- Email: [email protected]
Routine amniotic fluid α-fetoprotein measurement in 34,000 pregnancies
Routine amniotic fluid 34,000 pregnancies
a- fetoprotein
measurement in
Barbara F. Crandall, M.D., and Myles Matsumoto Los Angeles, California Of 34,000 initial amniotic fluid samples, 0.7% had an a-fetoprotein level ~+3 SO above the mean. The risk of an open neural tube defect or other serious fetal abnormality was 60% when a-fetoprotein levels measured ~+3 and 86% for levels ~+5 SO. In this series there were 72 open neural tube defects, and all were identified. The true false positive rate was 0.9 per 10,000 cases screened. (AM. J. OBSTET. GVNECOL. 149:744, 1984.)
Most prenatal diagnosis centers now measure a-fetoprotein routinely in all second-trimester amniotic fluid samples. l- 3 The second report of the United Kingdom Collaborative Study concluded that 98% of open neural tube defects could be detected in this way.l However, additional results are necessary to determine the number of false positives as well as the risk of a neural tube defect or other serious abnormality at specific a-fetoprotein levels and the indications for additional tests. This report describes the results of routine amniotic fluid a-fetoprotein assay in 34,000 pregnancies monitored by second-trimester amniocentesis. The fluids were derived from seven different genetic centers in California: 60% from two centers* and the remainder from five centers.t Pregnancy outcomes are known in at least 95% of cases. Material and methods
The indications for amniocentesis were divided into five categories: maternal age of 34 years or more; two elevated assays of maternal serum a-fetoprotein; previous birth of an infant with a chromosome abnormality; previous birth of an infant with a neural tube defect; other indications. Measurement of amniotic fluid a-fetoprotein was usually recommended when either
From the Departments of Psychiatry and Pediatrics, University of California (Los Angeles) Medical Center. This research was supported in part by Grant HD-00345 for Research Training in Mental Retardation, Grant HD-05615 for Developmental Biology in Mental Retardation, and Grant 8300043 of the Genetic Disease Section of the California State Department of Health Services. Receivedfor publication October 3, 1983; accepted March 1, 1984. Reprint requests: Barbara F. Crandall, M.D., Departments of Psychiatry and Pediatrics, University of California (Los Angeles) Medical Center, Los Angeles, CA 90024.
*University of California (Los Angeles) Medical Center and University of California (San Francisco) Medical Center. tPermanente Hospital, San Jose; Stanford Medical Center; Harbor/University of California (Los Angeles) Medical Center, Torrance; Children's Hospital, Oakland; Los Angeles County/University of Southern California Medical Center.
744
parent or a maternal first COUSIn had a neural tube defect, and measurement of maternal serum a-fetoprotein and sonography were recommended when more distant relatives were affected. These indications are included in the "other indications" category. All centers provided counseling and obtained family and pregnancy histories prior to the amniocentesis. Amniocentesis was preceded by sonography. Bloody samples were tested by a Kleihauer-Betke test or immunoelectrophoresis for the presence of fetal blood. a-Fetoprotein levels were measured by radioimmunoassay after appropriate dilution, and all samples were assayed in duplicate. 4 Interassay variation was less than 10%. Our normal range was established from clear amniotic fluid samples from pregnancies in which the outcome was known to be normal. Gestational dates were confirmed by sonography. At least 100 samples were used for each week of gestation from 14 to 24 weeks, and the mean and standard deviations were established; +3 SD above the mean is about two times the median, and +5 SD is between two and one-half and three times the median. Acetylcholinesterase gel electrophoresis was carried out as previously described by disc technique." This test was performed when a-fetoprotein levels measured ~+2 SD above the mean or when there was a higher risk for a neural tube defect such as a positive family history or two elevated assays of maternal serum a-fetoprotein. It was not in use for about the first 5000 amniocenteses although many of the samples with elevated amniotic fluid a-fetoprotein levels were tested retrospectively. Results
After 34,000 pregnancies were monitored by amniotic fluid a-fetoprotein assay, the elevated amniotic fluid a-fetoprotein results were analyzed (Table I): 225 individuals (0.7%) had amniotic fluid a-fetoprotein measuring ~+3 SD above the mean; the fetus was normal in 91 cases (40%) and abnormal or dead in 134 (60%). In 132 cases (0.4%) amniotic fluid a-fetoprotein
Routine amniotic fluid a-fetoprotein measurement
Volume 149 Number 7
745
Table I. Outcome of 225 pregnancies with elevated amniotic fluid a-fetoprotein (AFP) levels AFP
Outcome
No. of cases
Bloody*
Normal Abnormal
91
26t
134
311
I
2:+3 <+5 SD Clear 46:j: 18
I
AFP
Total
Bloody*
72
13
21
6
I
2:+5 SD Clear 6§ 107
I
Total 19 113
*Fetal blood contamination. tOne false positive test. :j:Twin pregnancy with death of one fetus. §Two false positive tests. IIOne open spina bifida, one omphalocele, and one Dandy-Walker cyst.
measured 2:+5 SD above the mean; the fetus was normal in 19 (14%) of these cases and abnormal in 113 (86%).
