False positive amniotic fluid alpha fetoprotein levels resulting from contamination with fetal blood: Results of an experiment 1‘. A. DORAN,

M.D.

I,. C. ALLEN,

PH.D.

B. B. K. PIRANI,

M.B.,

K. H. SHUMAK, Toronto,

Ontario,

CH.B.

M.D.

Camdu

The finding of an elevated level of alpha fetoprotein (AFP) in amnioticjuid is of value in the prenatal diagnosis of open neural tube defects. The present study was donv to determine the amount of fetal blood required in amniotic fluid to produce a significant and misleading increase in AFP. Fetal blood was obtained at hysterotomy, and measured 7folumes ulc’re added to amnioticJluid samples. Betke-Kleihauer tests, red cell coctnts, and AFP determinations were done. On the average, at I6 weeks’ gestation, contamination of 5 ml. of amnioticjuid with 22 pl offetal blood will result in an increase in AFP of 1.6 m‘g. per deciliter. Fetal cells in a much lower concentration can readily be detected by the Be&-Kleihauer technique. A Betke-Kleihazter test and red cell count should be dam on all blood-stained amniotic&d samples to determine the amount of fetal blood present. The contribution of the fetal blood AFP can. then be estimated and must be considered in the interpretation of the total amniotic fluid AFP result. (AM. J. OBSTET. GYNECOL. 127: 75’9, 1977.)~

HUMAN ALPHA FETOPROTEIN (AFP) is an alpha-l globulin* which is synthesized in the fetal liver, the yolk sac, and, in trace amounts, the gastrointestinal tract.‘, 3 AFP is detectable in fetal serum by about the sixth week of pregnancy, reaches a peak between 12 and 14 weeks, and thereafter declines steadily toward term.‘% ’ In normal pregnancies, AFP levels in amniotic fluid parallel those in the fetal serum, with a concentration gradient between fetal serum and amniotic fluid of about 150: 1. The highest concentration of amniotic fluid AFP is found at 13 to 14 weeks5; the level then decreases until, at term, the concentration in amniotic fluid is similar to that in the maternal serum.6V 7 In 1972, Brock and Sutcliffe” showed that the con-

From the Departments Clinical Biochemistry,

centration of amniotic fluid AFP was substantially raised in pregnancies in which the fetus had an open neural tube defect; their findings have been confirmed by many others.‘-12 The concentration of amniotic fluid AFP also has been reported to be elevated in cases of missed abortion,’ esophageal atresia,13 congenital nephrosis,” and several other disorders. In clinical practice, a common potential cause of elevation of amniotic fluid AFP is contamination with fetal blood. In our center, the incidence of blood-stained fluid in 405 amniocenteses was 5.4 per cent.15 As the level of AFP in fetal serum is 150 times that in amniotic fluid, even slight contamination of amniotic fluid with fetal blood can falsely elevate the AFP. Fig. 1 shows our normal range of AFP in 281 clear samples of amniotic fluid. In live of 15 blood-stained samples, AFP exceeded the normal range. BetkeKleihauer tests were not carried out on these amniotic fluid samples, but blood staining must have been due to varying proportions of fetal and maternal blood. Contamination with fetal blood could have been responsible for significant elevations of AFP in the five cases. The present study was designed to confirm that significant elevations of AFP could be caused by the small amounts of fetal blood which may be introduced into

of Obstetrics and Gynoecology, and Haematology, Toronto General

Hospital, Uniz~ersity of Toronto. Supported b Ontario Minislly of Hralth

Grant

No. PR 279. Presented by the Canadian Investigators in Reproduction at the Thirty-second Annual Meeting of the Societyof Obstetricians and Gynagrologisis of Canada, Toronto, Ontario, Canada, Juno 8-l 2, 1976. Reprint General

requests: Dr. T. A. Doran, NUW 4-104, Toronto Hospital, Toronto, Ontario, Canada M5G IL7.

