3920_Ch13_243-254 23/01/14 10:36 AM Page 243

CHAPTER 13
Amniotic Fluid
LEARNING OBJECTIVES
Upon completing this chapter, the reader will be able to:
13-1 State the functions of amniotic fluid. 13-8 Describe the analysis of amniotic fluid for the
detection of neural tube disorders.
13-2 Describe the formation and composition of amniotic
fluid. 13-9 Explain the physiologic significance of the lecithin-
sphingomyelin (L/S) ratio.
13-3 Differentiate maternal urine from amniotic fluid.
13-10 State the relationship of phosphatidyl glycerol to
13-4 State indications for performing an amniocentesis.
FLM.
13-5 Describe the specimen-handling and processing
13-11 Define lamellar bodies and describe their significance
procedures for testing amniotic fluid for bilirubin,
to FLM.
fetal lung maturity (FLM), and cytogenetic analysis.
13-12 Discuss the principle of and sources of error for the
13-6 Discuss the principle of the spectrophotometric analy-
L/S ratio, Amniostat-FLM, lamellar body count, and
sis for evaluation of hemolytic disease of the newborn.
Foam Stability Index for FLM.
13-7 Interpret a Liley graph.

KEY TERMS
Amniocentesis Hemolytic disease of the newborn Meconium
Amnion (HDN) Oligohydramnios
Amniotic fluid Lamellar bodies Polyhydramnios
Cytogenetic analysis Lecithin-sphingomyelin ratio Respiratory distress syndrome (RDS)
(L/S ratio)
Fetal lung maturity (FLM) Surfactants
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244 Part Three | Other Body Fluids

Although the testing of amniotic fluid is frequently associated

with cytogenetic analysis, the clinical laboratory also per-
forms several significant tests on amniotic fluid. Because am- Placenta
niotic fluid is a product of fetal metabolism, the constituents
Amniotic cavity
that are present in the fluid provide information about the Chorion
metabolic processes taking place during—as well as the
Umbilical cord
progress of—fetal maturation. When conditions that adversely
affect the fetus arise, the danger to the fetus must be measured Amnion
against the ability of the fetus to survive an early delivery. The
tests covered in this chapter are used to determine the extent
of fetal distress and fetal maturity (Table 13–1).

Physiology Figure 13–1 Fetus in amniotic sac.

