Scand J Haematol (1975) 15,333-338
Transplacental Passage of Foetal Blood Cells R. ZILLIACUS, M.B.,A. DE LA CHAPELLE, M.D., J. SCHRODER, PH.D., A. TIILIKAINEN, M.D.,E. KQHNE, M.D. & E. KLEIHAUER, M.D. Folkhalsan Institute of Genetics (Chief, A . de la Chapelle), Helsinki, Department of Serology and Bacteriology (Acting Chief, T . Kosunen), University of Helsinki, Finland and Department of Paediatrics (Chief, E. Kleihauer), University o f Ulm, West Germany
With the brightly fluorescent Y chromatin as a marker, the proportion of foetal lymphocytes in maternal blood was found to be 0.02 - 0.16 % in 7 of 11 primigravidae bearing a boy, but not a single XY mitosis was found among 112,000 leucoagglutininstimulated mitoses from the same women. The proportion of foetal lymphocytes did not change appreciably during pregnancy (first appearance detected at 15 weeks’ gestation), no’r were there any unequivocal foetal erythrocytes in spite of ABO and Rh compatibility. These findings suggest that foetal lymphocytes pass actively into the mother’s blood relatively early during pregnancy. The cells are protected from immunological elimination, and therefore may remain in the maternal blood for long periods. They are a valuable potential source of material for antenatal foetal diagnosis. Key words: foetal lymphocytes - foetal erythrocytes - placental passage maternal blood - Y chromatin - fluorescence
Accepted for publication September 10, 1975 Correspondence to: Dr. Albert de la Chapelle, Folkhalsan Institute of Genetics, P. 0. Box 819, 00101 Helsinki 10, Finland
Lymphocytes of foetal origin occur in the blood of most women during pregnancy and for long periods after delivery (Walknowska et a1 1969, de Grouchy & TrCbuchet 1971, Schroder & de la Chapelle 1972). Why these foetal cells survive has not yet been adequately explained. A correlation exists between the presence of cytotoxic HL-A antibodies and the absence or paucity of foetal lymphocytes in the maternal blood after
delivery (Schroder et a1 1974). Paternally derived foetal HL-A antigens are often masked in cord blood samples, and this might explain, at least in part, the maternal tolerance to histoincompatible foetal cells (Tiilikainen et a1 1974). Foetal lymphocytes in the mother’s blood are a potential source of material for antenatal diagnosis of foetal disease (Renwick 1971). Before attempts are made to purify
334
ZILLIACUS, D E LA CHAPELLE, SCHRODER, TILIKAlNEN et a1
these cells (Hulett et a1 1973), more must be known about their characteristics and the kinetics of their occurrence. We here report the results of such a study. The passage of foetal leucocytes and erythrocytes into the maternal circulation was monitored and related to maternal production of HL-A antibodies. MATERIALS AND METHODS Patients
The series of patients consisted of 30 consecutive primigravidae attending three maternity welfare centres. The patients and their families were informed about the purpose of the study and agreed to co-operate. Leucoagglutinin-stimulated lymphocyte cultures of peripheral blood from the women and the fathers of the expected children were prepared, and the mitoses and interphase cells were inspected in the fluorescence microscope after staining with Acrani1.B The purpose of this step was to exclude couples where the mother had autosomes with brightly fluorescent regions (Caspersson et a1 1970) or the father had an exceptionally small Y chromosome. These criteria had been evaluated and applied by us previously (Schroder et a1 1974). Of the 30 mothers initially selected, 9 had to be excluded because of strong autosomal fluorescence that might have interfered with the recognition