AMNIOTIC FLUID

Submitted to:
Ma’am Bailiza Shanecca Nawal, RMT
AUBF INSTRUCTOR

Submitted by:
Dalaygon, Zapirah Shane T.
Dormitorio, Simone len Marie L.
Ebina, Cristine C.
Maceren, Kyla Joyce
Maguinsawan, Angel Mae H.
Wenceslao, Rhea Mae A.

OCTOBER 31, 2023

 PHYSIOLOGY ● Third to half of pregnancy-
Amniotic fluid is present in the amnion. secretes a volume of lung liquid
Amnion necessary to expand the lungs
● membranous sac that surrounds with growth.
the fetus. ● Lung surfactants: lecithin,
● metabolically active sphingomyelin, and phosphatidyl
● involved in the exchanges of glycerol.
water and chemicals between ● Polyhydramnios
the fluid, the fetus, and the ○ An amniotic fluid volume
maternal circulation; greater than 1200 mL.
● produces peptides, growth ○ Failure of the fetus to
factors, and cytokines. begin swallowing results in
excessive accumulation of
➢ FUNCTIONS amniotic fluid
● provide a protective cushion for ○ An indication of fetal
the fetus distress, often associated
● allow fetal movement with neural tube disorders.
● stabilize the temperature to ○ Secondarily associated
protect the fetus from extreme with fetal structural
temperature changes anomalies, cardiac
● permit proper lung development arrhythmias, congenital
infections, or chromosomal
➢ VOLUME abnormalities.
● Increases in quantity throughout ● Oligohydramnios
pregnancy ○ An amniotic fluid volume
● Reaching a peak of less than 800 mL.
approximately 800 to 1200 mL ○ Possible cause is increased
during the third trimester fetal swallowing, urinary
● gradually decreases prior to tract deformities, and
delivery membrane leakage.
● First trimester- approximately 35 ○ May be associated with
mL of amniotic fluid is derived. congenital malformations,

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 premature rupture of that can be tested to determine
amniotic membranes, and the health or maturity of the fetus.
umbilical cord ● Biochemical markers to detect
compression, resulting in NTDs: Alpha-fetoprotein and
decelerated heart rate acetylcholinesterase.
and fetal death. ● The chemical composition of the
amniotic fluid changes when
fetal urine production begins.
● Increased concentrations -
creatinine, urea, and uric acid
● Decreased concentrations -
glucose and protein
● Electrolytes, enzymes, hormones,
and metabolic end products also
vary but are of little clinical
significance.

➢ CHEMICAL COMPOSITION
➢ MEASUREMENT OF AMNIOTIC
● Placenta: ultimate source of
FLUID CREATININE
amniotic fluid and solutes.
● Used to determine fetal age
● Amniotic fluid: Has a composition
● Before 36 weeks’ gestation = 1.5-
similar to that of the maternal
2.0 mg/dL
plasma and contains a small
● After 36 weeks’ gestation = > 2.0
amount of sloughed fetal cells
mg/dL
from the skin, digestive system,
and urinary tract.
● The fluid also contains
➢ DIFFERENTIATING MATERNAL
biochemical substances that are
URINE FROM AMNIOTIC FLUID
produced by the fetus, such as
● Differentiation between amniotic
bilirubin, lipids, enzymes,
fluid and maternal urine may be
electrolytes, urea, creatinine, uric
necessary to determine possible
acid, proteins, and hormones
premature membrane rupture or

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 accidental puncture of the ○ “Fern-like” crystals are
maternal bladder during seen in a positive screen
specimen collection. for amniotic fluid.
● pH test
CHEMICAL ANALYSIS ○ Normal vaginal pH = 4.5-
6.0
● Analytes: creatinine, urea,
○ Amniotic fluid pH = 7.1-7.3
glucose, and protein.
● Nitrazine test
● Amniotic fluid:
○ Screening test used to
○ Creatinine does not
detect the presence of
exceed 3.5 mg/dL;
amniotic fluid in vaginal
○ Urea does not exceed 30
secretions.
mg/dL
○ Strip with nitrazine dye -
● Urine:
turns blue from drop of
○ Creatinine = 10 mg/dL
vaginal fluid, suggesting
○ Urea = 300 mg/dL
the membranes have
● Measurement of glucose and
ruptured.
protein by a reagent strip is a less
● Biomarker testing
reliable indicator.
○ AmniSure ROM test

TESTS (Qiagen, Germantown,

● These tests are used to diagnose MD) - immunographic test

premature rupture of membranes that detects the presence

(PROM). of placental alpha

● Fern test microglobulin (PAMG-1)

○ Differentiate amniotic fluid protein in vaginal

from urine and other body discharge.