759

760

Doran

et al.

12 r

8

:

2

12

14

16

20

18

GESTATION

22

24

26

28

(WEEKS)

fluid alpha fetoprotein levels in 281 samples. n , Amniotic fluid contaminated blood. O-O, Upper and lower limits (-~2 SD.) of the normal range. O-O, Mean fetoprotein levels a; hifferent stages of gestation.

Fig. 1. Amniotic

Table

I. AFP

levels

in amniotic

fluid

and

fetal blood

ExPerimmt I

Gestation (weeks) Amniotic fluid AFP (mg./dl.) Fetal blood AFP (mg./dl.)

13 4.4

600

2

3

16 2.3

16 2.4

240

290

J

18 0.9 190

samples at the time of amniocentesis. Moreover, we wished to determine whether it was possible to detect fetal blood when it was present in the minimum concentration required to elevate AFP above the normal range. Therefore, we assayed amniotic fluid AFP before and after the addition of measured volumes of fetal blood. Betke-Kleihauer tests were performed on each sample to determine the minimum detectable volume of fetal blood.

Material and methods Amniotic fluid AFP was measured by electroimmunoassay (Laurell’6 rocket technique) with standards and antisera obtained from Behring Diagnostics. Standards up to 6.4 mg. per deciliter are used. The precision (5 1 S.D.) is 0.15 at a level of 2.2 mg. per deciliter and 0.21 at a level of 4.0 mg. per deciliter. Samples of

30

with alpha

fetal blood and of’amniotic fluid for experiments 1 to 4 were obtained at hysterotomy at IS. 16, 16. and 18 weeks’ gestation, respectively. In each of the four experiments, 1, 5, 10, 20, 25, and 50 ~1 of fetal blood were added to 5 ml. volumes of amniotic fluid. The lowest level of contamination at which blood was detected visually was 5 ~1 in 5 ml. of amniotic fluid. AFP was determined in duplicate on the amniotic fluid, fetal blood (diluted 1 : 100 in saline), and each of the contaminated samples. Amniotic fluid red cell counts were determined manually. The Betke-Kleihauer test’7 was used to count cells containing fetal hemoglobin.

Results The concentrations of AFP in amniotic fluid and fetal blood in each of the four experiments are shown in Table I. Table II shows the average increase in AFP concentration in each of the contaminated amniotic fluid samples. In experiments 1 and 2, in those samples with larger amounts (25 or 50 ~1) of fetal blood, the levels of’ AFP were too high to be measured by our method. In the four experiments, there is a linear relationship between the quantity of fetal blood added to the amniotic fluid and the average elevation of AFP above the bastline (Fig. 2).

Volume Number

19 7

False

positive

amniotic

alpha

fetoprotein

761

1200

1000

E

10 VOLUIE

Fig. 2. Relationship amniotic fluid and above the base line.

20

30

OF FETAL BLOOD (IL)

between average

40 IN 5 ML AflNIOTIC

50 FLUID

volume of fetal blood added elevation of alpha fetoprotein

200

to

0

the actual red cell count in samples to which only small volumes of fetal blood had been added differed from the expected counts. However, when all experiments were averaged, there was an approximately linear relationship between the red cell count and the volume of red cells added (Fig. 3). In experiments 2, 3, and 4, the Betke-Kleihauer test detected fetal cells when only 1 ~1 of fetal cells was added to 5 ml. of amniotic fluid. No fetal cells were observed at this level of contamination in experiment 1. Fetal cells were seen in all samples with higher levels of contamination. In each

experiment,

Comment Recently, there have been several reports of elevated amniotic fluid AFP in the presence of a normal fetus.“-“’ In one instance in which a normal fetus was aborted, the amniotic fluid was heavily contaminated with blood.” In another report,‘!’ there is no comment about contamination with blood. Milunsky and Atkins” reported that in 11 of a series of over 1,000 cases the levels of amniotic fluid were more than two standard deviations above the mean, although these patients had normal infants. In almost all of these samples, blood was visible after centrifugation, consistent with the possibility that fetal blood was present. Assuming total mixing of fetal blood with amniotic fluid,’ Ward and Stewart21 have calculated that fetal bleeding into amniotic fluid of only 1 ml. will increase the AFP to above the upper normal limit at 14 weeks’ gestation. Fetal bleeding of 2.5 and 5 ml. is required at 15i and 17 weeks, respectively, to give similar eleva-

