Lamellar Body Count As a Predictor of Neonatal Lung Maturity in High‐Risk

International Journal of Gynecology and Obstetrics (2005) 89,19—25

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CLINICAL ARTICLE Lamellar body count as a predictor of neonatal lung maturity in high-risk pregnancies

D.E.M. Abd El Aala,*, A.A. Elkhirshyb, S. Atwac, M.Y. El-Kabshc aDepartment of Obstetrics and Gynecology, Faculty of Medicine, Assiut University, Assiut, Egypt bDepartment of Pediatrics, Faculty of Medicine, Assiut University, Assiut, Egypt cDepartment of Clinical Pathology, Faculty of Medicine, Assiut University, Assiut, Egypt

Received 12 April 2004; received in revised form 20 December 2004; accepted 20 December 2004

KEYWORDS Abstract Lamellar body count; Neonatal lung Objectives: To compare the usefulness of a lamellar body count, a fluorescence maturity; polarization assay, and the foam stability index for predicting neonatal lung High-risk pregnancy maturity in high-risk pregnancies. Setting: This study was conducted at the Department of Obstetrics and Gynecology and the Department of Pediatrics, Assiut University Hospital. Design: A prospective clinical trail. Subjects and Methods: This study was performed after recruiting 73 pregnant women, 52 with high-risk pregnancies (25 had diabetes and 27 had premature labor) and 21 with a healthy full-term singleton pregnancy as controls. All women were delivered in the Department of Obstetrics and Gynecology of Assiut University Hospital. The newborns with respiratory distress syndrome (RDS) were admitted in the neonatal intensive care unit of the Department of Pediatrics. Amniotic fluid specimens were obtained near delivery. Apgar score, vital signs, anthropometric data, and complete clinical examination results were available for all newborns, and particular emphasis was placed on signs of RDS. Results: The incidence of RDS was 44.2% in the newborns of women who had experienced a high-risk pregnancy (of these, 82.6% were born preterm and 17.4% to diabetic mothers). We found that a lamellar body count is a good screening test for predicting neonatal lung maturity. It is as good as the fetal lung maturity assay by fluorescence polarization in some respects and better in others; moreover, it is better than the foam stability index test in all respects. A lamellar body count with cutoffs of 41Â103/AL and 18Â103/AL was a good predictor of low and high risks of RDS in newborns. Values between 19Â103/AL and 40Â103/AL were the best to predict an intermediate risk of RDS.

* Corresponding author. Tel: +20 105212137; fax: +20 882333327. E-mail address: [email protected] (D.E.M. Abd El Aal).

0020-7292/$ - see front matter D 2005 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijgo.2004.12.030 20 D.E.M. Abd El Aal et al.

Conclusion: Lamellar body count is a good screening test for predicting the degree of neonatal lung maturity. D 2005 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved.

