ANALYSIS OF URINE AND BODY FLUIDS
AMNIOTIC FLUID AMNIOTIC FLUID VOLUME
PHYSICAL CHARACTERISTICS
 The amniotic fluid is the fluid surrounding the
developing fetus that is found within the
amniotic sac contained in the mother's womb.
 It is clear pale yellow fluid
 Volume depends on gestation, 400ml at
mid pregnancy and reaches about
1000ml at 36-38 weeks.
AMNIOTIC FLUID COMPOSITION
 Amniotic fluid is found around the developing
fetus, inside a membraneous sac, called
AMNION.
 Amniotic fluid immediately begins to fill
the sac. In the early weeks of pregnancy,
amniotic fluid consists mainly of water
supplied by the mother. After about 20
weeks, fetal urine makes up most of the
fluid.
 Amniotic fluid also contains nutrients,
hormones and disease-fighting antibodies.
AMNIOTIC FLUID FUNCTION
o This fluid protects the developing fetus.
o Serves a key role in the exchange of water and
molecules between the fetus and the maternal
circulation.
o It helps the developing fetus to move in the
womb, which allows for proper bone growth
and muscle development.
o Helps prevent compression of the umbilical
cord.
o Contains Ig that also help in fighting pathogens.
PRIMARY FUNCTIONS
1. Cushion the fetus
2. Allows fetal movement
3. Stabilize temperature
4. Proper lung development
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o From fetal urine and lung fluid
o FETAL URINE: major contributor to the
amniotic fluid volume after the 1st trimester of
pregnancy.
AMIONITIC FLUID vs MATERNAL URINE
o The composition of the amniotic fluid changes
with gestation in early pregnancy it is similar to
maternal and fetal serum.
 98-99% of the amniotic fluid is water.
o A large number of dissolved substances such
as creatinine, urea, bile pigments, renin,
glucose, fructose, proteins (albumin and
globulin) lipids, hormones (estrogen and
progesterone), enzymes, minerals (Na+, K+, Cl-
).
o Suspended in it are some undissolved
material such as some fetal epithelial cells
o The amount of amniotic fluid increases until 28
to 32 weeks of pregnancy. After that time, the
level of fluid generally stays constant until the
baby is full term (37 to 40 weeks), when the
level begins to decline.
o In some pregnancies, however, there may be
too little or too much amniotic fluid.
Oligohydramnios - Having too little amniotic fluid
Polyhydramnios - Having too much amniotic fluid
OLIGOHYDRAMNIOS
o About 4% of pregnant women have
oligohydramnios.
o It can develop at any time during pregnancy,
although it is most common in the last
trimester.
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o 12 percent of women whose pregnancies last
about two weeks beyond their due dates (42
weeks gestation) develop oligohydramnios,
because the level of amniotic fluid tends to
decrease by that time in gestation.
Does oligohydramnios pose risks to mother or baby?
 The problems associated with
oligohydramnios differ depending on the stage
of the pregnancy.
 Oligohydramnios is more likely to have serious
consequences if it occurs in the first half of
pregnancy than if it occurs in the last trimester.
These consequences include :
Birth defects (too little amniotic fluid
early in pregnancy can lead to
compression of fetal organs, resulting
in lung and limb defects)
Miscarriage
Premature birth
Stillbirth
 When oligohydramnios occurs in the:
Second half of pregnancy, it may be
associated with poor fetal growth.
Near term, oligohydramnios may increase
the risk of complications of labor and
delivery, including compression of the
umbilical cord.
 This can deprive the baby of oxygen,
sometimes resulting in stillbirth. Women with
oligohydramnios are more likely than
unaffected women to need a CESAREAN
SECTION.
POLYHYDRAMNIOS
o About 1 percent of pregnant women have too
much amniotic fluid.
o Most cases are minor and result from a gradual
build-up of excess fluid in the second half of
pregnancy.
o However, a small number of women have a
