INVITATION ARTICLES - Prenatal Screening-Biochemical markers by Dr. Sreekumari

Birth of babies with congenital defects is the most traumatic experience to the expectant parents. The incidence of neural tube defects and chromosomal aneuploidy are quite high. Since a positive correlation was found with maternal age, early workers offered the choice of amniocentesis to detect a defective fetus. Amniocentesis posed a risk for the fetus which was as high as the chance of having an affected child, several groups worked on the development of non invasive prenatal screening tests.
All three types of aneuploidy have a strong correlation with maternal age above 35 years. Since the changing lifestyle has increased the maternal age above 35, reliable markers with high detection rate and low false positive are found to be essential.
Thus several biochemical markers were measured in maternal serum and have now been identified as reliable indices of neural tube defects and chromosomal aneuploidy. Most of these markers are normally present in maternal serum and their level varies with gestational age. Hence these markers are said to have temporality. So both the patient and requesting physician must have a clear idea that the result of a screening test is different from that of a diagnostic test. In order to confirm the diagnosis of a chromosomal disorder, invasive procedures like Chorionic villous sampling or amniocentesis should be done and cytogenetic studies on the cells to be carried out.
Unlike markers used in other diseases, the levels of biochemical markers used in prenatal diagnosis are expressed by the statistical term Multiple of Median (MOM). The markers are quantitated by any of the modern techniques like RIA/EIA/CLIA and then converted to MOM. MOM values normalize results for purposes of comparison between laboratories .Each laboratory has to use its own data to establish Median values for each marker (analyte) for each week (day) of gestation. Ideally the median is to be calculated with results from 200 samples. MOM values are obtained by dividing the actual value with the lab Median. Kit specific and population specific median values increase reliability and the MOM should be updated.
Since the 1990’s prenatal screening has become standard obstetric practice in all pregnancies at risk. Since maternal age is associated with errors in meiosis an increase in maternal age increased the risk of having children with chromosomal aneuploidy. This is one of the primary indications for prenatal screening.
The Triple Test (Triple screen)
The triple screen is done during the second trimester between 14 and 18 weeks. The markers used are AFP, uE3 and HCG. The pattern of changes and detection rates for Neural tube defects, Trisomy 21 and trisomy 18 are given in table.
Alpha fetoprotein is the major serum protein of the fetus synthesized by the fetal liver and yolk sac. There is a steady increase in AFP level in maternal serum from 10th week of gestation and reaches a peak by 25 weeks of gestation in unaffected pregnancy. Then the maternal serum alpha feto protein (MSAFP) steadily declines until term. In fetal serum and amniotic fluid, the AFP level reaches a peak by 9th week of gestation and then slowly falls till term. In NTD, the AFP is increased but in chromosomal aneuploidy it is decreased.
Human Chorionic Gonadotrophin is a glycoprotein hormone produced during normal pregnancy by the trophoblast and placenta. HCG appears in maternal serum by 6 to 8 weeks and reaches a peak by 10 weeks. By the second trimester it falls to a constant level by 18 to 20 weeks.
A marked increase about twice the normal was found in pregnancies with trisomy 21 during the second trimester. It was also noted that free beta HCG was increased during the 1st trimester in DS (MOM 2.4) even though total HCG remained normal (MOM 1.33). Both were increased during the second trimester in Trisomy 21(MOM 2 to 2.5). A hyperglycosylated variant (produced by cytotrophoblasts) was also found in Down’s syndrome. This is referred to as Invasive Trophoblast Antigen (ITA). The higher level of ITA is believed to be due to the defect in the conversion of cytotrophoblast to syncytiotrophoblasts. In trisomy18 the HCG levels remained lower than normal.
Unconjugated Estriol (uE3) is a steroid hormone produced by the fetoplacental unit .It is produced by the placenta but the fetal liver completes the synthesis. It is an estrogen with 3 hydroxyl groups and 3 organs –fetal adrenal, fetal liver and maternal liver are involved in the synthesis. Initially estriol was used as a measure of fetal wellbeing especially at term. Maternal serum uE3 levels rise by 8weeks of gestation and continue to increase through out pregnancy. A 25 % reduction in uE3 levels was found in maternal serum when the fetus had chromosomal aneuploidy.
Even though the Triple screen has a high detection rate, temporalities of the markers have to be emphasized. During the second trimester, AFP and uE3 increase in unaffected pregnancies, where as that of HCG declines. Yet another point which can give false positive results is the method of calculation of gestational age- by ultrasound or by LMP. This fallacy can be corrected using the USG results at 6 weeks of gestation for calculating correct gestational age.
In spite of these limitations, the triple screen has a high detection rate, 80% for neural tube defects and 55-60% for chromosomal aneuploidy and a false positive less than 5 %. When the cut off is around 1:270,false positive rises to 7 to 9%.The increase in maternal age with the changing lifestyle, career options and work pressure in educated and employed women have increased the need for additional markers.
The Quadruple Test (Quad screen)
Includes AFP.uE3, HCG and an additional marker Inhibin-A
Dimeric Inhibin A (DIA) is a glycoprotein produced by the placenta. It is a Dimer, but with dissimilar subunits alpha and beta. Inhibin A has the subunit make up ab-A and inhibin B a-b B .Inhibin A is measurable in maternal serum and has a feed back effect on FSH secretion.
The level increases in the first trimester until 10 weeks and then remains stable upto 25 weeks of gestation. Thereafter it increases to reach a peak by term. The DIA levels are increased in DS and remains elevated through out the second trimester unlike AFP and uE3 that increase and hCG that decreases during the testing period.
It is the least stable of the four analytes, since decomposition can occur during storage. Serum should be promptly separated and assay completed within 3 days. Whole blood should not be transported. 0.7 –2.5 microgram/L in unaffected pregnancy at second trimester.
Statistical modeling of different combinations of AFP, uE3, hCG and DIA indicates that the highest detection rates and lowest false positive rates are achieved when using all four markers corrected for maternal weight. DIA is an independent variable having no correlation with maternal age, race or Insulin dependent diabetes mellitus. The only factor which has a significant effect on DIA levels is maternal weight. There was no correlation with AFP and uE3, but significant correlation was found with hCG
Factors affecting the level of the Quad screen markers

  • Maternal Weight was found to have an inverse relation with the levels of all four markers.

