Clinical Biochemistry 39 (2006) 1 10

Review

Fetal lung maturity

David G. Grenache a,, Ann M. Gronowski b
a

University of North Carolina at Chapel Hill, School of Medicine, Department of Pathology and Laboratory Medicine, CB #7525, Chapel Hill, NC 27599, USA
b
Washington University School of Medicine, Department of Pathology and Immunology, Saint Louis, MO 63110, USA
Received 16 September 2005; received in revised form 19 October 2005; accepted 19 October 2005
Available online 21 November 2005

Abstract
Respiratory distress syndrome of the newborn infant caused by immaturity of the fetal lung continues to be a clinical problem. Measurement of
pulmonary surfactant production is the most effective way to evaluate pulmonary maturity. Since the first fetal lung maturity test was described
more than two decades ago, advances in methodology have produced diagnostically sensitive tests that are both rapid and precise. Unfortunately,
currently available tests continue to demonstrate low diagnostic specificity and remain poor predictors of fetal lung immaturity. We review the
background, methodology, pre-analytical and analytical concerns, and clinical performance of various fetal lung maturity assays, and discuss the
appropriate use and interpretation of these tests.
2005 The Canadian Society of Clinical Chemists. All rights reserved.
Keywords: Pulmonary surfactant; Fetal lung maturity; Amniotic fluid; Analytical tests

Contents
Fetal lung development . . . .
Pulmonary surfactant . . . . .
Respiratory distress syndrome
Tests of fetal lung maturity . .
Lecithin/sphingomyelin ratio .
Phosphatidylglycerol . . . . .
Foam stability. . . . . . . . .
Surfactant/albumin ratio . . .
Lamellar body count (LBC) .
Test use and interpretation . .
Summary . . . . . . . . . . .
References . . . . . . . . . .

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Fetal lung development

The development of the human lung is an exquisitely
regulated and highly coordinated process of branching
morphogenesis, angiogenesis, and alveolarization. It begins in
the 3-week-old embryo with the creation of a diverticulum from

Corresponding author. Fax: +1 919 966 9490.

E-mail address: [email protected] (D.G. Grenache).

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1
2
2
2
3
4
5
5
6
7
8
8

the caudal end of the foregut that divides into the two lung buds
[1]. These, in turn, undergo successive rounds of branching
morphogenesis to produce the lung lobes: three on the right and
two on the left.
Following branching morphogenesis, the canalicular phase
at 16 to 24 weeks of gestation is characterized by increased
angiogenesis and the differentiation of cuboidal epithelium into
the types I and II pneumocytes that will permit gas exchange
[2,3].

0009-9120/$ - see front matter 2005 The Canadian Society of Clinical Chemists. All rights reserved.
doi:10.1016/j.clinbiochem.2005.10.008

D.G. Grenache, A.M. Gronowski / Clinical Biochemistry 39 (2006) 110

In the final stage of development, the fetal lung is prepared

for its function as a gas exchange organ in an atmospheric
environment. The most important of these late-stage events are
the formation of alveoli and an integrated capillary network in
conjunction with an increased production of surfactant, a
complex mixture of phospholipids and proteins that functions to
decrease the surface tension at the alveolar airliquid interface
[3]. The process of alveoli formation continues after birth to
approximately 8 years of age and greatly increases the number
of functional alveoli [2].
Pulmonary surfactant
Surface tension in the hydrated lung works to oppose lung
inflation and promote the collapse of alveoli. Pulmonary
surfactant prevents atelectasis in the newborn infant by
decreasing alveolar surface tension through the creation of a
lipid-rich monolayer phase that separates alveolar gas from the
liquid surfaces of epithelial cells. This stabilizes alveoli and
allows lung volumes to be maintained at expiration. In the
absence of surfactant small alveoli collapse and larger alveoli
are over-inflated resulting in a stiff, non-compliant lung.
Pulmonary surfactant is synthesized by type II pneumocytes
and packaged into storage granules called lamellar bodies.
Lamellar bodies appear in the cytoplasm of pneumocytes at
approximately 24 weeks of gestation however the surfactant
content of amniotic fluid does not begin to increase until week
32 suggesting that lamellar body secretion of surfactant does not
coincide with their appearance [2].
As secreted from lamellar bodies, pulmonary surfactant is a
heterogenous mixture of approximately 90% phospholipids and
10% protein. The most abundant phospholipids are phosphotidylcholine (PC or lecithin) and phosphatidylglycerol (PG).
The majority of pulmonary PC has the saturated fatty acid
palmitate in both acyl positions and so is more accurately
described as dipalmitoylphosphatidylcholine [4]. Phosphatidylinositol (PI), phosphatidylethanolamine, and sphingomyelin
are much less abundant.
The synthesis of lecithin gradually increases from 28 weeks
of gestation until delivery with peak production occurring at
week 36 [5]. Similarly, PI production is evident at 28 weeks of
gestation but peaks between 32 and 35 weeks. The last
surfactant to appear in the fetal lung is PG, appearing at 36
weeks and increasing to term [6].
There are four surfactant proteins (SP) synthesized by type II
pneumocytes that provide important functional and regulatory
properties to pulmonary surfactant and are designated SP-A, -B,
-C, and -D. SP-A and SP-D are hydrophilic proteins that,
respectively, regulate surfactant secretion and recycling and
support immune responses in the lung [7]. The contribution of
SP-D to surfactant metabolism has also been reported [8]. SP-B
and SP-C are hydrophobic proteins that are required for proper
functioning of pulmonary surfactant. Both are packaged into
lamellar bodies and eventually secreted into the alveolar air
space along with phospholipids [9]. SP-B interacts with calcium
ions and SP-A to accelerate the formation of the phospholipid
monolayer at the airwater interface [10]. SP-C functions to

