lab test for fetus lung maturity

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laboratorymedicine>july 2001> number7> volume 32

Advances in fetal lung maturity

(FLM) testing have assisted the clinician

in determining the course of complicated

as well as uncomplicated pregnancies.

Fetal lung maturation is a complex process

involving a balance of physiologic, cellu-

lar, and biologic functions. During

intrauterine development, the fetus relies

on the exchange of gases through the pla-

centa. At birth, once the umbilical cord isclamped, 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

collapse of the pulmonary alveoli on end

expiration. Surfactant is produced by the

type II pneumocytes of the lung and ispackaged in concentrically layered struc-

tures called lamellar bodies. Consisting al-

most entirely of proteins and phospholipids,

they represent the storage form of

pulmonary surfactant.1

Lamellar bodies first appear in the am-

niotic fluid between 20 and 24 weeks of

gestation and are released into the alveolar

space. The dynamics of fluid turnover and

intrauterine fetal breathing movements

allow the components of surfactant to bereadily measured in the amniotic fluid.

Throughout the pregnancy, there is a grad-

ual increase in the amount of amniotic fluid.

It rises steadily with a peak volume of ap-

proximately 800 mL at 33 weeks and grad-

ually decreasing until delivery.2

Human surfactant is a complex sub-

stance composed of phospholipids, carbo-

hydrates, and a variety of proteins.

Surfactant as recovered from lungs of all

mammalian species contains 70% to 80%phospholipids, about 10% protein and about

10% neutral lipids, primarily cholesterol

[F1].3 With advancing gestation, the most

abundant phospholipid in surfactant is

lecithin (phosphatidylcholine), followed by

phosphatidylglycerol (PG), which appears

around the 35th week. Other phospholipids

include sphingomyelin, phosphatidylinosi-

tol, and phosphatidylethanolamine.

your la b foc us

CE Update [chemistry]

Laboratory Testing To Assess Fetal LungMaturity

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

Collection methods and test

(biochemical and biophysical) methods

Test utilization and optimization of

maternal/fetal outcome

[F1] C omposition of surfactant recovered by alveolar wash. Adapted from: Jobe.3

PhosphatidylcholineProtein

NeutralLipids

Phosphatidylinositol

Phosphatidylethanolamine

Phosphatidylglycerol


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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 theleading 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 TestingIndications 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 availablecan be divided into 2 groups, biochemical

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

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 theonly 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 specificcutoff value for maturity.

Lecithin/Sphingomyelin RatioThe 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-

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 thegold 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 extractionusing 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-

laboratoryme dicine>july 2001> number7> volume 32

94

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Tests To Assess Fetal Lung Maturity

Method Cost Turnaround Time Availability Labor Requirement

Lamellar Low Less than 10 min U niversal, on-site, S imple, no commercial k it required,

body count on demand no preanalytic treatment

Surfactant/ M oderate Approximately 40 min O n-site, on demand Simple, minor

albumin ratio preanalytic filtration step,

fully automated

P hosphatidylglycerol M o derate A pproximately 15 min On-site, on dem and S imple, visual slide

by slide method agglutination

Lecithin/sphingomyelin H igh 2-4 hr Usually not on site Dedicated personnel,

ratio high degree of

labor-intensiveness

T1


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laboratorymedicine>july 2001> number7> volume 32

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 areinterpreted 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 surfactantand 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 RatioFluorescence 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,

approximately 1.0 mL, and can be

performed in 40 minutes with minimal

specimen preparation. The cost to perform

the test is less than half the cost of perform-

ing an L/S ratio or TLC. It can be

performed using instruments already avail-

able in many laboratories that perform ther-

apeutic drug monitoring.Results are reported as milligrams

of surfactant per gram of albumin

(mg/g). Because the test is automated,

variation between laboratories is low.

Vaginal pool and amniocentesis speci-

mens can be used. However, gross blood

or meconium has an adverse effect on

the results, as do vaginal pool collect-

ions contaminated with urine.18

It has been shown that diabetic pa-

tients have the same S/A ratio results as

nondiabetic patients, making this test a

good predictor of FLM.19

Lamellar Body Counts

Lamellar body counts and lamellar

body number density have both been used

to assess the number of lamellar bodies in

the amniotic fluid. Characteristically, they

are small structures, approximately the size

of platelets, and they scatter light, often giv-

ing the fluid an opalescent appearance. Be-cause of their size, lamellar bodies can be

counted rapidly by using the platelet chan-

nel of most whole blood analyzers.

The methods for performing lamellar

body counts have varied across

institutions. Studies have shown this count

to be useful in the prediction of FLM; sev-

eral have shown it to be as reliable as other

FLM tests.20-23 The specimen requirement

is small, often less than 1.0 mL. With the

widespread use of these analyzers in labo-

ratories, lamellar body counts can be per-

formed in less than 10 minutes in most

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Sensitivity and Specificity of Fetal Lung Maturity Tests

Test Method Sensitivity Specificity

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

T2

[F2] Fetal lung maturity test algorithm. Uncontaminated means no gross blood, meconium,bilirubin, or urine. *Irvine Scientific's M AmnioS tat phosphatidylglycerol determination can beperformed at either of these 2 points as a supplementary test to further predict fetal lung maturity.