1. Introduction 2. Subjects and methods

Despite the development of artificial surfactants The study is a prospective clinical trial including and advances in respiratory support, respiratory 73 pregnant women and their newborns deliv- distress syndrome (RDS) continues to be a major ered in the obstetrics department of Assiut problem in newborns. As the successful establish- University Hospital from January 2002 to Decem- ment of adequate lung function at birth is depend- ber 2002. ent on the maturity of the respiratory system [1], There were 52 women at high risk for RDS, the ability to accurately predict neonatal lung 25 women with diabetes mellitus, and 27 maturity would be a helpful adjunct in the manage- women having preterm labor. Their newborns ment of pregnancies when premature delivery is were admitted to the neonatal intensive care expected or administration of glucocorticoids is unit of the Pediatrics Department. The control needed [2]. group included 21 pregnant women with a Lamellar bodies—1- to 5-Am lamellated struc- normal full-term singleton pregnancy who were tures synthesized by type 2 granular pneumocytes vaginally delivered and had no history of new- [3]—can be counted using commercial blood cell borns affected with RDS. Cases involving a dead analyzers [4]. These storage granules contain phos- fetus or a pregnant woman receiving cortico- pholipids, cholesterol, and several surfactant-spe- steroids, having hypertensive disease, or experi- cific proteins and become pulmonary surfactant [5]. encing premature rupture of membranes were Lamellar bodies first appear in the cytoplasm of excluded. fetal pneumocytes between the 20th and the 24th Detailed history taking, complete clinical and week of gestation [6]. The lamellar bodies abdominal sonographic examinations, and random become more numerous, larger, and are continu- blood sugar estimation were performed for each ously secreted into the fetal alveoli, and fetal participating woman. Amniotic fluid specimens breathing movements and exudation of lung fluid were obtained near delivery by amniocentesis carry these lamellar bodies into the amniotic fluid under sonographic guidance under complete asep- [7]. The phospholipid content and the laminated sis (1 case); transvaginal amniotomy (43 cases); structure of these particles change as the fetal or amniotomy during Cesarean delivery (29 lung is maturing [8]. The fetal lung maturity (FLM) cases). test consists in measuring the surfactant—albumin Each newborn received a complete clinical ratio in the amniotic fluid. Other means of examination, arterial blood gases analyses at predicting neonatal lung maturity include calcu- admission and daily thereafter, a chest radiograph lating the lecithin—sphingomyelin (L/S) ratio, (within 6—12 h when possible), and follow-up care measuring amniotic fluid levels of phosphatidyl- to monitor clinical progress. glycerol [9], performing the foam stability test A minimum of 5 mL of amniotic fluid was [10] or a fluorescence polarization assay [11], and obtained in a test tube. The specimens were well measuring amniotic fluid optical density at 650 mixed by inverting the collection tube 5—10 nm [12]; all of these tests measure some aspects times or placing it on a tube rocker for 2—5 of the pulmonary surfactant contained in the min, and its contents were transferred to a clear amniotic fluid and none is perfect for prediction test tube. Since blood and meconium may of lung maturity. The ideal test must be readily interfere with most tests, the condition of the available, inexpensive, and of good predictive specimen was then reported as uncontaminated, value. bloody, meconium-stained, xanthochromic, or The aim of this study was to compare obviously contaminated with mucus. The speci- lamellar body count, fetal lung maturity assay mens were finally divided into separate aliquots: by fluorescence polarization, and foam stability 0.5—1 mL for LBC, at least 1 mL for the index in the prediction of neonatal lung fluorescence polarization FLM assay, and at least maturity. 3—3.5 mL for the FSI test. Lamellar body count as a predictor of neonatal lung maturity in high-risk pregnancies 21

2.1. LBC Table 2 Comparison between newborns with respiratory distress syndrome (RDS) and newborns in the Lamellar body particles were directly counted control group according to lamellar body count (LBC), using the platelet channel of a standard hema- fetal lung maturity (FLM), and foam stability index tology cell counter (Coulter Counter Model STKS; (FSI) Coulter Electronics, Hialeah, Fla) or other Test Newborns Controls P Coulter Counter model hematology cell counters with RDS value 3 [13]. LBC Â10 /AL, meanFS.D. 20F22 70F33 0.00T FLM mg/g, meanFS.D. 20.6F9.9 58.8F13.3 0.00T FSI, dilution, mean 0.44 0.47 0.00T 2.2. FLM assay by fluorescence polarization T P b 0.001.

The FLM assay was done by using the TDx/Fetal Lung Maturity II (FLM II) assay kit with the Abbott reference cutoff levels are shown in Tables 2 TDx fluorescence polarization device (Abbott Labo- and 3. ratories, North Chicago, IL) [14]. 3.1. Performance characteristics of LBC at 2.3. FSI test different cutoff values

