amnoitic Fluid Analyis.pptx

Chapter nine
Amniotic Fluid
Sinte.ambachew@gmail.com 1

Chapter outline
• Introduction to amniotic fluid
• Routine laboratory assays
Collection of sample
Physical examination
Chemical analysis

Learning Objectives
Upon completion of this chapter the student will be able to:
1. State the functions of amniotic fluid
2. Describe the formation and composition of amniotic fluid
3. State indications for performing an amniocentesis
4. Describe the specimen-handling and processing procedures
for testing amniotic fluid for HDN, fetal lung maturity, and
cytogenetic analysis
5. Discuss the principle of the spectrophotometric analysis for
evaluation of hemolytic disease of the newborn
6. Interpret a Liley graph

Cont’….
7. Describe the analysis of amniotic fluid for the detection of
neural tube disorders
8. Explain the physiologic significance of the lecithin-
sphingomyelin (L/S) ratio
9. State the relationship of phosphatidyl glycerol to FLM
10. Discuss the principle of and sources of error for the L/S ratio,
Amniostat-FLM, Foam Stability Index, and microviscosity tests
for FLM
11. Describe the relationship of lamellar bodies to FLM and the
laboratory tests performed

Amniotic Fluid
• It is liquid that surrounds the fetus in the amniotic cavity
(amnion)
• Amniotic fluid
– Provides protective cushion for fetus
– Allow room for fetal movement, growth and development
– Stabilizes fetal temperature
– Permits proper lung development
– Exchanges water and chemicals among the fluid, fetus, and
maternal circulation
– Contains antibacterial activity
– Aids dilatation of the cervix during labour

Fetus in Amniotic Sac

Volume
Production: fetal urine, lung fluid, and maternal circulation
Absorption: from fetal swallowing and intramembranous flow
During the first trimester, 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
Lung fluid adds lung surfactants to amniotic fluid; used as a
measure of lung maturity
Physiology

• 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 problems
Cont’……

• 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, hormones, etc
 Tested to determine the health or maturity of the fetus
 Fetal age can be estimated by creatinine
– Fetal urine increases creatinine, urea, and uric acid
<36 weeks = 1.5 to 2.0 mg/dL
>36 weeks = >2.0 mg/dL
Chemical Composition

• Abnormal screening blood tests: maternal alpha fetal protein,
human chorionic gonadotropin (HCG), unconjugated estriol
• 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
Indications for Amniocentesis
Amniocentesis is recommended when

Risk groups
 Age of the mother over 35 years old
 Diabetic mother
 Mothers who have other children who have a genetic
problem
 Mothers who have had an abnormal serum triple screen test
(alpha-fetoprotein, estriol, and HCG) or are Rh sensitized
 Three or more miscarriages

• Amniocentesis: needle aspiration of fluid from amniotic sac
• It is a safe procedure, after the 14th week of gestation
• 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
Collection

• Perform all handling procedures immediately, and deliver to
laboratory promptly
Amber tubes to protect bilirubin integrity
• 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
Specimen Handling and Processing

What do you think of ??
Differentiation between amniotic fluid and maternal
urine may be necessary to determine possible
premature membrane rupture or accidental
puncture of the maternal bladder during specimen
collection.
So can you guess how to differentiate?

• Needed to determine premature membrane rupture or
accidental puncture of maternal bladder from amniocentesis
• Measure creatinine, urea, glucose, and protein
– Amniotic fluid has:- (Creatinine <3.5 mg/dL and Urea <30 mg/dL)
– 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
Maternal Urine versus Amniotic Fluid
fern-leaf

Gross examination of amniotic fluid color
COLOR SIGNIFICANCE
Colorless Normal
Blood-streaked Traumatic tap, abdominal trauma,
intra-amniotic hemorrhage
Yellow Hemolytic Disease of the Newborn
(HDN), Bilirubin
Dark green Meconium (first bowel movement)
Dark red-brown Fetal Death

• Hemolytic Disease of the Newborn (HDN)
– Most commonly Rh-negative mothers
– Other red blood cell (RBC) antigens can also
produce HDN
– Fetal cells with antigens inter maternal circulation
and cause production of maternal antibodies
– Maternal antibodies cross the placenta and destroy
fetal cells with the corresponding antigen
Fetal Distress

Antibodies Crossing the Placenta

• 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
semilogarithmic graph 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
Cont’….

