Comparison of Circulating Cell-Free DNA Extraction Methods for Downstream Analysis in Cancer Patients.
Author(s): Leest PV, Boonstra PA, Elst AT, Kempen LCV, Tibbesma M, Koopmans J, Miedema A, Tamminga M, Groen HJM, Reyners AKL, Schuuring E
Publication: Cancers (Basel), 2020, Vol. 12, Page
PubMed ID: 32414097 PubMed Review Paper? No
Purpose of Paper
This paper compared the cell-free DNA (cfDNA) yield, integrity, fragment size, and variant allele frequency (VAF) in EDTA plasma or citrated diagnostic leukapheresis plasma when different extraction methods were used.
Conclusion of Paper
The yield of cfDNA from plasma varied among the extraction kits with significantly higher yields obtained from EDTA plasma using the QIAamp Circulating Nucleic Acid (CNA) Kit than the Maxwell RSC ccfDNA Plasma Kit (RSC) or the Zymo Quick ccfDNA Serum & Plasma Kit (Zymo) and from citrated diagnostic leukapheresis plasma using the QIAamp MinElute ccfDNA Midi Kit (MinElute) rather than the CNA kit. Significantly more of the 137 bp and 420 bp fragments of β-actin were amplified per mL of EDTA plasma when extraction was with the CNA Kit than the RSC or Zymo kits, but the MinElute Kit yielded more 50-250 bp fragments and a nonsignificant trend toward more 137 bp, 420 bp, and 1950 bp fragments of β-actin from citrated diagnostic leukapheresis plasma than the CNA Kit. Although there was no significant difference in the copies of the 137 bp fragment per ng cfDNA among the traditional EDTA plasma extraction methods (CNA, Zymo, and RSC), but the high volume MinElute Kit yielded more copies of the 137 bp fragment per ng cfDNA than the CNA Kit from citrated diagnostic leukapheresis plasma. Further, the ratio of short to medium fragments using the bioanalyzer and 137 to 420 bp amplicons did not differ among traditional extraction methods (CNA, Zymo, and RSC), but there was a higher ratio of small to medium sized fragments when extraction from citrated diagnostic leukapheresis plasma was with the MinElute Kit rather than the CNA Kit.
More copies of mutant ctDNA per mL EDTA plasma or per ng of cfDNA were obtained in two patients’ specimens when extraction was with the CNA Kit but more copies cfDNA per mL plasma were found when extraction was with RSC in the other two patients’ specimens. However, a higher number of copies of mutant cfDNA per mL of citrated diagnostic leukapheresis plasma was found when extraction was with MinElute rather than the CNA Kit from specimens for all six patients. In three of four EDTA plasma specimens, the VAF was higher when extracted using the RSC than the CNA kit but the VAF was higher using the MinElute Kit rather than the CNA Kit in all six citrated diagnostic leukapheresis plasma specimens.
Studies
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Study Purpose
This study compared the cfDNA yield, integrity, fragment size, and VAF in EDTA when three different extraction methods were used. Plasma was collected from seven patients with gastrointestinal stromal tumor (GIST) into EDTA tubes and 11 patients with non-small cell lung carcinoma (NCSLC) into Streck cell-free BCT tubes. Plasma was obtained from GIST specimens by centrifugation for 10 min at 820 x g followed by 10 min at 16,000 x g within 4 h of blood collection. Plasma was obtained from NSCLC blood in BCT tubes by centrifugation for 10 min at 1600 x g followed by 10 min at 16,000 x g within 24 h of blood collection. cfDNA was extracted from plasma using the QIAamp Circulating Nucleic Acid (CNA) Kit, the Maxwell RSC ccfDNA Plasma Kit (RSC), and the Zymo Quick ccfDNA Serum & Plasma Kit according to the manufacturer’s recommendations. cfDNA was quantified using the Qubit dsDNA HS Assay Kit and integrity assessed using a fragment analyzer. Amplificability of cfDNA was assessed using multiplex ddPCR amplification of 137 bp, 420 bp, and 1950 bp fragments of β-actin. KRAS G12/G13, BRAF G466V, PDGFRA M844_D846del, TP53 H179R, TP53 R273H, and TP53 Y205C mutations were detected using mutation-specific ddPCR.