  • Diabetes- Insulin dependent (not GDM) AFP was found to be 40% lower than non diabetics

  • Twin pregnancy- MSAFP higher than those having single fetus. MOM > 4 significant

  • Racial differences were also noted, but not significant in our set up

Screening during the First trimester
Several workers suggested that a screening test done between 10 and 14 weeks of pregnancy (1st trimester) may be as accurate as that done during the second trimester. In this screening method, AFP, hCG and Pregnancy associated plasma protein-A(PAPP-A) are measured along with ultrasound examination for Nuchal Translucency(NT) This screening pattern is referred to as an OSCAR(One Stop Clinic for Assessment of Risk) .All the markers are measured in a single serum sample.
PAPP-A is a high molecular weight zinc containing metalloglycoprotein. It is produced by the trophoblast and its biological function is still unclear. In addition to being a marker of chromosomal aneuploidy, it is an indicator of early pregnancy failure and complications, Cornelia de Lange syndrome and acute coronary syndrome.
The level of PAPP-A was found to be significantly lower in pregnancy with Trisomy 21 compared to unaffected pregnancy (MOM 0.27 as against 1.01 in normal pregnancy)But PAPP-A levels when measured in second trimester the results are found to be normal. Persistently lower levels of PAPP-A in second trimester was indicative of Trisomy18.
Similarly total hCG was found to be a poor marker in the first trimester with an MOM of 1.33, but an adequate marker during the second trimester. Free beta hCG on the other hand is higher and has a relatively stable MOM (2) from 10 to 18 weeks.
The detection rate was 89% with 5% false positive.

Maternal serum marker(10-14 weeks)


Down’s syndrome

Trisomy -18






MOM 0.45

Total beta hCG





Free beta hCG




MOM 2.0

Hence the present suggestion is to combine the markers of first and second trimester in maternal serum. The suggested protocol is given

  • Measurements of NT and PAPP-A are made in the first trimester, but not interpreted or acted upon until the second trimester.

  • In the second trimester a second serum sample is drawn and quadruple test performed.

  • Results for all the six tests, NT, PAPP-A, AFP, uE3, hCG and DIA are combined into a single risk estimate for interpretation in the second trimester.

  • 85% detection rate for DS with only 1% false positive is achieved

  •  Information to be provided by Physician
    1. Specimen collection date
    2. 1st or second specimen
    3. date by LMP or gestational age by USG
    4. maternal age and weight
    5. relevant family and obstetric history
    6. presence of maternal Diabetes mellitus
    7. Maternal race if indicated
    8. Whether multiple pregnancy is present/suspected
    The report should contain
    1. MOM values for the measured analyte
    2. DS risk estimate along with risk for NTD or trisomy 18
    3. an interpretation as screen positive or screen negative
    4. information that suggests possible further action.
    Interpretation of screen results and calculation of risk
    It is seen that about 5% test screen positive, for DS, 3% for NTD and 0.2 % for Trisomy 18, but inaccurate dating can give an abnormal screening result. Ultrasound dating gives the best dating accuracy. The MOM values are adjusted according to the patient’s gestational age, maternal weight, insulin dependent diabetic status and twin pregnancy.
    The AFP, hCG, uE3 and DIA values are then compared to known data for affected and unaffected pregnancies and a likelihood ratio is calculated for each marker. The likelihood ratios are then combined with the patient’s age related risk for DS. The resulting number is the patient’s specific risk for DS. There are no reference intervals for each analyte.
    The detection rate and false positive rate are determined by the screen positive cut off level. Maternal serum marker levels are used to modify a pregnant woman’s age related risk (priori risk) to calculate the patient specific risk. A test is said to be positive if a woman’s fetal DS risk by maternal serum screening is the same as or greater than the fetal DS risk of a 35 year old woman. At 35 years, the risk during second trimester is 1/270 (with 1/350 at term). Using a risk of 1/270 as a cut off level, four marker screening will achieve a detection rate of 75 to 80% which is 15 to 20% above the DR of Triple screen with same false positive rate of 5%. However if the cut off is fixed at 1/150 then the false positive falls to 3%. This would ensure less patient anxiety and fewer amniocenteses.
    False positive rate signifies the proportion of women with test results falling at or above a specified MOM for AFP .But since several women are True positive, at present the term Initial positive rate (IPR) is used instead of FPR. The IPR can be used to assess whether Medians are appropriate since it will be shifted upward or downward if medians are incorrect.
    Follow up of Patients with Screen positive results:

    • Genetic counseling if patient is screen positive

    • For moderately elevated results (MOM 2-3 ) a second test

    • If second test is negative – Screen negative

    • If second test also gives elevated results, further testing

    • USG, Amniocentesis and analysis of AF for Acetyl Choline esterase to confirm NTD

    • Amniotic fluid AFP results may give false positive due to contamination by fetal blood, hence confirmed by Acetyl choline esterase .Ach-E is not normally present in AF, but appears in open neural tube defects since fetal CSF contains the enzyme

    • In cases of suspected Chromosomal aneuploidy, Fetal karyotyping.



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