Prediction of fetal lung immaturity using gestational age, patient characteristics and fetal lung maturity tests: a probabilistic approach
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- Wijnberger, L.D.E., de Kleine, M., Voorbij, H.A.M. et al. Arch Gynecol Obstet (2010) 281: 15. doi:10.1007/s00404-009-1033-0
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The lecithin/sphingomyelin (L/S) ratio and the lamellar body count (LBC) can be used to predict respiratory distress syndrome (RDS).
We performed a retrospective cohort study among consecutive women who underwent amniotic fluid sampling for the assessment of fetal lung maturity. Logistic regression was used to construct models for the prediction of RDS in three gestational age categories, with models based on clinical characteristics only, clinical characteristics and the LBC, and on clinical characteristics and L/S ratio.
When amniotic fluid was collected <30 weeks, the specificity of the LBC was 30% and the sensitivity 100%. Addition of the L/S ratio increased the specifity to 60%, for a sensitivity of 100%. When amniocentesis was performed between 30 and 33 weeks, addition of the L/S ratio only marginally improved the performance of the LBC.
At a gestational age <30 weeks, the L/S ratio has additional value over the LBC. Above 30 weeks of gestation, single use of the LBC seems sufficient.
KeywordsFetal lung maturityLamellar body countL/S ratioRespiratory distress sydrome (RDS)
The lecithin/sphingomyelin (L/S) ratio and the lamellar body count (LBC) are two invasive tests used to assess fetal lung maturity in pregnancies at risk for preterm delivery [1–3]. Many studies have reported on the accuracy of these tests. In a recent meta-analysis in which we summarized six studies in which both tests were compared, the LBC was found to be at least as accurate as the L/S ratio . However, accuracy is not the only issue that is of importance in establishing their value for clinical practice. In this context, Richardson and Heffner  recently stated that although tests for fetal lung maturity were mature, their interpretation was not. They hypothesized that due to the high risk of lung immaturity at low gestation, testing for fetal lung maturity was not useful before a gestational age of 32–33 weeks. On the other hand, they assumed the prevalence of RDS and other complications after a gestational age of 37 weeks to be low, thus implicating that testing was also not useful near term. They suggested that there is a need for studies in which pre-test probabilities, test performance and post-test probabilities are related. The aim of the present study was therefore to evaluate the contribution of L/S ratio and LBC in the prediction of fetal lung immaturity. To do so, we constructed prediction models using patient characteristics and then evaluated whether the prediction changed after adding results from L/S ratio and LBC.
The study was performed in two large teaching hospitals with a tertiary referral function for perinatal medicine in The Netherlands. We reviewed in retrospect the medical files of consecutive women who underwent amniotic fluid sampling for the assessment of fetal lung maturity. Amniotic fluid was collected by transabdominal amniocentesis. Exclusion criteria were an uncertain gestational age, fetal anomalies which could possibly interfere with the occurrence of RDS, and monoamniotic twin pregnancies. Women with ruptured membranes in whom amniotic fluid was collected vaginally, were also excluded from the study. When repeated sampling was performed in one woman, only the last sample before delivery was used for analysis. For each patient, we recorded the reason for admission, use of antenatal glucocorticoids and tocolytic drugs, ultrasound and Doppler findings, multiple pregnancy, gestational age at time of testing and at delivery, contamination of the samples with blood or meconium and neonatal outcome. Gestational age was calculated from the first day of the last menstrual period, or from first trimester ultrasonography. All samples were obtained for clinical purposes. Clinical management of pregnancies was based on the maternal and/or fetal condition and on the outcome of the fetal lung maturity tests. The occurrence of RDS was diagnosed according to clinical symptoms of respiratory stress and findings on chest radiographs . Clinical symptoms of respiratory stress included the need for continuous positive airway pressure for at least 24 h. Moreover, an experienced neonatologist, who was unaware of the outcome of the fetal lung maturity tests, reviewed all chest radiographs of the infants with reported RDS. Women of whom the outcome of the infant was unknown or of whom the infant died within 24 h after delivery without developing RDS, were excluded from the analysis.
Multiple regression analysis indicated that both the L/S ratio and the LBC varied statistically significant with gestational age. Both were statistically different in the patients who delivered more than 14 days after amniocentesis (both P values <0.01). However, the L/S ratio and the LBC differed not statistically significant between women who delivered within 48 h after amniocentesis, women that delivered between 48 h and 7 days after amniocentesis, and women who delivered between 7 and 14 days after amniocentesis .
In view of these data, we excluded women who delivered more than 14 days after the amniocentesis. The exclusion of these women introduces a bias called verification bias, since women with low fetal lung maturity test results were more likely to be excluded than women with normal or high fetal lung maturity test results . We controlled for verification bias by calculating for each women the probability of verification, i.e. the probability that a woman delivered within 2 weeks after amniotic fluid collection . This probability was calculated with a logistic regression model, in which the occurrence of verification was the dependent variable, and gestational age and the results of fetal lung parameters were the independent variables . This probability was used as a weight factor in the statistical calculations that are described later in the “Methods” section.
