A new methodology to preserve the original proportion and integrity of cell-free fetal DNA in maternal plasma during sample processing and storage.

Author(s): Fernando MR, Chen K, Norton S, Krzyzanowski G, Bourne D, Hunsley B, Ryan WL, Bassett C

Publication: Prenat Diagn, 2010, Vol. 30, Page 418-24

PubMed ID: 20306459 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of collection tube type and room temperature pre-centrifugation storage of whole blood on the quantity of total and fetal cell-free DNA in plasma. The effect of WGA on detection of SRY was also investigated. Blood was transported at ambient temperatures to the manufacturer of the Streck cell-free BCT DNA tubes within 2 h of collection. Aliquots were removed from each tube at each experimental time-point and centrifuged to obtain plasma. DNA was extracted from plasma using the NucleoSpin Plasma XS kit and stored frozen until analysis by real-time PCR. Half of the DNA from plasma stored as whole blood in BCT tubes for 14 days was amplified using the REPLI-g UltraFast kit before PCR while the other half was used for PCR directly. Total DNA levels were quantified by real-time PCR amplification of the Ras-association domain family 1 isoform A (RASSF1A) promoter. Fetal DNA was quantified by real-time PCR amplification of the RASSF1A promoter after digestion of unmethylated (maternal) RASSF1A with the restriction enzyme BstU1.

   Summary of Findings:

   The percentage of fetal DNA in maternal plasma was higher in specimens collected in the third trimester than the first trimester of pregnancy. Among first trimester specimens, a higher percentage of fetal DNA was found in plasma collected in BCT tubes than EDTA tubes (6.8% versus 4.05%), but for third trimester specimens, the percentage of fetal DNA was higher in plasma collected in EDTA tubes than BCT tubes (13.6% versus 12.6%), but significance was not determined. When first trimester blood was stored in EDTA tubes for 24 h or more prior to centrifugation, more cell-free DNA was isolated from plasma than when blood was centrifuged immediately (p<0.05), but the percentage of fetal DNA was significantly lower when blood was stored in EDTA tubes for 24 h or more before centrifugation than when centrifuged after 3 h (p<0.05). In contrast, there was no effect of storing blood in BCT tubes for 14 days before centrifugation on the amount of cell-free DNA in plasma or the percentage of fetal DNA compared to unstored blood, but there was slightly less DNA in plasma from blood collected and stored for 3 h in BCT tubes than EDTA tubes (p<0.05). WGA of the SRY region was successful in cell-free fetal DNA from blood stored for 14 days in BCT tubes before isolation of plasma, but the authors did not investigate if the same was true from blood stored in EDTA tubes.

   Biospecimens

   • Bodily Fluid - Blood
   • Bodily Fluid - Plasma

   Preservative Types

   • None (Fresh)
   • Streck/BCT

   Diagnoses:

   • Pregnant

   Platform:

   Analyte	Technology Platform
   DNA	Real-time qPCR
   DNA	Whole genome amplification

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Diagnosis/ patient condition	First-trimester of pregnancy Third-trimester of pregnancy
   Biospecimen Acquisition	Type of collection container/solution	K3EDTA tubes Streck cell-free BCT DNA tubes
   Storage	Time at room temperature	3 h 24 h 48 h 72 h 7 days 14 days
   Real-time qPCR Specific	Targeted nucleic acid	SRY Beta actin Total RASSF1A Methylated RASSF1A
   Whole genome amplification Specific	Nucleic acid amplification	Repli-G Amplified Not amplified
   Biospecimen Aliquots and Components	Centrifugation	Centrifugation delays investigated
   Biospecimen Preservation	Type of fixation/preservation	Blood collection tube additive None (fresh)

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