Review

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Biomarker tests for fetal lung maturity

The production of surfactant is a key step in fetal lung development. Surfactant decreases alveolar surface
tension, thereby preventing alveolar collapse and allowing efficient gas exchange. The lack of adequate
amounts of lung surfactant results in respiratory distress syndrome. Tests that assess surfactant
concentrations in amniotic fluid are good predictors of infants that will not develop respiratory distress
syndrome. The most frequently used test to assess fetal lung maturity (TDx FLM II) will not be available
after December 2011. Therefore, we review the currently available tests for fetal lung maturity including
lecithin:sphingomyelin ratio, phosphatidyl glycerol, surfactant:albumin ratio and lamellar body counts.
Herein, we discuss their clinical utility and consider a suitable replacement for the future.

Keywords: fetal lung maturity testing n lamellar body counts n respiratory distress Van Leung-Pineda1
syndrome n surfactant n TDx FLM II & Ann M Gronowski†1
1
Department of Pathology
Surfactant function in fetal where they are packaged into laminated storage & Immunology, Washington University
School of Medicine, 660 S. Euclid,
lung maturation granules called lamellar bodies. The lamellar Box 8118, St Louis, MO 63110, USA
„„ Lung development bodies are subsequently secreted by endocyto­ †
Author for correspondence:
Tel.: +1 314 362 0194
Fetal lung development can be divided into four sis. The secreted lam­ellar body unfolds to form Fax: +1 314 362 1491
broad stages: pseudoglandular, canalicular, sac­ tubular myelin and other large aggregates that are [email protected]
cular and alveolar. The pseudoglandular stage adsorbed into the expanded surface mono­layer.
spans weeks 5–17 in the embryo with the form­ Lamellar bodies are 1–5 µM or 1.7–7.3 fl [5,6] in
ation of two budding lungs. These undergo several diameter and appear around week 24 of gesta­
steps of branching morphogenesis that result in the tion in the cytosol of type II pneumocytes, but
production of three lung lobes on the right side and surfactant is not measurable in the amniotic fluid
two lung lobes on the left [1,2] . The second canalic­ until approximately week 32 [2] .
ular stage takes place between gestational weeks 2 The function of pulmonary surfactant is to
and 16 and is remarkable for the increased angio­ coat the alveolar epithelium and decrease its sur­
genesis and differentiation of type I and II pneu­ face tension. According to Laplaces’ law, there is
mocytes, which will be responsible for mediating an inverse relationship between surface tension
gas exchange [3] . The future lung parenchyma is and alveolar radius. Therefore, assuming their
also canalized by capillaries and surfactant first surface tensions are equal, a small alveolus will
appears during this period [1] . The saccular stage have greater inward force than a large alveolus.
comprises of weeks 24–38 and involves the forma­ Therefore, small alveoli are more likely to col­
tion of transitory clusters of widened spaces in the lapse. Surfactant reduces the surface tension on
peripheral airways [1] . The fourth alveolar stage all alveoli, but its effect is greater on the small
begins around gestational week 36 and involves alveoli [7] . Thus, surfactant compensates for the
the formation of alveoli. Alveolarization begins size differences between alveoli and ensures that
with low ridges on both sides of the saccular walls; smaller alveoli do not collapse. The end result
with increasing growth the saccules subdivide into is that adequate amounts of surfactant allow
smaller units named alveoli. During this stage, the alveoli to expand and contract during gas
there is increased production of pulmonary sur­ exchange without collapsing.
factant by type II pneumocytes [1] . Lung devel­ Surfactant is comprised of approximately 90%
opment and alveoli formation continues until lipids and 10% protein. In the lipid portion, leci­
approximately 8 years of age [4] . thin (phosphotidyl choline; 76–86%) and phos­
phatidylglycerol (PG; 6–13%) are most abun­
„„ Lung surfactant dant. Phosphatidylinositol and sphingomyelin
Surfactant is synthesized in the rough endo­plasmic are in lower abundance (4 and 2%, respectively).
reticulum of the alveolar type II pneumocytes. As illustrated in Table 1, the kinetics of produc­
They are transported to the multi­vesicular bodies tion differs between the different lipids. Lecithin

