AMNIOTIC FLUID ANALYSIS

Why examine amniotic fluid ? 1.detection of fetal lung maturity

2.HDN
3.neural tube defect
4.determination of fetal age
5.cytogenetic analysis
6.detection of hereditary ,teratology , and infectious disorders

Formation and physiology  Found in the amnion, a sac surrounding the fetus.

 Formation is regulated by balance between the production of fetal urine and lung fluid and the
absorption from fetal swallowing and intramembranous flow.

 Intramembranous flow is the absorption of amniotic fluid water and solutes into the fetal
vascular system.

Amnion Sac surrounding the fetus

Fetus in amniotic sac :

1st trimester 35 mL of amniotic fluid is derived primarily from maternal circulation (maternal plasma )

3-6 months of gestation  fetus secretes a volume of lung liquid necessary to expand the lungs with growth.

 During each episode of fetal breathing movement, secreted lung liquid enters the amniotic fluid,
as evidenced by lung surfactants that serve as index of fetal lung maturity.

 After the 1st trimester, FETAL URINE is the major contributor to the amniotic fluid volume

 At the time the fetal urine production occurs, fetal swallowing of the amniotic fluid begins and
regulates the increase in fluid from the fetal urine.

Polyhydramnios Failure of fetus to begin swallowing of amniotic fluid

results to excessive accumulation of amniotic fluid indication of fetal distress often associated
with neural tube disorders , may be 2o associated with fetal structural anomalies.

association :
neural tube disorder
cardiac arrythmias
congenital infection
chromosomal abnormalities
Oligohydramnios
Function of amniotic fluid
Amniocentesis
Indication of amniocentesis
Decrease amniotic fluid
Cause by:
increased fetal swallowing
urinary tract deformaties
membrane leakage
association:
umbilical cord compression
decelerated heart rate
fetal death
 Protective cushion for the fetus
 Allows movement
 Stabilize the temperature to protect the fetus from extreme temperature changes
 Permit proper lung development
 Exchanges of water and chemicals
 Needle aspiration of amniotic fluid
 May be safely done after the 14th week of gestation
 Done in 2nd trimester
o 16th week of gestation – for assessment of genetic defects/chromosome analysis
o Near the end of 2nd trimester – for assessment of intrauterine growth restriction
 Done in 3rd trimester for assessment of fetal pulmonary maturity or fetal hemolytic disease
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 as:
-sickle cell disease
-Tay-Sachs disease,
-hemophilia,
-muscular dystrophy,
-sickle cell anemia,
-Huntington chorea, and
-cystic fibrosis
 Elevated maternal serum a-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
SPECIMEN HANDLING AND PROCESSING
Specimen Handling and Processing o A maximum of 30 mL of amniotic fluid is collected in sterile syringes.
- The first 2 to 3 mL collected can be contaminated
by maternal blood, tissue fluid, and cells and are
discarded.
o Specimens should be transferred to sterile plastic containers and taken immediately to the
laboratory.
o Fluid to bilirubin analysis in cases of HDN must be protected from light at all times

Fetal lung maturity test placed on ice for delivery to lab and ref. prior to testing, or
keep frozen and tested within 72 hrs;
low speed centrifuge for no longer than 5 min (500 to 1000xg) to prevent loss of phospholipids
Cytogenetic studies maintained at room temperature or body temperature to prolong the life of cells needed for analysis
Chemical testing separate from cellular elements and debris ASAP
Volume The amount of amniotic fluid increases throughout pregnancy,
reaching a peak of approx. 1L during the third trimester, and then
gradually decreases prior to delivery;
major contributors are maternal circulation (1st trimester) and fetal urine (after 1st trimester).

DIFFERENCE OF AMNIOTIC FLUID MATERNAL URINE

Possible cause Premature membrane rapture Puncture of the bladder in
amniocentesis
Creatinine 3.5 mg/dL 10 mg/dL
Urea 30 mg/dL 300 mg/dL

COLOR
Colorless Normal
Blood -streaked Traumatic tap
Abdominal trauma
Intra-amniotic hemorrhage
Yellow HDN (bilirubin )
Dark-green Meconium
Dark red -brown Fetal death

Test for FLM

L/S (Lecithin/Sphingomyelin) Ratio REFERENCE METHOD

 LECITHIN – primary component of the surfactant that maintains alveolar stability

 SPHINGOMYELIN – serves as a control on which to base the rise of lecithin

 Up to the 26th week of gestation, the amount of lecithin is LESS than sphingomyelin.

 On the 36th week, both phospholipids are EQUAL

 After 36th weeks, LECITHIN INCREASES MARKEDLY while sphingomyelin remains constant or may
decrease

 Disadvantage: cannot be used when contaminated with blood or meconium

 L/S ratio> 2.0 = FLM

- Preterm delivery, if indicated, will be safe because can survive
outside the womb.

