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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., Comment: 10 pages