rapid build-up of fluid occurring as early as 16
weeks of pregnancy that usually results in very
early delivery.
What complications can polyhydramnios
cause for mother and baby?
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 Polyhydramnios may increase the risk of
pregnancy complications including :
Premature delivery
Placental abruption (the placenta partially
or completely peels away from the uterine
wall before delivery)
Stillbirth
Postpartum haemorrhage (severe
bleeding after delivery)
Fetal malposition (the baby is not lying in
a head-down position and may need to be
delivered by cesarean section)
o a major cause of fetal loss and death among
newborn babies.
o The first description of HDN is thought to be
in 1609 by a French midwife who delivered
twins— one baby was swollen and died
soon after birth, the other baby developed
jaundice and died several days later. For the
next 300 years, many similar cases were
described in which newborns failed to
survive.
o It was not until the 1950s that the
underlying cause of HDN was clarified;
namely, the newborn's red blood cells
(RBCs) are being attacked by antibodies
from the mother. The attack begins while
the baby is still in the womb and is caused
by an incompatibility between the mother's
and baby's blood.
HEMOLYTIC DISEASE OF THE NEWBORN
HDN is an alloimmune condition that develops in
fetus, when the IgG molecules produces by the
mother pass through the placenta.
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ANALYSIS OF URINE AND BODY FLUIDS
Among these antibodies are some which attack
the red blood cells in the fetal circulation.
The red cells are broken down and the fetus can
develop reticulocytosis and anemia.
This fetal disease ranges from mild to very
severe, and fetal death from heart failure
(hydrops) can occur.
When the disease is moderate or severe, many
erythroblasts are present in the fetal blood and
so these forms of the disease can be called
erythroblastosis fetalis (or erythroblastosis
foetalis)
PATHOPHYSIOLOGY
This disorder occurs when the fetus has a blood
group antigen that the mother does not
possess. The mother’s body forms an antibody
against that particular blood group antigen,
and hemolysis begins. The process of antibody
formation is called MATERNAL SENSITIZATION.
The fetus has resulting anemia from the
hemolysis of blood cells. The fetus
compensates by producing large numbers of
immature erythrocytes, a condition known as
erythroblastosis fetalis, haemolytic disease of
the newborn, or hydrops fetalis.
Hydrops refers to the edema and fetalis refers
to the lethal state of the infant.
In Rh incompatibility, the hemolysis usually
begins in utero. It may not affect the first
pregnancy but all pregnancies that follow will
experience this problem. In ABO
incompatibility, the hemolysis does not usually
begin until the birth of the newborn.
ETIOLOGY
Hemolytic disease occurs most frequently
when the mother does not have the Rh factor
present in her blood but the fetus has this
factor.
Another common cause of hemolytic disease is
ABO incompatibility. In most cases of ABO
incompatibility, the mother has blood type O
and the fetus has blood type A. It may also
occur when the fetus has blood type B or AB.
Hemolysis is occasionally caused by maternal
anemias, such as thalassemia or from other
blood group antigens (anti-D).
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SYMPTOMS
Hemolysis leads to elevated bilirubin levels.
After delivery, bilirubin is no longer cleared (via
the placenta) from the neonate’s blood and the
symptoms of jaundice (yellowish skin and
yellow discoloration of the whites of the eyes)
increase within 24 hours after birth.
Like any other severe neonatal jaundice, there
is the possibility of acute or chronic
kernicterus.
Profound anemia can cause:
- high output heart failure, with palor,
- enlarged liver and/or spleen,
- generalized swelling, and
- respiratory distress
