Amniocentesis test is performed on women to gather information about the fetus. Fetal maturity, fetal distress, and risk for respiratory distress syndrome can be assessed. Genetic and chromosomal abnormalities can be identified. Maternal-fetal Rh incompatibility can be diagnosed. The sex of the child can be ascertained. This is important for a mother carrying a sex-linked gene. Neural tube defects can also be recognized. The test is performed on mothers whose pregnancies are considered to be high risk. These may include diabetic mothers, very obese mothers, older mothers (over 35 to 40 years) especially if there is a family history of trisomy 21, mothers with repeated spontaneous abortions, mothers whose prior children have genetic defects, and mothers in a couple in which either the mother or the father is a carrier for genetic defects. This test is also done on women who have an abnormal obstetric ultrasound.
Amniocentesis involves the placement of a needle through the patient’s abdominal and uterine walls into the amniotic cavity to withdraw fluid for analysis. Studying amniotic fluid is vitally important in assessing the following:
1. Fetal maturity status, especially pulmonary maturity (when early delivery is preferred). Fetal maturity is determined by analysis of the amniotic fluid in the following manner:
a. Lecithin and sphingomyelin (L/S ratio). The measurement of the ratio of the lipids L/S ratio has emerged as the standard criterion test to evaluate fetal lung maturity. Lecithin is the major constituent of surfactant, an important substance required for alveolar ventilation. If surfactant is insufficient, the alveoli collapse during expiration. This results in atelectasis and respiratory distress syndrome (RDS), which is a major cause of death in immature babies. In the immature fetal lung, the sphingomyelin concentration in amniotic fluid is higher than the lecithin concentration. At 35 weeks of gestation, the concentration of lecithin rapidly increases, whereas the sphingomyelin concentration decreases. An L/S ratio of 2:1 (3:1 in mothers with diabetes) or greater is a highly reliable indication that the fetal lung, and therefore the fetus, is mature. In such a case the infant would be unlikely to develop RDS after birth. As the L/S ratio decreases, the risk of RDS increases. Unfortunately, the L/S ratio assay involves a long and labor-intensive thin layer chromatography separation of the lipids. An alternative test is an assay based on fluorescence depolarization, implemented on the TDx fluorescence polarimeter and is called TDx Fetal Lung Maturity (FLM) test. This test, which yields the ratio of surfactant to albumin (S/A ratio), is quite sensitive.
FLM results are less affected by other factors such as contaminated blood or meconium. A fluorescent phospholipid analogue (C6-NBD-PC) is added to amniotic fluid and its fluorescence polarization is measured with a TDx fluorescence polarimeter. Polarization values decrease during gestation in parallel with maturation of the pulmonary surfactant system. Polarization value can be used to predict the probability that a fetus will develop respiratory distress syndrome following birth. Infrared (IR) spectroscopy offers an alternative method to detect and quantitate the key surfactants. The infrared spectrum of amniotic fluid shows strong absorptions from protein such as albumin when compared with the surfactant lipids contributing subtle absorption differences to the overall profile.
b. Phosphatidylglycerol (PG). This is a minor component (about 10%) of lung surfactant phospholipids. However, because PG is synthesized almost entirely by mature lung alveolar cells, it is a good indicator of lung maturity. Because PG appears late in gestation, this test indicates a more mature surfactant than that found in the L/S ratio described previously. In healthy pregnant women, PG appears in amniotic fluid after 35 weeks of gestation, and levels gradually increase until term. An advantage of the PG assay is that it is not affected by contamination of amniotic fluid by blood or meconium. These two contaminants cause false-positive and false-negative results for the L/S ratio evaluation. In addition, the presence of PG in the amniotic fluid in the vagina after the membranes are ruptured indicates a low risk for RDS of the newborn. The simultaneous determination of the L/S ratio and the presence of PG is an excellent method of assessing fetal maturity based on pulmonary surfactant. c. Lamellar body count. This newer test to determine fetal maturity is also based on the presence of surfactant. Lamellar bodies are concentrically layered structures produced by type II pneumocytes. On cross section, these small (about 3 μm) structures look like an onion. These lamellar bodies represent the storage form of pulmonary surfactant. Because lamellar bodies and platelets are indistinguishable to cell counters, the lamellar body count is obtained by analyzing the amniotic fluid with a cell counter and recording the platelet count. Lamellar body results are calculated in units of particle density per microliter of amniotic fluid. Some researchers have recommended cutoffs of 30,000/μL and 10,000/μL to predict low and high risk for RDS, respectively. If the count is greater than 30,000/μL, the negative predictive value for RDS is 100% (i.e., there is a 100% chance that the infant’s lungs are mature enough to not experience RDS). If the lamellar body count is less than 10,000/μL, the probability of RDS is high (67%). Values between 10,000/μL and 30,000/mcL represent intermediate risk for RDS. At this time, not enough information is available on lamellar body count in diabetics to advocate its use in this high-risk group. There are several advantages of lamellar body counts. First, they are faster, more precise, and more objective, and they require less amniotic fluid than phospholipid analysis. Second, test results are not invalidated by the presence of blood or meconium. Third, the instrumentation required for this test is readily available, thus allowing it to be performed in all laboratories. d. Microviscosity. Microvisocity in lipid aggregates is dependent on the L/S ratio and the degree of saturation of fatty acid side chains. The pattern of change of amniotic fluid microviscosity during gestation parallels the expected development of the surfactant system. Amniotic fluid microviscosity is high during early gestation and abruptly and sequentially decreases between the 28th and 36th week of gestation. The measurements are an accurate reflection of the development of the surfactant system and thereby fetal lung maturity. With the development of more accurate testing such as FLM as described above, this testing is no longer routinely performed and is included here more for recent historical value.
