Prenatal diagnosis employs techniques like amniocentesis and chorionic villus sampling to determine the health of the unborn fetus. It is helpful for managing the pregnancy, determining outcomes, and planning for complications. It allows decisions about continuing the pregnancy and identifying conditions that could affect future pregnancies. Maternal serum screening measures markers like alpha-fetoprotein to screen for neural tube defects and other abnormalities by detecting higher than normal levels that cross the placenta.

1. Prof. M.C.Bansal
MBBS., MS., FICOG., MICOG.
Founder Principal & Controller,
Jhalawar Medical College & Hospital
Jjalawar.
MGMC & Hospital , sitapura ., Jaipur

2.  Prenatal diagnosis employs a variety of techniques to
determine the health and condition of an unborn
fetus. Without knowledge gained by prenatal
diagnosis, there could be an untoward outcome for the
fetus or the mother or both.
 Congenital anomalies account for 20 to 25% of
perinatal deaths.
 Specifically, prenatal diagnosis is helpful for:
 Managing the remaining weeks of the pregnancy
 Determining the outcome of the pregnancy
 Planning for possible complications with the birth
process
 Planning for problems that may occur in the newborn
infant
 Deciding whether to continue the pregnancy
 Finding conditions that may affect future pregnancies

3.  There are a variety of non-invasive and invasive
techniques available for prenatal diagnosis. Each of
them can be applied only during specific time periods
during the pregnancy for greatest utility. The
techniques employed for prenatal diagnosis include:
 Amniocentesis
 Chorionic villus sampling
 Fetal blood cells in maternal blood
 Maternal serum alpha-fetoprotein
 Maternal serum beta-HCG
 Maternal serum estriol
 Inhibin A
 Pregnancy associated plasma protein A

4. Amniocentesis
TIME TO PERFORM:-14 and 20 weeks gestation .
(Enough amniotic fluid is present for this to be
accomplished starting about 14 weeks gestation.)
However, an ultrasound examination always proceeds
amniocentesis in order to determine gestational age, the
position of the fetus and placenta, and determine if
enough amniotic fluid is present.
Within the amniotic fluid are fetal cells (mostly
derived from fetal skin).
After the amniotic fluid is extracted, the fetal cells are
separated from the sample. The cells are grown in a
culture medium, then fixed and stained for chromosome
analysis, biochemical analysis, and molecular biologic
analysis.

5.  In the third trimester of pregnancy, the amniotic fluid
can be analyzed for determination of fetal lung
maturity. This is important when the fetus is below
35 to 36 weeks gestation, because the lungs may not
be mature enough to sustain life. This is because the
lungs are not producing enough surfactant. After
birth, the infant will develop respiratory distress
syndrome from hyaline membrane disease.
 The amniotic fluid can be analyzed by fluorescence
polarization (fpol), for lecithin:sphingomyelin (LS)
ration, and/or for phosphatidyl glycerol (PG).

6. Chorionic Villus Sampling (CVS)
 CVS can be safely performed between 9.5 and 12.5 weeks
gestation.
 Sampling of cells from the placental chorionic villi.
These cells can then be analyzed by a variety of techniques.
 The most common test employed on cells obtained by CVS
is chromosome analysis to determine the karyotype of
the fetus.
 The cells can also be grown in culture for biochemical or
molecular biologic analysis
 Disadvantage :- invasive procedure, and it has a small but
significant rate of morbidity for the fetus; this loss rate is
about 0.5 to 1% higher than for women undergoing
amniocentesis. Rarely, CVS can be associated with limb
defects in the fetus. The possibility of maternal Rh
sensitization is present. There is also the possibility that
maternal blood cells in the developing placenta will be
sampled instead of fetal cells and confound chromosome
analysis.

7. Karyotyping
 Tissues must be obtained as fresh as possible for
culture and without contamination.
 A useful procedure is to wash the tissue samples in
sterile saline prior to placing them into cell culture
media.
 Tissues with the best chance for growth are those with
the least maceration: placenta, lung, diaphragm.

