Amniotic Fluid Analysis and L/S Ratio


To assist in identification of fetal gender, genetic disorders such as hemophilia and sickle cell anemia, chromosomal disorders such as Down syndrome, anatomical abnormalities such as spina bifida, alloimmune hemolytic disease of the newborn (HDN), and hereditary metabolic disorders such as cystic fibrosis. To assess for preterm infant fetal lung maturity to assist in evaluating for potential diagnosis of respiratory distress syndrome (RDS).

Patient Preparation
There are no food, fluid, activity, or medication restrictions unless by medical direction.

Normal Findings
Method: Macroscopic observation of fluid for color and appearance, immunochemiluminometric assay (ICMA) for α1-fetoprotein, electrophoresis for acetylcholinesterase, spectrophotometry for Cr, bilirubin, and chromatography for lecithin/sphingomyelin (L/S) ratio and phosphatidylglycerol, tissue culture for chromosome analysis, dipstick for leukocyte esterase, and automated cell counter for white blood cell count and lamellar bodies.

TestReference Value
Color Colorless to pale yellow
α1-Fetoprotein (AFP)Less than 2 MoM
Cr 1.8–4 mg/dL at term (159.1–353.6 micromol/L) (SI Units = Conventional Units × 88.4)
Bilirubin Less than 0.075 mg/dL in early pregnancy (Less than 1.28 micromol/L) (SI Units = Conventional Units × 17.1)
Less than 0.025 mg/dL at term (Less than 0.428 micromol/L) (SI Units = Conventional Units × 17.1)
Bilirubin ΔOD450Less than 0.05 ΔOD in early to mid-pregnancy (approximately 14–27 weeks, using the Queenan curve when gestational age is less than 27 weeks)
Less than 0.06 ΔOD in late pregnancy (approximately 28–36 weeks, using the Liley Chart when gestational age is equal to or greater than 27 weeks)
Less than 0.03 ΔOD at term (approximately 37–40 weeks, using the Liley Chart when gestational age is equal to or greater than 27 weeks)
L/S ratio
 Mature (nondiabetic)Greater than 2:1 in the presence of phosphatidyl glycerol
 Borderline1.5 to 1.9:1
 ImmatureLess than 1.5:1
PhosphatidylglycerolPresent at term
Chromosome analysisNormal karyotype
White blood cell countNone seen
Leukocyte esteraseNegative
Lamellar bodiesFindings and interpretive ranges vary depending on the type of instrument used
MoM = Multiples of the median.

Critical Findings and Potential Interventions

  • An L/S ratio less than 1.5:1 is predictive of RDS at the time of delivery.

Timely notification to the requesting health-care provider (HCP) of any critical findings and related symptoms is a role expectation of the professional nurse. A listing of these findings varies among facilities.

Infants known to be at risk for RDS can be treated with surfactant by intratracheal administration at birth.


(Study type: Body fluid, amniotic fluid collected in a clean amber (glass or plastic) container; related body system: Reproductive system.) Amniotic fluid is formed in the membranous sac that surrounds the fetus. The total volume of fluid at term is 500 to 2,500 mL. In amniocentesis, fluid is obtained by ultrasound-guided needle aspiration from the amniotic sac. This procedure is generally performed between 14 and 16 weeks’ gestation for accurate interpretation of test results, but it also can be done between 26 and 35 weeks’ gestation if fetal distress is suspected. Fluid is tested to identify fetal genetic and neural tube defects, infection, renal malfunction, lung maturity, and hemolytic diseases of the newborn. Examples of genetic defects commonly tested for and identifiable from a sample of amniotic fluid include sickle cell anemia, cystic fibrosis, and inborn errors of metabolism.

