Lamellar body counts: A consensus on protocol

Mark G. Neerhof, DO, James C. Dohnal, PhD, Edward R. Ashwood, MD, In-Sik Lee, MD, and Maurizio M. Anceschi, MD
Lamellar bodies, concentrically layered “packages” of phospholipid that represent the storage form of surfactant, can be counted in the platelet channel of most electronic cell counters. The lamellar body count has been used for more than a decade and performs as well as traditional phospholipid analysis as an assay for evaluating fetal lung maturity. It is preferable to phospholipid analysis because it is rapid, objective, and inexpensive and can be performed in any hospital laboratory. The current methodologies for specimen preparation vary widely among laboratories, most notably with respect to centrifugation, resulting in differences in maturity cutoffs used. Our goal was to establish a consensus regarding a standardized methodology for the lamellar body count. Institutions that previously had published their results with lamellar body counts were invited to contribute. The consensus of the four participating institutions includes the following: centrifugation is not a necessary step and should be abandoned, maturity is suggested by a count of 50,000/ L or greater, and immaturity is suggested by a count of 15,000/ L or lower. As the lamellar body count gains wider acceptance as a primary assay for assessing fetal lung maturity, the test must be performed uniformly and accurately, given the implications of acting on a falsely negative test resulting from improper methodology. (Obstet Gynecol 2001;97:318 –20. © 2001 by The American College of Obstetricians and Gynecologists.)

laboratories as an assay for evaluation of fetal lung maturity for just over a decade. During that time, numerous studies have evaluated the performance of the lamellar body count.2–11 In each of these studies, the lamellar body count was found to perform as well as traditional phospholipid analysis as a fetal lung maturity assay, although most of these studies were relatively small. In the report by Neerhof et al in this issue of Obstetrics & Gynecology, the collective experience of four laboratories with lamellar body counts is presented. The goals of that project were to provide a multicenter update on the clinical performance of the lamellar body count compared with traditional phospholipid analysis and, if lamellar body counts were found to compare favorably with traditional phospholipid analysis, to agree on a standardized methodology for lamellar body counts. All of the centers that previously had reported their results with lamellar body counts were invited to participate. The results of this study demonstrated that the lamellar body count performs as well as traditional phospholipid analysis in the assessment of fetal lung maturity, with a high negative predictive value as its most attractive operational characteristic. Consequently, those who were invited to participate in the multicenter update also were invited to contribute to the development of a consensus.

Toward Development of a Consensus
As the lamellar body count was introduced into clinical practice, no single protocol for performance of the assay was followed. Rather, several different protocols have developed that vary most notably with respect to centrifugation. Centrifugation initially was thought to be integral to protocols because it was believed that cellular debris in the AF fluid might interfere with normal functioning of the cell counter. Because most laboratories began evaluation of the lamellar body count by running it in parallel with phospholipid analysis, the centrifugation protocols used derived from pre-existing methodologies already being used for phospholipid analysis, which happen to vary widely. The centrifugation protocol affects the test result, with longer centrifugation times and higher rates resulting in lower lamellar body counts.6,12 As a consequence of the differences in centrifugation protocols among laboratories, the cutoffs for maturity that were established at these laboratories are widely discordant. This leads to confusion when results between institutions are compared or reported. Further, failure to adhere strictly to a centrifugation protocol also can lead to error. Some centers began omitting the centrifugation step altogether, realizing that, in the absence of obvious mucus or heavy meconium staining, processing noncentrifuged AF

Lamellar bodies are produced by type II alveolar cells in increasing quantities as gestation advances. They are composed almost entirely of phospholipid and represent the storage form of surfactant. Quantification of lamellar bodies produces an objective estimate of the quantity of surfactant in amniotic fluid (AF). Lamellar bodies are similar in size to platelets and can be counted in the platelet channel of most electronic cell counters. Thus, lamellar bodies can be quantified readily by running the AF sample through a cell counter and reading off the platelet channel, as described by Dubin in 1989.1 Lamellar body counts have been used by several
From the Departments of Obstetrics and Gynecology and Laboratory Medicine, 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.

