Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

Search BRD Papers

Workshop report on the extraction of foetal DNA from maternal plasma.

Author(s): Legler TJ, Liu Z, Mavrou A, Finning K, Hromadnikova I, Galbiati S, Meaney C, Hultén MA, Crea F, Olsson ML, Maddocks DG, Huang D, Fisher SA, Sprenger-Haussels M, Soussan AA, van der Schoot CE

Publication: Prenat Diagn, 2007, Vol. 27, Page 824-9

PubMed ID: 17604339 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to compare the reliability of RhD detection and cell-free fetal DNA (cffDNA) yield among manual and automated DNA extraction methods. EDTA plasma obtained from 252 RhD-negative pregnant women was pooled based on RhD cycle threshold value to create three pools with different cell-free fetal DNA concentrations. DNA was distributed by an unspecified method to each of the 12 laboratories in the Special Non-Invasive Advance in Fetal and Neonatal Evaluation Network of Excellence (SAFE-NoE) as well as to Qiagen. After arrival each laboratory extracted ccfDNA from each of the plasma pools in triplicate by their chosen method(s). Fetal DNA was identified and quantified based on amplification of RhD exons 5 and 7.

   Summary of Findings:

   While amplification of RhD was possible from all three pools with differing ccfDNA concentrations when DNA was extracted with 5 of the 6 manual methods and all 5 automated methods examined, amplification of RhD failed in 10 out of 11 reactions when specimens with intermediate or low cffDNA concentration were extracted using the QIAamp midi kit. Amplification of RhD also became unreliable when specimens were extracted using the QIAamp mini kit and  the centrifugation step to dry the column was omitted. Importantly, cffDNA yield was highest when extracted manually using the QIAamp DSP Virus kit (p<0.005). Of the automated methods evaluated, the QIAamp virus MDx kit on the BioRobot MDx resulted in the highest yields from specimens with intermediate or high fetal cell-free DNA concentrations, but the yield from the low concentration pool was at the detection limit. In contrast, both the Tecan workstation with magnetic tips and Magna Pure LC kits performed well for specimens with low concentrations of cell-free DNA.

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Pregnant

   Platform:

   Analyte	Technology Platform
   DNA	Real-time qPCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	QIAamp DSP Virus Kit High Pure PCR Template Preparation Kit QIAamp DNA Blood Mini Kit QIAamp DNA Blood Midi Kit CST genomic DNA purification kit Magna Pure LC kit QIAamp virus MDx kit on BioRobot MDx MagAttract virus mini M48 kit on BioRobot M48 EZ1 virus mini kit on BioRobot EZ1 In-house protocol using magnetic beads Tecan workstation with magnetic tips

   View more