Lamellar body counts: A
maturity for just over a decade. During that time,
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 phos-
pholipid 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 phospho-
lipid 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 re-
sults 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 accu-
rately, 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 Obste-
tricians and Gynecologists.)
Lamellar bodies are produced by type II alveolar cells in
increasing quantities as gestation advances. They are
composed almost entirely of phospholipid and repre-
sent 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 North-
western 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.
318 0029-7844/01/$20.00
PII S0029-7844(00)01134-0
laboratories as an assay for evaluation of fetal lung
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 matu-
rity assay, although most of these studies were rela-
tively 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 pre-
sented. The goals of that project were to provide a
multicenter update on the clinical performance of the
lamellar body count compared with traditional phos-
pholipid analysis and, if lamellar body counts were
found to compare favorably with traditional phospho-
lipid analysis, to agree on a standardized methodology
for lamellar body counts. All of the centers that previ-
ously had reported their results with lamellar body
counts were invited to participate. The results of this
study demonstrated that the lamellar body count per-
forms as well as traditional phospholipid analysis in the
assessment of fetal lung maturity, with a high negative
predictive value as its most attractive operational char-
acteristic. 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 cen-
trifugation. Centrifugation initially was thought to be
integral to protocols because it was believed that cellu-
lar debris in the AF fluid might interfere with normal
functioning of the cell counter. Because most laborato-
ries 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 centrifuga-
tion protocol affects the test result, with longer centrif-
ugation times and higher rates resulting in lower lamel-
lar body counts.6,12 As a consequence of the differences
in centrifugation protocols among laboratories, the cut-
offs for maturity that were established at these labora-
tories are widely discordant. This leads to confusion
when results between institutions are compared or
reported. Further, failure to adhere strictly to a centrif-
ugation protocol also can lead to error. Some centers
began omitting the centrifugation step altogether, real-
izing that, in the absence of obvious mucus or heavy
meconium staining, processing noncentrifuged AF
Obstetrics & Gynecology
specimens does not affect instrumentation adversely
and does not affect test performance.6,12 Centrifugation
therefore might cause confusion and error, is not nec-
essary, 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 non-
centrifuged 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 An-
geles. 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 us-
ing 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 noncen-
trifuged samples on the basis of the calculated effect of
centrifugation on the lamellar body count. He calcu-
lated 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 predic-
tive 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 con-
trast, 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
VOL. 97, NO. 2, FEBRUARY 2001
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: ⱖ50,000/␮L
Transitional: ⬎15,000 to ⬍50,000/␮L
Immature: ⱕ15,000/␮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 centrifu-
gation, 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, irre-
spective 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 false-
negative 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
Neerhof et al Lamellar Body Count Consensus 319
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, irrespec-
tive 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 in-
creased 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 intro-
duction 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 poten-
tial effects of contamination of AF with blood on lamel-
lar 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 exer-
cised 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 re-
quired to produce this effect likely would be a super-
vening indication for delivery. Conversely, hydramnios
could lead to a false-positive test result.
320 Neerhof et al Lamellar Body Count Consensus
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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.
Obstetrics & Gynecology