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specimens does not affect instrumentation adversely and does not affect test performance.6,12 Centrifugation therefore might cause confusion and error, is not necessary, and should be abandoned. Omission of this step saves time, further simplifies the assay, and makes interpretation of results uniform. With the centrifugation step omitted, a consensus regarding cutoffs is necessary. Both the considerable clinical experience of the centers currently using noncentrifuged samples and extrapolation based on the calculated effects of centrifugation on the lamellar body counts were considered to reach a consensus. Several centers have established cutoffs for maturity using noncentrifuged samples. Greenspoon et al8 found a 100% negative predictive value for a lamellar body count of 46,000/ L. Their lamellar body counts were performed at Cedars-Sinai Medical Center in Los Angeles. The laboratory there, which is the source of the original reports on lamellar body counts, currently uses a cutoff of 40,000/ L. The University of Utah also has considerable experience with lamellar body counts using noncentrifuged samples. Researchers there use a cutoff of 60,000/ L, arbitrarily set high to minimize the likelihood of a false-negative result. In a review of previous studies of lamellar body counts that used centrifugation, Dubin12 extrapolated cutoffs for noncentrifuged samples on the basis of the calculated effect of centrifugation on the lamellar body count. He calculated a maturity cutoff range of 43,000 – 49,000/ L for these previous reports and suggested a conservative cutoff of 50,000/ L for noncentrifuged samples. The clinical experience of the centers using noncentrifuged samples and extrapolation from reports of studies in which centrifugation was used lead us to suggest a maturity cutoff of 50,000/ L for the lamellar body count. The use of a single dichotomous cutoff for fetal lung maturity, set intentionally to maximize negative predictive value, leads to low positive predictive values for the lamellar body count. For this reason, a lower cutoff for the lamellar body count for predicting a high likelihood of respiratory distress syndrome (RDS) has been introduced at most centers. 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. For example, Neerhof et al report in this issue of Obstetrics & Gynecology that when immature lamellar body counts were obtained, the likelihood of RDS was high (45.5%) and traditional phospholipid analysis yielded mature results in only 19.4% of cases. In contrast, if the lamellar body count was in the transitional zone, the likelihood of RDS was much lower (12.5%) and traditional phospholipid analysis was mature in

Table 1. Protocol for Lamellar Body Counts
1. Mix the amniotic fluid sample by inverting the capped sample container five times. 2. Transfer the fluid to a clear test tube. 3. Inspect the specimen. Fluids containing obvious mucus or meconium should not be processed for a lamellar body count. 4. Place the test tube on a tube rocker for 2 min. 5. Flush the platelet channel; analyze the instrument’s diluent buffer until zero is obtained in two consecutive analyses. 6. Process the specimen through the cell counter and record the platelet channel as the lamellar body count. 7. Notify the physician if the associated hematocrit exceeds 1%. The hematocrit is obtained from the hematocrit channel of the cell counter. Interpretation Mature: 50,000/ L Transitional: 15,000 to 50,000/ L Immature: 15,000/ L

62.5% of cases. Thus, a stepwise approach 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 evaluating fetal lung maturity. Given the experience at the University of Utah, which uses 15,000/ L as a lower cutoff in noncentrifuged samples, and on the basis of extrapolation of results from centers that use centrifugation, we suggest that 15,000/ L be used as a lower cutoff for immaturity in noncentrifuged samples.13 Although the lamellar body count has proved to be a valuable assay for assessment of fetal lung maturity, there undoubtedly will be false-negative results, irrespective of the cutoffs that are chosen. This is simply a result of biologic variability and the vagaries of the diagnosis of RDS. With this in mind, we have suggested conservative cutoffs, to minimize the incidence of falsenegative results. Table 1 is a summary of a laboratory protocol for lamellar body counts. This protocol reflects a consensus of the four centers represented by the authors of this article.

Special Circumstances
Vaginal pool specimens containing obvious mucus should not be processed for lamellar body counts. Beyond potentially interfering with the cell counter, mucus also artificially increases the lamellar body count and decreases the lecithin-sphingomyelin ratio. Vaginal pool specimens that are obtained from patients with