Forty-eight of the samples with an elevated a-fetoprotein level contained fetal blood. When these were excluded, 177 of the clear amniotic fluid samples (0.5%) had a-fetoprotein levels of 2:+3 SD, and 113 (0.3%) were 2:+5 SD above the mean. In 125 (71%) of the former cases and 107 (95%) of the latter the fetus was dead or abnormal. Management of pregnancies with elevated levels of amniotic fluid a-fetoprotein. Initially a second amniocentesis was recommended whenever the amniotic fluid a-fetoprotein level measured 2:+3 SD above the mean. With the addition of the acetylcholinesterase test, this was usually avoided if the sample contained fetal blood and the a-fetoprotein level did not exceed 2:+5 SD, providing the sonogram and acetylcholinesterase test were normal. For levels 2:+5 SD, a second amniocentesis was usually recommended regardless of fetal blood contamination. Normal pregnancies with elevated levels of amniotic fluid aJetoprotein. Only one normal pregnancy with an initial
amniocentesis a-fetoprotein level of +3 to +5 SD had an elevated a-fetoprotein reading in the second amniocentesis. This sample contained fetal blood. The pregnancy was terminated. Of those cases with first amniocenteses a-fetoprotein levels of 2:+5 SD, one bloody and six clear amniotic fluid samples still showed elevated a-fetoprotein levels in the second amniocentesis. In five of these cases the levels were 2:+3 and ~+5, and the pregnancies were continued with normal outcomes; in two cases the levels remained 2:5 SD above the mean, and the pregnancies were terminated. These false positive tests will be discussed later. All had normal acetylcholinesterase tests, but three were retrospective tests. Acetylcholinesterase gel electrophoresis. The qualitative acetylcholinesterase test shows little change with gestational age, at least between 15 and about 24 weeks, and is much less affected by fetal blood contamination. 6 However, when the sample was very bloody, a faint or wide second band was sometimes noted on the gel.
There were eight false poslUve acetylcholinesterase tests, seven in bloody fluid and one in clear fluid. All were "weak" positives as described above, and a second amniocentesis was performed. The acetylcholinesterase result was clearly positive in one case of open spina bifida although the elevated amniotic fluid a-fetoprotein level appeared to be explained by fetal blood contamination. The positive acetylcholinesterase test led to the detection of another case of open spina bifida although the amniotic fluid a-fetoprotein measured +2 SD above the mean. Positive acetylcholinesterase tests also confirmed the presence of two encephaloceles with amniotic fluid a-fetoprotein levels of +2 SD above the mean and three with levels between +3 and +5 SD. The acetylcholinesterase test was normal in one case of congenital nephrosis and in another with amniotic bands, but abnormal in one case each of sacral teratoma and duodenal atresia. Fetal abnormalities Neural tube defects. Fetal abnormalities detected by elevated amniotic fluid a-fetoprotein levels are shown in Table II. There were 72 cases of open neural tube defect, including 28 cases of open spina bifida and 35 anencephalies. One cervical spina bifid a was identified by sonography; both the amniotic fluid a-fetoprotein and acetylcholinesterase measurements were normal, and a skin-covered defect was confirmed after elective abortion. Two closed occipital encephaloceles were detected at delivery; both the amniotic fluid a-fetoprotein and acetylcholinesterase measurements had been normal. No case of simple hydrocephalus had an elevated amniotic fluid a-fetoprotein level. There were three Dandy-Walker cysts~all identified by sonography; the elevated levels of amniotic fluid a-fetoprotein and positive acetylcholinesterase results in one case probably resulted from fetal blood contamination. The risks of an open neural tube defect were 31 % and 45% if the amniotic fluid a-fetoprotein levels measured 2:+3 SD and 2:+5 SD above the mean, respectively. Other abnormalities. In this series there were 20 omphaloceles; amniotic fluid a-fetoprotein levels were elevated in 19 and the acetylcholinesterase test was positive in about half, but the specific band was faint
746
Crandall and Matsumoto
August 1, 1984 Am. J. Obstet. Gynecol.
