In a pregnant woman with a singleton pregnancy and no prior abnormal screening, what is the triple test, how is a positive result defined, and what are the recommended next steps?

The Triple Test in Prenatal Screening

What is the Triple Test?

The triple test is a second-trimester maternal serum screening that measures three analytes—maternal serum alpha-fetoprotein (MSAFP), human chorionic gonadotropin (hCG), and unconjugated estriol (uE3)—to adjust a woman's age-based risk for Down syndrome (trisomy 21) and trisomy 18, with a detection rate of approximately 65% for Down syndrome. 1

Components and Timing

• The three markers measured are: 1

  • Maternal serum alpha-fetoprotein (MSAFP)
  • Human chorionic gonadotropin (hCG)
  • Unconjugated estriol (uE3)

• Optimal testing window is 16-18 weeks gestation, though testing can be performed between 15.0 and 20.9 weeks 1

• Accurate gestational age is critical—ultrasound dating significantly improves both sensitivity and specificity compared to last menstrual period (LMP) dating alone 1

How Results are Interpreted

Pattern Recognition for Different Conditions

For Down syndrome (Trisomy 21): 1

• AFP levels are lower than normal
• hCG levels are higher than normal
• uE3 levels are lower than normal

For Trisomy 18: 1

• All three markers are low
• Detection rate is at least 70% 1

Defining a Positive Result

• A positive screen for Down syndrome is defined as a calculated risk of ≥1:270 (or sometimes 1:200 depending on the laboratory) 1, 2, 3

• For trisomy 18, a positive screen requires: 3

  • AFP ≤0.75 multiples of the median (MoM)
  • uE3 ≤0.60 MoM
  • hCG ≤0.55 MoM

Detection Rates and Performance

• The triple test detects approximately 65% of Down syndrome cases with a false-positive rate of approximately 5-8% 1

• In women ≥35 years old, detection rates may reach 75-85% with false-positive rates of 21-27% 2, 3

• The test detects 70% or more of trisomy 18 cases 1

Critical Factors That Must Be Reported for Accurate Interpretation

The laboratory requires the following information to adjust MoM values: 1

• Gestational age (expressed as weeks and days, not rounded weeks)
• Maternal weight (AFP levels are higher in lighter women, lower in heavier women)
• Maternal race (Caucasian or Black/African American)
• Presence of insulin-dependent diabetes
• Number of fetuses (singleton vs. twin)
• Family history of neural tube defects

If gestational age is discrepant by ≥2 weeks after ultrasound examination, the test result must be reinterpreted with the corrected dates. 1

Recommended Next Steps After a Positive Result

Immediate Management

When the triple test is positive for Down syndrome or trisomy 18, genetic counseling should be offered immediately, followed by discussion of diagnostic testing options. 1

The recommended diagnostic pathway includes: 1

1. Genetic counseling to explain the specific risk calculation and limitations of screening
2. Amniocentesis for definitive karyotype analysis (the gold standard for diagnosis)
3. Targeted ultrasound examination to evaluate for structural abnormalities associated with aneuploidy

For Elevated MSAFP (Neural Tube Defect Screening)

If MSAFP is elevated (≥2.0-2.5 MoM in singletons, ≥4.0-5.0 MoM in twins), the following steps are recommended: 1

1. Verify gestational age with ultrasound—if dates are off by ≥2 weeks, reinterpret the result
2. Genetic counseling
3. Targeted ultrasound examination to evaluate for neural tube defects and ventral wall defects
4. Amniocentesis for amniotic fluid AFP and acetylcholinesterase if ultrasound is inconclusive

Important Clinical Caveats

Limitations of the Triple Test

• The triple test is a screening test, not a diagnostic test—it only modifies risk based on maternal age and does not provide a definitive diagnosis 1

• The test does NOT detect other aneuploidies such as trisomy 13 or Klinefelter syndrome (47,XXY), which would only be detected on routine karyotype analysis 1

• Approximately 30-35% of Down syndrome cases will be missed (false-negative rate) even with optimal screening 1, 3

• The false-positive rate is 5-8%, meaning most women with positive screens will have unaffected pregnancies 1, 3

Common Pitfalls to Avoid

Technical errors that reduce accuracy: 1

• Using LMP dating instead of ultrasound dating
• Failing to reinterpret results when gestational age is corrected
• Not adjusting for maternal weight, race, or diabetes
• Using singleton cutoffs for twin pregnancies

Clinical management errors: 1

• Inadequate pre-test counseling about the limitations of screening
• Not offering follow-up diagnostic testing after a positive screen
• Treating a positive screen as a diagnosis rather than a risk assessment

Modern Context

It is important to note that the quad screen (which adds inhibin-A as a fourth marker) has largely replaced the triple test in clinical practice, improving Down syndrome detection to approximately 75-80%. 1

More recently, noninvasive prenatal screening (NIPS/cell-free DNA) has become the preferred screening method, with detection rates exceeding 99% for trisomy 21 and significantly lower false-positive rates than traditional serum screening. 1