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ruptured membranes who have “free-flowing” fluid and do not have obvious mucus may be processed for lamellar body counts. The effect of meconium on lamellar body counts has been evaluated.1 Meconium has a marginal impact on lamellar body counts, increasing the count by a modest 5,000/ L. Even heavy meconium staining does not increase the count greatly. The incidence of RDS will be low in cases in which the AF is meconium stained. Further, the presence of meconium may provide a compelling reason to move toward delivery, irrespective of lung maturity status. Clinical judgment should be exercised if the lamellar body count is borderline mature in the setting of meconium-stained AF. The effect of contamination of AF with whole blood is biphasic.1,6,13 The lamellar body count is initially increased because the platelets in the blood are counted as lamellar bodies. This effect, however, is relatively small. Indeed, even addition of enough blood to produce an AF hematocrit of 2% (which, incidentally, is rare) led to only a 5% increase in lamellar body counts, which lasted for only approximately 20 minutes after introduction of blood. Over the next 2 hours, however, the procoagulant activity of AF causes coagulation, which traps both platelets and lamellar bodies and leads to a decrease in lamellar body counts. Because of the potential effects of contamination of AF with blood on lamellar body counts, the responsible physician should be notified if the AF hematocrit exceeds 1%. This most commonly will lead to a falsely decreased lamellar body count. In a subset of 104 diabetic women in the study by Neerhof et al in this issue of Obstetrics & Gynecology, lamellar body counts were found to perform as well as phospholipid analysis as a fetal lung maturity assay. However, the experience with lamellar body counts in diabetic patients is limited. Given the increased risk of RDS particularly in infants of patients with poorly controlled diabetes, clinical judgment should be exercised when any assay for evaluating fetal lung maturity is used in a diabetic patient. Lastly, AF volume may affect quantitative tests such as the lamellar body count. It is possible that the lamellar body count could be increased falsely in cases of oligohydramnios, leading to a false-negative test result. However, the degree of oligohydramnios required to produce this effect likely would be a supervening indication for delivery. Conversely, hydramnios could lead to a false-positive test result.

References
1. Dubin SB. Characterization of amniotic fluid lamellar bodies by resistive-pulse counting: Relationship to measures of fetal lung maturity. Clin Chem 1989;35:612– 6. 2. Anceschi MM, Piazze Garnica JJ, Rizzo G, Di Pirro G, Maranghi L, Cosmi EV. Density of amniotic fluid lamellar bodies: A comparison with classical methods for the assessment of fetal lung maturity. Prenat Neonat Med 1996;1:343– 8. 3. Fakhoury G, Daikoku NH, Benser J, Dubin NH. Lamellar body concentrations and the prediction of fetal pulmonary maturity. Am J Obstet Gynecol 1994;170:72– 6. 4. Pearlman ES, Baiocchi JM, Lease JA, Gilbert J, Cooper JH. Utility of a rapid lamellar body count in the assessment of fetal maturity. Am J Clin Pathol 1991;95:778 – 80. 5. Bowie LJ, Shammo J, Dohnal JC, Farrell E, Vye MV. Lamellar body number density and the prediction of respiratory distress. Am J Clin Pathol 1991;95:781– 6. 6. Ashwood ER, Palmer SE, Taylor JS, Pingree SS. Lamellar body counts for rapid fetal lung maturity testing. Obstet Gynecol 1993;81:619 –24. 7. Dalence CR, Bowie LJ, Dohnal JC, Farrell EE, Neerhof MG. Amniotic fluid lamellar body count: A rapid and reliable fetal lung maturity test. Obstet Gynecol 1995;86:235–9. 8. Greenspoon JS, Rosen DJD, Roll K, Dubin SB. Evaluation of lamellar body number density as the initial assessment in a fetal lung maturity test cascade. J Reprod Med 1995;40:260 – 6. 9. Lee IS, Cho YK, Kim A, Min WK, Kim KS, Mok JE. Lamellar body count in amniotic fluid as a rapid screening test for fetal lung maturity. J Perinatol 1996;16:176 – 80. 10. Dilena BA, Ku F, Doyle I, Whiting MJ. Six alternative methods to the lecithin/sphingomyelin ratio in amniotic fluid for assessing fetal lung maturity. Ann Clin Biochem 1997;34:106 – 8. 11. Lewis PS, Lauria MR, Dzieczkowski J, Utter GO, Dombrowski MP. Amniotic fluid lamellar body count: Cost-effective screening for fetal lung maturity. Obstet Gynecol 1999;93:387–91. 12. Dubin SB. Assessment of fetal lung maturity. Am J Clin Pathol 1998;110:723–32. 13. Dubin SB. Fetal lung maturity assessment by determination of the lamellar body number density. In: Weinstein RS, ed. Advances in pathology and laboratory medicine. Vol 7. St. Louis, Missouri: Mosby, 1994:495–514.

Address reprint requests to:

M. G. Neerhof, DO Evanston Northwestern Healthcare 2650 Ridge Avenue Evanston, IL 60201 E-mail: m-neerhof@northwestern.edu

Received July 3, 2000. Received in revised form September 22, 2000. Accepted October 12, 2000.
Copyright © 2001 by The American College of Obstetricians and Gynecologists. Published by Elsevier Science Inc.

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