1. AMNIOTIC FLUID
CHAPTER 13

2. Learning Objectives
Upon completing this chapter, the reader will be able to
State the functions of amniotic fluid.
Describe the formation and composition of amniotic fluid.
Differentiate maternal urine from amniotic fluid.
State indications for performing an amniocentesis.
Describe the specimen-handling and processing procedures for testing amniotic fluid for bilirubin, fetal lung maturity (FLM), and cytogenetic analysis.
Discuss the principle of the spectrophotometric analysis for evaluation of hemolytic disease of the newborn.

3. Learning Objectives (cont’d)
Interpret a Liley graph.
Describe the analysis of amniotic fluid for the detection of neural tube disorders.
Explain the physiologic significance of the lecithin-sphingomyelin (L/S) ratio.
State the relationship of phosphatidyl glycerol to FLM.
Define lamellar bodies and describe their significance to FLM.
Discuss the principle of and sources of error for the L/S ratio, Amniostat-FLM, lamellar body count, and Foam Stability Index for FLM.

4. Physiology Amnion: a membranous sac that surrounds the fetus
Amniotic fluid
Provides protective cushion for fetus
Allows fetal movement
Stabilizes fetal temperature exposure
Permits proper lung development
Exchanges water and chemicals among the fluid, fetus, and maternal circulation

5. Fetus in Amniotic Sac

6. Physiology
Volume
Production: fetal urine, lung fluid, and maternal circulation
During the first trimester, the approximately 35 mL of amniotic fluid is derived primarily from the maternal circulation
Increased amniotic fluid peak at 800 to 1200 mL in the third trimester is the result of fetal urine
Increased urine is regulated by increased fetal swallowing
Lung fluid adds lung surfactants to amniotic fluid; used as a measure of lung maturity

7. Physiology (cont’d) Polyhydramnios Oligohydramnios
Excess amniotic fluid from failure of fetus to swallow >1200 mL
Neural tube disorders, structural/chromosomal abnormalities, cardiac arrhythmias, infections
Oligohydramnios
Decreased amniotic fluid from increased fetal swallowing, membrane leakage <800 mL
Umbilical cord compression

8. Chemical Composition
Composition similar to maternal plasma with sloughed fetal cells
Cells are used for cytogenetic analysis
The fluid also contains biochemical substances that are produced by the fetus, such as bilirubin, lipids, enzymes, electrolytes, urea, creatinine, uric acid, proteins, and hormones
Fetal urine increases creatinine, urea, and uric acid
Fetal age can be estimated by creatinine
<36 weeks = 1.5 to 2.0 mg/dL
>36 weeks = >2.0

9. Maternal Urine versus Amniotic Fluid
Needed to determine premature membrane rupture or accidental puncture of maternal bladder from amniocentesis
Measure creatinine, glucose, protein, and urea
Amniotic fluid has <3.5 mg/dL creatinine and <30 mg/dL urea
Values as high as 10 mg/dL for creatinine and 300 mg/dL for urea may be found in urine
The presence of glucose, protein, or both is associated more closely with amniotic fluid
Fern test: specimen air dries on glass slide; examine microscopically for “fern-like” amniotic fluid crystals

10. Indications for Amniocentesis
Abnormal screening blood tests: maternal alpha fetal protein, human chorionic gonadotropin, unconjugated estriol
Abnormal chromosome analysis and history of genetic disorders
Abnormal ultrasound for fetal body measurements
In later pregnancy for possible early delivery
Fetal lung maturity, hemolytic disease of the newborn (HDN), infection

11. Indications for Amniocentesis (cont’d)
Collection of fetal epithelial cells in amniotic fluid indicate the genetic material of the fetus
Examined for chromosome abnormalities by
Karyotyping
Fluorescence in situ hybridization (FISH)
Fluorescent mapping spectral karyotyping
DNA testing
Biochemical substances that the fetus has produced
Analyzed by thin-layer chromatography

12. Collection Amniocentesis: needle aspiration of fluid from amniotic sac
Transabdominal amniocentesis
Maximum of 30 mL collected in sterile syringes
Discard first 2 to 3 mL for contamination
Protect specimens from light for bilirubin analysis for HDN at all times: amber tubes or black plastic tube covers

13. Specimen Handling and Processing
Perform all handling procedures immediately, and deliver to laboratory promptly
Amber tubes to protect bilirubin integrity
Deliver fetal lung maturity (FLM) tests on ice; refrigerate or freeze up to 72 hours if needed
Cytogenetic specimens kept at room temperature or 37°C to prolong cell life
Centrifuge or filter fluid for chemical tests to remove debris; filter only for FLM tests

14. Color and Appearance
Normal amniotic fluid is colorless, with slight to moderate turbidity from cells
Blood streaked: traumatic tap, abdominal trauma, intra-amniotic hemorrhage
Fetal versus maternal blood: use Kleihauer-Betke
Bilirubin: bright yellow
Meconium (first bowel movement): dark green
Fetal death: dark red-brown

15. Summary of Amniotic Fluid Color
Significance
Colorless
Normal
Blood streaked
Traumatic tap
Abdominal trauma
Intra-amniotic hemorrhage
Yellow
Hemolytic disease of the newborn (HDN)
Dark green
Meconium
Dark red-brown
Fetal death

