Short Report
Six alternative methods to the lecithin/sphingomyelin ratio
in amniotic fluid for assessing fetal lung maturity
B A Dilena ', F Ku 2, I Doyle- and M J Whiting!
From the Departments of I Biochemistry & Chemical Pathology and 3Human Physiology, Flinders
Medical Centre, Bedford Park, South Australia 5042, and 2Department of Chemical Pathology,
Women's and Children's Hospital, North Adelaide, South Australia 5006, Australia
Additional key phrases: fluorescence polarization;
lamellar bodies; lung surfactant apoproteins
During the last trimester of human fetal
gestation, lung surfactant is synthesized and
secreted into the alveolar space, and becomes
detectable in amniotic fluid. Pulmonary surfact-
ant is composed of phospholipids, neutral lipids
including cholesterol, and specific apoproteins,
and functions to lower surface tension and
facilitate gas exchange in the lung. A deficiency
of surfactant is associated with respiratory
distress syndrome, which can be life-threatening
in premature infants.
As a test for fetal lung maturity, the most
widely accepted method remains the analysis of
amniotic fluid for surfactant-derived lecithin
expressed as a ratio to sphingomyelin. 1 This tes~
has a high sensitivity and specificity, but
employs thin-layer chromatography which is
laborious and time-consuming. The potential
exists to estimate other components of lung
surfactant in amniotic fluid, such as the
apoprotein, fatty acid or phospholipid compon-
ents, or to measure physical properties of
surfactant-containing lamellar bodies, such as
their number or size. The aim of this study was
to examine six methods as more practical
alternatives to the lecithin/sphingomyelin (L/S)
ratio for the estimation of fetal lung maturity
from the analysis of amniotic fluid. In keeping
with current trends in clinical laboratory
practice, of particular interest were methods
that could be automated and/or took less than
1 h to produce a result.
Forty-four amniotic fluid specimens received
by two Adelaide hospitals (Queen Victoria
Maternity Hospital and the Flinders Medical
Centre) over a I year period were entered into
the study. Each of the samples was obtained
from an individual patient by transabdominal
Correspondence Dr MJ Whiting.
106
Ann Clin Biochem 1997; 34: 106-108
amniocentesis. Gestational ages, which were
estimated by obstetric clinical parameters,
ranged from 28 to 38 weeks. Four specimens
which contained blood or meconium were
discarded. The remaining 40 specimens were
analysed by a panel of tests, with the exception
of the L/S ratio, which was determined in only
31 specimens due to limited specimen volume.
The performance details of the tests are as
follows.
Lecithin to sphingomyelin (L/S) ratios were
determined using the Fetal-Tek 200 kit (Helena
Laboratories, Beaumont, Texas, USA), based
on the technique of Gluck et al,' The inter-assay
coefficient of variation was 8·3% at a L/S ratio
of 1·8, and 8·6% at a L/S ratio of 2·9.
Fluorescence polarization measurements were
done on an Abbott TDx FLX instrument
(Abbott Laboratories, Chicago, USA), using
the unit dose technique." The assay measures the
surfactant/albumin ratio after the partitioning of
a fluorescent dye between these two components
in amniotic fluid. The coefficients of variation at
ratios of 30,50 and 100mg/g were 6,7%,5·0%
and 2'5%, respectively. Lamellar body counts
were assessed on uncentrifuged fluid using the
platelet channel of a Coulter Counter Model JS,
as described by Fakhoury et al.' The coefficient
of variation at a count of 100000 particles/ul,
was 4·0%. Differential light scattering measure-
ments were done by the technique of Dubin,"
using a Shimadzu UV-160 double beam spectro-
photometer (Shimadzu Corporation, Tokyo,
Japan). At an absorbance reading of 0'056, the
coefficient of variations was < 4·0%. Surfactant
apoproteins A and B were assayed on uncen-
trifuged amniotic fluid by an ELISA method
using microtitre plates and specific polyclonal
rabbit antibodies directed against purified
human lung surfactant apoproteins A and B.5
The intra- and inter-assay coefficients of varia-
tion were 6·6% and 9·9% for apo-A and 6·0%
and 8·6% for apo-B, respectively. Samples for

palmitic acid measurement were centrifuged at
1500 x g for 10 min, before analysis by the
method of Warren et al" using gas-liquid
chromatography. The coefficient of variation at
a palmitate concentration of 0·07 mrnol/L was
14%. Whenever possible fresh specimens were
used, but some specimens were stored at - 20°C
for batch analysis. Statistical comparison of
means was by an unpaired t-test, while regres-
sion equations and correlation coefficients were
calculated by the method of least squares with a
Statview version 4·5 statistics package.
