Placental Function Testing Below is an overview of the techniques used to assess placental function

1. FETAL PLACENTAL FUNCTION TESTS
Suramya Babu
Bsc MLT

2. ● The pulmonary system is among the last of the fetal organ
systems to mature.
● Because the immature pulmonary system may not oxygenate
the neonate adequately, preterm birth can lead to significant
neonatal morbidity or mortality.
● Therefore, fetal lung maturity is sometimes assessed before
preterm delivery and can be a factor in determining the timing
of delivery in these cases

3. Lung Surfactants
● Normal lung function depends on a constant supply of lung
surfactants. It is produced by epithelial cells.
● It decreases surface tension of the aqueous layer of lung and
prevents collapse of lung alveoli.
● Constituent’s of surfactants are dipalmitoyl lecithin,
phosphatidyl glycerol, cholesterol, proteins A, B and C.
● During fetal life, the lung synthesizes sphingomyelin before
28th week ofgestation.
●

4. ● But as fetus matures, more lecithin is synthesized The lecithin-
sphingomyelin (LS) ratio of amniotic fluid is an index of fetal
maturity.
● A ratio of 2 indicates full lung maturity.
●
● Low levels of surfactant leads to respiratory distress syndrome
(RDS), which
● is a common cause of neonatal morbidity.

5. ●
● Fetal lung immaturity, or the Respiratory Distress Syndrome
(RDS), occurs most often when insufficient lung surfactant is
present

6. Respiratory distress syndrome (RDS)
● Having enough of these surfactants in the lungs at birth is
extremely important because that's
● when the baby has to make a transition from getting its oxygen
from mom to getting it from the air.
● If there's not enough surfactant then the alveoli may collapse and
the baby may have a difficult time breathing.
● When this does happen it's called respiratory distress syndrome
of the newborn, or just RDS.
●

7. ● Lung surfactants begin to be made around the 25th week of
pregnancy but there's usually not enough of it present to prevent
RDS until the 37th week.
● That means babies born prematurely are at greater risk of
developing RDS than those born at term.
● Because the fetus essentially "breathes" amniotic fluid in and out of
its lungs, the amount of surfactant in the lungs can be determined
by measuring surfactants in the amniotic fluid.
●
●

8. Fetal lung maturity tests
•Lecithin/Sphingomyelin Ratio
•Lamellar Body Count
•Fluorescent Polarization Test (FLM)
• Detection of phosphatidylglycerol (PG)
•Foam stability index (FSI )

9. Appropriate Specimen
● Amniotic fluid, obtained by amniocentesis, is the specimen of choice
because it does not contain sources of contamination that can
interfere with the various assays.
● Fluid may be obtained by transvaginal puncture of the bulging
membranes

10. Amniocnetesis
● Process in which amniotic fluid is sampled using a hollow needle
inserted into the uterus, to screen for abnormalities in the
developing fetus

11. Amniocnetesis
● There are 2 methods of collecting amnioticfluid, transabdominal
amniocentesis and vaginal pool collection.
● The latter method consists of aspirating amniotic fluid from a
vaginal pool collection in cases involving rupture of membranes.
● Both methods provide adequate fluid for testing provided that they
are relatively free of contaminating substances such as gross blood
or meconium

12. Lamellar Body Count
● In certain cells of the lungs, surfactants are packaged into granules
called lamellar bodies and secreted from the cells into the
alveoli. This test actually counts the number of lamellar bodies in
amniotic fluid.
● The higher the lamellar body count, the more likely it is that the
fetal lungs are mature.
●

13. ●Compared to thin-layer chromatography techniques, the lamellar
body count is faster, more objective, less labor intensive, less
technique dependent, and less expensive
●The lamellar body count was as accurate as TDx-FLM ll; both
performed best when interpreted according to gestational age
●Blood contamination can lead to false elevation of the lamellar body
count because platelets are counted as lamellar body; the effect of
meconium is minimal.

