Tests for monitoring fetal maturity via amniocentesis are also available. Bilirubin levels in erythroblastosis are discussed in chapter 11. Amniotic creatinine assay, amniotic epithelial cell stain with Nile blue sulfate, fat droplet evaluation, osmolality, and the Clemens shake test, alone or in combination, have been tried with varying and not entirely satisfactory results. Most current tests measure one or more components of alveolar surfactant. Surfactant is a substance composed predominantly of phospholipids; it is found in lung alveoli, lowers the surface tension of the alveolar lining, stabilizes the alveoli in expiration, and helps prevent atelectasis. Surfactant deficiency causes neonatal respiratory distress syndrome (RDS), formerly called “hyaline membrane disease.” The major phospholipid components of surfactant are phosphatidylcholine (lecithin, about 80%; range, 73%-88%), phosphatidylglycerol (PG, about 3%; range, 1.8%-4.2%), and sphingomyelin (about 1.6%). The current most widely used tests are the lecithin/sphingomyelin (L/S) ratio, assay of phosphatidylglycerol (PG), the foam stability index (FSI), and TDx fluorescent polarization.

Lecithin/Sphingomyelin (L/S) ratio. The L/S ratio has been the most widely accepted fetal maturity procedure. Lecithin (phosphatidylcholine), a phospholipid, is the major component of alveolar surfactant. There is a 60% or greater chance of RDS in uncomplicated pregnancies when the fetus is less than 29 weeks old; about 8%-23% at 34 weeks; 0%-2% at 36 weeks; and less than 1% after 37 weeks. In amniotic fluid, the phospholipid known as sphingomyelin normally exceeds lecithin before the 26th week; thereafter, lecithin concentration is slightly predominant until approximately the 34th week, when the lecithin level swiftly rises and the sphingomyelin level slowly decreases so that lecithin levels in the 35th or 36th week become more than twice sphingomyelin levels. After that happens it was originally reported (not entirely correctly), that there was no longer any danger of neonatal RDS. The L/S ratio thus became a test for fetal lung and overall maturity. Certain precautions must be taken. Presence of blood or meconium in the amniotic fluid or contamination by maternal vaginal secretions may cause a false increase in lecithin and sphingomyelin levels, so that “mature” L/S ratios are decreased and “immature” L/S ratios are increased. The amniotic fluid specimen must be cooled immediately, centrifuged to eliminate epithelial cells and other debris, and kept frozen if not tested promptly to prevent destruction of lecithin by certain enzymes in the amniotic fluid.

Evaluations in unselected amniocentesis patients have revealed that about 55% of neonates with immature L/S ratios using the 2.0 ratio cutoff point do not develop RDS and about 5% (literature range, 0%-17%) of neonates with a mature L/S ratio (ratio >2.0) develop RDS. Some have attempted to eliminate the falsely mature cases by changing the cutoff point to a ratio of 2.5, but this correspondingly increases the number of falsely immature results. In clinically normal pregnancies, only about 3% of neonates with a mature L/S ratio, using proper technique, develop RDS. In complicated pregnancies, especially those with maternal type I insulin-dependent diabetes, hypertension, or premature rupture of the amniotic membrane, about 15% (literature range, 3%-28%) of neonates with mature L/S ratios are reported to develop RDS. In other words, RDS can develop at higher L/S ratios in a relatively small number of infants. The wide range in the literature reflects differences in opinion among investigators as to the effect of diabetes on neonatal L/S ratios. Also, the L/S ratio can produce falsely mature results if contaminated by blood or meconium. It takes experience and careful attention to technical details to obtain consistently accurate L/S results.

Phosphatidylglycerol (PG). A number of other tests have been developed in search of a procedure that is more accurate in predicting or excluding RDS and that is also technically easy to perform. PG is a relatively minor component (about 10%) of lung surfactant phospholipids. However, PG is almost entirely synthesized by mature lung alveolar cells and therefore is a good indicator of lung maturity. In normal pregnancies PG levels begin to increase after about 30 weeks’ gestation and continue to increase until birth. It normally becomes detectable about the 36th week. In conditions that produce severe fetal stress, such as maternal insulin-dependent diabetes, hypertension, and premature membrane rupture, PG levels may become detectable as early as 30 weeks’ gestation. Most of the limited studies to date indicate that the presence of PG in more than trace amounts strongly suggests that RDS will not develop, whether the pregnancy is normal or complicated. Overall incidence of RDS when PG is present seems to be about 2% (range 0%-10%). It is considered to be a more reliable indicator of fetal lung maturity than the L/S ratio in complicated pregnancy. It may be absent in some patients with clearly normal L/S ratios and occasionally may be present when the L/S ratio is less than 2.0. PG assay is not significantly affected by usual amounts of contamination by blood or meconium.

