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Lamellar Body Counts Compared With Traditional Phospholipid Analysis as an Assay for Evaluating Fetal Lung Maturity

From the Department of Obstetrics and Gynecology, Northwestern University Medical School, Evanston Northwestern Healthcare, Evanston, Illinois; University of Utah School of Medicine, Salt Lake City, Utah; the College of Medicine, University of Ulsan, Asan Medical Center, Seoul, Korea; and the Second Institute of Gynecology and Obstetrics, "La Sapienza" University, Rome, Italy.

Address reprint requests to: Mark G. Neerhof, DO Evanston Northwestern Healthcare 2650 Ridge Avenue Evanston, IL 60201 E-mail: m-neerhof{at}northwestern.edu

	Abstract

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Objective: To compare lamellar body counts with the lecithin/sphingomyelin ratio and phosphatidylglycerol analysis in terms of assessment of risk of respiratory distress syndrome (RDS).

Methods: Lamellar body counts, lecithin-sphingomyelin ratios (L/Ss), and phosphatidylglycerol levels were assessed in 1611 amniotic fluid samples obtained at four clinical sites from pregnant women whose fetuses were at risk for RDS. Cases in which delivery occurred within 72 hours of sample collection (n = 833) were analyzed. Specific cutoffs for predicting the likelihood of RDS for both the lamellar body count and the L/S had been derived previously at each of the clinical sites based on receiver operating characteristic curves using unrelated samples, whereas phosphatidylglycerol was reported as either mature (present) or immature (absent). Standard clinical and radiographic criteria were used to diagnose RDS, and the diagnosis was confirmed by review of newborn records.

Results: One hundred (12.0%) of the 833 infants delivered within 72 hours of sample collection developed RDS. The negative predictive value of the lamellar body count (97.7%) was similar to that of the L/S (96.8%) and slightly better than that of phosphatidylglycerol analysis (94.7%) (P = .048). The lamellar body count performed as well as phospholipid analysis irrespective of gestational age or patient population.

Conclusion: The lamellar body count compares favorably with traditional phospholipid analysis as an assay for assessment of fetal lung maturity. Lamellar body counts are preferable because they are faster, more objective, less labor intensive, less technique dependent, and less expensive and because they can be performed with equipment available in every hospital laboratory.

Determination of fetal lung maturity status is central to obstetric management strategies. Gluck et al¹ first described evaluation of the amniotic fluid (AF) lecithin-sphingomyelin ratio (L/S) by thin-layer chromatography, in 1971. Phosphatidylglycerol analysis subsequently was added in response to the appreciated limitations of the L/S, particularly in diabetic patients.² Amniotic fluid phospholipid analysis by thin-layer chromatography continues to be commonly used for fetal lung maturity analysis. Although it is an effective means of evaluating fetal lung maturity status, phospholipid analysis is also labor intensive, costly, prone to subjective interpretation, and not available in all laboratories. Further, the L/S cannot be determined in fluids contaminated by blood or meconium. These limitations of phospholipid analysis have led to the consideration of alternatives for fetal lung maturity analysis.^3,4 These assays have not yet been widely accepted, for a variety of reasons, including cost, poor sensitivity or specificity, and a reluctance to change from a time-tested assay, given the fact that the clinical consequences of using an inferior test can have significant consequences in the newborn.

Lamellar bodies are concentrically layered "packages" of phospholipid that are produced by type II alveolar cells, representing the storage form of surfactant. They are present in AF in increasing quantities as gestation advances.^5,6 Lamellar bodies are similar in size to platelets and can be counted in the platelet channel of most electronic cell counters. In 1989, Dubin⁷ described a method for quantifying lamellar bodies using widely available commercial cell counters. Since that time, the lamellar body count has been used in several institutions, and the utility of the lamellar body count as an assay for determining pulmonary maturity has been evaluated in several retrospective and prospective studies.^5,6,8–15 The purpose of this study was to compare the predictive value of the lamellar body count with that of traditional phospholipid analysis as an assay for assessment of fetal lung maturity.

	Materials and Methods

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Between 1992 and 1999, AF samples were obtained from 1611 patients at four participating clinical sites who either were at risk of preterm delivery (preterm labor, premature rupture of membranes [PROM], or preeclampsia) or were anticipating elective delivery. Patients who delivered within 72 hours of collection of the AF sample and who had neonatal follow-up data available constituted the study group (n = 833).

