British Journal of Obstetrics and Gynaecology August 1978. V O 85. ~ pp 575-581

MATERNAL PLASMA ALPHA-FETOPROTEIN SCREENING FOR FETAL NEURAL TUBE DEFECTS BY

D. J. H. BROCK,Reader Department of Human Genetics, University of Edinburgh J. B. SCRIMGEOUR, Senior Lecturer J. STEVEN, Registrar

Department of Obstetrics and Gynaecology, Western GeneraI Hospital, Edinburgh 4 LILIASBARRON,Technician Department of Human Genetics, University of Edinburgh AND

MURIEL WATT,Research Assistant Department of Obstetrics and Gynaecology, Western General Hospital, Edinburgh 4

Summary Maternal plasma alpha-fetoprotein (AFP) screening for fetal neural tube defects (NTD) was used as a part of routine antenatal care in three hospitals over a 26 month period. Blood samples were obtained for plasma AFP measurement at 15 to 20 weeks gestation from 6377 women, representing 79 per cent of antenatal bookings. The outcome of pregnancy was ascertained in 96 per cent of patients: 13 cases of anencephaly and 7 of open spina bifida were detected by plasma screening and a further 3 cases of open NTD through the mother’s previous medical history and amniotic fluid determination. Four fetuses with open NTDs and four with closed NTDs were not detected by plasma AFP measurement and the detection efficiency for open NTDs was thus 83 per cent. Integration of screening into the existing pattern of antenatal care required only minor alterations in clinic schedules. Some extra time was needed for explanation of the objectives of the study, for ultrasound examination and for amniocentesis. Eight patients declined the offer of a plasma test, while only one refused an amniocentesis.

be at increased risk. Thus there is a case for screening all pregnancies for NTD by measuring AFP in maternal blood (Brock et a / , 1973; Leek et al, 1973) and there have been a number of reports about this, notably the United Kingdom Collaborative Study on AFP in Relation to Neural Tube Defects (1977). We now describe a prospective study in which maternal plasma AFP screening was integrated

ANENCEPHALY and open spina bifida may be diagnosed early in pregnancy by measurement of alpha-fetoprotein (AFP) in the amniotic fluid (Brock and Sutcliffe, 1972; Brock and Scrimgeour, 1972) of patients known to be at increased risk of bearing children with neural tube defects (NTD). However, more than 90 per cent of children with neural tube defects are born to mothers not thought to 515

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BROCK, SCRIMGEOUR, STEVEN, BARRON AND WATT

into routine antenatal care over a period of 26 months. METHODS Patients Our patients booked for confinement at the Western General Hospital (WGH) in North Edinburgh (1400 deliveries per annum), Dunfermline Maternity Hospital (DMH) (1400 deliveries per annum) and Forth Park Maternity Hospital (FPMH) (2400 deliveries per annum). At DMH, all patients with raised serum AFP levels were referred to WGH for ultrasound examination and if necessary, amniocentesis; the patients at other hospitals were not sent elsewhere. No extra medical staff were employed except for a research assistant with nursing qualifications who co-ordinated results and assisted with amniocentesis at WGH. AFP assays were done by a senior and junior technician, who sent out results to all three hospitals and took responsibility for ensuring that second blood samples were requested where necessary.

Organization Patients who were less than 20 weeks gestation at the time of booking were told about the nature of the plasma AFP test and asked to participate in the study. Those attending between 15 and 20 weeks gestation had a blood sample taken immediately. Patients who had completed less than 15 weeks of amenorrhea were given an appointment to return between 15 and 16 weeks gestation. Those who defaulted were sent an appointment for the next clinic, and if there was no response contacted by the sister in charge of the antenatal clinic, or by their General Practitioner, district midwife or health visitor. The duration of pregnancy was calculated in completed weeks from the onset of the last menstrual period. If dates were uncertain gestational age was revised by information gained from clinical examination or ultrasound scans. However, results recorded in the Tables are based on unrevised gestational ages. Patients who were more than 20 weeks pregnant at first booking were excluded from the study. Any patient, who was aged over 39

or who had a family history of NTD, was offered amniocentesis without preliminary plasma AFP estimation.

