section epub:type=”chapter” id=”c0014″ role=”doc-chapter”> After studying this chapter, the student should be able to: By far the most frequently performed tests on amniotic fluid in the clinical laboratory are used to assess fetal lung maturity (FLM) and fetal anemia resulting from an isoimmune disease, which are discussed in this chapter. The specialized laboratory techniques required to detect numerous and varied genetic and metabolic disorders using amniotic fluid are beyond the scope and intent of this text and therefore are not discussed. Amniotic fluid is the liquid medium that bathes a fetus throughout its gestation (Fig. 14.1). The amnion, a membrane composed of a single layer of cuboidal epithelial cells, surrounds the fetus and is filled with this fluid. Amniotic fluid protects the fetus while enabling fetal movement and plays an important role in numerous biochemical processes. Fetal cells and many biochemical compounds, such as electrolytes, nitrogenous compounds, proteins, enzymes, lipids, and hormones, are present in the amniotic fluid. Although studies have investigated many substances as potential biochemical markers of disease, few substances (e.g., phospholipids) have demonstrated reliable clinical utility and value. The dynamics of amniotic fluid formation and its composition change throughout fetal gestation. Initially, amniotic fluid is produced by the amnion and the placenta, and its composition is similar to that of a dialysate of plasma. However, as gestation progresses, the fetus plays more of an active role in the composition of the fluid. Water and solutes are exchanged between the fetus and its surrounding medium through several mechanisms: (1) intestinal absorption after fetal swallowing of amniotic fluid; (2) capillary exchange in the pulmonary system as the alveoli of the fetal lungs are bathed with amniotic fluid; and (3) fetal urination. Early in gestation (before keratinization of the skin), a transudate passes through the skin of the fetus and makes a small contribution to the amniotic fluid volume. Because of fetal respiration in utero, the fetal pulmonary surfactants produced by alveolar cells of the fetal lungs mix with and can be evaluated using amniotic fluid. In the later stages of pregnancy, fetal swallowing and urination play a major role in the volume and composition of the amniotic fluid. The fetus swallows amniotic fluid, removing water and electrolytes, and replaces them through urination with metabolic byproducts such as urea, creatinine, and uric acid. At the same time, a similar exchange occurs between the amniotic fluid and the maternal plasma. The maternal plasma removes metabolic waste products and replenishes them with water, nutrients, and electrolytes. This ongoing, dynamic equilibrium results in complete exchange of the amniotic fluid volume every 2 to 3 hours.1 The presence of a neural tube defect causes fetal cerebrospinal fluid to also contribute substances to the amniotic fluid. In such cases, alpha-fetoprotein and acetylcholinesterase are two biochemical markers used to identify these defects. The volume of amniotic fluid increases steadily throughout pregnancy, from approximately 25 to 50 mL at 12 weeks’ gestation to a volume of 800 to 1200 mL at 37 weeks’ gestation.2 Abnormally increased amounts of amniotic fluid (>1200 mL), termed polyhydramnios, are associated with decreased fetal swallowing and often indicate congenital fetal malformations. Abnormally decreased amounts of amniotic fluid (<800 mL), termed oligohydramnios, occur with congenital malformations and other conditions, such as premature rupture of the amniotic membranes. Amniotic fluid is collected transabdominally or vaginally with simultaneous ultrasonic examination. Use of real-time ultrasonography allows the clinician to identify a maternal tapping site that will yield amniotic fluid and at the same time avoid injury to the fetus or the placenta. Transabdominal amniocentesis is preferred because vaginal amniocentesis is associated with an increased risk of infection, can result in contamination of the fluid with vaginal cells and bacteria, and can adversely affect results obtained using FLM tests. Typically, amniocentesis is performed after 14 weeks’ gestation; however, the purpose of performing the procedure dictates when it is done (Table 14.1). For example, an amniocentesis to detect neural tube defects or genetic abnormalities is usually performed at 15 to 18 weeks’ gestation. This allows sufficient time for the performance of chromosomal and biochemical studies, which may include culturing of fetal cells, as well as time for consideration of pregnancy termination if the fetus is determined to be abnormal. Table 14.1 Amniocentesis later in pregnancy is primarily used to assess the pulmonary and overall health status of the fetus. Tests can determine the maturity of the fetal pulmonary system by analyzing surfactants in the amniotic fluid. If results indicate an immature fetal pulmonary system, elective delivery can be postponed and corticosteroids (betamethasone) that