Available rapid tests can be used to differentiate between amniotic fluid and other body fluids in a vaginal specimen collection. Nitrazine paper impregnated with an indicator dye will produce a color change indicative of vaginal pH. Normal vaginal pH is acidic (4.5–6), and the color of the paper will not change. Amniotic fluid has an alkaline pH (7.1–7.3), and the paper will turn blue. False-positive results occur in the presence of semen, blood, alkaline urine, vaginal infection, or antibiotic treatment. Amniotic fluid crystallization, or fern test, is the observation of a fern pattern when fluid air dries on a glass slide. The fern pattern is from the protein and sodium chloride content of the amniotic fluid. False-positive results occur in the presence of blood, urine, or cervical mucus. Both tests can produce false-negative results when a small amount of fluid is leaked. Result reliability is significantly diminished with the passage of time (greater than 24 hr). AmniSure is an immunoassay performed using a vaginal swab sample. This rapid test detects placental alpha microglobulin-1 (PAMG-1) protein, found in high concentrations in amniotic fluid. AmniSure does not have the high frequency of false-positive and false-negative results found with the pH and fern tests.

RDS is the most common problem encountered in the care of premature infants. RDS, also called hyaline membrane disease, results from a deficiency of phospholipid lung surfactants. The phospholipids in surfactant are produced by specialized alveolar cells and stored in granular lamellar bodies in the lung. In normally developed lungs, surfactant coats the surface of the alveoli. Surfactant reduces the surface tension of the alveolar wall during breathing. When there is an insufficient quantity of surfactant, the alveoli are unable to expand normally, and gas exchange is inhibited. Amniocentesis, a procedure by which fluid is removed from the amniotic sac, is used to assess fetal lung maturity.

Lecithin is the primary surfactant phospholipid, and it is a stabilizing factor for the alveoli. It is produced at a low but constant rate until the 35th wk of gestation, after which its production sharply increases. Sphingomyelin, another phospholipid component of surfactant, is also produced at a constant rate after the 26th wk of gestation. Before the 35th wk, the L/S ratio is usually less than 1.6:1. The ratio increases to 2 or greater when the rate of lecithin production increases after the 35th wk of gestation. Other phospholipids, such as phosphatidyl glycerol (PG) and phosphatidyl inositol (PI), increase over time in amniotic fluid as well. The presence of PG indicates that the fetus is within 2 to 6 wk of lung maturity (i.e., at full term). Simultaneous measurement of PG with the L/S ratio improves diagnostic accuracy. Production of phospholipid surfactant is delayed in mothers with diabetes. Therefore, caution must be used when interpreting the results obtained from a patient who is diabetic, and a higher ratio is expected to predict maturity.

HDN, also called erythroblastosis fetalis, is a condition that occurs after red blood cells (RBCs) from an Rh negative mother become sensitized by fetal RBCs from an Rh positive baby. Rh sensitization of the mother can result from a miscarriage, trauma such as a fall or blow to the abdominal area, after an invasive prenatal test (such as amniocentesis), or when the placenta detaches during birth. The mother’s immune system recognizes the baby’s RBCs as foreign and makes antibodies that cause the fetal RBCs to hemolyze. Bilirubin is a breakdown product of Hgb, the oxygen carrying protein in RBCs. Bilirubin measurements from amniotic fluid are used to screen for hemolysis in high risk situations, sometimes serial measurements are required to monitor elevated measurements or therapeutic interventions such as administration of RhIG, fetal transfusions, or a decision to deliver the baby.

Testing for common genetically transferred conditions can be performed on either or both prospective parents by blood tests, skin tests, or DNA testing. DNA testing can also be performed on the fetus, in utero, through the collection of fetal cells by amniocentesis or chorionic villus sampling. Genetics is the study and identification of genes, genetic mutations, and inheritance. For example, genetics provides some insight into the likelihood of inheriting a medical condition such as cystic fibrosis or of errors of amino acid metabolism. Knowledge of genetics assists in identifying those who may benefit from additional education, risk assessment, and counseling. Further information regarding inheritance of genes can be found in the study titled “Genetic Testing.” Counseling and written, informed consent are recommended and sometimes required before genetic testing.