8.  Sample Collection
 A karyotype will be done on the white blood cells which are
actively dividing (a state known as mitosis).
 During pregnancy, the sample can either be amniotic fluid
collected during an amniocentesis or a piece of the
placenta collected during a chorionic villi sampling test
(CVS). The amniotic fluid contains fetal skin cells
which are used to generate a karyotype.
 Separating the Cells
 In order to analyze chromosomes, the sample must contain
cells that are actively dividing (or in mitosis). In blood,
the white blood cells are actively dividing cells. Most fetal
cells are actively dividing. Once the sample reaches the
cytogenetics lab, the non-divided cells are separated from
the dividing cells using special chemicals.

9.  Growing Cells
 In order to have enough cells to analyze, the dividing
cells are grown in special media or a cell culture. This
media contains chemicals and hormones that enable
the cells to divide and multiply. This process of
“culturing” the cells can take 3 to 4 days for blood
cells, and up to a week for fetal cells.
 Synchronizing Cells
 Chromosome are long string of human DNA. In order
to see chromosomes under a
microscope, chromosomes have to be in their most
compact form. This compact form occurs at a specific
stage of mitosis called metaphase. In order to get all
the cells to this specific stage of cell division, the cells
are treated with a chemical which stops cell division at
the point where the chromosomes are the most
compact.

10.  Releasing the Chromosomes from their Cells
 In order to see these compact chromosomes under a
microscope, the chromosomes have to be out of the cells.
This is done by treating the cells with a special solution
that causes them to burst. This is done while the cells are
on a microscopic slide. The leftover debris from the white
blood cells is washed away, and the chromosomes are now
fixed (or stuck) to the slide.
 Staining the Chromosomes
 Chromosomes are naturally colorless. In order to be able to
tell one chromosome from another, a special dye called
Giemsa dye is applied to the chromosomes on the slide.
Giemsa dye stains regions of chromosomes that are rich in
the bases adenine (A) and thymine (T). When stained, the
chromosomes look like strings with light and dark bands.
Each chromosome has a specific pattern of light and dark
bands which enables cytogeneticist to tell one chromosome
from another. Each dark or light band actually
encompasses hundreds of different genes.

11.  The chromosomes may be stained with aceto- orcein, feulgen or a
basophilic dye such as toluidine blue or methylene blue if only the
general morphology is desired. If more detail is desired, the
chromosomes can be treated with various enzymes in combination
with stains to yield banding patterns on each chromosome
 Q-banding
Quinacrine stain
Fluorescence microscopy
 G-banding
Giemsa stain
Additional Conditions
a. Heat hydrolysis
b. Trypsin treatment
c. Giemsa at pH 9.0
 R-banding
Giemsa or acridine orange
Negative bands of Q and G reversed
Heat hydrolysis in buffered salt
 C-banding
Giemsa stain
Pretreatment with BaOH or NaOH followed by heat and salt.

12.  Analysis
 Once chromosomes are stained, the slide is put under the
microscope and the analysis of the chromosomes begins. A
picture is taken of the chromosomes and at the end of the
analysis, the total number of chromosomes will be known
and there will be a picture of the chromosomes arranged by
size.
 Counting Chromosomes
 The first step of the analysis is counting the chromosomes.
Most humans have 46 chromosomes. People with Down
syndrome have 47 chromosomes. It is also possible for
people to have missing chromosomes or more than one
extra chromosome. By looking at just the number of
chromosomes, it is possible to diagnose different
conditions including Down syndrome.

13.  Looking at the Structure
 In addition to looking at the total number of
chromosomes and the sex chromosomes, the
cytogeneticist will also look at the structure of the
specific chromosomes to make sure that there is no
missing or additional material, no structural
abnormalities like translocations and a variety of other
possible chromosome abnormalities.
 The Final Result
 In the end, the final karyotype test shows the total
number of chromosomes, the sex of the person being
studied, and if there are any structural abnormalities
with any of the individual chromosomes. A digital
picture of the chromosomes is generated with all of
the chromosomes arranged by number.

14. Spectral karyotype (SKY
technique)
 Spectral karyotyping is a
molecular cytogenetic technique used to
simultaneously visualize all the pairs
of chromosomes in an organism in different colors.
 Fluorescently labeled probes for each chromosome are
made by labeling chromosome-specific DNA with
different fluorophores. Because there are a limited
number of spectrally-distinct fluorophores, a
combinatorial labeling method is used to generate
many different colors.

