AMF

1. Amniotic Fluid

2. • Amniotic fluid is a clear, slightly yellowish liquid that
surrounds the unborn baby (fetus) during pregnancy. It is
contained in the amniotic sac.
• Amniotic fluid analysis is frequently associated with
cytogenetic analysis.
• The clinical laboratory also performs several significant tests
on amniotic fluid. Because amniotic fluid is a product of fetal
metabolism, the constituents that are present in the fluid
provide information about the metabolic processes taking
place during—as well as the progress of—fetal maturation.

3. Origin and Production
• The fluid is produced by the mother’s placenta during the first
trimester and the early part of the second trimester, until the
baby’s kidneys are mature enough to take over the task.
• The baby swallows the fluid as they “breathe” and then
excretes it again as urine, thus maintaining the constant
circulation of the fluid.
• The majority of the fetal waste is passed through the placenta
to be filtered by the mother’s kidneys so the fluid is not pure
waste material

4. Function
The primary functions of the fluid are:
• To provide a protective cushion for the fetus, Allow fetal
movement,
• Stabilize the temperature to protect the fetus from extreme
temperature changes.
• To permit proper lung development.
• Exchanges of water and chemicals, also take place between the
fluid, the fetus, and the maternal circulation.
• Helps with the uniform growth of the body parts and organs of
the baby.
• Assists with the proper bone and muscle development

5. Volume
Amniotic fluid volume is regulated by the balance
 Between the production of fetal urine and lung fluid
&
 The absorption from fetal swallowing and intra
membranous flow
Intramembranous flow is the absorption of amniotic
fluid water and solution to the fetal vascular system.

6. Volume
• The amount of amniotic fluid increases throughout pregnancy,
reaching a peak of approximately 1 L during the third
trimester, and then gradually decreases prior to delivery.
• The volume of amniotic fluid is positively correlated with the
growth of fetus. From the 10th to the 20th week it increases
from 25ml to 400ml approximately.
• From the 8th week, when the fetal kidneys begin to function,
fetal urine is also present in the AF.
• Then the relationship between AF and fetal growth stops. It
reaches the high peak of 800ml at the 28 week.
• The amount of fluid declines to roughly 400 ml at 32weeks
age.

7.  Polyhydramnios: An excessive amount of amniotic fluid in
 Multiple pregnancy (twins or triplets)
 Congenital anomalies
 Gestational diabetes
• Oligohydramnios: An abnormally small amount of amniotic
fluid in
• Ruptured membranes
• Placental dysfunction
• Fetal abnormalities.

8. Chemical Composition
• Water and solutes are the ultimate source of amniotic
fluid
• Amniotic fluid composition similar to that of the
maternal plasma and contains a small amount of
sloughed fetal cells from the skin, digestive system,
and urinary tract.
• Biochemical substances that are produced by the fetus,
such as bilirubin, lipids, enzymes, electrolytes,
nitrogenous compounds, and proteins that can be tested
to determine the health or maturity of the fetus.

9. • A portion of the fluid arises from:
• Fetal respiratory tract, fetal urine, the amniotic membrane, and
the umbilical cord.
• chemical composition of the amniotic fluid changes when fetal
urine production begins.
• The concentrations of creatinine, urea, and uric acid increase,
whereas glucose and protein concentrations decrease.
• The concentrations of electrolytes, enzymes, hormones, and
metabolic end products also vary (change) but are of little
clinical significance.

10. • Measurement of amniotic fluid creatinine has been
used to determine fetal age.
• Prior to 36 weeks’ gestation, the amniotic fluid
creatinine level ranges between 1.5 and 2.0 mg/dL.
• Greater than 36 weeks creatinine rises above 2.0
mg/dL

11. Specimen Collection
• Amniocentesis is recommended when:
• Screening blood tests such as abnormal maternal serum of:
• Alpha fetal protein [AFP]
• Human chorionic gonadotropin [hCG]
• unconjugated estriol [UE3])
• Fetal body measurements taken with ultrasonography
accurately estimate the gestational age and size of the
fetus

12. • Abnormality on the ultrasound need:
• 1- An amniocentesis and laboratory measurements of
fetal lung maturity.
• 2- Fetal epithelial cells in amniotic fluid:
• Indicate the genetic material of the fetus and the
biochemical substances that the fetus has produced.
• These cells can be separated from the fluid, cultured,
and examined for chromosome abnormalities

13. • Storage Instructions:
• If cell culture is requested the specimen should be
kept at 37oC.
• Freeze within 4 hours to transport to laboratory;
stable refrigerated up to 1 week.
• Protect from light.
• Avoid repeated freezing and thawing of the
specimen, which may cause the sample to precipitate,
resulting in a lower than expected value.

