AMNIOTIC FLUID
CHAPTER 13
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Learning Objectives
Upon completing this chapter, the reader will be able to
1.
2.
3.
4.
5.
State the functions of amniotic fluid.
Describe the formation and composition of amniotic fluid.
Differentiate maternal urine from amniotic fluid.
State indications for performing an amniocentesis.
Describe the specimen-handling and processing procedures
for testing amniotic fluid for bilirubin, fetal lung maturity
(FLM), and cytogenetic analysis.
6. Discuss the principle of the spectrophotometric analysis for
evaluation of hemolytic disease of the newborn.
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Learning Objectives (cont’d)
7. Interpret a Liley graph.
8. Describe the analysis of amniotic fluid for the detection of
neural tube disorders.
9. Explain the physiologic significance of the lecithinsphingomyelin (L/S) ratio.
10. State the relationship of phosphatidyl glycerol to FLM.
11. Define lamellar bodies and describe their significance to FLM.
12. Discuss the principle of and sources of error for the L/S ratio,
Amniostat-FLM, lamellar body count, and Foam Stability
Index for FLM.
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Physiology
• Amnion: a membranous sac that surrounds the fetus
• Amniotic fluid
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Provides protective cushion for fetus
Allows fetal movement
Stabilizes fetal temperature exposure
Permits proper lung development
Exchanges water and chemicals among the fluid, fetus,
and maternal circulation
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Fetus in Amniotic Sac
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Physiology
• Volume
– Production: fetal urine, lung fluid, and maternal
circulation
– During the first trimester, the approximately 35 mL of
amniotic fluid is derived primarily from the maternal
circulation
– Increased amniotic fluid peak at 800 to 1200 mL in the
third trimester is the result of fetal urine
– Increased urine is regulated by increased fetal swallowing
– Lung fluid adds lung surfactants to amniotic fluid; used as
a measure of lung maturity
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Physiology (cont’d)
• Polyhydramnios
– Excess amniotic fluid from failure of fetus to swallow
>1200 mL
• Neural tube disorders, structural/chromosomal
abnormalities, cardiac arrhythmias, infections
• Oligohydramnios
– Decreased amniotic fluid from increased fetal
swallowing, membrane leakage <800 mL
• Umbilical cord compression
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Chemical Composition
• Composition similar to maternal plasma with sloughed
fetal cells
– Cells are used for cytogenetic analysis
• The fluid also contains biochemical substances that are
produced by the fetus, such as bilirubin, lipids, enzymes,
electrolytes, urea, creatinine, uric acid, proteins, and
hormones
• Fetal urine increases creatinine, urea, and uric acid
• Fetal age can be estimated by creatinine
– <36 weeks = 1.5 to 2.0 mg/dL
– >36 weeks = >2.0
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Maternal Urine versus Amniotic Fluid
• Needed to determine premature membrane rupture or
accidental puncture of maternal bladder from amniocentesis
• Measure creatinine, glucose, protein, and urea
– Amniotic fluid has <3.5 mg/dL creatinine and <30 mg/dL urea
– Values as high as 10 mg/dL for creatinine and 300 mg/dL for urea
may be found in urine
– The presence of glucose, protein, or both is associated more closely
with amniotic fluid
• Fern test: specimen air dries on glass slide; examine
microscopically for “fern-like” amniotic fluid crystals
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Indications for Amniocentesis
• Abnormal screening blood tests: maternal alpha
fetal protein, human chorionic gonadotropin,
unconjugated estriol
• Abnormal chromosome analysis and history of
genetic disorders
• Abnormal ultrasound for fetal body measurements
• In later pregnancy for possible early delivery
– Fetal lung maturity, hemolytic disease of the newborn
(HDN), infection
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Indications for Amniocentesis (cont’d)
• Collection of fetal epithelial cells in amniotic fluid
indicate the genetic material of the fetus
– Examined for chromosome abnormalities by
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Karyotyping
Fluorescence in situ hybridization (FISH)
Fluorescent mapping spectral karyotyping
DNA testing
• Biochemical substances that the fetus has produced
– Analyzed by thin-layer chromatography
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Collection
• Amniocentesis: needle aspiration of fluid from
amniotic sac
– Transabdominal amniocentesis
• Maximum of 30 mL collected in sterile syringes
• Discard first 2 to 3 mL for contamination
• Protect specimens from light for bilirubin analysis
for HDN at all times: amber tubes or black plastic
tube covers
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Specimen Handling
and Processing
• Perform all handling procedures immediately, and
deliver to laboratory promptly
– Amber tubes to protect bilirubin integrity
• Deliver fetal lung maturity (FLM) tests on ice;
refrigerate or freeze up to 72 hours if needed
• Cytogenetic specimens kept at room temperature or
37°C to prolong cell life
• Centrifuge or filter fluid for chemical tests to remove
debris; filter only for FLM tests
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Color and Appearance
• Normal amniotic fluid is colorless, with slight to
moderate turbidity from cells
• Blood streaked: traumatic tap, abdominal
trauma, intra-amniotic hemorrhage
– Fetal versus maternal blood: use Kleihauer-Betke
• Bilirubin: bright yellow
• Meconium (first bowel movement): dark green
• Fetal death: dark red-brown
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Summary of
Amniotic Fluid Color
Color
Significance
Colorless
Normal
Blood streaked
Traumatic tap
Abdominal trauma
Intra-amniotic hemorrhage
Yellow
Hemolytic disease of the newborn
(HDN)
Dark green
Meconium
Dark red-brown
Fetal death
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Tests for Fetal Distress
• HDN
– Most commonly Rh-negative mothers
– Other red blood cell (RBC) antigens can also produce
