Amniotic Fluid Analysis and L/S Ratio

Synonym/acronym: N/A.

Common use

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, 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).

Specimen

Amniotic fluid (10 to 20 mL) collected in a clean amber glass or plastic container.

Normal findings

(Method: Macroscopic observation of fluid for color and appearance, immunochemiluminometric assay [ICMA] for α₁-fetoprotein, electrophoresis for acetylcholinesterase, spectrophotometry for creatinine and bilirubin, 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)

Test	Reference Value
Color	Colorless to pale yellow
Appearance	Clear
α₁-Fetoprotein	Less than 2 MoM*
Acetylcholinesterase	Absent
Creatinine	1.8–4 mg/dL at term
Bilirubin	Less than 0.075 mg/dL in early pregnancy
	Less than 0.025 mg/dL at term
Bilirubin ΔA₄₅₀	Less than 0.048 ΔOD in early pregnancy
	Less than 0.02 ΔOD at term
L/S ratio
Mature (nondiabetic)	Greater than 2:1 in the presence of phosphatidyl glycerol
Borderline	1.5 to 1.9:1
Immature	Less than 1.5:1
Phosphatidylglycerol	Present at term
Chromosome analysis	Normal karyotype
White blood cell count	None seen
Leukocyte esterase	Negative
Lamellar bodies	Findings and interpretive ranges vary depending on the type of instrument used

*MoM = Multiples of the median.

Description

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. Amniotic fluid is tested to identify genetic and neural tube defects, hemolytic diseases of the newborn, fetal infection, fetal renal malfunction, or maturity of the fetal lungs. Examples of genetic defects that are commonly tested for and can be identified from a sample of amniotic fluid include sickle cell anemia, cystic fibrosis, and inborn errors of metabolism. Several rapid tests are also used to differentiate amniotic fluid from other body fluids in a vaginal specimen when premature rupture of membranes (PROM) is suspected. A vaginal swab obtained from the posterior vaginal pool can be used to perform a rapid, waived procedure to aid in the assessment of PROM. Nitrazine paper impregnated with an indicator dye will produce a color change indicative of vaginal pH. Normal vaginal pH is acidic (4.5 to 6) and the color of the paper will not change. Amniotic fluid has an alkaline pH (7.1 to 7.3) and the paper will turn a blue color. False-positive results occur in the presence of semen, blood, alkaline urine, vaginal infection, or if the patient is receiving antibiotics. The amniotic fluid crystallization or Fern test is based on the observation of a fern pattern when amniotic fluid is placed on a glass slide and allowed to air dry. The fern pattern is due to the protein and sodium chloride content of the amniotic fluid. False-positive results occur in the presence of blood urine or cervical mucus. Both of these tests can produce false-negative results if only a small amount of fluid is leaked. The reliability of results is also significantly diminished with the passage of time (greater than 24 hr). AmniSure is an immunoassay that can be performed on a vaginal swab sample. It is a rapid test that detects placental alpha microglobulin-1 protein (PAMG-1), which is found in high concentrations in amniotic fluid. AmniSure does not demonstrate the high frequency of false-positive and false-negative results inherent with the pH and fern tests.

Respiratory distress syndrome (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 lecithin/sphingomyelin (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 diabetic mothers. Therefore, caution must be used when interpreting the results obtained from a diabetic patient, and a higher ratio is expected to predict maturity.

This procedure is contraindicated for

high alert 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 this 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 the membranes, premature labor, spontaneous abortion, and stillbirth.