of interphase Y chromatin, 1 because her husband's Y chromosome was very small, 1 because she only later told us that she had had a previous abortion (Zilliacus et a1 1975), and 1 who aborted during the study. This report deals with the remaining 18 women. They were aged 22-31 years. Methods Blood samples were taken from each mother on four occasions, i.e. at about 4, 6, and 8 months of gestation and within 2 days after delivery. Every sample (20 ml) was divided into three alimquots. From the first alimquot 5000 uncultured interphase lymphocytes and 5000 granulocytes were screened for Y chromatin as previously described (Schroder Lk de la Chapelle 1972, Schroder et a1 1974). Even when investigating the sample taken after delivery, the investigator was always unaware of the child's
sex. In addition, lymphocyte cultures set up from the same aliquot of blood were stimulated with leucoagglutinin (Weber et a1 1974), and slides were prepared. Because to find and examine thousands of mitoses is very time-consuming, and recognition of a brightly fluorescent Y chromosome in mitoses represents no problem (Caspersson et a1 1970), these slides were stored until the patient had delivered. Only slides from women with interphase cells containing Y chromatin or women who had given birth to a boy were stained with Acranil and examined by fluorescence microscopy for the presence of Y-chromosome-containing mitoses. The presence of foetal erythrocytes was evaluated from the second aliquot by the method described by Kleihauer (1974). From the third aliquot, serum was prepared and stored at -20" C for later determination of HL-A antibodies (Schroder et a1 1974). From a sample of cord blood the HL-A types were determined and in some cases ABO and Rh types as well (Tiilikainen et a1 1974). The child's karyotype was also determined and in all cases conformed with the phenotypic sex. RESULTS
Scoring of 330,000 uncultured interphase lymphocytes and the same number of granulocytes showed that cells containing a brightly fluorescent spot interpreted as Y chromatin occurred in 8 of the 18 women studied (Table I). Of these 8 women, 7 gave birth to a boy and one (case 12) to a girl. This means that 4 of the 11 women carrying a boy did not have cells containing Y chromatin in their blood. Of the 7 women bearing a girl, 6 did not have Y chromatin-positive cells. The seventh woman (case 12) will be discussed below. In 4 women, all of whom had Y chromatin-positive lymphocytes, a few granulocytes containing Y chromatin were seen (Table I). The proportion of lymphocytes containing Y chromatin varied between 0.02 and 0.16 % in the women bearing a boy. In one mother foetal cells were detected as early as at 15
0 0 0 0 0
0
0
0 0 0 0 0 0
0 0
-
-
-
-
-
-
-
0 0 0 0 0 0
0 0 0 0 0 0
2
0 0 0 0
0 0 0 0
6
0 0 0
3 3 2 2 2
-
-
-
-
-
0
0 0 0
0 0 0 0 0
1 0
0 0 0 0
0 0 0 0
10 0 0
0 0
0
2 0 0
2 2
i
8 3 2
-
Ly = No. of lymphocytes with Y chromatin per 5000 lymphocytes studied. G r = No. of granulocytes with Y chromatin per 5000 granulocytes studied. E = Foetal erythrocytes present or absent. * = Sample taken 4 weeks after delivery.
12 13 14 15 16 17 18
4 5 6 7 8 9 10 11
0 0 0
0
2 3
0 0 0 0 0 0 0
0
1
1
0 0 0 1 0 3 0 0 0 0
E
Ly
-
-
-
-
-
-
-
+
. = - = = (+) = C = I =
0
0
0 0
11 0 0
0 0" 0
4 5 1 0 1 3
E
not determined. negative. positive. weakly positive. compatible. incompatible.
0 0 0 0 0
1 0
0 0
0
0 0 1 0
1 2
Gr
1 1 1
Gr
1 I
LY
E
I I
E
1
Gr
Gr
Ly1
C C C
c
i
C C I I C
F F F F F F F
M M M M M M M M M M M
ABO and RhcomSex patibility of of child to child HL-A- mother antibodies
Sample IV within 48 h after delivery
Sample I11 3&38
Sample I1 24-3 1
Sample I 15-22
Ly
Patient no.
