Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

Search BRD Papers

Evaluation of automated techniques for extraction of circulating cell-free DNA for implementation in standardized high-throughput workflows.

Author(s): Lehle S, Emons J, Hack CC, Heindl F, Hein A, Preuß C, Seitz K, Zahn AL, Beckmann MW, Fasching PA, Ruebner M, Huebner H

Publication: Sci Rep, 2023, Vol. 13, Page 373

PubMed ID: 36611077 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to compare ccfDNA yield and fragment size and mitochondrial ccfDNA copy number between two automated ccfDNA extraction methods. Multiple methods were used to quantify ccfDNA.  Blood was collected from thirty women with breast cancer (mean age 62.4 years) into Streck Cell-free DNA BCT blood collection tubes. Within 24 h of collection, plasma was isolated by centrifugation at 1600 g for 10 min followed by 16,000 g for 10 min. Plasma aliquots were frozen at -80°C. ccfDNA was extracted from plasma using the EZ1&2 ccfDNA field test kit on a EZ2 Connect Instrument and the Maxwell RSC ccfDNA plasma kit with a Maxwell RSC instrument. ccfDNA was storedfrozen at -20°C until quantification using the QuantiFluor dsDNA System Kit and the Agilent Cell-free DNA ScreenTape Assay.  ALU was quantified by real-time PCR and fragment size was assessed by amplification of 60 and 247 bp ALU amplicons. Mitochondrial DNA was quantified by real-time PCR amplification of a 65 bp fragment of the mitochondrial genome.

   Summary of Findings:

   The yield of ccfDNA was higher when extraction was with the EZ2 than the Maxwell Kit (P<0.0001 by fluorometer, qPCR, and TapeStation), and yields were correlated between the two extraction methods regardless of the quantification method (r=0.9618 for fluorometer, r=0.9959 for qPCR, and r=0.9881 for TapeStation). Higher cfDNA yields were obtained using the EZ2 Kit than the Maxwell Kit from all thirty specimens (median of 2.9 ng or more).  The proportion of extracted DNA that was ccfDNA was modestly correlated between extraction methods (r=0.6207, P<0.0001) and no significant differences in the proportion of ccfDNA were found between extraction methods. ccfDNA isolated with the EZ2 Kit had more copies of the 60 bp fragment and fewer copies of the 247 bp fragment of ALU relative to  the Maxwell Kit (3.8 x10⁸ versus 2.9 x 10⁸ copies/mL, P<0.0001 and 1.6 x10⁸ versus 2.0 x 10⁸ copies/mL, P<0.0001, respectively).  Consequently, the percentage of long (247 bp) to short (60 bp) ALU fragments was higher when isolation was with the Maxwell Kit than the EZ2 Kit (P<0.0001) and this difference occurred in all thirty specimens.  The mean copy number of mitochondrial cfDNA was significantly higher when ccfDNA was isolated using the EZ2 Kit rather than the Maxwell Kit (median 15.9-fold, P<0.0001). The number of copies of mitochondrial DNA were not correlated between extraction methods (r=0.1363). Compared to the Maxwell Kit, the EZ2 Kit accommodated a wider range of plasma volumes (1-8 mL versus 1 mL) and had a shorter total runtime (66 versus 80 min) but had a longer hands-on time (30 min versus 10 min).

    

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Automated electrophoresis/Bioanalyzer
   DNA	Fluorometry
   DNA	Real-time qPCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	EZ1&2 ccfDNA field test kit Maxwell RSC ccfDNA plasma kit
   Real-time qPCR Specific	Length of gene fragment	60 bp 247 bp
   Real-time qPCR Specific	Targeted nucleic acid	ALU (nuclear) Mitochondrial DNA
   Fluorometry Specific	Technology platform	Fluorometry Real-time Tapestation

   View more