PG can be assayed in several ways, including gas chromatography, thin-layer chromatography (TLC), enzymatically, and immunologically. The TLC technique is roughly similar to that of the L/S ratio. Some report the visual presence or absence of PG, with or without some comment as to how much appears to be present (trace or definite). Some report a PG/sphingomyelin (PG/S) ratio. A PG/S ratio of 2.0 or more is considered mature. A commercially available enzymatic PG method (“PG-Numeric”) separates phospholipids from the other components of amniotic fluid (using column chromatography or other means), followed by enzymatic assay of glycerol in the phospholipid fraction. After several years there is still an insufficient number of published evaluations of this technique. Immunological methods are still restricted to a slide agglutination kit called Amniostat FLM-Ultra (improved second-generation test). Current small number of evaluations indicate that Amniostat FLM-Ultra detects about 85%-90% of patients who are positive for PG on TLC. The risk of RDS is about 1%-2% if the test is reactive (positive).

Foam stability index (FSI). The FSI is a surfactant function test based on the ability of surfactant to lower surface tension sufficiently to permit stabilized foaming when the amniotic fluid is shaken. This depends on the amount and functional capability of surfactant as challenged by certain amounts of the antifoaming agent ethanol. It is thought that the phospholipid dipalmitoyl lecithin is the most important stabilizing agent. The FSI is actually a modification of the Clemens shake test, which used a final amniotic fluid-ethanol mixture of 47.5% ethanol. The FSI consists of a series of seven tubes containing amniotic fluid with increasing percentages of ethanol in 1% increments from 44%-50%. The endpoint is the tube with the highest percentage of ethanol that maintains foam after shaking. An endpoint of the 47% tube predicts about a 4% chance of RDS and an endpoint in the 48% tube predicts less than 1% chance. Because even tiny inaccuracies or fluctuations of ethanol concentration can influence results considerably, and also the tendency of absolute ethanol to adsorb water, some problems were encountered in laboratories making their own reagents. To solve these problems a commercial version of the FSI called Lumidex was introduced featuring sealed tubes containing the 1% increments of ethanol to which aliquots of amniotic fluid are added through the rubber caps that seal the tubes. The FSI (or Lumidex) has been reported to be more reliable than the L/S ratio in predicting fetal lung maturity. At least two reports indicate that the FSI correctly demonstrates fetal lung maturity much more frequently than the L/S ratio in fetuses who are small for their gestational age. Drawbacks of the FSI method in general are interference (false positive) by blood, meconium, vaginal secretions, obstetrical creams, and mineral oil. A major drawback of the current Lumidex kit is a shelf-life of only 3 weeks without refrigeration. Although the shelf life is 3 months with refrigeration, it is necessary to stabilize the tubes at room temperature for at least 3 hours before the test is performed.

TDx-FLM fluorescent polarization. The TDx is a commercial instrument using fluorescent polarization to assay drug levels and other substances. It has been adapted to assay surfactant quantity indirectly by staining surfactant in amniotic fluid with a fluorescent dye and assaying surfactant (in mg/gm of albumin content) using the molecular viscosity of the fluid as an indicator of surfactant content. The assay in general produces results similar to the L/S ratio and a little better than the FSI. There is some difference in results depending on whether a single cutoff value is used, what that value is, and whether multiple cutoff values are applied depending on the situation. Test technical time is about 30 minutes. Specimens contaminated with meconium, blood, or urine (in vaginal pool material) interfere with the test.

Lamellar body number density. Surfactant is produced by alveolar type II pneumocytes in the form of a concentrically wrapped small structure about 3 microns in size that on cross-section looks like an onion and is called a lamellar body. It is possible to count the lamellar bodies using some hematology platelet counting machines, with the result calculated in units of particle density per microliter of amniotic fluid. In the very few evaluations published to date, results were comparable to those of the L/S ratio and FSI.

Amniocentesis laboratory problems. Occasionally, amniotic puncture may enter the maternal bladder instead of the amniotic sac. Some advocate determining glucose and protein levels, which are high in amniotic fluid and low in normal urine. To prevent confusion in diabetics with glucosuria, it has been suggested that urea and potassium levels be measured instead; these are relatively high in urine and low in amniotic fluid. Another potential danger area is the use of spectrophotometric measurement of amniotic fluid pigment as an estimate of amniotic fluid bilirubin content. Before 25 weeks’ gestation, normal pigment levels may be greater than those usually associated with abnormality.