Vol. 012_2024.07
NIPT - PPVs and False-Positives

NIPT - PPVs and False-Positives
Non-invasive prenatal testing (NIPT), also known as non-invasive prenatal screening (NIPS) or
cell-free DNA testing (cfDNA), is a screening test that screens for certain chromosomal
abnormalities in the fetus. This method was introduced commercially in 2011, following thediscovery of circulating feto-placental-derived cell-free DNA in maternal plasma in 1997[1].
According to the latest guideline from the American College of Medical Genetics and Genomics(ACMG) in 2023, NIPT is strongly recommended over traditional screening methods for all
pregnant patients with singleton and twin gestations for fetal trisomies 21, 18, and 13. ACMG alsostrongly recommended that NIPT be offered to patients with singleton gestations to screen for fetal
sex chromosome aneuploidy (SCA) [2].

NIPT assesses the small amounts of cell free DNA released from placental cells that are
flowing in the maternal plasma. These cell free DNA pieces comprises approximately 3-13% of totalmaternal plasma DNA. As NIPT does not directly assess fetal DNA and cannot directly determine thefetal chromosomal status, it is still strongly recommended that a high-risk NIPT result be confirmedwith a diagnostic test. False-positive and false-negatives occur, though rarely, due to technical andbiological factors that can influence the test results.
In this issue, we compliled information on PPVs and factors influencing NIPT results. Inaddition, several cases of false-positives NIPT for fetal trisomies, but where subsequent genetic
testing helped uncover the reasons affecting the original NIPT test are presented.

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NIPT - PPVs and False-Positives

2

Positive Predictive Values (PPVs) of NIPT
Positive Predictive Values (PPV) refers to the likelihood that an individual truly has a specificdisease following a positive test result. Multiple systematic reviews have demonstrated that NIPT isa highly accurate screening test for trisomy 21, 18 and 13 [3]. The PPVs of NIPT is highest fortrisomy 21 at 91.78%. For trisomy 18 and trisomy 13, due to the low natural prevalence rate andthe increased incidence of confined placental mosaicism (CPM), their PPVs are lower at 65.77% and37.23%, respectively. PPVs for SCA are lower than those observed for the common autosomal
trisomies. PPVs for SCA ranged from 30% for 45,X to 74% for 47,XXY or 47,XYY (Table 1) [3].

Condition

PPV (%)

95% CI

Trisomy 21

91.78

88.43-94.23

Trisomy 18
Trisomy 13
Monosomy X
47,XXX

65.77
37.23
29.52
53.95

45.29-81.68
26.08-49.93
22.72-37.36
40.58-66.77

47,XXY47,XYY
Overall SCAs

74.05
74.45
43.13

59.47-84.73
58.40-85.81
37.92-48.5

Table 1. The performance of
non-invasive prenatal testing(NIPT) in a general
unselected population forcommon trisomies and sex
chromosome aneuploidy(SCA). (Reference from ISPD
Position Statement_2023 [3])Factors that may influence NIPT resulting
As apparent from the above table, none of the screened conditions have a 100% PPV. Althoughcell-free DNA is the most sensitive and specific screening test for the common autosomal
aneuploidies, it still has the potential for false-positive and false-negative results. NIPT testing is
not equivalent to diagnostic testing. There are several factors that may affect the quantity andquality of fetal fraction, and NIPT results [4]: gestational age
 fetal or maternal chromosomal mosaicism
 multiple gestations (fetal demise or fetal reduction)
 confined placental mosaicism (CPM)maternal body mass index (BMI)
maternal medication exposurematernal malignancy

Below are a few examples in which the fetus does not have trisomy 21 even though the original
NIPT result indicated such. Subsequent genetic testing revealed the true presence of the extra
dosage of chromosome 21, and showed that the NIPT result can be influenced by placental
chromosomal abnormality, maternal chromosomal abnormality, and fetal microduplication.

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NIPT - PPVs and False-Positives

3【Case 1】Confined placental mosaicism (CPM)
Ms. W was 25-year-old. She chose to undergo NIPT at 13 weeks of pregnancy.
Ms. W’s testing process:13 wksNIPT•Result:
Result:• High risk for Trisomy 21 (Down syndrome)

17 wks•Result:
Result:• Normal male 46,XY

Amniocentesis withChromosomal
Karyotype Analysis•Result:
Result:Placental Analysis • 43% Mosaic Trisomy 21
After delivery

43% mosaic Trisomy 21

Placental Analysis - SNP microarray

When the fetal chromosomal analysis result from amniocentesis was found to be normal, thephysician and Ms. W discussed the potential of placental chromosomal abnormality influencing theNIPT result. Decision was made to collect different sections of the placenta post-delivery in order toinvestigate the possibility of placental mosaicism. A section of the placenta did in fact reveal mosaic
Trisomy 21 with a microarray result of arr(21)x3[0.43].