2. Sex of the fetus. Sons of mothers who are known to be carriers of X-linked recessive traits have a 50:50 risk of inheritance. It is important to note that amniocentesis is not done to determine the sex of the child just out of interest.
3. Genetic and chromosomal aberrations, such as hemophilia, Down syndrome, and galactosemia. Genetic and chromosomal studies performed on cells aspirated within the amniotic fluid can indicate the gender of the fetus (important in sex-linked diseases such as hemophilia) or any of the described genetic and chromosomal aberrations (e.g., trisomy 21).
4. Fetal status affected by Rh isoimmunization. Mothers with Rh isoimmunization have a series of amniocentesis procedures during the second half of pregnancy to assess the level of bilirubin pigment in the amniotic fluid. The quantity of bilirubin is used to assess the severity of hemolytic anemia in Rh-sensitized pregnancy. The higher the amount of bilirubin, the lower is the amount of fetal hemoglobin. Amniocentesis is usually initiated at 24 to 25 weeks. This allows assessment of the severity of the disease and the status of the fetus. Early delivery or blood transfusion may be indicated. It is important to take into consideration the volume of amniotic fluid because bilirubin concentration will be affected by total fluid volume.
5. Hereditary metabolic disorders, such as cystic fibrosis.
6. Anatomic abnormalities, such as neural tube closure defects (myelomeningocele, anencephaly, spina bifida). Increased levels of alpha-fetoprotein (AFP) in the amniotic fluid may indicate a neural crest abnormality. Decreased levels of AFP may be associated with increased risk of trisomy 21.
7. Fetal distress, detected by meconium staining of the amniotic fluid. This is caused by relaxation of the anal sphincter. In this case the normally colorless and pale, straw-colored amniotic fluid may be tinged with green. Other color changes may also indicate fetal distress. For example, a yellow discoloration may indicate a blood incompatibility. A yellow-brown opaque appearance may indicate intrauterine death. A red color indicates blood contamination from either the mother or the fetus.
Amniocentesis may be done on the premise that elective abortion could be performed if the fetus is severely defective. Chorionic villus sampling (CVS) may be even better than amniocentesis for karyotyping and genetic analysis. CVS can be performed earlier in the pregnancy than can amniocentesis. (The earliest one can obtain amniotic fluid is at about 12 to 14 weeks.) Thus with CVS a decision can be made concerning abortion much earlier in the pregnancy than with amniocentesis.
The timing of the amniocentesis varies according to the clinical circumstances. With advanced maternal age and if chromosomal or genetic aberrations are suspected, the test should be done early enough to allow a safe abortion. If information on fetal maturity is sought, performing the study during or after the thirty-fifth week of gestation is best. Placental localization by ultrasonography should be done before amniocentesis to avoid the needle passing into the placenta, possibly interrupting the placenta, and inducing bleeding or abortion.
When not to Perform Amniocentesis
Amniocentesis shouldn’t be used in the following conditions since it may lead to harmful complications:
- Mothers with Abruptio Placentae.
- Mothers with Placenta Previa.
- Mothers with a history of Premature Labor (before 34 weeks of gestation, unless the patient is receiving anti-labor medication).
- Mothers with an Incompetent Cervix.
Amniocentesis may lead to complications that may be harmful for both the baby and the mother if it is not performed properly. The following is a list of possible complications of Amniocentesis:
- Fetal Injury.
- Leak of Amniotic Fluid.
- Infection (Amnionitis).
