What is NIPT?

Fetal congenital malformations are the leading cause of perinatal mortality and morbidity, often stemming from chromosomal or genetic abnormalities such as aneuploidy, translocations, duplications, and deletions, which occur in 1 in 150 live births. Invasive diagnostic tests like chorionic villous sampling (CVS) and amniocentesis, followed by karyotyping or comparative genomic hybridization (CGH) microarray, are available but pose a small yet significant risk of miscarriage. Traditionally, pregnancies at higher risk of chromosomal abnormalities have been screened using combined or triple tests; however, further invasive testing is often required.¹

In this context, NIPT is a non-invasive test that allows for the detection of genetic conditions such as Down syndrome, Turner syndrome, Klinefelter syndrome, and triple X syndrome. NIPT can also determine the sex of the fetus with high accuracy.² It is based on the analysis of cfDNA in maternal blood, where the majority of the cfDNA originates from the mother herself, while the fetal component, cell-free fetal DNA (cffDNA), contributes approximately 10-20% of the total. cffDNA originates from the placenta and can be detected in maternal blood as early as 5 weeks of gestation, although NIPT is often done at 10 weeks of gestation. 1 The portion of fetal DNA is known as the fetal fraction (FF), and it varies among individuals. The FF is strongly linked to how reliable the NIPT results are.^1,3

How Does NIPT Work?

There are several methods of performing an NIPT. Some of the most common NIPT methodologies currently used are single-nucleotide polymorphism (SNP) analysis, massively parallel shotgun sequencing (MPSS), and targeted massively parallel sequencing (t-MPS).

Single-Nucleotide Polymorphism Analysis

SNP is the most common genetic variation in human DNA. It is a single letter difference in the genetic code that varies across individuals. Unlike traditional counting methods that simply measure the amount of cfDNA per chromosome, SNP-based tests examine the specific combinations of maternal and fetal alleles to accurately estimate FF, detect aneuploidies through allelic ratio shifts, and even assess inheritance patterns for single-gene disorders. This approach is especially useful in complex pregnancies, like twins or those involving a vanishing twin, where standard methods can be a struggle.⁴ Moreover, the SNP method has several advantages, such as it does not require a reference chromosome sequence and it is highly accurate in detecting vanishing twin, maternal mosaicism, and triploidy.^5,6

Massively Parallel Shotgun Sequencing

MPSS is based on the amplification and sequencing of fetal and maternal cffDNA present in the mother's plasma. This method analyzes the ratio of DNA fragments from specific maternal and fetal chromosomes in a maternal blood sample, then compares those ratios to the expected chromosomal distribution.⁷ Moreover, it does not require the separation of fetal and maternal DNA, and it does not require the enrichment of the FF. However, a major limitation of multiplexing in NIPT is its strong dependence on FF, requiring a minimum of 4% for accurate results, which is often difficult to acquire. cfDNA screen failures or no calls occur in up to 3% of the samples, which is often frustrating for parents. Fortunately, a study in 2019 found that sequencing shorter cfDNA fragments has the ability to increase FF. They found that, compared to FF results from fragments that are less than 120bps, FF decreased by 14.47% for fragments that are more than 141bps. Additionally, for dizygotic twins, 98.8% of the samples achieved an FF > 10%.⁸

Targeted Massively Parallel Sequencing

t-MPS is a similar method to MPSS; however, it contains one extra step. “t-MPS selectively amplifies only the chromosomal region of choice (i.e., chromosome 21, 18, or 13), and then calculates whether there is an excess for one particular chromosome relative to another. A benefit of the T-MPS methodology is the lower sequencing cost because this method avoids having to sequence all regions.”⁸ However, a key limitation is that it requires custom assay design and optimization for each specific target. This can be especially difficult when screening for new conditions or genes because the panel needs to not only be redesigned but also tested and clinically validated, which takes significant time and resources.⁹

Role of Norgen Biotek's Technology

Careful pre-analytical handling is critical to the accuracy of NIPT. From sample collection and stabilization to cfDNA isolation, every step directly impacts test reliability and can help reduce the risk of false positives. Norgen Biotek offers integrated solutions to support the entire NIPT workflow. The cfDNA/cfRNA Preservative Tubes are designed to prevent nucleic acid degradation and inhibit the release of maternal genomic DNA during transport and storage. This is particularly important when there are delays in processing, as it helps maintain a consistent and interpretable fetal DNA fraction.

Following plasma separation, cfDNA can be efficiently isolated using Norgen's Plasma/Serum Cell-Free Circulating DNA Purification Kits to maximize recovery of all fragment sizes of cffDNA. These silicon carbide, column-based kits eliminate the need for phenol/chloroform extraction and do not require carrier RNA, producing high-quality DNA that is immediately compatible with sensitive downstream applications such as massively parallel sequencing.

This study by Diefenbach et al. confirms Norgen's Plasma/Serum Cell-Free Circulating DNA Purification Kit's ability to purify small fragments of cfDNA with high recovery efficiency. The work evaluated several commercially available kits for isolating cfDNA from Plasma samples. The results demonstrated that Norgen is not only one of the top choices for recovering high-quality cfDNA but that it is the best at recovering high yields of short fragments of cfDNA (50- 200bp), which is essential for increasing FF and NIPT accuracy.¹⁰

What Does NIPT Test For?

NIPT is most commonly used to screen for trisomies, including trisomy 21 (Down syndrome), trisomy 18 (Edwards syndrome), and trisomy 13 (Patau syndrome). These conditions are caused by an extra copy of a chromosome and are associated with varying degrees of intellectual disability, birth defects, and often limited survival beyond infancy.

