The amount of amniotic fluid increases in quantity throughout

pregnancy, reaching a peak of approximately 800 to 1200 mL during the

MLS 065: ANALYSIS OF URINE AND BODY FLUIDS third trimester, and then gradually decreases prior to delivery

LESSON TITLE: AMNIOTIC FLUID

Polyhydramnios amniotic fluid volume greater than 1200 mL
TESTING OF AMNIOTIC FLUID
Oligohydramnios amniotic fluid volume less than 800 mL
➔ is frequently associated with cytogenetic analysis

AMNIOTIC FLUID
➔ is a product of fetal metabolism During the first trimester:
approximately 35 mL of amniotic fluid is derived primarily from the maternal circulation

During the latter third to half of pregnancy:

fetus secretes a volume of lung liquid necessary to expand the lungs with growth

During each episode of fetal respiratory movement:

secreted lung liquid enters the amniotic fluid, bathing the lungs and washing pulmonary
and alveolar contents such as lecithin, sphingomyelin, and phosphatidyl glycerol
into the amniotic fluid surrounding the fetus

LUNG SURFACTANTS
➔ serve as an index of fetal lung maturity

FETAL URINE
➔ major contributor to the amniotic fluid volume after the first trimester

Failure of the fetus to begin - indication of fetal distress

swallowing results in - often associated with neural tube
polyhydramnios disorders

Polyhydramnios - may be secondarily associated with

fetal structural anomalies,
cardiac arrhythmias, congenital
infections, or chromosomal
abnormalities

Oligohydramnios - possible causes are increased

PHYSIOLOGY fetal swallowing, urinary tract
deformities, and membrane
leakage
○ Amniotic fluid is present in the amnion - may be associated with congenital
malformations, premature
AMNION rupture of amniotic membranes,
➔ a membranous sac that surrounds the and umbilical cord compression,
fetus resulting in decelerated heart rate
➔ is metabolically active and is involved and fetal death
in the exchanges of water and chemicals
between the fluid, the fetus, and the maternal circulation
➔ produces peptides, growth factors, and cytokines CHEMICAL COMPOSITION

PLACENTA
PRIMARY FUNCTIONS OF THE AMNIOTIC FLUID
➔ is the ultimate source of amniotic fluid water and solutes

1. Provide a protective cushion for the fetus

2. Allow fetal movement ➢ AMNIOTIC FLUID HAS A COMPOSITION SIMILAR TO THAT OF
THE MATERNAL PLASMA and contains a small amount of
3. Stabilize the temperature to protect the fetus from extreme temperature
sloughed fetal cells from the skin, digestive system, and urinary tract
changes
4. Permit proper lung development. Sloughed fetal cells → provide the basis for cytogenetic analysis

Fluid → contains biochemical substances that are produced by the fetus

VOLUME
1. Bilirubin
2. Lipids
AMNIOTIC FLUID VOLUME
3. Enzymes
➔ is regulated by a balance between the production of fetal urine and 4. Electrolytes
lung fluid and the absorption from fetal swallowing and 5. Urea
intramembranous flow 6. Creatinine
7. Uric acid
INTRAMEMBRANOUS FLOW 8. Proteins
9. Hormones
➔ is the absorption of amniotic fluid water and solutes into the fetal
vascular system ➔ can be tested to determine the health or maturity of the fetus
 ○ Neural tube defects allow fetal cerebrospinal fluid to enter the
amniotic fluid directly
SPECIMEN COLLECTION
○ TWO BIOCHEMICAL MARKERS
1. Alpha-fetoprotein
2. Acetylcholinesterase
Amniocentesis → recommended for neural tube defects when screening blood tests:
➢ maternal serum alpha fetoprotein test are abnormal
➢ detect genetic disorders
Portion of the fluid - arises from the fetal respiratory tract, fetal ➢ evaluate the health of the fetus
urine, the amniotic membrane, and the
umbilical cord
FETAL BODY MEASUREMENTS TAKEN WITH ULTRASONOGRAPHY
➢ chemical composition of the amniotic fluid CHANGES when fetal ➔ accurately estimate the gestational age of the fetus
urine production BEGINS ➔ provide an assessment of the size and growth of the fetus
throughout pregnancy to diagnose and manage intrauterine
INCREASE - concentrations of creatinine, urea, and uric growth retardation
acid