Table II. Abnormalities detected at different amniotic fluid a-fetoprotein (AFP) levels* AFP +2 SD Abnormality
Open spina bifid a Anencephaly Encephalocele
No.
2
I
AChE+t
2
AFP:2':+3<+5SD No.
I
AFP :2':+5 SD AChE+t
No. detected
20 35t 4
20 27§ 211
28 35 9
18 16 7 9** 4#
13 IO 7 8 2
22 20 7 13 6
AChE+t
No.
7
7
3
3
4 3
0 I
I
72
Total open neural tube defects Fetal death Omphalocele Hydrops/ascites~ hygroma~
Cystic Other
Total other abnormalities* Total abnormalities
3# 2tt
68 140
*Prematurity, small gestational weight, congenital heart defect, cleft lip and palate, hip dislocation, clubfoot, inguinal hernia, and polydactyly are not included. t Positive acetylcholinesterase test. tBoth monoamniotic twins affected; counted as one case. §Eight cases prior to acetylcholinesterase test. IITwo cases prior to acetylcholinesterase test. ~AFP < + 2 SD in orie case. #One case with trisomy 21 **One case with trisomy 18 ttOne case with trisomy 18 and other with Dandy-Walker cyst. ttOne case each of congenital nephrosis, amniotic band syndrome (both acetylcholinesterase-negative), duodenal atresia, and sacral teratoma (both acetylcholinesterase-positive).
and usually easily distinguished from that seen in open neural tube defects. Except for one case of severe Kell sensitization, the cause of fetal hydrops or ascites was not known. One case of cystic hygroma had a normal amniotic fluid a-fetoprotein level but the acetylcholinesterase test was positive; the others all had elevated a-fetoprotein levels. The cystic hygromas were all associated with Turner's syndrome (45, X) except for one case each of trisomy 21 and trisomy 18. False positive and negative tests. There were three true false positive tests, which we defined as ones resulting in the termination of the pregnancy of a normal fetus. In one case the initial amniotic fluid a-fetoprotein level measured +4 SD and the sample contained fetal blood; a second amniocentesis resulted in an amniotic fluid a-fetoprotein level of about the same level. In the second case, both amniotic fluid samples were clear; the first measured above +9 SD and the second between +6 and +7 SD above the mean. Spina bifida occulta of the lower lumbar vertebrae and the entire sacrum was noted at autopsy; the kidneys were not examined. In the third case, the amniotic fluid sample was clear and the a-fetoprotein level measured :;::+9 SD and remained :;::+5 SD on the second amniocentesis. The acetylcholinesterase test was consistently negative. The pregnancy was terminated and no abnormalities were detected at autopsy although the kidneys were not examined microscopically. The iden-
tical findings recurred in a succeeding pregnancy, which this time was continued with an apparently normal outcome. a-Fetoprotein levels in the mother and child were normal. The incidence of true false positives in this series was 0.9 per 10,000. Acetylcholinesterase gel electrophoresis, which was added after the first two false positives, would have corrected both. It predicted correctly that the third case was not an open neural tube defect. There "!:IS no false negative tests for open neural tube defects. Recurrence risks for neural tube defects. In 911 (2.7%) of cases the indication for amu'iocentesis was a previous child with a neural tube defect. An open neural tube defect recurred in 1.8% of these women, and it was the same type in about half the cases. One case of encephalocele was most probably due to Meckel's syndrome. A second case of encephalocele preceded by two hydrocephalic births and a previous anencephalic birth lacked any other evidence of Meckel's syndrome. If both of these cases are excluded, the recurrence rate is 1.6%. In addition, two cases of simple hydrocephalus occurred in women with a previous anencephalic birth and were detected by sonography. Amniotic fluid a-fetoprotein levels and acetylcholinesterase tests were normal. Neural tube defects and maternal age. Of the 34,000 amniocenteses 29,031 were performed because of maternal age :;::35 years. Forty-three open neural tube de-
Volume 149 Number 7
fects were identified in this group; 19 anencephalies, 18 cases of open spina bifid a, and 6 encephaloceles. The risk for an open neural tube defect in this group is therefore 1.5 per 1000. This compares to 1.1 per 1000 in a younger group of women identified in a maternal serum a-fetoprotein screening program. 4 Comment