  • Assist in the diagnosis of (in utero) metabolic disorders, such as cystic fibrosis, or errors of lipid, carbohydrate, or amino acid metabolism.
  • Assist in the evaluation of fetal lung maturity when preterm delivery is being considered.
  • Detect infection secondary to ruptured membranes.
  • Detect fetal ventral wall defects.
  • Determine the optimal time for obstetric intervention in cases of threatened fetal survival caused by stresses related to maternal diabetes, toxemia, hemolytic diseases of the newborn, or postmaturity.
  • Determine fetal gender when the mother is a known carrier of a sex-linked abnormal gene that could be transmitted to male offspring, such as hemophilia or Duchenne muscular dystrophy.
  • Determine the presence of fetal distress in late-stage pregnancy.
  • Evaluate fetus in families with a history of genetic disorders, such as Down syndrome, Tay-Sachs disease, chromosome or enzyme anomalies, or inherited hemoglobinopathies.
  • Evaluate fetus in mothers of advanced maternal age (some of the aforementioned tests are routinely requested in mothers age 35 and older).
  • Evaluate fetus in mothers with a history of miscarriage or stillbirth.
  • Evaluate known or suspected hemolytic disease involving the fetus in an Rh-sensitized pregnancy, indicated by rising bilirubin levels, especially after the 30th wk of gestation.
  • Evaluate suspected neural tube defects, such as spina bifida or myelomeningocele, as indicated by elevated α1-fetoprotein (see study titled “Maternal Markers” for information related to triple-marker testing).
  • Identify fetuses at risk of developing RDS.

Interfering Factors


Women with a history of premature labor, incompetent cervix, or in the presence of placenta previa or abruptio placentae. There is some risk to having an amniocentesis performed, and the risk should be weighed against the need to obtain the desired diagnostic information. A small percentage (0.5%) of patients have experienced complications including premature rupture of membranes (PROM), premature labor, spontaneous abortion, and stillbirth.

Factors that may alter the results of the study

  • Bilirubin may be falsely elevated if maternal Hgb or meconium is present in the sample; fetal acidosis may also lead to falsely elevated bilirubin levels.
  • Bilirubin may be falsely decreased if the sample is exposed to light or if amniotic fluid volume is excessive.
  • Maternal serum Cr should be measured simultaneously for comparison with amniotic fluid Cr for proper interpretation. Even in circumstances in which the maternal serum value is normal, the results of the amniotic fluid Cr may be misleading. A high fluid Cr value in the fetus of a mother who is diabetic may reflect the increased muscle mass of a larger fetus. If the fetus is big, the Cr may be high, and the fetus still may have immature kidneys.
  • Contamination of the sample with blood or meconium or complications in pregnancy may yield inaccurate L/S ratios; fetal blood falsely elevates the L/S ratio.
  • α1-Fetoprotein and acetylcholinesterase may be falsely elevated if the sample is contaminated with fetal blood.
  • Karyotyping cannot be performed under the following conditions: (1) failure to promptly deliver samples for chromosomal analysis to the laboratory performing the test or (2) improper incubation of the sample, which causes cell death.