All methods were significantly correlated
(P < 0·00 I) with the L/S ratio, and correlation
coefficients, as determined by linear regression
analysis, ranged from 0·93 for TDx to 0·65 for
palmitate. Due to time differences between the
performance of the test and delivery, and the low
incidence of respiratory distress syndrome, it
was not possible to analyse the data in terms of
clinical outcome. Therefore, fetal lung maturity
was assigned on the basis of the L/S ratio, with a
L/S ratio> 2·0 indicating maturity and :s::; 2·0
indicating immaturity. Mean (SO) values were
determined for each of the six tests for mature
and immature groups, and the tests were ranked
in order of the statistical significance of the
difference between the group means, as shown in
Table I. The TDx fluorescence polarization
assay showed the greatest difference between
immature and mature groups. Highly significant
differences were also observed for lamellar body
counts, light scatter and surfactant apoprotein
B, but the difference between mean palmitate
concentrations of amniotic fluid in the two
groups was of borderline statistical significance.
Using the cut-off values shown in Table I, which
were selected to maximize the discrimination
between mature and immature groups, the
sensitivity, specificity, and positive and negative
predictive values of each test were determined.
All tests were highly sensitive in detecting
TABLE 1. Comparison of the mean (SD) for six fetal lung maturity tests in immature (L/S ratio ~2'O; n = 15) and
mature (L/S ratio> 2·(); n = 16) groups, ranked in order ofstatistical significance of the difference between the means.
A cut-off value selected to discriminate between mature and immature groups is also shown
Biochemical test Units
Fluorescence polarization (mg/g)
Lamellar body count (no/nl.)
Light scatter (Abs)
Surfactant apoprotein B (mgjL)
Surfactant apoprotien A (rng/L)
Palmitate (mmo1jL)
Fetal lung maturity 107
immature samples, as defined by a L/S ratio
:S::;2·0, with sensitivities between 90% to 100%.
The specificity or ability to reliably detect a
mature sample was much less, and varied
between 50% and 81%. The TDx assay showed
the best discrimination, with a sensitivity of
100% and a specificity of 81%. Using this assay,
all samples of less than 34 weeks' gestation were
immature. Specimens judged mature by the L/S
ratio often gave low results with many of the
other tests. This resulted in all tests having
higher predictive values for lung maturity than
for immaturity.
Of the six methods investigated, the closest
agreement to the L/S ratio was obtained with the
fluorescence polarization assay, which is avail-
able commercially. This automated assay has
undergone a multicentre evaluation and was
found to have a high sensitivity and specificity
similar to the L/S ratio, using a cut-off for
maturity set at a surfactant/albumin ratio of
50 mg/g.? Another recent report on the clinical
utility of the TDx assay concluded that
compared to the L/S ratio, a foam stability
index, and the presence of phosphatidylglycerol
in over 100 amniotic fluid samples, the TDx
assay had superior predictive values for im-
maturity and maturity." The findings of the
present study are consistent with these reports.v?