10.2217/BMM.10.109 © 2010 Future Medicine Ltd Biomarkers Med. (2010) 4(6), 849–857 ISSN 1752-0363 849
Review Leung-Pineda & Gronowski

Table 1. Phospholipid composition of lung surfactant and detection in

amniotic fluid.
Phospholipid Detection in amniotic fluid Composition (%) Ref.
Lecithin Detected at week 28 of gestation 76–86 [4,7,8]
Surges at week 36 of gestation
Continues to increase until delivery
Phosphatidylglycerol Detected at week 36 of gestation 6–13 [4,7,8]
Continues to increase until delivery
Phosphatidylinositol Detected at week 28 of gestation 4 [8]
Peaks at week 35 of gestation and decreases
until delivery
Sphingomyelin Detected at week 28 of gestation 2 [4,7,8]
Constant in last trimester of pregnancy

is synthesized around 28 weeks with a surge at noncompliant lungs and alveoli that are per­
36 weeks and continues to increase until deliv­ fused but not aerated, a condition named ven­
ery. Phosphatidylinositol production also starts tilation perfusion mismatch [7] . The resulting
at week 28, but peaks between 32 and 35 weeks, ischemia to the lung leads to a secondary defi­
and subsequently decreases. PG is the last lipid ciency in surfactant, perpetuating a vicious cycle
to appear in surfactant, starting at 36 weeks that leads to worsening of the condition [17] . In
and increasing until delivery. Sphingomyelin 2007, RDS was the seventh most common cause
is the least abundant phospholipid, comprising of infant death [18] .
­approximately 2% of total lipids [4,8] . Surfactant production increases with gesta­
Proteins found in surfactant include sur­ tional age; therefore, the risk of RDS is high­
factant protein (SP)‑A, ‑B, ‑C and ‑D. SP‑A est in premature births. The risk for develop­
and ‑D are hydrophilic proteins that regulate ing RDS is more than 60% at 29 weeks, 20%
immunity in the lung. The function of SP‑A at 34 weeks and less than 5% at 37 weeks or
concerns the formation of tubular myelin that later [13,19] . Prevention of premature birth is the
derives the surfactant multilayer, whereas SP‑D best way to avoid RDS. The American College
regulates surfactant homeostasis and the innate of Obstetricians and Gynecologists (ACOG)
immunity of the lungs [8–10] . SP‑B interacts with practice bulletin regarding fetal lung matu­
surfactant phospholipids and forms surfactant rity (FLM) does not recommend FLM testing
layers at the water–air boundary. Interaction of if delivery is mandated for fetal or maternal
SP‑B with ‑A is not required to form surfactant indications [20–22] . FLM testing should only
layers. Surfactant presents as multilayers on the be performed if delivery is desired, but could
surface of alveoli [9,11,12] . SP‑C stabilizes the safely be delayed. In addition, a mature FLM
physical properties of surfactant by promoting test result before gestational week 39 should
movement of lipid molecules [11] . be interpreted with caution, as complications
have been reported in premature neonates with
„„ Respiratory distress syndrome mature FLM test results [20–22] . If waiting for
Respiratory distress syndrome (RDS) of the term birth is not indicated, antenatal steroid
newborn, previously called hyaline membrane treatment can be administered to the mother
disease, is a syndrome caused in premature to accelerate fetal lung maturation [23] . From
infants by developmental insufficiency of sur­ animal models, cortico­steroid administration
factant production and structural immaturity of leads to an improvement in fetal lung mechan­
the lungs. It can also be caused by genetic defects ics, alterations in lung structure and increase of
that cause altered production of SPs [13,14] . For phospholipid synthesis [24–26] . Current ACOG
instance, a genetic deficiency in SP‑B results in recommendations suggest a single course of cor­
surfactant deficiency in newborns and is often ticosteroids for pregnant women at risk of pre­
fatal [15] . Another genetic deficiency that leads term birth between 24 and 34 weeks of gesta­
to RDS is mutations in the ATP-binding cas­ tion [27] . Furthermore, a recent article by Shanks
sette A3, which lead to the abnormal trans­ et al. used the same protocol on pregnancies over
port of lipids into lamellar bodies, resulting in 34 weeks of gestation with similar increases in
deficient or absent surfactant [16] . Insufficient lung maturity [28] . Neonatal treatment with exo­
surfactant causes collapse of small alveoli and genous surfactant through endotracheal delivery
over-inf lation of large alveoli, resulting in has proven to be effective in treating RDS [29,30] .