enhance the movement of lipid molecules that maintains the

physical properties of the surfactant [9]. A hereditary deficiency
of SP-B results in a deficiency of surfactant in full-term
newborns that is invariably fatal [11], while a deficiency of SP-C
is associated with interstitial lung disease [12].
Lamellar bodies are secreted from type II pneumocytes into
the alveoli via exocytosis where they become hydrated and
unravel to form an expanded membrane lattice called tubular
myelin. Tubular myelin functions as a reservoir for the
formation of the surfactant monolayer that is oriented with the
fatty acid chains extending toward the air space and the head
groups located in the aqueous phase.
During the respiratory cycle, when the monolayer is
compressed during exhalation, a nearly pure layer of lecithin
is formed, some of which is extruded and forms small liposomal
aggregates. These aggregates are removed from the alveoli
through endocytosis by pulmonary macrophages and type II
pneumocytes [13]. The majority of these aggregates are
recycled into lamellar bodies [13].
Respiratory distress syndrome
Respiratory distress that occurs in the newborn infant within
the first few hours of life is termed respiratory distress syndrome
(RDS). Because the increase in pulmonary surfactant occurs late
in gestation, RDS is nearly always associated with preterm
birth. It is the most common cause of respiratory failure in
neonates and is inversely related to the gestational age at the
time of birth such that the risk of RDS at 29 weeks is N60%,
20% at 34 weeks, and is b5% 37 or greater weeks [14]. In 2003,
infant death from RDS in the United States was 20 per 100,000
live births [15] which was slightly less than that reported for
2002.
The atelectasis caused by surfactant deficiency, in combination with small respiratory units, results in perfused but
unventilated alveoli leading to hypoxia, hypercapnia, and
acidosis. These conditions produce vasoconstriction of
pulmonary arteries that, together with increased right-to-left
shunting through the foramen ovale and ductus arteriosus,
decreases pulmonary blood flow. The resulting ischemic
injury to the lung creates a secondary surfactant deficiency
that exacerbates the disease [16]. Preventing premature birth is
the most effective way to prevent RDS. Alternatively,
administration of steroids to the mother can be used to
accelerate surfactant production [17]. Treatment of neonates
immediately after birth with exogenous surfactant delivered
endotracheally can be effective if early delivery cannot be
prevented [18].
Tests of fetal lung maturity
Understanding the physiology of fetal lung development,
which began in the 1950s, has lead to the development of tests
to assess fetal lung maturity in utero. Because fetal lung liquids
contribute to amniotic fluid, the amount of surfactant in fetal
lungs can be estimated by measuring the amount of surfactants
in amniotic fluid.

D.G. Grenache, A.M. Gronowski / Clinical Biochemistry 39 (2006) 110

Table 1
Number of laboratories performing fetal lung maturity testing according to the
College of American Pathologists [24,103]
Method

Number of laboratories

TDx FLM II
PG (AmnioStat-FLM)
L/S Ratio
PG (TLC)
Lamellar body count

469
285
125
79
59

To be clinically useful, tests for fetal lung maturity should

have a high diagnostic sensitivity for immaturity and a high
negative (mature) predictive value. As with all diagnostic tests,
maximizing sensitivity is achieved by sacrificing specificity
such that many infants with positive (immature) fetal lung
maturity test results are born without RDS. The most common
methods for assessing fetal lung maturity are shown in Table 1.
For this review, unless otherwise noted, the use of the term
positive result will be used to indicate an immature test result
while the term negative result will be used to indicate a
mature test result.
Lecithin/sphingomyelin ratio
First reported in 1971, the lecithin/sphingomyelin (L/S) ratio
was the first biochemical test for assessing the maturity of the
fetal lungs [19]. As discussed above, the major pulmonary
surfactant is lecithin and this normally increases in concentration from 28 weeks of gestation through birth. The L/S ratio
describes the relative change in the concentration of lecithin to
that of sphingomyelin in amniotic fluid as determined by thin
layer chromatography (TLC). Although low, the concentration
of amniotic fluid sphingomyelin remains largely constant
throughout the last trimester of pregnancy and therefore serves
as an internal standard against which the concentration of
lecithin can be compared. This is important, as the volume of
amniotic fluid is difficult to determine accurately. The L/S ratio
increases with increasing gestational age and correlates with the
maturity of the fetal lung [20].
Although numerous variations to the original TLC procedure
have been described, the essential elements remain the same.
The test requires a minimum of 3 to 4 mL of amniotic fluid that
is centrifuged briefly at low speed to remove debris while
preserving phospholipid content. The specimen is mixed with