HDN
– Fetal cells with antigens enter maternal circulation
and cause production of maternal antibodies
– Maternal antibodies cross the placenta and destroy
fetal cells with the corresponding antigen
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Antibodies Crossing the Placenta
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Tests for Fetal Distress
• Bilirubin from RBC destruction appears in the amniotic
fluid
• The amount of unconjugated bilirubin present correlates
with the amount of RBC destruction
• Spectrophotometric analysis of fluid optical density (OD)
between 365 and 550 nm is plotted on semilog paper
• Bilirubin causes OD rise at its maximum absorbance level
of 450 nm; difference between baseline and 450 nm peak
is the ΔA450
• Difference is plotted on a Liley graph
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Spectrophotometric Bilirubin Scan Showing
Bilirubin and Oxyhemoglobin Peaks
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Tests for Fetal Distress
• Liley graph
– Plots ΔA450 against gestational age
– Consists of three zones based on hemolytic severity
– Zone I: mildly affected fetus
– Zone II: requires careful monitoring
– Zone III: severely affected fetus, may require
induction of labor or intrauterine exchange
transfusion
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Liley Graph
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Tests for Fetal Distress
• Neural tube defects
– Alpha-fetoprotein (AFP) produced by the fetal liver
prior to 18 weeks’ gestation
– Increased levels in maternal blood or amniotic fluid
indicate possible anencephaly or spinal bifida
– Increased levels are found when skin fails to close
over neural tissue
– Measure maternal blood first, then amniotic fluid
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Tests for Fetal Distress (cont’d)
• Normal values based on weeks of gestation
(maximum AFP 12 to 15 weeks)
• Report multiples of the median (MoM)
– Median is laboratory’s reference level for a given week of
gestation
– More than two times the median (MOM) is abnormal
• Follow with fluid amniotic acetylcholinesterase
(AChE): more specific for neural disorders
– Do not perform on a bloody specimen
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Fetal Lung Maturity (FLM)
• Most common complication of early delivery is
respiratory distress syndrome (RDS)
• Lack of lung surfactant, which keeps the alveoli
open during inhaling and exhaling
• Surfactant decreases the surface tension on the
alveoli so they can inflate more easily
• Many laboratory tests are available for FLM
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Lecithin-Sphingomyelin
(L/S) Ratio
• Considered the reference method
• Lecithin is the primary component of the lung surfactants; increased
production occurs after the 35th week
• Sphingomyelin is produced at a constant rate after the 26th week and
serves as a control for the rise in lecithin
• L/S ratio is 1.6 prior to week 35 and rises to 2.0 or greater for alveolar
stability after week 35
• Therefore, preterm delivery is considered safe with an L/S ratio of 2.0 or
higher
• Test is performed using thin-layer chromatography
• Many laboratories have replaced the L/S ratio with the quantitative
phosphatidyl glycerol immunoassays and lamellar body density
procedures
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Phosphatidyl Glycerol
• Lung surface lipid phosphatidyl glycerol is also
needed for lung maturity
• Normally parallels lecithin, except in diabetics, so
must be included in L/S ratio
• Amniostat-FLM is an immunologic agglutination test
for PG using antibody specific for phosphatidyl
glycerol that can replace the L/S ratio (no special
equipment needed)
• Blood and meconium do not interfere with the test
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Foam Stability
• Performed at the bedside
• Amniotic fluid is mixed with 95% ethanol, shaken for
15 seconds, and allowed to sit undisturbed for 15
minutes
• A continuous line of bubbles around the outside
edge indicates the presence of a sufficient amount
of surfactant to maintain alveolar stability (alcohol is
an antifoaming agent, and fluid can overcome this)
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Foam Stability Index
• 0.5 mL amniotic fluid added to increasing
amounts of 95% ethanol
• Provides ethanol:fluid ratios of 0.42 mL to 0.55
mL in 0.01 mL increments, giving
semiquantitative measure of surfactant amount
• A value >47 indicates FLM
• Correlates well with L/S ratio and tests for
phosphatidyl glycerol
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Lamellar Bodies
• Lamellar bodies: storage form of surfactant
– Approximately 90% phospholipid and 10% protein
• Secreted by the type II pneumocytes of the fetal
lung to the aveolar space at about 24 weeks of
gestation
• Increase in amniotic concentration from 50,000 to
200,000/mL by the end of the third trimester
• OD of 150 at 650 nm correlates with L/S ratio of 2.0
and the presence of PG
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Lamellar Body Count (LBC)
• Lamellar body diameter ranges in size from 1.7 to 7.3 fL or 1 to 5 µm
• LBCs can be obtained using the platelet channel of automated
hematology analyzers
• Advantages of LBC
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Rapid turnaround time
Low reagent cost
Wide availability
Low degree of technical difficulty
Low volume of amniotic fluid required
Excellent clinical performance
• Specimens contaminated with meconium or mucous cannot be used
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Protocol for Performing LBC
1.
2.
3.
4.
5.
6.
Mix the amniotic fluid sample by inverting the capped sample container five
times.
Transfer the fluid to a clear test tube to allow for visual inspection.
Visually inspect the specimen. Fluids containing obvious mucus, whole
blood, or meconium should not be processed for an LBC.
Cap the tube and mix the sample by gentle inversion or by placing the test
tube on a tube rocker for 2 minutes.
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.
Process the specimen through the cell counter and record the platelet
channel as the LBC.
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