11/12/2023

Outline
• Formation and Composition of
Amniotic Fluid, and its functions
• Specimen Handling and
Amniotic Fluid Processing
• Indications for performing
amniocentesis
• Test for FLM, and the Clinical
Significance

Prepared by: Fe P. Lleno, RMT, MSMT

AMNIOTIC FLUID FUNCTIONS OF AMNIOTIC FLUID

Ø Provide a protective cushion for the fetus
• Found in the membranous sac that surrounds the Ø Allows fetal movement
fetus providing a cushion for protection (amnion) Ø Stabilize the temperature to protect the fetus from
• Formed from metabolism of fetal cells, transfer of extreme temperature changes
Ø Permits proper lung development
water across the placental membrane & in the 3rd
trimester by fetal urine

Formation and Volume VOLUME

During the first trimester:
• amniotic fluid is derived from MATERNAL CIRCULATION (35 ml)
• Regulated by the balance between the production of
fetal urine and lung fluid, and absorption from fetal After the first trimester: (app 1L)
swallowing and intramembranous flow. • URINE is the major contributor of amniotic fluid
• Fetal swallowing starts and regulates the increase in the fluid from the fetal urine
• Volume increases on the course of pregnancy.
• app 800-1200ml during the 3rd trimester On the latter third to half pregnancy
• Fetus secretes a volume of LUNG LIQUID which will enter the amniotic fluid

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VOLUME CHEMICAL COMPOSITION

POLYHYDRAMNIOS
Abnormal increase of the fluid volume • Similar to the composition of maternal plasma
Accumulation of amniotic fluid due to fetus’ failure to swallow the urine Indicator of: • Contains:
a.fetal distress • Sloughed fetal cells from the skin, digestive system, urinary tract
b.neural tube defects • Biochemical substances
c. Secondary effects like congenital infection, urinary tract deformities • Bilirubin, lipid, enzymes, nitrogen compounds, protein
OLIGOHYDRAMNIOS
 Abnormal decreased amniotic fluid AMNIOTIC FLUID CREATININE
Associated with: - Use to determine fetal age
- umbilical cord compression------ decelerated heart rate----- fetal death  - Prior to 36 weeks of gesttion, crea levels is 1.5-2.0 mg/dL
> 2.0 mg/dl creatinine = indicates > 36 weeks gestation

SPECIMEN COLLECTION
Indications:
Trans-abdominal amniocentesis
Diagnosis of genetic diseases 30 ml  amber bottle  protect from light
§ Early (14-16 weeks):
(Bilirubin)
§ Mid Trimester: Bile pigments level for HDN Discard first 3-5ml  contaminated with
Neural tube defects maternal blood, tissue fluid and cells
Performed at 14 weeks gestation
§  3rd
Renal maturity 15-18 weeks  genetic and chromosomal
Trimester:
Bile pigments level for HDN studies

SPECIMEN HANDLING AMNIOTIC FLUID TESTS

• FETAL LUNG MATURITY TEST Significance:
• Placed in ice for delivery in the laboratory Detection of HDN
• Can be kept frozen or in refrigerator temperature for 72 hours. Detection of Fetal-lung maturity
• Repeat freeze-thawing is not recommended Detection of Fetal-lung hypoxia
Detection of Fetal disorders
• FILTRATION is recommended for this method to prevent phospholipid
Detection of Cytogenic disorders
loss Detection of Chromosomal abnormalities
• Transport with ice if for phospholipid determination
CYTOGENETIC STUDIES
 Maintained at room temperature or at body temperature (25 or 37˚C)

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Description Normal Abnormal Clinical Correlation

Appearance Colorless to • Blood streak • Traumatic tap, bdominal Difference of Amniotic and Maternal Urine
pale yellow trauma, intra-amniotic
hemorrhage
Point of reference Amniotic Fluid Maternal Urine
• Yellow • Hemolytic disease of the
newborn (bilirubin)
• Dark green • Meconium
Color Pale yellow Pale yellow
• Dark red-brown • Fetal death
Creatinine 3.5mg/dl 10mg/dl
Volume 1,000-1,500mL • Polyhydramnios • Abnormal increase
at term (36 • Oligohydramnios • Abnormal decrease Urea 30mg/dl 300mg/dl
weeks)
Glucose Present Absent
Protein Present Absent