Immediately after arrival at the laboratory, amniotic fluid samples were centrifuged for 5 min at 450g. In one center the LBC was performed on a Technicon H*1 haematology analyser (Bayer Diagnostics, Tarrytown, USA) during the first 3 years and on a Cell-Dyn 4000 (Abbott Diagnostics, Santa Clara, USA) from 1999. The other center used only the Cell Dyn 1600 (Abbott Diagnostics, Santa Clara USA). Measurements on the Technicon H*1 were made with the laser cell counter (helium neon laser at 632.8 nm) with measurement on the basis of low angle scatter signal with fixed thresholds for counting between 2 μ and 30 μm (platelet channel). Measurements on the Cell-Dyn were made with the aperture impedance cell counter in the erythrocyte/platelet channel. The histogram of the platelets was used for the LBC. Both methods were calibrated against each other. In both centers the L/S ratio was determined by thin-layer chromatography according to Gluck et al.  and performed in duplicate. The variation between the measurements was about 15% for both the L/S ratio and the LBC. We tested several amniotic fluid samples in both centers to verify comparability of L/S and LBC results and did not find statistically significant differences.
We distinguished three categories of women based on the gestational age at amniocentesis: (1) <30 weeks; (2) between 30 and 33 weeks; (3) >33 weeks. In each category, we used stepwise logistic regression to construct a model for the prediction of RDS, using clinical characteristics, but not the results of L/S ratio and LBC. Clinical characteristics considered were reason for admission, use of antenatal glucocorticoids and tocolytic drugs, multiple pregnancy, ultrasound findings, diabetes type I, gestational diabetes, gender of the fetus, and gestational age at time of amniocentesis. The probability of verification was used as relative weight in this logistic model. Selection of variables is usually performed with a significance level of 0.05. However, the incorrect exclusion of a prognostic factor would be more deleterious than including too many factors . Therefore, multivariable analysis included all variables with a P value <0.30. We then added the LBC to the model in the three gestational age groups. Subsequently, we plotted the probability of RDS as predicted by the model without the LBC against the probability of RDS predicted by the model with the LBC added to the clinical characteristics. Similarly, the L/S ratio was forced into the models with the LBC, and we then plotted the probability predicted with the model with the LBC added to the clinical characteristics against the probability predicted with L/S ratio added to the clinical characteristics.
During the study period, 363 women met the inclusion criteria. From these women, 13 infants died within 24 h without developing RDS and from 16 infants we had an incomplete follow up because of transport to another hospital. There were 154 women who delivered within 48 h after amniocentesis, 82 who delivered within 48 h and 7 days, 49 who delivered within 7 and 14 days, and 49 who delivered more than 14 days after amniocentesis. For the analysis, we excluded the 49 women who delivered more than 14 days after amniocentesis. Thus, 285 women were available for analysis, of whom 61 infants (21%) developed RDS. In 31 women (11%) the L/S ratio and in 19 women (7%) the LBC was not determined.
The three logistic models that were used to calculate the probability of RDS
Model without fetal lung maturity tests
Model with LBC
Model with L/S ratio
Gestational age (per day)
Fetal growth (%)
This study reports on the contribution of L/S ratio and LBC to the prediction of fetal lung immaturity. To do so, we constructed prediction models using patient characteristics, and then evaluated whether the prediction changed after adding results from L/S ratio and LBC. In women who had an amniocentesis below 30 weeks, the specificity of the LBC was 30% for a 100% sensitivity. Use of the L/S ratio increased the specificity to 60%, for a sensitivity of 100%. In women who had amniocentesis between 30 and 33 weeks, the specificity of the LBC was 40% for a sensitivity of 96%. Addition of the L/S ratio improved the specificity only slightly. In women who had amniocentesis above 33 weeks, 15% could have been identified as being at low risk before amniocentesis. Addition of the LBC increased the specificity to 30%.
Although we had a relatively large sample size, and we used a P value as threshold for selection in the model of 0.10, other factors known to be associated with the development of RDS, such as maternal diabetes, being born from a multiple pregnancy and being a male fetus were not selected in our model. Individual patient data meta-analysis, in which data from individual studies are added, might be a solution to overcome this problem of statistical power.
When the results of fetal lung maturity tests are divided in ‘mature’ and ‘immature’ results, which is done in most studies on fetal lung maturity testing, the gradual maturation of the fetal lung is not taken into account. We divided our population in three categories based on gestational age and calculated the pre-test probability for RDS based on the gestational age. Unfortunately, the gestational age group >33 weeks covered a wide period with different risks for RDS. However, the number of patients and the number of infants with RDS were too low to divide this population into subgroups. Another limitation of our study is that the severity of RDS could not be taken into account.
The novel approach used in this study is the introduction of prediction models, rather than the use of fixed cut-off levels for LBC or L/S ratio. As a consequence, the interpretation of the LBC or L/S ratio is different at different gestational ages. As can be seen from the constructed logistic models as well as from the Figs. 1, 2 and 3, a lower gestational age results in a higher probability of RDS, and thus requires a relatively high LBC or L/S ratio for a similar post-test probability, as compared to a similar patient undergoing the tests at a higher gestational age .
