CLIN. CHEM.

23/12, 2250-2253(1977)
2250 CLINICAL CHEMISTRY, Vol. 23. No. 12, 1977
Laboratory Assessmentof Fetal LungMaturity:Lecithin/
SphingomyelinRatiovs. Cortisoland Foam-Test Assays
Daisy S. McCann,1 Lorraine S. Klrklsh,2 Mel L. Barclay,3 and A. James Blair, Jr.4
Values for amniotic fluid cortisol correlate fairly well with
the L/S ratio, but the latter is the better test for assessing
fetal lung maturity.
Additional K.yphrases: cortisol in amniotic fluid . L IS
ratios . foam test . plasma cart/so! in/ate pregnancy
Inter-test correlation
There is evidence that fetal production of cortisol
(hydrocortisone), which parallels lung maturation, is
reflected by the appearance and increase of cortisol in
amniotic fluid. Lung maturation is commonly assessed
by determining the lecithin/sphingomyelin (L/S) ratio
in the amniotic fluid. Drawbacks of this criterion in-
clude the somewhat empirical way the two lipids are
generally quantitated and the fact that in certain cases,
such as infants from poorly controlled diabetic mothers,
the rate at which the L/S ratio increases is accelerated,
so that it is not always a reliable index of lung matura-
tion. Consequently, alternative procedures are of in-
terest, and the possibility of substituting the measure-
ment of cortisol in amniotic fluid as an index to fetal
lung maturation seems attractive. Existing evidence is
conflicting: Fend and Tulchinsky (1) reported excellent
correlations (r = 0.83, n = 43) between the two measures
in normal pregnancy; Sivakumaran et al. (2) reported
a correlation as low as r = 0.37 (n = 114).
We undertook to compare total cortisol in amniotic
fluid with L/S ratios, plasma cortisol concentrations in
the mother, and amniotic-fluid foam tests in a series of
52 high-risk pregnancies.
1 Dept. of Medicine (Box 124), Wayne County General Hospital,
Eloise, Mich. 48132; and the Depth. of Biological Chemistry and In-
ternal Medicine, the University of Michigan, Ann Arbor, Mich.
48104.
2Dept. of Medicine, Wayne County General Hospital, Eloise, Mich.
48132.
Dept. of Obstetrics & Gynecology, Wayne County General Hos-
pital, Eloise, Mich. 48132; and Dept. of Obstetrics and Gynecology,
The University of Michigan, Ann Arbor, Mich. 48104.
4Dept. of Medicine, Wayne County General Hospital, Eloise, Mich.
48132; and Dept. of Internal Medicine, The University of Michigan,
Ann Arbor, Mich. 48104.
Received July 20, 1977; accepted Sept. 8, 1977.
Materials and Methods
Amniocentesis was performed where clinically indi-
cated to assist in the determination of fetal pulmonary
maturity. The cases selected for the procedure were
those about which there was some question of gesta-
tional age and in whom termination of gestation, either
by section or induction, was clinically indicated. The
specimen of amniotic fluid was placed on ice, brought
immediately to the laboratory, centrifuged, and L/S
ratios and foam tests were done without delay. Aliquots
for use in cortisol determination were stored frozen at
-10 #{176}C for later evaluation. Samples stained with
meconium or blood were rejected.
Blood was sampled from the antecubital vein of the
mother at the same time the amniotic fluid was sam-
pled. All L/S ratios were measured in quadruplicate by
the modified method of Gluck et al. (3). Plasma and
amniotic fluid cortisol were radioimmunoassayed, with
use of a commercially available immobilized antibody
produced against the 21-oxy-succinyl-derived conjugate
of this steroid (4). The sample volume is 0.1 ml of either
maternal plasma or amniotic fluid; a heat-denaturation
step frees cortisol from binding proteins. Before the
current study we had validated the radioimmunoassay
by comparing results with those of the classical fluoro-
metric technique (5) for estimation of free 11-hy-
droxycorticoids in human plasma.
Murphy et al. (6) reported conversion of maternal
cortisol to cortisone by the fetoplacental unit. Although
most of the available evidence suggests that very little
cortisol finds its way into the amniotic fluid from the
maternal circulation, either as such or as cortisone (7),
we checked potential interference from this metabolite
in our assay system. Trace amounts of tritiated cortisol
and cortisone were added to several specimens of am-
niotic fluid; the two steroids were then separated on a
Sephadex LH-20 column (8). The cortisone- and cor-
tisol-containing peaks were submitted separately to
cortisol radioimmunoassay.
Finally, in cases where enough sample was available,
specimens were submitted to a foam test, which has
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Fig. 3. Maternal plasma cortisol concentration (pg/liter) vs.
amniotic fluid L/S ratio
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Fig. 2. Amniotic fluid cortisol concentration (jig/liter) vs. L/S
ratio
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CLINICAL CHEMISTRY, Vol. 23, No. 12, 1977 2251
Fig. 1. Separation of amniotic fluid cortisol and cortisone by
Sephadex LH-20 chromatography. Trace quantities of [3H]-
cortisol and cortisone were addedto 2 ml of amniotic fluid after
alkalinization with 0.2 ml 1 mol/liter NaOH. The steroids were
extracted with two 7.5-mI aliquots of methylene dichloride. The
extracts were evaporated under nitrogen, the steroids taken up
in 0.3 ml solvent (CH2CI2:MeOH, 98:2) and applied to a 39 X 1
cm column of SephadexLH-20. The same solvent was used for
elution
been advocated as a rapid test for surf actant activity of
amniotic fluids (9).
