Effect of formaldehyde treatment on the recovery of cell-free fetal DNA from maternal plasma at different processing times.
Author(s): Zhang Y, Li Q, Hui N, Fei M, Hu Z, Sun S
Publication: Clin Chim Acta, 2008, Vol. 397, Page 60-4
PubMed ID: 18692490 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to determine the effects of formaldehyde fixation and refrigerated storage of blood prior to centrifugation on cell-free DNA (cfDNA) concentrations and DNAse activity in plasma and the number of intact cells in the cell pellet.
Conclusion of Paper
Refrigerated storage of blood before centrifugation led to increased maternal and decreased fetal cfDNA in plasma and fewer intact cells in the cell pellet. The addition of formaldehyde to the blood attenuated the effects of delayed centrifugation and inhibited plasma DNAse activity.
Studies
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Study Purpose
The purpose of this study was to determine the effects of formaldehyde fixation and refrigerated storage of blood prior to centrifugation on cfDNA concentrations and DNAse activity in plasma and the number of intact cells in the cell pellet. EDTA anticoagulated blood was collected from 24 women at the end of the third trimester of pregnancy (36-40 weeks). DNA was extracted from blood using the QIAamp DNA blood mini kit. Fetal DNA was quantified by amplification of sex determining region Y (SRY) and total DNA was quantified by amplification of β-globin.
Summary of Findings:
Maternal cfDNA levels increased and fetal cfDNA levels decreased when blood was refrigerated for 24 h (p<0.05) or 36 h (p<0.05), respectively, before centrifugation instead of processed immediately. Further, the cell pellet from blood centrifuged after delays of 24 h or more had fewer intact cells (p<0.05). While formaldehyde fixation of the blood had no effect on the number of intact cells or percentage of fetal DNA when centrifugation was delayed by 6 h, the addition of formaldehyde resulted in more intact cells and a higher percentage of fetal DNA when blood was refrigerated for 36 h before centrifugation (p<0.05) such that both the percentage of fetal DNA and intact cells were comparable to those in specimens centrifuged after 6 h. Importantly, despite refrigeration, plasma retained low levels of DNAse activity which was inhibited by the addition of formaldehyde.
Biospecimens
Preservative Types
- Other Preservative
Diagnoses:
- Pregnant
Platform:
Analyte Technology Platform DNA Real-time qPCR Protein Fluorometry Cell count/volume Light microscopy Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation Refrigeration
Formaldehyde
Real-time qPCR Specific Targeted nucleic acid SRY
Beta-globin
Storage Storage duration 0 h
6 h
24 h
36 h
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated