your lab focus

CE Update [chemistry]

Laboratory Testing To Assess Fetal Lung

Maturity
Darlynn J. Lafler, BS MT(ASCP)CLS, and Arturo Mendoza, MD
From the Sharp Mary Birch Hospital for Women Department of Pathology, San Diego, CA

On completion of this article, the reader should be able to describe clinical indications for fetal lung maturity testing, what laboratory tests
are used to determine fetal lung maturity, and which components are measured in each test.
Chemistry exam 0103 questions and the corresponding answer form are located after the Your Lab Focus section on page 397.

Indications for fetal lung maturity testing

Phosphatidylethanolamine
Collection methods and test
(biochemical and biophysical) methods Phosphatidylglycerol
Phosphatidylinositol
Test utilization and optimization of
maternal/fetal outcome

Advances in fetal lung maturity

(FLM) testing have assisted the clinician
in determining the course of complicated
as well as uncomplicated pregnancies. Neutral
Fetal lung maturation is a complex process Lipids
involving a balance of physiologic, cellu-
lar, and biologic functions. During
intrauterine development, the fetus relies
on the exchange of gases through the pla- Phosphatidylcholine
Protein
centa. At birth, once the umbilical cord is
clamped, the infants system must assume
responsibility for ventilation. The lung is
the last major organ to mature in the fetus,
and it is critical that it be adequately ma-
ture to handle this transition.
The production of pulmonary surfac-
tant is the critical step in this progression to
maturity. Surfactant is responsible for low-
ering the surface tension in the lungs fol-
lowing delivery and acts to prevent the [F1] Composition of surfactant recovered by alveolar wash. Adapted from: Jobe.3
collapse of the pulmonary alveoli on end
expiration. Surfactant is produced by the intrauterine fetal breathing movements Surfactant as recovered from lungs of all
type II pneumocytes of the lung and is allow the components of surfactant to be mammalian species contains 70% to 80%
packaged in concentrically layered struc- readily measured in the amniotic fluid. phospholipids, about 10% protein and about
tures called lamellar bodies. Consisting al- Throughout the pregnancy, there is a grad- 10% neutral lipids, primarily cholesterol 393
most entirely of proteins and phospholipids, ual increase in the amount of amniotic fluid. [F1].3 With advancing gestation, the most
they represent the storage form of It rises steadily with a peak volume of ap- abundant phospholipid in surfactant is
pulmonary surfactant.1 proximately 800 mL at 33 weeks and grad- lecithin (phosphatidylcholine), followed by
Lamellar bodies first appear in the am- ually decreasing until delivery.2 phosphatidylglycerol (PG), which appears
niotic fluid between 20 and 24 weeks of Human surfactant is a complex sub- around the 35th week. Other phospholipids
gestation and are released into the alveolar stance composed of phospholipids, carbo- include sphingomyelin, phosphatidylinosi-
space. The dynamics of fluid turnover and hydrates, and a variety of proteins. tol, and phosphatidylethanolamine.

laboratorymedicine> july 2001> number 7> volume 32

 your lab focus
Tests To Assess Fetal Lung Maturity
Method Cost
Lamellar Low
body count
Surfactant/ Moderate
albumin ratio
Phosphatidylglycerol Moderate
by slide method
Lecithin/sphingomyelin High
ratio
Premature infants may have multiple
complications, but surfactant deficiency can
be the most acute. Surfactant deficiency in
the neonate is the primary cause of respira-
tory distress syndrome (RDS) and is the
leading cause of morbidity and death in
preterm infants.4 Acute fetal distress typi-
cally occurs, primarily resulting in RDS or
hyaline membrane disease. The clinical
signs become apparent with neonatal grunt-
ing, chest wall retraction, and cyanosis and
are confirmed by radiographs of the infants
lungs. Infant RDS is the most expensive
inpatient condition billed for by US hospi-
tals according to the national statistics col-
lected by the US Agency for Healthcare
Research and Quality.5
Fetal Lung Maturity Testing
Indications for fetal lung maturity
testing vary and can include preeclampsia,
premature rupture of membranes, fetal
distress, or preterm labor with imminent
delivery. The clinical management of
these patients is enhanced by laboratory
determination of FLM. These tests can
provide timely information to the clinician
about whether to prevent a preterm deliv-
ery or to provide maternal drug therapy to
enhance lung maturity.
The fetal lung maturity tests available
can be divided into 2 groups, biochemical
394 and biophysical. Biochemical tests meas-
ure components of the surfactant and in-
clude the lecithin/sphingomyelin (L/S)
ratio and phosphatidylglycerol measure-
ments. Biophysical tests measure the
physical properties of the phospholipid
surfactants and include the surfactant/al-
bumin (S/A) ratio and lamellar body
Turnaround Time Availability
Less than 10 min Universal, on-site,
on demand
Approximately 40 min On-site, on demand
Approximately 15 min On-site, on demand
2-4 hr Usually not on site
counts. These tests rely on the premise
that the amniotic fluid accurately reflects
the fetal lung fluid components.
Demonstrated pulmonary maturity is
valuable to the clinician, but it is not the
only indicator on which a delivery is based.
Overall clinical circumstances and assess-
ment are considered. When testing indicates
lung immaturity, the delivery can be post-
poned if the clinical assessment is favorable
and glucocorticoids can be given to the
mother to help facilitate lung maturation.
There are 2 methods of collecting am-
niotic fluid, transabdominal amniocentesis
and vaginal pool collection. The latter
method consists of aspirating amniotic
fluid from a vaginal pool collection in
cases involving rupture of membranes.
Both methods provide adequate fluid for
testing provided that they are relatively
free of contaminating substances such as
gross blood or meconium.
Several tests are available, but the
focus is on the 4 most used; the L/S ratio,
phosphatidylglycerol determination, S/A
ratio, and lamellar body counts. Cost, turn-
around time, and labor requirements vary
with each assay and are outlined in [T1].
Comparison of laboratory results with clini-
cal evaluation of newborns is essential in
determining a given laboratorys specific
cutoff value for maturity.
Lecithin/Sphingomyelin Ratio
The first laboratory procedure for as-
sessment of fetal lung maturity was
described by Gluck and associates in 1971.6
Their evaluation of amniotic fluid
introduced the L/S ratio, a measurement of
the relative amount of lecithin and sphin-
laboratorymedicine> july 2001> number 7> volume 32
Labor Requirement
T1
Simple, no commercial kit required,
no preanalytic treatment
Simple, minor
preanalytic filtration step,
fully automated
Simple, visual slide
agglutination
Dedicated personnel,
high degree of
labor-intensiveness
gomyelin in amniotic fluid. This early lung
maturity test along with clinical correlations
served to make the L/S ratio the gold stan-
dard for fetal lung maturation.
Although it may be considered the
gold standard of FLM testing, the L/S ratio
is not without error. According to Ash-
wood,7 gold standards must have perfect
sensitivity and specificity. The L/S ratio has
neither. Studies performed by Herbert and
Chapman8 reported the sensitivity and
specificity of the L/S ratio to be 84.6% and
84.6%, respectively. An L/S ratio of 2.0 or
greater in a nonstressed pregnancy without
complications (such as maternal diabetes)
can be indicative of probable lung maturity
and a low likelihood of RDS.
Originally, pulmonary maturity in dia-
betic pregnancies was described as
delayed9,10; further studies suggested that
maturity is not delayed in diabetic pregnan-
cies that are well controlled. Although some
investigators have presented data to support
the notion that diabetes in pregnancy does
not effect pulmonary maturation,11,12 the
issue remains controversial.
Assessing the L/S ratio is labor inten-
sive, time consuming, and technique de-
pendent, taking up to several hours to
perform. It involves centrifugation of the
amniotic fluid and phospholipid extraction
using organic solvents followed by thin-
layer chromatography (TLC) with quantita-
tion by planometry or densitometry. Given
the current laboratory environment, it is
difficult and not cost-effective to accommo-
date highly specialized personnel to per-
form TLC around the clock.
Vaginal pool and amniocentesis speci-
mens can be used if they are not contami-