14. Lecithin/Sphingomyelin Ratio
● This was the first test of fetal lung maturity ever developed and is
more commonly known as the L/S ratio.
● It was first introduced by Gluck and colleagues in 1971
● It is based upon the observation that there is outward flow of
pulmonary secretions from the lungs into the amniotic fluid. This
process changes the phospholipid composition of amniotic fluid,
thereby enabling indirect assessment of fetal lung maturity
through evaluation of this fluid.
●
●
●

15. ● It's a measure of the ratio of two lung surfactants, lecithin and
sphingomyelin, that's determined using a technique known as
thin-layer chromatography.
● Lecithin is the most important lung surfactant and provides the
greatest surface tension-lowering properties of all the
surfactants. It increases dramatically in the last few weeks of
pregnancy.

16. ● Sphingomyelin is a minor lung surfactant and that amount of it in
the lungs stays about the same throughout pregnancy so it serves
as a good baseline against which the increasing amount of lecithin
can be compared. A ratio that is 2.5 or greater is usually used to
indicate lung maturity.

17. Fluorescent Polarization Test (FLM)
● This test measures the ratio of surfactant to albumin and so is
sometimes called the S/A ratio.
● The test relies on a technique known as fluorescence polarization
and is the most widely used fetal lung maturity test.
●

18. ● Advantages of this test are that it is a simple, automated, rapid test
that varies minimally between laboratories, and requires only a
small volume of amniotic fluid
● A disadvantage to the TDx-FLM ll method is the large
quantification scale.
● Values greater than 55 are regarded as mature and values less
than 40 are considered immature
● While values of 40 to 54 are considered "indeterminate" . Blood or
meconium in the amniotic fluid also affects results.

19. Detection of phosphatidylglycerol (PG)
● Phosphatidylglycerol (PG) is a minor constituent of
surfactant.
● It begins to increase appreciably in amniotic fluid after 35
weeks, several weeks after the rise in lecithin
● Because PG enhances the spread of phospholipids on the
alveoli, its presence indicates an advanced state of fetal lung
development and function.
●

20. ● Phosphatidylglycerol testing can be performed by thin-layer
chromatography, so it can be determined alone or in conjunction with
testing for the lecithin/sphingomyelin ratio.
● It may be reported qualitatively as positive or negative, where positive
represents an exceedingly low risk of respiratory distress, or in a
quantitative fashion, in which a value ≥0.5 is associated with a minimal
rate of respiratory distress .

21. ● An advantage of this test is that usually it is not affected by the
presence of blood or meconium
● A disadvantage is that the absence of phosphatidylglycerol,
especially before 36 weeks of gestation, is less predictive of the
occurrence of respiratory distress than immature results from
other tests

22. Foam stability index (FSI )
● The foam stability index (FSI) is a rapid predictor of fetal lung
maturity.
● It is based upon the ability of surfactant to generate stable foam
in the presence of ethanol .
● Ethanol is added to a sample of amniotic fluid to eliminate the
effects of nonsurfactant factors on foam formation.
● The mixture is then shaken and will demonstrate generation of a
stable ring of foam if surfactant is present.
● The foam stability index (FSI) is calculated by utilizing serial
dilutions of ethanol to quantitate the amount of surfactant present

23. ● Amniotic fluid samples should not be collected in silicone
tubes when this test is planned, as the silicone will produce
"false foam." The discriminating value indicative of lung
maturity is usually set at ≥47.

24. ● * When the test for estriol is done with alpha-fetoprotein
(AFP) and human chorionic gonadotropin (hCG), it is
called a triple screen test.

25. HEMOLYTIC DISEASE OF NEWBORN

26. ● HDN (also known as erythroblastosis fetalis) is a syndrome
associated with hemolysis in the foetus either in utero and / or
after delivery with consequent hyperbilirubinemia.
● It is commonly due to immune destruction of red cells of the
foetus as a result of maternal IgG antibodies which transmit
through placenta.