Management of patients with raised plasma AFP values A plasma AFP value above the 95th centile for the week in question was considered abnormal. Patients with raised levels were contacted by telephone or letter and arrangements made for a repeat blood sample and further explanation. Early in the study patients with AFP values above the estimated 99th centile were referred directly for ultrasound assessment. At the same time a second blood sample was taken, and the AFP value determined. Subsequently, all patients with values above the 95th centile had a second AFP assay before ultrasound examination was considered. If the AFP value on the second sample lay below the 95th centile no further action was taken. If the value remained above the 95th centile the patient was referred for ultrasound examination and possible amniocentesis. The biparietal diameter was measured and the result compared with the menstrual history. If a major discrepancy existed the result of ultrasound and clinical examination were given precedence. If ultrasound examination suggested the presence of twins, amniocentesis was not attempted. Patients who had vaginal bleeding at the time of the second plasma AFP determination had ultrasound examination to confirm fetal life. An amniocentesis was only done if a third plasma AFP value was abnormal a week after the bleeding stopped. Once mistaken gestational age, multiple pregnancy and threatened or missed abortion had been excluded, patients with raised plasma AFP levels were told that the chance of an open fetal NTD was about 1 in 10 and that amniocentesis was required if an accurate diagnosis was desired. If the results of amniocentesis done under ultrasound control suggested the presence of a fetus with an open NTD, termination of pregnancy by instillation of prostaglandin was explained and offered; if the offer was accepted early admission was arranged.

PLASMA AFP SCREENING

MATERNAL -1 4 0 0 ~PLASMA A F P



I

-

I

t-

stages of the study, examination of the morphology of rapidly adherent amniotic fluid cells was also used in diagnosing NTD (Gosden and Brock, 1977).

I

300

-’

577

15 16 17 18 19 20 21 GESTATION ( W E E K S )

FIG.1 Sample of reporting chart on which plasma AFP values were reported to consultant obstetricians.

Laboratory methods Plasma AFP was determined by radioimmunoassay methods (Brock et al, 1978). Results were reported on a form allowing the consultant some freedom of interpretation (Fig. 1). Amniotic fluid AFP was measured by the Laurel1 rocket technique (Brock and Sutcliffe, 1972), and compared with published normal ranges (Brock et al, 1975). In the latter

RESULTS The patients included in this study had plasma AFP estimations between 1st January, 1975 and 28th February, 1977; thus all pregnancies had ended before analysis of the results. Table I shows that the 6377 women included in the study represented approximately 79 per cent of antenatal bookings during the 26 month period. Since booking numbers in the three hospitals were recorded annually, the figures for incomplete years were estimated on a pro rata basis. The comparatively low ‘screened’ figure for FPMH in 1976 arose through difficulties in incorporating its ancillary clinics into the study. In the last six months of the study the FPMH ‘screened’ figure was a more satisfactory 86 per cent. Table I1 shows the results of plasma AFP testing in each of the three hospitals. The use of the 95th centile of the normal range as the initial cut-off point gave rather different proportions of ‘at risk’ pregnancies in the three hospitals, and these differences persisted after a second plasma AFP estimation and after exclusion of women deemed unsuitable for amniocentesis. Thus the amniocentesis rate ranged from 1 . 5 per cent at the WGH to 2.9 per cent at DMH. However, the proportion of women with an initially high serum AFP value who eventually had

TABLEI Proportion of antenatal bookings subjected to AFP screening 1975

Hospital

No,

Screened

booked WGH DMH FPMH All

1398 939* -

* Estimated on pro rata

NO.

1139 790 -

basis.

Complete trial

1976

Percent 82 84

No. booked 1393 1417 2121

Screened NO.

1246 1169 1294

Per cent 89 83 61

Screened

No.

booked 3024* 2592* 2474* 8090*

NO.

Per cent

2626 2150 1601 6377

87 83 65 79

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BROCK, SCRIMGEOUR, STEVEN, BARRON AND WATT

TABLE I1 Result of plasma AFP testing

All pregnancies High value (1st test) High value (both tests) Exclusions Underestimated gestation Missed abortion Threatened abortion Twins Refused amniocentesis N o amniocentesis Amniocentesis Abnormal amniotic fluid AFP Anencephaly Spina bifida Exornphalos Normal

WGH

DMH

FPMH

2626 99 (3.8%) 64 (2.4%)

2150 159 (7.4%) 103 (4.8%)

1601

10 3 4 6 1 1 39 (1.5%) 6 2 3 0 1

28 3 1 5 0 3 63 (2.9%) 5 2 2 1 0

5 1 1 0

81 (5.1%) 39 (2.4%)

0 0 32 (2.0%) 10 8 2 0 0

All Hospitals 6377 339 (5.3%) 206 (3.2%) 43 7 6 11 1 4 134 (2.1 %) 21 12 7* 1 1

* Two spina bifida pregnancies not terminated. amniocenteses was a constant 40 per cent in each hospital. With two successive high plasma AFP values the most common cause of exclusion from amniocentesis was an under-estimate of gestational age (43 patients). Other causes of exclusion are shown in Table 11. Of the 134 patients who had amniocenteses there were 21 with amniotic fluid AFP levels more than four SDs above the mean for that gestational week. Termination of pregnancy was done in 19 of these patients; 12 of the fetuses had anencephaly, 5 had spina bifida, one had an exomphalos and one was apparently normal. Two patients did not have a termination of pregnancy despite abnormal amniotic fluid AFP values. In addition one termination of pregnancy was done (without prior amniocentesis) after two serial plasma AFP values above the 95th centile and an ultrasound scan showing an incomplete cephalic outline; the fetus had anencephaly. The outcome of pregnancy (Table 111) was ascertained in 6122 patients (96.0 per cent); 238 patients (3.7 per cent) moved from the locality and a further 17 (0.3 per cent) could not be traced. The spontaneous abortion rate was higher in the WGH and DMH than in FPMH, while incidence of NTD was higher in the last hospital.

