Predominant hematopoietic origin of cell-free DNA in plasma and serum after sex-mismatched bone marrow transplantation.

Author(s): Lui YY, Chik KW, Chiu RW, Ho CY, Lam CW, Lo YM

Publication: Clin Chem, 2002, Vol. 48, Page 421-7

PubMed ID: 11861434 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 delayed centrifugation of blood, repeated aspiration of blood, and extra centrifugation steps on the levels of DNA in EDTA plasma from healthy individuals and to compare the concentration of DNA in EDTA plasma and serum from sex mismatched bone-marrow transplant recipients. DNA was extracted by the QIAamp blood kit and stored at -20 degrees C. Blood was processed by a single centrifugation at 3000 x g for 10 min (once), a second centrifugation at 16000 x g for 10 min (twice), or gentle shaking, a second centrifugation at 3000 x g, and a third centrifugation at 16000 x g for 10 min (three times). Additionally, some blood was processed as in the three times protocol after which the pellet was resuspended by pipetting and then recentrifuged at 16000 x g for 10 min (four times).

   Summary of Findings:

   When whole blood was stored for 6 h at room temperature prior to centrifugation (only serum time-point investigated), serum had an average DNA concentration that was 6-fold higher than in plasma (p<0.001), and most of this DNA was shown to be of hematopoietic origin. DNA concentration in plasma was not affected by repeatedly aspirating blood through a 23-gauge needle and syringe compared to no additional aspiration, storing whole blood at room temperature for 3 or 6 h prior to centrifugation instead of 0 h, or the number of centrifugation steps. There was no effect of the patients having a prior bone marrow transplant on the concentration of cell-free DNA in plasma, but the Y chromosome was detected in women who underwent a sex-mismatched bone marrow transplant.

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Serum
   • Bodily Fluid - Blood

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Normal
   • Other diagnoses

   Platform:

   Analyte	Technology Platform
   DNA	Real-time qPCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Time at room temperature	0 h 3 h 6 h
   Biospecimen Aliquots and Components	Blood and blood products	Plasma Serum
   Preaquisition	Diagnosis/ patient condition	Sex mismatched bone-marrow transplant recipient Normal
   Biospecimen Aliquots and Components	Biospecimen components	No further manipulation Repeatedly aspirated through 23 gauge needle with a syringe
   Biospecimen Aliquots and Components	Hemolysis	Fine needle aspiration-induced Not induced
   Real-time qPCR Specific	Targeted nucleic acid	Sex-determining region of Y chromosome Beta-globin
   Biospecimen Aliquots and Components	Centrifugation	Centrifugation delays investigated Different number of centrifugation steps compared

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