Professional Guidance on the Role of NIPT as a First Tier Screening Test

ACOG has endorsed the SMFM guidance (Consult Series 74) on cell free DNA screening for aneuploidies. Cell free DNA is the most sensitive and specific screening test for common aneuploidies. Of note, while acknowledging that cell free DNA has good screening performance for common aneuploidies, the updated guidance recommends that “After pretest counseling, every patient has the right to pursue or decline prenatal genetic screening and diagnostic testing.” For those patients who want more comprehensive genetic information regarding her pregnancy, invasive testing such as amniocentesis or CVS remains an option.

SUMMARY:

• Cell-free DNA screening, also known as noninvasive prenatal testing (NIPT) is the most sensitive and specific screening methodology for common fetal aneuploidies in all obstetric patients
• Microdeletions
  • Women who want information regarding microdeletions should be offered microarray testing using CVS or amniocentesis  
  • SMFM does not recommend routine general population screening for any microdeletion syndromes and “Patients who choose to undergo cfDNA screening for 22q11.2 deletion specifically should do so only after appropriate pretest counseling”
• Sex chromosome conditions (also known as SCAs or sex chromosome aneuploidies)
  • SMFM recommends that “screening for SCAs be made available to obstetrical patients as an “opt-in” consideration with appropriate pretest counseling”
• Twins
  • SMFM recommends NIPT as a first line screening test in twins for T21 (trisomy 21 or Down syndrome)
  • Testing for SCAs in not recommended in twins or higher order multiples due to lack of data
• Genome-wide copy number deletions and duplications
  • Prenatal screening is not currently recommended

What Is NIPT?

NIPT is a blood test that utilizes cell-free DNA technology (cfDNA) to predict the risk for fetal genetic disorders during pregnancy. In 2011, NIPT was introduced as a screen for T21. Today, NIPT cover the most common aneuploidies (T21, T13 and T18), as well as sex chromosomes and may also include some microdeletions and single gene genetic disorders

How Does It Work?

• DNA fragments (outside of cells) can be found floating in the blood of all individuals
• In every pregnant woman, there are fragments from
  • Her own DNA
  • Fragments of placental DNA (generally thought to derive from the outer cytotrophoblast rather than the inner mesenchymal layer)
• A maternal blood sample is obtained ≥10 weeks (with some labs offering testing beginning at 9 weeks) of pregnancy
  • Usually performed at 11 to 13 weeks
• Fetal Fraction
  • The percentage of fetal DNA found in maternal blood is known as the fetal fraction | Typical range 3 to 13% of maternal cfDNA
  • The fetal fraction is critical to the success of NIPT | Minimum required approximately 2 to 4%
  • Sensitivity drops with lower fetal fraction and if too low, test failure will result

Technologies

• Generally, NIPT utilizes next generation sequencing and bioinformatics algorithms to interrogate DNA fragments that have been extracted from maternal samples
  • SNP-based: Bioinformatic algorithms combine risk from individual targeted single nucleotide polymorphisms (SNPs) to differentiate maternal from placental DNA fragments
    • SNP is the only analysis that can report on zygosity and individual fetal fractions in the case of twins (dependent on laboratory)
  • Quantification method: Bioinformatic algorithms determine the amount of DNA from chromosomal regions of interest | E.g. if too much chromosome 21 DNA is detected, then a ‘screen positive’ result for T21 will be generated

What Disorders Are Included?

• T13, T18 and T21
  • Most effective at screening for T21
• Chromosomes X and Y (fetal sex usually available)
• Sex chromosome conditions (depending on the lab)
  • Monosomy X (Turner syndrome)
  • 47,XXX (Triple X syndrome)
  • 47,XXY (Klinefelter syndrome)
  • 47,XYY (Jacob’s syndrome)
• Microdeletions (also known as copy number variants or CNVs) are available with more extensive panels | CNVs occur in 0.4% of pregnancies and are not related to maternal age
  • 22q11.2 deletion syndrome (DiGeorge syndrome) | 1,3000 to 1,4000 live births | May be more prevalent prenatally and cases may be missed at birth (see ‘Related ObG Topics)
  • 1p36 deletion syndrome | 1 in 5,000 to 1 in 10,000 live births
  • 4p16.3 deletion syndrome (Wolf-Hirschhorn syndrome) | 1 in 50,000 live births
  • 5p15.2 deletion syndrome (Cri du Chat syndrome) | 1 in 20,000 to 1 in 50,000 live births
  • 15q11.2 deletion syndrome (Angelman syndrome/Prader-Willi syndrome) |1 in 12,000 to 20,000 (AS) | 1 in 10,000 to 30,000 (PWS)

SYNOPSIS:

NIPT is a screening test only and not diagnostic. Cell-free DNA (cfDNA) is also commonly used with an understanding that the DNA is derived from placenta and not the fetus. NIPT utilizes next generation sequencing and bioinformatics algorithms to look at the DNA fragments in the mother and fetus, as a way of determining the likelihood of certain genetic conditions in the fetus.  While there are multiple panels available, there is consensus regarding the clinical utility of NIPS screening for T13, T18 and T21. Patient education, especially around the concept of positive predictive value (PPV) is a priority. Calculator tools are available from professional societies (see ‘Learn More – Primary Sources’ below) or ideally laboratories should be able to provide obstetric professionals with real world test performance results.