28. Pathogenesis
● Due to blood group incompatibly between mother and foetus.
● The mother may be alloimunised to red cell antigen either following
trans placental haemorrhage (TPH) of incompatible foetal red cells
during pregnancy or labour or by transfusion of incompatible red
cells.
● If IgG Antibodies are formed they can cross the placenta during a
subsequent pregnancy in amounts sufficient to cause immune
destruction of incompatable foetal red cells.
●

29. Prevention of HDN
● Rh Ig (300 mg) is given intramuscularly with in 72 hrs to Rh – (D)
negative women who have
● Delivered an Rh (D) positive infant and have not developed
anti D in the serum.
● Undergone abortion or medical termination of pregnancy
● Undergone amniocentesis

31. Estriol (E3)
● Estriol is a natural steroidal estrogen and one of the three main
estrogens in the human body.
● Measurement of the total estriol has been shown to be useful in
monitoring the fetal function.
● Production of estriol by the ovaries is almost undetectable in non-
pregnant women.
● However, during pregnancy, estriol is synthesized in very high
amounts by the placenta and is by far the most produced estrogen in
the body.
●

32. Estriol
● Estriol ( E3) was first isolated by Edward Adelbert Doisy in
1923

33. The chemical structure of estriol: C18H24O3

34. Biosynthesis of Estriol

35. Estriol biosynthesis
● The mother provides cholesterol to the placenta, which
converts it to progesterone for release into the maternal and
fetal circulations.
● In the fetus, progesterone is converted to
dehydrepandrosterone (DHEA) or dehydroepiandrosterone
sulfate (DHEAS) by the fetal zone of the adrenal glands
● DHEA/DHEAS is then converted to 16-OH DHEA/16-OH
DHEAS in the fetal liver.
● This steroid then goes to the placenta and is aromatized to
estriol, the major estrogen secreted by the placenta as
unconjugated estriol (uE3).
● In the maternal circulation, E3 is transported to the liver,
where it is conjugated (cE3) into other forms (80%-90%)

36. Estriol in pregnant women:
● 1st trimester: ● Less than 38 ng/mL
● 2nd trimester:
●
● 38–140 ng/mL
●
● 3rd trimester:
● 31–460 ng/mL

37. High Estriol level Low Estriol level
● Ovarian stimulation used to treat
infertility
● Menopause.
● Cancer, such as cancer of the ovaries
, testicles , or adrenal glands
●
● Anorexia nervosa.
● Serious liver disease (cirrhosis). ● Problems with ovarian function
● Early puberty ● Problem with the fetus or placenta.
(Down syndrome, neural tube defects,
and adrenal abnormalities)
● Pregnancy with more than one fetus
(such as twins or triplets).

38. Estimation of the Estriol

39. Principle of the assay
● This is an ELISA (Enzyme-Linked Immunosorbent Assay)
for the quantitative analysis of Estriol levels in biological
fluid.
●
● This test operates on the basis of competition between
the enzyme conjugate and the Estriol in the sample, for a
limited number of binding sites on the antibody coated
plate.

40. Procedure
● The sample or standard solution is first added to the
microplate.
● diluted enzyme conjugate is added and the mixture is shaken
and incubated at room temperature for one hour.
● During the incubation, competition for binding sites is taking
place.
● The plate is then washed removing all the unbound material.
● The bound enzyme conjugate is detected by the addition of
substrate which generates an optimal color after 30 minutes.

41. Quantitative test
● Results may be obtained by measuring and comparing the
absorbance reading of the wells of the samples against the
standards with a microplate reader at 650 nm.
● The extent of color development is inversely proportional to
the amount of estriol in the sample or standard.
● For example, the absence of estriol in the sample will result
in a bright blue color, whereas
● The presence of estriol will result in decreased or no color
development.