Quantification Of Cell-free Fetal DNA In Maternal Plasma And Investigation Of The Impacts And Underlying Mechanisms Of Delay In Blood-Processing And Formaldehyde-treatment

Cell-free fetal DNA in the plasma of pregnant women has potential for non-invasive prenatal diagnosis. Detection of fetal DNA sequences in maternal plasma has been used for the prenatal assessment of sex-linked diseases, fetal RhD status and diagnosis of single gene disorders. Fetal DNA can be detected as early as 5 weeks of gestation, before the time period invasive testing is usually done. Early detection of fetal DNA could have a profound influence on the management of pregnancy.The application of fetal DNA technology in prenatal diagnosis has been limited by the low proportion of fetal DNA in a high background of maternal DNA. In normal pregnancies, fetal DNA represents only 5-7% of the total DNA in maternal plasma. The overwhelming amount of background maternal DNA in the samples has rendered the differentiation of more subtle genetic differences between mother and child (e.g. point mutations) considerably more difficult. Methods to increase the recovery of fetal DNA from maternal plasma are of great significance and constantly being explored.To date, multiple factors that may affect the analysis of plasma DNA have been explored, including use of different anticoagulants, centrifugation speed, and DNA extraction methods. Here, we confirmed that delay in processing can also be another factor affecting plasma DNA analysis. So we design the partⅠand PartⅡ.PartⅠImpact of Delay in Blood-Processing on Cell-free DNA Concentrations in Maternal Plasma. Objective: To investigate the impact of delay in blood-processing on cell-free DNA in maternal plasma. Methods: Aliquots of blood sample from each pregnant woman with male fetus were processed at different time (6h and 36h) after sampling. The SRY andβ-globin gene were quantified for fetal and total DNA in each plasma sample by real-time Quantitative PCR, Death of blood cells were assayed by Flow Cytometry after stained with AnnexinV/PI, Plasma DNase activity was assayed by radial enzyme-diffusion method and plasma LDH by rate method. Results: the concentration of total DNA remained constant during the first 6 h of storage, but markedly increased at 24 h and increased to a higher level at 36 h; the quantity of fetal DNA stayed relatively stable through 0-24 h of storage. A small decrease in the level of fetal DNA was detected at 24 h, but did not reach statistical significance. At 36 h, the decrease in the level of fetal DNA became more obvious, and reached statistical significance. Due to such a differential effect on the level of maternal and fetal DNA, the delay in blood processing led to a dramatic decline in the percentage of fetal DNA [from original baseline of 15.5% (8.9-25%) (median and IQR)] at Oh and 15.8% (8.0-21.9%) at 6h, declined to 13.3% (5.6-17.8%) at 24h and 4.9% (3.4-7.3%) at 36h.Conclusions: Undue delay(>24 h) in blood-processing impart a significant effect on the quantification of free DNA in maternal plasma. It resulted in maternal blood cell lysis and release of extra maternal free DNA into plasma, and a simultaneous decrease in the quantity of free fetal DNA due to hydrolysis by plasma DNase.PartⅡThe Impacts and Underlying Mechanisms of Formaldehyde-treatment on the Concentrations of Cell-free DNA in Maternal Plasma. Objective: To evaluate the effect of formaldehyde and investigate the underlying mechanism. Methods: Blood samples from pregnant women were treated or not treated with formaldehyde, and processed at different times. Total and fetal DNA in plasma was quantified by real-time polymerase chain reaction. Death and lysis of blood cells were assayed by trypan blue exclusion test. Plasma DNase activity was determined by the radial enzyme-diffusion method. Results: Formaldehyde addition showed no effect on the percentage of fetal DNA in samples processed 6h after blood collection. In samples processed at 36h, formaldehyde addition inhibited blood cell lysis and nuclease-mediated DNA degradation, thus markedly decreasing the concentration of total DNA and increasing the recovery of fetal DNA. The median (interquartile range) percentage of fetal DNA increased from 4.6% (3.8-6.8%) to 13.1% (10.3-17.0%). Conclusion: The effect of formaldehyde on the percentage of fetal DNA in maternal plasma depends on processing time and is associated with prevention of cell lysis and inhibition of plasma DNase activity.Overall, The data presented here suggest that within the first 6 h of blood sampling, no maternal blood cell lyses and releases extra maternal free DNA into plasma, meanwhile, the limited plasma DNase activity can not cause markedly change in the quantity of fetal DNA. Therefore, formaldehyde addition will offer less or no beneficial effect of fetal DNA enrichment under this situation. However, undue delay in sample processing could result in maternal blood cell lysis and release of extra maternal free DNA into plasma, and a simultaneous decrease in the quantity of free fetal DNA due to hydrolysis by plasma DNase. Under such situation, formaldehyde addition may confer advantages on the enrichment of fetal DNA through preventing cells from lysis and protect fetal DNA from degradation. Therefore, To maitain the percentage of free fetal DNA and facilitate its analysis, we emphasize the need for a short time delay between blood sampling and processing, blood sample should be stored at 4℃and processed rapidly (within 6 h) after sampling. In case that delay is anticipated, we recommend use of formaldehyde to keep cell lysis to a minimum and preserve the integrity of fetal DNA. Further optimization and standardization of blood processing protocol is still needed to provide a robust, clinically relevant platform for future application of circulating fetal DNA in noninvasive prenatal diagnosis.