18.  Neural tube defects can be distinguished from other
fetal defects (such as abdominal wall defects) by use of
the acetylcholinesterase test performed on amniotic
fluid obtained by amniocentesis
 If the acetylcholinesterase is elevated along with
MSAFP then a neural tube defect is likely. If the
acetylcholinesterase is not detectable, then some other
fetal defect is suggested

19. DNA Probes
 Fetal cells obtained via amniocentesis or CVS can be analyzed by
probes specific for DNA sequences.
 One method employs restriction fragment length
polymorphism (RFLP) analysis. This method is useful for
detection of mutations involving genes that are closely linked to
the DNA restriction fragments generated by the action of an
endonuclease.
 The DNA of family members is analyzed to determine
differences by RFLP analysis.
 In some cases, if the DNA sequence of a gene is known, a probe
to a DNA sequence specific for a genetic marker is available, and
the polymerase chain reaction (PCR) technique can be applied
for diagnosis.
 There are many genetic diseases, but only in a minority have
particular genes been identified, and tests to detect them
have been developed in some of these. Thus, it is not possible to
detect all genetic diseases. Moreover, testing is confounded by
the presence of different mutations in the same
gene, making testing more complex

20.  In RFLP analysis, the DNA sample is broken into
pieces (digested) by restriction enzymes and the
resulting restriction fragments are separated according
to their lengths by gel electrophoresis.
 Although now largely obsolete due to the rise of
inexpensive DNA sequencing technologies, RFLP
analysis was the first DNA profiling technique
inexpensive enough to see widespread application.
 In addition to genetic fingerprinting, RFLP was an
important tool in genome mapping, localization of
genes for genetic disorders, determination
of risk for disease, and paternity testing.

23. Maternal blood sampling for fetal
blood cells
 This is a new technique that makes use of the phenomenon
of fetal blood cells gaining access to maternal circulation
through the placental villi. Ordinarily, only a very small
number of fetal cells enter the maternal circulation in this
fashion (not enough to produce a positive Kleihauer-
Betke test for fetal-maternal hemorrhage). The fetal cells
can be sorted out and analyzed by a variety of techniques to
look for particular DNA sequences, but without the risks
that these latter two invasive procedures inherently have.
 Fluorescence in-situ hybridization (FISH) is one
technique that can be applied to identify particular
chromosomes of the fetal cells recovered from maternal
blood and diagnose aneuploid conditions such as the
trisomies and monosomy X.
 The problem with this technique is that it is difficult to get
many fetal blood cells. There may not be enough to reliably
determine anomalies of the fetal karyotype or assay for
other abnormalities.

24.  FISH (performed on fresh tissue or paraffin
blocks)
 In addition to karyotyping, fluorescence in situ
hybridization (FISH) can be useful. A wide variety of
probes are available. It is useful for detecting
aneuploid conditions (trisomies, monosomies).
 Fresh cells are desirable, but the method can be
applied even to fixed tissues stored in paraffin blocks,
though working with paraffin blocks is much more
time consuming and interpretation can be difficult
 The ability to use FISH on paraffin blocks means that
archival tissues can be examined in cases where
karyotyping was not performed, or cells didn't grow in
culture.

26. A metaphase cell positive for thebcr/abl rearrangement (associated
withchronic myelogenous leukemia) using FISH. The chromosomes can be
seen in blue. The chromosome that is labeled with green and red spots is the
one where the wrong rearrangement is present

27. Maternal serum alpha-fetoprotein
(MSAFP)
 The developing fetus has two major blood
proteins--albumin and alpha-fetoprotein (AFP).
 Since adults typically have only albumin in their
blood, the MSAFP test can be utilized to
determine the levels of AFP from the fetus.
Ordinarily, only a small amount of AFP gains
access to the amniotic fluid and crosses the
placenta to mother's blood.
 However, when there is a neural tube defect in the
fetus, from failure of part of the embryologic
neural tube to close, then there is a means for
escape of more AFP into the amniotic fluid.

28.  Neural tube defects include anencephaly .Also, if there
is an omphalocele or gastroschisis (both are defects in
the fetal abdominal wall), the AFP from the fetus will
end up in maternal blood in higher amounts.
 The blood taken is that from mom, but a sample can
be obtained for testing from amniotic fluid.
 The AFP test is not diagnostic. It can only be used to
test for the increased likelihood of an abnormality or
birth defect.
Alpha-Fetoprotein is a substance produced by the
fetus in utero. AFP stops being produced once the
baby is born. The AFP is excreted in the fetal urine
which crosses into the mother’s blood stream. This is
why AFP can be detected by a blood sample taken
from the pregnant mother.

