Lamellar body counts: a consensus on protocol.

Lamellar bodies, concentrically layered "packages" of phospholipid that represent the storage form of surfactant, can be counted in the platelet channel of most electronic cell counters. The lamellar body count has been used for more than a decade and performs as well as traditional phospholipid analysis as an assay for evaluating fetal lung maturity. It is preferable to phospholipid analysis because it is rapid, objective, and inexpensive and can be performed in any hospital laboratory. The current methodologies for specimen preparation vary widely among laboratories, most notably with respect to centrifugation, resulting in differences in maturity cutoffs used. Our goal was to establish a consensus regarding a standardized methodology for the lamellar body count. Institutions that previously had published their results with lamellar body counts were invited to contribute. The consensus of the four participating institutions includes the following: centrifugation is not a necessary step and should be abandoned, maturity is suggested by a count of 50,000/microL or greater, and immaturity is suggested by a count of 15,000/microL or lower. As the lamellar body count gains wider acceptance as a primary assay for assessing fetal lung maturity, the test must be performed uniformly and accurately, given the implications of acting on a falsely negative test resulting from improper methodology.

Amniotic fluid lamellar body count: a rapid and reliable fetal lung maturity test.

OBJECTIVE: To evaluate the lamellar body count as a predictor of fetal lung maturity. METHODS: We conducted a prospective clinical outcome study. Amniocentesis was performed for evaluation of fetal lung maturity status within 72 hours of delivery in 130 patients. A lamellar body count was performed on each specimen, and a lecithin-sphingomyelin ratio and lung phospholipid profile were performed when possible (insufficient sample or contamination in eight cases). Each infant was evaluated for evidence of respiratory distress syndrome (RDS). RESULTS: A lamellar body count exceeding 30,000/microL predicted pulmonary maturity correctly in all cases (negative predictive value 1.00). All 16 cases of RDS had counts of 30,000/microL or less. If the lamellar body count was less than 10,000/microL, the positive predictive value for RDS was 67%, and the likelihood of a mature result from chromatographic phospholipid analysis was low (one of 14, 7%). Values between 10,000-30,000/microL indicated intermediate risk (four of 39, 10%) for developing RDS. Phospholipid analysis indicated fetal lung maturity in 35 of 39 (90%) cases with lamellar body counts in the intermediate risk range. CONCLUSIONS: The lamellar body count compares favorably with traditional phospholipid testing in the prediction of fetal lung maturity. Phospholipid analysis is not needed with lamellar body counts greater than 30,000/microL or less than 10,000/microL, but may be of benefit for values in the intermediate risk range. Advantages of this test include speed, objectivity, small sample volume required, and universal availability of instrumentation.

Lamellar body number density and the prediction of respiratory distress.

The determination of amniotic fluid lamellar body number density (LBND) has recently been shown to correlate well with other established indicators of fetal lung maturity. The authors have compared the LBND with a fetal lung phospholipid profile in predicting the clinical outcome in 52 well-documented cases. If a cutoff of 30,000/microL was used to indicate fetal lung maturity, there were no false-negative results for the LBND whereas there was one for the fetal lung profile. On the other hand, this cutoff resulted in 22 false-positive results for the LBND, whereas there were only 7 false-positive results by the fetal lung profile. The number of false-positive results by the LBND can be decreased by using a separate cutoff of less than 10,000/microL to indicate high risk for development of respiratory distress, while leaving the cutoff for predicting mature lung at 30,000/microL. This resulted in only four false-positive results for the LBND; each of these were from the same patients who also had false-positive results by the fetal lung profile. Care must be taken to ensure that the particle counter used is properly calibrated and that the appropriate cutoffs for both lung maturity and immaturity are used.

Functional phospholipid/sphingomyelin ratio: an easy to interpret indicator of fetal lung maturity.

Data are presented which show that the functional phospholipid/sphingomyelin (FPL/S) ratio is a sensitive and specific prenatal indicator of fetal respiratory status. Phospholipids included in the ratio are acetone precipitable lecithin(L), phosphatidylglycerol(PG), phosphatidylethanolamine(PE), and sphingomyelin(S). These lipids have been shown to contribute to either the lowering of surface tension or to changes in microviscosity. Both of these properties contribute independently to the physiologic effectiveness of fetal lung surfactant. Although other phospholipid profiles have been described in the literature, the FPL/S ratio accounts for the major phospholipids which contribute to lung surfactant function with the added advantage of presenting the data in the form of a single easy to interpret number. Clinical correlates are also presented for the L/S ratio, fluorescence polarization assay (using 1,6-diphenyl-3,5-hexatriene), and absorbance at 650 nanometers.

