Improved fluorescence polarization assay for use in evaluating fetal lung maturity. III. Retrospective clinical evaluation and comparison with the lecithin/sphingomyelin ratio

We clinically evaluated, retrospectively, our improved fluorescence polarization assay for fetal lung maturity. The procedure requires 0.5 mL of amniotic fluid and a standard clinical laboratory fluorescence polarimeter (TDx Analyzer, Abbott Laboratories). We measured the L/S ratios for 93 freshly collected amniotic fluids, uncontaminated with blood or meconium, collected within three days of delivery. The fluids were stored frozen for eight to 32 months, then thawed and assayed for net fluorescence polarization. Fourteen of the infants developed respiratory distress syndrome; five, transient tachypnea of the newborn; and 74, no respiratory distress. The polarization assay and lecithin/sphingomyelin ratio had equivalent receiver operating characteristic curves, indicating no difference in their clinical performance. Although a prospective study with fresh amniotic fluid specimens will be necessary to establish a definitive reference range, the present study shows that this assay can be used to rapidly predict fetal lung maturity.     

Fetal lung maturity assessed by fluorescence polarization: evaluation of predictive value, correction for endogenous fluorescence, and comparison with L/S ratio

The steady-state polarization (or anisotropy) of the fluorescent dye 1,6-diphenyl-1,3-5-hexatriene in amniotic fluid samples and the lecithin/sphingomyelin ratio of the samples were correlated with development of respiratory distress syndrome in newborns. We found that clinical samples have a variable endogenous fluorescence that reduces the observed polarization (or anisotropy). This background is a major interference in the assessment of fetal lung maturity by the polarization method. Correction for this interference, by also measuring the blank fluorescence and anisotropy of the sample, provides a clinical tool with a lower coefficient of variation than that of the more time-consuming lecithin/sphingomyelin ratio. The clinical correlation for 17 cases of respiratory distress syndrome in a high-risk population (60 births; twins counted as a single birth) indicates that the two methods are equivalent for predicting immature fetal lung status.     

Degree of unsaturation of the fatty acid chains of phospholipids influences the fluorescence polarization: implications for evaluating fetal lung maturity

To study the effect of fatty acid chain saturation on the fluorescence polarization assay as a measure of fetal lung maturity, we used purified phospholipids isolated from human amniotic fluid and various commercial phospholipids. We found that the fluorescence polarization value decreased as the concentration of unsaturated fatty acids increased. In contrast, the lecithin/sphingomyelin ratio increases with increasing amounts of saturated lecithin, produced as the fetal lung matures. Since only saturated lecithins are surface active, the two indices of fetal respiratory status must reflect different properties of lung surfactant.     

Improved fluorescence polarization assay for use in evaluating fetal lung maturity. II. Analytical evaluation and comparison with the lecithin/sphingomyelin ratio

We assessed the analytical performance of an improved fluorescence polarization assay for use in evaluating fetal lung maturity and compared results with the lecithin/sphingomyelin ratio. During a three-month period 150 patients' samples were assayed by clinical laboratory personnel with both techniques. Values for the lecithin/sphingomyelin ratio correlate closely with net fluorescence polarization values (r = -0.85), less closely with net fluorescence intensity (r = 0.65). Background fluorescence intensity and polarization varied widely, indicating a need to correct measurements for endogenous fluorescence. Net fluorescence polarization values have a CV of 0.32% within-run, 1.07% between-day. A comparison of two amniotic fluid centrifugation procedures showed no significant difference in such values. For both methods, however, such values are slightly but significantly higher than those obtained for amniotic fluids without prior centrifugation. Short-term storage (less than 30 days) of uncentrifuged amniotic fluid samples at -20 degrees C does not significantly affect results.     