fluids. ○ Actim PROM

○ Used to evaluate (CooperSurgical, Trumbull,

premature rupture of the CT) - rapid immunoassay

membranes point-of-care test that

detects insulin-like growth
factor binding protein-1

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 (IGFBP-1), also known as ● Fetal body measurement taken
placental protein 12 with ultrasonography:
(PP12). ○ Accurately estimate the
○ ROM Plus (Clinical gestational age of the
Innovations, Murray, UT) - fetus and assess the size
detects both AFP and and growth of the fetus
IGFBP-1. throughout the pregnancy.
○ Abnormality on the
SPECIMEN COLLECTION, HANDLING, ultrasound can indicate
AND PROCESSING potential fetal
development problems
● AMNIOCENTESIS: recommended and the need for an
for cases of neural tube defects amniocentesis and
when screening blood tests are laboratory measurements
abnormal, to detect genetic of fetal lung maturity.
disorders, or to evaluate health of ● Fetal epithelial cell in amniotic
the fetus. fluid: indicate genetic material of
○ Safe procedure; the fetus and their produced
performed after the 14th biochemical substances.
week of gestation. ○ The cells are examined for
○ Fluid for chromosome chromosome
analysis: collected at abnormalities by
approximately 16 weeks’ karyotyping, fluorescence
gestation. in situ hybridization (FISH),
○ Tests for Intrauterine fluorescent mapping
growth retardation: spectral karyotyping (SKY),
performed near the end of and DNA testing.
the second trimester ○ Biochemical substances:
○ Tests for Fetal distress and analyzed by thin-layer
maturity: performed later chromatography (TLC) to
in the third trimester evaluate the health of the
fetus.

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 ➢ COLLECTION
➢ INDICATIONS FOR ● Amniotic fluid is obtained
AMNIOCENTESIS by needle aspiration into
the amniotic sac
At 15-18 weeks of gestation (early (AMNIOCENTESIS).
intervention): ● Transabdominal
amniocentesis: most
● Mother’s age of 35 or
frequently performed
older at delivery
procedure.
● Earlier pregnancy or child
● Maximum of 30 mL of
with a birth defect
amniotic fluid
● Elevated maternal serum
○ Discard the first 2 to
alpha-fetoprotein
3 mL, which are
● Three or more miscarriages
contaminated by
● Family history of
maternal blood,
chromosome
tissue fluid, and cells.
abnormalities like down
● Specimens should be
syndrome, or genetic
transferred to sterile plastic
diseases, such as sickle cell
containers and taken
disease, Tay-Sachs disease,
immediately to the
hemophilia, muscular
laboratory.
dystrophy, sickle cell
● Fluid for bilirubin analysis in
anemia, Huntington
cases of hemolytic disease
chorea, and cystic fibrosis
of the newborn (HDN)

At 20-42 weeks of gestation: MUST be protected from

light at all times.
● Fetal lung maturity and
fetal distress ➢ HANDLING AND PROCESSING
● Infection ● Special handling
● HDFN caused by Rh blood procedures should be
type incompatibility performed immediately
and the specimen

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 delivered promptly to the elements and debris as
laboratory. soon as possible to
● Specimen considerations prevent distortion of
for: chemical constituents by
○ Fetal lung maturity cellular metabolism or
(FLM) tests: place in disintegration.
ice for delivery to ○ Performed using
the laboratory and centrifugation or
kept refrigerated. filtration. Time and
○ Bilirubin testing: speed of
immediately centrifugation
protected from light depends on the test
(place in amber- and lab protocols.
colored tubes, wrap
with foil, or using a COLOR AND APPEARANCE
black plastic cover
COLOR SIGNIFICANCE
for the specimen
container) Colorless Normal

○ Cytogenetic studies
Blood-streaked Traumatic tap,
or microbial studies:
abdominal trauma,
processed
intra-amniotic
aseptically and
hemorrhage
maintained at room
temperature or Yellow Hemolytic disease of
body temperature the newborn (bilirubin)
(37 °C incubation)
Dark green Meconium
prior to analysis to
prolong the life of Dark red brown Fetal death
the cells.
● All fluid for chemical
● Kleihauer-Betke Test: Use to
testing should be
determine the source of blood
separated from cellular