DECREASE - glucose and protein concentrations Finding an abnormality - indicate potential fetal development
on the sound problems and indicate the need for an
LITTLE CLINICAL - concentrations of electrolytes, enzymes, amniocentesis and laboratory
SIGNIFICANCE hormones, and metabolic end products measurements of fetal lung maturity

Fetal epithelial cells in - indicate the genetic material of the

MEASUREMENT OF AMNIOTIC FLUID CREATININE amniotic fluid fetus and the biochemical substances
➔ has been used to determine fetal age that the fetus has produced

PRIOR TO 36 WEEKS’ GESTATION: FETAL EPITHELIAL CELLS can be separated from the fluid, cultured
Amniotic fluid creatinine level → ranges between 1.5 and 2.0 mg/dL and examined for chromosome abnormalities by:
1. Karyotyping
GREATER THAN 36 WEEKS: 2. Fluorescence in situ hybridization (FISH)
Amniotic fluid creatinine level → above 2.0 mg/dL 3. Fluorescent mapping spectral karyotyping (SKY)
4. DNA testing

DIFFERENTIATING MATERNAL URINE FROM AMNIOTIC FLUID THIN-LAYER CHROMATOGRAPHY

➔ analyzed the biochemical substances produced by the fetus to
DIFFERENTIATING BETWEEN AMNIOTIC FLUID AND MATERNAL URINE evaluate the health of the fetus
➢ Premature membrane rupture
➢ Accidental puncture of the maternal bladder during specimen collection

CHEMICAL ANALYSIS OF CREATININE, UREA, GLUCOSE AND PROTEIN

➔ aids in the differentiation

Creatinine does not exceed 3.5 mg/dL

AMNIOTIC FLUID
Urea does not exceed 30 mg/dL

Creatinine 10 mg/dL
URINE
Urea 300 mg/dL

MEASUREMENT OF GLUCOSE AND PROTEIN by a reagent strip

➔ is a less reliable indicator

★ Glucose and protein are NOT UNCOMMON urine constituents during pregnancy

Presence of glucose, protein or both - associated more closely

with amniotic fluid

FERN TEST
➔ also can differentiate amniotic fluid from urine and other body fluids
➔ a test used to evaluate premature rupture of the membranes

1. Vaginal fluid specimen is spread on a glass slide AMNIOCENTESIS

2. Allowed to completely air dry at room temperature ➔ is a safe procedure, particularly when performed after the 14th
3. Observed microscopically week of gestation

POSITIVE SCREEN FOR AMNIOTIC FLUID:

Fluid for chromosome analysis
★ presence of “fern-like” crystals due to the ➢ usually collected at approximately 16 weeks’ gestation
protein and sodium chloride content
 KLEIHAUER-BETKE TEST for fetal hemoglobin
Tests for intrauterine growth Tests for feta distress and maturity
➔ determine the source of the blood (maternal or fetal)
retardation ➢ performed later in the third
➢ performed near the end of trimester
the second trimester
Yellow color - presence of bilirubin
- indicative of red blood cell destruction
resulting from HDN
COLLECTION
Meconium - dark green, mucus-like material
- may be present in the amniotic fluid as a
➢ Amniotic fluid is obtained by needle aspiration into the amniotic sac, a result of fetal distres
procedure called amniocentesis
Very dark red-brown - associated with fetal death
Transabdominal amniocentesis → MOST FREQUENTLY PERFORMED

Vaginal amniocentesis → may also be performed; MECONIUM

→ method carries a greater risk of infection ➔ is usually defined as a newborn’s first bowel movement
➔ is formed in the intestine from the fetal intestinal secretions and
➢ maximum of 30 mL of amniotic fluid is collected in sterile syringes swallowed amniotic fluid

➢ First 2 or 3 mL collected can be contaminated by maternal blood,

tissue fluid, and cells → DISCARDED Fetal aspiration of meconium during fetal swallowing
is a concern when increased amounts are present in the fluid
➢ Specimens should be transferred to sterile plastic containers and
taken immediately to the laboratory

Fluid for bilirubin analysis

➢ Hemolytic disease of the newborn (HDN) → protected from light at all times

SPECIMEN HANDLING AND PROCESSING

○ Handling and processing of amniotic fluid vary with the tests

requested and with the methodology used by the laboratory
performing the test
○ Special handling procedures should be performed immediately
and the specimen delivered promptly to the laboratory