Weeks of gestation
TABLE I Findings in the series of 18 women
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336
ZILLIACUS, DE LA CHAPELLE, SCHRODER, TILIKAINEN et a1 TABLE I1 Number of mitoses studied
itive interphase lymphocytes not a single mitosis with a Y chromosome was detected (Table 11). Two women (cases 7 and 8) could not be studied. no. It is desirable to differentiate between 1 5000 5000 HbF-containing red blood cells of foetal and 2 4100 3400 5000 1500 3 5000 5000 maternal origin. The foetal cell type has a 4 5007 4146 2550 higher concentration of HbF. The elution of 5 1442 5006 99 6 2567 5001 2287 2000 HbF from red cells is dependent not only on 9 1247 2334 5003 pH but also on the intracellular HbF/HbA 10 2661 5042 3877 4700 11 5000 2400 800 ratio. We incubated the slides at pH 3.17, 12 4800 5000 5000 which leads to elution of 15-20 % HbF per cell with the HbA (Kleihauer & Kohne 1975, weeks of gestation. Inspection of Figure 1 personal information). In healthy pregnant indicates that during the period of time cov- women, erythrocytes that stain densely after ered by our investigation the proportion of this procedure can be identified with high cells containing Y chromatin was stable. In probability as cells of foetal origin. Using particular, there was no indication of a rise this method, we did not find a single erythrotowards term, nor was the proportion higher cyte that was unequivocally of foetal origin in early pregnancy. in any of the maternal blood samples (Table Among 112,000 mitoses from the blood I). Thus in these women foetal bleeding into of 10 women containing Y chromatin-pos- the maternal circulation could be excluded.
Figure 1. Kinetics of lymphocytes containing Y chromatin in the blood of 7 women. Dotted vertical line indicates first occasion on which any woman was tested (15 weeks). Short vertical line on each curve indicates time of delivery. Numbers 1-7 identify patients (Table I).
PASSAGE OF FOETAL CELLS
In the maternal serum samples taken within 2 days after delivery, antibodies against paternally derived foetal HL-A antigens were present in 3 cases only (1 strong, 2 weak; Table I). Since antibodies were not found in the serum samples taken from the women in question during pregnancy, antibody production must have occurred late in pregnancy or become discernible only after delivery. DISCUSSION
When lymphocytes containing Y chromatin are observed in the blood of a woman whose foetus is female (as in our case 12), the finding demands close consideration. We believe that the high incidence of cells scored as Y chromatin-positive clearly originates from misinterpretation of autosomal fluorescence in the mother’s own chromosomes (Caspersson et a1 1970), which we re-examined after she had delivered a girl. If this retrospective interpretation is correct she should have been excluded from the series at an early stage but erroneously was not. As there can be no hope of avoiding such errors, determination of foetal sex should not be performed by this method, as we have pointed out repeatedly (Schroder & de la Chapelle 1972, Schroder et a1 1974). Our data show that foetal lymphocytes may be present in the maternal circulation as early as the 15th week of gestation and that their number remains essentially constant throughout pregnancy. Thus there is no evidence that the proportion of foetal cells is high during early pregnancy and then declines, or that the number of foetal lymphocytes increases towards term, as is the case with foetal erythrocytes (Woodrow & Finn 1966, Bartsch 1972). Rather, our findings suggest that foetal lymphocytes pass Scnnd J Haematol(l975) 15
337
into the maternal blood relatively early in pregnancy and remain there in essentially unaltered numbers. This could be due either to a steady turnover or to absence of elimination (Tiilikainen et a1 1974)’ as lymphocytes have a very long life-span (Buckton et a1 1967). The proportions of foetal lymphocytes in the maternal blood found in this study (00.16 %) correspond very well with our previous findings, but not with those of Grosset et a1 (1974). These authors, who studied interphase nuclei of transformed lymphocytes after 5-6 days’ culture with phytohaemagglutinin, postulated preferential transformation of foetal cells in relation to maternal cells to explain the large numbers of foetal cells (0.2-1.5 %) in the maternal blood. We suggest, on the contrary, that the reason for the absence of XY mitoses in our study may be that foetal lymphocytes are prevented from being transformed and entering mitosis. This is not unlikely, since some of their HL-A antigens are masked (Tiilikainen et a1 1974) and in nylon wool columns they are known to behave as B lymphocytes (Schroder 1975). Moreover, factors in the maternal circulation may inhibit the transformation of foetal cells (Thorsby 1974, Gaugas 1974). As already pointed out (Jacobs & Smith 1969) misinterpretation may have marred some of the previous studies performed by conventional staining. Since many foetuses were ABO and Rhcompatible with their mothers, and since none of the mothers in the present series had any foetal erythrocytes in their blood, leakage or bleeding from the foetuses is excluded. If bleeding were responsible for the presence of foetal lymphocytes in the mother’s blood, there should be about 5000 times as many foetal erythrocytes as lymphocytes. So many 22
338
ZILLIACUS, D E LA CHAPELLE, SCHRODER, TILIKAINEN et a1
foetal erythrocytes would not escape detection. We conclude that foetal leucocytes must pass the placental barriers actively.
