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

High-throughput isolation of circulating tumor DNA: a comparison of automated platforms.

Author(s): van Dessel LF, Vitale SR, Helmijr JCA, Wilting SM, van der Vlugt-Daane M, Oomen-de Hoop E, Sleijfer S, Martens JWM, Jansen MPHM, Lolkema MP

Publication: Mol Oncol, 2019, Vol. 13, Page 392-402

PubMed ID: 30516338 PubMed Review Paper? No

Purpose of Paper

This paper investigated the effects of carrier RNA (cRNA) addition, extraction method, and blood collection tube type on the yield and fragment size of cell-free DNA (cfDNA). The effect of automated extraction method on genotyping results was also investigated.

Conclusion of Paper

The use of cRNA reduced the yield of small (136 bp) DNA without altering recovery of large DNA (? bp) and, while it increased the cfDNA yield as determined by Qubit, it did not alter yield by the PCR-based TERT assay. Overall, the CellSave tubes were found to be compatible with the automated extraction methods, but use of CellSave tubes resulted in significantly fewer 2000 bp fragments than when EDTA tubes were used, regardless of extraction kit. Further, blood from CellSave tubes produced lower cfDNA yields with the Maxwell kit and fewer copies of the 136 bp fragment when extracted using the QIAsymphony kit when compared to blood from EDTA tubes. Although overall cfDNA yields and copies of the different size fragment from the plasma of healthy patients were comparable between extraction methods, cfDNA yields from the plasma of cancer patients were higher when extraction was with QIAamp or QIAsymphony kits than the MAXwell kit. Use of the Maxwell kit resulted in fewer 136 bp fragments than the QIAamp kit and less of the 2000 bp fragment than the QIAsymphony kit. The number of wildtype and variant copies were more comparable to that in QIAamp when extraction was with QIAsymphony than the Maxwell kit. While significantly fewer wildtype copies were detected using the Maxwell than QIAsymphony kit, the variant allele frequency (VAF) was comparable between QIAsymphony and Maxwell kits.

Studies

  1. Study Purpose

    The purpose of this study was to determine if the use of cRNA during extraction with QIAsymphony or Maxwell increased the cfDNA recovery. Blood was collected from an unspecified number of healthy donors into either two CellSave tubes or one EDTA tube and one CellSave tube. All specimens were stored at room temperature until plasma isolation. Within 24 h (EDTA specimens) or 72 h (CellSave specimens), plasma was obtained by centrifugation at 1711 x g for 10 min at room temperature followed by 12 000 x g for 10 min at room temperature, pooled, and frozen at -80˚C until extraction. cfDNA was extracted from plasma aliquots using the QIAamp circulating nucleic acid kit, QIAsymphony SP Circulating DNA Kit, or the Maxwell RSC LV ccfDNA Plasma Custom Kit. The effect of cRNA concentration was tested by adding 0.25-4 µg to plasma along with synthetic DNA from a plant. Extracted cfDNA was quantified using Qubit Quant-iT dsDNA high Sensitivity assay and the TaqMan copy number reference assay TERT. DNA fragment size was investigated by digital PCR amplification of three beta-actin fragments (136 bp, 420 bp, and 2000 bp).

    Summary of Findings:

    Carrier RNA (cRNA) increased the cfDNA yield during extraction with QIAsymphony (P<0.001) and Maxwell RSC (P<0.001) as determined by Qubit, but this increase was not observed when quantified by the TERT qPCR assay. cRNA reduced the recovery of small (136 bp) DNA (P=0.001 for Maxwell and P<0.001 for QIAsymphony) without changing the recovery of large (420 and 2000 bp) cfDNA. cRNA increased the recovery efficiency for plant DNA (P=0.02 for Maxwell and P=0.04 for QIAsymphony) which was approximately 30% higher using the QIAsymphony than Maxwell kit, regardless of the addition of cRNA (P<0.001).

    Biospecimens
    Preservative Types
    • Frozen
    Diagnoses:
    • Normal
    Platform:
    AnalyteTechnology Platform
    DNA Fluorometry
    DNA Real-time qPCR
    DNA Digital PCR
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Analyte Extraction and Purification Analyte isolation method QIAsymphony SP Circulating DNA Kit
    Maxwell RSC LV ccfDNA Plasma Custom Kit
    With Carrier RNA
    No Carrier RNA added
    Digital PCR Specific Length of gene fragment 136 bp
    420 bp
    2000 bp
    Fluorometry Specific Technology platform Real-time PCR
  2. Study Purpose

    The purpose of this study was to determine if CellSave tubes are compatible with automated extraction methods. Blood was collected from 10 healthy donors into either an EDTA or a CellSave tube. All specimens were stored at room temperature until plasma isolation. Within 24 h (EDTA specimens) or 72 h (CellSave specimens), plasma was obtained by centrifugation at 1711 x g for 10 min at room temperature followed by 12 000 x g for 10 min at room temperature and frozen at -80˚C until extraction. cfDNA was extracted from plasma aliquots using the QIAsymphony SP Circulating DNA Kit with the addition of carrier RNA or the Maxwell RSC LV ccfDNA Plasma Custom Kit. Extracted cfDNA was quantified using Qubit Quant-iT dsDNA High Sensitivity Assay and the TaqMan Copy Number Reference Assay TERT. DNA fragment size was investigated by digital PCR amplification of three beta-actin fragments (136 bp, 420 bp, and 2000 bp).