Indications

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’s 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 week of gestation
Evaluate suspected neural tube defects, such as spina bifida or myelomeningocele, as indicated by elevated α₁-fetoprotein (see monograph titled “α₁-Fetoprotein” for information related to triple-marker testing)
Identify fetuses at risk of developing RDS

Potential diagnosis

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 is not usually 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 value of 0.28 to 0.46 OD at 28 to 31 weeks’ gestation indicates mild hemolytic disease, which probably will not affect the fetus; 0.47 to 0.9 OD indicates a moderate effect on the fetus; and 0.91 to 1 OD indicates a significant effect on the fetus. 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. Creatinine concentration greater than 2 mg/dL indicates fetal maturity (at 36 to 37 wk) if maternal creatinine 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 an insulin-dependent diabetic mother 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. Elevated α₁-fetoprotein levels and presence of acetylcholinesterase indicate a neural tube defect (see monograph titled “α₁-Fetoprotein”). Elevation of acetylcholinesterase is also indicative of ventral wall defects. Abnormal karyotype indicates genetic abnormality (e.g., Tay-Sachs disease, mental retardation, chromosome or enzyme anomalies, and inherited hemoglobinopathies). (See monograph titled “Chromosome Analysis, Blood.”) Elevated white blood cell count and positive leukocyte esterase are indicators of infection.

Critical findings

Note and immediately report to the health-care provider (HCP) any critically increased or decreased values and related symptoms.
It is essential that a critical finding be communicated immediately to the requesting HCP. A listing of these findings varies among facilities.
Timely notification of a critical finding for lab or diagnostic studies is a role expectation of the professional nurse. The notification processes will vary among facilities. Upon receipt of the critical finding the information should be read back to the caller to verify accuracy. Most policies require immediate notification of the primary HCP, hospitalist, or on-call HCP. Reported information includes the patient’s name, unique identifiers, critical finding, name of the person giving the report, and name of the person receiving the report. Documentation of notification should be made in the medical record with the name of the HCP notified, time and date of notification, and any orders received. Any delay in a timely report of a critical finding may require completion of a notification form with review by Risk Management.
Infants known to be at risk for RDS can be treated with surfactant by intratracheal administration at birth.

Interfering factors

Bilirubin may be falsely elevated if maternal hemoglobin 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 creatinine should be measured simultaneously for comparison with amniotic fluid creatinine for proper interpretation. Even in circumstances in which the maternal serum value is normal, the results of the amniotic fluid creatinine may be misleading. A high fluid creatinine value in the fetus of a diabetic mother may reflect the increased muscle mass of a larger fetus. If the fetus is big, the creatinine may be high, and the fetus may still 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.

Nursing Implications and Procedure

Potential nursing problems

Problem	Signs & Symptoms	Interventions
Fear (Related to fetal imperfections secondary to developmental abnormality)	Verbalization of fear; restlessness; increased tension; continuous questioning; increased blood pressure, heart rate, respiratory rate	Evaluate verbal and nonverbal indicators of fear; assess for the cause of fear; acknowledge the patient’s awareness of fear; explain all procedures with simple age and culturally appropriate language; administer proscribed mild tranquilizer; maintain a confident assured professional manner in all patient interactions; address concerns regarding care of disabled child; recommend support group and provide contact information
Spirituality (Related to anxiety associated with feral developmental abnormality; unexpected life changes)	Anger; stated feelings of lack of peace or serenity; stated feelings of alienation from others; stated feelings of hopelessness; request to meet with spiritual leader	Obtain a history of the patient’s religious affiliation; identify the patient’s willingness to meet with spiritual leader; encourage verbalization of concerns, feelings of fear and loneliness; acknowledge and support religious practices; accommodate a display of religious objects; facilitate communication between the patient, family, and religious leader
Knowledge (Related to insufficient information associated with diagnosed developmental abnormality; lack of familiarity or understanding with disease and treatment)	Lack of interest or questions; multiple questions; anxiety in relation to disease process and management; stating inaccurate information; frustration; confusion	Identify the primary learners and provide specific information that is culturally appropriate and to the correct literacy level; assess for the willingness and ability to learn; identify the patient’s priority for learning; identify and dispel any misconceptions associated with the developmental disability; identify the patient’s learning style; provide a quiet atmosphere for learning; allow the parents to be self-directed in their learning; provide sufficient time for questions and follow up; refer to a support group and social services as appropriate