detection du sexe du foetus. Ann Genet 14, 13337. Hulett H R, Bonner W A, Sweet R G & Herzenberg L A (1973) Development and application of a rapid cell sorter. Clin Chem 19, 813-16. Jacobs P A & Smith P G (1969) Practical and theoretical implications of fetavmaternal lymACKNOWLEDGEMENTS phocyte transfer. Lancet 2, 745. We thank all the patients, nurses, doctors and ad- Kleihauer E (1974) Determination of fetal hemoglobin: Elution technique. In R M Schmidt, ministrators who helped us to organize and carry T H S Huisman & H Lehmann (eds) The detecout this study. In particular we wish t o thank Protion of hemoglobinopathies, p 20. C R C Press, fessor Sakari Timonen, M.D. for his kind help. Dr. Ohio. Johan Fellman performed preliminary statistical J H (1971) The mapping of human chroRenwick calculations. mosomes. Ann Rev Genet 5 , 81-120. Supported by grants from the Finnish National Research Council for Medical and Natural Sci- Schroder J (1975) Are fetal cells in maternal blood mainly B lymphocytes? Scand J Immunol4,279ences, the Sigrid Jusklius Foundation, the Oskar 85. Oflund Foundation and the Finska LakaresallskaSchroder J & de la Chapelle A (1972) Fetal lympet. phocytes in the maternal blood. Blood 39, 15362. J, Tiilikainen A & de la Chapelle A Schroder REFERENCES (1974) Fetal leukocytes in the maternal circulation after delivery. I. Cytological aspects. TransBartsch F K (1972) Fetale Erythrozyten in miitterplantation 17, 346-54. lichen Blut und Immunprophylaxe der RhImmunisierung. Klinische und experimentelle Thorsby E (1974) The human major histocompatibility system. Transplant Rev 18, 51-129. Studie. Acta Obstet Gynecol Scand, Suppl 20. Buckton K E, Court Brown W M & Smith P G Tiilikainen A, Schroder J & de la Chapelle A (1967) Lymphocyte survival in men treated with (1974) Fetal leukocytes in the maternal circulaX-rays for ankylosing spondylitis. Nature 214, tion after delivery. 11. Masking of HL-A antigens. Transplantation 17, 355-60. 470-73. Caspersson T,Zech L, Johansson C, Lindsten J & Walknowska J, Conte F A & Grumbach M (1969) Practical and theoretical implications of fetal/ Hulten M (1970) Fluorescent staining of heteropycnotic chromosome regions in human intermaternal lymphocyte transfer. Lancet 1, 111922. phase nuclei. Exp Cell Res 61, 472-74. Weber T H, Skoog V T, Mattson A & LindahlGaugas J M (1974) Glycoproteins in pregnancy Kiessling K (1974) Kinetics of lymphocyte stimserum which interact with concanavalin A and may suppress lymphocyte transformation. Transulation in vitro by non specific mitogens. Exp plantation 18, 538-41. Cell Res 85, 351-61. Grosset L, Barrelet V & Odartchenko N (1974) Woodrow J C & Finn R (1966) Transplacental haemorrhage. Br J Haematol 12, 297-309. Antenatal fetal sex determination from maternal blood during early pregnancy. A m J Obstet Zilliacus R, de la Chapelle A, Schroder J & Tiilikainen A (1975) Massive invasion of fetal Gynecol 120, 60-63. lymphocytes into the mother’s blood at induced de Grouchy J & Trtbuchet C (1971) Transfusion abortion. Scand J Immunol 4, 601-05. foeto-maternelle de lymphocytes sanguins et
Transplacental Passage of Foetal Blood Cells R. ZILLIACUS, M.B.,A. DE LA CHAPELLE, M.D., J. SCHRODER, PH.D., A. TIILIKAINEN, M.D.,E. KQHNE, M.D. & E. KLEIHAUER, M.D. Folkhalsan Institute of Genetics (Chief, A . de la Chapelle), Helsinki, Department of Serology and Bacteriology (Acting Chief, T . Kosunen), University of Helsinki, Finland and Department of Paediatrics (Chief, E. Kleihauer), University o f Ulm, West Germany