methanol followed by lipid extraction into chloroform and

applied to a TLC plate along with controls and markers that will
show the locations of the various phospholipid components.
The plate is developed in a solvent system containing
chloroform, methanol, 2-propanol, triethylamine, and water.
The phospholipids are visualized by using cupric acetate and
phosphoric acid followed by charring. The lecithin and
sphingomyelin bands are quantified densitometrically and the
relative intensity of each band is expressed as a ratio.
Common to all the methodological variations is that TLC is
technique dependent [21]. As originally described, the test
included an acetone precipitation step to isolate the surfaceactive lecithins [19], a procedure that was the subject of much
dispute. Later studies demonstrated that precipitation was
incomplete [22] and could introduce uncertainty into the result.
Omitting this step did not effect result interpretation [23].
A commercially available method for performing the L/S
ratio is available (Fetal-Tek 200, Helena Laboratories, Beaumont, TX) and is used by slightly more than half of all
laboratories that determine the L/S ratio [24]. Still, there is poor
inter-laboratory precision even among those using this method.
Results from proficiency test specimens from laboratories using
either the Fetal-Tek 200 method or a home brew procedure that,
like the Fetal-Tek 200, does not contain an acetone precipitation
step and utilizes cupric acetate charring for visualization,
revealed a mean CV of 15.6% and 26.3% at a mean ratio of 1.5
and 3.9, respectively (n = 89 laboratories) [24,25].
As demonstrated by Gluck, the concentrations of lecithin and
sphingomyelin are nearly equivalent prior to 35 weeks of
gestation after which the lecithin concentration rises sharply
[19]. In a normal pregnancy, the L/S ratio can be expected to rise
from values less than 0.5 at 20 weeks gestation to 2.0 or greater,
depending on the method, at about 3536 weeks gestation. A
cutoff of 2.0 is frequently used as the decision point to indicate
fetal lung maturity. However, the absence of a single,
standardized procedure combined with the numerous analytical
variations of the original procedure requires that all laboratories
that perform the L/S ratio validate their method and their cutoff
with a clinical outcome study.
Many outcome studies have demonstrated that the L/S ratio
has good sensitivity but lacks specificity. As such, a high
number of immature results are observed with infants born
without RDS. Table 2 summarizes some of these studies
performed in the last 25 years. It is important to note the effect

Table 2
Outcome studies examining the L/S ratio for predicting fetal lung maturity
Reference

Year

RDS prevalence (%)

Cutoff

Sensitivity (%)

Specificity (%)

PPV (%) a

NPV (%) b

Anceschi [104]
Hagen [31]
Ashwood [105]
Lipshitz [50]
Lockitch [106]
Hamilton [107]
Plauch [37]
Golde [36]

1996
1993
1986
1984
1984
1984
1982
1980

41
140
88
205
47
131
311
74

9.8
20.7
15.9
5.4
17.0
7.6
6.4
5.4

2.0
2.0
2.0
2.0
2.0
2.5
2.0
2.0

100
83
84
100
63
100
90
50

81
60
87
87
97
77
74
93

36
53
57
31
83
26
19
29

100
86
97
100
92
100
99
97

a
b

Positive (immature) predictive value.

Negative (mature) predictive value.

