Medical analysis

Double Marker Study

By: Dr. Dipak Ladda M.D.

Introduction – Double marker

It helps to determine whether the unborn baby has any risk of developing neurological abnormalities or mental disorders.

It detects Down syndrome or Trisomy 21, Edward’s Syndrome of Trisomy 18, and Patau’s Syndrome of Trisomy 13, which cause mental and physical challenges.

Chromosomal Syndromes and Their Defects

What are tests done?

Combined First Trimester Screening

A double marker test consists of a blood sample and an ultrasonic examination. That’s why it is called as “Combined First Trimester Screening”.

Two Markers Examined

The two markers examined in the double marker test are free beta hCG (human chorionic gonadotropin) and PAPP-A- pregnancy-associated plasma protein A. For detailing please refer respective ppts of tests i.e. Free Beta HCG and PAPP-A.

How this test to be moved?

1. Timing: This test is conducted between 9th and 13th weeks of pregnancy. Timing is very important to get accurate results.
2. Patient Discussion: Discuss in details with patient i.e. Purpose of the test, benefits and potential risk of results.
3. Ultrasound Reports: Ultrasound examination reports are must.
4. Understanding Results: This test does not provide definite diagnosis but shows a risk in proportion for chromosomal abnormalities.
5. Further Testing: Your doctor may recommend further tests like amniocentesis or chorionic villous sampling to confirm suspected abnormalities.
6. Genetic Counseling: Genetic counseling for implications of findings.

Role of Ultrasound in Double Marker

The biochemical screening estimates are further improved by adding the CRL and NT measurements obtained from a level II Ultrasound scan. Needless to say the Ultrasound should be performed by an experienced Sonologist or Fetal Medicine Expert who has the training and experience in taking various fetal measurements like CRL, NT and others.

Significance of Ultrasound Examination

1. To confirm gestational age: Accurate dating ensures the correct interpretation of double marker test.
2. Nuchal Translucency Measurement (NT): Clear space in the tissue at the back of the fetus’s neck. Raised NT suggests the possibilities of chromosomal defect prompting further study.
3. CRL: Crown Rump Length (Top of head to bottom of torso length of fetus).
4. Structural Abnormality Detection: To detect any other gross structural abnormality.
5. Risk Calculation: Assesses in calculating combined risk for chromosomal abnormalities.
6. Genetic Counseling Support: Helps in comprehensive genetic counseling and explaining implications of the screening results.

Sample Collection & Transportation

Collection: Collect 3.0 ml blood in plain tube (Red capped).

Processing: Separate serum as early as possible and send to lab along with following information.

Required Information: Patient’s date of birth, weight and LMP. Ultrasound report along with fetus age and NT measurement.

Assay Methods & Reference Range

Refer ppts of Free Beta HCG and PAPP-A for assay methods.

Differences between Total & Free Beta HCG

Normal Reference Range – Free Beta HCG

Pathophysiology: PAPP-A

Placentation Impact: Low levels of PAPP-A possibly remains responsible for abnormal placentation, a concern for the development of preeclampsia during late gestation.

Protein Function: A (PAPP-A) – It is a protein produced by the placenta in early pregnancy. Abnormal levels are associated with an increased risk for chromosome abnormality.

Clinical Associations: Low maternal serum PAPP-A levels in the first trimester are associated with poor fetal growth and development of pregnancy induced hypertension, preeclampsia and stillbirth.

Adverse Outcomes: Very low PAPP-A levels are frequently related to adverse pregnancy outcome.

Normal Reference Range – PAPP A

MoM (Multiple of Median)

Definition: Multiple if Median: When multiple parameters show results deviating from their median value.

MoM Formula: Patient’s Results / Median Value for Gestational Age.

Median Value Calculation: Median Value for Gestational Age: It is calculated by taking mean from the reference population for the same gestational age.

MoM and Their Significance in Double Marker Study

Normal MoM Values: MoM for Free Beta HCG: 1.0; MoM for PAPP-A: 1.0

Increased Risk Pattern: Higher MoM for Free Beta HCG & Lower MoM for PAPP-A: Increased Risk of Down’s Syndrome.

Decreased Risk Pattern: Lower MoM for Free Beta HCG & Higher MoM for PAPP-A: Decreased Risk.

Responsibility of Doctor to Provide Following Information

1. Physical Measurements: Weight (kgs) & Height (cms) of patient.
2. Medical History: History of previous pregnancy helps to assess the risk.
3. Medications & Conditions: Things which can cause changes the hormonal levels; eg. Diabetes, HCG injections or any other medications.
4. IVF Considerations: For IVF conception; risk assessment is related with Date of Birth of Donor.
5. Ultrasound Accuracy: Accurate Ultrasound report is must. Total sensitivity and accuracy of the test results vary with ultrasound report.

Test Results

It is calculated by combining the biochemical results obtained (values of Free Beta HCG & PAPP-A) with the patient’s information along with CRL & NT value provided in previous page by using SsdwLab software or any other standard software.

All these parameters provide us MoM (Multiple of Median) values.

