S-100 protein and neuron-specific enolase in amniotic fluid as markers of abdominal wall and neural tube defects in the fetus

The aim of this study was to determine whether there is increased leakage of neuron-specific enolase (NSE) and S-100 protein into amniotic fluid in pregnancies with neural tube defects, since both these proteins are produced by neural tissue, and to compare the value of these substances for detecting such defects with that of the more conventional techniques of alpha-fetoprotein (AFP) and acetylcholinesterase (AChE) gel electrophoresis. Amniotic samples from 25 mid-pregnancies (15–17 weeks' gestation) with neural tube defects (14 with open spina bifida and 11 with anencephaly) and from seven mid-pregnancies with abdominal wall defects were compared with a control material consisting of 80 amniotic fluid samples from 80 consecutive mid-pregnancy amniocenteses, with normal karyotypes and AFP concentrations. All of the above cases of abnormalities were primarily detected through increased AFP levels in the amniotic fluid. Amniotic fluid samples from 13 pregnancies with fetuses with autosomal chromosomal abnormalities and seven amniotic fluid samples contaminated with blood were also included in the investigation. It is concluded from the results that the conventional AFP assay combined with AChE gel electrophoresis is the best method for screening amniotic fluid for neural tube defects and defects of the abdominal wall. Neither NSE nor S-100 assay alone proved to be superior for the detection of these cases in mid-trimester amniotic fluid. The S-100 assay, however, could give additional information in cases where AChE gel electrophoresis is not decisive; for example, in samples contaminated with blood.     

Alpha-fetoprotein and acetylcholinesterase activity in first-and early second-trimester amniotic fluid

Normal ranges of amniotic fluid alpha-fetoprotein (AFP) and acetylcholinesterase activity (AChE) are described for gestational weeks 11–14 using rocket gel immunoelectrophoresis for AFP quantitation and a monoclonal antibody (4F19) enzyme antigen immunoassay for AChE activity measurement. The normal ranges were established by the examination of 281 amniotic fluid samples from 281 normal pregnancies. AFP was found to increase from a median level of 14.0 MIU/1 at 11 weeks to a maximum at 13 weeks (median=18.0 MIU/l) (P<0.05), thereafter falling (not significant). No AChE test result exceeded 4.8 nkat/l. In addition, AFP and AChE values for three cases of fetal malformation, identified by the biochemical analyses of amniotic fluid, are given. These cases included two fetuses with a neural tube defect and one fetus with an abdominal wall defect. Amniocentesis was performed at 10, 11, and 14 weeks, respectively. The AFP and AChE values were all high.     

Amniotic fluid acetylcholinesterase measurement in the prenatal diagnosis of open neural tube defects. Second report of the collaborative acetylcholinesterase study

Seventeen centres from Australia, Britain, France, and the United States collaborated in a study to compare amniotic fluid acetylcholinesterase (AChE) determination by gel electrophoresis and amniotic fluid alpha-fetoprotein (AFP) measurement as diagnostic tests for open neural tube defects. The study was based on 32 642 women with singleton pregnancies (including 428 with open spina bifida and 238 with anencephaly) who had an amniocentesis at 13–24 weeks' gestation. The AChE test yielded a detection rate for open spina bifida of 99 per cent (95 per cent confidence interval 98–100 per cent), 98 per cent for anencephaly (95 per cent confidence interval 96–100 per cent), and a false-positive rate of 0.34 per cent (95 per cent confidence interval 0.28–0.40 per cent) excluding miscarriages, intrauterine death, and serious fetal abnormalities. The false-positive rate was 0.30 per cent among the 13 centres that used a specific AChE inhibitor in the test. Comparable rates for the AFP test were less favourable. (For example, the open spina bifida detection rate was 90 per cent and the false-positive rate was 0.46 per cent using the cut-off levels specified in the U.K. Collaborative AFP Study.) The AChE false-positive rate was lower in samples that were not bloodstained (0.16 per cent) than in those that were (2.4 per cent). It was higher in women who had an amniocentesis on account of a raised maternal serum AFP level (0.56 per cent) than in those who had one for other reasons (0.29 per cent). The best results were obtained by a combination of the two tests, an effective and economical policy being to perform the AFP measurement on all amniotic fluid samples and an AChE test on samples with AFP levels greater than or equal to 2.0 multiples of the normal median (about 5 per cent of all samples). Using this policy, the open spina bifida detection rate was 96 per cent and the false-positive rate was 0.14 per cent (0.06 per cent for samples that were not bloodstained and 1.2 per cent for those that were; 0.40 per cent for women with raised serum AFP levels and 0.09 per cent for other women). This policy offers a useful improvement to the prenatal diagnosis of open spina bifida.     