14. Indications for Performing Amniocentesis
• Mother’s age of 35 or more at delivery
• Family history of chromosome abnormalities, such as
trisomy 21 (Down syndrome).
• Parents carry an abnormal chromosome rearrangement
• Parent is a carrier of a metabolic disorder.
• Three or more miscarriages
• Fetal lung maturity

15. Collection
 Amniotic fluid is obtained by needle aspiration into the
amniotic sac, a procedure called amniocentesis. The
procedure most frequently performed is a transabdominal
amniocentesis. Using continuous ultrasound for guidance, the
physician locates the fetus and placenta to safely perform the
procedure.
 Amniocentesis is generally performed between 15 and 18
weeks of gestation for genetic studies
 Tests for fetal distress and maturity are performed later in
the third trimester.

16. Collection
• A maximum of 30 mL of amniotic fluid is
collected in sterile syringes.
• The first 2 or 3 mL collected can be
contaminated by maternal blood, tissue fluid,
and cells and are discarded.
• Fluid for bilirubin analysis in cases of
hemolytic disease of the newborn (HDN) must
be protected from light at all times.

18. Color and Appearance
• Normal amniotic fluid is colorless and may exhibit slight to
moderate turbidity from cellular debris, particularly in later
stages of fetal development.
• Blood-streaked fluid result of
• A traumatic tap,
• Abdominal trauma
• Intra-amniotic hemorrhage
• Yellow color
• The presence of bilirubin gives the fluid a yellow color and is
indicative of red blood cell destruction resulting from HDN.

20. Differentiating Maternal Urine From Amniotic Fluid
• Differentiation between amniotic fluid and
maternal urine necessary to determine possible
premature membrane rupture or accidental puncture
of the maternal bladder during specimen collection.
• Chemical analysis of creatinine and urea
• Levels of creatinine and urea are much lower in
amniotic fluid than in urine
• Amniotic fluid creatinine does not exceed 3.5 mg/dL
and urea does not exceed 30 mg/dL.
• Urine creatinine is 10 mg/dL and urea is 300 mg/dL

21. Testing amniotic fluid for genetic and congenital
disorders
• Amniocentesis is often performed to detect Down syndrome
and anencephaly prior to birth.
• Test of neural tube defects (NTD):
• 1-Alpha fetoprotein (AFP)
• 2-Acetylcholinesterase (AChE)
• AFP:
• Elevated alpha fetoprotein (AFP) in amniotic fluid and
maternal circulation in Fetal neural tube defects such as
anencephaly and spina bifida
• In normal fetal development, AFP peaks at about 16 weeks of
gestation and then declines gradually to term.

22. • Measurement of amniotic fluid AFP levels
• Elevated maternal serum levels and a family history of previous NTD
exists.
• Both serum and amniotic fluid AFP levels are reported in terms of
multiples of the median (MoM).
• The median is the laboratory’s reference level for a given week of
gestation.
• A value two times the median is considered (˃2MoM) for both
maternal serum and amniotic fluid
• Elevated amniotic fluid AFP levels are followed by measurement of
amniotic acetylcholinesterase(AChE).
• AChE:
• AChE is more specific for neural tube disorders than AFP
• AChE not performed on a bloody specimen, because blood
contains AChE.
• Amniotic fluid Ach= ˃5U/L in NTD

23. Fetal karyotype
• Retrieval of fetal somatic cells can be used to identify
genetic constitution of fetus.
• The problem of karyotyping is that it take 3 or more
weeks.
• Nowadays ,numerical abnormalities in the
chromosomes 21,18,13,X & Y can be detected more
rapidly by using chromosome specific DNA probes
tagged with fluorescent dyes, known as (FISH).
(90%specificity & gives results in 2days.)
*Fluorescence in situ hyperidization (FISH).