nated with gross blood or meconium, which
can cause interferences.
Even with commercially available
kits, variations can occur between labora-
tories secondary to labor-intensiveness
and tedious protocol.
Phosphatidylglycerol Determination
Phosphatidylglycerol generally appears
around the 35th week of gestation and can
appear earlier in infants with accelerated
lung maturity. Detectable amounts of PG in
amniotic fluid can be relied on to predict
more advanced lung maturity and the ab-
sence of RDS. With the inherent complexi-
ties of TLC, laboratories can opt for a
commercial kit, AmnioStat-FLM (Irvine
Scientific M), to make this determination.
This rapid slide test is a semiquantitative
immunologic agglutination test, results are
interpreted visually, and because the pres-
ence of PG is evidence that the lungs are
mature, subjectivity in analysis is minimal.
Some studies suggest that PG is
very useful when present but not as
helpful when absent.13-15 Strict adher-
ence to vendor guidelines for interpreta-
tion must be followed. Vaginal pool
specimens as well as amniocentesis
specimens can be used. Contamination
of amniotic fluid by blood or meconium
does not interfere with this test because
PG is predominantly found in surfactant
and lung tissue. However, if a vaginal
pool specimen is used and the mem-
branes have been ruptured for a
prolonged period, bacterial contamina-
tion can occur, and certain bacteria have
been shown to produce PG, thus giving
false-positive results.16,17 PG determina-
tion can be used to supplement S/A ratio
results as well as lamellar body counts.
Surfactant/Albumin Ratio
Fluorescence polarization is used to
determine the S/A ratio on the TDx System
(Abbott Diagnostics M). This is an auto-
mated assay that uses standardized reagents
and controls. A fluorescent dye is added to
the amniotic fluid that partitions between
the surfactant phospholipids and albumin.
The relative concentrations of surfactant
and albumin are measured against a stan-
dard curve of known S/A calibrators. The
test requires a minimum specimen volume,
your lab focus
Sensitivity and Specificity of Fetal Lung Maturity Tests
Test Method Sensitivity Specificity
T2
Surfactant/ 0.97 0.72
albumin ratio
Phosphatidylglycerol 0.93 0.55
by slide method
Lecithin/sphingomyelin 0.86 0.78
ratio
Adapted from Wong.19
approximately 1.0 mL, and can be Lamellar Body Counts
performed in 40 minutes with minimal Lamellar body counts and lamellar
specimen preparation. The cost to perform body number density have both been used
the test is less than half the cost of perform- to assess the number of lamellar bodies in
ing an L/S ratio or TLC. It can be the amniotic fluid. Characteristically, they
performed using instruments already avail- are small structures, approximately the size
able in many laboratories that perform ther- of platelets, and they scatter light, often giv-
apeutic drug monitoring. ing the fluid an opalescent appearance. Be-
Results are reported as milligrams cause of their size, lamellar bodies can be
of surfactant per gram of albumin counted rapidly by using the platelet chan-
(mg/g). Because the test is automated, nel of most whole blood analyzers.
variation between laboratories is low. The methods for performing lamellar
Vaginal pool and amniocentesis speci- body counts have varied across
mens can be used. However, gross blood institutions. Studies have shown this count
or meconium has an adverse effect on to be useful in the prediction of FLM; sev-
the results, as do vaginal pool collect- eral have shown it to be as reliable as other
ions contaminated with urine.18 FLM tests.20-23 The specimen requirement
It has been shown that diabetic pa- is small, often less than 1.0 mL. With the
tients have the same S/A ratio results as widespread use of these analyzers in labo-
nondiabetic patients, making this test a ratories, lamellar body counts can be per-
good predictor of FLM.19 formed in less than 10 minutes in most
Uncontaminated Uncontaminated vaginal
amniocentesis specimen pool specimen
Perform lamellar
body count
Mature result Immature result* Assess surfactant/
albumin ratio
Mature result Immature result