After due consideration of maternal & medical history, gestational age and machine on which parameters are calculated; the MoM values are calculated.

Interpretation of the results

Risk Assessment Graph: By considering age, biochemical parameters and NT value the graph is plotted; which provides the risk assessment for Trisomy 21 (Down’s Syndrome), Trisomy 18(Edward’s Syndrome) and Trisomy 13 (Patau’s Syndrome).

Risk Cut-off: A cut off of 1 in 250 is used to differentiate between Low and High risk.

Comments for the test

Test Platform & Software: This final section includes test platform, software used and disclaimers associated with screening test.

Screening vs Diagnostic: This is simple screening test and not a diagnostic one.

Clinical Decision: So; doctor must decide whether to consider additional tests for further evaluation or confirmation.

Conclusion

For more information on Double Marker Study and prenatal screening, please consult with your healthcare provider.

References:

• Snijders RJM, Sundberg K, Holzgreve W, Henry G, Nicolaides KH. Maternal age- and gestation-specific risk for trisomy 21. Ultrasound Obstet Gynecol. 1999;13:167–170.
• Wilson RD. Amended Canadian Guideline for prenatal diagnosis (2005) change to 2005—techniques for prenatal diagnosis. SOGC Clinical Practice Guidelines, No. 168. J Obstet Gynaecol Can. 2005;27:1048–1054.
• American College of Obstetricians and Gynecologists (ACOG). Practice Bulletin No. 226: Screening for Fetal Chromosomal Abnormalities. Obstet Gynecol. 2020;136(4):e48-e69.
• Nicolaides KH. Screening for fetal aneuploidies at 11 to 13 weeks. Prenat Diagn. 2011;31(1):7-15.
• Kagan KO, Wright D, Spencer K, et al. First-trimester screening for trisomy 21 by free beta-human chorionic gonadotropin and pregnancy-associated plasma protein-A: impact of maternal and pregnancy characteristics. Ultrasound Obstet Gynecol. 2008;31(5):493-502.
• Wright D, Spencer K, Kagan KO, et al. First-trimester sonographic markers for trisomy 21: a study of 12 188 fetuses. Ultrasound Obstet Gynecol. 2008;32(7):874-880.
• Spencer K, Bindra R, Farecq A, et al. First-trimester ultrasound screening for trisomy 21: the effect of nuchal translucency measurement on the detection rate and false-positive rate. Ultrasound Obstet Gynecol. 2003;22(2):147-152.
• Malone FD, Canick JA, Ball RH, et al. First-trimester or second-trimester screening, or both, for Down’s syndrome. N Engl J Med. 2005;353(19):2001-2011.
• Wald NJ, Rodeck C, Hackshaw AK, et al. SURUSS in perspective. Semin Perinatol. 2005;29(4):225-235.
• Breathnach FM, Malone FD. Screening for aneuploidy in first and second trimesters: is there an optimal approach? Curr Opin Obstet Gynecol. 2007;19(2):176-182.
• Jindal A, et al. Reference Centile Charts of PAPP-A and Free β-hCG in Indian Pregnant Women. Int J Sci Res. 2021.
• Cuckle HS, et al. First-trimester maternal serum screening for Down syndrome: a meta-analysis. Prenat Diagn. 2005;25(13):1160-1165.
• The Fetal Medicine Foundation. Screening for chromosomal abnormalities in the first trimester. [Online Resource].
• Journal of Surgery and Medicine. The relationship of PAPP-A and ß-HCG values with fetal gender and fetal birth weight: A single-center experience. 2025;9(7):93-97.
• Journal of Nursing Reports in Clinical Practice. The first-trimester screening to detect chromosomal abnormalities in pregnant women: A retrospective study. 2023.

FAQ:

• What is a double marker test? It is a combined screening for chromosomal abnormalities like Down, Edward’s, and Patau’s syndromes during early pregnancy.
• When is the test conducted? The test is conducted between the 9th and 13th weeks of pregnancy to ensure accurate results.
• What does the test detect? It detects risk factors for Trisomy 21 (Down’s), Trisomy 18 (Edward’s), and Trisomy 13 (Patau’s) chromosomal syndromes.
• Is it a diagnostic test? No, it is a screening test that identifies risk levels rather than providing a definite diagnosis.
• What markers are examined? The test examines levels of free beta-hCG and PAPP-A in the maternal blood sample.
• Why is ultrasound required? Ultrasound provides essential measurements, such as CRL and NT, which improve the accuracy of biochemical risk estimates.
• What is Nuchal Translucency (NT)? NT is a clear space behind the fetus’s neck; raised measurements may suggest a potential chromosomal defect.
• What does MoM represent? MoM (Multiple of Median) indicates how much a patient’s results deviate from the median value of the reference population.
• What information is needed? Required data includes the patient’s age, weight, last menstrual period (LMP), and the latest ultrasound report.
• What if the risk is high? High-risk results suggest the need for further diagnostic evaluations like amniocentesis or chorionic villous sampling.