Fluid for fetal lung maturity (FLM)

➢ tests should be placed in ice for delivery to the laboratory and kept refrigerated TESTS FOR FETAL DISTRESS

Specimens for bilirubin testing: Specimens for cytogenetic studies ○ The OLDEST ROUTINELY PERFORMED LABORATORY TEST ON
or microbial studies AMNIOTIC FLUID evaluates the severity of the fetal anemia produced
by HDN
➢ immediately protected from ➢ processed aseptically
light ➢ maintained at room
temperature or body Antibodies against other - capable of producing HDN
ACCOMPLISHED BY: temperature (37°C incubation) red cell antigens
⇨ Placing the specimens in prior to analysis to prolong the
amber-colored tubes life of the cells needed for Immunization of - may not be effective or even
⇨ Wrapping the collection tube in foil analysis RH-negative mothers performed in all cases
⇨ Use of a black plastic cover for the
specimen container
Initial exposure to - occurs during gestation, delivery
foreign red cell antigens of the placenta, or a previous
pregnancy when fetal red blood
All fluid for chemical testing cells enter into the maternal
➢ should be separated from cellular elements and debris as soon as possible to circulation and stimulate the
prevent distortion of chemical constituents by cellular metabolism or disintegration
mother to produce antibodies to
➢ performed using centrifugation or filtration the antigen

COLOR AND APPEARANCE When these antibodies present in the maternal circulation cross
the placenta into the fetal circulation and bind to the antigen on
the fetal cells, the cells are destroyed

Normal amniotic fluid - colorless

- may exhibit slight to moderate turbidity
from cellular debris (later stages of fetal destruction of - results in the appearance of the red blood
development) fetal red blood cell degradation product, unconjugated
cells bilirubin, in the amniotic fluid
Blood-streaked fluid - present as the result of a traumatic tap,
abdominal trauma, or intra amniotic MEASURING THE AMOUNT OF BILIRUBIN IN THE FLUID
hemorrhage
➔ Hemolysis taking place may be determined
➔ Danger this anemia presents to the fetus may be assessed
SPECTROPHOTOMETRIC ANALYSIS using serial dilutions CAN BE DETECTED BY:
➔ measure the amniotic fluid bilirubin 1. Maternal serum alpha-fetoprotein (MSAFP) blood test
2. High-resolution ultrasound
OPTICAL DENSITY (OD) of the fluid 3. Amniocentesis
➔ is measured intervals between 365 nm and 550 nm and the readings
plotted on semi logarithmic graph paper
Increased levels of - indicative of fetal neural tube
alpha-fetoprotein (AFP) in both defects, such as anencephaly
the maternal circulation and and spina bifida
Normal fluid - OD is highest at 365 nm and decreases
the amniotic fluid
linearly to 550 nm, illustrated by a straight
line
AFP (alpha-feto-protein)
Bilirubin is present - rise in OD is seen at 450 nm (wavelength ➔ is the major protein produced by the fetal liver during early
of bilirubin absorption) gestation (prior to 18 weeks)
➔ It is found in the maternal serum due to the combined
DIFFERENCE BETWEEN THE OD (theoretic baseline) AND THE OD (450 nm)
fetal-maternal circulations and in the amniotic fluid from
➔ represents the amniotic fluid bilirubin concentration
diffusion and excretion of fetal urine
DIFFERENCE IN OD → absorbance difference at 450 nm (∆A450), is then
plotted on a Liley graph to determine the severity of the hemolytic disease Increased levels - 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

MEASUREMENT OF AMNIOTIC FLUID AFP LEVELS

➔ is indicated when maternal serum levels are elevated or a family
history of previous neural tube defects exists

Elevated serum levels - possibility of a multiple pregnancy

also must be investigated

Normal values - are based on the week of gestational

age

Values falling in zone I - indicate no more than a mildly affected ○ Both serum and amniotic fluid AFP levels are reported in terms of
fetus multiples of the median (MoM)

Values falling in zone II - indicate moderate hemolysis and require Median → is the laboratory’s reference level for a given week of gestation
careful monitoring anticipating an early
delivery or exchange transfusion upon
delivery
Abnormal - value two times the median value
- greater than 2 MoM for both maternal serum and
Values falling in zone III - indicates severe hemolysis and suggests amniotic fluid
a severely affected fetus

