AMNIOTIC FLUID swallowing, and intramembranous

flow.
Amniotic fluid is the clear, slightly
- Intramembranous flow is the
yellowish liquid that surrounds the fetus
absorption of amniotic fluid water
during pregnancy. It is contained within
and solutes into the fetal vascular
the amniotic sac inside the uterus.
system.
Physiology - The amount of amniotic fluid
increases in quantity throughout
- Amniotic fluid is present in
pregnancy, reaching a peak of
amnion.
approximately 800 to 1200 mL
- Amnion: it is a membranous sac
during the third trimester and
surrounding the fetus,
then gradually decreases before
metabolically active, and involved
delivery.
in water exchanges and chemicals
between the fluid, the fetus, and Polyhydramnios: >1200 mL
the maternal circulation; it amniotic fluid
produces peptides, growth factors,
Oligohydramnios: <800 mL
and cytokines.
amniotic fluid
Function
- 35 mL of amniotic fluid during
- Provides a protective cushion for the first trimester is derived from
the fetus maternal circulation.
- Allows fetal movement - The fetus will secrete the volume of
- Stabilizes the temperature, lung liquid needed to expand the
protecting the fetus from extreme lungs with growth during the latter
temperature changes third to half of pregnancy.
- Permits proper lung development - Secreted lung liquid enters
amniotic fluid, bathing lungs and
washing pulmonary and alveolar
contents (lecithin,
sphingomyelin, and
phosphatidyl glycerol) into the
amniotic fluid surrounding the fetus
during each episode of fetal
respiratory movement.
- These lung surfactants serve as an
index of fetal lung maturity.
- Fetal urine is a major contributor
of amniotic fluid after the first
Volume
trimester.
- Amniotic fluid volume is regulated - At the time the fetal urine
by the balance between the production occurs, fetal swallowing
production of fetal urine and lung of amniotic fluid begins and
fluid, absorption from fetal regulates the increase in fluid from
fetal urine.
The fetus swallows the amniotic Tests for Fetal Well-Being and
fluid: Maturity