NIPT Screening Performance

Detection rates

• The following detection rates are based on recent meta-analysis (see ‘Learn More -Primary Resources’ below)
  • T21: >99% detection rate for T21
  • 98% detection rate for T18
  • 99% detection rate for T13
• Combined false positive rate of 0.13%
  • cfDNA is the most sensitive and specific screen for T21, T18 and T13
• Sensitivity and specificity are superior to standard screening for T21 and other common aneuploidies

Positive Predictive Value (PPV)

Trisomies

• NIPT generally has very high negative predictive values (NPV) | From ages 20 to 45, NPV is >99% (NSGC GSF calculator)
• PPV is also generally high but can vary based on age | Lower background risk will lower PPV
  • PPVs have been reported up to approximately 90% for T21 but tend to be lower for T18, T13 and monosomy X
  • Abnormal ultrasound will increase PPV

Microdeletions

• NIPT for microdeletion syndromes can have high sensitivity and specificity but still have very low PPV because the particular syndrome is a priori so rare | For example
  • Cri du Chat Syndrome (Using NSGC GSF calculator – see ‘Learn More – Primary Sources’ below)
    • Even if sensitivity and specificity are both >99%, the chance of a positive results being a true positive (i.e. PPV) is ≤1%
    • Negative predictive value will generally always be high for a rare disorder
  • 22q11.2 deletion syndrome (22q11.2DS) is the most common microdeletion syndrome, found in 1 in 3000 to 4000 live births | Appears to be more common prenatally with a prevalence of approximately 1 in 1000 of otherwise normal pregnancies (see ‘Learn More – Related Entries)
    • PPV in a low risk population (see ‘Learn More – Primary Sources’) has been reported to be approximately 1 in 20 and higher in pregnancies at higher risk (e.g. congenital heart disease consistent with 22q11.2DS)
    • While SMFM does not recommend routine screening for microdeletions, the guidance does specify that patients choosing screening for 22q11.2 deletion should first receive pretest counseling

Note: NIPT is a screening test and not diagnostic | Regardless of PPV, screen positive results require patients be offered invasive diagnostic testing to confirm results

• Some women may opt for a cfDNA screen after a positive serum analyte screen instead of an invasive test. This is a valid option for patients who do not want an invasive test

Reasons for a False Positive Result

• There is a high likelihood that a positive T21 NIPT screen is truly positive (approximately 90%)
• However, confirmatory testing is necessary because false positive results are possible
  • Confined placental mosaicism (since NIPT only looks at the placental DNA)
  • Vanishing twin that was aneuploid but surviving twin is normal
  • Maternal condition such as cancer (NIPT results will usually show multiple chromosomal aneuploidies)
  • Unknown

NIPT Test Failure

• Low fetal fraction
  • BMI: 10% of patients >250 lbs will have a fetal fraction <4% | May be caused by (1) dilutional effect or (2) elevation in maternal DNA due to inflammatory processes
  • Early Gestational age <9 weeks
• Other reasons for no result include
  • Laboratory failure | IVF pregnancy | Maternal drug exposure to LMWH | Racial background (e.g. Black and South Asian vs white) | Fetal aneuploidy (e.g. T13 or T18 and sex chromosome aneuploidies)

Follow-Up for ‘No Call Result’

• Inform patients that there is an increased risk of aneuploidy | Effect may be due to smaller placentas  
• Offer genetic counseling, detailed ultrasound evaluation and diagnostic testing
• Repeat screening “may be considered’
  • Success rate 75 to 80% (less with high BMI)
• Repeat screening is not advised for the following
  • Ultrasound anomalies present
  • Later gestational age where further delay may complicate access to reproductive options

Note: It is preferred that the laboratory report the fetal fraction

Is There Still a Role for Serum Screening?

• First‑ and second‑trimester serum screening is still useful
  • When a patient prefers it after counseling
  • When cfDNA is not an option
    • Vanishing twin | Maternal transplant or stem cell therapy | Maternal mosaicism/translocation
    • No insurance coverage
• Serum screening combined with first trimester ultrasound may have lower detection rates for trisomy 21 but it may detect genetic abnormalities that NIPT may miss
  • Pathogenic deletions/duplications
  • Single‑gene disorders
  • Placental abnormalities

Single Gene and NIPT

• NIPT also has the capability to identify single gene disorders
  • Ability to identify single gene variants that would likely not be detected with carrier testing
  • Generally serious autosomal dominant disorders that may be absent in the parents, but pathogenic variants may spontaneously occur in the fetus (de novo) such as Cornelia de Lange syndrome or Achondroplasia
  • Some of these conditions are associated with advanced paternal age (≥40 at time of conception)
• Single gene screening is clinically available, although currently not recommended by ACOG

Note: ACMG provides healthcare professionals with open access ‘ACT Sheets’ to guide next steps following a positive NIPT report (see ‘Learn More – Primary Sources’ below)  

Learn More – Primary Sources: 

SMFM Consult Series 74: Cell‐free DNA screening for aneuploidies: Updated guidance

ACOG Practice Advisory: Screening for Fetal Chromosomal Abnormalities

ACOG Committee Statement 24: Ethical Considerations for Genetic Testing and Counseling in Obstetrics and Gynecology

ACOG Practice Advisory: Cell-free DNA to Screen for Single-Gene Disorders

ACMG ACT Sheets and Algorithms

Analysis of cell‐free DNA in maternal blood in screening for aneuploidies: updated meta‐analysis

Screening for trisomies by cfDNA testing of maternal blood in twin pregnancy: update of The Fetal Medicine Foundation results and meta‐analysis

NIPT/Cell Free DNA Screening Predictive Value Calculator

MedlinePlus: What is noninvasive prenatal testing (NIPT) and what disorders can it screen for?

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