850 Biomarkers Med. (2010) 4(6) future science group

 Biomarker tests for fetal lung maturity Review
„„ Laboratory tests for FLM 33% on College of American Pathologists (CAP)
Tests for FLM assess the amount of surfactant proficiency testing material [33] . The commercial
in the amniotic fluid and use it as a surrogate method is the Fetal Tek 200 (Helena Laboratories,
reading of the amount of surfactant in the fetal Beaumont, TX, USA) and, according to CAP
lungs. The ideal FLM tests should have a high proficiency testing surveys, is currently used by
sensitivity for RDS and high predictive value more laboratories than the laboratory-developed
of a mature result. Such test characteristics are method (n = 50 vs 28) [33] . The commercial
designed to detect all neonates with RDS at risk method has slightly better precision with CVs
of a high number of false positives (neonates that ranging from 14 to 20%, but is still less precise
test positive for RDS but do not have RDS at than other FLM tests [33] .
birth). In this article, we will discuss the four Infants at 35–36 weeks of gestation have L:S
most frequently used FLM tests. ratios of 2.0 or greater; therefore, 2.0 is the usual
cutoff for lung maturity. However, owing to the
„„ Lecithin:sphingomyelin ratio low precision of the method, the individual labo­
Developed in the early 1970s, the lecithin:sphin­ ratory should determine the cutoff after perform­
gomyelin (L:S) ratio was the first biochemical ing its own clinical outcome study. The L:S ratio
test for FLM and, thus, was considered the gold has a high sensitivity for detecting lung imma­
standard for many years [31,32] . This test meas­ turity, with an expected low specificity (Table 2) .
ures the most abundant lipid (lecithin) and Sensitivity is best between 25 and 31 weeks of
the least abundant lipid (sphingomyelin) in gestation owing to the prevalence of RDS at this
lung surfactant. Lecithin increases with gesta­ time [34] . Both blood and meconium can adversely
tional age, starting around gestational week 28. affect results. Both lecithin and sphingo­myelin
Sphingomyelin remains constant during the last are present in blood, and the L:S ratio of blood
trimester of pregnancy, and serves as an internal is 1.3–1.5. Therefore, blood contamination can
control for the test. An increase in L:S ratio is falsely decrease a mature ratio and falsely increase
seen with lung maturation [32] . an immature ratio [35] . Amniotic fluid samples
Lecithin and sphingomyelin are separated by can be stored at ‑20°C for up to 12 months with
thin layer chromatography (TLC), after metha­ no effect on L:S ratio results [36] .
nol and chloroform extraction, and developed in
solvent containing methanol, 2‑propanol, chloro­ „„ Phosphatidylglycerol
form, triethylamine and water. The lipids are vis­ The measurement of PG in amniotic fluid was
ualized by exposure to cupric acetate, phosphoric developed as laboratories strived to improve the
acid and charring. The lecithin and sphingo­ specificity of the L:S ratio. By adding PG to the
myelin bands are quantified by densitometry and L:S ratio, the sensitivity was maintained while the
expressed as a ratio. test’s specificity was increased [37] . Two methods
This method requires a large sample volume are currently used to detect PG: quantitative TLC
(3–4 ml), is technically difficult and time con­ and qualitative agglutination. The TLC method
suming. In addition, although there is a com­ is similar to that described for detecting the L:S
mercial method available, many of the labo­ ratio and has the same disadvantages as described
ratories that perform this method still rely on previously. The AmnioStat-FLM® (Irvine
laboratory-developed protocols. This has lead to Scientific, Santa Ana, CA, USA) [38] is a slide
imprecision among laboratories as illustrated by agglutination test that uses anti-PG antibodies to
coefficients of variation (CV) ranging from 25 to agglutinate lamellar bodies containing PG into

Table 2. Fetal lung maturity methods: performance and interpretation.