Potential Medical Diagnosis: Clinical Significance of Results

  • Yellow, green, red, or brown fluid indicates the presence of bilirubin, blood (fetal or maternal), or meconium, which indicate fetal distress or death, hemolytic disease, or growth retardation.
  • Elevated bilirubin levels indicate fetal hemolytic disease or intestinal obstruction. Measurement of bilirubin usually is not performed before 20 to 24 weeks’ gestation because no action can be taken before then. The severity of hemolytic disease is graded by optical density (OD) zones. A trend of increasing values with serial measurements may indicate the need for intrauterine transfusion or early delivery, depending on the fetal age. After 32 to 33 weeks’ gestation, early delivery is preferred over intrauterine transfusion because early delivery is more effective in providing the required care to the neonate.
  • Cr concentration greater than 2 mg/dL (greater than 176.8 micromol/L) (SI Units = Conventional Units × 88.4)indicates fetal maturity (at 36–37 wk) if maternal Cr is also within the expected range. This value should be interpreted in conjunction with other parameters evaluated in amniotic fluid and especially with the L/S ratio because normal lung development depends on normal kidney development.
  • An L/S ratio less than 2:1 and absence of phosphatidylglycerol at term indicate fetal lung immaturity and possible respiratory distress syndrome. Other conditions that decrease production of surfactants include advanced maternal age, multiple gestation, and polyhydramnios. Conditions that may increase production of surfactant include hypertension, intrauterine growth retardation, malnutrition, maternal diabetes, placenta previa, placental infarction, and premature rupture of the membranes. The expected L/S ratio for the fetus of a mother who is diabetic is higher (3.5:1).
  • Lamellar bodies are specialized alveolar cells in which lung surfactant is stored. They are approximately the size of platelets. Their presence in sufficient quantities is an indicator of fetal lung maturity. Amniotic fluid lamellar body counts less than 15,000/microL are suggestive of immature lung development and predictive for increased risk of developing RDS; counts greater than 50,000/microL are predictive of mature lung development.
  • Elevated AFP levels and presence of acetylcholinesterase may indicate a neural tube defect (see study titled “Maternal Markers”) related to leakage from the open spinal cord into the amniotic fluid. Elevation of AFP and/or acetylcholinesterase is also indicative of ventral wall defects. The presence of AFP and acetylcholinesterase in amniotic fluid is abnormal; however, the test is not a sensitive marker for neural tube defects; false-positive results can be caused by contamination of the sample with fetal blood, which normally contains measurable levels of AFP and acetylcholinesterase. Abnormal results can be confirmed by testing the amniotic fluid for the presence of fetal Hgb (Hgb F). If Hgb F is detected, then the specimen is likely contaminated with fetal blood and the results are unreliable.
  • Abnormal karyotype indicates genetic abnormality (e.g., Tay-Sachs disease, intellectual disability, chromosome or enzyme anomalies, and inherited hemoglobinopathies). (See study titled “Chromosome Analysis, Blood.”)
  • Elevated white blood cell count and positive leukocyte esterase are indicators of infection.

Nursing Implications

Potential Nursing Problems Assessment and Nursing Diagnosis

ProblemsSigns and Symptoms
Fear (related to fetal imperfections secondary to developmental abnormality)Anxiety; restlessness; sleeplessness; increased tension; continuous questioning; increased blood pressure, heart rate, respiratory rate; apprehension; identifies cause of fear
Knowledge (related to insufficient information associated with diagnosed developmental abnormality, lack of familiarity or understanding of disease and treatment)Lack of interest or questions, multiple questions, anxiety related to disease and management, stating inaccurate information, frustration; confusion
Spirituality (related to anxiety associated with fetal developmental abnormality, unexpected life changes)Anger; stated feelings of lack of peace or serenity, alienation from others, hopelessness, request to meet with spiritual leader, lack of acceptance

Before the Study: Planning and Implementation

Teaching the Patient What to Expect

  • Explain that this procedure can assist in evaluation of fetal well-being.
  • Explain that an amniotic fluid sample is needed for the test. Address concerns about pain, and explain there may be some discomfort during the amniocentesis.
  • Amniocentesis takes about 20 to 30 min.
  • Partial disrobing will be required with removal of clothes below the waist before being assisted to a supine position on the examination table with the abdomen exposed.
  • Proper positioning may require that the head or legs be raised slightly to promote comfort and to relax the abdominal muscles. A pillow or rolled blanket may be placed under the right side to prevent hypertension caused by great-vessel compression with a large uterus.
  • Maternal and fetal baseline vital signs will be recorded. Maternal and fetal vital signs and uterine contractions should be monitored throughout the procedure. Fetal vital signs will be monitored using ultrasound. Protocols may vary among facilities.
  • Ultrasound will be used to assess the position of the amniotic fluid, fetus, and placenta.
  • Sterile technique is used during the procedure after cleaning the suprapubic area with an antiseptic solution and protecting the site with sterile drapes.
  • A local anesthetic is injected prior to amniocentesis and may cause a stinging sensation. A sensation of pressure may be experienced when a spinal needle is inserted through the abdominal and uterine walls. Focused and controlled breathing assist with relaxation during the procedure.
  • After the procedure, slight pressure is applied to the site after the fluid is collected and the needle is withdrawn. If there is no evidence of bleeding or other drainage, a sterile adhesive bandage is applied to the site.