The TDx assay is easily done in 30 min,
although some difficulty was experienced with
sampling of reagent from the unit dose cartridge
supplied as part of the TDx FLM kit. When
insufficient reagent was sampled by the instru-
ment, the specimen was lost and another
cartridge was required, leading to increased cost
of the test. This problem has now been
addressed by the manufacturer with the intro-
duction of batch reagents (FLM II).8
Two simple and rapid methods for the
evaluation of fetal lung maturity relate to
quantifying the lamellar bodies which contain
Immature Mature P-Value Cut-ofT
26 (14) 93 (51) <0.0001 60
16·6 (18'0) 74·8 (49'8) 0.0029 30
0·011 (0'016) 0·14 (0'15) 0.0038 0.035
\7·7 (8'7) 62·6 (56'5) 0.0068 35
1·1 (0'4) 3·1 (3'1) 0.0256 1.5
0·04 (0'02) 0·12 (0'16) 0.0523 0.07
Ann Clin Biochem 1997: 34
108 Dilena et al.
surfactant. In laboratories with access to
haematology equipment, passage of amniotic
fluid through a Coulter counter set for platelet
size gives an estimate of lamellar body number
per microlitre of fluid. In the present study, good
agreement was found between lamellar body
concentration determined by counting and the
LIS ratio. The cut-ofT value for deciding on lung
maturity depends on whether amniotic fluid is
centrifuged prior to counting. Another rapid
method which measures lamellar body concen-
tration is differential light scattering. The
difference in optical absorbance at 650 nm
between amniotic fluid diluted with distilled
water or glycerol is measured, thus removing
interference from absorbing chrornogens." Both
lamellar body counting and light scattering were
highly correlated with the LIS ratio, and with
each other (r=O'72), and the simplicity and
speed of their determination make these attrac-
tive tests for the routine laboratory.
Although much attention has centred on the
phospholipid components of pulmonary sur-
factant, the protein and lipid components of
surfactant may not develop in synchrony, and
measurement of surfactant apoproteins could
prove valuable. In contrast to the belief that
apoprotein A is the major surfactant-associated
protein, our data suggest that surfactant
apoprotein B concentrations in amniotic fluids
are much higher than previously realized. The
fact that our apoprotein B assay used EDTA
and detergents to dissociate and solubilize
proteins from surfactant components' could
explain the increased concentrations obtained
when compared to a previous study.? A practical
consequence of the high concentrations of
surfactant apoprotein B observed in mature
amniotic fluids is that it may be possible to
develop a rapid automated nephelometric assay
for its determination. Our results suggest that
surfactant apoprotein B deserves further exam-
ination as an easily measured marker for fetal
lung maturity.
In conclusion, although the LIS ratio in
amniotic fluid remains as the most widely
accepted biochemical method for the assessment
Ann Clin Biochem 1997: 34
of fetal lung maturity, alternative methods
which are highly correlated to the LIS ratio,
are now available. These newer methods include
fluorescence polarization or a test of lamellar
body concentration and have the distinct
advantage of reduced labour and short turn-
around time of less than one hour. Studies with
large numbers of patients are required to assess
whether these tests are better predictors of
respiratory distress syndrome.
REFERENCES
Gluck L, Kulovich MV, Borer RC, Brenner PH,
Anderson GG, Spellacy WN. Diagnosis of the
respiratory distress syndrome by amniocentesis.
Am J Obstet Gynecol 1971; 109: 440--5
2 Russell lC, Cooper CM, Ketchum CH, Torday IS,
Richardson OK, Holt lA, et al. Multicenter
evaluation of TDx test for assessing fetal lung
maturity. Clin Chem 1989; 35: 1005-10
3 Fakhoury G, Daikoku NH, Benser 1, Dubin NH.
Lamellar body concentrations and the prediction of
fetal pulmonary maturity. Am J Obstet Gynecol
1994; 170: 72-6
4 Dubin SB. Determination of lamellar body size,
number density and concentration by dilTerential
light scattering from amniotic fluid: physical sig-
nificance of A650. C/in Chem 1988; 34: 938-43
5 Yogalingam G, Doyle IR, Power IHT. Expression
and distribution of surfactant proteins and lysozyme
after prolonged hyperpnea. Am J Physiol 1996; 270:
L32D-301
6 Warren C, Holton IB, Allen IT. A method of
assessing amniotic fluid lecithin concentrations and
predicting foetal lung maturing by estimating total
palmitic acid concentrations using GLC. Ann Clin
Biochem 1974; 11: 31-5
7 Herbert WNP, Chapman IF, Schnoor MM. Role of
the TDx FLM assay in fetal lung maturity. Am J
Obstet Gynecol 1993; 168: 808-12
8 Moxness M, Lawson G, O'Brien 1, Burritt M.
Assessment of fetal lung maturity by the Abbott
FLM II assay and three other methods. Clin Chem
1995; 41: S95
9 Pryhuber GS, Hull WM, Fink I, McMahan Ml,
Whitsett lA. Ontogeny of surfactant proteins A and
B in human amniotic fluid as indices of fetal lung
maturity. Ped Res 1991; 30: 597--605
Acceptedfor publication 15 April 1996