Amniotic Fluid Analysis and L/S Ratio

General

Core Lab Study

Synonym/Acronym:
N/A

Rationale
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, 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).

This Core Lab Study is most commonly used to screen and assess risk for genetic disorders, HDN, and 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 white blood cell (WBC) count and lamellar bodies.

TestReference Value
Color Colorless to pale yellow
AppearanceClear
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 midpregnancy (approximately 14–27 wk, using the Queenan curve when gestational age is less than 27 wk)
Less than 0.06 ΔOD in late pregnancy (approximately 28–36 wk, using the Liley Chart when gestational age is equal to or greater than 27 wk)
Less than 0.03 ΔOD at term (approximately 37–40 wk, using the Liley Chart when gestational age is equal to or greater than 27 wk)
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 sequence variations)Normal karyotype
WBC 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.

Overview

(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 wk’ gestation for accurate interpretation of test results, but it also can be done between 26 and 35 wk’ 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 and 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 patients who have diabetes. Therefore, caution must be used when interpreting the results obtained from a patient who has diabetes, and a higher ratio is expected to predict maturity.

Studies used to identify HDN


HDN, also called erythroblastosis fetalis, is a condition that occurs most commonly after red blood cells (RBCs) from an Rh-negative patient become sensitized by fetal RBCs from an Rh-positive baby. Rh sensitization 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. HDN can also be caused by an ABO incompatibility between the biological mother (usually type O) and fetus (if type A or B). The patient’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 the biological father’s Rh type cannot be confirmed at the time the patient undergoes 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, Blood, and Prenatal Screening Tests.”

Genetics is the study and identification of genes, genetic sequence variations, 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.

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 diabetes in the patient, toxemia, hemolytic diseases of the newborn, or postmaturity.
  • Determine fetal gender when the biological 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 a patient of advanced age (some of the aforementioned tests are routinely requested in patients age 35 yr and older).
  • Evaluate fetus in a patient 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 “Prenatal Screening Tests” for information related to triple- and quad-marker testing).
  • Identify fetuses at risk of developing RDS.

Interfering Factors

  • Bilirubin may be falsely elevated if Hgb of the patient or meconium of the fetus 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 (of the fetus or patient), 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 wk’ 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 wk’ 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 age of the patient, multiple gestation, and polyhydramnios. Conditions that may increase production of surfactant include hypertension, delay in intrauterine growth, malnutrition, diabetes in the patient, placenta previa, placental infarction, and premature rupture of the membranes. The expected L/S ratio for the fetus of a patient who has diabetes 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 “Prenatal Screening Tests”) 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 WBC count and positive leukocyte esterase are indicators of infection.

Nursing Implications, Nursing Process, Clinical Judgement

Potential Problems: Assessment & Nursing Diagnosis/Analysis

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

Before the Study: Planning and Implementation


Teaching the Patient What to Expect

  • Review the procedure with the patient.
  • Explain that an amniocentesis can assist in evaluation of fetal well-being. A sample of amniotic fluid is needed for the test with the procedure taking about 20 to 30 min. Provide assurance that precautions to avoid injury to the fetus will be taken by locating the fetus with ultrasound.
  • Inform patient that removal of clothes below the waist is necessary (cultural approach to modesty will be implemented).
  • Discuss the importance of remaining still and breathing normally during the local anesthetic and procedure (to assist with relaxation).
  • Inform patient that some discomfort and stinging may be felt during the local anesthetic; a feeling of pressure and a slight pop may be felt when the needle is inserted through the abdominal and uterine walls.
  • 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 that may be elevated 20 to 30 degrees; 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 patient and fetal vital signs are taken and recorded; ultrasound is used to assess the position of the amniotic fluid, placenta, and fetus.
  • 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. Patient and fetal vital signs and uterine contractions are monitored throughout the procedure.
  • 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.
  • Organizational policy is followed for obtaining, labeling, and transporting samples.
  • Protocols may vary among facilities.

Potential Nursing Actions

  • Assess for a family history of genetic disorders.
  • Verify Rh type results of the patient; check for prior sensitization if Rh-negative (prenatal blood work includes an antibody screen [indirect Coombs antiglobulin test]). Administration of Rh-immune globulin may be required.
  • Instruct patients less than 20 wk’ 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 wk’ 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

  • Emotional/spiritual distress may be evident with outcome of test results.
  • Symptoms of emotional/spiritual distress may include anger, stated feelings of lack of peace or serenity, alienation from others, hopelessness, request to meet with spiritual leader, and lack of acceptance.
  • Interventions/actions related to distress include the following: Facilitate privacy for prayer, meditation, and reflection. Provide referrals to support group to help meet clinical challenges.

Fear

  • Interventions/actions related to fear include the following: 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.

Knowledge

  • Interventions/actions related to knowledge include the following: Provide simple straightforward information appropriate to age, culture, and literacy. Provide references for valid written or streaming services that can provide ongoing support. Be available to answer all questions.

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. Rh sensitization of the patient can result from fetal red blood cells (RBCs) mixing with blood of an RhD-negative patient carrying an RhD-positive fetus. In this case, the Rh-positive RBCs of the fetus can sensitize the patient’s immune system to stimulate production of anti-D antibodies.
  • RhIG or RhoGAM may be administered after amniocentesis to RhD-negative patients 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 (ABO or Rh), refer to the study titled “Coombs Antiglobulin, Indirect.”
  • Monitor for complications related to the procedure (e.g., premature labor, infection, leakage of amniotic fluid from needle site, development of spontaneous abortion, membrane rupture, placenta abruption, fetal injury, fetal hemorrhage).
  • 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.

Treatment Considerations

  • Compare fetal heart rate and life signs of the patient (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 Rh-negative patients to prevent Rh sensitization in the patient 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 an infant with developmental disabilities as well as for exploring other options (termination of pregnancy or adoption). It is also important to discuss problems the parents may experience (guilt, depression, anger) if fetal abnormalities are detected.