Uncontaminatedamniocentesis specimen

Uncontaminated vaginalpool specimen

Perform lamellarbody count

STOPSTOP

Assess surfactant/

albumin ratio

PerformLS ratio

Mature result

Mature result

Immature result*

Immature result

Result*


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laboratoryme dicine>july 2001> number7> volume 32

96

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 UtilizationThe 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 includediabetic 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 indicatesimmaturity. If any of the test results indi-

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 RevRespir Dis. 1978;117:541-550.

2. Brace RA, Wolf EJ. Characterization of normalgestation changes in amniotic fluid volume.Am JObstet 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 IntensiveCare for Low Birth Weight Infants: Costs andEffectiveness. 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 forHealthcare Research and Quality (AHRQ); 2000.AHRQ publication 00-0031.

6. Gluck L, Kulovich MV, Borer RC Jr, et al. Diagnosisof respiratory distress syndrome by amniocentesis.AmJ Obstet Gynecol. 1971;109:440-445.

7. Ashwood ER. Evaluating health and maturation of theunborn: the role of the clinical laboratory. Clin Chem.1992;38:1523-1529.

8. Herbert WNP, Chapman JF. Clinical and economicconsiderations associated with testing for fetal lungmaturity.Am J Obstet Gynecol. 1986;155:820-823.

9. Gluck L, Kulovich MV. Lecithin/sphingomyelin ratiosin amniotic fluid in normal and abnormal pregnancy.Am J Obstet Gynecol. 1973;115:547-552.

10. Whitfield CR, Sproule WB, Brudenell M. Theamniotic fluid L/S ratio in pregnancies complicated bydiabetes.J Obstet Gynecol Br Commonwealth.1973;80:918-922.

11. Fadel HE, Saad SS, Nelson GH, et al. Effect ofmaternal-fetal disorders on lung maturation, I:diabetes mellitus.Am J Obstet Gynecol.1986;155:544-553.

12. Mimouni F, Miodovnik M, Whitsett JA, et al.Respiratory distress syndrome in infants of diabeticmothers in the 1980s: no direct adverse effect ofmaternal diabetes with modern management.Am JObstet Gynecol. 1987;69:191-195.

13. Steinfield JD, Samuels P, Bulley MA, et al. The utilityof the TDx test in the assessment of fetal lungmaturity. Obstet Gynecol. 1992;79:460-464.

14. Towers CV, Garite TJ. Evaluation of the new

AmnioStat-FLM test for the detection ofphosphatidylglycerol in contaminated fluids.Am JObstet Gynecol. 1989;160:298-303.

15. Eisenbrey AB, Epstein E, Zak B, et al.Phosphatidylglycerol in amniotic fluid: comparison ofan ultrasensitive immunologic assay with TLC andenzymatic assay.Am J Clin Pathol. 1989;91:293-297.

16. Pastorek JG, Letellier RL, Gebbia K. Production ofphosphatidylglycerol-like substance by genital florabacteria.Am J Obstet Gynecol. 1988;159:199-202.

17. Lambers D, Bradley K, Leist P, et al. Ability of normalvaginal 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 DiagnosticDivision; 1996. List No. 7A76 66-7290/R4.

19. Wong S, Schenkel O, Qutishat A. Strategic utilizationof fetal lung maturity tests. Scand J Clin Lab Invest.1996;56:525-532.

20. Lafler D, Mendoza A, Poeltler D, et al. Coulter STKSvs Abbott Cell Dyn 3500 for counting lamellar bodiesin amniotic fluid.Lab Med. 1998;29:298-301.

21. Ashwood ER, Oldroyd RG, Palmer SE. Measuring thenumber of lamellar body particles in amniotic fluid.Obstet Gynecol. 1990;75:289-292.

22. Pearlman ES, Baiocchi JM, Lease JA, et al. Utility of arapid lamellar body count in the assessment of fetallung maturity.Am J Clin Pathol. 1991;95:778-780.

23. Ashwood ER, Palmer SE, Taylor JS, et al. Lamellarbody counts for rapid fetal lung testing. ObstetGynecol. 1993;81:619-624.

24. Dubin SB. Assessment of fetal lung maturity. practiceparameter.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 maturitycascade: a rapid and cost-effective method for fetallung maturity testing. Obstet Gynecol. 1986;67:619-622.

27. Lewis PS, Lauria MR, Dzieczkowski J, et al. Amnioticfluid lamellar body count: cost-effective screening forfetal lung maturity. Obstet Gynecol. 1999;93:387-391.

28. Greenspoon JS, Rosen DJD, Roll K, et al. Evaluationof lamellar body number density as the initialassessment in a fetal lung maturity test cascade.JReprod Med. 1995;40:260-266.

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