- It is absorbed through the TEST Reference Significance

gastrointestinal tract and Values at
Term
reexcreted by the kidneys from the Bilirubin scan A450 Hemolytic
blood into fetal urine and back to >.025 Disease of the
amniotic fluid. Newborn
- Failure of the fetus to begin Alpha- <2.0 MoM Neural tube
fetoprotein disorders
swallowing results in excessive
Lecithin- 2.0 Fetal lung
accumulation of amniotic fluid maturity
sphingomyelin
(polyhydramnios) and indicates ratio
fetal distress associated with Amniostat-fetal Positive Fetal lung
neural tube disorders. lung maturity maturity /
phosphatidyl
Polyhydramnios glycerol
Foam Stability 47 Fetal lung
- It may be secondarily associated Index maturity
with fetal structural anomalies, Optical density 0.150 Fetal lung
cardiac arrhythmias, congenital 650 nm maturity
infections, or chromosomal Lamellar body 32,000/ Fetal lung
abnormalities. count mL maturity
Chemical composition
Oligohydramnios
Placenta
- It can cause increased fetal
swallowing, urinary tract - It is the ultimate source of amniotic
deformities, and membrane fluid, water, and solutes.
leakage.
Amniotic fluid
- It may be associated with
congenital malformations, - It has a composition similar to the
premature rupture of amniotic maternal plasma and contains a
membranes, and umbilical cord small amount of sloughed fetal
compression, resulting in cells from the skin, digestive
decelerated heart rate and fetal system, and urinary tract.
death. - These cells provide the basis for
cytogenetic analysis.
Although the testing of amniotic fluid is
- The fluid also contains biochemical
frequently associated with cytogenetic
substances that are produced by
analysis, the clinical laboratory also
the fetus, such as bilirubin,
performs several significant tests on
lipids, enzymes, electrolytes,
amniotic fluid.
urea, creatinine, uric acid,
When conditions that adversely affect the proteins, and hormones that can
fetus arise, the danger to the fetus must be tested to determine the health
be measured against the ability of the or maturity of the fetus.
fetus to survive an early delivery.
Neural tube defects
 - Allows the fetal cerebrospinal fluid - Levels of creatinine and urea are
to enter the amniotic fluid directly. much lower in amniotic fluid than
- Alpha-fetoprotein and in urine.
acetylcholinesterase are the two
Amniotic fluid:
biochemical markers tested for
Creatinine = <3.5 mg/dL
these defects.
Urea = <30 mg/dL
The chemical composition of amniotic
Urine:
fluid changes when fetal urine production
Creatinine = >10 mg/dL
begins.
Urea = >300 mg/dL
- INCREASE: Creatinine, Urea, and
- The presence of glucose, protein,
Uric Acid
or both is associated more closely
- DECREASE: Glucose and Protein
with amniotic fluid.
- Vary but are of little
significance: Electrolytes, Fern Test
Enzymes, Hormones, and metabolic
- This test can also differentiate
end products.
amniotic fluid from urine and other
body fluids.
- It is a test used to evaluate
premature rupture of the
membranes.
- A vaginal fluid specimen is spread
Measurement of amniotic fluid
on a glass slide and allowed to
creatinine determines fetal age
completely air dry at room
- It ranges between 1.5 and 2.0 temperature; then it is observed
mg/dL before 36 weeks’ gestation microscopically.
- It then rises above 2.0 mg/dL, - The presence of “fern-like”
thereby providing a means to crystals due to the protein and
determine fetal age greater than sodium chloride content is a
36 weeks. positive screen for amniotic
fluid.
Differentiating Maternal Urine from
Amniotic fluid Specimen Collection
- Differentiation between amniotic Amniotic fluid is obtained by needle
fluid and maternal urine may be aspiration into the amniotic sac, a
necessary to determine possible procedure called amniocentesis.
premature membrane rupture of
accidental puncture of the - Transabdominal amniocentesis
maternal bladder during specimen is the most frequently performed
collection. procedure.
- Using continuous ultrasound for
Chemical analysis of creatinine, urea, guidance, the physician locates the
glucose, and protein aids in the fetus and placenta to safely
differentiation. perform the procedure.
 - A thin, hollow needle is inserted substances that the fetus has
through the mother’s abdomen into produced.
the mother’s uterus and into the - These cells can be separated from
amniotic sac to aspirate the the fluid, cultured, and examined
amniotic fluid. for chromosome abnormalities by:
- A maximum of 30 mL of amniotic - Karyotyping
fluid is collected in sterile syringes. - Fluorescence in situ
- The first 2 or 3 mL collected are hybridization (FISH)
discarded because the fluid may be - Fluorescent mapping Spectral
contaminated by maternal blood, Karyotyping (SKY)
tissue fluid, and cells. - DNA testing
- Specimens should be transferred to - Biochemical substances produced
sterile plastic containers and by the fetus can be analyzed by
taken immediately to the thin-layer chromatography to
laboratory. evaluate the health of the fetus.
- Fluid for bilirubin analysis in
Indications for Performing
cases of hemolytic disease of the
Amniocentesis
newborn (HDN) must be protected Amniocentesis may be indicated at 15 to
from light at all times. 18 weeks of gestation for the following
conditions to determine early treatment
or intervention:
- Mother’s age of 35 or older at delivery
- Family history of chromosome
abnormalities, such as trisomy 21 (Down
Syndrome)
- Parents carry an abnormal chromosome
rearrangement
- Earlier pregnancy or child with birth
defects
- Parent is a carrier of metabolic disorder
- Family history of genetic diseases such as
sickle cell disease, Tay-Sachs disease,
hemophilia, muscular dystrophy, sickle
cell anemia, Huntington chorea, and
cystic fibrosis
- Elevated maternal serum alpha-
Indications for Amniocentesis fetoprotein
- Abnormal triple marker screening test
- Amniocentesis is recommended for - Previous child with a neural tube disorder
neural tube defects when such as spina bifida, or ventral wall
screening blood tests such as the defects (gastroschisis)
- Three or more miscarriages
maternal serum alpha-
Amniocentesis is indicated later in the
fetoprotein test are abnormal or pregnancy (20 to 42 weeks) to evaluate:
to detect genetic disorders to - Fetal lung maturity
evaluate the health of the fetus. - Fetal distress
- HDN caused by Rh blood type
- Fetal epithelial cells in amniotic
incompatibility
fluid indicate the genetic material - infection
of the fetus and the biochemical
Specimen Handling and Processing hemoglobin and is important for
further case management.
- Fluid for fetal lung maturity
(FLM) tests should be placed in Yellow
ice for delivery to the laboratory
- The presence of bilirubin gives the
and kept refrigerated.
fluid a yellow color and is indicative
- Specimens for bilirubin testing
of red blood cell destruction
must be immediately protected
resulting from HDN.
from light. This can be
accomplished by placing the Dark green - Meconium
specimens in amber-colored tubes,
- It is usually defined as a newborn’s
wrapping the collection tube in foil,
first bowel movement.
or by use of a black plastic cover
- It is formed in the intestine from
for the specimen container.
fetal intestinal secretions and
- Specimens for cytogenetic
swallowed amniotic fluid.
studies or microbial studies
- It is a dark green, mucus-like
must be processed aseptically and
material
maintained at room temperature or
- It may be present in the amniotic
body temperature prior to analysis
fluid because of fetal distress.
to prolong the life of the cells
- Fetal aspiration of meconium
needed for analysis.
during fetal swallowing is a concern
- All fluid for chemical testing
when increased amounts are
should be separated from cellular
present in the fluid.
elements and debris as soon as
possible to prevent distortion of Very dark red-brown fluid is
chemical constituents by cellular associated with fetal death.
metabolism or disintegration. This
can be performed using
centrifugation or filtration. Tests for Fetal Distress