Identification of acetylcholinesterase and cholinesterase in amniotic fluid by immune absorption technique

A method for immunological detection of acetylcholinesterase (AChE) and cholinesterase (ChE) in amniotic fluid is described. By addition of a small amount of antihuman-erythrocyte membrane antibody or anti-pseudocholinesterase antibody to the sample before electrophoresis the two esterase bands on polyacrylamide gel (PAG) can be absorbed away. Similar staining results can also be obtained by specific inhibition of the two esterases with either BW 284C51 (AChE inhibitor) or Lysivane (ChE inhibitor). In cases with a faint AChE band and in cases with blood contamination the immune absorption technique makes interpretation easier. Nearly identical staining results have been obtained by the immune absorption technique and the inhibition technique in the following samples with an AChE band: 34 samples from pregnancies with severe fetal malformation or intrauterine death (2 cases), 4 fetal serum samples, 4 samples of cerebrospinal fluid, 4 samples of fetal erythrolysate and 4 samples of adult erythrolysate. It can be concluded that an antibody prepared against erythrocyte AChE cross-reacts with AChE in cerebrospinal fluid, and that this antibody can be used for demonstration of AChE in amniotic fluid.     

Isoelectric focusing pattern of human amniotic fluid α-fetoprotein

Isoelectric focusing (IEF) of amniotic fluid α-fetoprotein (AFP) in thin-layer polyacrylamide gels containing 8 M urea followed by immunoblotting reveals at least nine bands, band I lying next to the cathode. Compared with 298 amniotic fluid samples from normal pregnancies, we found that the density of band V was increased in seven cases of fetal death. In 16 amniotic fluid samples from pregnancies with open neural tube defects (ONTO), band V disappeared or was markedly decreased. In seven cases with elevated AFP and positive acetylcholines-terase (AChE) due to contamination with fetal blood, no difference in pattern was observed compared with samples from normal pregnancies. It is suggested that IEF of AFP and subsequent immunoblotting are an apparently diagnostic test for ONTD and intrauterine fetal death (IUFD).     

Predictive value of amniotic acetylcholinesterase analysis in the diagnosis of fetal abnormality in 3700 pregnancies

The value of acetylcholinesterase (AChE) analysis as an adjunctive test to amniotic alpha fetoprotein (amAFP) for the diagnosis of fetal abnormality has been investigated in a series of 3785 amniotic fluid samples. Quantitative analysis of AChE performed retrospectively on a selected group of 541 amniotic fluid samples failed to discriminate between normal and open neural tube defect pregnancies. Qualitative analysis of AChE by polyacrylamide gel (PAG) electrophoresis in the same series of 541 fluids correctly identified 251 of the 255 pregnancies with open neural tube defect and 29 of the 31 pregnancies with false positive amAFP results. The failure of the test to diagnose 4 cases of open neural tube defect was probably attributable to the age and condition of the stored AF samples. Routine diagnostic testing of AChE isoenzymes in a further 3244 AF samples successfully identified all 170 cases of open neural tube defect and 20 cases with other fetal defects. Thirteen fluids gave false positive AChE results (0.4 per cent) compared to 59 of the series in which there were false positive amAFP results (1.8 per cent). Six of the 13 false positive AChE cases had AChE bands of low intensity which would not be regarded as diagnostic of fetal abnormality, and in five the AChE band may have been the result of significant blood contamination. False positive AChE results contributed to the decision to abort three apparently normal fetuses, but a normal AChE result undoubtedly helped to save a number of pregnancies with false positive amAFP results. Our experience suggests that repeating the amniocentesis may help in resolving the rare diagnostic difficulty of a positive AChE result with or without an elevated amAFP in the absence of ultrasound evidence of fetal abnormality, particularly where there is blood contamination of the amniotic fluid sample.     