Measurement of fetal surfactant production by fluorescence polarization of amniotic fluid in complicated pregnancies

The results are presented of fluorescence polarization as a method for measurement of surfactant production in 159 specimens of amniotic fluid collected from pregnant women with diabetes, hypertension, Rh immunization, premature rupture of membranes (for more than 48 h prior to delivery) and intrauterine growth retardation (IUGR). The predictability of the development of respiratory distress syndrome has been assessed by this assay. Its specificity, sensitivity and overall accuracy were similar to the lecithin/sphingomyelin (L/S) ratio. The influence of the conditions detailed on fetal lung maturation was determined, lung development being enhanced until near term by Rh immunization, rupture of membranes and hypertension with IUGR.     

Combined enzymatic assay of phosphatidylglycerol and phosphatidylcholine in amniotic fluid

We present here a combined, quantitative enzymatic procedure for determining amniotic fluid phosphatidylglycerol and phosphatidylcholine and relate these findings to the assessment of fetal lung maturity. Under the assay conditions described phospholipase C specifically hydrolyses phosphatidylglycerol (PG) and phosphatidylcholine (PC) but not sphingomyelin, precluding the need for removal of sphingomyelin prior to analysis. Solvent extraction of the phospholipids from the amniotic fluid is, however, employed to avoid spurious elevation of PG and PC results by endogenous glycerol and choline. Of 45 amniocentesis fluids examined, 28 yielded detectable PG concentrations (greater than 0.5 mumol/l) and all but three of these exhibited PC concentrations in excess of 10 mumol/l. One case of respiratory distress occurred in an infant of 29 wk gestation with severe intrauterine growth retardation. Of the remaining 17 fluids in which PG was undetected enzymatically (less than or equal to 0.5 mumol/l), 14 also contained PC concentrations less than or equal to 10 mumol/l and all six cases of true respiratory distress syndrome came from within this sub-group. Strong correlations between the PC concentration and the lecithin:sphingomyelin ratio, r = 0.85 (p less than 0.001) and the PC and PG concentrations, r = 0.96 (p less than 0.001) were also found.     

羊水样本卵磷脂和鞘磷脂检测超高效液相色谱串联质谱方法的建立及应用

目的建立检测羊水中卵磷脂和鞘磷脂(L/S)的超高效液相色谱串联质谱(UPLC-MS/MS)方法,以便准确、高效地预测胎儿肺成熟度。方法收集孕32~39周孕妇分娩时的羊水样本23份。依据新生儿Apgar评分标准,有3例胎儿胎肺未成熟、20例胎儿胎肺成熟。另收集孕18周孕妇羊水样本7份作为基线对照,建立检测羊水中卵磷脂和鞘磷脂水平的UPLC-MS/MS方法,计算卵磷脂/鞘磷脂(L/S)比值,同时采用板层小体计数(LBC)法检测板层小体(LB),评价2种方法在预测胎肺成熟度中的价值。结果建立的检测羊水中卵磷脂和鞘磷脂的UPLC-MS/MS方法精密度良好,离子峰强度和保留时间均在可检测范围内,主成分分析(PCA)显示6个质控样本聚类良好。以L/S比值=10作为判断胎肺成熟度与不成熟的临界值,UPLC-MS/MS的敏感性和特异性均为100%。以LB=50×10^9/L作为判断胎肺成熟度与不成熟的临界值,LBC法的敏感性和特异性分别为80%和95%。结论建立了检测羊水中卵磷脂和鞘磷脂水平的UPLC-MS/MS方法,其结果可靠,可以准确、高效地预测胎肺成熟度。     

Phosphatidylglycerol in 261 samples of amniotic fluid from normal and diabetic pregnancies, as measured by one-dimensional thin-layer chromatography.

We describe a one-dimensional thin-layer chromatographic method for separating lecithin, sphingomyelin, phosphatidylglycerol, and other phospholipids. The occurrence of phosphatidylglycerol in relation to the lecithin/sphingomyelin ratio is reported for 261 amniotic fluid samples. This compound does not consistently appear until the ratio exceeds about 3.1, and occurs less often in samples from diabetic patients than in those from normal patients. The respiratory distress syndrome did not occur when phosphatidylglycerol was present in amniotic fluid although the reverse was not necessarily true. Thus the presence of phosphatidylglycerol offers additional assurance of pulmonary maturity.     