Method Reference intervals Assay time Sensitivity (%) Specificity (%) Ref.
L:S ratio ≥2.0 – mature 5h 50–100 60–97 [2]
PG Positive – mature ≤1 h 83–100 47–88 [2]
(Amniostat-FLM ® ) Negative – immature
S:A ratio ≥55 mg/g – mature ≤1 h 96–100 60–84 [2]
40–54 mg/g – indeterminate
≤39 mg/g – immature
LBC ≥50,000/µl – mature ≤1 h 83–100 54–100 [2,66]
16–49,000/µl – indeterminate
≤15,000/µl – immature
FLM: Fetal lung maturity; L:S: Lecithin:sphingomyelin; LBC: Lamellar body count; PG: Phospatidylglycerol; S:A: Surfactant:albumin.

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Review Leung-Pineda & Gronowski

macroscopic structures. The test is performed by are 55 mg/g or greater for mature and 39 mg/g or
mixing amniotic fluid with lecithin/cholesterol lower for immature. For the laboratory-developed
solution and buffer, to allow PG incorporation FPol, results are given in mPol units. The mPol
into lipid particles. An aliquot of the sample is units decrease with increasing maturity. This
then mixed with the anti-PG on a glass slide. is opposite to the TDx FLM II ratio, but there
Agglutination of the sample is compared with is a strong correlation between FPol and TDx
three controls: negative, low positive and high FLM II results, allowing conversion between
positive. The reported results are qualitative. the two [7,46] . Fpol less than 260 mPol indicates
The greatest advantage of AmnioStat-FLM over maturity, 260–290 mPol is indeterminate and
TLC is the short time needed to complete the over 290 mPol indicates immaturity. Amniotic
method (<30 min vs 3–5 h). Clinical sensitivity fluid samples have been demonstrated to be stable
for the TLC and AmmioStat-FLM methods is for 16 h at room temperature and 24 h at 4°C with
comparable [2,39] . no effect on S:A ratio results. Stability was studied
The main advantage of measuring PG is that up to 48 h, but values decreased after 16 h at room
it is not affected by blood or meconium [40] . temperature and 24 h at 4°C [45] . Freezing sam­
However, if the PG is performed by TLC and ples at -20°C has been demonstrated to introduce
expressed as a ratio of PG:S, the test is not valid a large random error to the TDx FLM II result,
in the presence of blood, since sphingomyelin is and is not recommended for any specimens [45] .
found in blood. This analyte has the disadvantage Numerous studies have demonstrated that the
of being the last lipid to increase in surfactant sensitivity of this method is excellent (95.7–100%)
(gestational week 36) (Table 1) . Other drawbacks (Table 2) , and the National Academy of Clinical
are that interpretation of the AmnioStat-FLM Biochemistry (NACB) has recommended its use
can be subjective and only one vendor (Irvine for routine ana­lysis of FLM [47] . The precision
Scientific) sells the test, which has caused of the TDx FLM II is relatively good, with CV