Surface fluids of the Lungs

Lecithin • Primary surface fluid Chemical Analysis
• Produced at relatively low & constant rate Description Normal Abnormal Clinical
until 35th week of gestation Correlation
Lecithin/Spingo Mature = • Immature = <1.5
myelin Ratio >2.0 • Transitional= 1.5-1.9
Saturated >500μg/L <500μg/L
Sphingomyelin Produced at constant rate after about 26th week Respiratory
phosphatidy-
of gestation choline distress

Phosphatidyl glycerol • Essential fluid for adequate lung maturity

• Liley graph
Amniotic Fluid Bilirubin
§ Measurement by spectrophotometric
• Plots ΔA450 against gestational
age
analysis & plotted in the Liley cur ve
§ Normal: Optical density will rise
• 3 zones based on hemolytic
at 365nm & will decrease linearly severity
550nm • Zone I: mildly affected fetus
§ (+) Bilirubin: Optical density will • Zone II: requires careful
be seen at 450 nm monitoring
§ Elevated in Hemolytic Disease of • Zone III: severely affected
the Newborn (HDN) / fetus, may require induction
Erythroblastosis fetalis of labor or intrauterine
exchange transfusion

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Chemical Analysis • HDN

Description Normal Abnormal Clinical Correlation • Most commonly Rh-negative mothers
Bilirubin 28 weeks: 28 weeks: Erythroblastosis
<0.075 >0.075 Hepatitis
• Other red blood cell (RBC)
antigens can also produce HDN
40 weeks: 40 weeks: Maternal infection • Fetal cells with antigens enter
<0.025 >0.025 Sickle cells maternal circulation and cause
Bilirubin Determination production of maternal antibodies
Lily’s Curve
• Represent the measurement • For establishment and determination • Maternal antibodies cross the
and reading of bilirubin from of antibody titer placenta and destroy fetal cells with
amniotic fluid plotted by the corresponding antigen
spectrophotometer

Neural Tube Defects

• Alpha FetoProtein (AFP): Determine neural tube disorders
• Major protein produced by the fetal liver & found in the
§ Leukocyte esterase reagent strip: Good
maternal serum
indicator for the presence of infection • Neural tube defects: Increased Maternal serum AFP & Very
§ WBC Count: >50/uL is Positive for infection high amniotic fluid AFP
• Acetylcholinesterase Test: Confirmatory test for neural tube
disorders

• anencephaly and spina bifida.

Neural tube defect Fetal Maturity Analysis

• Lecithin/Sphingomyelin Ratio (L/S ratio): Reference method to measure
fetal lung maturity
• Lecithin: Primary component of the phospholipids that make up the
majority of the alveolar lining & account for alveolar stability
• Sphingomyelin: Lipid that is produced at a constant rate after about the
26th weeks of gestation serving as a control on which to base the rise in
Lecithin.
• 2.0 L/S Ratio: Safe procedure for pre-term delivery
• Analytical errors:
• Over centrifugation, Blood contamination
• Imprecision of TLC, Meconium

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Fetal Maturity Analysis

• Phosphatidylglycerol & Phosphatidylinositol:
• Essential for adequate fetal lung maturity
• Amniostat FLM
• Immunologic agglutination test for phosphatidyl glycerol
• Provides rapid method for assessment of lung maturity
• Foam test:
• Mechanical screening test to measure the individual lung surface lipid
concentration
• Foam stability index: Good correlation with L/S Ratio
• Optical Density: 650nm :>0.250 (Normal for fetal lung maturity)
• Depend on longevity of bubbles (ethanol-antifoaming agent)
• Simple shake test
• Manual foam stability index