Samples were analyzed immediately after arrival at the laboratory. The lamellar body counts were performed according to established protocol at each institution; details of these protocols are outlined in Table 1. The procedure for lamellar body counts took 15 minutes or less. At each clinical site, lamellar body counts were reported as mature, immature, or transitional. The cutoff for predicting a high likelihood of fetal lung maturity and the cutoff for predicting a high likelihood of respiratory distress syndrome (RDS) had been derived previously from receiver operating characteristic (ROC) curves using unrelated samples at each institution, as previously described.^6,10,12,13

The respiratory status of each newborn was reviewed and documented. The diagnosis of RDS was based on the documented presence of all three of the following: physical signs (nasal flaring, grunting, retractions, tachypnea), supplemental oxygen requirement of more than 24 hours, and radiographic findings (reticulogranular opacification of lung fields with superimposed air bronchograms). Neonates classified as having transient tachypnea of the newborn were not considered to have RDS. Neonates who received surfactant for established RDS were included in this analysis as subjects with RDS. Eighteen neonates received surfactant prophylactically (ie, at birth) and were excluded from the analysis because of this confounding variable.

Sensitivity, specificity, and positive and negative predictive values were calculated for each of the assays. For the comparison of negative predictive values, all negative test results were analyzed categorically using the Fisher exact test.

	Results

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A total of 1611 AF samples were analyzed in the four participating institutions. No patient contributed more than one sample in this study. Of these, 833 patients delivered within 72 hours of collection and had neonatal follow-up data available. Of these 833 patients, 327 had been reported on in previous publications from the four institutions. A total of 198 patients were from Illinois, 290 were from Utah, 151 were from Italy, and 194 were from Korea. Mean (± standard deviation) gestational age at birth was 35.6 ± 2.8 weeks. Respiratory distress syndrome was diagnosed in 100 (12.0%) of the 833 infants. The incidence of RDS among the four clinical sites ranged from 11.1% to 12.4%.

The sensitivity, specificity, and positive and negative predictive values of the three tests in terms of prediction of lung maturity are shown in Table 2. The negative predictive value of the lamellar body count (97.7%) was not significantly different from that of the L/S (96.8%) but was slightly better than that of phosphatidylglycerol analysis (94.7%) (P = .048). One clinical site reported an uncharacteristically poor predictive value for phosphatidylglycerol analysis (Table 3). When the phosphatidylglycerol data from this site were excluded, the negative predictive value of the lamellar body count was similar to that of phosphatidylglycerol analysis (97.1%).

To account for a potential independent effect of gestational age on test performance, data obtained from neonates delivered before 34 completed weeks were reanalyzed separately and the predictive values were calculated (Table 4). As expected, the incidence of RDS was greater in this subgroup than in the entire group (39.7% versus 12.0%). The range in incidence of RDS among clinical sites in this subgroup was 30.6–44.1%. At earlier gestational ages, the negative predictive values of the tests studied were as follows: lamellar body count, 90.7%; L/S, 91.9%; and phosphatidylglycerol analysis, 75.0%. There was no significant difference between the negative predictive values of the lamellar body count and the L/S, but both outperformed phosphatidylglycerol analysis in this regard. Exclusion of data from the clinical site with poor predictive values for phosphatidylglycerol analysis in this subanalysis demonstrated that the negative predictive value of the lamellar body count was similar to that of phosphatidylglycerol analysis (91.7%).

One hundred four of the study subjects had diabetes. Of the infants of this subset of patients, nine had RDS, and their gestational ages ranged from 33 to 37 weeks. All nine of these samples were obtained by amniocentesis. Among the diabetic patients, there were three false-negative lamellar body counts, four false-negative L/Ss, and one false-negative phosphatidylglycerol analysis result. Two were falsely negative for both the lamellar body count and the L/S, and one was falsely negative for both the lamellar body count and phosphatidylglycerol analysis. In the diabetic subset, the negative predictive values of the lamellar body count, the L/S, and phosphatidylglycerol analysis were 94.6, 93.8, and 98.2%, respectively.

Thirty of the AF samples were collected from vaginal pools in patients with PROM. Of the infants of these women, eight had RDS. There were no false-negative lamellar body counts or L/Ss for vaginal pool specimens. There was one false-negative phosphatidylglycerol analysis result.

