Testing amniotic fluid for assessment of fetal lung maturity

INTRODUCTION — The pulmonary system is among the last of the fetal organ systems to mature, both functionally and structurally. Fetal sex appears to play a role in this process; respiratory problems near term are slightly more common among male compared with female fetuses [1-3].

Because the immature pulmonary system may not oxygenate the preterm neonate adequately, preterm birth can lead to significant neonatal morbidity or mortality. Laboratory tests can be performed on amniotic fluid before iatrogenic preterm birth to provide an indirect assessment of the likelihood of lung maturity (direct tests of fetal lung function are not possible), and can be a factor in planning the time of induction or cesarean birth. Although once commonly performed, fetal lung maturity testing is no longer available in many countries.

This topic will discuss testing of amniotic fluid for assessment of fetal lung maturity. Clinical manifestations, diagnosis, treatment, sequelae, and prevention of neonatal pulmonary immaturity are reviewed separately. (See "Respiratory distress syndrome (RDS) in the newborn: Clinical features and diagnosis" and "Respiratory distress syndrome (RDS) in preterm infants: Management".)

WHEN IS FETAL LUNG MATURITY TESTING PERFORMED? — In most clinical settings, testing for fetal lung maturity is not performed because (1) delaying delivery because of lung immaturity would place the pregnant patient or fetus at significant risk, or (2) the fetus would benefit from delaying delivery, even if lung maturity is documented, and delaying delivery does not place the pregnant patient at significant risk, or (3) a course of antenatal corticosteroids can be given, which will benefit the fetus with immature lungs and has no proven harms [4]. (See "Antenatal corticosteroid therapy for reduction of neonatal respiratory morbidity and mortality from preterm delivery".)

These assessments may be ambiguous, so information about lung maturity sometimes may be helpful in the balance [5]. The information may also be helpful in estimating the level of newborn care that will be required. Thus, a test for fetal lung maturity may be performed before semielective but medically indicated births <39 weeks when this information significantly impacts assessment of the balance between the maternal-fetal risks of continuing the pregnancy versus the maternal-fetal risks of preterm birth, but this is an infrequent occurrence. In the United States, testing for fetal lung maturity is now rarely performed, and there is no clinical scenario where it is required.

When should testing be avoided?

●Tests for fetal lung maturity are generally not performed before 32 weeks of gestation, given the high prevalence of fetal pulmonary immaturity and the lower predictive value of a mature test result at this gestational age.

●Tests for fetal lung maturity are not performed in pregnancies with a reliable gestational age ≥39 weeks. At this gestational age, fetal lung maturity can be inferred; test results are not more predictive of respiratory outcome than gestational age alone.

●Suboptimal gestational age assessment is not a good reason to perform a test for fetal lung maturity. The American College of Obstetricians and Gynecologists (ACOG) considers gestational age assessment unreliable (ie, suboptimally dated pregnancy) if no ultrasound examination was performed before 22+0 weeks of gestation confirming or revising the estimated due date [6]. ACOG recommends not performing amniocentesis for fetal lung maturity testing in suboptimally dated pregnancies to guide timing of delivery as this decision should be based on the best clinical estimate of gestational age and standard indications for intervention rather than results from fetal lung maturity testing [6].

●Fetal lung maturity testing is not performed to justify a planned repeat cesarean birth or labor induction for patient convenience at 37 or 38 weeks of gestation rather than at ≥39 weeks. Even when lung maturity test results suggest a low risk of respiratory problems, neonates delivered at 37 or 38 weeks are at higher risk of adverse outcome than those delivered at 39 to 40 weeks of gestation without fetal lung maturity testing [7,8]. A fetal lung maturity test consistent with maturity is not an appropriate indication for early delivery in the absence of appropriate clinical indications [9].

FETAL MATURITY TESTS

Overview — Fetal lung maturity is evaluated by biochemical tests or biophysical tests. Biochemical tests measure the concentration of particular components of pulmonary surfactant. Biophysical tests evaluate the surface-active effects of these phospholipids.

The choice of test (table 1) should be based upon availability, presence or absence of contaminants (see 'Blood, meconium' below), and clinician preference. Although randomized trials comparing multiple tests have not been performed, controlled studies suggest that no test in table 1 performed significantly better than the others [4,10-13]. All are better at predicting the absence, rather than the presence, of respiratory distress. All perform less well at earlier gestational ages, which should be taken into account when interpreting results.

If the first test performed on an amniotic fluid sample is immature, we suggest not performing a second different test on the same sample, as medicolegal issues may arise if the neonate is delivered after discordant results and develops respiratory problems.

Availability — The lamellar body count is the only commercially available test for assessing fetal lung maturity in the United States. The other tests described below are no longer available in commercial laboratories but may be available in some hospital laboratories. Some of these may also be available in other parts of the world.