Seventy-two open neural tube defects were detected in this series of 34,000 mid-trimester amniocenteses. All the cases of anencephaly showed marked elevation of a-fetoprotein (~+5 SD). Eight of 28 cases of open spina bifida (29%) had lower levels of a-fetoprotein, 7 between +3 and +5 SD and one at +2 SD above the mean. All of those tested by acetylcholinesterase gel electrophoresis were clearly positive. There were therefore no false negative tests for open neural tube defects. There were 3 true false positives; two would have been corrected by the acetylcholinesterase test, which also excluded a neural tube defect in the third. A positive acetylcholinesterase test is not specific for a neural tube defect, and other abnormalities identified by a-fetoprotein levels may also yield a positive result. These can frequently be distinguished from neural tube defects by the darkness of the specific band but are usually diagnosed by sonography. Congenital nephrosis has given a consistently negative acetylcholinesterase test. The identification of neural tube defects and certain other fetal abnormalities by sonography is usually highly accurate in expert hands. However, low-lying or "flat" open spina bifida anomalies such as a myeloschisis may prove particularly difficult to identify, and sonography is best used in conjunction with biochemical studies. In our experience, a-fetoprotein assay is the best test for the detection of neural tube defects, particularly if a large number of samples are being tested. However, the a-fetoprotein level is more sensitive to fetal blood contamination and changes in gestational age than the acetylcholinesterase level. Acetylcholinesterase can be measured quantitatively or examined qualitatively. Most reports suggest that the former fails to discriminate clearly between normal and abnormal levels. B • 7 The qualitative test may be included routinely for all amniotic fluid samples if a slab gel technique is used. Nevertheless, we have found interpretation of the bands to be less equivocal with disc electrophoresis although this has a more limited application for a large number of cases. As a compromise, we include the test when a-fetoprotein levels measure ~+2 SD or when there is an increased risk for neural tube defects. Our
Routine amniotic fluid a-fetoprotein measurement
747
experience suggests that in this way it would be very unlikely to miss an open neural tube defect. The risk of a serious abnormality or fetal demise after the first elevated amniotic fluid a-fetoprotein reading was 60% if the level measured ~+3 SD and 86% for levels ~+5 SD above the mean. Fetal blood contamination explained the elevated a-fetoprotein level in the 43% of cases with normal outcomes. When the acetylcholinesterase test and sonogram are normal, we have avoided a second amniocentesis in many cases if the a-fetoprotein measured between +3 and +5 SDs. THis decision, however, depended on adequate sonographic visualization and an unequivocal acetylcholinesterase test. A repeat amniocentesis was usually recommended when a-fetoprotein levels measured ~+5 SD regardless of the initial sonographic interpretation and the acetylcholinesterase test results. We wish to thank the following physicians and their staffs for supplying amniotic fluid samples and followup data: Drs. Mitchell Golbus, University of California (San Francisco) Medical Center, John Mann, Permanente Hospital, San Jose, Howard Cann, Stanford Medical Center, Michael Kaback, Harbor/University of California (Los Angeles) Medical Center, Torrance, Sanford Sherman, Oakland Children's Hospital, Miriam Wilson, County/University of Southern California Medical Center, and Cindy Curry, Valley Children's Hospital, Fresno; we also thank Drs. T. Lebherz, Lidia Rubinstein, A. Ketupanya, Dennis Sarti, and Ms. K. Yamazaki, of the University of California (Los Angeles) Prenatal Diagnostic Center, Ms. Sash a Biletsky, and the Hoffman-LaRoche Company. REFERENCES 1. Second report of the United Kingdom Collaborative Study: amniotic fluid alphafetoprotein measurement in antenatal diagnosis of anencephaly and open spina bifida in early pregnancy. Lancet 2:651-662, 1979. 2. Milunsky A. Prenatal detection of neural tube defects: experience with 20,000 pregnancies. JAM A 1980;244:27312735. 3. Brock DjH. Scrimgeour lB. Alphafetoprotein assay in all amniocentesis samples. Lancet 1976; 1: 1404. 4. Crandall BF, Robertson RD, Lebherz TB, King W, Schroth PC. Maternal serum alpha-fetoprotein screening for the detection of neural tube defects. West j Med 1983;138: 524-530. 5. Crandall BF, Kasha W, Matsumoto M. Prenatal diagnosis of neural tube defects: experiences with acetylcholinesterase gel electrophoresis. Am j Med Genet 1982; 12:361366. 6. Smith AD, Wald Nj, Cuckle HS, Stiratt GM, Bobrow M, Langercrantz H. Amniotic fluid acetylcholinesterase as a possible diagnostic test for neural tube defects in early pregnancy. Lancet 1979; 1:685-688. 7. Chubb IW, Pilowsky PM, Springell Hj, Pollard AC. Acetylcholinesterase in human amniotic fluid: an index of fetal neural development? Lancet 1979; 1:688-690.
a- fetoprotein
measurement in
Barbara F. Crandall, M.D., and Myles Matsumoto Los Angeles, California Of 34,000 initial amniotic fluid samples, 0.7% had an a-fetoprotein level ~+3 SO above the mean. The risk of an open neural tube defect or other serious fetal abnormality was 60% when a-fetoprotein levels measured ~+3 and 86% for levels ~+5 SO. In this series there were 72 open neural tube defects, and all were identified. The true false positive rate was 0.9 per 10,000 cases screened. (AM. J. OBSTET. GVNECOL. 149:744, 1984.)
