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Effects of blood-processing protocols on fetal and total DNA quantification in maternal plasma.

Author(s): Chiu RW, Poon LL, Lau TK, Leung TN, Wong EM, Lo YM

Publication: Clin Chem, 2001, Vol. 47, Page 1607-13

PubMed ID: 11514393 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 centrifugation, Percoll gradient and filtration steps on the quantification of maternal and fetal cell-free DNA in EDTA plasma. Blood was processed within 2 h of venipuncture. Plasma was stored in polypropylene tubes at -20 degrees C until analysis.

   Summary of Findings:

   When plasma was isolated by centrifugation on a Percoll gradient with no further purification, the concentration of beta-globin DNA was higher than when the plasma was isolated by a single centrifugation at 1600 x g, alone or in combination with a centrifugation at 16000 x g and/or filtration, or when further purified after Percoll gradient by centrifugation at 16000 x g or filtration. Further, significantly higher beta-globin concentrations were found in plasma isolated by centrifugation on a Percoll gradient, with or without further purification, than in plasma isolated by centrifugation at 1600 x g and 16000 x g steps with filtration between the two centrifugations (p<0.05). Significantly higher beta-globin concentrations were also found in plasma isolated by centrifugation at 800 x g without further purification then when plasma was subsequently filtered (p<0.05). When plasma was obtained from the same women on three different days, the standard deviation of the beta-globin concentration was significantly higher in plasma obtained by a single centrifugation at 800 x g than when filtered after centrifugation (p<0.05). However, there was no effect of blood processing protocol on the concentration of SRY in plasma.

   Biospecimens

   • Bodily Fluid - Blood
   • 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)
   Biospecimen Aliquots and Components	Filtration	0.2 uM filtered Unfiltered
   Biospecimen Acquisition	Time of biospecimen collection	Day 1 Day 2 Day 3
   Real-time qPCR Specific	Targeted nucleic acid	SRY Beta-globin
   Biospecimen Aliquots and Components	Blood processing method	Percoll-gradiant separation Centrifugation Centrifugation and filtration Percoll-gradient separation and filtration
   Biospecimen Aliquots and Components	Centrifugation	Different number of centrifugation steps compared Multiple speeds compared

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