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Maternal serum screening

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Title: Maternal serum screening


1
  • MATERNAL SERUM SCREENING
  • M.PRASAD NAIDU
  • MSC MEDICAL BIOCHEMISTRY,
  • Ph.D.RESEARCH SCHOLAR

2
  • Second trimester biochemical screeningBCS
    started in the 1970s when it was found that
    fetal neural tube defectsNTDs were associated
    with increase in maternal serum alpha feto
    proteinMSAFP.
  • 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 medianMOM
    85 of NTDs would be screened in with a
    threshold of 0.5 MOM approx 33 of DS fetus would
    be screened in.

3
  • With the addition of two other analytes Estriol
    which is low in DS Human chorionic
    gonadotropinhCG 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.

4
  • Woman with elevated serum AFP levels were offered
    Diagnostic amniotic fluid AFP testingAFAFP.
  • 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.

5
  • 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 ßhCG AND Ultrasound marker
    Nuchal TranslucencyNT 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.

6
  • Suggested protocol for screening
  • Measurement of nuchal translucencyNT PAPP-A
    in the 1st 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 DIA are combined into a single risk
    estimate for interpretation in the 2nd trimester.
  • 85 detection rate for Downs Syndrome with only
    1 false positive is achieved.

7
  • TRIPLE SCREEN TEST
  • 1.ALPHA FETO PROTEIN
  • In 1956,Bergstrand czar described a protein in
    fetal serum,located in the a1 region on
    electrophoresissubsequently labelled as a1-Feto
    ProteinAFP 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

8
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.
9
  • The molecular weight and structure of AFP is
    similar to that of albuminabout 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 _at_room temperature in
    serum as long as a week.

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  • Maximum concncentration of AFP in fetal
    serum3,000,000ng/ml reaches by 9 wk gestation
    decreases to 20,000ng/ml _at_ 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 _at_ 25 wk
    gestation,decline slowly till term.

12
  • After birth MSAFPdecreases to less than 2ng/ml.
  • In infant,serum AFP level decreases exponentially
    to reach adult level by 10th month of life.

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

15
  • METHODS FOR DETERMINING a-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,
  • A monoclonal bead assay
  • A microparticles immuno assay
  • A polyclonal bead assay

16
  • 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.

17
  • 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
18
  • 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 - gt or Equal to 10.0
    MOM
  • CLINICAL SIGNIFICANCE OF AFP
  • Predicting the risk of open NTDs.
  • Managing certain neoplasms.

19
  • HUMAN CHORIONIC GONADOTROPHINhCG
  • It is a glycoprotein hormone with molecular
    weight of 36 to 40 kd that is biologically
    immunologically similer to LHLeutenizing
    hormone but with a longer half life.
  • Produced during normal pregnancy by the
    trophoblast placenta.
  • hCG is a hetero dimer having a ß subunits of
    which the ß subunit is specific for hCG.
  • All the glycoprotein hormoneshCG,LH,FSH,TSH
    have a similer biological activity which is
    characteristic of the ß subunit component.

20
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 a subunit carbohydrate components are
    required for expression of the biological
    activity unique to the ß subunit.
  • The 28-30 AA on the C-terminal end of the ß
    subunit of hCG are unique compared with LH.

21
  • 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.

22
  • 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.

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  • Free ß-hCG was increased during the 1st trimester
    in Trisomy 21DS even though total hCG remained
    normal.
  • _at_16 wk gestation hCG median level in normal
    pregnancy is 20,000 40,000 IU / L .

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METHODS FOR DETERMINING hCG
  • QUALITATIVE TESTS
  • HOME TEST KITS MOST COMMONLY USED
    PREGNANCY TESTS.
  • RADIO IMMUNO ASSAY.
  • IMMUNO ENZYMATIC ASSAYIEMA.
  • IMMUNO RADIOMETRIC ASSAYIRMA.
  • QUANTITATIVE TEST By IMMUNOCHROMATOGRAPHY
  • CLINICAL SIGNIFICANCE OF hCG
  • Diagnosis dating of pregnancy.
  • Predicting the risk of Trisomy 21 18.
  • Managing certain neoplasms.

27
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
  • Fetal adrenal - Cholesterol
  • Fetal liver -DHEAsDehydroEpiAndrosteroneSul
    fate
  • Placenta - Estriol
  • Only a minor amount 9of the hormone
    circulates in plasma unconjugated.

28
  • 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

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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 .

31
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
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THE QUADRUPLE TESTQUAD TEST
  • This includes AFP, Ue3, hCG an additional
    marker INHIBIN-A .
  • Dimeric Inhibin-ADIA is a glycoprotein produced
    by the placenta.
  • It is a dimer , but with dissimilar subunits a
    ß.
  • 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.

34
  • 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

35
  • FACTORS AFFECTING THE LEVEL OF THE QUAD SCREEN
  • Maternal weight was found to have an inverse
    relation with the levels of all four markers.
  • In Diabetes mellitus,AFP was found to be 40
    lower than in non Diabetics.
  • In twin pregnancy, AFP was Higher than those
    having singlet fetus.

36
RATES OF DETCTION OF DOWNs SYNDROME
MATERNAL AGEYEARS TRIPLE TEST TRIPLE TEST QUADRUPLE TEST QUADRUPLE TEST
MATERNAL AGEYEARS DETECTION RATE FALSE POSITIVE RATE DETECTION RATE FALSE POSITIVE RATE
15 - 34 58 3.7 69 4.1
gt35 88 19 91 17
lt15 69 4.9 77 5.2
37
Pregnancy associated plasma protein A PAPP-A
  • Measured in the 1st 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 .

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  • First trimester Ultrasound findings in Down
    syndrome pregnancies
  • INDICATIONS.
  • 1.Advanced maternal age(gt35)
  • 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.

40
  • 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.

41
  • 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.

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  • 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.

44
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 should be done .
  • If second test is negative, screen is taken as
    negative.
  • If second test is also gives elevated results
    further diagnostic testing to be done.
  • Ultra sonography, Amniocentesis Analysis of
    amniotic fluid for Acetyl choline esterase to
    confirm neural tube defects.

45
  • 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.

46
  • 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 .

47
KEY POINTS
  • In 1956, a fetal-specific protein
    (alpha-fetoprotein or AFP ) was discovered in
    fetal serum.
  • Elevated AFP in second-trimester amniotic fluid
    is strong indicator of the presence of a fetal
    open neural tube defect (NTD).
  • AFP levels in maternal serum can be used as a
    screening (but not diagnostic) test for open
    NTDs in the second trimester.

48
  1. AFP measurements in both amniotic fluid and
    maternal serum very with gestation.
  2. They are routenely expressed as a multiple of
    median (MOM) AFP value found in unaffected
    pregnancies of the same gestational age.
  3. 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.

49
  1. In 1984, reduced levels of maternal serum AFP in
    the second trimester were reported in Down
    syndrome pregnancies.
  2. Second trimester multiple marker screening is
    also able to identify 60 of Trisomy 18
    pregnancies.
  3. At about the same time, ultrasound measurements
    of nuchal translucency (NT) thickness gt 5 mm (at
    between 11 and 13 completed gestational weeks )
    were found to be the best single marker for Down
    syndrome.
  4. 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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