Function
from fetal swallowing and intramembranous flow. Intramem-
Amniotic fluid is present in the amnion, a membranous sac branous flow is the absorption of amniotic fluid water and
that surrounds the fetus (Fig. 13–1). The amnion is metaboli- solutes into the fetal vascular system.1 The amount of amniotic
cally active and is involved in the exchanges of water and fluid increases in quantity throughout pregnancy, reaching a
chemicals between the fluid, the fetus, and the maternal cir- peak of approximately 800 to 1200 mL during the third
culation; and produces peptides, growth factors, and cytokines. trimester, and then gradually decreases prior to delivery. An
The primary functions of the amniotic fluid are to provide a amniotic fluid volume greater than 1200 mL is called polyhy-
protective cushion for the fetus, allow fetal movement, stabilize dramnios, whereas amniotic fluid volume less than 800 mL is
the temperature to protect the fetus from extreme temperature termed oligohydramnios. In either case, some analytes will be
changes, and permit proper lung development. falsely low or falsely high.2 During the first trimester, the
Volume approximately 35 mL of amniotic fluid is derived primarily
from the maternal circulation. During the latter third to half of
Amniotic fluid volume is regulated by a balance between the pregnancy, the fetus secretes a volume of lung liquid necessary
production of fetal urine and lung fluid and the absorption 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
Table 13–1 Tests for Fetal Well-Being and Maturity glycerol into the amniotic fluid surrounding the fetus.2 These
lung surfactants serve as an index of fetal lung maturity.1 After
Reference
the first trimester, fetal urine is the major contributor to the
Values at
amniotic fluid volume. At the time that fetal urine production
Test Term7 Significance
occurs, fetal swallowing of the amniotic fluid begins and
Bilirubin scan ⌬ A450 >.025 Hemolytic regulates the increase in fluid from the fetal urine. The fetus
disease of the swallows amniotic fluid, which is absorbed through the gas-
newborn trointestinal tract and reexcreted by the kidneys from the blood
Alpha- <2.0 MoM Neural tube into fetal urine and back into amniotic fluid.2
fetoprotein disorders Failure of the fetus to begin swallowing results in excessive
accumulation of amniotic fluid (polyhydramnios) and is an
Lecithin- ≥ 2.0 Fetal lung
indication of fetal distress, often associated with neural tube dis-
sphingomyelin maturity
orders. Polyhydramnios may be secondarily associated with fetal
ratio
structural anomalies, cardiac arrhythmias, congenital infections,
Amniostat- Positive Fetal lung or chromosomal abnormalities.1 Increased fetal swallowing, uri-
fetal lung maturity/phos- nary tract deformities, and membrane leakage are possible causes
maturity phatidyl glycerol of decreased amniotic fluid (oligohydramnios). Oligohydramnios
Foam Stability ≥ 47 Fetal lung maturity may be associated with congenital malformations, premature rup-
Index ture of amniotic membranes, and umbilical cord compression,
Optical density ≥ 0.150 Fetal lung maturity resulting in decelerated heart rate and fetal death.1
650 nm
Chemical Composition
Lamellar body ≥ 32,000/mL Fetal lung maturity
count The placenta is the ultimate source of amniotic fluid water and
solutes. Amniotic fluid has a composition similar to that of the
3920_Ch13_243-254 23/01/14 10:36 AM Page 245
maternal plasma and contains a small amount of sloughed fetal
cells from the skin, digestive system, and urinary tract. These
cells provide the basis for cytogenetic analysis. The fluid also
contains biochemical substances that are produced by the
fetus, such as bilirubin, lipids, enzymes, electrolytes, urea, cre-
atinine, uric acid, proteins, and hormones that 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. Alpha-fetoprotein and acetylcholinesterase are
two biochemical markers tested for these defects.2
A portion of the fluid arises from the fetal respiratory tract,
fetal urine, the amniotic membrane, and the umbilical cord.
As would be expected, the chemical composition of the amni-
otic fluid changes when fetal urine production begins. The con-
centrations of creatinine, urea, and uric acid increase, whereas
glucose and protein concentrations decrease. Concentrations
of electrolytes, enzymes, hormones, and metabolic end prod-
ucts also vary but are of little clinical significance. 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. It then rises above
2.0 mg/dL, thereby providing a means of determining fetal age
greater than 36 weeks.3
Differentiating Maternal Urine From
Amniotic Fluid
Differentiation between amniotic fluid and maternal urine may
be necessary to determine possible premature membrane rup-
ture or accidental puncture of the maternal bladder during
specimen collection. Chemical analysis of creatinine, urea, glu-
cose, and protein aids in the differentiation. Levels of creatinine
and urea are much lower in amniotic fluid than in urine. Cre-
atinine does not exceed 3.5 mg/dL and urea does not exceed
30 mg/dL in amniotic fluid, whereas values as high as
10 mg/dL for creatinine and 300 mg/dL for urea may be found
in urine.4 Measurement of glucose and protein by a reagent
strip is a less reliable indicator, because glucose and protein
are not uncommon urine constituents during pregnancy. How-
ever, under normal circumstances, the presence of glucose,
protein, or both is associated more closely with amniotic fluid.
The fern test also can differentiate amniotic fluid from
urine and other body fluids. It is a test used to evaluate pre-
mature rupture of the membranes. In the fern test, a vaginal
fluid specimen is spread on a glass slide and allowed to com-
pletely air dry at room temperature; then it is observed micro-
scopically. The presence of “fern-like” crystals due to the
protein and sodium chloride content is a positive screen for
amniotic fluid.5
Specimen Collection
Indications for Amniocentesis
Amniocentesis is recommended for neural tube defects
when screening blood tests such as the maternal serum alpha-
fetoprotein test are abnormal or to detect genetic disorders or
Chapter 13 | Amniotic Fluid 245
to evaluate the health of the fetus. Fetal body measurements
taken with ultrasonography accurately estimate the gestational
age of the fetus and provide an assessment of the size and
growth of the fetus throughout pregnancy to diagnose and
manage intrauterine growth retardation. Finding an abnormal-
ity on the ultrasound could indicate potential fetal develop-
ment problems and indicate the need for an amniocentesis and
laboratory measurements of fetal lung maturity.
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 by
karyotyping, fluorescence in situ hybridization (FISH), fluo-
rescent mapping spectral karyotyping (SKY), and DNA testing.
Biochemical substances produced by the fetus can be analyzed
by thin-layer chromatography to evaluate the health of the
fetus. (Table 13–2)
In general, amniocentesis is a safe procedure, particularly
when performed after the 14th week of gestation. Fluid for
chromosome analysis is usually collected at approximately
16 weeks’ gestation and tests for intrauterine growth retarda-
tion are performed near the end of the second trimester,
whereas tests for fetal distress and maturity are performed later
in the third trimester.2
Table 13–2 Indications for Performing Amniocentesis
Amniocentesis may be indicated at 15 to 18 weeks’
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 defect
• Parent is a carrier of a metabolic disorder
• Family history of genetic diseases such a sickle cell dis-
ease, Tay-Sachs disease, hemophilia, muscular dystrophy,
sickle cell anemia, Huntington chorea, and cystic fibrosis
• Elevated maternal serum alpha-fetoprotein
• Abnormal triple marker screening test
• Previous child with a neural tube disorder such as spina
bifida, or ventral wall defects (gastroschisis)
• Three or more miscarriages
Amniocentesis is indicated later in the pregnancy
(20 to 42 weeks) to evaluate:
• Fetal lung maturity
• Fetal distress
• HDN caused by Rh blood type incompatibility
• Infection
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246 Part Three | Other Body Fluids
Collection
Amniotic fluid is obtained by needle aspiration into the am-
niotic sac, a procedure called amniocentesis. The procedure
most frequently performed is a transabdominal amniocente-
sis. Using continuous ultrasound for guidance, the physician
locates the fetus and placenta to safely perform the procedure.
A thin, hollow needle is inserted through the mother’s ab-
domen into the mother’s uterus and into the amniotic sac to
aspirate the amniotic fluid. Vaginal amniocentesis may also
be performed; however, this method carries a greater risk of
infection.
A maximum of 30 mL of amniotic fluid is collected in ster-
ile syringes. The first 2 or 3 mL collected can be contaminated
by maternal blood, tissue fluid, and cells and are discarded.
Specimens should be transferred to sterile plastic containers
and taken immediately to the laboratory. Fluid for bilirubin
analysis in cases of hemolytic disease of the newborn (HDN)
must be 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. However, in all circumstances, special han-
dling 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. Specimens for bilirubin
testing must be immediately protected from light. This can be
accomplished by placing the specimens in amber-colored
tubes, wrapping the collection tube in foil, or by use of a black
plastic cover for the specimen container. Specimens for cyto-
genetic studies or microbial studies must be processed asepti-
cally and maintained at room temperature or body temperature
(37°C incubation) prior to analysis to prolong the life of the
cells needed for analysis.
All fluid for chemical testing should be separated from
cellular elements and debris as soon as possible to prevent
distortion of chemical constituents by cellular metabolism or
disintegration. This can be performed using centrifugation or
filtration. The time and speed of centrifugation also depends
on the test and lab protocol.
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 may be pres-
ent as the result of a traumatic tap, abdominal trauma, or intra-
amniotic hemorrhage. The source of the blood (maternal or
fetal) can be determined using the Kleihauer-Betke test for fetal
hemoglobin and is important for further case management.
The presence of bilirubin gives the fluid a yellow color and
is indicative of red blood cell destruction resulting from HDN.
Meconium, which is usually defined as a newborn’s first bowel
movement, is formed in the intestine from fetal intestinal
secretions and swallowed amniotic fluid. It is a dark green,
mucus-like material. It may be present in the amniotic fluid as
a result of fetal distress. Fetal aspiration of meconium during
fetal swallowing is a concern when increased amounts are pres-
ent in the fluid. A very dark red-brown fluid is associated with
fetal death (Table 13–3).
Tests for Fetal Distress
Hemolytic Disease of the Newborn
The oldest routinely performed laboratory test on amniotic
fluid evaluates the severity of the fetal anemia produced by
HDN. The incidence of this disease has been decreasing rapidly
since the development of methods to prevent anti-Rh antibody
production in postpartum mothers. However, antibodies
against other red cell antigens are also capable of producing
HDN, and immunization of Rh-negative mothers may not be
effective or even performed in all cases. Initial exposure to for-
eign red cell antigens occurs during gestation, delivery of the
placenta, or a previous pregnancy when fetal red blood cells
enter into the maternal circulation and stimulate the mother
to produce antibodies to the antigen. 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. The destruction of fetal red blood cells
results in the appearance of the red blood cell degradation
product, unconjugated bilirubin, in the amniotic fluid. By
measuring the amount of bilirubin in the fluid, the extent of
hemolysis taking place may be determined, and the danger this
anemia presents to the fetus may be assessed (Fig. 13–2).
Amniotic fluid bilirubin is measured by spectrophotometric
analysis using serial dilutions. As illustrated in Figure 13–3, the
optical density (OD) of the fluid is measured in intervals be-
tween 365 nm and 550 nm and the readings 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. The difference between the OD of the theoretic
baseline and the OD at 450 nm represents the amniotic fluid
Table 13–3 Amniotic Fluid Color
Color Significance
Colorless Normal
Blood-streaked Traumatic tap, abdominal trauma,
intra-amniotic hemorrhage
Yellow Hemolytic disease of the newborn
(bilirubin)
Dark green Meconium
Dark red-brown Fetal death
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Chapter 13 | Amniotic Fluid 247