With the brightly fluorescent Y chromatin as a marker, the proportion of foetal lymphocytes in maternal blood was found to be 0.02 - 0.16 % in 7 of 11 primigravidae bearing a boy, but not a single XY mitosis was found among 112,000 leucoagglutininstimulated mitoses from the same women. The proportion of foetal lymphocytes did not change appreciably during pregnancy (first appearance detected at 15 weeks’ gestation), no’r were there any unequivocal foetal erythrocytes in spite of ABO and Rh compatibility. These findings suggest that foetal lymphocytes pass actively into the mother’s blood relatively early during pregnancy. The cells are protected from immunological elimination, and therefore may remain in the maternal blood for long periods. They are a valuable potential source of material for antenatal foetal diagnosis. Key words: foetal lymphocytes - foetal erythrocytes - placental passage maternal blood - Y chromatin - fluorescence
Accepted for publication September 10, 1975 Correspondence to: Dr. Albert de la Chapelle, Folkhalsan Institute of Genetics, P. 0. Box 819, 00101 Helsinki 10, Finland
Lymphocytes of foetal origin occur in the blood of most women during pregnancy and for long periods after delivery (Walknowska et a1 1969, de Grouchy & TrCbuchet 1971, Schroder & de la Chapelle 1972). Why these foetal cells survive has not yet been adequately explained. A correlation exists between the presence of cytotoxic HL-A antibodies and the absence or paucity of foetal lymphocytes in the maternal blood after
delivery (Schroder et a1 1974). Paternally derived foetal HL-A antigens are often masked in cord blood samples, and this might explain, at least in part, the maternal tolerance to histoincompatible foetal cells (Tiilikainen et a1 1974). Foetal lymphocytes in the mother’s blood are a potential source of material for antenatal diagnosis of foetal disease (Renwick 1971). Before attempts are made to purify
334
ZILLIACUS, D E LA CHAPELLE, SCHRODER, TILIKAlNEN et a1
these cells (Hulett et a1 1973), more must be known about their characteristics and the kinetics of their occurrence. We here report the results of such a study. The passage of foetal leucocytes and erythrocytes into the maternal circulation was monitored and related to maternal production of HL-A antibodies. MATERIALS AND METHODS Patients
The series of patients consisted of 30 consecutive primigravidae attending three maternity welfare centres. The patients and their families were informed about the purpose of the study and agreed to co-operate. Leucoagglutinin-stimulated lymphocyte cultures of peripheral blood from the women and the fathers of the expected children were prepared, and the mitoses and interphase cells were inspected in the fluorescence microscope after staining with Acrani1.B The purpose of this step was to exclude couples where the mother had autosomes with brightly fluorescent regions (Caspersson et a1 1970) or the father had an exceptionally small Y chromosome. These criteria had been evaluated and applied by us previously (Schroder et a1 1974). Of the 30 mothers initially selected, 9 had to be excluded because of strong autosomal fluorescence that might have interfered with the recognition of interphase Y chromatin, 1 because her husband's Y chromosome was very small, 1 because she only later told us that she had had a previous abortion (Zilliacus et a1 1975), and 1 who aborted during