Prospective clinical evaluation of an improved fluorescence polarization assay for predicting fetal lung maturity

We performed a prospective clinical evaluation of our newly developed fluorescence polarization procedure to predict fetal lung maturity (Clin Chem 1986;32:248-54). Net fluorescence polarization was measured at 34 degrees C after a 6.5-min incubation of amniotic fluid with fluorophore. For the 26 cases of neonatal respiratory distress syndrome encountered in 196 deliveries, the net polarization exceeded 0.287 for 22 (85%) of these, and exceeded 0.260 for all 26. The specificity of the polarization assay equaled or exceeded the specificity of the lecithin/sphingomyelin ratio for all sensitivities greater than 70%. Neither assay was a good predictor of the clinical severity of respiratory distress. For a separate group of 21 amniotic fluid specimens clinically contaminated with blood or meconium, the discriminatory power of the polarization assay was decreased, but six of seven respiratory-distress cases still had polarization values greater than 0.260. We conclude that this fluorescence polarization assay is a better overall predictor of fetal lung maturity than is the lecithin/sphingomyelin ratio, and that polarization values less than 0.260 are associated with little risk of respiratory distress.     

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.     

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.     

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. 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.     

Quantitative determination of phosphatidylglycerol in amniotic fluid by enzymatic assay

Phosphatidylglycerol (PG) was extracted from 54 human amniotic fluids for the assessment of fetal lung maturity. The PG values were derived from an enzymatic assay involving initial conversion of PG to glycerol by phospholipase C and alkaline phosphatase with subsequent analysis of the glycerol formed. This method proved to be reliable when compared with a method for two-dimensional thin layer chromatographic (2D TLC) analysis of amniotic fluid phospholipids. The results revealed that in all but one of 27 amniotic fluids in which no PG was detected by 2D TLC, enzymatic PG concentrations were less than or equal to 1.5 mumol/l and out of these, from 10 newborn infants delivered within 72 h of sampling, 4 developed respiratory distress syndrome (RDS). Conversely, in all but one of 27 amniotic fluids found to contain PG by 2D TLC, enzymatic PG concentrations were greater than 1.5 mumol/l and except for one subject from non-identical twins, no infants developed RDS.     

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.     

A calibrated fluorescence polarization assay for assessment of fetal lung maturity

The mechanism of a new fluorescence polarization method for measuring phospholipid in amniotic fluid is described. The fluorescence polarization of a surfactant fluorescent dye solubilized in amniotic fluid correlates significantly with the L/S ratio. Model studies indicate that this results primarily from partitioning of the dye between endogenous albumin, which causes a high fluorescence polarization, and dispersed phospholipid, which causes a much lower polarization. The fluorescence polarization can be compared with a calibration curve to give the ratio of phospholipid to albumin in the sample. This procedure may prove useful in antenatal assessment of fetal lung maturity.     

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.     

Determination of amniotic fluid palmitic acid concentration for the estimation of fetal lung maturity

Palmitic acid concentrations in amniotic fluid (AF) were determined in 135 patients with normal and pathological pregnancies between the 27th and 42nd week of gestation. There was a sharp rise in the mean palmitic acid concentration after the 34th weeks of gestation from 2.7 μg/ml to 9.9 μg/ml at term. This increase is almost identical with the rise of AF-lecithin. It was found that between 70% and 100% of AF-palmitic acid originates from lecithin. 65 patients were delivered within 24 h after amniotic fluid sampling. 7 infants of these patients developed a respiratory distress syndrome (RDS). In all cases with RDS AF-palmitic acid concentration was far below 5 μg/ml. Assuming an AF-palmitic acid concentration > 5 μg/ml for characterising fetal lung maturity (= no RDS), there were no false negative results, but 16% false positive results. However, the determination of AF-palmitic acid concentration seems to be a most reliable method for the assessment of fetal lung maturity.     

Amniotic fluid phosphatidylglycerol: a predictor of fetal lung maturity using conventional one-dimensional thin-layer chromatography

We present a retrospective comparative analysis of 209 amniotic fluid samples and the neonatal outcome. The presence of phosphatidylglycerol in 159 transabdominal amniotic fluid samples and the associated lung status indicated a 98% prediction rate for absence of respiratory distress syndrome with 1.8% false-positive results corrected to 0%. Twenty-nine vaginal pool samples were 72% predictive of outcome with false-positive results corrected to 14%. These cases were mild, and if a phosphatidylglycerol level of greater than 3% is used to indicate lung maturity, the corrected false-positive rate is 0%. False-negative results are also corrected and discussed. We believe that predicting neonatal respiratory distress syndrome by phosphatidylglycerol determination, using readily available conventional one-dimensional thin-layer chromatography, is clinically reliable and can aid in determining the maturity of the lung regardless of site of fluid collection.     

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.     

Noninvasive fetal lung maturity prediction based on ultrasonic gray level histogram width

The aim of this work was to noninvasively predict fetal lung immaturity with the ultrasonic gray level histogram width (GLHW), a form of clinical tissue characterization. The study included 22 fetuses in which infant respiratory distress syndrome (IRDS) developed post-delivery, and 25 fetuses without IRDS development. Independent receiver operating characteristic (ROC) analysis of fetal lung-to-liver GLHW ratios, fetal weights, gestational ages and the product of GLHW ratios by gestational ages for this cohort indicated that optimal thresholds for these parameters to differentiate immature from mature were 0.94, 1.750 g, 31 weeks and 29, respectively. With the optimal decision threshold of 0.94, the GLHW ratio provided sensitivity and specificity of 0.86 and 0.72, respectively. The corresponding values for gestational age were 0.77 and 0.68, 0.77 and 0.60 for fetal weight versus 0.96 and 0.72 for the product of GLHW ratio by fetal age, which was comparable with invasive amniotic fluid tests. The areas under the ROC curve for these parameters were 0.82, 0.82, 0.70 and 0.91. We found that GLHW is a noninvasive, stable and reliable measure of fetal lung maturity using commercial scanners.     