NIPT also screens for sex chromosome aneuploidies, such as Turner syndrome (monosomy X), Klinefelter syndrome (47, XXY), Triple X syndrome (47, XXX), and Jacobs syndrome (47, XYY). These conditions may affect physical development, fertility, or learning, though they can sometimes be mild or asymptomatic.

Trisomy 21 (Down Syndrome)

Down syndrome, also known as trisomy 21, is the most common chromosomal aneuploidy in live births, occurring in approximately 1 in 700 newborns worldwide. It results from the presence of an extra copy of chromosome 21 and is associated with a range of clinical features, including mild to moderate intellectual disability, distinct facial characteristics, hypotonia, and an increased risk for congenital heart defects, thyroid dysfunction, and early-onset Alzheimer's disease.¹¹

NIPT is a highly accurate and non-invasive method for detecting trisomy 21, providing reliable results as early as 10 weeks into pregnancy. A clinical trial from 2015 evaluated the performance of cfDNA-based NIPT against first-trimester screening in over 15,000 pregnant women. They found that NIPT identified 100% of Down syndrome cases while standard screening only identified 78.9%. False positive rates for cfDNA-based NIPT were also low, at 0.06% compared to 5.4% with standard screening. These results highlight why NIPT has quickly become the preferred method for Down syndrome screening in both high- and average-risk pregnancies. It enables earlier detection, fewer false alarms, and greater confidence in results.¹²

Trisomy 18 (Edwards Syndrome)

Trisomy 18, also known as Edwards Syndrome, occurs when a fetus acquires an extra copy of chromosome 18. This genetic change disrupts normal development and leads to multiple severe congenital abnormalities, including heart defects, growth restriction, and abnormalities of the head, hands, and feet. Most affected pregnancies result in miscarriage or stillbirth, and of those born alive, the majority do not survive beyond the first year of life. Trisomy 18 occurs in about 1 in every 5,000 live births and is more common in pregnancies of people over 35. While there is no cure, early diagnosis through screening methods like NIPT can help guide decisions and provide supportive care planning.^{13, 14}

Trisomy 13 (Patau Syndrome)

Trisomy 13, or Patau syndrome, is caused by the presence of an additional copy of chromosome 13. It is associated with multiple major congenital abnormalities, including structural brain defects (e.g., holoprosencephaly), congenital heart defects, cleft lip and/or palate, polydactyly, and renal abnormalities. Trisomy 13 is rare in live births, occurring in approximately 1 in 5,000 births. The fetal loss rate is also extremely high, with a 97% failure for conceptions and infant death within four months post-birth.¹⁴

NIPT and Monogenic Disorders

While NIPT is used for detecting chromosomal aneuploidies, its potential for detecting monogenic disorders is an emerging field of research. Monogenic disorders such as sickle cell anemia, cystic fibrosis, and polycystic kidney disease are genetic diseases caused by mutations in a single gene. Unlike complex disorders that involve multiple genes and environmental factors, monogenic conditions result from a clear, identifiable alteration in just one gene, which can be inherited or occur spontaneously. Although NIPT is highly accurate in its detection of chromosomal disorders, detecting single-gene mutations remains challenging due to the small proportion of fetal DNA in maternal circulation and the need for deep sequencing.² In this context, NGS panels can detect maternally inherited monogenic disorders using the relative haplotype dosage (RHDO) method, which analyzes allele ratios in maternal plasma across haplotype blocks to infer fetal inheritance patterns.¹⁵

Who Should Consider NIPT?

NIPT is particularly beneficial for individuals in the following categories:

• Pregnant women aged 35 or older.
• Those with a history of chromosomal abnormalities.
• Cases involving positive results from other prenatal screenings.
• High-risk pregnancies that are identified through medical history or ultrasound findings.⁶

Future of NIPT

Although reliable, NIPT is currently not a diagnostic test, and invasive tests remain the gold standard. However, the future of NIPT is promising, with ongoing research and technological advancements enhancing its scope and accuracy. A key focus is expanding NIPT's ability to detect a broader range of genetic abnormalities beyond common aneuploidies. Furthermore, the integration of cutting-edge technologies such as artificial intelligence and machine learning is set to revolutionize NIPT. These innovations will significantly enhance data analysis, improving both the accuracy and efficiency of test results.¹⁷

Conclusion: A Safer, Smarter Path to Prenatal Care

NIPT has redefined the landscape of prenatal care by offering a safer, earlier, and more accurate approach to screening for chromosomal conditions that can affect fetal development and health outcomes. Through the analysis of cell-free fetal DNA in maternal blood, NIPT enables early detection of somatic aneuploidies such as trisomy 21, 18, and 13, as well as sex chromosome abnormalities. Compared to traditional screening methods, NIPT significantly reduces false positive rates and limits the need for invasive procedures like amniocentesis, providing expectant families with greater confidence and peace of mind.

As technology continues to advance, NIPT is expanding beyond its original scope. Researchers are actively exploring its application in screening for monogenic disorders, genome-wide copy number variations, and even epigenetic markers, all through a single non-invasive blood draw.

The accuracy of NIPT depends not only on analytical performance but also on proper sample collection, preservation, and nucleic acid isolation. High-quality pre-analytical handling is essential for reliable results. Technologies like Norgen Biotek's stabilization tubes and cfDNA purification kits help protect sample integrity at every step, supporting consistent fetal fraction and high-yield recovery suitable for downstream analysis.

NIPT has emerged as a cornerstone of modern prenatal screening. It empowers healthcare providers and families with early, actionable insights and reduces the physical and emotional burden associated with more invasive testing. As it continues to evolve, NIPT is poised to lead the way in advancing precision medicine during pregnancy, improving outcomes for both parents and babies.

If you're looking for high-quality tools to enhance your NIPT research or clinical practice, explore Norgen Biotek's comprehensive solutions for nucleic acid preservation and isolation.