Effects of Preanalytical Factors on the Molecular Size of Cell-Free DNA in Blood.
Author(s): Chan K, Yeung S, Lui W, Rainter T, Lo YM
Publication: Clin Chem, 2005, Vol. 51, Page 781
PubMed ID: 15708950 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to investigate the impact of (1) clotting, (2) delayed separation of blood cells from plasma, (3 and 4) freeze thaw cycling of both plasma and DNA samples, and (5) prolonged frozen storage on the integrity of circulating DNA in plasma collected from healthy volunteers.
Conclusion of Paper
The integrity (yield and DNA size) of circulating DNA was impacted by clotting (serum versus plasma), sample storage at room temperature or 4 degrees C for 6 h or more, and repeated freeze/thaw cycling of plasma but not DNA samples. The authors recommend processing blood samples within 6 h, aliquoting samples, and extracting DNA prior to long term frozen storage.
Studies
-
Study Purpose
To determine if clotting (serum vs plasma) affects the integrity of circulating DNA.
Summary of Findings:
Fresh serum samples had a significantly greater DNA yield (leptin gene) compared to plasma samples. Further, the ratio of amplicons of differing size (201 bp/ 105 bp) was greater in serum samples, indicating an increase in the size of circulating DNA.
Biospecimens
Preservative Types
- None (Fresh)
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA Real-time qPCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Blood and blood products Plasma
Serum
Real-time qPCR Specific Length of gene fragment 105 bp
201 bp
-
Study Purpose
The purpose of this study was to determine if whole blood storage for 0, 6, or 24 h at 4 degrees C or room temperature adversely impacts the integrity of circulating DNA in plasma.
Summary of Findings:
DNA yield increased significantly after whole blood storage for 24 h at 4 degrees C or room temperature. Storage at room temperature, but not 4 degrees C, also increased the relative expression of long/short PCR amplicons.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA Real-time qPCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage temperature 0 degrees C
Room temperature
Storage Storage duration 0 h
6 h
24 h
-
Study Purpose
The purpose of this study was to determine if the number of freeze thaw cycles plasma samples are subjected to affects the integrity of circulating DNA.
Summary of Findings:
Plasma samples that underwent three freeze thaw cycles had significantly decreased PCR amplicon size ratios (201 bp / 105 bp), suggesting possible DNA fragmentation.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA Real-time qPCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Freeze/thaw cycling 0 cycles
1 cycle
3 cycles
Real-time qPCR Specific Length of gene fragment 105 bp
201 bp
-
Study Purpose
To determine if the number of freeze thaw cycles of DNA samples extracted from plasma affects the integrity of circulating DNA.
Summary of Findings:
DNA concentration and relative expression of amplicon sizes were not affected by freeze thaw cycling up to three times.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA Real-time qPCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Freeze/thaw cycling 0 cycles
1 cycle
3 cycles
Real-time qPCR Specific Length of gene fragment 105 bp
201 bp
-
Study Purpose
To determine if plasma storage at -80 degrees C for 2 weeks affects the integrity of circulating DNA.
Summary of Findings:
DNA yield and integrity were unaffected by prolonged plasma sample storage at -80 degrees C.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA Real-time qPCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Real-time qPCR Specific Length of gene fragment 105 bp
201 bp