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.

Proportion of hemoglobin S in blood, as determined from solubility measurements.

We exploited the concentration dependence of hemoglobin S on its solubility in concentrated phosphate buffer to determine the percentage of hemoglobin S or hemoglobin A in whole blood or hemolysates. A sample is incubated for 1 h at 37 degrees C in "SickleQuik" (General Diagnostics) tubes, and the absorbance of the aqueous phase is then measured at 555 nm and 650 nm, the latter wavelength to correct for turbidity. The fractional hemoglobin S or hemoglobin A content is read from standard-curve data on mixtures of hemoglobin A and hemoglobin S. Such curves can be prepared with the use of hemoglobin A alone, because under our conditions all hemoglobin S is precipitated. The curve is reproducible for up to three weeks. Day-to-day CVs ranged from 1.8% at 10% hemoglobin S to 8.1% at 68% hemoglobin S. For similar concentration ranges, day-to-day CVs for electrophoresis, with densitometry, ranged from 4.9% to 18%. Results on 29 patients are presented. We recommend the method for rapid quantification of hemoglobin S percentage for patients with sickle cell disease in acute-care situations.

Total surface-active phospholipid/sphingomyelin ratio as an index of fetal lung maturity.

As an index of fetal lung maturity, the ratio of total surface-active phospholipid (phosphatidylethanolamine + phosphatidylinositol + phosphatidyglycerol + lecithin) to sphingomyelin performs as well as or better than any other indicator that we tested, showing 92% sensitivity and 98% specificity. The sensitivity and specificity, respectively, of the other methods were: (a) lecithin/sphingomyelin ratio, 92% and 95%; (b) alkaline phosphatase/glutamyltransferase ratio, 100% and 49%; (c) microviscosity, 75% and 97%; and (d) adsorbance at 650 nm, 50% and 73%. The procedure requires 2 h or less and its simplicity allows it to be offered on a 24 h/day basis without necessitating special training.

The molecular weights of three forms of glycophorin A in sodium dodecyl sulfate solution.

The molecular weights of each of the three major forms of glycophorin A, as defined by polyacrylamide gel electrophoresis, have been determined by sedimentation equilibrium in sodium dodecyl sulfate using 2H2O to blank out the contribution of the detergent according to Reynolds and Tanford (Reynolds, J.A. and Tanford, C. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 4467--4470). The molecular weights were: form 1, 65 700; form 2, 31 100; form 4, 27 900. These data establish form 1 as a dimer, and form 2 and form 4 as monomers. 2. The relative sizes of the three forms were estimated by determining mobilities in sodium dodecyl sulfate polyacrylamide gels of gradulated gel concentrations at an ionic strength in which all three species coexist (I = 0.2). Ferguson plots show that the three species have identical net surface charge densities but differing retardation coefficients (--Kr) as follows: form 1, --0.057; 'form 4', --0.038; form 2, --0.025. From these data it is concluded that the hydrodynamic radius of form 1 is greatest, 'form 4' is intermediate, and form 2 is least.

The interaction of dodecyl and tetradecyl sulfate with proteins during polyacrylamide gel electrophoresis.

1. The affinity of tetradecyl sulfate for many unfolded proteins is greater than that of dodecyl sulfate. 2. It is the presence of tetradecyl sulfate that results in the staining of proteins by pinacryptol yellow seen by Stoklosa and Latz (Stoklosa, J.T. and Latz, H.W. (1974) Biochem. Biophys. Res. Commun. 58, 74--79), as some tetradecyl sulfate remains associated with proteins during electrophoresis at room temperature (as opposed to dodecyl sulfate which, within the limit of detection, is completely removed). 3. Tetradecyl sulfate has a greater capacity to dissociate protein aggregates which consist of identical peptide chains, such as Glycophorin dimers and bovine serum albumin dimers, than does dodecyl sulfate.