Summary of Findings:
The yield of cfDNA from plasma varied among the extraction kits with significantly higher yields obtained using the CNA than RSC or Zymo kits (P<0.001 and P<0.01, respectively). Importantly, the highest amount of cfDNA was obtained in 18 of 21 specimens using the CNA Kit and the lowest amount of cfDNA was obtained in 14 of 21 specimens using the RSC Kit. Significantly more of the 137 and 420 bp fragments of β-actin were amplified per mL of plasma when extraction was with the CNA than RSC (P<0.05 and P<0.01, respectively) or Zymo (P<0.0001 and P<0.001, respectively) kits and significantly higher amplification of the 1950 bp fragment was observed when extraction was with the CNA Kit than the RSC Kit (P<0.001). However, there was no significant difference in the copies of the 137 bp fragment when normalized to amount of cfDNA input. Further, the ratio of short to medium fragments using the bioanalyzer and 137 to 420 bp amplicons did not differ among extraction methods, but the ratio of 137 to 1950 bp fragments was significantly higher when extraction was with the RSC than the Zymo Kit (P<0.05), indicating a higher percentage of gDNA when extraction was with the Zymo Kit.
More copies of mutant ctDNA per mL plasma or per ng cfDNA were obtained when extraction was with the CNA Kit in two patients’ specimens but more copies ctDNA per mL plasma were found when extraction was with RSC for the other two patients’ specimens. However, the VAF was higher when extracted using RSC than the CNA Kit in three of four specimens.
Biospecimens
Preservative Types
- None (Fresh)
- Streck/BCT
Diagnoses:
- Neoplastic - Sarcoma
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Automated electrophoresis/Bioanalyzer DNA Digital PCR DNA Fluorometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Analyte Extraction and Purification Analyte isolation method QIAamp Circulating Nucleic Acid (CNA) Kit
Maxwell RSC ccfDNA Plasma Kit (RSC)
Zymo Quick ccfDNA Serum & Plasma Kit
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Study Purpose
This study compared yield, integrity, and amplificability of cfDNA extracted from plasma using the MinElute ccfDNA Midi Kit with that using the QIAamp CNA Kit. Citrate-dextrose blood from six NSCLC patients was collected by aphereisis using the continuous mononuclear cell (cMNC) protocol. Diagnostic leukapheresis plasma was stored at -80°C within 30 min of collection. Plasma was thawed and centrifuged at 1600 x g for 10 min before extraction with the QIAamp MinElute ccfDNA Midi Kit and the QIAamp Circulating Nucleic Acid (CNA) Kit according to the manufacturer’s recommendations. cfDNA was quantified using the Qubit dsDNA HS Assay Kit and integrity assessed using a fragment analyzer. Amplificability of cfDNA was assessed using multiplex ddPCR amplification of 137 bp, 420 bp, and 1950 bp fragments of β-actin. KRAS G12/G13, BRAF G466V, PDGFRA M844_D846del, TP53 H179R, TP53 R273H, and TP53 Y205C mutations were detected using mutation-specific ddPCR.
Summary of Findings:
There was 3-fold more ng cfDNA per mL of plasma when the CNA Kit was used rather than the MinElute Kit (P<0.001). Importantly, more cfDNA was obtained using the CNA method than the MinElute method from each of the six patients. Analysis using the fragment analyzer showed more 50-250 bp fragments in cfDNA extracted using the MinElute Kit compared to the CNA kit. A non-significant trend toward more amplifiable copies per mL of plasma of the 137 bp, 420 bp, and 1950 bp fragments of β-actin were observed when extraction was with the CNA Kit. However, more copies of the 137 bp fragment per ng cfDNA were present when extraction was with MinElute rather than the CNA Kit (P<0.0001) and similar increases were noted for the 420 bp and 1950 bp fragments. Interestingly, while the fragment analyzer showed a higher ratio of small to medium sized fragments when extraction was with the MinElute Kit (3.10 versus 1.81, P<0.05), no differences in the ratio of the 137 bp to the 420 bp or 1950 bp amplicon were found. A higher number of copies of mutant cfDNA per mL of plasma was found when extraction was with MinElute rather than the CNA Kit and there was a higher VAF in all four specimens when extraction was with MinElute.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Fluorometry DNA Digital PCR DNA Automated electrophoresis/Bioanalyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Analyte Extraction and Purification Analyte isolation method QIAamp MinElute ccfDNA midi Kit
QIAamp Circulating Nucleic Acid (CNA) Kit