16. Tests for Fetal Distress
HDN
Most commonly Rh-negative mothers
Other red blood cell (RBC) antigens can also produce HDN
Fetal cells with antigens enter maternal circulation and cause production of maternal antibodies
Maternal antibodies cross the placenta and destroy fetal cells with the corresponding antigen

17. Antibodies Crossing the Placenta

18. Tests for Fetal Distress
Bilirubin from RBC destruction appears in the amniotic fluid
The amount of unconjugated bilirubin present correlates with the amount of RBC destruction
Spectrophotometric analysis of fluid optical density (OD) between 365 and 550 nm is plotted on semilog paper
Bilirubin causes OD rise at its maximum absorbance level of 450 nm; difference between baseline and 450 nm peak is the ΔA450
Difference is plotted on a Liley graph

19. Spectrophotometric Bilirubin Scan Showing Bilirubin and Oxyhemoglobin Peaks

20. Tests for Fetal Distress
Liley graph
Plots ΔA450 against gestational age
Consists of three zones based on hemolytic severity
Zone I: mildly affected fetus
Zone II: requires careful monitoring
Zone III: severely affected fetus, may require induction of labor or intrauterine exchange transfusion

21. Liley Graph

22. Tests for Fetal Distress
Neural tube defects
Alpha-fetoprotein (AFP) produced by the fetal liver prior to 18 weeks’ gestation
Increased levels in maternal blood or amniotic fluid indicate possible anencephaly or spinal bifida
Increased levels are found when skin fails to close over neural tissue
Measure maternal blood first, then amniotic fluid

23. Tests for Fetal Distress (cont’d)
Normal values based on weeks of gestation (maximum AFP 12 to 15 weeks)
Report multiples of the median (MoM)
Median is laboratory’s reference level for a given week of gestation
More than two times the median (MOM) is abnormal
Follow with fluid amniotic acetylcholinesterase (AChE): more specific for neural disorders
Do not perform on a bloody specimen

24. Fetal Lung Maturity (FLM)
Most common complication of early delivery is respiratory distress syndrome (RDS)
Lack of lung surfactant, which keeps the alveoli open during inhaling and exhaling
Surfactant decreases the surface tension on the alveoli so they can inflate more easily
Many laboratory tests are available for FLM

25. Lecithin-Sphingomyelin (L/S) Ratio
Considered the reference method
Lecithin is the primary component of the lung surfactants; increased production occurs after the 35th week
Sphingomyelin is produced at a constant rate after the 26th week and serves as a control for the rise in lecithin
L/S ratio is 1.6 prior to week 35 and rises to 2.0 or greater for alveolar stability after week 35
Therefore, preterm delivery is considered safe with an L/S ratio of 2.0 or higher
Test is performed using thin-layer chromatography
Many laboratories have replaced the L/S ratio with the quantitative phosphatidyl glycerol immunoassays and lamellar body density procedures

26. Phosphatidyl Glycerol
Lung surface lipid phosphatidyl glycerol is also needed for lung maturity
Normally parallels lecithin, except in diabetics, so must be included in L/S ratio
Amniostat-FLM is an immunologic agglutination test for PG using antibody specific for phosphatidyl glycerol that can replace the L/S ratio (no special equipment needed)
Blood and meconium do not interfere with the test

27. Foam Stability Performed at the bedside
Amniotic fluid is mixed with 95% ethanol, shaken for 15 seconds, and allowed to sit undisturbed for 15 minutes
A continuous line of bubbles around the outside edge indicates the presence of a sufficient amount of surfactant to maintain alveolar stability (alcohol is an antifoaming agent, and fluid can overcome this)

28. Foam Stability Index
0.5 mL amniotic fluid added to increasing amounts of 95% ethanol
Provides ethanol:fluid ratios of 0.42 mL to 0.55 mL in 0.01 mL increments, giving semiquantitative measure of surfactant amount
A value >47 indicates FLM
Correlates well with L/S ratio and tests for phosphatidyl glycerol

29. Lamellar Bodies Lamellar bodies: storage form of surfactant
Approximately 90% phospholipid and 10% protein
Secreted by the type II pneumocytes of the fetal lung to the aveolar space at about 24 weeks of gestation
Increase in amniotic concentration from 50,000 to 200,000/mL by the end of the third trimester
OD of 150 at 650 nm correlates with L/S ratio of 2.0 and the presence of PG

30. Lamellar Body Count (LBC)
Lamellar body diameter ranges in size from 1.7 to 7.3 fL or 1 to 5 µm
LBCs can be obtained using the platelet channel of automated hematology analyzers
Advantages of LBC
Rapid turnaround time
Low reagent cost
Wide availability
Low degree of technical difficulty
Low volume of amniotic fluid required
Excellent clinical performance
Specimens contaminated with meconium or mucous cannot be used

31. Protocol for Performing LBC
Mix the amniotic fluid sample by inverting the capped sample container five times.
Transfer the fluid to a clear test tube to allow for visual inspection.
Visually inspect the specimen. Fluids containing obvious mucus, whole blood, or meconium should not be processed for an LBC.
Cap the tube and mix the sample by gentle inversion or by placing the test tube on a tube rocker for 2 minutes.
Flush the platelet channel; analyze the instrument’s diluents buffer until a background count deemed acceptable by the laboratory is obtained in two consecutive analyses.
Process the specimen through the cell counter and record the platelet channel as the LBC.