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 (FLM) 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 alpha1-fetoprotein, electrophoresis for acetylcholinesterase, chromatography for lecithin/sphingomyelin (L/S) ratio and phosphatidylglycerol, cell culture for chromosome analysis, dipstick for leukocyte esterase, and automated cell counter for WBC count and lamellar bodies.

TestReference Value
ColorColorless to pale yellow
Alpha1-Fetoprotein (AFP)Less than or equal to 2 MoM
Acetylcholinesterase (in conjunction with AFP indicative of possible neural tube defect)Absent
Bilirubin ΔOD450 (indicative of HDN)Less 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 (indicative of FLM)
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 analysis (indicative of inherited genetic mutations)Normal karyotype
White blood cell count (indicative of infection)None seen
Leukocyte esterase (indicative of infection)Negative
Lamellar bodies (indicative of FLM)Findings 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.)

Evaluation of amniotic fluid
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, 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.

Studies that evaluate lung maturity
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.

Studies used to identify HDN
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.

The inheritance pattern of the RhD antigen is autosomal dominant; homozygotes (DD) will always pass the RhD antigen gene on to their offspring and heterozygotes (Dd) will pass the RhD antigen gene to their offspring with a probability of 50%. RhD genotyping is recommended when a father’s Rh type is unknown (e.g., paternity cannot be positively confirmed at the time the mother undergoes her prenatal blood work). For additional information regarding RhD genotyping, refer to the study titled “Coombs antiglobulin, indirect.”

Studies used to identify inherited disorders
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. The American College of Obstetricians and Gynecologists recommends chromosomal microarray instead of a fetal karyotype in patients with a pregnancy demonstrating one or more major structural abnormalities on ultrasound when undergoing chorionic villus tissue sampling or amniocentesis. Microarray can also be considered when undergoing invasive diagnostic testing (chorionic villus tissue sampling or amniocentesis) with a normal fetus. For additional information refer to the studies titled “Biopsy Chorionic Villus, Chromosome Analysis, and Maternal Markers.”

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 alpha1-fetoprotein (see study titled “Maternal Markers” for information related to triple- and quad-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.
  • 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.
  • Alpha1-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.
  • 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, Nursing Process, Clinical Judgement

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

  • Discuss how the amniocentesis procedure can assist in evaluation of fetal well-being.
  • Explain that a sample of amniotic fluid is needed for the test and that the procedure takes about 20 to 30 min.
  • Review the procedure with the patient.
  • Provide assurance that precautions to avoid injury to the fetus will be taken by locating the fetus with ultrasound.
  • Explain that removal of clothes below the waist will be required.
  • Address cultural issues regarding modesty.
  • Address concerns about pain, and explain there may be some discomfort during the amniocentesis.
  • Direct the patient to breathe normally and to avoid unnecessary movement during the local anesthetic and the procedure.
  • Explain that a local anesthetic is injected prior to amniocentesis and may cause a stinging sensation.
  • Advise that a sensation of pressure may be experienced when a needle is inserted through the abdominal and uterine walls. A slight pop may be felt.
  • Discuss how focused and controlled breathing assist with relaxation during the procedure.
  • Confirm that informed consent has been obtained prior to the procedure.

Procedural Information

  • HCP-requested equipment is assembled (amniocentesis tray, extra needles for replacement in the event of needle contamination, and foil-covered, amber-colored, or other light-resistant containers for specimen collection to prevent bilirubin breakdown.
  • The patient is assisted to the supine position on the examination table which may be elevated 20° to 30°; alternatively, the patient may be assisted to a slight left lateral decubitus position or dorsal lithotomy position to promote comfort and to relax the abdominal muscles, as directed by HCP and as clinically indicated.
  • Baseline maternal and fetal vital signs are taken and recorded.
  • Ultrasound is used to assess the position of the amniotic fluid, fetus, and placenta.
  • A local anesthetic is applied prior to amniocentesis.
  • After the suprapubic area is cleaned with an antiseptic solution the site is protected with sterile drapes; sterile technique is used during the procedure.
  • Maternal and fetal vital signs and uterine contractions are monitored throughout the procedure.
  • Organizational policy is followed for obtaining, labeling, and transporting samples.
  • After the fluid is collected, slight pressure is applied to the insertion site and the needle is withdrawn.
  • If there is no evidence of bleeding or other drainage a sterile adhesive bandage is applied to the site.
  • Protocols may vary among facilities.

Potential Nursing Actions

  • Verify maternal Rh type results.
  • Check for prior sensitization if Rh-negative (prenatal bloodwork includes an antibody screen [indirect coombs antiglobulin test]). Administration of Rh-immune globulin may be required.
  • Assess for a family history of genetic disorders.
  • 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.
  • 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.

After the Study: Implementation & Evaluation Potential Nursing Actions

Treatment Considerations

  • Use active listening to understand the nature of the fear.
  • Assess effectiveness of coping strategies.
  • Use simple easily understandable language when addressing fear concerns.
  • Provide referrals for support groups as applies to the specific clinical situation.


  • Provide simple straight forward information that is appropriate to age, culture, and literacy.
  • Reference valid written or streaming services that can provide ongoing support.
  • Be available to answer all questions.


  • Facilitate support of spiritual leaders.
  • Facilitate privacy for prayer, meditation, and reflection as desired.
  • Support desire to find meaning in clinical challenges with support groups or other referrals.

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. In this case the Rh positive RBCs of the fetus can sensitize the mother’s immune system to stimulate production of maternal anti-D antibodies.
  • 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 “Coombs Antiglobulin, Indirect.
  • Monitor for complications related to the procedure (e.g., premature labor, infection, leakage of amniotic fluid).
  • Advise reporting 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 for development of serious complications to include spontaneous abortion, membrane rupture, placenta abruption, fetal injury, fetal hemorrhage, and maternal disease transmission.
  • Observe/assess the abdominal 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.

Clinical Judgement

  • Consider how to mitigate the emotional trauma associated with an undesirable result thereby assisting the family to cope with the reality of a negative outcome.

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.