  The risk of false positive results in noninvasive prenatal diagnosis focused
on fetal gender and RhD status determination could be a problem in clinical
routine. This is because these tests are based on detection of presence of DNA
sequences with high population frequency and so there is the risk of sample
contamination during sample collection and processing. In our study the
different fragmentation of fetal and maternal DNA molecules present in maternal
circulation was utilized in identification of contaminated samples.
Amplification of Y-chromosome specific assays different in size was tested on
circulating DNA samples.
  Of the four tested assays two shorter (84 and 177 bp) showed expected qPCR
efficiency and have comparable amplification profiles. The difference in Ct
values between these two assays was found to be statistically significant in
comparison of fetal male and normal male samples (p<0.0001) as well as in
blinded pilot study performed on 10 artificially contaminated and 10
non-contaminated samples (F=34.4, p<0.0001) that were all identified correctly.
  Our results showed that differently sized assays performed well in detection
of external contamination of samples in noninvasive prenatal fetal gender test
and could be of help in clinical laboratories to minimize the risk of false
positive results.