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 (maternal or fetal) for fetal
Alpha <2.0 MoM Neural tube
hemoglobin.
Fetoprotein disorders
● Presence of bilirubin: indicative of
red blood cell destruction.
● Meconium: defined as the Lecithin ≥ 2.0 Fetal lung

newborn’s first bowel movement. Sphingomye maturity

○ Formed in the intestine lin ratio

from fetal intestinal

Amniostat Positive Fetal lung
secretions and swallowed
Fetal lung maturity/ph
amniotic fluid.
maturity osphatidylgl
○ Its presence may be due
ycerol
to fetal distress.
○ Fetal aspiration of Foam Fetal lung
meconium during fetal Stability ≥ 47 maturity
swallowing is a concern Index
when increased amounts
Optical ≥ 0.150 Fetal lung
are present in the fluid.
density 650 maturity
nm
TESTS FOR FETAL WELL-BEING AND
MATURITY Lamellar ≥ Fetal lung
body count 32,000/mL maturity

Test Reference Significance

Values at
Term TESTS FOR FETAL DISTRESS

Bilirubin D Hemolytic
➢ HEMOLYTIC DISEASE OF THE FETUS
scan A450 >.025 disease of
AND NEWBORN
the
● Antibodies against other
newborn
red cell antigens are also
capable of producing
HDN.

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● Initial exposure to foreign ● The optical density (OD) of
red cell antigens occurs the fluid is measured in
during gestation, delivery intervals between 365 nm
of the placenta, or a and 550 nm.
previous pregnancy when ● Normal fluid: OD is highest
fetal red blood cells enter at 365 nm and decreases
into the maternal linearly to 550 nm.
circulation and stimulate ● Bilirubin-present fluid: OD
the mother to produce is seen at 450 nm because
antibodies to the antigen. this is the wavelength of
● When these antibodies maximum bilirubin
present in the maternal absorption.
circulation cross the ● Absorbance difference at
placenta into the fetal 450 nm (ΔA450): The
circulation and bind to the difference between the
antigen on the fetal cells, OD of the theoretic
the cells are destroyed. baseline and the OD at
● The destruction of fetal red 450 nm represents the
blood cells results in the amniotic fluid bilirubin
appearance of the red concentration.
blood cell degradation ● Specimen considerations:
product—unconjugated ○ Protected from light
bilirubin—in the amniotic (marked decreased
fluid, which is measured to values if exposed
determine the extent of for 30 minutes)
hemolysis. ○ Centrifuge
● Amniotic fluid bilirubin is immediately to
measured by remove particulate
spectrophotometric interference.
analysis using serial ○ Meconium: cause
dilutions. falsely low ΔA450
values

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 ○ Blood: generally anticipating an early
unacceptable delivery or exchange
because maximum transfusion upon delivery.
absorbance of ○ Zone III: severe hemolysis
oxyhemoglobin and suggests a severe
occurs at 410 nm effect on the fetus.
and can interfere Intervention through
with the bilirubin induction of labor or
absorption peak. intrauterine exchange
Can be removed transfusion must be
by extraction with considered.
chloroform if QUEENAN CURVE
necessary. ● Modified Liley graph, shows
○ Control: prepared ΔA450 values from 14 to 40
by diluting weeks’ gestation, providing an
commercial earlier prediction of a possible
chemistry control hemolytic crisis.
sera 1 to 10 with ● ΔA450 values in the lowest zone
normal saline and indicate a Rh negative
treating it in the (unaffected) fetus. The
same manner as indeterminate and Rh positive
the patient (affected) zones indicate
specimen. increasing hemolytic severity.
LILEY GRAPH
● The values of (ΔA450) is plotted ➢ NEURAL TUBE DEFECTS
on this graph. ● Alpha-fetoprotein (AFP) is
● Divided into three zones: the major protein
○ Zone I: more than a mild produced by the fetal liver
effect on the fetus. during early gestation
○ Zone II: moderate (prior to 18 weeks).
hemolysis and require ● Production of maximal AFP
careful monitoring, by fetus: between 12 and

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 15 weeks’ gestation, after serum and amniotic
which levels in amniotic fluid.
fluid begin to decline. ● Elevated amniotic fluid
● Increased levels of alpha- AFP levels are followed by
fetoprotein (AFP) in both measurement of amniotic
the maternal circulation acetylcholinesterase
and the amniotic fluid can (AChE). The test is more
be indicative of fetal specific for NTDs than AFP,
neural tube defects, such provided it is not
as anencephaly and spina performed on a bloody
bifida. specimen, because blood
● Detected by maternal contains AChE.
serum alpha-fetoprotein
(MSAFP) blood test, high- TESTS FOR FETAL MATURITY
resolution ultrasound, and
amniocentesis. ➢ FETAL LUNG MATURITY
● Both serum and amniotic ● Respiratory distress
fluid AFP levels are syndrome (RDS): caused
reported in terms of by an insufficiency of lung
multiples of the median surfactant production and
(MoM). structural immaturity of the
○ The median is the fetal lungs.
laboratory’s ● Surfactant normally
reference level for a appears in mature lungs
given week of and allows the alveoli (air
gestation. sacs of the lung) to
○ A value two times remain open throughout
the median value is the normal cycle of
considered inhalation and exhalation.
abnormal (greater ● If the surfactant
than 2 MoM) for concentrations are too
both maternal