Modified lamellar body phospholipid assay compared with L/S ratio and phosphatidylglycerol assay for assessment of fetal pulmonary status

We used a modified lamellar body phospholipid (LB-PL) assay to evaluate the effect of sample processing and to evaluate the clinical efficacy of LB-PL determinations. Within-run and between-run CVs for the modified LB-PL assay respectively ranged from 2.9 to 6.7% and from 3.4 to 14.3%. Freezing and storage at -20 degrees C did not affect results for LB-PL (n = 12). Statistically significant amounts of lamellar bodies were lost on centrifugation, ranging from 20% at 150 X g to 54% at 1000 X g for 5 min. The LB-PL content, lecithin/sphingomyelin ratio, and phosphatidylglycerol content of 194 samples of amniotic fluid obtained within three days of delivery were compared for the assessment of fetal pulmonary maturity. Twenty-three neonates developed respiratory problems at or shortly after birth. With uncontaminated samples (n = 160), the LB-PL assay exhibited better specificity than the lecithin/sphingomyelin ratio (p less than 0.01, Chi square) or the phosphatidylglycerol assay (p less than 0.001, Chi square) for samples obtained after 36 weeks of gestation. Assay sensitivities did not differ significantly. Differences among assay predictive values were not significant.     

Multicenter evaluation of TDx test for assessing fetal lung maturity

"TDx Fetal Lung Maturity," an automated assay that measures the relative concentrations of surfactant and albumin in amniotic fluid, was compared with the lecithin/sphingomyelin (L/S) ratio and phosphatidylglycerol determination at five clinical sites. A total of 695 amniotic fluid samples were analyzed, of which 312 were followed by delivery of the infant within three days of sample collection. Of these 312, 24 developed respiratory distress syndrome and seven developed transient tachypnea. With the cutoff for maturity set at a surfactant/albumin value of 50 mg/g, the assay showed a sensitivity of 0.96 and a specificity of 0.88 for all samples, compared with a sensitivity of 0.96 and a specificity of 0.83 for the L/S ratio. The combination of rapid assay (30 min), accurate results, and uniformity among testing centers makes the TDx assay a very promising method.     

Studies on serum apolipoproteins and lipids in amniotic fluid and neonatal urine

Amniotic fluid and neonatal urine were examined for the presence of lipids and serum apolipoproteins. Human apolipoproteins A-I, A-II, and ApoD found principally in serum high density lipoproteins were identified in both neonatal urine and amniotic fluid. A lecithin/sphingomyelin ratio of greater than 5 associated with fetal lung maturity was accompanied by the disappearance of A-II from amniotic fluid. Dissimilarities of total fatty acid composition of amniotic fluid when compared to cord serum or neonatal urine indicate other tissue sources for fatty acids found in amniotic fluid. In addition, the presence of serum apolipoproteins and lipids in both amniotic fluid and neonatal urine suggests that a least a portion of these constituents could be derived from fetal urine.     

Is the L/S ratio really a lecithin/sphingomyelin ratio?

Fetal lung maturity is commonly assessed by determining the ratio of lecithin/sphingomyelin in centrifuged amniotic fluid. In a variety of chromatographic systems currently used for the routine determination of the lecithin/sphingomyelin ratio, including the systems recommended in the original procedure, at least one and frequently two additional phospholipids, normally present in amniotic fluid, tend to chromatograph between or overlapping with lecithin and/or sphingomyelin. These phospholipids have been tentatively identified as phosphatidylserine and phosphatidylinositol. The extra phospholipids contribute significantly to the routine lecithin/sphingomyelin ratio with considerable variation between individual cases. Treatment of dried lipid extracts with cold acetone before chromatography, as suggested in the original lecithin/sphingomyelin ratio procedure, does not remove the interfering phospholipids.     

