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A novel method for extracting circulating cell-free DNA from whole blood samples and its utility in the non-invasive prenatal test.

Author(s): Zhong H, Zeng L, Tao M, Ye Y, Wang Y, Hou L, Wu M, Liu H, Zhang H, Tang M

Publication: Prenat Diagn, 2022, Vol. 42, Page 1173-1181

PubMed ID: 35818872 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to compare yield, fragment size, and the specificity and sensitivity for trisomy detection in cfDNA that was either isolated directly from whole blood or extracted from plasma of pregnant women. EDTA blood was obtained from 366 pregnant women (12-24 weeks gestation) and within 8 h was either processed to plasma or used directly for cfDNA extraction. Plasma was obtained by centrifugation (speed and duration not specified) and cfDNA was extracted using the QIAamp Circulating Nucleic Acid Kit or the MGIEasy Circulating DNA Isolation Kit. cfDNA was extracted directly from whole blood using a modification of the magnetic bead-based Whole Blood Cell‐Free DNA Extraction Kit. cfDNA concentrations were quantified using a Qubit dsDNA HS Assay. Fragment length was evaluated by digital PCR amplification of 136 and 420 bp fragments of β-actin. To investigate the effects of extraction method on DNA yield and fragment size, DNA was extracted from whole blood of 22 pregnant women directly by the magnetic bead-based method and from matched plasma using the QIAamp Kit.  To investigate the effects of extraction on detection of tumor DNA, a single blood specimen was spiked with 5% and 0.5% DNA from an EGFR‐T790M tumor cell-line and was divided and used for direct extraction with the magnetic bead-based method or plasma was isolated and DNA was extracted with the MGIEasy Kit. To investigate if direct extraction from whole blood could be implemented as non-invasive prenatal testing, sequencing was conducted on 302 matched cfDNA extracts from whole blood and from plasma with the MGIEasy Kit using the MGISEQ‐2000RS high‐throughput system and used for detection of T21, T18 and T13.

   Summary of Findings:

   cfDNA yield was higher when cfDNA was extracted from plasma using the QIAamp Kit than extraction directly from whole blood using the magnetic-bead based method (4.90±0.50 ng/mL versus 4.34±0.41 ng/mL, P=0.015). The number of copies of the 136 bp fragment of β-actin was also higher in cfDNA samples isolated from plasma using the QIAamp Kit than when cfDNA was isolated directly from whole blood, but the difference was not significant (P=0.064); and, there was no effect on the 420 bp fragment (P=0.534).  The detected mutation ratio was higher than expected for both extraction workflows (cfDNA extracted directly from whole blood; cfDNA extracted from plasma), but detected levels were similar. The sensitivity and specificity of trisomy detection were the same for both cfDNA extraction workflows (cfDNA isolated directly from blood or from plasma). Trisomy detection using either plasma or whole blood for cfDNA extraction had a sensitivity of 100% and a specificity of 99.65%, with 282 of the 283 negative samples and 19/19 positive samples detected.

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Blood

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Pregnant

   Platform:

   Analyte	Technology Platform
   DNA	Fluorometry
   DNA	Next generation sequencing
   DNA	Digital PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Blood and blood products	Plasma Whole blood
   Analyte Extraction and Purification	Specimen format	cfDNA extracted from blood cfDNA extracted from plasma

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