intermittent supply problems over the years. for CAP proficiency testing ­ranging from 4.9 to
6.0% [33] .
„„ Surfactant:albumin ratio As mentioned previously, the prevalence of
The surfactant:albumin (S:A) ratio is the most RDS decreases with gestational age. This sug­
frequently performed FLM test in the USA [2,33] . gests that a single cutoff across all gestational
The principle of the method is based on the parti­ ages is not appropriate. Recently, two groups
tioning of a fluorescent dye between the surfactant have published tables that report risk of RDS
and albumin in amniotic fluid [20,41] . Fluorescent across various gestational age-stratified FLM val­
polarization is high when the dye binds to albu­ ues [48,49] . McElrath et al. provided the absolute
min and low when the dye binds to surfactant. risk for gestational age-stratified TDx FLM II
The current methodology uses PC‑16 dye on results [48] . The caveat with absolute risk is that
the Abbott TDx platform (Abbott Laboratories, if the prevalence of RDS in a particular insti­
Abbott Park, IL, USA) and is sold commercially as tution is different from the RDS prevalence in
the TDx/TdxFLx® FLM II (TDx FLM II) [42,43] . the study, then the estimates cannot be adopted
A laboratory-developed version of this test exists, without a complex statistical conversion. By
which can be performed on the Abbott TDx plat­ contrast, Parvin et al. published both the abso­
form. The main difference is that the laboratory- lute risk and relative risks associated with TDx
developed test uses NBD-phosphatidylcholine FLM II ratios by gestational week [49] . For the
as the fluorescent dye [44] and the test is known relative risk estimation, these authors established
as the fluorescent polarization test (FPol) [7] . a table that assigns an odds ratio (OR) of 1.0 to
The concentration of surfactant in the sample is a FLM result of 70 mg/g at 37 gestational weeks
extrapolated from a standard curve made with as a reference point. In this table, an OR greater
calibrators with known S:A ratios. Results are than 1.0 implies a higher risk of RDS and an
reported as milli­grams of surfactant per gram of OR of less than 1.0 denotes lower risk of RDS.
albumin for the TDx FLM II, and ratios increase The advantage of relative risk over absolute
with increasing lung maturity. Amniotic fluid risk is its independence from RDS prevalence.
samples are filtered through a glass filter prior to Therefore, the OR estimates can be adopted
testing. Centrifugation has been demonstrated across institutions regardless of differences in
to significantly decrease results; however, cen­ RDS ­prevalence [49] .
trifuged samples can be resuspended without The advantages of the S:A ratio are: excellent
causing significant variation [45] . Manufacturer- sensitivity, short turnaround time, simplicity,
recommended cutoff ratios for the TDx FLM II quantitative results and, furthermore, there are