The prenatal manifestations are known as:
- hydrops fetalis
- in severe forms this can include petechiae
and purpura
- the infant may be stillborn or die shortly
after birth
DIAGNOSIS
The diagnosis of HDN is based on history and
laboratory findings:
Blood tests done on the newborn baby
 Biochemistry test for jaundice
 Peripheral blood morphology shows
increased reticulocytes. Erythroblasts
(a.k.a nucleated RBC) occur in moderate
and severe disease.
 Positive direct Coombs test (might be
negative after fetal interuterine blood
transfusion)
Blood tests done on the mother
 Positive indirect Coombs test
COOMBS TEST
o Detects Rh incompatibility between
mother and fetus.
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ANALYSIS OF URINE AND BODY FLUIDS
o This test uses antibodies that bind to anti-
D antibodies. The test is named for ROBIN
COOMBS, who first developed the
technique of using antibodies that are
targeted against other antibodies.
DIRECT COOMBS TEST: Diagnoses HDN
The direct Coombs test detects maternal anti-
D antibodies that have already bound to fetal
RBCs.
First, a sample of fetal RBCs is washed to
remove any unbound antibody (Ig). When the
test antibodies (anti-Ig) are added, they
agglutinate any fetal RBCs to which maternal
antibodies are already bound.
This is called the direct Coombs test because
the anti-Ig binds “directly” to the maternal anti-
D Ig that coats fetal RBCs in HDN.
INDIRECT COOMBS TEST: Used in the
prevention of HDN
The indirect Coombs test finds anti-D
antibodies in the mother’s serum. If these
were to come into contact with fetal RBCs they
would hemolyse them and hence cause HDN.
By finding maternal anti-D before fetal RBCs
have been attacked, treatment can be giben to
prevent or limit the severity of HDN.
For this test, the mother’s serum is incubated
with Rh D-positive RBCs. If any anti-D is present
in the mother’s serum, they will bind to the
cells. The cells are then washed to remove all
free antibodies. When anti-Ig antibodies are
added, they will agglutinate any RBCs to which
maternal antibodies are found.
This is called the indirect Coombs test because
the anti-Ig finds “indirect” evidence of harmful
maternal antibodies, requiring the addition of
fetal RBCs to show the capacity of maternal
anti-D to bind fetal RBCs.
MANAGEMENT
BEFORE birth, options for treatment include:
 Intrauterine transfusion or early induction
of labor when the pulmonary maturity has
been attained, fetal distress is present, or
35 to 37 weeks of gestation have passed.
 the mother may also undergo plasma
exchange to reduce the circulating levels of
antibody by as much as 75%.
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AFTER birth, treatment depends on the severity
of the condition, but could include:
 temperature stabilization and monitoring
 phototheraphy
 transfusion with compatible packed red
blood
 exchange transfusion with a blood type
compatible with both the infant and the
mother
 sodium bicarbonate for correction of
acidosis and/or assisted ventilation
Rhesus-negative mothers who have had a
pregnancy with/are pregnant with a rhesus-
positive infant are given Rh immune globulim
(RhIg) at 28 weeks during pregnancy, at 34
weeks, and within 72 hours after delivery to
prevent sensitization to the D antigen.
AMNIOCENTESIS
o Amniocentesis is used to determine the
health of an unborn baby.
o Amniotic fluid contains cells that are
normally shed from the fetus. Samples of
these cells are obtained by withdrawing
some amniotic fluid.
Specimen Collection
o Procedure: AMNIOCENTESIS
o Maximum of 30 mL is collected in sterile
syringes
o 2nd trimester: assess genetic defects ex.
Trisomy 21, Down Syndrome
o 3rd trimester: fetal lung maturity (FLM), fetal
hemolytic disease (HDN)
Specimen Handling
o Placed on ice for delivery
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o Refrigerated or frozen (3 days)
o Filtration: prevents loss of phospholipids