Rh– Rh– Rh–

Baby 1 Rh Baby 2 Rh+

Rh+ antibody

1st Pregnancy 2nd Pregnancy

Rh+ fetus Rh+ fetus

Fetal blood enters IgG antibodies

maternal circulation cross placenta
via placenta to destroy
fetal Rh+ RBCs
IgG antibodies remain
Figure 13–2 Rh antibodies crossing the placenta. in mother’s circulation

As mentioned, specimens must be protected from light at

0.6 Oxyhemoglobin Bilirubin
peak at 410 peak at 450 all times. Markedly decreased values will be obtained with as
0.5 little as 30 minutes of exposure to light. Contamination of the
fluid by cells, hemoglobin, meconium, or other debris will in-
0.4
Absorbance

terfere with the spectrophotometric analysis. Specimens should

0.3 No be immediately centrifuged to remove particulate interference.
rm
sca al Specimens contaminated with meconium will cause falsely low
0.2 n
⌬ A450 values and are not acceptable for spectrophotometric
0.1 analysis. Specimens that are contaminated with blood are gen-
erally unacceptable because maximum absorbance of oxyhe-
moglobin occurs at 410 nm and can interfere with the bilirubin
365 410 450 500 550 absorption peak (see Fig. 13–3). This interference can be re-
Wavelength (nm) moved by extraction with chloroform if necessary.7 A control
may be prepared by diluting commercial chemistry control sera
Figure 13–3 Spectrophotometric bilirubin scan showing bilirubin
and oxyhemoglobin peaks.
1 to 10 with normal saline and treating it in the same manner
as the patient specimen. Bilirubin and protein levels approxi-
mate those in amniotic fluid and can be varied by using low
or high control sera.8
bilirubin concentration. This difference in OD, referred to as
the absorbance difference at 450 nm (⌬ A450), is then plotted
on a Liley graph to determine the severity of the hemolytic
Neural Tube Defects
disease (Fig. 13–4).6 Neural tube defects (NTD) are one of the most common birth
Notice that the Liley graph plots the ⌬ A450 against gesta- defects in the United States. It can be detected by maternal
tional age and is divided into three zones that represent the serum alpha-fetoprotein (MSAFP) blood test, high-resolution
extent of hemolytic severity. Values falling in zone I indicate ultrasound, and amniocentesis. Increased levels of alpha-feto-
no more than a mildly affected fetus; those in zone II indicate protein (AFP) in both the maternal circulation and the amniotic
moderate hemolysis and require careful monitoring anticipat- fluid can be indicative of fetal neural tube defects, such as anen-
ing an early delivery or exchange transfusion upon delivery, cephaly and spina bifida. AFP is the major protein produced by
whereas a value in zone III indicates severe hemolysis and sug- the fetal liver during early gestation (prior to 18 weeks). It is
gests a severely affected fetus. Intervention through induction found in the maternal serum due to the combined fetal-maternal
of labor or intrauterine exchange transfusion must be consid- circulations and in the amniotic fluid from diffusion and excre-
ered when a ⌬ A450 is plotted in zone III. tion of fetal urine. Increased levels are found in the maternal
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248 Part Three | Other Body Fluids

1.00
0.90
0.80
0.70
0.60
0.50
0.40
Severely affected fetus
Zone III intervention required
0.30
∆ O.D. Change in optical density

0.20

Zone II
0.10
0.09
0.08 Moderately affected fetus
0.07 requiring close monitoring
0.06
0.05
0.04

0.03
Zone I
0.02
Nonaffected or mildly
affected fetus
0.01
20 22 24 26 28 30 32 34 36 38 40
Gestational age in weeks Figure 13–4 Example of a Liley graph.

