streck/BCT
    Diagnoses:
    • Neoplastic - Carcinoma
    • Normal
    • Neoplastic - Melanoma
    Platform:
    AnalyteTechnology Platform
    DNA Digital PCR
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Storage Storage duration 1-3 h
    <24 h
    Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated

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