D.G. Grenache, A.M. Gronowski / Clinical Biochemistry 39 (2006) 110

that RDS prevalence has on the predictive values of the test and
emphasizes, again, the need for individual laboratories offering
the test to determine their own performance characteristics.
Amniotic fluid specimens analyzed for the L/S ratio are best
collected via amniocentesis rather than from vaginal pools that
are likely to contain mucus and bacteria. When performed on
specimens collected via amniocentesis and from vaginal pools
from the same patients, the L/S ratio was significantly lower in
vaginal pool specimens [26].
Contamination of amniotic fluid with either blood or
meconium can influence the L/S ratio. The L/S ratio of serum
varies between 1.3 and 1.5 [27] so the presence of blood can
decrease a mature result and increase an immature result. A
mature result obtained from a bloody specimen remains
clinically useful, however an immature result does not [28].
Although meconium contains neither lecithin nor sphingomyelin, it produces an L/S ratio of 1.1 to 3.6 and therefore has the
potential to complicate L/S result interpretation from meconium-contaminated amniotic fluid specimens [29].
Specimens are stable for 24 h at room temperature and the
L/S ratio was shown to be unaffected in specimens stored
frozen at 20C for 12 months [30].
Recognizing that the incidence of RDS decreases with
increasing gestational age, Hagen, et al. demonstrated the
influence that this covariate has on the predictive ability of the
L/S ratio [31]. As would be expected, the sensitivity and
positive predictive value was highest between 25 and 31 weeks
of gestation and both decreased to 0% at 36 weeks. This
concept of combining fetal lung maturity test results with
gestational age has continued to be investigated (see below).
Phosphatidylglycerol
Despite its low relative contribution to total pulmonary
surfactant, PG has also been used as an indicator of fetal lung
maturity. As previously stated, the appearance of PG occurs
several weeks after that of lecithin [6] and is therefore
considered to be a late marker of lung maturity. This was first
suggested in a report which noted that PG appeared when the
L/S ratio was N2.0 and may be necessary for functional
maturity [32]. It was the poor specificity of the L/S ratio that
prompted Gluck and colleagues to modify the test in an effort
to quantify PG and improve the accuracy of the L/S ratio [33].
The modified test employed two-dimensional thin-layer
chromatography to determine the L/S ratio as well as the
relative amounts of disaturated lecithin, phosphotidylinositol
and phosphatidylglyercol in amniotic fluid. This lung profile
offered advantages over the L/S ratio by enhancing the
accuracy of a ratio N2.0 and decreasing the rate of falseimmature results [3337]. One study of 358 amniotic fluid
specimens reported that the presence of PG at N2% in
conjunction with an L/S ratio N2.0 was 100% predictive of the
absence of RDS in 292 infants compared to 71% when the L/S
ratio was N2.0 but PG undetectable (n = 28) [35]. These
authors also noted that 6 infants were born without RDS when
PG was N2% despite an L/S ratio that was b2.0. While this and
other studies lead to the belief that the presence of PG was a

clear indicator that RDS would not occur, others reported data
that did not support this belief [38,39]. Still, the detection of
PG remains a very strong indicator that RDS will not occur.
A major advantage of PG as a marker of lung maturity is that
it is not present in blood or meconium [37]. Thus, the detection
of PG in amniotic fluid specimens contaminated with blood or
meconium remained a valid finding even when the results of the
L/S ratio were called into question.
Drawbacks to the use of the lung profile included its lengthy
analytical time (35 h), variations in technique that affected its
reproducibility, and the expression of PG and PI as a percentage
of the total phospholipids [40]. This last point is particularly
relevant in that the result is dependent upon the total amount of
phospholipids applied to the plate. Underestimation of PG in
specimens containing large quantities of non-surfactant phospholipids (such as those contaminated with blood or meconium)
remained a possibility.
An immunochemical approach to the detection of PG was
reported in 1983 and is marketed as the Amniostat-FLM (Irvine
Scientific, Santa Ana, CA) [41]. This slide-agglutination test
utilizes polyclonal anti-PG antibodies to agglutinate the
microscopic PG-containing lamellar bodies into macroscopic
clusters. The test is simple to perform and is accomplished by
mixing equal volumes of amniotic fluid and a lecithin/
cholesterol reagent with a buffer solution. An aliquot of this
mixture is combined with anti-PG antibody reagent on a glass
slide, mixed, and rotated on a serological rotator at 60 rpm for 9
min. Agglutination is determined by visible inspection and
indicates the presence of PG. Results for this test are reported as
either negative (immature) or low or high positive
(mature). Because this nomenclature is opposite that used for
other tests of fetal lung maturity (with positive meaning
immature), some reports mistakenly utilize the term specificity
in place of the more accurate term of sensitivity (likewise for
predictive values). A low positive result is one that produced
small agglutinates with definite background clearing while a
high positive result consists of obvious agglutinates of varying
size with a distinctly clear background [42]. Interpretations are
facilitated by the inclusion of a negative, a low positive (0.5 g/
mL of PG) and a high positive (2.0 g/mL of PG) control
specimen provided by the manufacturer for comparison with the
test sample.
A significant advantage of the Amniostat-FLM test is its
ability to be performed rapidly, typically b30 min, compared to
the labor-intensive TLC methods. Also, as previously mentioned, specimens contaminated with blood or meconium are
still suitable for analysis. Its major drawback, like that of the
lung profile, is the late appearance of PG in gestation such that
an immature result is frequently associated with a high
proportion of infants that do not develop RDS (low predictive
value of an immature result).
The analytical sensitivity for the detection of PG by the
Amniostat-FLM is not as great as detection by TLC [43,44].
One study reported that PG was detected in 50% of specimens
by 34 weeks of gestation using TLC compared with 36 weeks
when using Amniostat-FLM [43]. Another study noted that
Amniostat-FLM was consistently positive only when the PG