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 Biomarker tests for fetal lung maturity Review
commercially available quality control (QC) to affect LBC results. Whole blood, with a red
materials and external proficiency testing to blood cell count of more than 31,000/µl, leads
maintain the precision and accuracy of the assay. to a decrease in LBC, presumably caused by the
Disadvantages include interference caused by lamellar bodies being trapped in a fibrin matrix.
blood and meconium. In addition, only a single The opposite is seen in samples contaminated
vendor provides the assay. Owing to its numer­ with meconium, as these will give a falsely ele­
ous advantages, the TDx FLM II is currently the vated LBC, caused by contaminants in the meco­
most frequently used FLM test. However, Abbott nium that are detected in the platelet channel [53] .
Laboratories plan to phase out the TDx testing Centrifugation lowers the LBC, necessitating the
platform and the TDx FLM II reagents in late use of different cutoffs [39] .
2011. In a letter sent by Abbott in June 2010, The majority of studies have been per­
the company announced the retirement of the formed using the Beckman–Coulter (Brea,
TDx platform. In addition, Abbott reported that CA, USA) brand of hematological analyzers.
their efforts to adapt the TDx FLM II test to Beckman–Coulter and Sysmex (Kobe, Japan)
their ARCHITECT system were not successful analyzers use the conventional impedance (also
owing to instrument constraints and they would called resistive-pulse) method, whereas ADVIA®
no longer offer the test once the IMx platforms (Bayer HealthCare, Tarrytown, NY, USA)
were retired. Abbott recommended pursuing systems use the optical light scatter method,
alternative tests for FLM testing. and Abbott’s Cell-dyn utilizes a combination
of both methods. Szallasi et al. compared the
„„ Lamellar body counts Coulter Gen‑S™ to three other analyzers: the
As described previously, pulmonary surfactant is Sysmex XE-2100™, the ADVIA 120 and the
produced and stored in the cytoplasm of type II Cell-dyn 3500. Their results demonstrated dif­
pneumocytes, which are packaged into lamel­ ferences in the concordance between the Coulter
lar bodies that are then secreted into the alveo­ Gen‑S and Sysmex (86%), ADVIA (78%) and
lar space. Once in the alveolar space, lamellar Cell-dyn (66%) [54,55] . These data suggest that
bodies deliver surfactant to the alveolar surface. the consensus cutoff of 50,000/µl is probably ade­
Owing to the similarity in size of lamellar bodies quate for the Sysmex and Advia analyzers [53,54] .
(1–5 µM or 1.7–7.3 fl) [5] to platelets (2–4 µM or However, new cutoffs for the Cell-dyn should
5–7 fl) [50] they can be detected in most automated be established. Studies by Szallasi and Roiz-
hematological cell counters. Hernandez suggest that a cutoff of 80,000/µl
Several outcome studies have demonstrated would be appropriate [54,56] . Regardless of the
that the lamellar body count (LBC) performs instrument, it is recommended that each indi­
well compared with other FLM methods [51,52] , vidual laboratory validate their instrument prior
with a sensitivity of 83–100% (Table 2) . In a study to implementing the LBC test. This will be
of 833 samples, Neerhof et al. demonstrated that discussed in more detail later in the article.
the negative predictive value of LBC (97.7%) This test shares many of the advantages of the
was comparable to the L:S ratio (96.8%) and S:A ratio, in that it is a sensitive, rapid, quantita­
better than PG (94.7%) [51] . In addition, in a tive and simple method that only requires 1 ml
head-to-head comparison of the LBC method of sample. The CAP began proficiency testing
(using the Neerhof consensus cutoff of 50,000/µl) for LBCs in 2010 [57] . However, we are unaware
and the TDx FLM II, Haymond et al. demon­ of any commercial LBC QC material. Similar to
strated that the LBC had slightly better sensitivity the S:A ratio, LBCs can also be affected by blood
than the TDx FLM II (92 vs 83%, respectively) and meconium. However, one great advantage of
with comparable specificity (60 vs 65%, respec­ this method is that the instrumentation is already
tively) and negative predictive value (99 vs 98%, available in most clinical laboratories.
respectively) [52] .
Lamellar bodies have been demonstrated to be Conclusion & future perspective
stable when stored at 4°C for up to 33 days with „„ Trends in FLM test ordering
an imprecision of 1.2–15.1%. However, sample In 2009, Tita et al. looked at adverse respira­
freezing and thawing can cause a significant tory outcomes in elective cesarean deliveries
decrease in LBC. This is believed to be caused by performed before week 39 of gestation. The
the fragmentation of lamellar bodies into smaller results showed that adverse outcomes increased
particles (<1 µM) that are not detected by the by a factor of 1.3–4.2 in neonates delivered elec­
hematology counter in the platelet window [53] . tively before 39 weeks [58] . These data support
In addition, blood and meconium are also known recom­mendations to delay elective delivery until

