AMNIOTIC FLUID

Amniotic fluid is a product of fetal

metabolism and the constituents that are
present in the fluid provides information
about the metabolic processes taking place
during – as well as the progress of - fetal
maturation
Volume
Regulated by balance between production
of fetal urine & lung fluid and the
absorption from fetal swallowing and
intramembranous flow.

Intramembranous flow – absorption of AF

water and sloutes into the fetal vascular
system

PHYSIOLOGY AF reaches a peak volume of 1L during the

third trimester and decreases prior to
Function delivery
Amnion – membranous sac that surrounds
the fetus; this is where the amniotic fluid is 1st trimester – 35ml of AF is derived from
located maternal circulation
After the 1st trimester – fetal urine is the
Primary functions of amniotic fluid: major contributor to AF volume
- protective cushion Fetal swallowing begins once fetus starts
- allow fetal movement producing urine
- temperature stability
- permits proper lung development Polyhydramnios – excess AF volume; may
be an indication of fetal distress associated
Exchanges of water and chemicals also take with neural tube disorders, anomalies,
place between the fluid, fetus and maternal arrhythmias, infections, chromosomal
circulation abnormalities (due to failure of fetus to
begin swallowing)

Oligohydramnios – decreased AF volume;

associated with umbilical cord compression
(due to increased fetal swallowing, urinary
tract deformities, membrane leakage)
Chemical composition SPECIMEN COLLECTION
Placenta – ultimate source of AF water and
solutes Indications for amniocentesis
Composition similar to that of maternal - recommended when screening blood tests
plasma & contains small amount of are abnormal
sloughed fetal cells from the skin, digestive > Alpha fetoprotein test
system, urinary tract – the cells provide > triple screening test (AFP, hCG,
basis for cytogenetic analysis unconjugated estriol)
Fluid also contains bilirubin, lipids, enzymes, > Quadruple screening test (triple test +
electrolytes, nitrogenous compounds, inhibin A)
proteins from fetus – used to
determine/test fetal health and maturity Fetal body measurements taken with
ultrasound can detect intrauterine growth
A portion of fluid arises from fetal restrictions and any other abnormality
respiratory tract, urine, amniotic membrane which could indicate the need for
and umbilical cord amniocentesis

When fetal urine production begins: Fetal epithelial cells – indicate genetic
- increases – creatinine, urea, uric acid material of the fetus and biochemical
- decreases – glucose, protein substances produced
- cells can examined for chromosome
AF creatinine level (can be used to abnormalities by FISH, SKY and DNA testing
determine fetal age) - biochemical substances analyzed by
- before 36wks AOG – 1.5-2mg/dL fluorescence polarization and thin layer
- >36 wks AOG - >2mg/dL chromatography

Differentiating maternal urine from Indicated at 15-18wks AOG for the ff

amniotic fluid conditions:
- necessary to determine possible Mother’s age 35 or more at delivery
premature membrane rupture from Family history of chromosome
accidental puncture of the maternal bladder abnormalities
during specimen collection Parents carry an abnormal
chromosome rearrangement
Creatinine & urea – lower in AF Earlier pregnancy or child with birth
Amniotic fluid defect
- creatinine (not exceed 3.5mg/dl) Parent is a carrier of a metabolic
- urea (not exceed 30mg/dl) disorder
Urine History of genetic diseases
- creatinine 10mg/dl Elevated maternal serum AFP
- urea 300mg/dl Abnormal triple marker screening
test
Glucose & protein – less reliable, but when Previous child with a neural tube
present associated more closely with AF disorder
Fern test – “fern like” crystals – (+) AF Three or more miscarriages
Evaluation of amniocentesis indicated later COLOR & APPEARANCE
at 20-42wks AOG to evaluate for:
Fetal ung maturity
Fetal distress
Hemolytic disease of the newborn
(Rh incompatibility)
Infection
Collection
Amniocentesis – obtained by needle
aspiration
Most frequently a transabdominal
amniocentesis is done
Vaginal amniocentesis – greater risk of
infection
Amniocentesis is a safe procedure when
performed after 14wks AOG TESTS FOR FETAL DISTRESS

Maximum 30ml fluid Hemolytic Disease of the Newborn

- first 2-3ml is discarded (contaminated by
maternal blood, tissue fluid and cells)
- fluid for bilirubin analysis must be
protected from light at all times (use amber
colored tubes or black plastic cover)

