AMNIOTIC FLUID CHAPTER 13

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.

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.

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

Fetus in Amniotic Sac

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

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

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

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

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

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

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

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

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

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

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

Antibodies Crossing the Placenta

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

Spectrophotometric Bilirubin Scan Showing Bilirubin and Oxyhemoglobin Peaks

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

Liley Graph

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

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

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

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

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

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)

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

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

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

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.