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 low, the alveoli will concentration remains
collapse, causing RDS. constant.
● The amount of surfactant ● The L/S ratio will rise to 2.0
in fetal lungs can be or higher as the lecithin
estimated by measuring production increases to
the amount of surfactants prevent alveolar collapse.
in amniotic fluid. ● Preterm delivery is
considered to be a
➢ LECITHIN-SPHINGOMYELIN RATIO relatively safe procedure
● The reference method to when L/S ratio reaches 2.0.
which tests of FLM are ● Falsely elevated results are
compared. encountered in fluid
● Lecithin is produced at a contaminated with blood
relatively low and constant or meconium because
rate until the 35th week of these substances contain
gestation, at which time a lecithin and sphingomyelin.
noticeable increase in its
production occurs, ➢ PHOSPHATIDYL GLYCEROL
resulting in the stabilization ● The presence of another
of the fetal lung alveoli. lung surface lipid,
● Sphingomyelin is a lipid phosphatidyl glycerol (PG),
that is produced at a is also essential for
constant rate after about adequate lung maturity
26 weeks’ gestation; and can be detected
therefore, it can serve as a after 35 weeks’ gestation.
control on which to base The production of PG
the rise in lecithin. normally parallels that of
● After 35 weeks of gestation, lecithin, but its production
the lecithin concentration is delayed in cases of
increases while the maternal diabetes.
sphingomyelin ● TLC lung profile: lecithin,
sphingomyelin, and PG to

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 provide an accurate and meconium, which
measurement of FLM. reduces surface tension
● Immunologic and yields false mature
agglutination test: index.
Aminostat-FLM (Irving Procedure for Foam/Shake test:
Scientific, Santa Ana, CA)
uses antisera containing
polyclonal anti-PG
antibodies that are
specific for PG-containing
Procedure for FSI:
lamellar bodies in the
amniotic fluid. The test is
not affected by blood or
meconium.

➢ FOAM STABILITY INDEX

- A mechanical screening
test, called the “foam” or
➢ LAMELLAR BODIES
“shake” test, was used to
● Surfactant is composed of
determine the presence of
approximately 90%
individual lung-surface
phospholipid and
lipid concentrations.
10% protein and is
- In this test, the presence of
packaged into layered
bubbles indicates that a
storage granules called
sufficient amount of
lamellar bodies.
phospholipid is available
● Lamellar bodies are
to reduce the surface
densely packed layers of
tension of the fluid even in
phospholipids that
the presence of alcohol,
represent a storage form
an antifoaming agent.
of pulmonary surfactant.
- Affected by specimens
contaminated with blood

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● Secreted by the type II nm, which rules out
pneumocytes of the fetal interference from
lung at about 24 weeks of hemoglobin, but not other
gestation and are contaminants, such as
absorbed into the alveolar meconium.
spaces to provide LAMELLAR BODY COUNT
surfactant. ➔ Obtained using the platelet
● At about 26 weeks of channel of automated
gestation, they enter the hematology analyzers using
amniotic fluid and either optical or impedance
increase in concentration methods for counting
from 50,000 to 200,000 per ◆ Advantages of lamellar
microliter. body counting:
● As the fetal lung matures, ● Rapid turnaround
increased production of time
lamellar bodies is reflected ● Low reagent cost
by an increase in amniotic ● Wide availability
fluid phospholipids and the ● Low degree of
L/S ratio. technical difficulty
● Therefore, the number of ● Low volume of
lamellar bodies present in amniotic fluid
the amniotic fluid required
correlates with the amount ● Excellent clinical
of phospholipid present in performance
the fetal lungs. ◆ Specimen considerations:
● Its presence increases the ● Amniotic fluid must
optical density of amniotic not contain whole
fluid. blood, meconium,
● Specimens are and mucus.
centrifuged at 2000 g for ● Blood: initially raises
10 minutes and examined the LBC because of
using a wavelength of 650 the presence of

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 platelets and then
can lower the LBC
as lamellar bodies
are trapped in the
fibrin strands.
● Meconium and
mucus: false-
increase of LBC.
● Stored at 2-8 C but
never frozen.
◆ Reporting of results:
● Units of lamellar
bodies per
microliter
● FLM indication: >
50,000/uL
● Immature: <
15,000/uL
● Intermediate:
values in between
Procedure for LBC:

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