D.G. Grenache, A.M. Gronowski / Clinical Biochemistry 39 (2006) 110

Table 3
Outcome studies examining the detection of PG by Amniostat-FLM for
predicting fetal lung maturity
Reference

Year N

Garite [41]
Hamilton
[107]
Lockitch
[106]
Weinbaum
[43]
Halvorsen
[108]
Eisenbrey
[109]
Towers [45]
a
b

Sensitivity Specificity PPV

(%)
(%)
(%) a

NPV
(%) b

1983 74 10.8
1984 150 8.0

100
91.7

80
73.2

38
22.9

100
99

1984

49 16.3

100

68

39

100

1985

91 12.1

100

47.5

20.8

100

100

87.7

26.3

100

85.3

50

1985 119

RDS
prevalence
(%)

4.2

1989

40 15.0

1989

67

4.5

83.3
100

50

8.6

96.7
100

Positive (immature) predictive value.

Negative (mature) predictive value.

concentration was N15% by TLC [44]. Regardless, the clinical

performance of the Amniostat-FLM was on par with that of the
lung profile and had the advantage of being a rapid test. The
results of various outcome studies using the Amniostat-FLM
test are summarized in Table 3. Overall, the Amniostat-FLM
demonstrated excellent sensitivity for an immature result and a
high predictive value for maturity. It also performed well with
vaginal pool specimens as well as those contaminated with
blood or meconium [45].
In 2003, the Amniostat-FLM test was unavailable from the
manufacturer for approximately one year, however it has since
returned to the market.

0.5 mL of centrifuged (1000 g for 3 min) amniotic fluid to

various volumes of 95% ethanol such that the final ethanol
volume ranged from 42 to 55%. The tubes were then shaken for
30 seconds and allowed to settle for 15 seconds before
examining the meniscus for a stable ring of foam. The result
was reported as the highest volume of ethanol that supported a
stable ring [48]. In 1982, the FSI was marketed commercially as
the Lumadex Foam Stability Index Test by Beckman Instruments (now Beckman Coulter, Fullerton, CA) but was
discontinued in 1997 and is no longer available [49].
Tests of foam stability proved clinically effective at
predicting RDS with the FSI having the advantage over the
shake test due to its use of multiple ethanol volumes. Using a
FSI of 47 or 48% to indicate pulmonary maturity, the risk of
RDS increased with decreasing FSI [4853]. Like other tests of
lung maturity, the FSI had excellent sensitivity (98%) but
frequently produced false-immature results from infants who
did not develop RDS (specificity 85%).
Like Amniostat-FLM, the greatest technical advantage of the
FSI over TLC methods was the ease and speed at which it could
be performed. However, the test was not without its
disadvantages. The evaluation of the meniscus for a stable
ring of bubbles was a subjective assessment and amniotic fluid
specimens contaminated with blood or meconium could
produce false-mature results [49]. Additionally, the use of
hygroscopic ethanol also presented a challenge as it readily
absorbed water from the air and decreased the relative
percentage of ethanol in the reagent. This would present a
considerable challenge to laboratories that wish to continue to
offer this test as a home brew assay.
Surfactant/albumin ratio

Foam stability
A biophysical property of surfactant (rather than a biochemical one) was successfully exploited in the early search for a
rapid, low-cost test of lung maturity. The ability of surfactant to
maintain a stable foam is a function of the surface tension at the
airsolvent interface. In 1972, Clements et al. reported a
procedure that involved adding amniotic fluid to an equal
volume of 95% ethanol followed by shaking and observing the
meniscus for the presence of a ring of bubbles [46]. This shake
test proved comparable to the L/S ratio when it was made semiquantitative through the use of amniotic fluid serially diluted
into the ethanol [40,46].
A year after the shake test was first described, Edwards and
Baillie proposed a modification of the procedure through the
use of 100% ethanol altering the final volume of ethanol to
range from 47.5 to 50% [47]. Whereas the method by Clements
et al. occasionally produced a false-mature result in an infant
that subsequently developed RDS, the Edwards and Baillie
method demonstrated 100% sensitivity.
In an effort to characterize the effect that different
concentrations of ethanol have on the formation of a stable
foam and, therefore, the clinical performance of the shake test,
Sher et al. designed a semi-quantitative test referred to as the
foam stability index (FSI) [48]. This was performed by adding