    Summary of Findings:

    cfDNA yields were significant lower from CellSave tubes than EDTA tubes when extracted using the Maxwell kit (P=0.008), but there was no difference in cfDNA yield between CellSave and EDTA tubes when extracted using the QIAsymphony kit. The number of 2000 bp fragments of beta-actin was significantly lower in specimens in Cell Save tubes than EDTA tubes, regardless of extraction method (P=0.008 for both extraction methods), but the 136 bp fragment was only significantly lower in CellSave than EDTA tubes when extracted using QIAsymphony (P=0.04). Recovery of plant DNA was unaffected by collection tube type.

    Biospecimens
    Preservative Types
    • Frozen
    Diagnoses:
    • Normal
    Platform:
    AnalyteTechnology Platform
    DNA Digital PCR
    DNA Real-time qPCR
    DNA Fluorometry
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Biospecimen Acquisition Type of collection container/solution EDTA tube
    CellSave tube
    Digital PCR Specific Length of gene fragment 136 bp
    420 bp
    2000 bp
    Fluorometry Specific Technology platform Real-time PCR
    Analyte Extraction and Purification Analyte isolation method QIAsymphony SP Circulating DNA Kit
    Maxwell RSC LV ccfDNA Plasma Custom Kit
  3. Study Purpose

    The purpose of this study was to investigate if extraction method affects cfDNA yield, fragment size, and variant detection in plasma from healthy donors and cancer patients. Blood was collected from ten healthy donors and ten patients with a metastatic cancer (5 colorectal cancer, 1 non-small cell lung cancer, and 4 melanoma) into CellSave tubes. After less than 72 h at room temperature, plasma was obtained by centrifugation at 1711 x g for 10 min at room temperature followed by 12 000 x g for 10 min at room temperature and frozen at -80˚C until extraction. cfDNA was extracted from plasma aliquots using the QIAamp circulating nucleic acid kit, QIAsymphony SP Circulating DNA Kit with the addition of carrier RNA, or the Maxwell RSC LV ccfDNA Plasma Custom Kit. Extracted cfDNA was quantified using Qubit Quant-iT dsDNA High Sensitivity Assay and the TaqMan Copy Number Reference Assay TERT. DNA fragment size was investigated by digital PCR amplification of three beta-actin fragments (136 bp, 420 bp, and 2000 bp). Genotyping was performed using TaqMan SNP genotyping assays.

    Summary of Findings:

    Although overall cfDNA yields from the plasma of healthy patients were comparable between extraction methods, cfDNA yields from the plasma of cancer patients were higher when extraction was with QIAamp or QIAsymphony kits than with the MAXwell kit (P=0.002, both). Recovery of plant DNA was also significantly higher using the QIAamp or QIAsymphony kits than the MAXwell kit (P<0.001, both). Fragment size analysis showed comparable amounts of each fragment between extraction methods in plasma from healthy patients, but less of the 136 bp fragment was observed in cancer patient specimens extracted with the MAXwell kit than the QIAamp kit (P<0.01) and less of the 2000 bp fragment when extracted using the MAXwell kit than the QIAsymphony kit (P=0.002). The number of wildtype and variant copies were more comparable to that in QIAamp when extraction was with QIAsymphony kit than the Maxwell kit. While significantly fewer wildtype copies were detected using the Maxwell kit than QIAsymphony kit (P<0.001), the variant allele frequency (VAF) was comparable between QIAsymphony and Maxwell kits.

    Biospecimens
    Preservative Types
    • Frozen
    Diagnoses:
    • Neoplastic - Carcinoma
    • Normal
    • Neoplastic - Melanoma
    Platform:
    AnalyteTechnology Platform
    DNA Digital PCR
    DNA SNP assay
    DNA Real-time qPCR
    DNA Fluorometry
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Analyte Extraction and Purification Analyte isolation method QIAsymphony SP Circulating DNA Kit
    Maxwell RSC LV ccfDNA Plasma Custom Kit
    QIAamp circulating nucleic acid kit
    Digital PCR Specific Length of gene fragment 136 bp
    420 bp
    2000 bp
    Fluorometry Specific Technology platform Real-time PCR

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