Color and Appearance Hemolytic Disease of the Newborn

Normal amniotic fluid: colorless

- It may exhibit slight to moderate

turbidity from cellular debris,
particularly in the later stages of
fetal development

Blood-streaked fluid

- It may be present as the result of a

traumatic tap, abdominal trauma,
or intra-amniotic hemorrhage.
- The source of blood (maternal or
fetal) can be determined using the
Kleihauer-Betke test for fetal
Hemolytic Disease of the Newborn
 - The oldest routinely performed Zone II – indicates moderate hemolysis
laboratory test on amniotic fluid and requires careful monitoring
evaluates the severity of the fetal anticipating an early delivery or
anemia produced by HDN. exchange transfusion upon delivery.
- Amniotic fluid bilirubin is
Zone III – indicates severe hemolysis
measured by spectrophotometric
and suggests a severely affected fetus,
analysis using serial dilutions.
intervention through induction of labor or
intrauterine exchange transfusion must
be considered.

- The optical density (OD) of the

fluid is measured in intervals
between 365 nm and 550 nm,
and the readings are plotted on
semi-logarithmic graph paper.
- In normal fluid, the OD is highest at
365 nm and decreases linearly to
550 nm, illustrated by a straight
line.
- When bilirubin is present, a rise in
OD is seen at 450 nm because this
is the wavelength of maximum
bilirubin absorption.
Neural Tube Defects
- The difference between the OD of
the theoretic baseline and the OD - Neural tube defects (NTD) are one
at 450 nm represents the amniotic of the most common birth defects
fluid bilirubin concentration. in the United States.
- This difference in OD is referred to - It can be detected by maternal
as the absorbance difference at serum alpha-fetoprotein
450 nm (A450) and is then (MSAFP) blood test, high-
plotted on a Liley graph to resolution ultrasound, and
determine the severity of the amniocentesis.
hemolytic disease.
Alpha-fetoprotein
The Liley graph plots the A450 against
- It is the major protein produced by
gestational age and is divided into three
the fetal liver during early
zones that represent the extent of
gestation (prior to 18 weeks).
hemolytic severity.
- It is found in maternal serum due
Zone I – indicates no more than a mildly to the combined fetal-maternal
affected fetus circulations and in the amniotic
 fluid from diffusion and excretion of
fetal urine.
- Increased levels are found in the
maternal serum and amniotic fluid
when the skin fails to close over
the neural tissue, as occurs in
anencephaly and spina bifida. Test for Fetal Maturity