Pretest

Positively identify the patient using at least two unique identifiers before providing care, treatment, or services.
Patient Teaching: Inform the parent this procedure/test can assist in providing a sample of fluid that will allow for evaluation of fetal well-being.
Obtain a history of the patient’s complaints, including a list of known allergens, especially allergies or sensitivities to latex or anesthetics.
Obtain a history of the patient’s reproductive system, previous pregnancies, symptoms, and results of previously performed laboratory tests and diagnostic and surgical procedures. Include any family history of genetic disorders such as cystic fibrosis, Duchenne’s muscular dystrophy, hemophilia, sickle cell disease, Tay-Sachs disease, thalassemia, and trisomy 21. Obtain maternal Rh type. If Rh-negative, check for prior sensitization. A standard dose of Rh₁(D) immune globulin RhoGAM IM or Rhophylac IM or IV is indicated after amniocentesis; repeat doses should be considered if repeated amniocentesis is performed.
Note any recent procedures that can interfere with test results.
Record the date of the last menstrual period and determine the pregnancy weeks’ gestation and expected delivery date.
Obtain a list of the patient’s current medications, including herbs, nutritional supplements, and nutraceuticals (see Effects of Natural Products on Laboratory Values online at DavisPlus).
Review the procedure with the patient. Warn the patient that normal results do not guarantee a normal fetus. Assure the patient that precautions to avoid injury to the fetus will be taken by localizing the fetus with ultrasound. Address concerns about pain and explain that during the transabdominal procedure, any discomfort associated with a needle biopsy will be minimized with local anesthetics. If the patient is less than 20 weeks’ gestation, instruct her to drink extra fluids 1 hr before the test and to refrain from urination. The full bladder assists in raising the uterus up and out of the way to provide better visualization during the ultrasound procedure. Patients who are at 20 weeks’ gestation or beyond do not need to drink extra fluids and should void before the test, because an empty bladder is less likely to be accidentally punctured during specimen collection. Encourage relaxation and controlled breathing during the procedure to aid in reducing any mild discomfort. Inform the patient that specimen collection is performed by a health-care provider (HCP) specializing in this procedure and usually takes approximately 20 to 30 min to complete.
Sensitivity to social and cultural issues, as well as concern for modesty, is important in providing psychological support before, during, and after the procedure.
Note that there are no food, fluid, or medication restrictions unless by medical direction.
Make sure a written and informed consent has been signed prior to the procedure and before administering any medications.

Intratest

Potential complications:
Hemorrhage from highly vascular tissue or infection following amniocentesis. Instruct the patient to look for excessive bleeding, redness of skin, fever, or chills and to notify the HCP if these symptoms occur. An additional risk with amniocentesis is maternal Rh sensitization by fetal RBCs in the case of an Rh-negative mother carrying an Rh-positive fetus. RhIG (Rh immune globulin) or RhoGam may be administered after amniocentesis to Rh-negative mothers to prevent formation of Rh antibodies.
Avoid the use of equipment containing latex if the patient has a history of allergic reaction to latex.
Ensure that the patient has a full bladder before the procedure if gestation is 20 wk or less; have patient void before the procedure if gestation is 21 wk or more.
Positively identify the patient, and label the appropriate collection containers with the corresponding patient demographics, date, time of collection, and site location.
Have patient remove clothes below the waist. Assist the patient to a supine position on the examination table with the abdomen exposed. Drape the patient’s legs, leaving the abdomen exposed. Raise her head or legs slightly to promote comfort and to relax the abdominal muscles. If the uterus is large, place a pillow or rolled blanket under the patient’s right side to prevent hypertension caused by great-vessel compression. Instruct the patient to cooperate fully and to follow directions. Direct the patient to breathe normally and to avoid unnecessary movement during the local anesthetic and the procedure.
Record maternal and fetal baseline vital signs, and continue to monitor throughout the procedure. Monitor for uterine contractions. Monitor fetal vital signs using ultrasound. Protocols may vary among facilities.
Have emergency equipment readily available.
Observe standard precautions, and follow the general guidelines in Patient Preparation and Specimen Collection. Positively identify the patient, and label the appropriate specimen container with the corresponding patient demographics, initials of the person collecting the specimen, date, and time of collection.
Assess the position of the amniotic fluid, fetus, and placenta using ultrasound.
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), sterile drapes, sterile gloves, and foil-covered or amber-colored specimen collection containers.
Cleanse suprapubic area with an antiseptic solution, and protect with sterile drapes. A local anesthetic is injected. Explain that this may cause a stinging sensation.
Insert a 22-gauge, 5-in. spinal needle through the abdominal and uterine walls. Explain that a sensation of pressure may be experienced when the needle is inserted. Explain to the patient how to use focused and controlled breathing for relaxation during the procedure.
Apply slight pressure to the site after the fluid is collected and the needle is withdrawn. If there is no evidence of bleeding or other drainage, apply a sterile adhesive bandage to the site.
Monitor the patient for complications related to the procedure (e.g., premature labor, allergic reaction, anaphylaxis).

