Implementing prenatal diagnosis based on cell-free fetal DNA: accurate identification of factors affecting fetal DNA yield.

Author(s): Barrett AN, Zimmermann BG, Wang D, Holloway A, Chitty LS

Publication: PLoS One, 2011, Vol. 6, Page e25202

PubMed ID: 21998643 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 storage of whole blood at 4 degrees C or room temperature in cell-stabilizing tubes or K3EDTA tubes on quantification of maternal, fetal and y-chromosomal DNA in plasma. Blood collected into K3EDTA tubes was stored at 4 degrees C (0, 4, 8, 24 h) or room temperature (0, 8, 24 or 72 h) before centrifugation. Blood collected into cell stabilizing tubes was stored for 0, 24 or 72 h at room temperature before centrifugation. All plasma was centrifuged twice and transferred to a new tube for storage at -80 degrees C until DNA extraction. Unless otherwise specified, DNA was extracted using the QIAamp circulating nucleic acid kit.

   Summary of Findings:

   When whole blood was stored at 4 degrees C in K3EDTA tubes prior to processing, the resultant plasma had more total DNA (chromosome 18) after 24 h (p=0.016), a higher percentage of the long sequence of the Y chromosome (%Long) after 8 h (p=0.02), and a lower percentage of fetal DNA after 24 h (p=0.019). However, comparable numbers of copies of the Y chromosome were found in plasma that was isolated immediately and plasma from whole blood that was stored at 4 degrees C in K3EDTA tubes. Storage of whole blood in K3EDTA tubes at room temperature led to similar increases in plasma DNA and %Long DNA as in specimens stored at 4 degrees C, but larger changes were noted when whole blood was stored for 72 h before processing as opposed to 24 h. When whole blood was stored in cell-stabilizing tubes at room temperature before centrifugation, DNA , %Long DNA and the percentage of circulating cell free DNA were stable, but a slightly lower Y chromosome copy number was found when whole blood was stored for 24 h at room temperature in cell stabilizing tubes than when whole blood was not stored (p=0.04) or stored for 72 h. Extraction of DNA using the QIAamp circulating nucleic acid kit allowed for gender determination in 11 of 14 blood specimens that were inconclusive when DNA had been extracted using QIAamp MinElute Virus Spin.

   Biospecimens

   • Bodily Fluid - Blood
   • Bodily Fluid - Plasma

   Preservative Types

   • None (Fresh)
   • Streck/BCT

   Diagnoses:

   • Pregnant

   Platform:

   Analyte	Technology Platform
   DNA	Real-time qPCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage duration	0 h 4 h 8 h 24 h 72 h
   Storage	Storage temperature	4 degrees C Room temperature
   Biospecimen Acquisition	Type of collection container/solution	Stabilizing Streck tube EDTA tube
   Real-time qPCR Specific	Targeted nucleic acid	DYS14 Chromosome 18
   Analyte Extraction and Purification	Analyte isolation method	QIAamp circulating nucleic acid kit QIAamp MinElute Virus Spin
   Biospecimen Aliquots and Components	Centrifugation	Centrifugation delays investigated
   Biospecimen Preservation	Type of fixation/preservation	None (fresh) Blood collection tube additive Refrigeration

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