The measurement of the surfactant to albumin ratio by

fluorescence polarization is now the most common quantitative
method for assessing fetal lung maturity (Table 1). The method
is based on the competitive binding of a fluorescent probe to
albumin and pulmonary surfactant in amniotic fluid. Polarization is high when the probe is bound to albumin and low when
bound to surfactant [54]. The degree of net polarization is
correlated to fetal lung maturity. The original method was
developed by Shinitzky in 1976 [55] and used diphenylhexatrience (DPH) as the probe and an UV fluorescence spectrophotometer. As originally described, the assay was technically
difficult and required expensive equipment. In the mid 1980s
Russell [56] and Tait [57] improved the method using the
Abbott TDx (Abbott Laboratories, Abbott Park, IL), an
instrument common in most U.S. clinical laboratories. While
Taits method utilized the fluorescent dye NBD-PC, Russells
method used the more stable PC-16 dye. Russells method was
commercialized by Abbott Laboratories into the TDx FLM
assay that was later modified in 1995 to the TDx FLM II assay
(FLM). The NBD-PC method is still used by some laboratories
with the TDx instrument as a home brew fetal lung maturity
assay. The TDx FLM results are reported in mg surfactant/g
albumin (values increase with increased maturity) and the
NBD-PC results are reported in mPol units (values decrease

D.G. Grenache, A.M. Gronowski / Clinical Biochemistry 39 (2006) 110

with increased maturity). The two methods have a very strong,

non-linear correlation [5].
The more commonly performed TDx FLM II assay utilizes
uncentrifuged amniotic fluid samples that have been passed
through a glass filter. The manufacturer recommended cutoff
ratios are 55 mg/g for mature and 39 mg/g for immature.
Results between these two values are reported as indeterminate.
Although centrifugation of samples is not recommended, those
that have been accidentally centrifuged can be resuspended. In a
study by Grenache et al, 89% of centrifuged samples were
within 20% of baseline values after resuspension by gentle
vortex [58].
TDx FLM II measurements have been shown to be stable in
amniotic fluid for 24 h at 4C and 16 h at room temperature
[58]. Results from samples stored at 20C can be highly
variable and decrease over time. Blood contamination has been
shown to increase results from samples with FLM ratios b39
mg/g [58]. Although this change was statistically significant it
does not appear to be clinically significant as no results were
changed from an immature to a mature interpretation [58].
Studies that have examined the use of vaginal pool samples
with the TDx-FLM II assay demonstrated that the surfactant to
albumin ratio from vaginal pool samples were lower than those
from amniotic fluid samples [59,60]. Both of these reports
concluded that mature results from vaginal pool samples predict
the absence of RDS with a high degree of accuracy. However,
amniocentesis may be needed in cases where vaginal pool
samples yield transitional or immature results.
Several studies have examined the clinical utility of the TDx
FLM II assay and have shown that it has excellent sensitivity
[6164]. These studies are shown in Table 4. In a study of 185
women (15 RDS, 170 non-RDS), Fantz et al. [61] reported that
using the manufacturers recommended cutoff of 55 mg/g, the
TDx FLM II assay had a sensitivity of 100% and specificity of
72%. The authors further suggested that decreasing the cutoff to
45 mg/g would maintain 100% sensitivity while increasing
the specificity to 84%. These conclusions are in agreement with
a smaller study by McManamon et al. [62]. No direct
comparisons of the newer TDx FLM II assay and L/S ratio
have been reported, but older studies using the original TDX
FLM assay suggest that the TDx FLM assay had better
sensitivity than the L/S ratio [31].
The National Academy of Clinical Biochemistry (NACB)
published Standards of Laboratory Practice Guidelines for the
Evaluation and Management of the newborn Infant in 1998

[65]. In these guidelines, the NACB recommended that the

surfactant to albumin ratio be used for routine analysis of fetal
lung maturity due to its good analytical precision, availability,
and clinical validity. They further recommended that this
method be available 24 h a day with a turn around time of b90
min [65].
Lamellar body count (LBC)
As previously mentioned, surfactant is stored in the
cytoplasm of the alveolar type II pneumocyte in the form of
lamellar bodies that are secreted into the alveolar space where
they unravel and deposit a layer of surfactant along the alveolar
surface. They can also pass into the amniotic cavity and hence
are found in amniotic fluid. The similarity of lamellar body size
(1.7 to 7.3 fL [66] or 15 m [67]) to platelet size (57 fL or 2
4 m [68]) permits the use of a standard automated hematologic
cell counter to quantify the number of lamellar bodies in
amniotic fluid. Most whole blood counters are designed to
count platelets with a 220 fL size gate [5]. This technique,
known as the lamellar body count (LBC), is used to estimate
surfactant production in utero and thus predict the degree of
fetal lung maturity.
Amniotic fluid specimens for LBC should be mixed gently
(2 min on a tube rocker), but not centrifuged [69]. Originally,
centrifugation was thought to be necessary to remove cellular
debris, however centrifugation has been shown to lower the
LBC [21,70]. As a result, caution should be used when
interpreting published cutoffs for maturity since the various
studies often used different centrifugation protocols. Consensus
cutoffs, for uncentrifuged samples, were reported by Neerhof et
al. [69] with counts N50,000/L suggesting maturity and
b15,000/L suggesting immaturity. Specimens containing
obvious mucus should not be used [69]. Meconium has a
small effect on LBC, increasing the count by 5000 /L
[69,71]. Blood contamination resulting in a hematocrit N1% has
been reported to initially increase LBC results by the addition of
platelets, but as clotting occurs, the LBC decreases, apparently
due to the trapping of lamellar bodies in the clot matrix [7073].
Lamellar body counts have been shown to be stable for at
least 2 weeks in samples stored at 4C [66,74,75] and 10 days
for samples stored at room temperature. The effect of freezing
on the LBC is not clear, with one report indicating no change
after freezing [74] and another report indicating a 15% decrease
in LBC after a single or repeated freeze thaw cycles [75]. In

