BACKGROUND: Analysis of cell free fetal (cff) DNA in maternal plasma is used 
routinely for non invasive prenatal diagnosis (NIPD) of fetal sex determination, 
fetal rhesus D status and some single gene disorders. True positive results rely 
on detection of the fetal target being analysed. No amplification of the target 
may be interpreted either as a true negative result or a false negative result 
due to the absence or very low levels of cffDNA. The hypermethylated RASSF1A 
promoter has been reported as a universal fetal marker to confirm the presence 
of cffDNA. Using methylation-sensitive restriction enzymes hypomethylated 
maternal sequences are digested leaving hypermethylated fetal sequences 
detectable. Complete digestion of maternal sequences is required to eliminate 
false positive results.
METHODS: cfDNA was extracted from maternal plasma (n = 90) and digested with 
methylation-sensitive and insensitive restriction enzymes. Analysis of RASSF1A, 
SRY and DYS14 was performed by real-time PCR.
RESULTS: Hypermethylated RASSF1A was amplified for 79 samples (88%) indicating 
the presence of cffDNA. SRY real time PCR results and fetal sex at delivery were 
100% accurate. Eleven samples (12%) had no detectable hypermethylated RASSF1A 
and 10 of these (91%) had gestational ages less than 7 weeks 2 days. Six of 
these samples were male at delivery, five had inconclusive results for SRY 
analysis and one sample had no amplifiable SRY.
CONCLUSION: Use of this assay for the detection of hypermethylated RASSF1A as a 
universal fetal marker has the potential to improve the diagnostic reliability 
of NIPD for fetal sex determination and single gene disorders.