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. The possibility of a mul-
tiple pregnancy also must be investigated when serum levels
are elevated. Normal values are based on the week of gesta-
tional age, as the fetus produces maximal AFP between 12 and
15 weeks’ gestation, after which levels in amniotic fluid begin
to decline. 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 value is considered
abnormal (greater than 2 MoM) for both maternal serum and
amniotic fluid. Elevated amniotic fluid AFP levels are followed
by measurement of amniotic acetylcholinesterase (AChE).
The test is more specific for neural tube disorders than AFP,
provided it is not performed on a bloody specimen, because
blood contains AChE.4
Tests for Fetal Maturity
Fetal distress, whether caused by HDN or other conditions,
forces the obstetrician to consider a preterm delivery. At this
point, fetal maturity must be assessed.
Fetal Lung Maturity
Respiratory distress syndrome (RDS) is the most frequent
complication of early delivery and is the seventh most common
cause of morbidity and mortality in the premature infant.9
This disease is caused by an insufficiency of lung surfactant
production and structural immaturity of the fetal lungs. Sur-
factant normally appears in mature lungs and allows the alveoli
(air sacs of the lung) to remain open throughout the normal
cycle of inhalation and exhalation. Surfactant keeps the alveoli
from collapsing by decreasing surface tension and allows them
to inflate with air more easily. If the surfactant concentrations
are too low, the alveoli will collapse, causing RDS. The inci-
dence of RDS decreases with increasing gestational age and
lung maturity.9 Therefore, 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.9 Several laboratory
tests are available to measure FLM.
Lecithin-Sphingomyelin Ratio
The reference method to which tests of FLM are compared is
the lecithin-sphingomyelin (L/S) ratio. Lecithin is the pri-
mary component of the surfactants (phospholipids, neutral
lipids, and proteins) that make up the alveolar lining and
account for alveolar stability.
Lecithin is produced at a relatively low and constant rate
until the 35th week of gestation, at which time a noticeable
increase in its production occurs, resulting in the stabilization
of the fetal lung alveoli. Sphingomyelin is a lipid that is pro-
duced at a constant rate after about 26 weeks’ gestation; there-
fore, it can serve as a control on which to base the rise in
lecithin. Both lecithin and sphingomyelin appear in the am-
niotic fluid in amounts proportional to their concentrations
in the fetus.10 Prior to 35 weeks’ gestation, the L/S ratio is usu-
ally less than 1.6 because large amounts of lecithin are not
being produced at this time. After 35 weeks’ gestation, the
3920_Ch13_243-254 23/01/14 10:37 AM Page 249
lecithin concentration increases while the sphingomyelin con-
centration remains constant. The L/S ratio will rise to 2.0 or
higher as the lecithin production increases to prevent alveolar
collapse. Therefore, when the L/S ratio reaches 2.0, a preterm
delivery is usually considered to be a relatively safe procedure.
Falsely elevated results are encountered in fluid contaminated
with blood or meconium because both these substances con-
tain lecithin and sphingomyelin.
Quantitative measurement of lecithin and sphingomyelin
is performed using thin-layer chromatography (TLC). Because
the procedure is labor intensive and subject to high coefficients
of variation, many laboratories have replaced the L/S ratio with
the quantitative phosphatidyl glycerol immunoassays and
lamellar body density procedures.11
Phosphatidyl Glycerol
The presence of another lung surface lipid, phosphatidyl glyc-
erol (PG), is also essential for adequate lung maturity and can
be detected after 35 weeks’ gestation. The production of PG
normally parallels that of lecithin, but its production is delayed
in cases of maternal diabetes. In this circumstance, respiratory
distress occurs in the presence of an L/S ratio of 2.0. Therefore,
a thin-layer chromatography lung profile must include lecithin,
sphingomyelin, and PG to provide an accurate measurement
of FLM.12
Development of an immunologic agglutination test for PG
has provided a more rapid and easy to perform method for as-
sessment of fetal maturity that does not require a laboratory to
be equipped to perform thin-layer chromatography. The
Aminostat-FLM (Irving Scientific, Santa Ana, CA) uses antisera
containing polyclonal anti-PG antibodies that are specific for
PG-containing lamellar bodies in the amniotic fluid. The size
of the agglutinates is read macroscopically and the results are
reported as either negative, indicating pulmonary immaturity,
or low positive or high positive, indicating pulmonary maturity.
The test is not affected by specimen contamination with blood
and meconium.13 Studies have shown good correlation with
thin-layer chromatography but with a slightly higher incidence
of false-negative results that may need to be followed up with
further testing.14,15
Foam Stability Index
Until the development of biochemical techniques to measure
the individual lung-surface lipid concentrations, a mechanical
screening test, called the “foam” or “shake” test, was used to
determine their presence. Because it can be performed at the
bedside or in the laboratory, the test is still in use. Amniotic
fluid is mixed with 95% ethanol, shaken for 15 seconds, and
allowed to sit undisturbed for 15 minutes. At the end of this
time, the surface of the fluid is observed for the presence of a
continuous line of bubbles around the outside edge. The pres-
ence of bubbles indicates that a sufficient amount of phospho-
lipid is available to reduce the surface tension of the fluid even
in the presence of alcohol, an antifoaming agent.