Potential Nursing Actions

  • Verify maternal Rh type results. If Rh-negative, check for prior sensitization. Assess for a family history of genetic disorders. Record the date of the last menstrual period and determine the pregnancy weeks’ gestation and expected delivery date.

Make sure a written and informed consent has been signed prior to the procedure and before administering any medications.

  • Instruct patients less than 20 weeks’ gestation to drink extra fluids 1 hr before the test and to refrain from urination. A full bladder raises the uterus up and out of the way for easier visualization during the ultrasound procedure.
  • Instruct patients 20 weeks’ gestation or more to void before the test. An empty bladder is less likely to be accidentally punctured during specimen collection.
  • As required, assemble the necessary equipment, including an amniocentesis tray with solution for skin preparation, local anesthetic, 10- or 20-mL syringe, needles of various sizes (including a 22-gauge, 5-in. spinal needle), or as requested by the HCP. Also obtain sterile drapes, sterile gloves, and foil-covered or amber-colored specimen collection containers or other light-resistant containers to prevent bilirubin breakdown. Extra needles should be available for replacement in the event of needle contamination.
  • To decrease anxiety explain that there may be a feeling of cramping when the needle is inserted and pulling as the fluid sample is removed.

After the Study: Potential Nursing Actions

Avoiding Complications

  • Hemorrhage or infection can occur following amniocentesis. Instruct the patient to observe for and report excessive bleeding, redness of skin, fever, or chills. Maternal Rh sensitization can result from fetal red blood cells (RBCs) mixing with blood of an RhD-negative mother carrying an RhD-positive fetus. RhIG or RhoGAM may be administered after amniocentesis to RhD-negative mothers to prevent formation of Rh antibodies. The RhD antigen is the most potent RBC antigenic initiator of in vivo hemolysis. For additional information on prevention of hemolytic disease of the newborn refer to the study titled, “Indirect Antiglobulin Testing.”
  • Monitor the patient for complications related to the procedure (e.g., premature labor, infection, leakage of amniotic fluid). The patient should report moderate to severe abdominal pain or cramps, change in fetal activity, increased or prolonged leaking of amniotic fluid from abdominal needle site, vaginal bleeding that is heavier than spotting, and chills or fever. Observe/assess the amniocentesis site for bleeding, inflammation, or hematoma formation per facility protocol.

Treatment Considerations

  • Compare fetal heart rate and maternal life signs (i.e., heart rate, blood pressure, pulse, and respiration) with baseline values and closely monitor every 15 min for 30 to 60 min after the amniocentesis procedure or per facility protocol.
  • Administer, as ordered, standard RhIG or RhoGAM dose to maternal Rh-negative patients to prevent maternal Rh sensitization should the fetus be Rh-positive.
  • Instruct the patient to rest until all symptoms have disappeared before resuming normal levels of activity.
  • Explain that mild cramping, leakage of small amounts of amniotic fluid, and vaginal spotting can occur for up to 2 days following the procedure.

Followup Evaluation and Desired Outcomes

  • Acknowledges contact information provided for counseling related to pregnancy termination and genetic counseling or access to support groups in relation to diagnosed developmental disability if a chromosomal abnormality is determined.
  • Describes the care and lifestyle changes necessary to support healthy development of the disabled infant.
  • Accepts that normal results do not guarantee a healthy fetus. Provide a nonjudgmental, nonthreatening atmosphere for discussing the risks and difficulties of delivering and raising a developmentally challenged infant as well as for exploring other options (termination of pregnancy or adoption). It is also important to discuss problems the mother and father may experience (guilt, depression, anger) if fetal abnormalities are detected.

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