MATERNAL SERUM SCREENING M.PRASAD NAIDUMSC MEDICAL BIOCHEMISTRY,Ph.D.RESEARCH SCHOLAR

•Second trimester biochemical screening[BCS] started in the 1970’s when it was found that fetal neural tube defects[NTD’s] were associated with increase in maternal serum alpha feto protein[MSAFP].

•Such measurements were offered to pregnant woman for screening purposes.

•While the screening protocols for NTD’s were being refined, it was noted that MSAFP tended to be low in fetal down’s syndrome.

•With a cut off of 2.0 multiples of median[MOM] 85% of NTD’s would be screened in & with a threshold of 0.5 MOM approx 33% of DS fetus would be screened in.

With the addition of two other analytes Estriol [which is low in DS] & Human chorionic gonadotropin[hCG] [which is increased in DS] the sensitivity of biochemical screening for DS rose to approx 65% across all ages & was over 85% in those above 35 years of age.

The most recent addition to the biochemical screening regimen taking the above “Triple screen” to “Quad screen” is inhibin A.

This increases the sensitivity of the combined test by approx 8%.

•Woman with elevated serum AFP levels were offered Diagnostic amniotic fluid AFP testing[AFAFP].

•Initially the presence of open spina bifida could be confirmed only by ultrasound examination of the fetal spine.

•Now the recommendation is to perform the Triple marker screen test on all pregnant woman’s between 14 & 20 weeks gestation to assess the risk for neural tube defects, trisomy 21 & trisomy 18.

A further important breakthrough occurred with the identification of two biochemical markers [Pregnancy associated plasma protein A {PAPP-A} ; Free beta subunit of human chorionic gonadotrophin {βhCG} ] AND Ultrasound marker [Nuchal Translucency{NT}] as markers for down’s syndrome & trisomy 18 in the First trimester.

When used together these markers perform better than second trimester screening and have the added advantage of early detection.

These tests may also aid in the asessment of risk for obstetric complications such as pre-eclampsia,abruption,preterm labour & IUGR.

Suggested protocol for screening

1. Measurement of nuchal translucency[NT] & PAPP-A in the 1st trimester, but not interpreted or acted upon until the second trimester.

2. In the second trimester a second serum sample is drawn and Quadruple test performed.

3. Results for all the six tests , NT, PAPP-A, AFP, uE3 , hCG & DIA are combined into a single risk estimate for interpretation in the 2nd trimester.

4. 85% detection rate for Down’s Syndrome with only 1% false positive is achieved.

•TRIPLE SCREEN TEST •1.ALPHA FETO PROTEIN

• In 1956,Bergstrand & czar described a protein in fetal serum,located in the α1 region on electrophoresis[subsequently labelled as α1-Feto Protein[AFP] that was not present in maternal serum.

• It is this unique protein that serves as a marker for leakage of fetal serum into the amniotic fluid & which is therefore helpful in diagnosing open fetal lesions.

•AFP is the major serum protein of fetus synthesized by the fetal yolk sac & fetal liver

• Gene located on chromosome 4,is also part of a family of genes that also encodes for albumin & vitamin D-binding protein. .The protein is composed of carbohydrate & a single polypeptide chain containing 591 amino acids.

The molecular weight and structure of AFP is similar to that of albumin[about 69kd],but antibodies rised against AFP have virtual no cross reactivity.

This characteristic was critical in allowing the development of a veriety of antibody based assays for reliably measuring AFP in amniotic fluid & maternal serum.

The protein is very stable @room temperature in serum as long as a week.

•Maximum concncentration of AFP in fetal serum~3,000,000ng/ml reaches by 9 wk gestation decreases to 20,000ng/ml @ term.

•Maternal serum AFP first detectable [~5ng/ml] at about 10 wk gestation.•The concentration increases at a rate of 15% per week to a peak at about ~180ng/ml @ 25 wk gestation,decline slowly till term.

After birth MSAFPdecreases to less than 2ng/ml.

In infant,serum AFP level decreases exponentially to reach adult level by 10th month of life.

MULTIPLES OF MEDIAN [MOM]•To simplify interpretation of the result , each patient AFP result expressed as a Multiples of Median[MOM].

•Screening programmes should determine the AFP medians for each week of gestation from 14 to 20 weeks using at least 100 patients at each week.

METHODS FOR DETERMINING α-Feto Protein.

•TRADITIONALLY MEASURED BY RADIO IMMUNO ASSAY [RIA]

•NEWER METHODS USE IMMUNO ENZYMATIC ASSAYS [IEMA]

•Because of it’s lower detection limits , better precision , speed, avoidance of radiation & ease of automation.

•The FDA has licenced three immuno assay AFP kits for use in maternal serum screening for neural tube defects,

1. A monoclonal bead assay

2. A microparticles immuno assay

3. A polyclonal bead assay

•Each assay uses a sandwich design

•A solid phase antibody captures the AFP present in serum, then, after washing a second enzyme labelled antibody is added.After a second wash that removes unbound labelled antibody, substrate is added to produce a coloured product.