When pulmonary surfactant is in sufficient con- Using the cutoff value of 15Â103/AL to indicate centration in the amniotic fluid, the fluid is able pulmonary maturity, LBC predicted 18 cases of RDS to form a highly stable surface film that can with 5 false-negatives (for a sensitivity of 78.26% support the structure of foam. Other substances and a negative predictive value [NPV] of 90.90%); in the fluid, including proteins, bile salts, and this 15Â103/AL cutoff value resulted in no false- salts of free fatty acids, are also capable of positives (for a specificity and a positive predictive forming stable foam. But these can be excluded value [PPV] of 100%). from the film by ethanol, which competes with Using 18Â103/AL as the cutoff value to indicate these other substances for a position in the pulmonary maturity, LBC sensitivity, NPV, specific- surface film [13]. ity, and PPV were found to be the same as with a cutoff value of 15Â103/AL, as in no case were LBC values between 15 and 18Â103/AL. 3. Results Using 41Â103/AL as the cutoff value predicted all RDS cases with no false-negatives, which Of the 72 cases, there were 5 cases of provided a sensitivity and an NPV significantly congenital anomalies, 3 in the preterm group higher than with a cutoff value of 15Â103/AL (1 of anencephaly, 1 of heart disease, and 1 of (100% vs. 78.26%, P=0.00 and 100% vs. 90.90, multiple anomalies) and 2 in the infants of P=0.01, respectively) but resulted in seven false- diabetic mothers (IDM) group (1 of achondropla- positives, for a specificity and PPV less than with a sia and 1 of heart disease). The percentage of RDS in the preterm and IDM groups is shown in Table 3 Comparison between the results of tests for Table 1. Comparison results for the three lung lung maturity (LBC, FLM, FSI) and the presence or maturity tests (LBC, FLM, and FSI) and the absence of respiratory distress syndrome (RDS) presence or absence of RDS according to according to cutoff valuesa Test Interpretation RDS No RDS LBC, Â103/AL V15 18 (100) 0 (0) 16—49 5 (36) 9 (64) z50 0 (0) 20 (100) Table 1 Presence or absence of respiratory distress FLM, mg/g V39 22 (96) 1 (4) syndrome (RDS) in preterm infants (PT) and infants of 40—54 1 (10) 9 (90) diabetic mothers (IDM)a z55 0 (0) 19 (100) FSI, dilution V0.44 16 (100) 0 (100) RDS status PT (n=27) IDM (n=25) P value 0.45, 0.46 7 (70) 3 (30) Yes 19 (83%) 4 (17%) 0.002a z0.47 0 (0) 26 (100) No 8 (28%) 21 (72%) 0.01a Abbreviations: FLM, fetal lung maturity assay test; LBC, P value 0.03 0.001 lamellar body count; FSI, foam stability index test. a Values are given as number (percentage) unless otherwise a Values are given as number (percentage) unless otherwise indicated. indicated. 22 D.E.M. Abd El Aal et al.