Table IV details the relative success of the study: 20 fetuses with NTD (13 with anencephaly and 7 with spina bifida) were detected by plasma AFP screening. A further two fetuses with spina bifida and one with anencephaly were detected by amniotic fluid AFP determination in patients with a previous history of children with NTDs. Plasma AFP values in these three mothers were, in fact, above the 95th centile. Three fetuses with open spina bifida and one with anencephaly were missed by plasma AFP screening, thus giving a detection efficiency for open NTDs of 83 per cent. A further four fetuses with closed NTDs (3 with meningocoele spina bifida and one with an encephalocele) were also missed. Problem patients Three difficult problems arose during the study. The first such patient (para 0 1) had three sequential plasma AFP values above the 95th centile. Amniocentesis at 19 weeks and again at 20 weeks gave amniotic fluid AFP concentrations which were just above 4 SDs above the mean. The patient decided to continue with the pregnancy and had an infant with a severe lumbar myelocoele who survived for 4 days. The second patient was a primigravida and again three sequential plasma AFP values were above the 95th centile. Amniocentesis

+

PLASMA AFP SCREENlNG

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TABLE I11 Outcome of pregnancies involved in trial Hospital All Hospital

WGH

DMH

FPMH

No.

Per cent

No.

Percent

No.

Per cent

No.

Per cent

‘Normal’ singleton Twins Stillbirths Neonatal deaths Spontaneous and missed abortions Neural tube defects Exomphalos Total ascertained

2414 24 11 5

96, I 0.96 0.44 0.20

1978 21 9 6

96.3 1.02 0.44 0.29

1496 16 11 4

96.2 1.03 0.71 0.26

5888 61 31 15

96.2 1.00 0.51 0.25

48 10 0 2512

1.91 0.40

33 7 1 2055

1.61 0.34

14 14 0 1555

0.90 0.90

95 31 1 6122

1.55 0.51

Moved Lost Total screened

102 12 2626

94 1 2150

42 4 1601

238 17 6377

Notes: ‘Normal’ singleton includes all birth defects except neural tube defects and exomphalos. Twins include livebirths, stillbirths and neonatal deaths, but not spontaneous abortions. There were no neural tube defects among the twins. Percentages are based on ascertained outcomes and relate only to patients passing through the trial.

TABLE IV Relative success of screening trial Total pregnancies tested Outcome of pregnancy known Spina bifida detected by plasma AFP Anencephaly detected by plasma AFP Open NTD missed by plasma AFP Closed NTD missed by plasma AFP Plasma detection efficiency for open NTD Plasma detection efficiency for all NTD NTD detected by amniotic fluid AFP and family history

6377 6122 7 13 4 4 83 % (20/24) 71 = (20/28) 3

at 22 weeks showed an abnormally high amniotic fluid AFP concentration. At 23 weeks, amniography using Myodil revealed no apparent lesion. The patient elected to continue with the pregnancy and at 36 weeks delivered a male child with sacral myelomeningocoele which was operated on with good result. The third patient had two children and once more three sequential plasma AFP values were above the 95th centile. Amniotic fluid AFP levels at 19 weeks and again at 20 weeks were just above 4 SDs. The patient and her husband requested termination but the fetus was normal.

Further details of the first and third patients have been reported elsewhere (Brock and Gosden, 1977).

DISCUSSION To assess the practical value of maternal plasma AFP screening for fetal NTD as a part of routine antenatal care we included three hospitals of different character in the study. Previous patterns of antenatal care were altered minimally but it was necessary to explain the objectives of the study and the implications of a high plasma AFP value to patients. Because it was essential to obtain a blood sample between the 15th and 20th weeks most patients had to make an extra visit to the hospital or ancillary clinic. It was also necessary to increase the amount of time devoted to ultrasound scanning, both to exclude underestimated gestational age and to prepare patients for amniocentesis. The proportion of patients booking at the three hospitals who accepted AFP screening (Table 11) was remarkably high, particularly if the low numbers for FPMH in 1976 are discounted. The women who did not participate

