BACKGROUND: Discovery of short cell free fetal DNA (cffDNA) fragments in 
maternal plasma has created major changes in the field of prenatal diagnosis. 
The use of cffDNA to set up noninvasive prenatal test is limited due to the low 
concentration of fetal DNA in maternal plasma therefore, employing a high 
efficiency extraction method leads to more accurate results. The aim of this 
study was to evaluate the efficiency of Triton/Heat/Phenol (THP) protocol in 
comparison with the QIAamp DNA Blood mini Kit for cffDNA purification.
METHODS: In order to evaluate the efficiency of THP protocol, DNA of Rhesus D 
(RhD) negative pregnant women's plasma was collected, then real-time PCR for RHD 
exon 7 was performed. The Ct value data of real time PCR obtained by two 
different methods were compared and after delivery serology test on cord blood 
was done to validate the real time PCR results.
RESULTS: The results indicated significant differences between two extraction 
methods (p=0.001). The mean±SD of Ct-value using THP protocol was 33.8±1.6 and 
36.1±2.47 using QIAamp DNA Blood mini Kit.
CONCLUSION: Our finding demonstrated that THP protocol was more effective than 
the QIAamp DNA Blood mini Kits for cffDNA extraction and lead to decrease the 
false negative results.