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2001, Volume 12, Number 2
by Richard S. Bak, Ph.D.
The lungs are the last of the organ systems to mature sufficiently to support extrauterine life. Fetal lung immaturity, or the respiratory distress syndrome (RDS), occurs most often when insufficient lung surfactant is present. Methods for measuring lung surfactant and thus, fetal pulmonary maturity, have been available for more than 20 years, and a number of new tests have been introduced. This article reviews these tests and provides the rationale for the Warde Medical Laboratorys new Fetal Lung Maturity Test.
Appropriate Specimen for Fetal Lung Maturity Determinations
The importance of obtaining an appropriate specimen cannot be overemphasized. Amniotic fluid, obtained by amniocentesis, is the specimen of choice because it does not contain sources of contamination that can interfere with the various assays. Fluid may be obtained by transvaginal puncture of the bulging membranes, but it should not be grossly contaminated with vaginal secretions as might occur with aspiration of a vaginal pool after spontaneous rupture of the membrane. Vaginal pool specimens are adequate for testing only when the fluid has been in the vagina a short time and the sample is quickly chilled to 2 to 4 º C after collection (1). Blood contamination affects most of the fetal lung maturity tests because plasma contains a high concentration of most phospholipids. The exception is phosphatidylglycerol (PG) which is absent from plasma.
The first widely accepted direct test for assessment of fetal pulmonary status was the L/S ratio. This test evaluates a change in the relative amounts of lecithin (phosphatidyl choline) and sphingomyelin (a phospholipid of unknown origin) in amniotic fluid samples as gestational age increases. Until about 32-33 weeks of gestation, the concentration of these two substances are quite similar; thereafter the concentration of lecithin increases significantly compared with the relatively constant concentration of sphingomyelin. In the absence of complications, the ratio of these two components reaches 2.0 at about 35 weeks gestation. Infants delivered after attaining an L/S ratio of 2.0 or higher rarely develop RDS. This value of 2.0 has become the commonly accepted standard value indicating maturity in the fetus of a non-diabetic woman.
Determination of the L/S ratio involves thin-layer chromatography after organic extraction. This methodology is quite cumbersome and labor intensive. Correspondingly, the coefficient of variation at the maturity criterion of 2.0 is about 25%. Thus, for a specimen with a true L/S ratio of 2.0, the measured value could vary between 1.5 and 2.5, a range that nearly spans the complete clinical spectrum.
Contamination of samples with blood tends to produce falsely elevated levels for very immature samples and falsely lowered values for very mature samples. Meconium, vaginal secretion, and maternal urine contamination can also produce false results.
Because of the wide coefficient of variation and the potential for false positive and negative results, we no longer recommend use of the L/S ratio test.
Phosphatidylglycerol is a minor constituent of surfactant that generally appears several weeks after the increase in lecithin concentration. Because PG enhances the spread of phospholipids on the alveolar surface, its presence indicates an advanced state of fetal pulmonary maturity.
Phosphatidylglycerol determination can be accomplished by either thin-layer chromatography or by slide agglutination.
An advantage of PG determination in assessing fetal maturity is the fact that blood, meconium, or other contaminants do not generally affect it. This characteristic allows PG determination by using vaginal pool samples from patients who have spontaneous rupture of membranes. A disadvantage of using PG for assessing fetal maturity is its relatively late appearance in pregnancy. Compared with other tests, an immature result (negative PG) is associated with a greater proportion of infants who will not develop RDS.
Foam Stability Index
In this biophysical test, the generation of a stable ring of foam by shaking amniotic fluid and ethanol in a test tube demonstrates the presence of surface-active material in amniotic fluid. The determination of a stable ring is a visual, and somewhat subjective, process. This test is also associated with more false predictions of immaturity than other fetal lung maturity tests.
The presence of blood or meconium also interferes with the results of this test.
Lamellar Body Counts
Specialized alveolar cells synthesize pulmonary surfactant and package it into laminated storage granules called lamellar bodies. As gestation progresses, these bodies become more numerous and are continually secreted into the fetal alveoli and eventually into the amniotic fluid. Lamellar body particles are the size of small platelets and can be measured directly using the platelet channel of a standard hematology cell counter.
On specimens obtained by amniocentesis, the precision of this assay is 5 to 10 % (2). However, 1% blood contamination can decrease counts by 20% and meconium-stained fluids and vaginal pool fluids containing obvious mucus can have highly erroneous results.
Fluorescent Polarization Test (FLM)
Fetal maturity testing using fluorescent polarization measures the ratio of surfactant to albumin. High values indicate high levels of surfactant and lung maturity, while low values indicate lung immaturity.
The advantages of this technique are that it is simple and automated and much more precise than the L/S ratio. Precision at the medical decision point is ± 3%.
Blood and meconium will also interfere with this procedure. However, a free flowing or carefully tapped vaginal sample, free of visible blood or meconium is acceptable for analysis
Effect of Diabetes on Fetal Lung Maturity
Some investigators have reported altered fetal lung maturity results and an increased rate of RDS in newborns of diabetic patients. Others have reported no difference in either test results or clinical outcome (3). The possible answer to this debate is that poorly controlled diabetes clearly delays pulmonary maturation (4), while well controlled diabetes does not delay maturation nor appear to affect fetal lung maturity test results.