A prospective study of amniotic fluid cholinesterases: Comparison of quantitative and qualitative methods for the detection of open neural tube defects

The value of quantitative and qualitative methods of cholinesterase (ChE) analysis in the detection of open neural tube defect (NTD) has been assessed in a prospective survey of 1495 mid-trimester amniotic fluids. Using a quantitative method the mean ChE values were much lower in fluids from pregnancies of normal outcome but it was not possible to discriminate these fluids completely from those associated with NTD pregnancies. particularly when the specimens were contaminated with blood. Similarly, measurement of acetylcholinesterase (AChE) activity alone by three different methods also failed to eliminate the overlap between the two groups. In contrast, polyacrylamide gel electrophoresis revealed only a single band of ChE activity in 1408 out of 1410 fluids from pregnancies with a normal outcome whilst amniotic fluids from all 60 cases of open NTD. 6 out of 7 cases of exomphalos and 3 out of 4 cases of intra-uterine death gave the characteristic second faster-running AChE band. A qualitative gel method which requires the same amount of ChE activity to be loaded from each amniotic fluid is an effective method for pre-natal diagnosis of NTDs.     

A comparison of amniotic fluid alpha-fetoprotein and acetylcholinesterase in the prenatal diagnosis of open neural tube defects and anterior abdominal wall defects

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 percent (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 ⩾ 8·5 nkat/1 for clear samples and blood-stained samples becoming clear after centrifugation, and ⩾ 25·0 nkat/1 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).     

False-positive amniotic fluid acetylcholinesterase analysis in the third trimester

Thirty-two third-trimester amniotic fluid samples were studied according to the indication for amniocentesis, result of acetylcholinesterase (AChE) analysis, and outcome, in order to address the issue of the effectiveness of AChE testing late in gestation. The results indicate that third-trimester AChE analysis is less effective than second trimester in distinguishing open neural tube defects (ONTDs) and ventral wall defects (VWDs) from other abnormalities. False-positive results occurred in cases of isolated hydrocephaly (four of seven cases), polyhydramnios, and intrauterine growth retardation (IUGR). Caution is recommended in interpreting third-trimester AChE tests, particularly when neither an ONTD nor a VWD is observed by ultrasound.     

Amniotic fluid acetylcholinesterase measurements: Comparing immunochemical and polyacrylamide gel techniques

In the present study, a recently reported immunochemical technique for measuring acetylcholinesterase (AChE) in amniotic fluid utilizing the 4F19 antibody was compared with the widely utilized polyacrylamide gel technique to determine whether the immunochemical assay provided an advantage in separating unaffected pregnancies from those associated with open spina bifida (OSB) and open ventral wall defects (OVWD). The study included (1) 73 amniotic fluid samples from unaffected pregnancies [alpha-fetoprotein (AFP) < 2 MoM] with no visible gel AChE band, (2) nine bloodstained samples from unaffected pregnancies (AFP 2·2–4·0 MoM) with visible gel AChE bands, (3) 18 samples associated with OSB (AFP 2·2–7·0 MoM) with visible gel AChE bands, and (4) 20 samples associated with OVWD (AFP 3·2–53·5 MoM) with visible gel AChE bands. The immunochemical assay produced ranges of measurements in the four respective categories as follows: (1) 2–60 arbitrary units (AU): (2) 14–69 AU, (3) 61–593 AU, and (4)22–476 AU. Eight of the nine unaffected pregnancies with visible gel AChE bands had immunochemical measurements below the highest measurement for the samples with no visible AChE band (60 AU), as did five out of 20 OVWD pregnancies. Two of the OSB cases had values of 61 and 62 AU. These data indicate that the 4F19 specific monoclonal antibody to AChE is capable of distinguishing unaffected from affected pregnancies with reasonable reliability but that more work needs to be done to establish the extent of overlap between the unaffected and affected populations.     