Table 4 Comparison between performance characteristics of LBC vs. FLM and FSI tests at different cutoff points Cutoff values Sensitivity Specificity PPV NPV LBC V15 78.26% 100% 100% 90.90% V18 78.26% 100% 100% 90.90% z41 100% 86% 76.67% 100% z50 100% 74% 63.89% 100% FLM 95.65% 96.55% 95.65% 96.55% P value V15 0.00 0.08 0.04 0.07 V18 0.00 0.08 0.04 0.07 z41 0.04 0.005 0.00 0.08 z50 0.04 0.00 0.00 0.08 FSI 65.22% 100% 100% 78.38% P value V15 0.04T 1 1 0.01 V18 0.04 1 1 0.01 z41 0.00 0.00 0.00 0.00 z50 0.00 0.00 0.00 0.00 Abbreviations: FLM, fetal lung maturity assay test; LBC, lamellar body count; FSI, foam stability index; PPV, positive predictive value; NPV, negative predictive value. T P b 0.001. cutoff value of 15Â103/AL (86% vs. 100%, P=0.00 mg/g cutoff value (96.5% vs. 100%, P=0.06 and and 76.67% vs. 100%, P=0.00, respectively). 95.6% vs. 100%, P=0.06, respectively). Using a cutoff value of 50Â103/AL, LBC pre- Using a cutoff value of 41 mg/g, it predicted all dicted all RDS cases without false-negatives (100% cases of RDS, with one false-negative (for a sensi- sensitivity and 100% NPV), but resulted in more tivity of 100% and a NPV of 100%). One false-positive than false-positives than with a cutoff of 41Â103/ provided a specificity of 96.6% and a PPV of 95.8%. AL (13 false-positives), for a specificity and a PPV A cutoff value of 55 mg/g predicted all cases of significantly less than with a cutoff of 41Â103/AL RDS, with 10 false-positives, for a specificity and a (74% vs. 86%, P=0.03 and 63.89% vs. 76.67%, negative predictive value significantly lower than P=0.04, respectively) (Table 4). with the 41 mg/g cutoff value (65.5% vs. 96.6%, P=0.00) (Table 5). 3.2. Performance characteristics of FLM test at different cutoff values 3.3. Comparison between the performance characteristics of LBC vs. the FLM assay and The FLM assay with a cutoff value of 30 mg/g FSI test at different cutoff points predicted 19 cases of RDS with 4 false-negatives (for a sensitivity of 82.6% and a NPV of 89.2%), with The FSI test at a cutoff value of 0.44 predicted 15 no false-positives (for a specificity of 100% and a cases of RDS with 8 false-negatives (for a sensitivity PPV of 100%). of 65.22% and a NPV of 78.38%), without false- Using a cutoff value of 39 mg/g, the FLM assay positives (for a specificity of 100% and a PPV of predicted 22 cases of RDS, with 1 false-negative for 100%). Using 15 or 18Â103/AL as the LBC cutoff a sensitivity and an NPV significantly higher than values, the sensitivity of LBC was significantly with a cutoff value of 30 mg/g (95.6% vs 82.6%, lower than that of the FLM assay and significantly P=0.004 and 96.5% vs. 89.2%, P=0.02, respectively). higher than that of the FSI test (78.26% vs. 95.65%, One false-positive did not result in a specificity and P=0.00 and 78.26% vs. 65.22%, P=0.04, respec- a PPV significantly different from those at the at 30 tively). Also, the NPV of LBC was significantly higher than that of the FSI test (90.90% vs. Table 5 Performance characteristics of the fetal 78.38%, P=0.01) and not significantly different from lung maturity assay (FLM) at different cutoff values that of the FLM assay (90.90% vs. 96.55%, P=0.07). Cutoff Sensitivity Specificity PPV NPV Although the specificity and PPV of LBC at this values (%) (%) (%) (%) cutoff value was the same as those of the FSI test, V30 82.6 100 100 89.2 the PPV of LBC was significantly higher than that of V39 95.6 96.5 95.6 96.5 FLM assay (100% vs. 95.65%, P=0.04) and the z41 100 96.6 95.8 100 specificity was not significantly different (100% z55 100 65.5 69.6 100 vs. 96.55%, P=0.08). Abbreviations: FLM, fetal lung maturity assay test; NPV, Using 41Â103/AL as the LBC cutoff, the sensi- negative predictive value; PPV, positive predictive value. tivity and NPV of LBC were significantly higher than Lamellar body count as a predictor of neonatal lung maturity in high-risk pregnancies 23 those of the FSI test (100% vs. 65.22%, P=0.00 and maturity [16], and LBC appears to be a good 100% vs. 78.38%, P=0.00, respectively), but screening test [7]. resulted in a specificity and a PPV significantly The incidence of RDS in this study was 44.2% (23 lower than those of the FSI test (86% vs. 100%, cases), which is higher than that recorded by P=0.00 and 76.67% vs. 100%, P=0.00, respec- Dalence et al. (10%) [7] and Ashwood et al. (11%) tively). At the same cutoff point, the sensitivity [13]. This difference most probably reflects the of LBC was significantly higher than that of the high-risk characteristics of our patients and the FLM assay (100% vs. 95.65%, P=0.04), but the NPV lack of antenatal care and high rate of premature was not significantly different from that of the labor in our area, which is in agreement with a FLM assay (100% vs. 96.55%, P=0.08), and the report by Kaplan et al. [17]. The authors consider specificity and PPV of LBC were significantly gestational age as a very important clinical pre- lower than those of the FLM assay (86% vs. dictor in the interpretation of the fluorescence 96.55%, P=0.005 and 76.67% vs. 95.65%, P=0.00, polarization FLM assay. respectively). In this study, only four newborns (16%) in the Using 50Â103/AL as the LBC cutoff value, the IDM group (25 mothers) had RDS. This is in not in specificity and PPV of the FLM assay were agreement with Greenough and Roberton [18], significantly higher that those of LBC (96.55% who reported that infants of diabetic mothers vs. 74%, P=0.00 and 95.65% vs. 63.86%, P=0.00, have an increased incidence of RDS. But our respectively). Although the sensitivity of LBC results are in agreement