24. Fetal lung maturity
 laboratory tests to determine the maturity of
the fetal lungs:
 1-Lecithin:Sphingomyelin Ratio
 2-Phosphatidylglycerol
 3-Shake test & Foam stability
 4-Lamellar Bodies

25. FETAL LUNG MATURITY
 Respiratory distress is the most frequent complication of early
delivery.
 Laboratory tests to determine the maturity of the fetal
lungs:
 1- Lecithin:Sphingomyelin Ratio and Phosphatidylglycerol
 Fetal lung surfactants include three phospholipids:
 (Lecithin, sphingomyelin, and phosphatidyl glycerol)
 It is produced by type II pneumocytes in the form of lamellar
bodies.
 The ratio of lecithin to sphingomyelin is used to assess fetal
lung maturity.

26. • Up until the 33rd week of gestation, the levels of
these two L/S are relatively equal.
• After 34 weeks of gestation, the level of
sphingomyelin decreases, whereas the level of
lecithin increases significantly.
• L/S ratio≥ 2.0 usually indicate maturity, and ratio
˂1.5 indicate immaturity.
• Replaced the L/S ratio with the more cost-
effective phosphatidyl glycerol immunoassays,
fluorescence polarization, and lamellar body
density procedures.

27. • FETAL LUNG MATURITY

28. • FETAL LUNG MATURITY
 4-Shake test &Foam stability
• Both tests utilize dilutions of amniotic fluid in 95% ethanol
and look for formation of a persistent ring of foam / bubbles,
an indication of total surfactant concentration.
 Shake test: crude, fast, cheap and can be performed at
bedside. Physician can make an immediate decision
regarding safety of early delivery of infant.
 1:2 dilution (amniotic fluid : 95% ethyl alcohol), shake 15
seconds, If a complete ring of foam persists 15 minutes =
positive test.

29.  FETAL LUNG MATURITY
 FOAM STABILITY
 This is a screening test for fetal lung surfactant in amniotic
fluid. In this test, a fixed amount of amniotic fluid is mixed
with an increasing volume of 95% ethanol in a series of tubes
with alcohol concentrations ranging from 0.43 to 0.55.
 The mixtures are shaken vigorously for 30 seconds, and the
contents are allowed to settle for 15 seconds and the samples
are examined for an uninterrupted ring of foam in the tube.
 The highest concentration of 95% ethanol that is able to
support a ring of foam is known as the foam stability index.
The principle of the test is that more surfactant is needed to
maintain the foam in greater concentrations of ethanol and
more fetal lung surfactant is needed to support fetal lung
function at birth.
 Foam stability index= ≥0.47 indicate fetal lung maturity

30. • FETAL LUNG MATURITY
• 5-Lamellar Bodies
• Fetal lung surfactants are produced by fetal type II
pneumocytes of the fetal lung and are stored as
lamellar bodies after about 20 weeks of gestation.
• Lamellar bodies are about the size of small platelets.
• Lamellar bodies are storage forms of lung phospholipids
and they enter the fetal lungs and the amniotic fluid at
about 20–24 weeks of gestation.
• Lamellar bodies affect the optical density of amniotic
fluid and a measurement of the optical density of 0.150
at 650 nm has been shown to correlate with an L/S ratio
of 2.0 and to correlate with the presence of phosphatidyl
glycerol.

31. • Lamellar body counts provide a reliable estimate of fetal lung maturity.
Lamellar body counts can be performed easily with many hematology
analyzers using the platelet count channel.
Lamellar body counts of approximately 35,000 per microliter correspond to adequate
fetal lung surfactant levels.

32. HEMOLYTIC DISEASE OF THE NEWBORN
• HDN, also known as erythroblastosis fetalis
• caused when mother develops antibodies to an antigen on the fetal
erythrocytes and these maternal antibodies cross the placenta
to destroy many fetal red blood cells (RBCs).
• Most frequently, HDN is caused by the sensitization of an Rh
negative mother to fetal Rh antigen.
• The destruction of these fetal RBCs results in the appearance
of elevated level of bilirubin in the amniotic fluid.
• The measurement of amniotic fluid bilirubin is performed by
spectrophotometric analysis.
• The optical density (OD) of the fluid is measured in intervals
between 365 nm and 550 nm and the readings plotted on
semi logarithmic graph paper.

33. Infection
• Evidence is mounting of the importance of
microorganisms in the amniotic fluid contributing to
the incidence of preterm delivery and spontaneous
abortion.
• cytomegalovirus (CMV), toxoplasmosis, bacterial
infections, herpes, candidiasis, trichomonas's and
even bacterial vaginosis have been linked to preterm
birth.
• Gram stain, wet mount, culture, and molecular tests
may be used on amniotic fluid to look for potential
infectious agents.