the study. This report deals with the remaining 18 women. They were aged 22-31 years. Methods Blood samples were taken from each mother on four occasions, i.e. at about 4, 6, and 8 months of gestation and within 2 days after delivery. Every sample (20 ml) was divided into three alimquots. From the first alimquot 5000 uncultured interphase lymphocytes and 5000 granulocytes were screened for Y chromatin as previously described (Schroder Lk de la Chapelle 1972, Schroder et a1 1974). Even when investigating the sample taken after delivery, the investigator was always unaware of the child's
sex. In addition, lymphocyte cultures set up from the same aliquot of blood were stimulated with leucoagglutinin (Weber et a1 1974), and slides were prepared. Because to find and examine thousands of mitoses is very time-consuming, and recognition of a brightly fluorescent Y chromosome in mitoses represents no problem (Caspersson et a1 1970), these slides were stored until the patient had delivered. Only slides from women with interphase cells containing Y chromatin or women who had given birth to a boy were stained with Acranil and examined by fluorescence microscopy for the presence of Y-chromosome-containing mitoses. The presence of foetal erythrocytes was evaluated from the second aliquot by the method described by Kleihauer (1974). From the third aliquot, serum was prepared and stored at -20" C for later determination of HL-A antibodies (Schroder et a1 1974). From a sample of cord blood the HL-A types were determined and in some cases ABO and Rh types as well (Tiilikainen et a1 1974). The child's karyotype was also determined and in all cases conformed with the phenotypic sex. RESULTS
Scoring of 330,000 uncultured interphase lymphocytes and the same number of granulocytes showed that cells containing a brightly fluorescent spot interpreted as Y chromatin occurred in 8 of the 18 women studied (Table I). Of these 8 women, 7 gave birth to a boy and one (case 12) to a girl. This means that 4 of the 11 women carrying a boy did not have cells containing Y chromatin in their blood. Of the 7 women bearing a girl, 6 did not have Y chromatin-positive cells. The seventh woman (case 12) will be discussed below. In 4 women, all of whom had Y chromatin-positive lymphocytes, a few granulocytes containing Y chromatin were seen (Table I). The proportion of lymphocytes containing Y chromatin varied between 0.02 and 0.16 % in the women bearing a boy. In one mother foetal cells were detected as early as at 15
0 0 0 0 0
0
0
0 0 0 0 0 0
0 0
-
-
-
-
-
-
-
0 0 0 0 0 0
0 0 0 0 0 0
2
0 0 0 0
0 0 0 0
6
0 0 0
3 3 2 2 2
-
-
-
-
-
0
0 0 0
0 0 0 0 0
1 0
0 0 0 0
0 0 0 0
10 0 0
0 0
0
2 0 0
2 2
i
8 3 2
-
Ly = No. of lymphocytes with Y chromatin per 5000 lymphocytes studied. G r = No. of granulocytes with Y chromatin per 5000 granulocytes studied. E = Foetal erythrocytes present or absent. * = Sample taken 4 weeks after delivery.
12 13 14 15 16 17 18
4 5 6 7 8 9 10 11
0 0 0
0
2 3
0 0 0 0 0 0 0
0
1
1
0 0 0 1 0 3 0 0 0 0
E
Ly
-
-
-
-
-
-
-
+
. = - = = (+) = C = I =
0
0
0 0
11 0 0
0 0" 0
4 5 1 0 1 3
E
not determined. negative. positive. weakly positive. compatible. incompatible.
0 0 0 0 0
1 0
0 0
0
0 0 1 0
1 2
Gr
1 1 1
Gr
1 I
LY
E
I I
E
1
Gr
Gr
Ly1
C C C
c
i
C C I I C
F F F F F F F
M M M M M M M M M M M
ABO and RhcomSex patibility of of child to child HL-A- mother antibodies
Sample IV within 48 h after delivery
Sample I11 3&38
Sample I1 24-3 1
Sample I 15-22
Ly
Patient no.
