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Amniotic fluid samples received for routine prenatal diagnosis of open neural tube defects were used for a study to compare amniotic fluid acetylcholinesterase (AChE) determination using a monoclonal antibody (4F19) enzyme antigen immunoassay and amniotic fluid alpha-fetoprotein (AFP) measurement as diagnostic tests for open neural tube defects. The study was based on 9964 women with singleton pregnancies and known outcome (including 6 with anencephaly and 18 with open spina bifida) having an amniocentesis at 14-23 weeks of gestation. The AChE immunoassay yielded detection rates for anencephaly of 100 per cent (95 per cent confidence interval (CI) 54.07-100 per cent), for open spina bifida of 100 per cent (95 per cent CI 81.47-100 per cent), for anterior abdominal wall defects of 20 per cent (95 per cent CI 0.51-71.64 per cent), and a false-positive rate of 0.22 per cent (95 per cent CI 0.14-0.34 per cent) excluding anencephaly, open spina bifida, and anterior abdominal wall defects. For similar detection rates the false-positive rate of the AFP test was significantly higher, 0.74 per cent (95 per cent CI 0.58-0.94 per cent). On the basis of these findings, it is recommended that the technically simple AChE immunoassay should be used on all samples; the AFP test should only be used on the 0.5 per cent of the samples with concentrations of AChE activity > or = 8.5 nkat/l for clear samples and blood-stained samples becoming clear after centrifugation, and > or = 25.0 nkat/l for blood-stained samples that are discoloured after centrifugation; an AFP cut-off level of 2.0 MOM is recommended for this policy. Thereby, the detection rates for anencephaly, open spina bifida, and anterior abdominal wall defects would be 100, 100, and 20 per cent, respectively (95 per cent CIs 54.07-100, 81.47-100, and 0.51-71.64 per cent, respectively), and the false-positive rate would be 0.08 per cent (95 per cent CI 0.03-0.16 per cent) (excluding anencephaly, open spina bifida, and anterior abdominal wall defects).

The bimodal approach for quality specification in prenatal screening for neural tube defects has been used. The method for assessing specifications has been the expenses for the society. It has been shown that the cut-off values and analytical bias are of importance. It is recommended to use a reference serum for local calibration to keep the level within defined limits when changing to a new batch of reagents.

Stored amniotic fluid samples collected in Oxford and East Birmingham as part of the Collaborative Acetylcholinesterase Study were assayed for the presence of acetylcholinesterase (AChE) using a monoclonal antibody (4F19) enzyme antigen immunoassay. These results were compared with the results of a gel AChE which had been performed earlier. A total of 5689 samples from singleton pregnancies were analysed (including 36 with anencephaly, 77 with open spina bifida and 17 with anterior abdominal wall defects). The gel test yielded detection rates of 97% for anencephaly, 99% for open spina bifida and 94% for abdominal wall defects; the false positive rate (excluding pregnancies associated with serious abnormalities, miscarriages and intrauterine deaths) was 0.24%. The monoclonal test yielded similar results; using appropriate cut-off values to allow for differences in acetylcholinesterase levels in blood stained and clear samples, a similar false-positive rate of 0.22% was associated with detection rates of 97%, 95% and 71% respectively for the three types of defect. Although the detection rates and the false positive rate were slightly higher for the gel test, a result that might be explained by a decrease in AChE activity caused by storage of the samples, the monoclonal test has the advantages of requiring less interpretative expertise, it can be performed on a larger number of samples a day and it is not affected by contamination with fetal calf serum.

The percentage of alpha-fetoprotein (AFP) not reacting with concanavalin A (con A) was determined by crossed line affino-immunoelectrophoresis in amniotic fluid from 25 pregnancies with neural tube defects (NTD) and other fetal abnormalities giving rise to elevated AFP levels, and from 128 pregnancies with normal outcome. The percentage of con A non-reactive AFP is significantly lower in the presence of fetal abnormalities (mean 3.4%, range: 0.0-6.3, n = 25), compared to the percentage found in normal pregnancies (mean 17.2%, range: 6.6-35.8%, n = 128). In amniotic fluid samples from normal pregnancies, the percentage of con A non-reactive AFP in 84 cases with total AFP levels lying within the 95% reference interval was not significantly different from the percentage found in 44 cases with levels above the 95% reference interval. The percentage of non-reactive AFP in fetal serum and cerebrospinal fluid was of the same magnitude as found in amniotic fluids of pregnancies with fetal abnormalities. It is concluded that analysis of the percentage of con A non-reactive AFP by crossed line affino-immunoelectrophoresis is a simple, reliable and apparently diagnostic test for NTD and other abnormalities with leakage of fetal serum or cerebrospinal fluid into the amniotic fluid. The test should therefore be used in all cases with only marginally elevated AFP levels, so false positives may be avoided.