29.  MSAFP may be performed between the 14th and 22nd
weeks of pregnancy, however it seems to be most
accurate during the 16th to 18th week. Your levels of
AFP vary during pregnancy so accurate pregnancy
dating is imperative for more reliable screening
results.
 All pregnant women should be offered the MSAFP
screening, but it is especially recommended for:
 Women who have a family history of birth defects
 Women who are 35 years or older
 Women who used possible harmful medications or
drugs during pregnancy
 Women who have diabetes

30.  NORMAL VALUES:-
 Adults: <15 ng/mL (15 mcg/L)
 Fetal blood (first trimester): Peak 200-400 mg/dL (2-4 g/L)
 Pregnancy (2nd trimester):
 14 weeks' gestation: Median 25.6 ng/mL (25.6 mcg/L)
 15 weeks' gestation: Median 29.9 ng/mL (29.9 mcg/L)
 16 weeks' gestation: Median 34.8 ng/mL (34.8 mcg/L)
 17 weeks' gestation: Median 40.6 ng/mL (40.6 mcg/L)
 18 weeks' gestation: Median 47.3 ng/mL (47.3 mcg/L)
 19 weeks' gestation: Median 55.1 ng/mL (55.1 mcg/L)
 20 weeks' gestation: Median 64.3 ng/mL (64.3 mcg/L)
 21 weeks' gestation: Median 74.9 ng/mL (74.9 mcg/L)
 The MSAFP is typically reported as multiples of the mean
(MoM).
 The greater the MoM, the more likely a defect is present
 The multiple of the median (MoM) value is adjusted for
maternal weight, race, diabetes mellitus, and twin pregnancy

31.  However, the MSAFP can be elevated for a variety of
reasons which are not related to fetal neural tube or
abdominal wall defects, so this test is not 100%
specific.
 The most common cause for an elevated MSAFP is
a wrong estimation of the gestational age of the
fetus.

32.  Using a combination of MSAFP screening and
ultrasonography, almost all cases of anencephaly can
be found and most cases of spina bifida.
 The MSAFP can also be useful in screening for Down
syndrome and other trisomies.
 The MSAFP tends to be lower when Down syndrome
or other chromosomal abnormalities is present.

33. Maternal serum beta-HCG
 The hormone human chorionic gonadotropin (better
known as hCG) is produced during pregnancy. It is
made by cells that form the placenta, which nourishes
the egg after it has been fertilized and becomes
attached to the uterine wall.
 Levels can first be detected by a blood test about 11
days after conception and about 12 - 14 days after
conception by a urine test.
 In general the hCG levels will double every 72 hours.
The level will reach its peak in the first 8 - 11 weeks
of pregnancy and then will decline and level off for the
remainder of the pregnancy.

34.  An hCG level of less than 5mIU/ml is considered
negative for pregnancy, and anything above
25mIU/ml is considered positive for pregnancy.
 The hCG hormone is measured in milli-
international units per milliliter (mIU/ml).
 A transvaginal ultrasound should be able to show at
least a gestational sac once the hCG levels have
reached between 1,000 - 2,000mIU/ml.
 There are two common types of hCG tests. A
qualitative hCG test detects if hCG is present in the
blood. A quantitative hCG test (or beta hCG)
measures the amount of hCG actually present in the
blood.

35.  Guideline to hCG levels during pregnancy:
 hCG levels in weeks from LMP (gestational age)* :
 3 weeks LMP: 5 - 50 mIU/ml
 4 weeks LMP: 5 - 426 mIU/ml
 5 weeks LMP: 18 - 7,340 mIU/ml
 6 weeks LMP: 1,080 - 56,500 mIU/ml
 7 - 8 weeks LMP: 7, 650 - 229,000 mIU/ml
 9 - 12 weeks LMP: 25,700 - 288,000 mIU/ml
 13 - 16 weeks LMP: 13,300 - 254,000 mIU/ml
 17 - 24 weeks LMP: 4,060 - 165,400 mIU/ml
 25 - 40 weeks LMP: 3,640 - 117,000 mIU/ml
 Non-pregnant females: <5.0 mIU/ml
 Postmenopausal females: <9.5 mIU/ml