In a recent multicenter study, 2 of 13 diabetic patients with a mature Fluorescent Polarization Test (FLM) and absent PG delivered infants who developed mild RDS. Neither required intubation or prolonged oxygen therapy (5). This study concluded that diabetes mellitus did not affect the medical decision limit of the fluorescent polarization assay. This study also showed that the combination of a mature FLM value and an immature PG value occurred 37.5 % of the time. None of the infants who were delivered with this panel of results developed severe RDS. Indeed, in almost 30% of diabetic pregnancies, the detection of clinically significant amounts of PG may not occur at all (6). Awaiting its detection may prolong pregnancy and possibly jeopardize the health of the mother and fetus.
Although the L/S ratio was the first test to assess fetal lung maturity, other tests have been developed that are more rapid and are more precise at the medical decision point. Table 1 lists the manufacturer s data on the relative risk of developing respiratory distress syndrome given an immature, intermediate, or mature FLM result (7).
(delivery within 72 hours)
|(<40 mg/G)||(40-54 mg/G)||(>54 mg/G)|
Several clinical studies have also demonstrated that the FLM assay is as good as, or is superior to, the L/S ratio (8,9,10). Based on these studies and their own personal experiences, Ashwood published standards for fetal lung maturity testing in which he recommends the fluorescent polarization assay as the best initial test (11). Dubin, in his published clinical practice parameter, recommends either fluorescent polarization or lamellar body counts as the acceptable first test and finds it arguable whether the L/S ratio adds any clinical value either as a supplemental or final test (12). Finally, Hagen (8) found the predictive value of a mature result by fluorescent polarization to be 96%.
Because of the strong predictive values of a single mature result, little additional information will be gained by the performance of a battery of tests. When multiple tests are performed, discordant results are sometimes found, which is not surprising because these tests measure different analytes, some of which appear at different times in the lung maturation process.
As to the predictive value of an immature result, it is well known that none of the fetal lung maturity tests can accurately predict Respiratory Distress Syndrome. It may be helpful to know that models have been developed to predict the probability of RDS. One such model predicts a 32% chance of developing RDS at a fluorescent polarization value of 39 mg/G and a 50% chance of developing RDS at a value of 24 mg/G (13).
We believe that the best single assay for fetal lung maturity is the Fluorescent Polarization Test. Thus, effective April 1, 2001, Warde Medical Laboratory will offer this test on a routine and STAT basis. We will also offer the phosphatidylglycerol (PG) assay on a routine and STAT basis. L/S ratios and other fetal lung maturity tests will be available, if requested, with a 24 to 48 hour turn around time, from a referral laboratory.
A satisfactory amniotic fluid specimen is essential for accurate assessment of fetal lung maturity.
The FLM is much more precise than the L/S ratio at the medical decision point and is perhaps, the best single assessment of fetal lung maturity.
The PG test is a useful test in cases where a fetal lung maturity test must be performed and the only specimen available is one that is grossly contaminated.
- Dombrowski RA, Mackenna J, Brame RG. Comparison of amniotic fliud lung maturity profiles in paired vaginal and amniocentesis specimens. Am J. Obstet Gynecol. 1981; 140: 461-464
- Grenspan JS, Rosen DJO, Roll K, et al. Evaluation of lamellar body number density as the initial assessment in a fetal lung maturity test cascade. J Reprod Med. 1995; 40: 260-266
- Kjos SL, Walther FJ, Montero M, Paul RH, Diaz F, Stabler M. Prevalence and etiology of infants of diabetic mothers: predictive value of fetal lung maturation tests. Am J Obstet Gynecol 1990; 163: 898-903
- Piper JM, Langer O. Does maternal diabetes delay fetal pulmonary maturity? Am J. Obstet Gynecol 1993; 168: 783-786
- Livingston EG, Herbert WNP, Hage ML, Chapman JF, Stubbs TM. Use of the TDx-FLM assay in evaluating fetal lung maturity in an insulin-dependent diabetic population. Obstet Gynecol 1995;86:826-829
- Ojomo EO, Coustan DR. Absence of evidence of pulmonary maturity at amniocentesis in term infants of diabetic mothers. Am J Obstet Gynecol 1990;163:954-957
- Fetal Lung Maturity II package insert. Abbott Diagnostics, Abbott Park, Ill (1996)
- Hagen E, Links JC, Arias F. A comparison of the accuracy of TDx FLM assay, lecithin-sphyingomyelin ratio, and phosphatidylglycerol in the prediction of neonatal respiratory distress syndrome. Obstet Gynecol. 1993; 82: 1004-1008
- Herbert WNP, Chapman JF, Schnoor MM. Role of the TDx FLM assay in fetal lung maturity. Am J Obstet Gynecol 1993; 168: 808-812
- Bender TM, Stone LR, Amenta JS. Diagnostic power of lecithin/sphingomyelin ratio and fluorescent polarization assays for respiratory distress syndrome compared by relative operating characteristic curves. Clin Chem; 1994, 40: 541-545
- Ashwood ER. Standards of laboratory practice: evaluation of fetal lung maturity. Clin Chem 1997; 43: 211-214
- Dubin S. Assessment of Fetal Lung Maturity, Am J. Clin. Path 1998; 110: 723-732
- Tanasejevic MJ, Wybenga DR, Richardso D, Greene MF, Lopez R, Winkelman JW,. A predictive model for fetal lung maturity employing gestational age and test results. Am J Clin Path 1994; 102: 788-793
- American College of Obstetricians and Gynecologists. Committee on Educational Bulletins: Assessment of Fetal lung Maturity. ACOG Educational Bulletin No. 230 Washington,DC, American College of Obstetricians and Gynecologists, 1996
- Ashwood ER, Clinical Chemistry of Pregnancy, in Tietz Textbook of Clinical Chemistry, 3rd ed. W.B. Saunders Company, Philadelphia, 1999, 1736-1735