Specimen Handling
CYTOGENIC STUDIES:
o Processed aseptically
o Room temperature or body temperature
(37°C)
HEMOLYTIC DISEASE OF THE NEWBORN (HDN)
o For bilirubin analysis
o Protect from light

Color and Appearance

 COLORLESS - NORMAL
 BLOOD-STREAKED
o Traumatic tap, abdominal trauma, intra-
amniotic hemorrhage TEST FOR NEURAL TUBE DEFECTS
 YELLOW
o HDN (bilirubin) 1. SPINA BIFIDA (“split spine”)
 DARK-GREEN  Birth defect where there is
o Meconium incomplete closing of the
o 1st fetal bowel movement backbone and membranes
 DARK RED-BROWN around the spinal cord.
o Fetal death
2. ANENCEPHALY
 The absence of a major portion of the
brain, skull, and scalp that occurs during
TEST FOR HDN embryonic development

1. O.D. 450 3. SCREENING TEST: AFP (alpha-fetoprotein)

 Absorbance of amniotic fluid at 365 – 550 nm
 HDN: > 450 nm (maximum absorbance of
bilirubin)
 Results are plotted on a Liley graph
 Interferences: cells, meconium, debris and
hemoglobin (absorbance at 410 nm)
2. LILEY GRAPH
 Zone 1: mildly affected fetus
 Zone 2: moderate hemolysis
 Zone 3: severe hemolysis
 NV: < 2 MoM
 Increased in NTD, decreased in Down
Syndrome
4. CONFIRMATORY TEST: Acetylcholinesterase
Fetal lung maturity test:
 Lecithin – sphingomyelin ratio:
 Measurment of the lecithine/sphingomyelin
ratio.
 This test is done to assess the maturation of
the fetal lungs.
 A lecithin- sphingomyelin ratio of 2 or greater
is associated with fetal pulmonary system
maturity.

TEST FOR FETAL LUNG MATURITY

o Reference method; Thin layer

chromatography ( TLC )
o Ratio of ≥ 2: mature fetal lung
o Lecithin: responsible for alveolar stability

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o Sphingomyelin: control (due to constant o Measurment of bilirubin indicates the degree
production) of fetal red blood cell destruction.
o CANNOT be done on specimen
contaminated by blood or meconium Studies of the cells obtained from the
amniotic fluid permit:
TEST FOR FLM
1. AMNIOSTAT-FLM 1. Chromosomal analysis of the cells which
 Immunologic test for phosphatidylglycerol can be performed to investigate:
 Production of phosphatidylglycerol is delayed  Diagnosis of sex of the fetus
among diabetic mothers  Detection of
 Not affected by blood or meconium chromosomal
abnormalities
2. FOAM TEST e.g. (Down’s
 Amniotic fluid + 95% ETOH shake for 15 Syndrome)
sec stand for 15 mins  DNA studies
 (+) foam/bubbles
2. The cells may be cultured and analyzed for
3. MICROVISCOSITY enzymes, or for other materials that may
 Presence of phospholipids decrease indicate genetically transmitted disease.
microviscosity
 Measured by fluorescence polarization Who is indicated amniocentesis?
o That the pregnancy is 35 years or
4. LAMELLAR BODY COUNT more.
 Type II pneumocytes produce alveolar o Family history of genetic alterations
surfactants stored in the form of lamellar
bodies
 32 000/uL lamellar body count: adequate When we will have an amniocentesis?
FLM o Usually made between 15 to 18 weeks,
reaching even to 11 or 12.
5. O.D. 650 nm
 Absorbance of ≥ 0.150 corresponds to an L/S
ratio of at least 2 and presence of
phosphatidylglycerol

FETAL DISTRESS TESTING: What are the risks of amniocentesis?

o Erythroblastosis fetalis is caused when mother
develops antibodies to an antigen on the fetal
erythrocytes and these antibodies cross the
placenta to destroy many fetal RBCs.
o Abortion: about 1 in 200 to 400
women aborted (higher risk if done
in the first quarter)
o Uterine infection: 1 in 1000
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Backbone that did not close properly

Extra chromosome

Differentiation of amniotic fluid from maternal

urine:
o Differentiation between
amniotic fluid and maternal
urine may be necessary to
determine accidental puncture
of the maternal bladder during
specimen collection.

o Chemical analysis of creatinine, urea, glucose,

and protein aids in the differentiation.
o Levels of creatinine and urea are much lower
in amniotic fluid than in urine.
o while glucose and protein tend to be higher in
the amniotic fluid than in urine.

DIFFERENTIATION:
FERN TEST
 Used to evaluate premature rupture of the
membranes
 Vaginal fluid specimen
 (+) fern-like crystals
E
N
D

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