Measurement of amniotic fluid AFP levels - Fetal distress, whether caused by

is indicated when maternal serum HDN or other conditions, forces the
levels are elevated, or a family obstetrician to consider a preterm
history of previous neural tube delivery. At this point, fetal
defects exists. maturity must be assessed.

- Normal values are based on the Fetal Lung Maturity

week of gestational age, as the
Respiratory Distress Syndrome
fetus produces maximal AFP
(RDS)
between 12 and 15 weeks’
gestation, after which levels of - It is the most frequent complication
amniotic fluid begin to decline. of early delivery and is the seventh
- Both serum and amniotic fluid AFP most common cause of morbidity
levels are reported in terms of and mortality in premature infants.
multiples of median (MoM). - This disease is caused by an
- The median is the laboratory’s insufficiency of lung surfactant
reference level for a given week of production and structural
gestation. immaturity of the fetal lungs.
- > 2 MoM is considered abnormal
Surfactant
for both maternal serum and
amniotic fluid. - Surfactant normally appears in
mature lungs and allows the alveoli
Elevated amniotic fluid AFP levels are
(air sacs of the lungs) to remain
followed by measurement of amniotic
open throughout the normal cycle
acetylcholinesterase (AChE).
of inhalation and exhalation.
- The test is more specific for - It keeps the alveoli from collapsing
neural tube disorders than AFP, by decreasing surface tension and
provided it is not performed on a allows them to inflate with air more
bloody specimen, because blood easily.
contains AChE. - If the surfactant concentrations are
too low, the alveoli will collapse,
causing RDS.