Table 4
Outcome studies examining the utility of TDx FLM II method for predicting fetal lung maturity
Reference

Year

RDS prevalence (%)

Cutoff

Sensitivity (%)

Specificity (%)

PPV (%) a

NPV (%) b

Fantz [61]
Fantz [61]
Kaplan [63]
McManamon [62]
Dunston-Boone [64]

2002
2002
2002
1998
1997

185
185
303
94
50

8.1
8.1
N/A c
12.1
6.0

55
45
55
55
40

100
100
95.7
100
100

72.0
84.0
70.0
71.4
60.0

24
36
36.4
29.4
13.6

100
100
98.9
100
100

a
b
c

Positive (immature) predictive value.

Negative (mature) predictive value.
True prevalence could not be calculated as study was non-consecutive.

D.G. Grenache, A.M. Gronowski / Clinical Biochemistry 39 (2006) 110

general, samples should be used immediately after arrival in the

laboratory and freezing is not recommended.
The majority of published studies have utilized the Coulter
brand of hematology analyzer. The Coulter method utilizes a
conventional impedance approach and counts particles by
detecting changes in electrical resistance when a particle passes
through an aperture. A variety of electronic cell counting
instruments, each using a slightly different method for cell
enumeration, are now available and many have been employed
to determine the LBC. Recently, Szallasi et al. [76] compared
the LBC from four different hematology analyzers and found
dramatic differences when they assessed concordance with the
Coulter Gen-S counter (Beckman Coulter). Concordance
ranged from 86% with the Sysmex XE-2100 (Sysmex
Corporation, Kobe, Japan) and 78% with the ADVIA 120
(Bayer HealthCare, Diagnostics Division, Tarrytown, NY), to
66% with the Cell-dyne 3500 (Abbott Laboratories). These
results suggest that analyzer-specific cutoffs are required and
should be confirmed with outcome-based studies by each
laboratory offering the test.
Recently, one group has published a report establishing
cutoffs using the ADVIA 120 in 88 specimens collected within
72 h of delivery [72]. The data suggest a cutoff for maturity of
35,400/L with a sensitivity of 100%, specificity of 67.6%,
predictive value of an immature result of 36.8%, and predictive
value of a mature result of 100%. However, it should be noted
that this study used both fresh and frozen amniotic fluid samples
and, as stated earlier, freezing has been shown to decrease the
LBC by an average of 18% [75]. As most samples in a clinical
setting are utilized fresh, the use of frozen samples may have
falsely lowered the predicted cutoff. Still other groups have
established cutoffs for the LBC using a combination of two
different instruments [77,78]! Caution is advised when interpreting data from the literature as factors such as centrifugation,
freezing, and instrumentation can all affect the enumeration of
lamellar bodies.
Many outcomes studies [70,72,7784] and one metaanalysis [85] have examined the clinical utility of the LBC.

These studies demonstrate that the performance of the LBC is

equal to or better than the L/S ratio. No studies have directly
compared LBC to the surfactant to albumin ratio, but reports
suggest that they are very similar in their clinical utility with
extremely high sensitivity. Many of the studies reporting
outcomes data with the LBC are shown in Table 5. Seven out
of ten of these studies report a sensitivity of 100% with
specificities that range from 54 to 100% (median = 70%).
Test use and interpretation
In 1996 the American College of Obstetrics and Gynecology
(ACOG) recommended a cascade or sequential approach to
testing for fetal lung maturity [54]. This was recommended
because a mature result on any of the commonly used tests for
fetal lung maturity is strongly predictive of the absence of RDS
(99%) and little additional information is to be gained by the
performance of multiple assays. This type of approach has also
been suggested by others [80,82,8689]. It is recommended that
a rapid method, such as the TDx FLM II or LBC be performed
first and if that result is clearly mature or clearly immature then
no further testing is required. Results that are indeterminate or
close to the recommended cutoffs require additional testing of
the sample.
For many years, the reliability of fetal lung maturity testing
in diabetic mothers has been debated. The incidence of RDS has
been reported to be higher in infants of diabetic mothers [90],
however the mechanism responsible for this association is not
understood. Improvements in glycemic control and ultrasound
dating have lessened this debate. In the last several years there
have been reports that in mothers with good glycemic control
and accurate gestational dating, fetal lung maturity testing is no
more necessary than it is for mothers without diabetes [9193].
Testing should only be performed in patients with poorly
controlled diabetes or in patients with well-controlled diabetes
but inadequate gestational dating. The NACB recommends that
a single reference interval for the surfactant to albumin ratio be
used for both diabetic and non-diabetic patients [65]. The