Improved thin-layer chromatographic determination of phospholipids in gastric aspirate from newborns, for assessment of lung maturity

This one-dimensional thin-layer chromatographic method is used for assay of phospholipids in the gastric aspirate of newborns. The solvent mixture (chloroform/hexane/methanol/glacial acetic acid/water, 12/7/4/3/0.3 by vol) completely resolves lecithin, sphingomyelin, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, and phosphatidylglycerol. The method is simple, precise, inexpensive, and rapid (chromatographic development takes less than 25 min) and gives high chromatographic resolution. We used this method to determine the lecithin/sphingomyelin densitometric ratio (L/S ratio) and the phosphatidylglycerol percentage in 200 samples of gastric aspirate and found an L/S ratio of 2.5 to be a satisfactory cutoff value for distinguishing fetal lung maturity and immaturity. We confirmed that the presence of phosphatidylglycerol excluded the possibility of respiratory distress.     

Fetal lung maturity tests on the 10,000 X g pellet

Centrifugation has a profound effect on tests of fetal lung maturity performed on amniotic fluid. We have investigated the effect of a 700 X g centrifugal force for 10 min and a 10,000 X g force for 20 min on a battery of tests. While 91% of the OD650 was removed by the 10,000 X g centrifugal force, the supernatant fraction retained 34% and 38% of the L/S ratio and enzymatic lecithin respectively, when compared to the sample before centrifugation. Phosphatidylglycerol, when present in an amniotic fluid, was always absent from the 10,000 X g supernatant but present in the pellet formed by this centrifugal force. The pellet after 10,000 X g was unsuitable for OD650 and L/S ratio determinations but contained 63% of the enzymatic lecithin. When the pellet tests were subjected to a clinical trial, respiratory immaturity did not occur when phosphatidylglycerol was present or when the 10,000 X g pellet may be a useful means of detecting amniotic fluid surfactant and thus determining fetal lung maturity.     

Prediction of fetal lung maturity by use of the Lumadex-FSI test

We analyzed amniotic fluid from 91 pregnancies (estimated gestational age range 31 to 41 weeks), using the Lumadex-FSI Fetal Lung Maturity test (Beckman Instruments), and also determining phosphatidylglycerol content, the lecithin/sphingomyelin ratio, and foam stability index by the "shake test," and compared results with newborn outcome. Five of 64 babies born within 72 h of testing developed hyaline membrane disease. Except for the lecithin/sphingomyelin ratio, the predictive value of a negative test was 100% but that of a positive test was less than 50%. Use of all four tests did not offer diagnostic advantage over the use of Lumadex-FSI alone. For the laboratory that infrequently assesses fetal lung maturity, we believe the sealed cassette format of the Lumadex-FSI will permit better quality assurance than the shake test. The Lumadex-FSI test is intended to provide a graded estimate of positive risk for hyaline membrane disease, but more data from different centers and patient populations are needed to establish reliable predictive values.     

Automated enzymatic measurement of lecithin, sphingomyelin, and phosphatidylglycerol in amniotic fluid

We describe methods for automated enzymatic measurement of lecithin, sphingomyelin, and phosphatidylglycerol in amniotic fluid. Phospholipase C (EC 3.1.4.3) and sphingomyelin phosphodiesterase (EC 3.1.4.12) are reacted with lecithin and sphingomyelin, respectively, to liberate phosphocholine. Phosphocholine is then reacted with alkaline phosphatase, choline oxidase, peroxidase, and 4-aminoantipyrine to form a colored complex, for which the absorbance at 500 nm is measured with a centrifugal analyzer. Phosphatidylglycerol is hydrolyzed by phospholipase D (EC 3.1.4.4) to form glycerol, which is subsequently reacted with ATP and NAD+ in the presence of glycerol kinase and glycerol-3-phosphate dehydrogenase to yield NADH. The absorbance of the NADH formed is measured at 340 nm. These methods provide a simple, rapid, and accurate alternative to thin-layer chromatography for determination of phospholipids in amniotic fluid for assessment of fetal lung maturity.     