Lamellar body count — Lamellar body counts are a direct measurement of surfactant production by type-II pneumocytes. A standard hematology analyzer (eg, Coulter LH 750, Coulter Ac.T diff, Sysmex XE-2100) can be used for quantification because of the similar size of lamellar bodies and platelets. Values less than 15,000 per microliter are almost always associated with lung immaturity; values ≥50,000 per microliter strongly suggest lung maturity [14-16]. However, there is no consensus on the optimal threshold for predicting lung maturity; values greater than 30,000 to 40,000 per microliter, as well as higher levels, have been suggested (table 1) [17-19]. As with all tests of fetal lung maturity, gestational age affects the positive predictive value (see 'Gestational age' below). Gestational-age specific lamellar body count cutoffs for risk assessment of fetal lung maturity are available [13,20].

The lamellar body count has been used as an initial screening test, followed by the lecithin/sphingomyelin ratio where available when the lamellar body count is neither clearly mature (≥50,000 per microliter) nor immature (<30,000 per microliter).

Compared with thin-layer chromatography techniques, the lamellar body count is faster, more objective, less labor intensive, less technique dependent, and less expensive [10]. In meta-analyses, the lamellar body count performed as well or slightly better than the lecithin/sphingomyelin ratio in predicting respiratory distress [21,22]. The lamellar body count performs as well as the formerly available TDx-FLM II (see 'Surfactant/albumin ratio' below); both perform best when interpreted according to gestational age [13].

Blood contamination can lead to false elevation of the lamellar body count because platelets are counted as lamellar bodies; the effect of meconium is minimal.

All laboratories need to establish their own cutoffs for lamellar body count since hematology analyzers vary in sensitivity and gain settings. Establishing internal quality control is also important for this test. The Clinical and Laboratory Standards Institute (CLSI) provides guidance for the use of automated cell counting to count lamellar bodies in amniotic fluid. The document "Assessment of Fetal Lung Maturity by the Lamellar Body Count (C58)" provides guidelines for the use of automated cell counting to perform lamellar body counts, describes methods to assist in test verification and validation, and describes methods to select an appropriate maturity cutoff. Although C58 is no longer being reviewed through the CLSI Consensus Document Development Process, the document was technically valid of as July 2020 and is still available online because of its value to the laboratory community.

Phosphatidylglycerol — Phosphatidylglycerol (PG) is a minor constituent of surfactant. It begins to increase appreciably in amniotic fluid after 35 weeks, several weeks after the rise in lecithin [23]. Because PG enhances the spread of phospholipids in the alveoli, its presence indicates an advanced state of fetal lung development and function.

PG testing can be performed by thin-layer chromatography, so it can be determined alone or in conjunction with testing for the lecithin/sphingomyelin ratio. It may be reported qualitatively as positive or negative, where positive represents an exceedingly low risk of respiratory distress, or quantitatively, where a thin-layer chromatography value >2 percent is associated with a minimal rate of respiratory distress (table 1)[24].

Because thin-layer chromatography is a complicated and time-consuming technique, a rapid, semiquantitative immunologic slide agglutination test (AmnioStat-FLM) and several enzymatic assays were developed and have been validated as acceptable alternative techniques [25-28]. The slide agglutination test is the most common method for testing for PG; however, it appears to be less sensitive for detecting fetal lung maturity than thin-layer chromatography [29].

An advantage of testing for PG is that blood or meconium usually does not affect test results [4]. A disadvantage is that the absence of PG, especially before 36 weeks of gestation, is less predictive of the occurrence of respiratory distress than immature results from other tests.

Lecithin/sphingomyelin ratio — The lecithin/sphingomyelin ratio is based upon the observation that there is outward flow of lung secretions from the lungs into the amniotic fluid. This process changes the phospholipid composition of amniotic fluid, thereby enabling indirect assessment of fetal lung maturity through evaluation of this fluid.

The concentrations of lecithin and sphingomyelin in amniotic fluid are approximately equal until 32 to 33 weeks of gestation, at which time the concentration of lecithin begins to increase significantly while the sphingomyelin concentration remains approximately the same. The measurement of sphingomyelin serves as a constant comparison for control of the relative increases in lecithin because the volume of amniotic fluid cannot be accurately measured clinically.

Determining the lecithin/sphingomyelin ratio involves thin-layer chromatography after organic solvent extraction. It is a technically difficult test to perform and interpret; care at each step of sample handling is critical for consistent results [30]. The sample should be kept on ice or refrigerated if transport to a laboratory is required (most laboratories cannot perform this test). Improper storage conditions can change the lecithin/sphingomyelin ratio since amniotic fluid contains enzymes that can be affected by temperature [31]. The amniotic fluid sample must be well mixed before testing. It takes several hours to perform the test and may take 24 hours when both transport and testing time are considered, which is another disadvantage of this method.

