To perform his experiments, how did Mendel prevent pea flowers from self-pollinating and control their cross-pollination? He cut away the pollen-bearing male parts of a flower and dusted that flower with pollen from another plant.

1. Chapter five
Amniotic Fluid

2. Acknowledgements
• Addisa Ababa University
• Jimma University
• Hawassa University
• Haramaya University
• University of Gondar
• American Society for Clinical Pathology
• Center for Disease Control and Prevention-Ethiopia

3. Chapter outline
• Introduction to Cerebrospinal fluid
• Routine laboratory assays
• Collection of sample
• Chemical analysis
• Morphological Examination
• Microbiological Examination
• Serological Examination

4. 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 bilirubin, fetal lung maturity (FLM),
and cytogenetic
analysis.
5 Discuss the principle of the spectrophotometric analysis for
evaluation of hemolytic disease of the newborn.
6 Interpret a Liley graph.

5. Learning Objectives cont’d
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

6. Amniotic Fluid
• It is liquid that surrounds the fetus in the amniotic cavity
– The primary function is to provide protective cushion for the fetus,
allow for movement, and regulate temperature.
• Amniocentesis is the fluid collection procedure in which a
sample of the amniotic fluid surrounding a fetus is removed
by means of a fine needle inserted through the abdomen and
into the uterus of the pregnant woman.

7. Function of Amniotic Fluid
• Amniotic fluid is present in the amnion, a membranous sac
• that surrounds the fetus.
• The primary functions of the fluid are to provide a protective
cushion for the fetus, allow fetal movement, stabilize the
temperature to protect the fetus from extreme temperature
changes, and to permit proper lung development.
• Exchanges of water and chemicals also take place between
the fluid, the fetus, and the maternal circulation

9. Amniotic Fluid cont’d
• testing of amniotic fluid is frequently associated with
cytogenetic analysis, the clinical laboratory also
performs several significant tests on amniotic fluid.
• Because amniotic fluid is a product of fetal
metabolism, the constituents that are present in the
fluid provide information about the metabolic
processes taking place during—as well as the progress
of—fetal maturation.
• When conditions that adversely affect the fetus arise,
the danger to the fetus must be measured
• The tests used to determine the extent of fetal distress
and fetal maturity

10. Volume of Amniotic Fluid
• regulated by a balance b/n the production of fetal urine and
lung fluid and the absorption from fetal swallowing and
intramembranous flow.
• The amount of amniotic fluid increases throughout pregnancy,
reaching a peak of 1 L during the third trimester, and then
decreases prior to delivery. During the first trimester, the
approximately 35 mL (from the maternal circulation).

11. Volume of Amniotic Fluid cont’d
• During each episode of fetal breathing
movement, secreted lung liquid enters the
amniotic fluid, as evidenced by lung surfactants
that serve as an index of fetal lung maturity.
• After the first trimester, fetal urine is the major
contributor to the amniotic fluid volume.
• At the time that fetal urine production occurs,
fetal swallowing of the amniotic fluid begins and
regulates the increase in fluid from the fetal
urine.

12. Volume of Amniotic Fluid cont’d
• Failure of the fetus to begin results in excessive
accumulation of amniotic fluid (polyhydramnios) and
is an indication of fetal distress, often associated with
neural tube disorders.
• Polyhydramnios may be secondarily associated with
fetal structural anomalies, cardiac arrhythmias,
congenital infections, or chromosomal abnormalities.
• Increased fetal swallowing, urinary tract deformities,
and membrane leakage are possible causes of
decreased amniotic fluid (oligohydramnios).

13. Fetus in amniotic sac

15. Amniotic Fluid cont’d…
• Sample collection
– Collected by experienced professional
– A maximum of 30mL of fluid can be collected.
– The first 2 or 3 mL should be discarded because they
may contain maternal blood, tissue fluid, and maternal
cells.
– Sterile plastic syringes and conical centrifuge tubes
should be used for the collection and transportation of
the amniotic fluid.
– Specimens for cytogenetic studies are maintained at
25 - 37 0C incubation prior to analysis to prolong the
life of the cells needed for analysis.
– Do not freeze, refrigerate, or centrifuge.

16. Amniotic Fluid specimen
– Specimens for fetal lung maturity tests should be
placed in ice for delivery to the laboratory and
refrigerated prior to testing.
– The specimen will be centrifuged or filtered before
analysis.
– Specimens for bilirubin analysis in cases of Hemolytic
Disease of the Newborn, must be protected from light.
– The sample should be collected in an amber-colored
tube.
– The appearance of the amniotic fluid can indicate
presence of certain chemicals. For example, degree
of redness indicates presence of hemoglobin.

17. Color and Appearance
• Normal amniotic fluid is colorless and may exhibit slight to
moderate turbidity particularly in later stages of fetal
development.
• 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, may be present in the amniotic fluid as
the result of fetal intestinal secretions. It produces a dark
green color.
• Fetal aspiration of meconium during fetal swallowing is a
concern when increased amounts are present in the fluid. A
very dark red-brown fluid is associated with fetal death

18. Color

19. Gross examination of amniotic fluids 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

20. Amniotic Fluid testing
• Laboratory testing of amniotic fluid involves analysis of
bilirubin, alpha-fetoprotein and a variety of tests for fetal lung
maturity (FLM).
• During the third trimester of pregnancy but less than 35 to 36
weeks gestation, fluid collected from the amniocentesis
procedure is analyzed to evaluate fetal lung maturity.
• Limitations:
– Contamination of the amniotic fluid specimen by
blood or meconium invalidates the FLM results.

21. Chemical Composition
• The placenta is the ultimate source of amniotic fluid
water
• and solutes. Amniotic fluid has a composition similar to
that of the 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, nitrogenous compounds, and
proteins that can be tested to determine the health or
maturity of the fetus.

22. Amniotic Fluid testing
• Bilirubin Scan in Amniotic Fluid
• Hemolysis and bilirubin assessed by optical density of
amniotic fluid at 450 nm spectrophotometricaly
• Change in Absorbance due to bilirubin
• Clinical significance
– evaluate fetal hemolysis in hemolytic disease of
newborn

23. Hemolytic Disease of the Newborn
• amniotic fluid evaluates the severity of the fetal anemia
produced by HDN.
• 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 foreign red cell antigens occurs during
gestation and delivery of the placenta 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.

24. Hemolytic Disease of the Newborn
• 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

25. Rh antibodies crossing the placenta

26. Summary for Amniotic Fluid
• Mother and unborn child testing
– Health care 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

27. 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?

28. 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.
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