Circulating cell-free DNA undergoes significant decline in yield after prolonged storage time in both plasma and purified form.
Author(s): Yuwono NL, Boyd MAA, Henry CE, Werner B, Ford CE, Warton K
Publication: Clin Chem Lab Med, 2022, Vol. , Page
PubMed ID: 35643514 PubMed Review Paper? No
Purpose of Paper
This paper compared the stability of cell-free DNA (cfDNA) stored as extracted cfDNA or in plasma for up to 2 years and investigated the effects of plasma volume, sequential elution and hemolysis on cfDNA levels. cfDNA was quantified by real-time PCR amplification of ALU and TP53, and integrity was investigated by Qubit and amplification of 155 and 247 bp fragments of ALU. Plasma was collected from healthy women.
Conclusion of Paper
Although no differences in cfDNA levels were found between specimens that were not stored (fresh) and those stored for ≤1 month at -80°C as either plasma or extracted cfDNA, significant decreases in Qubit quantified cfDNA concentrations, TP53 and ALU levels, and the ratio of ALU 115/247 bp were found when plasma or extracted cfDNA was stored for 2 years. Extraction of cfDNA from 5 mL rather than 1 mL of plasma increased the level of TP53 but did not alter the levels of ALU. Concentrations of TP53 and ALU declined with each sequential elution, but the third elution still contained an average of 19.6% of the qPCR amplicons. The average standard deviation in the levels of ALU between replicate extractions was 12%. Levels of L1PA2 and ALU were comparable between hemolyzed and non-hemolyzed plasma.
Studies
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Study Purpose
This study compared the stability of cfDNA stored as extracted cell-free DNA (cfDNA) or in plasma for up to 2 years and investigated the effects of plasma volume, sequential elution and hemolysis on cfDNA levels. Blood was collected from 25 female volunteers in K2EDTA tubes. Plasma was separated by centrifugation at 2,500 g for ≥10 min at 4°C followed by 3,500 g for 10 min at 4°C. The resultant plasma was aliquoted and stored at -80°C. cfDNA was extracted from plasma using the QIAamp Circulating Nucleic Acid Kit with carrier RNA. cfDNA levels were quantified by real-time PCR amplification of TP-53 (105-bp), and ALU (115 bp) and using the Qubit dsDNA HS Assay Kit. The integrity of the DNA was investigated by calculating the ratio of 115 to 247 bp amplicons ALU. Effects of hemolysis were investigated by comparing cfDNA levels in seven pairs of matched non-hemolyzed (mean absorbance at 414 nm = 0.18 ±0.04) and spontaneously hemolyzed (mean absorbance at 414 nm = 0.36 ±0.10) plasma specimens that had been stored at -80°C for 16 months. The effect of storing plasma or cfDNA for 1 day, 1 week, or 1 month at -80°C was investigated by storing plasma/cfDNA from three patients and comparing values to baseline (values from analysis on day 0). The effect of storing plasma or cfDNA for 3 months, 6 months, 1 year and 2 years at -80°C was investigated by storing plasma/cfDNA from 10 patients and comparing values to values from matched plasma stored for 1 week or 6 months (ALU) and freshly extracted cfDNA, respectively. To test if plasma and elution volume alters cfDNA concentration, cfDNA levels were compared between extractions from 1 mL and 5 mL plasma eluted into 45 µL and 100 µL, respectively. To test if sequential elution could increase cfDNA yield, cfDNA was quantified in 25, 20 and 20 µL sequential elutions that were collected in separate tubes. The reproducibility of the extraction procedure was investigated by extracting cfDNA in triplicate from the plasma specimens of 12 women.
Summary of Findings:
There were no changes in levels of ALU or TP53, Qubit-quantified cfDNA levels, or the ratio of short to long ALU amplicons (115/247 bp) following 1 month of storage of plasma or extracted cfDNA at -80°C. However, significant effects of storage of extracted cfDNA or plasma for 2 years were found. Compared to plasma stored for 1 week, plasma stored for 1 year had significantly lower Qubit-quantified cfDNA concentrations (P<0.001) and when stored for 2 years a significant decline in TP53 concentration (P<0.0001). Similarly, there was significantly less ALU from plasma stored for 2 years and a lower ratio of ALU 115/247 bp in specimens stored for 1 year (but not 2 years) than plasma stored for 6 months (P<0.01 and P<0.0001, respectively). Compared to freshly extracted cfDNA, extracted cfDNA stored for 1 or 2 years had lower concentrations of TP53 (P<0.001, both) and a lower ratio of ALU 115/247 (P<0.05, both); extracted cfDNA stored for 1 year had a lower Qubit-quantified cfDNA concentration (P<0.0001); and extracted cfDNA stored for 2 years had significantly less ALU (P<0.01). Isolation of cfDNA from 5 mL rather than 1 mL of plasma increased the level of TP53 (P=0.0005) but did not alter levels of ALU. Concentrations of TP53 and ALU declined with each sequential elution, but the third elution still contained an average of 19.6% of the qPCR amplicons. The average standard deviation in the levels of ALU between replicate extractions was 12%. Levels of L1PA2 and ALU were comparable between hemolyzed and non-hemolyzed plasma.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA Real-time qPCR DNA Fluorometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Aliquot size/volume 1 mL
5 mL
Storage Storage conditions As plasma
As extracted cfDNA
Analyte Extraction and Purification Rehydration of dried sample/specimen First elution
Second elution
Third elution
Biospecimen Aliquots and Components Hemolysis Absent
Present
Storage Storage duration 0 days
1 day
1 week
1 month
3 months
6 months
1 year
2 years