Most prenatal diagnosis centers now measure a-fetoprotein routinely in all second-trimester amniotic fluid samples. l- 3 The second report of the United Kingdom Collaborative Study concluded that 98% of open neural tube defects could be detected in this way.l However, additional results are necessary to determine the number of false positives as well as the risk of a neural tube defect or other serious abnormality at specific a-fetoprotein levels and the indications for additional tests. This report describes the results of routine amniotic fluid a-fetoprotein assay in 34,000 pregnancies monitored by second-trimester amniocentesis. The fluids were derived from seven different genetic centers in California: 60% from two centers* and the remainder from five centers.t Pregnancy outcomes are known in at least 95% of cases. Material and methods
The indications for amniocentesis were divided into five categories: maternal age of 34 years or more; two elevated assays of maternal serum a-fetoprotein; previous birth of an infant with a chromosome abnormality; previous birth of an infant with a neural tube defect; other indications. Measurement of amniotic fluid a-fetoprotein was usually recommended when either
From the Departments of Psychiatry and Pediatrics, University of California (Los Angeles) Medical Center. This research was supported in part by Grant HD-00345 for Research Training in Mental Retardation, Grant HD-05615 for Developmental Biology in Mental Retardation, and Grant 8300043 of the Genetic Disease Section of the California State Department of Health Services. Receivedfor publication October 3, 1983; accepted March 1, 1984. Reprint requests: Barbara F. Crandall, M.D., Departments of Psychiatry and Pediatrics, University of California (Los Angeles) Medical Center, Los Angeles, CA 90024.
*University of California (Los Angeles) Medical Center and University of California (San Francisco) Medical Center. tPermanente Hospital, San Jose; Stanford Medical Center; Harbor/University of California (Los Angeles) Medical Center, Torrance; Children's Hospital, Oakland; Los Angeles County/University of Southern California Medical Center.
744
parent or a maternal first COUSIn had a neural tube defect, and measurement of maternal serum a-fetoprotein and sonography were recommended when more distant relatives were affected. These indications are included in the "other indications" category. All centers provided counseling and obtained family and pregnancy histories prior to the amniocentesis. Amniocentesis was preceded by sonography. Bloody samples were tested by a Kleihauer-Betke test or immunoelectrophoresis for the presence of fetal blood. a-Fetoprotein levels were measured by radioimmunoassay after appropriate dilution, and all samples were assayed in duplicate. 4 Interassay variation was less than 10%. Our normal range was established from clear amniotic fluid samples from pregnancies in which the outcome was known to be normal. Gestational dates were confirmed by sonography. At least 100 samples were used for each week of gestation from 14 to 24 weeks, and the mean and standard deviations were established; +3 SD above the mean is about two times the median, and +5 SD is between two and one-half and three times the median. Acetylcholinesterase gel electrophoresis was carried out as previously described by disc technique." This test was performed when a-fetoprotein levels measured ~+2 SD above the mean or when there was a higher risk for a neural tube defect such as a positive family history or two elevated assays of maternal serum a-fetoprotein. It was not in use for about the first 5000 amniocenteses although many of the samples with elevated amniotic fluid a-fetoprotein levels were tested retrospectively. Results
After 34,000 pregnancies were monitored by amniotic fluid a-fetoprotein assay, the elevated amniotic fluid a-fetoprotein results were analyzed (Table I): 225 individuals (0.7%) had amniotic fluid a-fetoprotein measuring ~+3 SD above the mean; the fetus was normal in 91 cases (40%) and abnormal or dead in 134 (60%). In 132 cases (0.4%) amniotic fluid a-fetoprotein
Routine amniotic fluid a-fetoprotein measurement
Volume 149 Number 7
745
Table I. Outcome of 225 pregnancies with elevated amniotic fluid a-fetoprotein (AFP) levels AFP
Outcome
No. of cases
Bloody*
Normal Abnormal
91
26t
134
311
I
2:+3 <+5 SD Clear 46:j: 18
I
AFP
Total
Bloody*
72
13
21
6
I
2:+5 SD Clear 6§ 107
I
Total 19 113
*Fetal blood contamination. tOne false positive test. :j:Twin pregnancy with death of one fetus. §Two false positive tests. IIOne open spina bifida, one omphalocele, and one Dandy-Walker cyst.
measured 2:+5 SD above the mean; the fetus was normal in 19 (14%) of these cases and abnormal in 113 (86%).