395
STOP Perform
STOP
Result* LS ratio
[F2] Fetal lung maturity test algorithm. Uncontaminated means no gross blood, meconium,
bilirubin, or urine. *Irvine Scientific's M AmnioStat phosphatidylglycerol determination can be
performed at either of these 2 points as a supplementary test to further predict fetal lung maturity.
laboratorymedicine> july 2001> number 7> volume 32
 your lab focus
cases. The test is quick and inexpensive
with no commercial kit required.
Amniocentesis collections appear to
be the most studied specimens in the liter-
ature as they are generally free of contami-
nating substances. Vaginal pool
collections, however, can be more of a
challenge to the instrumentation owing to
mucus strands and other contaminating
substances, which could potentially clog
some instrument apertures.
Platelet channels and methodologies
for counting platelets are not standardized.
Owing to the variety of whole blood ana-
lyzers and counting methods, this test is
dependent upon the method and analyzer
used. Institutions may find it necessary to
establish their own instrument-specific
cutoff values for lung maturity.
Test Utilization
The importance of collecting a satis-
factory specimen cannot be overempha-
sized. Vaginal pool collections tend to be
more contaminated with mucus or blood,
making interpretations difficult with vari-
ous test methodologies. According to
Dubin,24 use of vaginal pool material is to
be discouraged. The specimen of choice is
one collected via amniocentesis.
The overall sensitivity and specificity
of the L/S ratio, S/A ratio, and PG determi-
nation are listed in [T2]; the data include
diabetic patients.19 Lamellar body count
maturity ranges vary owing to instrument
variability and processing techniques.
However, just recently Neerhof and associ-
ates25 have attempted to standardize the
methodology for counting lamellar bodies
and maturity ranges.
A cascade or algorithm approach
[F2] to testing in which the most rapid
and inexpensive test is performed first
has been examined by some investiga-
tors.26-28 With this scheme, further testing
occurs only if the initial test indicates
immaturity. If any of the test results indi-
396 cate lung maturity, the sequence is termi-
nated and no further testing is performed.
The high predictive value of a mature
profile (negative test result, absence of
RDS) is demonstrated by all of the tests
discussed, but immature results (positive
test result, immature lungs) have a lower
predictive value.
The laboratory, in conjunction with
clinicians, can develop testing strategies that
use the most rapid and cost-effective test
first for their institution. Some important
criteria to consider when selecting FLM
testing include cost, availability, reliability,
and reproducibility. Correlation with neona-
tal clinical outcome is essential to
effectively predict cutoff values for matu-
rity, particularly with lamellar body counts
owing to the variability in methodologies.
The gold standard should be a test
that predicts lung maturity with clinical
correlation showing the absence of RDS.
Prudent use of FLM testing can minimize
or prevent maternal morbidity and mortal-
ity in a pregnancy complicated by pre-
eclampsia or other life-threatening
conditions. Thus, an FLM test indicating
fetal lung maturity in a complicated preg-
nancy requires further evaluation if a deci-
sion for delivery is to be made. While no
test is 100% accurate, a combination of
clinical evaluation and laboratory studies
will optimize maternal and fetal outcome.
1. Hook GER, Gilmore LB, Tombropoulos EG, et al.
Fetal lung lamellar bodies in amniotic fluid. Am Rev
Respir Dis. 1978;117:541-550.
2. Brace RA, Wolf EJ. Characterization of normal
gestation changes in amniotic fluid volume. Am J
Obstet Gynecol. 1989;161:382-388.
3. Jobe AH. Fetal lung development, tests for maturation,
induction of maturation, and treatment. In: Creasy
RK, Resnik R, eds. Maternal Fetal Medicine.
Principles and Practice. 3rd ed. Philadelphia, PA:
Saunders; 1994:427.
4. Office of Technology Assessment. Neonatal Intensive
Care for Low Birth Weight Infants: Costs and
Effectiveness. Health Technology Case Study 38.