GeneralCondition

CPM scenario

Picture Source : Genetics Education Canada – Knowledge Organization (GEC-KO)

Take Home Message:
This is a case of confined placental
mosaicism (CPM) affecting the NIPT
result and leading to a false positive
fetal trisomy scenario.

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NIPT - PPVs and False-Positives

4【Case 2】Fetal chr.21 microduplication
Mrs. K was 38 years old. She was pregnant with her second baby, and chose to have NIPT at 12
weeks of pregnancy.
Mrs. K’s testing process:12 wksNIPT•Result:
Result:• High risk for Trisomy 21 (Down syndrome)

18 wksAmniocentesis with
Chromosomal Karyotype+ Microarray Analysis•Result:
Result:• Karyotype: Normal male 46,XY• Microarray: 21q22, ~3Mb duplication

After consultation of the high-risk NIPT result, Mrs. K decided to undergo an amniocentesis withfetal chromosomal karyotype and microarray analysis. The microarray test showed a malechromosome complement with a gain of ~3Mb at 21q22 region. Luckily, there were no known
diseases or triplosensitivity genes within this region. Fetal risk for clinical abnormality is low. After
genetic counseling, Mrs. K understood and continued her pregnancy.
Microarray is a useful tool in detecting microdeletions/microduplications that are beyond theresolution of chromosomal analysis. If invasive testing is elected, a microarray analysis could also be
considered in addition to karyotype analysis to offer a more comprehensive evaluation of fetal
chromosomal abnormality.
Fetal analysis – Amniotic fluid SNP microarray

21q22, ~3Mb microduplication

Take Home Message:
This case shows how a fetal
microduplication can affect
the NIPT result. Although thefetus does not haveautosomal trisomy, the fetus
carries a microduplication on
the same chromosome that
influenced the NIPT result.

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GGA Genetic Counseling Newsletter – Vol. 012
NIPT - PPVs and False-Positives

5【Case 3】Maternal chr.21 microduplication
Thirty-year-old Ms. C chose to have NIPT at 12 weeks and 3 days of pregnancy.
Ms. C’s testing process:12 wks + 3 days
NIPT

•Result:
Result:• High risk for Trisomy 21 (Down syndrome)

17 wksAmniocentesis with
Chromosomal Karyotype +Microarray Analysis•Result:
Result:• Karyotype: Normal female 46,XX• Microarray: Normal female
20 weeks

Maternal blood
Microarray Analysis•Result:
Result:• Maternal microarray:
21q21, ~4Mb duplication

Mrs. C screened high risk for fetal trisomy on her NIPT, but amniotic fluid karyotype analysis andSNP microarray suggested a chromosomally normal fetus. For scientific investigation, the physician
and the testing laboratory, and with Ms. C consent, performed maternal peripheral blood
microarray analysis to determine whether maternal genetic background affected the NIPT result.
The maternal microarray revealed a gain of ~4Mb at chromosome 21q21 region. Although, thematernal microduplication at chromosome 21 was not passed down to the fetus, it influenced theNIPT result, leading to a false-positive NIPT outcome.
As this was Ms. C's first pregnancy and she planned to have more children in the future, thefindings had implication for future pregnancy planning. Ms. C was counseled that for future
pregnancies, NIPT may not be suitable for her to screen for fetal chromosomal abnormalities(especially for trisomy 21) due to her own chromosome 21 microduplication.

21q21, ~4Mb microduplication
MotherFetus

Take Home Message:
This case shows that maternal
chromosomal background can affect
the NIPT result. The high-risk NIPT
result reflected maternal genetics, butnot fetal status.
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GGA Genetic Counseling Newsletter – Vol. 012
NIPT - PPVs and False-Positives

6Counseling Notes
NIPT detects not only cell free DNA from placenta, but also maternal DNA

Although NIPT is the most sensitive and specific screening test for thecommon fetal aneuploidies, it still requires follow-up diagnostic, such askaryotype analysis and microarray, confirmation to determine the fetal
chromosomal status.

Factors which may affect the quantity and quality of fetal fraction, and NIPT
results:
 gestational age
 fetal or maternal mosaicism
 multiple gestations (including fetal demise or fetal reduction)
 confined placental mosaicism (CPM) maternal body mass index (BMI) maternal medication exposure maternal malignancy



Follow-up diagnostic tests using different platforms and tissues may increase
the chances of identifying the true factors affecting the NIPT results.

Maternal genetic background abnormalities may affect NIPT thus mask
fetal genetic status. Pregnant woman who personally carries achromosomal abnormality may need to consider other prenatal tests toassess fetal chromosomal status.

References: [1] Lancet 1997 Aug 16;350(9076):485-7, PMID: 9274585; [2] Genet Med. 2023 Aug;25, PMID: 37378664; [3]Prenat Diagn. 2023 Jun;43(7):814-828, PMID: 37076973; [4] Obstet Gynecol. 2020 Oct;136(4):e48-e69., PMID: 32804883

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