- Premature Labor.
- Maternal Hemorrhage with possible Maternal Rh Isoimmunization.
- Amniotic Fluid Embolism.
- Abruptio Placentae.
- Inadvertent damage to the bladder or intestines.
Causes of Amniocentesis False Findings
- Fetal blood contamination can cause falsely elevated AFP levels.
- Hemolysis of the specimen can alter results.
- Contamination of the specimen with meconium or blood may result in inaccurate L/S ratios.
Amniocentesis procedure procedure takes approximately 20 to 30 minutes. Due to the fear of fetal injury, many mothers are extremely anxious during this procedure. The mother’s fears need to be addressed, expressed, and discussed before the procedure.
The discomfort associated with amniocentesis is usually described as a mild uterine cramping that occurs when the needle contacts the uterus. Some mothers may complain of a “pulling” sensation as the amniotic fluid is withdrawn.
The following are the basic steps taken to perform Amniocentesis:
- The mother blood pressure and the Fetal Heart Rate need to be evaluated before starting the procedure.
- Before 20 weeks of gestation, the bladder may be kept full to support the uterus. After 20 weeks, the bladder may be emptied to minimize the chance of puncture.
- The placenta is localized by ultrasound examination in order to permit selection of a site that will avoid placental puncture. Then the mother is placed in
- The skin overlying the chosen site, which is often determined by obstetric ultrasonography, is prepared and usually anesthetized locally.
- A needle with a stylet is inserted through the midabdominal wall and directed at an angle toward the middle of the uterine cavity.
- The stylet is then removed and a sterile plastic syringe attached.
- After 5 to 10 mL of amniotic fluid is withdrawn, the needle is removed. This fluid volume is naturally replaced by newly formed amniotic fluid within 3 to 4 hours after the procedure.
- The specimen is placed in a light-resistant container to prevent breakdown of bilirubin.
- The site is covered with an adhesive bandage.
- If the amniotic fluid is bloody, the physician must determine whether the blood is maternal or fetal in origin. Kleihauer-Böetke stain is used to recognize Fetal Hemoglobin (Hemoglboin F), it will stain fetal cells pink if blood belongs the fetus is found.
- Amniotic fluid volume is calculated by injecting a known concentration of solute (such as para-aminohippuric acid [PAH]) into the amniotic fluid to distribute throughout the amniotic fluid. Amniotic fluid is then withdrawn, and the PAH concentration is determined.
Amniocentesis Analysis and Indications
Hemolytic Disease of the Newborn: This may be apparent as increased bilirubin in the amniotic fluid. The fetal hemolysis causes free heme to form. This is then catabolized to bilirubin.
Rh Isoimmunization: A rising anti-Rh antibody titer in an Rh-negative woman would indicate potential for erythroblastosis fetalis (Rh-positive fetus). The higher the bilirubin in the amniotic fluid, the greater is the risk to the fetus.
A High AFP level most commonly indicates Neural Tube Closure Defects including Myelomeningocele, Anencephaly, and Spina Bifida. However, Abdominal Wall Closure Defects (e.g., Gastroschisis, Omphalocele) and Sacrococcygeal Teratoma can also cause High AFP Levels. Neoplasms associated with neural tube defects may also be associated with increased AFP levels. Blood levels of AFP are also increased with these abnormalities.
Meconium staining: This is evidence of fetal distress and is noted as greenish staining of the amniotic fluid.
Immature Fetal Lungs: This may occur with premature labor, maternal hypertension, or placental injuries. The risk of RDS increases as evidence of fetal lung immaturity increases. Fetal lung maturity is diminished in diabetic mothers. This is also noted in hydrops fetalis.
The genetic defects of many diseases can be recognized through gene recognition and karyotyping. Other genetic defects causing metabolic disorders can be recognized by the results of protein analysis of the amniotic fluid. Genetic defects that can be detected by studying the amniotic fluid include:
- Hereditary Metabolic Disorders (e.g., Cystic Fibrosis, Tay-Sachs Disease, Galactosemia).
- Genetic or Chromosomal Aberrations (e.g., Sickle Cell Anemia, Thalassemia, Down Syndrome).
- Sex-linked Disorders (e.g., Hemophilia).
Polyhydramnios: This occurs in patients who have diabetes. When polyhydramnios (>2000 mL) is present, the risk of congenital aberrations increases significantly.
Oligohydramnios: This is recognized as less than 300 mL of amniotic fluid at 25 weeks gestation. It is associated with fetal renal diseases. Near term, it is associated with early membrane rupture, intrauterine growth restriction, or significant postterm pregnancy.