with those of Kjos et was significantly higher than that of FLM assay al. [19], who reported that, with improvements in (100% vs. 95.65%, P=0.04), the NPV of LBC was maternal diabetic control during pregnancy, deliv- not significantly different from that of the FLM ery can be delayed until 39—40 weeks of gesta- assay (100% vs. 96.55%, P=0.08). It was also tion. Most studies reported normal surfactant found that the sensitivity and NPV of LBC at patterns in the newborns of women with diabetes, this cutoff point was significantly higher than and that RDS cases are now extremely rare in those of the FSI assay (100% vs. 65.22%, P=0.00 these newborns. and 100% vs. 78.38%, P=0.00, respectively), but To predict the lung maturity of a newborn, we that the specificity and PPV were significantly can use certain tests (LBC, fluorescence polar- higher than those of LBC (100% vs. 74%, P=0.00 ization FLM assay, and FSI). The result values of and 100% vs. 63.89%, P=0.00, respectively) these tests will be lower in the amniotic fluid of the (Table 4). newborns expected to have RDS. In this study, it was found that LBC, fluorescence polarization FLM assay, and FSI test results were significantly lower in cases of RDS compared with controls, which is in 4. Discussion agreement with Dalence et al. [13], Alvarez et al. [15], and Sher and Statland [10]. Fetal lung surfactant is synthesized by type 2 Regarding LBC, we interpreted our results alveolar cells, packaged in the form of lamellar using cutoff values of 15Â103/ALorlessto bodies, and transported to the alveolar space. indicate lung immaturity (presence of RDS), Before delivery, a steady efflux of fluid between 50Â103/AL or greater to indicate full lung the alveoli and the amniotic cavity results in an maturity (no RDS), and a transitional zone 16 equilibrium of the concentration of surfactant in to 49Â103/AL as an intermediate risk to develop the amniotic fluid and the alveoli. Therefore, RDS, as recommended by Neerhof et al. [20] in the amniotic fluid level of fetal surfactant their protocol. No cases with RDS were detected indicates the level of surfactant in the alveoli. with a cutoff value of 41Â103/AL or greater, and After delivery, the initial passage of air into the the lowest value at which newborns were found alveoli initiates the unraveling of the lamellar to have RDS was 18Â103/AL or less. Therefore, bodies and the formation of a monolayer of according to the previously mentioned data, we surfactant at the air—liquid interface of the determined two cutoff values for LBC to predict alveoli. The surface tension-lowering effect of neonatal lung maturity (18 and 41Â103/AL), and the surfactant monolayer will prevent the col- this will be more accurate for detection of cases lapse of the alveoli and the development of RDS with RDS. [15]. Regarding the fluorescence polarization FLM The management of pregnancies at risk for assay, we have interpreted our results using a neonatal RDS would be enhanced by a rapid, cutoff value of 39 mg/g or less to indicate lung accurate, and objective test predicting fetal lung immaturity (presence of RDS), 55 mg/g or greater 24 D.E.M. Abd El Aal et al. to indicate lung maturity (no RDS), and a transi- decision threshold, with no cases of RDS at a tional zone of 40—54 mg/g as indicating an higher LBC. They did not evaluate a lower cutoff intermediate risk of developing RDS, as recommen- value indicating a high risk for RDS. Our study, ded by Herbert et al [14]. however, recommends a lower cutoff value The lowest cutoff value for the fluorescence (41Â103/AL). polarization FLM assay was 30 mg/g or less when We are in agreement with Dalence et al. [16] RDS was found and 41 mg/g or higher when no RDS who determined two cutoff values to indicate low was found. We detected two cutoff points for the and high risk for RDS; the differences between fluorescence polarization FLM assay to predict their recommended values of 30Â103/ALand neonatal lung maturity (30 and 41mg/g) and they 10Â103/AL and ours to predict low and high risk will be more accurate in the detection of cases with for RDS is due to a different centrifugation RDS. protocol. Also, we are in agreement with Dubin Regarding the FSI test, we interpreted our [2] who recommends a cutoff value of 40Â103/AL, results using an FSI of 0.44 or less to indicate lung but he, too, did not evaluate a lower cutoff immaturity (presence of RDS), 0.47 or greater to indicating a high risk for RDS. indicate lung maturity (no RDS), and 0.45—0.46 to Both LBC and the FLM assay are good predictors indicate an intermediate risk for the development of lung maturity. However, the LBC is inexpensive, of RDS. The three levels that we used as cutoff faster, and requires a smaller sample of amniotic values were close to those recommended by Sher fluid than the FLM assay. and Statland [10]. In most clinical settings, the most important function of a fetal lung maturity test is to predict References accurately the absence of RDS [16]. It was further noted that the ability to accurately predict the [1] Stoll BJ, Kliegman RM. Respiratory tract disorders. Nelson absence of RDS (negative predictive value) is the text book of pediatrics. 16th ed. Philadelphia7 W.B. most important characteristic in fetal lung matur- Saunders Co.;, 2000. p. 496–510. ity analysis [20]. Regarding the specificity and [2] Dubin SB. Characterization of amniotic fluid lamellar bodies PPV of lower cutoff values for LBC (15 and by resistive-pulse counting: relationship to measures of 3 A fetal lung maturity. 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