promote lung development can be given, or other attempts can be made to suppress premature labor. In late pregnancy, amniocentesis may be performed to assess fetal status owing to toxemia, diabetes mellitus, or isoimmunization by Rhesus (Rh) factor. At times, these conditions necessitate early termination of a pregnancy and the delivery of a premature infant. Using aseptic technique, a physician pierces the abdominal and uterine walls with a long, sterile needle and aspirates approximately 10 to 20 mL of amniotic fluid into several sterile syringes. A series of numbered syringes (usually two or three) are used to prevent contamination of the entire collection with blood that can be encountered initially. The blood can result from piercing a blood vessel in the abdominal wall, uterus, placenta, umbilical cord, or fetus. Ideally, the amniotic fluid shows no evidence of blood. Immediately after its collection, the amniotic fluid should be carefully transferred into sterile plastic containers for transport to the laboratory. Glass containers should be avoided because cells will adhere to glass. Amber-colored containers or aluminum foil should be used to protect the fluid from light; this prevents photo-oxidation of bilirubin, if present. When cytogenetic or microbial studies are to be performed, amniotic fluid must be processed aseptically. Transportation of amniotic fluid specimens to the laboratory should occur as soon as possible to ensure the preservation of cellular and biochemical constituents. Note that storage temperature and handling (e.g., centrifugation) will vary with the tests requested and the protocols used by the laboratory performing the test. Specimens for cell culture, chromosomal studies, and microbial or viral culture must be maintained at body or room temperature. For FLM testing, amniotic fluid should be analyzed as soon as possible; if testing will be delayed, it should be refrigerated at 2 to 8°C.3 When bilirubin analysis is requested, the specimen must be protected from light at the bedside by wrapping the collection tube in foil or by using an amber-colored container. Specimens for bilirubin testing can be refrigerated or frozen, depending on the requirements of the testing laboratory. Depending on the FLM tests performed, amniotic fluid may (L/S ratio) or may not (lamellar body count [LBC]) be centrifuged. Note that the speed and duration of centrifugation will alter the composition of the amniotic fluid supernatant and pellet significantly. Low centrifuge speeds are used to recover fetal cells from amniotic fluid for cell culture. For spectrophotometric assays (e.g., bilirubin), a high speed is used to maximally clear the supernatant of particulates and turbidity. Another approach used to remove residual turbidity is to filter the amniotic fluid; however, this can significantly reduce the amount of sample available for testing. At times it may be necessary to determine whether the fluid collected is amniotic fluid or whether it is urine aspirated from the bladder. Physical examination alone cannot distinguish between these fluids because they can have the same appearance. However, their chemical compositions are distinctly different for several analytes. Amniotic fluid contains glucose and a significant amount of protein (approximately 2–8 g/L), and, until late pregnancy, the creatinine concentration is similar to that of normal plasma. In contrast, urine has essentially no protein or glucose and contains characteristically high concentrations of urea and creatinine (50–100 times those of plasma). Therefore these chemical constituents can be used to positively determine the identity of the fluid collected. For example, if a reagent strip test were used, positive results for glucose and protein would identify the fluid as amniotic fluid. However, because diabetes and renal disease can cause protein and glucose to be present in urine, the creatinine or urea concentration of the fluids should also be determined. It should be noted that in late pregnancy (37 weeks’ gestation or longer), the use of creatinine levels to distinguish between urine and amniotic fluid is more challenging. Fetal renal function has begun and contributes creatinine to the amniotic fluid. At this stage, the creatinine concentration in amniotic fluid can be two to three times that of normal plasma or up to 3.9 mg/dL (345 μmol/L). If a creatinine value greater than 4 mg/dL (354 μmol/L) is obtained, this indicates that the fluid is either urine or amniotic fluid contaminated with urine. The physical examination of amniotic fluid should take place immediately after its receipt in the laboratory. This examination consists of a visual assessment of the color and turbidity of the fluid. Normally, amniotic fluid is colorless or pale yellow. Distinctive yellow or amber coloration is associated with the presence of bilirubin; a green color indicates the presence of meconium. Meconium is a gelatinous or mucus-like material that forms in the fetal intestine as a result of swallowed amniotic fluid and fetal