Fetal fraction determination

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.

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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%


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


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.


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.

Bibliography
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