Relative concentration of AFP in maternal serum & amniotic fluid

Gestational age in weeks

Concentration of MSAFPng /ml

Concentration of AFAFPng/ml

15

16

17

18

19

20

24.1

30.1

33.4

41.5

48.0

55.5

16.08

13.04

11.02

9.09

8.13

6.62

•MSAFP a cut off point of 2.5 MOM & values below 0.5 MOM are abnormal for Elevated & lowered values.•AFAFP a cut off point of 2.0 MOM is used to identify elevated AFAFP & 1.0 MOM for lowered values. 1. MILD - 2.0 to 4.9 MOM

2. MODERATE - 5.0 to 9.9 MOM3. VERY HIGH - > or Equal to 10.0 MOM

CLINICAL SIGNIFICANCE OF AFPPredicting the risk of open

NTD’s.Managing certain neoplasms.

HUMAN CHORIONIC GONADOTROPHIN[hCG]It is a glycoprotein hormone with molecular

weight of 36 to 40 kd that is biologically & immunologically similer to LH[Leutenizing hormone] but with a longer half life.

Produced during normal pregnancy by the trophoblast & placenta.

hCG is a hetero dimer having α & β subunits of which the β subunit is specific for hCG.

All the glycoprotein hormones[hCG,LH,FSH,TSH] have a similer biological activity which is characteristic of the β subunit component.

It is because of this similarity that hCG seems to have a stimulatory effect on the maternal thyroid in early pregnancy when hCG levels arehighest.

The α subunit & carbohydrate components are required for expression of the biological activity unique to the β subunit.

The 28-30 AA on the C-terminal end of the β subunit of hCG are unique compared with LH.

In spontaneous pregnancy, hCG can be detected by the 9th day after the LH surge.

This initial detection in maternal blood has been found to correlate with the implantation of the blastocyst & specifically with the moment that lacunae receive maternal blood.

hCG appears in maternal serum in significant quantity by 6-8 wks & reaches a peak by 10th wk of gestation.

By the second trimester it falls to a constant level by 18-20 wks.

A marked increase of total hCG about twice the normal value was found in pregnancies with Trisomy 21 during the 2nd trimester.

Free β-hCG was increased during the 1st trimester in Trisomy 21[DS] even though total hCG remained normal.

@16 wk gestation hCG median level in normal pregnancy is 20,000 – 40,000 IU / L .

METHODS FOR DETERMINING hCG QUALITATIVE TESTS1. HOME TEST KITS : MOST COMMONLY USED

PREGNANCY TESTS.2. RADIO IMMUNO ASSAY.3. IMMUNO ENZYMATIC ASSAY[IEMA].4. IMMUNO RADIOMETRIC ASSAY[IRMA].

QUANTITATIVE TEST : By IMMUNOCHROMATOGRAPHY

CLINICAL SIGNIFICANCE OF hCG5. Diagnosis & dating of pregnancy.6. Predicting the risk of Trisomy 21 & 18.7. Managing certain neoplasms.

UNCONJUGATED ESTRIOL [uE3]

Estriol as it’s name implies, is an estrogen with 3 hydroxyl groups [at position 3,16, & 17 ].

3 organs involved in the biosynthesis1.Fetal adrenal - Cholesterol2.Fetal liver -

DHEAs[DehydroEpiAndrosteroneSulfate]3.Placenta - Estriol

Only a minor amount [9%]of the hormone circulates in plasma unconjugated.

Maternal serum uE3 levels rise by 8 weeks of gestation & continue to increase throught the pregnancy.

A 25% reduction uE3 levels was found when the fetus had chromosomal aneuploidy.

The concentration typical for the 2nd trimester of pregnancy, 0.30 – 1.5 μg / L

METHODS OF DETERMINING UNCONJUGATED ESTRIOL

By ULTRA SENSITIVE RADIO IMMUNO ASSAY METHOD

The determination of uE3 is the most difficultThe analyte has a concentration lower than & is lower in molecular weight than AFP & hCG .

The Triple screen has a high detection rate, 80% for neural tube defecs & 55-60% for chromosomal aneuploidy & a false positive less than 5 % .Conditions associated with abnormal

maternal serum screening results

condition AFP hCG uE3NTD’s VERY

HIGH- VERY LOW

TRISOMY 21[DOWN’S

SYNDROME]

LOW HIGH LOW

TRISOMY 18[EDWARDS

SYNDROME]

LOW LOW VERY LOW

THE QUADRUPLE TEST[QUAD TEST]This includes AFP, Ue3, hCG & an additional

marker INHIBIN-A .

Dimeric Inhibin-A[DIA] is a glycoprotein produced by the placenta.

It is a dimer , but with dissimilar subunits α & β.

Inhibin-A is measurable in maternal serum & has a feedback effect on FSH secretion.

The level increases in the 1st trimester until 10 wks & then remains stable upto 25wks of gestation.

There after it increases to reach a peak by term.

The DIA levels are increased in DS & remains elevated throught the second trimester.

DIA is an independent variable having no correlation with maternal age, race, diabetes mellitus.

Referance value is 0.7 – 2.5 μg / L . In unaffected pregnancy at second trimester.