Laboratory tests must be performed to

determine the maturity of the fetal lungs.
The amount of surfactant in fetal lungs
can be estimated by measuring the
amount of surfactants in amniotic fluid.
Lecithin-Sphingomyelin Ratio (L/S) layer chromatography (TLC). Because
the procedure is labor intensive and
- It is the reference method to which
subject to high coefficients of variations,
tests to FLM are compared.
many laboratories have replaced the L/S
Lecithin ratio with the quantitative phosphatidyl
glycerol immunoassays and lamellar
- It is the primary component of the
body density procedures.
surfactants (phospholipids, neutral
lipids, and proteins) that make up
the alveolar lining and account for
alveolar stability.
- It is produced at a relatively low
and constant rate until the 35th
week of gestation, at which time a
noticeable increase in its Phosphatidyl Glycerol (PG)
production occurs, resulting in the
- It is another lung surface lipid, and
stabilization of the fetal lung
it is also essential for adequate
alveoli.
lung maturity and can be detected
Sphingomyelin after 35 weeks of gestation.
- The production of PG normally
- It is a lipid that is produced at a
parallels that of lecithin, but its
constant rate after about 26 weeks
production is delayed in cases of
of gestation; therefore, it can serve
maternal diabetes.
as a control on which to base the
- Respiratory distress occurs in the
rise in lecithin.
presence of an L/S ratio of 2.0.
Before 35 weeks of gestation, the L/S Therefore, a thin-layer
ratio is usually < 1.6 because large chromatography lung profile must
amounts of lecithin are not being include lecithin, sphingomyelin,
produced at this time. and PG to provide an accurate
measurement of FLM.
After 35 weeks of gestation, the lecithin
concentration increases while the The Aminostat-FLM uses an antisera
sphingomyelin concentration remains containing polyclonal anti-PG antibodies
constant. that are specific for PG-containing
lamellar bodies in the amniotic fluid.
The L/S ratio will rise to 2.0 or higher as
the lecithin production increases to - The size of the agglutinates is read
prevent alveolar collapse. macroscopically, and the results
are reported as either negative,
Therefore, when the L/S ratio reaches
indicating pulmonary
2.0, preterm delivery is usually
immaturity, or high positive,
considered to be a relatively safe
indicating pulmonary maturity.
procedure.
Foam Stability Index
Quantitative measurement of lecithin and
sphingomyelin is performed using thin-
 - A mechanical screening test called - They are secreted by the type II
the “foam” or “shake” test was pneumocytes of the fetal lung at
used to determine the presence of about 24 weeks of gestation and
lung-surface lipid concentrations. increase in concentration from
50,000 to 200,000/mL by the end
Procedure: Foam Shake Test
of the third trimester.
1. Mix equal parts of amniotic fluid - As the fetal lung matures,
with 95% ethanol. increased lamellar body production
2. Vigorously shake for 15 seconds. is reflected by an increase in
3. Allow to sit undisturbed for 15 amniotic fluid phospholipids and
minutes. the L/S ratio. Therefore, the
4. Observe for the presence of a number of lamellar bodies present
continuous line of bubbles around in the amniotic fluid correlates with
the outside edge. the amount of phospholipid present
in the fetal lungs.
The presence of bubbles indicates that a
sufficient amount of phospholipid is Lamellar body diameter is similar to that
available to reduce the surface tension of of small platelets ranging in size from 1.7
the fluid even in the presence of alcohol, to 7.3 fL, or 1 to 5 um; therefore, lamellar
an antifoaming agent. body counts (LBC) can be obtained using
the platelet channel of automated
Procedure: Foam Stability Index
hematology analyzers using either
1. Add 0.5 mL of amniotic fluid to optical or impedance methods for
tubes containing increasing counting.
amounts of 95% ethanol ranging
Procedure: Lamellar Body Count
from 0.42 to 0.55 mL in 0.01 mL
increments. 1. Mix the amniotic fluid sample by
2. Vigorously shake for 15 seconds. inverting the capped sample
3. Allow to sit undisturbed for 15 container five times.
minutes. 2. Transfer the fluid to a clear test
4. Observe for the presence of a tube to allow for visual inspections.
continuous line of bubbles around 3. Visually inspect the specimen.
the outside edge. Fluids containing obvious mucus,
5. Values 47 indicate fetal lung whole blood, or meconium should
maturity. not be processed for an LBC.
4. Cap the tube and mix the sample
Lamellar Bodies
by gentle inversion or by placing
- It is a surfactant that is composed the test tube on a tube rocker for 2
of approximately 90% phospholipid minutes
and 10% protein and is packaged 5. Flush the platelet channel; analyze
into layered storage granules. the instrument’s diluents buffer
- Lamellar bodies are densely packed until a background count deemed
layers of phospholipids that acceptable by the laboratory is
represent a storage form of obtained in two consecutive
pulmonary surfactant. analyses.
 6. Process the specimen through the
cell counter and record the platelet
channel as the LBC.

The advantages of lamellar body

counting include:

- Rapid turnaround time

- Low reagent cost
- Wide availability
- Low degree of technical difficulty
- Low volume of amniotic fluid
required
- Excellent clinical performance

Amniotic fluid specimens containing

whole blood, meconium, and mucus
should not be used.

Blood – initially raises the LBC because

of the presence of platelets and then can
lower the LBC as lamellar bodies are
trapped in the fibrin strands.

Meconium and mucus – can cause a

false increase in the LBC and should not
be used for testing.

Specimens may be stored at 2 – 8

degrees Celsius but never frozen.

A consensus protocol for non-centrifuged

samples considers LBCs greater than
50,000/uL an indication of FLM and
values below 15,000/uL as immature.