Individual laboratories should calculate a threshold value for predicting lung maturity by correlating their test results with clinical outcome as the variation within and between laboratories can be considerable. Empirically, the risk of respiratory distress is exceedingly low when the lecithin/sphingomyelin ratio is greater than 2.0 (table 1) [32].

Optical density at 650 nm — An indirect measurement of lamellar bodies can be obtained by measuring the optical density of amniotic fluid at a wavelength of 650 nanometers. It is based upon the concept that increasing opalescence is due to increasing numbers of lamellar bodies. An optical density reading ≥0.15 is used as the indicator of lung maturity [33].

Foam stability index — The foam stability index (FSI) assesses total surfactant activity. It is a rapid predictor of fetal lung maturity based upon the ability of surfactant to generate stable foam in the presence of ethanol [34]. Ethanol is added to a sample of amniotic fluid to eliminate the effects of nonsurfactant factors on foam formation. The mixture is then shaken and will generate a stable ring of foam if surfactant is present. The FSI is calculated by utilizing serial dilutions of ethanol to quantitate the amount of surfactant present [35].

Amniotic fluid samples for FSI should not be collected in silicone tubes, as the silicone will produce "false foam." The discriminating value indicative of lung maturity is usually set at ≥47. A positive result virtually excludes the risk of respiratory distress; however, a negative test often occurs with mature lungs [36]. The presence of blood or meconium interferes with results of the FSI.

Surfactant/albumin ratio — The surfactant/albumin ratio also assesses total surfactant activity. The TDx-FLM II was the commercially available test for measuring the surfactant/albumin ratio, but the manufacturer retired the analytical systems for TDx-FLM II testing and ended production of the reagent required to perform it.

TDx-FLM II was based on the principle of fluorescence polarization and used an automated analyzer to quantitate the competitive binding of a fluorescent probe to both surfactant and albumin in a sample of amniotic fluid; thus, it was a true direct measurement of surfactant concentration [37]. An elevated surfactant/albumin ratio correlated with fetal lung maturity; the threshold for maturity was 55 mg of surfactant per gram albumin [38]. Test performance compared favorably with the well-established lecithin/sphingomyelin ratio and PG tests (table 1) and was gestational-age dependent (table 2) [12,39,40].

The advantage of the TDx-FLM II was that it was a simple, automated, rapid test that varied minimally between laboratories and required only a small volume of amniotic fluid [4]. A disadvantage was the wide indeterminate range: Values greater than 55 were considered mature, values less than 40 were considered immature, while values of 40 to 54 were considered "indeterminate" [4,39]. Blood or meconium in the amniotic fluid also affected results.

FACTORS THAT MAY AFFECT INTERPRETATION

Gestational age — For each test, the ability to predict respiratory distress is influenced by the prevalence of respiratory distress in the population tested; thus, the predictive value varies with gestational age [13,41,42]. For example, in one analysis, the risk of respiratory distress after a TDx-FLM II of 60 mg/g at 29 and 37 weeks of gestation was 16 and 1 percent, respectively [41]. In another analysis, the risk of respiratory distress after a lamellar body count of 60,000 per microliter at 32 and 37 weeks of gestation was 11 and 1.2 percent, respectively [20].

Gestational age is an important factor for predicting readiness for neonatal life and absence of neonatal morbidity, even when fetal lung maturity is documented prenatally. In a retrospective cohort study of neonatal outcomes of newborns with mature fetal lung indices, the rate of composite, adverse neonatal outcome with delivery at 34+0 to 36+6 weeks, 37+0 to 38+6 weeks, and ≥39 weeks was 21, 13, and 4 percent, respectively [43]. In a study that compared outcomes of early-term births after positive fetal lung maturity testing (n = 180) with full-term births (n = 47,957), the primary composite outcome (death, ventilator for ≥2 days, continuous positive airway pressure, proven sepsis, pneumonia or meningitis, treated hypoglycemia, hyperbilirubinemia [phototherapy], five minute Apgar <7) was approximately fourfold higher in the early-term births [8].

Blood, meconium — Phosphatidylglycerol (PG) determination generally is not affected by blood, meconium, or other contaminants; its ability to predict lung maturity is the same whether or not contamination is present [33]. This is an advantage for assessing fetal lung maturity status since these substances are commonly found in amniotic fluid.

The surfactant/albumin ratio is usually reliable if mature since contaminants tend to cause falsely immature findings, although the degree and direction of interference are not well defined [44-48].

By contrast, the presence of blood or meconium can interfere with interpretation of the lecithin/sphingomyelin ratio [49]. Bloody samples give false information because blood contains sphingomyelin and decreases extraction of lecithin by cold acetone techniques (falsely low "L" and falsely high "S") [50]. Therefore, if blood or other particulate matter is present in the amniotic fluid sample, a low-speed, short centrifugation should be used to remove the cellular component [30]; however, this does not guarantee an accurate result, especially when there is a lot of blood or meconium.

