205

Clinica Chimica A&a, 70 fEiTBf 205~-2ft8 @ EIsetier Scientific ~blisbi~g Company, Amsterdam

- Printed in The Netbertands

SHORT COMMUNICATION CCA 1935

A SIMPLEMETHOD FOR THE %U~~TATION ~-~ETU~ROTE~ THRGUGHG~ PREGN~C~

D. BROCKLEHURST Area llepartment RN2 SLT (U.K.)

OF ~~0~~

FLUID

and C.E. WILDE

of Clinical Chemistry,

Dancaster

Royal infirmary, Doncoster

Summary A simple method of Amicon membrane cone concentration followed by radial ~un~if~sion is describedand evaluatedfor the detection of amniotic fluid ~-fe~p~tein from X3-46 weeks gestation. Introduction The measurementof amniotic fluid cu-fetoproteinhas now become established as a guide to the diagnosis of fetal neural tube defects in patients at risk f 1 f . ~thuu~ there are a number of sensitivecliques for its me~u~ment e.g. radiuimmunoassay (RIA) f2] and counter current electrophoresis [3f the simplestand most generallyapplicablemethod is radial immunodiffusion (RID). Commercially availableimmunodiffusion plates are, however, only sensitiveto about 15 mg/l so that normal levelscannot be quantitatedafter about 22 weeks gestation and even at 16 weeks, concentrations of less than 15 mg/l are not uncommon. Levels of a-fetoprotein in affected pregnancies also decrease after 22 weeks gestation often approaching condensations which are undetectable by RID. A simple method (with a maximum sensitivityof 0.15 mgfll which enables amniotic fluid ~-fetoprote~ to be measuredt~ughout pregnancy is described. Materials BehringwerkeM Par&en cu-fetoproteinRID plates.and standards.Amicon (High ~y~ombe, Bucks) Centriflo CF 56 membrane cones flower retention limit 50 606 daltons),

206

Method Each amniotic fluid was centrifuged at 4500 X g for 20 min to remove cell debris. This was stored at 4°C for subsequent Kleihauer testing for fetal red cells should an abnormal level of a-fetoprotein be detected. Amicon centriflo CF 50 membrane cones containing 5 ml amniotic fluid were cent~fug~ at 1000 X g for 20 min to concentrate the ~-fetoprotein (mol. wt. 70 000-72 000) within the cone by a factor of ten. Occasionally concentration was increased to one hundred fold by increasing the time of centrifugation, when very low a-fetoprotein levels were encountered. The concentrates containing o-fetoprotein were adjusted when necessary to 500 ~1 with the filtered amniotic fluid by means of an adjustable Finn-pipette and 5 ~1 of the concentrated sample was applied to the immunodiffusion plate in the normal way together with the original sample and standards. After a 48 h incubation at room temperature the a-fetoprotein concentrations were assayed by measuring precipitin ring diameter in the normal manner. R@sults

To assess precision an amniotic fluid sample with a level of cu-fetoprotein close to the limit of sensitivity of the RID plates was chosen. The fluid was assayed ten times and then ahquots of the same fluid were concentrated ten fold and then assayed. The RID plates were read by two different operators. The results of this study are shown in Table I. At the limit of sensitivity of the RID plate the coefficient of variation (C.V.) was 15% and the two operators gave means which were significantly different (P < 0.001). After concentration and assay within a more favourable part of the response curve their results were similar with an improved C.V. of 6%. If the sample was concentrated 100 fold, it was possible to measure down to 0.15 mg/l at a C.V. of 15%; lower concentrations were only measured with unacceptably poor precision. Fig. 1 shows the levels of cu-fetoprotein, measured after concentration, in thirty six normal pregnancies and two patients who first presented later than TABLE I WITHIN BATCH PRECISION OF RADIAL I~MUNODIFFUSION PROTEIN WITH AND WITHOUT CONCENTRATION Operator

FOR AMNIOTIC

Amniotic fluid (mg/I) Unconcentrated

Concentrated X 10 (corrected)

l(N=lO) Mean Standard deviation Coefficient of variation

28.3 4.3 15

20 1.26 6.3

2(N= 10) Mean Standard deviation Coefficient of variation

16.6 2.6 15

18.6 1.02 5.5

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WEEKS GESTATION Fig. 1. Radial immunodiffusion or-fetoprotein levels in normal and affected patients (13-39 weeks gestation). n? normal pregnancies; spina bifida myelomeningeocele patients: 0, patient 1; X, patient 2.

25 weeks gestation and who were subsequently shown to have fetuses with neural tube malformations. The mean value in normal pregnancies after 25 weeks gestation was 0.6 mg/l (range 0.07-1.6 mg/l), well below the limit of sensitivity of the RID method. Patient 1 was a 17 year old prima gravida with a family history of spina bifida. Amniotic fluid was taken at 26,30 and 32 weeks. Although the c-fetoprotein in the first sample was measurable without concentration, the levels in subsequent samples could only be accurately quantitated after concentration, despite being well above the reference range at this gestational age. Similarly patient 2, first tested at 34 weeks, had an abnormal level of a-fetoprotein which could only be measured accurately after concentration. Both patients had stillbirths; patient 1 after induction at 32 weeks; patient 2 at 36 weeks following a spontaneous delivery. Discussion Concentration of amniotic fluid has increased the sensitivity of a commercially available a-fetoprotein RID method by up to 100 fold. This has enabled levels to be measured simply and accurately in late gestation. The method has reduced operator bias and improved precision by making it possible to read on a more favourable part of the RID response curve. The observed reference range compares well with published ranges for a sensitive radioimmunoassay [2]. This communication shows that the method can be used for the detection of neural tube defects in the third trimester of pregnancy and that even in affected pregnancies the level of cu-fetoprotein falls to relatively low levels,

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confirming the observation of Stewart et al. [4] . This finding contrasts with the rising amniotic fluid ol-fetoprotein levels reported in fetal distress due to other causes [5] and emphasises the importance of accurate quantitative measurement at this stage of pregnancy. Acknowledgements We wish to thank the obstetricians at this hospital, and in particular C.E. Peaker, for permission to publish results of patients under their care. References 1 2 3 4 6

Leading article (1974) Lancet i. 907 Se~~ala. M. and Ruoslahti. E. (1972) Am. J. Obstet. Gynaecol. 114, 696-598 Kahn. J. (1970) J. Clin. Pathol. 23.733-735 Stewart, C.R., Milford Ward. A. and Lorbar. J. (1976) Br. J. Obstet. Gynecol. 82. 257-261 Seppala. M. and Ruoslahti. E. (1973) Lancet i, 155

Miss

A simple method for the quantitation of amniotic fluid alpha-fetoprotein throughout pregnancy.

205 Clinica Chimica A&a, 70 fEiTBf 205~-2ft8 @ EIsetier Scientific ~blisbi~g Company, Amsterdam - Printed in The Netbertands SHORT COMMUNICATION CC...
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