Lecithin Sphingomyelin Ratio

Specimen Collection

A sample of amniotic fluid may be obtained by performing an amniocentesis after 34 weeks of gestation or collecting fluid from the vaginal canal in cases of preterm premature rupture of membranes (PPROM). For amniocentesis, ultrasound is used to locate an adequately sized pocket of amniotic fluid away from the fetus. Local anesthesia may be applied at the sampling site. A stylet is inserted through the abdomen under ultrasound guidance, and a syringe is attached to collect the sample.[8] In PPROM, the sample may be collected from fluid pooling in the vagina. However, the presence of blood in the sample or vagina invalidates L/S ratio results.[9]

Amniotic fluid samples collected for FLM testing should be processed promptly to maintain accuracy and reliability.[10] If left untreated at room temperature, amniotic fluid undergoes degradation of key components, particularly lecithin and sphingomyelin, which are essential for calculating the L/S ratio.[11] These phospholipids can break down over 48 hours, reducing their measurable concentrations. This degradation can compromise test integrity, leading to inaccurate FLM assessments and potentially inappropriate clinical decisions.[12]

Proper handling and storage protocols must be followed to prevent degradation. Centrifugation of the amniotic fluid sample is essential if a delay between sample collection and testing is expected. This process separates the fluid into its components, removing cellular debris and particulate matter that could contribute to lecithin and sphingomyelin breakdown. After centrifugation, the supernatant—the clear fluid layer—may be stored under controlled conditions to maintain stability. For long-term storage, centrifuged samples should be frozen at -20 °C, preserving integrity without significantly altering the L/S ratio. Studies have shown that frozen samples remain stable for weeks or months, making freezing an effective preservation method.[13]

If a short delay, eg, a few hours to a day, is expected, refrigerated storage at 2 °C to 8 °C can slow degradation. However, freezing remains the preferred method for longer storage. Avoiding repeated freezing and thawing is crucial, as these cycles can further degrade biomarkers.

Procedures

TLC is used to analyze the L/S ratio in amniotic fluid.[14] This method employs a stationary phase made of silica gel and a mobile phase consisting of either a chloroform-methanol-water or chloroform-methanol-ammonium hydroxide mixture. The separation process in TLC relies on the differential adsorption of phospholipids, which vary in polarity. Both 1- and 2-dimensional TLC systems may be utilized, with the choice depending on the need to effectively separate the key phospholipids lecithin, sphingomyelin, and phosphatidylglycerol from each other and other phospholipid components in amniotic fluid.[15]

Once phospholipids are separated via TLC and identified on the chromatogram using an indicator, the L/S ratio may be assessed through various methods. One common approach involves evaluating the chromatogram using transmission or reflection densitometry. In this process, the separated and charred compounds on the TLC plate are quantified densitometrically using a scanner. The L/S ratio is then calculated based on recorder tracings, which correspond proportionally to lecithin and sphingomyelin concentrations on the plate.[16] High-performance liquid chromatography (HPLC) is another reliable method for quantifying lecithin and sphingomyelin and determining the L/S ratio in amniotic fluid.[17]

Laboratory performance in L/S ratio determination is evaluated by ensuring results remain within ±3 standard deviations of the peer-group mean, maintaining consistency and reliability. Values outside this range indicate potential errors requiring investigation into reagents, calibration, technique, or sample handling. Participation in proficiency testing and external quality assessment helps laboratories align with clinical standards. Since the L/S ratio is critical for assessing FLM, accurate results prevent misguiding clinical decisions, such as unnecessary preterm delivery or missed corticosteroid therapy, optimizing patient care.[18]

Indications

Historically, indications for amniocentesis to assess FLM included maternal comorbidities, uterine and placental complications, and obstetric concerns. Conditions such as diabetes, chronic hypertension, preeclampsia, placenta previa, preterm labor, PPROM, and fetal heart rate abnormalities were among the factors considered. However, updated guidelines no longer recommend testing the L/S ratio or performing other FLM assessments for these indications. One possible exception involves uncertainty in gestational dating. Clinicians may consider FLM testing if pregnancy dating is unreliable and delivery is planned between 32 and 39 weeks of gestation.[19]

Potential Diagnosis

The L/S ratio evaluates FLM to assess the risk of RDS in neonates, particularly in preterm infants younger than 39 weeks, as the risk of RDS increases with decreasing gestational age. Measuring surfactant levels informs delivery timing and interventions such as corticosteroid therapy, helping to prevent unnecessary preterm births and improve neonatal outcomes. Accurate testing ensures informed clinical decisions and enhances patient care.

Normal and Critical Findings

The normal L/S ratio ranges from 2.0 to 2.5, indicating appropriate fetal lung development. An L/S ratio below 2.0 suggests fetal lung immaturity. For patients with poorly controlled diabetes, an L/S ratio of 3.0 was once considered due to concerns that elevated maternal glucose levels could delay fetal lung maturation. However, subsequent findings showed no significant difference in the L/S ratio between patients with and without diabetes. Some institutions continue to use 3.0 as the cutoff for pregnancies complicated by poorly controlled diabetes.[20]

FLM test results should be promptly communicated to the ordering clinicians to facilitate timely decision-making, particularly when early delivery is being considered. Laboratory reports should provide detailed information about the sample condition, noting any contamination, hemolysis, or improper handling that may affect test accuracy and interpretation.

Laboratories performing L/S ratio determination should validate their reference intervals through clinical outcome studies, correlating FLM test results with neonatal respiratory outcomes to establish institution-specific reference ranges. Alternatively, laboratories may compare their methodologies with data from published clinical outcome studies to ensure alignment with established medical evidence and best practices.[21]

Interfering Factors

The presence of meconium and blood in the sample can alter the L/S ratio. Meconium contamination can invalidate the final result.[22] Blood in the sample can lower the L/S ratio, leading to misinterpretation of the test result. If the initial L/S ratio is less than 2.0, the presence of blood would further reduce the value, reinforcing the interpretation of immature fetal lungs. Conversely, if the initial result is greater than 2.0, the sample would still be interpreted as mature despite the decrease in the measured value.[23]

Complications

The birth of a neonate before 39 weeks presents substantial respiratory risks due to incomplete lung development. Major complications include RDS resulting from surfactant deficiency, bronchopulmonary dysplasia (BPD) contributing to chronic lung disease, and pulmonary hypertension impairing oxygen exchange. These neonates frequently require respiratory support, including oxygen therapy, mechanical ventilation, and surfactant replacement, to enhance outcomes.[24]

Patient Safety and Education

Amniocentesis for L/S ratio assessment is generally safe but carries rare risks, including maternal sepsis, intrauterine rupture, maternal-fetal hemorrhage, and fetal heart rate abnormalities. Though uncommon, these complications may lead to infection, preterm labor, alloimmunization, or fetal distress, requiring careful monitoring. Due to these potential risks, amniocentesis is typically not recommended for patients with a history of preterm labor, incompetent cervix, placenta previa, or abruptio placentae. Providing a thorough explanation of the procedure's indications, benefits, and possible risks to the patient and family is essential.

Clinical Significance

Historically, the L/S ratio was a parameter routinely used to guide clinicians in determining the optimal timing of delivery to reduce the risk of neonatal RDS. Evidence of fetal lung immaturity based on this biochemical quantifier supported the administration of glucocorticoids to promote lung maturation. Other FLM testing methods have been developed since the introduction of the L/S ratio, including lamellar body count, phosphatidylglycerol test, foam stability test, and surfactant-albumin ratio. However, the necessity of FLM testing has declined in recent years. In many cases, determining the L/S ratio is not required if a maternal or fetal indication warrants delivery before term.