Oligohydramnios and polyhydramnios — The effect of amniotic fluid volume (oligohydramnios, polyhydramnios) on test results has not been studied extensively [51,52]. Theoretically, tests that are expressed as a ratio or proportion of two solutes released into the amniotic fluid should remain accurate independent of amniotic fluid volume, while tests that reflect the concentration of a substance in the amniotic fluid (eg, lamellar body count, PG) may be affected by amniotic fluid volume.

Vaginal pool samples — In pregnant patients with intact membranes, amniotic fluid is obtained by amniocentesis. (See "Diagnostic amniocentesis", section on 'Third-trimester amniocentesis'.)

In pregnant patients with ruptured membranes, a syringe can be used to aspirate amniotic fluid pooled in the posterior vaginal fornix. Vaginal pool specimens can also be collected using a sterile sponge.

However, the lecithin/sphingomyelin ratio can be higher in the vaginal pool than in fluid obtained by amniocentesis [53], and false-positive PG have been reported in vaginal pool samples due to bacterial contamination [54,55].

Antenatal corticosteroids — Standard tests for predicting fetal lung maturity may be less reliable for predicting absence of fetal lung maturity in patients who have received a course of antenatal corticosteroids to enhance fetal lung maturation. (See "Antenatal corticosteroid therapy for reduction of neonatal respiratory morbidity and mortality from preterm delivery".)

Maternal diabetes mellitus — Most experts use the same threshold value for lung maturity for pregnant people with and without diabetes (preexisting [pregestational] or gestational diabetes) [56-60].

Twin pregnancy — If amniocentesis to determine fetal lung maturity is performed, some authors believe that amniocentesis of only one twin is adequate if the gestation is ≥36 weeks [61,62], but others test both twins in all cases because pulmonary maturity can be asynchronous [63]. When only one sac is sampled, it is reasonable to sample the sac of the fetus less likely to be mature. As an example, a larger, nonpresenting male fetus would be less likely to have achieved lung maturity than a smaller, presenting female fetus.

ALTERNATIVES TO TESTING AMNIOTIC FLUID — Quantitative ultrasound fetal lung maturity assessment, Doppler assessment, and magnetic resonance imaging (MRI) are some of the investigative techniques used for noninvasive assessment of fetal lung maturity in pilot studies [64-70]. None of these techniques is appropriate for clinical use.

SUMMARY AND RECOMMENDATIONS

●Overview – Fetal lung maturity testing before delivery is rarely performed. It is not useful as a component of delivery timing decision-making in pregnancies with well-documented gestational age ≥39 weeks (because lung maturity is likely), pregnancies less than 32 weeks of gestation (because lung immaturity is likely), and when delivery is obstetrically/medically indicated (because delaying delivery will place the pregnant patient or fetus at significant risk). If a pregnancy is suboptimally dated (no ultrasound examination before 22+0 weeks), timing of delivery should be based on the best clinical estimate of gestational age and standard indications for intervention; fetal lung maturity testing is not recommended as a component of decision-making. (See 'When is fetal lung maturity testing performed?' above.)

●Indications – A test for fetal lung maturity may be performed before semielective but medically indicated births <39 weeks when this information significantly impacts assessment of the balance between the maternal-fetal risks of continuing the pregnancy versus the maternal-fetal risks of preterm birth. This is an infrequent occurrence. (See 'When is fetal lung maturity testing performed?' above.)

●Testing

•Fetal lung maturity can be assessed by testing for components of fetal lung secretions in amniotic fluid. No test performs significantly better than another, and all are better at predicting the absence, rather than the presence, of respiratory distress. (See 'Fetal maturity tests' above.)

•The choice of test should be based upon availability, presence or absence of contaminants, and physician preference. A summary of the different tests available and the assets and liabilities associated with each is shown in the table (table 1). The lamellar body count is the test generally used for assessing fetal lung maturity in the United States. The other tests in the table are no longer available in commercial laboratories, but may be available in some hospital laboratories. (See 'Fetal maturity tests' above.)

•We suggest performing only one test for fetal lung maturity on an amniotic fluid sample. (See 'Fetal maturity tests' above.)

●Interpretation – Gestational age is an important factor in interpreting tests of fetal lung maturity. All tests perform less well at earlier gestational ages, which should be taken into account when interpreting results. Gestational age-specific tables for prediction of respiratory distress have been published. (See 'Gestational age' above.)

Blood or meconium in amniotic fluid affects some test results (table 1). Oligohydramnios, polyhydramnios, and source of amniotic fluid (vaginal pool versus amniocentesis) can also affect results. The same threshold value for lung maturity can be used for both nondiabetic and diabetic patients (pregestational or gestational diabetes). (See 'Factors that may affect interpretation' above.)