1 Department of Transfusion Medicine, 2 Forensic Medicine, 3 Human Genetics, 4 Immunology and 5 Paediatrics, University of Kiel Medical School, Michaelisstrasse 5, D-24105 Kiel, Germany
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Abstract |
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Key words: blood chimerism/dizygotic triplets/in-vitro fertiliz-ation
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Introduction |
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The growth of the genetically different cell line of the other fetus is associated with special immunological phenomena in blood chimeras caused by the induction of specific immuno-tolerance during fetal development (Tippett, 1983). This tolerance results in the failure to express the regular antibodies against A or B blood group antigens of the `donor' twin cells and the mutual acceptance of organ grafts (Billingham et al., 1953
).
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Case report |
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Samples of blood, hair roots and buccal swabs from the triplets and their parents were taken for further serological, flow cytometric and molecular genetic analysis. Red cells were typed with conventional, commercially available test sera for the following blood group antigens: ABO1, Rhesus1, Kell1, P12, MNSs2, Lewis2, Duffy2, Kidd2, and Lutheran2 (1Micro Typing System, Fa DiaMed, Cressier, CH; 2 tube test). The gel test revealed two erythrocyte populations of blood groups A and AB in similar proportions in each of the three infants. Likewise, the separated A-positive/B-negative erythrocyte population of the children showed a negative response for the Rhesus E-antigen using a gel test (Lapierre et al., 1990). No haemagglutinins were detected in the triplets' sera. The phenotype of the mother's erythrocytes was A1, CCD.ee, the father's B, ccD.Ee (Table I
).
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Amplification of specific DNA sequences by means of PCR demonstrated the presence of Y chromosomes (Figure 2) in the peripheral leukocytes of the girl (Suttorp et al., 1993
). The conventional analysis of the chromosomes revealed the karyotype 46,XY in four of seven evaluated metaphases from the girl's cells. The karyotype 46,XX was found in one of six metaphases in cells from one of the boys, while the other showed a regular male karyotype in all six metaphases analysed. Neither the parents' nor the children's cells showed any structural chromosome anomalies. The distribution of the female and male cells was further analysed by chromosomal interphase in-situ hybridization. Using X and Y specific probes detecting the DYZ3 and the DXZ1 locus, XX (green/green) and XY (green/red) fluorescence of the chromosomes was seen in all leukocyte preparations (200 cells were analysed) from each of the children (Siebert et al., 1998
). Similar to the red cell population, the leukocytes were also equally distributed. The girl showed 84% XY cells, 16% were of her own XX. The boys had 92/89% XY cells; 8/11% were XX (Figure 3
).
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Discussion |
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DNA analysis of blood cells and non-haematological tissue (e.g. buccal cells, hairs and nails) was necessary to detect blood chimerism and distinguish it from mosaicism (Dauber et al., 1999). In cases of unlike sex, PCR analysis and fluorescent in-situ hybridization (FISH) techniques using Y and X specific probes can reveal chimerism and proportions of nucleated male and female cells (Suttorp et al., 1993
). The triplets showed additional erythrocyte antigens of paternal origin. HLA typing was not helpful in our cases, as all three children were HLA identical.
Blood chimerism may be a pitfall in forensic investigations like paternity testing and crime cases. On the one hand, the application of only two or three DNA probes can fail to detect chimerism (Hansen and Sondervang, 1993); on the other hand, if additional bands are found, they can be falsely interpreted as an artificial cell mixture. These details have been discussed elsewhere from the viewpoints of forensic medicine (Milde et al., 1999
).
DNA fingerprinting using several single locus probes demonstrated the simultaneous presence of both paternal and maternal alleles, a finding that can only be interpreted as a DNA mixture derived from two zygotes (Hansen and Sondervang, 1993). Typing of serum groups and the DNA analysis of hairs and buccal swabs demonstrated that the two boys, who had previously been thought to be dizygotic because three quadricellular embryos had been transferred, were actually monozygotic. Evidently in cases of multiple birth following IVF it is worthwhile to clarify zygocity, even if the number of transferred embryos is identical to the number of fetuses that had developed.
We assume that this blood cell chimerism was the result of an intensive reciprocal transfer of haematopoetic stem cells between the triplets. Some transfused cells settled down in the bone marrow of the host and produced blood cells of the donor line, a phenomena that is well known from animal work (Owen, 1945) and first described and supposed in the human (Dunsford et al., 1953
). Other types of spontaneous blood chimerism, such as cell mosaicism, whole body chimerism or maternalfetal transfusion could be excluded. Further subtle forms of chimerism were described (Van Dijk et al., 1996
). Their cases showed a low percentage of donor red blood cells, some of these forms were transient and were possibly caused by maternalfetal transfusions. We assume that our results indicate that the triplets are immunologically tolerant to their sibling(s) of the opposite sex and that the chimerism will probably remain stable for their whole life.
An explanation for dystrophic embryonal growth following IVF could be the development of placental anastomoses between fetal blood vessels. Apart from chimerism vascular communications can produce a fetalfetal transfusion syndrome also in dichorionic placentae (Robertson et al., 1983; French et al., 1998). A dichorionic diamniotic placentation in dizygotic twins with vascular anastomoses followed by amelia, cutis aplasia and blood chimerism has been demonstrated (Phelan et al., 1998
). In our report the anaemia and dystrophy observed in the female triplet could have been caused by blood losses because of an unidirectional strong placental blood flow to the two monozygotic siblings (Bajoria, 1998
).
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Acknowledgments |
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Notes |
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References |
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Submitted on October 4, 1999; accepted on January 27, 2000.