Results
For a series of 71 plasmas assayed both by the present
procedure and the conventional fluorometric procedure,
the coefficient of correlation, r, was 0.91. As anticipated,
because the fluorometric method is less specific, abso-
lute values were somewhat lower for the radioimmu-
noassay: a mean value of 169.3 128.0 pg/liter was ob-
tained by the present procedure, 219.4 152.3 pg/liter
by fluorometry. Reproducibility (CV) for our procedure
is about 13.9%, a figure based on results for a plasma
pool assayed 10 separate times.
The cross reactivity with cortisone of the antibody
used in our study was 1%, which implies that the limit
of sensitivity of the cortisol assay for cortisone is about
1 mg/liter. Cortisone concentrations in amniotic fluid
were low enough that we saw no interference from this
compound in our amniotic-fluid cortisol measurements.
When amniotic fluid was fractionated on a LH-20 col-
umn before radioimmunoassay, virtually all of the free
cortisol measured in the intact specimen was recovered
from the appropriate peak (Figure 1), and no displace-
ment of label was observed with material from the cor-
tisone peak.
Values for cortisol in amniotic fluid ranged from 15.0
to 127.5 gig/liter, with a mean and standard deviation
of 56.3 30.3 zg/liter. L/S ratios ranged from 1.1 to 5.9,
with a mean of 3.02 0.97. The linear coefficient of
correlation between the two parameters was statistically
significant (r = 0.489, P .01 with n = 50; Figure 2).
Cortisol concentrations in the maternal plasma
ranged, for this group of high risk pregnancies, from 120
to 590 pg/liter, with a mean of 341 108. The mean
value is close to that we obtained for a comparable group
of 30 normal primigravid controls, 333 117 pig/liter.
There was no significant correlation between the ma-
ternal plasma cortisol concentration and the amniotic
fluid L/S ratio (r = 0.084, n = 51; Figure 3); there was
a low correlation between the cortisol concentrations
in the maternal plasma and the amniotic fluid (r =0.286,
P .05, n = 47; Figure 4).
Results of the foam test, despite its apparently sim-
plistic approach to the problem of lung maturity, cor-
relate quite nicely with L/S ratios. The appearance of
bubbles after shaking serial dilutions of the amniotic
fluid is scored from 0 to +5. Correlation between these
scores and the L/S ratios were r = 0.648, P .01, n = 33
(Figure 5).
Discussion
We validated our procedure both against the con-
ventional fluorometric procedure and against a com-
mercially pre-analyzed plasma pool. The method is
quick, simple, reproducible, and, used as described,
measures total cortisol. The absolute values obtained
100
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2
20 50 15 ISO ISO
N. 33
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2252 CLINICALCHEMISTRY, Vol. 23, No. 12, 1977
A_go, Ri4 CtA_
Fig. 4. Maternal plasma cortisol concentration vs. amniotic fluid
cortisol concentration (both in tg/liter)
for cortisol in amniotic fluid, 56.3 30.3 pg/liter, com-
pare favorably with those of Fend and Tulchinsky (1),
who reported values of 31.8 1.5 (SEM) pig/liter be-
tween weeks 20 and 34 and 72.4 3.8 gig/liter for weeks
35 through 40 of the gestational period. Substantially
lower values are obtained by the so-called competitive
protein-binding methods, around 20 pig/liter in the last
week or two before onset of labor (10-12). References
11 and 12 specifically designate the measurement of
unconjugated cortisol. Gennser et al. (13), using a ra-
dioimmunoassay procedure for unconjugated cortisol,
report values as high as 28 zg/liter, with increases to
about 35 ig/ liter at delivery.
Statistical correlation between other measures of fetal
lung maturation and amniotic cortisol has generally
been good, with one striking exception: Sivakumaran
et al. (2) reported a correlation of r = 0.37 between their
amniotic fluid cortisol and L/S ratios. However, their
cortisol values are uniquely high, with means at 144
big/liter between weeks 35-36 to 202 /Lg/liter between
weeks 37-40 of gestation. Fend and Tulchinsky (1) re-
ported a coefficient of rank correlation as high as 0.83
for their series of 48 samples of amniotic fluid obtained
at various stages of normal pregnancy. Sharp-Cageorge
et al. (14) found r = 0.55 for the correlation between
palmitic/stearic acid ratios vs. amniotic fluid cortisol.
With a linear correlation our own data yielded r = 0.489,
P .01; substitution of a product moment method for
grouped data increased r to 0.79.
Further inspection of our data suggests that in no
instance was an amniotic fluid cortisol value of more
than 50 igfliter associated with a low L/S ratio. How-
ever, as many as 18 amniotic cortisol concentrations
below 50 rig/liter were associated with L/S ratios greater
than 2.0. Because deliveries in this series were based on
L/S ratios, and because no problems were encountered
in any of the infants delivered after the L/S ratios ex-
ceeded 2, some apparently low amniotic-fluid cortisol
concentrations were associated with healthy infants
born without signs of respiratory distress.
0 I 2 S 4 5
Fig. 5. Amniotic fluid LIS ratios vs. foam test scores
The low correlation between maternal plasma and
amniotic-fluid cortisol values was not unexpected, be-
cause most of the available evidence suggests that am-
niotic-fluid cortisol reflects fetal adrenal activity rather
than maternal cortisol concentrations (7). Maternal
cortisol is known to cross the placenta (15, 16) and the
low correlation that does obtain between maternal
plasma and amniotic-fluid cortisol may therefore be
related to regulatory effects on the fetal pituitary.
As reported by others (9, 17) the foam test offers a
simple, inexpensive approach to assessment of lung
maturity. Again, no false positives were recorded in our
series, i.e., in no case were high scores on the foam test
associated with low L/S ratios. However, some of the +2
and +3 scores might be difficult to interpret in the ab-
sence of a confirming L/S ratio.
Supported by the Medical Staff Research & Education Fund,
Wayne County General Hospital.
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