➢ Intervention through induction of labor or intrauterine exchange ○ Elevated amniotic fluid AFP levels are followed by measurement of
transfusion must be considered when a ∆A450 is plotted in zone III amniotic acetylcholinesterase (AChE)

AMNIOTIC ACETYLCHOLINESTERASE (AChE)

○ Contamination of the fluid by cells, hemoglobin, meconium, or
other debris will interfere with the spectrophotometric analysis ➔ is more specific for neural tube disorders than AFP, provided it is
not performed on a bloody specimen, because blood contains
○ Specimens should be immediately centrifuged to remove AChE
particulate interference

TESTS FOR FETAL MATURITY

Markedly decreased - obtained with as little as 30 minutes of
values exposure to light
RESPIRATORY DISTRESS SYNDROME (RDS)
Falsely low ∆A450 - specimens contaminated with meconium ➔ is the most frequent complication of early delivery and is the
- NOT ACCEPTABLE FOR seventh most common cause of morbidity and mortality in the
SPECTROPHOTOMETRIC ANALYSIS premature infant
➔ this disease is caused by an insufficiency of lung surfactant
specimens that are contaminated with blood → GENERALLY UNACCEPTABLE production and structural immaturity of the fetal lungs
★ maximum absorbance of oxyhemoglobin occurs at 410 nm
★ can interfere with the bilirubin absorption peak SURFACTANT
➔ normally appears in mature lungs and allows the alveoli (air sacs
➢ this interference can be removed by extraction with chloroform if necessary
of the lung) to remain open throughout the normal cycle of
inhalation and exhalation
➔ keeps the alveoli from collapsing by decreasing surface tension
NEURAL TUBE DEFECTS
and allows them to inflate with air more easily

➔ are one of the most common birth defects in the United States
 IMMUNOLOGIC AGGLUTINATION TEST for PG
If the surfactant concentrations are too low,
➔ provided a more rapid and easy to perform method for assessment of
the alveoli will collapse, causing RDS
fetal maturity that does not require a laboratory to be equipped to
perform thin-layer chromatography
LECITHIN-SPHINGOMYELIN (L/S) RATIO
Aminostat-FLM (Irving Scientific, Santa Ana. CA)
➔ uses antisera containing polyclonal anti-PG antibodies that are
➔ is the reference method to which tests of FLM are compared specific for PG-containing lamellar bodies in the amniotic fluid

LECITHIN
➔ is the primary component of the surfactants (phospholipids, neutral Size of the agglutinates → read microscopically
lipids, and proteins) that make up the alveolar lining and account for
alveolar stability Results are reported as either:
➔ is produced at a relatively low and constant rate until the 35th week of
LOW POSITIVE or - indicating pulmonary maturity
gestation, at which time a noticeable increase in its production occurs,
HIGH POSITIVE
resulting in the stabilization of the fetal lung alveoli
NEGATIVE - indicating pulmonary immaturity
SPHINGOMYELIN
➔ is a lipid that is produced at a constant rate after about 26 weeks’ ➔ the test is not affected by specimen contamination with
gestation; therefore, it can serve as a control on which to base the blood and meconium
rise in lecithin
➔ Studies have shown good correlation with thin-layer
chromatography but with a slightly higher incidence of
Prior to 35 weeks’ gestation: false-negative results that may need to be followed up with
L/S ratio is usually less than 1.6 further testing
★ large amounts of lecithin are NOT BEING PRODUCED at this time

FOAM STABILITY INDEX

After 35 week’s gestation:
Lecithin concentration increases, Sphingomyelin concentration remains constant
“FOAM” or “SHAKE” TEST
➔ mechanical screening test
L/S ratio will rise to 2.0 or higher ➔ was used to determine the presence of individual lung-surface
★ lecithin production increases to PREVENT ALVEOLAR COLLAPSE lipid concentrations
➔ can be performed at the bedside or in the laboratory, the test is
L/S ratio reaches 2.0 → PRETERM DELIVERY (relatively safe procedure) still in use

