MATERNAL

SERUM
SCREENING
M.PRASAD NAIDU
MSC MEDICAL BIOCHEMISTRY,
Ph.D.RESEARCH SCHOLAR
Second trimester biochemical screening[BCS] started
in the 1970s when it was found that fetal neural tube
defects[NTDs] 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 NTDs were being
refined, it was noted that MSAFP tended to be low in
fetal downs syndrome.

With a cut off of 2.0 multiples of median[MOM] 85% of
NTDs 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 womans 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 {hCG} ] AND
Ultrasound marker [Nuchal Translucency{NT}]
as markers for downs 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 1
st
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 2
nd
trimester.

4. 85% detection rate for Downs 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 1 region on
electrophoresis[subsequently labelled as 1-
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 10
th

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 -Feto Protein.

TRADITIONALLY MEASURED BY RADIO IMMUNO ASSAY [RIA]

NEWER METHODS USE IMMUNO ENZYMATIC ASSAYS [IEMA]
Because of its 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
MSAFP
ng /ml
Concentration of
AFAFP
ng/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 MOM
3. VERY HIGH - > or Equal to
10.0 MOM
CLINICAL SIGNIFICANCE OF AFP
Predicting the risk of open NTDs.
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 & subunits
of which the subunit is specific for hCG.

All the glycoprotein
hormones[hCG,LH,FSH,TSH] have a similer
biological activity which is characteristic of
the 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 subunit & carbohydrate components
are required for expression of the biological
activity unique to the subunit.

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

In spontaneous pregnancy, hCG
can be detected by the 9
th
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 10
th
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 2
nd
trimester.

Free -hCG was increased
during the 1
st
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 TESTS
1. 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 hCG
1. Diagnosis & dating of pregnancy.
2. Predicting the risk of Trisomy 21 & 18.
3. Managing certain neoplasms.


UNCONJUGATED ESTRIOL [uE3]
Estriol as its name implies, is an estrogen with 3
hydroxyl groups [at position 3,16, & 17 ].
3 organs involved in the biosynthesis
1. Fetal adrenal - Cholesterol
2. 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
2
nd
trimester of pregnancy, 0.30
1.5 g / L
METHODS OF DETERMINING UNCONJUGATED
ESTRIOL
By ULTRA SENSITIVE RADIO IMMUNO ASSAY METHOD

The determination of uE3 is the most difficult
The 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 uE3
NTDs
VERY
HIGH
- VERY LOW
TRISOMY 21
[DOWNS
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 & .

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

The level increases in the 1
st
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 g / 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 DOWNs
SYNDROME
MATERNAL
AGE[YEAR
S]
TRIPLE
TEST
QUADRUP
LE TEST
DETE
CTION
RATE
FALSE
POSITI
VE
RATE
DETE
CTION
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 1
st
trimester as an early marker
for Downs 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 weeksgestation,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 POINTS
1. 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 NTDs 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 womans 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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