Discrepant karyotypes after second- and third-trimester combined placentacentesis/amniocentesis

Cytogenetic data are presented from a total of 1306 consecutive pregnancies with successful diagnosis obtained from both chorionic villi after short-time culture (CVS-SC) and amniotic fluid cell cultures (AC); samples had been taken simultaneously at combined placentacentesis (placental biopsy) and amniocentesis during the second (92·8 per cent) and third (7·2 per cent) trimesters. Concordant results were obtained in 1218 pregnancies with a normal karyotype and in 62 pregnancies with an aberrant fetal karyotype. Discrepant, i. e. false-positive and false-negative, results were found in 26 cases (2 per cent). From these data the accuracy of CVS-SC, defined as the proportion of all correct diagnoses, is calculated to be 98 per cent. Three non-mosaic and 14 mosaic false-positive results obtained after CVS-SC could not be confirmed by AC. Related to 1235 true normal fetal karyotypes, the specificity of CVS-SC, i.e. the proportion of normal karyotypes correctly diagnosed, amounts to 98·6 per cent. In nine pregnancies, an aberrant fetal karyotype detected after AC was missed by CVS-SC. The sensitivity of CVS-SC, i.e. the proportion of abnormal fetuses correctly diagnosed (62 out of 71), amounts to 87·3 per cent in our study group.     

Prenatal diagnosis of citrullinemia: Elevated levels of citrulline in the amniotic fluid in the three affected pregnancies

In three pregnancies at risk for citrullinemia affected fetuses were predicted both by strongly increased levels of citrulline in the amniotic fluid and by the reduced incorporation of ¹⁴C-citrulline into TCA-precipitable material in cultured amniotic fluid cells. The prenatal diagnoses of affected fetuses were confirmed after termination of the pregnancies by direct and indirect assays of argininosuccinate synthetase in the fetal livers and fibroblasts respectively. Measurement of the citrulline concentration in amniotic fluid appears to be a valuable adjunct in the prenatal diagnosis of citrullinemia.     

Biamnial elevated alpha-fetoprotein and positive acetylcholinesterase in twins,one with anencephaly

Anencephaly in twin B was accompanied by elevated amniotic fluid alpha-fetoprotein (AFP) and a positive acetylcholinesterase (AChE) band on gel electrophoresis in both twin sacs, although twin A was normal. AChE results did not help distinguish the false positive AFP in this set of twins, implying that AChE may diffuse transamniotically as has been previously postulated for AFP. In light of the low concordance rate for neural tube defects in twins, patient counselling in this situation must include the information that AFP and AChE may be falsely elevated in normal twin when the other twin has a neural tube defect.     

Maternal serum alpha-fetoprotein and fetal triploidy

Fetal triploidy is commonly found in early pregnancy. The majority of these pregnancies spontaneously abort in the first trimester. Occasionally, the pregnancy progresses to the second and third trimesters. We reviewed the maternal serum alpha-fetoprotein (MSAFP), amniotic fluid alpha-fetoprotein (AFP), amniotic fluid acetylcholinesterase (ACHE), fetal pathology, and placental pathology in sex second-trimester pregnancies complicated by fetal triploidy. Four of these patients had MSAFP values greater than 7.5 multiples of the median (MoM). Five of six pregnancies had MSAFP values greater than 2.25 MoM. All five of these patients had a partial mole. Four patients had amniotic fluid AFP values greater than 2.0 MoM. Two fetuses had associated neural tube defects. These were the only patients with positive amniotic fluid ACHE. None of the other patients had fetuses with anomalies that are known to be associated with an elevated MSAFP. The elevated MSAFP appeared to be related to the presence of a partial mole. Two of the five cases with an MSAFP greater than 2.25 MoM did not have sonographic evidence of a significant anomaly. Therefore, karyotyping can be of benefit in evaluating patients with elevated MSAFP.     

Determination of maternal serum acetylcholinesterase in pregnancies with fetal neural tube defects

We have investigated the occurrence of acetylcholinesterase (AChE) (E.C.3.1.1.7) in fetal serum, amniotic fluid and maternal serum using an immuno-chemical assay-technique employing both polyclonal and monoclonal antibodies. Fetal serum had increased amounts of AChE, which is due to an increase in the 10.5S form of the enzyme. This form was also found in amniotic fluids of pregnancies with a fetal neural tube defect (NTD), but not in normal amniotic fluid. The increase in amniotic fluid AChE was however, not reflected in the maternal serum.     