Falsely elevated results - encountered in fluid contaminated with PROCESS:

blood or meconium (these substances ⇨ Amniotic fluid is mixed with 95% ethanol,
contain lecithin and sphingomyelin) ⇨ Shaken for 15 seconds
⇨ Allowed to sit undisturbed for 15 minutes
⇨ Surface of the fluid is observed for the presence of a continuous
THIN-LAYER CHROMATOGRAPHY (TLC) line of bubbles around the outside edge
➔ quantitative measurement of lecithin and sphingomyelin

REPLACED THE L/S RATIO WITH THE: Presence of bubbles - indicates that a sufficient amount of
1. Quantitative phosphatidyl glycerol phospholipid is available to reduce the
2. Immunoassays surface tension of the fluid even in the
3. Lamellar body density procedures presence of alcohol, an antifoaming
agent

PHOSPHATIDYL GLYCEROL (PG)

MODIFICATION OF THE FOAM TEST
⇨ uses 0.5 mL of amniotic fluid added to increasing amounts of 95%
➔ presence of another lung surface lipid
ethanol, providing a gradient of ethanol/fluid ratios ranging from
➔ is also essential for adequate lung maturity and can be detected after 0.42 mL to 0.55 mL in 0.01-mL increments, which can be used to
35 weeks’ gestation provide a semiquantitative measure of the amount of surfactant
present

Production of PG normally parallels that of lecithin, but

its production is delayed in cases of maternal diabetes
Value of 47 or higher - indicates FLM

Respiratory distress - occurs in the presence of an L/S ratio of 2.0 FOAM STABILITY INDEX
➔ has shown good correlation with the L/S ratio and tests for
phosphatidyl glycerol
THIN-LAYER CHROMATOGRAPHY LUNG PROFILE must include:
➔ the test cannot be used with contaminated amniotic fluid
➢ Lecithin
because blood and meconium also reduce surface tension, yielding
➢ Sphingomyelin
a falsely mature index result
➢ PG
➔ provide an accurate measurement of FLM
 CONSENSUS PROTOCOL for performing LBC
➔ has been published by CLSI
LAMELLAR BODIES
RESULTS ARE REPORTED IN:
➔ Surfactant is composed of approximately 90% phospholipid and 10% ➢ units of lamellar bodies per microliter
protein and is packaged into layered storage granules ○ should be accompanied by the laboratory’s established
➔ are densely packed layers of phospholipids that represent a storage values for maturity and the instrument that was used
form of pulmonary surfactant
➔ are secreted by the type II pneumocytes of the fetal lung at about 24
weeks of gestation and are absorbed into the alveolar spaces to CONSENSUS PROTOCOL FOR NON CENTRIFUGED SAMPLES:
provide surfactant
➔ enter the amniotic fluid at about 26 weeks of gestation and increase in LBCs greater than 50,000/uL - indication of FLM
concentration from 50,000 to 200,000 per microliter by the end of the
third trimester Values below 15,000/uL - immature

As the fetal lung matures, increased lamellar body production is reflected by an

➔ results in between these two values are considered
increase in amniotic fluid phospholipids and the L/S ratio indeterminate and further testing using alternate
methods is recommended

Number of lamellar bodies present in the amniotic fluid correlates with the amount
of phospholipid present in the fetal lungs

Presence of lamellar bodies - increases the OD of the amniotic fluid

○ Specimens are centrifuged at 2000 g for 10 minutes and

examined using a wavelength of 650 nm, which rules out
interference from hemoglobin but not other contaminants, such as
meconium

OD of 0.150 - has been shown to correlate well with an L/S

ratio of greater than or equal to 2.0 and the
presence of phosphatidylglycerol

LAMELLAR BODY COUNT (LBC)

➔ can be obtained using the platelet channel of automated hematology

analyzers using either optical or impedance methods for counting

Lamellar body diameter is similar to that of small platelets

ranging in size from 1.7 to 7.3 fL, or 1 to 5um

ADVANTAGES OF LAMELLAR BODY COUNTING

1) rapid turnaround time

2) low reagent cost
3) wide availability
4) low degree of technical difficulty
5) low volume of amniotic fluid required
6) excellent clinical performance

○ Amniotic fluid specimens containing whole blood, meconium,

and mucus should NOT BE USED

Presence of platelets - blood initially raises the LBC

Lamellar bodies are trapped - lower the LBC

in the fibrin strands

Meconium and mucus - cause a false increase in the LBC

- SHOULD NOT BE USED FOR TESTING

○ Specimens may be stored at 2°C to 8°C but NEVER FROZEN