Washington, DC: Office of Technology Assessment,
US Congress; 1987. Publication OTA-HCS-38.
5. Hospitalization in the United States, 1997.
Heathcare Cost and Utilization Project (HCUP)
Fact Book No. 1. Rockville, MD: Agency for
Healthcare Research and Quality (AHRQ); 2000.
AHRQ publication 00-0031.
6. Gluck L, Kulovich MV, Borer RC Jr, et al. Diagnosis
of respiratory distress syndrome by amniocentesis. Am
J Obstet Gynecol. 1971;109:440-445.
7. Ashwood ER. Evaluating health and maturation of the
unborn: the role of the clinical laboratory. Clin Chem.
1992;38:1523-1529.
8. Herbert WNP, Chapman JF. Clinical and economic
considerations associated with testing for fetal lung
maturity. Am J Obstet Gynecol. 1986;155:820-823.
9. Gluck L, Kulovich MV. Lecithin/sphingomyelin ratios
in amniotic fluid in normal and abnormal pregnancy.
Am J Obstet Gynecol. 1973;115:547-552.
10. Whitfield CR, Sproule WB, Brudenell M. The
amniotic fluid L/S ratio in pregnancies complicated by
diabetes. J Obstet Gynecol Br Commonwealth.
1973;80:918-922.
11. Fadel HE, Saad SS, Nelson GH, et al. Effect of
maternal-fetal disorders on lung maturation, I:
diabetes mellitus. Am J Obstet Gynecol.
1986;155:544-553.
laboratorymedicine> july 2001> number 7> volume 32
12. Mimouni F, Miodovnik M, Whitsett JA, et al.
Respiratory distress syndrome in infants of diabetic
mothers in the 1980s: no direct adverse effect of
maternal diabetes with modern management. Am J
Obstet Gynecol. 1987;69:191-195.
13. Steinfield JD, Samuels P, Bulley MA, et al. The utility
of the TDx test in the assessment of fetal lung
maturity. Obstet Gynecol. 1992;79:460-464.
14. Towers CV, Garite TJ. Evaluation of the new
AmnioStat-FLM test for the detection of
phosphatidylglycerol in contaminated fluids. Am J
Obstet Gynecol. 1989;160:298-303.
15. Eisenbrey AB, Epstein E, Zak B, et al.
Phosphatidylglycerol in amniotic fluid: comparison of
an ultrasensitive immunologic assay with TLC and
enzymatic assay. Am J Clin Pathol. 1989;91:293-297.
16. Pastorek JG, Letellier RL, Gebbia K. Production of
phosphatidylglycerol-like substance by genital flora
bacteria. Am J Obstet Gynecol. 1988;159:199-202.
17. Lambers D, Bradley K, Leist P, et al. Ability of normal
vaginal flora to produce detectable phosphat-
idylglycerol in amniotic fluid in vitro. Obstet Gynecol.
1995;85:651-655.
18. Fetal Lung Maturity II Reagent [package insert].
Abbott Park, IL: Abbott Laboratories Diagnostic
Division; 1996. List No. 7A76 66-7290/R4.
19. Wong S, Schenkel O, Qutishat A. Strategic utilization
of fetal lung maturity tests. Scand J Clin Lab Invest.
1996;56:525-532.
20. Lafler D, Mendoza A, Poeltler D, et al. Coulter STKS
vs Abbott Cell Dyn 3500 for counting lamellar bodies
in amniotic fluid. Lab Med. 1998;29:298-301.
21. Ashwood ER, Oldroyd RG, Palmer SE. Measuring the
number of lamellar body particles in amniotic fluid.
Obstet Gynecol. 1990;75:289-292.
22. Pearlman ES, Baiocchi JM, Lease JA, et al. Utility of a
rapid lamellar body count in the assessment of fetal
lung maturity. Am J Clin Pathol. 1991;95:778-780.
23. Ashwood ER, Palmer SE, Taylor JS, et al. Lamellar
body counts for rapid fetal lung testing. Obstet
Gynecol. 1993;81:619-624.
24. Dubin SB. Assessment of fetal lung maturity. practice
parameter. Am J Clin Pathol. 1998;110:723-732.
25. Neerhof MG, Dohnal JC, Ashwood ER, et al.
Lamellar body counts: a consensus on protocol.
Obstet Gynecol. 2001;97:318-320.
26. Garite TJ, Freeman RK, Nageotte MP. Fetal maturity
cascade: a rapid and cost-effective method for fetal
lung maturity testing. Obstet Gynecol. 1986;67:619-
622.
27. Lewis PS, Lauria MR, Dzieczkowski J, et al. Amniotic
fluid lamellar body count: cost-effective screening for
fetal lung maturity. Obstet Gynecol. 1999;93:387-391.
28. Greenspoon JS, Rosen DJD, Roll K, et al. Evaluation
of lamellar body number density as the initial
assessment in a fetal lung maturity test cascade. J
Reprod Med. 1995;40:260-266.
InterNetConnect
American Academy of Family Physicians fetal
lung maturity page:
<www.aafp.org/afp/970800ap/briefs3.html>
ARUPs Guide to Laboratory Testing fetal
lung maturity page:
<www.aruplab.com/guides/clt/tests/
clt_a211.htm#1149122>