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

Phosphatidylethanolamine Phosphatidylinositol Phosphatidylglycerol

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, cellular, and biologic functions. During intrauterine development, the fetus relies on the exchange of gases through the placenta. 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 mature to handle this transition. The production of pulmonary surfactant is the critical step in this progression to maturity. Surfactant is responsible for lowering the surface tension in the lungs following 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 is packaged in concentrically layered structures called lamellar bodies. Consisting almost entirely of proteins and phospholipids, they represent the storage form of pulmonary surfactant.1 Lamellar bodies first appear in the amniotic fluid between 20 and 24 weeks of gestation and are released into the alveolar space. The dynamics of fluid turnover and

Neutral Lipids

Protein

Phosphatidylcholine

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

intrauterine fetal breathing movements allow the components of surfactant to be readily measured in the amniotic fluid. Throughout the pregnancy, there is a gradual increase in the amount of amniotic fluid. It rises steadily with a peak volume of approximately 800 mL at 33 weeks and gradually decreasing until delivery.2 Human surfactant is a complex substance composed of phospholipids, carbohydrates, 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, phosphatidylinositol, and phosphatidylethanolamine.

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Tests To Assess Fetal Lung Maturity
Method Lamellar body count Surfactant/ albumin ratio Phosphatidylglycerol by slide method Lecithin/sphingomyelin ratio Cost Low Moderate Turnaround Time Less than 10 min Approximately 40 min Availability Universal, on-site, on demand On-site, on demand 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

Moderate High

Approximately 15 min 2-4 hr

On-site, on demand Usually not on site

Premature infants may have multiple complications, but surfactant deficiency can be the most acute. Surfactant deficiency in the neonate is the primary cause of respiratory distress syndrome (RDS) and is the leading cause of morbidity and death in preterm infants.4 Acute fetal distress typically occurs, primarily resulting in RDS or hyaline membrane disease. The clinical signs become apparent with neonatal grunting, 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 hospitals according to the national statistics collected 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 delivery or to provide maternal drug therapy to enhance lung maturity. The fetal lung maturity tests available can be divided into 2 groups, biochemical and biophysical. Biochemical tests measure components of the surfactant and include the lecithin/sphingomyelin (L/S) ratio and phosphatidylglycerol measurements. Biophysical tests measure the physical properties of the phospholipid surfactants and include the surfactant/albumin (S/A) ratio and lamellar body

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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 assessment are considered. When testing indicates lung immaturity, the delivery can be postponed 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 amniotic 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, turnaround time, and labor requirements vary with each assay and are outlined in [T1]. Comparison of laboratory results with clinical evaluation of newborns is essential in determining a given laboratorys specific cutoff value for maturity. Lecithin/Sphingomyelin Ratio The first laboratory procedure for assessment 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 standard 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 Ashwood,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 diabetic pregnancies was described as delayed9,10; further studies suggested that maturity is not delayed in diabetic pregnancies 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 intensive, time consuming, and technique dependent, taking up to several hours to perform. It involves centrifugation of the amniotic fluid and phospholipid extraction using organic solvents followed by thinlayer chromatography (TLC) with quantitation by planometry or densitometry. Given the current laboratory environment, it is difficult and not cost-effective to accommodate highly specialized personnel to perform TLC around the clock. Vaginal pool and amniocentesis specimens can be used if they are not contami-

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nated with gross blood or meconium, which can cause interferences. Even with commercially available kits, variations can occur between laboratories 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 absence of RDS. With the inherent complexities 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 presence 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 adherence to vendor guidelines for interpretation 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 membranes have been ruptured for a prolonged period, bacterial contamination can occur, and certain bacteria have been shown to produce PG, thus giving false-positive results.16,17 PG determination 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 automated 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 standard curve of known S/A calibrators. The test requires a minimum specimen volume,
Sensitivity and Specificity of Fetal Lung Maturity Tests
Test Method Surfactant/ albumin ratio Phosphatidylglycerol by slide method Lecithin/sphingomyelin ratio
Adapted from Wong.19

Sensitivity 0.97 0.93 0.86

Specificity 0.72 0.55 0.78

T2

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 performing an L/S ratio or TLC. It can be performed using instruments already available in many laboratories that perform therapeutic 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 specimens can be used. However, gross blood or meconium has an adverse effect on the results, as do vaginal pool collections contaminated with urine.18 It has been shown that diabetic patients 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 giving the fluid an opalescent appearance. Because of their size, lamellar bodies can be counted rapidly by using the platelet channel 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; several 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 laboratories, lamellar body counts can be performed in less than 10 minutes in most

Uncontaminated amniocentesis specimen

Uncontaminated vaginal pool specimen

Perform lamellar body count

Mature result Immature result*

Assess surfactant/ albumin ratio

Mature result Immature result

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STOP STOP
Result*

Perform 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.

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cases. The test is quick and inexpensive with no commercial kit required. Amniocentesis collections appear to be the most studied specimens in the literature as they are generally free of contaminating 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 analyzers 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 satisfactory specimen cannot be overemphasized. Vaginal pool collections tend to be more contaminated with mucus or blood, making interpretations difficult with various 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 determination 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 associates25 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 investigators.26-28 With this scheme, further testing occurs only if the initial test indicates immaturity. If any of the test results indicate lung maturity, the sequence is terminated 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 neonatal clinical outcome is essential to effectively predict cutoff values for maturity, 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 mortality in a pregnancy complicated by preeclampsia or other life-threatening conditions. Thus, an FLM test indicating fetal lung maturity in a complicated pregnancy requires further evaluation if a decision 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. 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 phosphatidylglycerol 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;67:619622. 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>

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