Influence of delay in blood-processing on the level of cell-free fetal DNA in maternal plasma sample / 第二军医大学学报

ABSTRACT

Objective: To investigate the influencing factors of cell-free fetal DNA level in the maternal plasma during blood-processing. Methods: Aliquots of blood samples from pregnant women with male fetus were processed at 6 h and 36 h after sampling. The SRY and β-globin genes and the total DNA level were quantified by real-time quantitative PCR. Death of white blood cells was assayed by flow cytometry after stained with Annexin V/PI. The plasma DNase activity was assayed by radial enzyme-diffusion method and plasma lactate dehydrogenases (LDH) by rate method. Results: A 36 hour delay in blood-processing led to a significant increase in the total DNA and decrease in the fetal DNA (SRY gene) in the maternal plasma. The ratio of fetal DNA decreased from (10.3±5.6) % at 6 h after sampling to (3.0±2.1) % at 36 h after sampling under 4°C (P< 0.05). No dead cells were identified in the blood sample 6 h after sampling; however, apoptosis and necrosis of white blood cells were identified 36 h after sampling. The activity of LDH at 36 h was significantly higher than that at 6 h (P<0.05). Radial enzyme-diffusion result showed that, though greatly decreased at 4°C, the DNase was still able to degrade DNA. Conclusion: Delay in blood-processing can lead to increase of the total free DNA in maternal plasma but decrease of fetal DNA, which might be related to the death of white blood cells and degradation of fetal DNA by plasma DNase, so the extraction of fetal DNA should be done as early as possible after sampling (within 6 h).