10

20

30

VOLUME OF FETAL BLOOD (FL)

50

40

IN 5 ML AMNIOTIC FLUID

Fig. 3. Relationship between volume of fetal blood added amniotic fluid and average fetal red blood cell count. Table II. Increase from contamination

1

0.6

5

1.0 1.4

10 20 25 50

to

in amniotic fluid AFP resulting with fetal blood

2.2 -

Numbers in parentheses *Not measured.

0.0 0.1

0.1

0.6 1.3 1.7 -

1.0

0.5 -*

0.2 0.4 0.6 1.3

0.2 0.5 0.9 1.6

(4) (4) (4) (3)

1.9

1.5

1.7 (3)

3.8

2.6

3.2 (2)

are the

numbers

of experiments.

tions. They suggested that amniotic fluid samples should be screened for the presence of fetal erythrocytes by the Betke-Kleihauer technique. In our laboratory, the mean amniotic fluid AFP level at 16 weeks’ gestation is 2.5 mg. per deciliter, and a value greater than 4.1 mg. per deciliter is considered abnormal. In the average case, therefore, an increase in AFP of 1.6 mg. per deciliter would lead to an “abnormal” AFP level. From Fig. 2, it can be seen that such an increase would occur if there were fetal bleeding of about 22 ,ul per 5 ml. of amniotic fluid. Thus, if 15 ml. of amniotic fluid were removed at amniocentesis, a 1.6

762

Doran

et al.

mg. per deciliter elevation of AFP above the base line would occur if 66 ~1 of fetal blood were present. If the base-line AFP le\;el were in the upper normal range. a much smaller quantity of fetal blood could result in a false positive AFP value. We have found that small concentrations of fetal red cells can be detected by the Betke-Kleihauer rechnique. In practice, most contaminated samples would be expected to contain both fetal and maternal cells, but, since maternal cells can also be enumerated by the Betke-Kleihaucr technique, it is possible to determine the percentage of’ fetal cells present in any mixture. By performing a red cell count and the BetkeKleihauer test. one can thus determine the number of fetal red cells present in any amniotic fluid sample. From the fetal red cell count. one can estimate the total amount of fetal blood present in the sample by assuming a normal fetal hematocrit and mean cell volume.

The contribution of this amount of fetal blood (jvith ;LII average serum AFP level) to the total amniotic fluid AFP can be derived from Fig. 9. It is obviously important when one is performing an amniocentesis to avoid blood staining of the amniotic, fluid not only for safety and successful amniotic, fluid cell culture but also for correct interpretation of’ AFP results. Preliminar>localization of the plan-enta bv ultrasonography should be mandatory in order To kwp the incidence of blood-stained samples Io\v. However. when a blood-stained sample is obtained. an estimate 01 the amount of fetal blood present and its contribution to total AFP will be of value to the clinician. We acknowledge the participation in these esprriments of Sheila A. Haddad, Blood Transfusion Laboratory, Toronto General Hospital, and F. K. Ho. Clinical Biochemistry Laboratorp. Toronto General Hospital.

REFERENCES 1.

Bergstrand, C. G., and Czar, B.: Demonstration of a new protein fraction in serum from the human fetus, Stand. I. Clin.