SPECIMEN HANDLING AND PROCESSING

Fluid for fetal lung maturity – placed in ice

for delivery to lab and refrigerated up to
72h prior to testing or kept frozen and
tested within 72h (frozen specimens should
be thoroughly mixed); filtration
recommended to prevent loss of
phospholipids Destruction of fetal RBCs results in
appearance of its degradation product –
Fluid for cytogenetic studies – maintained at unconjugated bilirubin
room temperature / body temperature - measured by spectrophotometric analysis
prior to analysis to prolong life of cells - optical density measured 365nm-550nm
normal – OD highest at 365nm and
Fluid for chemical testing – separated from decreases linearly to 550nm
cellular elements & debris as soon as bilirubin present – rise in OD is seen at
possible to prevent distortion of chemical 450nm (wavelength of maximum bilirubin
constituents by cellular metabolism or absorption)
disintegration (by centrifugation / filtration)
 Neural Tube Defects

AFP – major protein produced in fetal liver

during early gestation
- increased levels can be indicative of NTD
(anencephaly, spina bifida)
- found both in maternal serum and AF
- measurement indicated when maternal
serum levels are elevated or a family history
of previous NTD
- maximal AFP (12-15wks aog)

Oxyhemoglobin absorbance is at 410nm MoM (mulitples of median)

- a value 2x the median is abnormal
and can interfere with bilirubin absorption
peak (interference can be removed by
Amniotic acetylcholinesterase (AChE)
chloroform)
– measure after if with elevated AFP levels
- more specific to NTD
absorbance difference at 450nm (A450) –
difference in OD – is then plotted on a Liley - provided on performed on bloody
specimen since blood contains AChE
graph to determine severity of hemolytic
disease
TESTS FOR FETAL LUNG MATURITY

Fetal lung maturity

Respiratory Distress Syndrome (RDS) – most
frequent complication of early delivery;
cause of morbidity and mortality in a
premature infant
- lack of lung surfactant

Lecithin – Sphingomyelin ratio

Lecithin – primary component of surfactant
(phospholipids, neutral lipids, proteins)
- produced low and constant rate until
35wks aog – where production increases –
Zone 1 – no more than a mildly affected maturation of lungs
fetus
Zone 2 – require monitoring Sphingomyelin – lipid produced at constant
Zone 3 – severely affected fetus rate after 26wks aog

<35wks AOG – L/S ratio <1.6

>2.0 to prevent alveolar collapse
Falsely elevated – fluid contaminated with
blood or meconium
Thin – layer chromatography – quantitative
measurement of L/S ratio
Replaced with more cost effective
phosphatidyl glycerol immunoassays,
fluorescence polarization, lamellar body
density procedures

Amniostat-FLM
Phosphatidyl glycerol – production parallels Microviscosity Fluorescence Polarization
that of lecithin; delayed in cases of maternal Assay
DM (+) phospholipids – decreases viscosity
principle: fluorescence polarization
Thin layer chromatography
Immunologic agglutination test – rapid TDx/TDxFLx Fetal Lung Maturity II assay –
method reagent system for quantitative
Uses antisera specific for phosphatidyl measurement of the ratio of surfactant to
glycerol and not affected by presence of albumin in amniotic fluid
blood or meconium - measures the polarization of a fluorescent
dye that combines with both surfactant and
Foam Stability albumin
- “foam or shake” test
can be done at bedside / laboratory dye bound to surfactant – longer
fluorescence time, low polarization
dye bound to albumin – decreased
fluorescence time, high polarization

surfactant:albumin (mg/g)
</= 39mg/g – immature
40-54mg/g – further evaluation
55mg/g – mature

(sufficient amount of phospholipid is - test requires 1ml of AF, filtered to prevent

available to reduce surface tension of fluid sedimentation of lips and reporting falsely
even in the presence of alcohol as an decreased result
antifoaming agent) -Contaminated specimens with blood,
- not used with contaminated AF because meconium, maternal urine and icteric
blood and meconium reduce surface samples should not be used
tension
Lamellar bodies and Optical density
Lamellar bodies – lamellated phospholipids
that represent storage form of surfactant
Type II pneumocytes – produce surfactant
and stored in form of lamellar bodies at
20wks aog

Lamellar bodies
>correlates with amount of phospholipid
present in fetal lungs

Optical density
> increases OD of AF
Centrifuged at 2000g for 10mins and at a
wavelength of 650nm (rules out
interference from Hb)
OD of 0.150 (correlates with L/S ratio of 2)

Lamellar body counts (platelet channel of

hematology analyzers)
> Diameter – small platelets
> stored at 4C are stable up to 10 days

Resistance pulse counting

- easily performed
- samples must be free of particle
contamination
>/= 32,000/uL = adequate FLM

ADVIA hematology system – measures two

light scarrer angles of particles based on cell
volume and refractive index
>/= 35,400 – FLM

Sysmex XE-2100 – detects direct current

and radiofrequency impedance thought to
reflect intracellular changes

Cell-dyn 3500 – combines optical scatter

and impedance