36.  What can a low hCG level mean?
 A low hCG level can mean any number of things and
should be rechecked within 48-72 hours to see how
the level is changing. A low hCG level could indicate:
 Miscalculation of pregnancy dating
 Possible miscarriage or blighted ovum
 Ectopic pregnancy

37.  What can a high hCG level mean?
 A high level of hCG can also mean a number of things
and should be rechecked within 48-72 hours to
evaluate changes in the level. A high hCG level can
indicate:
 Miscalculation of pregnancy dating
 Molar pregnancy
 Multiple pregnancy

38. Maternal serum estriol
 The amount of estriol in maternal serum is dependent
upon a viable fetus, a properly functioning placenta,
and maternal well-being.
 The substrate for estriol begins as
dehydroepiandrosterone (DHEA) made by the fetal
adrenal glands. This is further metabolized in the
placenta to estriol. The estriol crosses to the maternal
circulation and. is excreted by the maternal kidney
in urine or by the maternal liver in the bile
 The measurement of serial estriol levels in the third
trimester will give an indication of general well-being
of the fetus.

39.  If the estriol level drops, then the fetus is threatened
and delivery may be necessary emergently.
 Estriol tends to be lower when Down syndrome is
present and when there is adrenal hypoplasia with
anencephaly.
 When it is used this way, each sample should be drawn
at the same time each day.

40. Inhibin-A
 Inhibin is secreted by the placenta and the corpus
luteum.
 Inhibin A is made by the placenta during pregnancy.
 The level of inhibin A in the blood is used in a
maternal serum quadruple screening test. Generally
done between 15 and 20 weeks
 Inhibin-A can be measured in maternal serum.
 An increased level of inhibin-A is associated with an
increased risk for trisomy 21.
 A high inhibin-A may be associated with a risk for
preterm delivery.

41. Pregnancy-associated plasma
protein A (PAPP-A)
 Pregnancy-associated plasma protein A,
pappalysin 1, also known as PAPPA, is a protein used
in screening tests for Down syndrome
 Low levels of PAPP-A as measured in maternal serum
during the first trimester may be associated with fetal
chromosomal anomalies including trisomies 13, 18,
and 21.
 In addition, low PAPP-A levels in the first trimester
may predict an adverse pregnancy outcome, including
a small for gestational age (SGA) baby or stillbirth.
 A high PAPP-A level may predict a large for
gestational age (LGA) baby.

42. Triple" or "Quadruple" screen
 Combining the maternal serum assays may aid in
increasing the sensitivity and specificity of detection
for fetal abnormalities.
 The classic test is the triple screen for alpha-
fetoprotein (MSAFP), beta-HCG, and estriol (uE3).
The "quadruple screen" adds inhibin-A.

43. TRIPLE TEST
 The triple test, also called triple screen, the
Kettering test or the Bart's test, is an investigation
performed during pregnancy in the second trimester
to classify a patient as either high-risk or low-risk for
chromosomal abnormalities (and neural tube defects).
 The Triple test measures serum levels
of AFP, estriol, and beta-hCG, with a
70% sensitivity and 5% false-positive rate.

44.  The triple test measures the following three levels in the
maternal serum:
 alpha-fetoprotein (AFP)
 human chorionic gonadotropin (hCG)
 unconjugated estriol (UE3)
AFP UE3 hCG ASSOCIATED
CONDITIONS
LOW LOW HIGH DOWN
SYNDROME
LOW LOW LOW TRISOMY
18(EDWARD
SYNDROME)
HIGH N/A N/A NEURAL TUBE
DEFECT LIKE
SPINA BIFIDA,
MULTIPLE
GESTATIONS,OMP
HALOCELE

45.  Quadruple test
 A test of levels of dimeric inhibin A (DIA) is
sometimes added to the other three tests, under the
name "quadruple test.” Other names used include
"quad test", "quad screen", or "tetra screen." Inhibin A
(DIA) will be found high in cases of Trisomy 21 and
low in cases of Trisomy 18.

46.  Biochemical Analysis
 Tissues can be obtained for cell culture or for
extraction of compounds that can aid in identification
of inborn errors of metabolism. Examples include:
 long-chain fatty acids (adrenoleukodystrophy)
 amino acids (aminoacidurias)

47. Thank you….

48. THANK YOU