**4.6. Factors explaining false positive and false negative results**

**4.4. Mosaicism**

414 Next Generation Sequencing - Advances, Applications and Challenges

societies.

**4.5. Twins**

trisomy 21 [43].

In mosaic autosomal trisomies, the detection with NIPT is less effective compared to complete fetal trisomies. The major reason is that the representation of the fetal chromosome is only partial. The detection of a fetal mosaicism is dependent on the fetal fraction and on the percentage of abnormal cells in the mosaic. There have been two relevant studies investigating the ability of detecting mosaicisms showing far less sensitive results for mosaic aneuploidies with NGS. Since cell-free "fetal" DNA stems from the trophoblast, a confined placental mosaicism can be a reason for a false positive result. Also, maternal mosaicism can lead to false positive results. On the other hand, mosaicisms can be missed since it is more difficult to detect due to the lower percentage of abnormal cells [38]. However, mosaicism is found in approxi‐ mately 0.25% of pregnancies in women undergoing amniocentesis and conventional karyo‐ typing [39]. Finally, if NIPT is positive for a trisomy, the distinction of mosaic versus complete trisomy can only be made after karyotyping. This shows the importance of confirmation of the findings detected by NIPT through an invasive procedure as recommended by the professional

Most of the approaches using whole genome NGS and targeted NGS offer an analysis for twin pregnancies. The analysis, however, is more complex since maternal blood then carries the cell-free DNA from three individuals. For monozygotic twins that usually carry the same genetic information, the analysis can be made analogue to singletons. In dizygotic twins it is likely that only one fetus is affected from an aneuploidy. NGS relies on a small increase of reads identified for the trisomic chromosome. The total cell-free fetal DNA fraction is larger compared to singleton pregnancies most likely due to a larger placental volume [40] and this would be an advantage for NGS compared to singletons. However, this advantage is reduced by the fact that in most cases only half of the fetal DNA fraction stems from the aneuploid fetus. Furthermore, it is possible that the cell-free-DNA, which is found in the maternal circulation, is not equally released half by half from each of the two fetuses. So the aneuploid fetal fraction could be lower compared to the euploid fetus [41]. To circumvent the mistakes of the total fetal fraction, the lower fetal fraction is used for the risk assessment. A consequence

of this policy is that the rate of non-reporting will be higher for twin pregnancies.

The published data from twin pregnancies now count almost one thousand analyzed twin pregnancies [40,42–47]. The SNP-targeted approach does not yet offer twin analysis. The most recent analysis on 515 twin pregnancies showed a test failure rate of 5.6% compared to 1.7% in singletons. The median lower individual fetal fraction was lower than in singletons (8.7% versus 11.7%). Among the 351 pregnancies with complete follow-up and with a test result, there were no false positives among 334 euploid fetuses. All 5 cases of trisomy 18 were detected, but there was 1 false negative case of trisomy 21 among the 12 pregnancies discordant for

The analysis for twins, however, will not reach a diagnostic level with NGS from maternal blood since it will never be able to tell which one of the fetuses is affected until this information

is acquired via separate analysis of each twin through an invasive procedure.

Even though NIPT is the best available screening test for the detection of the three most common aneuploidies trisomy 21,13, and 18, the method of analyzing cell-free DNA in maternal blood by NGS, false negative, as well as false positive results are possible. To understand the technology, one has to keep in mind two essential things: first, cell-free "fetal" DNA stems from the trophoblast rather than from the fetus itself [7], and second, the cell-free DNA analysis of maternal and fetal cell-free DNA in NIPT uses maternal blood as the DNA source for the analysis. As known from chorionic villous sampling for many years, there is the phenomenon of feto-placental mosaicism in which only the cytotrophoblast but not the fetus is affected by the aneuploid cell line or vice versa [48]. If only the cytotrophoblast is affected, this would lead to a false positive result while a false negative NIPT result is expected if only the fetus but not the trophoblast is affected from the aneuploid cell line.

Another potential cause for a false positive result could stem from cell-free DNA from an unrecognized vanishing twin [42,49]. Fetal aneuploidy is a common reason for early fetal loss and has been described as a reason for a false positive NIPT result [42]. In fact, an additional fetal haplotype was identified in 0.42% of over 30,000 routine NIPT samples from a SNP-based assay [49].

If an abnormal karyotype is present in the mother, this might lead to a false positive result. False positive findings have been reported associated with maternal malignancies [50] or with maternal X-chromosome abnormalities in otherwise healthy women [51]. As mentioned before, the depth of sequencing and a low fetal fraction can be the causes of false negative results due to the counting technology.
