AUBF: ANALYSIS OF URINEAND OTHER BODY FLUIDS

LECTURE 1: AMNIOTIC FLUID

MRS. CHARI K. WACHAYNA, RMT

1st SEMESTER | S.Y. 2024-2025

AMNIOTIC FLUID

 Present in amnion
 Product of fetal metabolism
 Give information on progress of fetal maturation and the metabolic processes taking place in the fetus
a. Measures the ability of the fetus to survive early delivery in case of fetal distress
b. Associated with cytogenetics analysis

FUNCTION

 Provides a protective cushion for the fetus

 Allows movement of fetus

PRENATAL

 280 days= 40 weeks=9 months and 7 days

 Age of Gestation (AOG)
 Ovulation: 10-14 days
 Last Menstrual Period (LMP)
 Expected Date of Confinement (EDC)

PHYSIOLOGY

 Exchange of water and chemicals takes place between the fluid, fetus and maternal circulation
 1st trimester: 35mL of amniotic fluid is derived primarily from maternal circulation.
o Composition
a) Same as maternal plasma plus
b) Small amount of sloughed fetal cells- these cells are basis for cytogenetics analysis
 After 1st trimester: fetal urine is the major contributor to the AF volume
 During 3rd trimester: a peak of 1 liter increases the amount of AF throughout the pregnancy
 Gradually decreases prior to delivery
 Fetal swallowing of AF: increase in fluid from fetal urine
 Failure of fetal swallowing leads to Hydramnios

Slightly increased risk of pregnancy and birth complications:

a. Before 37 weeks: giving birth prematurely

b. Premature Rapture of Membrane (PROM): Bag of water breaking early
c. Prolapsed umbilical cord: problem with the position of the umbilical cord
d. Heavy bleeding after the baby is born because the womb has stretched
e. The baby might have health condition

DISORDER

 Hydramnios: excessive accumulation of AF

 Polyhydramnios: fetal distress associated with neural tube defect
 Causes
a) A twin or multiple pregnancy
b) Gestational diabetes: diabetic mother
c) Gut atresia: blockage in the baby’s gut
d) Infection during pregnancy
e) Rhesus disease: the baby’s blood cells being attacked by the mother’s blood cells
 Oligohydramnios: decreased AF
 Diagnose
a) Ultrasound examination and may be described:
- qualitatively (reduced amniotic fluid volume)
- quantitative (amniotic fluid index < 5cm, single deepest pocket <2cm)

 Causes
a) Increased fetal swallowing
b) Urinary tract deformities
 c) Membrane leakage

MATERNAL URINE

 To determine Premature Membrane Rupture (PMR) from accidental maternal bladder puncture during
specimen collection.
 Creatine, Urea, Glucose and Carbon dioxide, Hydrogen, Oxygen and Nitrogen (CHON) will aid in differentiation.

Creatinine (mg/dL) Urea (mg/dL)

Amniotic Fluid >3.5 30
Urine 10 300

Note:
1. Chemical composition of AF changes when fetal urine production begins.

Increased
a. Creatinine: determine fetal age - Prior to 36 wks AOG: 1.5-2.0 mg/dL
- after 36 wks AOG: >2.0 mg/dL
b. Urea
c. Uric Acid

2. Changes in electrolytes, enzymes, hormones, metabolic end production but of little significance.

SPECIMEN COLLECTION: Amniocentesis- needle aspiration

 Transabdominal: most frequently performed

 Transvaginal: increased risk of infection
 Done after the 14th weeks (general collection)
 On 16th week of gestation: Chromosome analysis
 On 3rd trimester: test for fetal distress and maturity
 30 mL AF: maximum volume collected in sterile syringes
 Discard first 2-3mL because it contaminated with:
a) Maternal blood
b) Tissue fluid
c) Cells
 Bilirubin analysis: Hemolytic Diseases Fetal Newborn (HDN

SPECIMEN HANDLING

1. Fetal Lung Maturity (FLM): deliver in ice; refrigerate prior to testing

 Note: a. Low speed centrifugation for not >5min to prevent loss of phospholipids
b. Filtration is recommended to prevent phospholipids
2. Cytogenetics Specimen: store at Room Temperature or Body Temperature
: incubate at 37degree C to prior to prolong life of cells needed.
3. Chemical Test: centrifuge specimen immediately to prevent distortion of chemical constituent by cellular
metabolism or disintegration.

