Maternal serum screening

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

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• 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.

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• 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.

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• 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.

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• 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.

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• 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.

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• 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

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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.
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• 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.

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• 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.

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• 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

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• 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.

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• 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
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• 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.

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• 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.

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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.

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• 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.

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• 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.

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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.

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• 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 .

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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.

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• 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

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• 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.

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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
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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.

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• 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.

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• 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.

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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.

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• 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.

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• 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 .

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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.

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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.

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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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