Fetal Maturity Analysis • Amniotic particle counting

• TDx-FLM: Utilize fluorescence polarization between lipids & (Lamellar bodies)
albumin for fetal lung maturity • Fetal maturity:> 32,000
• Measurement of microviscosity >70 mg/g particles /microliter

Lamellar Bodies Count

• Creatinine: Detects fetal age
-The count correlates with the amount
• >2.0 mg/dL: indicates approximately 36 weeks of phospholipids present in fetal lung
• Renal maturity

• Optical density: Examined at 650 nm

• Normal value: >0.250

FETAL RED CELLS & MATERNAL RED CELLS: Tests for Fetal Well Being
and Maturity
• Kleihauer-Betke Stain
Test Normal Values Significance
• Fetal RBC: Red, refractile, large cells, resistant to alkali
at Term
• Maternal RBC: Uncolored ghost cells or dull red, poorly Bilirubin scan Δ A450 > .025 HDN
stained, sensitive to alkali Alpha-fetoprotein <2.0 MoM Neural tube disorders
L/S ratio ≥2.0 Fetal lung maturity
Amniostat Positive Fetal lung/phosphatidyl
• If maternal blood is present = colorless when stained glycerol
Foam Stability Index ≥47 Fetal lung maturity
• If fetal blood is present = purple – pink when stained Microviscosity (FLM II-TDx) ≥55 mg/g Fetal lung maturity
Optical density 650 nm ≥0.150 Fetal lung maturity
Lamellar body count ≥32,000/mL Fetal lung maturity

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FEMALE FERTILITY STUDIES

• Study of ovulatory & menstrual cycle
• Follicular / Proliferative phase
• hyperplasia of uterus/endometrium

PREGNANCY TEST • Ovulatory phase

• oocyte is already matured
• Increased body temperature
• Increased cervical mucus
• Weight gain
• Pelvic pain
• Irritability

FERTILIZATION
Luteal/Secretory phase
•  ovary luteolysis
• Ovum must be fertilized within 1-2 days
• If the ovum is not fertilized, luteolysis occurs
 Menstruation : regular, periodic, physiologic extrusion of
blood, mucus & cellular debris from the • Ampulla of fallopian tube: where fertilization takes place
uterus • Within 6-8 days
• Zygote will migrate to the upper portion of the uterus
 Menarche:
- 1st menstruation • HCG works when progesterone is decreased
 Menopause:
- cessation of menstruation

HUMAN CHORIONIC GONADOTROPIN (HCG)

FERTILIZATION
• Estrogen • Detectable 10-14 days after conception
• Increased in Follicular phase • Dimeric Glycoprotein
• Follicle stimulating hormone & Luteinizing hormone • Secreted by syncytiotrophoblast
• Increased after ovulation • Properties:
• Progesterone • Polypeptide chains
• Increased in luteal phase • Alpha subunits: 14,000 Daltons
• Decreased progesterone = Increased HCG • Beta subunits: 22,000, specific
• Peak levels
• 60-80 days after the last menses then decreases concentration:
100,000mIU/mL or 80,000 ng/mL

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IMMUNOLOGIC ASSAYS
IMPORTANCE OF PREGNANCY TESTS • Hemagglutination Inhibition (HAI)
• RBC covered/coated with HCG incubated with anti-HCG serum + urine
• 150-4,000mIU/mL (sensitivity)

• Confirm normal pregnancy. • Latex Agglutination Inhibition (LAI)

• HCG coated particle of latex + urine
• Evaluate abnormal pregnancy. •  500-350,000 mIU/mL
• (+)Pregnancy: no agglutination or milky suspension
• Determine if there has been complete or incomplete
abortion. • Direct Latex Agglutination
• Antibody to HCG coated on RBC or on latex particles
• Differentiate Gestational Trophoblastic disease • (+)Pregnancy: presence of agglutination

(myoma/H-mole) & other pelvic disease. • Enzyme Immunoassay; Complement fixation; Sol Particle Immunoassay

End of Lecture

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