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11.

eitlin,

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D., and Boesman, M.: Serum alpha fetoprotein,

albumin and globulin in human conceptus. J. Clin. Invest. 45: 1826, 1966. Gitlin. D., Perricelli. A., and Gitlin, G. M.: Synthesis of 01 fetoprotein by liver, yolk sac and gastro-intestinal tract of human conceptus, Cancer Res. 32: 979, 1972. Brock, D. J. H.: The molecular nature of alpha fetoprotein in anencephaly and spina bifida, Clin. Chem. Acta 57: 315, 1974. Brock, D. J. H.: The prenatal diagnosis of neural tube defects, Obstet. Gynecol. Survey 31: 32, 1976. SeppZi, M.. and Ruoslahti, E.: Alpha fetoprotein in amniotic fluid: an index of gestational age, AM. .J. OBSTET. GYNECOL. 114: 595, 1972. Sepp&, M.. and Ruoslahti, E.: Radioimmunoassay of maternal serum alphafetoprotein during pregnancy and delivery, AM. J. O&XET. ~YNECOL. 112:-LfO8,-1972.’ Brock. D. I. H., and Sutcliffe. R. G.: Aloha fetoarotein in the anten&al diagnosis of anencephal; and sp&a bifida. Lancet 2: 197, 1972. Milunsky, A., and Alpert, E.: The value of alpha fetoprotein in the prenatal diagnosis of neural tube defects, J. Pediatr. 84: 889, 1974. Lorber, J.. Stewart, C. R.. and Ward. A. M.: Alpha fetoprotein in antenatal diagnosis of anencephaly and spina bifida, Lancet 2: 1187. 1973. Nevin, N. C.. Nesbitt, S., and Thompson, W.: Myelocele and alpha feroprotein in amniotic fluid, Lancet 1: 1383, 1973.

12.

Field, B.. Mitchell, G., Garett. W., and Kerr. C.: Amniotic

alpha fetoprotein levels and anencephaly. Lancct 52: 798. 1973. 13. Sepp&i, M.: Increased alpha fetoprotein in amniotic fluid associated with congenital oesophageal atresia. Obstet. Gynecol. 42: 613, 1973. 14. Kjessler. B., Johansson, S. G. O., Sherman, M., Gusta!son, K.-H.. and Hultquist. G.: Alpha-fetoprotein in antenatal diagnosis of congenital nephrosis. Lancet 1: 432. 1975. 15. Malone, R. M., Doran, T. A.. Benzie. R. J.. Rudd. N. L.,

Gardner, H. A., Hunter, A.. Miskin, M., Lowden, J. ri., B.: Prenatal genetic diagnosis-advantage5 II of a team approach. In preparation. and Youson.

,I

16. Laurell. C. B.: Electroimmunbassav. Stand. 1. Clin. Lab. Invest. (Suppl. 29) 124: 21. 1972. -’ 17. Dacie, J. \‘., and Lewis, S. M.: Practical Haematology, ed. 4. London, 1968, J, & A. Churchill, Ltd., p. 107. 18. Campbell, S., Pryse-Davis, J., Coltart, T. M.. Seller, M. J., and Singer, J. D.: Ultrasound in the diagnosis of spina bifida. Lancet 1: 1065, 1975. 19. Field, B.. and Kerr, C.: Antenatal diagnosis of neural tube defects, Lancet 2: 324. 1975. 20. Milunsky. A., and Atkins, L.: Prenatal diagnosis of geetic disorders, in Milunsky. A., editor: The Prevention of’ Genetic Disease and Mental Retardation, Philadelphia, 1973. W. B. Saunders Company, pp. 233-234. 21. Ward, A. M.. and Stewart. C. R.: False positive results in antenatal diagnosis of neural tube disorders, Lanret 2: 345, 1974.

False positive amniotic fluid alpha fetoprotein levels resulting from contamination with fetal blood: results of an experiment.

False positive amniotic fluid alpha fetoprotein levels resulting from contamination with fetal blood: Results of an experiment 1‘. A. DORAN, M.D. I,...
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