Weeks of gestation
TABLE I Findings in the series of 18 women
5;
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om*
v
8
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wl
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336
ZILLIACUS, DE LA CHAPELLE, SCHRODER, TILIKAINEN et a1 TABLE I1 Number of mitoses studied
itive interphase lymphocytes not a single mitosis with a Y chromosome was detected (Table 11). Two women (cases 7 and 8) could not be studied. no. It is desirable to differentiate between 1 5000 5000 HbF-containing red blood cells of foetal and 2 4100 3400 5000 1500 3 5000 5000 maternal origin. The foetal cell type has a 4 5007 4146 2550 higher concentration of HbF. The elution of 5 1442 5006 99 6 2567 5001 2287 2000 HbF from red cells is dependent not only on 9 1247 2334 5003 pH but also on the intracellular HbF/HbA 10 2661 5042 3877 4700 11 5000 2400 800 ratio. We incubated the slides at pH 3.17, 12 4800 5000 5000 which leads to elution of 15-20 % HbF per cell with the HbA (Kleihauer & Kohne 1975, weeks of gestation. Inspection of Figure 1 personal information). In healthy pregnant indicates that during the period of time cov- women, erythrocytes that stain densely after ered by our investigation the proportion of this procedure can be identified with high cells containing Y chromatin was stable. In probability as cells of foetal origin. Using particular, there was no indication of a rise this method, we did not find a single erythrotowards term, nor was the proportion higher cyte that was unequivocally of foetal origin in early pregnancy. in any of the maternal blood samples (Table Among 112,000 mitoses from the blood I). Thus in these women foetal bleeding into of 10 women containing Y chromatin-pos- the maternal circulation could be excluded.
Figure 1. Kinetics of lymphocytes containing Y chromatin in the blood of 7 women. Dotted vertical line indicates first occasion on which any woman was tested (15 weeks). Short vertical line on each curve indicates time of delivery. Numbers 1-7 identify patients (Table I).
PASSAGE OF FOETAL CELLS
In the maternal serum samples taken within 2 days after delivery, antibodies against paternally derived foetal HL-A antigens were present in 3 cases only (1 strong, 2 weak; Table I). Since antibodies were not found in the serum samples taken from the women in question during pregnancy, antibody production must have occurred late in pregnancy or become discernible only after delivery. DISCUSSION
When lymphocytes containing Y chromatin are observed in the blood of a woman whose foetus is female (as in our case 12), the finding demands close consideration. We believe that the high incidence of cells scored as Y chromatin-positive clearly originates from misinterpretation of autosomal fluorescence in the mother’s own chromosomes (Caspersson et a1 1970), which we re-examined after she had delivered a girl. If this retrospective interpretation is correct she should have been excluded from the series at an early stage but erroneously was not. As there can be no hope of avoiding such errors, determination of foetal sex should not be performed by this method, as we have pointed out repeatedly (Schroder & de la Chapelle 1972, Schroder et a1 1974). Our data show that foetal lymphocytes may be present in the maternal circulation as early as the 15th week of gestation and that their number remains essentially constant throughout pregnancy. Thus there is no evidence that the proportion of foetal cells is high during early pregnancy and then declines, or that the number of foetal lymphocytes increases towards term, as is the case with foetal erythrocytes (Woodrow & Finn 1966, Bartsch 1972). Rather, our findings suggest that foetal lymphocytes pass Scnnd J Haematol(l975) 15
337
into the maternal blood relatively early in pregnancy and remain there in essentially unaltered numbers. This could be due either to a steady turnover or to absence of elimination (Tiilikainen et a1 1974)’ as lymphocytes have a very long life-span (Buckton et a1 1967). The proportions of foetal lymphocytes in the maternal blood found in this study (00.16 %) correspond very well with our previous findings, but not with those of Grosset et a1 (1974). These authors, who studied interphase nuclei of transformed lymphocytes after 5-6 days’ culture with phytohaemagglutinin, postulated preferential transformation of foetal cells in relation to maternal cells to explain the large numbers of foetal cells (0.2-1.5 %) in the maternal blood. We suggest, on the contrary, that the reason for the absence of XY mitoses in our study may be that foetal lymphocytes are prevented from being transformed and entering mitosis. This is not unlikely, since some of their HL-A antigens are masked (Tiilikainen et a1 1974) and in nylon wool columns they are known to behave as B lymphocytes (Schroder 1975). Moreover, factors in the maternal circulation may inhibit the transformation of foetal cells (Thorsby 1974, Gaugas 1974). As already pointed out (Jacobs & Smith 1969) misinterpretation may have marred some of the previous studies performed by conventional staining. Since many foetuses were ABO and Rhcompatible with their mothers, and since none of the mothers in the present series had any foetal erythrocytes in their blood, leakage or bleeding from the foetuses is excluded. If bleeding were responsible for the presence of foetal lymphocytes in the mother’s blood, there should be about 5000 times as many foetal erythrocytes as lymphocytes. So many 22
338
ZILLIACUS, D E LA CHAPELLE, SCHRODER, TILIKAINEN et a1
foetal erythrocytes would not escape detection. We conclude that foetal leucocytes must pass the placental barriers actively.