MSIA - At 14 – 16 wk ---- 150 to 200 pg / ml

AFIA - At 14 – 16 wk ---- 800 to 1200 pg / ml

FACTORS AFFECTING THE LEVEL OF THE QUAD SCREEN

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

2. In Diabetes mellitus,AFP was found to be 40% lower than in non Diabetics.

3. In twin pregnancy, AFP was Higher than those having singlet fetus.

RATES OF DETCTION OF DOWN’s SYNDROME

MATERNAL

AGE[YEARS]

TRIPLE TEST

QUADRUPLE TEST

DETECTIO

N RATE

FALSE POSITI

VE RATE

DETECTIO

N RATE

FALSE POSITI

VE RATE

15 - 34 58 3.7 69 4.1>35 88 19 91 17<15 69 4.9 77 5.2

Pregnancy associated plasma protein – A [PAPP-A]

Measured in the 1st trimester as an early marker for Down’s Syndrome.

PAPP-A is a high molecular weight Zinc containing metalloprotein.

It is produced by the trophoblast.In addition to being a marker of

chromosomal aneuploidy , it is an indicator of early pregnancy failure & complications.

The level of PAPP-A was found to be significantly lower in pregnancy with trisomy 21 compared to unaffected pregnancy.

Persistently lower levels of PAPP-A in second trimester is indicative of Trisomy 18 .

First trimester Ultrasound findings in Down syndrome pregnancies

INDICATIONS.

1.Advanced maternal age(>35)2.Prior pregnancy with a chromosomal disorder

3.Family H/o mental retardation or Birth defects.

In his initial description of the syndrome that bears his name, Langdon Down described skin which was so deficient in elasticity that it appeared to be too large for the body.This was perticularly evident in the neck area of newborns.

Since that time it has been clearly demomstrated that,as early as 10 weeks’ gestation,the fetal neck area is expanded in Down syndrome.Although all fetuses demonstrate a small amount of fluid in the posterior nuchal area(called nuchal translucency(NT)) at between 10 & 13 weeks’gestation,fetuses with Down syndrome will,on everage,have a larger amount.

NT is defined as the maximum fluid-filled space between the skin of the posterior fetal neck area & the underlying strutures.

This area can be measured by transabdominal ultrasound in 95% of cases.

Scond trimester ultrasound markers for Down syndrome.

An increased nuchal fold is the most distinctive second trimester marker.

The distance between the external surface of the occipital bone & the external surface of the skin is measured.

About 35% of Down syndrome fetuses have a nuchal skinfold measurement that is greater than 6mm compared with only 0.7% of unaffected fetuses.

FOLLOW-UP OF PATIENTS WITH SCREEN POSITIVE RESULTS

1. Genetic counseling if patient is screen positive.

2. For moderately elevated results [ MOM 2-3 ] a second test should be done .

3. If second test is negative, screen is taken as negative.

4. If second test is also gives elevated results further diagnostic testing to be done.

5. Ultra sonography, Amniocentesis & Analysis of amniotic fluid for Acetyl choline esterase to confirm neural tube defects.

6. Amniotic fluid AFP results may give false positive due to contamination by fetal blood,Hence confirmed by acetyl choline esterase.

7. Acetyl choline esterase is not normally present in amniotic fluid but appears in open neural tube defects.

8. In cases of suspected chromosomal aneuploidy, fetal karyotyping is to be done.

ACETYL CHOLINE ESTERASE

AChE is a neuronally derived protein.

Measurements of AChE in amniotic fluid also used to significantly improve the ability to distinguish between affected & unaffected pregnancies.

DETERMINED BY GEL-ELECTROPHORESIS.This approach has not only proved to be highly sensitive at detecting open neural tube defects [99% anencephaly cases & 98% of open spina bifida cases with positive AFP results ].

KEY POINTS1. In 1956, a fetal-specific protein

(alpha-fetoprotein or AFP ) was discovered in fetal serum.

2. Elevated AFP in second-trimester amniotic fluid is strong indicator of the presence of a fetal open neural tube defect (NTD).

3. AFP levels in maternal serum can be used as a screening (but not diagnostic) test for open NTD’s in the second trimester.

4. AFP measurements in both amniotic fluid and maternal serum very with gestation.

5. They are routenely expressed as a multiple of median (MOM) AFP value found in unaffected pregnancies of the same gestational age.

6. Beginning in the 1970s, a woman’s age was used as a determinant in screening for Down syndrome, with those aged 35 and older being offered amniocentesis and karyotyping.

7. In 1984, reduced levels of maternal serum AFP in the second trimester were reported in Down syndrome pregnancies.

8. Second trimester multiple marker screening is also able to identify 60% of Trisomy 18 pregnancies.

9. At about the same time, ultrasound measurements of nuchal translucency (NT) thickness > 5 mm (at between 11 and 13 completed gestational weeks ) were found to be the best single marker for Down syndrome.

10.Combining [NT ]measurement with biochemical markers (combined testing ) in the first trimester yields equivalent performance to second trimester quadruple marker testing.

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