 L/S ratio < 1.5 – patient will develop respiratory distress syndrome

Phosphatidylglycerol/  Another LUNG SURFACTANT essential for lung maturity

Phosphatidylinositol  Performed by TLC
 May be used in place of L/S ratio
 PRODUCTION PARALLELS THAT OF LECITHIN BUT DELAYED IN DIABETIC MOTHERS

Amniostat – FLM Uses antisera specific for phosphatidylglycerol and not affected by specimen contamination with
blood or meconium
Foam/Shake Test  Done by shaking amniotic fluid w/ 95% ethanol for 15 seconds
 (+) presence of bubbles for 15 minutes
 Foam Stability Index
Microviscosity
Lamellar bodies and Optical Density
Test for Fetal Distress
Infection of mother and fetus
Bilirubin analysis
Rh antibodies crossing the placenta:
 SEMIQUANTITATIVE MEASURE OF AMOUNT OF SURFACTANTS PRESENT
 Amniotic fluid is reacted w/ varying amounts of 95% ethanol
 Value of >47 = FLM
 Value of <47 – immature lungs
 PRESENCE OF PHOPHOLIPIDS DECREASES THE MICROVISCOSITY OF AMNIOTIC FLUID
 This change in microviscosity can be measured using FLUORESCENCE POLARIZATION by Abbott
TDx analyzer
 ALBUMIN is used as INTERNAL STANDARD
 >55 mg/g = FLM
 Surfactant responsible for FLM are produced and secreted by the type II pneumocytes of the
fetal lung in the form of LAMELLAR BODIES
 Lamellar bodies enter the alveolar spaces to provide surfactant and also enter the amniotic fluid
 The number of lamellar bodies CORRELATES with amount of phospholipids present in fetal lungs
and they are counted using RESISTANCE PULSE COUNTING (in the platelet channel of the
hemocytometer)
- >32,000/mL = FLM
 Presence of lamellar bodies INCREASES OD of amniotic fluid
 Specimens are centrifuged at 2000 g for 10 min. and examined using a wavelength of 650 nm
- OD of 0.150 correlate w/ a L/S ratio of >2.0 and
presence of phosphatidylglycerol
>50/mL = infection
 For the evaluation of HDN
 Performed by SPECTROPHOTOMETRY and plotted on a LILEY CURVE
 In the course of normal pregnancy, bilirubin pigment in amniotic fluid DECREASES
 If there is maternal antibody crossing the placenta and destroying the fetal cells, bilirubin
INCREASES
 Oxyhemoglobin will peak at 410 nm while bilirubin at 450 nm
 Increased OD at 450 nm = HDN
 LILEY GRAPH
- plots the change in OD at 450 nm versus gestational age in
weeks
- Zone 1 – NON-AFFECTES/MILDLY AFFECTED – observe fetus for stress
- Zone 2 – MODERATELY AFFECTED –requiring close monitoring and treatment
- Zone 3 – SEVERELY AFFECTED – intervention is required (deliver/treat)
 Alpha-fetoprotein (AFP)  For detection of NEURAL TUBE DEFECTS (spina bifida, anencephaly)

 AFP is the major protein produced by the fetal liver during early gestation (prior to 18 weeks)
and found in maternal serum due to the combined circulation and in amniotic fluid by excretion
in fetal urine.

 Normal values are based on the week of gestational age, as the fetus produces maximal AFP
between 12 and 15 weeks’ gestation, after which levels in amniotic fluid begin to decline.

 MoM>2.0 is ABNORMAL
- both serum and amniotic fluid AFP levels are reported in terms of multiples of median (MoM)
- The median is the laboratory’s reference level in a given week of gestation.

Acetylcholinesterase level  Elevated in amniotic fluid in NEURAL TUBE DEFECTS because cholinesterase is a component of a
nerve tissue.
 Confirmatory after elevated AFP levels are demonstrated because it is more specific provided it
is not performed on a bloody specimen, because blood contains AChE

SPINA BIFIDA:

ANENCEPHALY No brain formation

Test for fetal age :

Creatinine concentration  Rises as the baby nears TERM
 Measured by serum method (Jaffe’s reaction)
 1.5 to 2.0 mg/dL = prior to 36th week of gestation
 >2.0 mg/dL = PREGNANCY IS OVER 36 weeks
NORMAL AMNIOTIC FLUID BILIRUBIN: Δ A450 > 0.025
AFP: <2.0 MoM
L/S ratio: ≥2.0
AMNIOSTAT-FLM: POSITIVE
FOAM STABILITY INDEX: ≥47
MICROVISCOSITY: ≥55mg/g
OPTICAL DENSITY: ≥0.150
LAMELLAR BODY COUNT: ≥32,000/mL
CREATIINE (for age): >2.0 mg/dL
Kleihauer-Betke Test used to determine the source of the blood (maternal or fetal) in a bloody specimen for further case
management.