AMNIOTIC FLUID COLOR

Color Significance
Colorless Normal
Blood-streaked: determine source of blood Traumatic tap, abdominal trauma, intra amniotic hemorrhage
Yellow HDN (bilirubin)
Dark Green Meconium
- Newborn’s first bowel movement
- Present in the AF as a result of fetal intestinal secretion
- Significant when increased amount present in AF
Dark red- brown Fetal Death

KLEIHAUER-BETKE (Acid Elution)

 Test for fetal Hgb

 Utilized to determine if there is fetal blood in maternal circulation, with a threshold of 5mL
 Stains: Erlich acid Hematoxylin and counterstained with Erythrosine.
 - Fetal RBCs= stained bright pink- red
- Maternal RBCs= ghost cells
 Procedure: to determine the % of RBCs containing fetal hemoglobin
1. Determine the average no. of red cells per field
2. No. of Hgb F RBC/field= no. of Hgb F RBC counted / no. of fields counted
3. No. RBC with Hgb F= no. HGB F RBC/ field x 100 / average no. of RBC/ field

TEST FOR FETAL DISTRESS

 HDN: antibodies (against fetal RC) present in maternal circulation cross the placenta causing the RC
destruction leading to increased bilirubin level.
 Bilirubin: a. RBC degradation product
b. anemia: measured to assess degree of hemolysis
c. maximally absorbed at 450 nm
 Spectrophotometric: 365-550 nm
 Normal fluid: OD is at its peak at 365 nm, decreases linearity at 550 nm
 Difference in Od is plotted Liley graph
 Zones of Liley Graph

Zone 1 mild anemia 14 gm%

Zone 2 Moderate anemia 13.9 – 8 gm%
Zone 3 Severe anemia <8 gm% : death in 7-10
days

 Formula: change in absorbance reading at 450 nm = absorbance reading at 450nm – absorbance reading at
theoretic baseline
 Notes:
a. Specimen must be kept in amber bottle at all times to prevent decreased bilirubin level.
b. Prevent contamination from cells, hemoglobin, meconium, other debris- interferes with reading
c. Centrifuge immediately to remove particulate interference.

NEURAL TUBE DEFECT

- The skin fails to close over the neural tissue

- Increased AFP in both maternal circulation and AF
 Anencephaly: congenital absence of all or a major part of the brain
 Spina bifida: congenital cleft of the spinal column with hernial protrusion of the meninges and sometimes the
spinal cord.

1. Alpha-fetoprotein
- Major CHON produced by fetal liver prior to 18 wks gestation
- Maximal production between 12 and 15 wks AOG, then begins to decline.
 Found in: a) maternal serum – due to combined fetal maternal circulation
b) AF- from diffusion and excretion of fetal urine
 Measurements of AFP in AF: Indications
1. Increase amount of AFP in maternal circulations
2. Family history of previous neural defect
3. Possible multiple pregnancy
 Test: a) automated by Access Immunoassay System
b)If elevated, measure Amniotic Acetylcholinesterase (AChE)

2. Acetylcholinesterase (AChE)
- More specific for neural tube defect
- Do not use bloody specimen, it contains AChE

TEST FOR FETAL LUNG MATURITY (FLM)

 Perform in case of forced preterm delivery in fetal distress maybe due to HDN or other conditions.
 Fetal Lung Maturity (FLM): respiratory distress is the most frequent preterm delivery complication.

I. LECITHIN- SPHINGOMYELIN (L/S) RATIO

  Lecithin
- The primary component of the surfactants (phospholipids, lipids, CHON) that make up the alveolar
lining and account for alveolar stability
- Produced at low but constant rate up to 35 wks gestation
- At 36 wks AOG increases production resulting to fetal lung alveolar stability.
 Alveoli
- tiny air sacs in the lungs that take up the oxygen that we breathe in
- they’re microscopic
- the workhorse of the respiratory system
- there are about 480 million alveoli, located at the end of the bronchial tubes.
 Pulmonary Surfactant: is a mixture of lipids and proteins which is secreted into the alveolar space by
epithelial type II cells.
 Surfactant: to lower the surface tension at the air/liquid interface within alveoli of the lung.
 Sphingomyelin: a) after 26 wks AOG – lipid produced at a constant rate
b)serves as control to base the rise of Lecithin.
 Note: both Sphingomyelin and Lecithin appear in the AF with the same proportion as the concentration in the
fetus
 < 1.6 L/S ratio prior to 36 wks AOG
 >2 L/S ratio at 36 wks AOG due to increase production of lecithin
 Significance: if L/S ratio is 2= preterm delivery is safe
 False elevation of L/S ratio: blood and meconium contamination due to presence of L and S in those
substances