A modification of the foam test uses 0.5 mL of amniotic
fluid added to increasing amounts of 95% ethanol, providing a
Chapter 13 | Amniotic Fluid 249
gradient of ethanol/fluid ratios ranging from 0.42 mL to 0.55 mL
in 0.01-mL increments, which can be used to provide a semi-
quantitative measure of the amount of surfactant present. A value
of 47 or higher indicates FLM. The Foam Stability Index has
shown good correlation with the L/S ratio and tests for phos-
phatidyl glycerol. The test cannot be used with contaminated am-
niotic fluid because blood and meconium also reduce surface
tension, yielding a falsely mature index result.
Lamellar Bodies
Surfactant is composed of approximately 90% phospholipid
and 10% protein and is packaged into layered storage granules
called lamellar bodies.9,18 Lamellar bodies are densely packed
layers of phospholipids that represent a storage form of pul-
monary surfactant. They are secreted by the type II pneumo-
cytes of the fetal lung at about 24 weeks of gestation and are
absorbed into the alveolar spaces to provide surfactant. They
enter the amniotic fluid at about 26 weeks of gestation and
increase in concentration from 50,000 to 200,000 per micro-
liter by the end of the third trimester. As the fetal lung ma-
tures, increased lamellar body production is reflected by an
increase in amniotic fluid phospholipids and the L/S ratio.19
Therefore, the number of lamellar bodies present in the am-
niotic fluid correlates with the amount of phospholipid pres-
ent in the fetal lungs.
The 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
PROCEDURE 13-1
Foam Shake Test
1. Mix equal parts of amniotic fluid with 95% ethanol.
2. Vigorously shake for 15 seconds.
3. Allow to sit undisturbed for 15 minutes.
4. Observe for the presence of a continuous line of
bubbles around the outside edge.
PROCEDURE 13-2
Foam Stability Index
1. Add 0.5 mL of amniotic fluid to tubes containing
increasing amounts of 95% ethanol ranging from
0.42 to 0.55 mL in 0.01-mL increments.
2. Vigorously shake for 15 seconds.
3. Allow to sit undisturbed for 15 minutes.
4. Observe for the presence of a continuous line of
bubbles around the outside edge.
5. Values ≥ 47 indicate fetal lung maturity.
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250 Part Three | Other Body Fluids
HISTORICAL NOTE
Microviscosity: Fluorescence
Polarization Assay
The fluorescence polarization assay is no longer performed
because the manufacturer (Abbott Laboratories, Abbott
Park, IL) discontinued the instrument and reagents in
2011. It was the most widely used test in evaluating FLM
and was considered an excellent screening tool for FLM
because the results correlated well with an L/S ratio of
2.0.16 The fluorescence polarization assay for FLM meas-
ured the surfactant to albumin (S/A) ratio to measure FLM.
The test was based on the principle that phospholipids de-
crease the microviscosity of the amniotic fluid. This
change in microviscosity was measured using the principle
of fluorescence polarization using the TDX/TDxFLx FLM
II Assay System (Abbott Laboratories, Abbott Park, IL).
The assay measured the polarization of a fluorescent dye
that combined with both surfactants and albumin in the
amniotic fluid. The fluorescent dye bound to surfactant
had a longer fluorescence lifetime and a low polarization,
whereas dye bound to albumin had a decreased fluores-
cence lifetime and a high polarization. The recorded
changes in polarization produced a surfactant/albumin
ratio expressed in milligrams surfactant to grams albumin
that was compared with a fetal lung maturity II assay
calibration standard curve using calibrators of known sur-
factant/albumin content. A surfactant/albumin ratio of
55 mg/g indicated FLM, whereas values between 40 mg/g
and 54 mg/g were indeterminate and ratios less than or
equal to 39 mg/g indicated immature lungs.17
The test required 1.0 mL of amniotic fluid, and speci-
mens contaminated with blood, meconium, suspected ma-
ternal urine, and visibly icteric sample could not be used.
contaminants, such as meconium. An 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 phosphatidyl glycerol.20
Lamellar Body Count
Lamellar body diameter is similar to that of small platelets
ranging in size from 1.7 to 7.3 fL, or 1 to 5␮m; therefore,
lamellar body counts (LBCs) can be obtained using the
platelet channel of automated hematology analyzers using ei-
ther optical or impedance methods for counting.18 Some au-
tomated cell counters use both impedance and optical
methods for counting. Because the various hematology ana-
lyzers count lamellar bodies differently and require different
specimen preparation, cutoff values for FLM vary, making it
necessary to establish analyzer-specific LBC clinical decision
limits.18 The advantages of lamellar body counting include:
(1) rapid turnaround time, (2) low reagent cost, (3) wide
availability, (4) low degree of technical difficulty, (5) low
volume of amniotic fluid required, and (6) excellent clinical
performance.18
Amniotic fluid specimens containing whole blood, meco-
nium, 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.18 Meconium and mucus cause a false increase in the
LBC and should not be used for testing. Specimens may be
stored at 2°C to 8°C but never frozen. A consensus protocol
for performing LBC has been published by CLSI and is de-
scribed in Procedure 13–3. Results are reported in units of
lamellar bodies per microliter and should be accompanied by
the laboratory’s established values for maturity and the instru-
ment that was used. A consensus protocol for noncentrifuged
samples considers LBCs greater than 50,000/␮L an indication
of FLM and values below 15,000/␮L as immature. Results in
between these two values are considered indeterminate and
further testing using alternate methods is recommended.21
