Amniotic fluid provides protection and nutrients for the developing fetus. Analysis of amniotic fluid obtained through amniocentesis can be used to assess fetal lung maturity, detect fetal distress, and screen for genetic and infectious disorders. Tests of amniotic fluid measure surfactant levels, bilirubin levels, and concentrations of proteins like alpha-fetoprotein to evaluate lung development and identify issues like hemorrhage or neural tube defects. Precise collection and rapid processing of amniotic fluid specimens is important for obtaining accurate test results.

1. AMNIOTIC FLUID

2. Utilities of Analysis:
• Determination of fetal lung maturity
• Detection of fetal distress
• Cytogenetic analysis
• Detection of hereditary, teratology
and infectious disorders
• Determination of fetal age

3. Formation and Physiology:
• formation in the amnion is regulated by
balance between the production of fetal
urine and lung fluid and the absorption
from fetal swallowing and
intramembranous flow
• Functions:
(1) provides a protective cushion for the
fetus
(2) allows fetal movement
(3) stabilizes the temperature to protect
the fetus from extreme temperature
changes
(4) permit proper lung development.

4. Volume:
Approximately 35 mL during the 1st
trimester, peaks during the 3rd trimester
(approx.1 L) and gradually decreases prior
to deliver; major contributors are
maternal circulation (1st trimester) and
fetal urine (after the 1st trimester)
Polyhydramnios
Excessive accumulation of amniotic fluid
usually resulting from the failure of the
fetus to begin swallowing; indicates fetal
distress often associated with neural tube
disorders

5. Oligohydramnios
Decreased amniotic fluid due
primarily to increased fetal
swallowing urinary tract
deformities, and membrane leakage
Composition:
Same as that of maternal plasma
plus a small amount of sloughed fetal
cells, biochemical substance
produced by the fetus, and a portion
from the fetal respiratory tract, fetal
urine, the amniotic membrane, and
the umbilical cod

6. 1. Specimen Considerations
a. Amniocentesis
• Needle aspiration of amniotic fluid from
the amniotic as; may be transabdominal
or transvaginal; safety performed after
the 14th week of gestation , volume
collected:

7. 2. Specimen handling
a. Fluid for FLM tests
• Transported in ice and refrigerated up
to 72 hours prior to testing or kept
frozen and tested within 72 hours;
filtration or low-speed centrifugation is
recommended
b. Fluid for cytogenetic studies
• Maintained at room temperature
or incubated at 37°C prior to
analysis

8. c. Fluid for chemical testing
• Separated from cellular elements and
debris ASAP to prevent distortion of
chemical constituents by cellular
metabolism or disitegration.
d. Fluid for bilirubin analysis
• placed in amber bottles or
containers covered with a black
plastic.

9. Gross Examination
Appearance Significance
Colorless with Normal
slight to moderate
turbidity
Blood- streaked Traumatic tap, abdominal
trauma,intra-amniotic
hemorrhage
Yellow HDN
dark- green Meconium
Dark red- brown Fetal Death

10. Tests for Lung Maturity
1. Lecithin/ sphingomyelin ratio
a. Method:____________________________
b. Principles: Lecithin is produced at a relatively low and constant rate until the
35th week of gestation while sphingomyelin is produced at a constant rate
after about 26 week’ gestation and therefore conserve as a control on which
to base the rise in lecithin. Prior to 35 week’ gestation, L/S ratio is ˂1.6 and
rises to >2.0 when lecithin production increases.
2. Amniostat-FLM
a. Method:________________________
b. Principle: the test uses antisera for phosphatidly glycerol and is
affectected by specimen contamination with blood and meconium.

11. 3. Foam stability index
a. method:_____________________________
b. Principle: a semiquatitative measure of the amount of surfactant is
done by adding 0.5 mL of amniotic fluid to increasing amounts of 95%
ethanol (0.42 mL to 0.55 mL in 0.01-mL increments), shaken for 15
seconds, and allowed to sit undisturbed for 15 minutes. If a sufficient
amount of phospholipid is present, a continuous line of bubbles will be
observed even in the presence of alcohol, an anti-foaming agent.
4. Microviscosity
a. Method:______________________
b. Principle: Phospholipids decrease the microviscosity of amniotic
fluid and the change is detected by determining the surfactant to
albumin ratio (mg/g) based on the polarization of a fluorescent dye
that combines (internal standard, decreased fluorescence lifetime
and high polarization).

12. 5. Lamellar body count
a. Method:___________________________
b. Principle: Lamellar bodies (lamellated phospholipids that represent
a storage from of surfactants secreted by the type II pneumocytes of
the fetal lung)range in size from 1.7 to 7.3 fL, and therefore can be
counted using the platelet channel of hematology analyzers.
6. Optical density at 650 nm
a. Method:_________________________
b. Principle : the increase in OD of the amniotic fluid caused by the
presence of lamella bodies in determined by centrifuging the
specimen at 2000 g for 10 min and reading the absorbance at
650 nm.

13. Table 24. Tests for fetal lung maturity
Normal Significance
values
L/S ratio ≥2.0 FLM
Amniostat-FLM Positive FLM/phosphotidyl glycerol
Foams Stability index ≥47 FLM
Microviscosity ≥55 mg/ g FLM
Lamellar body count ≥32,00/ mL FLM
OD at 650 nm ≥0.150 FLM
Bilirubin scan A 450 less HDN
.025
Alpha Fetoprotein Less than Neural tube disorder
2.0 MoM

14. Test for Fetal Distress
1. Bilirubin assay
a. Method: _____________________
b. Principle: the optical density of amniotic fluid is normally
highest at 365 nm and decreases linearly to 550 nm except when
bilirubin is present where a rise in OD is seen at 450 nm. The
ᴧ ᴧ450 is then plotted on a liley graph to determine the severity
of HDN and the need for interventions.
2. Alpha fetoprotein
a. Method:_________________
b. Principle: The Test is based on the measurement of the
neural tube defects using an automated immunoassay
method: results are reported in terms of multiples of the
median with a value >2 MoM considered abnormal

15. 3. Acetylcholinesterase
a. Method: ____________________
b. Principle: Ache is an enzyme derived primarily from the
neural tissue and is normally absent in amniotic fluid. Its
presence in amniotic fluid in conjunction with elevated AFP
values is highly diagnostic of NTDs.

16. Other Tests
1. Differentiation of amniotic fluid from maternal urine
• Creatinine is ˂3.5 mg/ dL and urea is ˂30
mg/dL in amniotic fluid, whereas high as 10
mg/ dL creatinine and 300 mg/dL urea may
be found in urine
2. Determination of fetal age
• AF creatinine level ranges from 1.5 to 2.0
mg/ dL prior to 36 weeks’ gestation and rises
above 2.0 mg/dL thereafter, providing a
means of determining fetal age as >36 weeks
3. Kleihauer-Betke test
• used to determine the source of the
blood (maternal or fetal) in a bloody
specimen for further case management.