Forty-eight of the samples with an elevated a-fetoprotein level contained fetal blood. When these were excluded, 177 of the clear amniotic fluid samples (0.5%) had a-fetoprotein levels of 2:+3 SD, and 113 (0.3%) were 2:+5 SD above the mean. In 125 (71%) of the former cases and 107 (95%) of the latter the fetus was dead or abnormal. Management of pregnancies with elevated levels of amniotic fluid a-fetoprotein. Initially a second amniocentesis was recommended whenever the amniotic fluid a-fetoprotein level measured 2:+3 SD above the mean. With the addition of the acetylcholinesterase test, this was usually avoided if the sample contained fetal blood and the a-fetoprotein level did not exceed 2:+5 SD, providing the sonogram and acetylcholinesterase test were normal. For levels 2:+5 SD, a second amniocentesis was usually recommended regardless of fetal blood contamination. Normal pregnancies with elevated levels of amniotic fluid aJetoprotein. Only one normal pregnancy with an initial
amniocentesis a-fetoprotein level of +3 to +5 SD had an elevated a-fetoprotein reading in the second amniocentesis. This sample contained fetal blood. The pregnancy was terminated. Of those cases with first amniocenteses a-fetoprotein levels of 2:+5 SD, one bloody and six clear amniotic fluid samples still showed elevated a-fetoprotein levels in the second amniocentesis. In five of these cases the levels were 2:+3 and ~+5, and the pregnancies were continued with normal outcomes; in two cases the levels remained 2:5 SD above the mean, and the pregnancies were terminated. These false positive tests will be discussed later. All had normal acetylcholinesterase tests, but three were retrospective tests. Acetylcholinesterase gel electrophoresis. The qualitative acetylcholinesterase test shows little change with gestational age, at least between 15 and about 24 weeks, and is much less affected by fetal blood contamination. 6 However, when the sample was very bloody, a faint or wide second band was sometimes noted on the gel.
There were eight false poslUve acetylcholinesterase tests, seven in bloody fluid and one in clear fluid. All were "weak" positives as described above, and a second amniocentesis was performed. The acetylcholinesterase result was clearly positive in one case of open spina bifida although the elevated amniotic fluid a-fetoprotein level appeared to be explained by fetal blood contamination. The positive acetylcholinesterase test led to the detection of another case of open spina bifida although the amniotic fluid a-fetoprotein measured +2 SD above the mean. Positive acetylcholinesterase tests also confirmed the presence of two encephaloceles with amniotic fluid a-fetoprotein levels of +2 SD above the mean and three with levels between +3 and +5 SD. The acetylcholinesterase test was normal in one case of congenital nephrosis and in another with amniotic bands, but abnormal in one case each of sacral teratoma and duodenal atresia. Fetal abnormalities Neural tube defects. Fetal abnormalities detected by elevated amniotic fluid a-fetoprotein levels are shown in Table II. There were 72 cases of open neural tube defect, including 28 cases of open spina bifida and 35 anencephalies. One cervical spina bifid a was identified by sonography; both the amniotic fluid a-fetoprotein and acetylcholinesterase measurements were normal, and a skin-covered defect was confirmed after elective abortion. Two closed occipital encephaloceles were detected at delivery; both the amniotic fluid a-fetoprotein and acetylcholinesterase measurements had been normal. No case of simple hydrocephalus had an elevated amniotic fluid a-fetoprotein level. There were three Dandy-Walker cysts~all identified by sonography; the elevated levels of amniotic fluid a-fetoprotein and positive acetylcholinesterase results in one case probably resulted from fetal blood contamination. The risks of an open neural tube defect were 31 % and 45% if the amniotic fluid a-fetoprotein levels measured 2:+3 SD and 2:+5 SD above the mean, respectively. Other abnormalities. In this series there were 20 omphaloceles; amniotic fluid a-fetoprotein levels were elevated in 19 and the acetylcholinesterase test was positive in about half, but the specific band was faint
746
Crandall and Matsumoto
August 1, 1984 Am. J. Obstet. Gynecol.