PROCEDURE 13-3
Lamellar Body Count18
1. Mix the amniotic fluid sample by inverting the
capped sample container five times.
2. Transfer the fluid to a clear test tube to allow for
visual inspection.
3. Visually inspect the specimen. Fluids containing
obvious mucus, whole blood, or meconium should
not be processed for an LBC.
4. Cap the tube and mix the sample by gentle inversion or
by placing the test tube on a tube rocker for 2 minutes.
5. Flush the platelet channel; analyze the instrument’s
diluents buffer until a background count deemed
acceptable by the laboratory is obtained in two
consecutive analyses.
6. Process the specimen through the cell counter and
record the platelet channel as the LBC.
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References
1. Ross, MG, Brace, RA, and the NIH Workshop Participants,
National Institute of Child Health and Development Conference
summary: Amniotic fluid biology—basic and clinical aspects.
J Maternal-Fetal Med 10:2–19, 2001.
2. Clinical and Laboratory Standards Institute (CLSI): Analysis of
Body Fluids in Clinical Chemistry: approved guideline, CLSI
Document C49-A, Wayne, PA, 2007, CLSI.
3. Weiss, RR, et al: Amniotic fluid uric acid and creatinine as meas-
ures of fetal maturity. Obstet Gynecol 44(2):208–214, 1974.
3920_Ch13_243-254 23/01/14 10:37 AM Page 251
4. Wenk, RE, and Rosenbaum, JM: Examination of amniotic fluid.
In Henry, JB (ed): Clinical Diagnosis and Management by
Laboratory Methods. WB Saunders, Philadelphia, 1996.
5. Heron, HJ: The use of the Fern test to differentiate amniotic
fluid from urine. Triangle Jul:20:60–62, 1963.
6. Liley, AW: Liquor amnii analysis in the management of the
pregnancy complicated by Rhesus sensitization. Am J Obstet
Gynecol 82:1359, 1961.
7. Spinnato, JA, et al: Amniotic bilirubin and fetal hemolytic
disease. Am J Obstet Gynecol 165(4):1030–1035, 1991.
8. McDonald, OL, and Watts, MT: Use of commercially prepared
control sera as quality control materials for spectrophotometric
bilirubin determinations in amniotic fluid. Am J Clin Pathol
84(4):513–517, 1985.
9. Grenache, D: Fetal lung maturity testing: what labs need to
know now. Medical Lab Observer (MLO), February 2012.
10. Gluck, L, et al: Diagnosis of the respiratory distress syndrome
by amniocentesis. Am J Obstet Gynecol 109(3):440–445, 1971.
11. Dubin, SB: Assessment of FLM: Practice parameter. Am J Clin
Pathol 110:723–732, 1998.
12. Kulovich, MV, Hallman, MB, and Gluck, L: The lung profile:
Normal pregnancy. Am J Obstet Gynecol 135:57–60, 1979.
13. Eisenbrey, AB, et al: Phosphatidyl glycerol in amniotic fluid:
Comparison of an “ultrasensitive” immunologic assay with TLC
and enzymatic assay. Am J Clin Pathol 91(3):293–297, 1989.
Study Questions
1. Which of the following is not a function of amniotic fluid?
A. Allows movement of the fetus
B. Allows carbon dioxide and oxygen exchange
C. Protects fetus from extreme temperature changes
D. Acts as a protective cushion for the fetus
2. What is the primary cause of the normal increase in amni-
otic fluid as a pregnancy progresses?
A. Fetal cell metabolism
B. Fetal swallowing
C. Fetal urine
D. Transfer of water across the placenta
3. Which of the following is not a reason for decreased
amounts of amniotic fluid?
A. Fetal failure to begin swallowing
B. Increased fetal swallowing
C. Membrane leakage
D. Urinary tract defects
4. Why might a creatinine level be requested on an amniotic
fluid?
A. Detect oligohydramnios
B. Detect polyhydramnios
C. Differentiate amniotic fluid from maternal urine
D. Evaluate lung maturity
Chapter 13 | Amniotic Fluid 251
14. Chapman, JF: Current methods for evaluating FLM. Lab
Med 17(10):597–602, 1986.
15. Saad, SA, et al: The reliability and clinical use of a rapid
phosphatidyl glycerol assay in normal and diabetic pregnan-
cies. Am J Obstet Gynecol 157(6):1516–1520, 1987.
16. Winn-McMillan, T, and Karon, BS: Comparison of the
TDx-FLM II and lecithin to sphingomyelin ratio assays in
predicting fetal lung maturity. Am J Obstet Gynecol
193,778–782, 2005.
17. TDx/TDxFLx Fetal Lung Maturity II (FLM II) package insert.
Reference 7A76. Abbott Laboratories, Diagnostics Division,
Abbott Park, IL, April 2003.
18. Clinical and Laboratory Standards Institute. Assessment of fetal
lung maturity by the lamellar body count; approved guideline,
CLSI document C58-A. Wayne, PA, CLSI, 2011.
19. Khazardoost, S, et al: Amniotic fluid lamellar body count and
its sensitivity and specificity in evaluating of fetal lung maturity.
J Obstet Gynaecol 25(3):257–259, 2005.
20. Sbarra, AJ, et al: Correlation of amniotic fluid optical density at
650 nm and lecithin/sphingomyelin ratios. Obstet Gynecol
48:613, 1976.
21. Lu Ji, Gronowski, AM, Eby, C,: Lamellar Body Counts Performed
on Automated Hematology Analyzers to Assess Fetal Lung Matu-
rity. LabMedicine 39(7): 419–423, 2008.
5. Amniotic fluid specimens are placed in amber-colored tubes
prior to sending them to the laboratory to prevent the
destruction of:
A. Alpha-fetoprotein
B. Bilirubin
C. Cells for cytogenetics
D. Lecithin
6. How are specimens for FLM testing delivered to and stored
in the laboratory?
A. Delivered on ice and refrigerated
B. Immediately centrifuged
C. Kept at room temperature
D. Delivered in a vacuum tube
7. Why are amniotic specimens for cytogenetic analysis incu-
bated at 37°C prior to analysis?
A. To detect the presence of meconium
B. To differentiate amniotic fluid from urine
C. To prevent photo-oxidation of bilirubin to biliverdin
D. To prolong fetal cell viability and integrity
8. Match the following colors in amniotic fluid with their
significance.
___ A. Colorless 1. Fetal death
___ B. Dark green 2. Normal
___ C. Red-brown 3. Presence of bilirubin
___ D. Yellow 4. Presence of meconium
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252 Part Three | Other Body Fluids

