Controls to validate plasma samples for cell free DNA quantification.
Author(s): Pallisgaard N, Spindler KL, Andersen RF, Brandslund I, Jakobsen A
Publication: Clin Chim Acta, 2015, Vol. 446, Page 141-6
PubMed ID: 25896958 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to determine if cellular contamination of plasma specimens can be detected by real-time PCR and to establish cut-off values for cell-free DNA analysis.
Conclusion of Paper
Copy number of rearranged immunoglobulin H (IgH) was more sensitive than beta-2-microglobulin (B2M) to blood contamination of plasma. Using rearranged IgH copy number, the authors were able to determine 7 of 100 plasma specimens were contaminated with blood cells and establish that a threshold of ≤0.1% copies of the rearranged IgH per B2M was required for accurate cell-free DNA quantification.
Studies
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Study Purpose
The purpose of this study was to determine the effects of plasma contamination with blood cells on the levels of the B-cell-specific rearranged IgH and B2M. The authors used this information to determine contamination cut-off values for analysis of cell-free DNA in plasma. Leftover blood specimens from lactose intolerance screening from 76 patients that were stored at -20°C for less than 4 weeks were used to determine the ratio of IgH to B2M in blood specimens. A single plasma pool created from the plasma of 3 volunteers was used to determine the effect of blood contamination of plasma. Additionally, EDTA plasma was obtained from healthy individuals was used to investigate if blood contamination could be detected in plasma specimens. All plasma specimens were obtained by a single centrifugation and were stored at -80°C until use. Cell-free DNA was isolated using the QIAsymphony virus/bacteria midi kit and DNA was isolated from whole blood using the MaxWell 16 Blood DNA kit.
Summary of Findings:
On average, there was 0.4% as many copies of the rearranged immunoglobulin H (IgH) gene as the beta-2-microglobulin (B2M) gene in blood specimens, indicating that 0.4% of the DNA from blood comes from B-cells. The ratio of rearranged IgH to B2M exhibited extensive variation among the 76 blood specimens (>10-fold) variation in the ratio). Blood contamination of plasma was detectable by an increase in rearranged IgH copy number when plasma was spiked with as little as 0.3 µL blood in 1200 µL plasma, while contamination was first detectable by B2M when 1-3 µL of blood were added to 1200 µL of plasma. Using rearranged IgH copy number, the authors were able to determine that 7 of 100 plasma specimens were contaminated and established a threshold of ≤0.1% copies of the rearranged IgH per B2M for accurate cell-free DNA quantification.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Not specified
- Normal
Platform:
Analyte Technology Platform DNA Real-time qPCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Biospecimen components 0.1-1000 µL blood added
No blood added
Real-time qPCR Specific Targeted nucleic acid IgH
B2M
Biospecimen Aliquots and Components Blood and blood products Red blood cells
Plasma