Workshop report on the extraction of foetal DNA from maternal plasma.
OBJECTIVE
Cell free foetal DNA (cff DNA) extracted from maternal plasma is now recognized as a potential source for prenatal diagnosis but the methodology is currently not well standardized. To evaluate different manual and automated DNA extraction methods with a view to developing standards, an International Workshop was performed.


METHODS
Three plasma pools from RhD-negative pregnant women, a DNA standard, real-time-PCR protocol, primers and probes for RHD were sent to 12 laboratories and also to one company (Qiagen, Hilden, Germany). In pre-tests, pool 3 showed a low cff DNA concentration, pool 1 showed a higher concentration and pool 2 an intermediate concentration.


RESULTS
The QIAamp DSP Virus Kit, the High Pure PCR Template Preparation Kit, an in-house protocol using the QIAamp DNA Blood Mini Kit, the CST genomic DNA purification kit, the Magna Pure LC, the MDx, the M48, the EZ1 and an in-house protocol using magnetic beads for manual and automated extraction were the methods that were able to reliably detect foetal RHD. The best results were obtained with the QIAamp DSP Virus Kit. The QIAamp DNA Blood Mini Kit showed very comparable results in laboratories that followed the manufacturer's protocol and started with > or = 500 microL plasma. One participant using the QIAamp DNA Blood Midi Kit failed to detect reliably RHD in pool 3.


CONCLUSIONS
This workshop initiated a standardization process for extraction of cff DNA in maternal plasma. The highest yield was obtained by the QIAamp DSP Virus Kit, a result that will be evaluated in more detail in future studies.
Legler
Tobias J
TJ
Department of Transfusion Medicine, University of Göttingen, Göttingen, Germany. tlegler@med.uni-goettingen.de
Liu
Zhong
Z
Mavrou
Ariadni
A
Finning
Kirstin
K
Hromadnikova
Ilona
I
Galbiati
Silvia
S
Meaney
Cathy
C
Hultén
Maj A
MA
Crea
Francesco
F
Olsson
Martin L
ML
Maddocks
Deborah G
DG
Huang
Dorothy
D
Fisher
Sylvia Armstrong
SA
Sprenger-Haussels
Markus
M
Soussan
Aicha Ait
AA
van der Schoot
C Ellen
CE
D001331:Automation; D004247:DNA / Q000097:blood* / Q000302:isolation & purification*; D005260:Female; D005333:Fetus / Q000378:metabolism; D005820:Genetic Testing / Q000379:methods* / Q000592:standards; D006801:Humans; D033261:Maternal-Fetal Relations; D009035:Mothers; D016133:Polymerase Chain Reaction / Q000379:methods; D011247:Pregnancy; D011296:Prenatal Diagnosis / Q000379:methods / Q000592:standards
D004247:DNA