9. A significant rise in the OD of amniotic fluid at 450 nm
indicates the presence of which analyte?
A. Bilirubin
B. Lecithin
C. Oxyhemoglobin
D. Sphingomyelin
10. Plotting the amniotic fluid OD on a Liley graph represents
the severity of hemolytic disease of the newborn. A value
that is plotted in zone II indicates what condition of the
fetus?
A. No hemolysis
B. Mildly affected fetus
C. Moderately affected fetus that requires close monitoring
D. Severely affected fetus that requires intervention
11. The presence of a fetal neural tube disorder may be
detected by:
A. Increased amniotic fluid bilirubin
B. Increased maternal serum alpha-fetoprotein
C. Decreased amniotic fluid phosphatidyl glycerol
D. Decreased maternal serum acetylcholinesterase
12. True or False: An AFP MoM value greater than two times
the median value is considered an indication of a neural
tube disorder.
13. When severe HDN is present, which of the following tests
on the amniotic fluid would the physician not order to
determine whether the fetal lungs are mature enough to
withstand a premature delivery?
A. AFP levels
B. Foam stability index
C. Lecithin/sphingomyelin ratio
D. Phosphatidyl glycerol detection
14. True or False: Prior to 35 weeks’ gestation, the normal L/S
ratio is less than 1.6.
15. When performing an L/S ratio by thin-layer chromatography,
a mature fetal lung will show:
A. Sphingomyelin twice as concentrated as lecithin
B. No sphingomyelin
C. Lecithin twice as concentrated as sphingomyelin
D. Equal concentrations of lecithin and sphingomyelin
16. True or False: Phosphatidyl glycerol is present with an L/S
ratio of 1.1.
17. A rapid immunologic test for FLM that does not require
performance of thin-layer chromatography is:
A. AFP levels
B. Amniotic acetylcholinesterase
C. Aminostat-FLM
D. Bilirubin scan
18. Does the failure to produce bubbles in the Foam Stability
Index indicate increased or decreased lecithin?
A. Increased
B. Decreased
19. The presence of phosphatidyl glycerol in amniotic fluid
fetal lung maturity tests must be confirmed when:
A. Hemolytic disease of the newborn is present
B. The mother has maternal diabetes
C. Amniotic fluid is contaminated by hemoglobin
D. Neural tube disorder is suspected
20. A lamellar body count of 50,000 correlates with:
A. Absent phosphatidyl glycerol and L/S ratio of 1.0
B. L/S ratio of 1.5 and absent phosphatidyl glycerol
C. OD at 650 nm of 1.010 and an L/S ratio of 1.1
D. OD at 650 nm of 0.150 and an L/S ratio of 2.0