During a 6-year period 19,647 consecutively received amniotic fluid samples were analyzed for the alpha-fetoprotein (AFP) concentration. In samples with an AFP concentration above the 95% upper normal limit the fraction of AFP non-reactive with concanavalin A (con A) was determined by crossed affinity immunoelectrophoresis. Of 436 samples from normal pregnancies with a moderate elevation of AFP (between the 95% limit and 2.5 times the median) the con A fraction was normal in 92 to 98% of the cases, depending on the gestational age. At significantly elevated AFP concentrations (above 2.5 times the median), the fractions were normal in 78% of the 50 cases with a normal outcome. In 80 of 89 cases of fetal malformations, abnormally low fractions were found, and in all 19 cases of abdominal wall defects and congenital nephrosis the fractions were low. No significant difference was found in the sensitivity for detection of fetal malformations by con A analysis compared to quantitative analysis for acetylcholinesterase (AChE). However, significantly more false positive results were found in normal pregnancies for the con A analysis compared to the AChE analysis. Both analyses performed significantly better than ultrasound scanning in terms of sensitivity. It is concluded that AChE is the test of choice in prenatal diagnosis of fetal neural tube defects. However, a sensitivity of 1.00 and a predictive value of a negative test of 1.00 were found for the con A analysis for fetal abdominal wall defects. The AchE analysis performed less well in this context.

D2 is a glycoprotein existing in both membrane-bound and soluble forms. Employing a specific rabbit antibody against purified human brain D2, we developed an enzyme-linked immunosorbent assay (ELISA) for the quantification of D2 and applied it to amniotic fluids from 87 normal and 36 pathological pregnancies. With a cut-off point of 150 ng D2/ml, no false positive D2 values were obtained in any of the amniotic fluids from normal fetuses, although the alpha-fetoprotein concentrations were slightly increased in 13 cases. No false negative D2 values were found in any of the 18 investigated amniotic fluids from fetuses with anencephaly. Of 8 amniotic fluids from fetuses with spina bifida, 2 false negative D2 values were found. No false negative alpha-fetoprotein values were found in any of the cases with neural tube defects in this study. In 10 amniotic fluids from fetuses with other malformations, 5 samples showed raised D2 concentrations. The D2 level in sera from 10 women carrying normal fetuses and 16 women carrying malformed fetuses was also determined, but no statistically significant difference in D2 level was found in the pathological sera when compared with normal sera. It was concluded that the determination of D2 concentrations in amniotic fluid by means of the D2-ELISA may be used as an additional test in the screening of fetal malformations in early pregnancy.

The D2-protein is a neuronal membrane protein which has an immunochemically detectable soluble derivative in cerebrospinal fluid. We used rocket-on-line immunoelectrophoresis with an antiserum against rat synaptosomal membranes for demonstration of the human D2-protein in fetal cerebrospinal fluid and in amniotic fluids from pregnancies with fetal neural tube defects. D2 was neither found in normal amniotic fluids nor in cases of amniotic fluids from pregnancies with other malformations. Analysis of D2 in amniotic fluids may thus have a future role in the antenatal diagnosis of fetal neural tube defects.

The glial fibrillary acidic protein (GFAP) is the subunit protein of intermediate filaments in astrocytes and closely related cell types. By means of an enzyme immunoassay we have determined the concentration of GFAP in amniotic fluids from normal pregnancies and from pregnancies complicated by various fetal malformations. The group of 20 cases of fetal anencephaly had a significantly higher mean amniotic fluid GFAP concentration (115 micrograms/l +/- 133.6 (S.D.), range 6-378 micrograms/l) than the control group of 117 normal pregnancies (13 micrograms/l +/- 5.5 (S.D.), range 0-31 micrograms/l), (P less than 0.001). Two cases of fetal encephalocele likewise had very high amniotic fluid GFAP concentrations. None of the other cases of fetal malformations investigated, including 12 cases of spina bifida, had increased amniotic fluid GFAP concentrations. We conclude that determination of the amniotic fluid GFAP concentration may give additional information in the prenatal diagnosis of fetal nervous system malformations.

Concanavalin A (con A) and lens culinaris agglutinin (LCA) microheterogeneity pattern of AFP (crossed affinity immunoelectrophoresis), alpha-2-macroglobulin and synaptic membrane protein D-2 (rocket immunoelectrophoresis) and qualitative (polyacrylamide gel electrophoresis) and quantitative (enzyme kinetic reaction rate) acetylcholinesterase were analysed in 87 consequtive samples from normal pregnancies and 37 abnormal samples (fetal neural tube defect or abdominal wall defect). Very few false positive results were obtained in normal pregnancies with any of the tests. In all cases of neural tube defects the correct result was obtained with qualitative acetylcholinesterase analysis, whereas only 2/3 of the abdominal wall defects were correctly predicted. Testing with con A or LCA was less optimal in neural tube defects, whereas all abdominal wall defects could be predicted correctly. Acetylcholinesterase in the quantitative test and protein D-2 did not decrease the rate of false results. Determination of the alpha-2-macroglobulin concentration performed well in the present study, but is not recommended because of the very high susceptibility to contamination of amniotic fluid with fetal or maternal blood.