Testosterone levels in midtrimester maternal and fetal plasma and amniotic fluid

Testosterone was measured in maternal plasma (58 samples), amniotic fluid (71 samples) and fetal plasma (55 samples) in 79 patients between 15 and 23 weeks' gestation. Maternal plasma testosterone levels were unrelated to fetal sex. Amniotic fluid testosterone was significantly higher in male than female fetuses but did not reliably predict fetal sex. A correct diagnosis of fetal sex was made by testosterone assay of pure fetal plasma in 39 out of 40 males and in 15 out of 15 females using 1.70 nmol/1 as the cut-off value. This investigation is not the method of choice for routine fetal sexing but may be of value in fetuses suspected of having certain endocrine disorders.     

Routine chromosome analysis on fetal blood microaliquots obtained at fetoscopy

A routine study of the fetal karyotype was performed on samples obtained at 64 fetoscopic procedures. In 13 cases only pure amniotic fluid was available for the cultures, while in the remaining 51 cases the chromosome analysis was carried out on PHA-stimulated lymphocyte microcultures set up with any excess fetal blood above the requirements for globin-chain synthesis. Karyotype could be determined on fetal lymphocytes in 44 cases (86 per cent). All the fetuses were chromosomally normal. This experience shows that cytogenetic analysis using microaliquots of fetal blood is a relatively simple technique which should be introduced into routine prenatal diagnosis by fetoscopy.     

Deceased co-twin as a cause of false positive amniotic fluid AFP and AChE

A pregnancy was terminated because of persistently elevated amniotic fluid AFP (+10 S.D.) and an AChE band of low intensity on gel electrophoresis. No fetal anomalies were detected by ultrasonographic examination. Autopsy revealed an apparently normal fetus of about 20 weeks gestation. Attached to the placenta was a small sac containing a fetus papyraceus co-twin of about 8–9 weeks gestation. The small deceased co-twin and its gestational sac were not detected prenatally despite multiple ultrasonographic examinations. The difficulty in the interpretation of apparently conflicting results is emphasized.     

Alpha-fetoprotein and acetylcholinesterase are not predictive of fetal junctional epidermolysis bullosa,Herlitz variant

Junctional epidermolysis bullosa, Herlitz variant (junctional EB-Herlitz) is a lethal autosomal recessive skin disorder currently amenable to prenatal diagnosis only by direct analysis of fetal skin. However, elevated levels of alpha-fetoprotein, as well as the presence of acetylcholinesterase in amniotic fluid, have been associated with other severe fetal genodermatoses. Fetal skin samplings were performed in ten pregnancies at risk for fetal junctional EB-Herlitz, with three fetuses affected on the basis of electron microscopic detection of blisters within the lamina lucida and abnormal hemidesmosomes. In neither affected nor unaffected pregnancies were maternal serum or amniotic fluid alpha-fetoprotein levels elevated. Moreover, alphafetoprotein levels in both maternal serum and amniotic fluid were not statistically different comparing affected and unaffected fetuses. Acetylcholinesterase was not present in the amniotic fluid samples of the three affected pregnancies. Unlike other severe fetal genodermatoses, neither alpha-fetoprotein nor acetylcholinesterase was predictive of junctional EB-Herlitz.     

Origin of raised maternal serum alpha-fetoprotein levels in second-trimester oligohydramnios

Concanavalin A (Con A) subtyping of alpha-fetoprotein (AFP) revealed higher concentrations of AFP non-reactive with Con A in sera of 12 pregnant women with second-trimester oligohydramnios and raised total serum AFP levels than in sera of 42 pregnant women with raised total serum AFP levels and a normal amniotic fluid volume. This suggests that in oligohydramnios the origin of excess AFP in the maternal compartment is amniotic fluid. It is proposed that oligohydramnios and the associated raised maternal serum AFP levels are caused by damage of the fetal membranes prior to 16 weeks of gestation resulting in leakage of amniotic fluid to the decidual tissue and resorption in the maternal circulation.