Fetal fraction determination

The fetal fraction (FF), i.e. the amount of fetal cfDNA detected in the plasma sample analysed compared to the total cfDNA, is an important parameter to determine for the performing of NIPT tests, since with very low FF quantities the aneuploidies may not be detectable, and therefore produce false negatives results.
To reduce such risk, some NIPT methods currently in use utilize a cut-off of 4% of fetal fraction. With FF values lower than this cut-off, an inconclusive result is reported due to low quantity of fetal DNA and a new sample is required.

The FF≥4% limit needed to report a result was determined using a statistical model 2,4 and not through validation studies that determine the effective Limit of Detection (LOD) of the NIPT method, i.e. the lowest FF to which an aneuploidy can be detected.
A recent study 5,6 conducted by Genoma, had the aim of determining the actual LOD of Prenatalsafe test for trisomy 21, 18 and 13 and to evaluate the test performance at low fetal fractions (<4%) on clinical samples.
The study demonstrated that the main chromosomal aneuploidies can be reliably detected by Prenatalsafe test with FF≥2% (Picture 1). This allowed to reduce the incidence of inconclusive results due to low fetal fraction, from 8.7% (if a cut-off of 4% of FF was used), at 2.2% (using a cut-off of 2% of FF) (Picture 2). Furthermore, if the FF cut-off> 4% was used, 23.4% of the aneuploidies would not have been detected (Tables 1 and 2).

The same study showed that the incidence of aneuploidies is 6 times higher in samples with a low fetal fraction (2%<FF<4%) compared to those with FF>4%. An increased incidence of chromosomal aneuploidies, 4 to 10 times, was also reported by other studies in samples with an inconclusive result due to low fetal fraction (<4%) (Picture 3) 7,8. Therefore, by using the FF cut-off> 4%, samples with the highest risk of aneuploidy are excluded from the analysis.

In conclusion, the cut-off value of the FF must be related to the actual LOD of each NIPT method with respect to the specific technology used.

The study shows the importance of a NIPT technology able to analyse samples with low FF (2%<FF<4%), for:
• reducing the incidence of the blood samples;
• quickly allowing the identification of pregnancies at high risk of aneuploidies, with a low fetal fraction.


The importance of using a NIPT technology able to detect aneuploidies in samples with low Fetal Fraction
The most recent scientific literature and the Italian and International guidelines 1 agree to pay particular attention to the samples with a low fetal fraction (<%), as they resulted at greater risk (from 4 to 10 times) of chromosomal aneuploidies.

The high resolution sequencing technology used to perform Prenatalsafe test, allows detecting the aneuploidies even in samples with a low Fetal Fraction, unlike other low-resolution NIPT methods, which instead require a quantity of fetal DNA> 4%.

Figura 1
Figure 1: Determination of the LOD for Trisomy 21 in plasma samples with experimentally reconstructed FF. The LOD (that is the lowest FF with a detectable aneuploidy) is highlighted by the red dashed vertical line. The green circles represent the samples with aneuploidy detected; the red circles, instead, represent the samples in which the aneuploidy was not detected due to the low FF. The LOD for trisomy 21 was at FF≥2%

Figura 2
Figure 2: Distribution of the fetal fraction in samples investigated in the study with FAST 4.5 protocol 4,5

Figura 3
Figura 3: The incidence of aneuploidies increases from 4 to 10 times in samples with a low fetal fraction. Using a 4% cut-off for the FF, samples at increased risk of aneuploidy are excluded from the analysis. Requesting a new sampling increases the time to diagnose any aneuploidies and is effective in about 50% of the retested samples.

Tabella 1
Table 1. Aneuploidies detected by fetal cfDNA analysis and follow-up results by invasive prenatal diagnosis. Using a 4% cut-off for the FF, the 23,4% of aneuploidies would not have been detected.

1. Ministero della Salute. Linee-Guida Screening prenatale non invasivo basato sul DNA (Non Invasive Prenatal Testing – NIPT). Maggio 2015 - Committee Opinion No. 640: Cell-free DNA Screening for Fetal Aneuploidy. Obstet Gynecol 2015; 126:e31-7. - Society for Maternal-Fetal Medicine (SMFM) Publications Committee #36: Prenatal aneuploidy screening using cell-free DNA. Am J Obstet Gynecol 2015;212:711-6. - Benn P, Borrell A, Chiu RW, et al. Position statement from the Chromosome Abnormality Screening Committee on behalf of the Board of the International Society for Prenatal Diagnosis. Prenat Diagn 2015; 35:725-34. 2. Ashoor G, Poon L, Syngelaki A, et al. Fetal fraction inmaternal plasma cell-free DNA at 11-13 weeks' gestation: effect of maternal and fetal factors. Fetal Diagn Ther 2012; 31:237-43. 3. Canick JA, Palomaki GE, Kloza EM, et al. The impact of maternal plasma DNA fetal fraction on next generation sequencing tests for common fetal aneuploidies. Prenat Diagn 2013;33:667-74. 4. Benn P, Cuckle H. Theoretical performance of non-invasive prenatal testing for chromosome imbalances using counting of cell-free DNA fragments in maternal plasma. Prenat Diagn. 2014; 34:778-83 5. Fiorentino F, Spinella F, Bono S, Pizzuti F, Mariano M, Polverari A, Duca S, Cottone G, Nuccitelli A, Sessa M, Baldi M. Feasibility of noninvasive prenatal testing for common fetal aneuploidies in maternal serum with low levels circulating fetal cell-free DNA fraction. Prenat Diagn 2015; 35 Suppl. 1: pag 1 6. Bono S, Pizzuti F, Mariano M, Polverari A, Duca S, Cottone G, Nuccitelli A, Sessa M, Spinella F, Baldi M, Fiorentino F. Massively Parallel Sequencing (MPS) reliably identifies trisomy 21, 18, and 13 in maternal plasma with low-level fetal cell-free DNA fractions. Poster presentation from the European Society of Human Genetics (ESHG) meeting Glasgow 2015. 7. Pergament E, Cuckle H, Zimmermann B, et al. Single-nucleotide polymorphism-based noninvasive prenatal screening in a high-risk and low-risk cohort. Obstet Gynecol. 2014;124 (2 Pt 1):210-8. 8. Norton ME, Jacobsson B, Swamy GK, et al. Cell-free DNA analysis for non invasive examination of trisomy. N Engl J Med. 2015;372:1589-97.