Cholesterol in amniotic fluid, determined by gas chromatography.

Cholesterol was extracted from amniotic fluid, saponified, converted to its trimethylsilyl derivative, and gas chromatographed, with cholesteryl acetate as the internal standard. The method is sufficiently accurate and precise for use with the range of concentrations of cholesterol found in amniotic fluid (5 to 48 mg/litre). Total cholesterol was measured in amniotic fluids collected at different stages of gestation. No significant trend or change was observed nor was cholesterol in the amniotic fluid and the mother's serum correlated at any stage of gestation. Thus we conclude that cholesterol is not a useful indicator of fetal age or maturity. Cholesterol concentrations in amniotic fluid from complicated pregnancies were within the range found for normal pregnancies.     

Failure to detect an effect of prolactin on pulmonary surfactant and adrenal steroids in fetal sheep and rabbits.

Recent reports have indicated an association between low cord prolactin (PRL) and the occurrence of respiratory distress syndrome in premature infants, and it is reported that PRL administration increases the lecithin content of fetal rabbit lung. We administered 1 mg ovine PRL to 32 rabbit fetuses on day 24 of gestation and evaluated lung phospholipid synthesis and content on day 26. Compared with diluent-injected littermates, PRL had no effect on the rate of choline incorporation into lecithin, tissue content of phospholipid and disaturated lecithin, or plasma corticoids. However, both choline incorporation and corticoids were increased in all animals undergoing surgery compared with unoperated controls. We also infused PRL (1 mg/day, i.v.) into three fetal sheep continuously over five periods of 5-8 days. Although supraphysiologic concentrations of PRL were achieved in plasma and amniotic fluid, there was no effect of this treatment on the flux of tracheal fluid surfactant or on plasma concentrations of corticoids of dehydroepiandrosterone sulfate. Thus, in this study, we failed to detect either a stimulation of the surfactant system or an adreno-corticotropic effect by PRL as previously postulated. This suggests that the relationship between PRL and respiratory distress sundrome is an indirect association.     

Evaluation of the shake test to predict respiratory distress syndrome

An analysis of the relationship of amniotic fluid shake test titers and the subsequent fetal lung maturity as evinced by the development of the respiratory distress syndrome (RDS) has been conducted. Over a four-year period, 131 amniotic fluid samples were tested within 48 hours of delivery. RDS was diagnosed in 16 infants. When the shake test was positive at a 1:2 dilution, one child developed a mild case of RDS (2.3%). If the test was postivie at a 1:4 dilution, none developed RDS. RDS occurred in 15% of the cases with a positive test at a 1:1 dilution and in 30% of the cases with a negative test. This test has proved to be an excellent screening method for predicting fetal lung maturity if it is positive at 1:2 or greater. If the result is positive at a 1:1 dilution, or negative, other methods must be used for assessment. Its advantages are its rapidity, simplicity, and low cost.     

延期妊娠318例临床分析

目的探讨延期妊娠对围生儿的影响及其临床处理对策。方法收集安徽医科大学第一附属医院2002年1月至2004年4月间收治的318例延期妊娠的临床资料,并随机抽取同期我院收治的318例足月妊娠资料,两组相对比。结果延期妊娠组羊水过少、羊水污染、胎儿宫内窘迫、新生儿窒息、剖宫产等发生率较足月妊娠明显增高(P<0.01),两组间巨大儿、低体重儿发生率无显著差异(P>0.05)。结论延期妊娠时胎盘功能减退、胎儿宫内环境差、胎儿对缺氧耐受性降低、高危程度增加,故应积极采取措施,适时计划分娩,产前产时需严密监护,以确保母婴安全。     

The evaluation of fetal lung maturity by amniotic fluid analysis.

The frequent necessity for termination of pregnancy before the spontaneous onset of labor requires that we be able to accurately predict fetal lung maturity. We have used amniotic fluid studies for evaluation of fetal lung maturity and have found that (1) a "fat" cell concentration of 30% or more, or (2) a creatinine concentration of 2.0 mg/100 ml or more, or (3) a lecithin:spingomyelin (L:S) ratio of 2.0 or greater all correlated well with fetal maturity. Since each of these studies is open to a variety of possible errors, the use of several different ones adds reliability to the estimation of fetal lung 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.     

The value of various investigations of amniotic fluid in the estimation of fetal maturity (author's transl)]

The amniotic fluid lecithin-spingomyelin (L/S) ratio, creatinine and uric acid concentrations, percentage of organe-stained fat cells and the foam test by Clements and coworkers have been compared in 82 samples of amniotic fluid from 66 patients. The specimens were obtained by transabdominal amniocentesis or amniotomy between the 24th and the 42nd week of pregnancy. The determination of the L/S ratio and the foam test seem to be reliable methods of estimating pulmonary surfactant and, hence, of predicting the respiratory distress syndrome (RDS) in the newborn infant. There was no RDS with an L/S ratio greater than 1.6 to 1.8. Positive foam tests also seem to be a valuable indicator of pulmonary maturity, no cases of RDS being found. However, false negative foam tests are not rare. The amniotic fluid concentration of creatinine correlated well with gestational age and birth weight. The determination of uric acid in the amniotic fluid is an unreliable test of fetal maturity on account of the large scatter. The percentage of orange-stained cells did not rise above 10% before the 39th week of pregnancy in most cases.