Table II. Abnormalities detected at different amniotic fluid a-fetoprotein (AFP) levels* AFP +2 SD Abnormality
Open spina bifid a Anencephaly Encephalocele
No.
2
I
AChE+t
2
AFP:2':+3<+5SD No.
I
AFP :2':+5 SD AChE+t
No. detected
20 35t 4
20 27§ 211
28 35 9
18 16 7 9** 4#
13 IO 7 8 2
22 20 7 13 6
AChE+t
No.
7
7
3
3
4 3
0 I
I
72
Total open neural tube defects Fetal death Omphalocele Hydrops/ascites~ hygroma~
Cystic Other
Total other abnormalities* Total abnormalities
3# 2tt
68 140
*Prematurity, small gestational weight, congenital heart defect, cleft lip and palate, hip dislocation, clubfoot, inguinal hernia, and polydactyly are not included. t Positive acetylcholinesterase test. tBoth monoamniotic twins affected; counted as one case. §Eight cases prior to acetylcholinesterase test. IITwo cases prior to acetylcholinesterase test. ~AFP < + 2 SD in orie case. #One case with trisomy 21 **One case with trisomy 18 ttOne case with trisomy 18 and other with Dandy-Walker cyst. ttOne case each of congenital nephrosis, amniotic band syndrome (both acetylcholinesterase-negative), duodenal atresia, and sacral teratoma (both acetylcholinesterase-positive).
and usually easily distinguished from that seen in open neural tube defects. Except for one case of severe Kell sensitization, the cause of fetal hydrops or ascites was not known. One case of cystic hygroma had a normal amniotic fluid a-fetoprotein level but the acetylcholinesterase test was positive; the others all had elevated a-fetoprotein levels. The cystic hygromas were all associated with Turner's syndrome (45, X) except for one case each of trisomy 21 and trisomy 18. False positive and negative tests. There were three true false positive tests, which we defined as ones resulting in the termination of the pregnancy of a normal fetus. In one case the initial amniotic fluid a-fetoprotein level measured +4 SD and the sample contained fetal blood; a second amniocentesis resulted in an amniotic fluid a-fetoprotein level of about the same level. In the second case, both amniotic fluid samples were clear; the first measured above +9 SD and the second between +6 and +7 SD above the mean. Spina bifida occulta of the lower lumbar vertebrae and the entire sacrum was noted at autopsy; the kidneys were not examined. In the third case, the amniotic fluid sample was clear and the a-fetoprotein level measured :;::+9 SD and remained :;::+5 SD on the second amniocentesis. The acetylcholinesterase test was consistently negative. The pregnancy was terminated and no abnormalities were detected at autopsy although the kidneys were not examined microscopically. The iden-
tical findings recurred in a succeeding pregnancy, which this time was continued with an apparently normal outcome. a-Fetoprotein levels in the mother and child were normal. The incidence of true false positives in this series was 0.9 per 10,000. Acetylcholinesterase gel electrophoresis, which was added after the first two false positives, would have corrected both. It predicted correctly that the third case was not an open neural tube defect. There "!:IS no false negative tests for open neural tube defects. Recurrence risks for neural tube defects. In 911 (2.7%) of cases the indication for amu'iocentesis was a previous child with a neural tube defect. An open neural tube defect recurred in 1.8% of these women, and it was the same type in about half the cases. One case of encephalocele was most probably due to Meckel's syndrome. A second case of encephalocele preceded by two hydrocephalic births and a previous anencephalic birth lacked any other evidence of Meckel's syndrome. If both of these cases are excluded, the recurrence rate is 1.6%. In addition, two cases of simple hydrocephalus occurred in women with a previous anencephalic birth and were detected by sonography. Amniotic fluid a-fetoprotein levels and acetylcholinesterase tests were normal. Neural tube defects and maternal age. Of the 34,000 amniocenteses 29,031 were performed because of maternal age :;::35 years. Forty-three open neural tube de-
Volume 149 Number 7
fects were identified in this group; 19 anencephalies, 18 cases of open spina bifid a, and 6 encephaloceles. The risk for an open neural tube defect in this group is therefore 1.5 per 1000. This compares to 1.1 per 1000 in a younger group of women identified in a maternal serum a-fetoprotein screening program. 4 Comment
