Prenatal Screening (Combined Test, NIPT, Quadruple Test)

Prenatal screening estimates the chance that a pregnancy is affected by a chromosomal condition (mainly trisomy 21, trisomy 18 and trisomy 13) or an open neural tube defect, so that women can choose diagnostic testing. It does not diagnose.

Three approaches are in use: the first trimester combined test (maternal age, nuchal translucency on ultrasound, and serum pregnancy associated plasma protein A and free beta human chorionic gonadotrophin at 11 weeks to 13 weeks 6 days), the second trimester quadruple test (alpha fetoprotein, total or free beta human chorionic gonadotrophin, unconjugated oestriol and inhibin A at 15 to 22 weeks),

and non-invasive prenatal testing (NIPT), which analyses cell free DNA in maternal plasma, mostly from placental trophoblast, from 10 weeks.

Sequential, integrated and contingent strategies combine these, and the anomaly scan at 18 to 22 weeks screens for structural defects.

Trisomy 21 affects about 1 in 700 live births, trisomy 18 about 1 in 5000 and trisomy 13 about 1 in 10 000, with risk rising from about 1 in 1500 at age 25 to 1 in 350 at 35 and 1 in 100 at 40. Detection rates at a 5% false positive rate are about 85 to 90% for trisomy 21 with the combined test, 80% for the quadruple test, and over 99% for NIPT with a false positive rate under 0.1%.

Because of this performance, NIPT is used as a first line test in many programmes, or as a contingent test for women with an intermediate result on the combined test.

Components. Nuchal translucency, pregnancy associated plasma protein A, free beta human chorionic gonadotrophin, alpha fetoprotein, unconjugated oestriol, inhibin A, cell free DNA (NIPT), and maternal age, weight, ethnicity and conception method as modifiers.

Indications. Offer to every pregnant woman, with pre-test counselling that explains the aim, limits, the possibility of a high chance result, and the option to decline. NIPT is also used after an increased risk combined or quadruple result, after an abnormal ultrasound marker, and in women with advanced maternal age. Late booking after 14 weeks uses the quadruple test.

Not for women who would not act on the result, and not a substitute for diagnostic testing when ultrasound shows major structural anomalies or a nuchal translucency of 3.5 mm or more.

Specimen. Serum from 5 mL of clotted blood for biochemical markers, with accurate gestational age from a dating scan (crown rump length of 45 to 84 mm for the combined test). Nuchal translucency measured by accredited sonographers in the mid-sagittal plane.

NIPT needs 10 mL of maternal blood in cell stabilising tubes, processed within 3 to 5 days, and a foetal fraction of at least 4%. Record maternal weight, diabetes, smoking, assisted conception, egg donation, twin pregnancy, previous affected pregnancy, and ethnicity.

Nuchal translucency. Fluid filled space behind the foetal neck. Increases with crown rump length, with the 95th centile about 2.1 to 2.5 mm and 99th centile about 3.5 mm. Raised in trisomies 21, 18 and 13, Turner syndrome, triploidy, congenital heart disease, diaphragmatic hernia, skeletal dysplasias and many genetic syndromes. A value of 3.5 mm or more with a normal karyotype needs a detailed scan, echocardiography and microarray.

Pregnancy associated plasma protein A. Low in trisomy 21 (median about 0.4 multiples of the median, MoM), trisomy 18 and 13 (about 0.2 MoM), and Cornelia de Lange syndrome. Also low in pregnancies at risk of foetal growth restriction, pre-eclampsia, stillbirth and miscarriage. Values fall with raised maternal weight (haemodilution) and are corrected for it.

Free beta human chorionic gonadotrophin. Raised in trisomy 21 (about 2.0 MoM), and low in trisomies 18 and 13 (about 0.2 MoM). Very high values also occur in molar pregnancy, twins and triploidy of paternal origin.

Alpha fetoprotein. Raised at 2.0 to 2.5 MoM or more in open spina bifida (about 85 to 90% detection), anencephaly (over 95%), abdominal wall defects (gastroschisis, exomphalos), foetal death, twins, wrong dates and placental causes. Low (about 0.75 MoM) in trisomy 21 and (about 0.65 MoM) in trisomy 18.

Unconjugated oestriol. Low in trisomies 21 and 18 (about 0.7 and 0.4 MoM), steroid sulphatase deficiency (X-linked ichthyosis), Smith-Lemli-Opitz syndrome, adrenal hypoplasia, anencephaly and maternal corticosteroid use.

Inhibin A. Raised in trisomy 21 (about 2.0 MoM), and low or normal in trisomy 18.

Cell free DNA (NIPT). Reports high chance or low chance for trisomies 21, 18 and 13, and often sex chromosome conditions (monosomy X, XXX, XXY, XYY) and foetal sex. Sensitivity is about 99% for trisomy 21, 97% for trisomy 18 and 90 to 95% for trisomy 13.

Positive predictive value depends on the prevalence in the tested woman: about 80 to 90% for trisomy 21 in a woman of average age, 50 to 70% for trisomy 18, 30 to 50% for trisomy 13 and 25 to 40% for monosomy X. Microdeletion panels (22q11.2) have lower predictive values and limited validation.

No result occurs in 1 to 5%, mostly from low foetal fraction (raised maternal weight, early gestation, aneuploidy), and carries an increased chance of an abnormal karyotype.

Interpretation.