Post-Test

Inform the patient that a report of the results will be made available to the requesting HCP, who will discuss the results with the patient.
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. Protocols may vary among facilities.
Observe/assess for delayed allergic reactions, such as rash, urticaria, tachycardia, hyperpnea, hypertension, palpitations, nausea, or vomiting. Immediately report symptoms to the appropriate HCP.
Observe/assess the amniocentesis site for bleeding, inflammation, or hematoma formation.
Instruct the patient in the care and assessment of the amniocentesis site.
Instruct the patient to report any redness, edema, bleeding, or pain at the amniocentesis site.
Instruct the patient to expect mild cramping, leakage of small amounts of amniotic fluid, and vaginal spotting for up to 2 days following the procedure. Instruct the patient to 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 either chills or fever.
Instruct the patient to rest until all symptoms have disappeared before resuming normal levels of activity.
Administer standard RhoGAM dose to maternal Rh-negative patients to prevent maternal Rh sensitization should the fetus be Rh-positive.
Recognize anxiety related to test results. Discuss the implications of abnormal test results on the patient’s lifestyle. Provide teaching and information regarding the clinical implications of the test results, as appropriate. Encourage the family to seek appropriate counseling if concerned with pregnancy termination and to seek genetic counseling if a chromosomal abnormality is determined. Decisions regarding elective abortion should take place in the presence of both parents. 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.
Depending on the results of this procedure, additional testing may be performed to evaluate or monitor progression of the disease process and determine the need for a change in therapy. Evaluate test results in relation to the patient’s symptoms and other tests performed.
Patient Education
- Reinforce information given by the patient’s HCP regarding further testing, treatment, or referral to another HCP.
- Inform the patient that it may be 2 to 4 wk before all results are available.
- Answer any questions or address any concerns voiced by the patient or family.
- Instruct the patient in the use of any ordered medications.
Expected Patient Outcomes
- Knowledge
- The patient states understanding of the importance of adhering to the therapy regimen provided by the HCP.
- The patient states understanding of the significant side effects and systemic reactions associated with the prescribed medication.
- Skills
- The patient accurately describes care necessary to support the health of the developmentally disabled infant.
- The patient accurately describes the lifestyle changes that will be necessary to provide care for the developmentally disabled infant.
- Attitude
- The patient complies with the request to review the literature provided by a pharmacist regarding prescribed medications.
- The patient agrees to meet with support group in relation to diagnosed developmental disability.

Related Monographs

Related tests include α₁-fetoprotein, antibodies anticardiolipin, blood groups and antibodies, chromosome analysis, fetal fibronectin, glucose, ketones, Kleihauer-Betke test, lupus anticoagulant antibodies, newborn screening, potassium, US biophysical profile obstetric, and UA.
Refer to the Reproductive System table at the end of the book for related tests by body system.

Amniotic Fluid Analysis and L/S Ratio