• Liley graph
Plots ΔA450 against gestational age
Consists of three zones based on hemolytic severity
1. Zone I: mildly affected fetus
2. Zone II: requires careful monitoring
3. Zone III: severely affected fetus, may require
induction of labor or intrauterine exchange
transfusion
Cont’….

Liley Graph

• Are congenital birth defects in the spinal cord, brain, and
spine.
Caracterized by elevation of Alpha-fetoprotein (AFP) in
maternal and fetal circulation
AFP is produced by the fetal liver prior to 18 weeks’
gestation
Increased levels in maternal blood or amniotic fluid
indicate possible neural tube defects (anencephaly or
spinal bifida)
Neural tube defects

• 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
Cont’….
Neural tube defects…

A multiple of the median (MoM) is a measure of how far an individual test result deviates
from the median. MoM is commonly used to report the results of medical screening tests,
particularly where the results of the individual tests are highly variable.
MoM was originally used as a method to normalize data from participating laboratories of
Alpha-fetoprotein (AFP) so that individual test results could be compared.
As an example, Alpha-fetoprotein (AFP) testing is used to screen for a neural tube defect
(NTD) during the second trimester of pregnancy. If the median AFP result at 16 weeks of
gestation is 30 ng/mL and a pregnant woman's AFP result at that same gestational age is
60 ng/mL, then her MoM is equal to 60/30 = 2.0. In other words, her AFP result is 2 times
higher than "normal."

• 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
Fetal Lung Maturity (FLM)

• Considered the reference method (standard for fetal lung maturation)
• 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
Lecithin-Sphingomyelin (L/S) Ratio

• 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)
Foam Stability

• 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 0.150 at 650 nm correlates with L/S ratio of 2.0 and the
presence of phosphatidyl glycerol
Lamellar Bodies

• 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
– 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
Lamellar Body Count (LBC)

Summary for Amniotic Fluid
• Mother and unborn child testing
– Healthcare provider makes decisions about care
and treatment
– Assess chemical changes in mother and fetus
– Understanding the stage of the fetus
– An understanding of the developing physiology of
the fetus is used to predict outcome by assessing
chemical changes in the mother and fetus

Exercises
1. What may cause yellow color in amniotic fluids
2. Mention the limitation of chemical tests in amniotic fluid for
specimen transport
3. Describe the principle of bilirubin test in amniotic fluid
4. Describe the clinical significance of amniotic fluid tests
5. Describe the function of amniotic
6. What is the primary cause of the normal increase in amniotic
fluid as a pregnancy progresses?
7. What is the reason for decreased amounts of amniotic fluid?

References:
• Urinalysis and body fluids / Susan King Strasinger, 5th ed. 2008
• District laboratory practice in tropical countries. 2nd ed. Part I. Monica
Cheesbrough, 2005
• Text book of urinalysis and body fluids. Doris LR, Ann EN, 1983
• Urinalysis and body fluids: A color text and atlas. Karen MR, Jean JL. 1995
• Clinical chemistry: Principles, procedures, correlation. 3rd ed. Michael L. Bishop
et al. 1996
• Tietz Text book of clinical chemistry. 3rd ed. Carl AB, Edward RA, 1999
• Clinical chemistry: Theory, analysis, correlation 4th ed. Lawrence AK. 2003
• ASCP Document
• Urinalysis lecture note . Mistire W. , Dawite Y.
36
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