Pre-analytical conditions in non-invasive prenatal testing of cell-free fetal RHD.
Author(s): Clausen FB, Jakobsen TR, Rieneck K, Krog GR, Nielsen LK, Tabor A, Dziegiel MH
Publication: PLoS One, 2013, Vol. 8, Page e76990
PubMed ID: 24204719 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to determine the effects of transport duration and temperature and the use of an automated pipetting system on the detection and measurement of circulating cell-free DNA (ccfDNA) and circulating cell-free fetal DNA (ccffDNA) in plasma from pregnant women.
Conclusion of Paper
CcffDNA was detected in plasma regardless of blood transport duration, but total ccfDNA increased in the first 2-4 days of transport and then plateaued. Slightly less ccffDNA was obtained from plasma when the average ambient temperature during blood transport was 5-10°C than when it was -5-0°C or 15-20°C. Further, glyceraldehyde 3-phosphate dehydrogenase GAPDH cycle threshold (CT) values were higher (24-30 versus 21-24), when the average ambient temperature during transport was lower (2.4°C versus 10.3°C). However, the ccffDNA CT values were not different between these groups. When the PCR was set-up using the QIAgility pipetting system rather than QiaSymphony, 1.2-fold more DNA was detected.
Studies
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Study Purpose
The purpose of this study was to determine the effects of transport duration and temperature and the use of an automated pipetting system on the detection and measurement of ccfDNA and ccffDNA in plasma from pregnant women. EDTA blood from pregnant (23-28 weeks gestation) Rhesus blood group D (RhD) negative women was transported by an unspecified method at ambient temperature to a central site within Denmark. At the central laboratory, blood was centrifuged and DNA was extracted from plasma using the QiaSymphony kit. The effect of transport time was studied using 110 specimens, and the effect of transport temperature was studied using 1939 specimens. Real time qPCR of RhD was used to quantify ccffDNA while GAPDH was used to quantify total ccfDNA.
Summary of Findings:
CcffDNA was detected in plasma regardless of blood transport duration, but total ccfDNA increased in the first 2-4 days of transport and then plateaued. Slightly less ccffDNA was obtained from plasma when the average ambient temperature during blood transport was 5-10°C than when it was -5-0°C (p<0.05) or 15-20°C (p<0.05). Further, GAPDH CT values were higher (21-24 versus 24-30) when the average ambient temperature was lower (2.4°C versus 10.3°C). However, the ccffDNA CT values were not different between these groups. When the PCR was set-up using the QIAgility pipetting system rather than QiaSymphony, 1.2-fold more DNA was detected.
Biospecimens
Preservative Types
- None (Fresh)
Diagnoses:
- Pregnant
Platform:
Analyte Technology Platform DNA Real-time qPCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Between site transportation method Unspecified transport method
Storage Specimen transport duration/condition 2 days
3 days
4 days
5 days
6 days
7 days
-5-0°C
0-5°C
5-10°C
10-15°C
15-20°C
20-25°C
Real-time qPCR Specific Technology platform QIAgility pipetting system
QiaSymphony pipetting system