	Discussion

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The lamellar body count performed as well as traditional phospholipid analysis in the evaluation of fetal lung maturity status. In the context of clinical management, the ability to predict the absence of RDS accurately (negative predictive value) is the most important characteristic in fetal lung maturity analysis. The negative predictive value of the lamellar body count was similar to that of the L/S and exceeded that of phosphatidylglycerol analysis. However, this latter finding is weakened by the fact that there was phosphatidylglycerol testing discordance at one clinical site.

Using a single dichotomous cutoff for fetal lung maturity, intentionally set high to maximize negative predictive value, resulted in a low positive predictive value for the lamellar body count (25.1%) as well as for the L/S (32%) and phosphatidylglycerol analysis (15%). For this reason, a lower (immature) cutoff for the lamellar body count, which predicts a high likelihood of RDS, was introduced at each center. As would be expected, the positive predictive value of the immature cutoff is considerably higher than that of the mature cutoff (45.5 versus 25.1%). The clinical advantage of introducing a lower cutoff is that it further refines the probability of RDS as well as the likelihood that further testing will yield results consistent with fetal lung maturity. In this study, when immature lamellar body counts were obtained, the likelihood of RDS was high (45.5%) and phospholipid analysis yielded results consistent with fetal lung maturity for either the L/S or phosphatidylglycerol analysis in only 19.4% of cases. In contrast, if the lamellar body count was in the transitional zone, the likelihood of RDS was lower (12.5%), and the L/S or phosphatidylglycerol analysis result was mature in 62.5% of cases. An accurate estimation of RDS risk permits more informed clinical judgment. Further, a stepwise approach to lung maturity analysis can be used in which an immature or mature lamellar body count can stand on its own, whereas the transitional values could be refined further by phospholipid or alternative second-line analysis. Omission of further testing in cases in which the risk of RDS is high and the likelihood of mature results from additional testing is low could save both time and cost. This feature makes the lamellar body count particularly attractive as a primary assay for evaluation of fetal lung maturity.

There is considerable variability among laboratories that perform lamellar body assays regarding centrifugation of the AF sample, as demonstrated in Table 1. Therefore, we considered introducing a correction factor that would account for the variation in centrifugation protocols, to normalize data from the four clinical sites. However, because of the potential of introducing error into the analysis, we did not add a correction factor. Further, the purpose of this study was to evaluate the collective clinical experience with lamellar body counts to date, and that experience shows that the lamellar body count is a valuable assay for assessing fetal lung maturity. The lamellar body count was tested at a wide range of gestational ages in several populations of patients, populations that also varied with respect to race and socioeconomic status. The lamellar body count performed as well as phospholipid analysis in all populations of patients and irrespective of gestational age.

Steroid use was not documented uniformly at each site. Steroid exposure might affect test performance of fetal lung maturity assays. This would be most concerning if steroid use were to lead to a falsely increased negative predictive value. However, a subanalysis of findings for patients for whom steroid data were available revealed that steroid use did not affect the negative predictive value of any of the lung maturity assays, including the lamellar body count. Therefore, this concern does not appear to be justified.

Every assay for assessing fetal lung maturity should be used with caution in patients with diabetes, as well as on AF samples collected from vaginal pools. In diabetic patients, the lamellar body count functioned as well as phospholipid analysis. However, the experience with diabetic patients remains limited; consequently, lamellar body counts should be interpreted with caution, particularly in patients with poorly controlled diabetes, in whose infants the risk of RDS is increased significantly. Similarly, the lamellar body count performed well in the case of vaginal pool specimens, but the experience with these samples is limited. Mucus can increase lamellar body counts artificially. Cellular debris also can alter the L/S (cellular material has a low L/S).⁴ Therefore, samples obtained from vaginal pools should not be processed either for lamellar body counts or for L/S if they contain obvious mucus.

Quantitative tests such as the lamellar body count can be affected by AF volume. In cases of oligohydramnios, it is possible that the lamellar body count could be falsely increased, leading to a false-negative test result, although the degree of oligohydramnios required to have this effect likely would be a supervening indication for delivery. Conversely, hydramnios could lead to a false-positive test result.

We conclude that the lamellar body count is superior to traditional phospholipid analysis as an assay for evaluating fetal lung maturity. The lamellar body count performs as well as phospholipid analysis, particularly in terms of negative predictive value. Lamellar body counts are preferable to phospholipid analysis because they are faster, less labor intensive, less technique dependent, less expensive, and more objective and because they can be performed easily in any laboratory with an electronic cell counter.

	Footnotes

 
PII S0029-7844(00)01133-9

Received July 3, 2000. Received in revised form September 21, 2000. Accepted October 12, 2000.

	References

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