Seventy-two open neural tube defects were detected in this series of 34,000 mid-trimester amniocenteses. All the cases of anencephaly showed marked elevation of a-fetoprotein (~+5 SD). Eight of 28 cases of open spina bifida (29%) had lower levels of a-fetoprotein, 7 between +3 and +5 SD and one at +2 SD above the mean. All of those tested by acetylcholinesterase gel electrophoresis were clearly positive. There were therefore no false negative tests for open neural tube defects. There were 3 true false positives; two would have been corrected by the acetylcholinesterase test, which also excluded a neural tube defect in the third. A positive acetylcholinesterase test is not specific for a neural tube defect, and other abnormalities identified by a-fetoprotein levels may also yield a positive result. These can frequently be distinguished from neural tube defects by the darkness of the specific band but are usually diagnosed by sonography. Congenital nephrosis has given a consistently negative acetylcholinesterase test. The identification of neural tube defects and certain other fetal abnormalities by sonography is usually highly accurate in expert hands. However, low-lying or "flat" open spina bifida anomalies such as a myeloschisis may prove particularly difficult to identify, and sonography is best used in conjunction with biochemical studies. In our experience, a-fetoprotein assay is the best test for the detection of neural tube defects, particularly if a large number of samples are being tested. However, the a-fetoprotein level is more sensitive to fetal blood contamination and changes in gestational age than the acetylcholinesterase level. Acetylcholinesterase can be measured quantitatively or examined qualitatively. Most reports suggest that the former fails to discriminate clearly between normal and abnormal levels. B • 7 The qualitative test may be included routinely for all amniotic fluid samples if a slab gel technique is used. Nevertheless, we have found interpretation of the bands to be less equivocal with disc electrophoresis although this has a more limited application for a large number of cases. As a compromise, we include the test when a-fetoprotein levels measure ~+2 SD or when there is an increased risk for neural tube defects. Our
Routine amniotic fluid a-fetoprotein measurement
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experience suggests that in this way it would be very unlikely to miss an open neural tube defect. The risk of a serious abnormality or fetal demise after the first elevated amniotic fluid a-fetoprotein reading was 60% if the level measured ~+3 SD and 86% for levels ~+5 SD above the mean. Fetal blood contamination explained the elevated a-fetoprotein level in the 43% of cases with normal outcomes. When the acetylcholinesterase test and sonogram are normal, we have avoided a second amniocentesis in many cases if the a-fetoprotein measured between +3 and +5 SDs. THis decision, however, depended on adequate sonographic visualization and an unequivocal acetylcholinesterase test. A repeat amniocentesis was usually recommended when a-fetoprotein levels measured ~+5 SD regardless of the initial sonographic interpretation and the acetylcholinesterase test results. We wish to thank the following physicians and their staffs for supplying amniotic fluid samples and followup data: Drs. Mitchell Golbus, University of California (San Francisco) Medical Center, John Mann, Permanente Hospital, San Jose, Howard Cann, Stanford Medical Center, Michael Kaback, Harbor/University of California (Los Angeles) Medical Center, Torrance, Sanford Sherman, Oakland Children's Hospital, Miriam Wilson, County/University of Southern California Medical Center, and Cindy Curry, Valley Children's Hospital, Fresno; we also thank Drs. T. Lebherz, Lidia Rubinstein, A. Ketupanya, Dennis Sarti, and Ms. K. Yamazaki, of the University of California (Los Angeles) Prenatal Diagnostic Center, Ms. Sash a Biletsky, and the Hoffman-LaRoche Company. REFERENCES 1. Second report of the United Kingdom Collaborative Study: amniotic fluid alphafetoprotein measurement in antenatal diagnosis of anencephaly and open spina bifida in early pregnancy. Lancet 2:651-662, 1979. 2. Milunsky A. Prenatal detection of neural tube defects: experience with 20,000 pregnancies. JAM A 1980;244:27312735. 3. Brock DjH. Scrimgeour lB. Alphafetoprotein assay in all amniocentesis samples. Lancet 1976; 1: 1404. 4. Crandall BF, Robertson RD, Lebherz TB, King W, Schroth PC. Maternal serum alpha-fetoprotein screening for the detection of neural tube defects. West j Med 1983;138: 524-530. 5. Crandall BF, Kasha W, Matsumoto M. Prenatal diagnosis of neural tube defects: experiences with acetylcholinesterase gel electrophoresis. Am j Med Genet 1982; 12:361366. 6. Smith AD, Wald Nj, Cuckle HS, Stiratt GM, Bobrow M, Langercrantz H. Amniotic fluid acetylcholinesterase as a possible diagnostic test for neural tube defects in early pregnancy. Lancet 1979; 1:685-688. 7. Chubb IW, Pilowsky PM, Springell Hj, Pollard AC. Acetylcholinesterase in human amniotic fluid: an index of fetal neural development? Lancet 1979; 1:688-690.