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.     

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).     

First-trimester amniotic fluid and extraembryonic coelomic fluid acetylcholinesterase electrophoresis

Acetylcholinesterase (AChE) gel electrophoresis was performed on samples of amniotic fluid and extraembryonic coelomic fluid obtained by high resolution transvaginal ultrasound-guided amniocentesis from 38 women between 8 and 12 weeks of pregnancy. AChE was positive in 33 per cent (12/36) of the amniotic fluid samples; the percentage of positive results decreased as gestation advanced. AChE was positive in 32 per cent (9/28) of the extraembryonic coelomic fluid samples. In 81 per cent (21/26) of matched samples, the AChE results were identical in the two fluids. Amniotic fluid and extraembryonic coelomic fluid AChE electrophoresis cannot be used to diagnose neural tube defects prior to 12 weeks of gestation.     

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).     

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.     

Measurement of cholinesterases in amniotic fluid using a naphthyl acetate as substrate: A possible initial test for prenatal diagnosis of open neural tube defects

A quantitative method for cholinesterases in amniotic fluid using the non-specific substrate α naphthyl acetate and the cholinesterase-specific inhibitor, eserine, is described. This assay was used to test 671 samples of amniotic fluid. The diagnoses for fetal ONTDs, based on the levels of AChE + ChE, were compared with those made for the same samples by the AFP method. Correct diagnoses were made by both methods with amniotic fluid from 35 women carrying fetuses with ONTDs and 631 carrying normal fetuses. There were five false-positive test results for normal fetuses by both methods when the cut-off points were 5 standard deviations above the mean for AFP and above the upper limit of the normal range (7. 5 milliunits) for cholinesterase (AChE + ChE). None of the false-positive samples from either method had the acetylcholinesterase band of activity characteristic of ONTDs after gel electrophoresis. In addition to the above 671 samples, 37 pregnancies with serious fetal abnormalities other than ONTDs were tested. Two were identified by both the AFP and AChE + ChE methods, two more by AFP assay and one other by the AChE + ChE assay.     

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.     

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.     

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.     

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.     

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.     

Amniotic fluid acetylcholinesterase in the prenatal diagnosis of open neural tube defects and abdominal wall defects: A comparison of gel electrophoresis and a monoclonal antibody immunoassay

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.     

Prenatal diagnosis of spina bifida aperta after first-trimester valproate exposure

In the context of a prospective study on the adverse effects of anti-epileptic drugs on fetal outcome, we evaluated our experience with prenatal diagnosis by ultrasonography and α- fetoprotein (AFP) determination in amniotic fluid. We compared these results with AFP values in maternal serum obtained prior to amniocentesis. From November 1985 to July 1990, amniocentesis at 16–18 weeks of gestation was performed in 267 pregnancies of 237 different women using anti-epileptic drugs. Among 92 pregnancies with maternal valproic acid use, five (including one concordantly affected monozygotic twin-pair) were terminated because of a spina bifida aperta, all prenatally diagnosed by AFP determination and acetylcholinesterase electrophoresis in amniotic fluid. The maternal serum AFP level was raised (⩾ 2.5 multiples of the median (MOM) for singleton pregnancies and ⩾ 4.5 MOM for twin pregnancies) in only two of these five affected pregnancies. We emphasize that maternal serum AFP levels may be unreliable for prenatal screening for fetal neural tube defects in women taking valproate and recommend that amniocentesis and fetal ultrasound examination should be offered directly.     

The synaptic membrane D2-protein in amniotic fluid from pregnancies with fetal neural tube defects

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.     

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.     

S-100 protein in amniotic fluid of anencephalic fetuses

S-100 protein, which is found essentially in the astrocytes of the nervous system, was assayed in amniotic fluids by Particle Counting Immuno Assay. It was present in 19 cases of anencephaly out of 26, in 1 case of open spina bifida out of 5 and in each of the 4 cases of fetal death, whereas it was not detected in the 48 control amniotic fluids collected between the 16th and the 35th week of gestation. Thirty-one amniotic fluids from fetuses with other congenital malformations were devoid of detectable S-100. The presence of S-100 in amniotic fluid of anencephalic fetuses can presumably be considered as a biological sign of necrosis of the exencephalic brain and seems specific to damage of the central nervous system accompanied by neural tube defect.     

Alpha-fetoprotein and acetylcholinesterase in twins discordant for neural tube defect

Twins concordant for elevated alpha-fetoprotein (AFP) and acetylcholinesterase (AChE) and discordant for neural tube defect (NTD) and sex are reported. A literature review reveals instances of termination of twin pregnancies with one normal and one abnormal fetus, partly based on concordant high AFP and positive AChE (although discordant on ultrasound examination). The levels of AFP and AChE in twin pregnancies are probably a function of the number of layers of fetal membranes separating twin sacs: dichorionic, diamniotic membranes allow transfer of AFP; monochorionic, diamniotic membranes allow transfer of both AFP and AChE. Cautious interpretation of biochemical findings and reliance on high resolution ultrasonography are suggested.     

First-trimester biochemical screening for fetal chromosome abnormalities and neural tube defects

Alpha-fetoprotein (AFP), unconjugated oestriol (UE3), intact human chorionic gonado-trophin (intHCG), and the free β subunit of chorionic gonadotrophin (FβHCG) were investigated in a series of 21 chromosomally abnormal and 14 open neural tube defect pregnancies ascertained from a series of 14 000 prospectively collected maternal serum samples at 6–14 weeks' gestation. In 16 cases of Down's syndrome, significant reductions were found for AFP (0.65 multiples of the normal median) and UE3 (0.67 MOM). IntHCG levels were unaltered (0.97 MOM) but a significant increase was found for FβHCG (1.96 MOM). Significant correlations were found for AFP and UE3 in the controls and for int HCG and FβHCG in both the control and the Down's syndrome pregnancies. In a group of five trisomy 18 pregnancies, median MOMs were for AFP 0. 71 , for UE3 0. 34 , for intHCG 0. 27 , and for FβHCG 0.15. None of 13 pregnancies with open neural tube defects at 8-13 weeks gestation had elevated maternal serum AFP levels, whereas matched second-trimester samples from the same pregnancies at 16-18 weeks gestation all had significantly elevated AFP levels. Thus, biochemical screening for chromosome abnormalities may be practicable in the first trimester using free β human chorionic gonadotrophin in combination with AFP and maternal age. However, a separate screening protocol using AFP at 15-18 weeks gestation would still be required for effective detection of neural tube defects.     

The impact of maternal serum alpha fetoprotein screening on open neural tube defect births in North-East Scotland

Over the three years period 1980–1982, 18 256 pregnancies in the Grampian Region of N-E Scotland including the islands of Orkney and Shetland were screened for raised levels of maternal serum alpha fetoprotein (MSAFP) in the second trimester. Thirty six cases of fetal open neural tube defect in singletons were detected (18 anencephaly and 18 spina bifida). Four additional cases of open spina bifida were associated with normal MSAFP levels although two of these were detected by amniotic fluid AFP measurement when amniocentesis was carried out because of previous NTD history. A further three cases of open spina bifida and two of anencephaly occurred in unscreened pregnancies. The MSAFP screening programme alone was thus instrumental in reducing the birth incidence of open neural tube defects by 36 out of 45 cases (80 per cent) in singletons.