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Review Leung-Pineda & Gronowski

39 weeks of gestation. As the numbers of elective testing. We speculate that this test is performed,
deliveries have decreased, this has resulted in a in large part, to provide confirmatory evidence
decrease in the volume of FLM tests [59–61] . In of lung maturity for legal purposes in cases of
a survey of Maternal Fetal Medicine Fellows, elective delivery. With the newest recommenda­
McGinnis et al. demonstrated that the major­ tions to avoid elective delivery before 39 weeks,
ity of physicians (60%) had decreased their it is feasible that FLM tests could be phased
order for FLM tests [60] . In the same survey, out owing to a lack of evidence for improving
the most common reason given for decreased patient care.
FLM testing was that physicians felt these tests In terms of popularity, the S:A ratio test (TDx
were not needed for patient care. The authors FLM II) is the most frequently performed [61] .
also noted that many physicians have changed When the Maternal Fetal Medicine Fellows
their practice, preferring to wait until 39 weeks were asked about which tests they would order
of gestation for delivery [60] . instead of the TDx FLM II when it is retired
A separate, more recent survey of Maternal in December 2011, respondents indicated a
Fetal Medicine Fellows regarding FLM test­ preference for the L:S ratio and LBC [61] .
ing demonstrated that FLM test utilization is
declining [61] . However, this decrease is gradual „„ Replacement of the S:A ratio
and does not suggest that FLM testing will soon Faced with the imminent removal of the TDx
be obsolete. Furthermore, although both surveys FLM II from the market, clinicians and medi­
demonstrated that the decline in FLM testing cal laboratory professionals need to agree upon
was mainly caused by physicians not needing and implement a suitable replacement. Ideally,
them for patient care [60,61] , Grenache et al. the replacement would have many of the same
demonstrated that 92% of physicians were not features as the S:A ratio: high sensitivity, quick
willing to eliminate FLM testing [61] . turnaround time, simplicity, high precision,
To our knowledge, no studies have directly quantitative results with QC and external pro­
addressed the impact of FLM testing on improv­ ficiency testing available. As mentioned previ­
ing patient care. Interestingly, except for the ously, the L:S ratio and the LBC were the tests
USA, most of the world does not perform FLM favored by physicians. However, in a survey

Table 3. Fetal lung maturity methods: advantages and disadvantages.

Analyte/method Advantages Disadvantages
L:S ratio by TLC – High sensitivity – Time consuming
– Quantitative – Considerable skill required
– QC/PT available – Large sample volume required
– Imprecise
– Wide gray zone
– Blood and meconium interference
PG by TLC – High sensitivity – Analyte is late marker of maturity
– Enhances specificity of L:S ratio – Time consuming
– Not affected by blood and meconium – Considerable skill required
– QC/PT available – Large sample volume required
– Imprecise
PG by Amniostat® – Rapid – Analyte is late marker of maturity
– Simple – Single vendor
– Not affected by blood and meconium – Qualitative result
– QC/PT available – Subjective interpretation
S:A ratio by – Widely available platform – Single vendor (not available after
fluorescent – Rapid December 2011)
polarization – Quantitative – Blood and meconium interference
– Standards available
– QC/PT available
LBC – Widely available platform – Blood and meconium interference
– Rapid – No commercial QC available
– Quantitative
– Low sample volume
– PT available
L:S: Lecithin/sphingomyelin; LBC: Lamellar body count; PG: Phosphatidylglycerol; PT: Proficiency testing reagents;
QC: Quality control reagents; S:A: Surfactant:albumin; TLC: Thin layer chromatography.