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BROCK, SCRIMGEOUR, STEVEN, BARRON AND WATT

were (1) those who booked after 20 weeks gestation, (2) those who booked early but miscarried before 15 weeks and (3) those who would not contemplate a termination of pregnancy. The method of reporting the results (Fig. 1) allowed the consultant some flexibility in interpretation but he did know that a single and two sequential plasma AFP values above the 95th centile respectively indicated 5 per cent and 10 per cent risk of fetal NTD. Several consultants delegated responsibility for recalling patients for a second blood sample to the nursing staff and only intervened when further procedures were necessary. This arrangement worked very efficiently. The policy of serial sampling is inconvenient for staff and patients and requires some justification. However, it did reduce by 40 per cent (Table 11) the number of patients who needed ultrasound scans and amniocentesis. The plasma detection efficiency of 83 for open NTD (Table IV) accords well with the projections of the UK Collaborative Study (1977) which had an 88 per cent detection rate at the 95th centile of the normal range, if screening was done between 16 and 18 weeks of pregnancy. Many of our plasma AFP measurements were made at 15 weeks when the detection efficiency is known to be somewhat lower. With only 79 per cent of our patients having plasma AFP measurement, this means that the reduction in birth incidence of NTD during the period of the study was of the order of two thirds. Two other prospective studies of maternal AFP screening for NTD have been published. In a Swedish project (Kjessler et 02, 1977), 7158 patients were examined between 14 and 20 weeks. Of the 7 NTD encountered, five (three anencephaly, two spina bifida) had serum AFP values which were more than three standard deviations above the mean for the appropriate week, while two (one anencephaly, one spina bifida) had normal values. Only one termination of pregnancy was done. In the other study (Clarke et 01, 1977), 5339 consecutive patients were screened in three maternity units in London but only 1423 of them booked between 16 and 22 weeks. Within this group there were ten fetuses with

anencephaly and two with spina bifida with plasma AFP levels above the 95th centile and one fetus with spina bifida and a normal maternal plasma AFP value. There were seven terminations of pregnancy. The high proportion of NTD among these patients suggests some pre-selection, as does the high ratio of anencephaly to spina bifida. The major purpose of this study was to determine whether or not antenatal plasma AFP screening was acceptable and effective. Effectiveness was shown by the 83 per cent detection rate for open NTDs. Acceptability is more difficult to assess. In the 26 months of the study eight patients declined the offer of plasma AFP testing, while only one patient out of 135 refused an amniocentesis. It is our view that facilities and finance should be found to provide an antenatal maternal plasma AFP screening service.

ACKNOWLEDGEMENTS This study would not have been possible without the active participation ofthe Consultant Obstetricians at WGH (Dr F. R. Clark), DMH (Dr R. G. Whitelaw, Dr G. R. Brown) and FPMH (Dr I. M. Duthie, Dr M. Hill, Dr A. Herriot and D r E. J. Fairlie). We thank them and also Miss L. Arthur, Mrs S . Wiles, Mrs P. Jelen, Dr C. M. Gosden, Miss S . Brown and Miss A. Henderson for valuable assistance. The study was supported by a grant from the Medical Research Council.

REFERENCES Brock, D. J. H., and Gosden, C. (1977): British Medical Journal, 2, 934. Brock, D. J. H., and Scrirngeour, J. B. (1972): Lancet, 2, 1252. Brock. D. J. H.. and Sutcliffe. R. G. (1972): Lancet, 2,’ 197. Brock, D. J. H., Bolton, A. E., and Monoghan, J. M. (1973): Lancet, 2, 923. Brock, D. J. H., Scrirngeour, J. B., and Nelson, M. M. (1975): Clinical Genetics, 7 , 163. Brock, D. J. H., Barron, L., Manson, J., and McCrae, W. N. (1978): Clinica chimica acta, 82, 101. Clarke, P. C., Gordon, Y . B., Kitau, M. J., Chard, T., and Letchworth, A. T. (1977): British Journal of Obstetrics and Gynaecology, 84, 565.

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Gosden, C., and Brock, D. J. H. (1977): Lancet, 1, 919.

Leek, A. E., Ruoss, C. F., Kitau, M. J., and Chard, T.

Kjessler, B., Johansson, S. G . O., Lidbjork, G . , and Sherman, M. S. (1977): Acta obstetricia et

UK Collaborative Study on Alpha-fetoprotein in Relation to Neural Tube Defects (1977): Lancet,

(1973): Lancer, 2, 385.

gynecologica Scandinavica, Supplement 69, 20.

1, 1323.

Maternal plasma alpha-fetoprotein screening for fetal neural tube defects.

British Journal of Obstetrics and Gynaecology August 1978. V O 85. ~ pp 575-581 MATERNAL PLASMA ALPHA-FETOPROTEIN SCREENING FOR FETAL NEURAL TUBE DEF...
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