intestinal secretions. Biliverdin is responsible for its dark green color. Normally, full-term infants do not have a bowel movement in utero but excrete meconium as their first bowel movement after birth. However, fetal distress can cause premature release of meconium into the amniotic fluid. Blood contamination causes amniotic fluid to appear anywhere from pale pink to red. If blood is present in an amniotic fluid sample, the specimen should be centrifuged immediately to remove any intact red blood cells before hemolysis occurs. Hemolysis causes the formation of oxyhemoglobin, which can interfere with several biochemical tests. All amniotic fluid is turbid to some degree depending on the stage of pregnancy. Early in pregnancy, little particulate matter is present and the fluid is not very turbid. As pregnancy progresses, increased amounts of fetal cells, hair, and vernix are sloughed and remain suspended in the amniotic fluid. Two techniques that can be used to remove the particulate matter causing the turbidity are centrifugation and filtration. When premature delivery is anticipated or desired because of fetal distress or other complications, ensuring that the fetus will be viable outside the mother’s uterus is important. To assess fetal maturity and potential viability, tests that evaluate the functional status of the fetal lungs predominate because the pulmonary system is one of the last organ systems to mature. Note that FLM testing on amniotic fluid when gestation is less than 32 weeks is not performed because all FLM test results will indicate immaturity.4 Respiratory distress syndrome (RDS) is the most com-mon cause of death in the newborn and is a primary concern when a preterm delivery is imminent. RDS results from insufficient production of surfactant at the alveolar surfaces in the newborn’s lungs. Normally, alveolar epithelial cells of the lungs (type II pneumocytes) produce and secrete phospholipids (90%) and proteins (10%) in the form of lamellar bodies.5 These lamellar bodies release their “surface active” compounds, also known as surfactants, into the alveolar air space. Surfactants act in two ways: they alter the surface tension of the alveoli, preventing their collapse during expiration, and they reduce the amount of pressure needed to reopen them during inspiration. Gluck and associates discovered the correlation between FLM and the concentrations of specific phospholipids in amniotic fluid.6 More recently, it has been recognized that the probability of developing RDS is best determined by using two factors: the results of FLM tests and the gestational age of the fetus at the time of testing.4 From several studies, a table has been developed to guide clinicians in making individualized risk-benefit decisions for preterm delivery using gestational age and the FLM value.7 The American College of Obstetrics and Gynecology (ACOG) recommends a sequential or “cascade” approach to FLM testing. With this approach, a “mature” result using any of the common FLM tests is strongly predictive for the absence of RDS.8 In other words, a series of FLM tests can be performed until a mature result is obtained or all testing options have been used (Table 14.2). A rapid test, such as a LBC, should be performed first. In late pregnancy (> 35 weeks’ gestation), the rapid immunochemical test used to detect phosphatidylglycerol (PG) can be used. Additional testing is required when initial test results (1) are indeterminate, (2) are at the cutoff value, or (3) indicate immaturity. The cutoff values for FLM tests have been selected to reduce the risk of delivering infants with immature lungs. However, a “mature” result from an FLM test does not completely eliminate the possibility of RDS. In other words, the predictive value of a negative test (i.e., a mature result) is high (95% to 100%) for all available FLM tests; however, the predictive value of a positive test (i.e., an immature result and the presence of RDS) is low (23–61%) and varies with the FLM test used.4 Table 14.2
Amniotic Fluid Analysis
Key Terms1
Physiology and Composition
Function
Formation
Volume
Specimen Collection
Timing of and Indications for Amniocentesis
When to Perform Amniocentesis
Indications
14–18 weeks
Mother ≥35 years
Parent has known chromosomal abnormality
Previous child with chromosomal abnormality
Previous child with neural tube defect
Parent is carrier of a metabolic disorder
Elevated maternal alpha-fetoprotein (suspect neural tube defect)
20–42 weeks
Assessment of fetal distress due to
Assessment of fetal lung maturity
Collection and Specimen Containers
Specimen Transport, Storage, and Handling
Differentiation From Urine
Physical Examination
Color
Turbidity
Chemical Examination
Tests to Determine Fetal Lung Maturity
Test
Principle
Effects of Blood and Meconium
Advantages
Disadvantages
Lamellar body counts (LBC)
Automated cell counter (Hematology analyzers) enumerates lamellar bodies in amniotic fluid using the platelet channel
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Amniotic Fluid Analysis
Learning Objectives