Case Studies and Clinical Situations

1. Amniocentesis is performed on a woman believed to be c. What information did this test provide for the physician?
in approximately the 31st week of gestation. This is the d. Why did the physician prefer an Aminostat-FLM to an
second pregnancy for this Rh-negative woman with dia- L/S ratio in this situation?
betes. Spectrophotometric analysis of the fluid shows a
2. Amniocentesis is performed following a maternal serum
∆ A450 of 0.3.
AFP level of 2.2 MoM at 15 weeks’ gestation.
a. Based on the Liley graph, should the physician
a. What fetal condition is suspected?
consider inducing labor?
b. If the amniotic fluid AFP is 2.5 MoM, what additional
b. What else must the physician consider prior to inducing test could be performed?
labor?
c. In what situation would this additional test not be
The physician decides to induce labor based on a performed?
positive Aminostat-FLM.
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Chapter 13 | Amniotic Fluid 253

3. How might a dark green amniotic fluid affect the results 5. Amniocentesis is performed on a woman whose last two
of the following tests? pregnancies resulted in stillbirths due to hemolytic disease
a. Foam Stability Index of the newborn. A screening test performed at the hospital
is positive for bilirubin, and the specimen is sent to a
b. L/S ratio
reference laboratory for a bilirubin scan. Physicians are
c. Aminostat-FLM concerned when the report comes back negative. What
d. OD650 factors would be considered in evaluating this result.
4. How might a blood-streaked amniotic fluid affect the a. Correct specimen was sent
results of the following tests? b. Specimen was refrigerated
a. L/S ratio c. Specimen was exposed to light
b. AChE d. Specimen reached the reference lab within 30 mins
c. Bilirubin analysis
d. Aminostat-FLM