Objective: Cell-free fetal DNA (cff-DNA) is fetal DNA circulating in the maternal blood stream. The most studies have focused on how to extract this fetal source but what is less discussed in articles is determine the factors that affect the quality of cff-DNA. As this issue is so important in diagnosis fetal monogenic hereditary diseases, in this article the factors that can affect the quality of cff-DNA were evaluated. Method: From 6 pregnant women with gestational age of 11-15 weeks, after signing consent form, 12 ml blood was taken in k3 EDTA tube and the effect of storage time, temperature and centrifuge method, on the quality of cff-DNA were evaluated in 12 different modes according to Taguchi algorithm. Firstly DNA fraction was determined by Nanodrop then based on difference between length of fetal and maternal DNA base pairs, Real Time PCR was used to confirm the cff-DNA fraction. Results: The results demonstrated, in the samples that stored at 4ºc and their plasma was separated in two steps (5min 800g and 5min 1600g), maternal DNA didn’t affect cff-DNA fraction quantity, up to 24h; but by increasing incubation time or/and temperature, maternal DNA degraded to fragments as same size as fetal DNA, therefor fetal fraction was increased falsely. Conclusion: To have a cff-DNA with the minimum maternal DNA interferes, plasma must be separated as quickly as possible and samples must be stored in 4ºc. In conditions, which, samples must be transferred to reference lab, instead of whole blood, plasma should be transferred.