For Non-Medicos:

Overview

The Double Marker Study, also known as the Combined First Trimester Screening, is a prenatal screening test used to assess the risk of chromosomal abnormalities in an unborn baby. It is performed between the 9th and 13th weeks of pregnancy and combines biochemical markers in the mother’s blood with ultrasound findings to calculate risk levels.

This test helps detect three major chromosomal syndromes:

1. Down Syndrome (Trisomy 21)
2. Edward’s Syndrome (Trisomy 18)
3. Patau’s Syndrome (Trisomy 13)

The test measures Free Beta hCG and PAPP-A (Pregnancy-Associated Plasma Protein A), and combines these results with NT (Nuchal Translucency) and CRL (Crown-Rump Length) obtained from a specialized ultrasound. The Double Marker Study does not diagnose abnormalities—it only provides a risk proportion. High-risk results may require confirmation through amniocentesis or chorionic villous sampling, followed by genetic counseling.

Symptoms

The test itself does not screen for physical symptoms in the mother; rather, it screens for potential developmental problems in the baby. The abnormalities indicated by this test are associated with specific fetal symptoms described in the PDF.

Symptoms Related to Trisomy 13 (Patau Syndrome):

1. Cleft palate
2. Extra fingers or toes
3. Low muscle tone
4. Small head
5. Possible severe defects in internal organs

Symptoms Related to Trisomy 18 (Edward’s Syndrome):

1. Craniofacial malformations
2. Abnormal hands and feet
3. Skeletal malformations
4. Congenital heart defects
5. Cryptorchidism
6. Feeding difficulty
7. Under-developed lungs
8. Joint and bone abnormalities
9. Hearing loss
10. Cleft lip
11. Eye defects

Symptoms Related to Down Syndrome (Trisomy 21):

1. Characteristic facial features
2. Learning difficulties
3. Birth defects
4. Vision and hearing problems
5. Possible heart abnormalities

These symptoms highlight why early screening is essential for pregnancy planning and management.

Causes

According to the PDF, chromosomal abnormalities detected by the Double Marker Study arise from genetic issues leading to abnormal fetal development.

Primary Causes Detected by the Test:

1. Trisomy 13 (Patau’s Syndrome): Caused by an extra chromosome 13, leading to severe structural and organ defects.
2. Trisomy 18 (Edward’s Syndrome): Caused by an extra chromosome 18, resulting in multiple malformations and developmental challenges.
3. Trisomy 21 (Down Syndrome): Caused by an extra chromosome 21, the most common trisomy associated with cognitive and physical challenges.

Biochemical Causes Indicated by Markers:

1. Low PAPP-A: Associated with abnormal placentation, poor fetal growth, pregnancy-induced hypertension, preeclampsia, and possible stillbirth.
2. Abnormal Free Beta hCG levels: Indicate variations in trophoblastic function and increased likelihood of chromosomal abnormalities.

Technical Causes (Affecting Interpretation):

1. Incorrect gestational age
2. Inaccurate NT or CRL measurements
3. Missing ultrasound reports
4. Incomplete patient information (age, weight, LMP)

Risk Factors

The PDF identifies several factors that may increase the likelihood of abnormal Double Marker results or influence risk calculation.

Maternal Risk Factors:

1. Advanced maternal age
2. History of chromosomal abnormalities in previous pregnancies
3. Medical conditions such as diabetes
4. Medication use, including hormonal treatments (e.g., hCG injections)
5. IVF pregnancies, where donor age affects the calculation
6. Inaccurate or incomplete medical history

Test-Related Risk Factors:

1. Errors in ultrasound measurements (NT, CRL)
2. Incorrect gestational age interpretation
3. Low PAPP-A levels in first trimester
4. High Free Beta hCG MoM values
5. Calculation biases due to incorrect patient data, such as weight or date of birth

Syndrome-Specific Risk Patterns (MoM Values):

1. High Free Beta hCG MoM + Low PAPP-A MoM = Increased risk of Down Syndrome
2. Opposite pattern = Decreased risk

These factors emphasize the need for precise data entry and quality imaging.

Prevention

Prevention in the context of the Double Marker Study focuses on accurate testing, proper data collection, and early pregnancy monitoring, as highlighted in the PDF.

Clinical Prevention Measures:

1. Conduct the test strictly between 9–13 weeks for reliable results.
2. Provide complete patient information, including weight, height, LMP, and medical history.
3. Ensure accurate NT and CRL ultrasound measurements by a trained fetal medicine expert.
4. Discuss the purpose, benefits, and implications of the test in detail with the patient.
5. Recommend further diagnostic tests (CVS or amniocentesis) when risk is high.
6. Offer genetic counseling to help families understand the results.

Sample Collection Prevention:

1. Collect 3 mL blood in a plain red-capped tube.
2. Separate serum early and send it with:
   1. Patient DOB
   2. Weight
   3. LMP
   4. Ultrasound report containing fetal age & NT measurement

Interpretation Prevention:

1. Use standard software such as SsdwLab to generate MoM values.
2. Always combine biochemical markers, maternal information, and ultrasound data for complete risk assessment.
3. Remember that this is a screening, not a diagnostic test.