854 Biomarkers Med. (2010) 4(6) future science group

 Biomarker tests for fetal lung maturity Review
of clinical laboratories, only 18% perform the is a problem, as 77% of physicians surveyed by
L:S ratio test and 13% perform LBC [61] . This Grenache et al. preferred results within 12 h of
indicates that, to replace the TDx FLM II, the collection [61] .
majority of laboratories will have to implement Grenache et al. identified the following per­
a new method for assessing FLM. ceived obstacles for the implementation of the
As shown in Table 2 , all FLM tests have a high LBC: lack of QC material; concern that amnio­
sensitivity for fetal lung immaturity. Several tic fluid samples will damage the analyzer; the
clinical outcome studies have compared the per­ need for validation of this noncommercial assay;
formances of the LBC method to the L:S and decreased demand for FLM testing; and a lack
S:A ratios [51,52,62–64] . The conclusion from these of familiarity with the test [61] . We address each
studies was that the LBC method performed as of these concerns in turn:
well as the S:A ratio and better than the L:S ƒƒ Although we are unaware of any commercially
ratio. With their accuracy being similar, we available QC material for LBC testing,
move forward to looking at turnaround time, CAP has introduced surveys for proficiency
simplicity and precision. Table 3 shows that the testing in 2010 [57] . Furthermore, at our insti­
L:S ratio method can take 5 h to perform in tution we dilute the low platelet QC material
skilled hands, requiring a considerable sample at a 1:1 ratio and use it as QC with every
volume (3–4 ml of sample) and suffers from LBC performed.
imprecision owing to the skill needed for the
method. Owing to the technical difficulty of the ƒƒ
To our knowledge, amniotic fluid has not led
method, it has been recommended that labora­ to any hematology instrument malfunction [66] .
tories with less than 15 test requests per week do ƒƒ
Although laboratory validation is required, a
not implement the L:S ratio [65] . By contrast, the consensus protocol has been published with
LBC has a turn­around of less than 1 h, requires guidelines for sample processing to avoid
only 1 ml of sample and is performed by an auto­ preanalytical variables [66] . Furthermore, a
mated analyzer, requiring no specialized skill. validation protocol is available, which demon­
In regards to LBC precision, Lockwood et al. strates precision and linearity, and also sample
tested two different instruments with inter-assay storage and sample rejection caused by
CVs of 1.9–5.1% for the Coulter LH750 and contaminants [53] .
1.3–7.1% for the Sysmex XE-2100 [53] . These
ƒƒ
Although FLM testing has decreased, we do
are definite improvements on CVs reported for
not foresee its extinction as physicians still
the L:S ratio [33] . An alternative for laborato­
require it for adequate patient care [61] .
ries and physicians who would still like the L:S
ratio as their primary FLM test is to send it to ƒƒ
Owing to the lack of familiarity with this test
a reference laboratory. However, the drawback we urge laboratory technicians to educate ­both
is a turnaround time in excess of 12 h, which themselves and physicians.

Executive summary
ƒƒ Respiratory distress syndrome is the seventh leading cause of infant mortality and is caused by lack of adequate amounts of pulmonary
surfactant when babies are born prematurely. It is the most common cause of adverse respiratory outcomes in newborns and incidence
decreases with increasing gestational age.
ƒƒ Fetal lung maturity (FLM) tests should be performed between 32 and 39 weeks of gestation and no FLM testing should be performed if
delivery is the only option. Testing before 32 weeks should be avoided as the risk of other comorbidities caused by preterm birth are too
high. The incidence of respiratory distress syndrome at 39 weeks is minimal.
ƒƒ Ordering of FLM tests is declining, the major reason is that physicians now wait until 39 weeks of gestation for elective delivery.
Significant respiratory complications have been observed in elective cesareans before 39 weeks. Most physicians feel that FLM testing
is not needed for patient care, but are not willing to forego these tests. The FLM tests have a high sensitivity for immaturity but a poor
positive (immature) predictive value.
ƒƒ The most popular FLM test is the surfactant:albumin (S:A) ratio (TDx FLM II). It has high sensitivity, short turnaround time, high
precision and is not technically challenging to perform. It is only provided by a single vendor, and reagents and platforms will be
discontinued soon.
ƒƒ Currently, the lamellar body count method is the best replacement for the S:A ratio. It uses instrumentation available in most
laboratories and shares many of the advantages of the S:A ratio.
ƒƒ Proficiency testing is now available for the College of American Pathologists (CAP).
ƒƒ The validation protocol has been published.
ƒƒ Although FLM testing has been declining in recent years, it is unlikely it will become obsolete in the near future.
ƒƒ With the discontinuation of the TDx FLM II, the lamellar body count method is primed to take its place.

future science group www.futuremedicine.com 855

Review Leung-Pineda & Gronowski

In conclusion, we propose that the LBC Financial & competing interests disclosure
test would be a suitable substitute for the TDx AM Gronowski has received consulting fees from Abbott
FLM II, based on its widely available instrumen­ Laboratories. The authors have no other relevant affilia-
tation, high sensitivity, quick turnaround time tions or financial involvement with any organization or
and automated methodology. We also encourage entity with a financial interest in or financial conflict with
laboratory technicians to begin educating physi­ the subject matter or materials discussed in the manuscript
cians regarding the features and advantages of apart from those disclosed.
the LBC method, and how it is the best test to No writing assistance was utilized in the production of
replace the TDx FLM II. this manuscript.

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