In studies on fetal lung maturity testing, analysis is often limited to women who delivered within 72 h after amniotic fluid collection. Thereafter, it is thought that the test result is not a reliable measure of the actual state of fetal lung maturity anymore. However, as the decision to accept delivery is likely to depend on the results of the tests, the number of cases with a test result indicating mature fetal lungs, will be over represented if women who delivered after 72 h are excluded. The bias introduced by excluding women who delivered after a certain interval after amniocentesis is called verification bias. In our previous study, only women who delivered more than 2 weeks after amniotic fluid collection appeared to have a lower L/S ratio . To correct for verification bias, we calculated the probability for verification for the included women and used this probability as relative weight in the logistic model to calculate the risk for RDS [8, 9].
If we want to assess whether an amniocentesis to obtain information on fetal lung maturity is useful, we should consider both the prevalence of RDS as well as the additional information obtained from either the LBC or the L/S ratio. From the prediction models that were developed in this study, we can obtain the predicted prevalence of RDS-obtained without testing, the predicted prevalence of RDS-obtained after testing with the LBC and the predicted prevalence of RDS-obtained after testing with the L/S ratio. If we assume an arbitrarily chosen threshold probability of RDS, and decide to postpone delivery in case the probability of RDS is over the threshold, then we can discriminate four categories of patients: (1) patients in whom delivery is delayed in the absence of fetal lung maturity (true-positives, ‘wait correctly’); (2) patients in whom delivery is delayed in the presence of fetal lung maturity (false-positives, ‘wait incorrectly’); (3) patients in whom delivery is allowed in the absence of fetal lung maturity (false-negatives, ‘deliver incorrectly’); and (4) patients in whom delivery is allowed in the presence of fetal lung maturity (true-negatives, ‘deliver correctly’).
Classification of patients at risk for pre-term delivery as true-positive, false-negative, false-positive, and true-negative, for gestational age <30 weeks, and based on three prognostic models: (a) model without fetal lung maturity testing (b) model with LBC and (c) model with L/S ratio
(a) No test
(c) L/S ratio
Classification of patients at risk for pre-term delivery as true-positive, false-negative, false-positive, and true-negative, for gestational age 30–33 weeks, and based on three prognostic models: (a) model without fetal lung maturity testing (b) model with LBC and (c) model with L/S ratio
(a) No test
(c) L/S ratio
Classification of patients at risk for pre-term delivery as true-positive, false-negative, false-positive, and true-negative, for gestational age >33 weeks, and based on three prognostic models: (a) model without fetal lung maturity testing (b) model with LBC and (c) model with L/S ratio
(a) No test
(c) L/S ratio
Recently, Karcher et al.  reported on the accuracy of the lamellar body count and surfactant-to-albumin ratio in relation to gestational age in the prediction of RDS. In contrast to our study, they only recruited women at a gestational age >34 weeks. Consequently, the prevalence of RDS in their study was only 6%. This hampers comparison with our study. Moreover, the authors did not correct for verification bias, as we did in the current study.
In summary, we can state that if one wants to assess fetal lung maturity below 30 weeks the L/S ratio is the procedure of choice, and that the value of the LBC is limited in these patients. At a gestational age between 30 and 33 weeks, assessment of fetal lung maturity with the LBC is useful. As can be derived from Fig. 2, a probability of RDS below 15% virtually rules out the probability of RDS, whereas at higher probabilities additional performance of the L/S ratio is useful. At a gestational age above 33 weeks, the value of fetal lung maturity assessment is debatable due to the low prevalence of RDS.
Whether invasive testing for fetal lung maturity is useful, will depend on the effectiveness of expectant management as compared to immediate delivery in fetuses of which lung maturity tests have indicated mature lungs. While invasive fetal lungs test predicts the presence of mature lungs correctly, immediate delivery does not improve the outcome for mother and infant as compared to expectant management, then the fetal lung test might be accurate, but it will not be of value for the patient.
The value of the fetal lung maturity tests will also depend on the individual clinical situation, for example on the risk of intrauterine death in case delivery is postponed, and on the risk of deterioration of the condition of the mother in case of severe pre-eclampsia. The clinical situations will differ in women with fetal growth restriction in singleton pregnancies, in fetal growth restriction in twin pregnancies, and in women with severe pre-eclampsia. Such situations should be addressed in decision analytic models as well as in new clinical studies on the subject.
Another potential indication for invasive fetal lung testing might be that testing might prevent unnecessary or even harmful treatment with glucocorticoids. A restrictive use of these drugs may be achieved by obtaining information on the status of the fetal lung maturation. This is especially important in elective (medically indicated) preterm deliveries. In those cases, timing of delivery might depend on information of the fetal lung maturation. In such cases glucocorticoid administration may be reserved for those cases in which fetal lung testing has indicated immaturity. This strategy should be studied in decision analytic models and possibly also in randomised controlled trials.
Conflict of interest statement
None of the authors have a conflict of interest.
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