Sonographically detected free-floating particles in amniotic fluid predict a mature lecithin-sphingomyelin ratio

Amniocentesis for the determination of fetal lung maturity is associated with some morbidity, and so the prediction of a mature fetus by noninvasive means could be useful in managing certain high-risk patients. In the first portion of this study, 467 amniotic fluid specimens were classified into one of five groups based on the turbidity and particulate matter present. The mean lecithin: sphingomyelin (L/S) ratios and the proportion of mature L/S values increased with increasing turbidity of the amniotic fluid. In the second portion of this study, the presence of amniotic fluid free-floating particles (FFPs) detected by real-time ultrasound was correlated with fetal lung maturity. When FFPs were present, the L/S was uniformly mature, but when FFPs were absent, the L/S was mature in 74% of patients (P less than 0.01). Among the patients in whom the L/S ratios were mature, 39/110 were associated with positive FFPs (sensitivity = 35%). This preliminary study suggests that the presence of FFPs on real-time ultrasound could be used to confirm fetal lung maturity.     

Amniotic fluid alkaline phosphatase, gamma-glutamyltransferase, and 5'-nucleotidase activity from 13 to 40 weeks' gestation, and alkaline phosphatase as an index of fetal lung maturity

Reference ranges for amniotic fluid alkaline phosphatase, gamma-glutamyltransferase, and 5-nucleotidase are described from 13 to 40 weeks' gestation. Gamma-glutamyltransferase and 5-nucleotidase activities peak early in the second trimester and then decrease to low values. Alkaline phosphatase shows a similar pattern of activity from 13 to 29 weeks' gestation, but thereafter activity increases to term; this late increase is mainly related to the heat-labile particulate form of alkaline phosphatase. Total and heat-labile alkaline phosphatase alone or expressed as a ratio with gamma-glutamyltransferase can be used with or as an alternative to lecithin/sphingomyelin ratios in the investigation of fetal lung maturity. A total alkaline phosphatase activity of 0.36 mukat/L and an alkaline phosphatase/gamma-glutamyltransferase ratio greater than 2 indicate pulmonary maturity.     

Interference by endogenous glycerol in an enzymatic assay of phosphatidylglycerol in amniotic fluid

We investigated the possibility of interference by endogenous glycerol with the enzymatic measurement of phosphatidylglycerol in amniotic fluid. Phosphatidylglycerol is an important indicator of fetal lung maturity. The concentrations of glycerol and phosphatidylglycerol in amniotic fluid were measured by using a coupled enzymatic assay with and without phospholipase D (EC 3.1.4.4). The precision of the assay was acceptable (within-run CV = 1.2%, between-run CV = 4.8%). Endogenous glycerol content was demonstrated to be approximately 10-20 times that of phosphatidylglycerol. This high proportion of endogenous glycerol in amniotic fluid would preclude the accurate enzymatic determination of amniotic fluid phosphatidylglycerol unless the glycerol is first removed. Nor can the actual phosphatidylglycerol concentration be determined by subtracting the endogenous glycerol concentration from the total glycerol, which includes that glycerol derived from phosphatidylglycerol. With a usual range of 9 +/- 7 mumol/L, the error for a given phosphatidylglycerol measurement of +/- 6.6 mumol/L (+/- 2 SD) clearly is too high for this assay to be clinically useful. There was no correlation between concentration of endogenous glycerol or apparent phosphatidylglycerol in amniotic fluid and the lecithin/sphingomyelin ratio of the sample.     

Improved fluorescence polarization assay for use in evaluating fetal lung maturity. I. Development of the assay procedure

We describe a fluorescence polarization assay for use in predicting fetal lung maturity, which is suitable for the TDx Analyzer (an automated fluorescence polarimeter). The assay requires 0.5 mL of amniotic fluid and approximately 30 min, and involves the fluorophore 1-palmitoyl-2(6-[(7-nitro-2, 1, 3-benzoxadiazol-4-yl)amino]caproyl) phosphatidylcholine. Reproducible polarization measurements depend on proper regulation of incubation time and temperature, but variations in the concentrations of fluorescent probe and amniotic fluid have little effect on measured polarization and therefore little effect on assay precision. Working solutions of the fluorescent probe are stable for at least nine months when stored at -20 degrees C and pH 5. Interferences include erythrocytes, serum, bilirubin, meconium, and lidocaine.