II. THIN-LAYER CHROMATOGRAPHY

- Quantitative measurement of L/S
- Tedious and subject to high coefficient variation

III. PHOSPHATIDYL GLYCEROLIMMUNOASSAYS

 Phosphatidyl Glycerol
- A lung surface lipid also essential for adequate lung maturity and its production parallels that of
lecithin
- Delayed production in diabetic mother so a 2.0 L/S ratio might cause respiratory distress
- Always perform TLC lung profile that includes L, S and phosphatidyl glycerol
 Foam Stability: Foam or Shake Test
- Determines the concentration of lung surface lipids
- Performed at bedside
- Procedure:
a) Mix equal parts of AF with 95% ethanol
b) Vigorously shake for 15 sec
c) Allow to stand undisturbed for 15 min
d) Observe for the presence of a continuous line of bubbles around the outside edge
 Measurement of Surfactant Function: Shake Test
1) It evaluates the ability of pulmonary surfactant to generate a stable foam in the presence of ethanol
2) Ethanol: a non- foaming competitive surfactant, eliminates the contributions of protein, bile salts and
salts of free fatty acid to the formation of a stable foam
3) At the ethanol concentration of 47.5%, stale bubbles that form after shaking are due to amniotic fluid
lecithin
4) Positive tests, a compete ring of bubbles at the meniscus with a 1:2 dilution of amniotic fluid, are
rarely associated with neonatal RDS
5) It Is a screening test that gives useful information if mature
 Note:
 The presence of bubbles indicates sufficient amount of phospholipids that can reduce the surface
tension of the fluid
 Not reliable if contaminated with blood and meconium
 Foam Stability Index: semiquantitative measure of the surfactant present.
 Procedure:
a) Add 0.5mL AF to tubes containing increasing amounts of 95% ethanol ranging from 0.42-0.55
mL in 0.01mL increments
b) Follow same procedures as Foam Test
c) Values >47 indicate FLM
IV. FLOURESCENCE POLARIZATION
 Microviscosity
- Presence of phospholipids decreases the microviscosity of AF
- The change in microviscosity is measured using Fluorescence Polarization Principle
- Fluid used should be filtered instead centrifuge to prevent sedimentation of lipids
- Do not use contamination fluid
 Abbott TDx Analyzer
- It measures the polarization of the fluorescent dye that combines to both surfactant and albumin
- Dye bound to surfactant exhibits low polarization
- Dye bound to the recorded change in polarization produces a surfactant/ albumin (mg/g) ratio
- Ratio is compared to a standard curve that includes phosphatidyl glycerol ranging from 0-160 mg/g
- Ratio of >70 means FLM
 Note: Albumin – is used as an internal standard since it has a constant level throughout the pregnancy like
sphingomyelin

V. LAMELLAR BODY DENSITY

 Lamellar Bodies and Optical Density
- The surfactants responsible for FLM produced and secreted by type II pneumocytes of the fetal lung in
the form of lamellar bodies
- The lamellar bodies enter the alveolar spaces to provide surfactant also enters the AF
- The amount of Lamellar bodies present in the AF correlates with the amount of phospholipid present in
fetal lung.
- The presence of Lamellar bodies increases the OD of the AF
- Specimens are centrifuged at 2,000 rpm for 10 min then examined using 650 nm wavelength to rule
out interference due to Hgb but not meconium
- An OD of 0.150 correlates well will L/S ratio of >2.0 and the presence of phosphatidyl glycerol.
- Adequate FLM
 Coulter Cell Counter
- Counts lamellar bodies using the platelet channel
- It is easy to perform
- Samples must be free from contaminations such as blood and meconium
- Normal: >32,000 particles/mL= adequate FLM

VI. AMNIOSTAT – FLM

 Immunologic agglutination test that uses antisera specific for Phosphatidyl glycerol
 Advantage: its not affected by contamination with blood and meconium.