• Reports give a chance as a ratio, for example 1 in 50 (high chance) or 1 in 5000 (low chance), compared with a programme cut off (commonly 1 in 150 to 1 in 300 at the time of screening, programme specific). A high chance is not a diagnosis, and a low chance does not exclude an affected pregnancy.
• Typical patterns: trisomy 21 gives raised nuchal translucency, low pregnancy associated plasma protein A and high free beta human chorionic gonadotrophin in the first trimester, and low alpha fetoprotein and oestriol with high human chorionic gonadotrophin and inhibin A in the second. Trisomy 18 gives low values for every marker. Trisomy 13 gives low pregnancy associated plasma protein A and low human chorionic gonadotrophin.
• Marker levels are corrected to MoM for gestation, maternal weight, ethnicity, smoking, diabetes, number of foetuses and method of conception (in vitro fertilisation lowers pregnancy associated plasma protein A and raises human chorionic gonadotrophin slightly).
• A high chance result is followed by counselling and a diagnostic test: chorionic villus sampling (from 11 to 14 weeks, procedure related loss about 0.2 to 0.5%) or amniocentesis (from 15 weeks, about 0.1 to 0.3%), with rapid aneuploidy testing and microarray.
• NIPT results that are discordant with ultrasound or that show unexpected findings should be confirmed by diagnostic testing before any irreversible decision.
• Isolated markers (raised nuchal translucency, low pregnancy associated plasma protein A, raised alpha fetoprotein) with normal screening for aneuploidy still define a group at higher risk of structural anomaly, growth restriction or stillbirth, and need detailed scan and follow up growth assessment.

Critical values and findings needing immediate action.

• Nuchal translucency of 3.5 mm or more, or cystic hygroma: diagnostic testing with microarray, detailed anatomy scan and foetal echocardiography.
• High chance for trisomy 21, 18 or 13 by any method: genetic counselling within days, because options include diagnostic testing and decisions depend on gestation and local law.
• Alpha fetoprotein of 2.5 MoM or more: confirm dates, exclude twins, and arrange a detailed scan to look for neural tube and abdominal wall defects; amniocentesis for acetylcholinesterase if the scan is inconclusive.
• Failed NIPT with raised nuchal translucency or abnormal scan: diagnostic testing rather than repeat NIPT.
• Cell free DNA result showing multiple chromosome abnormalities, or sex chromosome result not matching scan: consider maternal causes (maternal mosaicism, malignancy), and arrange maternal assessment.
• Very low pregnancy associated plasma protein A (under 0.2 MoM): serial growth scans and uterine artery Doppler assessment.

Interferences and pitfalls.

• Wrong gestational age is the commonest cause of false results for every marker, so dating by crown rump length is needed.
• Maternal factors change marker levels: raised weight lowers concentrations (haemodilution) and low weight raises them, insulin treated diabetes lowers alpha fetoprotein, and smoking lowers human chorionic gonadotrophin but raises pregnancy associated plasma protein A. Women of African ancestry have higher alpha fetoprotein and human chorionic gonadotrophin, and ethnic corrections are required.
• Twin pregnancies have reduced biochemical performance and need nuchal translucency based risks for each foetus; vanishing twins can cause false positives in NIPT.
• Confined placental mosaicism (about 1 to 2% of chorionic villus samples) is the main cause of false positive NIPT, and amniocentesis is more reliable than villus sampling for resolving it. Maternal chromosomal mosaicism, maternal copy number variants, benign fibroids, previous organ or stem cell transplant from a male donor, and undiagnosed maternal cancer also cause discordant NIPT.
• NIPT has lower accuracy in low foetal fraction, in twin pregnancies, with egg donation, and in the presence of maternal obesity.
• Screening positive predictive values are lower than patients expect; many women overestimate a high chance result as a diagnosis. Good counselling about the difference between screening and diagnosis is as important as the test.
• Screening does not detect most genetic disorders, structural anomalies, triploidy (except biochemical patterns), microdeletions, or single gene conditions. A normal result does not guarantee a healthy baby.
• Access to ultrasound, accredited nuchal translucency measurement, quality assured biochemistry and NIPT is limited in many settings; where only second trimester biochemical screening or ultrasound is available, programmes should state its performance.
• Cell free DNA findings can reveal unexpected maternal conditions (cancer, mosaic aneuploidy), and policies on reporting are needed.
• Disclosure of foetal sex raises the risk of sex selective termination, so some programmes withhold it.

Special groups.

• Women over 35 or 40: higher baseline risk, so the same screening result gives a higher chance than in younger women; NIPT or diagnostic testing is often chosen directly.
• Twin pregnancies: combined test with nuchal translucency is used; the quadruple test is not used, and NIPT is validated for dichorionic twins but with lower sensitivity.
• Pregnancy after in vitro fertilisation or egg donation: use the egg age based risk and corrected marker values.
• Previous affected pregnancy or parental translocation carrier: offer diagnostic testing.
• Obese women: higher failure rate with NIPT, and ultrasound is technically more difficult.
• Late presenters (after 14 weeks): quadruple test or NIPT; second trimester ultrasound markers.
• Regions: in many parts of Africa and Asia, access to first trimester ultrasound and NIPT is limited, and programmes may rely on maternal age, quadruple test at selected centres and the anomaly scan; maternal age risk and consanguinity counselling are used to prioritise referral.

Follow up tests. Chorionic villus sampling or amniocentesis with rapid aneuploidy testing (quantitative fluorescent PCR), karyotype and chromosomal microarray, detailed anomaly scan at 18 to 22 weeks, foetal echocardiography for raised nuchal translucency, amniotic fluid alpha fetoprotein and acetylcholinesterase for suspected neural tube defects,

exome sequencing for normal microarray with multiple anomalies, serial growth scans and uterine artery Doppler for low pregnancy associated plasma protein A, maternal assessment for discordant cell free DNA, parental karyotype, and genetic counselling.