detection du sexe du foetus. Ann Genet 14, 13337. Hulett H R, Bonner W A, Sweet R G & Herzenberg L A (1973) Development and application of a rapid cell sorter. Clin Chem 19, 813-16. Jacobs P A & Smith P G (1969) Practical and theoretical implications of fetavmaternal lymACKNOWLEDGEMENTS phocyte transfer. Lancet 2, 745. We thank all the patients, nurses, doctors and ad- Kleihauer E (1974) Determination of fetal hemoglobin: Elution technique. In R M Schmidt, ministrators who helped us to organize and carry T H S Huisman & H Lehmann (eds) The detecout this study. In particular we wish t o thank Protion of hemoglobinopathies, p 20. C R C Press, fessor Sakari Timonen, M.D. for his kind help. Dr. Ohio. Johan Fellman performed preliminary statistical J H (1971) The mapping of human chroRenwick calculations. mosomes. Ann Rev Genet 5 , 81-120. Supported by grants from the Finnish National Research Council for Medical and Natural Sci- Schroder J (1975) Are fetal cells in maternal blood mainly B lymphocytes? Scand J Immunol4,279ences, the Sigrid Jusklius Foundation, the Oskar 85. Oflund Foundation and the Finska LakaresallskaSchroder J & de la Chapelle A (1972) Fetal lympet. phocytes in the maternal blood. Blood 39, 15362. J, Tiilikainen A & de la Chapelle A Schroder REFERENCES (1974) Fetal leukocytes in the maternal circulation after delivery. I. Cytological aspects. TransBartsch F K (1972) Fetale Erythrozyten in miitterplantation 17, 346-54. lichen Blut und Immunprophylaxe der RhImmunisierung. Klinische und experimentelle Thorsby E (1974) The human major histocompatibility system. Transplant Rev 18, 51-129. Studie. Acta Obstet Gynecol Scand, Suppl 20. Buckton K E, Court Brown W M & Smith P G Tiilikainen A, Schroder J & de la Chapelle A (1967) Lymphocyte survival in men treated with (1974) Fetal leukocytes in the maternal circulaX-rays for ankylosing spondylitis. Nature 214, tion after delivery. 11. Masking of HL-A antigens. Transplantation 17, 355-60. 470-73. Caspersson T,Zech L, Johansson C, Lindsten J & Walknowska J, Conte F A & Grumbach M (1969) Practical and theoretical implications of fetal/ Hulten M (1970) Fluorescent staining of heteropycnotic chromosome regions in human intermaternal lymphocyte transfer. Lancet 1, 111922. phase nuclei. Exp Cell Res 61, 472-74. Weber T H, Skoog V T, Mattson A & LindahlGaugas J M (1974) Glycoproteins in pregnancy Kiessling K (1974) Kinetics of lymphocyte stimserum which interact with concanavalin A and may suppress lymphocyte transformation. Transulation in vitro by non specific mitogens. Exp plantation 18, 538-41. Cell Res 85, 351-61. Grosset L, Barrelet V & Odartchenko N (1974) Woodrow J C & Finn R (1966) Transplacental haemorrhage. Br J Haematol 12, 297-309. Antenatal fetal sex determination from maternal blood during early pregnancy. A m J Obstet Zilliacus R, de la Chapelle A, Schroder J & Tiilikainen A (1975) Massive invasion of fetal Gynecol 120, 60-63. lymphocytes into the mother’s blood at induced de Grouchy J & Trtbuchet C (1971) Transfusion abortion. Scand J Immunol 4, 601-05. foeto-maternelle de lymphocytes sanguins et