Table 5
Outcome studies examining the utility of LBC for predicting fetal lung maturity
Reference

Year

RDS
Prevalence (%)

Method

Centrifuge

Cutoff
(per L)

Sensitivity
(%)

Chapman [72]
Roiz-Hernandez [83]
Piazze [84]
Beinlich [82]
Greenspoon [80]
Lee [81]
Dalence [78]

2004
2002
1999
1999
1995
1996
1995

88
264
105
68
70
170
130

15.9
14.9
18
7.0
11.3
8.2
12.3

None
None
300 g
300 g
None
500 g
276 g

35,400
57,000
20,000
30,000
46,000
50,000
30,000

100
92
94
83
100
100
100

Fakhoury [79]
Ashwood [70]
Bowie [77]

1994
1993
1991

56
247
56

14.3
11.3
9.6

Advia 120
CellDyne 3900
Coulter
Sysmex K800
Coulter JT
Coulter STKR
Coulter STKR AND
Coulter S+IV AND
Sysmex 780
Coulter 660
Coulter STKR
Coulter ST AND
Coulter STKR AND
Sysmex

30,000
55,000
30,000

100
100
100

a
b

Positive (immature) predictive value.

Negative (mature) predictive value.

10 min
5 min
3 min
5 min

500 g 5 min
400 g 2 min
1000 g 5 min

PPV
(%) a

NPV
(%) b

68
71
72
67
89
73
64

37
36
47
50
54
31
28

100
98
98
100
100
100
100

100
59
54

100
24
27

100
100
100

Specificity
(%)

D.G. Grenache, A.M. Gronowski / Clinical Biochemistry 39 (2006) 110

ACOG emphasizes that no mature result can completely

eliminate the risk of RDS. Therefore, the risk of adverse
outcome with delivery on the basis of lung maturity assessment
must be weighed against the potential risk of poor outcome by
permitting the pregnancy to continue [54].
Data regarding the use of fetal lung maturity assessment in
twin gestations is sparse. A 1999 report by Whitworth et al. [94]
indicated that in diamniotic twin gestations 32 weeks L/S
ratios should be obtained from both amniotic sacs. It would be
safest to assume the same is true for assessment using TDx FLM
II as well. McElrath et al. reported that beyond 31 weeks, twin
gestations appear to have TDx FLM II results 22 mg/g higher
than that of singleton pregnancies [95]. It is unclear if these are
false mature results or represent a real maturational difference
between twin and singleton gestations.
Traditionally, cutoffs for fetal lung maturity testing have
been selected so as to maximize sensitivity and the predictive
value of a mature result in order to reduce the risk of delivering
infants with immature lungs. A potential problem with this
approach is that the same cutoff is applied to all gestational ages
despite the fact that the risk of RDS decreases with increasing
gestational age. In 1994, Tanasijevic et al. reported a predictive
model for the probability of fetal lung maturity that combined
gestational age and the original TDx FLM assay [96]. This
approach individualized TDx FLM cutoffs for different
gestational ages and allowed the prediction of the probable
risk of RDS associated with a given TDx FLM result.
The concept of reporting the probability of RDS based on
gestational age and test result has been revisited several times
recently [63,63,97100], with all emphasizing the need for an
easy and practical approach to predicting fetal lung maturity. As
a result, two groups [101,102] have published risk tables that
report risk of RDS at various gestational age-stratified TDx
FLM II ratios. The paper by McElrath et al. provides only
absolute risk [101] and promotes the use of a regression line set
at a 15% probability of RDS resulting in a sensitivity of b50%.
Parvin et al. published tables of both absolute and relative risk
[102]. The benefit of relative risk is that it can be applied across
institutions regardless of RDS prevalence. In contrast to
McElraths group, Parvin et al. promote the use of a regression
line set at a 4.3% risk of RDS and corresponds to 100%
sensitivity and 74% specificity. These types of risk tables will
produce consistent sensitivities and specificities across the
gestational age range and can help physicians individualize the
risk-benefit decisions based on gestational age and TDx FLM II
results.
Summary
Fetal lung maturity tests have contributed greatly to the
clinical management of preterm infants. From an analytical
perspective there have been ongoing improvements in methodology and technique since the introduction of the L/S ratio
nearly 25 years ago. These improvements have resulted in
automated assays that, compared to the L/S ratio are more
precise, rapid to perform, less technique dependent, have a
reduced turn around time, and can be made available 24 h a

day. Still, fetal lung maturity tests continue to be poor

predictors of immaturity. Testing strategies that incorporate
gestational age into predictors of RDS risk are practical
approaches to addressing the dilemma of indeterminate
results.
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